Methods of treating cancer using a PKA activator and a CDK inhibitor

Abstract

The present disclosure provides, inter alia, methods for treating or delaying progression of cancer in an individual, or increasing sensitivity of a cancer to treatment, comprising administering to the individual an effective amount of a protein kinase A (PKA) activator; and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor, or a salt thereof. Kits, compositions, and uses related thereto are also provided.

Description

METHODS OF TREATING CANCER USING A PKA ACTIVATOR AND A CDKINHIBITORCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 575,422, filed on April 5, 2024, which is hereby incorporated by reference in its entirety.FIELD

[0002] Provided herein are methods (e.g., for treating or delaying progression of cancer in an individual or increasing sensitivity of a cancer to treatment) comprising administering to the individual an effective amount of a protein kinase A (PKA) activator or salt thereof; and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK 12) inhibitor or salt thereof. Kits, compositions, and uses related thereto are also provided.BACKGROUND

[0003] Cancer is a leading cause of death worldwide, with almost 10 million deaths attributed to cancer in 2020 alone. Outcomes for cancer treatment have improved and mortality rates declined with advances in cytotoxic chemotherapy, radiation, and surgery, but there is still a need for more effective, safer, and more targeted approaches to treatment.

[0004] Cyclin-dependent kinases (CDKs) are major regulators of cell cycle progression (e.g., CDKs 1, 2, 4, and 6) as well as transcription, due to their regulation of RNA polymerase II C-terminal domain (CTD) phosphorylation (e.g., CDKs 7-9, 12, and 13). See, e.g., Xiao, L. et al. (2023) Cancer Biol Ther 24(1) :2219470; Liang, S. et al. (2020) Cells 9(6): 1483; and Liu, H. et al. (2021) Cancer Res 81( 1): 18-26. In particular, CDK12 is known to phosphorylate Ser2 of the carboxy-terminal domain (CTD) of RNA polymerase II (RNA pol II), which modulates the processivity of the transcriptional elongation process (Krajewska, M. et al. Nat Commun. 10(1 ): 1757 (2019)). In addition, CDK12 inhibition is thought to cause a homologous recombination (HR) defect by down regulating the expression of certain HR genes (Quereda V, et al. Cancer Cell. 36(5):545-558.e7 (2019)). The HR process has also been shown to be essential to repair the DNA damage generated by poly(ADP-ribose) polymerase inhibitors (PARPis), such as Olaparib (Scully R, et al. Nat Rev Mol Cell Biol. 20(11 ):698-714 (2019)). CDKs including CDK9 and CDK12 have become therapeutic targets for new cancer treatments. However, the precise mechanisms by whichthey contribute to tumorigenesis and cancer progression are not fully understood, and there is still a need to improve the efficacy and safety of new cancer therapies, including CDK inhibitors.

[0005] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.SUMMARY

[0006] The present disclosure relates, inter alia, to methods of treating or delaying progression of cancer, as well as methods of increasing sensitivity of a cancer to a treatment comprising a CDK9 or CDK12 inhibitor. These methods (along with kits, compositions, and uses of the present disclosure) are based at least in part on the surprising findings demonstrated herein that DHE, which was developed and approved for treatment of migraines, activates the protein kinase A (PKA) pathway and displays synergistic anti-tumor activity with CDK12 and CDK9 inhibitors. The data disclosed herein demonstrate that the surprising combination of CDK9 or CDK 12 inhibitors and PKA activators such as DHE is an unexpected treatment for cancers such as acute myeloid leukemia (AML), particularly in cancer cells (e.g., AML cells) that are not sensitive, or are less sensitive, to CDK9 or CDK12 monotherapy. Without wishing to be bound to theory, it is hypothesized that the synergy between the cyclic AMP (cAMP) / PKA pathway and CDK9 / 12 inhibition is based on regulation of RNA polymerase II CTD phosphorylation at Ser2, leading to effects on transcriptional elongation and cell death.

[0007] In certain aspects, provided herein is a method for treating or delaying progression of cancer in an individual, comprising administering to the individual an effective amount of a protein kinase A (PKA) activator or salt thereof; and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor, or a salt thereof. In other aspects, provided herein is a method for increasing sensitivity of a cancer in an individual to a treatment comprising a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK 12) inhibitor, said method comprising administering to the individual an effective amount of a protein kinase A (PKA) activator or salt thereof; and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK 12) inhibitor, or a salt thereof. In some embodiments, the PKA activator is dihydroergotamine (DHE) or a salt thereof.

[0008] In some embodiments according to any of the embodiments described herein, the cancer is acute myeloid leukemia (AML). In some embodiments, the cancer is lung cancer (e.g., lung adenocarcinoma), osteosarcoma, or ovarian cancer (e.g., serous ovarian cancer).In some embodiments, the cancer is refractory or resistant to treatment with a CDK9 or CDK12 inhibitor in the absence of the PKA activator. In some embodiments, the cancer is refractory or resistant to monotherapy with a CDK9 or CDK12 inhibitor. In some embodiments, the methods further comprise administering to the individual an effective amount of a poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) or a salt thereof. In some embodiments, the cancer is refractory or resistant to treatment with a PARP inhibitor in the absence of a PKA activator. In some embodiments, the cancer is refractory or resistant to treatment with a PARP inhibitor and a CDK9 or CDK12 inhibitor in the absence of a PKA activator. In some embodiments, the PARP inhibitor is olaparib, rucaparib, niraparib, talazoparib, fuzuloparib, veliparib, IMP4927, pamiparib, AZD5305, or a salt thereof.

[0009] In some embodiments according to any of the embodiments described herein, the CDK9 inhibitor is a small molecule inhibitor of CDK9 activity. In some embodiments, the CDK9 inhibitor is a CDK9 degrader. In some embodiments, the CDK9 degrader is a proteolysis targeting chimera (PROTAC). In some embodiments, the CDK9 inhibitor is a selective CDK9 inhibitor. In some embodiments, the CDK9 inhibitor is a CDK2 / 9 inhibitor. In some embodiments, the CDK9 inhibitor is nucleic acid that inhibits CDK9 expression. In some embodiments, the CDK9 inhibitor is an antisense molecule, dsRNA, siRNA, shRNA, ribozyme, gRNA, or triple helix molecule. In some embodiments, the CDK9 inhibitor is NVP-2, JSH-150, A-1467729, A-1592668, CAN508, LDC000067, LY2857785, CDKI-73 (LS-007), FIT-039, atuveciclib (BAY 1,143,572), MC180295, 21e, AZD4573, KB-0742, LZT-106, VIP152 (BAY 1,251,152), SNS-032 (BMS-387032), 11c, B03, PROTAC 2, Compound 45, flavopiridol, zotiraciclib (ZTR, TG02), seliciclib (CYC202), AT7519, TP- 1287, voruciclib, roniciclib (BAY 1,000,394), fadraciclib (CYC065), or a salt thereof. In some embodiments, cells of the cancer overexpress CDK9. In some embodiments, the CDK12 inhibitor is a small molecule inhibitor of CDK12 activity. In some embodiments, the CDK12 inhibitor is a CDK12 degrader. In some embodiments, the CDK12 degrader is a proteolysis targeting chimera (PROTAC). In some embodiments, the CDK12 inhibitor is a CDK7 / 12 inhibitor. In some embodiments, the CDK12 inhibitor is a CDK9 / 12 inhibitor. In some embodiments, the CDK12 inhibitor is a CDK12 / 13 inhibitor. In some embodiments, the CDK12 inhibitor is nucleic acid that inhibits CDK12 expression. In some embodiments, the CDK12 inhibitor is an antisense molecule, dsRNA, siRNA, shRNA, ribozyme, gRNA, or triple helix molecule. In some embodiments, the CDK12 inhibitor is a cyclin K inhibitor. In some embodiments, the CDK12 inhibitor is dinaciclib (SCH-727965), BSJ-4-116, THZ1, E9,THZ531, SR-4835, PP-C8, CDK12-IN-3, procaterol, BSJ-01-175, or a salt thereof. In some embodiments, cells of the cancer overexpress CDK12.

[0010] In some embodiments according to any of the embodiments described herein, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual in the same composition. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual in different or separate compositions. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual simultaneously. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual intermittently. In some embodiments, the individual is a human.

[0011] In other aspects, provided herein are kits or articles of manufacture comprising a protein kinase A (PKA) activator or a salt thereof; and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or a salt thereof. In some embodiments, the kits comprise a pharmaceutical composition comprising the PKA activator or salt thereof and a pharmaceutically acceptable carrier. In some embodiments, the kits comprise a pharmaceutical composition comprising a CDK9 or CDK12 inhibitor or salt thereof and a pharmaceutically acceptable carrier. In some embodiments, the kits comprise a pharmaceutical composition comprising the PKA activator or a salt thereof, the CDK9 or CDK12 inhibitor or salt thereof, and a pharmaceutically acceptable carrier. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated in one composition. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated in separate compositions. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated for intermittent administration. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated for simultaneous administration. In some embodiments, the kits further comprise instructions for administering the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof to an individual in need thereof. In some embodiments, the individual has cancer.

[0012] In other aspects, provided herein are compositions (e.g., pharmaceutical compositions) comprising a protein kinase A (PKA) activator or salt thereof for use in a method of treating or delaying progression of cancer in an individual, said method comprising administering to the individual an effective amount of the composition incombination with a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor. In other aspects, provided herein are compositions (e.g., pharmaceutical compositions) comprising a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or a salt thereof for use in a method of treating or delaying progression of cancer in an individual, said method comprising administering to the individual an effective amount of the composition in combination with a protein kinase A (PKA) activator. In other aspects, provided herein are compositions (e.g., pharmaceutical compositions) comprising a protein kinase A (PKA) activator or salt thereof and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof for use in a method of treating or delaying progression of cancer in an individual, said method comprising administering to the individual an effective amount of the composition. In other aspects, provided herein are compositions (e.g., pharmaceutical compositions) comprising a protein kinase A (PKA) activator or salt thereof for use in any one of the methods disclosed herein. In other aspects, provided herein are compositions (e.g., pharmaceutical compositions) comprising a cyclin- dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof for use in any one of the methods disclosed herein. In other aspects, provided herein are compositions (e.g., pharmaceutical compositions) comprising a protein kinase A (PKA) activator or salt thereof and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof for use in any one of the methods disclosed herein.

[0013] In other aspects, provided herein is the use of a protein kinase A (PKA) activator or salt thereof in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament is to be administered in combination with a cyclin- dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof (e.g., according to any one of the methods disclosed herein). In other aspects, provided herein is the use of a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament is to be administered in combination with a protein kinase A (PKA) activator or salt thereof (e.g., according to any one of the methods disclosed herein). In other aspects, provided herein is the use of a protein kinase A (PKA) activator or salt thereof and a cyclin-dependent kinase 9 (CDK9) or cyclin- dependent kinase 12 (CDK12) inhibitor or salt thereof in the manufacture of a medicament for treating or delaying progression of cancer in an individual (e.g., according to any one of the methods disclosed herein).

[0014] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1A shows a grid matrix depicting cell viability estimates derived from a potency assay combining CDK12 PROTAC degrader BSJ-4-116 (BSJ) at 0.0, 0.0469, 0.0938, 0.188, 0.375, 0.75, 1.5, 3.0, or 6.0 pM concentration with dihydroergotamine (DHE) at 0.0, 0.195, 0.391, 0.781, 1.6, 3.1, 6.2, 12.5, or 25.0 pM concentration. The first row and first column show the single agent responses on cell viability, and the rest of the grid displays cell viability where the two compounds are mixed at the indicated concentrations.

[0016] FIG. IB shows a synergy grid calculated with the Bliss independence model from the viability matrix data in FIG. 1A. Positive scores represent synergy, and negative scores represent antagonism.

[0017] FIG. 2A shows a grid matrix depicting cell viability estimates derived from a potency assay combining CDK12 / 13 inhibitor THZ531 at 0.0, 0.0469, 0.0938, 0.188, 0.375, 0.75, 1.5, 3.0, or 6.0 pM concentration with DHE at 0.0, 0.195, 0.391, 0.781, 1.6, 3.1, 6.2, 12.5, or 25.0 pM concentration. The first row and first column show the single agent responses on cell viability, and the rest of the grid displays cell viability where the two compounds are mixed at the indicated concentrations.

[0018] FIG. 2B shows a synergy grid calculated with the Bliss independence model from the viability matrix data in FIG. 2A. Positive scores represent synergy, and negative scores represent antagonism.

[0019] FIG. 3A shows IC50 dose response curves in Kasumi-1 cells for BSJ alone or BSJ in combination with DHE. BSJ was used at 0.0, 0.0469, 0.0938, 0.188, 0.375, 0.75, 1.5, 3.0 and 6.0 pM, and DHE was used at 0.0, 6.25 and 12.5 pM.

[0020] FIG. 3B shows IC50 dose response curves in KG-1A cells for BSJ alone or BSJ in combination with DHE. BSJ was used at 0.0, 0.0469, 0.0938, 0.188, 0.375, 0.75, 1.5, 3.0 and 6.0 pM, and DHE was used at 0.0, 6.25 and 12.5 pM.

[0021] FIG. 4 shows the potency effects of BSJ alone and in combination with DHE in acute myeloid leukemia (AML) cells. The calculated log2 (IC50) of the AML cells THP-1, KG-1A, OCI-AML3, Kasumi-1, MOLM-13, AML-193, MV4-11, HL-60, and SKNO-1treated with BSJ alone or in combination with 12.5 |aM DHE are shown. Survival was evaluated using potency assays and normalized to cells treated with vehicle control (DMSO).

[0022] FIG. 5A shows a phosphokinase array that compares the phosphorylation of CREB S133, eNOS SI 177, ERK1 / 2 T202 / Y204, T185 / Y187 and GSK3a / p S21 / S29 in cell lysates of KG-1A cells treated with vehicle control (DMSO; lower) or 10 pM DHE (upper).

[0023] FIG. 5B shows a Western blot showing the phosphorylation levels of CREB S133, total CREB protein, and P-actin (control) in cell lysates of KG-1A cells treated with vehicle control (0.1% DMSO), 10 pM DHE, 0.2 pM BSJ, or 10 pM DHE + 0.2 pM BSJ.

[0024] FIG. 6A shows RNA-seq results profiling transcriptome-wide changes in gene expression of KG-1A (lower) and Kasumi-1 cells (upper) treated with 10 pM DHE, 0.2 pM BSJ, or the combination of 10 pM DHE with 0.2 pM BSJ. Fold changes are depicted with respect to their respective DMSO controls.

[0025] FIG. 6B shows a quantitative analysis comparing gene expression alterations between DHE + BSJ treated KG-1A and Kasumi-1 cells as log2 fold-changes relative to their respective DMSO controls.

[0026] FIG. 7A shows a Western blot showing phosphorylation levels at Ser2 of the RNA pol II CTD, total RNA pol II protein levels, CDK12, cleaved PARP, and P-actin in cell lysates of KG-1A and Kasumi-1 cells treated with vehicle (0.1% DMSO), 10 pM DHE, 0.2 pM BSJ, or 10 pM DHE + 0.2 pM BSJ.

[0027] FIG. 7B shows a Western blot showing phosphorylation levels of CREB S133, phosphorylation levels at Ser2 of the RNA pol II CTD, total RNA pol II protein, and total CREB protein in KG-1A cells treated with vehicle (0.1% DMSO), 10 pM DHE, 0.2 pM BSJ, 0.25 pM THZ531, 0.5 pM THZ531, 1 pM THZ531, 0.2 pM BSJ + 10 pM DHE, 0.25 pM THZ531 + 10 pM DHE, 0.5 pM THZ531 + 10 pM DHE, or 1 pM THZ531 + 10 pM DHE.

[0028] FIG. 8 shows the results of potency assays using KG-1A cells treated with vehicle control (DMSO), 6.25 pM DHE, 0.38 pM BSJ, 1.6 pM Olaparib (PARPi), 6.25 pM DHE + 1.6 pM Olaparib, 6.25 pM DHE + 0.38 pM BSJ, 0.38 pM BSJ + 1.6 pM Olaparib, and 6.25 pM DHE + 0.38 pM BSJ + 1.6 pM Olaparib. Numbers indicate calculated excess over Bliss (EOB), a measure of synergy comparing viability from the drug combination against that expected from the effect of each individual drug.

[0029] FIG. 9A shows a grid matrix depicting cell viability estimates derived from a potency assay combining CDK9 inhibitor NVP2 at 0.0, 0.00137, 0.00412, 0.0123, 0.037, 0.111, 0.333, 1.0, or 3.0 pM concentration with DHE at 0.0, 0.195, 0.391, 0.781, 1.6, 3.1, 6.2, 12.5, or 25.0 pM concentration. The first row and first column show the single agentresponses on cell viability and the rest of the grid displays cell viability where the two compounds are mixed at the indicated concentrations.

[0030] FIG. 9B shows a synergy grid calculated with the Bliss independence model from the viability matrix data in FIG. 9A. Positive scores represent synergy, and negative scores represent antagonism.

[0031] FIG. 10 shows a Western blot showing MYC protein levels, RNA pol II total and CTD Ser2 phosphorylation levels and CREB total and S133 phosphorylation levels in KG-1A cells treated with vehicle control (0.1% DMSO), 10 pM DHE, 0.2 pM BSJ, 10 pM DHE + 0.2 pM BSJ, 0.01 pM NVP2, 0.05 pM NVP2, 0.2 pM NVP2, 0.01 pM NVP2 + 10 pM DHE, 0.05 pM NVP2 + 10 pM DHE, and 0.2 pM NVP2 + 10 pM DHE.DETAILED DESCRIPTION

[0032] The present disclosure provides methods (e.g., for treating or delaying progression of cancer in an individual or increasing sensitivity of a cancer to treatment) comprising administering to the individual an effective amount of a protein kinase A (PKA) activator; and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor, or a salt thereof, as well as kits, compositions, and uses related thereto. These methods (along with kits, compositions, and uses of the present disclosure) are based at least in part on the surprising finding demonstrated herein that the PKA activator DHE, which was developed and approved for treatment of migraines, displayed synergistic anti-tumor activity with CDK12 and CDK9 inhibitors. The data disclosed herein demonstrate that the surprising combination of CDK9 or CDK12 inhibitors and DHE is an unexpected treatment for cancers such as acute myeloid leukemia (AML), particularly in cancer cells (e.g., AML cells) that are not sensitive, or are less sensitive, to CDK9 or CDK12 monotherapy. Without wishing to be bound to theory, it is hypothesized that the synergy between DHE and CDK inhibitors is based on activation of the cyclic AMP (cAMP) / protein kinase A (PKA) pathway and regulation of RNA polymerase II CTD phosphorylation at Ser2, leading to effects on transcriptional elongation and cell death.I. General Techniques

[0033] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in theart, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).II. Definitions

[0034] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a molecule” optionally includes a combination of two or more such molecules, and the like.

[0035] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0036] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0037] As used herein, “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. In some embodiments, the cancer is acute myeloid leukemia (AML).

[0038] The terms “tumor cell” or “cancer cell” used either in the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that normally manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings alone may be insufficient to meet this definition. In some embodiments, the cancer or tumor cell is an AML cell.

[0039] As used herein, a “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Non-limiting examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; saltforming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0040] A “pharmaceutically acceptable salt” is a salt form that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to the subject. See generally Berge et al. (1977) J. Pharm. Sci. 66, 1. Particular pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of subjectswithout undue toxicity, irritation, or allergic response. Pharmaceutically acceptable salts include, without limitation, acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, oxalic acid, propionic acid, succinic acid, maleic acid, tartaric acid and the like. These salts may be derived from inorganic or organic acids. Non-limiting examples of pharmaceutically acceptable salts include, without limitation, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne- 1,4-dioates, hexyne- 1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene- 1- sulfonates, naphthalene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, y-hydroxybutyrates, glycolates, tartrates, and mandelates. In some embodiments, pharmaceutically acceptable salts are formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base. Salts derived from pharmaceutically acceptable organic non-toxic bases include, without limitation, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2- diethylaminoethanol, tromethamine, trimethamine, dicyclohexylamine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N- ethylglucamine, N- methylglucamine, theobromine, purines, piperazine, piperidine, N- ethylpiperidine, polyamine resins, amino acids such as lysine, arginine, histidine, and the like. Examples of pharmaceutically acceptable base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. In some embodiments, the organic non-toxic bases are L-amino acids, such as L-lysine and L- arginine, tromethamine, N-ethylglucamine and N- methylglucamine. Acceptable inorganic bases include, without limitation, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like. Lists of other suitable pharmaceutically acceptable salts are found inRemington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa., 1985.

[0041] A “package insert” refers to instructions customarily included in commercial packages of medicaments that contain information about the indications customarily included in commercial packages of medicaments that contain information about the indications, usage, dosage, administration, contraindications, other medicaments to be combined with the packaged product, and / or warnings concerning the use of such medicaments, etc.

[0042] As used herein, “delaying progression” of a disease means to defer, hinder, slow, retard, stabilize, and / or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed. A method that “delays” development of cancer is a method that reduces probability of disease development in a given time frame and / or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of subjects. Cancer development can be detectable using standard methods, including, but not limited to, computerized axial tomography (CAT scan), Magnetic Resonance Imaging (MRI), ultrasound, clotting tests, arteriography, biopsy, urine cytology, and cystoscopy. Development may also refer to cancer progression that may be initially undetectable and includes occurrence, recurrence, and onset.

[0043] As used herein, the term “effective amount” or “therapeutically effective amount” of a substance is at least the minimum concentration required to effect a measurable improvement or prevention of a particular disorder. An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the substance to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. In reference to cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and / or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation in cancer. In some embodiments, an effective amount is an amount sufficient to delay development of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. In some embodiments, an effective amount is an amount sufficient to reduce recurrence rate in the individual. An effective amount can beadministered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (z.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; (vii) reduce recurrence rate of tumor, and / or (viii) relieve to some extent one or more of the symptoms associated with the cancer. An effective amount can be administered in one or more administrations. For purposes of this disclosure, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0044] A “subject,” “patient ” or “individual” includes a mammal, such as a human or other animal, and typically is human. In some embodiments, the subject, e.g., patient, to whom the therapeutic agents and compositions are administered, is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent, a dog, a cat, a farm animal, such as a cow or a horse, etc.

[0045] As used herein, the term “treatment” refers to clinical intervention designed to have beneficial and desired effects to the natural course of the individual or cell being treated during the course of clinical pathology. For the purpose of this disclosure, desirable effects of treatment include, without limitation, decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with cancer are mitigated or eliminated, including, but are not limited to, reducing the proliferation of (or destroying) cancerous cells, increasing cancer cell-killing, decreasing symptoms resulting from the disease, preventing spread of diseases, preventing recurrence of disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging survival of individuals.

[0046] By ‘ ‘in combination with,” it is not intended to imply that the therapy or the therapeutic agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of the disclosure. The pharmaceutical compositions or agents can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents. In general, each agent will be administered at a dose and / or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutic agent utilized in combination can be administered together in a single composition or administered separately in different or separate compositions. The particular combination to employ in a regimen will take into account compatibility of the applicable pharmaceutical composition with the additional therapeutically active agent, therapeutic modality and / or the desired therapeutic effect to be achieved.

[0047] As used herein, the term “inhibitor” or “antagonist” refers to biological or chemical substance that interferes with or otherwise reduces the physiological and / or biochemical action of another biological or chemical molecule. In some embodiments, the inhibitor or antagonist specifically binds to the other molecule. In some embodiments, the inhibitor or antagonist inhibits or antagonizes a signaling component upstream or downstream of the molecule in a cellular signaling pathway.

[0048] As used herein, the term “activator” or “agonist” refers to biological or chemical substance that enhances, stimulates, potentiates or leads to the potentiation of, or otherwise increases the physiological and / or biochemical action of another biological or chemical molecule. In some embodiments, the activator or agonist specifically binds to the other molecule. In other embodiments, the activator or agonist activates or agonizes a signaling component upstream or downstream of the molecule in a cellular signaling pathway.III. Methods

[0049] In one aspect, provided herein are methods for treating or delaying progression of cancer, e.g., in an individual. In another aspect, provided herein are methods for increasing sensitivity of a cancer in an individual to a treatment comprising a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor. In some embodiments, the individual has or has been diagnosed with cancer. In some embodiments, the methods comprise administering to the individual an effective amount of a protein kinase A (PKA)activator or salt thereof and (e.g., in combination with) a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof.

[0050] In some embodiments, the PKA activator is dihydroergotamine (DHE) or a pharmaceutically acceptable salt thereof. Dihydroergotamine (DHE) was developed as a synthetic drug for treatment of acute migraines based on ergotamine (used since the Middle Ages for treatment of migraines but whose use was limited due to unwanted side effects) and was approved for treatment of migraines in 1946. DHE has been shown to restore expression of NR4A and its gene signature in AML cells via RNA polymerase II (Boudreaux, S.P. et al. (2019) Leukemia 33(l):52-63). The instant application demonstrates that DHE activates the PKA pathway and promotes phosphorylation of cAMP-response element binding protein (CREB) S133. The combination of DHE and the CDK12 PROTAC degrader BSJ-4-116 (BSJ) was also found to promote a similar transcriptional response to that of BSJ and forskolin, a known PKA activator.

[0051] DHE is an ergot alkaloid (in particular, a 9,10alpha-dihydro derivative of ergotamine) that is an agonist for serotonin, adrenergic and dopamine receptor mediated signaling and is an approved medication for migraine treatment. In particular, DHE is thought to act as an agonist for certain 5-HT receptors e.g., 5-HTIB, 5-HTID, and 5-HTIF) in addition to binding dopaminergic, cholinergic, and adrenergic receptors. The chemical structure of DHE is provided below.

[0052] In some embodiments, the DHE is DHE 45® (Valeant), a dihydroergotamine mesylate salt formulated for injection. In some embodiments, the DHE is MIGRANAL® (Valeant), a dihydroergotamine mesylate salt formulated for nasal spray.

[0053] In some embodiments, the PKA activator is forskolin, sumatriptan, cabergoline, dobutamine, or salbutamol.

[0054] In some embodiments, the methods comprise administering a CDK9 inhibitor or salt thereof. In some embodiments, the CDK9 inhibitor is a small molecule inhibitor of CDK9 activity, e.g., CDK9 kinase activity. In some embodiments, the CDK9 inhibitor is a selective CDK9 inhibitor. In some embodiments, the CDK9 inhibitor is a CDK2 / 9 inhibitor.

[0055] In some embodiments, the CDK9 inhibitor is a CDK9 degrader, including but not limited to a proteolysis targeting chimera (PROTAC). As is known in the art, PROTACs refer to molecules comprising a ligand binding to a protein of interest e.g., CDK9, CDK12, or an associated cyclin such as cyclin T or cyclin K) linked with an E3 ubiquitin ligase- binding ligand, thereby bringing the protein of interest into proximity with the ubiquitin ligase, resulting in the protein of interest being marked for degradation by the proteasome via ubiquitin chain formation. See, e.g., Sakamoto, K.M. et al. (2001) Proc Natl. Acad Sci USA 98(15):8554-8559 and Bekes, M. et al. (2022) Nat Rev Drug Discov 21(3): 181-200.

[0056] In some embodiments, the CDK9 inhibitor is a nucleic acid, e.g., an antisense molecule, dsRNA, siRNA, shRNA, ribozyme, gRNA, or triple helix molecule, that inhibits CDK9 expression. For example, the nucleic acid may inhibit transcription, translation, and / or post-transcriptional stability of an mRNA molecule encoding CDK9 (or an associated cyclin, such as cyclin T or cyclin K), e.g., via RNA interference (RNAi) or translational inhibition, or it may inhibit expression of and / or direct the modification of DNA or RNA encoding CDK9 (or an associated cyclin, such as cyclin T or cyclin K), resulting in mutation ordecreased / eliminated expression, e.g., via RNA editing or CRISPR-Cas9-mediated gene editing. In some embodiments, the nucleic acid may promote G4 (G-quadruplex structure involving folded guanine tetrads) formation and / or stabilization in a promoter of CDK9 (or an associated cyclin, such as cyclin T or cyclin K), leading to downregulated transcription.

[0057] In some embodiments, the CDK9 inhibitor is an inhibitor of cyclin T (T1 / T2) or cyclin K, e.g., an inhibitor of cyclin T (T1 / T2) or cyclin K transcription, translation, or expression.

[0058] Examples of CDK9 inhibitors include, without limitation, NVP-2, JSH-150, A- 1467729, A-1592668, CAN508, LDC000067, LY2857785, CDKI-73 (LS-007), FIT-039, atuveciclib (BAY 1,143,572), MC180295, 21e, AZD4573, KB-0742, LZT-106, VIP152 (BAY 1,251,152), SNS-032 (BMS-387032), 11c, B03, PROTAC 2, Compound 45, flavopiridol (alvocidib), zotiraciclib (ZTR, TG02), seliciclib (CYC202), AT7519, TP-1287, voruciclib, roniciclib (BAY 1,000,394), fadraciclib (CYC065), or a salt thereof. See, e.g., Xiao, L. et al. (2023) Cancer Biol Ther 24(1) :2219470.

[0059] In some embodiments, the methods comprise administering a CDK12 inhibitor or salt thereof. In some embodiments, the CDK12 inhibitor is a small molecule inhibitor of CDK12 activity, e.g., CDK12 kinase activity. In some embodiments, the CDK12 inhibitor is a selective CDK12 inhibitor. In some embodiments, the CDK12 inhibitor is a CDK7 / 12, CDK9 / 12, or CDK12 / 13 inhibitor.

[0060] In some embodiments, the CDK12 inhibitor is a CDK12 degrader, including but not limited to a proteolysis targeting chimera (PROTAC).

[0061] In some embodiments, the CDK12 inhibitor is a nucleic acid, e.g., an antisense molecule, dsRNA, siRNA, shRNA, ribozyme, gRNA, or triple helix molecule, that inhibits CDK9 expression. For example, the nucleic acid may inhibit transcription, translation, and / or post-transcriptional stability of an mRNA molecule encoding CDK12 (or an associated cyclin, such as cyclin K), e.g., via RNA interference (RNAi) or translational inhibition, or it may inhibit expression of and / or direct the modification of DNA or RNA encoding CDK12 (or an associated cyclin, such as cyclin K), resulting in mutation or decreased / eliminated expression, e.g., via RNA editing or CRISPR-Cas9-mediated gene editing. In some embodiments, the nucleic acid may promote G4 (G-quadruplex structure involving folded guanine tetrads) formation and / or stabilization in a promoter of CDK12 (or an associated cyclin, such as cyclin K), leading to downregulated transcription.

[0062] In some embodiments, the CDK12 inhibitor is an inhibitor of cyclin K, e.g., an inhibitor of cyclin K transcription, translation, or expression.

[0063] Examples of CDK12 inhibitors include, without limitation, dinaciclib (SCH- 727965), BSJ-4-116, THZ1, E9, THZ531, SR-4835, PP-C8, CDK12-IN-3, procaterol, BSJ- 01-175, or a salt thereof. See, e.g., Liang, S. et al. (2020) Cells 9(6): 1483; and Liu, H. et al. (2021) Cancer Res 81(1): 18-26.

[0064] In some embodiments, the cancer is acute myeloid leukemia (AML). AML is associated with a defect in myeloid cell differentiation that causes immature blasts to accumulate, leading to transformed leukemia-initiating cells (LICs). However, the genetic makeup of AML cells and properties of the LIC population is heterogeneous among patients. One potential target pathway is the silencing of the NR4A nuclear receptors, which leads to upregulation of MYC (Boudreaux, S.P. et al. (2012) Oncogenesis l(7):el9). In some embodiments, the cancer is lung cancer (e.g., lung adenocarcinoma), osteosarcoma, or ovarian cancer (e.g., serous ovarian cancer).

[0065] In some embodiments, the cancer is refractory or resistant to treatment with a CDK9 or CDK12 inhibitor in the absence of the PKA activator. In some embodiments, the cancer is refractory or resistant to monotherapy with a CDK9 or CDK12 inhibitor, e.g., a CDK9 or CDK12 monotherapy.

[0066] In some embodiments, cells of the cancer overexpress CDK9 and / or CDK12. In some embodiments, cells of the cancer are characterized by increased CDK9 and / or CDK12 activity. Upregulation of CDK9 and CDK12 expression and / or activity has been noted in many cancers; see, e.g., Mandal, R. et al. (2021) Cancers (Basel) 13(9):2181 and Liang, S. et al. (2020) Cells 9(6): 1483.

[0067] In some embodiments, the methods of the present disclosure further comprise administering to the individual an effective amount of a poly(ADP-ribose) polymerase (PARP) inhibitor (PARPi) or a salt thereof. As is known in the art, PARPis are under investigation as anti-cancer agents due to their ability to inhibit PARP and thus block DNA repair (e.g., repair of single- stranded DNA breaks), causing lethality in cells deficient in homologous repair (HR). The HR process has been shown to be essential to repair the DNA damage generated by PARPis (Scully R, el al. Nat Rev Mol Cell Biol. 20(11):698-714 (2019)). Examples of PARPis include, without limitation, olaparib (LYNPARZA®, Merck / Astra Zeneca), rucaparib (RUBRACA®, pharmaand GmbH), niraparib (ZEJULA®, GSK), talazoparib (TALZENNA®, Pfizer), fuzuloparib (fluzoparib, AiRuiYi®, Jiangsu Hengrui), veliparib (ABT-888), IMP4927, pamiparib (PARTRUVIX™, BeiGene), AZD5305, or a salt thereof. See, e.g., Lu, X. et al. (2024) Genes Dis 1 l(l):306-320.

[0068] In some embodiments, the cancer is refractory or resistant to treatment with a PARP inhibitor in the absence of a PKA activator. In some embodiments, the cancer is refractory or resistant to treatment with a PARP inhibitor and a CDK9 or CDK12 inhibitor in the absence of a PKA activator.

[0069] In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual in the same composition. For example, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof can be formulated as one composition.

[0070] In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual in different or separate compositions.

[0071] In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual simultaneously. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual simultaneously in one composition. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual simultaneously in different or separate compositions.

[0072] In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual intermittently. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual intermittently in different or separate compositions.

[0073] In some embodiments, intermittent administrations are about 1-30 minutes apart, about 30-60 minutes apart, about 60-120 minutes apart, about 120-240 minutes apart, about 240-480 minutes apart, about 480-720 minutes apart, about 720-960 minutes apart or about 960-1440 minutes apart. In some embodiments, intermittent administrations are about 1-2 days apart, 2-3 days apart, 3-4 days apart, 4-5 days apart, 5-6 days apart, or 6-7 days apart.

[0074] In some embodiments, administration of a PKA activator or salt thereof and a CDK9 or CDK12 inhibitor or salt thereof leads to increased cancer cell death, e.g., as compared to administration of the PKA activator or salt thereof, or the CDK9 or CDK12 inhibitor or salt thereof alone. In some embodiments, administration of a PKA activator or salt thereof and a CDK9 or CDK12 inhibitor or salt thereof leads to a synergistic increase in cancer cell death, e.g., as compared to a probabilistic additive expectation of combining the two agents. In some embodiments, synergy is assayed using a Bliss independence model andrefers to an observed level of cell death greater than what is expected from the Bliss reference model based on administration of both agents.

[0075] In some embodiments, administration of a PKA activator or salt thereof and a CDK9 or CDK12 inhibitor or salt thereof and a PARP inhibitor or salt thereof leads to increased cancer cell death, e.g., as compared to administration of the PKA activator or salt thereof, or the CDK9 or CDK12 inhibitor or salt thereof, or the PARP inhibitor or salt thereof alone, or as compared to any combination of two of the three agents. In some embodiments, administration of the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof and the PARP inhibitor or salt thereof leads to a synergistic increase in cancer cell death, e.g., as compared to a probabilistic additive expectation of combining the three agents. In some embodiments, synergy is assayed using a Bliss independence model and refers to an observed level of cell death greater than what is expected from the Bliss reference model based on administration of all three agents.

[0076] In some embodiments, administration of the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof leads to increased CREB S133 phosphorylation, as compared to CREB S133 phosphorylation after administration of either single agent. Assays for monitoring CREB S133 phosphorylation are known in the art, and exemplary and nonlimiting assays are described below. In some embodiments, administration of the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof leads to decreased RNA polymerase II CTD phosphorylation (e.g., at Ser2), as compared to RNA polymerase II CTD phosphorylation (e.g., at Ser2) after administration of either single agent. Assays for monitoring RNA polymerase II CTD phosphorylation (e.g., at Ser2) are known in the art, and exemplary and non-limiting assays are described below. In some embodiments, administration of the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof leads to increased cleavage of PARP, as compared to PARP cleavage after administration of either single agent. Assays for monitoring PARP cleavage are known in the art, and exemplary and non-limiting assays are described below. In some embodiments, administration of the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof leads to decreased MYC expression, as compared to MYC expression after administration of either single agent. Assays for monitoring MYC expression are known in the art, and exemplary and non-limiting assays are described below.

[0077] In some embodiments, the individual is a human.IV. Articles of Manufacture or Kits

[0078] In another aspect, provided herein are articles of manufacture or kits comprising a protein kinase A (PKA) activator or salt thereof and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof. In some embodiments, the kits comprise a pharmaceutical composition comprising the PKA activator and a pharmaceutically acceptable carrier and / or a pharmaceutical composition comprising a CDK9 or CDK12 inhibitor or a salt thereof and a pharmaceutically acceptable carrier.

[0079] In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated in the same composition. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated in different or separate compositions. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated for intermittent administration. In some embodiments, the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated for simultaneous administration.

[0080] In some embodiments, the kits further comprise instructions for administering the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof to an individual in need thereof, e.g., according to any one of the methods disclosed herein. In some embodiments, the individual has or has been diagnosed with cancer.

[0081] In other aspects, provided herein are compositions comprising a protein kinase A (PKA) activator or salt thereof for use in combination with a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof, e.g., according to any one of the methods disclosed herein. In other aspects, provided herein are compositions comprising a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof for use in combination with a PKA activator or salt thereof, e.g., according to any one of the methods disclosed herein. In other aspects, provided herein are compositions comprising a protein kinase A (PKA) activator or salt thereof and a cyclin- dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof, e.g., for use according to any one of the methods disclosed herein.

[0082] In other aspects, provided herein are uses of a protein kinase A (PKA) activator or salt thereof in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament is to be administered in combination with a cyclin- dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof, e.g., according to any one of the methods disclosed herein. In other aspects, provided hereinare uses of a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or a salt thereof in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament is to be administered in combination with a protein kinase A (PKA) activator or salt thereof, e.g., according to any one of the methods disclosed herein. In other aspects, provided herein are uses of a protein kinase A (PKA) activator or salt thereof and a cyclin-dependent kinase 9 (CDK9) or cyclin- dependent kinase 12 (CDK12) inhibitor or salt thereof in the manufacture of a medicament for treating or delaying progression of cancer in an individual, e.g., according to any one of the methods disclosed herein.

[0083] In some embodiments, the article of manufacture or kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating cancer and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a PKA activator described herein or a CDK9 inhibitor, CDK12 inhibitor, or salt thereof described herein. The label or package insert indicates that the composition is used for treating the condition of choice (e.g., cancer) according to any of the methods described herein. Alternatively, or additionally, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0084] In one aspect, the composition may further comprise a pharmaceutically acceptable carrier, excipient, binder, or diluent. A pharmaceutically-acceptable excipient is a substance that is non-toxic and otherwise biologically suitable for administration to a subject. Such excipients facilitate administration of the compounds described herein and are compatible with the active ingredient. Examples of pharmaceutically-acceptable excipients include stabilizers, lubricants, surfactants, diluents, anti-oxidants, binders, coloring agents, bulking agents, emulsifiers, or taste-modifying agents. In some embodiments, pharmaceutical compositions according to the embodiments are sterile compositions. Pharmaceutical compositions may be prepared using compounding techniques known or that become available to those skilled in the art. Sterile compositions are also contemplated by theembodiments, including compositions that are in accord with national and local regulations governing such compositions.

[0085] The pharmaceutical compositions and compounds described herein may be formulated as solutions, emulsions, suspensions, dispersions, or inclusion complexes such as cyclodextrins in suitable pharmaceutical solvents or carriers, or as pills, tablets, lozenges, suppositories, sachets, dragees, granules, powders, powders for reconstitution, or capsules along with solid carriers according to conventional methods known in the art for preparation of various dosage forms. Pharmaceutical compositions provided herein may be administered by a suitable route of delivery, such as oral, parenteral, rectal, nasal, or topical route, or by inhalation. In some embodiments, the compositions are formulated for intravenous, oral, or subcutaneous administration.

[0086] Non-limiting examples of pharmaceutically acceptable salts include, without limitation, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne- 1,4-dioates, hexyne- 1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene- 1- sulfonates, naphthalene-2-sulfonates, phenylacetates, mesylates phenylpropionates, phenylbutyrates, citrates, lactates, y-hydroxybutyrates, glycolates, tartrates, and mandelates.

[0087] The specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.EXAMPLES

[0088] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.Example 1: Selection of drug combination candidates

[0089] Using a high-throughput combination drug screening platform (see, e.g., WO2022251085A1) consisting of a physical matrix plate containing >9,000 nano wells arrayed in a 96x96 grid, along with a microfluidics-based emulsified droplet delivery system, compounds were screened for potential drug-drug synergies. Within the wells of the plate, live cells (e.g. cancer cells) were deposited, and droplets containing various drug compounds were added with the delivery system. Droplets can be added to a matrix plate both row and column-wise, facilitating combination drug profiling. After several days of incubation, acute myeloid leukemia (AML) cells within each well were counted following cell staining and plate imaging. These counts were converted to viabilities, or the fraction of observed live cells relative to what is expected from within-plate controls (i.e. non-drug treated wells).From this viability data, both single agent (i.e. the effects of one drug alone) and combination (the effects of two drugs) responses were extracted and used to estimate drug-drug synergies.

[0090] Drug-drug synergy can be estimated with several established reference models.The Bliss independence model was used (Bliss Ann. Appl. Biol. 26:585-615 (1939)), whereby observed combination viabilities were compared against a probabilistic additive expectation of the amount of cell killing between the two agents. Mathematically, if the observed killing is greater than what is expected from the Bliss reference model, then the interaction is synergistic; if the observed killing is less than the expectation, then the interaction is antagonistic; otherwise, if the observed killing approximately equals the expectation, then the interaction is additive. These actual numerical differences between observation and expectation were used as Bliss scores from which successful synergistic combinations were quantified from the high-throughput combination drug screening platform and other cell viability -based experiments.Example 2: Synergistic effects of CDK12 inhibitors and dihydroergotamine

[0091] Using the high-throughput combination drug screening platform and synergy estimate as described in Example 1, possible drug-drug synergies between CDK12 inhibitors and dihydroergotamine (DHE) were observed. DHE is an ergot alkaloid that is an agonist for serotonin, adrenergic and dopamine receptor mediated signaling and is an approved medication for migraine treatment.

[0092] Potency assays using CellTiter-Glo® cell viability assay kit (Promega, G7570) were performed using the combination of CDK12 PROTAC degrader BSJ-4-116 (BSJ) (Selleckchem, 2519823-34-6) and DHE (Selleckchem, S5751) on KG-1A cells at the following concentrations: 0.0, 0.0469, 0.0938, 0.188, 0.375, 0.75, 1.5, 3.0, and 6.0 pM for BSJ; 0.0, 0.195, 0.391, 0.781, 1.6, 3.1, 6.2, 12.5, and 25.0 pM for DHE. Results from these potency assays are shown in FIG. 1A, and synergy estimates using the Bliss independence model are shown in FIG. IB. Treatment with DHE alone had minimal impact on KG-1A cell survival. However, the combination of BSJ and DHE synergistically killed these cells over multiple doses (mean excess over Bliss [EOB] = 0.15, p = 1.6e-6; optimized EOB = 0.37, p = 1.3e-7).

[0093] The combination of the CDK12 / 13 inhibitor THZ531 (Selleckchem, S6595) and DHE on KG-1A cells was also tested at the following concentrations: 0.0, 0.0469, 0.0938, 0.188, 0.375, 0.75, 1.5, 3.0, and 6.0 pM for THZ531; 0.0, 0.195, 0.391, 0.781, 1.6, 3.1, 6.2, 12.5, and 25.0 pM for DHE. Results from these potency assays are shown in FIG. 2A, and synergy estimates using the Bliss independence model are shown in FIG. 2B. Treatment with DHE alone had a minimal impact on KG-1A cell survival, while the combination of DHE and THZ531 synergistically killed these cells over multiple doses (mean EOB = 0.11, p = 1.3e-4; optimized EOB = 0.43, p = 1.7e-5). Overall, the combination of DHE with either of these two CDK12 inhibitors showed high overall synergy scores.

[0094] The BSJ and DHE combination was also tested in potency assays using KG-1A cells and another AML cell line, Kasumi-1. For each cell line, BSJ was used at 0.0, 0.0469, 0.0938, 0.188, 0.375, 0.75, 1.5, 3.0 and 6.0 pM, and DHE was used at 0.0, 6.25 and 12.5 pM. Results from these potency assays are shown in FIG. 3A and FIG. 3B. In both cell lines, the combination of BSJ and DHE demonstrated synergy as shown with the shift of the IC50 dose response curves to the left.Example 3: Synergistic effects of CDK12 inhibitors and DHE on an expanded panel of AML cells

[0095] The synergistic effects of CDK12 inhibitor BSJ and DHE were tested in potency assays in a panel of AML cells including THP-1, KG-1A, OCI-AML3, Kasumi-1, MOLM- 13, AML-193, MV4-11, HL-60 and SKNO-1 cell lines using the same methods described in Example 2 above. Results from these tests are shown in FIG. 4, where the IC50 of BSJ on each of the cell lines was measured in the presence of 12.5 pM DHE or vehicle control. Similar to the results from Example 2, the addition of DHE enhanced the potency of BSJ,particularly in KG-1A cells, and also at a lesser extent in 0CI-AML3, Kasumi-1, MOLM-13, AML-193, MV4-11, HL-60 and SKNO-1 cells. Taken together, these data demonstrate the surprising combination of CDK12 inhibitors and DHE as an unexpected treatment for AML, particularly in AML cells that are not / less sensitive to CDK12 monotherapy.Example 4: Synergistic effects of BS J and DHE on KG-1A cells are through the activation of the PKA pathway

[0096] Phosphokinase arrays were used to quantify proteomic markers of pathways targeted by DHE in KG-1A cells. Extracts from KG-1A cells treated with 10 pM DHE or vehicle control (DMSO) were hybridized to commercial membranes on which specific phospho-antibodies were spotted (R&D Systems, ARY003C). The results from these arrays are shown in FIG. 5A and showed that DHE significantly enhanced the phosphorylation of CREB S133, while it decreased the phosphorylation of eNOS SI 177, ERK1 / 2 T202 / Y204, T185 / Y187 and GSK3a / p S21 / S29. These results suggest that DHE activates the cAMP / PKA pathway, as PKA is known to phosphorylate CREB at SI 33 and to activate PP2A phosphatase, the latter of which is responsible for dephosphorylating eNOS SI 177, ERK1 / 2 T202 / Y204, T185 / Y187 and GSK3a / p S21 / S29 (Qi Y, Li L, Wei Y, Ma F. Biomed Pharmacother. 173:116398 (2024)).

[0097] The effects of DHE, BSJ, and the combination of DHE + BSJ were investigated on CREB S133 phosphorylation in KG-1A cells. KG-1A cells were treated with vehicle control (0.1% DMSO), 10 pM DHE, 0.2 pM BSJ, or 10 pM DHE + 0.2 pM BSJ. Cell lysates were obtained from each of these conditions and Western blots of these lysates were performed using CREB S133 phospho-specific (ThermoFisher, YH4021321), CREB (ThermoFisher, YC349426), and P-actin (Cell Signaling Technology, 3700S) antibodies. As shown in FIG. 5B, the addition of either DHE or BSJ alone did increase CREB S133 phosphorylation, while the overall CREB protein levels did not change. Strikingly, the combination of DHE and BSJ markedly increased CREB S133 phosphorylation. To support these mechanistic findings, studies using a known PKA activator, forskolin (Seamon KB, Padgett W, Daly JW. Proc Natl Acad Sci U SA. 78(6):3363-7 (1981)) + BSJ, and DHE + BSJ, using qPCR transcriptional profiling of markers that are upregulated upon activation of the cAMP / PKA pathway, were performed. Results from these studies show that the transcriptional activation of these markers were consistent and similar between the forskolin + BSJ treated cells and the DHE + BSJ treated cells (data not shown). These data combined with the cell potency assay resultsstrongly suggests that the synergistic cell killing effects of DHE and BSJ is affected through the activation of the PKA pathway.Example 5: Transcriptome comparison between the effects of BSJ and DHE on KG-1A and Kasumi-1 cells

[0098] The potency assays from Example 3 showed that KG-1A cells had the strongest differential sensitivity between BSJ monotherapy and BSJ + DHE co-treatment, while Kasumi-1 cells demonstrated sensitivity to BSJ monotherapy and modest synergy in the BSJ + DHE co-treatment condition. RNA-seq was used to profile transcriptome-wide changes in gene expression induced by 10 pM DHE, 0.2 pM BSJ or the combination of 10 pM DHE and 0.2 pM BSJ in both KG-1A and Kasumi-1 cells.

[0099] As seen in FIG. 6A, treatment with DHE alone induced a small number of expression changes in both KG-1A and Kasumi-1 cells. In contrast, treatment with BSJ alone induced widespread transcriptional changes in Kasumi-1 cells, but not in KG-1A cells. This is consistent with the lower observed potency of BSJ treatment alone in KG-1A cells from the results in Example 2. Interestingly, co-treatment with both BSJ and DHE induced widespread changes in both cell lines, which is again consistent with the potency assay results for both cell lines in Example 2. Overall, these combination-induced transcriptional changes were highly quantitatively consistent between the two cell lines (r= 0.778, where r is the Pearson correlation coefficient) as shown in the comparison in FIG. 6B.Example 6: Inhibition of the RNA polymerase II elongation by combining DHE with CDK12 inhibitors in AML cells

[0100] CDK12 is known to phosphorylate Ser2 of the carboxy-terminal domain (CTD) of RNA polymerase II (RNA pol II), which modulates the processivity of the transcriptional elongation process (Krajewska, M. et al. Nat Commun. 10(1): 1757 (2019)). The effects of DHE, BSJ, and DHE + BSJ in combination on RNA pol II phosphorylation at Ser2 in KG-1A and Kasumi-1 cells were investigated. Cells were treated with vehicle control (0.1% DMSO), 10 pM DHE, 0.2 pM BSJ, or 10 pM DHE + 0.2 pM BSJ. Cell lysates were obtained from each of these conditions, and Western blots of these lysates were performed using RNA pol II Ser2 phospho-specific (ThermoFisher, YF3945241A), CTD RNA pol II (ThermoFisher, YF3945001) CDK12 (ThermoFisher, 11973S), cleaved PARP (Cell Signaling Technology, 9541T), and P-actin (Cell Signaling Technology, 3700S) antibodies.

[0101] FIG. 7A shows Western blot experiments demonstrating that DHE does not impact Ser2 CTD phosphorylation in either KG-1A or Kasumi-1 cells. BSJ appears to degrade CDK12 in both KG-1A and Kasumi-1. However, Ser2 phosphorylation is only impacted in Kasumi-1 cells with BSJ alone, and co-treatment with DHE as a modifier is necessary to reduce Ser2 phosphorylation in KG-1A cells. Moreover, the reduction of Ser2 phosphorylation in the presence of the combination correlates with the appearance of cleaved PARP, a marker of cell death. These observations are consistent with the general transcriptional patterns observed in the RNA-seq studies described in Example 5 above. Combining DHE with the alternative CDK12 inhibitor THZ531 produced similar findings in KG-1A cells with respect to impacting Ser2 CTD phosphorylation and also CREB S133 phosphorylation (FIG. 7B). Taken together, these data show that the combination of DHE and CDK12 inhibitors inhibits transcriptional elongation and results in cell death.Example 7: Synergistic effects of DHE and BSJ can be further enhanced by the addition of a poly(ADP-ribose) polymerase inhibitor (PARPi)

[0102] It has been shown in the literature that CDK12 inhibition causes a homologous recombination (HR) defect by down regulating the expression of certain HR genes (Quereda V, et al, Cancer Cell. 36(5):545-558.e7 (2019)). The HR process has also been shown to be essential to repair the DNA damage generated by PARPi, such as Olaparib (Scully R, et al. Nat Rev Mol Cell Biol. 20(ll):698-714 (2019)).

[0103] Potency assays were performed using vehicle control (DMSO), 6.25 pM DHE, 0.38 pM BSJ, 1.6 pM Olaparib (PARPi) (MedchemExpress, 763113-22-0), 6.25 pM DHE + 1.6 pM Olaparib, 6.25 pM DHE + 0.38 pM BSJ, 0.38 pM BSJ + 1.6 pM Olaparib, and 6.25 pM DHE + 0.38 pM BSJ + 1.6 pM Olaparib on KG-1A cells. Results from the potency assays are shown in FIG. 8. The combination of DHE with BSJ showed synergy that was consistent with what was observed from previous experiments. The combination of DHE and Olaparib showed mild synergy, while the combination of BSJ with Olaparib showed no synergy. This latter result suggests that KG-1A cells may be particularly insensitive to this combination, which has been shown to be a successful combination in other cell lines from prior studies (Quereda V, et al. Cancer Cell. 36(5):545-558.e7 (2019)). However, the triple combination with BSJ, Olaparib, and DHE showed strong synergy in these cells, suggesting that cells insensitive to BSJ + Olaparib can be sensitized to this combination with the addition of DHE.Example 8: Synergistic effects of CDK9 inhibitor and DHE

[0104] CDK9 is also known to phosphorylate Ser2 of the RNA pol II CTD (Egloff S. Cell Mol Life Sci. 78(14):5543-5567 (2021)). Based on the results in Example 6, it was investigated whether there were also synergistic effects with a CDK9 inhibitor and DHE on AML cells (KG-1A). The CDK9 inhibitor NVP2 (Medchem Express, 1263373-43-8) and DHE in KG-1A cells was tested in potency assays at the following concentrations: 0.0, 0.195, 0.391, 0.781, 1.6, 3.1, 6.2, 12.5, and 25.0 pM for DHE; 0.0, 0.00137, 0.00412, 0.0123, 0.037, 0.111, 0.333, 1.0, and 3.0 pM for NVP2. Results from these potency assays are shown in FIG. 9A, and synergy estimates using the Bliss independence model are shown in FIG. 9B. Taken together, these data show that synergy was observed in the combination of CDK9 inhibitor, NVP2, and DHE (mean EOB = 0.15, p = 3.8e-8; optimized EOB = 0.17, p = 9.1e- 9).

[0105] The effects of the combination of DHE + NVP2 and DHE + BSJ on MYC protein levels, CTD Ser2 phosphorylation levels and CREB S133 phosphorylation levels in KG-1A cells were also investigated. KG-1A cells were treated with vehicle control (0.1% DMSO), 10 pM DHE, 0.2 pM BSJ, 10 pM DHE + 0.2 pM BSJ, 0.01 pM NVP2, 0.05 pM NVP2, 0.2 pM NVP2, 0.01 pM NVP2 + 10 pM DHE, 0.05 pM NVP2 + 10 pM DHE, and 0.2 pM NVP2 + 10 pM DHE. Cell lysates from KG-1A cells from these conditions were used in a Western blot, and results are shown in FIG. 10.

[0106] Results of using DHE and BSJ alone and DHE + BSJ in combination were consistent with previous experiments, showing how the combination affects RNA pol II ser2 phosphorylation. In addition, MYC protein expression (using the anti-MYC antibody from SantaCruz Biotechnology, SC-40) was shown to be reduced when BSJ was combined with DHE. The combination of DHE and NVP2 cooperatively reduced CTD Ser2 phosphorylation and MYC expression while enhancing CREB S133 phosphorylation. These data are similar to the effects seen with the DHE and BSJ combination, suggesting that combination of CDK12 and DHE combination as well as the combination of CDK9 and DHE have synergistic effects on killing AML cells, and that both combinations are acting through a cAMP / PKA activation pathway.Example 9: Effects of DHE and CDK inhibitors in cancer cell lines and normal fibroblasts

[0107] BSJ was further tested in combination with DHE in lung cancer (NCI-H2030 and NCI-H3122), osteosarcoma (U2OS), and ovarian cancer (SKOV3) cell lines, as well as in normal fibroblasts. The combination was mildly synergistic in the NCI-H2030 and U2OSlines, but not so in the other cancer lines and fibroblasts. The CDK12 / 13 inhibitor THZ531 was also tested in combination with DHE in these same lines, as well as in the lung cancer cell lines PC9, HCC827, and HCC78. This combination was synergistic in the lung cancer lines NCI-H2030, PC9, and HCC78 and the osteosarcoma line U2OS, mildly synergistic in NCI-H3122 (lung) and SKOV3 (ovarian), and not synergistic in the HCC827 lung line and normal fibroblasts. CDK9 inhibitor NVP-2 was also tested in combination with DHE in the lung cancer lines NCI-H2030 and NCI-H3122 and found not to be synergistic. Synergy summary results for these additional experiments performed with potency assays are displayed in Table 1.Table 1. Synergy statistics for cancer and non-cancer cell lines tested in potency assays combining DHE (Drugl ) with either CDK12 PROTAC degrader BSJ-4-116, CDK12 / 13 inhibitor THZ531 , or CDK9 inhibitor NVP2 (Drug2).Lung Adenocarcinoma (LUAD); Serous Ovarian Cancer (SOC); Not Applicable (N / A)

[0108] Table 1 shows a summary of synergy statistics for additional cancer and non-cancer cell lines run in potency assays combining DHE (Drugl) with either CDK12 PROTAC degrader BSJ-4-116, CDK12 / 13 inhibitor THZ531, or CDK9 inhibitor NVP2 (Drug2). ‘ExcessOverBliss’ and ‘p-value’ indicate the mean excess over Bliss and associated p-value calculated from the potency assay data.

Claims

CLAIMSWhat is claimed is:

1. A method for treating or delaying progression of cancer in an individual, comprising administering to the individual an effective amount of: a protein kinase A (PKA) activator or salt thereof; and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor, or salt thereof.

2. A method for increasing sensitivity of a cancer in an individual to a treatment comprising a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor, said method comprising administering to the individual an effective amount of: a protein kinase A (PKA) activator or salt thereof; and the cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor, or salt thereof.

3. The method of claim 1 or claim 2, wherein the PKA activator is dihydroergotamine (DHE) or a salt thereof.

4. The method of any one of claims 1-3, wherein the cancer is acute myeloid leukemia (AML).

5. The method of any one of claims 1-4, wherein the cancer is refractory or resistant to treatment with a CDK9 or CDK12 inhibitor in the absence of the PKA activator.

6. The method of claim 5, wherein the cancer is refractory or resistant to monotherapy with a CDK9 or CDK12 inhibitor.

7. The method of any one of claims 1-6, further comprising administering to the individual an effective amount of a poly(ADP-ribose) polymerase (PARP) inhibitor or salt thereof.

8. The method of claim 7, wherein the cancer is refractory or resistant to treatment with a PARP inhibitor in the absence of a PKA activator.

9. The method of claim 8, wherein the cancer is refractory or resistant to treatment with a PARP inhibitor and a CDK9 or CDK12 inhibitor in the absence of a PKA activator.

10. The method of any one of claims 7-9, wherein the PARP inhibitor is olaparib, rucaparib, niraparib, talazoparib, fuzuloparib, veliparib, IMP4927, pamiparib, AZD5305, or a salt thereof.

11. The method of any one of claims 1-10, wherein the CDK9 inhibitor is a small molecule inhibitor of CDK9 activity.

12. The method of any one of claims 1-10, wherein the CDK9 inhibitor is a CDK9 degrader.

13. The method of claim 12, wherein the CDK9 degrader is a proteolysis targeting chimera (PROTAC).

14. The method of any one of claims 1-10, wherein the CDK9 inhibitor is a selective CDK9 inhibitor.

15. The method of any one of claims 1-10, wherein the CDK9 inhibitor is a CDK2 / 9 inhibitor.

16. The method of any one of claims 1-10, wherein the CDK9 inhibitor is nucleic acid that inhibits CDK9 expression.

17. The method of claim 16, wherein the CDK9 inhibitor is an antisense molecule, dsRNA, siRNA, shRNA, ribozyme, gRNA, or triple helix molecule.

18. The method of any one of claims 1-10, wherein the CDK9 inhibitor is NVP-2, JSH- 150, A-1467729, A-1592668, CAN508, LDC000067, LY2857785, CDKI-73, FIT-039, atuveciclib (BAY 1,143,572), MC180295, 21e, AZD4573, KB-0742, LZT-106, VIP152 (BAY 1,251,152), SNS-032 (BMS-387032), 11c, B03, PROTAC 2, Compound 45, flavopiridol, zotiraciclib (ZTR, TG02), seliciclib (CYC202), AT7519, TP-1287, voruciclib, roniciclib (BAY 1,000,394), fadraciclib (CYC065), or a salt thereof.

19. The method of any one of claims 1-18, wherein cells of the cancer overexpress CDK9.

20. The method of any one of claims 1-10, wherein the CDK12 inhibitor is a small molecule inhibitor of CDK12 activity.

21. The method of any one of claims 1-10, wherein the CDK12 inhibitor is a CDK12 degrader.

22. The method of claim 21, wherein the CDK12 degrader is a proteolysis targeting chimera (PROTAC).

23. The method of any one of claims 1-10, wherein the CDK12 inhibitor is a CDK7 / 12 inhibitor.

24. The method of any one of claims 1-10, wherein the CDK12 inhibitor is a CDK9 / 12 inhibitor.

25. The method of any one of claims 1-10, wherein the CDK12 inhibitor is a CDK12 / 13 inhibitor.

26. The method of any one of claims 1-10, wherein the CDK12 inhibitor is nucleic acid that inhibits CDK12 expression.

27. The method of claim 26, wherein the CDK12 inhibitor is an antisense molecule, dsRNA, siRNA, shRNA, ribozyme, gRNA, or triple helix molecule.

28. The method of any one of claims 1-10, wherein the CDK12 inhibitor is a cyclin K inhibitor.

29. The method of any one of claims 1-10, wherein the CDK12 inhibitor is dinaciclib (SCH-727965), BSJ-4-116, THZ1, E9, THZ531, SR-4835, PP-C8, CDK12-IN-3, procaterol, BSJ-01-175, or a salt thereof.

30. The method of any one of claims 1-10 and 20-29, wherein cells of the cancer overexpress CDK12.

31. The method of any one of claims 1-30, wherein the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual in the same composition.

32. The method of any one of claims 1-30, wherein the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual in different compositions.

33. The method of any one of claims 1-32, wherein the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual simultaneously.

34. The method of any one of claims 1-30 and 32, wherein the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are administered to the individual intermittently.

35. The method of any one of claims 1-34, wherein the individual is a human.

36. A kit, comprising:(a) a protein kinase A (PKA) activator or salt thereof; and(b) a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof.

37. The kit of claim 36, comprising a pharmaceutical composition comprising the PKA activator or salt thereof and a pharmaceutically acceptable carrier.

38. The kit of claim 36 or claim 37, comprising a pharmaceutical composition comprising a CDK9 or CDK12 inhibitor or salt thereof and a pharmaceutically acceptable carrier.

39. The kit of any one of claims 36-38, wherein the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated in one composition.

40. The kit of any one of claims 36-38, wherein the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated in separate compositions.

41. The kit of any one of claims 36-38 and 40, wherein the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated for intermittent administration.

42. The kit of any one of claims 36-39, wherein the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof are formulated for simultaneous administration.

43. The kit of any one of claims 36-42, further comprising instructions for administering the PKA activator or salt thereof and the CDK9 or CDK12 inhibitor or salt thereof to an individual in need thereof.

44. The kit of claim 43, wherein the individual has cancer.

45. A composition comprising a protein kinase A (PKA) activator or salt thereof for use in a method of treating or delaying progression of cancer in an individual, said method comprising administering to the individual an effective amount of the composition in combination with a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof.

46. A composition comprising a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or a salt thereof for use in a method of treating or delaying progression of cancer in an individual, said method comprising administering to the individual an effective amount of the composition in combination with a protein kinase A (PKA) activator or salt thereof.

47. A composition comprising a protein kinase A (PKA) activator or salt thereof and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof for use in a method of treating or delaying progression of cancer in an individual, said method comprising administering to the individual an effective amount of the composition.

48. Use of a protein kinase A (PKA) activator or salt thereof in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament is to be administered in combination with a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof.

49. Use of a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament is to be administered in combination with a protein kinase A (PKA) activator or salt thereof.

50. Use of a protein kinase A (PKA) activator or salt thereof and a cyclin-dependent kinase 9 (CDK9) or cyclin-dependent kinase 12 (CDK12) inhibitor or salt thereof in the manufacture of a medicament for treating or delaying progression of cancer in an individual.

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