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WO2025137355A1 - Liaison par substitution pour le criblage de composés thérapeutiques - Google Patents

Liaison par substitution pour le criblage de composés thérapeutiques Download PDF

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Publication number
WO2025137355A1
WO2025137355A1 PCT/US2024/061138 US2024061138W WO2025137355A1 WO 2025137355 A1 WO2025137355 A1 WO 2025137355A1 US 2024061138 W US2024061138 W US 2024061138W WO 2025137355 A1 WO2025137355 A1 WO 2025137355A1
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myc
cancer
atf4
gcn1
inhibitor
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Sarki Abba ABDULKADIR
Mihai Ioan TRUICA
Gary E. SCHILTZ
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Northwestern University
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Northwestern University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • c-MYC oncogene
  • MYC is the most common oncogene involved in human cancers and is overexpressed in up to half of all cancers. Therefore, developing c-MYC inhibitors is among the most attractive potential anti-cancer strategies.
  • c-MYC is currently regarded as “undruggable.” Thus, there remains an unmet need to develop effective small molecule compounds with biological activities toward IDPs, including MYC protein for the treatment of diseases.
  • the method may include treating a subject for a cancer responsive to MYC inhibition, the method comprising administering a MYC inhibitor to the subject.
  • Another aspect of the technology provides for a method for treating a subject for a cancer, the method comprising determining the binding of an MYC inhibitor to an intrinsically disordered region of MYC and one or both of a distal N-terminal region of MYC ( ⁇ C) and an intrinsically disordered region of GCN1 and administering the MYC inhibitor if the MYC inhibitor binds to the intrinsically disordered region of MYC and one or both of the distal N-terminal region of MYC ( ⁇ C) and the intrinsically disordered region of GCN1.
  • Another aspect of the technology provides for a method for screening a compound for biological activity toward an intrinsically disordered protein (IDP), the method comprising determining a binding affinity of the compound to a proxy protein; and evaluating the biological activity of the compound toward the IDP based on the binding affinity of the compound to the proxy protein, wherein the proxy protein is less disordered than the IDP and comprises a binding domain with at least 80% sequence identity to an intrinsically disordered region (IDR) of the IDP; wherein the compound binds to the binding domain of the proxy protein.
  • Another aspect of the technology provides for a method of identifying a cancer responsive to MYC inhibition, the method comprising determining a MYC inhibitor Response Signature (MiRS) score for the cancer.
  • MiRS MYC inhibitor Response Signature
  • Another aspect of the technology provides for a method for treating a subject for a cancer responsive to ATF4 activation or induction, the method comprising administering a GCN1 ligand that activates or induces expression of ATF4.
  • Another aspect of the technology provides for a method for sensitizing a subject having cancer to MYC inhibition treatment, the method comprising activating or inducing expression of ATF4 in the subject.
  • BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows that MYCi activates the ATF4-ISR pathway.
  • n 3 repeats, representative blot shown
  • n Western blot for Attorney Docket No.702581.02595 (NU2023-153-02) MYC and ATF4 protein in PC3 cells treated for 24 hours with 435, 436 or MYCi975 at 10 ⁇ M.
  • o Volcano plot displaying differentially bound ATF4 peaks after 16-hour treatment with 10 ⁇ M of MYCi975 in 22Rv1 cells.
  • FIG. 3 shows that (a) Western blots shows ATF4 level in MEF eIF2 ⁇ S/S and eIF2 ⁇ A/A cells after MYCi975 treatment for 24 hrs. (b) MycCaP cells were treated with PERK (10 ⁇ M) and GCN2 (5 ⁇ M) inhibitor alone or in presence of MYCi for 24 hrs. (c) GCN1 identified in both proteomics of MYCI-bound protein and by PHI-blast of MycHot region.
  • H3K27ac regions overlapping with sites of ATF4-gain or MYC-loss are indicated by black solid bars in the adjacent single-column heatmaps.
  • the fraction of H3K27ac sites containing overlapping ATF4-gain or MYC-loss peaks in the H3K27ac decrease or H3K27ac increase clusters are summarized in the bar charts above the ATF4- gain/MYC-loss single-column heatmaps.
  • H3K27ac regions overlapping with increased ATF4 are highlighted towards the right.
  • FIG. 1 Violin plots of significantly differentially induced H3K27ac sites after 16 hours of 10 ⁇ M MYCi treatment in 22RV1 cells. The two plots are comparing the log2FC of MYCi-induced H3K27ac gain at sites that overlap with increased MYCi-induced ATF4 chromatin binding versus sites that do not.
  • Figure 5 shows (a) Alpha-fold model of MycHot and surrounding region in GCN1. (b) Docking of 975 to GCN1. (c) Deletion mutant MYC pulldown by biotin-975. (d, e) Alpha-fold models of MYC and C-Linker-G MYC showing C region helices coming near the MycHot region to form a potential pocket. (f).
  • FIG. 1 BLI for binding of full length MYC (FL) and C region containing N terminal 1-353 amino acids of MYC.
  • FIG. 1 BLI data for MYC proteins.
  • Figure 6 shows that MYCi-induced ATF4 mediates anti-tumor efficacy and remodeling of the TIME.
  • Figure 7 shows (a) Cell viability of shATF4 MycCaP cells after treatment with Doxycyline (Dox.) (500ng/ml) and MYCi (5 ⁇ M) for 72hrs. Statistical analysis performed with two-sided Student’s t test. (b, c) Inducible knockdown of ATF4 with Doxycycline (500 Attorney Docket No.702581.02595 (NU2023-153-02) ng/mL) in 22Rv1 cells attenuates ATF4 induction and MYCi975 efficacy.
  • Dox. 500ng/ml
  • MYCi 5 ⁇ M
  • Figure 10 shows ATF4 rescue with MYCi-975.
  • FIG 10 shows the effect of GCN1 Knockout on ATF4 Induction in PC12 Cells Treated with MYCi-975.
  • ATF4 induction was assessed in PC12 cells following GCN1 knockout (KO) using pLentiCRISPR v2.
  • Cells were treated with MYCi-975 (20 ⁇ M) for 24 and 48 hours.
  • the results demonstrate an attenuation of ATF4 induction in GCN1 KO cells compared to Cas9 control, indicating the role of GCN1 in mediating the cellular response to MYCi-975.
  • Cells were processed for western blot with indicated antibodies.
  • Figure 11 shows ATF4 rescue with MYCi-975.
  • ATF4 Attenuation in 22Rv1 cells with GCN1 Knockout (KO) and MYC Knockdown (KD) Treated with MYCi-975 was evaluated in 22Rv1 cells with simultaneous GCN1 KO and MYC KD, treated with 10 ⁇ M MYCi-975. The results demonstrate a significant attenuation of ATF4 compared to control cells, highlighting the combined impact of GCN1 KO and MYC KD on MYCi-975-mediated ATF4 induction. Cells were processed for western blot with indicated antibodies.
  • Figure 12 shows that acetylation of lysine residues 148 and 157 enhances MYC C- region stability in silico and increases MY Ci binding in BLI assay.
  • pLDDT predicted local distance difference test
  • pLDDT score ranges for models of unacetylated and acetylated MYC at residues in the C-region where average pl DDT score becomes "high" (> 70) in acetylated models.
  • PEE Predicted aligned error
  • Modeling MYCi Attorney Docket No.702581.02595 (NU2023-153-02) binding to GCN1 and to MYC suggests that an additional distal portion of the MYC protein contributes to MYCi binding.
  • the present disclosure confirmed this by binding studies using full-length and MYC mutants. MYCi binding to GCN1 disrupts ribosomes and activates the eIF2 ⁇ /ATF4 pathway. ATF4 activation by MYCi promotes cancer cell death and remodeling of the tumor immune microenvironment. The present studies indicate that bifunctional activity of MYC inhibitors is important for their efficacy.
  • MCYi may also bind to a distal N-terminal region of MYC and that binding to the IDR and distal N-terminal region improves binding.
  • the disclosed technology allows for the screening of therapeutic compounds to identify those capable of modulating the biological activity of an IDP by a compound by evaluating the compounds’ ability to modulate the activity of a proxy protein.
  • a proxy protein is a protein that share sequence similarity to an IDR of the IDP.
  • the proxy protein may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an IDR of the IDP.
  • Percentage of sequence identity'' or “percent similarity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or peptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical means that a polynucleotide or peptide comprises a sequence that has at least 80% sequence identity.
  • percent identity can be any integer from 80% to 100%. More preferred embodiments include at least: 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs such as BLAST using standard parameters.
  • the method for screening the compound may comprise determining a binding affinity of the compound to a proxy protein and evaluating the biological activity of the compound toward the IDP based on the binding affinity of the compound to the proxy protein.
  • the proxy protein is less disordered than the IDP and comprises a binding domain with at least 80% sequence identity to an intrinsically disordered region (IDR) of the IDP.
  • IDR intrinsically disordered region
  • the biological activity of interest may comprise inhibition, activation, modulation, or regulation of the IDP by the compound. Determination of the binding activity may include methods known in the art, such as molecular modeling, pulldown assay, biolayer Interferometry (BLI) or a combination thereof.
  • GCNI is an exemplary proxy protein.
  • the IDR may comprise a basic helix-loop-helix (bHLH) domain.
  • MYC is an exemplary IDP.
  • the compound may be an inhibitor of the IDP.
  • the IDP may be MYC protein and the compound a MYC inhibitor such as any othe MYC inhibitors disclosed herein.
  • the disclosed technology allows for identifying diseases or disorders responsive to the inhibition of an IDP.
  • cancers responsive to MYC inhibition may be identified by determining a MYC inhibitor Response Signature (MiRS) score for the cancer.
  • the MiRS can be used to identify patients that can benefit from MCYi administration.
  • the MiRS score may be determined by gene set variance analysis of a MYC inhibition responsive gene, such as any of the genes identified herein.
  • Exemplary genes may include, but are not limited to, ADM2, AIG1, AKNA, ANK2, ARHGEF2, ATF3, BBC3, BMF, CCNE2, CHAC1, DDIT3, DDIT4, ERN1, FYN, GADD45A, GDF15, GINS2, GTPBP2, HMOX1, KDM7A, MCM4, NUPR1, PPP1R15A, RELN, RRM2, TCP11L2, THSD7A, TK1, TR1B3, UHRF1, UNG, and YPEL2.
  • the MYC inhibition responsive gene is a MYC target or a ATF4 target.
  • a positive MiRS score indicates a cancer responsive to MYC inhibition.
  • the MiRS score may be greater than 0.0, 0.5, 1.0, 1.5, or 2.0.
  • Screening of compounds by the methods disclosed herein can be used to identify candidate compounds for use in therapies or identifying diseases or conditions that are responsive to the screened compounds.
  • MYCi screened by the present methods can be used to identify compounds having the desired properties to be effective in treatment or identify diseases or conditions that are responsive to the MYCi.
  • Attorney Docket No.702581.02595 NU2023-153-02
  • the disclosed technology provides for a method for treating a subject for a cancer responsive to MYC inhibition, the method comprising administering a MYC inhibitor to the subject.
  • the MYC inhibitor Response Signature (MiRS) score for the cancer is greater than 0.
  • the method further comprises determining the MiRS score for the cancer.
  • the MiRS score may be determined by gene set variance analysis of a MYC inhibition responsive gene.
  • the MYC inhibition responsive genes may include, but are not limited to, ADM2, AIG1, AKNA, ANK2, ARHGEF2, ATF3, BBC3, BMF, CCNE2, CHAC1, DDIT3, DDIT4, ERN1, FYN, GADD45A, GDF15, GINS2, GTPBP2, HMOX1, KDM7A, MCM4, NUPR1, PPP1R15A, RELN, RRM2, TCP11L2, THSD7A, TK1, TR1B3, UHRF1, UNG, and YPEL2.
  • the MYC inhibition responsive gene is a MYC target or a ATF4 target.
  • the cancer has acetylated MYC.
  • the method may further comprise screening the cancer for acetylated MYC.
  • the acetylated MYC has an acetylated lysine in an extended MYC Box II domain.
  • K148 or K157 is acetylated.
  • the MYCi may bind to an intrinsically disordered region of MYC and one or both of a distal N-terminal region of MYC ( ⁇ C) and an intrinsically disordered region of GCN1.
  • the IDR of MYC may comprise a MycHot motif.
  • the MYCi may activate ATF4 or induce ATF4 expression.
  • the MYCi may comprise an MYCi disclosed herein.
  • the disclosed technology provides for a method for treating a subject for a cancer, the method comprising administering a MYC inhibitor that binds to an intrinsically disordered region of MYC and one or both of a distal N-terminal region of MYC ( ⁇ C) and an intrinsically disordered region of GCN1.
  • the method may comprise determining the binding of an MYC inhibitor to an intrinsically disordered region of MYC and one or both of a distal N-terminal region of MYC ( ⁇ C) and an intrinsically disordered region of GCN1.
  • the IDR of MYC may comprise a MycHot motif.
  • the MYCi may activate ATF4 or induce ATF4 expression.
  • the MYCi may comprise an MYCi disclosed herein.
  • the MYC may be acetylated.
  • the acetylated MYC may have an acetylated lysine in an extended MYC Box II domain.
  • K148 or K157 is acetylated.
  • the disclosed technology provides for a method for treating a subject for a cancer responsive to ATF4 activation or induction, the method comprising administering a Attorney Docket No.702581.02595 (NU2023-153-02) GCN1 ligand that activates or induces expression of ATF4.
  • the GCN1 ligand binds an intrinsically disordered region of GCN1.
  • the disclosed technology provides for a method for sensitizing a subject having cancer to MYC inhibition treatment, the method comprising activating or inducing expression of ATF4 in the subject.
  • Activating or inducing expression of ATF4 in the subject may comprise administering to the subject a GCN1 ligand that activates or induces expression of ATF4.
  • the GCN1 ligand binds an intrinsically disordered region of GCN1.
  • the GCN1 ligand may be an MYC inhibitor, such as a MYC inhibitor disclosed herein.
  • a “subject” may be interchangeable with “patient” or “individual” and means an animal, which may be a human or non-human animal, in need of treatment.
  • a “subject in need of treatment” may include a subject having a disease, disorder, or condition that is responsive to therapy with a compound as disclosed herein, including MYC inhibitors.
  • a “subject in need of treatment” may include a subject having a cell proliferative disease, disorder, or condition such as cancer.
  • cancers includes, but are not limited to, breast cancer, multiple myeloma, non-small cell lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and leukemia.
  • the cancer may have an MiRS score that is greater than 0.
  • the cancer tissue may possess elevated levels of lysine- acetylated MYC (e.g., K148-Ac MYC), compared to normal tissues.
  • the phrase “effective amount” shall mean that drug dosage that provides the specific pharmacological response for which the drug is administered in a significant number of subjects in need of such treatment.
  • the compounds as disclosed herein include MYC inhibitors, such as those targeting c-MYC.
  • MYC inhibitors include, for example, the compounds disclosed in U.S. Patent Nos. 11,142,504 and 11,420,957, U.S. Publication Nos. 2020/0390894 and 2021/0395206, and international patent application PCT/US2023/071380, the contents of all of which are incorporated herein by reference in their entirety.
  • Examples include, but are not limited to, Attorney Docket No.702581.02595 (NU2023-153-02) .
  • Table No. MYC i nhibitors Structure Function Reference 1 10058-F4 MYC-MAX dimerization PMID: inhibitor 17046567 2 10074-G5 MYC-MAX dimerization PMID: inhibitor 20801893 Covalent ligand that targets 3 EN4 cysteine 171 (C171) of MYC PMID: to Inhibit MYC 32966806 transcriptional activity 4 Mycro 3 MAX dimerization inhibitor.
  • the formulae of the compounds and molecules disclosed herein should be interpreted as encompassing salts, esters, amides, or solvates thereof of the compounds and molecules.
  • the compounds may exhibit one or more biological activities.
  • the compounds may inhibit binding of the Myc/Max complex to DNA (e.g., in a DNA gel shifting assay).
  • the compounds inhibit binding of the Myc/Max complex to DNA by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than about 100 ⁇ M, 50 ⁇ M, 10 ⁇ M, 1 ⁇ M, 0.1 ⁇ M, 0.05 ⁇ M, 0.01 ⁇ M, 0.005 ⁇ M, 0.001 ⁇ M, or less.
  • the compounds may not produce significant DNA damage (e.g., in an rH2AX staining assay at a concentration greater than about 0.001 ⁇ M, 0.005 ⁇ M , 0.01 ⁇ M, 0.1 ⁇ M, 1.0 ⁇ M, 10 ⁇ M, 100 ⁇ M, or higher).
  • the compounds may inhibit the growth of cells that express c-Myc (preferably by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than about 100 ⁇ M, 50 ⁇ M, 10 ⁇ M, 1 ⁇ M, 0.1 ⁇ M, 0.05 ⁇ M, 0.01 ⁇ M, 0.005 ⁇ M, 0.001 ⁇ M, or less).
  • the compounds may not inhibit the growth of cells that do not express c-Myc (preferably by not more than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2% or less at a concentration of greater than about 0.001 ⁇ M, 0.005 ⁇ M, 0.01 ⁇ M, 0.5 ⁇ M, 0.1 ⁇ M, 1.0 ⁇ M, 10 ⁇ M, and 100 ⁇ M or higher). Concentration ranges also are contemplated herein, for example, a concentration range bounded by end-point concentrations selected from 0.001 ⁇ M, 0.005 ⁇ M, 0.01 ⁇ M, 0.5 ⁇ M, 0.1 ⁇ M, 1.0 ⁇ M, 10 ⁇ M, and 100 ⁇ M.
  • the compounds may be effective in inhibiting cell proliferation of cancer cells, including cancer cells that express c-MYC and whose proliferation is inhibiting by inhibiting the biological activity of c-MYC.
  • the compounds may be effective in inhibiting cell proliferation of one or more types of cancer cells including: multiple myeloma cells, such as MM.1S cells; leukemia cells, such as CCRF-CEM, HL-60(TB), MOLT-4, RPMI-8226 and SR; non-small lung cancer cells, such as A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460 and NCI-H522; colon cancer cells, such as COLO 205, HCC-2998, HCT-116, HCT-15, HT29, KM12 and SW-620; CNS: SF-268, SF-295, SF-539, Attorney Docket No.702581.02595 (NU
  • Cell proliferation and inhibition thereof by the presently compounds may be assessed by cell viability methods disclosed in the art including colorimetric assays that utilize dyes such as MTT, XTT, and MTS to assess cell viability.
  • the compounds Preferably, have an IC 50 of less than about 10 ⁇ M, 5 ⁇ M, 1 ⁇ M, 0.5 ⁇ M, 0.01 ⁇ M, 0.005 ⁇ M, 0.001 ⁇ M or lower in the selected assay.
  • the compounds may activate ATF4 or induce ATF4 expression. Activation of ATF4 or induction of ATF4 expression may be determined by methods known in the art, such as Western blot analysis.
  • the compounds may be formulated as anti-cancer therapeutics, including hematologic malignancies, breast, lung, myeloma, pancreas and prostate malignancies.
  • the compounds also may be formulated as anti-inflammation therapeutics.
  • the compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents.
  • the pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg).
  • the pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to 100 mg/kg body weight (preferably about 0.5 to 20 mg/kg body weight, more preferably about 0.1 to 10 mg/kg body weight).
  • the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 ⁇ M, 0.005 ⁇ M, 0.01 ⁇ M, 0.5 ⁇ M, 0.1 ⁇ M, 1.0 ⁇ M, 10 ⁇ M, and 100 ⁇ M (e.g., 0.1 ⁇ M - 1.0 ⁇ M).
  • the compounds and pharmaceutical compositions comprising the compounds may be administered in methods of treating a subject in need thereof.
  • a subject in need thereof may include a subject having a cell proliferative disease, Attorney Docket No.702581.02595 (NU2023-153-02) disorder, or condition such as cancer (e.g., cancers such as multiple myeloma, leukemia, lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer).
  • cancer e.g., cancers such as multiple myeloma, leukemia, lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer.
  • the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject.
  • a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35
  • the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject.
  • a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg,
  • Minimal and/or maximal doses of the compounds may include doses falling within dose ranges having as end-points any of these disclosed doses (e.g., 2.5 mg – 200 mg).
  • a minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng/kg body weight of the subject.
  • a maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng/kg body weight of the subject.
  • Minimal and/or maximal dose levels of the compounds for achieving therapy in the disclosed methods of treatment may include dose levels falling within ranges having as end- points any of these disclosed dose levels (e.g., 500 – 2000 ng/kg body weight of the subject).
  • the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized.
  • Exemplary solid dosage forms include, but are not limited to, Attorney Docket No.702581.02595 (NU2023-153-02) tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast melt dosage form, controlled release dosage form, lyophilized dosage form, delayed release dosage form, extended release dosage form, pulsatile release dosage form, mixed immediate release and controlled release dosage form, or a combination thereof.
  • the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier.
  • the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.
  • the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents.
  • Filling agents may include lactose monohydrate, lactose anhydrous, and various starches;
  • binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCCTM).
  • Suitable lubricants may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.
  • colloidal silicon dioxide such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.
  • sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame.
  • sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame.
  • flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like.
  • preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.
  • Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing.
  • diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.
  • Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.
  • MYC inhibitors 7 chemically distinct small-molecule MYC inhibitors to activate ATF4. These include 5 MYC/MAX dimerization inhibitors (10058-F4, 10074-G5, Mycro 3, KJ-Pyr-9, MYCMI-6); an inhibitor of MYC/MAX/DNA complex formation (KS-3716); and a covalent inhibitor that targets cysteine 171 (C171) of MYC (EN4) (7-13). We found that all these compounds can induce ATF4 protein expression with variable potencies in MycCaP and PC3 prostate cancer cells. Thus, ATF4 activation is a common feature of chemically diverse MYC binding small molecules.
  • MYCi induces ATF4 in a partially MYC-dependent manner
  • MYC suppression attenuated induction of ATF4 by MYCi975 as well as by the ER tress inducer thapsigargin ( Figure 4, panel a).
  • ATF4 induction by MYCi975 was attenuated in PC3 cells after MYC depletion by siRNA, as well as in MYC-null rat fibroblasts HO.15 ( Figure 4, panels b,c).
  • MYCi induction of ATF4 is only partially dependent on MYC expression.
  • MYC transcriptionally regulates ATF4 reduced levels of ATF4 mRNA in MYC-deficient cells may result in suboptimal ATF4 induction by MYCi in these cells.
  • EIF2 ⁇ eukaryotic initiation factor 2 ⁇
  • GCN1 shows a striking sequence similarity to MYC in this region which is even better than N-MYC and L-MYC in the C-terminal portion FALRDQIPELENNE (SEQ ID NO: 14) ( Figure 3, panel d), and several amino acids conserved between MYC and GCN1 in this region are divergent in MAX, the MYC homodimerizing partner which does not bind MYCi (1).
  • the intrinsically disordered region of GCN1 has 42.9% identity with 14 residues overlap, a score of 30.0, and gap frequency 0.0%. This region of GCN1 has been shown to be involved in binding to ribosomes. We therefore examined whether MYCi975 affects ribosome profiles.
  • MYC mutants were next generated in which K148/K157 were mutated to glutamines and tested for binding to MYCi975 in the BLI assay ( Figure 12, panel d).
  • the MYC lysine-to-arginine mutant was used as a control.
  • MycCaP Pten-KO cells are generated from the parental MycCaP cells as previously described (1).
  • Mouse Embryonic fibroblast (MEF) cells – MEF S/S and MEF A/A cells were provided by Prof. Randal J. Kaufman (Sanford Burnham Prebys Medical Discovery Institute).
  • TGR-1 and HO.15.19 rat fibroblast cells were a gift from Prof. John Sedivy (Brown University). All cells were authenticated and tested as mycoplasma-free several times throughout the studies.
  • TGR-1 and HO.15.19 cells were cultured in DMEM (Gibco #11965118) with Attorney Docket No.702581.02595 (NU2023-153-02) 10% calf serum (Fisher Scientific, #SH3007303). All cells were cultured in 1% Penicillin- Streptomycin (10,000U/ml, Life Technologies) and 5% CO2 in humidified incubator at 37 0 C.
  • the virus was generated as follows: Lentiviral shRNA plasmids were constructed by inserting target oligonucleotides into Tet-pLKO-puro (Addgene, #21915) plasmid. Plasmid DNA was extracted using a DNA extraction kit (Vazyme, DC112–01). The lentivirus was packaged by transfecting the plasmids with packaging vectors (psPAX and pMD2.G) and Lipofectamine 2000 (Invitrogen, #11668-019) in Opti-MEM media (Gibco) into HEK293T cells.
  • packaging vectors psPAX and pMD2.G
  • Lipofectamine 2000 Invitrogen, #11668-019
  • Opti-MEM media Gibco
  • the virus supernatant was collected, filtered with a 0.45 ⁇ m strainer, concentrated with PEG6000 (Sigma-Aldrich, #81253), resolved in PBS and then aliquoted and stored for subsequent transfection.
  • Cells were infected with viruses and selected for 72 hours with puromycin (2 ⁇ g/mL, Sigma-Aldrich, #P8833).
  • the following oligonucleotide sequences were used: GCCAAGCACTTCAAACCTCAT (SEQ ID NO: 15) for the shATF4 sequence employed for the human cell line 22Rv1 and CGGACAAAGATACCTTCGAGT (SEQ ID NO: 16) for the mouse cell line MycCaP.
  • MYC Human c-MYC (NM_002467.6 ORF clone) was introduced into backbone vector pET-28a(+)-TEV (GenScript) with an N-terminal hexa-histidine (His 6) tag separated by a TEV (Tobacco Etch Virus) protease digestion site and expressed in bacteria BL21-Gold (DE3) strain (Agilent #230132).
  • plasmid 50ng/ ⁇ L concentration in nuclease-free water
  • the cells were placed in 42 °C water bath for 30 seconds, after which were Attorney Docket No.702581.02595 (NU2023-153-02) further incubated on ice for 2 minutes.
  • LB pre-heated Luria Broth
  • the falcon tubes were quickly spun down at 1000g for 4 minutes and the pellet concentrated using 100 ⁇ L of the supernatant.
  • the solution was then spread on LB agar plates with 50 ⁇ g/mL working Kanamycin concentration and incubated at 37 °C overnight. Single colonies were picked from the plates and grown overnight in 5 mL of LB medium with 50 ⁇ g/mL of Kanamycin at 37 °C and shaken at 200 rpm. Next day, culture volume was scaled up 6x (30mL total volume) with fresh LB containing 50 ⁇ g/mL of Kanamycin and also left overnight under same conditions.
  • Lysis was allowed to proceed for 1 hour (shaken at 260rpm) at room temperature, followed by centrifugation for 30 minutes at 10,000 g at room temperature.
  • the supernatant was collected and mixed with Ni-NTA agarose slurry (Qiagen #30210), using 1 mL slurry per 4 mL of cleared supernatant, for a total of 3 mL Ni-NTA slurry.
  • the supernatant-slurry mixture was placed back on rocker at 260 rpm for 1 more hour at room temperature, after which the protein was purified using 5 mL polypropylene columns (Qiagen #34964) with a pH gradient elution as instructed in QIAexpressionist (protocols 17, page 90).
  • His-tagged Myc protein (439 amino acids long, UniProt accession number P01106-1) was immobilized on a Ni-NTA sensor by incubating the sensor in 200 ⁇ l of peptide solution (1 ⁇ g/mL) for five minutes.
  • Kinetics assay consisted of the following steps: equilibration in buffer (50 mM HEPES-KOH, pH 7.4, 150 mM NaCl) for three minutes to establish a baseline signal, association with different concentrations of MYCi for six minutes, followed Attorney Docket No.702581.02595 (NU2023-153-02) by dissociation in the same buffer as the baseline. Data analysis was done using ForteBio Data Analysis 7.0. Western Blot analysis Western blot was performed as described (3).
  • the membranes were blocked for 1 h at room temperature with 5% blotting-grade blocker non-fat dry milk (Bio-Rad) or 2% BSA, followed by overnight 4 °C incubation with the appropriate primary antibody and 1 h room temperature incubation with an anti-rabbit or anti-mouse IgG (H + L)-HRP conjugate (Bio-Rad) secondary antibody. Blots were imaged using Supersignal West Femto Maximum Sensitivity Substrate detection system (Thermo Scientific #34096) and the ChemiDoc Imaging System (Bio-Rad #12003153).
  • c-Myc (Y69) (Abcam #ab32072, 1:1000), MYCN (Proteintech #10159-2-AP, 1:1000), eIF2 ⁇ S51 (Cell Signaling #9721, 1:1000), eiF2 ⁇ (Cell Signaling #9722, 1:1000), CHOP (Cell Signaling #2895, 1:1000), GAPDH (Cell Signaling #3683, 1:5000) and Actin (Cell Signaling #5125, 1:5000). Quantification analyses were performed by Biorad ChemiDoc Imager and Bio-Rad Image Lab software. Small molecule inhibitors Small molecule inhibitors were purchased from MedChem express.
  • the inhibitors used are: 10058-F4 (#HY-12702), 10074-G5 (#HY-100996), Mycro 3 (#HY-100669), KJ- Pyr-9 (#HY-19735), MYCMI-6 (#HY-124675), KSI-3716 (#HY-12703), EN4 (#HY- 134761), PERKi/GSK2656157 (#HY-13820) and GCN2i/GCN2-IN-1 (#HY-100877).
  • ICD- ATP secretion assay MycCaP shATF4 cells were plated into 6-well plates at approximately 1500 cells/well in triplicate.
  • Splenic CD1dhiCD5+ B cells were determined using V450-conjugated antimouse CD19, PE-conjugated anti-CD5, Alexa fluor 647-conjugated CD1d (Biolegend, San Diego, CA).
  • splenocytes were stained with FITC-conjugated anti-mouse CD4 and APC-conjugated anti- mouse CD25 Abs, fixed, permeabilized, and subsequently stained with PE-conjugated anti- mouse Foxp3 Ab (eBioscience, San Diego, CA).
  • Migration assay BMDMs (5x10 4 ) in media containing 2% FBS were seeded on the top chamber of Transwell with a pore size of 8 ⁇ m (Corning cat# 353097). The conditioned media of Myc- CaP cells were added into the bottom chamber. After 24 hours, the chambers were fixed with 70% ethanol for 5 minutes and stained with crystal violet. Cells on the upper surface of the transwell membrane were wiped with a cotton swab. Cell migration was quantified by counting the migrated cells in four random fields under a microscope. ATAC seq Cells were plated in 10 cm 2 plates and treated with 10 ⁇ M MYCi975 or DMSO (0.2%) for 48 hrs.
  • ATAC-seq libraries were generated as previously described with slight modifications (4, 5). Treated and control cells were trypsinized and 1 million cells were washed in ice-cold PBS. Cells were pelleted at 500g for 5 min at 4°C and resuspended in 1 ml of lysis reaction mix (0.1% Tween-20, 0.1% IGEPAL (Sigma-Aldrich, cat#: I8896), 0.01 Attorney Docket No.702581.02595 (NU2023-153-02) % Digitonin (Promega, cat#: G9441) in ATAC resuspension buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 3 mM MgCl2).
  • the lysis reaction was carried out on ice for 3 min, then 4 ml of wash buffer (0.1% Tween-20 in ATAC resuspension buffer) was added and mixed end- over-end. Nuclei were pelleted at 500g for 10 min at 4°C. The pellet was resuspended in 125 ⁇ l of ice-cold PBS and the nuclei were counted and inspected for quality.
  • DNA was isolated using the Zymo DNA clean and concentrator (cat#: D4013) and amplified for 9 cycles with New England Biosystems High Fidelity 2X PCR Master Mix (NEB, cat#: M0541S) as described (4).
  • PCR amplified ATAC-seq libraries were purified using the Zymo kit (cat#: D4013) and Ampure XP beads (Cat#A63880) were used for 0.6X-1.8X size selection. Library distribution was analyzed with an Agilent 2100 Bioanalyzer. Paired-end sequencing was performed (2x42bp) using an Illumina NextSeq 500.
  • Lysis Buffer 1 50 mM HEPES-KOH, pH 7.6, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% IGEPAL-CA630, 0.25% Triton X-100 supplemented with 1x Complete Protease Inhibitor cocktail [Roche]) and incubated with end-over-end mixing for 10 minutes at 4 C.
  • Chromatin was recovered by centrifugation (2000g, 10 min, 4 C), resuspended in 4mls of Lysis Buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x Complete Protease Inhibitor cocktail) and extracted with end-over-end mixing for Attorney Docket No.702581.02595 (NU2023-153-02) 10 minutes at 4 C.
  • Lysis Buffer 2 10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x Complete Protease Inhibitor cocktail
  • the insoluble chromatin was recovered by centrifugation (2000g, 10 min, 4 C), resuspended in 0.9 ml of Lysis Buffer 3 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Sodium Deoxycholate, 0.5% Sarkosyl, 1x Complete Protease Inhibitor cocktail) and transferred to pre-chilled 1.5ml microcentrifuge tubes on ice.
  • Lysis Buffer 3 10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Sodium Deoxycholate, 0.5% Sarkosyl, 1x Complete Protease Inhibitor cocktail
  • Chromatin was sheared by sonication in an ice-water bath using a Misonix micro-tip equipped sonicator at setting 5 ( ⁇ 5W RMS output power) for 13 cycles of 15 seconds sonication followed by a 45 second cooling interval.
  • the sonicated chromatin was adjusted to 1% Triton X-100 from a 10% stock solution and debris removed by centrifugation at 20,000g at 4°C for 20 minutes.
  • the protein concentration of solubilized chromatin was determined by BCA assay. Approximately 200 g of chromatin was used for H3K27ac ChIP and 400 g of chromatin was used for ATF4 and MYC ChIP.
  • Libraries were prepared from less than 5ng of ChIP-enriched or input DNA using KAPA Hyper Prep Kit (Roche #KK8502) according to manufacturer’s instructions except adapter ligations were extended from 15 to 60 minutes at room temperature. A post-adapter ligation, pre- amplification double-sided size selection (0.7-0.9X) step using Ampure XP beads (Beckman- Coulter #A63880) was included. Library concentration was determined by fluorometry using Qubit high-sensitivity dsDNA assay kit (Thermo Fisher Scientific #Q32851) and library size Attorney Docket No.702581.02595 (NU2023-153-02) was determined using an Agilent 2100 Bioanalyzer.
  • ChIP-seq peaks were identified using HOMER (v4.10) with settings -tbp 1 and either -style factor (for ATF4/MYC ChIP-seq) or - style histone (H3K27ac ChIP-seq). Regions of differential ATF4/MYC chromatin binding or H3K27ac deposition were determined using the R/Bioconductor package DiffBind (v3.4.11). Problematic genomic regions of known artifactual signal/noise were excluded from differential affinity analysis using the built-in DiffBind function dba.blacklist.
  • ChIP- seq e.g., ATF4, MYC, H3K27ac
  • Peak calling was performed using MACS2 (v2.2.6) (9) with the DNase-seq enriched cut site method (--nomodel --shift -75 --extsize 150 --nolambda -p 0.01). Peaks from each ATAC-seq dataset (DMSO vs. 48-hr MYCi975) were used as input for differential transcription factor activity analysis (DAStk) using default settings (12).
  • RNA-seq For paired-end RNA-seq, reads were aligned to either human (hg38) or mouse (mm10) reference genomes using the STAR aligner (9) (v2.7.5, settings: --alignIntronMin 20 --alignIntronMax 500000).
  • Count matrices were generated using featureCounts from the Subread package (v1.6.1, settings: -g gene_id -t gene -p -s 2) with either human (hg38.ncbiRefSeq.gtf, http://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/genes/) or mouse (mm10.ncbiRefSeq.gtf, http://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/genes/) annotation files.
  • RNA levels in 24-hour MYCi975 treated samples versus DMSO controls were determined using DESeq2 (10) with default settings.
  • GSEA (11) analysis of RNA-seq each gene was assigned a rank metric [-log10(FDR)Xlog2(foldchange)] and this pre-ranked list was used as input for GSEA hallmarks v7.4 database.
  • GSVA scoring and MiRS generation Cell line baseline transcriptomic data were obtained from the Cancer Cell Line Encyclopedia (CCLE) hosted on the Cancer Dependency Portal (DepMap) (12) for human cancer cell lines as raw counts and from the Tumor Immune Syngeneic Mouse (TISMO) (13) portal for murine lines as TPM normalized counts.
  • CCLE Cancer Cell Line Encyclopedia
  • DepMap Cancer Dependency Portal
  • TISMO Tumor Immune Syngeneic Mouse
  • RNAseq experiments performed in 3 different cell lines (MycCaP, PC3 and 22Rv1), treated with 8 ⁇ M (PC3) or 10 ⁇ M (MycCaP, 22Rv1) MYCi975 for 24 hours.
  • RNA levels were determined by comparing MYCi vs DMSO treated conditions to obtain significantly differentially expressed genes.
  • the genes that are significantly differentially expressed and in the same direction (upregulated/downregulated) in all three cell lines are termed the MYCi response genes and serve as the baseline pool of genes from which candidate genesets were obtained for GSVA analysis.
  • the candidate genesets were derived from the baseline set of 1881 genes by applying log2 fold change filters.
  • the GSVA rank sum algorithm outputs scores which depend on the matrix of normalized counts as input, as well as the gene set this matrix is scored against.
  • PEE Predicted aligned error
  • Nuclear extracts from 22RV1 cells were pre-cleared with streptavidin beads (Thermo, 88817) for 1 hr at 4C°.100 ⁇ g nuclear extract was applied to each sample and incubated with 10 ⁇ M of Biotin975, 10 ⁇ M of D-Biotin or DMSO on a rotator over night at 4C°. Next day, 60 ⁇ l of streptavidin beads was added to each sample and further rotated for 1 hr at 4C°. Beads were washed, then eluted with 2x sample buffer and boiled at 95C° for 5 min.
  • ATAC-seq A Method for Assaying Chromatin Accessibility Genome-Wide. Curr Protoc Mol Biol.2015;109:2191- 99. 5. Corces MR, Trevino AE, Hamilton EG, Greenside PG, Sinnott-Armstrong NA, Vesuna S, et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods.2017;14(10):959-62. 6. Vatapalli R, Sagar V, Rodriguez Y, Zhao JC, Unno K, Pamarthy S, et al. Histone methyltransferase DOT1L coordinates AR and MYC stability in prostate cancer. Nat Commun.2020;11(1):4153.
  • Small- Molecule MYC Inhibitors Suppress Tumor Growth and Enhance Immunotherapy. Cancer Cell.2019;36(5):483-97 e15.
  • SEQUENCES MYC WT Amino acid sequence, region underlined is C region, region double underlined is MYCHot. In italics is G region, removed in MYC G (SEQ ID NO:1).
  • Amino acids 120-191 are the extended MYC Box II (bolded). Lysines K148 and K157 are italicized.
  • Attorney Docket No.702581.02595 (NU2023-153-02) MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQSELQPPAPSEDIWKKFELLPTPPLS PSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETF IKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAAS ECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTT APPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKR SFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQ

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Abstract

L'invention concerne une méthode de traitement d'un sujet atteint d'un cancer. La méthode peut comprendre le traitement d'un sujet atteint d'un cancer en réponse à l'inhibition de MYC, la méthode comprenant l'administration d'un inhibiteur de MYC au sujet si le score de signature de réponse à l'inhibiteur de MYC (MiRS) pour le cancer est supérieur à 0.
PCT/US2024/061138 2023-12-19 2024-12-19 Liaison par substitution pour le criblage de composés thérapeutiques Pending WO2025137355A1 (fr)

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WO2023178074A1 (fr) * 2022-03-14 2023-09-21 Northwestern University Inhibition de myc utilisant des polymères en brosse à haute densité
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Title
HAN HUIYING; JAIN ATUL D.; TRUICA MIHAI I.; IZQUIERDO-FERRER JAVIER; ANKER JONATHAN F.; LYSY BARBARA; SAGAR VINAY; LUAN YI; CHALME: "Small-Molecule MYC Inhibitors Suppress Tumor Growth and Enhance Immunotherapy", CANCER CELL, vol. 36, no. 5, 31 October 2019 (2019-10-31), US , pages 483 - 497, XP085906223, ISSN: 1535-6108, DOI: 10.1016/j.ccell.2019.10.001 *
HURD MATTHEW, PINO JEFFREY, JANG KAY, ALLEVATO MICHAEL M., VORONTCHIKHINA MARINA, ICHIKAWA WATARU, ZHAO YIFAN, GATES RYAN, VILLALP: "MYC acetylated lysine residues drive oncogenic cell transformation and regulate select genetic programs for cell adhesion-independent growth and survival", GENES & DEVELOPMENT, vol. 37, 1 October 2023 (2023-10-01), US , pages 865 - 882, XP093331868, ISSN: 0890-9369, DOI: 10.1101/gad.350736.123 *
TRUICA MIHAI I., BURNS MICHAEL C., HAN HUIYING, ABDULKADIR SARKI A.: "Turning Up the Heat on MYC: Progress in Small-Molecule Inhibitors", CANCER RESEARCH, vol. 81, no. 2, 15 January 2021 (2021-01-15), pages 248 - 253, XP093331870, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-20-2959 *

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