Treatment of high-grade serous carcinoma

Abstract

The present invention provides methods of treating high-grade serous carcinoma in an immunocompetent patient in need thereof, comprising administering to the immunocompetent patient in need thereof an effective amount of a SIK2 / 3 inhibitor or a pharmaceutically acceptable salt thereof, wherein the effective amount is lower than the amount needed to directly kill the tumor cells. The methods of treating high-grade serous carcinoma in an immunocompetent patient in need thereof may further comprise administration of an immune checkpoint inhibitor.

Description

TREATMENT OF HIGH-GRADE SEROUS CARCINOMACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 582,188, filed September 12, 2023, which is incorporated herein by reference.GOVERNMENT INTERESTS

[0002] This invention was made with government support under R03 CA262839-01A1 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0003] The present invention is in the field of treating patients with high-grade serous carcinoma. The present invention is in the field of use of SIK2 / 3 inhibitors to sensitize an immunosuppressive tumor microenvironment.BACKGROUND

[0004] In the United States in 2022, it is estimated that there were 19,800 new cases and 12,800 deaths from ovarian cancer, with approximately 70-80% of patients diagnosed in the advanced stage of the disease [M. Fischetti, et al., Expert Opinion on Pharmacotherapy (2023), 24(1): 65-72], Highgrade serous carcinoma (HGSC) is the most aggressive and common ovarian carcinoma type, accounting for more than 75% of all ovarian tumors. The current treatment standard for HGSC is cytoreductive surgery (to achieve status of no residual tumor) combined with doublet chemotherapy of carboplatin and paclitaxel [M. Luyckx, et al., “Chapter 6 - Recurrent High Grade Serous Ovarian Cancer Management” Ovarian Cancer (2022), 87-103], Even with such aggressive treatment, most patients will relapse, especially those with stages III-IV HGSC who failed to benefit from an optimal upfront surgery. Despite early responses to chemotherapy, the median progression-free survival of these patients is 13.8 months. A significant number of women with HGSC are intrinsically refractory to standard platinum-based chemotherapy. In addition, the accumulation of fluid, or ascites, occurs more frequently in patients with advanced-stage ovarian cancer than any other type of cancer, and the presence of ascites indicates the poorest outcomes for patients with advanced-stage ovarian cancer [B. Rickard, et al., Cancers (2021), 13(17): 4318], Malignant ascites in ovarian cancer increases resistance to chemotherapy, immunomodulators, and growth factors in the tumor environment, aswell as metastatic potential [M. Luyckx, et al., “Chapter 6 - Recurrent High Grade Serous Ovarian Cancer Management” Ovarian Cancer (2022), 87-103],

[0005] In the United States in 2023, approximately 20 clinical trials are active for HGSC, many of which are testing chemotherapy or combinations thereof, PARP inhibitors, surgery, or diagnostics. Most patients with HGSC do not have BRCA or related mutations, and even for patients who have BRCA-deficient HGSC, there is still a significant number who are unresponsive to PARP inhibitors or acquire resistance. Cyclin El (CCNE1) amplification, one of the most common genetic alterations in epithelial ovarian cancer, has been identified as a predictive biomarker of poor chemotherapy response [J.W. Gorski. Diagnostics (Basel) (2020), 10(5): 279], CCNE1 amplification and homologous recombination pathway deficiency are unlikely to co-occur, so patients with CCNE1 amplification are unlikely to benefit from PARP inhibitors. From the approximately 24 completed US clinical trials that targeted HGSC, only chemotherapy and PARP inhibitors have been FDA approved for ovarian cancer. Further, HGSC is almost always unresponsive to immunotherapies such as immune checkpoint inhibitors [P.A. Konstantinopoulos, et al., "Clinical and translational advances in ovarian cancer therapy ” Nature Cancer (2023)].

[0006] Salt-inducible kinases 1-3 (SIK1-SIK3) are serine / threonine kinases that belong to the AMP- activated protein kinase (AMPK) family and have been implicated in a number of signaling pathways relevant for energy metabolism, inflammation, immune responses, and sleep and circadian rhythms [A. Jagannath, et al., Physiological Review (2023), 103(3): 2231-2269], The SIK signaling pathway is highly context-dependent among cell types and disease settings. Both SIK2 and SIK3 expression correlate with poor outcomes in ovarian cancer. The only ongoing US clinical trial testing SIK2 / 3 inhibitors in gynecological cancers, including ovarian cancer, only tests efficacy at doses high enough to directly kill patient tumors, with doses ranging from 100 to 400 mg twice daily [see NCT04711161 and S. Fu, et al., Journal of Clinical Oncology (2022), 40(16 suppl): TPS5616-TPS5616], In the application herein, the applicant has shown that SIK2 / 3 inhibitors at doses lower than necessary to directly kill tumors can treat HGSC via immune T cell activation despite the presence of ascites. By treating at doses low enough to not significantly inhibit T cell growth, the applicant has also shown synergy of SIK2 / 3 inhibitors with immune checkpoint inhibitors in HGSC.SUMMARY

[0007] The present application provides a method of treating high-grade serous carcinoma (HGSC) comprising the administration to an immunocompetent patient in need of such treatment an effectiveamount of a SIK2 / 3 inhibitor. The present application further provides a method of treating HGSC comprising the administration to an immunocompetent patient in need of such treatment an effective amount of a SIK2 / 3 inhibitor and an immune checkpoint inhibitor.

[0008] The present application provides a method of treating HGSC in an immunocompetent patient in need thereof, the method comprising administering to the immunocompetent patient an effective amount of a compound means of inhibiting SIK2 / 3 and an immune checkpoint inhibitor means. The present application provides a method of treating HGSC in an immunocompetent patient in need thereof, the method comprising administering to the immunocompetent patient an effective amount of a SIK2 / 3 inhibitor and an immune checkpoint inhibitor.

[0009] The present application provides a method of treating HGSC in an immunocompetent patient in need thereof, the method comprising administering to the immunocompetent patient an effective amount of compound means of inhibiting SIK2 / 3 and a pharmaceutically acceptable carrier, wherein the effective amount is lower than the amount needed to directly kill the tumor cells. The present application provides a method of treating HGSC in an immunocompetent patient in need thereof, the method comprising administering to the patient an effective amount of a SIK2 / 3 inhibitor or a pharmaceutically acceptable salt thereof, wherein the effective amount is lower than the amount needed to directly kill the tumor cells.

[0010] The present application provides a method of treating HGSC in an immunocompetent patient in need thereof, the method comprising administering to the immunocompetent patient an effective amount of compound means of inhibiting SIK2 / 3 and a pharmaceutically acceptable carrier, wherein the effective amount is lower than the amount needed to directly kill the tumor cells, and an adoptive cellular therapy means. The present application provides a method of treating HGSC in an immunocompetent patient in need thereof, the method comprising administering to the patient an effective amount of a SIK2 / 3 inhibitor or a pharmaceutically acceptable salt thereof, wherein the effective amount is lower than the amount needed to directly kill the tumor cells, and an adoptive cellular therapy.

[0011] The present application provides a method of treating HGSC in an immunocompetent patient in need thereof, the method comprising administering to the immunocompetent patient an effective amount of a compound means of inhibiting SIK2 / 3 and an adoptive cellular therapy means. The present application provides a method of treating HGSC in an immunocompetent patient in need thereof, the method comprising administering to the immunocompetent patient an effective amount of a SIK2 / 3 inhibitor and an adoptive cellular therapy.

[0012] The present application provides a method of sensitizing ovarian cancer cells in an immunocompetent patient via the surrounding tumor microenvironment to treatment by an immune checkpoint inhibitor means or immune checkpoint inhibitor comprising administering a compound means of inhibiting SIK2 / 3, a SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof. The present application further provides a method of modifying a tumor microenvironment in an immunocompetent patient, the method comprising administering a compound means of inhibiting SIK2 / 3 and a pharmaceutically acceptable carrier to increase ITNy in the tumor microenvironment. The present application further provides a method of modifying a tumor microenvironment in an immunocompetent patient, the method comprising administering a compound means of inhibiting SIK2 / 3 and a pharmaceutically acceptable carrier to increase granzyme B in the tumor microenvironment. The present application further provides a method of modifying a tumor microenvironment in an immunocompetent patient, the method comprising administering a compound means of inhibiting SIK2 / 3 and a pharmaceutically acceptable carrier to increase CD80 and / or CD86 expression on dendritic cells in the tumor microenvironment.

[0013] The present application provides a composition for use in treating HGSC in an immunocompetent patient in need thereof, comprising: compound means for inhibiting SIK2 / 3 and a pharmaceutically acceptable carrier; wherein said composition is suitable for use in the treatment of treating HGSC.

[0014] The present application provides a SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor for use in treating HGSC in an immunocompetent patient in need thereof. The present application provides an effective amount of a SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof, for use in the treatment of HGSC in an immunocompetent patient in need thereof, wherein the effective amount of the SIK2 / 3 inhibitor is lower than the amount needed to directly kill the tumor cells. The present application further provides an effective amount of a SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof, for use in the treatment of HGSC in an immunocompetent patient in need thereof, wherein the effective amount of the SIK2 / 3 inhibitor is lower than the amount needed to directly kill the tumor cells, wherein the treatment further comprises an adoptive cellular therapy. The present application provides a SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof, and an adoptive cellular therapy for use in treating HGSC in an immunocompetent patient in need thereof.

[0015] The present application provides use of a SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor in the manufacture of a medicament for HGSC inan immunocompetent patient in need thereof. The present application provides use of a SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof, and an adoptive cellular therapy in the manufacture of a medicament for HGSC in an immunocompetent patient in need thereof. The present application provides use of an effective amount of a SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof, wherein the effective amount of the SIK2 / 3 inhibitor is lower than the amount needed to directly kill tumor cells, in the manufacture of a medicament for HGSC in an immunocompetent patient in need thereof. The present application provides a pharmaceutical composition for the treatment of HGSC, comprising a SIK2 / 3 inhibitor and an immune checkpoint inhibitor. The present application provides a pharmaceutical composition for the treatment of HGSC, comprising a SIK2 / 3 inhibitor and an adoptive cellular therapy. The present application provides a pharmaceutical composition for the treatment of HGSC, comprising a SIK2 / 3 inhibitor at an effective amount, wherein the effective amount of the SIK2 / 3 inhibitor is lower than the amount needed to directly kill tumor cells. The present application provides a pharmaceutical composition for use in the treatment of HGSC, comprising a SIK2 / 3 inhibitor and an immune checkpoint inhibitor. The present application provides a pharmaceutical composition for use in the treatment of HGSC, comprising a SIK2 / 3 inhibitor and an adoptive cellular therapy. The present application provides a pharmaceutical composition for use in the treatment of HGSC, comprising a SIK2 / 3 inhibitor at an effective amount, wherein the effective amount of the SIK2 / 3 inhibitor is lower than the amount needed to directly kill tumor cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 . Addition of HGSC human patient cell-free ascites fluid induces activation of SIK 1 - 3 signaling in ex vivo co-culture of human primary T cells from healthy donors (A) as soon as 1 hour and (B) which persists after 16 hours.

[0017] Figure 2. (A) SIK inhibition causes increased ZFNy concentration in human primary T cells in the presence of HGSC human patient cell-free ascites fluid in vitro compared to control. (B) YKL- 05-099 diminishes SIK signaling, as shown by decreased phosphorylation of class Ila HDACs 4, 5, 7 and CRTC2 in vitro in human Jurkat T cells.

[0018] Figure 3. Cre-induced genetic Sik2Sik3 DKO (A) increases ZFNy in vitro in murine T cells, which is suppressed by addition of human patient ovarian cancer cell-free ascites fluid. (B-D) Stimulation of fFNy production in vitro in murine T cells is also suppressed by addition of human patient ovarian cancer cell-free ascites fluid.

[0019] Figure 4. Schematic of experimental setup for in vivo FVB / PPM mouse studies.

[0020] Figure 5. SIK2 / 3 inhibition demonstrates in vivo efficacy in treating HGSC in PPM inoculated immunocompetent FVB mice.

[0021] Figure 6. Schematic of experimental setup for in vivo BeCon / B6 mouse studies.

[0022] Figure 7. SIK2 / 3 inhibition demonstrates in vivo efficacy in treating HGSC in BeCon inoculated immunocompetent B6 mice. Spleens shown in (G) are not shown to scale with mice shown in (H).

[0023] Figure 8. Schematic of experimental setup for in vivo BeCon / Sik2Sik3 DKO B6 mouse studies.

[0024] Figure 9. (A-B) Cre-induced Sik2Sik3 DKO demonstrates in vivo efficacy in increasing lifespan in HGSC BeCon inoculated immunocompetent B6 mice. (C) T cell subpopulations in mice with Sik2Sik3 DKO Cre-induction demonstrate increased T cell cytotoxicity and effector function. (D) Sik2Sik3 DKO, but not Sikl KO or Sik2 KO alone, leads to an increase in cytotoxic T cell function in vitro.

[0025] Figure 10. PPM inoculated immunodeficient mice show little to no improvement with SIK2 / 3 inhibitor dosing in vivo.

[0026] Figure 11. SIK2 / 3 inhibitor dose study in vitro in (A) human primary T cells from healthy donors, (B) human serous carcinoma cell lines, and (C-E) human patient-derived HGSC organoid lines.

[0027] Figure 12. fFNy concentrations measured in vitro in human patient-derived HGSC organoid / immune cell co-culture models exposed to YKL-05-099 vs. control (DMSO).

[0028] Figure 13. Cre-induced Sik2Sik3 DKO in in vitro cultured mouse immune cells demonstrates an upregulated PD-1 / PD-L1 axis at the protein level.

[0029] Figure 14. SIK2 / 3 inhibition and anti-PD-1 treatment demonstrate in vivo synergy in treating HGSC in PPM inoculated immunocompetent FVB mice.

[0030] Figure 15. SIK2 / 3 inhibition and anti-PD-1 treatment demonstrate in vivo efficacy in treating HGSC in BeCon inoculated immunocompetent B6 mice. Spleens shown in (B) are not shown to scale with mice shown in (A).

[0031] Figure 16. (A-B) YKL-05-099, SK-124, and ARN-3236 promote IFNy production in mouse T cells in vitro. (C) SIK2 / 3 inhibitor ARN-3236 demonstrates in vivo efficacy in increasing lifespan in HGSC PPM inoculated immunocompetent FVB mice.

[0032] Figure 17. SIK2 / 3 inhibition improves antigen-specific anti-tumor T cell responses.

[0033] Figure 18. SIK2 / 3 inhibition or Sik2Sik3 DKO improves innate anti -tumor activity via a direct effect on DCs.DETAILED DESCRIPTION

[0034] As used herein, the term High-Grade Serous Carcinoma, or HGSC, refers to ovarian cancer that is classified as grade three and arises from the serous membrane in the epithelial layer in the abdominopelvic cavity. Ovarian cancer is typically diagnosed by a blood test for certain biomarkers (cancer antigen-125 (CA-125), carcinoembryonic antigen (CEA), and CA 19-9) in conjunction with histology techniques, which are also used to distinguish between HGSC and low-grade serous carcinoma [See, e.g., C.A. Penn and R.D. Alvarez, “Current Issues in the Management of Patients With Newly Diagnosed Advanced-Stage High-Grade Serous Carcinoma of the Ovary” JCO Oncology Practice (2023), 19(3): 116-122], Tumor protein 53 (Tp53) overexpression and / or mutation is recognized as a genetic risk factor for HGSC. HGSC is often characterized by the presence of ascites.

[0035] As used herein, the term “SIK2 / 3 inhibitor” refers to an inhibitor of SIK2 and SIK3. The SIK2 / 3 inhibitor need not inhibit SIK2 and SIK3 equally. Some known SIK2 / 3 inhibitors and their reported SIK2 and SIK3 IC50 values in nanomolar are shown in Table 1.reference or in A. Jagannath, et al., Physiological Review (2023), 103(3): 2231-2269. n.a. indicates not available.

[0036] The phrase “compound means of inhibiting SIK2 / 3” refers to organic compounds i) having a combination of atoms that result in a molecular weight of less than 1000 g / mol, ii) that inhibit SIK2 and SIK3 by binding one or more binding sites through direct molecular interaction between the compound and SIK2 and SIK3, and iii) having an IC50 against SIK2 or SIK3 of 1 pM or less. Preferred compound means have an IC50 of 0.4 pM or less, or 40 nM or less, or even 4.0 nM or less. Structures having this function include compounds found in Table 1 and their pharmaceutically acceptable salts thereof. The references cited in Table 1 are incorporated by reference in their entirety. Preferably, the compound means of inhibiting SIK2 / 3 are YKL-05-099, ARN-3236, ARN-3261, Bosutinib, Crenolanib, Dasatinib, G-5555, GLPG3312, GLPG3970, MRIA9, MRT199665, MRT67307, SK- 124, Tenalisib, WH-04-023, YKL-06-061, YKL-06-062, YKL-05-096, YKL-05-093, or a pharmaceutically acceptable salt thereof. More preferably, the compound means of inhibiting SIK2 / 3 are YKL-05-099, ARN-3236, ARN-3261, Bosutinib, Crenolanib, Dasatinib, GLPG3312, GLPG3970, MRIA9, SK-124, or a pharmaceutically acceptable salt thereof. A determination of the SIK2 or SIK3 IC50 of a compound is carried out by conventional techniques found in T.B. Sundberg, et al., ACS Chem Biol. (2016), 11(8): 2105-2111. Functional equivalents would also result in inhibition of SIK2 / 3 to a degree sufficient to inhibit SIK2 / 3 while not significantly inhibiting growth of T cells, where ‘significantly inhibiting growth of T cells’ is defined as a dose that preserves approximately 75% of T cell growth (and preferably 80%, more preferably 85%, and even more preferably 90% of T cell growth), as shown in the dose study in Figure 11 A and Tables 5 and 6. For example, for SIK2 / 3 inhibitor YKL-05-099, a preferred daily dose in a patient would not exceed 100 mg. This dose is less than half the lowest proposed dose in the only ongoing SIK2 / 3 inhibitor clinical trial in gynecological cancers. Those skilled in the art will appreciate how to conduct the dosage study outlined herein. Stated differently, for each of the molecules that inhibit SIK2 / 3, a skilled artisan can determine what dose or amount constitutes an effective amount that is lower than the amount needed to directly killthe tumor cells for a particular SIK2 / 3 inhibitor by conducting an in vitro dose study similar or identical to that shown in Figure 11 A and Tables 5 and 6 for that molecule and finding the dose that preserves approximately 75% of T cell growth (and preferably 80%, more preferably 85%, and even more preferably 90% of T cell growth). Those skilled in the art will appreciate that a dose Tower than the amount needed to directly kill the tumor cells’ may directly kill an insignificant number of tumor cells but is not a high enough dose to directly kill a significant number of tumor cells. A dose that directly kills a significant number of tumor cells is a dose that directly kills 50% or more of tumor cells in vitro, as illustrated in Figures 11B-E. Preferably, a dose that directly kills a significant number of tumor cells is a dose that directly kills 25% or more of the tumor cells in vitro. Those skilled in the art will appreciate that ‘directly kill the tumor cells’ means via a non-immune cell-based killing, i.e., by inhibiting SIK2 / 3 in the tumor cells. This dosage of the SIK2 / 3 inhibitor will be lower than needed to directly kill a significant number of tumor cells but will treat HGSC via immune T cell activation despite the presence of ascites. Further, this lower dosage of the SIK2 / 3 inhibitor found via the dose study similar or identical to that shown in Figure 11A and Tables 5 and 6 may also be a dosage that will synergistically work with immune checkpoint inhibitors in HGSC. This lower dosage of the SIK2 / 3 inhibitor may also be a dosage that will work with an adoptive cellular therapy in HGSC. As used herein, “SIK2 / 3 inhibitor” and “compound means of inhibiting SIK2 / 3” are interchangeable expressions of the compounds useful in the present methods.

[0037] As used herein, “an immune checkpoint inhibitor” is an antibody that inhibits an immune checkpoint protein or ligand thereof. As used herein, “an immune checkpoint inhibitor means” is a means for inhibiting an immune checkpoint protein or ligand thereof and is well understood in the art. As used herein, the terms “immune checkpoint inhibitor” and “immune checkpoint inhibitor means” are interchangeable expressions of the compounds useful in the present methods. The immune checkpoint inhibitor is an anti-PD-1, anti-PD-Ll, or anti-CTLA4 antibody. In a preferred embodiment, the anti-PD-lF, anti-PD-Ll, or anti-CTLA4 antibody is pembrolizumab, nivolumab, cemiplimab, dostarlimab, tezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, or a pharmaceutically acceptable salt thereof. FDA approved anti-PD-1 antibodies are known in the art and can be made and used according to US8354509B2 (pembrolizumab - Merck), US8008449B2 (nivolumab - Bristol-Myers Squibb), US9987500B2 (cemiplimab - Regeneron Pharmaceuticals), and US9815897B2 (dostarlimab - AnaptysBio, GlaxoSmithKline). FDA approved anti-PD-Ll antibodies are known in the art and can be made and used according to US8217149B2 (atezolizumab - Roche, Genentech), US9624298B2 (avelumab - Merck), US8779108B2 (durvalumab - AstraZeneca). FDAapproved anti-CTLA4 antibodies are known in the art and can be made and used according to US6984720B1 (ipilimumab - Bristol-Myers Squibb) and US6682736B1 (tremelimumab - AstraZeneca).

[0038] As used herein, “an adoptive cellular therapy” or “cell therapy” is an immune cell therapy composition comprising cells capable of tumor antigen recognition and effector function either from a patient or from a donor and is well understood in the art, such as in V. Albarran et al., Front. Immunol. (2024), 15: 1352805 and D. Olson et al., J Clin Onco (2023), 41(18): 3397 - 3407. As used herein, “an adoptive cellular therapy means” or “cell therapy means” is an immune cell means for tumor antigen recognition and effector function. As used herein, the terms “an adoptive cellular therapy”, “a cell therapy”, “an adoptive cellular therapy means”, and “a cell therapy means” are interchangeable expressions of the compositions useful in the present methods. In a preferred embodiment, the adoptive cellular therapy is a T cell therapy. In a preferred embodiment, the T cell therapy is a chimeric antigen receptor (CAR) T cell therapy. T cell and CAR T therapies are known in the art and can be made and used as in A. Haas et al., Molecular Therapy (2019), 27(11): 1919- 1929; L. Kandalaft et al., Journal of Translational Medicine (2012), 10(157): 1-10; and M. Koneru et al., Journal of Translational Medicine (2015), 12(102): 1-11.

[0039] As used herein, the term “treating” (or “treat” or “treatment”) refers to restraining, slowing, stopping, or reversing the progression or severity of an existing symptom, condition, or disorder.

[0040] As used herein, the phrase "effective amount” of a SIK2 / 3 inhibitor means an amount of a compound of the invention that is sufficient to inhibit SIK2 / 3 while not significantly inhibiting growth of T cells to achieve the objectives of the invention, which is the treatment of HGSC in one or more doses. In embodiments, an effective amount of a SIK2 / 3 inhibitor is unexpectedly lower than the amount required for direct killing of HGSC tumor cells. Such lower doses are expected to result in fewer side effects for the immunocompetent patient. In embodiments, an effective amount of a SIK2 / 3 inhibitor is the amount necessary to significantly inhibit SIK2 / 3 such that T cell TFNy production increases and T cell GzmB production increases. The amount of an immune checkpoint inhibitor is sufficient to inhibit an immune checkpoint protein or ligand thereof. The amount of an immune checkpoint inhibitor is well-known in the art. The amount may be the same or different from the effective amount of the SIK2 / 3 inhibitor. More particularly, an effective amount provides improvement in important cancer endpoints, Overall Survival (OS), Disease-Free Survival (DFS), Objective Response Rate, Complete Response Rate or Progression Free Survival (PFS). See Dept, of Health and Human Services, Food and Drug Admin, Clinical Trial Endpoints for the Approval ofCancer Drugs and Biologies: Guidance for Industry (2018); E.A. Eisenhauer, et al., New Response Evaluation Criteria in Solid Tumours: Revised RECIST Guideline (Version 1.1), 45 Eur. J. Cancer 228 (2009).

[0041] As used herein, the phrase “administering” (or “administer” or “administration”) refers to the act of the attending physician or caregiver, prescribing the agent for administration and thereby causing the application of an agent to a subject, through ingestion, infusion, injection, or any other means, whether self-administered or administered by a clinician or other qualified caregiver. Two or more compounds of the invention may be administered in combination or separately, and if separately, simultaneously or sequentially over a period of time as determined by a qualified caregiver. If sequentially, the SIK2 / 3 inhibitor is to be given first. In a preferred embodiment, the SIK2 / 3 inhibitor is injected intraperitoneally at or adjacent to the tumor site.

[0042] As used herein, the phrase “pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for human pharmaceutical or therapeutic use. Pharmaceutically acceptable carriers can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. The term carrier may also encompass any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. Pharmaceutical compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the SIK2 / 3 inhibitor based on the weight of the total composition including the carrier.

[0043] As used herein, "patient" refers to a mammal, preferably human. The patient may or may not have received previous treatment for the instant or other indications, such as chemotherapy, surgery, radiation, or PARP inhibitors. The patient must be immunocompetent.

[0044] As used herein, the “tumor microenvironment” is a dynamic environment that surrounds the tumor. This includes but is not limited to the surrounding blood vessels, immune cells, fibroblasts, signaling molecules and the extracellular matrix ascites. The tumor microenvironment may play a role in the growth or apoptosis of the tumor. Collection of samples by standard laparoscopy or debulking surgery techniques can be paired with measurement of signaling molecules in the tumor microenvironment by a variety of procedures known in the art, such as single-cell RNA sequencing [I. Vazquez-Garcia et al., Nature (2022), 612(7941): 778-786],

[0045] The present invention provides a method for treating HGSC in an immunocompetent patient in need thereof comprising administering an effective amount of a SIK2 / 3 inhibitor as a monotherapy without concurrent therapy of a checkpoint inhibitor. Further, the present invention also provides a SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof, for use in therapy, in particular for the treatment of HGSC. In a preferred embodiment, the SIK2 / 3 inhibitor is selected from the group comprising YKL-05-099, ARN-3236, ARN-3261, Bosutinib, Crenolanib, Dasatinib, G-5555, GLPG3312, GLPG3970, MRIA9, MRT199665, MRT67307, SK-124, Tenalisib, WH-04-023, YKL- 06-061, YKL-06-062, YKL-05-096, YKL-05-093, and a pharmaceutically acceptable salt thereof. In a further preferred embodiment, the SIK2 / 3 inhibitor is YKL-05-099, ARN-3236, ARN-3261, Bosutinib, Crenolanib, Dasatinib, GLPG3312, GLPG3970, MRIA9, SK-124, or a pharmaceutically acceptable salt thereof. In a more preferred embodiment, the SIK2 / 3 inhibitor is YKL-05-099, or a pharmaceutically acceptable salt thereof. SIK2 / 3 inhibitors are known in the art and can be made and used according to the patents, patent applications, and references listed in Table 1.

[0046] In the present methods, the SIK2 / 3 inhibitors may be administered as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts and common methodologies for preparing them are well known in the art. See, e.g., P. Stahl, et al., Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd 30 Revised Edition Wiley-VCR (2011); S. M. Berge, et al., "Pharmaceutical Salts" Journal of Pharmaceutical Sciences (1977), 66(1).

[0047] The present invention provides a method of sensitizing the tumor microenvironment in an immunocompetent patient to treatment by an immune checkpoint inhibitor or a pharmaceutically acceptable salt thereof by inhibiting SIK2 / 3. The present invention also provides an immune checkpoint inhibitor for use in therapy, in particular for the treatment of HGSC in combination with a SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof.

[0048] SIK2 / 3 inhibitors are preferably formulated as pharmaceutical compositions administered by a variety of routes. Such pharmaceutical compositions and processes for preparing the same are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy (I) [A. Gennaro, et al., eds., 21st ed., Mack Publishing Co., (2005)]. SIK2 / 3 inhibitors, anti-PD-1 antibodies, anti-PD-Ll antibodies and other compounds included in the methods of the present invention, or salts thereof, may be prepared by a variety of procedures known in the art or are commercially available.

[0049] The pharmaceutical compositions of the present application include those suitable for any acceptable route of administration. Acceptable routes of administration include oral, parenteral, or intraperitoneal administration.

[0050] In some embodiments, the pharmaceutical composition may be formulated for oral administration. Compositions suitable for oral administration can be presented as discrete units such as capsules or tablets each containing a predetermined amount (e.g., a unit dose amount, which when administered as one or more doses, preferably 1 or 2 doses, provides an effective amount) of the active ingredient.

[0051] Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions or infusion solutions which can contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, saline (e.g., 0.9% saline solution) or 5% dextrose solution, immediately prior to use.

[0052] The effective dose of SIK2 / 3 can vary with the severity of the disease, the route of administration, the sex, age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician.EXAMPLESExample 1: Ovarian Cancer Ascites Activate SIK Signaling In VitroCo-culture of human primary T cells and cell-free malignant ascites from patients with OvarianCancer

[0053] Collection and analysis of ovarian cancer (OvCa) specimens were classified as surgical discard and remained completely de-identified. Malignant ascites samples were centrifuged for 10 minutes at 1,300 revolutions per minute (rpm). Ascites supernatants were collected, depleted of cells by passing through a 0.22-pm filter, and stored in small aliquots at -80°C until use. For human primary T cell assays, whole blood was collected from anonymous healthy donors. Healthy donor peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (GE Healthcare). Pan-T cells were enriched by negative selection using the human pan-T Cell Isolation Kit (Miltenyi Biotec; > 95% purity confirmed via flow cytometry). Pan-T cells were activated by anti- CD3 / CD28 Dynabeads™ (Life Technologies) at a 2: 1 cell-to-bead ratio and were cultured overnight in complete Roswell Park Memorial Institute medium (RPMI 1640 supplemented with 10% FBS, penicillin-streptomycin, L-glutamine and -mercaptoethanol) at 37°C with 5% CO2. Cell-free OvCamalignant ascites were added to the T cell culture 20 hours later for a 10% final concentration. In cases where small molecule drugs were tested, the compound solution was added 16 hours after T cell activation (4 hours prior to addition of ascites). At 48 hours (one day after the addition of ascites), co-culture supernatants were collected for IFNy measurement as an indicator of T cell anti-tumor capability via an enzyme-linked immunosorbent assay (ELISA) readout (Life Technologies) using a BioTek automated microplate washer and a PerkinElmer Envision plate reader.Immunoblots

[0054] Human purified T cells (purity > 95%) were collected in complete RMPI medium as a singlecell suspension and activated with anti-CD3 / CD28 T cell activation Dynabeads™ for 3 days before the co-cultures with or without cell-free ovarian cancer ascites as indicated in Figure 1. Jurkat human T cell lines were cultured for Figure 2B. At assay conditions, the cells were spun down, and the cell pellets were washed twice with ice-cold IX phosphate-buffered saline (PBS) solution before resuspension into IX radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with proteinase inhibitors on ice for 20 minutes. The homogenates were centrifuged at 13,000 rpm for 10 minutes at 4°C, and the supernatant was carefully collected. Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific), and lysates containing an equivalent amount of protein were mixed with 4X lithium dodecyl sulfate (LDS) sample buffer for sample preparation (Invitrogen). The samples were heated at 70°C for 10 minutes, and equal amounts of protein were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS- PAGE) using Bis-Tris gels (Invitrogen) and transferred onto polyvinylidene difluoride (PVDF) membranes following a standard protocol.

[0055] Anti-phosphorylated(p)SIK 1 / 2 / 3 (Abeam #abl99474), anti-p histone deacetylase (HDAC)4 / 5 / 7 (Cell Signaling Technology #3443 S), anti-p cAMP regulated transcriptional coactivator 2 (CRTC2) (Abeam #ab203187), anti-P-Actin (#4967S, Cell Signaling Technology) and goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) (Thermo Fisher Scientific) were used. SuperSignal™ West Pico and Femto chemiluminescent substrates (Thermo Fisher Scientific) were used to image blots in a ChemiDoc MP Imaging System instrument (BioRad).

[0056] The results in Figure 1 show that addition of HGSC human patient cell-free ascites fluid induces activation of SIK signaling in e vivo co-culture of human primary T cells from healthy donors. Activation of SIK is marked by higher levels of phosphorylated SIK1 (at Tyrosine (T) 182), SIK2 (at T175), and SIK3 (at T163) shown by Western blotting upon addition of cell-free ascites fluid. Actin Beta (ACTB) is shown as a reference gene (denominator) for Western blotting. Activationof SIK signaling is observed as soon as 1 hour (Figure 1A) and persists after 16 hours (Figure IB). The applicant further discovered that expression of SIK 1, 2, and 3 correlates with immune suppression using three hundred and three available The Cancer Genome Atlas (TCGA) ovarian cancer samples.Example 2: SIK2 / 3 Inhibition Activates T cell Anti-Tumor Function in the Presence of Ascites and Decreases SIK Signaling In VitroCell Lines and Culture Conditions

[0057] Jurkat cells (human CD4 T cell line purchased from ATCC) were cultured in complete RPMI medium as above with a cell concentration maintained at ~5 x 105 / ml. Murine ovarian cancer cell lines PPM-luciferase (PPM-Luc: Trp53 Pten MycC)l, on FVB background), BeCon (ID8- Defb29 Vegf-'A, on C57BL / 6 (B6) background), and human serous carcinoma cell lines OVCAR8 (ATCC), CAOV-3 (ATCC), and SKOV3 (ATCC) cells were cultured in complete Dulbecco’s Modified Eagle Medium (DMEM) consisting of DMEM media supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl (GlutaMAX™), 1 mM sodium pyruvate, 55 pM 2-mercaptoethanol, 10 mM N-2 -hydroxy ethylpiperazine-N-2-ethane sulfonic acid (HEPES), 50 pg / ml gentamicin, lOO U / ml penicillin, and lOO pg / ml streptomycin (all from Coming or Gibco Life Technologies, except FBS from Atlanta Biologicals). Cell lines were cultured in an incubator at 37°C with 5% CO2. All cell lines routinely tested negative for mycoplasma contamination using a Mycoplasma Detection Kit (Lonza) and maintained in medium containing anti-mycoplasma reagent Plasmocin® (Invivogen) for prophylactic purposes.Statistics

[0058] For graphs, data are shown as mean ± standard error of the mean (s.e.m.) or standard deviation (s.d.) as indicated. Unless otherwise indicated, statistical differences were evaluated using a two-tailed unpaired Student's / -test, assuming equal sample variance. P < 0.05 was considered significant. Graphs were produced and statistical analyses were performed using GraphPad Prism or R.

[0059] The results in Figure 2A show that SIK inhibitors increase IFNy concentration in human primary T cells in the presence of HGSC human patient cell-free ascites fluid, a suppressive tumor microenvironment, in vitro compared to control (no exposure). Data are representative of four independent experiments at different doses (10 pM, 2 pM, 0.4 pM, 0.08 pM) performed in triplicate. The results in Figure 2B show that YKL-05-099 diminishes SIK signaling via decreased phosphorylation of class Ila HDACs - HDAC4 at Serine (S)246, HDAC5 at S259, HDAC7 at SI 55,and CRTC2 at S171 in human Jurkat T cells. Actin Beta (ACTB) is shown as a reference gene (denominator) for Western blotting.Example 3: Induced Genetic Knock Out of SIK2 / 3 and P / I Stimulation Activates Mouse T cell Anti-Tumor Function in the Presence of Ascites, which Suppress Activation In VitroEx vivo activation of primary mouse T cells

[0060] Purified pan-T cells from mouse spleens were activated with anti-CD3 / CD28 antibodies for 2-5 days in complete RPMI medium with recombinant mouse IL -2 (10 pg / ml; BioLegend) for ex vivo T cell experiments. In some assays indicated, 0.05-0.5 pM of YKL-05-099 (SIK2 / 3 inhibitor; MedChemExpress, HY-101147) was added to the culture and dimethyl sulfoxide (DMSO) was used as the control.Quantitative real time PCR

[0061] Cells were pre-enriched (Miltenyi Biotec) and then sorted to > 95% purity and RNA was isolated using the RNeasy RNA purification mini (for 5 x IO3cells or beyond) or micro (for < 5 x 105cells) kit (Qiagen) according to the manufacturer’s protocol. From purified RNA, cDNA was synthesized using the qScript cDNA synthesis kit (Quanta Biosciences). Quantitative real time PCR (qRT-PCR) was performed in triplicate in a 384-well plate using Taqman Probes (Thermo Fisher)- based detection on an ABI QuantStudio 6 Flex qRT-PCR machine. For the analysis of mRNA abundance, the derived values were normalized to those of P-Actin (P-ACTIN for human materials) using a deltaCt method. Taqman probe information is in Table 2.Table 2. Taqman gene expression assays used for qRT-PCR analysis shown in Figure 3A.Flow cytometry and cell sorting

[0062] Cell surface staining of single-cell suspensions was performed using the following fluorophore-conjugated antibodies purchased from BioLegend, Thermo Fisher, BD Biosciences, Tonbo Biosciences, Cell Signaling Technology, and R&D Systems. Unless otherwise indicated,mouse T cells were defined as CD3+TCRp+CD4+(or CD8+) CD19' F4 / 8O'NK1.1‘ cells. All analyses were performed on viable populations by exclusion of dead cells using a fixable viability dye (Thermo Fisher). Intracellular cytokine detection was performed by first stimulating T cells with phorbol myristate acetate and lonomycin (P / I) in the presence of the protein transport inhibitor monensin and brefeldin A (BioLegend) for 3-5 hours, and then fixed, permeabilized (supplier: Thermo Fisher Scientific, BDB554722; manufacturer: BD Cell Analysis, 554722), and intracellularly stained with anti-ZFNy, TNFa, IL-2, etc. (BioLegend). Flow cytometry data acquisition was performed on a CytoFLEX LX (Beckman Coulter). Cell sorting was performed on Aria II cytometers (BD Biosciences). For experiments involving qRT-PCR, cell populations were sorted to > 95% purity. Data were analyzed with FlowJolO software (Tree Star). The results in Figure 3 show Cre-induced Sik2Sik3 double knockout (DKO) in mouse T cells enhances immune T cell function in vitro at both the genetic and protein level. The results in Figure 3A show Cre-induction of Sik2Sik3 DKO in murine T cells in vitro increases IFNy transcription at the mRNA level relative to Actin Beta (bAct), and addition of human patient ovarian cancer cell-free ascites fluid suppresses this transcription. The results in Figure 3B show SIK2 / 3 DKO in mouse T cells enhances anti -tumor T cell function in vitro at the protein level as shown by increasing amounts of IFNy protein produced compared to wildtype littermates. The results in Figures 3C and 3D show the same result analyzed as a percentage of T cells that produce IFNy and the amount of IFNy produced per T cell, respectively. The results in Figures 3B-D further show that stimulation of IFNy production by P / I is suppressed by addition of human ovarian cancer cell-free ascites fluid in vitro.Example 4: SIK2 / 3 Inhibition and Induced SIK2 / 3 Genetic Knock Out Show Efficacy in Treating Models of HGSC in Immunocompetent Mice, but SIK2 / 3 Inhibition shows Little to No Efficacy in Treating Models of HGSC in Immunocompromised MiceMice

[0063] Mice were bred at Dana-Farber Cancer Institute in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). The following strains were used in this study: FVB / NJ (The Jackson Laboratory; strain # 001800), C57BL6 / J (The Jackson Laboratory; strain #000664), Rag2 ~'~H2rg (Taconic: model # 4111), NSG (The Jackson Laboratory; strain # 005557), Sik2r iSik3f / f, UBC-Cre-ERT2 (The Jackson Laboratory; strain # 007001), and CD4-CreERT2(The Jackson Laboratory; strain # 022356) Sik2f / fSik3f / fUBC-Cre-ERT2, Siklf / f UBC-Cre-ERT2, Sik2f / f UBC-Cre-ERT2, Sik2f / fSik3f / fCD4-CreERT2 and Sik2f / fSik3f / fCD8a-Cre animals and littermatecontrols were generated by breeding at Dana-Farber Cancer Institute, all on the B6 genetic background. Experiments were conducted using 8-16 week old, female littermate mice.Ovarian Cancer Mouse Models

[0064] 0.5-1 x 105BeCon cells or 1.0 x 106PPM-luc cells were orthotopically inoculated to host mice via intraperitoneal (i.p.) injections. In the BeCon model, the wildtype B6 recipient mice (or SIK KO mice as indicated) were randomly allocated to different treatment groups. The BeCon genetic model mirrors human ascites and progression in HGSC more closely but produces fewer tumors compared to the PPM model [See J.R. Cubillos-Ruiz, et al., Cell (2015), 161(7): 1527-38 and L. Ding, et al., Cell reports (2018), 25(11): 2972-2980], The BeCon model is metastatic and aggressive, resistant to chemotherapy and immune checkpoint blockade, and unresponsive to PARP inhibition due to the lack of BRCA mutations [M. Song, et al. Nature (2018), 562: 423-428, Z. Zhao, et al., Biomacromolecules (2022), 23(10): 4379-4387], The PPM genetic model has the Trp53 / Pten / Myc mutations most common in HGSC and is an appropriate metastasis model. In the PPM-luc model, the engraftment was confirmed by in vivo bioluminescence imaging (BLI) at day 6 post tumor cell injection, and the wildtype FVB (or immunodeficient NSG mice as indicated) recipient mice were randomized into different treatment groups. YKL-05-099 or ARN-3236 (Medchem Express, HY- 120856) at 6 mg / kg or the vehicle control administration started at day 7 post tumor cell inoculation via daily i.p. injections (5 times per week skipping the weekend). In some assays and starting at day 7, anti-PD-1 was administrated twice a week at 200 pg / i.p., alone or in combination with YKL-05- 099. Mice were euthanized by CO2 inhalation before the humane endpoint at the indicated time, and organs, including tumors, ascites, tumor-draining lymph nodes (TDLNs) and spleens, were harvested for flow cytometry or co-detection by indexing (CODEX) analyses. Freshly obtained tumor samples were frozen and embedded in OCT (Sakura Finetek USA) via a bath of isopentane (Millipore Sigma) and liquid nitrogen, and 6 pm thick sections of frozen blocks were acquired and mounted to coverslips (Electron Microscopy Science) coated with poly-L-lysine (Sigma Aldrich). CODEX samples were prepared as in S. Black et al., Nat Protoc. (2021), 16(8): 3802-3835 and stained with antibody-DNA barcode conjugations as shown in Table 3. CODEX sample imaging was done by connecting a CODEX robot (Akoya Biosciences) to a Zeiss Axio Observer 7 microscope (Carl Zeiss microscopy) that was equipped with a Calibri 7 light source (405, 493, 575, 654, 761 nm excitations) and Prime BSI camera (Photometries). A Plan-Apochromat 20x / 0.8 NA M27 objective was used to collect 16- bit four-channel images and a 10 ms exposure time for DAPI, 300 ms for AF647 and AF750. 112 HE DAPFGFP / Cy3 / Cy5 / Cy7 filter was used for all channels and definite focus was used to keep samplesfocused during the imaging process. 200-500 tiles were collected for each sample depending on the sample size.Table 3. Antibody -DNA barcode conjugations used for staining of CODEX samples. Antibodies are described in the left table, and DNA barcodes are described in the right table.

[0065] For survival metrics analyses, the humane endpoint for BeCon model is either approaching 5 grams of body weight gain or body condition scores (BCS) declining to less than 2.5 when examined by a rodent veterinarian. PPM-luc solid tumors and BeCon malignant ascites were weighed at the endpoint to indicate the extent of disease progression. In some assays, peripheral blood was drained at the indicated time point for serum cytokine measurement or immune profiling via flow cytometry. In assays where PPM-luc was inoculated, tumor burden was measured up to twice a week in a blinded manner by BLI using a Xenogen IVIS-200 Spectrum camera, and the quantifications were shown as the geometric mean of the luciferase signal intensity (in photons / second) in the indicated treatment group during the indicated time course. In some assays, T cells were depleted from the wildtype FVB mice by administration of both CD4- and CD8-depleting monoclonal antibodies (BioXCell) twice a week at 200 pg / i.p. starting one week prior to tumor cell injections. T cell depletion was confirmed by flow cytometry of peripheral blood during this time and at the endpoint when harvesting spleens, lymph nodes, and tumors. To induce depletion of Sik2 and Sik3 via Cre activation in the UBC-Cre- ERT2Sik2Sik3-floxed mice, tamoxifen was administrated at 1 mg / i.p. on day 7, 9, and 11 post tumor cell injections. The in vivo knockout efficacy was confirmed by qRT-PCR, immunoblots, and flow cytometry of a tdTomato Cre reporter. The Sik2Sik3 DKO model depletes SIK from the host immune cell, but the tumor remains SIK-competent.

[0066] The schematic shown in Figure 4 shows the experimental setup for the mouse experiments in Figures 5, 10, and 14. The results in Figure 5 show SIK2 / 3 inhibition demonstrates in vivo efficacy in treating HGSC after there was a detectable tumor burden in PPM inoculated immunocompetent FVB mice, a model that is known to be resistant to immune checkpoint inhibitors and PARP inhibition. Figure 5A shows a photo of tumors dissected on day 24 after PPM inoculation and day 17of daily treatments of YKL-05-099 or vehicle (DMSO) of a representative mouse. The results in Figures 5B-D show corresponding mouse tumor decreases as B) PPM tumor weight in grams, C) percentage of tumor weight to body weight, and D) surface area of the largest tumor in millimeters squared (mmA2), with four replicates. The results in Figure 5E show weight changes for YKL-05-099 and vehicle dosed mice. The results in Figure 5F show the corresponding increase in probability of survival and median survival days of mice treated with YKL-05-099 compared to vehicle. The results in Figure 5G show the corresponding in vivo tumor burden measurements as geometric mean of the bioluminescence imaging signal total flux in photons per second (sec) at day 24 which show that YKL-05-099 treatment decreases tumor burden compared to vehicle. The results in Figures 5H-I show corresponding CODEX measurements from tumors harvested at day 24 which show that YKL-05- 099 treatment (six replicates) compared to vehicle (four replicates) elevated Cleaved Caspase 3 measured as H) percentage in tumor microenvironment (TME) and I) number of cells per mmA2 in TME, indicating induction of tumor apoptosis as a result of YKL-05-099 treatment. Corresponding CODEX CD31 measurements as J) percentage in TME and I) number of cells per mmA2 in TME, indicate suppression of tumor angiogenesis as a result of YKL-05-099 treatment.

[0067] The schematic shown in Figure 6 shows the experimental setup for the mouse experiments shown in Figure 7. The results in Figure 7 show that SIK2 / 3 inhibition demonstrates in vivo efficacy in treating HGSC in BeCon inoculated immunocompetent B6 mice. The results in Figure 7A show that daily YKL-05-099 treatment compared to vehicle (DMSO) starting 7 days post-BeCon tumor inoculation increases IFNy concentration in peripheral blood. The results in Figure 7B show the corresponding cytokine IL-10 concentration in peripheral blood on days 10, 17, and 24, which decreases as a result of YKL-05-099 treatment, indicating a decrease in immune suppression. The results of Figures 7C-D and H show that YKL-05-099 treatment compared to vehicle (DMSO) leads to a decrease in ascites measured as weight in grams (Figure 7C), percentage of body weight (Figure 7D), and visually in photos of mice (Figure 7H) on day 24. The results of Figures 7E-G show that YKL-05-099 treatment compared to vehicle leads to a decrease in spleen size measured as weight in grams (Figure 7E), percentage of body weight (Figure 7F), and visually in photos of the spleen (Figure 7G) on day 24. The reduced splenomegaly indicates slowing disease progression. The results in Figure 71 show the corresponding increase in probability of survival and median survival days of mice treated with YKL-05-099 compared to vehicle. Figure 7J shows the increase in percentage of cytotoxic T cells (including both CD8+and CD4+cytotoxic T cells) within total immune cells (CD45+cells) from ascites samples on day 24 of mice treated with YKL-05-099 compared to vehicle. Figure 7K showscorresponding single-cell RNAseq results demonstrating that SIK2 / 3 inhibition increases the percentage of infiltrated cytotoxic CD4+and CD8+T cells within CD45+cells in ascites samples compared to vehicle (Ctrl). Figure 7L shows corresponding granzyme B (GzmB) and IFNy flow cytometry measurements from tumor-draining mesenteric lymph nodes (MLN) from mice on day 24. Results demonstrate that SIK2 / 3 inhibition increases both CD8+and CD4+T cell cytotoxicity (shown as an increase in GzmB staining) as well as anti-tumor cytokine production (shown as an increase in IFNy staining).

[0068] The schematic shown in Figure 8 shows the experimental setup for the mouse experiments shown in Figures 9A-C. The results in Figures 9A-C show Cre-induced Sik2Sik3 DKO demonstrates in vivo efficacy in increasing lifespan in HGSC in BeCon inoculated immunocompetent B6 mice. The induction of the Sik2Sik3 DKO allows for the mice to grow and develop with SIK2 and SIK3 and only lose SIK2 and SIK3 8-10 days after BeCon inoculation. The results in Figure 9A show that Sik2Sik3 DKO decreases ascites accumulation compared to wildtype littermates, as shown by the size of the mice at day 25. The results in Figure 9B show the corresponding increase in probability of survival and median survival days of mice when Sik2Sik3 DKO is induced. Note that these results, together with the results of the experiments shown in Figures 5 and 7, indicate that the SIK2 / 3 inhibitor’s direct effect on the tumor cells is not essential for therapeutic benefit. Figure 9C shows Sik2Sik3 DKO increases T cell infiltration and activates T cell cytotoxicity and effector function. The Sik2Sik3 DKO mice show higher percentages of T cell infiltration in the peripheral blood (PB) at day 19, including both CD4+and CD8+subpopulations, compared to their wildtype littermate mice, consistent with increased T cell anti-tumor responses. Figure 9D shows the fold change in IFNy levels measured by flow cytometry in mouse splenic T cells upon tamoxifen induction of Sik2Sik3KO, Sik2 KO, or Sikl KO relative to non-induced T cells. Data is pooled from four experiments. Only the Sik2 / 3 double KO T cells (not the Sikl or Sik2 single KO) resulted in elevated IFNy compared with their wildtype counterparts. This data shows SIK2 / 3 DKO, but not SIK1 KO or SIK2 KO alone, leads to an increase in cytotoxic T cell function in T cells, similar to treatment of wildtype cells by SIK2 / 3 inhibitors.

[0069] The results in Figure 10 compared to Figure 5 show loss of in vivo efficacy in treating HGSC with a SIK2 / 3 inhibitor after detectable tumor burden in PPM inoculated FVB mice when mice are no longer immunocompetent due to genetic depletion of both CD4+and CD8+T cells. Figure 10 also shows little to no in vivo efficacy in treating HGSC with a SIK2 / 3 inhibitor after detectable tumor burden in NSG mice, which are missing T, B, and NK cells. The results in Figure 10A show a photoof tumors at day 38 after PPM inoculation and day 28 of daily dosing of YKL-05-099 or vehicle of a representative mouse. The number and size of tumors between YKL-05-099 or vehicle dosed immunodeficient mice are comparable, indicating unchanged tumor burden, in contrast to the photo in Figure 5 A. The results in Figures 10B-C show the corresponding probability of survival and median survival days of mice dosed with YKL-05-099 compared to vehicle, which shows little to no change in both immunodeficient PPM inoculated NSG (Figure 10B) and FVB, CD4", CD8" (Figure IOC) mice. The results in Figures 10D-E show the corresponding in vivo tumor burden measured as geometric mean of the bioluminescence imaging signal total flux in photons per second (sec) which show that YKL-05-099 dosing caused little to no change in tumor burden compared to vehicle in both immunodeficient PPM inoculated NSG (Figure 10D) and FVB, CD4’, CD8‘ (Figure 10E) mice.Example 5: SIK2 / 3 Inhibitors Show Efficacy in Activating T cell Anti-tumor Response at Doses Low Enough to Not Significantly Inhibit T cell Growth or Directly Kill Tumor CellsHuman Serous Carcinoma and HGSC organoid lines

[0070] The organoid lines used in Figure 11 (from patients 17-116, 17-39, and 18-47) are described in S.J. Hill, et al., Cancer Discovery (2018), 8(11): 1404-1421, which is incorporated by reference herein. Co-culture system of HGSC organoid lines and immune cells used in Figure 12 and Tables 4- 6 were derived directly from surgery discard tissues (from patients 1, 2, and 3) as described previously in S.J. Hill, et al., Cancer Discovery (2018), 8(11): 1404-1421 and C. Wan, et al., Cancer Research (2021), 81(1): 158-173, which are incorporated by reference herein.CellTiter-Glo® assays to measure impact of compounds on tumor and T cell growth rate

[0071] Ovarian cancer cells were seeded in 96-well plates at a density of 1500 / well and allowed to adhere overnight. Cells were then dosed with the indicated concentrations of YKL-05-099 or vehicle control for 3 days (SK-OV-3) or 6 days (OVCAR8, CAOV-3) Growth rate was measured by CellTiter-Glo® 2.0 Cell Viability Assay from Promega according to the manufacturer’s protocols. Ovarian cancer organoid cell cultures were dosed with serial dilutions of YKL-05-099 for 5 days before growth rate measurement. For human primary T cell culture, 1500 purified pan-T cells from healthy PBMC donors were suspended in complete RPMI media supplemented with 50 U / mL recombinant human IL-2 and then dosed with the indicated concentrations of YKL-05-099 or vehicle control for 4 days before growth rate measurement.

[0072] Figure 11 shows results quantifying percent growth 5 days post YKL-05-099 exposure at various doses in A) human primary T cells from healthy donors and B) human serous carcinoma cell lines compared to vehicle (DMSO-ctrl). Data show dose responses to SIK2 / 3 inhibition in two healthyhuman donors (Figure 11 A) and three human serous carcinoma cell lines - 0VCAR8, CAOV-3, SK- OV-3 (Figure 11B). Results in Figures 11C-E show growth rate dose responses in HGSC organoid lines derived from human patients. Data points on each graph represent the growth rate of each organoid line at each dose tested (0.005 pM, 0.01 pM, 0.05 pM, 0.1 pM, 1 pM, and 5 pM). Solid lines are drawn to determine the YKL-05-099 dose required to inhibit 50% of cell growth (i.e., GR50), which is approximately 5 pM (Figure 11 C), 2 pM (Figure 1 ID), and 0.5 pM (Figure 1 IE). A dose of 0.625 pM has minimal effect on tumor growth in human ovarian serous carcinoma cell lines. Human primary T cell growth is not significantly affected by a dose of 0.625 pM.

[0073] Results in Figure 12 show TFNy concentrations (pg / ml) measured via ELISA in human patient-derived HGSC organoid / immune cell co-culture supernatants collected 96 hours post exposure with control (DMSO) or 0.1 pM YLK-05-099. Human patients whose tumor samples were used to derive HGSC organoids for the co-culture experiments described in Figure 12 are further characterized in Table 4.Table 4. Characterization of human patients whose tumor samples were used to derive HGSC organoids for the co-culture experiments shown in Figure 12.

[0074] Tables 5 and 6 show corresponding results from flow cytometry experiments of patient 3- derived organoids measuring immune cell, T-cell, and T-cell subpopulations in co-culture supernatants collected 48 hours (Table 5) or 96 hours (Table 6) post exposure at various doses of SIK2 / 3 inhibitor. At both time points, a dose of 0.1 pM YKL-05-099 does not significantly affect the various immune cell population numbers compared to control (Ctrl).Table 5. T cell, CD8+T cell, CD4+T ce 1, and CD45+cell numbers measured by flow cytometry in human patient 3 -derived HGSC organoid / immune cell co-culture supernatants collected 48 hours post exposure with YKL-05-099 or Ctrl.Table 6. T cell, CD8+T cell, CD4+T cell, and CD45+cel numbers measured by flow cytometry in human patient 3-derived HGSC organoid / immune cell co-culture supernatants collected 96 hours post exposure with YKL-05-099 or without (Ctrl).Example 6: SIK2 / 3 Inhibition and anti-PD-1 Treatment Demonstrate In Vivo Synergy in Treating HGSC Models in Immunocompetent MiceEx vivo growing of mouse bone marrow-derived macrophages (BMM)

[0075] Sik2f / f Sik3f / f UBC-Cre-ERT2 mice and their Cre-negative littermate control mice were euthanized, and their bone marrow cells were harvested from the femurs. The bone marrow cells were cultured in DMEM containing 10% FBS, 55 pM 2-mercaptoethanol and 20 ng / ml macrophage colony-stimulating factor (M-CSF) for 7 days to generate BMM (here they were M2-like macrophages due to polarization under M-CSF; while granulocyte macrophage colony-stimulating factor (GM-CSF) is known for Ml -like macrophage differentiation) for flow cytometry analyses of PD-L1 expression. 4-Hydroxytamoxifen (4OHT) was applied at day 3 of cell culture to induce Cre expression for Sik2 Sik3 DKO in the UBC-Cre-ERT2 mice. At day 7, the adherent cells (BMM) werefirst dissociated by gentle trypsinization, and the single-cell suspensions were then stained with antimouse PD-L1 (BioLegend) for flow cytometry.

[0076] Results in Figure 13 show Sik2Sik3 DKO in in vitro cultured mouse immune cells demonstrate an upregulated PD-1 / PD-L1 axis at the protein level. The results in Figures 13A and 13B show increased percentage of CD8+or CD4+T cells, respectively, that express PD-1 protein upon induction of Sik2Sik3 DKO compared to wildtype littermates as measured by flow cytometry. The results in Figure 13C show increased PD-L1 expression at the protein level in in vitro mouse bone marrow-derived macrophages (BMM) upon induction of Sik2Sik3 DKO compared to wildtype littermates as measured by geometric mean fluorescence intensity in flow cytometry. Applicant shows that SIK2 / 3 inhibition reshapes the immune transcriptomic landscape in HGSC ovarian cancer and specifically increases T cell infiltration in vivo.

[0077] The results in Figure 14 show that SIK2 / 3 inhibition and anti -PD-1 treatment demonstrate in vivo synergy in treating HGSC in PPM inoculated immunocompetent FVB mice. Figure 14A shows a photo of tumors dissected at day 38 after PPM inoculation and day 28-31 of daily treatments of YKL-05-099 with (bottom- ‘Comb’) and without (top- ‘YKL-05-099’) twice weekly treatment of anti -PD-1 of a representative mouse. The number of tumors for the mouse receiving the twice weekly treatment of anti-PD-1 is much smaller than for the mouse not receiving twice weekly treatment of anti-PD-1. The results in Figures 14B-C show corresponding mouse tumor decreases as PPM tumor weight in grams (Figure 14B) and percentage of tumor weight to body weight (Figure 14C) on day 38. The results in Figure 14D show weight changes at day 38 for mice receiving the twice weekly treatment of anti-PD-1 and those that did not. The results in Figure 14E show the corresponding decrease in probability of survival and median survival days of mice treated with and without twice weekly treatment of anti-PD-1. Figure 14E also shows comparisons with a vehicle replacing YKL- 05-099 treatment with (Comb) and without (YKL-05-099) twice weekly treatment of anti-PD-1, which shows little improvement between vehicle only and anti-PD-1 only treatment. Further, although a significant improvement is shown between vehicle-only and YKL-05-099-only treatment, a significant improvement is also shown between YKL-05-099-only treatment and YKL-05-099 treatment combined with anti-PD-1 treatment. This result is greater than additive for YKL-05-099 only treatment plus anti-PD-1 only treatment. The results in Figure 14F show corresponding in vivo tumor burden measured as a geometric mean of the bioluminescence imaging signal total flux in photons per second (sec).

[0078] The results in Figure 15 show that SIK2 / 3 inhibition and anti-PD-1 treatment demonstrate in vivo efficacy in treating HGSC in BeCon inoculated immunocompetent B6 mice. Treatment started on day 7 after BeCon inoculation. The results of Figures 15 A and 15C show that daily YKL-05-099 treatment compared to vehicle (Ctrl) leads to a decrease in ascites visually in photos of mice (Figure 15 A) and measured as weight in grams (Figure 15C) on day 27 and that combination of YKL-05-099 with anti-PD-1 treatment every three days (Comb) leads to a further decrease. The results of Figures 15B and 15D show that YKL-05-099 treatment compared to vehicle leads to a decrease in spleen size visually in photos of the spleen (Figure 15B) and measured as weight in grams (Figure 15D) on day 27 and that combination of YKL-05-099 with anti-PD-1 treatment every three days lead to a further decrease. Although Figure 15 shows a significant improvement between vehicle-only and YKL-05- 099-only treatment, a significant improvement is also shown between YKL-05-099-only treatment and YKL-05-099 treatment combined with anti-PD-1 treatment. This result is greater than additive compared to YKL-05-099 only treatment plus anti-PD-1 only treatment since the BeCon model is known to be resistant to treatment by anti-PD-1. Per C-S Chae et al., Cancer Discov (2022), 12(8): 1904-1921, anti-PD-1 only treatment of BeCon / B6 mice “did not improve the observed therapeutic benefit” (see their Figure 7A). Note that by day 27, some vehicle dosed mice did not survive (see Figure 71) and that the divergence between vehicle, YKL-05-099 only treated, and YKL-05-099 combined with anti-PD-1 treated would be expected to increase beyond day 27. The observed efficacy from our combination SIK2 / 3 inhibitor and anti-PD-1 treated BeCon / B6 mice is synergistic.Example 7: SIK2 / 3 Inhibitors Activate T cell Anti-Tumor Function In Vitro and SIK2 / 3 Inhibitor ARN-3236 Shows Efficacy in Treating Models of HGSC in Immunocompetent MiceEx vivo activation of primary mouse T cells

[0079] Purified pan-T cells from C57BL6 mouse spleens were enriched by MACS pan-T cell isolation kit (Miltenyi) and activated with anti-CD3 / CD28 antibodies for 3 days in complete RPMI medium with recombinant mouse IL -2 (100 ng / ml) for ex vivo T cell experiments. After 3 days, 0.5- 5 pM of YKL-05-099, ARN-3236, or SK-124 (from M. Wein, MGH, see Table 1) were added to the culture for 20 hours and dimethyl sulfoxide (DMSO) was used as the control. This was followed by 3-4 hours treatment with IX protein transport inhibitor cocktail (eBioscience). Cells were then subjected to flow cytometry analysis. For IFNy staining, T cells were fixed and permeabilized using BD Fix / Perm and BD Perm / Wash buffer, respectively.

[0080] The results in Figures 16A-B show that SIK inhibitors ARN-3236 and SK-124, in addition to YKL-05-099, cause increased IFNy concentration in mouse splenic primary T cells in vitrocompared to DMSO as measured via ELISA. The results in Figures 16A and 16B show the same result analyzed as a percentage of T cells that produce IFNy and the amount of IFNy produced per IFNy-producing T cell, respectively. Figure 16B shows the same experiments but measures geometric mean fluorescence intensity via flow cytometry. Data are representative of three independent experiments at different concentrations of SIK2 / 3 inhibitor (5 pM, 1 pM, 0.5 pM).

[0081] The schematic shown in Figure 4 shows the experimental setup for the mouse experiments in Figure 16C. The results in Figure 16C show the corresponding in vivo tumor burden measurements as geometric mean of the bioluminescence imaging signal total flux in photons per second (sec) at day 21 after PPM inoculation and day 10 of daily dosing of 6 mg / kg ARN-3236 or vehicle (DMSO). These results show that ARN-3236 dosing decreases tumor burden compared to vehicle. YKL-05- 099 and ARN-3236 at 6 mg / kg were well tolerated in both the PPM / FVB and BeCon / B6 mice. Histopathology of FVB and B6 mice that received 6 mg / kg YKL-05-099 revealed no abnormalities in major organs or any evidence of toxicity.Example 8: SIK2 / 3 inhibition with T cell adoptive transfer reduces HGSC tumor burdenAdoptive Transfer Models

[0082] BeCon cells, as above, were transduced with the ovalbumin (OVA) antigen, which is recognized by TCR transgenic OT1 cells [Y. Shinkai et al., Cell (1992) 68(5): 855-67, K. Hogquist et al., Cell (1994) 76(1): 17-27], OT1 CD8 T cells were harvested from the spleen of transgenic OT- I mice from Taconic Biosciences (Model No. 2334-F). Introduction of OVA antigen on BeCon cells was confirmed by a zsGreen-OVA reporter, as in Y. Ito, et al., Cancer Discov. (2023), 13(5): 1186- 1209. For in vitro experiments, the BeCon-OVA cells were cultured overnight in the presence or absence of 0.1 pM YKL-05-099, and with or without 0.1 pM antigen-specific OT1 cells. In vitro BeCon-OVA tumor cell apoptosis was measured by flow cytometry Annexin V assay, as described in Biolegend Annexin V Apoptosis Detection Kit (catalog # 640914). For in vivo experiments, wildtype B6 mice were inoculated with BeCon-OVA in the presence or absence of OT1 cell transfer (2 million per mouse) the day before and 6 mg / mL ARN-3236 daily starting at day 7.

[0083] The results in Figure 17A show in vitro BeCon-OVA tumor cell apoptosis measured by flow cytometry, where data are representative of two independent experiments and n = 6 technical replicates. 0.1 pM YKL-05-099 did not induce apoptosis of BeCon-OVA cells in culture, but it significantly enhanced OT1 cell-mediated cytotoxicity. The schematic in Figure 8 shows the experimental setup one day after OT1 cell transfer for the mouse experiments shown in Figures 17B- D. The results in Figures 17B-C show flow cytometry analyses of peripheral blood from the vehicleor ARN-3236 dosed mice, with n = 4 or 5 mice per group. Figure 17B shows the increase in OT1 cells within total immune cells (CD45+cells) from peripheral blood samples on day 20 post tumor inoculation of mice dosed with ARN-3236 compared to vehicle. Figure 17C shows the increase in OT1 cells within cytotoxic T cells (CD8+cells) from peripheral blood samples on day 20 post tumor inoculation of mice dosed with ARN-3236 compared to vehicle. The results in Figure 17D show the corresponding increase in probability of survival and median survival days of mice, comparing vehicle (n = 8), ARN-3236 (n = 4), OT1 cell transfer (n =4), and ARN-3236 with OT1 (n = 5). OT1 cell transfer only significantly extended median survival of BeCon-OVA-bearing mice, though not as much as SIK2 / 3 inhibitor only. Further, combination of OT1 cell transfer with SIK2 / 3 inhibitor led to prolonged survival with a median of 75 days. These data indicate that SIK2 / 3 inhibition together with T cell adoptive transfer reduces tumor burden.Example 9: SIK2 / 3 Inhibition or SIK2 / 3 DKO Activates the T cell microenvironment by Reprograming Dendritic CellsEx vivo growing of mouse bone marrow-derived dendritic cells (BMDCs)

[0084] Sik2f / f Sik3f / f UBC-Cre-ERT2 mice and their Cre-negative littermate control mice were euthanized, and their bone marrow cells were harvested from the femurs. The bone marrow cells were cultured in complete DMEM supplemented with 20 ng / ml GM-CSF and IL-4 for 7 days, and then BMDCs were purified from suspended cells by CDl lc enrichment using magnetic beads. 4OHT at 0.3 iiM final concentration was introduced at day 3 of cell culture to induce Cre expression for Sik2 Sik3 double KO in the UBC-Cre-ERT2 samples. The single-cell suspensions of BMDCs were either stained for surface activation markers for flow cytometry or pulsed with OVA antigen and co-cultured with carboxyfluorescein succinimidyl ester (CFSE) pre-labeled OT1 T cells, made per manufacturer’s protocol (Thermo Fisher Scientific Cat. No. C34554, C34570), for functional readouts in antigen processing / presentation.

[0085] Figure 18A shows pathway enrichment analysis of differentially expressed genes in an ascites dendritic cell (DC) population from BeCon / B6 mice with 6 mg / kg YKL-05-099 or DMSO, as in Figure 8. A total of 1078 differentially expressed genes were measured in the ascites DCs, with 460 being upregulated and 618 downregulated. DC genes associated with antigen processing and presentation had the highest upregulated differential expression gene ratio [analysis as in J. Reimand et al., Nat Protoc. (2019), 14(2): 482-517], scRNA-seq analyses show that SIK2 / 3 inhibition did not significantly increase the percentage of tumor-infiltrated DCs (10-20% in the total CD45+population) in the TME of BeCon / B6 mice. Figures 18B-D show quantifications of flow cytometry measurementsof the upregulated activation markers CD80 and CD86 in Sik2Sik3KO BMDCs and non-induced wildtype littermate BDMCs. Data are representative of three independent experiments. Figures 18B and 18C show the geometric mean fluorescence intensity measuring CD80 and CD86 expression, respectively, and Figure 18D shows the percentages of activated DCs that expressed both CD80 and CD86. Sik2Sik3KO BMDCs expressed significantly higher levels of activation markers CD80 and CD86 compared to the littermate control / wildtype BMDCs (Figures 18B-D). Figure 18E shows quantifications of DC functional assays measuring OT1 cell proliferation by flow cytometry, where full-length OVA protein was applied at the indicated doses and Sik2Sik3KO versus wildtype BMDCs were compared. Data are representative of two independent experiments. Sik2Sik3 DKO BMDCs were more active in triggering antigen-dependent T cell proliferation than wildtype BMDCs (Figure 18E). T cell priming via professional antigen-presenting cells such as DCs is critical in establishing potent T cell responses. These data indicate that SIK2 / 3 inhibition or Sik2Sik3 DKO in DCs can enhance antigen presentation, suggesting SIK2 / 3 inhibition or Sik2Sik3 DKO can establish a potent T cell response by improving DC antigen presentation.

Claims

CLAIMSWhat is claimed:

1. A method of treating high-grade serous carcinoma (HGSC) in an immunocompetent patient in need thereof, the method comprising administering to the immunocompetent patient an effective amount of a SIK2 / 3 inhibitor and an immune checkpoint inhibitor.

2. The method of claim 1 wherein the immune checkpoint inhibitor is an anti-PD-1, anti-PD-Ll, or anti-CTLA4 antibody.

3. The method of claim 2 wherein the antibody is an anti-PD-1 antibody.

4. The method of claim 3 wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, and dostarlimab.

5. A method of treating HGSC in an immunocompetent patient in need thereof, the method comprising administering to the patient an effective amount of a SIK2 / 3 inhibitor or a pharmaceutically acceptable salt thereof, wherein the effective amount is lower than the amount needed to directly kill the tumor cells.

6. The method of claim 5 wherein the method further comprises administering to the patient an adoptive cellular therapy.

7. The method of claims 1, 2, 3, 4, 5, or 6 wherein the SIK2 / 3 inhibitor is selected from the groupBosutinib, Crenolanib, Dasatinib,8. The method of claim 7 wherein the SIK2 / 3 inhibitor is selected from the group consisting of:pharmaceutically acceptable salt thereof.

10. A method of sensitizing ovarian cancer cells in an immunocompetent patient via the surrounding tumor microenvironment to treatment by an immune checkpoint inhibitor comprising administering a SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof.

11. The method of claim 10 wherein the tumor microenvironment in an immunocompetent patient comprises ascites.

12. The method of claim 10 or 11, further comprising administering an immune checkpoint inhibitor.

13. A SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof, and an immune checkpoint inhibitor for use in treating HGSC in an immunocompetent patient in need thereof.

14. The use of claim 13, wherein the immune checkpoint inhibitor is an anti-PD-1, anti-PD-Ll, or anti-CTLA4 antibody.

15. The use of claim 14, wherein the antibody is an anti-PD-1 antibody.

16. The use of claim 15, wherein the anti-PD-1 antibody is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, and dostarlimab.

17. A SIK2 / 3 inhibitor, or a pharmaceutically acceptable salt thereof, for use in the treatment of HGSC in an immunocompetent patient in need thereof, wherein an effective amount of the SIK2 / 3 inhibitor is lower than the amount needed to directly kill the tumor cells.

18. The use of claim 17, where the treatment further comprises an adoptive cellular therapy.

19. The use of claims 13-17 wherein the SIK2 / 3 inhibitor

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