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WO2025174794A1 - Methods and materials for treating cancer - Google Patents

Methods and materials for treating cancer

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Publication number
WO2025174794A1
WO2025174794A1 PCT/US2025/015464 US2025015464W WO2025174794A1 WO 2025174794 A1 WO2025174794 A1 WO 2025174794A1 US 2025015464 W US2025015464 W US 2025015464W WO 2025174794 A1 WO2025174794 A1 WO 2025174794A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
endx
compound
mammal
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/015464
Other languages
French (fr)
Inventor
Matthew P. Goetz
Shahrzad JALALI
John R. HAWSE IV
Elizabeth S. Bruinsma
Matthew J. SCHELLENBERG
Swaathi JAYARAMAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
Original Assignee
Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Filing date
Publication date
Application filed by Mayo Foundation for Medical Education and Research, Mayo Clinic in Florida filed Critical Mayo Foundation for Medical Education and Research
Publication of WO2025174794A1 publication Critical patent/WO2025174794A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • one or more endoxifen (ENDX) compounds can be administered to a mammal (e.g., a human) having an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal.
  • a mammal e.g., a human
  • an ER- cancer e.g., an ER- breast cancer such as a TNBC
  • one or more ENDX compounds and one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell can be administered to a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal.
  • an ER- cancer e.g., an ER- breast cancer such as a TNBC
  • Breast cancer is the most common cancer worldwide with over 2.3 million new cases and 685,000 deaths in 2020 (Arnold, Morgan et al.2022).
  • Breast cancer displays substantial heterogeneity in prognosis and therapy responses and is categorized into four primary subtypes based on an analysis of its molecular characteristics and immunohistochemical markers: luminal A breast cancers and luminal B breast cancers (both characterized by ER expression), HER2-positive breast cancers, and TNBCs (characterized by the absence of ER, the absence of progesterone receptor (PR), and without HER2 amplification).
  • TNBC exhibit aggressive clinical behavior and high rates of death.
  • ENDX also referred to as 4-hydroxy-N-desmethyltamoxifen
  • a mammal e.g., a human
  • an ER- cancer e.g., an ER- breast cancer such as a TNBC
  • the methods can include, or consist essentially of, administering an ENDX compound to a mammal having an ER- cancer.
  • the mammal can be a human.
  • the mammal can be a female mammal.
  • the mammal can be a pre-menopausal female human.
  • the ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a Attorney Docket No. 07039-2296WO1 / 2023-600 leukemia, or a lymphoma.
  • the ER- cancer can be a breast cancer.
  • the breast cancer can be a triple negative breast cancer.
  • the ENDX compound can be a Z-ENDX compound.
  • this document features methods for treating a mammal having an ER+ cancer or an ER- cancer.
  • the methods can include, or consist essentially of, administering, to mammal having an ER+ cancer or an ER- cancer, (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ER+ cancer or said ER- cancer.
  • the mammal can be a human.
  • the mammal can be a female mammal.
  • the mammal can be a pre-menopausal female human.
  • the mammal can have ER- cancer, and said ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate Attorney Docket No. 07039-2296WO1 / 2023-600 cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma.
  • the ER- cancer can be a breast cancer.
  • the breast cancer can be a triple negative breast cancer.
  • the mammal can have ER+ cancer, and said ER+ cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, or a lung cancer.
  • the ENDX compound can be a Z-ENDX compound.
  • the Z-ENDX compound can be a Z-ENDX salt.
  • the Z-ENDX salt can be Z-ENDX hydrochloride.
  • the method can include administering from about 20 mg/day to about 360 mg/day of said ENDX compound to said mammal.
  • this document features uses of a composition comprising an ENDX compound to treat a mammal having an ER- cancer.
  • the mammal can be a human.
  • the mammal can be a female mammal.
  • the mammal can be a pre-menopausal female human.
  • the ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma.
  • the ER- cancer can be a breast cancer.
  • the breast cancer can be a triple negative breast cancer.
  • the ENDX compound can be a Z-ENDX compound.
  • the Z- ENDX compound can be a Z-ENDX salt.
  • this document features ENDX compounds for use in the preparation of a medicament to treat an ER- cancer.
  • the ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma.
  • the ER- cancer can be a breast cancer.
  • the breast cancer can be a triple negative breast cancer.
  • the ENDX compound can be a Z-ENDX compound.
  • the Z-ENDX compound can be a Z-ENDX salt.
  • the Z-ENDX salt can be Z-ENDX hydrochloride.
  • this document features ENDX compounds for use in the treatment of an ER- cancer.
  • the ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin Attorney Docket No.
  • the ER- cancer can be a breast cancer.
  • the breast cancer can be a triple negative breast cancer.
  • the ENDX compound can be a Z-ENDX compound.
  • the Z-ENDX compound can be a Z-ENDX salt.
  • the Z-ENDX salt can be Z-ENDX hydrochloride.
  • this document features uses of a composition comprising (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ER+ cancer or said ER- cancer to treat a mammal having an ER+ cancer or an ER- cancer.
  • the mammal can be a human.
  • the mammal can be a female mammal.
  • the mammal can be a pre-menopausal female human.
  • the mammal can have ER- cancer, and said ER- cancer be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma.
  • the ER- cancer can be a breast cancer.
  • the breast cancer can be a triple negative breast cancer.
  • the mammal can have ER+ cancer, and said ER+ cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, or a lung cancer.
  • the ENDX compound can be a Z-ENDX compound.
  • the Z-ENDX compound can be a Z-ENDX salt.
  • the Z-ENDX salt can be Z-ENDX hydrochloride.
  • the composition can include from about 20 mg to about 360 mg of said ENDX compound.
  • the agent can be abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetani
  • the ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma.
  • the ER- cancer can be a breast cancer.
  • the ER- breast cancer can be a triple negative breast cancer.
  • the ER+ cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, or a lung cancer.
  • Figure 1E In the inhibited state, many hydrophobic lipid binding residues are occluded by interaction with the catalytic domain.
  • Figure 1F Molecular details of the interdomain contacts between the C1a domain, kinase domain and C-terminal tail, and the three phosphorylation sites: activation loop (pT500), turn (pT641), and hydrophobic (pS660) with surrounding structures, and the bound AMPPNP nucleotide.
  • Figures 2A – 2C Structure of PKC ⁇ I reveals an ordered active conformation.
  • Figure 2A) Two PKC ⁇ I crystal forms with 1 or 2 PKC ⁇ I molecules per asymmetric unit (ASU) were aligned by their kinase domain.
  • ASU asymmetric unit
  • N-term 1, N- term 2, and N-term 3 Two domain arrangements corresponding to the active and inhibited state are common amongst the PKC ⁇ I molecules.
  • the regulatory domain lipid-binding residues are in a plane for the activated state (upper) while the pseudosubstrate occupies the active site in the inhibited state.
  • Figure 2B Recombinant PKC ⁇ protein with mutations engineered to disrupt the C1b-kinase and C2-kinase domain interactions.
  • Figure 2C Mutant PKC ⁇ were assayed for kinase activity in vitro using the FRET probe CKAR. Error bars represent standard deviation.
  • FIG. 3A Lipid-lever model of PKC activation. A steric clash between the phospholipid bilayer and kinase domain drives PKC ⁇ I/II into its active conformation.
  • Figure 3B In vitro CKAR assay showing the kinase activity of PKC ⁇ I in the indicated concentrations 5 of PDBu or 40 ⁇ g/mL PS/DAG lipid micelles. Error bars represent standard Attorney Docket No. 07039-2296WO1 / 2023-600 deviation.
  • Figure 3C Limited proteolysis with elastase used to probe change in PKC ⁇ I/II conformation in the presence of the indicated ligands, with quantification of cleavage product (*) as a percentage of protein indicated at the bottom of each gel.
  • Figures 4A – 4C Molecular basis for differential lipid affinities between PKC ⁇ I and PKC ⁇ II.
  • Figure 4A Comparison between the structural architecture of PKC ⁇ I and PKC ⁇ II inactive conformations and the relative locations of the lipid binding residues and pseudosubstrate.
  • Figure 4B Position of the 5 C1b domain differs between PKC ⁇ I and PKC ⁇ II with respect to the C1a and kinase domains.
  • FIG 4C Intramolecular details of the residue interactions that lead to differential placement of the C1b domain, and the effect of this placement on the susceptibility of PKC ⁇ I ( ⁇ I), PKC ⁇ II ( ⁇ II), and PKC ⁇ I F648A (FA) to proteolytic cleavage by elastase (*).
  • Endoxifen is an allosteric inhibitor of PKC ⁇ I.
  • FIG. 7A Sequence alignment of conventional PKCs with domains coloured as indicated. Sequences shown include, from top to bottom, PKC ⁇ (SEQ ID NO:1), PKC ⁇ (SEQ ID NO:2), PKC ⁇ I (SEQ ID NO:3), and PKC ⁇ II (SEQ ID NO:4).
  • the Attorney Docket No. 07039-2296WO1 / 2023-600 altered C-terminal tails derived from an alternative splicing event are coloured pink (PKC ⁇ I) or salmon (PKC ⁇ II).
  • Figure 7B Domain arrangement and second messenger sensitivity of the three PKC families.
  • Figure 9B Domain–swapped arrangement of PKCBII with an adjacent identical monomer.
  • Figure 9C Molecular details of the N-terminal pseudosubstrate side chains and their specific interactions with the C1b and kinase domains labeled by residues and color-coded (hydrogen bonding, salt bridge, hydrophobic/Van der Waals).
  • Figure 10A Ion exchange and size exclusion chromatograms of PKC ⁇ I purification and SDS-PAGE analysis of indicated fractions.
  • Figure 10B Mass spectrometry of purified PKC ⁇ I protein detects phosphorylation at T500 and T642.
  • the polypeptide sequence in the top spectrum is SEQ ID NO:5 and the polypeptide sequence in the bottom spectrum is SEQ ID NO:6.
  • Figure 10C Individual copies of PKC ⁇ I within each crystal form.
  • Figure 10D Experimental 2F0 – Fc map of phosphorylation sites on PKC ⁇ I in each crystal form.
  • Figure 10E Experimental 2F0 – Fc map contoured at 1 ⁇ for the PKC ⁇ I C1a – C1b linker region and C1b – C2 linker region.
  • Figure 11A Pseudosubstrate of the adjacent monomer is present in the active site of crystal form 1.
  • Figure 12B 70 A distance between N-terminal tail and active site prevents re- engagement of the pseudosubstrate.
  • Figure 12C Modeled mutations that disrupt C2-kinase and C1b-kinase domain interactions in the active conformation of PKC ⁇ I.
  • Figure 12D Aligned crystal structures of the C1a domain with PDBu (PDB 7KNJ) and PKC ⁇ I or PKC ⁇ II in their auto-inhibited conformation shows PDBu binding does not disrupt the inactive conformation.
  • Figure 12E Insertion of C1a domain hydrophobic residues in the phospholipid bilayer encourages kinase domain to disengage from the pseudosubstrate to prevent unfavourable charge interactions at the lipid bilayer.
  • Figure 13D Confocal live cell microscopy images showing dose-dependent cellular localization of YFP-tagged PKC ⁇ I in the presence of indicated drug treatment.
  • Figure 14A Cells grown in CSS medium were treated with vehicle control or the indicated ENDX concentrations for 48 hours. Cell Attorney Docket No. 07039-2296WO1 / 2023-600 viability was assessed by the crystal violet assay.
  • FIG. 14B Cells were co-treated with vehicle control or the indicated ENDX concentrations, IncuCyte Annexin V green and NucLight red reagents in CSS medium for 48 hours.
  • the apoptosis (%) graphs are presented as the green object count (which correspond to cells that are stained with the IncuCyte green fluorescence Annexin V reagent) divided by the red object count (which correspond to the total number of cells in the culture that are stained with the IncuCyte red fluorescence Nuclight Rapid Red Cell Labeling reagent that labels the nucleus of all cells without perturbing cell function or biology) and displayed as percentage using the IncuCyte S3 analysis software.
  • FIG. 15 A schematic depicting the strategy used for quantitative proteomic and phosphoproteomic profiling of ENDX-treated MCF7AC1 cells. All experiments were performed in triplicate. Cells were treated with vehicle control or Z-endoxifen (ENDX) as specified dosages for 24 hours. After the ENDX treatment, cells were harvested and lysed in 8 M urea buffer, followed by trypsin digestion, desalting and tandem mass tag (TMT) labeling.
  • ENDX Z-endoxifen
  • Figures 16B – 16C Volcano plots showing the total number of phosphosites, and the percentage that are upregulated (right side) and downregulated (left side) (Fold change (FC)
  • Figure 16E Venn diagram indicating the overlap of upregulated and downregulated phosphosites in ENDX-treated cells relative to vehicle-treated cells.
  • Figure 16F Heatmap indicating relative abundance of the phosphosites analyzed in the ENDX- treated cells relative to vehicle-treated cells.
  • Motifs shown include, from top to bottom, an AKT substrates motif (SEQ ID NO:7), a MAPK/CDK substrates motif (SEQ ID NO:8), and a CK2 substrates motif (SEQ ID NO:9).
  • Figure 19A) MCF7AC1 cells in CSS medium were treated for 24 hours with vehicle control or 0.01, 0.1 and 5 ⁇ M ENDX.
  • FIG 19C Serum starved MCF7AC1 cells were pretreated with vehicle control and 0.01, 0.1 and 5 ⁇ M ENDX for two hours followed by the addition of 100 nM insulin for one hour as indicated. IB assay of pAKT substrates and ⁇ -actin are shown.
  • Figures 19D and 19E In vitro kinase assay showing % PKC ⁇ 1 kinase activity in the presence of different concentrations of ENDX ( Figure 19D) and TAM ( Figure 19E). The broad-spectrum kinase inhibitor staurosporine serves as a positive control. The IC 50 concentration of ENDX, TAM and staurosporine are indicated. F igures 20A – 20F.
  • IB assays of pPKC ⁇ 1 Ser661 , PKC ⁇ 1, pAKT Ser473 , AKT, p-AKT substrates and ⁇ -actin are shown.
  • Figure 20C Serum starved MCF7AC1 cells were pretreated with vehicle control or 1 ⁇ M ENZA for two hours followed by the addition of 200 nM PMA for 30 minutes as indicated.
  • IB assays of pPKC ⁇ 1 Ser661 , PKC ⁇ 1, pAKT Ser473 , AKT and ⁇ -actin are shown.
  • FIG 20D Serum starved MCF7AC1 cells were pretreated with vehicle control, 0.01, 0.1 and 5 ⁇ M ENDX, 0.1 ⁇ M TAM or 0.1 ⁇ M ICI followed by the addition of 100 nM insulin for one hour as indicated. IB assays of pPKC ⁇ 1 Ser661 , PKC ⁇ 1 and ⁇ -actin are shown.
  • Figure 20E MCF7AC1 cells in CSS medium were transfected with non-targeting (siNT) or PKC ⁇ -targeting (siPKC ⁇ ) siRNAs for 48 hours. IB assays of PKC ⁇ 1, pAKT Ser473 and ⁇ -actin are shown.
  • FIG 22A MCF7AC1 caAKT cells were grown in FBS medium in the absence (-) or presence (+) of cumate for 48 hours. IB assay of C-terminally hemagglutinin tagged AKT (AKT-HA), endogenous AKT and ⁇ -actin.
  • Figure 22B MCF7AC1 caAKT cells grown in FBS medium in the (-) or (+) of cumate for 48 hours. IB assay of pAKT-substrates (SEQ ID NO:10) and ⁇ -actin.
  • FIG 23A Activation of PKC ⁇ 1 Ser661 by the PKC agonist PMA and/or insulin phosphorylates AKT Ser473 resulting in the activation of p-AKT downstream substrates, which mediates cell survival.
  • Figure 23B ENDX binds to PKC ⁇ 1 and facilitates PKC ⁇ 1 protein degradation, resulting in the attenuation of phosphorylation of AKT Ser473 as well as downstream p-AKT substrates, leading to induction of apoptosis.
  • Figures 24A – 24C Effects of ENDX on the global protein expression in MCF7AC1 cells.
  • Figure 24A Stacked barplot showing the number of total proteins and the percentage that are upregulated (red) and downregulated (blue) (Fold change (FC)
  • Figure 24B Venn diagram indicating the overlap of the total proteins in the 0.01, 0.1 and 5 ⁇ M ENDX treated cells relative to vehicle treated cells.
  • Figure 24C Venn diagram indicating the overlap of the list of the phosphosites and the list of the total proteins that are altered by ENDX treatment regardless of the concentration.
  • FIG. 25A MCF7AC1 xenograft tumors were treated with control, letrozole, tamoxifen (TAM) and 25 mg/kg and 75 mg/kg ENDX for four weeks in vivo.
  • IB assay of pAKTSer473, pAKTThr308, AKT, p-AKT substrates are shown with ⁇ - actin as a loading control.
  • FIG 25B Serum starved T47D cells were pretreated with vehicle control or 0.01, 0.1 and 5 ⁇ M ENDX and 0.1 ⁇ M tamoxifen (TAM) and ICI-182780 (ICI) for two hours followed by the addition of 100 nM insulin treatment for one hour. IB assay of pAKTSer473, pAKTThr308, AKT and ⁇ -actin.
  • ENDX binds to PKC ⁇ 1.
  • FIG. 27B MCF7AC1 cells in CSS medium in the absence (-) or presence (+) of doxycycline (Dox) for 72 hours. IB assay of PKC ⁇ 1 and ⁇ -actin are shown. The histogram indicates the percentage (%) of PKC ⁇ 1 protein levels remaining in dox induced cells compared to noninduced cells.
  • Figure 27C IB assay of basal PKC ⁇ 2 protein expression Attorney Docket No. 07039-2296WO1 / 2023-600 and ⁇ -actin in MCF7AC1 and K562 (a positive control for PKC ⁇ 2) cells.
  • Figure 27D IB assay of the relative protein expression of PKC family members and ⁇ -actin in siNT and siPKC ⁇ 1 transfected MCF7AC1 cells.
  • Figure 28D The effects of ENDX pretreatment for two hours followed by treatment in the presence or absence of 20 nM PMA for 20 minutes on the protein expression levels of pPKC ⁇ 1Ser661, PKC ⁇ 1 and ⁇ -actin in MDAMB231 and HEK293F cells.
  • Figures 29A – 29J Effects of the pan-AKT inhibitor MK-2206 or ENDX on phenotypes in ER ⁇ + breast cancer cells.
  • Figure 29A) MCF7AC1 cells were treated with vehicle control or 0.01, 0.1 and 5 ⁇ M MK-2206 in CSS medium for six days. Cell viability is assessed by the crystal violet assay.
  • FIG 29B MCF7AC1 cells were co-treated with vehicle control or 0.01, 0.1 and 5 ⁇ M MK-2206 and IncuCyte Annexin V green and NucLight rapid red reagents in CSS medium for 48 hours.
  • the apoptosis graphs are presented as the green object count divided by the red object count and displayed as percentage using the IncuCyte S3 analysis software. Cells were plated at a density of 2000 cells per well.
  • Figure 29C MCF7AC1 cells were treated with vehicle control or 0.01, 0.1 and 5 ⁇ M MK-2206 in CSS medium for 24 hours. IB assay of pAKTSer473, AKT, PARP, cleaved PARP and ⁇ -actin.
  • FIG 29D MCF7AC1 xenograft protein lysates were treated with the indicated drugs for four weeks. IB assay of cPARP, PARP and ⁇ -actin.
  • Figure 29E Attorney Docket No. 07039-2296WO1 / 2023-600 T47D cells were cultured in FBS versus CSS medium for six days. Cell viability is assessed by the crystal violet assay.
  • Figure 29F Parental T47D and T47D-LTED cells were processed for protein lysates. IB assay of ER ⁇ , pAKTSer473 and ⁇ -actin.
  • Figure 29G T47D-LTED cells were treated with vehicle control or 0.01, 0.1 and 5 ⁇ M MK-2206 in CSS medium for six days.
  • FIG. 29H T47D-LTED cells were co-treated with vehicle control or 0.01, 0.1 and 5 ⁇ M MK-2206 and IncuCyte Annexin V green and NucLight rapid red reagents in CSS medium for 48 hours. Percentage of cells undergoing apoptosis was calculated as mentioned in Figure 29B.
  • Figure 29I T47D-LTED cells were treated with vehicle control or 0.01, 0.1 and 5 ⁇ M MK-2206 in CSS medium for 24 hours. IB assay of pAKTSer473, AKT, PARP, cleaved PARP and ⁇ -actin.
  • Figure 29J T47D-LTED cells were treated with vehicle control or 0.01, 0.1 and 5 ⁇ M ENDX in CSS media for six days. Cell viability is assessed by the crystal violet assay.
  • Figures 29A, 29B, 29E, 29G, 29H, and 29J data represent mean of six wells per treatment performed as biological duplicates ⁇ s.d. *, p ⁇ 0.05, ****, p ⁇ 0.0001 by one-way ANOVA for Figures 29A, 29B, 29G, 29H, and 29I and unpaired test for Figure 29E.
  • Figure 30 Unlike ENDX, TAM and ICI do not induce apoptosis.
  • MCF7AC1 cells were co-treated with vehicle control or 5 ⁇ M ENDX, 0.1 ⁇ M TAM and 0.1 ⁇ M ICI and IncuCyte Annexin V green and NucLight rapid red reagents in CSS medium for 48 hours. The percentage (%) of cells undergoing apoptosis is calculated as described in Example 2. Cells were plated at a density of 2000 cells per well. Data represents mean of six wells per treatment performed as biological duplicates ⁇ s.d. *, p ⁇ 0.05 by one-way ANOVA. Figures 31A – 31C. ENDX neither induces apoptosis nor inhibit growth of ER- breast cancer cells.
  • FIG. 31A MDAMB231 cells grown in CSS medium were were co-treated with vehicle control or the indicated ENDX concentrations, IncuCyte Annexin V green and NucLight red reagents in CSS medium for 48 hours.
  • the apoptosis graphs are presented as the green object count divided by the red object count and displayed as percentage using the IncuCyte S3 analysis software.
  • Cells were plated at a density of 2000 cells per well. Data represents the mean of six wells per treatment performed as biological duplicates ⁇ s.d. ****, p ⁇ 0.0001 by one-way ANOVA.
  • Figure 31B Effects of ENDX on cell proliferation at the indicated concentrations on day seven of treatment. Cell viability was assessed by the crystal Attorney Docket No.
  • IFN ⁇ expression can be used as a favorable prognostic marker in breast cancer.
  • Kaplan-Meier (KM) survival analysis shows the correlation between the gene expression level of IFN ⁇ and overall survival (OS) in different subtypes of breast cancer tumors. Logrank P ⁇ 0.05 denotes significance.
  • Endoxifen increased the expression of Granzyme B and IL-2, but not perforin, in CD8 + and CD4 + T cells. PBMCs were treated with endoxifen (2 ⁇ M) or enzastaurin (2 ⁇ M) for 5 days.
  • FIGS 34A – 34C CyTOF analysis showing that endoxifen altered the expression of immune cells markers.
  • PBMCs were isolated from normal donors and treated with/without endoxifen (2 ⁇ M) for 5 days. Cells were then treated with brefeldin for 4 hours, followed by staining with PBMC enhanced panel of immune markers and CyTOF analysis.
  • Figure 34A) tSNE maps show the clusters of the cells that were altered in endoxifen treated cells.
  • Figure 34B Bar graphs represent the number of the cells in each cluster.
  • FIG 34C Heat map shows the expression of each of 36 immune markers within each cluster or population (Pop).
  • Figures 35A – 35B Pre-treatment of PBMCs with endoxifen enhanced their ability to suppress the proliferation of MCF7 tumor cells.
  • PBMCs were treated with endoxifen (2 ⁇ M) or enzastaurin (2 ⁇ M) for 5 days, followed by washing the cells and removing the drug and co-culturing with MCF7AC1 tumor cells in vitro. Cell proliferation was monitored by IncuCyte for a duration of 5 days ( Figures 35A and 35B, left panels).
  • Figures 35A and 35B left panels.
  • ENDX compounds can be administered to a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal.
  • a mammal e.g., a human
  • an ER- cancer e.g., an ER- breast cancer such as a TNBC
  • cancers that can be ER+ and that can be treated as described herein include, without limitation, breast cancers, ovarian cancers, endometrial cancers, and lung cancers.
  • cancers that can be ER- and that can be treated as described herein include, without limitation, breast cancers (e.g., TNBCs), ovarian cancers, endometrial cancers, brain and/or central nervous system cancers (e.g., gliomas and glioblastomas), bone cancers (e.g., osteosarcomas and Ewing sarcomas, biliary tract cancers, thyroid cancers, lung cancers (e.g., mesotheliomas and non-small cell lung cancers), colorectal cancers, head and neck cancers, stomach cancers, pancreatic cancers, kidney cancers (e.g., clear cell renal carcinomas and rhabdoid cancers), liver cancers (e.g., hepatocellular carcinomas), prostate cancers, testicular
  • the methods described herein can include identifying a mammal (e.g., a human) as having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). Any appropriate method can be used to identify a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). For example, imaging techniques, biopsy techniques, and molecular techniques (e.g., molecular techniques to detect RNA expression or protein expression such as immunohistochemistry) can be used to Attorney Docket No.
  • a mammal having an ER+ cancer and/or an ER- cancer can be administered or instructed to self-administer one or more (e.g., one, two, three, four, or more) ENDX compounds described herein.
  • An ENDX compound can be any type of ENDX compound.
  • an ENDX compound can be a trans isomer of ENDX (E-ENDX) or a cis isomer of ENDX (Z-ENDX).
  • An effective amount (e.g., a therapeutically effective amount) of a composition containing one or ENDX compounds described herein can be any amount that can treat a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) as described herein without producing significant toxicity to the mammal.
  • an effective amount of one or more ENDX compounds can be about 40 milligrams (mg) or more.
  • an effective amount of one or more ENDX compounds can be a plasma concentration of from about 300 nM to about 3000 nM (e.g., from about 300 nM to about 2500 nM, from about 300 nM to about 2000 nM, from about 300 nM to about 1500 nM, from about 300 nM to about 1000 nM, from about 300 nM to about 750 nM, from about 300 nM to about 500 nM, from about 500 nM to about 3000 nM, from about 750 nM to about 3000 nM, from about 1000 nM Attorney Docket No.
  • 07039-2296WO1 / 2023-600 to about 3000 nM from about 1500 nM to about 3000 nM, from about 2000 nM to about 3000 nM, from about 2500 nM to about 3000 nM, from about 500 nM to about 2500 nM, from about 750 nM to about 2000 nM, from about 1000 nM to about 1500 nM, from about 500 nM to about 1500 nM, from about 1000 nM to about 2000 nM, or from about 1500 nM to about 2500 nM).
  • an effective amount of one or more ENDX compounds can be from about 20 mg/day to about 360 mg/day (e.g., from about 20 mg/day to about 340 mg/day, from about 20 mg/day to about 300 mg/day, from about 20 mg/day to about 250 mg/day, from about 20 mg/day to about 200 mg/day, from about 20 mg/day to about 150 mg/day, from about 20 mg/day to about 100 mg/day, from about 20 mg/day to about 50 mg/day, from about 50 mg/day to about 360 mg/day, from about 100 mg/day to about 360 mg/day, from about 150 mg/day to about 360 mg/day, from about 200 mg/day to about 360 mg/day, from about 250 mg/day to about 360 mg/day, from about 300 mg/day to about 360 mg/day, from about 50 mg/day to about 350 mg/day, from about 100 mg/day to about 300 mg/day, from about 150 mg/day to about 250 mg/day, from about 50 mg/day to about 150 mg/
  • an effective amount of one or more ENDX compounds can be as described elsewhere (see, e.g., Goetz et al., J. Clin. Oncol., 35:3391-3400 (2017)).
  • one or more ENDX compounds described herein can be administered to a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) together with one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell.
  • An agent that can alter the antigens presented on the surface of a cancer cell can be any appropriate type of molecule (e.g., small molecules and polypeptides such as antibodies).
  • an agent that can alter the antigens presented on the surface of a cancer cell can be a CDK4/6 inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a cell cycle checkpoint inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be an ALK inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a RET inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be an interferon. In some Attorney Docket No.
  • an agent that can alter the antigens presented on the surface of a cancer cell can be an anthracyclin.
  • an agent that can alter the antigens presented on the surface of a cancer cell can be a platinum-based agent.
  • an agent that can alter the antigens presented on the surface of a cancer cell can be a HDAC inhibitor.
  • an agent that can alter the antigens presented on the surface of a cancer cell can be a tyrosine kinase inhibitor.
  • an agent that can alter the antigens presented on the surface of a cancer cell can be a proteasome inhibitor.
  • agents that can alter the antigens presented on the surface of a cancer cell include, without limitation, abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti- CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cab
  • ENDX compounds described herein can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal having an ER+ and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC).
  • a therapeutically effective amount of one or more ENDX compounds described herein can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • a pharmaceutical Attorney Docket No. 07039-2296WO1 / 2023-600 composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.
  • a composition e.g., a pharmaceutically acceptable composition
  • ENDX compounds described herein and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein
  • a composition containing one or more ENDX compounds described herein (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be designed for oral, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), or inhaled.
  • a composition containing one or more ENDX compounds described herein (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be administered systemically by an oral administration to or inhalation by a mammal (e.g., a human).
  • one or more ENDX compounds can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC)) to reduce the size of the cancer present within a mammal.
  • a mammal e.g., a human
  • an ER+ cancer and/or an ER- cancer e.g., an ER- breast cancer such as a TNBC
  • the materials and methods described herein can be used to reduce the number of cancer cells present within a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • the materials and methods described herein can be used to reduce the size (e.g., volume) of one or more tumors present within a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • one or more ENDX compounds can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC)) to improve survival of the mammal.
  • a mammal e.g., a human
  • an ER+ cancer and/or an ER- cancer e.g., an ER- breast cancer such as a TNBC
  • disease-free survival e.g., relapse-free survival
  • progression-free survival can be improved using the materials and methods described herein.
  • the materials and methods described herein can be used to improve the survival of a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • one or more ENDX compounds described herein (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be used as the sole active agent(s) to treat a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC).
  • a composition including one or more ENDX compounds (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell) can include the one or more ENDX compounds (and, optionally, the one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell) as the sole active agent(s) to treat a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC).
  • a mammal e.g., a human having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC).
  • one or more ENDX compounds described herein can be administered to a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) together with one or more (e.g., one, two, three, four, or more) additional agents/therapies used to treat an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC).
  • an anti- cancer agent can be a targeted therapy.
  • one or more ENDX compounds (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell) are used with one or more additional agents treat a cancer
  • the one or more additional agents can be administered at the same time (e.g., in a single composition) or independently.
  • one or more ENDX compounds (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell) can be administered first, and the one or more additional agents administered second, or vice versa.
  • therapies that can be used to treat cancer include, without limitation, surgery, radiation therapy, carbon ion therapy, and proton therapy.
  • the one or more additional therapies can be performed at the same time or independently of the administration of one or more ENDX compounds (and, optionally, the one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell).
  • the one or more ENDX compounds (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell) can be administered before, during, and/or after the one or more additional therapies are performed.
  • an ER+ cancer and/or an ER- cancer within a mammal can be monitored to evaluate the effectiveness of the cancer treatment.
  • Any appropriate method can be used to determine whether or not a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) is treated.
  • imaging techniques or laboratory assays can be used to assess the number of cancer cells and/or the size of a tumor present within a mammal.
  • imaging techniques or laboratory assays can be used to assess the location of cancer cells and/or a tumor present within a mammal.
  • PKCs are ubiquitously expressed throughout the body, yet only a variable subset of the PKC enzymes is present in each tissue type.
  • the four canonical PKC family members ( ⁇ , ⁇ I, ⁇ II, and ⁇ ) are activated upon interacting with the plasma membrane in response to diacylglycerol (DAG) and Ca 2+ signals.
  • DAG diacylglycerol
  • PKC ⁇ , ⁇ I, ⁇ II, and ⁇ are >65% identical in sequence ( Figure 7A), they perform distinct cellular functions and display varied sensitivity to lipid signals (Kedei et al., Cancer Res., 64:3243-3255 (2004); and Steinberg, Physiol. Rev., 88:1341-1378 (2008)).
  • PKC ⁇ I and PKC ⁇ II isoforms are expressed from the same gene and differ by only a 50 aa C-terminal segment that is derived from a regulated alternative splicing event (Figure 7A) (Chalfant et al., J. Biol. Chem., 270:13326-13332 (1995)), suggesting that even subtle sequence differences can impart altered activity on each isozyme.
  • Figure 7A regulated alternative splicing event
  • Staurosporine and derivatives which target the highly conserved ATP binding site, have demonstrated limited or no antitumor activity in multiple different cancers despite nanomolar affinity and are prone to off-target toxicity (Mina et al., Invest. New Drugs, 27:565-570 (2009); Clemons et al., Breast Cancer Res. Treat., 124:177-186 (2010); Millward et al., Br. J. Cancer, 95:829-834 (2006); Robertson et al., J. Clin. Oncol., 25:1741-1746 (2007); Attorney Docket No.
  • the final, purified PKC ⁇ II contains the phosphorylation sites (T500- activation loop, T641-turn motif, and S660-hydrophobic motif) (Figure 7D) and migrates as a single band on phos-tag SDS-PAGE ( Figure 1B), indicating that the YFP-tagged purified PKC was fully phosphorylated at all three sites. Crystals of PKC ⁇ II in the presence of a non- hydrolysable ATP analogue AMPPNP were obtained and a 3.3A structure was solved ( Figures 1C and 1D; Table 1).
  • Both chains contained a kinase active site occupied by the PS sequence, but chain A formed an intramolecular complex, and chain B formed a domain-swapped complex with the kinase domain of an adjacent monomer through a nearly identical molecular interface (Figure 9B).
  • Figure 9B the structure revealed the complete molecular architecture of the inhibited, inactive PKC ⁇ II.
  • a complex network of inter-domain interactions comprised the inactive state of PKC ⁇ II ( Figures 1E and 1F).
  • the N-terminal PS residues (aa 19-30) were positioned in the substrate binding site with the A25 sidechain in a position equivalent to a Ser/Thr in a true substrate ( Figure 1F), with specific recognition of the hydrophobic and positively charged residues contained within PKC substrate motifs ( Figure 9C).
  • the C1b domain interacted with the kinase domain and PS, which sequestered the hydrophobic lipid-binding residues and buttressed the PS in the kinase active site ( Figure 1E).
  • the C1a domain interacted with the kinase domain and C-terminal extension through a small hydrophobic patch and salt bridges such that most of its lipid binding residues and the DAG binding pocket remained solvent- accessible (Figure 1F).
  • the kinase domain contained three phosphorylated residues (pT500, pT641, and pS660), which were necessary for catalytic activity (Figure 1F).
  • pT500 served a structural role mediated by salt bridges and a hydrogen bond to surrounding residues as well as to PS residues R27 and N30. The latter suggests pT500 formed part of the substrate binding site and explains both the requirement of T500 phosphorylation for kinase activity and the strong counter-selection against negatively charged residues at the +2 and +5 Attorney Docket No. 07039-2296WO1 / 2023-600 substrate positions.
  • pT641 formed salt bridges to two lysines located in the ATP-binding ⁇ - hairpin and ⁇ -helix active-site motifs, as well as an additional hydrogen bond to S111 of the C1b domain.
  • pS660 was located distal from the active site and played a structural role in stabilizing the C-terminal V5 domain by interacting with Q411 and R336 residues. Phosphorylation of these three residues also regulated the affinity of PKC for lipid membranes despite being located outside of the lipid–binding domains.
  • the pT500–PS and pT641–C1b interactions stabilized the inter-domain interfaces that sequestered the lipid binding surfaces of the C1a and C1b domains in an inaccessible conformation.
  • pT500 and pT641 also mediated molecular interactions that maintain PKC in its inactive state and in turn reduced the lipid binding affinity of the fully phosphorylated protein.
  • Crystal structures of PKC ⁇ I reveal active and inactive states PKC ⁇ I and PKC ⁇ II isoforms differed by only 50 C-terminal amino acids ( Figure 7A) derived from an alternative splicing event that is responsive to signals such as extracellular glucose levels and signaling by AKT2 kinase. Although the 50 residues were derived from different exons, many of the residues, including the two phosphorylation sites (pT642 and pS661) present in this region, were conserved.
  • each kinase domain formed an interface with three N-terminal domain modules (Figure 11D), and two of them were comprised of similar molecular interactions (Figure 2A; N-term 1 & 2), while the third interface was variable amongst the three asymmetric units (N-term 3).
  • the interface with N-term 2 describes a conformation that was similar to PKC ⁇ II ( Figure 1) but with an altered location for the C1b domain, which was attributed to the auto-inhibited conformation for the PKC ⁇ I enzyme.
  • PKCs can be activated by a phospholipid bilayer containing DAG or an agonist such as phorbol 12,13-dibutyrate (PDBu).
  • PDBu phorbol 12,13-dibutyrate
  • Figure 3B PKC ⁇ kinase activity
  • the lipid membrane acted as a lever that can pry PS from the catalytic domain and disrupt the interdomain interactions that preserve the inhibited state via a mechanism referred to herein as a “lipid-lever”.
  • the kinase domain occluded some of the lipid-binding surface centered around F43 ( Figure 1F) in the inactive conformation, providing a mechanistic basis by which a fully folded kinase domain moderated membrane binding by the C1 domains.
  • the lipid-lever mechanism is likely shared across PKC isoforms as the PS was always found immediately N-terminal to a C1 domain across the family of PKCs ( Figure 7B).
  • PKC ⁇ II The C1b domain only contacted pT641 and F633 of the PKC ⁇ II V5 domain, yet PKC ⁇ I contained spatially equivalent residues (pT642 and F634) at the same locations ( Figure 7A).
  • PKC ⁇ II F114 of the C1b domain packed against helix ⁇ B, whereas this helix was shifted Attorney Docket No. 07039-2296WO1 / 2023-600 in PKC ⁇ I which occluded the F114 binding pocket.
  • the ⁇ B shift can be attributed to PKC ⁇ I F648, which corresponds to V647 in PKC ⁇ II.
  • PKC ⁇ I F648A was less sensitive to elastase, consistent with a model whereby a smaller residue yields an enzyme with enhanced stability of the inactive state.
  • the more potent lipid binding of the PKC ⁇ I isoform was attributed to a shift in the position of helix ⁇ B that displaced the C1b from its docked position.
  • Endoxifen as an allosteric regulator of PKC ⁇ P KCs can be inhibited by tamoxifen (TAM) (Gundimeda et al., J. Biol.
  • PKC ⁇ expressing culture was spun down at 5000xg for 10 minutes, followed by resuspension of pelleted cells in 1X PBS containing 0.1X Roche EDTA-free protease inhibitor cocktail (Sigma). Centrifugation step was repeated to obtain cell pellet that was used directly for protein prep or frozen down at - 80oC for storage.
  • Thawed HEK293F cells expressing PKC ⁇ were lysed at 4oC with a lysis buffer solution consisting of 50 mM Tris pH 7.4, 300 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM ⁇ -glycerol phosphate, 1 mM TCEP, 2 mM benzamidine, 2 ⁇ g/mL leupeptin, 0.5 mM sodium orthovanadate, 0.5% CHAPS, and 1:100 protease inhibitor cocktail (Sigma P8849).
  • a lysis buffer solution consisting of 50 mM Tris pH 7.4, 300 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM ⁇ -glycerol phosphate, 1 mM TCEP, 2 mM benzamidine, 2 ⁇ g/mL leupeptin, 0.5 mM sodium orthovanadate, 0.5% CHAPS, and 1:100 prote
  • Lysate was sonicated with a Branson sonicator at 50% power for 10 seconds (x2) and spun down in a Lynx 4000 centrifuge (Thermo Fisher) at 15,000 g, 4oC for 10 minutes.
  • the clarified lysate was bound and recycled 3x over a GFP-enhancer nanobody linked NHS Sepharose (Cytiva 45002965) resin bed equilibrated in lysis buffer. Protein bound resin was washed in 3x resin volume with the same lysis buffer.
  • TEV cleavage of the YFP tag was completed overnight by incubating the resin with 1.5x resin volume TEV cleavage buffer containing 50 mM Tris pH7.4, 50 mM NaF, 5 mM sodium pyrophosphate, Attorney Docket No. 07039-2296WO1 / 2023-600 10 mM ⁇ -glycerol phosphate, 1 mM TCEP, 0.5 mM sodium orthovanadate, and 0.25% CHAPS, supplemented with 0.09 mg/mL TEV protease.
  • Cleaved protein was eluted from the resin using buffer without TEV protease, and the presence of protein in eluted fractions was monitored using a Coomassie-stained SDS-PAGE gel.
  • PKC ⁇ protein was polished on an AKTA go FPLC system (Cytiva) to remove TEV protease and further purify protein for biochemical assays and crystallization.
  • TEV eluted protein was diluted (1:3) with a low salt buffer containing 50 mM Tris pH 8.0 and 1 mM TCEP prior to being loaded onto a HiTrap Q HP anion exchange column (Cytiva).
  • Concentrated protein was subjected to final purification on a Superdex 200 Increase 10/300 GL column (Cytiva) using 20mM Tris pH 8.0, 100mM NaCl, 2mM MgCl 2 , and 1mM TCEP at a flow rate of 0.5 mL/min. All FPLC elution fractions were monitored for protein presence using Coomassie-stained SDS-PAGE gel.
  • Fractions containing PKC were pooled and concentrated to 10 mg/mL using a 10K centrifugal filter (Sartorius, Vivaspin) for crystallization, or buffer exchanged into storage buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM MgCl 2 , 0.5 mM TCEP, 25% (v/v) glycerol) and stored at -80 oC prior to use in kinase assays.
  • Typical yield of final, purified PKC ⁇ I/II proteins was 3 to 6 mg per litre of culture.
  • Lambda Phosphatase Reactions To generate dephosphorylated PKC, 7 ⁇ g PKC protein were incubated at 37°C for 2 hours with 1400U Lambda Phosphatase (New England BioLabs P0753S) and its 1x reaction buffer according to the manufacturer’s protocol. As a control, 7 ⁇ g PKC protein was incubated under the same conditions in the absence of phosphatase. After the incubation period, loading dye was added and the reactions were heated to 75°C for 5 minutes prior to visualization via 8% SDS-PAGE with 0 ⁇ M PhosTag or 40 ⁇ M Phostag (APExBIO). Attorney Docket No.
  • X-ray diffraction datasets were collected at the NE-CAT beamlines (24-C and 24-E) at the Advanced Photon Source. X-ray diffraction data were processed and scaled using the HKL2000 suite. Structures were solved via molecular replacement using the C1b, C2, and kinase domains from PDB entry 3PFQ as search models using the PHENIX-PHASER.
  • CKAR Protein expression was induced by the addition of 50 ⁇ M of Attorney Docket No. 07039-2296WO1 / 2023-600 isopropylthio- ⁇ -galactoside (IPTG, Goldbio) for 18 hours at 16°C.
  • IPTG, Goldbio isopropylthio- ⁇ -galactoside
  • the bacterial cell pellet was harvested by centrifugation at 6000 g for 20 minutes at 4°C, and frozen at -80°C.
  • CKAR was concentrated and buffer exchanged into PKC Storage Buffer (20 mM Tris pH 8.0, 100 mM NaCl, 2mM MgCl 2 , 1mM TCEP, and 25%(v/v) glycerol) using an Amicon 10K concentrator (Millipore). The final CKAR product was quantified using A520nm and an extinction coefficient of 70,000 M-1cm-1 for YFP.
  • CKAR Kinase Assay Lipid vesicles were prepared fresh by dissolving 10 mg/mL bovine brain phosphatidylserine (PS) (Avanti) or/and 1 mg/mL 1,2-dioleoyl-glycerol (DAG) (Avanti) in chloroform. Lipids were mixed at the indicated ratio and chloroform was evaporated with a stream of dry air, then lipids were redissolved in water to a final concentration of 40 ⁇ g/mL, vortexed for 60 seconds, and sonicated 3 times for 30 seconds at 10% power on a Branson 250 sonifier with a microtip (12840498). Concentrated PKC protein was diluted into storage Attorney Docket No.
  • FRET Forster Resonance Energy Transfer
  • Z’-LYTE Kinase Assay Kinase activity was measured using the Z’-LYTE Kinase Assay Kit – Ser/Thr 7 Peptide (ThermoFisher) following the manufacturer’s protocol.
  • the reaction mix contained 250 mM HEPES pH 7.5, 50 mM MgCl 2 , 5 mM EGTA, 0.05% Brij-35, 40 ⁇ g/mL PS:DAG lipids, PKC protein at the indicated concentration, drug serial dilution at the respective concentration, 40 ⁇ M ATP and 2 ⁇ M Z’-LYTE Ser/Thr 7 peptide substrate.
  • the peptide substrate and ATP mixture were added last to initiate the reaction.
  • PKA DNA encoding PKA catalytic domain was transformed into Rosetta2 cells (EMD) and inoculated in a 2 L culture of terrific broth (Research Products International) supplemented with 300 ⁇ L Antifoam 204, 100 ⁇ g/mL carbenicillin, and 34 ⁇ g/mL chloramphenicol. Culture was grown using a LEX-48 Bioreactor (Epiphyte) at 37°C and protein expression was induced with 100 ⁇ M IPTG overnight at 16°C. E.
  • coli culture was pelleted, resuspended in lysis buffer (20 mM Tris pH 7.5, 300 mM NaCl, 10 mM imidazole, and 0.5 mM TCEP) supplemented with 10 mg of lysozyme (GoldBio) and 20 ⁇ L of saturated PMSF in ethanol (GoldBio).
  • Cell lysate was incubated on ice for 30 minutes with occasional mixing and then sonicated by a Branson 250 sonicator at 80% power in five 15-second intervals. Clarified lysate was centrifuged at 25,000g for 30 minutes and passed over a Ni-NTA column equilibrated in lysis buffer.
  • Ni-NTA resin was washed with column volumes lysis buffer and eluted with lysis buffer supplemented with 250 mM imidazole. Protein containing fractions were detected by a color change in a Bradford Assay (99 ⁇ L bradford reagent, 1 ⁇ L elution fraction), pooled and precipitated with two volumes of 4 M ammonium sulfate followed by centrifugation at 25,000g for 30 minutes at 4°C. Precipitated protein was dissolved in 3 mL Milli-Q water and loaded onto a Superdex S200 size exclusion column equilibrated in 20 mM Tris pH 7.5, 300 mM NaCl, and 0.5 mM TCEP.
  • Concentrated protein was run over a Superdex 200 increase (Cytiva) equilibrated in running buffer (20 mM Tris pH 7.5, 300 mM NaCl, and 0.5 mM TCEP). Purified protein was analyzed by SDSPAGE gel and buffer exchanged during Attorney Docket No. 07039-2296WO1 / 2023-600 Amicon centrifugation to PKC storage buffer (20 mM Tris pH 8.0, 100 mM NaCl, 2 mM MgCl2, 1mM TCEP, and 25% (v/v) glycerol).
  • Live Cell Imaging MCF7AC1 cells that stably expressed YFP-PKC ⁇ 1 were generated by transfecting plasmid DNA using Lipofectamine 2000 (Thermofisher) according to the manufacturer’s instructions, and then following the protocol described in the Expression and purification of PKC ⁇ I and PKC ⁇ II section for generating a stable expression cell line.
  • YFP+ cells were seeded in 35-mm glass bottom microwell dishes (MatTek Corporation) for at least 24 hours. Subsequently, cells were incubated with ENDX followed by 15 minutes of PMA incubation as indicated in figure legends.
  • Example 2 Endoxifen Downregulates AKT Phosphorylation in ER ⁇ + Breast Cancer Endoxifen (ENDX), a secondary tamoxifen (TAM) metabolite, is a potent antiestrogen exhibiting estrogen receptor alpha (ER ⁇ ) binding at nanomolar concentrations.
  • ENDX a secondary tamoxifen
  • ER ⁇ estrogen receptor alpha
  • Phase 1/2 clinical trials identified clinical activity of Z-ENDX, in endocrine-refractory metastatic breast cancer as well as ERa+ solid tumors, raising the possibility that ENDX may have a second, ERa-independent, mechanism of action.
  • This Example describes the identification of PKC ⁇ 1 as a ENDX target whose engagement results in inhibition of AKT signaling and induction of apoptosis. Attorney Docket No.
  • ENDX at 5 M inhibits growth and induces apoptosis in estrogen deprived ER ⁇ + breast cancer cells
  • ENDX concentrations ranging from 0 – 10 ⁇ M were used to evaluate dose dependent effects of ENDX on cell viability under estrogen deprived conditions, i.e., in medium containing charcoal-stripped serum (CSS), to evaluate ENDX effects that may extend beyond ER ⁇ inhibition.
  • ENDX concentrations ⁇ 2.5 ⁇ M significantly reduced cell viability ( Figure 14A) and induced apoptosis in these cells ( Figure 14B).
  • ENDX concentration-dependent effects on the phosphoproteome of ER ⁇ + breast cancer cells It was then sought to identify additional protein targets of ENDX that may contribute to its anticancer effects in estrogen deprived conditions.
  • MCF7AC1 cells were treated with 0.01, 0.1, and 5 ⁇ M ENDX concentrations achieved in various clinical settings for 24 hours in CSS medium and subjected to TMT labeling-based LC-MS/MS mass spectrometry analysis to evaluate changes in the global protein expression and the phosphoproteome relative to vehicle treated cells (Figure 15). Assessment of the total proteome identified and quantified 8,894 unique proteins (accession number: PXD035007).
  • Figure 17 shows the top enriched upstream kinases predicted to regulate the protein phosphorylation changes induced by ENDX at different concentrations.
  • Casein kinase CSNK1A1
  • SRPK1, SRPK2 serine/arginine-rich protein-specific kinase
  • MAPK8 mitogen-activated protein kinases
  • ENDX downregulated phosphosites are enriched for PKC ⁇ , CDK1 and AKT1 target sequences It was postulated that the observed ENDX effects on cluster 1 phosphosites were due to effects on kinase mediators of these phosphorylation events.
  • NetworKIN and RoKAI kinase prediction tools were used. Using the 325 phosphosites from cluster 1 as an input, these two tools collectively identified protein kinase C beta (PKC ⁇ ) and cyclin-dependent kinase 1 (CDK1) followed by AKT1 and PKC /PKC ⁇ as the top five most frequently predicted kinases involved (Figure 18C; Table 4).
  • motif enrichment analysis identified that RXXpS, pS/pTP and pSXXE as prevalent motifs in the regulated cluster 1 phosphosites. These motifs mapped to the AKT, MAPK/CDK and CK2 kinase substrate motifs, respectively ( Figure 18D).
  • PKCb siRNA recapitulated both the signaling and growth inhibitory effects of ENDX.
  • Attorney Docket No. 07039-2296WO1 / 2023-600 To determine whether the observed effects of ENDX on PKC ⁇ 1 degradation and AKT Ser473 phosphorylation inhibition are dependent on the presence of ER ⁇ , ENDX effects on PKC ⁇ 1 degradation and AKT Ser473 phosphorylation in the ER negative (ER-) MDAMB231 breast cancer cells and nonbreast HEK293F cells, a human embryonic kidney cell line, both of which express higher amounts of PKC ⁇ 1 compared to ER+ MCF7AC1 cells, were additionally evaluated (Figure 28A).
  • ER ⁇ overexpression also resulted in increased AKT Ser473 phosphorylation that remained unaffected by ENDX pretreatment in these cells.
  • MDAMB231 and HEK293F cells were pretreated with or without 5 ⁇ M ENDX for two hours followed by 0 or 200 nM PMA treatment for 20 minutes and effects on PKC ⁇ 1 were evaluated.
  • MCF7 human breast cancer cells stably transfected with the aromatase gene were cultured in phenol-red free IMEM medium (Gibco #A10488-01) supplemented with 10% fetal bovine serum (FBS) (Gemini #900-108), 600 ⁇ g/mL geneticin (G418) (Gibco #10131-027) and 1% Antibiotic-Antimycotic (AA) (Gibco #15240-062).
  • MCF7AC1 cells were cultured in IMEM medium containing 10% charcoal-stripped serum (CSS) (Hyclone #SH30068), 600 ⁇ g/mL G418 and 1% AA.
  • T47D cells were cultured in DMEM/F12 medium (Corning #16-405-V) containing 10% FBS and 1% AA.
  • T47D-long-term estrogen deprived (LTED) cells were cultured in DMEM/F12 medium containing 10% CSS and 1% AA.
  • HA hemagglutinin
  • MCF7AC1 caAKT catalytically active AKT expressing MCF7AC1 cells
  • IMEM IMEM containing 10% FBS, 600 ⁇ g/mL G418, 1% AA and 0.5 ⁇ g/mL puromycin (Gibco #A11138-03).
  • the ER- MDAMB231, BT549 and MDAMB436 breast cancer cells were cultured in DMEM/F12 medium containing 10% FBS and 1% AA.
  • the HEK293F cells were cultured in DMEM medium (Corning #34722014) containing 10% FBS and 1X penicillin- streptomycin (Sigma #P0781).
  • the doxycycline-inducible ER ⁇ -expressing MDAMB231 cell line was established using the T-REx TM system (InVitrogen) and were maintained in DMEM/F12 medium containing 10% FBS 1% AA, 5 mg/L Blasticidin S (Sigma #15205) and 500 mg/L Zeocin (InVivoGen #ant-zn-5b).
  • the Z-endoxifen hydrochloride utilized in this study was synthesized. Estrogen deprived MCF7AC1 were treated with vehicle control or ENDX (National Cancer Institute) for 24 hours.
  • MCF7AC1 cells were maintained in serum-free medium for 24 hours prior to pretreatment with vehicle or ENDX for two hours followed by treatment with 20 or 200 nM PMA for 20 minutes.
  • PMA phorbol 12-myristate 13-acetate
  • MCF7AC1 and T47D cells were maintained in serum-free medium for 24 hours prior to pretreatment with vehicle control or drugs for two hours followed by treatment with 100 nM insulin for one hour.
  • Attorney Docket No. 07039-2296WO1 / 2023-600 Proliferation assay Cells were plated at a density of 2000 cells per well.
  • Cell viability of (i) vehicle or drug treated MCF7AC1 and T47D-LTED cells in CSS medium, (ii) siNT or siPKC ⁇ - transfected MCF7AC1 cells in CSS medium, and (iii) T47D cells in FBS versus CSS medium were analyzed by crystal violet staining assay after six days of treatment or siRNA transfection. Cell viability was calculated as the average absorbance of the drug treated cells divided by the average absorbance of the vehicle treated cells x 100. Apoptosis assay MCF7AC1 and T47D-LTED cells were plated at a density of 2000 cells per well in CSS medium for 24 hours.
  • MCF7AC1 caAKT cells were plated at a cell density of 2000 cells per well in CSS medium in the absence or presence of cumate for 48 hours and then co-treated with vehicle or drug, Annexin V green and NucLight rapid red reagents in the absence or presence of cumate for an additional 48 hours in CSS medium.
  • the apoptosis graphs are presented as the green object count (which correspond to cells that are stained with the IncuCyte green fluorescence Annexin V reagent) divided by the red object count (which correspond to the total number of cells in the culture that are stained with the IncuCyte red fluorescence Nuclight Rapid Red Cell Labeling reagent that labels the nucleus of all cells without perturbing cell function or biology) and displayed as percentage using the IncuCyte S3 analysis software.
  • Protein sample preparation for mass spectrometry-based quantitative proteomics analysis i). Cell lysis and in-solution trypsin digestion.
  • TMT Tandem Mass Tag
  • the lyophilized tryptic peptides were reconstituted in 150 ⁇ L 100 mM triethylammonium bicarbonate (TEABC) and measured with peptide BCA assay (Thermo Scientific).1 mg peptides from each sample in a final volume of 100 uL 1 mM TEABC were mixed with 1 mg ⁇ g TMTpro reagent that was dissolved in 20 ⁇ L anhydrous acetonitrile. After 1 hour incubation at RT, 10 ⁇ L of 5% hydroxylamine was added and incubated for 15 minutes at room temperature to quench the labeling reaction. Peptides labeled by different TMT reagents were then mixed and dried with Speed-Vac.
  • TEABC triethylammonium bicarbonate
  • the dried TMT-labeled peptides were reconstituted in 20 mM ammonium formate and fractionated by high-pH reversed-phase liquid chromatography on Dionex Ultimate 3000 (Thermo Scientific). Peptides (12 mg) were separated on a 4.6 mm ⁇ 50 cm ⁇ 3.5 ⁇ m Xbridge column (Waters) with a 2-hour gradient from 2 to 40% mobile phase B (MPB). Mobile phase A was composed of 20 mM ammonium formate in water, and MPB was composed of 20 mM ammonium formate in 80% acetonitrile. A total of 96 fractions were collected and concatenated into 24 fractions.
  • the eluted phosphopeptides were desalted with C18 Stage Tips and Speed-Vac dried. ( iv). LC-MS/MS analysis.
  • the peptide fractions were loaded on a 2 cm trap column (Acclaim PepMap 100, C 18 , 5 ⁇ m particle size, 100 ⁇ m i.d.100 ⁇ pore size, Thermo Scientific, San Jose, CA) using 0.1% formic acid with a flow rate 20 ⁇ L/minute for 4 minutes.
  • the peptides were separated on a 50 cm analytical column (Acclaim PepMap 100, C 18 , 2 ⁇ m particle size, 75 ⁇ m i.d.100 ⁇ pore size, Thermo Scientific, San Jose, CA) with a 135 minute gradient from 3% to 40% acetonitrile in 0.1% formic acid at a flow rate of 0.3 ⁇ L/minute.
  • the spray voltage was set to 2.3 kV while capillary temperature was set to 275°C.
  • the samples were analyzed on an Orbitrap Fusio Lumos mass spectrometer (Thermo Scientific, Bremen, Germany). The MS instrument was operated in data-dependent acquisition mode.
  • a survey full scan MS (from 350–1,500 m/z) was acquired in the Orbitrap with resolution 120,000 at m/z 200 with a maximum AGC target value of 800,000 ions.
  • the data-dependent MS/MS was carried out using Top Speed method with a duty cycle of 2 seconds. Singly charged precursor ions were excluded while precursor ions with charge states 2-7 were sequentially isolated and fragmented in the higher-energy collisional dissociation (HCD) cell using 34% normalized collision energy (NCE).
  • the maximum ion injection time for MS and MS/MS were set to 50 ms. Fragment ion spectra were detected in Orbitrap mass analyzer with a resolution 30,000 at m/z 200.
  • Search parameters included trypsin as a protease with full specificity and a maximum of two allowed missed cleavages; carbamidomethylation of cysteine and TMTpro tag (+304.207 ⁇ Da) on lysine residues or peptide N-terminus as a fixed modification; oxidation at methionine and phosphorylation at serine/threonine/tyrosine as variable modifications.
  • the precursor tolerance was set at 10 ppm, while the fragment match tolerance was set to 0.02 Da.
  • the PSMs, peptides and proteins were filtered at 1% false discovery rate cut-off calculated using target-decoy database searches.
  • the probability of an identified phosphorylation of specific Ser/Thr/Tyr residue on each identified phosphopeptide was determined from the PhosphoRS algorithm.
  • Phosphoproteome data analysis The intensities of TMT reporter ions were normalized based on the average total phosphopeptide intensity detected in each TMT-labeling channel. Differentially phosphorylated sites were identified with an empirical Bayesian moderated t-statistics test as implemented in the R limma package. Multiple comparison correction was performed with Benjamini-Hochberg procedure. Phosphorylation sites with log2 fold change > 1.5 and unadjusted p-value ⁇ 0.05 were selected for downstream analysis. DAVID, an integrated online functional annotation tool, was used to annotate the functions of the differentially modulated phosphoproteins.
  • Kyoto Encyclopedia of Genes and Genomes (KEGG) database was selected to identify enriched signaling pathways.
  • the ggplot package in R was used to generate the bubble plot depicting the enriched pathways.
  • the Fuzzy C-means clustering showing the dynamic regulation patterns of phosphosites were generated using ggplot and mfuzz packages in R.
  • Kinase substrate enrichment analysis KSEA
  • PhosphoSitePlus PhosphoSitePlus
  • NetworKIN NetworKIN
  • RoKAI datasets were used to predict upstream kinases of regulated phosphosites, as described below.
  • MoMo program with motif-x algorithm were used to identify the motifs enriched in the ENDX-regulated phosphosites.
  • Equal amounts of protein lysates were separated on 10% Criterion gels (Bio-Rad #3450112), transferred to PVDF membranes (Bio-Rad #1620177), blocked in TBST-5% milk and probed with primary antibodies listed in Table 6, at the indicated dilutions.
  • Membranes were incubated with HRP-conjugated anti-rabbit (CST #7074) or anti-mouse (CST #7076) secondary antibodies and visualized using chemiluminescent West Pico (Thermo Scientific #34580) or West Femto (Thermo Scientific #34096) reagents and a Li-Cor Odyssey ® XF imager.
  • Protein lysates from the MCF7AC1 xenograft model were obtained as described elsewhere (Jayaraman et al., Breast Cancer Res. 22:51 (2020)). Quantitation of the protein bands signal intensity was performed using the National Institute of Health (NIH) ImageJ image analysis software (imagej.nih.gov/ij///index.html). For the quantitation of the phospho protein levels, total protein levels were first normalized to ⁇ -actin loading control and these values were used to normalize the phospho protein levels and compared change in protein expression levels relative to vehicle control normalized to 1.0. For the quantitation of total protein levels, the total protein levels were normalized to ⁇ -actin and change in expression Attorney Docket No.
  • IC 50 concentrations of these drugs in inhibiting PKC kinase activity are provided in Table 5.
  • Affinity measurements by Surface Plasma Resonance (SPR) Binding assays were performed at 25°C on a Biacore T200 biosensor (GE healthcare).
  • Purified PKC ⁇ 1 protein were immobilized on a CM5 S sensor chip using amino coupling and immobilization buffer (10 mM HEPES, 150 mM NaCl, pH 7.4, P200.01% (w/w)) and acetate pH 5.0 at a flow rate of 10 ⁇ L/minute and reaching 10,000-12,000 resonance units (RUs).
  • ENDX at concentrations ranging from 0- 8000 nM in phosphate buffer (Gibco) with 2% DMSO (v/v) and 0.01% (w/w) P20 were run over the chips at a flow rate of 50 ⁇ L/minute for 30 seconds.
  • Binding kinetics were derived from sensograms using Biacore BIA evaluation software (GE). Sensograms were subtracted for background Attorney Docket No. 07039-2296WO1 / 2023-600 contributions, and affinity constants were derived using a steady state affinity fitting of a 1:1 interaction model.
  • a SMART vector inducible human PRKCB mCMV-TurboGFP lentiviral shRNA was used for the generation of doxycycline (dox)-inducible PRKCB gene silencing.
  • MCF7AC1 cells grown in 6-well plate in IMEM medium containing 10% FBS, 600 ⁇ g/mL G418 and 1% amino acids and at a confluency of 50% were infected with the lentivirus at a multiplicity of infection (MOI) of 5.0 in the presence of 1 ⁇ g/mL polybrene.
  • MOI multiplicity of infection
  • vector pCDNA3.1+ containing an N-terminal Attorney Docket No. 07039-2296WO1 / 2023-600 SRCMyr signal AKT ORF and C-terminal hemagglutinin (HA)-tag was used.
  • the constitutively active AKT (caAKT)-HA insert was excised using NheI/EcoRV restriction digest, gel purified and cloned into the NheI//PmeI site of an SBI (System biosciences, Palo Alto, CA) vector with modified restriction sites. Sanger sequencing was performed by Azenta/Genewiz, (South Plainfield, NJ) to confirm in frame sequence.
  • caAKT-HA construct After viral transduction of the caAKT-HA construct into MCF7AC1 cells, cells were selected beginning 48 hours later for a mixed population (MCF7AC1 caAKT cells) using puromycin (Invitrogen) 0.5 ⁇ g/mL for several weeks. Expression of caAKT-HA was induced by adding 60 ⁇ g/mL cumate (Sigma Aldrich 268402, 4-Isopropylbenzoic acid) for 48 hours prior to drug treatment.
  • Example 3 ENDX and Immune Function in Patients with ER+ Breast Cancer ENDX upregulates the expression of IFN , IL-2 and Granzyme B expression by CD4+ and CD8+ T cells in vitro
  • ENDX upregulates the expression of IFN , IL-2 and Granzyme B expression by CD4+ and CD8+ T cells in vitro
  • the ability of PKC ⁇ 1 to bind ENDX was linked to decreases in PKC ⁇ 1 protein levels and reduction in Akt activity, resulting in the induction of Attorney Docket No. 07039-2296WO1 / 2023-600 apoptosis (see, Example 2 and Jayaraman et al., NPJ Breast Cancer, 9(1):101 (2023)).
  • ENDX is highly expressed in immune cells, the potential impact of ENDX on immune function was investigated.
  • PBMC peripheral blood mononuclear cells
  • TILs tumor-infiltrating lymphocytes
  • ENDX primes immune cells to regulate ER+ breast cancer cell line growth
  • PBMCs were pre-treated with either 2 ⁇ M of ENDX or enzastaurin for 5 days, and subsequently co-cultured with either MCF7AC1 cells sensitive to ENDX or MCF7 cells resistant to ENDX, followed by monitoring real-time tumor proliferation using IncuCyte over 5 days (Figure 35A).
  • Example 4 Z-Endoxifen (ENDX) mediated CD4+ and CD8+ T cell alterations at physiological achievable drug concentrations
  • ENDX Z-Endoxifen
  • This Example demonstrates that endoxifen negatively regulated inhibitory receptors PD-1 and TIM-3 on CD4+ and CD8+ T cells.
  • a flow cytometry gating strategy for of CD4+ and CD8+ T cells is shown in Figure 36.
  • Endoxifen negatively regulated inhibitory receptors PD-1 and TIM-3, T cell exhaustion markers that are upregulated in poor prognosis ER+/HER2- breast cancer, on CD4+ and CD8+ T cells Figure 37. These data were not seen with fulvestrant, a pure antiestrogen ( Figure 37).
  • Example 7 Exemplary Embodiments Embodiment 1.
  • a method for treating a mammal having an estrogen receptor negative (ER neg ) cancer wherein said method comprises administering, to said mammal, an endoxifen (ENDX) compound.
  • Embodiment 2. The method of embodiment 1, wherein said mammal is a human.
  • Embodiment 3. The method of any one of embodiments 1-2, wherein said mammal is a female mammal.
  • Embodiment 4. The method of any one of embodiments 1-3, wherein said mammal is a pre-menopausal female human.
  • Embodiment 5 The method of any one of embodiments 1-4, wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.
  • Embodiment 6 The method of embodiment 5, wherein said ERneg cancer is a breast cancer. Attorney Docket No.
  • Embodiment 7 The method of embodiment 6, wherein said breast cancer is a triple negative breast cancer.
  • Embodiment 8. The method of any one of embodiments 1-7, wherein said ENDX compound is a Z-ENDX compound.
  • Embodiment 9. The method of embodiment 8, wherein said Z-ENDX compound is a Z-ENDX salt.
  • Embodiment 10. The method of embodiment 9, wherein said Z-ENDX salt is Z-ENDX hydrochloride.
  • Embodiment 11 The method of any one of embodiments 1-10, wherein said method comprising administering from about 20 milligrams per day (mg/day) to about 360 mg/day of said ENDX compound to said mammal.
  • Embodiment 13 The method of embodiment 12, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti- PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, forit
  • Embodiment 14 A method for treating a mammal having an ERpos cancer or an ERneg cancer, wherein said method comprises administering, to said mammal, (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ER pos cancer or said ER neg cancer.
  • Embodiment 15. The method of embodiment 14, wherein said mammal is a human.
  • Embodiment 16 The method of any one of embodiments 14-15, wherein said mammal is a female mammal.
  • Embodiment 17 The method of any one of embodiments 14-16, wherein said mammal is a pre-menopausal female human.
  • ER neg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.
  • ER neg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer,
  • Embodiment 20 The method of embodiment 19, wherein said breast cancer is a triple negative breast cancer.
  • Embodiment 21 The method of any one of embodiments 14-17, wherein said mammal has said ER pos cancer, and wherein said ER pos cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer.
  • Embodiment 22 The method of any one of embodiments 14-21, wherein said ENDX compound is a Z-ENDX compound.
  • Embodiment 23 The method of embodiment 22, wherein said Z-ENDX compound is a Z-ENDX salt.
  • Embodiment 24 The method of embodiment 19, wherein said breast cancer is a triple negative breast cancer.
  • Embodiment 21 The method of any one of embodiments 14-17, wherein said mammal has said ER pos cancer, and wherein said ER pos cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a
  • Embodiment 25 The method of any one of embodiments 14-24, wherein said method comprising administering from about 20 mg/day to about 360 mg/day of said ENDX compound to said mammal.
  • Embodiment 26 The method of any one of embodiments 14-24, wherein said method comprising administering from about 20 mg/day to about 360 mg/day of said ENDX compound to said mammal.
  • any one of embodiments 14-25 wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabo
  • Embodiment 27 The use of a composition comprising an ENDX compound to treat a mammal having an ER neg cancer.
  • Embodiment 28 The use of embodiment 27, wherein said mammal is a human.
  • Embodiment 29 The use of any one of embodiments 27-28, wherein said mammal is a female mammal.
  • Embodiment 30 The use of any one of embodiments 27-29, wherein said mammal is a pre-menopausal female human.
  • Embodiment 31 The use of any one of embodiments 27-29, wherein said mammal is a pre-menopausal female human.
  • any one of embodiments 27-30 wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.
  • Embodiment 32 The use of embodiment 31, wherein said ERneg cancer is a breast cancer.
  • Embodiment 33 The use of embodiment 31, wherein said ERneg cancer is a breast cancer.
  • composition further comprises an agent that can alter the antigens presented on the surface of a cancer cell of said ER neg cancer.
  • an agent that can alter the antigens presented on the surface of a cancer cell of said ER neg cancer.
  • Embodiment 39 The use of embodiment 38, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti- PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritini
  • Embodiment 40 An ENDX compound for use in the preparation of a medicament to treat an ER neg cancer.
  • Embodiment 43 The ENDX compound of embodiment 42, wherein said ERneg cancer is a breast cancer.
  • Embodiment 45. The ENDX compound of any one of embodiments 40-44, wherein said ENDX compound is a Z-ENDX compound.
  • Embodiment 46. The ENDX compound of embodiment 45, wherein said Z-ENDX compound is a Z-ENDX salt.
  • Embodiment 47. The ENDX compound of embodiment 46, wherein said Z-ENDX salt is Z-ENDX hydrochloride. Attorney Docket No. 07039-2296WO1 / 2023-600 Embodiment 48.
  • Embodiment 53 The use of embodiment 52, wherein said ERneg cancer is a breast cancer.
  • Embodiment 57 wherein said Z-ENDX salt is Z-ENDX hydrochloride.
  • Embodiment 59 The use of any one of embodiments 48-58, wherein said composition comprises from about 20 mg to about 360 mg of said ENDX compound.
  • Embodiment 60 The use of any one of embodiments 48-58, wherein said composition comprises from about 20 mg to about 360 mg of said ENDX compound.
  • any one of embodiments 48-59 wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cab
  • Embodiment 61 An ENDX compound and an agent comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the preparation of a medicament to treat an ER pos cancer or ER neg cancer.
  • Embodiment 62 An ENDX compound and an agent comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the treatment of an ER neg cancer.
  • Embodiment 63 An ENDX compound and an agent comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the treatment of an ER neg cancer.
  • Embodiment 64 The ENDX compound of embodiment 63, wherein said ERneg cancer is a breast cancer.
  • Embodiment 65 The ENDX compound of embodiment 63, wherein said ERneg cancer is a breast cancer.
  • the ENDX compound of any one of embodiments 61-66, wherein said ENDX compound is a Z-ENDX compound.
  • the ENDX compound of embodiment 67, wherein said Z-ENDX compound is a Z-ENDX salt.
  • Embodiment 70 The ENDX compound of any one of embodiments 61-69, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib

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Abstract

This document provides method and materials for treating cancer. In some cases, methods and materials for treating estrogen receptor negative (ER-) cancers (e.g., ER- breast cancers such as triple-negative breast cancers (TNBCs)) are provided. For example, one or more endoxifen (ENDX) compounds can be administered to a mammal (e.g., a human) having an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal. For example, one or more ENDX compounds and one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell can be administered to a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal.

Description

Attorney Docket No. 07039-2296WO1 / 2023-600 METHODS AND MATERIALS FOR TREATING CANCER CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application Serial No. 63/552,505, filed on February 12, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application. STATEMENT REGARDING FEDERAL FUNDING This invention was made with government support under CA116201 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2296WO1_SL.xml.” The XML file, created on December 10, 2024, is 389,866 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. TECHNICAL FIELD This document relates to methods and materials for treating cancer. In some cases, this document provides methods and materials for treating cancers including estrogen positive (ERpos or ER+ or ER+) cancers and/or estrogen receptor negative (ERneg or ER- or ER-) cancers (e.g., ER- breast cancers such as triple-negative breast cancers (TNBCs)). For example, one or more endoxifen (ENDX) compounds can be administered to a mammal (e.g., a human) having an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal. For example, one or more ENDX compounds and one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell can be administered to a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal. Attorney Docket No. 07039-2296WO1 / 2023-600 BACKGROUND Breast cancer is the most common cancer worldwide with over 2.3 million new cases and 685,000 deaths in 2020 (Arnold, Morgan et al.2022). Breast cancer displays substantial heterogeneity in prognosis and therapy responses and is categorized into four primary subtypes based on an analysis of its molecular characteristics and immunohistochemical markers: luminal A breast cancers and luminal B breast cancers (both characterized by ER expression), HER2-positive breast cancers, and TNBCs (characterized by the absence of ER, the absence of progesterone receptor (PR), and without HER2 amplification). TNBC exhibit aggressive clinical behavior and high rates of death. SUMMARY This document provides methods and materials for treating cancer (e.g., ER+ cancers and/or ER- cancers such as ER- breast cancers (e.g., TNBCs)). For example, one or more ENDX (also referred to as 4-hydroxy-N-desmethyltamoxifen) compounds can be administered to a mammal (e.g., a human) having an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal. For example, one or more ENDX and one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell can be administered to a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal. As demonstrated herein, ENDX can be used as an allosteric inhibitor of a protein kinase C beta 1 isoform (PKC 1) polypeptide, thereby altering AKT signaling. Also as demonstrated herein, ENDX exhibits a dose dependent targeting of a PKC 1 polypeptide. In general, one aspect of this document features methods for treating a mammal having an ER- cancer. The methods can include, or consist essentially of, administering an ENDX compound to a mammal having an ER- cancer. The mammal can be a human. The mammal can be a female mammal. The mammal can be a pre-menopausal female human. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a Attorney Docket No. 07039-2296WO1 / 2023-600 leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The ENDX compound can be a Z-ENDX compound. The Z- ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. The method can include administering from about 20 milligrams per day (mg/day) to about 360 mg/day of said ENDX compound to said mammal. The method also can include administering to said mammal an agent that can alter the antigens presented on the surface of a cancer cell of said ER- cancer. The agent can be abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, or TQB-3602. In another aspect, this document features methods for treating a mammal having an ER+ cancer or an ER- cancer. The methods can include, or consist essentially of, administering, to mammal having an ER+ cancer or an ER- cancer, (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ER+ cancer or said ER- cancer. The mammal can be a human. The mammal can be a female mammal. The mammal can be a pre-menopausal female human. The mammal can have ER- cancer, and said ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate Attorney Docket No. 07039-2296WO1 / 2023-600 cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The mammal can have ER+ cancer, and said ER+ cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, or a lung cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. The method can include administering from about 20 mg/day to about 360 mg/day of said ENDX compound to said mammal. The agent can be abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU- D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, or TQB-3602. In another aspect, this document features uses of a composition comprising an ENDX compound to treat a mammal having an ER- cancer. The mammal can be a human. The mammal can be a female mammal. The mammal can be a pre-menopausal female human. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The ENDX compound can be a Z-ENDX compound. The Z- ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. Attorney Docket No. 07039-2296WO1 / 2023-600 The composition can include from about 20 mg to about 360 mg of said ENDX compound. The composition also can include an agent that can alter the antigens presented on the surface of a cancer cell of said ER- cancer. The agent can be abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, or TQB-3602. In another aspect, this document features ENDX compounds for use in the preparation of a medicament to treat an ER- cancer. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. In another aspect, this document features ENDX compounds for use in the treatment of an ER- cancer. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin Attorney Docket No. 07039-2296WO1 / 2023-600 cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. In another aspect, this document features uses of a composition comprising (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ER+ cancer or said ER- cancer to treat a mammal having an ER+ cancer or an ER- cancer. The mammal can be a human. The mammal can be a female mammal. The mammal can be a pre-menopausal female human. The mammal can have ER- cancer, and said ER- cancer be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The mammal can have ER+ cancer, and said ER+ cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, or a lung cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. The composition can include from about 20 mg to about 360 mg of said ENDX compound. The agent can be abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, Attorney Docket No. 07039-2296WO1 / 2023-600 sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU- D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, or TQB-3602. In another aspect, this document features ENDX compounds and agents comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the preparation of a medicament to treat an ER+ cancer or ER- cancer. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The ER- breast cancer can be a triple negative breast cancer. The ER+ cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, or a lung cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. The agent abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti- PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU- D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, TQB-3602. In another aspect, this document features ENDX compounds and agents comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the treatment of an ER- cancer. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary Attorney Docket No. 07039-2296WO1 / 2023-600 tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The ER- breast cancer can be a triple negative breast cancer. The ER+ cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, or a lung cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. The agent abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti- CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU- D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, TQB-3602. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Attorney Docket No. 07039-2296WO1 / 2023-600 The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-F. Structure of PKCȕII reveals architecture of the auto-inhibited state. Figure 1A) PKCȕI/II domain diagram with phosphorylation sites (phospho-S/T) indicated by circles. Figure 1B) 8% Acrylamide gel with or without Phos-tag 5 of purified PKCȕII and purified PKCȕII treated with phosphatase. Figure 1C) PKCȕII crystal grown using sitting drop vapor diffusion method. Figure 1D) Overall structure of the PKCȕII inactive state with the domains coloured as in Figure 1A. Figure 1E) In the inhibited state, many hydrophobic lipid binding residues are occluded by interaction with the catalytic domain. Figure 1F) Molecular details of the interdomain contacts between the C1a domain, kinase domain and C-terminal tail, and the three phosphorylation sites: activation loop (pT500), turn (pT641), and hydrophobic (pS660) with surrounding structures, and the bound AMPPNP nucleotide. Figures 2A – 2C. Structure of PKCȕI reveals an ordered active conformation. Figure 2A) Two PKCȕI crystal forms with 1 or 2 PKCȕI molecules per asymmetric unit (ASU) were aligned by their kinase domain. Their crystal-packing environments are shown (N-term 1, N- term 2, and N-term 3). Two domain arrangements corresponding to the active and inhibited state are common amongst the PKCȕI molecules. The regulatory domain lipid-binding residues are in a plane for the activated state (upper) while the pseudosubstrate occupies the active site in the inhibited state. Figure 2B) Recombinant PKCȕǿ protein with mutations engineered to disrupt the C1b-kinase and C2-kinase domain interactions. Figure 2C) Mutant PKCȕǿ were assayed for kinase activity in vitro using the FRET probe CKAR. Error bars represent standard deviation. Figures 3A – 3C. Allosteric activation of PKC is driven by binding to a phospholipid membrane. Figure 3A) Lipid-lever model of PKC activation. A steric clash between the phospholipid bilayer and kinase domain drives PKCȕI/II into its active conformation. Figure 3B) In vitro CKAR assay showing the kinase activity of PKCȕI in the indicated concentrations 5 of PDBu or 40 ^g/mL PS/DAG lipid micelles. Error bars represent standard Attorney Docket No. 07039-2296WO1 / 2023-600 deviation. Figure 3C) Limited proteolysis with elastase used to probe change in PKCȕI/II conformation in the presence of the indicated ligands, with quantification of cleavage product (*) as a percentage of protein indicated at the bottom of each gel. Figures 4A – 4C. Molecular basis for differential lipid affinities between PKCȕI and PKCȕII. Figure 4A) Comparison between the structural architecture of PKCȕI and PKCȕII inactive conformations and the relative locations of the lipid binding residues and pseudosubstrate. Figure 4B) Position of the 5 C1b domain differs between PKCȕI and PKCȕII with respect to the C1a and kinase domains. Figure 4C) Intramolecular details of the residue interactions that lead to differential placement of the C1b domain, and the effect of this placement on the susceptibility of PKCȕI (ȕI), PKCȕII (ȕII), and PKCȕI F648A (FA) to proteolytic cleavage by elastase (*). Figures 5A – 5F. Endoxifen is an allosteric inhibitor of PKCȕI. Figure 5A) In vitro Z’-LYTE kinase assay shows differences in kinase activity of unstimulated (- lipids) compared to active (+ lipids) PKCȕI and PKCȕII in response to ENDX (E) and TAM (T) inhibition. The associated IC50 values are reported. Data 5 are mean ± SD for each data point (n=4 independent experiments). Figure 5B) In vitro Z’-LYTE kinase assay with 7.5 nM unstimulated PKCȕI (- lipids) is more sensitive to inhibition by ENDX than 5nM activated PKCȕI (+ lipids) and 0.3 nM PKCȕI catalytic domain. Data are mean ± SD for each data point (n=4 independent experiments). Figure 5C) In vitro kinase assay (CKAR) on the catalytic domain of PKCȕI and PKC ȕII reveals that ENDX as a non-competitive inhibitor with respect to ATP in contrast to the known ATP-competitive inhibitor ENZA. Error bars represent standard deviation (n=4 independent experiments). Figure 5D) SAXS scattering plot of PKCȕI in the presence of the indicated ligands. Figure 5E) Limited proteolysis demonstrates dose-dependent conformational alteration of PKCȕI by ENDX and not TAM. Data are presented as mean ± SD. Figure 5F) Confocal microscopy data shows plasma membrane recruitment of YFP-PKCȕI in the presence of the indicated drug treatments. Figure 6. Overall model of PKC allosteric regulation. Figures 7A – 7D. Figure 7A) Sequence alignment of conventional PKCs with domains coloured as indicated. Sequences shown include, from top to bottom, PKCȖ (SEQ ID NO:1), PKCĮ (SEQ ID NO:2), PKCȕI (SEQ ID NO:3), and PKCȕII (SEQ ID NO:4). The Attorney Docket No. 07039-2296WO1 / 2023-600 altered C-terminal tails derived from an alternative splicing event are coloured pink (PKCȕI) or salmon (PKCȕII). Figure 7B) Domain arrangement and second messenger sensitivity of the three PKC families. Figure 7C) Schematic diagram of YFP-PKCȕII protein expression and affinity isolation using the anti-YFP nanobody system. Chromatograms and corresponding Coomassie-stained gels of PKCȕII from FPLC purification steps. Figure 7D) Western blots with the indicated phospho-specific PKCȕII antibodies detects phosphorylation at all three expected sites (T500 – activation, T642 – turn, and S660 – hydrophobic) in the purified protein. Figures 8A – 8D. Figure 8A) Experimental 2F0 – Fc map contoured at 1ı indicating continuous electron density for the PKCȕII C1a – C1b linker region and C1b – C2 linker region. Figure 8B) Experimental 2F0 – Fc map contoured at 1ı for residues 298 – 312 in the PKCȕII C2-kinase domain linker region. Figure 8C) Experimental 2F0 – Fc of the three phosphorylation sites on PKCȕII. Figure 8D) Location of Zn2+ ions (gray spheres) coordinated within the C1a and C1b domains, as well as a fifth Zn2+ ion that is part of the crystal lattice. Figures 9A – 9C. Figure 9A) Measured length of the disordered region between C2 and kinase domain used to define pairing of N-terminal and kinase domains that belong to the same chain. Figure 9B) Domain–swapped arrangement of PKCBII with an adjacent identical monomer. Figure 9C) Molecular details of the N-terminal pseudosubstrate side chains and their specific interactions with the C1b and kinase domains labeled by residues and color-coded (hydrogen bonding, salt bridge, hydrophobic/Van der Waals). Figures 10A – 10E. Figure 10A) Ion exchange and size exclusion chromatograms of PKCȕI purification and SDS-PAGE analysis of indicated fractions. Figure 10B) Mass spectrometry of purified PKCȕI protein detects phosphorylation at T500 and T642. The polypeptide sequence in the top spectrum is SEQ ID NO:5 and the polypeptide sequence in the bottom spectrum is SEQ ID NO:6. Figure 10C) Individual copies of PKCȕI within each crystal form. Figure 10D) Experimental 2F0 – Fc map of phosphorylation sites on PKCȕI in each crystal form. Figure 10E) Experimental 2F0 – Fc map contoured at 1ı for the PKCȕI C1a – C1b linker region and C1b – C2 linker region. Attorney Docket No. 07039-2296WO1 / 2023-600 Figures 11A – 11E. Figure 11A) Pseudosubstrate of the adjacent monomer is present in the active site of crystal form 1. Figure 11B) Linkers between C1a and C1b domain and C2 and kinase domains are not observed in PKCȕI crystal form 1. Figure 11C) PKCȕI active site adopts a more closed conformation when occupied by AMPPNP nucleotide and remains open in its absence. Figure 11D) Local symmetry environment of the three copies of PKCȕI reveals similar molecular interactions. Figure 11E) AlphaFold-predicted structure of PKCȕI in its active form. Figures 12A – 12E. Figure 12A) Ligands coordinate in crystal form 1 and crystal form 2 of PKCȕI. DAG binding site of PKCȕI is occupied by glycerol molecules in crystal form 2. Figure 12B) 70 A distance between N-terminal tail and active site prevents re- engagement of the pseudosubstrate. Figure 12C) Modeled mutations that disrupt C2-kinase and C1b-kinase domain interactions in the active conformation of PKCȕI. Figure 12D) Aligned crystal structures of the C1a domain with PDBu (PDB 7KNJ) and PKCȕI or PKCȕII in their auto-inhibited conformation shows PDBu binding does not disrupt the inactive conformation. Figure 12E) Insertion of C1a domain hydrophobic residues in the phospholipid bilayer encourages kinase domain to disengage from the pseudosubstrate to prevent unfavourable charge interactions at the lipid bilayer. Figures 13A – 13D. Figure 13A) Differential scanning fluorimetry of 1 ^g full-length (FL), catalytic (CAT) domain or the N-terminal (NT) domain of PKCȕI or of PKCȕII with ddH2O as negative control or 40 ^M ENDX. Presented melt curve and derivative melt plots are shown for each independent measurement. Average melting temperatures (TM) and standard deviations of each condition are reported. Figure 13B) In vitro Z’-LYTE kinase assay of other PKC isoforms and their associated IC50 values. Data are mean ± SD. Figure 13C) Confocal live cell microscopy images showing time-dependent cellular localization of YFP-tagged PKCȕI in the presence of indicated drug treatment. Figure 13D) Confocal live cell microscopy images showing dose-dependent cellular localization of YFP-tagged PKCȕI in the presence of indicated drug treatment. Figures 14A – 14B. Z-endoxifen (ENDX) effects on cell viability and apoptosis in estrogen deprived ERĮ+/HER2- MCF7AC1 cells. Figure 14A) Cells grown in CSS medium were treated with vehicle control or the indicated ENDX concentrations for 48 hours. Cell Attorney Docket No. 07039-2296WO1 / 2023-600 viability was assessed by the crystal violet assay. Figure 14B) Cells were co-treated with vehicle control or the indicated ENDX concentrations, IncuCyte Annexin V green and NucLight red reagents in CSS medium for 48 hours. The apoptosis (%) graphs are presented as the green object count (which correspond to cells that are stained with the IncuCyte green fluorescence Annexin V reagent) divided by the red object count (which correspond to the total number of cells in the culture that are stained with the IncuCyte red fluorescence Nuclight Rapid Red Cell Labeling reagent that labels the nucleus of all cells without perturbing cell function or biology) and displayed as percentage using the IncuCyte S3 analysis software. Data represents the mean of six wells per treatment performed as biological duplicates ± s.d. **, p 0.01; ***, p 0.001; ****, p 0.0001 by one-way ANOVA. Figure 15. A schematic depicting the strategy used for quantitative proteomic and phosphoproteomic profiling of ENDX-treated MCF7AC1 cells. All experiments were performed in triplicate. Cells were treated with vehicle control or Z-endoxifen (ENDX) as specified dosages for 24 hours. After the ENDX treatment, cells were harvested and lysed in 8 M urea buffer, followed by trypsin digestion, desalting and tandem mass tag (TMT) labeling. The labeled peptides were fractionated, and phosphopeptides were enriched with immobilized metal affinity chromatography (IMAC) approach. Both fractionated peptides and IMAC-enriched phosphopeptides were analyzed by Orbitrap Lumos mass spectrometer. Figures 16A – 16F. Effects of ENDX on the phosphoproteome of MCF7AC1 cells. Figure 16A) A pie chart showing the distribution of identified phosphorylation sites. Figures 16B – 16C) Volcano plots showing the total number of phosphosites, and the percentage that are upregulated (right side) and downregulated (left side) (Fold change (FC) |1.5|; p value < 0.05) in cells treated for 24 hours in CSS medium with 0.01 (Figure 16B), 0.1 (Figure 16C), or 5 M (Figure 16D) ENDX relative to vehicle-treated cells, as detected by mass spectrometry analysis. Figure 16E) Venn diagram indicating the overlap of upregulated and downregulated phosphosites in ENDX-treated cells relative to vehicle-treated cells. Figure 16F) Heatmap indicating relative abundance of the phosphosites analyzed in the ENDX- treated cells relative to vehicle-treated cells. The hierarchical clustering of phosphosites is shown on the left. Attorney Docket No. 07039-2296WO1 / 2023-600 Figures 17A – 17C. Kinase enrichment analysis predicts AKT signaling is regulated by high-dose ENDX. 210 phospho-regulated proteins from 0.01 M (Figure 17A), 224 phospho-regulated proteins from 0.1 M (Figure 17B), and 347 phospho-regulated proteins from 5 M (Figure 17C) ENDX treated cells were respectively used for KEA3 upstream kinase analysis. Left panels: Integrated rankings of most enriched kinases across libraries based on the MeanRank. The stacking bar chart shows the summation of the MeanRank derived from the libraries used for the KEA analysis. The libraries are color-coded. Right panels: Kinase co-regulatory networks constructed from kinase-kinase interactions between top-ranked kinase results for the integrated rankings. Directed edges indicate interactions supported by kinase-substrate evidence. Figures 18A – 18D. Downregulated phosphosites following ENDX treatment are enriched for PKCȕ, CDK1, and AKT target sequences. Figure 18A) fuzzy c-means clustering showing the classification of ENDX treatment effects on the phosphoproteome into three regulatory clusters. Cluster 1 represents phosphosites downregulated by ENDX in a dose- dependent manner. Cluster 2 represents phosphosites that are upregulated by ENDX at all concentrations examined. Cluster 3 represents phosphosites downregulated at 0.01 M concentration but mostly unaffected at the 0.1 and 5 M concentrations. Figure 18B) Molecular and cellular pathways potentially impacted by ENDX. Kyoto Encyclopedia of Genes and Genomes (KEGG) database analysis of the phosphosites altered by ENDX in cluster 1 showing the top biological pathways associated with these phosphosites. Figure 18C) Enriched phosphorylation motifs identified in cluster 1 phosphosites. Figure 18D) Graph showing the frequency of kinases known to phosphorylate cluster 1 phosphosites that were depleted by ENDX as assessed using NetworKIN and RoKAI prediction tools. Motifs shown include, from top to bottom, an AKT substrates motif (SEQ ID NO:7), a MAPK/CDK substrates motif (SEQ ID NO:8), and a CK2 substrates motif (SEQ ID NO:9). Figures 19A – 19E. ENDX specifically downregulates pAKTSer473 at 5 μM and inhibits PKC 1 kinase activity. Figure 19A) MCF7AC1 cells in CSS medium were treated for 24 hours with vehicle control or 0.01, 0.1 and 5 μM ENDX. Immunoblot assays of pAKTSer473, pAKTThr308, AKT and p-AKT substrates are shown with ȕ-actin as a loading Attorney Docket No. 07039-2296WO1 / 2023-600 control. Figure 19B) Serum starved MCF7AC1 cells were pretreated with vehicle control, 0.01, 0.1, 5 μM ENDX, 0.1 μM tamoxifen (TAM) or 0.1 μM fulvestrant (ICI-182780) followed by the addition of 100 nM insulin for one hour as indicated. IB assays of pAKTSer473, pAKTThr308, AKT and ȕ-actin are shown. Figure 19C) Serum starved MCF7AC1 cells were pretreated with vehicle control and 0.01, 0.1 and 5 μM ENDX for two hours followed by the addition of 100 nM insulin for one hour as indicated. IB assay of pAKT substrates and ȕ-actin are shown. Figures 19D and 19E) In vitro kinase assay showing % PKCȕ1 kinase activity in the presence of different concentrations of ENDX (Figure 19D) and TAM (Figure 19E). The broad-spectrum kinase inhibitor staurosporine serves as a positive control. The IC50 concentration of ENDX, TAM and staurosporine are indicated. Figures 20A – 20F. Role of PKC 1 in mediating ENDX inhibition of AKTSer473 phosphorylation. Figure 20A) Serum starved MCF7AC1 cells were treated with vehicle control or 20 and 200 nM PMA for 20 minutes. IB assays of pPKCȕ1Ser661, PKCȕ1, pAKTSer473, AKT, p-AKT substrates and ȕ-actin are shown. Figure 20B) Serum starved MCF7AC1 cells were pretreated with vehicle control or 0.01, 0.1 and 5 μM ENDX for two hours followed by the addition of 200 nM PMA for 20 minutes as indicated. IB assays of pPKCȕ1Ser661, PKCȕ1, pAKTSer473, AKT, p-AKT substrates and ȕ-actin are shown. Figure 20C) Serum starved MCF7AC1 cells were pretreated with vehicle control or 1 μM ENZA for two hours followed by the addition of 200 nM PMA for 30 minutes as indicated. IB assays of pPKCȕ1Ser661, PKCȕ1, pAKTSer473, AKT and ȕ-actin are shown. Figure 20D) Serum starved MCF7AC1 cells were pretreated with vehicle control, 0.01, 0.1 and 5 μM ENDX, 0.1 μM TAM or 0.1 μM ICI followed by the addition of 100 nM insulin for one hour as indicated. IB assays of pPKCȕ1Ser661, PKCȕ1 and ȕ-actin are shown. Figure 20E) MCF7AC1 cells in CSS medium were transfected with non-targeting (siNT) or PKCȕ-targeting (siPKCȕ) siRNAs for 48 hours. IB assays of PKCȕ1, pAKTSer473 and ȕ-actin are shown. The histogram indicates the percentage (%) of PKCȕ1 and pAKTSer473 protein levels remaining upon PKCȕ1 knockdown in siPKC -treated cells relative to siNT-treated cells from two biological replicates ± s.d. The vertical lines indicate that different lanes of the same blot were juxtaposed to remove intervening lanes. Figure 20F) MCF7AC1 cells were treated with siNT or siPKCȕ1 in CSS medium for six days. Cell viability was assessed by crystal violet assays. Attorney Docket No. 07039-2296WO1 / 2023-600 Data represents the mean of six wells per treatment performed as biological triplicates ± s.d. *, p 0.05; **, p 0.01; ***, p < 0.001 by one sample t-test. Figures 21A – 21D. ENDX replicates the effects of the pan-AKT inhibitor, MK-2206, on apoptosis. Figures 21A and 21C) MCF7AC1 (Figure 21A) and T47D-LTED (Figure 21C) cells were co-treated with vehicle control or 0.01, 0.1 and 5 μM ENDX or MK-2206 in the presence of IncuCyte Annexin V green and NucLight red reagents in CSS medium for 48 hours. The apoptosis (%) graph was generated as described in Figure 14B. Data represents the mean of six wells per treatment performed as biological duplicates ± s.d. ***, p 0.001; ****, p < 0.0001 by one-way ANOVA. Figures 21B and 21D) MCF7AC1 (Figure 21B) and T47D-LTED (Figure 21D) cells in CSS medium were treated with vehicle control or 0.01, 0.1 and 5 μM ENDX for 48 hours (Figure 21B) and 24 hours (Figure 21D), respectively. IB assay of pAKTSer473, AKT, PARP, cleaved PARP and ȕ-actin are shown. Figures 22A – 22C. Expression of catalytically active AKT diminishes ENDX ability to induce apoptosis. Figure 22A) MCF7AC1caAKT cells were grown in FBS medium in the absence (-) or presence (+) of cumate for 48 hours. IB assay of C-terminally hemagglutinin tagged AKT (AKT-HA), endogenous AKT and ȕ-actin. Figure 22B) MCF7AC1caAKT cells grown in FBS medium in the (-) or (+) of cumate for 48 hours. IB assay of pAKT-substrates (SEQ ID NO:10) and ȕ-actin. Figure 22C) MCF7AC1caAKT cells grown in CSS medium were treated for 48 hours in the (-) or (+) of cumate, following which cells were co-treated with vehicle control or 5 μM ENDX and IncuCyte Annexin V green and NucLight red reagents for 48 hours. The percentage (%) of cells undergoing apoptosis was calculated as indicated in Fig.1B. Data represents the mean of six wells per treatment performed as biological duplicates ± s.d. ns: nonsignificant. **, p < 0.01 by one-way ANOVA. Figures 23A – 23B. Summary of ENDX anticancer effects in ERĮ+ breast cancer cells. Figure 23A) Activation of PKCȕ1Ser661 by the PKC agonist PMA and/or insulin phosphorylates AKTSer473 resulting in the activation of p-AKT downstream substrates, which mediates cell survival. Figure 23B) ENDX binds to PKCȕ1 and facilitates PKCȕ1 protein degradation, resulting in the attenuation of phosphorylation of AKTSer473 as well as downstream p-AKT substrates, leading to induction of apoptosis. Attorney Docket No. 07039-2296WO1 / 2023-600 Figures 24A – 24C. Effects of ENDX on the global protein expression in MCF7AC1 cells. Figure 24A) Stacked barplot showing the number of total proteins and the percentage that are upregulated (red) and downregulated (blue) (Fold change (FC) |1.5|; p value < 0.05) in 0.01, 0.1 and 5 μM ENDX treated cells relative to vehicle treated cells for 24 hours in CSS medium, as identified by mass spectrometry analysis. Figure 24B) Venn diagram indicating the overlap of the total proteins in the 0.01, 0.1 and 5 μM ENDX treated cells relative to vehicle treated cells. Figure 24C) Venn diagram indicating the overlap of the list of the phosphosites and the list of the total proteins that are altered by ENDX treatment regardless of the concentration. Figures 25A – 25B. ENDX effects on AKT phosphorylations in ERĮ+ breast cancer models in vivo and in vitro. Figure 25A) MCF7AC1 xenograft tumors were treated with control, letrozole, tamoxifen (TAM) and 25 mg/kg and 75 mg/kg ENDX for four weeks in vivo. IB assay of pAKTSer473, pAKTThr308, AKT, p-AKT substrates are shown with ȕ- actin as a loading control. Figure 25B) Serum starved T47D cells were pretreated with vehicle control or 0.01, 0.1 and 5 ^M ENDX and 0.1 ^M tamoxifen (TAM) and ICI-182780 (ICI) for two hours followed by the addition of 100 nM insulin treatment for one hour. IB assay of pAKTSer473, pAKTThr308, AKT and ȕ-actin. Figures 26A – 26D. ENDX binds to PKCȕ1. Figures 26A and 26B) SPR sensograms (relative units, RU) of ENDX binding at the indicated concentrations to immobilized PKCȕ1. Figures 26C and 26D) Dot plots showing the affinity binding of ENDX to PKCȕ1 corresponding to sensograms in Figures 26A and 26B, respectively. Figures 27A – 27D. PKCȕ1 knockdown by different strategies and the effects of PKCȕ1 knockdown on PKC family members. Figure 27A) MCF7AC1 cells in CSS medium were transfected with non-targeting (siNT) or PKCȕ1-targeting (siPKCȕ1) siRNAs for 72 hours. IB assay of PKCȕ1 and ȕ-actin are shown. The histogram indicates the percentage (%) of PKCȕ1 protein levels remaining in siPKCȕ1-transfected cells compared to siNT- transfected cells. Figure 27B) MCF7AC1 cells in CSS medium in the absence (-) or presence (+) of doxycycline (Dox) for 72 hours. IB assay of PKCȕ1 and ȕ-actin are shown. The histogram indicates the percentage (%) of PKCȕ1 protein levels remaining in dox induced cells compared to noninduced cells. Figure 27C) IB assay of basal PKCȕ2 protein expression Attorney Docket No. 07039-2296WO1 / 2023-600 and ȕ-actin in MCF7AC1 and K562 (a positive control for PKCȕ2) cells. Figure 27D) IB assay of the relative protein expression of PKC family members and ȕ-actin in siNT and siPKCȕ1 transfected MCF7AC1 cells. For Figures 27A and 27B, data represent mean of six wells per treatment performed as biological triplicates ± s.d. *, p < 0.05; ****, p < 0.0001 by one sample t test. Figures 28A – 28D. Effects of ENDX on PKCȕ1, phospho-PKCB1Ser661 and phospho-AKTSer473 expression levels in ERĮ- breast cancer cells. Figure 28A) Basal expression of PKCȕ1 and ERĮ in the indicated cell lines. K562 cells serves as the positive control for PKCȕ1. Figure 28B) The effects of ENDX pretreatment for two hours followed by treatment in the presence or absence of 100 nM insulin for one hour on the protein expression levels of PKCȕ1, pAKTSer473, AKT and ȕ-actin in MDAMB231 and HEK293F cells. Figure 28C) The effects of ENDX pretreatment for two hours followed by treatment in the presence or absence of 100 nM insulin for one hour on the protein expression levels of PKCȕ1, pAKTSer473, AKT, ERĮ and ȕ-actin in MDAMB231-ERĮ cells. Dox was added 48 hours prior to the treatments to allow for induction of ERĮ protein expression. Figure 28D) The effects of ENDX pretreatment for two hours followed by treatment in the presence or absence of 20 nM PMA for 20 minutes on the protein expression levels of pPKCȕ1Ser661, PKCȕ1 and ȕ-actin in MDAMB231 and HEK293F cells. Figures 29A – 29J. Effects of the pan-AKT inhibitor MK-2206 or ENDX on phenotypes in ERĮ+ breast cancer cells. Figure 29A) MCF7AC1 cells were treated with vehicle control or 0.01, 0.1 and 5 ^M MK-2206 in CSS medium for six days. Cell viability is assessed by the crystal violet assay. Figure 29B) MCF7AC1 cells were co-treated with vehicle control or 0.01, 0.1 and 5 ^M MK-2206 and IncuCyte Annexin V green and NucLight rapid red reagents in CSS medium for 48 hours. The apoptosis graphs are presented as the green object count divided by the red object count and displayed as percentage using the IncuCyte S3 analysis software. Cells were plated at a density of 2000 cells per well. Figure 29C) MCF7AC1 cells were treated with vehicle control or 0.01, 0.1 and 5 ^M MK-2206 in CSS medium for 24 hours. IB assay of pAKTSer473, AKT, PARP, cleaved PARP and ȕ-actin. Figure 29D) MCF7AC1 xenograft protein lysates were treated with the indicated drugs for four weeks. IB assay of cPARP, PARP and ȕ-actin. Figure 29E) Attorney Docket No. 07039-2296WO1 / 2023-600 T47D cells were cultured in FBS versus CSS medium for six days. Cell viability is assessed by the crystal violet assay. Figure 29F) Parental T47D and T47D-LTED cells were processed for protein lysates. IB assay of ERĮ, pAKTSer473 and ȕ-actin. Figure 29G) T47D-LTED cells were treated with vehicle control or 0.01, 0.1 and 5 ^M MK-2206 in CSS medium for six days. Cell viability is assessed by the crystal violet assay. Figure 29H) T47D-LTED cells were co-treated with vehicle control or 0.01, 0.1 and 5 ^M MK-2206 and IncuCyte Annexin V green and NucLight rapid red reagents in CSS medium for 48 hours. Percentage of cells undergoing apoptosis was calculated as mentioned in Figure 29B. Figure 29I) T47D-LTED cells were treated with vehicle control or 0.01, 0.1 and 5 ^M MK-2206 in CSS medium for 24 hours. IB assay of pAKTSer473, AKT, PARP, cleaved PARP and ȕ-actin. Figure 29J) T47D-LTED cells were treated with vehicle control or 0.01, 0.1 and 5 ^M ENDX in CSS media for six days. Cell viability is assessed by the crystal violet assay. For Figures 29A, 29B, 29E, 29G, 29H, and 29J, data represent mean of six wells per treatment performed as biological duplicates ± s.d. *, p ^ 0.05, ****, p ^ 0.0001 by one-way ANOVA for Figures 29A, 29B, 29G, 29H, and 29I and unpaired test for Figure 29E. Figure 30. Unlike ENDX, TAM and ICI do not induce apoptosis. MCF7AC1 cells were co-treated with vehicle control or 5 ^M ENDX, 0.1 ^M TAM and 0.1 ^M ICI and IncuCyte Annexin V green and NucLight rapid red reagents in CSS medium for 48 hours. The percentage (%) of cells undergoing apoptosis is calculated as described in Example 2. Cells were plated at a density of 2000 cells per well. Data represents mean of six wells per treatment performed as biological duplicates ± s.d. *, p ^ 0.05 by one-way ANOVA. Figures 31A – 31C. ENDX neither induces apoptosis nor inhibit growth of ER- breast cancer cells. Figure 31A) MDAMB231 cells grown in CSS medium were were co-treated with vehicle control or the indicated ENDX concentrations, IncuCyte Annexin V green and NucLight red reagents in CSS medium for 48 hours. The apoptosis graphs are presented as the green object count divided by the red object count and displayed as percentage using the IncuCyte S3 analysis software. Cells were plated at a density of 2000 cells per well. Data represents the mean of six wells per treatment performed as biological duplicates ± s.d. ****, p ^ 0.0001 by one-way ANOVA. Figure 31B) Effects of ENDX on cell proliferation at the indicated concentrations on day seven of treatment. Cell viability was assessed by the crystal Attorney Docket No. 07039-2296WO1 / 2023-600 violet assay. Figure 31C) immunoblot of basal PKCȕ1 protein expression in the indicated cell lines. Figures 32A – 32B. Figure 32A) Endoxifen increased the expression of IFNȖ by CD8+ cells. Peripheral blood mononuclear cells (PBMC) were isolated from normal donors’ apheresis cones and treated with endoxifen (2μM) or Enzastaurin (2μM) for 5 days. Control cells were left untreated. Cells were then treated with brefeldin for 4 hours, followed by staining with fluorochrome-conjugated antibodies for surface and intracellular markers and flow cytometry analysis. Bar graphs and representative dot plots depict the percentage of IFNȖ+ CD8+ cells. n=8, *p<0.02 denotes significant difference. Figure 32B) IFNȖ expression can be used as a favorable prognostic marker in breast cancer. Kaplan-Meier (KM) survival analysis shows the correlation between the gene expression level of IFNȖ and overall survival (OS) in different subtypes of breast cancer tumors. Logrank P<0.05 denotes significance. Figure 33. Endoxifen increased the expression of Granzyme B and IL-2, but not perforin, in CD8+ and CD4+ T cells. PBMCs were treated with endoxifen (2μM) or enzastaurin (2μM) for 5 days. Cells were then treated with brefeldin for 4 hours to stop secretion for 4 hours. Cells were stained with viable cell markers and Fluorochrome- conjugated surface and intracellular antibodies and analyzed by flow cytometry. Figures 34A – 34C. CyTOF analysis showing that endoxifen altered the expression of immune cells markers. PBMCs were isolated from normal donors and treated with/without endoxifen (2μM) for 5 days. Cells were then treated with brefeldin for 4 hours, followed by staining with PBMC enhanced panel of immune markers and CyTOF analysis. Figure 34A) tSNE maps show the clusters of the cells that were altered in endoxifen treated cells. Figure 34B) Bar graphs represent the number of the cells in each cluster. Figure 34C) Heat map shows the expression of each of 36 immune markers within each cluster or population (Pop). Figures 35A – 35B. Pre-treatment of PBMCs with endoxifen enhanced their ability to suppress the proliferation of MCF7 tumor cells. PBMCs were treated with endoxifen (2μM) or enzastaurin (2μM) for 5 days, followed by washing the cells and removing the drug and co-culturing with MCF7AC1 tumor cells in vitro. Cell proliferation was monitored by IncuCyte for a duration of 5 days (Figures 35A and 35B, left panels). In a similar assay set- Attorney Docket No. 07039-2296WO1 / 2023-600 up, MCF7AC1 or MCF7 endoxifen-resistant tumor cells were pretreated with abemaciclib (500 nM) for 5 days and then co-cultured with endoxifen-pretreated PBMCs, followed by IncuCyte analysis (Figures 35A and 35B, middle and left panels). Line graphs represent the proliferation of tumor cells over a duration of 5 days using live-cell analysis by IncuCyte. Figures 36A-36D. CD3+ T cells were isolated via negative selection from healthy control donor PBMCs according to EasySep kit instructions (STEMCELL Cat. #19051) and cultured for 3-days under activating conditions using CD3/CD28 Dynabeads (ThermoFisher Scientific Cat.11131D). Figure 36A) Unstimulated cells at day-3 of in vitro culture. Figures 36B-36D) Stimulated cells at day-3 of in vitro culture. Figure 36B) No ENDX. Figure 36C) 2^M ENDX, Figure 36D) 5^M ENDX. Figures 37A-37H. Gating strategy from Fig.36 implemented to analyze CD4+ and CD8+ T cell subsets. Fulvestrant (FUL). Figure 37A) Frequency (%) of TIM-3+ CD4+ T cells. Figure 37B) Surface expression of TIM-3 (Geometric (g) MFI) on TIM-3+ CD4+ T cells. Figure 37C) Frequency (%) of TIM-3+ CD8+ T cells. Figure 37D) Surface expression of TIM-3 (gMFI) on TIM-3+ CD8+ T cells. Figures 37E-37F) CD4+ T cells. Figure 37E) Frequency (%) of PD-1+ cells. Figure 37F) Left: histogram of PD-1 expression. Right: PD-1 surface expression (gMFI) on PD-1+ CD4+ T cells. Figures 37G-37H) CD8+ T cells. Figure 37G) Frequency (%) of PD-1+ cells. Figure 37H) Left: histogram of PD-1 expression. Right: PD-1 surface expression (gMFI) on PD-1+ CD8+ T cells.. Figures 38A-38E. CD3+ T cells were isolated via negative selection from healthy control donor PBMCs according to EasySep kit instructions (STEMCELL Cat. #19051) and cultured for 5-hours under activating conditions using CD3/CD28 Dynabeads (ThermoFisher Scientific Cat.11131D) or PMA (50ng/mL) and Ionomycin (1^g/mL). Figure 38A) Representative gating strategy from unstimulated CD3+ negative selected cells. Figure 38B) Separate CD3 staining showing purity of EasySep T cell negative selection kit. Figure 38C) Plots showing CD4+ and CD8+ T cell gates under CD3/CD28 Dynabeads (left) or PMA/Iono (right) activation methods. Figures 38D-38E) Representative gating for effector cytokines IL- 2, IFN-Ȗ, and TNF-Į. Figure 38D) CD4+ T cells. Figure 38E) CD8+ T cells. Figures 39A-39B. Frequency of IFN-Ȗ+ (left), TNF-Į+ (middle), IL-2+ (right) cells. Figure 39A) CD4+ T cells. Figure 39B) CD8+ T cells. Attorney Docket No. 07039-2296WO1 / 2023-600 DETAILED DESCRIPTION This document provides methods and materials for treating cancers (e.g., ER+ cancers and/or ER- cancers such as ER- breast cancers (e.g., TNBCs)). For example, one or more ENDX compounds can be administered to a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal. When treating a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) as described herein, the mammal can have any type of cancer. In some cases, a cancer treated as described herein can include one or more solid tumors. In some cases, a cancer treated as described herein can be a blood cancer. In some cases, a cancer treated as described herein can be a primary cancer. In some cases, a cancer treated as described herein can be a metastatic cancer. In some cases, a cancer treated as described herein can be a refractory cancer. In some cases, a cancer treated as described herein can be a relapsed cancer. Examples of cancers that can be ER+ and that can be treated as described herein include, without limitation, breast cancers, ovarian cancers, endometrial cancers, and lung cancers. Examples of cancers that can be ER- and that can be treated as described herein include, without limitation, breast cancers (e.g., TNBCs), ovarian cancers, endometrial cancers, brain and/or central nervous system cancers (e.g., gliomas and glioblastomas), bone cancers (e.g., osteosarcomas and Ewing sarcomas, biliary tract cancers, thyroid cancers, lung cancers (e.g., mesotheliomas and non-small cell lung cancers), colorectal cancers, head and neck cancers, stomach cancers, pancreatic cancers, kidney cancers (e.g., clear cell renal carcinomas and rhabdoid cancers), liver cancers (e.g., hepatocellular carcinomas), prostate cancers, testicular cancers, skin cancers (e.g., melanomas), leukemias, and lymphomas (e.g., acute myeloid leukemias (AMLs)). In some cases, the methods described herein can include identifying a mammal (e.g., a human) as having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). Any appropriate method can be used to identify a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). For example, imaging techniques, biopsy techniques, and molecular techniques (e.g., molecular techniques to detect RNA expression or protein expression such as immunohistochemistry) can be used to Attorney Docket No. 07039-2296WO1 / 2023-600 identify mammals (e.g., humans) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). Any type of mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) can be treated as described herein. Examples of mammals that can have an ER- cancer (e.g., an ER- breast cancer such as a TNBC) and can be treated with one or more ENDX compounds as described herein include, without limitation, humans, non- human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats,. In some cases, a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) can be a female. For example, a premenopausal female having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) can be treated with one or more ENDX compounds as described herein. In some cases, a human having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) can be treated with one or more ENDX compounds as described herein. A mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) can be administered or instructed to self-administer one or more (e.g., one, two, three, four, or more) ENDX compounds described herein. An ENDX compound can be any type of ENDX compound. For example, an ENDX compound can be a trans isomer of ENDX (E-ENDX) or a cis isomer of ENDX (Z-ENDX).
Attorney Docket No. 07039-2296WO1 / 2023-600 In some cases, an ENDX compound can have one of the following structures: In In some cases, an ENDX compound can be in the form of a salt. For example, an ENDX compound can be in the form of a hydrochloride salt (e.g., a Z-ENDX hydrochloride salt). Any appropriate amount (e.g., any appropriate dose) of one or more ENDX compounds can be administered to a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). An effective amount (e.g., a therapeutically effective amount) of a composition containing one or ENDX compounds described herein can be any amount that can treat a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) as described herein without producing significant toxicity to the mammal. In some cases, an effective amount of one or more ENDX compounds can be about 40 milligrams (mg) or more. In some cases, an effective amount of one or more ENDX compounds can be a plasma concentration of from about 300 nM to about 3000 nM (e.g., from about 300 nM to about 2500 nM, from about 300 nM to about 2000 nM, from about 300 nM to about 1500 nM, from about 300 nM to about 1000 nM, from about 300 nM to about 750 nM, from about 300 nM to about 500 nM, from about 500 nM to about 3000 nM, from about 750 nM to about 3000 nM, from about 1000 nM Attorney Docket No. 07039-2296WO1 / 2023-600 to about 3000 nM, from about 1500 nM to about 3000 nM, from about 2000 nM to about 3000 nM, from about 2500 nM to about 3000 nM, from about 500 nM to about 2500 nM, from about 750 nM to about 2000 nM, from about 1000 nM to about 1500 nM, from about 500 nM to about 1500 nM, from about 1000 nM to about 2000 nM, or from about 1500 nM to about 2500 nM). In some cases, an effective amount of one or more ENDX compounds can be from about 20 mg/day to about 360 mg/day (e.g., from about 20 mg/day to about 340 mg/day, from about 20 mg/day to about 300 mg/day, from about 20 mg/day to about 250 mg/day, from about 20 mg/day to about 200 mg/day, from about 20 mg/day to about 150 mg/day, from about 20 mg/day to about 100 mg/day, from about 20 mg/day to about 50 mg/day, from about 50 mg/day to about 360 mg/day, from about 100 mg/day to about 360 mg/day, from about 150 mg/day to about 360 mg/day, from about 200 mg/day to about 360 mg/day, from about 250 mg/day to about 360 mg/day, from about 300 mg/day to about 360 mg/day, from about 50 mg/day to about 350 mg/day, from about 100 mg/day to about 300 mg/day, from about 150 mg/day to about 250 mg/day, from about 50 mg/day to about 150 mg/day, from about 100 mg/day to about 200 mg/day, from about 150 mg/day to about 250 mg/day, or from about 200 mg/day to about 300 mg/day). In some cases, an effective amount of one or more ENDX compounds can be as described elsewhere (see, e.g., Goetz et al., J. Clin. Oncol., 35:3391-3400 (2017)). In some cases, one or more ENDX compounds described herein can be administered to a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) together with one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell. An agent that can alter the antigens presented on the surface of a cancer cell can be any appropriate type of molecule (e.g., small molecules and polypeptides such as antibodies). In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a CDK4/6 inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a cell cycle checkpoint inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be an ALK inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a RET inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be an interferon. In some Attorney Docket No. 07039-2296WO1 / 2023-600 cases, an agent that can alter the antigens presented on the surface of a cancer cell can be an anthracyclin. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a platinum-based agent. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a HDAC inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a tyrosine kinase inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a proteasome inhibitor. Examples of agents that can alter the antigens presented on the surface of a cancer cell include, without limitation, abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti- CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU- D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602. Examples of therapies that can alter the antigens presented on the surface of a cancer cell include, without limitation, radiation therapies and oncolytic virus therapies. One or more ENDX compounds described herein (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal having an ER+ and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). For example, a therapeutically effective amount of one or more ENDX compounds described herein can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. A pharmaceutical Attorney Docket No. 07039-2296WO1 / 2023-600 composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules. A composition (e.g., a pharmaceutically acceptable composition) including one or more ENDX compounds described herein (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be administered locally or systemically. A composition containing one or more ENDX compounds described herein (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be designed for oral, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), or inhaled. For example, a composition containing one or more ENDX compounds described herein (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be administered systemically by an oral administration to or inhalation by a mammal (e.g., a human). When being administered orally, a composition containing one or more ENDX compounds described herein (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be in the form of a pill, tablet, or capsule. In some cases, one or more ENDX compounds (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC)) to reduce the size of the cancer present within a mammal. For example, the materials and methods described herein can be used to reduce the number of cancer cells present within a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, the materials and methods described herein can be used to reduce the size (e.g., volume) of one or more tumors present within a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. Attorney Docket No. 07039-2296WO1 / 2023-600 In some cases, one or more ENDX compounds (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC)) to improve survival of the mammal. For example, disease-free survival (e.g., relapse-free survival) can be improved using the materials and methods described herein. For example, progression-free survival can be improved using the materials and methods described herein. In some cases, the materials and methods described herein can be used to improve the survival of a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, one or more ENDX compounds described herein (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be used as the sole active agent(s) to treat a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). For example, a composition including one or more ENDX compounds (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell) can include the one or more ENDX compounds (and, optionally, the one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell) as the sole active agent(s) to treat a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). In some cases, one or more ENDX compounds described herein (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be administered to a mammal (e.g., a human) having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) together with one or more (e.g., one, two, three, four, or more) additional agents/therapies used to treat an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). In some cases, an anti- cancer agent can be a targeted therapy. In some cases, an anti-cancer agent can be a hormone therapy. In some cases, an anti-cancer agent can be an immunotherapeutic agent. Examples of anti-cancer agents include, without limitation, trametinib, dabrafenib, binimetinib, selumntinib, vemurafenib, encorafenib, cobimetinib, goserelin, leuprolide, tamoxifen, Attorney Docket No. 07039-2296WO1 / 2023-600 letrozole, anastrozole, exemestane, bevacizumab, rucaparib, and any combinations thereof. In cases where one or more ENDX compounds (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell) are used with one or more additional agents treat a cancer, the one or more additional agents can be administered at the same time (e.g., in a single composition) or independently. In some cases, one or more ENDX compounds (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell) can be administered first, and the one or more additional agents administered second, or vice versa. Examples of therapies that can be used to treat cancer include, without limitation, surgery, radiation therapy, carbon ion therapy, and proton therapy. In cases where one or more ENDX compounds (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell) are used in combination with one or more additional therapies used to treat cancer, the one or more additional therapies can be performed at the same time or independently of the administration of one or more ENDX compounds (and, optionally, the one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell). For example, the one or more ENDX compounds (and, optionally, one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell) can be administered before, during, and/or after the one or more additional therapies are performed. In certain instances, an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) within a mammal can be monitored to evaluate the effectiveness of the cancer treatment. Any appropriate method can be used to determine whether or not a mammal having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) is treated. For example, imaging techniques or laboratory assays can be used to assess the number of cancer cells and/or the size of a tumor present within a mammal. For example, imaging techniques or laboratory assays can be used to assess the location of cancer cells and/or a tumor present within a mammal. The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. Attorney Docket No. 07039-2296WO1 / 2023-600 EXAMPLES Example 1: Allosteric Regulation and Pharmaceutical Targeting of Protein Kinase Cȕ Protein Kinase C (PKC) enzymes are a family of ten kinases that mediate signal transduction in a wide range of cellular pathways, and altered PKC activity is implicated in many disease states, including cancer and neurodegenerative diseases (Leitges et al., Science, 273:788-791 (1996); Tagawa et al., Hum. Mol. Genet., 24:540-558 (2015); Zarate et al., CNS Drugs, 23:569-582 (2009); Geraldes et al., Circ. Res., 106:1319-1331 (2010); Palaniyandi et al., Cardiovasc. Res., 82:229-239 (2009); Garg et al., Oncogene, 33:5225-5237 (2014); and Sadeghi et al., Int. J. Mol. Sci., 22:5527 (2021)). PKCs are ubiquitously expressed throughout the body, yet only a variable subset of the PKC enzymes is present in each tissue type. The four canonical PKC family members (Į, ȕI, ȕII, and Ȗ) are activated upon interacting with the plasma membrane in response to diacylglycerol (DAG) and Ca2+ signals. Although PKCĮ, ȕI, ȕII, and Ȗ are >65% identical in sequence (Figure 7A), they perform distinct cellular functions and display varied sensitivity to lipid signals (Kedei et al., Cancer Res., 64:3243-3255 (2004); and Steinberg, Physiol. Rev., 88:1341-1378 (2008)). PKCȕI and PKCȕII isoforms are expressed from the same gene and differ by only a 50 aa C-terminal segment that is derived from a regulated alternative splicing event (Figure 7A) (Chalfant et al., J. Biol. Chem., 270:13326-13332 (1995)), suggesting that even subtle sequence differences can impart altered activity on each isozyme. Despite decades of investigation, the molecular mechanisms of PKC regulation and activation are not known. In spite of the considerable interest in developing drug candidates that inhibit (or activate) PKCs to treat an array of diseases including a variety of cancers, a strategy of using ATP40 competitive inhibitors has, thus far, been unsuccessful. Staurosporine and derivatives, which target the highly conserved ATP binding site, have demonstrated limited or no antitumor activity in multiple different cancers despite nanomolar affinity and are prone to off-target toxicity (Mina et al., Invest. New Drugs, 27:565-570 (2009); Clemons et al., Breast Cancer Res. Treat., 124:177-186 (2010); Millward et al., Br. J. Cancer, 95:829-834 (2006); Robertson et al., J. Clin. Oncol., 25:1741-1746 (2007); Attorney Docket No. 07039-2296WO1 / 2023-600 Macedo et al., Cancer Res., 68:3516-3522 (2008); and Kreisl et al., Neuro. Oncol., 12:181- 189 (2010)). This Example demonstrates that endoxifen (ENDX) can act as an allosteric inhibitor of PKC polypeptides. Results It was sought to improve on the established insect cell PKC expression system (Mukai et al., Methods Mol. Biol., 233:21-34 (2003)) by using HEK293F cells and the YFP- tag system (Schellenberg et al., Protein Sci., 27:1083-1092 (2018)) to generate recombinant PKCȕII (Figure 7C). The final, purified PKCȕII contains the phosphorylation sites (T500- activation loop, T641-turn motif, and S660-hydrophobic motif) (Figure 7D) and migrates as a single band on phos-tag SDS-PAGE (Figure 1B), indicating that the YFP-tagged purified PKC was fully phosphorylated at all three sites. Crystals of PKCȕII in the presence of a non- hydrolysable ATP analogue AMPPNP were obtained and a 3.3A structure was solved (Figures 1C and 1D; Table 1).
Attorney Docket No. 07039-2296WO1 / 2023-600 Table 1. X-ray diffraction data collection and structure refinement statistics Continuous electron density was visible for all four structured domains and the PS, as well as linker residues between the C1a, C1b, and C2 domains (Figure 8A), and portions of the linker between the C2 domain and the kinase domain (Figure 8B). Density was observed for the three phosphorylated residues required for proper PKC folding and enzyme function (Figure 8C), C1 domain Zn2+ ions (Figure 8D), and an active–site engaged Mg-AMPPNP (Figure 1F). Altogether, 91% and 94% of the residues in each of two chains respectively were observed, which represented the most complete picture of any PKC isoform to date. Attorney Docket No. 07039-2296WO1 / 2023-600 Molecular basis of PKCȕII autoinhibition and regulation by phosphorylation All domains (PS, C1a, C1b, C2, and kinase) were assigned to each of two chains based on the maximum length that the disordered residues of unobserved linkers could span (Figure 9A). Both chains contained a kinase active site occupied by the PS sequence, but chain A formed an intramolecular complex, and chain B formed a domain-swapped complex with the kinase domain of an adjacent monomer through a nearly identical molecular interface (Figure 9B). Thus, the structure revealed the complete molecular architecture of the inhibited, inactive PKCȕII. A complex network of inter-domain interactions comprised the inactive state of PKCȕII (Figures 1E and 1F). The N-terminal PS residues (aa 19-30) were positioned in the substrate binding site with the A25 sidechain in a position equivalent to a Ser/Thr in a true substrate (Figure 1F), with specific recognition of the hydrophobic and positively charged residues contained within PKC substrate motifs (Figure 9C). The C1b domain interacted with the kinase domain and PS, which sequestered the hydrophobic lipid-binding residues and buttressed the PS in the kinase active site (Figure 1E). The C1a domain interacted with the kinase domain and C-terminal extension through a small hydrophobic patch and salt bridges such that most of its lipid binding residues and the DAG binding pocket remained solvent- accessible (Figure 1F). This configuration was supported through site-directed mutagenesis that showed F43A, D382K, or E655K mutations disrupted the C1a-Kinase interface and resulted in increased phospholipid interactions or a more extended conformation. A conserved PKC domain architecture consisting of a PS sequence immediately adjacent to a C1 domain in all PKC isozymes (Figure 7B) suggested the same regulatory mechanism was common to all PKC isoforms. The C2 domain interacted with the C1a and C1b domains such that its Ca2+ binding site and lipid binding residues were accessible for interaction with the plasma membrane. The kinase domain contained three phosphorylated residues (pT500, pT641, and pS660), which were necessary for catalytic activity (Figure 1F). pT500 served a structural role mediated by salt bridges and a hydrogen bond to surrounding residues as well as to PS residues R27 and N30. The latter suggests pT500 formed part of the substrate binding site and explains both the requirement of T500 phosphorylation for kinase activity and the strong counter-selection against negatively charged residues at the +2 and +5 Attorney Docket No. 07039-2296WO1 / 2023-600 substrate positions. pT641 formed salt bridges to two lysines located in the ATP-binding ȕ- hairpin and Į-helix active-site motifs, as well as an additional hydrogen bond to S111 of the C1b domain. pS660 was located distal from the active site and played a structural role in stabilizing the C-terminal V5 domain by interacting with Q411 and R336 residues. Phosphorylation of these three residues also regulated the affinity of PKC for lipid membranes despite being located outside of the lipid–binding domains. The pT500–PS and pT641–C1b interactions stabilized the inter-domain interfaces that sequestered the lipid binding surfaces of the C1a and C1b domains in an inaccessible conformation. Thus, in addition to the structural and substrate-binding roles, pT500 and pT641 also mediated molecular interactions that maintain PKC in its inactive state and in turn reduced the lipid binding affinity of the fully phosphorylated protein. Crystal structures of PKCȕI reveal active and inactive states PKCȕI and PKCȕII isoforms differed by only 50 C-terminal amino acids (Figure 7A) derived from an alternative splicing event that is responsive to signals such as extracellular glucose levels and signaling by AKT2 kinase. Although the 50 residues were derived from different exons, many of the residues, including the two phosphorylation sites (pT642 and pS661) present in this region, were conserved. To determine the molecular basis of isoform- specific differences recombinant PKCȕI (Figure 10A) was generated, and it was co- crystalized with AMPPNP in two crystal forms (Figures 2A and 10C; Table 1). Electron density was observed for all four domains (C1a, C1b, C2, and kinase) together with phosphorylation at the corresponding ȕI residues T500, T642, and S661 (Figure 10B and 10D), but the PS of only one monomer was visibly bound to the adjacent kinase domain through a domain swap in crystal form 1 (Figure 11A). Similar to PKCȕǿǿ, the hinge region between the C2 and kinase domain as well as the N-terminal 18 amino acids were disordered. The linkers between the C1a, C1b, and C2 domains of PKCȕI were visible (Figure 10E) except for a gap of 12–13 amino acids linking the C1a and C1b domains in crystal form 1 (Figure 11B). However, this gap was small enough that it did affect the ability to unambiguously assign the domains to individual chains. AMPPNP nucleotide was only present in the monomer with a bound PS, and the active site was slightly more open when the Attorney Docket No. 07039-2296WO1 / 2023-600 nucleotide was absent (Figure 11C). Nonetheless, all monomers contained the DFG motif and helix ĮC “in” conformations which indicated the kinase domains were in the activated conformation. To identify the biological unit of PKCȕI from the crystal lattice, the kinase domain from the three asymmetric units were aligned to explore the local symmetry environment and to identify common interaction modalities present amongst the domain arrangements. Each kinase domain formed an interface with three N-terminal domain modules (Figure 11D), and two of them were comprised of similar molecular interactions (Figure 2A; N-term 1 & 2), while the third interface was variable amongst the three asymmetric units (N-term 3). The interface with N-term 2 describes a conformation that was similar to PKCȕII (Figure 1) but with an altered location for the C1b domain, which was attributed to the auto-inhibited conformation for the PKCȕI enzyme. The interface with N-term 1 positioned the C1b and C2 domains, so they interacted with the kinase domain distal from the active-site in an unexpected conformation. AlphaFold also predicted a very similar structural arrangement (Figure 11E), which prompted the exploration of possible functional roles for this second PKC conformation. PKCȕI active conformation An unresolved mystery about PKC enzymes is how the activated state prevents the PS from re-engaging the active site. The domain arrangement comprised of the interface with N-term 1 cannot be attributed to any previously described structure of PKC isozymes and has a few striking differences compared to the inactive conformation. No PS was bound within the kinase domain of crystal form 2, and the active site appeared to be unobstructed and available to engage substrate. The C1b and C2 domains interacted with the kinase domain on the side opposite the active site, and the C1a domain was buttressed against the C1b and C2 domains (Figure 2A). The C1a and C1b domains contained a bound glycerol molecule in the DAG binding site (Figure 12A). Calcium was not included in the crystallization condition. The lipid binding surfaces of the C1a, C1b, and C2 domains aligned in the same plane, as would be the case were they bound or embedded in a plasma membrane. Furthermore, the PS Attorney Docket No. 07039-2296WO1 / 2023-600 would be anchored ~70A away from the active site and prevented from re-engaging the active site in this conformation (Figure 12B). To test the hypothesis that PKC can form an active conformation upon binding to the lipid membrane, a site-directed mutagenesis strategy was employed, and a panel of mutants was generated to disrupt the observed interfaces between the kinase and C1b/C2 domains (Figure 2B). Kinase activity stimulated by Ca2+ and DAG-containing phospholipids were assayed in vitro using the FRET–based CKAR substrate. All of the mutants that disrupted the C2-kinase and C1b-kinase interfaces impaired the kinase activity of PKCȕI (Figures 2C and 12C). Since these mutations did not affect residues involved in the active-site or lipid binding, impaired kinase activity was attributed to disruption of the interfaces between the lipid binding domains and the kinase domain observed in the crystal structure. These findings indicated that PKCȕI took on a defined and ordered conformation upon association with DAG-containing membranes that unmasked the kinase active site to activate PKCȕI. The Lipid-Lever mechanism of PKC activation Another fundamental question about the mechanism of PKC activation is the role of the phospholipid membrane. In the presence of Ca2+, PKCs can be activated by a phospholipid bilayer containing DAG or an agonist such as phorbol 12,13-dibutyrate (PDBu). However, in the absence of a phospholipid bilayer, PDBu and Ca2+ cannot activate PKCȕ kinase activity (Figure 3B). This suggested that it was not the DAG or phorbol agonist that was the activating ligand per se, but somehow the membrane itself drove a conformational change in PKC. The DAG/phorbol-binding cleft of the C1a domain is solvent-accessible, and PDBu binding would not disrupt the inactive conformation (Figure 12D), ruling out a direct competition mechanism and suggesting the phospholipid membrane itself is critical for driving the conformational change that activates PKC. Limited proteolysis was used to probe the conformation of PKCȕI and increased elastase cleavage was observed in the presence of Ca2+ and DAG-containing micelles but not in the presence of Ca2+ and PDBu, even at 100 ^M (Figure 3C), indicating that phorbol binding alone was insufficient to activate PKCȕI. Modelling of the C1a domain engaged in a lipid bilayer revealed that the C1a-PSkinase domain architecture observed in the crystal structure would necessitate that the Attorney Docket No. 07039-2296WO1 / 2023-600 kinase domain become embedded into the membrane as well (Figure 3A). When the hydrophobic residues of the C1a domain engaged the membrane lipids, a clash between the charged kinase domain and hydrophobic membrane would prevent the kinase domain from remaining bound to the C1a and PS (Figure 12E). In this way, the lipid membrane acted as a lever that can pry PS from the catalytic domain and disrupt the interdomain interactions that preserve the inhibited state via a mechanism referred to herein as a “lipid-lever”. The kinase domain occluded some of the lipid-binding surface centered around F43 (Figure 1F) in the inactive conformation, providing a mechanistic basis by which a fully folded kinase domain moderated membrane binding by the C1 domains. The lipid-lever mechanism is likely shared across PKC isoforms as the PS was always found immediately N-terminal to a C1 domain across the family of PKCs (Figure 7B). Molecular basis of isoform-specific differences between the splice variants PKCȕI and PKCȕII One well–characterized difference between PKCȕI and ȕII is the difference in affinity for phospholipid membranes (Kedei et al., Cancer Res., 64:3243-3255 (2004); and Truebestein et al., J. Mol. Biol., 428:121-141 (2016)), yet these splice variants contained identical lipid-binding domains, suggesting that a mechanism such as the lipid-lever links the variant C-termini to altered phospholipid binding. The crystal structures of PKCȕI and PKCȕII were examined to identify the molecular basis of isoform-specific effects. The active conformation did not contain any interactions between the unique C-terminal extension and lipid-binding domains, so the inhibited conformation of PKCȕI (crystal form 1) was compared with that of PKCȕII (Figure 4A). The most significant difference was the position of the C1b domain. In PKCȕII, the C1b domain was sequestered through extensive contact with the catalytic domain but in contrast was displaced with its lipid-binding residues exposed in PKCȕI (Figure 4B). This suggested a model whereby enhanced accessibility of C1b lipid-binding residues contributed to a stronger lipid interaction of the PKCȕI isoform. The C1b domain only contacted pT641 and F633 of the PKCȕII V5 domain, yet PKCȕI contained spatially equivalent residues (pT642 and F634) at the same locations (Figure 7A). In PKCȕII, F114 of the C1b domain packed against helix ĮB, whereas this helix was shifted Attorney Docket No. 07039-2296WO1 / 2023-600 in PKCȕI which occluded the F114 binding pocket. The ĮB shift can be attributed to PKCȕI F648, which corresponds to V647 in PKCȕII. The larger F648 residue shifted helix ĮB closer to the PS and occluded the pocket occupied by F114 in PKCȕII, thus preventing C1b from making close contact with this region (Figure 4C). To evaluate this model, a PKCȕI F648A mutant was engineered and the conformational state of PKCȕI (WT and F648A) and PKCȕII was probed using limited proteolysis. In the presence of Ca2+ and activating lipids elastase cleavage was observe at a site corresponding to the hinge region (Figure 4C). PCKȕI was more sensitive to elastase than PKCȕII, indicating that PKCȕI more easily adopted the active conformation in the presence of lipids. PKCȕI F648A was less sensitive to elastase, consistent with a model whereby a smaller residue yields an enzyme with enhanced stability of the inactive state. Thus, the more potent lipid binding of the PKCȕI isoform was attributed to a shift in the position of helix ĮB that displaced the C1b from its docked position. Endoxifen as an allosteric regulator of PKCȕ PKCs can be inhibited by tamoxifen (TAM) (Gundimeda et al., J. Biol. Chem., 271:13504-13514 (1996)) and more potently by its metabolite endoxifen (ENDX) (Ali et al., Bioorg. Med. Chem. Lett., 20:2665-2667 (2010)). The effects of ENDX and TAM on PKCȕI kinase activity were assessed in vitro in the presence and absence of activating lipids and it was found that ENDX was uniquely able to inhibit PKCȕI at clinically achievable concentrations (IC50 = 1.49 ^M; Goetz et al., J. Clin. Oncol., 35:3391-3400 (2017)), while TAM required much higher concentrations (IC50 = 5.9 ^M, p<0.001), beyond that which is achievable with the FDA approved 20 mg/day dose (Figure 5A). Both ENDX and TAM were less effective inhibitors in the presence of Ca2+ and activating phospholipid micelles, similar to when using a truncated, constitutively active PKCȕI consisting of only the kinase domain (Figure 5B). Additionally, differential scanning fluorimetry (DSF) analysis showed that both the N-terminal regulatory domain and the kinase domain bound to ENDX (Figure 13A), and ENDX exhibited distinctly non-competitive inhibition with ATP (Figure 5C) in contrast to the established ATP-competitive PKC inhibitor enzastaurin (Faul et al., Bioorg. Med. Chem. Lett., 13:1857-1859 (2003)). Collectively, these data suggested ENDX inhibits PKCȕI via a unique mechanism through binding to both the N-terminal and kinase domains. Furthermore, Attorney Docket No. 07039-2296WO1 / 2023-600 ENDX inhibited several PKC enzymes, albeit with a range of IC50 values, whereas the closely related AGC kinase PKA was completely unaffected by ENDX up to 100 ^M, suggesting specificity for PKC enzymes (Figure 13B). The effect of ENDX on the solution conformation of PKCȕI was evaluated using Small-Angle X-ray Scattering (SAXS) and it was found that ENDX but not TAM decreased the Rg and Dmax of PKCȕI protein (Figure 5D). A limited proteolysis assay that monitors cleavage at a sensitive site located within the linker between the C1b and C2 domains by elastase with increasing concentrations of ENDX revealed that ENDX but not TAM caused a noticeable structural change with an apparent midpoint of 3.5 ^M, as evidenced by a decrease in band intensity on SDS-PAGE gel (Figure 5E). Collectively, these data demonstrated that ENDX is a non-ATP-competitive inhibitor and an allosteric regulator of PKCȕI, with the potential to target additional PKC enzymes at clinically achievable concentrations. Live-cell imaging of MCF7AC1 cells expressing YFP-tagged PKCȕI was performed to examine whether ENDX also affects PKCȕI translocation to the plasma membrane. It was found that in the presence of sub-saturating amounts of the PKC agonist phorbol myristate acetate (PMA), ENDX increased PKCȕI localization to the membrane in response to PMA in both a time-dependent and dose-dependent manner (Figures 5F, 13C, and 13D). Furthermore, localization was mitigated by the addition of the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP1/2) inhibitor NSC117079. These data suggested that ENDX can induce dephosphorylation of PKCȕǿ by PHLPP1/2, which in turn enhances recruitment to the plasma membrane (Figure 5F). Collectively, these results indicated that ENDX altered the conformation of PKC in a way that inhibited kinase activity but also promoted recruitment to the plasma membrane via a PHLPP1/2-mediated mechanism. Materials and Methods Reagents, cell lines, antibodies, plasmids HEK293F cells (Thermofisher) and MCF7AC1 cells were grown in DMEM with 10% (v/v) Fetal Bovine Serum (Gibco), 50 U/^L penicillin, 50 ^g/mL streptomycin, and 0.5 mmol/L sodium pyruvate at 37°C in 5% CO2 atmosphere for adherent growth. Suspension cultures of HEK293F cells were grown in CDM4HEK (Cytiva) media supplemented with 20 Attorney Docket No. 07039-2296WO1 / 2023-600 mM glutamine, 12 U/^L penicillin, and 12 ^g/mL streptomycin at 37°C in 8% CO2 atmosphere at 135 rpm in a Multitron shaker incubator (HT Infors) for suspension growth. Antibodies used in this study: Anti PKCȕ phospho-T500 (Abcam ab5817) Anti-phospho T642 (Abcam ab75657), and anti-phospho S661 (Abcam 192184). DNA encoding PKC constructs was cloned into pMCentr2 (DNASU) and recombined into mammalian protein expression vector pcDNA6.2/NYFP-Dest using LR Clonase II (Invitrogen). Mutant PKCȕI plasmids were generated using the Quickchange kit (Stratagene). Expression and purification of PKCȕI and PKCȕII Recombinant plasmid was mixed with 1 mg/mL polyethyleneimine pH 7.5 (Polysciences, Inc.) in a 1:3 ratio to form a DNA-PEI mixture used to transfect HEK293F cells in suspension culture and grown in HyClone HyCell Transfx-H (Cytiva) medium supplemented with 200 mM Glutamine (Thermofisher) and 0.075% Pluronic (Gibco 24040- 032). Transfected cells were grown for 72 hours at 37ºC, while cell count and protein expression (GFP) level were monitored every 24 hours. PKCȕ expressing culture was spun down at 5000xg for 10 minutes, followed by resuspension of pelleted cells in 1X PBS containing 0.1X Roche EDTA-free protease inhibitor cocktail (Sigma). Centrifugation step was repeated to obtain cell pellet that was used directly for protein prep or frozen down at - 80ºC for storage. Thawed HEK293F cells expressing PKCȕ were lysed at 4ºC with a lysis buffer solution consisting of 50 mM Tris pH 7.4, 300 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM ȕ-glycerol phosphate, 1 mM TCEP, 2 mM benzamidine, 2 ^g/mL leupeptin, 0.5 mM sodium orthovanadate, 0.5% CHAPS, and 1:100 protease inhibitor cocktail (Sigma P8849). Lysate was sonicated with a Branson sonicator at 50% power for 10 seconds (x2) and spun down in a Lynx 4000 centrifuge (Thermo Fisher) at 15,000 g, 4ºC for 10 minutes. The clarified lysate was bound and recycled 3x over a GFP-enhancer nanobody linked NHS Sepharose (Cytiva 45002965) resin bed equilibrated in lysis buffer. Protein bound resin was washed in 3x resin volume with the same lysis buffer. TEV cleavage of the YFP tag was completed overnight by incubating the resin with 1.5x resin volume TEV cleavage buffer containing 50 mM Tris pH7.4, 50 mM NaF, 5 mM sodium pyrophosphate, Attorney Docket No. 07039-2296WO1 / 2023-600 10 mM ȕ-glycerol phosphate, 1 mM TCEP, 0.5 mM sodium orthovanadate, and 0.25% CHAPS, supplemented with 0.09 mg/mL TEV protease. Cleaved protein was eluted from the resin using buffer without TEV protease, and the presence of protein in eluted fractions was monitored using a Coomassie-stained SDS-PAGE gel. Following purification, PKCȕ protein was polished on an AKTA go FPLC system (Cytiva) to remove TEV protease and further purify protein for biochemical assays and crystallization. TEV eluted protein was diluted (1:3) with a low salt buffer containing 50 mM Tris pH 8.0 and 1 mM TCEP prior to being loaded onto a HiTrap Q HP anion exchange column (Cytiva). A salt gradient was introduced by the gradual addition of buffer supplemented with 1M NaCl at a flow rate of 5 mL/min. Gradient fraction elution continued to a final of 60% or 600mM NaCl. Eluted fractions containing PKCȕ were combined and concentrated to 0.5 mL using a 10K cut off centrifugal filter (Sartorius,Vivaspin), pretreated overnight with 3% PEG 3350 (Jena Biosciences) at 4ºC to avoid protein loss through binding to filter. Concentrated protein was subjected to final purification on a Superdex 200 Increase 10/300 GL column (Cytiva) using 20mM Tris pH 8.0, 100mM NaCl, 2mM MgCl2, and 1mM TCEP at a flow rate of 0.5 mL/min. All FPLC elution fractions were monitored for protein presence using Coomassie-stained SDS-PAGE gel. Fractions containing PKC were pooled and concentrated to 10 mg/mL using a 10K centrifugal filter (Sartorius, Vivaspin) for crystallization, or buffer exchanged into storage buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP, 25% (v/v) glycerol) and stored at -80 ºC prior to use in kinase assays. Typical yield of final, purified PKCȕI/II proteins (77kDa) was 3 to 6 mg per litre of culture. Lambda Phosphatase Reactions To generate dephosphorylated PKC, 7 ^g PKC protein were incubated at 37°C for 2 hours with 1400U Lambda Phosphatase (New England BioLabs P0753S) and its 1x reaction buffer according to the manufacturer’s protocol. As a control, 7 ^g PKC protein was incubated under the same conditions in the absence of phosphatase. After the incubation period, loading dye was added and the reactions were heated to 75°C for 5 minutes prior to visualization via 8% SDS-PAGE with 0 ^M PhosTag or 40^M Phostag (APExBIO). Attorney Docket No. 07039-2296WO1 / 2023-600 Crystallization and structure determination of PKCȕI and PKCȕII Crystals of PKCȕI and PKCȕǿǿ were grown using the sitting-drop vapor diffusion method by mixing 200 nL of precipitant with 200 nL of protein mixture (PKCȕI/II with 1 mM AMPPNP). Crystals of PKCȕI crystal form 1 and 2 grew in 200–300 mM sodium citrate pH 7–8 and 8–10% (w/v) PEG3350. Crystals of PKCȕII grew in 100–200 mM magnesium chloride, 0.1 M MES pH 6.5, and 6–10% (w/v) PEG8000. Crystals grew over a period of 2–6 weeks at 4°C and were transferred to a cryoprotectant containing crystallization condition supplemented with 25% (v/v) glycerol, then flash frozen in liquid nitrogen. For manganese soaks, the cryoprotectant also contained 1 mM MnCl2. X-ray diffraction datasets were collected at the NE-CAT beamlines (24-C and 24-E) at the Advanced Photon Source. X-ray diffraction data were processed and scaled using the HKL2000 suite. Structures were solved via molecular replacement using the C1b, C2, and kinase domains from PDB entry 3PFQ as search models using the PHENIX-PHASER. The C1a domain and other missing regions were built manually using COOT, followed by refinement against the high-resolution datasets with PHENIX to produce the final models. Limited Proteolysis 0.4 mg/mL PKC protein was digested with 1.4 ^g/mL elastase (Promega) in the presence and absence of activating factors (40 ^g/mL lipids and 82 ^M Ca2+). Additional reaction components, like drugs or phorbol dibutyrate, were added in the concentrations indicated. Reactions were incubated at room temperature for 45 minutes. Protease activity was quenched by adding SDS reducing dye and heating at 70°C for 10 minutes. The full mixture was loaded onto a SurePAGE Bis-Tris 4-12% gel (Genscript) and stained with Coomassie blue to visualize proteins. Expression and purification of CKAR pRSET-B-CKAR (Addgene) was transformed into JM109 DE3 E. coli (Promega). Cultures of pRSET-B-CKAR in JM109 DE3 were grown in 2 L of Terrific Broth (Research Products International), supplemented with 300 ^L of antifoam 204 (Sigma-Aldrich) in a LEX-48 Bioreactor (Epiphyte3) at 37°C, until they reached an optical density (OD600) between 3.0 and 4.0. CKAR Protein expression was induced by the addition of 50 ^M of Attorney Docket No. 07039-2296WO1 / 2023-600 isopropylthio-ȕ-galactoside (IPTG, Goldbio) for 18 hours at 16°C. The bacterial cell pellet was harvested by centrifugation at 6000 g for 20 minutes at 4°C, and frozen at -80°C. Cell pellets were thawed and lysed in Ni running buffer (20 mM Tris pH 7.5, 300 mM NaCl, 0.5 mM TCEP, 10 mM imidazole) supplemented with 0.1 mg/mL of lysozyme and a 1/2000 dilution of ethanol saturated phenylmethyl sulfonyl fluoride (PMSF, Goldbio) on ice for 30 minutes. Lysate was sonicated at 80% power for three 30-second intervals using a Branson 250 sonifier. Sonicated lysate was centrifuged at 25,000 g for 30 minutes and soluble fraction was passed over Ni-NTA resin (Qiagen) pre-equilibrated with lysis buffer. The resin was washed six times, each with 1 column volume of lysis buffer and CKAR was eluted with Ni running buffer supplemented with 250 mM imidazole. Protein was precipitated out of solution by the addition of 2x volume of 4 M ammonium sulfate and pellets were collected after centrifugation at 25,000 g for 30 minutes. The protein pellets were redissolved in 2 mL of milli-Q water and polished on a Superdex 20016/60 column (Cytiva) in 20 mM Tris pH 7.5, 300 mM NaCl, and 0.5 mM TCEP buffer. Protein elution was monitored by absorbance at 280 nm, 460 nm, and 520 nm to identify fractions with full- length CKAR and both YFP and CFP modules. CKAR was further polished by ion exchange chromatography on a Hi-Trap Q HP column (Cytiva) using a 0-50% gradient between low salt (20 mM Tris pH 7.5, 3 mM DTT) and high salt buffers (20 mM Tris pH 8.0, 1M NaCl). CKAR was concentrated and buffer exchanged into PKC Storage Buffer (20 mM Tris pH 8.0, 100 mM NaCl, 2mM MgCl2, 1mM TCEP, and 25%(v/v) glycerol) using an Amicon 10K concentrator (Millipore). The final CKAR product was quantified using A520nm and an extinction coefficient of 70,000 M-1cm-1 for YFP. CKAR Kinase Assay Lipid vesicles were prepared fresh by dissolving 10 mg/mL bovine brain phosphatidylserine (PS) (Avanti) or/and 1 mg/mL 1,2-dioleoyl-glycerol (DAG) (Avanti) in chloroform. Lipids were mixed at the indicated ratio and chloroform was evaporated with a stream of dry air, then lipids were redissolved in water to a final concentration of 40 ^g/mL, vortexed for 60 seconds, and sonicated 3 times for 30 seconds at 10% power on a Branson 250 sonifier with a microtip (12840498). Concentrated PKC protein was diluted into storage Attorney Docket No. 07039-2296WO1 / 2023-600 buffer containing 20 mM Tris pH 8.0, 100 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 25%(v/v) glycerol and 1 mg/mL BSA to create a 10X stock based on final assay concentration. Kinase reaction mixtures (100 ^L) contained 20 mM HEPES pH 7.5, 2 mM MgCl2, 1 ^M CKAR, 10 nM PKCȕ protein and indicated concentrations of ENDX and ATP. Reaction mixtures were pre-heated at 30°C and ATP was mixed in last to initiate the reaction, just prior to placing the reaction plate in a CLARIOstarPlus plate reader (BMG Labtech). Forster Resonance Energy Transfer (FRET) was measured every 5 minutes using a 434-16 excitation filter with 476-16 and 528-16 emission filters at 30°C. Control reactions without PKC were used as a background measurement to correct for fluorophore decay throughout the experiment. The FRET ratio was calculated by dividing the emission measurement at 476 nm by 528 nm emission. PKC kinase activity was plotted as the change in FRET ratio over time (dFRET/dT). Z’-LYTE Kinase Assay Kinase activity was measured using the Z’-LYTE Kinase Assay Kit – Ser/Thr 7 Peptide (ThermoFisher) following the manufacturer’s protocol. The reaction mix contained 250 mM HEPES pH 7.5, 50 mM MgCl2, 5 mM EGTA, 0.05% Brij-35, 40 ^g/mL PS:DAG lipids, PKC protein at the indicated concentration, drug serial dilution at the respective concentration, 40 ^M ATP and 2 ^M Z’-LYTE Ser/Thr 7 peptide substrate. The peptide substrate and ATP mixture were added last to initiate the reaction. For reactions without lipids, the 40 ^g/mL lipids were substituted with milli-Q H2O. Reactions were incubated at room temperature for 1 hour. The development solution, created using the manufacturer's instructions, was added and mixed with the kinase reaction prior to placing the plate in the CLARIOstarPlus plate reader (BMG Labtech). The extent of phosphorylation of the peptide substrate was calculated through the ratio of the coumarin emission at 445 nm to the fluorescein emission at 520 nm after a 1-hour incubation with development solution. Kinase activity was plotted as percent activity to the corresponding drug concentration. To determine inhibitor IC50 values, data were fitted to a 4-parameter dose-response model using Graphpad Prism 9. In cases where the IC50 could not be accurately determined due to precipitation of ENDX at very high concentrations, the model was constrained to a bottom value of 0 % and Attorney Docket No. 07039-2296WO1 / 2023-600 the resulting estimated IC50 value was reported. Extra sum of F squares analysis was used to calculate P values between different drug treatments. Expression and purification of PKA DNA encoding PKA catalytic domain (Addgene 14921) was transformed into Rosetta2 cells (EMD) and inoculated in a 2 L culture of terrific broth (Research Products International) supplemented with 300 ^L Antifoam 204, 100 ^g/mL carbenicillin, and 34 ^g/mL chloramphenicol. Culture was grown using a LEX-48 Bioreactor (Epiphyte) at 37°C and protein expression was induced with 100 ^M IPTG overnight at 16°C. E. coli culture was pelleted, resuspended in lysis buffer (20 mM Tris pH 7.5, 300 mM NaCl, 10 mM imidazole, and 0.5 mM TCEP) supplemented with 10 mg of lysozyme (GoldBio) and 20 ^L of saturated PMSF in ethanol (GoldBio). Cell lysate was incubated on ice for 30 minutes with occasional mixing and then sonicated by a Branson 250 sonicator at 80% power in five 15-second intervals. Clarified lysate was centrifuged at 25,000g for 30 minutes and passed over a Ni-NTA column equilibrated in lysis buffer. Ni-NTA resin was washed with column volumes lysis buffer and eluted with lysis buffer supplemented with 250 mM imidazole. Protein containing fractions were detected by a color change in a Bradford Assay (99 ^L bradford reagent, 1 ^L elution fraction), pooled and precipitated with two volumes of 4 M ammonium sulfate followed by centrifugation at 25,000g for 30 minutes at 4°C. Precipitated protein was dissolved in 3 mL Milli-Q water and loaded onto a Superdex S200 size exclusion column equilibrated in 20 mM Tris pH 7.5, 300 mM NaCl, and 0.5 mM TCEP. Fractions were analyzed by SDS-PAGE gel and PKA containing fractions were pooled and diluted 1:5 in Milli-Q water. The pH of the dilution was adjusted to pH 6 by addition of MES powder and loaded onto a Resource 15S cation exchange column (Cytiva) equilibrated in running buffer (20 mM NaH2PO4 pH 6.0, 0.1 mM TCEP) and eluted with a linear gradient of 0-100% 1 M NaCl in 20 mM NaH2PO4 pH 6.0. To increase the pH in PKA–containing fractions 20 mM Tris pH 8.0 was added to pooled protein and protein was then concentrated by Amicon ultrafiltration (Millipore). Concentrated protein was run over a Superdex 200 increase (Cytiva) equilibrated in running buffer (20 mM Tris pH 7.5, 300 mM NaCl, and 0.5 mM TCEP). Purified protein was analyzed by SDSPAGE gel and buffer exchanged during Attorney Docket No. 07039-2296WO1 / 2023-600 Amicon centrifugation to PKC storage buffer (20 mM Tris pH 8.0, 100 mM NaCl, 2 mM MgCl2, 1mM TCEP, and 25% (v/v) glycerol). Live Cell Imaging MCF7AC1 cells that stably expressed YFP-PKCȕ1 were generated by transfecting plasmid DNA using Lipofectamine 2000 (Thermofisher) according to the manufacturer’s instructions, and then following the protocol described in the Expression and purification of PKCȕI and PKCȕII section for generating a stable expression cell line. For microscopy YFP+ cells were seeded in 35-mm glass bottom microwell dishes (MatTek Corporation) for at least 24 hours. Subsequently, cells were incubated with ENDX followed by 15 minutes of PMA incubation as indicated in figure legends. For the PHLPP inhibitor study, cells were treated with NSC117079 (MedChem Express) for the last 45 minutes of the total incubation prior to imaging. Successively, NucRed Live 647 Reagent (Invitrogen) was added for live cell nuclear staining and visualized using a Zeiss-LSM 780 confocal microscope. Confocal images were processed using Carl-Zeiss Blue/Black ZEN 3.0 SR software. Quantification And Statistical Analysis Standard statistical analyses were used to distinguish significant from non-significant results as indicated. Example 2: Endoxifen Downregulates AKT Phosphorylation in ERĮ+ Breast Cancer Endoxifen (ENDX), a secondary tamoxifen (TAM) metabolite, is a potent antiestrogen exhibiting estrogen receptor alpha (ERĮ) binding at nanomolar concentrations. Phase 1/2 clinical trials identified clinical activity of Z-ENDX, in endocrine-refractory metastatic breast cancer as well as ERa+ solid tumors, raising the possibility that ENDX may have a second, ERa-independent, mechanism of action. This Example describes the identification of PKCȕ1 as a ENDX target whose engagement results in inhibition of AKT signaling and induction of apoptosis. Attorney Docket No. 07039-2296WO1 / 2023-600 Results ENDX at 5 M inhibits growth and induces apoptosis in estrogen deprived ERĮ+ breast cancer cells ENDX concentrations ranging from 0 – 10 μM were used to evaluate dose dependent effects of ENDX on cell viability under estrogen deprived conditions, i.e., in medium containing charcoal-stripped serum (CSS), to evaluate ENDX effects that may extend beyond ERĮ inhibition. ENDX concentrations ^ 2.5 μM significantly reduced cell viability (Figure 14A) and induced apoptosis in these cells (Figure 14B). These findings suggested that higher plasma concentrations of ENDX may elicit cytotoxic, and not just cytostatic, effects in ERĮ+ breast cancer cells. ENDX concentration-dependent effects on the phosphoproteome of ERĮ+ breast cancer cells It was then sought to identify additional protein targets of ENDX that may contribute to its anticancer effects in estrogen deprived conditions. To this end, MCF7AC1 cells were treated with 0.01, 0.1, and 5 μM ENDX concentrations achieved in various clinical settings for 24 hours in CSS medium and subjected to TMT labeling-based LC-MS/MS mass spectrometry analysis to evaluate changes in the global protein expression and the phosphoproteome relative to vehicle treated cells (Figure 15). Assessment of the total proteome identified and quantified 8,894 unique proteins (accession number: PXD035007). The impact on global total protein expression induced by ENDX treatment for 24 hours in estrogen deprived cells was limited, with only 25, 34, and 65 total proteins differentially altered by treatment with 0.01, 0.1, and 5 μM ENDX, respectively, compared to vehicle treated cells (based on criteria of |1.5|-fold change and p value of < 0.05) (Table 2). Although ENDX impact on the total proteome was limited, ENDX at 5 μM downregulated two-fold more total proteins compared to 0.1 μM concentration (44 versus 22) and four-fold more total proteins compared to the 0.01 μM concentration (44 versus 11) (Figure 24A). Also, the number of total proteins uniquely downregulated by 5 M ENDX (35) was greater than the number of total proteins uniquely downregulated by 0.01 M (3) and 0.1 M (9) concentrations (Figure 24B). Attorney Docket No. 07039-2296WO1 / 2023-600 Table 2. Accession Gene Description Log FC FC Relative fold P value change 6 1 14 56 71 5 79 7 37 3 1 6 3 98 43 8 80 32 8 9 - 93 7 24 04 - Attorney Docket No. 07039-2296WO1 / 2023-600 Q9C0D6 FHDC1 FH2 domain-containing protein 1 -1.1 0.466516 -2.143546925 0.00428 Q8NE31 FAM13C Protein FAM13C -1.04 0.486327 -2.056227653 0.0349 57 03 3 1 9 1 04 4 98 46 5 52 48 7 15 1 4 52 4 21 6 4 - 5 Attorney Docket No. 07039-2296WO1 / 2023-600 member 11 Q96PQ0 SORCS2 VPS10 domain-containing receptor 1.52 2.86791 2.867910496 0.0123 35 82 - 84 - 77 81 97 97 17 78 - 24 6 93 23 79 59 30 7 5 83 Attorney Docket No. 07039-2296WO1 / 2023-600 O43147 SGSM2 Small G protein signaling modulator -0.822 0.565657 -1.767855062 0.00083 2 2 32 01 9 62 7 79 5 03 1 62 92 8 27 47 5 9 9 6 - 39 9 9 6 - Attorney Docket No. 07039-2296WO1 / 2023-600 Q9NZ53 PODXL2 Podocalyxin-like protein 2 0.616 1.53262 1.53261996 0.00501 P27449 ATP6V0C V-type proton ATPase 16 kDa 0.618 1.534746 1.534746096 0.00457 6 54 61 4 - 6 8 14 - 99 - 10 ENDX displayed a much greater impact on the phosphoproteome compared to the total proteome during the course of the 24-hour treatment. Phosphoproteomic analyses identified 14,715 unique phosphosites derived from 4,480 proteins (accession number: PXD035007). Out of all sites, 10,046 (82%) were phospho-Serine (pS) sites, 2,042 (17%) were phospho-Threonine (pT) sites, and 134 (1%) were phospho-Tyrosine (pY) sites, as shown in Figure 16A. In MCF7-AC1 cells, treatment with 0.01 μM ENDX resulted in the downregulation of 109 phosphosites and upregulation of 132 phosphosites (|1.5|-fold change and a p value of < 0.05) (Figure 16B). Similarly, treatment with 0.1 μM ENDX led to the Attorney Docket No. 07039-2296WO1 / 2023-600 downregulation of 94 phosphosites and the upregulation of 150 phosphosites (Figure 16C). Finally, 5 μM ENDX treatment downregulated 341 phosphosites and upregulated 164 phosphosites (Figure 16D). Remarkably, ENDX at 5 μM downregulated three-fold more phosphosites compared to 0.1 μM (341 versus 94) and 0.01 μM (341 versus 109, Figures 16B-16D). In addition, the number of phosphosites uniquely downregulated by 5 μM ENDX (289) was also three-fold greater than the number uniquely downregulated by 0.01 μM (87) and 0.1 μM (59, Figure 16E). Heat-map analysis suggested an ENDX concentration- dependent downregulation for a subset of these phosphosites (Figure 16F). A comparison of the phosphosite list with the total protein list altered by ENDX revealed minimal overlap (Figure 24C). In order to identify protein phosphorylation signaling pathways regulated by ENDX, a kinase enrichment analysis (KEA3) was performed, which integrated multiple databases covering kinase-substrate interactions (KSI), kinase-protein interactions (KPI), and interactions supported by co-expression and co-occurrence data to infer the overrepresentation of upstream kinases whose putative substrates were among the phosphorylated proteins altered by ENDX treatments. Three separate analyses were performed for 210 proteins, 224 proteins, and 347 proteins differentially phosphorylated in cells treated with 0.01 μM, 0.1 μM, and 5 μM ENDX, respectively. Figure 17 shows the top enriched upstream kinases predicted to regulate the protein phosphorylation changes induced by ENDX at different concentrations. Casein kinase (CSNK1A1), serine/arginine-rich protein-specific kinase (SRPK1, SRPK2), and mitogen-activated protein kinases (MAPK1 and MAPK8) were identified as putative kinases regulated by low-dose (0.01 and 0.1 μM) ENDX (Figures 17A-17B). This analysis suggested that proteins (MTOR, RPS6K and AKT1) involved in the AKT signaling pathway can be regulated by high-dose (5 μM) ENDX (Figure 17C). In addition to recognizing potential upstream kinases, based on the effects of ENDX on phosphorylation, fuzzy-C mean clustering was also performed to identify the regulation patterns induced by different ENDX concentrations. This analysis identified three clusters depicting three different regulatory patterns (Figure 18A). Cluster 1 represented 325 phosphosites downregulated by ENDX in a dose-dependent manner, cluster 2 represented Attorney Docket No. 07039-2296WO1 / 2023-600 201 phosphosites upregulated by ENDX at all concentrations and cluster 3 represented 73 phosphosites downregulated at 0.01 M concentration but mostly unaffected at the 0.1 and 5 M concentrations (Table 3).
Attorney Docket No.07039-2296WO1 / 2023-600 Table 3. prt_site_ 0.01 μM S54 S54 S199 T202 S52 S54 S199 T202 S52 S53 S329 S1549 S134 T260 S443 T446 S451 S132 S309 S322 S939 S419 LSVSRVGsAAQTRAM ATP5F1A 25 16.22 10.63 12.64 9.96 13.26 6.17 14.43 8.66 6.61 9.54 5.85
Attorney Docket No.07039-2296WO1 / 2023-600 S76 T330 T123 S126 S197 S201 S80 S585 S769 S699 S703 S138 S190 S23 S203 S86 S100 T21 S315 S754 T261 T261 T261 T86 Y189 S419 Y774 S489 WEKTGSHsEPQARGD FAM120A 51 1.29 2.04 2.03 3.59 2.10 0.94 3.29 0.92 1.03 1.07 1.08
Attorney Docket No.07039-2296WO1 / 2023-600 T34 S324 S962 S488 S148 S266 S746 T846 Y848 S623 S187 S908 T466 S113 S56 T97 T46 T45 S105 S105 S116 S102 S113 S41 S103 S42 S102 S113 KLNKKAAsGEAKPKA HIST1H1E 70 57.15 22.38 31.55 48.56 32.09 20.40 33.75 28.92 17.76 23.16 16.03
Attorney Docket No.07039-2296WO1 / 2023-600 S41 S106 S114 S57 T98 S113 S37 S39 S56 T97 S113 S56 T97 S113 S56 T97 S113 S56 T97 S113 S37 S39 S56 T97 S113 S56 T97 S113 ELAKHAVsEGTKAVT HIST1H2BL 65 30.89 12.16 27.40 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80
Attorney Docket No.07039-2296WO1 / 2023-600 S56 T97 S113 S56 T97 S113 S56 T97 S113 S37 S39 S56 T97 T46 T31 S48 Y52 S37 S39 S37 S39 S113 S37 S39 S56 T97 S113 S56 VHPDTGIsSKAMGIM HIST2H2BF 66 20.59 13.90 17.66 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80
Attorney Docket No.07039-2296WO1 / 2023-600 T97 T46 S113 S37 S39 S56 T46 S185 S397 S398 S400 S121 S121 S365 T110 S391 S68 S68 S383 S434 S48 S63 S48 S63 S325 S48 S63 S24 QELISNAsDALDKIR HSP90AB4P 96 32.56 19.82 31.16 29.57 24.59 14.78 34.76 16.58 11.98 19.62 7.80
Attorney Docket No.07039-2296WO1 / 2023-600 S109 S98 S67 S423 S258 S274 S25 T713 S731 S570 S193 S159 S138 S128 S118 S130 S97 T11 S319 S323 T425 S93 S48 S93 S191 S211 S181 S176 TLNNKFAsFIDKVRF KRT6A 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27
Attorney Docket No.07039-2296WO1 / 2023-600 S176 S176 S104 S143 S138 S145 S153 S162 Y327 S196 S177 S124 S155 S104 Y267 S291 S58 S178 T865 S144 S140 S301 S1616 S214 S507 S410 S1384 T1425 TSTDQPVtPEPTSQA MDC1 131 13.39 11.85 10.71 15.71 12.98 6.92 11.39 5.71 9.91 7.18 7.91
Attorney Docket No.07039-2296WO1 / 2023-600 S1119 Y47 S1406 S732 T117 S145 S282 S106 S44 S29 S43 S948 S259 T65 S232 S415 S259 S512 S150 T256 S437 S34 S778 S255 S93 S78 S120 S329 ATGEGGAsDLPEDPD RNF113A 159 3.73 1.23 2.89 2.46 1.10 1.12 1.01 1.11 1.24 1.29 1.30
Attorney Docket No.07039-2296WO1 / 2023-600 S77 S233 S206 S6 S82 S35 S73 S138 S1630 T1185 S45 S63 S34 S2 S216 S451 S258 S180 S1822 S1824 S67 S342 S41 S40 S1039 S425 S91 S224 PKDGSNKsGAEEQGP TGOLN2 184 21.62 24.22 28.94 19.81 21.67 25.45 26.32 30.21 14.98 15.55 17.01
Attorney Docket No.07039-2296WO1 / 2023-600 S266 S271 S393 T109 T223 S277 T109 T223 S277 T109 T223 S277 T109 T223 S277 T109 T223 S277 T109 T223 S277 T109 T223 S277 T162 T48 T223 S48 DLQLDRIsVYYNEAT TUBB 193 6.95 4.80 9.45 11.19 9.78 4.77 7.76 5.62 2.17 4.61 4.44
Attorney Docket No.07039-2296WO1 / 2023-600 S288 Y541 S680 S407 S569 S356 S356 S544 S47 S46 S46 S46 S45 S114 S156 S45 S63 S345 S1182 S1184 Y483 T484 S82 0.1 μM S1410 S404 S423 T217 RSQSAAVtPSSTTSS ADRM1 215 8.37 9.91 6.95 10.76 8.93 15.45 11.13 10.63 8.38 11.98 13.40 13.97
Attorney Docket No.07039-2296WO1 / 2023-600 S62 S100 S669 S96 S592 S227 S413 S728 S733 S134 S165 S168 S643 S280 S240 T177 S793 S12 T417 T418 S164 S756 S533 S320 S560 S381 S413 S420 SEPIPHPsNELRGLN DBF4 243 3.35 2.25 4.82 4.93 5.85 4.68 4.89 5.34 4.13 4.61 5.96
Attorney Docket No.07039-2296WO1 / 2023-600 S287 S109 S111 S160 S265 S460 S454 S370 S196 T55 S1194 S66 T148 S150 S161 S369 S270 S276 S436 S461 S135 S2107 S13 S23 S10 S620 S508 S9 SGRPRTTsFAESCKP GSK3B 271 27.16 34.23 28.07 30.39 36.84 41.26 47.84 37.77 46.58 40.53 48.42
Attorney Docket No.07039-2296WO1 / 2023-600 T427 S784 S454 S1003 T33 T104 S17 S730 T259 S177 S64 S324 S592 S696 S42 T474 T821 S824 S357 S649 S1307 S1317 S69 T1867 S674 S1299 S53 S295 DFYPSPSsPAAGSRT NFIB 302 2.45 0.97 0.92 4.93 4.39 2.62 4.56 3.59 5.27 4.07 2.92
Attorney Docket No.07039-2296WO1 / 2023-600 T709 S203 T883 S899 T888 S904 T887 S903 T884 S900 T884 S900 T884 S900 T914 S930 T883 S899 T884 S900 T884 S900 T884 S900 T884 S900 T879 S895 QNVYIPGsNATLTNA PCDHGB1 306 17.50 16.35 18.81 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10
Attorney Docket No.07039-2296WO1 / 2023-600 T883 S899 T881 S897 T875 S891 T875 S891 T882 S898 T881 S897 T886 S902 T890 S906 T896 S912 S1909 S1097 T657 T707 T8 S527 S545 S78 S2079 S277 TVGTPIAsVPGSTNT PSMD1 316 8.62 8.48 9.74 11.09 9.05 16.09 12.32 13.08 11.77 10.72 10.51
Attorney Docket No.07039-2296WO1 / 2023-600 S98 S773 S2628 S583 T208 S350 S501 S9 S452 S503 S528 T77 S212 T14 T473 T473 S534 T171 S417 T478 S297 S709 T479 S484 S1323 S17 S837 T714 KTKFICVtPTTCSNT STAT3 343 1.29 2.04 2.99 5.95 3.38 3.37 3.88 3.32 3.82 3.43 3.79
Attorney Docket No.07039-2296WO1 / 2023-600 S281 S823 S153 S307 S403 S1585 S154 S8 S130 S42 S779 S782 S1978 S883 S884 S164 S61 S189 S293 S1053 T168 S2801 S426 S1348 S1352 S551 S301 LLTSEEDsGFSTSPK ZNF148 370 5.41 4.09 5.50 7.07 5.44 7.95 7.30 9.11 6.91 8.26 4.72 5.20
Attorney Docket No.07039-2296WO1 / 2023-600 S412 S452 5 μM E S166 S332 S5237 T444 T1023 S675 S782 S557 S381 S332 S878 S250 S512 T85 T172 S14 S184 S896 S470 S1257 S597 S348 S435 S358 S177 DKILPPPsPWPKSSI GGA2 397 2.45 3.58 2.51 0.98 0.87 2.81 3.71 4.33 2.38 3.43 4.33
Attorney Docket No.07039-2296WO1 / 2023-600 S3016 S1160 S600 S180 S253 S832 S897 S12 S148 S775 S639 S156 S1030 S107 S145 S153 S1329 S411 S648 S116 S537 S77 S710 S973 T978 S449 S564 S971 STVEEPVsPMLPPSA REST 425 2.83 4.60 2.60 2.77 0.96 3.46 1.77 3.22 3.72 2.25 4.33
Attorney Docket No.07039-2296WO1 / 2023-600 S608 S330 S21 S1021 S1441 T148 S136 S13 S707 S509 S222 S263 S267 S24 S46 S29 S31 S158 S284 S480 RGQAEEEsPSQEETV ZBTB37 445 5.15 6.34 5.02 2.87 3.80 3.66 4.40 3.54 3.13 4.44 3.22 6.50
Attorney Docket No. 07039-2296WO1 / 2023-600 Given the interest in ENDX dose-dependent effects and the mechanistic basis for induction of apoptosis at 5 μM ENDX, cluster 1 was focused on. KEGG pathway enrichment analysis using DAVID, an online gene functional annotation tool, identified viral carcinogenesis, systemic lupus erythematosus, phagosome, PI3K-AKT signaling pathway and gap junction as the top five biological pathways impacted by ENDX (Figure 18B). ENDX downregulated phosphosites are enriched for PKCȕ, CDK1 and AKT1 target sequences It was postulated that the observed ENDX effects on cluster 1 phosphosites were due to effects on kinase mediators of these phosphorylation events. To identify these kinases, NetworKIN and RoKAI kinase prediction tools were used. Using the 325 phosphosites from cluster 1 as an input, these two tools collectively identified protein kinase C beta (PKCȕ) and cyclin-dependent kinase 1 (CDK1) followed by AKT1 and PKC /PKCĮ as the top five most frequently predicted kinases involved (Figure 18C; Table 4). Further, motif enrichment analysis identified that RXXpS, pS/pTP and pSXXE as prevalent motifs in the regulated cluster 1 phosphosites. These motifs mapped to the AKT, MAPK/CDK and CK2 kinase substrate motifs, respectively (Figure 18D).
Attorney Docket No. 07039-2296WO1 / 2023-600 Table 4. Attorney Docket No. 07039-2296WO1 / 2023-600 ENDX at 5 μM downregulates AKTSer473 phosphorylation in ERĮ+ breast cancer cells Pathway analysis studies identified PI3K-AKT signaling as one of the top biological pathways targeted by ENDX (Figure 18B), with AKT1 frequently predicted as a top kinase for phosphosites downregulated by ENDX (Figure 18C). Therefore, the effect of ENDX on AKTSer473 phosphorylation was examined in estrogen deprived MCF7AC1 cells. Immunoblot assays revealed that ENDX at 5 μM, but not at 0.01 and 0.1 μM, attenuated AKTSer473 phosphorylation compared to vehicle treated cells (Figure 19A). Given that phosphorylation of AKT is initiated at Thr308 followed by phosphorylation at Ser473 for full AKT activation (Cicenas, Int. J. Biol. Markers, 23:1-9 (2008)), the effects of ENDX on AKTThr308 phosphorylation were also evaluated. While 0.01 and 0.1 μM concentrations of ENDX stimulated AKTThr308 phosphorylation, ENDX at 5 μM did not alter AKTThr308 phosphorylation under estrogen deprived conditions (Figure 19A). Because commercially available phosphosite specific antibodies for predicted AKT-mediated phosphorylations in cluster 1 were limited, to an alternate approach where we evaluated ENDX effects on the levels of total phospho-AKT substrates was used. A commercially available antibody that specifically recognizes the RXXpS/pT AKT motif in AKT substrates was used to determine whether downregulation of AKTSer473 phosphorylation by 5 M ENDX had a global impact on the phosphorylation of AKT substrates. Consistent with reduced AKTSer473 phosphorylation, ENDX at 5 M, but not 0.01 and 0.1 M, also reduced the phosphorylation of AKT substrates compared to vehicle treated estrogen deprived MCF7AC1 cells (Figure 19A; arrows). The effects of ENDX on AKTSer473 and AKTThr308 phosphorylations were next evaluated in the endocrine-sensitive MCF7AC1 xenograft model in vivo. Treatment of mice harboring MCF7AC1 tumors with high-dose ENDX (75 mg/kg) but not low-dose ENDX (25 mg/kg), TAM or letrozole, attenuated AKTSer473 phosphorylation and reduced the levels of AKT phosphorylated substrates but had no appreciable impact on AKTThr308 phosphorylation compared to control treatment (Figure 25A). These data support a dose and concentration dependent effect of ENDX on AKT signaling both in vitro and in vivo. Next, it was examined whether ENDX can block ligand stimulated AKTSer473 phosphorylation. Insulin, a known activator of AKTSer473 phosphorylation, robustly stimulated AKTSer473 phosphorylation in serum starved MCF7AC1 cells. Pretreatment with Attorney Docket No. 07039-2296WO1 / 2023-600 ENDX at 5 M, but not at 0.01 and 0.1 M, for two hours prior to insulin stimulation blocked AKTSer473 phosphorylation (Figure 19B). Insulin treatment also stimulated AKTThr308 phosphorylation, albeit modestly compared to AKTSer473 phosphorylation in serum starved MCF7AC1 cells (Figure 19B). Interestingly, pretreatment with ENDX at 5 M was also able to block AKTThr308 phosphorylation (Figure 19B). Additionally, ENDX at 5 μM also diminished insulin-stimulated phosphorylation of AKT substrates (Figure 19C). In contrast, treatment with clinically attainable concentrations of 0.1 μM TAM or 0.1 μM ICI, failed to inhibit insulin stimulated AKTSer473 and AKTThr308 phosphorylations (Figure 19B), an effect also observed with ERĮ-targeting 0.1 μM concentration of ENDX. The ability of ENDX at 5 μM, but not at 0.01 or 0.10 μM, to block insulin-stimulated AKTSer473 phosphorylation was also observed in the ERĮ+/HER2- T47D breast cancer cells under serum starved conditions, with TAM and ICI again failing to block insulin-stimulated AKTSer473 phosphorylation (Figure 25B). However, contrary to the observation in serum starved MCF7AC1 cells (Figure 19B), insulin induced stimulation of AKTThr308 phosphorylation was not blocked by ENDX at 5 μM in serum starved T47D cells (Figure 25B). Collectively, these findings suggest that ENDX attenuates AKT signaling primarily through attenuation of AKTSer473 phosphorylation in ERĮ+ breast cancer cells at clinically relevant 5 μM concentration, a unique effect not observed with other SERM’s at clinically relevant concentrations. ENDX inhibits PKC 1 kinase activity and binds to PKCȕ1 To examiner wheter ENDX might mediate its effects on AKT through PKC 1, it was first sought to evaluate ENDX effects on PKC 1 kinase activity. To this end, the concentration-dependent effects of ENDX on a kinase panel composed of 12 PKC isoforms were evaluated, since multiple PKC family members including PKCȕ were identified in the kinase prediction analysis (Figure 18C). While ENDX inhibited the kinase activity of PKC 1 with an IC50 concentration of 360 nM (Figure 19D), ENDX did not inhibit other PKC family members as potently (Table 5). TAM, a known PKC inhibitor, also inhibited PKC 1 kinase activity, but at higher concentrations (IC50 = 4.9 M) (Figure 19E), a concentration not achievable with the 20 mg/day dose. These findings suggest that ENDX may inhibit PKC 1 kinase activity in vitro. Attorney Docket No. 07039-2296WO1 / 2023-600 Table 5. PKC family kinase Symbol ENDX IC50* (M) Staurosporine IC50 (M) PKC alpha PKCa >5.00E-05 <1.00E-09 PKC beta 2 PKCb2 4.33E-05 <1.00E-09 It was next determined whether ENDX directly bound PKCȕ1. By employing surface plasmon resonance (SPR), a widely used method for assessing protein-ligand interactions and using a wide range of ENDX concentrations (100 - 8000 nM), we demonstrate that ENDX binds PKC 1 (Figures 26A and 26B). Taken together, these findings establish PKCȕ1 as a potential ENDX substrate. ENDX downregulates AKTSer473 phosphorylation through PKC 1 inhibition in ERĮ+ breast cancer cells To determine the role of PKC 1 in mediating ENDX effects on AKTSer473 phosphorylation, it was first assessed whether PKC 1 activation can impact AKTSer473 phosphorylation. In serum starved MCF7AC1 cells, the PKC agonist phorbol myristyl acetate (PMA) stimulated PKC 1Ser661 auto-phosphorylation and AKTSer473 phosphorylation, which was associated with increased levels of AKT substrate phosphorylation (Figure 20A). It was next evaluated the effects of ENDX on PKCȕ1 under PMA-stimulated conditions. Pretreatment with ENDX at 0.01, 0.1 and 5 M had either no (0.01, 0.1 M) or minimal (5 M) effect to block PMA-stimulated PKC 1Ser661 phosphorylation, respectively. In contrast, only ENDX 5 M robustly reduced PKCȕ1 total protein levels, which correlated with reduced AKTSer473 phosphorylation and AKT substrate phosphorylation (Figure 20B). In contrast, while treatment with the potent and selective ATP competitive PKC kinase Attorney Docket No. 07039-2296WO1 / 2023-600 inhibitor enzastaurin reduced PKCȕ1Ser661 phosphorylation, it neither impacted the expression of PKC 1 nor downregulated AKTSer473 phosphorylation (Figure 20C). Further, in insulin treated MCF7AC1 cells, ENDX pretreatment also reduced PKCȕ1 total protein levels, with no effects on PKC 1Ser661 phosphorylation. In contrast, both TAM and ICI pretreatments failed to diminish PKCȕ1 total protein expression in insulin treated MCF7AC1 cells (Figure 20D), which correlated with the lack of attenuation of AKTSer473 phosphorylation (Figure 19B). Given that ENDX robustly blocked PMA- and insulin-stimulated AKTSer473 phosphorylation and additionally targeted PKCȕ1 for degradation (Figure 19B, Figure 20B), it was sought to determine the effects of downregulating PKCȕ1 protein expression on AKTSer473 phosphorylation using three different approaches to silence PKCȕ1 expression in MCF7AC1 cells. In the first approach, a commercially available siRNA that targets an mRNA sequence common to both PKCȕ1 and PKCȕ2 isoforms (siPKCȕ) was used (Figure 20E). In the second, a custom designed siRNA from Dharmacon that specifically targets nucleotides 2049-2067 of the PKCȕ1 mRNA, a target sequence that is unique and distinct from PKCȕ2, was used (Figure 27A). In the third approach, a doxycycline (dox)-inducible SMART vector inducible human PRKCB mCMV-TurboGFP shRNA for PRKCB gene silencing (shPKCȕ1dox) was utilized (Figure 27B). Of these approaches, siPKCȕ resulted in the greatest reduction in PKCȕ1 protein levels (Figure 20E). Accordingly, it was sought to assess the biological effects of PKCȕ1 knockdown using siPKCȕ. Even though PKCb2 is theoretically targeted by this reagent, PKCȕ2 protein expression was undetectable in MCF7AC1 cells (Figure 27C), indicating that effects on expression of PKCb2 are unlikely to contribute to effects of siPKCȕ. Reduction in PKCȕ1 protein expression by siPKCȕ resulted in a 51% reduction in AKTSer473 phosphorylation levels compared to non-targeting (siNT) control at 48 hours (Figure 20E). PKCȕ siRNA did not affect the expression of other PKC family members (Figure 27D), suggesting that the decrease in AKTSer473 phosphorylation was due to PKCȕ1 alone. Downregulation of PKCȕ1 protein levels also significantly inhibited growth of MCF7AC1 cells (Figure 20F). Thus, PKCb siRNA recapitulated both the signaling and growth inhibitory effects of ENDX. Attorney Docket No. 07039-2296WO1 / 2023-600 To determine whether the observed effects of ENDX on PKCȕ1 degradation and AKTSer473 phosphorylation inhibition are dependent on the presence of ERĮ, ENDX effects on PKCȕ1 degradation and AKTSer473 phosphorylation in the ER negative (ER-) MDAMB231 breast cancer cells and nonbreast HEK293F cells, a human embryonic kidney cell line, both of which express higher amounts of PKCȕ1 compared to ER+ MCF7AC1 cells, were additionally evaluated (Figure 28A). Pretreatment with 5 μM ENDX for two hours followed by treatment with 100 nM insulin for one hour did not impact PKCȕ1 protein levels in either cell line (Figure 28B). While insulin treatment induced AKTSer473 phosphorylation in ER- cells, pretreatment with ENDX did not inhibit this phosphorylation (Figure 28B). To determine whether the addition of ERĮ to the MDAMB231 cell line could facilitate ENDX effects on PKCȕ1, doxycycline (dox)-induced ERĮ protein expression was performed in MDAMB231 cells and ENDX effects on PKCȕ1 were evaluated using the above-mentioned experimental conditions. While forced expression of ERĮ in dox-induced cells modestly decreased PKCȕ1 protein levels compared to nondox-induced cells, ENDX pretreatment displayed no impact on PKCȕ1 protein expression in the presence of ERĮ (Figure 28C). ERĮ overexpression also resulted in increased AKTSer473 phosphorylation that remained unaffected by ENDX pretreatment in these cells. Additionally, MDAMB231 and HEK293F cells were pretreated with or without 5 μM ENDX for two hours followed by 0 or 200 nM PMA treatment for 20 minutes and effects on PKCȕ1 were evaluated. Treatment with PMA had minimal effects on PKCȕ1 phosphorylation and ENDX pretreatment in the presence of PMA displayed no impact on PKCȕ1 protein expression (Figure 28D). Taken together, these data demonstrate that in ER- cells, while AKT signaling may be further activated by insulin, PMA does not result in meaningful activation of PKCȕ1, suggesting that activation of AKT signaling in ER- cells may not be mediated through PKCȕ1 nor blocked by ENDX. ENDX at 5 M replicates apoptotic effects of the pan-AKT inhibitor MK-2206 in estrogen deprived ERĮ+ breast cancer cells To assess the impact of AKT inhibition on estrogen deprived MCF7AC1 cells, MK- 2206 was administered at a variety of concentrations. These studies showed that growth was Attorney Docket No. 07039-2296WO1 / 2023-600 inhibited at MK-2206 concentrations 0.1 μM, AKTSer473 phosphorylation was reduced at MK-2206 concentrations ^ 1 μM, and apoptosis as manifested by both annexin V binding and caspase-mediated PARP1 cleavage was induced at 5 μM MK-2206 (Figure 21A, Figures 29A-29C). In these same cells, treatment with ENDX at 5 μM induced apoptosis (Figure 21A). Lower (0.01 and 0.1 μM) and higher (5 μM) ENDX concentrations were also evaluated and it was demonstrated that only the 5 μM concentrations reduced AKTSer473 phosphorylation and increased PARP cleavage (Figure 21B). Consistent with these in vitro finding, in the MCF7AC1 xenograft model, in vivo treatment with ENDX at 75 mg/kg but not at 25 mg/kg also increased PARP cleavage (Figure 29D). It was next asked whether ENDX had similar effects on T47D cells. Unlike MCF7AC1 cells, parental T47D cells failed to proliferate in CSS medium (Figure 29E). Therefore, ENDX effects on the growth of the ERĮ+/HER2- LTED T47D cell line model (T47D-LTED) that proliferates well in CSS medium were examined. Evaluation of basal protein expression revealed reduced ER levels and a modest decrease in AKTSer473 phosphorylation in the T47D-LTED cell line compared to the parental T47D cell line (Figure 29F). As noted with MCF7AC1 cells, treatment of T47D-LTED cells with MK-2206 significantly inhibited growth starting at 0.1 μM (Figure 29G). At 1 and 5 μM MK-2206, but not 0.1 μM, reduced AKTSer473 phosphorylation and increased apoptosis, as indicated by both annexin V staining and increased PARP cleavage were observed (Figures 29H and 29I), phenocopying the reported biological effects of MK-2206. Treatment of T47D-LTED cells with 5 μM ENDX likewise attenuated AKTSer473 phosphorylation, inhibited growth, and induced apoptosis as manifested by annexin V binding and PARP1 cleavage (Figures 21C and 21D, and Figure 29J), replicating the MCF7AC1 response. ENDX (5 μM) was then compared with clinically attainable concentrations of TAM and ICI (0.1 μM) in terms of their effects on apoptosis. Unlike ENDX, both TAM and ICI failed to induce apoptosis (Figure 30). Attorney Docket No. 07039-2296WO1 / 2023-600 Expression of constitutively active AKT attenuates ENDX-induced apoptosis in ERĮ+ breast cancer cells To confirm the role of AKT inhibition in ENDX-induced apoptosis, a cumate inducible expression system was utilized to overexpress a C-terminally HA-tagged constitutively active AKT in MCF7AC1 cells (MCF7AC1caAKT cells). Immunoblot assays with an anti-HA antibody confirmed cumate induced expression of caAKT (Figure 22A) that was associated with increased phosphorylation of AKT substrates (Figure 22B, arrows). While ENDX at 5 μM induced apoptosis in the absence of cumate in this cell model, expression of caAKT significantly diminished the ability of ENDX to induce apoptosis (Figure 22C). Taken together, these findings established that ENDX not only inhibited proliferation, but at higher concentrations, induced apoptosis of ERĮ+ breast cancer cells, and this may occur in part through inhibition of PKCȕ1 and the resulting decrease in AKT kinase activity. Evaluation of ENDX dose response effects (0-10 μM) on apoptosis in MDAMB231 cells cultured in CSS medium showed that ENDX did not induce apoptosis in these cells until it reached the highest concentration of 10 μM (Figure 31A). Concurrent studies performed to evaluate ENDX effects on growth of MDAMB231 cells at the above-mentioned concentrations, revealed that ENDX did not inhibit growth until it reached concentrations of ^ 7.5 μM (Figure 31B). Given the lack of MDAMB231 response to ENDX treatment, ENDX response in the ER- BT549 and MDAMB436 breast cancer cells was evaluated. Similar to MDAMB231 cells, ENDX did not inhibit growth of these cells until concentrations > 7.5 μM (Figure 31B). Immunoblot confirmed basal expression of PKCȕ1 in all three cell lines (Figure 31C). Collectively, the lack of an effect of ENDX on apoptosis in ER- cells was consistent with the null findings regarding ENDX’s effects on AKT signaling in ER- cells, and suggested that unlike ER+ cells, ENDX did not elicit pharmacodynamic effects on apoptosis through PKCȕ1 targeting. Attorney Docket No. 07039-2296WO1 / 2023-600 Methods Cell culture MCF7 human breast cancer cells stably transfected with the aromatase gene (MCF7AC1) were cultured in phenol-red free IMEM medium (Gibco #A10488-01) supplemented with 10% fetal bovine serum (FBS) (Gemini #900-108), 600 ^g/mL geneticin (G418) (Gibco #10131-027) and 1% Antibiotic-Antimycotic (AA) (Gibco #15240-062). To maintain an estrogen deprived state, MCF7AC1 cells were cultured in IMEM medium containing 10% charcoal-stripped serum (CSS) (Hyclone #SH30068), 600 ^g/mL G418 and 1% AA. T47D cells were cultured in DMEM/F12 medium (Corning #16-405-V) containing 10% FBS and 1% AA. T47D-long-term estrogen deprived (LTED) cells were cultured in DMEM/F12 medium containing 10% CSS and 1% AA. C-terminally hemagglutinin (HA)- tagged, catalytically active AKT expressing MCF7AC1 (MCF7AC1caAKT) cells were cultured in IMEM containing 10% FBS, 600 ^g/mL G418, 1% AA and 0.5 μg/mL puromycin (Gibco #A11138-03). The ER- MDAMB231, BT549 and MDAMB436 breast cancer cells were cultured in DMEM/F12 medium containing 10% FBS and 1% AA. The HEK293F cells were cultured in DMEM medium (Corning #34722014) containing 10% FBS and 1X penicillin- streptomycin (Sigma #P0781). The doxycycline-inducible ERĮ-expressing MDAMB231 cell line was established using the T-RExTM system (InVitrogen) and were maintained in DMEM/F12 medium containing 10% FBS 1% AA, 5 mg/L Blasticidin S (Sigma #15205) and 500 mg/L Zeocin (InVivoGen #ant-zn-5b). The Z-endoxifen hydrochloride utilized in this study was synthesized. Estrogen deprived MCF7AC1 were treated with vehicle control or ENDX (National Cancer Institute) for 24 hours. For the phorbol 12-myristate 13-acetate (PMA) (LC Laboratories, #P-1680) experiments, MCF7AC1 cells were maintained in serum-free medium for 24 hours prior to pretreatment with vehicle or ENDX for two hours followed by treatment with 20 or 200 nM PMA for 20 minutes. For the insulin (Sigma-Aldrich, #I0516) experiments, MCF7AC1 and T47D cells were maintained in serum-free medium for 24 hours prior to pretreatment with vehicle control or drugs for two hours followed by treatment with 100 nM insulin for one hour. Attorney Docket No. 07039-2296WO1 / 2023-600 Proliferation assay Cells were plated at a density of 2000 cells per well. Cell viability of (i) vehicle or drug treated MCF7AC1 and T47D-LTED cells in CSS medium, (ii) siNT or siPKCȕ- transfected MCF7AC1 cells in CSS medium, and (iii) T47D cells in FBS versus CSS medium were analyzed by crystal violet staining assay after six days of treatment or siRNA transfection. Cell viability was calculated as the average absorbance of the drug treated cells divided by the average absorbance of the vehicle treated cells x 100. Apoptosis assay MCF7AC1 and T47D-LTED cells were plated at a density of 2000 cells per well in CSS medium for 24 hours. Cells were then co-treated for 48 hours with vehicle or drug, IncuCyte Annexin V green reagent (#4642, 1:300), an early-stage apoptosis marker, and IncuCyte NucLight rapid red reagent (#4717, 1:500), a dye that stains all cell nuclei red, in CSS medium. MCF7AC1caAKT cells were plated at a cell density of 2000 cells per well in CSS medium in the absence or presence of cumate for 48 hours and then co-treated with vehicle or drug, Annexin V green and NucLight rapid red reagents in the absence or presence of cumate for an additional 48 hours in CSS medium. The apoptosis graphs are presented as the green object count (which correspond to cells that are stained with the IncuCyte green fluorescence Annexin V reagent) divided by the red object count (which correspond to the total number of cells in the culture that are stained with the IncuCyte red fluorescence Nuclight Rapid Red Cell Labeling reagent that labels the nucleus of all cells without perturbing cell function or biology) and displayed as percentage using the IncuCyte S3 analysis software. Protein sample preparation for mass spectrometry-based quantitative proteomics analysis (i). Cell lysis and in-solution trypsin digestion. Following treatment of MCF7AC1 cells with vehicle control or 0.01, 0.1, or 5 μM ENDX for 24 hours in CSS media, cells were harvested and lysed in 8 M urea buffer (8 M urea, 20 mM HEPES pH 8.0, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM ȕ-glycerophosphate, and 5 mM sodium fluoride), followed by sonication, and centrifugation at 15,000 x g at 4°C for 20 minutes to clear cell debris. BCA Protein Assay was used to measure the protein Attorney Docket No. 07039-2296WO1 / 2023-600 concentration.2 mg of protein lysates from each treatment condition was used for digestion with trypsin. Briefly, the protein lysates were reduced with 5 mM dithiothreitol at 37°C for 1 hour and alkylated with 10 mM iodoacetamide at room temperature in dark for 30 minutes. The protein lysates were then diluted in 20 mM HEPES pH 8.0 to a final concentration < 2 M urea and digested with TPCK-treated trypsin (Worthington Biochemical Corp. Lakewood, NJ) overnight at room temperature. Digested peptides were acidified with 20% trifluoroacetic acid (TFA) to a final concentration of 1% TFA. The tryptic peptides were desalted using SepPak C18 cartridge (Waters Corporation, Milford, MA). Eluted peptides were lyophilized and stored at -80°C prior to TMT labeling. (ii). Tandem Mass Tag (TMT) labeling of peptides and basic reversed-phase liquid chromatography (bRPLC) fractionation. The lyophilized tryptic peptides were reconstituted in 150 μL 100 mM triethylammonium bicarbonate (TEABC) and measured with peptide BCA assay (Thermo Scientific).1 mg peptides from each sample in a final volume of 100 uL 1 mM TEABC were mixed with 1 mg μg TMTpro reagent that was dissolved in 20 μL anhydrous acetonitrile. After 1 hour incubation at RT, 10 ^L of 5% hydroxylamine was added and incubated for 15 minutes at room temperature to quench the labeling reaction. Peptides labeled by different TMT reagents were then mixed and dried with Speed-Vac. The dried TMT-labeled peptides were reconstituted in 20 mM ammonium formate and fractionated by high-pH reversed-phase liquid chromatography on Dionex Ultimate 3000 (Thermo Scientific). Peptides (12 mg) were separated on a 4.6 mm × 50 cm × 3.5 ^m Xbridge column (Waters) with a 2-hour gradient from 2 to 40% mobile phase B (MPB). Mobile phase A was composed of 20 mM ammonium formate in water, and MPB was composed of 20 mM ammonium formate in 80% acetonitrile. A total of 96 fractions were collected and concatenated into 24 fractions. A 20-^g equivalent of each fraction was set aside for global proteome analysis, and the rest of each sample was concentrated into 12 fractions and dried before phosphopeptide enrichment. (iii). Phosphopeptide enrichment. Each fraction was reconstituted in 1 mL of 80% acetonitrile in 0.1% TFA. Phosphopeptides were enriched using an immobilized metal affinity chromatography (IMAC) approach. In brief, nickel-nitrilotriacetic (Ni-NTA) Attorney Docket No. 07039-2296WO1 / 2023-600 superflow agarose beads were stripped of nickel with 100 mM EDTA, incubated with 10 mM FeCl3 solution and equilibrated in 80% ACN/0.1%TFA.10 ^L IMAC beads were mixed with each fractionated peptide in 80% acetonitrile/0.1% TFA and rotated for 30 minutes at room temperature. Subsequently, incubated IMAC beads were washed with 500 ^L 80% ACN/0.1%TFA four times and 500 ^L 0.1% FA one time. Phosphopeptides were eluted from IMAC beads with 200 ^L of 500 mM dibasic sodium phosphate (pH 7.0) for three times. The eluted phosphopeptides were desalted with C18 Stage Tips and Speed-Vac dried. (iv). LC-MS/MS analysis. The peptide fractions were loaded on a 2 cm trap column (Acclaim PepMap 100, C18, 5 μm particle size, 100 μm i.d.100 Å pore size, Thermo Scientific, San Jose, CA) using 0.1% formic acid with a flow rate 20 μL/minute for 4 minutes. The peptides were separated on a 50 cm analytical column (Acclaim PepMap 100, C18, 2 μm particle size, 75 μm i.d.100 Å pore size, Thermo Scientific, San Jose, CA) with a 135 minute gradient from 3% to 40% acetonitrile in 0.1% formic acid at a flow rate of 0.3 μL/minute. The spray voltage was set to 2.3 kV while capillary temperature was set to 275°C. The samples were analyzed on an Orbitrap Fusio Lumos mass spectrometer (Thermo Scientific, Bremen, Germany). The MS instrument was operated in data-dependent acquisition mode. A survey full scan MS (from 350–1,500 m/z) was acquired in the Orbitrap with resolution 120,000 at m/z 200 with a maximum AGC target value of 800,000 ions. The data-dependent MS/MS was carried out using Top Speed method with a duty cycle of 2 seconds. Singly charged precursor ions were excluded while precursor ions with charge states 2-7 were sequentially isolated and fragmented in the higher-energy collisional dissociation (HCD) cell using 34% normalized collision energy (NCE). The maximum ion injection time for MS and MS/MS were set to 50 ms. Fragment ion spectra were detected in Orbitrap mass analyzer with a resolution 30,000 at m/z 200. Dynamic exclusion was enabled one event of fragmentation followed by exclusion of the precursor for next 45 seconds within 7 ppm of the selected m/z. For all measurements with the Orbitrap detector, a lock-mass ion from ambient air (m/z 445.120025) was used for internal calibration. Attorney Docket No. 07039-2296WO1 / 2023-600 Mass spectrometry data analysis Proteome Discoverer software suite (v 2.5; Thermo Fisher Scientific, San Jose, CA) was used for quantitation and database searches. The MS/MS data were searched using the SEQUEST search algorithm against a Human Uniport protein database supplemented with frequently observed contaminants. Search parameters included trypsin as a protease with full specificity and a maximum of two allowed missed cleavages; carbamidomethylation of cysteine and TMTpro tag (+304.207ௗDa) on lysine residues or peptide N-terminus as a fixed modification; oxidation at methionine and phosphorylation at serine/threonine/tyrosine as variable modifications. The precursor tolerance was set at 10 ppm, while the fragment match tolerance was set to 0.02 Da. The PSMs, peptides and proteins were filtered at 1% false discovery rate cut-off calculated using target-decoy database searches. The probability of an identified phosphorylation of specific Ser/Thr/Tyr residue on each identified phosphopeptide was determined from the PhosphoRS algorithm. Phosphoproteome data analysis The intensities of TMT reporter ions were normalized based on the average total phosphopeptide intensity detected in each TMT-labeling channel. Differentially phosphorylated sites were identified with an empirical Bayesian moderated t-statistics test as implemented in the R limma package. Multiple comparison correction was performed with Benjamini-Hochberg procedure. Phosphorylation sites with log2 fold change > 1.5 and unadjusted p-value < 0.05 were selected for downstream analysis. DAVID, an integrated online functional annotation tool, was used to annotate the functions of the differentially modulated phosphoproteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was selected to identify enriched signaling pathways. The ggplot package in R was used to generate the bubble plot depicting the enriched pathways. The Fuzzy C-means clustering showing the dynamic regulation patterns of phosphosites were generated using ggplot and mfuzz packages in R. Kinase substrate enrichment analysis (KSEA), PhosphoSitePlus, NetworKIN, and RoKAI datasets were used to predict upstream kinases of regulated phosphosites, as described below. To identify the motifs enriched in the ENDX-regulated phosphosites, MoMo program with motif-x algorithm were used. Attorney Docket No. 07039-2296WO1 / 2023-600 Upstream kinase prediction analysis The 325 phosphosites in Cluster 1 was provided as an input in the NetworKIN and RoKAI kinase prediction tools. While NetworKIN predicted upstream kinases for 32 (10%) of the 325 phosphosites, RoKAI predicted upstream kinases for 14 (4%) of the 325 phosphosites. For the remaining 279 phosphosites no upstream kinase predictions were provided by these prediction tools. Taken together, both NetworKIN and RoKAI predicted a total of 46 upstream kinases for only 14% of the phosphosites in Cluster 1 and these kinases were graphed in the X-axis of Figure 16A. The number of counts in the Y-axis refers to the total number of phosphosites substrates for which the given kinase is predicted as a potential upstream kinase. For example, PKCB is predicted as the upstream kinases for 5 phosphosites, CDK1 is predicted as the upstream kinase for 4 phosphosites and AKT1 is predicted as the upstream kinase for 3 phosphosites, respectively in Cluster 1. Immunoblot analysis Protein lysates were prepared using the RIPA lysis buffer system (ChemCruz #sc- 24948) and quantified using the DCTM Protein Assay reagents (Bio-Rad #5000112). Equal amounts of protein lysates were separated on 10% Criterion gels (Bio-Rad #3450112), transferred to PVDF membranes (Bio-Rad #1620177), blocked in TBST-5% milk and probed with primary antibodies listed in Table 6, at the indicated dilutions. Membranes were incubated with HRP-conjugated anti-rabbit (CST #7074) or anti-mouse (CST #7076) secondary antibodies and visualized using chemiluminescent West Pico (Thermo Scientific #34580) or West Femto (Thermo Scientific #34096) reagents and a Li-Cor Odyssey® XF imager. Protein lysates from the MCF7AC1 xenograft model were obtained as described elsewhere (Jayaraman et al., Breast Cancer Res. 22:51 (2020)). Quantitation of the protein bands signal intensity was performed using the National Institute of Health (NIH) ImageJ image analysis software (imagej.nih.gov/ij///index.html). For the quantitation of the phospho protein levels, total protein levels were first normalized to ȕ-actin loading control and these values were used to normalize the phospho protein levels and compared change in protein expression levels relative to vehicle control normalized to 1.0. For the quantitation of total protein levels, the total protein levels were normalized to ȕ-actin and change in expression Attorney Docket No. 07039-2296WO1 / 2023-600 levels was compared relative to vehicle control normalized to 1.0. All blots were derived from the same experiment and processed in parallel. Table 6. y For the evaluation of ENDX effects on the kinase activity of the protein kinase C (PKC) family members and tamoxifen (TAM) effects on the kinase activity of PKC beta 1 (PKC 1), the drug compounds were tested in a 10-dose IC50 mode with three-fold serial dilution starting at 50 M, in the presence of 10 M ATP. Staurosporine, a broad-spectrum kinase inhibitor and a positive control, was also tested as described above. The IC50 concentrations of these drugs in inhibiting PKC kinase activity are provided in Table 5. Affinity measurements by Surface Plasma Resonance (SPR) Binding assays were performed at 25°C on a Biacore T200 biosensor (GE healthcare). Purified PKCȕ1 protein were immobilized on a CM5 S sensor chip using amino coupling and immobilization buffer (10 mM HEPES, 150 mM NaCl, pH 7.4, P200.01% (w/w)) and acetate pH 5.0 at a flow rate of 10 μL/minute and reaching 10,000-12,000 resonance units (RUs). ENDX at concentrations ranging from 0- 8000 nM in phosphate buffer (Gibco) with 2% DMSO (v/v) and 0.01% (w/w) P20 were run over the chips at a flow rate of 50 μL/minute for 30 seconds. Binding kinetics were derived from sensograms using Biacore BIA evaluation software (GE). Sensograms were subtracted for background Attorney Docket No. 07039-2296WO1 / 2023-600 contributions, and affinity constants were derived using a steady state affinity fitting of a 1:1 interaction model. siRNA transfection MCF7AC1 cells were maintained in CSS medium and transfected with non-targeting siRNA (siNT) (SI03650325) or a pool of two different siRNA’s targeting total PKCȕ (Hs_PRKCȕ1_6 SI00605948 and Hs_PRKCȕ1_4 SI00042273) (siPKCȕ) (Qiagen) at 5 nM concentration in the presence of lipofectamine RNAiMAX transfection reagent (13778-075, Invitrogen) for up to 48 hours for IB analysis and for up to six days for proliferation assays in biological triplicates. The custom made siPKCȕ1 siRNA (sense: 5’-AAGCCAAAAGCUAGAGACAUU- 3’ (SEQ ID NO:446); antisense: 5’-UGUCUCUAGCUUUUGGCUUUU-3’ (SEQ ID NO:447)) or the siGENOME non-Targeting siRNA Pool #1 (#D-001206-13-20) from Dharmacon were transfected into MCF7AC1 cells maintained in CSS medium at 40 nM concentration in the presence of DharmaFECT 1 (Dharmacon #T-2001-03) for up to 72 hours for IB assay. A SMART vector inducible human PRKCB mCMV-TurboGFP lentiviral shRNA (Horizon Discovery #V3SH7675-01EG5579) was used for the generation of doxycycline (dox)-inducible PRKCB gene silencing. MCF7AC1 cells grown in 6-well plate in IMEM medium containing 10% FBS, 600 ^g/mL G418 and 1% amino acids and at a confluency of 50% were infected with the lentivirus at a multiplicity of infection (MOI) of 5.0 in the presence of 1 μg/mL polybrene. After 48 hours, 10 mg/mL puromycin (Gibco #A11138-03) was added to cells at a dilution of 1 μL per 10 mL to allow for the selection of lentivirus- transfected cells. The expression of the shRNA sequence was induced in the presence of 1 μg/mL of doxycycline (Sigma-Aldrich #D3072) for 72 hours for IB assay. The target PRKCB gene sequence used is CAGTGTTGATGGCTGGTTT (SEQ ID NO:448; Horizon Discovery #V3IHSMCG_9696026). Molecular cloning Molecular cloning was performed for the generation of catalytically active AKT- expressing MCF7AC1 cells. For this purpose, vector pCDNA3.1+ containing an N-terminal Attorney Docket No. 07039-2296WO1 / 2023-600 SRCMyr signal AKT ORF and C-terminal hemagglutinin (HA)-tag was used. The constitutively active AKT (caAKT)-HA insert was excised using NheI/EcoRV restriction digest, gel purified and cloned into the NheI//PmeI site of an SBI (System biosciences, Palo Alto, CA) vector with modified restriction sites. Sanger sequencing was performed by Azenta/Genewiz, (South Plainfield, NJ) to confirm in frame sequence. After viral transduction of the caAKT-HA construct into MCF7AC1 cells, cells were selected beginning 48 hours later for a mixed population (MCF7AC1caAKT cells) using puromycin (Invitrogen) 0.5 μg/mL for several weeks. Expression of caAKT-HA was induced by adding 60 μg/mL cumate (Sigma Aldrich 268402, 4-Isopropylbenzoic acid) for 48 hours prior to drug treatment. Statistical analysis Differences in cell proliferation in the drug treated MCF7AC1 and T47D-LTED cells compared to vehicle treated cells, the % of apoptosis in the drug treated MCF7AC1 and T47D-LTED cells compared to vehicle treated cells and the % of apoptosis in the vehicle or 5 μM ENDX treated MCF7AC1caAKT cells in the presence and absence of cumate were analyzed by one-way ANOVA. Differences in the % of PKCȕ1 and AKTSer473 protein levels remaining upon PKCȕ1 knockdown in the siNT versus siPKCȕ or siPKCȕ1 transfected MCF7AC1 cells and in the doxycycline-induced versus noninduced MCF7AC1 cells as well as differences in cell proliferation in the siNT versus siPKCȕ transfected MCF7AC1 cells were analyzed by one sample t-test. Comparison of proliferation rates of T47D cells cultured in FBS medium versus CSS medium were analyzed by unpaired t-test. All statistical analysis was performed in Graphpad Prism imaging software (Version 9). A p value of < 0.05 was considered statistically significant. Example 3: ENDX and Immune Function in Patients with ER+ Breast Cancer ENDX upregulates the expression of IFN , IL-2 and Granzyme B expression by CD4+ and CD8+ T cells in vitro In multiple ER+ cell lines, the ability of PKCȕ1 to bind ENDX was linked to decreases in PKCȕ1 protein levels and reduction in Akt activity, resulting in the induction of Attorney Docket No. 07039-2296WO1 / 2023-600 apoptosis (see, Example 2 and Jayaraman et al., NPJ Breast Cancer, 9(1):101 (2023)). Given that PKCȕ1 is highly expressed in immune cells, the potential impact of ENDX on immune function was investigated. Using peripheral blood mononuclear cells (PBMC) from apheresis cones derived from healthy premenopausal female donors, concentrations of ENDX (0.1 uM), tamoxifen (1 uM), and fulvestrant (0.1 uM) known to solely target the ER were evaluated. When using these concentrations, no effect on immune cell proliferation or function was observed. Based on data demonstrating that ENDX inhibits PKCȕ1 kinase and targets PKCȕ1 protein for degradation at concentrations of > 1.5 uM, 2 ^M ENDX concentrations and its effects on PBMCs were evaluated. As a control, the PBMCs were also treated with enzastaurin, a potent and selective serine/threonine kinase inhibitor of PKCȕ1 previously studied in phase II metastatic BC setting which did not demonstrate sufficient antitumor activity for further development (Mina et al., Invest. New Drugs, 27(6): 565-570 (2009)).5-day treatment of PBMCs with 2 μM ENDX significantly (p=0.02) increased IFNȖ expression compared to untreated or 2 μM enzastaurin-treated cells (Figure 32A). The expression of several cytokines by PBMCs following treatment with ENDX or enzastaurin was additionally assessed. The findings revealed that ENDX significantly induced the expression of GranzymeB and IL-2 in both CD4+ and CD8+ T cells. However, it had no impact on perforin expression (Figure 33). Conversely, enzastaurin treatment led to a reduction in the expression of these cytokines, suggesting distinct regulatory mechanisms (Figure 33). Within the context of the tumor microenvironment, tumor-infiltrating lymphocytes (TILs) constitute a predominant source of IFNȖ secretion, thereby playing a pivotal role in tumor immune surveillance and cytotoxicity. While both pro- and anti-tumorigenic roles are attributed to IFNȖ, its gene expression profiles have been correlated with enhanced overall survival outcomes across distinct subtypes of BC (Figure 32B), most likely via distinct mechanisms. These in vitro data showing increased IFNȖ secretion in response to ENDX suggested the drug may exhibit an immune modulatory role in patients receiving this drug. Attorney Docket No. 07039-2296WO1 / 2023-600 ENDX modulates the phenotypic profile of immune cells The influence of ENDX on IFNȖ, GranzymeB, and IL-2 prompted expansion of the analysis and the drug’s regulatory impact on various immune markers, encompassing immune checkpoint molecules, across different T cell populations was examined. To accomplish this, peripheral blood mononuclear cells (PBMCs) from healthy donors were subjected to treatment with ENDX (2 μM). Simultaneously, these cells were stimulated with CD3/CD28 Dynabeads for 5 days. Subsequently, a panel consisting of 37 immune markers was employed for cellular staining, followed by analysis utilizing mass cytometry (CyTOF) on the Fluidigm® Helios™ platform. The acquired data using FlowJo-v10.8.1 software detected notable changes in specific cell subpopulations in response to drug treatment (Figure 34). The data provide further evidence that ENDX treatment increased the population of CD4+ and CD8+ T cells exhibiting a memory phenotype, as evidenced by the expression of specific markers such as CD28, CD45RO, CCR4, CXCR3, CD25, CD11a, CD38, and TIGIT. Conversely, there was a decrease in the CD8+ T cell population characterized by low CD45RO expression but high levels of CD25, CD27, CD28, CCR7, CXCR3, and TIM3. These findings provide further in vitro support demonstrating that ENDX alters the phenotypic profile of immune cells, thereby potentially contributing to their function. ENDX primes immune cells to regulate ER+ breast cancer cell line growth To investigate the potential impact of ENDX-induced alterations in immune cell functionality on their anti-tumor efficacy, PBMCs were pre-treated with either 2 μM of ENDX or enzastaurin for 5 days, and subsequently co-cultured with either MCF7AC1 cells sensitive to ENDX or MCF7 cells resistant to ENDX, followed by monitoring real-time tumor proliferation using IncuCyte over 5 days (Figure 35A). In a series of parallel experiments, tumor cells were subjected to prior treatment with abemaciclib, a potent CDK4/6 inhibitor, after which they were co-cultured with PBMCs, followed by a real-time proliferation assay using IncuCyte (Figure 35). It was investigated whether synergistic immune killing may be present when ER+ tumor cells are co-cultured with PBMCs that have been treated with both ENDX and abemaciclib. The data indicated that co-culture of PBMCs previously exposed to ENDX along with either aromatase expressing MCF7AC1 cells or Attorney Docket No. 07039-2296WO1 / 2023-600 MCF7 ENDX-resistant cells, resulted in a significant decrease in tumor cell proliferation as compared to untreated control cells (Figure 35B). In contrast, PBMCs exposed to enzastaurin demonstrated no discernible impact on proliferation. Furthermore, when either MCF7AC1 or MCF7 ENDX-resistant cells were pre-treated with abemaciclib (500 nM) before ENDX exposed PBMCs, nearly complete inhibition of tumor growth was observed, demonstrating the effectiveness of this combination therapy in reducing BC cell proliferation (Figure 35, middle and right panels). Example 4: Z-Endoxifen (ENDX) mediated CD4+ and CD8+ T cell alterations at physiological achievable drug concentrations This Example demonstrates that endoxifen negatively regulated inhibitory receptors PD-1 and TIM-3 on CD4+ and CD8+ T cells. A flow cytometry gating strategy for of CD4+ and CD8+ T cells is shown in Figure 36. Endoxifen negatively regulated inhibitory receptors PD-1 and TIM-3, T cell exhaustion markers that are upregulated in poor prognosis ER+/HER2- breast cancer, on CD4+ and CD8+ T cells (Figure 37). These data were not seen with fulvestrant, a pure antiestrogen (Figure 37). These data suggest that endoxifen may have the ability to activate the immune system by eradicating a subset of T cells associated with immune exhaustion. A flow cytometry gating strategy for T cell effector cytokines is shown in Figure 38. T cell IFN-Ȗ+, TNF-Į+, and IL-2+ expression was preserved in presence of endoxifen (Figure 39). Example 5: Treating ER- Cancer A human identified as having an ER- cancer (e.g., an ER- breast cancer such as a TNBC) is administered from about 20 mg/day to about 360 mg/day of one or more ENDX compounds (e.g., a Z-ENDX such as Z-ENDX hydrochloride). The administered one or more ENDX compounds can reduce the number of cancer cells present in the human. Example 6: Treating ER- Cancer A human identified as having an ER+ cancer and/or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) is administered one or more ENDX compounds (e.g., a Z- Attorney Docket No. 07039-2296WO1 / 2023-600 ENDX such as Z-ENDX hydrochloride) together with one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell (e.g., abemaciclib) of the ER+ cancer and/or the ER- cancer. The administered one or more ENDX compounds and one or more agents/therapies that can alter the antigens presented on the surface of a cancer cell can reduce the number of cancer cells present in the human. Example 7: Exemplary Embodiments Embodiment 1. A method for treating a mammal having an estrogen receptor negative (ERneg) cancer, wherein said method comprises administering, to said mammal, an endoxifen (ENDX) compound. Embodiment 2. The method of embodiment 1, wherein said mammal is a human. Embodiment 3. The method of any one of embodiments 1-2, wherein said mammal is a female mammal. Embodiment 4. The method of any one of embodiments 1-3, wherein said mammal is a pre-menopausal female human. Embodiment 5. The method of any one of embodiments 1-4, wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. Embodiment 6. The method of embodiment 5, wherein said ERneg cancer is a breast cancer. Attorney Docket No. 07039-2296WO1 / 2023-600 Embodiment 7. The method of embodiment 6, wherein said breast cancer is a triple negative breast cancer. Embodiment 8. The method of any one of embodiments 1-7, wherein said ENDX compound is a Z-ENDX compound. Embodiment 9. The method of embodiment 8, wherein said Z-ENDX compound is a Z-ENDX salt. Embodiment 10. The method of embodiment 9, wherein said Z-ENDX salt is Z-ENDX hydrochloride. Embodiment 11. The method of any one of embodiments 1-10, wherein said method comprising administering from about 20 milligrams per day (mg/day) to about 360 mg/day of said ENDX compound to said mammal. Embodiment 12. The method of any one of embodiments 1-11, said method further comprising administering to said mammal an agent that can alter the antigens presented on the surface of a cancer cell of said ERneg cancer. Embodiment 13. The method of embodiment 12, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti- PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, Attorney Docket No. 07039-2296WO1 / 2023-600 REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602. Embodiment 14. A method for treating a mammal having an ERpos cancer or an ERneg cancer, wherein said method comprises administering, to said mammal, (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ERpos cancer or said ERneg cancer. Embodiment 15. The method of embodiment 14, wherein said mammal is a human. Embodiment 16. The method of any one of embodiments 14-15, wherein said mammal is a female mammal. Embodiment 17. The method of any one of embodiments 14-16, wherein said mammal is a pre-menopausal female human. Embodiment 18. The method of any one of embodiments 14-17, wherein said mammal has said ERneg cancer, and wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. Embodiment 19. The method of embodiment 18, wherein said ERneg cancer is a breast cancer. Attorney Docket No. 07039-2296WO1 / 2023-600 Embodiment 20. The method of embodiment 19, wherein said breast cancer is a triple negative breast cancer. Embodiment 21. The method of any one of embodiments 14-17, wherein said mammal has said ERpos cancer, and wherein said ERpos cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer. Embodiment 22. The method of any one of embodiments 14-21, wherein said ENDX compound is a Z-ENDX compound. Embodiment 23. The method of embodiment 22, wherein said Z-ENDX compound is a Z-ENDX salt. Embodiment 24. The method of embodiment 23, wherein said Z-ENDX salt is Z-ENDX hydrochloride. Embodiment 25. The method of any one of embodiments 14-24, wherein said method comprising administering from about 20 mg/day to about 360 mg/day of said ENDX compound to said mammal. Embodiment 26. The method of any one of embodiments 14-25, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5- fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, Attorney Docket No. 07039-2296WO1 / 2023-600 abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602. Embodiment 27. The use of a composition comprising an ENDX compound to treat a mammal having an ERneg cancer. Embodiment 28. The use of embodiment 27, wherein said mammal is a human. Embodiment 29. The use of any one of embodiments 27-28, wherein said mammal is a female mammal. Embodiment 30. The use of any one of embodiments 27-29, wherein said mammal is a pre-menopausal female human. Embodiment 31. The use of any one of embodiments 27-30, wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. Embodiment 32. The use of embodiment 31, wherein said ERneg cancer is a breast cancer. Embodiment 33. The use of embodiment 32, wherein said breast cancer is a triple negative breast cancer. Attorney Docket No. 07039-2296WO1 / 2023-600 Embodiment 34. The use of any one of embodiments 27-33, wherein said ENDX compound is a Z-ENDX compound. Embodiment 35. The use of embodiment 34, wherein said Z-ENDX compound is a Z- ENDX salt. Embodiment 36. The use of embodiment 35, wherein said Z-ENDX salt is Z-ENDX hydrochloride. Embodiment 37. The use of any one of embodiments 27-36, wherein said composition comprises from about 20 mg to about 360 mg of said ENDX compound. Embodiment 38. The use of any one of embodiments 27-37, said composition further comprises an agent that can alter the antigens presented on the surface of a cancer cell of said ERneg cancer. Embodiment 39. The use of embodiment 38, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti- PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, Attorney Docket No. 07039-2296WO1 / 2023-600 bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602. Embodiment 40. An ENDX compound for use in the preparation of a medicament to treat an ERneg cancer. Embodiment 41. An ENDX compound for use in the treatment of an ERneg cancer. Embodiment 42. The ENDX compound of any one of embodiments 40-41, wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. Embodiment 43. The ENDX compound of embodiment 42, wherein said ERneg cancer is a breast cancer. Embodiment 44. The ENDX compound of embodiment 43, wherein said breast cancer is a triple negative breast cancer. Embodiment 45. The ENDX compound of any one of embodiments 40-44, wherein said ENDX compound is a Z-ENDX compound. Embodiment 46. The ENDX compound of embodiment 45, wherein said Z-ENDX compound is a Z-ENDX salt. Embodiment 47. The ENDX compound of embodiment 46, wherein said Z-ENDX salt is Z-ENDX hydrochloride. Attorney Docket No. 07039-2296WO1 / 2023-600 Embodiment 48. The use of a composition comprising (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ERpos cancer or said ERneg cancer to treat a mammal having an ER+ cancer or an ERneg cancer. Embodiment 49. The use of embodiment 48, wherein said mammal is a human. Embodiment 50. The use of any one of embodiments 48-49, wherein said mammal is a female mammal. Embodiment 51. The use of any one of embodiments 48-50, wherein said mammal is a pre-menopausal female human. Embodiment 52. The use of any one of embodiments 48-51, wherein said mammal has said ERneg cancer, and wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. Embodiment 53. The use of embodiment 52, wherein said ERneg cancer is a breast cancer. Embodiment 54. The use of embodiment 53, wherein said breast cancer is a triple negative breast cancer. Embodiment 55. The use of any one of embodiments 48-51, wherein said mammal has said ERpos cancer, and wherein said ERpos cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer. Attorney Docket No. 07039-2296WO1 / 2023-600 Embodiment 56. The use of any one of embodiments 48-55, wherein said ENDX compound is a Z-ENDX compound. Embodiment 57. The use of embodiment 56, wherein said Z-ENDX compound is a Z- ENDX salt. Embodiment 58. The use of embodiment 57, wherein said Z-ENDX salt is Z-ENDX hydrochloride. Embodiment 59. The use of any one of embodiments 48-58, wherein said composition comprises from about 20 mg to about 360 mg of said ENDX compound. Embodiment 60. The use of any one of embodiments 48-59, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5- fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602. Attorney Docket No. 07039-2296WO1 / 2023-600 Embodiment 61. An ENDX compound and an agent comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the preparation of a medicament to treat an ERpos cancer or ERneg cancer. Embodiment 62. An ENDX compound and an agent comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the treatment of an ERneg cancer. Embodiment 63. The ENDX compound of any one of embodiments 61-62, wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. Embodiment 64. The ENDX compound of embodiment 63, wherein said ERneg cancer is a breast cancer. Embodiment 65. The ENDX compound of embodiment 64, wherein said breast cancer is a triple negative breast cancer. Embodiment 66. The ENDX compound of any one of embodiments 61-62, wherein said ERpos cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer. Embodiment 67. The ENDX compound of any one of embodiments 61-66, wherein said ENDX compound is a Z-ENDX compound. Embodiment 68. The ENDX compound of embodiment 67, wherein said Z-ENDX compound is a Z-ENDX salt. Attorney Docket No. 07039-2296WO1 / 2023-600 Embodiment 69. The ENDX compound of embodiment 68, wherein said Z-ENDX salt is Z-ENDX hydrochloride. Embodiment 70. The ENDX compound of any one of embodiments 61-69, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602. OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 07039-2296WO1 / 2023-600 WHAT IS CLAIMED IS: 1. A method for treating a mammal having an estrogen receptor negative (ERneg) cancer, wherein said method comprises administering, to said mammal, an endoxifen (ENDX) compound. 2. The method of claim 1, wherein said mammal is a human. 3. The method of claim 1, wherein said mammal is a pre-menopausal female human. 4. The method of claim 1, wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. 5. The method of claim 4, wherein said ERneg cancer is a breast cancer. 6. The method of claim 5, wherein said breast cancer is a triple negative breast cancer. 7. The method of claim 1, wherein said ENDX compound is a Z-ENDX compound. 8. The method of claim 1, wherein said method comprising administering from about 20 milligrams per day (mg/day) to about 360 mg/day of said ENDX compound to said mammal. 9. The method of claim 1, said method further comprising administering to said mammal an agent that can alter the antigens presented on the surface of a cancer cell of said ERneg cancer. Attorney Docket No. 07039-2296WO1 / 2023-600 10. The method of claim 9, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti- PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU- D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602. 11. A method for treating a mammal having an ERpos cancer or an ERneg cancer, wherein said method comprises administering, to said mammal, (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ERpos cancer or said ERneg cancer. 12. The method of claim 11, wherein said mammal is a human. 13. The method of claim 11, wherein said mammal is a pre-menopausal female human. 14. The method of claim 11, wherein said mammal has said ERneg cancer, and wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. Attorney Docket No. 07039-2296WO1 / 2023-600 15. The method of claim 14, wherein said ERneg cancer is a breast cancer. 16. The method of claim 15, wherein said breast cancer is a triple negative breast cancer. 17. The method of claim 11, wherein said mammal has said ERpos cancer, and wherein said ERpos cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer. 18. The method of claim 11, wherein said ENDX compound is a Z-ENDX compound. 19. The method of claim 11, wherein said method comprising administering from about 20 mg/day to about 360 mg/day of said ENDX compound to said mammal. 20. The method of claim 11, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti- PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU- D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602. Attorney Docket No. 07039-2296WO1 / 2023-600 21. The use of a composition comprising an ENDX compound to treat a mammal having an ERneg cancer. 22. The use of claim 21, wherein said mammal is a human. 23. The use of claim 21, wherein said mammal is a pre-menopausal female human. 24. The use of claim 21, wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. 25. The use of claim 24, wherein said ERneg cancer is a breast cancer. 26. The use of claim 22, wherein said breast cancer is a triple negative breast cancer. 27. The use of claim 21, wherein said ENDX compound is a Z-ENDX compound. 28. The use of claim 21, wherein said composition comprises from about 20 mg to about 360 mg of said ENDX compound. 29. The use of claim 21, said composition further comprises an agent that can alter the antigens presented on the surface of a cancer cell of said ERneg cancer. 30. The use of claim 29, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti- PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, Attorney Docket No. 07039-2296WO1 / 2023-600 brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU- D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602. 31. An ENDX compound for use in the preparation of a medicament to treat an ERneg cancer. 32. An ENDX compound for use in the treatment of an ERneg cancer. 33. The ENDX compound of any one of claims 31-32, wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. 34. The ENDX compound of claim 33, wherein said ERneg cancer is a breast cancer. 35. The ENDX compound of claim 34, wherein said breast cancer is a triple negative breast cancer. 36. The ENDX compound of any one of claims 31-32, wherein said ENDX compound is a Z-ENDX compound. Attorney Docket No. 07039-2296WO1 / 2023-600 37. The use of a composition comprising (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ERpos cancer or said ERneg cancer to treat a mammal having an ER+ cancer or an ERneg cancer. 38. The use of claim 37, wherein said mammal is a human. 39. The use of claim 37, wherein said mammal is a pre-menopausal female human. 40. The use of claim 37, wherein said mammal has said ERneg cancer, and wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. 41. The use of claim 40, wherein said ERneg cancer is a breast cancer. 42. The use of claim 41, wherein said breast cancer is a triple negative breast cancer. 43. The use of claim 37, wherein said mammal has said ERpos cancer, and wherein said ERpos cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer. 44. The use of claim 37, wherein said ENDX compound is a Z-ENDX compound. 45. The use of claim 37, wherein said composition comprises from about 20 mg to about 360 mg of said ENDX compound. Attorney Docket No. 07039-2296WO1 / 2023-600 46. The use of claim 37, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti- PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU- D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602. 47. An ENDX compound and an agent comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the preparation of a medicament to treat an ERpos cancer or ERneg cancer. 48. An ENDX compound and an agent comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the treatment of an ERneg cancer. 49. The ENDX compound of any one of claims 47-48, wherein said ERneg cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and/or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma. 50. The ENDX compound of claim 49, wherein said ERneg cancer is a breast cancer. Attorney Docket No. 07039-2296WO1 / 2023-600 51. The ENDX compound of claim 50, wherein said breast cancer is a triple negative breast cancer. 52. The ENDX compound of any one of claims 47-48, wherein said ERpos cancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer. 53. The ENDX compound of any one of claims 47-48, wherein said ENDX compound is a Z-ENDX compound. 54. The ENDX compound of any one of claims 47-48, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, I-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5- fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602.
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