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WO2024119113A1 - Compositions et méthodes pour thérapies ciblant le splicéosome - Google Patents

Compositions et méthodes pour thérapies ciblant le splicéosome Download PDF

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WO2024119113A1
WO2024119113A1 PCT/US2023/082139 US2023082139W WO2024119113A1 WO 2024119113 A1 WO2024119113 A1 WO 2024119113A1 US 2023082139 W US2023082139 W US 2023082139W WO 2024119113 A1 WO2024119113 A1 WO 2024119113A1
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tumor
spliceosome
cells
pladienolide
modulator
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Israel CAÑADAS
Xinpei JIANG
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Institute Cancer Research D/b/a Research Institute Of Fox Chase Cancer Center
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Institute Cancer Research D/b/a Research Institute Of Fox Chase Cancer Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to the field of cancer treatments. More specifically, the invention provides methods and compositions for inducing necroptosis and apoptosis in cancer cells using spliceosome-targeted therapies.
  • SCLC small cell lung cancer
  • SCLCs are immune “COLD” cancers categorized by low immune infiltrates and evasion from the host immune responses. 20,57 Patients with SCLC have 5-year overall survival of approximately 5% 2 ' 8 .
  • immunotherapy has shown a durable response in tumor 9 , but only a small fraction of SCLC patients respond.
  • SCLC is a heterogenous and highly proliferative malignancy with a high prevalence of somatic mutations 17 18 , resistance to apoptosis 19 , and high tumor mutational burden (TMB) 20 .
  • TMB tumor mutational burden
  • High TMB cancers are associated with high neoantigen frequency, however, SCLCs exhibit low to zero expression of neoantigens 21 " 23 .
  • Lack of antigen presentation results in poor immune checkpoint blockade (ICB) therapies and through immune evasion, 24 while elevated antigen expression directly correlates with higher efficacy of ICB treatment 25 .
  • ICB immune checkpoint blockade
  • spliceosome modulator In accordance with the present invention, methods for treating a subject having tumors comprising administering to said subject at least one spliceosome modulator are provided. In certain embodiments, administration of these modulators reduces tumor cell proliferation or induces tumor cell killing which exceeds that observed in tumor cells not treated with the modulator.
  • the spliceosome modulator is selected from a SF3b inhibitor, an SF3B1 inhibitor, or a TIME modulator.
  • the spliceosome modulator is selected from pladienolide D, pladienolide B, pladienolide A, pladienolide C, pladienolide E, pladienolide F, pladienolide G, H3B-88OO, indisulam, herboxidiene, spliceostatin, sudemycin, FR901463, FR901464, FR901465, splicostatins A-G, thailanstatins A- C, meamycins, E7107, FD-895, herboxidiene, GEX1A, GEX1Q1-5, RQN-18690A (18- deoxyherboxidiene), sudemycins Cl, sudemycins DI, sudemycins Fl, sudemycins E, sudemycins D6, isoginkgetin, madrasin, tetrocarcin A, N-palmitoyl-L-leucine, psoromic acid, clotri
  • the method further comprises administering at least one additional cancer treatment or therapeutic agent.
  • this additional therapeutic agent or therapy is immunotherapy, TNF therapy, chemotherapy, radiotherapy, and/or a PD-L1 inhibitor.
  • the tumor is a recalcitrant tumor or a COLD cancer.
  • the tumors treated using the methods disclosed herein may be a lung cancer, such as Small Cell Lung Cancer (SCLC).
  • SCLC Small Cell Lung Cancer
  • the modulators arc administered in a pharmaceutically acceptable carrier via route selected from systemic, oral, intraperitoneal, intravenous, intracerebral, intratumoral and topical administration.
  • Another aspect of the invention includes methods for inducing ZB Pl -dependent necroptosis in tumor cells comprising contacting the cells with at least one spliceosome modulator, thereby reducing tumor cell proliferation or inducing tumor cell killing which exceeds that observed in tumor cells not treated with the modulator.
  • Yet another aspect of the invention includes methods of generating Z-nucleic acids (Z- NA) and A-RNA species comprising, a) contacting a tumor cell with at least one spliceosome modulator, and b) separating the Z-NA and A-RNA molecules from the cells.
  • Z- NA Z-nucleic acids
  • A-RNA species comprising, a) contacting a tumor cell with at least one spliceosome modulator, and b) separating the Z-NA and A-RNA molecules from the cells.
  • Another aspect of the invention includes methods of identifying endogenous Z-NA and A-RNA biomarkers, the methods comprising a) treating tumor cells with a spliceosome modulator, b) releasing immunogenic nucleic acids produced by said cells, and c) characterizing the immunogenic nucleic acids of step b).
  • the immunogenic nucleic acids identified by these methods are enriched when compared to an untreated control, and thereby provide new biomarkers for said tumor cells.
  • the methods further comprise compiling the biomarkers into a biomarker profile for said tumor cells.
  • nucleic acids encoding the biomarkers identified herein are used RNA based silencing methods for treating a patient with an aggressive tumor.
  • the method further comprises administering at least one chemotherapy or radiotherapy, thereby inducing tumor cell killing which exceeds that observed in tumor cells treated with the spliceosome modulator alone.
  • the at least one chemotherapy, or radiotherapy and at least one spliceosome modulator act synergistically.
  • the method further comprises administering at least one immunotherapy, thereby inducing tumor cell killing which exceeds that observed in tumor cells treated with the spliceosome modulator alone.
  • the at least one immunotherapy and at least one spliceosome modulator act synergistically.
  • the at least one spliceosome modulator is selected from a SF3b inhibitor, an SF3B 1 inhibitor, or a TIME modulator.
  • the spliceosome modulator is selected from at least one of pladienolide D, pladienolide B, pladienolide A, pladienolide C, pladienolide E, pladienolide F, pladienolide G, H3B-88OO, indisulam, herboxidiene, spliceostatin, sudemycin, FR901463, FR901464, FR901465, splicostatins A-G, thailanstatins A-C, meamycins, E7107, FD-895, herboxidiene, GEX1A, GEX1Q1-5, RQN-18690A (18-deoxyherboxidiene), sudemycins Cl, sudemycins DI, sudemycins Fl, sudemycins
  • the tumor is a recalcitrant tumor or a COLD cancer.
  • the tumors treated using the methods disclosed herein may be a lung cancer, such as Small Cell Lung Cancer (SCLC).
  • SCLC Small Cell Lung Cancer
  • the modulators are administered in a pharmaceutically acceptable carrier via route selected from systemic, oral, intraperitoneal, intravenous, intracerebral, intratumoral and topical administration.
  • Another aspect of the invention includes methods for inducing ZB Pl -dependent necroptosis in tumor cells comprising contacting the cells with at least one spliceosome modulator and at least one immunotherapy, thereby reducing tumor cell proliferation or inducing tumor cell killing which exceeds that observed in tumor cells treated with the modulator alone.
  • the at least one immunotherapy and at least one spliceosome modulator act synergistically.
  • Yet another aspect of the invention comprises methods for inducing ZB Pl -dependent necroptosis in Small Cell Lung Cancer (SCLC) tumor cells, the method comprising administering to said subject effective amounts at least one spliceosome modulator and at least one immunotherapy, thereby reducing tumor cell proliferation or inducing tumor cell killing which exceeds that observed in tumor cells treated with the modulator alone, wherein said spliceosome modulator is selected from pladienolide B, H3B-88OO, sudemycin D6, and indisulam; and said immunotherapy is a PD-1 inhibitor or PD-L1 inhibitor.
  • the immunotherapy and the at least one spliccosomc modulator act synergistically.
  • the spliceosome modulator is plabienolide B administered a) via intratumoral administration at a dose of 0-200nM; b) via intraperitoneal administration at a dose of 0-20mg/kg; or c) via systemic or intravenous administration at a dose of 2-20mg/kg.
  • the spliceosome modulator is H3B-88OO administered orally at a dose of 0-40mg.
  • the spliceosome modulator is sudemycin D6 administered via systemic or intravenous administration at a dose of 14mg/kg-50mg/kg.
  • the spliceosome modulator is indisulam administered via systemic or intravenous administration at a dose of 12.5mg/kg-100mg/kg.
  • the SCLC is a COLD cancer and said modulators are administered in a pharmaceutically acceptable carrier via route selected from systemic, oral, intraperitoneal, intravenous, intracerebral, intratumoral and topical administration, said method optionally comprising administration of a radiotherapy.
  • the method further comprises administering at least one TNFR1, TRADD, FADD, or CASP8 inhibitor.
  • Fig. 1 Schematic of Pladienolide B (PlaB)-mediated antiviral response and schematic diagram showing effects of treatment protocol hypotheses. PlaB induces multiple A-RNAs and Z-NAs that in turn trigger both necroptosis and apoptosis in targeted cells.
  • PlaB induces multiple A-RNAs and Z-NAs that in turn trigger both necroptosis and apoptosis in targeted cells.
  • Fig. 2A-2C SF3B1 inhibition dramatically reduces SCLC cell survival and RIPK1 depletion rescues PlaB mediated cytotoxicity.
  • Fig. 2A SCLC cell lines (H446 and H82) and immortalized fibroblasts (FC 1010 and HS68) cell survival curves after 72 hours of PlaB treatment.
  • Fig. 2B RIPK1 depletion rescues H82 and H446 cell survival after 18 hours of PlaB treatment.
  • Fig. 2C Western blot showing RIPK1 protein depletion. Mean ⁇ SEM, Welch’s t test *p ⁇ 0.05, **p ⁇ 0.01.
  • Fig. 3A-3D SF3B1 inhibition induces a potent IFN response and increases expression of IFN-regulated proteins.
  • PlaB induces potent IFN response in SCLC cell lines, H446 and H82, in a dose-dependent manner.
  • Fig. 3C and Fig. 3D 48 hours of 0.5nM of PlaB treatment effectively upregulates antigen and PD-L1 expression in H446 and H82 SCLC cell lines. Mean ⁇ SEM, two-tailed unpaired Student’s t test *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • Fig. 4A-4C SF3B 1 perturbation increases dsRNA and Z-nucleic acid levels in treated cells.
  • Fig. 4A Low dose of PlaB and indisulam induces accumulation of A-RNA and Z-Nucleic acids (mouse J2 and rabbit Z22 antibodies respectively) in H446.
  • Fig. 4B Quantification of A- RNA and Z-Nucleic acids.
  • Fig. 4C Western blot showing induction of cleaved-PARP, pH2AX, and ZBP1, and the reduction of RIPK1 protein levels. Plots of signal intensity are mean ⁇ SEM, two-tailed unpaired Student’s t test **p ⁇ 0.01, ****p ⁇ 0.0001.
  • Fig. 5A-5E SF3B1 perturbation upregulates A-RNA and Z-nucleic acids in MEFs.
  • Fig. 5A 5nM of PlaB induces A-RNA and Z-Nucleic acids in AZBP1 MEF cells.
  • Fig. 5B Quantification of A-RNA and Z-Nucleic acids.
  • Fig. 5C RIPK3 inhibitor, GSK-843, successfully rescue PlaB mediated necroptosis in ZBP1 reconstituted MEF.
  • Fig. 5D Western blot showing induction of pMLKL only in MEF expressing full-length ZBPE
  • Fig. 5E MEF AZa and RHIM-A mutants avoid PlaB-mediated necroptosis.
  • Plots of signal intensity are mean ⁇ SEM, two-tailed unpaired Student’s t test *p ⁇ 0.05, ***p ⁇ 0.001, ****p ⁇ 0.0001. 18 hours of PlaB treatment for IF, WB and rescue experiments. IF: 5nM PlaB, WB and rescue experiments: 2.5nM PlaB.
  • Fig. 6 Spliceosome Perturbation induces necroptosis.
  • Fig. 7 Western Blot analysis showing pTBKl protein depletion.
  • Fig. 8A-8B Spliceosome Perturbation induces RIPK1 -dependent apoptosis.
  • Fig. 8A H82 and H446 cells were treated with PlaB, PlaB+Z-vad, PlaB+R3i, and PlaB+Z-Vad+R3i.
  • Fig. 9A-9E SF3B1-KO model data showing that spliceosome perturbation induces necroptosis.
  • FIG. 9A Western blot showing SF3B 1 KO in a hypertriploid human cell line (H446).
  • FIG. 9B SF3B1KO induces accumulation of Z-NA in H446 cells.
  • FIG. 9C Quantification of A-RNA and Z-NA in SF3B1 KO H446 cells and wt H446 cells.
  • Fig. 9D SF3B1 knockout induces potent IFN response in SCLC cell line, H446.
  • FIG.9E Treatment of cells with HLA ABC and PD-L1 antibodies.
  • H446 EV or EV refers to H446 cells treated with an empty vector.
  • Fig. 10 Spliceosome inhibition induces necroptosis in the Primary Lung Fibroblast of mouse model.
  • Fig. 11A-11B Analysis of Z-NA and A-RNA sources.
  • Fig. 11A A-Nucleic acid and A- Nucleic acid accumulation in MEF cells after treatment with PlaB with or without Dnasel, RnaseA, or Rnase H.
  • Fig. 11B Quantification of A-RNA and Z-Nucleic acids in treated cells.
  • MEF EV refers to MEF cells treated with an empty vector.
  • Fig. 12 Treatment of necroptosis competent SCLC model.
  • Fig. 13 Comparison of necroptosis in RPPM631 cells with and without SF3B1 knockout.
  • RPPM631 EV or EV refers to RPPM631 cells treated with an empty vector.
  • Fig. 14 Analysis of ZBP1- dependent Necroptosis in RPPM631 cells with and without SF3B1 knockout.
  • EV refers to RPPM631 cells treated with an empty vector.
  • Fig. 15A-15D CytoToxicity analysis of PlaB in TNFR1-KO and TRADD-KO H82 cells (Fig. 16A) and H446 cells (Fig. 16B). CytoToxicity analysis of PlaB in FADD-KO and CASP8- KO H82 cells (Fig. 16C) and H446 cells (Fig. 16D). EV refers to cells treated with an empty vector.
  • Fig. 16 Analysis of the effect of spliceosome inhibition on necroptosis in WT MEF cells.
  • EV refers to cells treated with an empty vector.
  • Fig. 17 Analysis of combination therapy in mouse model using PlaB and aPD-1. Detailed Description of the Invention
  • the spliceosome is a dynamic multiprotcin complex that diversifies the transcriptomc and proteome 26 and removes introns from precursor (pre-) mRNA to generate mature mRNA 27 ' 29 .
  • Large-scale sequencing studies have identified cancers that exhibit deregulated splicing producing irregular transcriptomes and proteomes associated with disease progression 30 ' 33 .
  • Some highly proliferative tumors with MYC hyper- activation have deregulated RNA repertoire and increased burden on splicing components resulting in spliceosome dependencies.
  • SF3B1 the largest subunit of the core spliceosome factor 3b (SF3b) complex, plays an integral part of RNA splicing fidelity 28,38 , and is essential in tumors 39 ' 44 .
  • splicing factor deregulation can result in loss of intron retention in tumors 30 , and that intron retention is the mechanism for tumor-suppressor inactivation 45 .
  • Intron retention in cancers is dynamic 46 , diversifies cancer transcriptome 43 , establishes inter- and intra-tumoral heterogeneity 47,48 , and enhances neoantigens to promote sternness and aggressiveness 49,50 .
  • Double-stranded (ds)RNA molecules can also trigger an innate immune response. Pattern recognition receptors recognize these A-RNA and initiate production of type I interferons (IFNs) to activate antiviral responses 51 ' 55 . Viral mimicry is an antiviral response triggered by endogenous A-RNA, Z-NA, and transposable elements.
  • IFNs type I interferons
  • the data presented herein indicate that SCLCs respond favorably to spliceosome inhibition, especially inhibition targeting the splicing core component, SF3B1.
  • the data demonstrate inhibition following treatment with small molecule inhibitors, such as Pladienolide B (PlaB).
  • PlaB Pladienolide B
  • spliceosome perturbation triggers a robust anti-tumor response through accumulation of double- stranded (ds)RNA (A-RNA) and, strikingly, the induction of uncommon left-handed Z-Nucleic acids (Z-NAs).
  • ds double- stranded
  • Z-NAs uncommon left-handed Z-Nucleic acids
  • IFN tumor-intrinsic antiviral interferon
  • spliceosome inhibition also induces necroptosis in necroptotic competent mouse embryonic fibroblast (MEF) through activation of Z-DNA Binding Protein 1 (ZBP1) by Z-NAs.
  • the data provided herein display three novel features.
  • Second, the data presented herein shows that spliccosomc inhibition in SCLCs induces tumor-intrinsic immunogenicity, antigenicity, and PD-L1 expression that are known to play crucial roles in immune checkpoint blockade (ICB) therapies.
  • ICB immune checkpoint blockade
  • a compound "selected from the group consisting of” refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds.
  • an isolated, or biologically pure molecule is a compound that has been removed from its natural milieu.
  • isolated and biologically pure do not necessarily reflect the extent to which the compound has been purified.
  • An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route.
  • agent and “test compound” denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • Biological macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, peptide/DNA complexes, and any nucleic acid-based molecule which encoded the proteins described herein.
  • compound refers to the compounds discussed herein and includes precursors and derivatives of the compounds, and pharmaceutically acceptable salts of the compounds, precursors, and derivatives.
  • phrases "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO.
  • the phrase when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the functional and novel characteristics of the sequence.
  • delivery refers to the introduction of foreign molecule (i.e., miRNA encoding the polypeptide of interest) into cells.
  • administration means the introduction of a foreign molecule into a cell.
  • delivery means the introduction of a foreign molecule into a cell.
  • the spliceosome is a multi-megadalton complex of ribonucleoprotein (snRNP) particles, which are each composed of one or more uridine-rich small nuclear RNAs and several proteins.
  • snRNP ribonucleoprotein
  • the snRNA components of the spliceosome promote the two transesterification reactions of splicing, among other functions.
  • Initial RNA transcripts (pre-mRNA) of most eukaryotic genes are retained in the nucleus until non-coding intron sequences are removed by the spliceosome to produce mature messenger RNA (mRNA).
  • mRNA messenger RNA
  • the splicing that occurs can vary, so the synthesis of alternative protein products from the same primary transcript can be affected by tissue- specific or developmental signals.
  • spliceosome refers to this ribonucleoprotein complex that removes introns from one or more RNA segments, such as pre-mRNA segments.
  • splicing modulator refers to compounds that have anti-cancer activity by interacting with components of the spliceosome.
  • a splicing modulator alters the rate or form of splicing in a target cell.
  • Splicing modulators that function as inhibitory agents, for example, are capable of decreasing uncontrolled cellular proliferation.
  • Such modulators may be natural compounds or synthetic compounds.
  • Non-limiting examples of splicing modulators and categories of such modulators include pladienolide (e.g., pladienolide D or pladienolide B), pladienolide derivatives (e.g., pladienolide D or pladienolide B derivatives), indisulam, indisulam derivatives, herboxidiene, herboxidiene derivatives, spliceostatin, spliceostatin derivatives, sudemycin, or sudemycin derivatives.
  • pladienolide e.g., pladienolide D or pladienolide B
  • pladienolide derivatives e.g., pladienolide D or pladienolide B derivatives
  • indisulam indisulam derivatives
  • herboxidiene herboxidiene derivatives
  • spliceostatin e.g., spliceostatin derivatives
  • sudemycin or sudemycin derivatives.
  • the terms “derivative” and “analog” when referring to a splicing modulator, or the like, means any such compound that retains essentially the same, similar', or enhanced biological function or activity as the original compound but has an altered chemical or biological structure.
  • the splicing modulator is a pladienolide or pladienolide derivative.
  • a “pladienolide derivative” refers to a compound which is structurally related to a member of the family of natural products known as the pladienolides and which retains one or more biological functions of the starting compound. Pladienolides were first identified in the bacteria Streptomyces platensis (Mizui et al. (2004) J Antibiot. 57:188-96) as being potently cytotoxic and resulting in cell cycle arrest in the G1 and G2/M phases of the cell cycle (e.g., Bonnal et al. (2012) Nat Rev Drug Dis 11:847-59). There are seven naturally occurring pladienolides, pladienolide A-G (Mizui et al. (2004) J Antibiot.
  • WO 2003/099813 describe exemplary methods for synthesizing E7107 (Dl l) (Compound 45 of WO 2003/099813) from Pladienolide D (11107D of WO 2003/099813).
  • Dl l Compound 45 of WO 2003/099813
  • Pladienolide D 11107D of WO 2003/099813
  • inhibitors refer to a decrease or cessation of any event (such as protein ligand binding) or to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic.
  • To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to a reference. It is not necessary that the inhibition or reduction be complete.
  • “reduce” or “inhibit” refers to the ability to cause an overall decrease of 20% or greater.
  • “reduce” or “inhibit” refers to the ability to cause an overall decrease of 50% or greater.
  • “reduce” or “inhibit” refers to the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater. Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
  • modulate refers to the ability of a compound to change an activity in some measurable way as compared to an appropriate control.
  • activities can increase or decrease as compared to controls in the absence of these compounds.
  • an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound.
  • a decrease in activity is preferably at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound.
  • a compound that increases a known activity is an “agonist”.
  • One that decreases, or prevents, a known activity is an “antagonist”.
  • inhibitor refers to an agent that slows down or prevents a particular chemical reaction, signaling pathway or other process, or that reduces the activity of a particular reactant, catalyst, or enzyme.
  • the splicing modulators may act by binding to the SF3b spliceosome complex.
  • SF3b inhibitors include, without limitation, SF3B 1 inhibitors, isoginkgetin, madrasin, tetrocarcin A, N-palmitoyl-E-leucine, psoromic acid, clotrimazole, NSC635326, and napthazarin.
  • the splicing modulator acts by binding to SF3B1, the largest subunit of the core spliceosome factor 3b (SF3b) complex.
  • SF3B1 inhibitor refers to a class of agents that inhibit the action of SF3B1.
  • Exemplary SF3B1 inhibitors include, without limitation, pladienolide (e.g., pladienolide D or pladienolide B or pladienolides A-G), pladienolide derivatives (e.g., pladienolide D or pladienolide B derivatives), indisulam, indisulam derivatives, H3B-8800, H3B-88OO derivatives, FR901463, FR901464, FR901465, splicostatins A-G, thailanstatins A-C, meamycins, E7107, FD-895, herboxidiene, GEX1 A, GEX1Q1-5, RQN-18690A (18-deoxyherboxidiene), sudemycins Cl , sudemycins DI , sudcmycins Fl, sudemycins E, and sudemycins D6 .
  • pladienolide e.g., pladienolide D or pladienolide B or
  • the splicing modulators remodel the tumor- immune microenvironment (TIME).
  • TIME tumor- immune microenvironment
  • TIME refers to the normal cells, molecules, and blood vessels that surround and protect or attach to a tumor cell.
  • a tumor can change its TIME and the TIME can affect how a tumor grows and spreads.
  • These cells include lymphocytes with tumor suppressor effects, such as CD8+ T cells and natural killer cells, as well as some tumor-promoting cells with immunosuppressive functions, such as regulatory T cells and myeloid-derived suppressor cells.
  • the spliceosome modulators of the present invention work to remodel the TIME to enhance tumor suppression, cell apoptosis, and cell necroptosis.
  • apoptosis refers to an active process of cell death. Typically, the process requires ATP, involves new RNA and protein synthesis, and culminates in the activation of endogenous endonucleases that degrade the DNA of the cell, thereby destroying the genetic template required for cellular hemostasis. Apoptosis is observed in controlled deletion of cells during metamorphosis, differentiation, and general cell turnover and appears normally to be regulated by receptor-coupled events. For these reasons, apoptosis has been called “programmed cell death” or “cell suicide.” While every cell likely has the genetic program to commit suicide, it is usually suppressed. Under normal circumstances, only those cells no longer required by the organism activate this self-destruction program.
  • Apoptotic cell death is characterized by plasma membrane blebbing, cell volume loss, nuclear condensation, and endonucleolytic degradation of DNA at nucleosome intervals. Loss of plasma membrane integrity is a relatively late event in apoptosis, unlike the form of cell death termed necrosis, which can be caused by hypoxia and exposure to certain toxins and which is typically characterized early-on by increased membrane permeability and cell rupture.
  • Necroptosis refers to a regulated, caspase-independent cell death, that can be an alternative way to eliminate apoptosis-resistant cancer cells.
  • the core necroptotic pathway consisting of a receptor-interacting protein kinase 1 (RIP1 or RIPK1) — receptorinteracting protein kinase 3 (RIP3 or RIPK3) — mixed lineage kinase domain-like protein (MLKL) complex, also called the ‘necrosome’.
  • the necrosome initiates downstream effector functions such as generation of a reactive oxygen species (ROS) burst, plasma membrane permeabilization, and cytosolic ATP reduction that further drives irreversible necroptosisexecuting mechanisms.
  • ROS reactive oxygen species
  • Provided herein arc methods of treating patients by modulating necroptosis comprising administering to a patient a disclosed antisense oligonucleotide.
  • the spliceosome modulators increase concentrations of A-RNA and Z-Nucleic acids.
  • the structure of double- stranded DNA (dsDNA) in nature can be broadly categorized into 3 major forms, namely compact right-handed A-DNA, loose right-handed B- DNA and the unique left-handed Z-DNA conformation.
  • the nucleoside bases in Z-DNA adopt alternating syn- and anti-conformation bases, giving rise to its distinctive left-handed double helical structure with zigzag backbone (thus its name).
  • dsRNA double- stranded RNA
  • Z-DNA/Z-RNA exist at higher energy configuration and thus are energetically unstable on their own.
  • ZBP1 Z-DNA-binding protein 1
  • DAI DNA-dependent activator of IFN-regulatory factors
  • DLM1 Tumor stroma and activated macrophage protein
  • ZBP1 sensing activates NLRP3 inflammasome complex that leads to PAN-optosis (pyroptosis, apoptosis, and necroptosis) process.
  • preventing refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.
  • a caregiver e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals
  • a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the disclosed compounds.
  • subject includes, but is not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity.
  • the subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.
  • the subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans).
  • arthropod e.g., insects and crustaceans.
  • a patient refers to a subject afflicted with a disease or disorder.
  • patient includes human and veterinary subjects.
  • treatment and “treating” is meant the medical management of a subject with the intent to cure, ameliorate, or stabilize, a pathological condition or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • treatment while intended to cure, ameliorate, or stabilize, a disease, pathological condition, or disorder, need not actually result in the cure, ameliorization, or stabilization.
  • the effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and/or quantitative terms.
  • characteristics or features of a disease, pathological condition, or disorder and/or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.
  • tumor As used herein, the terms “tumor”, “tumor growth” or “tumor tissue” can be used interchangeably, and refer to an abnormal growth of tissue resulting from uncontrolled progressive multiplication of cells and serving no physiological function.
  • a solid tumor can be malignant, e.g. tending to metastasize and being life threatening, or benign.
  • TMB tumor mutational burden
  • Cancer treatments which may be used in combination with the spliceosome modulators provided herein include, without limitation, surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, and/or hormone therapy.
  • chemotherapeutic agents include, without limitation alkylating agent, anti-metabolic antineoplastic agent, anti-tumor antibiotic, anti-tumor botanical, platinum compound antineoplastic agent, hormonal balance antineoplastic agent, and miscellaneous antineoplastic agent, wherein therapeutical agent used in said targeted therapy is selected from the group consisting of rituximab, bevacizumab, trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, and sorafenib, wherein therapeutical agent used in said immunotherapy is selected from the group consisting of PD-1 inhibitor, PD-L1 inhibitor and CTLA4 inhibitor; more preferably, said alkylating agent is selected from the group consisting of cyclophosphamide, ifo
  • drug response means any biological response in an organism that is the result of exposure to the drug.
  • Drug responses can be favorable, such as when a patient's disease is eradicated by treatment with the drug, or unfavorable, such as when a patient enters a coma upon treatment with a drug.
  • treatment using two or more substances improves the therapy using either one of the substances alone, by maximizing efficacy, reducing toxicity, and addressing interpatient variability, as well as delaying and/or overcoming innate or acquired resistance.
  • the cancer is a COLD cancer or a cancer with a low tumor mutated burden (TMB).
  • TMB tumor mutated burden
  • the cancer is small cell lung cancer (SCLC).
  • SCLC small cell lung cancer
  • the methods include administration of an effective amount of at least one spliceosome modulator to a subject in need thereof.
  • the spliceosome modulator is selected from a small molecule inhibitor, such as SF3B1 inhibitors, anti-SF3Bl antibodies, nucleotide aptamers, soluble receptors, or other compounds.
  • SF3B1 inhibitors include without limitation pladienolide (e.g., pladienolide D or pladienolide B), pladienolide derivatives (e.g., pladienolide D or pladienolide B derivatives), herboxidiene, herboxidiene derivatives, spliceostatin, spliceostatin derivatives, sudemycin, or sudemycin derivatives.
  • the symptoms of the cancer are reduced, as compared to a control.
  • the methods include administration of an additional chemotherapeutic agent or chemotherapy.
  • the additional therapy is an immune checkpoint blockade therapy, such as anti-PD-Ll antibodies.
  • the additional therapy is surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, and/or hormone therapy.
  • the chemotherapeutic agents is alkylating agent, anti-metabolic antineoplastic agent, anti-tumor antibiotic, anti-tumor botanical, platinum compound antineoplastic agent, hormonal balance antineoplastic agent, and miscellaneous antineoplastic agent, wherein therapeutical agent used in said targeted therapy is selected from the group consisting of rituximab, bevacizumab, trastuzumab, imatinib, dinoxetine, cetuximab, nilotinib, and sorafenib, wherein therapeutical agent used in said immunotherapy is selected from the group consisting of PD-1 inhibitor, PD-L1 inhibitor and CTLA4 inhibitor; more preferably, said alkylating agent is selected from the group consisting of cyclophosphamide, ifosfamide and thiotepa, said anti-metabolic antine
  • the method of treatment effectively suppresses symptoms associated with cancer. Symptoms of vary according to the location and type of cancer being treated. In certain embodiments, symptoms of cancer include, fatigue, weight loss, lumps, pain coughing, wheezing, new or unusual growth, discoloration, and no symptoms at all. In certain embodiments, the treatment reduces the risk of relapse.
  • treatment or inhibition may be assessed by inhibition of disease progression, inhibition of tumor growth, reduction of primary tumor, relief of tumor-related symptoms, inhibition of tumor secreted factors, delayed appearance of primary or secondary tumors, slowed development of primary or secondary tumors, decreased occurrence of primary or secondary tumors, slowed or decreased severity of secondary effects of disease, arrested tumor growth and regression of tumors, increased Time To Progression (TTP), increased Progression Free Survival (PFS), increased Overall Survival (OS), among others.
  • OS as used herein means the time from treatment onset until death from any cause.
  • TTP refers to the time from treatment onset until tumor progression; TTP does not include deaths.
  • Time to Remission means the time from treatment onset until remission, for example, complete or partial remission.
  • PFS means the time from treatment onset until tumor progression or death.
  • PFS rates will be computed using the Kaplan-Meier estimates.
  • Event- free survival means the time from study entry until any treatment failure, including disease progression, treatment discontinuation for any reason, or death.
  • Relapse-free survival means the length of time after the treatment ends that the patient survives without any signs or symptoms of that cancer.
  • ORR Overall response rate
  • ORR means the sum of the percentage of patients who achieve complete and partial responses.
  • Complete remission rate refers to the percentage of patients achieving complete remission (CR).
  • Duration of response is the time from achieving a response until relapse or disease progression.
  • Duration of remission is the time from achieving remission, for example, complete or partial remission, until relapse.
  • complete inhibition is referred to herein as prevention or chemoprevention.
  • prevention includes either preventing the onset of clinically evident cancer altogether or preventing the onset of a preclinically evident stage of a cancer.
  • the compounds described herein can be formulated for enteral, parenteral, topical, or systemic administration.
  • the compounds can be combined with one or more pharmaceutically acceptable carriers and/or excipients that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.
  • the carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. Typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the compounds are known by those skilled in the art. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained- release formulations and the like.
  • the compounds described herein can be formulated for parenteral administration.
  • parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion.
  • Parenteral formulations can be prepared as aqueous compositions using techniques known in the ail.
  • such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • the compositions may be packaged in solutions of sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent.
  • the components of the composition are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or concentrated solution in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to injection.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof.
  • polyols e.g., glycerol, propylene glycol, and liquid polyethylene glycol
  • oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.)
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants.
  • isotonic agents for example, sugars or sodium chloride.
  • Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.
  • Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface-active agents.
  • Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.
  • the formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • the formulation may also contain an antioxidant to prevent degradation of the active agent(s).
  • the formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution.
  • Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.
  • Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.
  • the compounds described herein can be administered in an effective amount to a subject that is in need of alleviation or amelioration from one or more symptoms associated with tumor growth.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “effective amount.” However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.
  • the dosages or amounts of the compounds described herein are large enough to produce the desired effect in the method by which delivery occurs.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross -reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the subject and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician based on the clinical condition of the subject involved.
  • the dose, schedule of doses and route of administration can be varied.
  • compositions are administered in an effective amount and for a period of time effect to reduce one or more symptoms associated with the disease to be treated.
  • the “effective amount” for a composition having anti-cancer cell proliferation properties may vary.
  • an effective amount includes without limitation about 0.001 to about 25 mg/kg subject body weight.
  • the range of effective amount is 0.001 to 0.01 mg/kg body weight.
  • the range of effective amount is 0.001 to 0.1 mg/kg body weight.
  • the range of effective amount is 0.001 to 1 mg/kg body weight.
  • the range of effective amount is 0.001 to 10 mg/kg body weight.
  • the range of effective amount is 0.001 to 20 mg/kg body weight.
  • the range of effective amount is 0.01 to 25 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 0.1 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 1 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 10 mg/kg body weight. In another embodiment, the range of effective amount is 0.01 to 20 mg/kg body weight. In another embodiment, the range of effective amount is 0.1 to 25 mg/kg body weight. In another embodiment, the range of effective amount is 0.1 to 1 mg/kg body weight. In another embodiment, the range of effective amount is 0.1 to 10 mg/kg body weight. In another embodiment, the range of effective amount is 0.1 to 20 mg/kg body weight.
  • the range of effective amount is 1 to 25 mg/kg body weight. In another embodiment, the range of effective amount is 1 to 5 mg/kg body weight. In another embodiment, the range of effective amount is 1 to 10 mg/kg body weight. In another embodiment, the range of effective amount is 10 to 20 mg/kg body weight. In another embodiment, the range of effective amount is 20 to 30 mg/kg body weight. In another embodiment, the range of effective amount is 30 to 40 mg/kg body weight. In another embodiment, the range of effective amount is 40 to 50 mg/kg body weight. In another embodiment, the range of effective amount is 1 to 50 mg/kg body weight. Still other doses falling within these ranges are expected to be useful.
  • the range of effective amount is O.OOlmg to 10g. In another embodiment, the range of effective amount is 0.01 mg to 1 g. In another embodiment, the range of effective amount is 0.01 mg to 100 mg. In another embodiment, the range of effective amount is 0.1 mg to 100 mg. Tn another embodiment, the range of effective amount is 0.1 mg to 500 mg.
  • the range of effective amount is 1 mg to 100 mg. In another embodiment, the range of effective amount is 10 mg to 500 mg. In another embodiment, the range of effective amount is 10 mg to 750 mg. In another embodiment, the range of effective amount is 0.01 mg to 100 mg. In another embodiment, the range of effective amount is 1 mg to 500 mg.
  • the spliceosome inhibitor is administered via intertumoral administration.
  • the effective amount of the spliceosome inhibitor may be between 0-200nM, 0-150nM, O-lOOnM, 0-50nM, 0-25nM, 25nm-200nM, 25-150nM, 25-100nM, 25-50nM, 50nM-200nM, 50-150nM, 50-100nM, 100-200nM, 100-150nM, 150nM-200nM, about 25nM, about 50nM, about lOOnM, about 150nM or about 200nM.
  • PlaB is administered via intratumural administration at any one of the doses above.
  • the spliceosome inhibitor is administered via intraperitoneal administration.
  • the effective amount of the spliceosome inhibitor may be between 2-20mg/kg, 0-20mg/kg, about Img/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about lOmg/kg, about l lmg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, or about 20mg/kg.
  • PlaB is administered via intraperitoneal administration at anyone of the doses above.
  • the spliceosome inhibitor is administered via systemic or intravenous administration.
  • the effective amount of the spliceosome inhibitor may be between 2-10mg/kg, 2-20mg/kg, 0-20mg/kg, 14-50mg/kg, 12.5-100mg/kg, or at least about Img/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about lOmg/kg, about l lmg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/
  • PlaB is administered via systemic or intravenous administration at any one of the doses above. In certain embodiments, PlaB is administered at a dose of 2- 20mg/kg. In certain embodiments, sudemycin D6 is administered via systemic or intravenous administration at anyone of the doses above. In certain embodiments, sudemycin D6 is administered at a dose of 14mg/kg-50mg/kg. In certain embodiments, indisulam is administered via systemic or intravenous administration at anyone of the doses above. In certain embodiments, indisulam is administered at a dose of 12.5mg/kg- lOOmg/kg.
  • the spliceosome inhibitor is administered via oral administration.
  • the effective amount of the spliceosome inhibitor may be between 2- lOmg/kg, 2-20mg/kg, 0-20mg/kg, 0-40mg/kg, 2-40mg/kg, 10-40mg/kg, 10-20mg/kg, 20- 40mg/kg, about Img/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about lOmg/kg, about 1 Img/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, or about 20mg/kg, about 21mg/kg, about 22mg/kg, or about 20mg/
  • the effective amount of the spliceosome inhibitor is reduced by 75%, 85%, 90%, 95%, or greater when compared to solo treatment.
  • the effective amount can be reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53,
  • the effective amount of the secondary therapy is reduced by 75%, 85%, 90%, 95%, or greater when compared to solo treatment.
  • the effective amount can be reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • kit which may contain a spliceosome modulator, a pharmaceutically acceptable carrier, instructions for use, a container, a vessel for administration, or any combination thereof.
  • These Examples provide novel insights into how spliceosome modulation confer antitumor response to remodel the tumor-immune microenvironment (TIME) and elucidate an unexplored mechanism of aberrant splicing triggering ZBP1 -dependent necroptosis and ZBP1- independent apoptosis to re-activate host anti-tumor immunity to combat SCLC malignancy.
  • TIME tumor-immune microenvironment
  • the Examples characterize novel A-RNA and Z-NA species from perturbed splicing and determine their genomic locations to better understand regions of the genome harboring potent immunogenic signals.
  • ICB therapy could only minimally extend the overall survival in SCLC.
  • the findings disclosed herein describe how spliceosome-targeted therapies and combinatorial therapies, such as spliceosome modulation with immunotherapy provide a more efficacious and personalized treatment for SCLC patients.
  • the findings show that spliceosome perturbation induces necroptosis in the TIME potently enough to activate anti-tumor response. Furthermore, the data elucidate the mechanism of spliceosome inhibition induced cell death which can be used to advantage to identify RNA biomarkers, create RNA therapeutics, and design combinatorial therapies for SCLC patients.
  • RNA immunoprecipitation RIP
  • RNA-Seq total RNA-sequencing
  • combinatorial therapy spliceosome inhibition and ICB therapy
  • ICB therapy is used to reveal tumor-immune interplay, elucidate TIME remodeling, assess synergy between ICB therapy and spliceosome modulation, and test whether necroptosis is induced in vivo.
  • the experiments performed herein were designed to identify splicing perturbation induced immunogenic A-RNA and Z-NA species that are targets of ZBP1, determine if spliceosome perturbation synergizes with ICB therapy, and further clarify the mechanism of SF3B1 inhibition induced cell death in necroptosis incompetent tumors.
  • the data presented herein is further utilized for the development of targeted and combinatorial therapies to enhance anti-tumor immunity in SCLCs or other immunologically “COLD” tumors.
  • the central hypothesis of the application is that spliceosome inhibition can induce immunogenic A-RNA and Z-NA species in both cancer cells and normal cells to induce a more immunogenic tumor- immune microenvironment (TIME) and ultimately improve the response to cancer immunotherapies .
  • TIME tumor- immune microenvironment
  • A-RNA and Z-NA was further analyzed in MEF cells.
  • Cells were treated with PlaB alone, or in combination with dNasel (dsDNA), rNaseA(dsRNA at [high]), or dNaseH (DNA/RNA hybrid). After treatment, cells treated with PlaB had an increase in both A-RNAs and Z-nAs. Cells treated with PlaB and dNasel, rNaseA, or RnaseH still showed an increase in A-RNAs and Z-nAs, however, the increase in A-RNAs was not as robust as PlaB alone. (FIG. 11A-11B).
  • PlaB-mediated cell death was completely rescued by RIPK3 inhibitor GSK-843, R3i (Fig. 5C). Furthermore, spliccosomc inhibition triggered pMLKL (necroptosis) only in MEF Flag-ZBPl cells. However, western blot analysis showed that pMLKL was not triggered in MEFs AZBP1 and MEF, AZa, and MEF RA, which ultimately induces membrane rupture and immunogenic cell death (Figs. 5D and 5E). We also determined that MEFs AZBP1, WT, and Flag-ZBPl cells treated with PlaB began dying within 24hours after treatment. With MEF Flag-ZBPl cells showing only 20% survival after 24 hours (Fig. 16).
  • necroptosis in primary lung fibroblasts showed that spliceosome inhibition also triggered pMLKL (necroptosis) in the primary lung fibroblast. (Fig. 10)
  • Example II Characterization of Spliceosome Inhibition induced endogenous A-RNAs and Z-nAs in SCLC cells and MEFs
  • MEF AZBP1 cells and PlaB are utilized for the following experiments.
  • MEF AZBP1 cells are divided into two experimental conditions in the absence and presence of PlaB, and then subsequently further divided into six groups: DMSO isotype control (immunoglobulin (Ig)G), DMSO J2, DMSO Z22, PlaB IgG, PlaB J2, and PlaB Z22. All conditions and doses of reagents used are the same as previously described in Fig. 5A.
  • RIP-Scq is performed using J2 and Z22 antibodies to capture all native A-RNA and Z- NA species regardless of polyadenylation status to allow unbiased interrogation of immunogenic species that are degraded, immature, coding, and noncoding.
  • RNA contamination especially ribosomal (r)RNA which can comprise greater than 85% of the total RNA 91 , and has higher conserved secondary structures 92 .
  • all samples undergo rRNA depletion using commercially available rRNA removal kit to allow for precise interrogation of less abundant A-RNA and Z-NA transcripts.
  • FCCC Fox Chase Cancer Center
  • RNA species that are enriched in IgG control are removed from each individual condition to precisely seek those targets in the presence of PlaB that have potent immunogenic properties that can be exploited therapeutically and future vaccine development to increase treatments for metastatic SCLCs.
  • RIP qRT-PCR RIP qRT-PCR
  • SYBR Green SYBR Green
  • the genes and genome locations of PlaB induced A-RNA and Z-NA are elucidated by mapping results above to the human genome. Genes of origin for A-RNA overlap with Z-NA, and the genomic regions that harbor immunogenic Z-NAs are targeted.
  • A-RNA and Z-NA species are obtained after RIP to allow for multiple rounds of experiments.
  • A-RNA and Z-NA responses are specific to SF3B1 inhibition, direct aberrant splicing induced immunogenic species are elucidated after multiple rounds of filtering.
  • Intron-retained mRNA, degraded or immature RNA, IncRNA, DNA/RNA hybrids, and other uncommon RNA species are specifically targeted.
  • some accumulated endogenous nucleic acids in are expressed in SCEC cell lines.
  • bulk J2- and Z22-enriched species are immuno-stimulatory and have unique structures that overlap and are derived from specific genome locations.
  • SF3B 1 inhibition was further explored in H446 cells with SF3B 1 knocked out.
  • the SF3B1 knock out was confirmed using Western Blot Analysis.
  • Fig. 9A Our data shows that SF3B1-KO cells exhibit enhanced Z-NA accumulation, thereby causing ZBP1- dependent necroptosis.
  • Fig. 9B We then used immune-fluorescence (IF) technique to determine if both Z-NA and A-RNA were increased in the SF3B1-KO cells.
  • IF immune-fluorescence
  • SF3B1 inhibition was directly compared to SF3B1 knockout in the mouse SCEC cell line RPPM63E RPPM631 cells were treated with 25nM PLAB and analyzed for their accumulation of Z-NA and A-RNA. PlaB treatment caused an increase in both Z-NA and A- RNA accumulation. (Fig. 12) Next, SF3B1-KO RPPM631 cells were generated and analyzed using immunc-fluorcsccncc technique. The knock-out cells also showed an increase in the accumulation of Z-NA and A-RNA (Fig. 13).
  • Example III Effect of Spliceosome inhibition on TIME remodeling and synergy with anti- PD1 immunotherapy
  • SCLC is an immunologically ‘COLD’ tumor characterized by an immuno-suppressive TME 21 ' 23 .
  • Our data shows that SCLC are hyper-sensitive to spliceosome perturbation (Figure 2).
  • IFN antiviral response Figs. 3A and 3B
  • HLA-ABC HLA-ABC
  • PD-L1 protein expression Figs. 3C and 3D.
  • This is a phenotype that responds favorable to ICB therapies 20,93 ' 95 .
  • Currently, whether tumor-intrinsic features can modulate immune-features in the TIME to facilitate ICB is unexplored.
  • SCLC remains a devastating disease and only a very small fraction of SCLC patients respond to these therapies 10 ' 15 .
  • Tumors are treated intratumorally with 2.5nM PlaB 2 weeks after tumor engraftment, and tumors are collected between 1500 and 2000 mm 3 for downstream intracellular staining flow cytometry and IHC immune profiling.
  • flow cytometry analysis of tumor- associated immune tumor chunks are homogenized into mononuclear cells using previously described methods 98 . Then, cells then are fixed with 2% formaldehyde in PBS for 10 minutes at 37°C, washed and permeabilized with ice-cold 90% methanol for 30 minutes, and washed for staining per manufacturer’s instructions. For surface stains, staining is performed prior to permeabilization.
  • B cells CD19+
  • CD3+/CD4+ T cells CD3+/CD8+ T cells
  • CD3+/Foxp3+ regulatory T cells CD3+CD56+
  • monocytes CD14+CD16+, CD14+CD16-, CD14-CD16+.
  • the tumor tissue is fixed in 10% formalin at 4°C overnight, and subsequently dehydrated in 70% ethanol and embedded in paraffin, then sectioned at even intervals. Slides are deparaffinized and hydrated using xylene, graded ethyl alcohol, and dH2 ⁇ D followed by antigen retrieval and stained with hematoxylin and eosin or appropriate immune cell specific antibodies.
  • tumors are stained with PDGFRa, Z- NAs, and pMLKL following protocol described previously 80 .
  • the above-mentioned experiments allowed us to interrogate the impact of spliceosome inhibition on the TIME, and confirm whether necroptotic fibroblasts in the TIME potentiate anti-tumor response.
  • the status of the TIME is critical for tumor progression, immune surveillance, and response to immunotherapy 99 .
  • the effect of immune infiltrates recruited by spliceosome perturbation on further mitigation of tumor development alone or in combination with anti-PD-1 immunotherapy is also assessed.
  • 5 x 10 6 tumor cells are engrafted subcutaneously into C57BL/6J mice described above. Once tumor reaches 150-250mm 3 it is intratumorally injected with 2.5nM PlaB. Animals arc co-trcatcd with 200pg of anti-PD-1 or IgG intraperitoneally starting on day 7 after tumor challenge and twice a week thereafter for 6 weeks for a total of 12 doses. Tumor volumes is measured using calipers twice a week until 7-week timeframe is reached, Tumor volume across treatment conditions is plotted and analyzed using PRIMS v9.
  • mice and 5 male mice are tested per condition.
  • the minimum number of animals required are used to accomplish these experiments to ensure scientific rigor based on G-power statistical analyses to achieve a power of 0.8, p-value 0.05, and anticipated effect size of 0.8. All statistical comparison between conditions are performed utilizing Prism v9. To ensure scientific rigor, all statistical tests with a p-value of ⁇ 0.05 are considered significant.
  • the data is presented as the average ⁇ SEM.
  • RPP and RPM SCLC tumor models can be pretreated for 72 hours in vitro with PlaB. Pre-treated cells are then inoculated into mice without further PlaB treatment. Additionally, H3B-8800, an enhanced analog of PlaB, can be used for in vivo experiments.
  • mice This combination therapy was tested in RPP631 cells in mice.
  • the mice were injected with 8 x 10 6 RPP631 cells.
  • the mice were injected with PlaB or a vehicle intra-tumorally and IgG or aPD-1 intra-peritoneally. The tumors were then measured over 24 days. (Fig. 17). These results show that mice treated with PlaB and IgG or PlaB and anti- PD-1 had significantly reduced tumor size when compared to tumors treated with IgG or anti- PD1 only.
  • Example IV Spliceosome Inhibition induces cell death in necroptosis incapable tumors
  • spliceosome perturbation in SCLC drastically reduces cell survival (Fig. 2A)
  • induces Z-NAs and apoptosis Figs. 4A and 4C
  • RIPK1-K0 rescues SCLC cell lines from PlaB induced cytotoxicity Fig. 2B.
  • ZBP1 and AD ARI are the only two known proteins harbor Za and interact with Z-NAs 104 " 109 .
  • Our data shows an increase of ZBP1 (Fig.
  • TNFR1 and TRADD - KO SCLC cell lines are generated herein for subsequent studies.
  • CRISPR-Cas9 KO to effectively knockout TNFR1 and TRADD in SCLC cells.
  • cell lysates are collected and western blot analysis is performed. The following experiments are performed using the TNFR1 and TRADD - KO SCLC cells generated above.
  • ZBP1 senses Z-NAs and has an important role in necroptosis 78 " 84 , since ZBP1 and RIPK3 are downregulated in SCLC cells, the hypothesis that knocking out TNF signaling mediated apoptosis will partially or fully rescue SCLC cell lines from PlaB induced cytotoxicity is tested.
  • H82 and H446 WT and TNFR1 and TRADD-KO cells generated above are treated with DMSO or 2.5nM PlaB for 18 hrs. Then, the cells are counted using 0.4% trypan blue solution assessing total viable cells and the percent of cell survived with the H82 and H446 WT cells is compared. (FIG. 15A-15B) This allows us to confirm whether TNF is partially or fully responsible for PlaB- mediated cell death in SCLC cell lines.
  • TNF sensing can at least partially rescue spliceosome perturbation mediated cell death.
  • RNA-Seq and pathway analyses for WT vs RIPK1-K0 cells treated with DMSO and PlaB to examine pathways that are downregulated and upregulated to precisely determine the pathways that have protective function against PlaB treatment.
  • Example V Administration of Spliceosome Inhibitors Alone and in Combination Therapies in Human Patients
  • a preferred embodiment of the invention comprises clinical application of the information described herein to a patient. This can occur after a patient arrives in the clinic and presents with cancer symptoms or after confirmation of a cancer diagnosis.
  • the derived therapeutic dose of the spliceosome inhibitors disclosed herein for human would be in the ranges described above based on response rate and given at least once daily.
  • One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • the spliceosome inhibitor is administered via intertumoral administration.
  • the effective amount of the spliceosome inhibitor may be between 0-200nM, 0-150nM, O-lOOnM, 0- 50nM, 0-25nM, 25nm-200nM, 25-150nM, 25-100nM, 25-50nM, 50nM-200nM, 50-150nM, 50- lOOnM, 100-200nM, 100-150nM, 150nM-200nM, about 25nM, about 50nM, about lOOnM, about 150nM or about 200nM.
  • PlaB is administered via intratumural administration at anyone of the doses above.
  • the spliceosome inhibitor is administered via intraperitoneal administration.
  • the effective amount of the spliceosome inhibitor may be between 2-20mg/kg, 0-20mg/kg, about Img/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about lOmg/kg, about 1 Img/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, or about 20mg/kg.
  • PlaB is administered via intraperitoneal administration at anyone of the doses above.
  • the spliceosome inhibitor is administered via systemic or intravenous administration.
  • the effective amount of the spliceosome inhibitor may be between 2-10mg/kg, 2-20mg/kg, 0-20mg/kg, about Img/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about lOmg/kg, about 1 Img/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, or about 20mg/kg.
  • PlaB is administered via systemic or intravenous administration at anyone of the doses above. In certain patients, PlaB is administered at a dose of 2-20mg/kg.
  • sudemycin D6 is administered via systemic or intravenous administration at anyone of the doses above. In certain patients, sudemycin D6 is administered at a dose of 14mg/kg-50mg/kg.
  • indisulam is administered via systemic or intravenous administration at anyone of the doses above. In certain patients, indisulam is administered at a dose of 12.5mg/kg- lOOmg/kg.
  • the spliceosome inhibitor is administered via oral administration.
  • the effective amount of the spliceosome inhibitor may be between 2- lOmg/kg, 2-20mg/kg, 0-20mg/kg, 0-40mg/kg, 2-40mg/kg, 10-40mg/kg, 10-20mg/kg, 20- 40mg/kg, about Img/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about lOmg/kg, about 1 Img/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, or about 20mg/kg, about 21mg/kg, about 22mg/kg, or about 20mg/
  • the spliceosome modulator may be selected from pladienolide D, pladienolide B, pladienolide A, pladienolide C, pladienolide E, pladienolide F, pladienolide G, H3B-8800, indisulam, herboxidiene, spliceostatin, sudemycin, FR901463, FR901464, FR901465, splicostatins A-G, thailanstatins A-C, meamycins, E7107, FD-895, hcrboxidicnc, GEX1A, GEX1Q1-5, RQN-18690A (18-dcoxyhcrboxidicnc), sudcmycins Cl, sudemycins DI, sudemycins Fl, sudemycins E, sudemycins D6, isoginkgetin, madrasin, tetrocarcin A, N-palmitoyl-L-leucine
  • the treatment protocol can also optionally include administration of effective amounts of one or more of additional therapeutic agents that treat or inhibit cancer.
  • the treatment protocol can also optionally include co-administration of additional chemotherapeutic s.
  • treatment using two or more substances improves the therapy when compared to using either one of the substances alone, by maximizing efficacy, reducing toxicity, and addressing interpatient variability, as well as delaying and/or overcoming innate or acquired resistance.
  • the combination therapies may be effective to reduce the effective amount of at least one of the spliceosome inhibitor and the second therapy.
  • the effective amount of the spliceosome inhibitor and/or second therapy is reduced by 75%, 85%, 90%, 95%, or greater when compared to solo treatment.
  • the effective amount can be reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
  • Baessmann I Becker C, de Wilde B, Vandesompele J, Bohm D, Ansen S, Gabler F, Wilkening I, Heynck S, Heuckmann JM, Lu X, Carter SL, Cibulskis K, Banerji S, Getz G, Park KS, Rauh D, Griitter C, Fischer M, Pasqualucci L, Wright G, Wainer Z, Russell P, Petersen I, Chen Y, Stoelben E. Ludwig C, Schnabel P. Hoffmann H, Muley T. Brockmann M.
  • Torres A Wang MS, Korbel JO, Menon R, Chun SM, Kim D, Wilkerson M, Hayes N, Engelmann D, Piitzer B, Bos M, Michels S, Vlasic I, Seidel D, Pinther B, Schaub P, Becker C, Altmiiller J, Yokota J, Kohno T, Iwakawa R, Tsuta K, Noguchi M, Muley T, Hoffmann H, Schnabel PA, Petersen I, Chen Y, Soltermann A, Tischler V.
  • H3B-8800 an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers. Nat Med. 2018 May;24(4):497-504. doi: 10.1038/nm.4493. Epub 2018 Feb 19. PMID: 29457796; PMCID: PMC6730556.
  • Spliceosome-targeted therapies trigger an antiviral immune response in triple-negative breast cancer.
  • Gallinger S Hruban RH, Maitra A, lacobuzio-Donahue CA, Schulick RD, Wolfgang CL, Morgan RA, Lawlor RT, Capelli P, Corbo V, Scardoni M, Tortora G, Tempero MA, Mann KM, Jenkins NA, Perez-Mancera PA, Adams DJ, Largaespada DA, Wessels LF, Rust AG, Stein LD.
  • Tuveson DA Copeland NG, Musgrove EA, Scarpa A, Eshleman JR, Hudson TJ, Sutherland RL, Wheeler DA, Pearson JV, McPherson JD, Gibbs RA, Grimmond SM.
  • Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature. 2012 Nov 15 ;491(7424): 399-405. doi: 10.1038/naturel 1547. Epub 2012 Oct 24. PMID: 23103869; PMCID: PMC3530898.
  • Exome sequencing identifies recurrent mutations of the splicing factor SF3B 1 gene in chronic lymphocytic leukemia. Nat Genet. 2011 Dec 11 ;44(l):47-52. doi: 10.1038/ng.l032. PMID: 22158541.
  • McPherson R Neale BM, Palotie A, Purcell SM, Saleheen D, Scharf JM, Sklar P, Sullivan PF, Tuomilehto J, Tsuang MT, Watkins HC, Wilson JG, Daly MJ, MacArthur DG; Exome Aggregation Consortium. Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016 Aug 18;536(76161:285-91. doi: 10.1038/naturel9057. PMID: 27535533; PMCID: PMC5018207.

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Abstract

L'invention concerne des compositions et des méthodes pour le traitement de cancers à l'aide d'inhibiteurs de splicéosome.
PCT/US2023/082139 2022-12-01 2023-12-01 Compositions et méthodes pour thérapies ciblant le splicéosome Ceased WO2024119113A1 (fr)

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