Methods of treating liposarcoma using a pyrazole compound

Abstract

Provided herein are methods of treating, preventing, or ameliorating one or more symptoms of liposarcoma using a pyrazole having the formula (I).

Description

METHODS OF TREATING LIPOSARCOMA USING A PYRAZOLE COMPOUNDPRIORITY

[0001] The application claims priority to U.S. Provisional Application No. 63 / 568,085, filed March 21, 2024, which is incorporated by reference in its entirety.FIELD

[0002] This disclosure relates to methods of treating, preventing, or ameliorating one or more symptoms of liposarcoma using a pyrazole compound.BACKGROUND

[0003] The tumor suppressor, p53, is a transcription factor that induces cell cycle arrest and activates apoptotic cell death through target gene activation in response to DNA damage and cellular stress. (Fields S, Jang SK, Presence of apotent transcription activating sequence in the p53 protein, Science. Aug 31 1990; 249(4972): 1046-9; doi: 10.1126 / science.2144363; Kastenhuber ER, Lowe SW. Putting p53 in Context. Cell. Sep 7 2017; 170(6): 1062-1078. doi: 10.1016 / j.cell.2017.08.028.) While p53 is mutated in about half of solid tumors, cancers that have a wild type p53 must adopt other mechanisms to inhibit p53 activity.

[0004] Casein kinases are serine / threonine kinases that phosphorylate proteins to mediate normal biological functions and malignant transformation. (Schittek et al., Mol. Cancer 2014, 13, 231-245.) Casein kinase 1 alpha (CKla) functions as a tumor inducer in several cancers through negative regulation of Wnt / p-catenin signaling and p53. (Ebert & Krbnke, N. Engl. J. Med. 2018, 379, 1873-1874.) CKla phosphorylates [3-catenin at serine 45, leading to ubiquitination and degradation of P-catenin. (Schittek et al., Mol. Cancer 2014, 13, 231-245; Ebert & Krbnke, N. Engl. J. Med. 2018, 379, 1873-1874; Elyada et al., Nature 2011, 470, 409-413.) CKla also phosphorylates murine double minute X (MDMX) at serine 289, resulting in enhanced binding of MDMX to p53. (Wu et al., Mol. Cell. Biol. 2012, 32, 4821-4832.) Additionally, a complex of CKla and mouse double minute 2 homolog (MDM2) inhibits p53. (Elyada et al., Nature 2011, 470, 409-413.) Thus, enhanced inhibition of CKla with subsequent p53 activation has the potential to be effective in treating a wide array of cancers.

[0005] Super-enhancers (SEs) are large clusters of transcriptional enhancers that normally drive expression of genes that define cell identity. (Hnisz et al., Cell 2013 155(4): 934-947.) Tumor cells acquire SEs at key oncogenes like c-Myc and at genes that produce the hallmark features of cancer. (Hnisz et al. 2013.) Given the dependency on high levels of gene expression, tumor cells are particularly sensitive to disturbances in transcription. Transcriptional kinases are essential components of the transcription apparatus. In particular, cyclin dependent kinase 7 (CDK7) phosphorylates the carboxy terminal domain (CTD) of ribonucleic acid polymerase II (RNA Pol II) at Ser5 and Ser7, enabling transcriptional initiation, and CDK9 phosphorylates RNA Pol II CTD at Ser2 to facilitate transcription elongation. (Ferguson and Gray, Nat. Rev. Drug. Discov., 2018 17(5): 353-377.)

[0006] The dual mechanism of action of preventing the transcription of key oncogenic genes via the inhibition of CDK7 and CDK9 and activating p53 via inhibition of CKla may result in tumor responses in patients with a wide variety of tumor types.

[0007] MDM2 is a negative regulator of p53 that promotes p53 ubiquitination and subsequent degradation. (Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of p53. Nature. 1997 / 05 / 01 1997;387(6630):296-299. doi:10.1038 / 387296a0.) One mechanism for inhibiting wild type p53 is to amplify the gene expression of MDM2 and thus increase the turnover of p53. Mutations that result in increased expression of MDM2 may be in transcription factors or DNA sequences, e.g., selecting for changes in promoter or enhancer function that increase MDM2 transcription. Tumors that utilize Super Enhancer- mediated transcription have been characterized as “transcriptionally addicted” due to their need for high rates of expression of proteins with short half-lives, such as MDM2. SE- mediated transcription regulates MDM2 expression in some tumors.

[0008] Another mechanism that increases MDM2 is amplification of its gene copy number. In liposarcoma, MDM2 gene amplification is observed in almost all patients and activates p53-independent growth and stress response genes. (Bevill SM, Casani-Galdon S, El Farran CA, et al. Impact of supraphysiologic MDM2 expression on chromatin networks and therapeutic responses in sarcoma. Cell Genom. Jul 12 2023;3(7): 100321. doi: 10.1016 / j.xgen.2023.10032). Furthermore, a higher copy number of MDM2, e.g., >8, is associated with decreased survival rates.

[0009] Multiple biopharmaceutical companies have developed MDM2 inhibitors. For example, Rain Oncology tested milademetan in MDM2-amplified solid tumors (clinicaltrials.gov NCT05012397). This multicenter, single-arm, open-label basket study was designed to evaluate the safety and efficacy of milademetan in patients with advanced or metastatic solid tumors refractory or intolerant to standard-of-care therapy that exhibit wildtype (WT) TP53 and MDM2 copy number (CN) > 8 using prespecified biomarker criteria. Interim results for this study showed intriguing anti-tumor activity. Boehringer-Ingelheim presented data at the American Society of Oncology meeting in 2023 for its MDM2 inhibitor, BI-907828 also known as brigimadlin, in patients with biliary cancer. [Yamamoto et al, J Clin Oncol 2023 41:4_suppl, 543], About 5-8% of biliary cancers have amplified MDM2. In two parallel studies, brigimadlin was either given as monotherapy, 45 mg every 3 weeks, or at lower doses in combination with an anti-PDl antibody. In addition, brigimadlin has been evaluated in an MDM2-amplified basket study of solid tumors. It demonstrated clinical activity in both well-differentiated liposarcoma and dedifferentiated liposarcoma (LoRusso et al, The MDM2-p53 Antagonist Brigimadlin (BI 907828) in Patients with Advanced or Metastatic Solid Tumors: Results of a Phase la, First-in-Human, Dose-Escalation Study. Cancer Disc. 2023; 13: 1802-13 doi: 10.1158 / 2159-8290.CD-23-0153).

[0010] Despite intriguing clinical activity, direct MDM2 inhibitors have proven to be challenging to develop due to hematologic toxicity. For example, milademetan was evaluated at various doses and regimens to obviate undue toxicity. A phase 3 study in liposarcoma was conducted using the optimized regimen (260 mg for 3 consecutive days followed by 11 days off treatment; cycles repeated every 14 days). [NCT04979442.] This randomized, multicenter, open-label study was designed to evaluate the safety and efficacy of milademetan compared to trabectedin in patients with unresectable or metastatic dedifferentiated liposarcoma that had progressed on 1 or more prior systemic therapies. The study was terminated early. A significant contributing factor to the lack of efficacy was the high rate of hematologic toxicity which led to treatment interruptions and patient discontinuations. Rates of grade 3 / 4 hematologic toxicity were significant and reported as follows: 39.5% of patients with thrombocytopenia; 25.5% of patients with neutropenia; and 18.6% of patients with anemia.

[0011] Similarly, brigimadlin is dosed infrequently with a long treatment holiday in an attempt to overcome toxicity. As monotherapy, 45 mg is administered only on the first dayof a 21-day cycle. (Macarulla et al., Efficacy and safety of brigimadlin (BI 907828), an MDM2- -p53 antagonist, in patients (pts) with advanced biliary tract cancer: Data from two phase la / Ib dose-escalation / expansion trials, Journal of Clinical Oncology Volume 42, Number 3_supp). Early results for this agent reported the following rates of Grade 3 / 4 hematologic toxicity: 19% thrombocytopenia; 20% neutropenia; 10% anemia. While these data appear to be improved compared to milademetan, there remains a need to provide better tolerated agents that can achieve continuous pharmacologic coverage of the drug target.

[0012] Sirimadlin (HDM 201) is a direct MDM2 inhibitor that was evaluated in 115 solid tumor patients and 93 patients with hematologic malignancies. [Stein et al Clin Cancer Res 2022;28:870-81 doi: 10.1158 / 1078-0432.CCR-21-1295] Grade 3 / 4 Adverse Events (AEs) suspected to be treatment related were observed in 52 (45%) patients with solid tumors and 66 (71%) patients with hematologic tumors. Hematologic toxicities were the most common grade 3 / 4 AE for all indications and were more frequent in patients with hematologic tumors compared with those with solid tumors. A total of 50 patients (43.5%) with solid tumors and 38 patients (40.9%) with hematologic tumors had at least one AE leading to dose adjustment or interruption. Importantly, researchers concluded that thrombocytopenia is an on-target effect of MDM2 inhibition.

[0013] Therefore, despite promising anti-tumor activity of targeted MDM2 inhibitors, there remains a pressing need for agents that inhibit MDM2 by a mechanism that does not cause unacceptable Grade 3 / 4 toxicity, particularly hematologic toxicities including thrombocytopenia. In addition, there remains a need for agents that can be dosed continuously rather than with long holiday periods, such as 1, 2 or 3 weeks between doses. A need exists for a better tolerated agent with more frequent dosing that provides more uniform exposure and improved pharmacology.

[0014] Liposarcomas are a group of soft tissue sarcomas of adipocytic origin. They include well differentiated, dedifferentiated, myxoid / round cell, and pleomorphic subtypes. Collectively, they account for 10-20% of all soft tissue sarcomas. The well differentiated / de- differentiated subtype is characterized by MDM2 amplification, usually with co-amplification of CDK4 on chromosome 12ql 3- 15. They are almost exclusively TP53 wild type. (Somaiah N, Beird HC, Barbo A, et al., Targeted next generation sequencing of well- differentiated / dedifferentiated liposarcoma reveals novel gene amplifications and mutations, Oncotarget. Apr 13 2018; 9(28): 19891 - 19899; doi: 10.18632 / oncotarget.24924.) Althoughthey can arise anywhere in the body, they typically arise in the retroperitoneum. Well differentiated liposarcoma is generally a lower-grade disease, albeit one that can be difficult to achieve local control, and is generally managed surgically. De-differentiated liposarcoma has a higher metastatic potential and, when metastatic, is treated with systemic therapies. Despite the use of cytotoxic chemotherapies like doxorubicin and gemcitabine-based regimens, outcomes for patients with metastatic dedifferentiated liposarcoma are abysmal, with 5-year survival rate only 12.1%. (Lin F, Duan J, Lin Y, et al., Survival and risk factors in patients with liposarcoma with distant metastasis, Am J Transl Res. 2020;12(5):2071- 2082.). Management of metastatic or surgically unresectable well differentiated or dedifferentiated LPS remains purely palliative. More recently, additional chemotherapeutic agents such as trabectedin and eribulin have been approved for treatment of advanced liposarcoma, and drugs targeting CDK4 / 6 have demonstrated activity and are actively being studied but none are currently used as a standard or care treatment (e.g. NCT04967521). (Demetri GD, von Mehren M, Jones RL, et al., Efficacy and Safety of Trabectedin or Dacarbazine for Metastatic Liposarcoma or Leiomyosarcoma After Failure of Conventional Chemotherapy: Results of a Phase III Randomized Multicenter Clinical Trial, J Clin Oncol. Mar 10 2016;34(8):786-93; doi: 10.1200 / jco.2015.62.4734; Schoffski P, Chawla S, Maki RG, et al., Eribulin versus dacarbazine in previously treated patients with advanced liposarcoma or leiomyosarcoma: a randomised, open-label, multicentre, phase 3 trial, The Lancet. 2016;387(10028): 1629-1637; doi: 10.1016 / S0140-6736(l 5)01283-0.)

[0015] Therefore, in view of the difficulty of treating liposarcoma and the poor long-term outcomes, a need exists for a treatment that suppresses oncogenes, causes tumor regression, and improves outcomes for patients with liposarcoma.SUMMARY

[0016] In one aspect, this disclosure provides methods of method of treating liposarcoma in a subject, comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of Compound A22,or a pharmaceutically acceptable salt thereof.

[0017] In some embodiments, the liposarcoma is well differentiated. In some embodiments, the liposarcoma is de-differentiated.

[0018] In some embodiments, the patient has TP53wt. In some embodiments, the subject over-expresses MDM2 or has a >4 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

[0019] In some embodiments, the liposarcoma is refractory. In some embodiments, the liposarcoma is metastatic. In some embodiments, the liposarcoma is drug-resistant.

[0020] In some embodiments, the subject has failed a prior therapy. In some embodiments, the subject is a human.

[0021] In some embodiments, the compound is administered orally. In some embodiments, the compound is administered as a tablet or capsule. In some embodiments, the therapeutically effective amount ranges from about 0.001 to about 10 mg / kg per day. In some embodiments, the therapeutically effective amount ranges from about 15 to about 35 mg per day. In some embodiments, the therapeutically effective amount is about 1, about 2, about 3, about 5, about 8, about 10, about 11, about 14, about 15, about 17, about 20, about 21, about 25, about 30, or about 35 mg per day.

[0022] In some embodiments, the compound is administered in one or more cycles. In some embodiments, the compound is administered in a 28-cycle. In some embodiments, the compound is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days per week. In some embodiments, the compound is administered for 3 days or 5 days per week. In some embodiments, the compound is administered on Days 1, 2, and 3 in a week. In some embodiments, the compound is administered on Days 1, 3, and 5 in a week. In some embodiments, the compound is administered on three non-consecutive days in a week. In some embodiments, the compound is administered in a 28-day cycle for 3 days per week. In some embodiments, the compound is administered on three non-consecutive days per week. In some embodiments, the compound is administered on Days 1, 3, and 5 per week. In some embodiments, the compound is administered in a 28-day cycle for 5 days per week. In some embodiments, the compound is administered in a 28-day cycle on Days 1, 2, 3, 4, and 5 per week.

[0023] In some embodiments, Compound A22 is formulated as a p-toluenesulfonate salt. In some embodiments, Compound A22 is formulated as a di-p-toluenesulfonate salt. In some embodiments, the therapeutically effective amount is about 21 mg. In certain embodiments, the therapeutically effective amount is about 30 mg. In still other embodiments, the therapeutically effective amount is about 35 mg.

[0024] In another aspect, this disclosure provides methods of inhibiting CDK7 in a subject having liposarcoma, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of Compound A22, or a pharmaceutically acceptable salt thereof.

[0025] In some embodiments, the liposarcoma is well differentiated. In some embodiments, the liposarcoma is de-differentiated.

[0026] In some embodiments, Compound A22 is formulated as a -toluenesulfonate salt. In some embodiments, Compound A22 is formulated as a di- -toluenesulfonate salt. In some embodiments, the therapeutically effective amount is about 21 mg. In certain embodiments, the therapeutically effective amount is about 30 mg. In still other embodiments, the therapeutically effective amount is about 35 mg.

[0027] In some embodiments, the liposarcoma has been resistant to a CDK4 / 6 inhibitor.

[0028] In some embodiments, the subject has TP53wt.

[0029] In some embodiments, the subject over-expresses MDM2 or has a >4 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

[0030] In some embodiments, administering comprises administering about 21 mg of Compound A22 three times per week. In other embodiments, administering comprises administering about 30 mg of Compound A22 three times per week. In still other embodiments, administering comprises administering about 35 mg of Compound A22 three times per week.

[0031] In a further aspect, this disclosure provides methods of treating liposarcoma in a subject having amplified expression of MDM2 or having a >4 copy number of the MDM2 gene, comprising administering to the subject a therapeutically effective amount of Compound A22, or a pharmaceutically acceptable salt thereof.

[0032] In some embodiments, the liposarcoma is well differentiated. In some embodiments, the liposarcoma is de-differentiated.

[0033] In some embodiments, Compound A22 is formulated as a -toluenesulfonate salt. In some embodiments, Compound A22 is formulated as a di- -toluenesulfonate salt. In some embodiments, the therapeutically effective amount is about 21 mg. In certain embodiments, the therapeutically effective amount is about 30 mg. In still other embodiments, the therapeutically effective amount is about 35 mg.

[0034] In some embodiments, the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

[0035] In some embodiments, administering comprises administering about 21 mg of Compound A22 three times per week. In some embodiments, administering comprises administering about 30 mg of Compound A22 three times per week. In some embodiments, administering comprises administering about 35 mg of Compound A22 three times per week.

[0036] In another aspect, this disclosure provides methods of inhibiting MDM2 expression in a subject having liposarcoma, comprising administering to the subject an effective amount of Compound A22, or a pharmaceutically acceptable salt thereof.

[0037] In some embodiments, the liposarcoma is well differentiated. In some embodiments, the liposarcoma is de-differentiated.

[0038] In some embodiments, Compound A22 is formulated as a -toluenesulfonate salt. In some embodiments, Compound A22 is formulated as a di- -toluenesulfonate salt. In some embodiments, the effective amount is about 21 mg. In certain embodiments, the effective amount is about 30 mg. In still other embodiments, the effective amount is about 35 mg.

[0039] In some embodiments, the subject over-expresses MDM2 or has a >4 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

[0040] In some embodiments, administering comprises administering about 21 mg of Compound A22 three times per week. In other embodiments, administering comprises administering about 30 mg of Compound A22 three times per week. In further embodiments, administering comprises administering about 35 mg of Compound A22 three times per week.

[0041] In another aspect, this disclosure provides methods of inhibiting CDK9 in a subject having liposarcoma, comprising administering to the subject an effective amount of Compound A22, or a pharmaceutically acceptable salt thereof.

[0042] In some embodiments, the liposarcoma is well differentiated. In some embodiments, the liposarcoma is de-differentiated.

[0043] In some embodiments, Compound A22 is formulated as a -toluenesulfonate salt.In some embodiments, Compound A22 is formulated as a di- -toluenesulfonate salt. In some embodiments, the effective amount is about 21 mg. In certain embodiments, the effective amount is about 30 mg. In still other embodiments, the effective amount is about 35 mg.

[0044] In some embodiments, the liposarcoma has been resistant to a CDK4 / 6 inhibitor. In some embodiments, the subject has P53wt.

[0045] In some embodiments, the subject over-expresses MDM2 or has a >4 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

[0046] In some embodiments, administering comprises administering about 21 mg of Compound A22 three times per week. In further embodiments, administering comprises administering about 30 mg of Compound A22 three times per week. In other embodiments, administering comprises administering about 35 mg of Compound A22 three times per week.

[0047] In another aspect, this disclosure provides methods of inhibiting CKla in a subject having liposarcoma, comprising administering to the subject an effective amount of Compound A22, or a pharmaceutically acceptable salt thereof. In a further aspect, this disclosure provides methods of inhibiting CKla and CDK9 in a subject having liposarcoma, comprising administering to the subject Compound A22, or a pharmaceutically acceptable salt thereof.

[0048] In some embodiments, Compound A22 is formulated as a -toluenesulfonate salt.In some embodiments, Compound A22 is formulated as a di- -toluenesulfonate salt. In some embodiments, the effective amount is about 21 mg. In certain embodiments, the effective amount is about 30 mg. In still other embodiments, the effective amount is about 35 mg.

[0049] In some embodiments, the liposarcoma has been resistant to a CDK4 / 6 inhibitor. In some embodiments, the subject has P53wt.

[0050] In some embodiments, the subject over-expresses MDM2 or has a >4 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

[0051] In some embodiments, administering comprises administering about 21 mg of Compound A22 three times per week. In further embodiments, administering comprises administering about 30 mg of Compound A22 three times per week. In other embodiments, administering comprises administering about 35 mg of Compound A22 three times per week.

[0052] In yet another aspect, this disclosure provides a pharmaceutical composition comprising compound A22,

[0053] or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, for use in the treatment of a patient diagnosed with liposarcoma.

[0054] In some embodiments, the patient has TP53wt. In some embodiments, the treatment comprises administration of the pharmaceutical composition to the patient on three non-consecutive days in a week. In some embodiments, administration occurs for one or more cycles. In some embodiments, a cycle is 28 days. In some embodiments, the pharmaceutical composition is orally administered to the patient. In some embodiments, the pharmaceutical composition is formulated as an oral unit dosage form.

[0055] In some embodiments, the oral unit dosage form comprises about 21 mg of Compound A22. In other embodiments, the oral unit dosage form comprises about 30 mg of Compound A22. In further embodiments, the oral unit dosage form comprises about 35 mg of Compound A22.

[0056] In yet another aspect, this disclosure provides a method of inducing apoptosis in a liposarcoma cancer cell, comprising contacting the cell with an effective amount of Compound A22, or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture oftwo or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.BRIEF DESCRIPTION OF THE FIGURES

[0057] FIG. 1 shows the results of ranking of genes according to average dependency scores in liposarcoma cell lines.

[0058] FIG. 2 shows that MDM2 and CDK4. CSNK1A1 was identified as LPS-specific dependents.

[0059] FIG. 3 shows the results of siRNA-mediated knockdown of CSNK1A1 performed in LPS cell lines.

[0060] FIG. 4 shows that CKla depletion results in an increase in p53 expression and PARP cleavage.

[0061] FIG. 5 shows the results of a panel of LPS cell lines was treated with Compound A22.

[0062] FIG. 6 shows the results of treatment cell lines (LP6, LPS141, LPS853, and T778) with Compound A22 significantly reduced cell viability in the majority of the cell lines (LP6, LPS141, and T778) with IC50 less than lOOnM.

[0063] FIG. 7 shows the inhibitory effects of Compound A22 on multiple biomarkers.

[0064] FIG. 8 shows that Compound A22 induced apoptosis in LP6 cells in a dosedependent manner as reflected by Annexin V staining.

[0065] FIG. 9 shows the results of LPS3 and LPS27 liposarcoma PDX mouse models treated with Compound A22.

[0066] FIG. 10 shows the results of LPS3 and LPS27 liposarcoma PDX mouse models treated with Compound A22.

[0067] FIG. 11 shows that Compound A22 was well-tolerated and did not affect body weight between the two mice treatment groups.

[0068] FIG. 12 depicts data showing siRNA-mediated knockdown of CSNK1 Al reduced the IC50 of all three CDK9 inhibitors in cell viability assays.

[0069] FIG. 13. depicts data showing that CKla depletion significantly potentiated the effects of suboptimal doses of three CDK9 inhibitors on cell survival.DETAILED DESCRIPTION

[0070] To facilitate understanding of the disclosure set forth herein, a number of terms are defined below.

[0071] Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, biochemistry, biology, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, or context dictates otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0072] The term “subject” refers to an animal, including, but not limited to, a primate (e.g., human), cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms “subject” and “patient” are used interchangeably herein in reference, for example, to a mammalian subject, such as a human subject. In one embodiment, the subject is a human.

[0073] The terms “treat,” “treating,” and “treatment” are include alleviating, improving, inhibiting the progression of, or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating, improving, inhibiting the progression of, or eradicating the cause(s) of the disorder, disease, or condition itself. Treating includes curing, improving, or at least partially ameliorating the disorder, disease, or condition.

[0074] The terms “prevent,” “preventing,” and “prevention” are meant to include a method of delaying and / or precluding the onset of a disorder, disease, or condition, and / or its attendant symptoms; barring a subject from acquiring a disorder, disease, or condition; or reducing a subject’s risk of acquiring a disorder, disease, or condition.

[0075] The terms “alleviate” and “alleviating” refer to easing or reducing one or more symptoms (e.g., pain) of a disorder, disease, or condition. The terms can also refer toreducing adverse effects associated with an active ingredient. Sometimes, the beneficial effects that a subject derives from a prophylactic or therapeutic agent do not result in a cure of the disorder, disease, or condition.

[0076] The term “contacting” or “contact” is meant to refer to bringing together of a therapeutic agent and cell or tissue such that a physiological and / or chemical effect takes place as a result of such contact. Contacting can take place in vitro, ex vivo, or in vivo. In one embodiment, a therapeutic agent is contacted with a cell in cell culture (in vitro to determine the effect of the therapeutic agent on the cell. In another embodiment, the contacting of a therapeutic agent with a cell or tissue includes the administration of a therapeutic agent to a subject having the cell or tissue to be contacted.

[0077] The term “therapeutically effective amount” or “effective amount” is meant to include the amount of a compound that, when administered, is sufficient to treat, prevent development of, or alleviate a disorder, disease, or condition, or one or more of the symptoms of the disorder, disease, or condition being treated. In some contexts, the term “therapeutically effective amount” or “effective amount” also refers to the amount of a compound that is sufficient to elicit a biological or medical response of a biological molecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician.

[0078] The term “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of a subject (e.g., a human or an animal) without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Remington: The Science and Practice of Pharmacy, 22nd ed.;Allen Ed.: Philadelphia, PA, 2012; Handbook of Pharmaceutical Excipients, 8th ed.; Sheskey et al., Eds.; The Pharmaceutical Press: 2017; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009.

[0079] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0080] The term “solvate” refers to a complex or aggregate formed by one or more molecules of a solute, e.g., a compound provided herein, and one or more molecules of a solvent, which are present in stoichiometric or non-stoichiometric amount. Suitable solvents include, but are not limited to, water, methanol, ethanol, ^-propanol, isopropanol, and acetic acid. In certain embodiments, the solvent is pharmaceutically acceptable. In one embodiment, the complex or aggregate is in a crystalline form. In another embodiment, the complex or aggregate is in a noncrystalline form. Where the solvent is water, the solvate is a hydrate. Examples of hydrates include, but are not limited to, a hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and pentahydrate.Pyrazole Compound

[0081] Compound A22 of this disclosure, (lr,4r)-Nl-(5-chloro-4-(5-(cyclopropylmethyl)- l-methyl-lH-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-l,4-diamine, has the following structure:A22.

[0082] Compound A22 is a CKla, CDK7, and CDK9 inhibitor. Minzel et aL, Cell 2018, 775, 1-15. The Compound can be prepared according to the procedures described in Minzel el al., Cell ' 2018, 775, 1-15; or U.S. Pat. Appl. Publ. No. 2018 / 0214447 Al. The entire contents of those disclosures are incorporated herein by reference.

[0083] Compound A22 may also be referred to as compound A51 or BTX-A51.

[0084] In some embodiments, Compound A22 is formulated as a non-hygroscopic crystalline salt of (lr,4r)-Nl-(5-chloro-4-(5-(cyclopropylmethyl)-l-methyl-lH-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-l,4-diamine with an acid, or a pharmaceutically acceptable solvate thereof. In some embodiments, Compound A22 is formulated as a non-hygroscopic crystalline tosylate salt. In some embodiments, Compound A22 is formulated as a non- hygroscopic crystalline ditosylate salt.

[0085] In another embodiment, the disclosure provides Compound A22,chloro-4-(5-(cyclopropylmethyl)- 1 -methyl- U / -pyrazol-4-yl)pyrimidin-2-yl)cyclohexane- 1 ,4- diamine, or a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.

[0086] In another embodiment, Compound A22 is provided as a -toluenesulfonate of (lr,4r)-A1-(5-chloro-4-(5-(cyclopropylmethyl)-l-methyl-lJ7-pyrazol-4-yl)pyrimidin-2- yl)cyclohexane-l,4-diamine, or a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically solvate or hydrate.

[0087] In another embodiment, Compound A22 is provided as di- -toluenesulfonate of (lr,4r)-A1-(5-chloro-4-(5-(cyclopropylmethyl)-l-methyl-lJ7-pyrazol-4-yl)pyrimidin-2- yl)cyclohexane-l,4-diamine, or a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically solvate or hydrate. The -toluenesulfonate and di- -toluenesulfonate salts are described in U.S. Pub. No. 2022 / 0332702, the entire contents of which are incorporated herein by reference. 0332702, the entire contents of which are incorporated herein by reference. The term “ / ?-toluenesulfonate” is used interchangeably with the term “tosylate.” Thus, in some embodiments Compound A22 is provided as a tosylate salt. In some embodiments, Compound A22 is provided as a ditosylate salt.

[0088] In certain embodiments, a compound described herein is isolated or purified. In certain embodiments, a compound described herein has a purity of at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% by weight.

[0089] The compounds described herein are intended to encompass all possible stereoisomers unless a particular stereochemistry is specified. Where a compound described herein contains an alkenyl group, the compound may exist as one or mixture of geometric cisltrans (or ZZE) isomers. Where structural isomers are interconvertible, the compound may exist as a single tautomer or a mixture of tautomers. This can take the form of proton tautomerism in the compound that contains, for example, an imino, keto, or oxime group; orso-called valence tautomerism in the compound that contain an aromatic moiety. It follows that a single compound may exhibit more than one type of isomerism.

[0090] Compound A22 be enantiomerically pure, such as a single enantiomer or a single diastereomer, or be stereoisomeric mixtures, such as a mixture of enantiomers, e.g., a racemic mixture of two enantiomers; or a mixture of two or more diastereomers. As such, one of ordinary skill in the art will recognize that administration of a compound in its (R) form is equivalent, for compounds that undergo epimerization in vivo, to administration of the compound in its (5) form. Conventional techniques for the preparation / isolation of individual enantiomers include synthesis from a suitable optically pure precursor, asymmetric synthesis from achiral starting materials, or resolution of an enantiomeric mixture, for example, chiral chromatography, recrystallization, resolution, diastereomeric salt formation, or derivatization into diastereomeric adducts followed by separation.

[0091] Compound A22 can also be provided as a pharmaceutically acceptable salt. Salts of Compound A22 are described in U.S. Pub. No. 2022 / 0332702, the entire contents of which are incorporated herein by reference.

[0092] Compound A22 may also be provided as a prodrug, which is a functional derivative of a compound, and is readily convertible into the parent compound in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have enhanced solubility in pharmaceutical compositions over the parent compound. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis.

[0093] The compounds described herein can be prepared, isolated, or obtained by any method known to one of ordinary skill in the art, for example, by following the procedures described in U.S. Pat. No. 10,376,511.Pharmaceutical Compositions

[0094] In some embodiments, provided herein are pharmaceutical compositions, comprising Compound A22, or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or anisotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof; and a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises the -toluenesulfonate or di- -toluenesulfonate salt of Compound A22.

[0095] A pharmaceutical composition provided herein can be formulated in various dosage forms, including, but not limited to, dosage forms for oral, parenteral, and topical administration. The pharmaceutical composition can also be formulated as modified release dosage forms, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated-, fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art. See, e.g., Remington: The Science and Practice of Pharmacy, supra, Modified-Release Drug Delivery Technology, 2nd ed.; Rathbone et al., Eds.; Drugs and the Pharmaceutical Sciences 184; CRC Press: Boca Raton, FL, 2008.

[0096] In one embodiment, a pharmaceutical composition provided herein is formulated in a dosage form for oral administration. In some embodiments, the pharmaceutical composition is a tablet or a capsule. In another embodiment, a pharmaceutical composition provided herein is formulated in a dosage form for parenteral administration. In yet another embodiment, a pharmaceutical composition provided herein is formulated in a dosage form for intravenous administration. In yet another embodiment, a pharmaceutical composition provided herein is formulated in a dosage form for intramuscular administration. In yet another embodiment, a pharmaceutical composition provided herein is formulated in a dosage form for subcutaneous administration. In still another embodiment, a pharmaceutical composition provided herein is formulated in a dosage form for topical administration.

[0097] A pharmaceutical composition provided herein can be provided in a unit-dosage form or multiple-dosage form. A unit-dosage form, as used herein, refers to physically discrete a unit suitable for administration to a subject, and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of an active ingredient(s) (e.g., a compound provided herein) sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical excipient(s). Examples of a unit-dosage form include, but are not limited to, an ampoule, syringe, and individually packaged tablet and capsule. A unitdosage form may be administered in fractions or multiples thereof. A multiple-dosage form is a plurality of identical unit-dosage forms packaged in a single container to be administeredin a segregated unit-dosage form. Examples of a multiple-dosage form include, are not limited to, a vial, bottle of tablets or capsules, or bottle of pints or gallons.

[0098] A pharmaceutical composition provided herein can be administered at once or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the subject being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the subject’s need and the professional judgment of the person administering or supervising the administration of the pharmaceutical composition.

[0099] In some embodiments, a pharmaceutical composition provided herein comprises Compound A22, or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof; and sugar beads, talc, and povidone.

[0100] In some embodiments, a pharmaceutical composition provided herein comprises Compound A22, or a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof; and sugar beads, talc, and povidone.

[0101] In some embodiments, a pharmaceutical composition provided herein comprises Compound A22 or a pharmaceutically acceptable salt; and sugar beads, talc, and povidone. In some embodiments, a pharmaceutical composition comprises a tosylate salt of Compound A22. In some embodiments, a pharmaceutical composition comprises a ditosylate salt of Compound A22. In some embodiments, the pharmaceutical composition is formulated as a capsule. In some embodiments, the pharmaceutical composition is formulated as a tablet.

[0102] In some embodiments, a pharmaceutical composition provided herein comprises Compound A22 or a pharmaceutically acceptable salt in an amount ranging from about 0.1 to about 50, from about 0.2 to about 40, from about 0.5 to about 35, or from about 0.5 to about 30 mg per capsule or tablet. In certain embodiments, a pharmaceutical composition provided herein comprises Compound A22 or a pharmaceutically acceptable salt in an amount ranging from about 0.1 to about 50 mg per capsule or tablet. In certain embodiments, apharmaceutical composition provided herein comprises Compound A22 or a pharmaceutically acceptable salt in an amount ranging from about 0.2 to about 35 mg per capsule or tablet. In certain embodiments, a pharmaceutical composition provided herein comprises Compound A22 or a pharmaceutically acceptable salt in an amount ranging from about 0.5 to about 30 mg per capsule or tablet. In certain embodiments, a pharmaceutical composition provided herein comprises Compound A22 or a pharmaceutically acceptable salt in an amount ranging from about 0.5 to about 5 mg per capsule.

[0103] In certain embodiments, a pharmaceutical composition provided herein comprises Compound A22 or a pharmaceutically acceptable salt in an amount of about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 6, about 8, about 10, about 12, about 15, about 17, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40 mg per capsule or tablet or unit oral dosage form. In certain embodiments, a pharmaceutical composition provided herein comprises Compound A22 or a pharmaceutically acceptable salt in an amount of about 0.5, about 1, or about 2 mg per capsule or tablet or unit oral dosage form. In some embodiments, a pharmaceutical composition comprises about 21 mg of Compound A22 per capsule or tablet or unit oral dosage form. In some embodiments, a pharmaceutical composition comprises about 30 mg of Compound A22 per capsule or tablet or unit oral dosage form. In some embodiments, a pharmaceutical composition comprises about 35 mg of Compound A22 per capsule or tablet or unit oral dosage form.

[0104] In yet another embodiment, a pharmaceutical composition provided herein comprises a -toluenesulfonate salt of Compound A22 or a di- -toluenesulfonate salt of Compound A22; and sugar beads, talc, and povidone. In one embodiment, the pharmaceutical composition is formulated as a capsule or tablet or unit oral dosage form.

[0105] In certain embodiments, a pharmaceutical composition provided herein comprises a -toluenesulfonate salt of Compound A22 in an amount ranging from about 0.1 to about 50, from about 0.2 to about 20, from about 0.5 to about 10, or from about 0.5 to about 5 mg per capsule. In certain embodiments, a pharmaceutical composition provided herein comprises a -toluenesulfonate salt of Compound A22 in an amount ranging from about 0.1 to about 50 mg per capsule. In certain embodiments, a pharmaceutical composition provided hereincomprises a -toluene sulfonate salt of Compound A22 in an amount ranging from about 0.2 to about 40 mg per capsule. In certain embodiments, a pharmaceutical composition provided herein comprises a -toluenesulfonate salt of Compound A22 in an amount ranging from about 0.5 to about 30 mg per capsule. In certain embodiments, a pharmaceutical composition provided herein comprises a -toluenesulfonate salt of Compound A22 in an amount ranging from about 0.5 to about 25 mg per capsule.

[0106] In certain embodiments, a pharmaceutical composition provided herein comprises a -toluenesulfonate salt of Compound A22 in an amount of about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 6, about 8, about 10, about 12, about 15, about 17, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40 mg per capsule or tablet or unit oral dosage form. In certain embodiments, a pharmaceutical composition provided herein comprises a p- toluenesulfonate salt of Compound A22 in an amount of about 21, 30, or 35 mg per capsule or tablet or unit oral dosage form.

[0107] In still another embodiment, a pharmaceutical composition provided herein comprises a di- -toluenesulfonate salt of Compound A22; and sugar beads, talc, and povidone. In one embodiment, the pharmaceutical composition is formulated as a capsule.

[0108] In certain embodiments, a pharmaceutical composition provided herein comprises a di- -toluenesulfonate salt of Compound A22 in an amount ranging from about 0.1 to about 50, from about 0.2 to about 20, from about 0.5 to about 10, or from about 0.5 to about 5 mg per capsule. In certain embodiments, a pharmaceutical composition provided herein comprises a di- -toluenesulfonate salt of Compound A22 in an amount ranging from about 0.1 to about 50 mg per capsule. In certain embodiments, a pharmaceutical composition provided herein comprises a di- -toluenesulfonate salt of Compound A22 in an amount ranging from about 0.2 to about 40 mg per capsule. In certain embodiments, a pharmaceutical composition provided herein comprises a di- -toluenesulfonate salt of Compound A22 in an amount ranging from about 0.5 to about 30 mg per capsule. In certain embodiments, a pharmaceutical composition provided herein comprises a di- - toluenesulfonate salt of Compound A22 in an amount ranging from about 0.5 to about 25 mg per capsule.

[0109] In certain embodiments, a pharmaceutical composition provided herein comprises a di- / ?-toluenesulfonate salt of Compound A22 in an amount of about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 6, about 8, about 10, about 12, about 15, about 17, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40 mg per capsule or tablet or unit oral dosage form. In certain embodiments, a pharmaceutical composition provided herein comprises a di- -toluenesulfonate salt of Compound A22 in an amount of about 21, 30, or 35 mg per capsule or tablet or unit oral dosage form.

[0110] In certain embodiments, a pharmaceutical composition provided herein is formulated as an immediate-release capsule with a size of, e.g., size 1.Methods of Treatment

[0111] This disclosure provides methods of treating liposarcoma in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of Compound A22. This disclosure is based on the surprising discovery of the dependence of liposarcomas on CKla and the observation that inhibition of CKla acts synergistically with inhibition of CDK9 in treating liposarcoma tumors. The methods of this disclosure are based on the discovery that Compound A22 inhibits CKla, inducing p53 activation and apoptosis, and also inhibits CDK7 and CDK9, which inhibits MDM2 expression and MCL1 expression, thereby also activating p53 and inducing apoptosis. Prior to this application, a major limitation for exploiting the direct inhibition of MDM2 activity has been the lack of well- tolerated treatments for inhibiting MDM2 and activating P53. This disclosure is also based on the discovery of the novel, synergistic and disparate mechanisms of Compound A22 and its surprising clinical tolerability for patients with liposarcoma.

[0112] In some embodiments, the liposarcoma is well differentiated. In some embodiments, the liposarcoma is de-differentiated. In some embodiments, the liposarcoma is refractory. In some embodiments, the liposarcoma is metastatic. In some embodiments, the liposarcoma is drug-resistant. In some embodiments, surgery and / or prior therapy has failed to stop progression of the liposarcoma.

[0113] In some embodiments, the subject has TP53VI . In some embodiments, the subject the subject over-expresses MDM2. In some embodiments, the subject has a >4 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene. In some embodiments, the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene. In some embodiments, the subject overexpresses MDM2 or has a >10 copy number of the MDM2 gene.

[0114] In certain embodiments, the liposarcoma is drug-resistant. In certain embodiments, the liposarcoma is resistant to CDK4 / 6 inhibitors.

[0115] In certain embodiments, the subject has failed a prior therapy. In other embodiments, the subject has failed more than one prior therapy. In some embodiments, a prior therapy has failed to stop progression of the liposarcoma.

[0116] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0117] A method provided herein encompasses treating a subject regardless of patient’s age, although some diseases are more common in certain age groups.

[0118] In certain embodiments, the therapeutically effective amount of Compound A22 is ranging from about 0.001 to about 10 mg / kg per day, from about 0.002 to about 5 mg / kg per day, from about 0.005 to about 2 mg / kg per day, from about 0.01 to about 1 mg / kg per day, or from about 0.01 to about 0.5 mg / kg per day. In one embodiment, the therapeutically effective amount of Compound A22 ranges from about 0.001 to about 10 mg / kg per day. In another embodiment, the therapeutically effective amount of Compound A22 ranges from about 0.002 to about 5 mg / kg per day. In yet another embodiment, the therapeutically effective amount of Compound A22 ranges from about 0.005 to about 2 mg / kg per day. In yet another embodiment, the therapeutically effective amount of Compound A22 ranges from about 0.01 to about 1 mg / kg per day. In yet another embodiment, the therapeutically effective amount of a Compound A22 ranges from about 0.01 to about 0.5 mg / kg per day. In still another embodiment, the therapeutically effective amount of Compound A22, is about 0.01, about 0.02, about 0.03, about 0.05, about 0.08, about 0.1, about 0.12, about 0.15, about 0.17, about 0.2, or about 0.25 mg / kg per day.

[0119] In certain embodiments, the therapeutically effective amount of Compound A22 ranges from about 0.1 to about 200 mg per day, from about 0.2 to about 100 mg per day, from about 0.5 to about 50 mg per day, or from about 1 mg every other day to about 20 mg per day. In one embodiment, the therapeutically effective amount of Compound A22 ranges from about 0.1 to about 200 mg per day. In another embodiment, the therapeutically effective amount of Compound A22 ranges from about 0.2 to about 100 mg per day. In yet another embodiment, the therapeutically effective amount of Compound A22 ranges from about 0.5 to about 50 mg per day. In yet another embodiment, the therapeutically effective amount of Compound A22 ranges from about 1 to about 20 mg per day. In yet another embodiment, the therapeutically effective amount of Compound A22, is about 1, about 2, about 3, about 5, about 8, about 10, about 11, about 14, about 15, about 17, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 mg per day.

[0120] In certain embodiments, the therapeutically effective amount of Compound A22 ranges from about 1 to 500, from about 2 to 250, from about 5 to about 105, from about 10 to about 90, or from about 20 to about 65 mg per week. In one embodiment, the therapeutically effective amount of Compound A22 ranges from about 1 to 500 mg per week. In another embodiment, the therapeutically effective amount of Compound A22 ranges from about 2 to 250 mg per week. In yet another embodiment, the therapeutically effective amount of Compound A22 ranges from about 5 to about 105 per week. In yet another embodiment, the therapeutically effective amount of Compound A22 ranges from about 10 to about 90 mg per week. In some embodiments, the therapeutically effective amount of Compound A22 ranges from about 10 to about 63 or 65 mg per week. In still another embodiment, the therapeutically effective amount of Compound A22, is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105 mg per week.

[0121] In certain embodiments, the compound is administered at a dose of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 40 mg, about 42 mg, about 45 mg, about 48 mg, about 51 mg, about 52 mg, about 55 mg, about 58 mg, about 60 mg, about 62 mg, about63mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 75 mg, about 78 mg, about 80, about 81 mg, about 84 mg, about 87 mg, about 90 mg, about 93 mg, about 96 mg, about 99 mg, about 102 mg, or about 105 mg per week. In certain embodiments, the compound is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days per week.

[0122] It is understood that the administered dose of Compound A22 can also be expressed in units other than mg / kg every other day. For example, doses for parenteral administration can be expressed as mg / m2per day. One of ordinary skill in the art would readily know how to convert doses from mg / kg per day to mg / m2per day to given either the height or weight of a subject or both. For example, a dose of 1 mg / m2per day for a 65 kg human is approximately equal to 58 mg / kg per day.

[0123] Depending on the disease to be treated and the subject’s condition, Compound A22 may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, CIV, intracistemal injection or infusion, subcutaneous injection, or implant), inhalation, nasal, vaginal, rectal, sublingual, or topical (e.g., transdermal or local) routes of administration.

[0124] In one embodiment, Compound A22, is administered orally. In some embodiments, Compound A22 is administered as tablet or capsule. In another embodiment, Compound A22, is administered parenterally. In yet another embodiment, Compound A22, is administered intravenously. In yet another embodiment, Compound A22, is administered intramuscularly. In yet another embodiment, Compound A22, is administered subcutaneously. In still another embodiment, Compound A22, is administered topically.

[0125] A compound described herein, e.g., Compound A22, can be delivered as a single dose such as, e.g., a single bolus injection, or oral tablets or pills; or over time such as, e.g., continuous infusion over time or divided bolus doses over time. Compound A22, can be administered repetitively if necessary, for example, until the subject experiences stable disease or regression, or until the subject experiences disease progression or unacceptable toxicity. Stable disease or lack thereof is determined by a method known in the art such asevaluation of subject’s symptoms, physical examination, visualization of the cancer that has been imaged using X-ray, CAT, PET, or MRI scan and other commonly accepted evaluation modalities.

[0126] Compound A22, can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), and three times daily (TID). In addition, the administration can be continuous, z.e., every day, or intermittently. The term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of Compound A22, is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days. In one embodiment, Compound A22 is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days per week. In certain embodiments, Compound A22 is administered for 3 days or 5 days per week. In some embodiments, Compound A22 is administered for 3 non-consecutive days per week. In certain embodiments, Compound A22 is administered on days 1, 2, and 3 in a week. In some embodiments, Compound A22 is administered on days 1, 2, 3, 4, and 5 in a week. In some embodiments, Compound A22 is administered on days 1, 3, and 5 in a week.

[0127] It will be understood, however, that the specific dose level and frequency of dosage for any particular subject can be varied and will depend upon a variety of factors including the activity of the specific compound employed, e.g., Compound A22, the metabolic stability and length of action of the compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

[0128] In certain embodiments, Compound A22, is cyclically administered to a subject to be treated. Cycling therapy involves the administration of the compound for a period of time, followed by a rest for a period of time, and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and / or improves the efficacy of the treatment.

[0129] Consequently, in one embodiment, Compound A22, is administered for a cycle of about one week, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about eight weeks, or about ten weeks, with a rest period of 0 days to aboutfour weeks. In one embodiment, Compound A22, is administered for a cycle of three weeks, four weeks, five weeks, or six weeks with a rest period of 0, 1, 3, 5, 7, 9, 12, or 14 days. In some embodiments, Compound A22 is administered for a cycle of 21-42 days. In some embodiments, Compound A22 is administered for a cycle of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days. In some embodiments, a cycle has no rest period. In some embodiments, Compound A22 is administered for consecutive cycles with no rest period between cycles. In some embodiments, Compound A22 is administered for multiple cycles with a rest period. In certain embodiments, the rest period is 7 days. In certain embodiments, the rest period is 14 days. In certain embodiments, the rest period is a period that is sufficient for bone marrow recovery. In certain embodiments, no rest period is required between cycles. The frequency, number, and length of dosing cycles can be increased or decreased.

[0130] In one embodiment, Compound A22, is administered for three weeks in a 28-day cycle with a 7-day rest period. In one embodiment, in a 28-day cycle with a 7-day rest period, Compound A22, is administered every day for five days of a week. In another embodiment, in a 28-day cycle with a 7-day rest period, Compound A22, is administered on Days 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 15, 16, 17, 18, and 19. In one embodiment, in a 28-day cycle with a 7-day rest period, Compound A22, is administered every other day for three days of a week. In another embodiment, in a 28-day cycle with a 7-day rest period, Compound A22, is administered on Days 1, 3, 5, 8, 10, 12, 15, 17, and 19.

[0131] In some embodiments, Compound A22 is administered 3 days per week for four weeks in a 28-day cycle. In some embodiments, Compound A22 is administered for multiple cycles. In some embodiments, when Compound A22 is administered for multiple cycles, there is a rest period between cycles. In some embodiments when Compound A22 is administered for multiple cycles, there is no rest period between cycles.

[0132] In certain embodiments, the subject is treated with Compound A22, from about 1 to about 50, from about 2 to about 20, from about 2 to 10, or from about 4 to about 8 cycles. In certain embodiments, the subject is treated with Compound A22 from about 1 to about 50 cycles. In certain embodiments, the subject is treated with Compound A22 from about 2 to about 20 cycles. In certain embodiments, the subject is treated with Compound A22 from about 2 to 10 cycles. In certain embodiments, the subject is treated with Compound A22, from about 4 to about 8 cycles. In some embodiments, Compound A22 is administered witha rest period between cycles. In some embodiments, Compound A22 is administered with no rest period between cycles.

[0133] In one embodiment, provided herein is a method of inhibiting the growth of a cell, comprising contacting the cell with an effective amount of Compound A22, or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.

[0134] In another embodiment, provided herein is a method of modulating the activity of CKla in a cell, comprising contacting the cell with Compound A22, or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.

[0135] In yet another embodiment, provided herein is a method of inducing apoptosis in a cell, comprising contacting the cell with Compound A22, or an enantiomer, a mixture of enantiomers, a diastereomer, a mixture of two or more diastereomers, a tautomer, a mixture of two or more tautomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.

[0136] In certain embodiments, the cell is a liposarcoma cell. In certain embodiments, the cell is a relapsed or refractory liposarcoma cell. In certain embodiments, the cell is a relapsed liposarcoma cell. In certain embodiments, the cell is a refractory liposarcoma cell. In certain embodiments, the cell is a metastatic liposarcoma cell. In certain embodiments, the cell is a drug-resistant liposarcoma cell.Embodiments1. A method of treating liposarcoma in a subject, comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of Compound A22,A22, or a pharmaceutically acceptable salt thereof.2. The method of embodiment 1, wherein the liposarcoma is well differentiated.3. The method of embodiment 1, wherein the liposarcoma is de-differentiated.4. The method of any one of embodiments 1 to 104, wherein the patient has TP53VI .5. The method of any one of embodiments 1 to 2, wherein the subject over-expresses MDM2 or has a >4 copy number of the MDM2 gene.6. The method of any one of embodiments 1 to 5, wherein the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene.7. The method of any one of embodiments 1 to 6, wherein the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene.8. The method of any one of embodiments 1 to 7, wherein the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.9. The method of any one of embodiments 1 to 8, wherein the liposarcoma is refractory.10. The method of any one of embodiments 1 to 9, wherein the liposarcoma is metastatic.11. The method of any one of embodiments 1 to 10, wherein the liposarcoma is drugresistant.12. The method of any one of embodiments 1 to 11, wherein the subject has failed a prior therapy.13. The method of any one of embodiments 1 to 12, wherein the subject is a human.14. The method of any one of embodiments 1 to 13, wherein the compound is administered orally.15. The method of any one of embodiments 1 to 14, wherein the compound is administered as a tablet or capsule.16. The method of any one of embodiments 1 to 15, wherein the therapeutically effective amount ranges from about 0.001 to about 10 mg / kg per day.17. The method of any one of embodiments 1 to 16, wherein the therapeutically effective amount ranges from about 15 to about 35 mg per day.18. The method of any one of embodiments 1 to 17, wherein the therapeutically effective amount is about 1, about 2, about 3, about 5, about 8, about 10, about 11, about 14, about 15, about 17, about 20, about 21, about 25, about 30, or about 35 mg per day.19. The method of any one of embodiments 1 to 18, wherein the compound is administered in one or more cycles.20. The method of any one of embodiments 1 to 19, wherein the compound is administered in a 28-cycle.21. The method of any one of embodiments 1 to 20, wherein the compound is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days per week.22. The method of any one of embodiments 1 to 21, wherein the compound is administered for 3 days or 5 days per week.23. The method of any one of embodiments 1 to 22, wherein the compound is administered on Days 1, 2, and 3 in a week.24. The method of any one of embodiments 1 to 23, wherein the compound is administered on Days 1, 3, and 5 in a week.25. The method of any one of embodiments 1 to 22, wherein the compound is administered on three non-consecutive days in a week.26. The method of any one of embodiments 1 to 25, wherein the compound is administered in a 28-day cycle for 3 days per week.27. The method of embodiment 26, wherein the compound is administered on three non-consecutive days per week.28. The method of embodiment 27, wherein the compound is administered on Days 1, 3, and 5 per week29. The method of any one of embodiments 1 to 22, wherein the compound is administered in a 28-day cycle for 5 days per week.30. The method of embodiment 29, wherein the compound is administered in a 28- day cycle on Days 1, 2, 3, 4, and 5 per week.31. The method of any one of embodiments 1 to 30, wherein the compound is formulated as a -toluenesulfonate salt or a di- -toluenesulfonate salt.32. The method of any one of embodiments 1 to 31, wherein the therapeutically effective amount is about 21 mg.33. The method of any one of embodiments 1 to 31, wherein the therapeutically effective amount is about 30 mg.34. The method of any one of embodiments 1 to 31, wherein the therapeutically effective amount is about 35 mg.35. A method of inhibiting CDK7 in a subject having liposarcoma, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of Compound A22, or a pharmaceutically acceptable salt thereof.36. The method of embodiment 35, wherein the liposarcoma is well differentiated.37. The method of embodiment 35, wherein the liposarcoma is de-differentiated.38. The method of any one of embodiments 35 to 37, wherein the compound is formulated as a -toluenesulfonate salt or a di- -toluenesulfonate salt.39. The method of any one of embodiments 35 to 38, wherein the liposarcoma has been resistant to a CDK4 / 6 inhibitor.40. The method of any one of embodiments 35 to 39, wherein the subject has TP53VI .41. The method of any one of embodiments 35 to 40, wherein the subject overexpresses MDM2 or has a >4 copy number of the MDM2 gene.42. The method of any one of embodiments 35 to 41, wherein the subject overexpresses MDM2 or has a >6 copy number of the MDM2 gene.43. The method of any one of embodiments 35 to 42, wherein the subject overexpresses MDM2 or has a >8 copy number of the MDM2 gene.44. The method of any one of embodiments 35 to 43, wherein the subject overexpresses MDM2 or has a >10 copy number of the MDM2 gene.45. The method of any one of embodiments 35-44, wherein administering comprises administering about 21 mg of Compound A22 three times per week.46. The method of any one of embodiments 35-44, wherein administering comprises administering about 30 mg of Compound A22 three times per week.47. The method of any one of embodiments 35-44, wherein administering comprises administering about 35 mg of Compound A22 three times per week.48. A method of treating liposarcoma in a subject having amplified expression of MDM2 or having a >4 copy number of the MDM2 gene, comprising administering to the subject Compound A22, or a pharmaceutically acceptable salt thereof.49. The method of embodiment 48, wherein the liposarcoma is well differentiated.50. The method of embodiment 48, wherein the liposarcoma is de-differentiated51. The method of any one of embodiments 48 to 50, wherein the compound is formulated as a -toluenesulfonate salt or a di- -toluenesulfonate salt.52. The method of any one of embodiments 48 to 49, wherein the subject overexpresses MDM2 or has a >6 copy number of the MDM2 gene.53. The method of any one of embodiments 48 to 52, wherein the subject overexpresses MDM2 or has a >8 copy number of the MDM2 gene.54. The method of any one of embodiments 48 to 53, wherein the subject overexpresses MDM2 or has a >10 copy number of the MDM2 gene.55. The method of any one of embodiments 48-54, wherein administering comprises administering about 21 mg of Compound A22 three times per week.56. The method of any one of embodiments 48 to 54, wherein administering comprises administering about 30 mg of Compound A22 three times per week.57. The method of any one of embodiments 48 to 54, wherein administering comprises administering about 35 mg of Compound A22 three times per week.58. A method of inhibiting MDM2 expression in a subject having liposarcoma, comprising administering to the subject Compound A22, or a pharmaceutically acceptable salt thereof.59. The method of embodiment 58, wherein the liposarcoma is well differentiated.60. The method of embodiment 58, wherein the liposarcoma is de-differentiated.61. The method of any one of embodiments 58 to 61, wherein the compound is formulated as a -toluenesulfonate salt or a di- -toluenesulfonate salt.62. The method of any one of embodiments 58 to 59, wherein the subject overexpresses MDM2 or has a >4 copy number of the MDM2 gene.63. The method of any one of embodiments 58 to 62, wherein the subject overexpresses MDM2 or has a >6 copy number of the MDM2 gene.64. The method of any one of embodiments 58 to 63, wherein the subject overexpresses MDM2 or has a >8 copy number of the MDM2 gene.65. The method of any one of embodiments 58 to 64, wherein the subject overexpresses MDM2 or has a >10 copy number of the MDM2 gene.66. The method of any one of embodiments 58-65, wherein administering comprises administering about 21 mg of Compound A22 three times per week.67. The method of any one of embodiments 58-65, wherein administering comprises administering about 30 mg of Compound A22 three times per week.68. The method of any one of embodiments 58-65, wherein administering comprises administering about 35 mg of Compound A22 three times per week.69. A method of inhibiting CDK9 in a subject having liposarcoma, comprising administering to the subject Compound A22, or a pharmaceutically acceptable salt thereof.70. The method of embodiment 69, wherein the liposarcoma is well differentiated.71. The method of embodiment 69, wherein the liposarcoma is de-differentiated.72. The method of any one of embodiments 69 to 71, wherein the compound is formulated as a -toluenesulfonate salt or a di- -toluenesulfonate salt.73. The method of any one of embodiments 68 to 70, wherein the liposarcoma has been resistant to a CDK4 / 6 inhibitor.74. The method of any one of embodiments 68 to 70, wherein the subject has P53VI7.75. The method of any one of embodiments 68 to 74, wherein the subject overexpresses MDM2 or has a >4 copy number of the MDM2 gene.76. The method of any one of embodiments 68 to 75, wherein the subject overexpresses MDM2 or has a >6 copy number of the MDM2 gene.77. The method of any one of embodiments 68 to 76, wherein the subject overexpresses MDM2 or has a >8 copy number of the MDM2 gene.78. The method of any one of embodiments 68 to 77, wherein the subject overexpresses MDM2 or has a >10 copy number of the MDM2 gene.79. The method of any one of embodiments 69 to 78, wherein administering comprises administering about 21 mg of Compound A22 three times per week.80. The method of any one of embodiments 69 to 78, wherein administering comprises administering about 30 mg of Compound A22 three times per week.81. The method of any one of embodiments 69 to 78, wherein administering comprises administering about 35 mg of Compound A22 three times per week.82. A method of inhibiting CKla in a subject having liposarcoma, comprising administering to the subject Compound A22, or a pharmaceutically acceptable salt thereof.83. A method of inhibiting CKla and CDK9 in a subject having liposarcoma, comprising administering to the subject Compound A22, or a pharmaceutically acceptable salt thereof.84. The method of any one of embodiments 82 to 83, wherein the compound is formulated as a -toluenesulfonate salt or di- -toluenesulfonate salt.85. The method of any one of embodiments 82 to 84, wherein the liposarcoma has been resistant to a CDK4 / 6 inhibitor.86. The method of any one of embodiments 82 to 84, wherein the subject has P53VI7.87. The method of any one of embodiments 82 to 86, wherein the subject overexpresses MDM2 or has a >4 copy number of the MDM2 gene.88. The method of any one of embodiments 82 to 87, wherein the subject overexpresses MDM2 or has a >6 copy number of the MDM2 gene.89. The method of any one of embodiments 82 to 88, wherein the subject overexpresses MDM2 or has a >8 copy number of the MDM2 gene.90. The method of any one of embodiments 82 to 89, wherein the subject overexpresses MDM2 or has a >10 copy number of the MDM2 gene.91. The method of any one of embodiments 82-90, wherein administering comprises administering about 21 mg of Compound A22 three times per week.92. The method of any one of embodiments 82-90, wherein administering comprises administering about 30 mg of Compound A22 three times per week.93. The method of any one of embodiments 82-90, wherein administering comprises administering about 35 mg of Compound A22 three times per week.94. A pharmaceutical composition comprising compound A22,or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, for use in the treatment of a patient diagnosed with liposarcoma.95. The pharmaceutical composition for use according to embodiment 94, wherein the patient has P53wt.96. The pharmaceutical composition for use according to any one of embodiments 94 to 95, wherein the treatment comprises administration of the pharmaceutical composition to the patient on three non-consecutive days in a week.97. The pharmaceutical composition for use according to any one of embodiments 94 to 96, wherein the administration occurs for one or more cycles.98. The pharmaceutical composition for use according to any one of embodiments 94 to 97, wherein the cycle is 28 days.99. The pharmaceutical composition for use according to any one of embodiments 94 to 98, wherein the pharmaceutical composition is orally administered to the patient.100. The pharmaceutical composition for use according to any one of embodiments 94 to 99, wherein the pharmaceutical composition is formulated as an oral unit dosage form.101. The pharmaceutical composition for use according to any one of embodiments 94 to 100, wherein the oral unit dosage form comprises about 21 mg of Compound A22.102. The pharmaceutical composition for use according to any one of embodiments 94 to 100, wherein the oral unit dosage form comprises about 30 mg of Compound A22.103. The pharmaceutical composition for use according to any one of embodiments 94 to 100, wherein the oral unit dosage form comprises about 35 mg of Compound A22.104.

[0137] The disclosure will be further understood by the following non-limiting examples.EXAMPLES

[0138] As used herein, the symbols and conventions used in these processes, schemes, and examples, regardless of whether a particular abbreviation is specifically defined, are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society, the Journal of Medicinal Chemistry, or the Journal of Biological Chemistry. Specifically, but without limitation, the following abbreviations may be used in the examples and throughout the specification: g (grams); mg (milligrams); mL (milliliters); mL (microliters); mM (millimolar); mM (micromolar); mmol (millimoles); h (hour or hours); and min (minutes).Example 1: Materials and MethodsCell Lines

[0139] Liposarcoma cell lines used in this study include LP6, which was developed at Dana Farber Cancer Institute (DFCI), and 94T778, which was purchased from ATCC. LPS853 and LPS141 were provided by Adrian Marino-Enriquez and Jonathan Fletcher. Lipo224, Lipo246, and Lipo963 were provided by Raphael Pollock. p53-CRISPR-KO LP6 or LPS853 cells were generated using lentivirus vectors packaged from pLentiCRISPR_Puro_sgTP53, a gift from William Kaelin (Addgene plasmid # 187819 ; http: / / n2t.nct / addgene: 187819 ; RRID:Addgene_187819). Cells were routinely tested by a mycoplasma PCR detection Kit (ABM, Cat. No. G238) to guarantee they were free ofmycoplasma contamination. All cells were maintained in a humidified incubator with 95% air plus 5% CO2 at 37°C and were cultured in DMEM F12 (Life Technologies # 10565042) supplemented with 10% fetal bovine serum, 1% penicillium: streptomycin solution (10,000 units / mL of penicillin and 10,000 pg / mL of streptomycin, Gibco).Mice

[0140] All in vivo studies were conducted at Dana-Farber Cancer Institute with the approval of the Institutional Animal Care and Use Committee in an AAALAC accredited vivarium. The PDX model, LPS3, was generated from surgical resection tissue of a liposarcoma patient who consented to research use of material under an IRB-approved protocol and previously described. Tumors were grown and serially passaged via subcutaneous implantation in female NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ (NSG) mice from The Jackson Laboratories (ME). For efficacy studies, tumor fragments dipped in Matrigel were implanted into 8-week-old NSG mice. Tumors were allowed to establish to 70.5 ± 17.4 mm3 in size before randomization into various treatment groups (Studylog, CA software) with n=8 / group as: vehicle control (1% methylcellulose in water with 0.01% Tween 80 and 0.02% PEG400), 15 mg / kg Compound A22 (MedchemExpress, NJ) administered every other day. Mice were treated orally for 3 weeks. Tumor volumes were determined from caliper measurements by using the formula, Tumor volume = (length x width2) / 2. Tumor volumes and body weights were measured twice weekly.Western Blot Analysis

[0141] Cells in culture dishes were washed with PBS, harvested by cell scrapers, and centrifuged to obtain the cell pellets, which were subsequently lysed with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-4O, 0.5% sodium deoxycholate, and 0.1% SDS, Boston BioProducts) supplemented with protease and phosphatase inhibitor cocktails (Calbiochem). Lysates were incubated on ice for 15 mins and vortexed at the highest setting every 5 mins and were then centrifuged at 15,000 g for 15 min. Protein concentration was measured using a Pierce-BCA assay kit. Proteins were separated by gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% milk, immunoblotted with protein-specific primary antibodies overnight at 4oC on a shaker, and then washed with TBST and probed with horseradish peroxidase (HRP) conjugated anti-mouse or anti-rabbit secondary antibodies (GE Healthcare) for 1 hour at room temperature. Finally, the blots were washed with TBST and developed using X-ray films to detect chemiluminescence. Theintensity of the chemiluminescence was analyzed to reveal relative expression levels of specific proteins.RNA-Seq

[0142] RNA was extracted from cultured cells treated with vehicle or JQ1 using the RNeasy Mini Kit (Qiagen). Prior to RNA sequencing, RNA was treated with DNase using RNase-Free DNase Set (Qiagen). rRNA depletion was performed from lOOng of purified RNA using QIAseq FastSelect rRNA HMR reagents according to manufacturer’s protocol. Libraries were prepared using Roche Kapa Biosystems RNA HyperPrep sample preparation reagents on a Beckman Coulter Biomek i7. Finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced with paired-end 150bp reads on an Illumina NovaSeq 6000at the Dana-Farber Cancer Institute Molecular Biology Core Facilities. Sequenced reads were aligned to the UCSC hg38 reference genome assembly and gene counts were quantified using STAR (v2.7.3a) [PMID: 23104886], Differential gene expression testing was performed by DESeq2 (vl.22.1). RNAseq analysis was performed using the VIPER snakemake pipeline.GSEA analysis

[0143] To identify significant activated and repressed pathways within the Hallmark gene sets, we performed an unbiased Gene Set Enrichment Analysis (GSEA) using the DESeq2- generated log2fold change (log2FC) values from our RNA-seq data derived from samples treated with Compound A22 with differing TP53 status (single-guide non-targeting (sgNT) vs knockout (KO)). Starting with raw gene counts, we normalized the data and conducted differential expression analysis using DESeq2 without applying any log2FC thresholds to include all genes in the analysis. The resulting log2FC values were ranked and input into the cluster Profiler package's GSEA function, utilizing Hallmark gene sets sourced from the Molecular Signatures Database (MSigDB) via the msigdbr package. GSEA was performed separately for each experimental condition to determine enrichment scores. Pathways with normalized enrichment scores (NES) greater than zero were classified as activated, while those with NES less than zero were considered repressed. Significant pathways were identified between group comparisons based on adjusted p-values (FDR q-value < 0.05) andare presented based on TP53 status modulating differential responses in genetic signatures following Compound A22 treatment. qPCR

[0144] mRNA was extracted from cultured cells using the PureLink RNA Mini Kit (Life Technologies of Thermo Fisher Scientific). cDNA was generated by using the iScript cDNA synthesis kit from BIO-RAD. SYBR Green super-mix for running qPCR was from Applied Biosystems. qPCR was performed using primers for human BAX, MDM2, p21,BBC3(PUMA), MYC, andACTB. The primer sequences are as follows:

[0145] SYBR green signal was detected using the QuantStudio™ 3 Real-Time PCR System from Applied Biosystems. Relative expression of each gene was analyzed using the 2-AACT method.Flow cytometry

[0146] For cell cycle analysis, cells were harvested by trypsinization and washed with PBS. The cell pellets were resuspended and fixed with 80% ethanol (-20°C) for 30 minutes on ice, washed with PBS, and then stained with propidium iodide / RNase solution (BD Biosciences) for 15-30 minutes in flow tubes covered with aluminum foil on a rack. Prior to loading the tubes to a cytometer for cell cycle analysis, the cells were filtered. Cell cycle analysis was performed using a BD Fortessa cytometer with FACS Diva software (BD Biosciences). For the Edu Labeling, cells were pulsed with 10 pM EdU for 30 min prior to harvest. Trypsinized cells were washed with PBS and fixed in 4% formaldehyde in PBS for 15 min at room temperature. Cells were then washed three times with 1% BSA in PBS andpermeabilized in 70% ethanol at -20°C for 30 min to overnight. After three washes with 1% BSA in PBS, incorporated EdU was labeled with a CLICK reaction (2 mM CuSO4, 100 pM THPTA, 100 mM sodium ascorbate, and 2 pM Calfluor 647 Azide in PBS) by rotating for 30 min at room temperature. Samples were then washed thrice with 1% BSA in PBS and stained with a 1 pg / ml DAPI, 100 ng / ml RNase A staining solution. Samples then were analyzed on Fortessa analyzers (BD Biosciences) and 50,000 total events were captured for each sample. Annexin V apoptosis assays were performed by utilizing flow cytometry following the manufacturer’s instructions (BD Biosciences) to quantify apoptosis induced by Compound A22.High Throughput Dynamic BH3 profiling

[0147] HTDBP was performed on cell lines as previously described (10). Briefly, cells were plated in 3764BC (Coming) one day prior to drug treatment. Drugs (Compound A22) were added using the D300e digital drug dispenser. Upon drug treatment, cells were incubated at 37°C for 24 hours. Subsequently, medium was washed from plates using the BioTek 406EL plate washer (BioTek). Medium was replaced with PBS. A 2* concentrated BH3 profiling buffer was added to cells with appropriate levels of digitonin (0.001% for mouse cells and 0.002% for human cells) and with multiple concentrations of the synthetic BIM or PUMA BH3 peptides. Fixatives were neutralized using a Tris-Glycine buffer, and cells were subsequently stained with antibodies in a Tween 20 permeabilizing solution. Cells were stained overnight, and before imaging, stain solution was washed using the BioTek plate washer.

[0148] Nuclei were stained with Hoechst 33342 (Invitrogen). Cytochrome c was measured using the Cytochrome c-Alexa Fluor 647 antibody (BioLegend). All imaging was performed on the IXM4 high-content widefield microscope (Molecular Devices). A 10x objective was used to perform all imaging. A 4',6-diamidino-2-phenylindole (DAPI) filter cube was used to measure Hoechst 33342 staining, and a Cy5 cube to measure Cytochrome c-Alexa Fluor 647 antibody staining.

[0149] Image analysis was performed in MetaMorph using the multi wavelength cellscoring module and the adaptive background correction module to segment cells on the basis of an intensity above local background. This results in an approximate single-cell segmentation and the area of cytochrome c intensity. Cells are scored as being positive ornegative on the basis of the area. All subsequent data analysis was performed in Excel or GraphPad Prism.Colony formation assay

[0150] For long term colony formation assay, 2000 cells per well were seeded in 6-well plates and treated with a specific drug, or DMSO as labelled. The cells were incubated for 10 days to allow colony formation in the presence of drug or DMSO. Colonies were fixed using fixation solution (75% methanol + 25% Acetic acid) for 20 minutes, stained with 1% Crystal Violet in water for 15min, washed in tap water, and quantified using ImageJ software.Growth inhibition assay

[0151] For short term growth inhibition assay, LPS cells (1000 cells / well) were seeded in 96-well plates and incubated for one day prior to DMSO or drug treatment. The cells were incubated for 4 days in the presence of DMSO or drug. Cell viability was quantified using CellTiter-Glo reagent from Promega following the manufacturer’s protocol. Relative cell viability percentage was calculated by first subtracting background (CellTiter-Glo reagent in medium) from the drug-treated wells and then normalizing to the DMSO-treated control.After obtaining the relative cell viability values, IC50 of drug response were calculated using Graphpad Prism software. BLISS Synergy / antagonism analysis was performed using Combenefit software.DepMap analysis for liposarcoma specific dependencies

[0152] Through the DepMap portal, a two-class comparison was performed using the CRISPR (DepMap Public 24Q2+Score, Chronos) dataset to compare LPS cell lines (LPS 141, 93T449, LPS510, LPS27, 94T778, LPS853, 95T1000, and LPS6) with all other cell lines. A volcano plot was generated by plotting the effect size on the x axis and the significance (-loglO(q)) on the y axis. A waterfall plot was generated by plotting the gene rank on the x axis and mean gene effect on the y axis.Statistical analysis

[0153] Two-tail Student’s t test or ANOVA were performed for statistical analysis. A P value < 0.05 was considered significant for our study (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Statistical details were also described in the relevant figure legends. Statistical analyses were performed using the software Graphpad Prism.

[0154] KEY RESOURCES TABLE

[0155] Example 2: Evaluation of TargetsCKla is essential to liposarcoma versus non-liposarcoma cell lines

[0156] Using the DepMap database, genes were ranked according to the average dependency score across 8 liposarcoma (“LPS”) cell lines. Surprisingly, results found that CSNK1A1, the gene encoding CKla, is essential to LPS which has not previously been demonstrated. Essential and previously known, pathognomonic LPS-amplified genes, MDM2 and CDK4, were also identified (FIG. 1). A chronos score of 0 indicated that the growth of the cell line was not dependent on the gene, and a larger negative value indicated greater dependence.

[0157] Next, dependencies were evaluated across 8 liposarcoma cell lines compared to all other types of cancers to identify LPS-specific dependencies. In addition to MDM2 and CDK4, CSNK1A1 was identified as an LPS-specific dependency (FIG. 2).

[0158] Further analyses of the DepMap database demonstrated that CSNK1A1 is expressed in all the LPS cell lines to a similar level as non-LPS cells. Examination of a panel of LPS cell lines demonstrated that CKla protein is readily expressed. To validate whether CKla is essential to LPS, siRNA-mediated knockdown of CSNK1A1 was performed in LPScell lines. The results are shown in FIG. 3 and determined that CKla depletion significantly reduced LPS cell colony formation.

[0159] Since CKla is a negative regulator of p53, we next analyzed p53 protein levels in a panel of LPS cell lines. As expected due to high MDM2 amplification, p53 protein is only slightly detected in the 6 LPS cell lines examined. Upon CKla depletion, p53 expression and PARP cleavage, an indicator of apoptosis, were significantly increased (FIG. 4).

[0160] Consistently, CKla-depleted cells are highly positive for annexin-V staining, an established marker of apoptosis. To investigate the role of p53 in PARP cleavage following CKla depletion, we generated CRISPR / Cas9-mediated TP53 knockout LP6 cells. Loss of TP 53 significantly attenuated PARP cleavage induced by CKla depletion, suggesting apoptosis is largely mediated by p53 activation. This was further confirmed by RT qPCR analyses of p53 target genes demonstrating that the expression of pro-apoptotic BAX and BBC 3 (PUMA) was induced upon CKla depletion.CKla depletion is synergistic with CDK9 inhibition in LPS cells

[0161] In addition to CSNK1 Al, DepMap analysis identified other negative regulators of p53 as top essential genes in LPS including MDM2, AURKA, USP7, and CDK9. The transcriptional kinase CDK9 is a critical regulator of the expression of MDM2 and MDMX and has been shown to indirectly suppress the function of p53.

[0162] According to the DepMap database, CDK9 is highly expressed in LPS cell lines. In a panel of six LPS cell lines, CDK9 is readily detectable by Western blot. The expressions of MCL1, MYC, and MDM2 followed the general pattern of CDK9 expression levels, suggesting that these genes may be transcriptionally regulated by CDK9 in LPS similar to a previous report in AML (Minzel et al., 2018).

[0163] Next, the effects of CDK9 inhibitor treatment of LPS cells was determined. Treatment with the CDK9 inhibitors, AZD4573, KB-0742, and flavopiridol, significantly reduced Ser2- / Ser5-phosphorylation of RNA Pol II CTD and expression of MDM2 and MCL1, suggesting that CDK9 inhibition suppresses RNA Pol II activity in LPS cells. CDK9 inhibitor treatment upregulated p53 expression and induced PARP cleavage, suggesting induction of cell death. CDK9 inhibitor-induced apoptosis was confirmed by Annexin V staining analyzed via flow. Cell viability assays demonstrated that the LPS cell line, LP6, issensitive to all three inhibitors of CDK9 (AZD4573, KB-0742, and flavopiridol), whereas Nutlin-3, an inhibitor of MDM2, reduced cell viability at much higher doses. Similar responses were also observed in LPS141 cells while LPS853 cells responded but were less sensitive to the treatment. In summary, these data suggest that CDK9 in LPS is targetable, and its inhibition is a viable approach of suppressing LPS cell growth and inducing apoptosis.

[0164] Since CSNK1 Alis an essential gene for liposarcoma cells and CKla is a negative regulator of p53, the depletion of CKla potentiates the effects of CDK9 inhibitors. In both LP6 and LPS853 cells, siRNA-mediated knockdown of CSNK1 Al reduced the IC50 of all three CDK9 inhibitors in cell viability assays (FIG. 12). PARP cleavage was enhanced upon CKla depletion. Furthermore, through colony formation assays, we found that CKla depletion significantly potentiated the effects of suboptimal doses of three CDK9 inhibitors on cell survival (FIG. 13). These unexpected data imply that co-targeting of CDK9 and CKla is a useful strategy for liposarcoma therapy.Inhibition of CDK7 suppresses LPS cell growth

[0165] In addition to CDK9, transcriptional CDK was also identified as essential in LPS. CDK7 phosphorylates and enhances the activity of CDK9 and CDK7 inhibitors are being investigated for the treatment of various cancers. However, their effect on LPS remains unknown.Example 3: Effect of Compound A22 in LPS Cells

[0166] A panel of LPS cell lines was treated with Compound A22. Compound A22 significantly suppressed colony formation of all cell lines tested (LP6, LPS853, LPS141, and T778) at nanomolar concentrations. Results are shown in FIG. 5.

[0167] Treatment with Compound A22 significantly reduced cell viability in the majority of the cell lines (LP6, LPS141, and T778) with IC50 less than lOOnM. Results are shown in FIG. 6. The IC50 values were as shown in Table 1 :Table 1

[0168] At the molecular level, we determined that Compound A22 inhibited the activity of CDK7, CDK9, and CKla as indicated by reduced phosphorylation of RNA polymerase II at pSer7 / 5 CTD, reduced pSer2 / 5 CTD, increased p53 levels leading to PARP cleavage (FIG. 7). Moreover, Compound A22 suppressed the expression of target genes (MCL1, MYC, and MDM2) that are transcriptionally regulated by CDK7 / 9.

[0169] In LP6 cells, Compound A22 induced apoptosis in a dose-dependent manner as reflected by Annexin V staining. Results are shown in FIG. 8.

[0170] Additionally, using cells from PDX models (LPS27 and LPS3), it was determined that Compound A22 enhanced Delta priming at nanomolar concentrations.Example 4

[0171] To assess the in vivo efficacy of Compound A22, LPS3 and LPS27 liposarcoma PDX mouse models were established. Mice were treated with vehicle control or Compound A22 at 15 mg / kg QOD for 10 doses (n = 8 / group). Compound A22 effectively suppressed tumor growth and tumor suppression was observed in less than a week post-treatment (FIGS. 9 and 10). Correspondingly, survival was significantly improved in the Compound A22 group. During the treatment course, Compound A22 was well tolerated as there were no clear differences in body weight between the two treatment groups (FIG. 11).

[0172] Tumors from both groups were harvested for pharmacodynamic assessments. Consistent with our in vitro results, p53 levels were significantly increased by Compound A22 treatment whereas pSer7 / 5 CTD, pSer2 / 5 CTD, MDM2, MCL-1, and Myc levels were markedly reduced. Moreover, p21, a major target regulated by p53, was also increased by Compound A22 treatment. By immunohistochemistry (H4C) analyses, we further confirmed the alterations in p53, MDM2, MCL-1, and p21 in the tumor tissues. Ki67, a marker of cell proliferation, was significantly reduced by Compound A22 treatment. These results suggest that Compound A22 was successfully delivered to the PDX tumors and exerted its tumorsuppressive effects through inhibition of CKla, CDK7, and CDK9 .Example 5A Pilot Study of Compound A22 in Patients with Metastatic and / or RecurrentLiposarcomas Characterized by MDM2 amplifications

[0173] This is a single-arm pilot trial with primary objective to confirm the safety and tolerability of Compound A22 in patients with metastatic and / or recurrent liposarcomas and to confirm the recommended dose schedule is safe in this population for further clinical development. This study relies on data from prior Phase 1 and lb trials performed in patients with advanced, refractory acute myeloid leukemia as well as another trial studying patients with advanced solid tumors.

[0174] Based on preliminary clinical pharmacodynamic data from Cohorts 1-7 in the study of Compound A22 in acute myeloid leukemia, Compound A22 exposure appears to increase with dose in a roughly proportional manner with peak drug concentrations at the highest evaluated dose (42 mg) of up to 289 ng / mL. Thus, based on previous studies, Compound A22 is administered orally at a dose of 21 mg, 3 times per week (either Monday, Wednesday, Friday or Tuesday, Thursday, Saturday) for 4 weeks in 28-day cycles in this study.

[0175] Study drug administration is performed on an outpatient basis. No investigational or commercial anticancer agents or therapies other than those described below are administered with the intent to treat the participant's malignancy.

[0176] The participant is requested to maintain a medication diary of each dose of medication. Participants provide the medication diary to clinic staff at the end of each cycle.

[0177] During this study, disease status of participants is re-evaluated every 2 cycles (approx, every 8 weeks). In addition to a baseline scan, confirmatory scans are obtained 8 weeks following initial documentation of an objective response. The window for imaging scans is + / - 3 days from the designated timepoint.Study Population

[0178] Study participants have histologically-confirmed metastatic and / or recurrent liposarcoma and have the subtypes of well-differentiated and / or dedifferentiated liposarcoma, which are pathognominically associated with MDM2 amplifications.

[0179] Participants must have an Eastern Cooperative Oncology Group (ECOG) performance status <2.

[0180] Participants must have adequate organ and marrow function as defined by the following metrics resulted within 7 days of study enrollment: WBC >3000 / mm3, platelets >75,000pl, ANC >1500pl, Hgb >9g / dl, Creatinine <1.5 x ULN or measured CrCl of >50ml / m2 / 1.73 m2, total bilirubin <2 x ULN, AST / ALT <3 x ULN.

[0181] Participants must have measurable disease, defined as at least one lesion that can be accurately measured in at least one dimension (longest diameter to be recorded for non- nodal lesions and short axis for nodal lesions) as >20 mm (>2 cm) by chest x-ray or as >10 mm (>1 cm) with CT scan, MRI, or calipers by clinical exam. Evaluation of measurable disease is described further below.

[0182] Participants must have recovered from toxicity related to prior therapy to grade <=1 (defined by CTCAE v5.0) (except alopecia and neuropathy, or immunotherapy related hypothyroidism).

[0183] As the effect of this study drug on the developing human fetus is not known, women of child-bearing potential and men must agree to use at least 2 methods of contraception (abstinence; hormonal or barrier method of birth control) for the study and at least 2 months after completion.

[0184] Female participants of childbearing potential must have a negative serum pregnancy test within 7 days of study enrollment.

[0185] Participants must have the ability to understand and the willingness to sign a written informed consent document and must be at least 18 years old.

[0186] Subjects with current evidence of active and uncontrolled infection, NYHA Class III-IV CHF, documented Child's class B-C cirrhosis, or uncontrolled medical disease which in the opinion of the investigator or the sponsor could compromise safety and / or assessment of efficacy are excluded from the study. Subjects with active infection with hepatitis C virus (HCV) or hepatitis B virus (HBV); subjects who are positive for hepatitis B core antibody, hepatitis B surface antigen, or hepatitis C antibody must have a negative PCR result before enrollment. Subjects who have a positive PCR result are excluded.

[0187] Subjects who have had a major surgical procedure or open surgical biopsy within 28 days of first dose of study drug are excluded from the study.

[0188] Subjects with active central nervous system (CNS) disease involvement, or prior history of NCI CTCAE Grade >3 drug-related CNS toxicity are excluded from the study. Subjects with known CNS metastases that are treated and stable (without evidence of CNS toxicity) and are not requiring systemic steroids are allowed to be enrolled.

[0189] Subjects with known psychiatric or substance abuse disorders that would interfere with cooperation with the requirements of the trial are excluded from the study. Subjects who had myocardial infarction within 12 months of screening are excluded from the study. Subjects who are using any other concurrent investigational agents or anticancer agents, excluding hormonal therapy for breast or prostate cancer, are exclude from the study.Pregnant women are excluded from this study because there is an unknown but potential risk for adverse events. There is also an unknown risk in nursing infants and breastfeeding should be discontinued if the mother is enrolled in the study.Evaluation and Measurement of Response to Compound A22

[0190] Response and progression are evaluated in this study using the standard international criteria proposed by the Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version 1.1). Changes in the largest diameter (unidimensional measurement) of the tumor lesions and the shortest diameter in the case of malignant lymph nodes are used in the RECIST criteria.

[0191] Only those participants who have measurable disease present at baseline, have received at least one cycle of therapy, and have had their disease re-evaluated are considered evaluable for target disease response. The response of these participants is classified according to the definitions stated below. Participants who exhibit objective disease progression prior to the end of cycle 1 are also considered evaluable.

[0192] Participants who have lesions present at baseline that are evaluable but do not meet the definitions of measurable disease, have received at least one cycle of therapy, and have had their disease re-evaluated are considered evaluable for non-target disease. The response assessment is based on the presence, absence, or unequivocal progression of the lesions.

[0193] Measurable lesions are defined as those that can be accurately measured in at least one dimension (longest diameter to be recorded) as >20 mm by chest x-ray or >10 mm withCT scan, MRI, or calipers by clinical exam. All tumor measurements are recorded in millimeters (or decimal fractions of centimeters). Tumor lesions that are situated in a previously irradiated area might or might not be considered measurable. Previously irradiated lesions that have progressed after radiation are allowed as measurable disease.

[0194] To be considered pathologically enlarged and measurable, a malignant lymph node must be >15 mm in short axis when assessed by CT scan (CT scan slice thickness recommended to be no greater than 5 mm). At baseline and in follow-up, only the short axis is measured and followed.

[0195] All other lesions (or sites of disease), including small lesions (longest diameter <10 mm or pathological lymph nodes with >10 to <15 mm short axis), are considered non- measurable disease. Bone lesions, leptomeningeal disease, ascites, pleural / pericardial effusions, lymphangitis cutis / pulmonitis, inflammatory breast disease, abdominal masses (not followed by CT or MRI), and cystic lesions are all considered non-measurable.

[0196] Cystic lesions that meet the criteria for radiographically defined simple cysts are not considered as malignant lesions (neither measurable nor non-measurable) since they are, by definition, simple cysts. ‘Cystic lesions’ thought to represent cystic metastases can be considered as measurable lesions, if they meet the definition of measurability described above. However, if non-cystic lesions are present in the same participant, these are preferred for selection as target lesions for purposes of this study.

[0197] All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, are identified as target lesions and recorded and measured at baseline. Target lesions are selected on the basis of their size (lesions with the longest diameter), are representative of all involved organs, and are those that lend themselves to reproducible repeated measurements. In some instances, the largest lesion does not lend itself to reproducible measurement. In such instances, the next largest lesion which can be measured reproducibly is selected. A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions is calculated and reported as the baseline sum diameters. If lymph nodes are included in the sum, then only the short axis is added into the sum. The baseline sum diameters are used as reference to further characterize any objective tumor regression in the measurable dimension of the disease.

[0198] All other lesions (or sites of disease) including any measurable lesions over and above the 5 target lesions are identified as non-target lesions and are recorded at baseline. Although this study does not require measurements of these non-target lesions, but the presence, absence, or unequivocal progression of each is noted throughout follow up.Methods for Evaluation of Disease

[0199] Measurements are taken and recorded in metric notation using a ruler, calipers, or a digital measurement tool. Baseline evaluations are performed as closely as possible to the beginning of treatment and not more than 4 weeks before the beginning of the treatment.

[0200] The same method of assessment and the same technique is used to characterize each identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is preferred to evaluation by clinical examination unless the lesion(s) being followed cannot be imaged but are assessable by clinical exam.

[0201] Clinical lesions are only considered measurable when they are superficial (e.g., skin nodules and palpable lymph nodes) and >10 mm in diameter as assessed using calipers (e.g., skin nodules). In the case of skin lesions, documentation by color photography, including a ruler to estimate the size of the lesion, is recommended.

[0202] Lesions on chest x-ray are acceptable as measurable lesions when they are clearly defined and surrounded by aerated lung. However, measurement by CT is preferred. This study has defined measurability of lesions on CT scan based on the assumption that CT thickness is 5mm or less. If CT scans have slice thickness greater than 5 mm, the minimum size of a measurable lesion should be twice the slice thickness. MRI is also acceptable in certain situations (e.g., for body scans).

[0203] Use of MRI to measure lesions remains a complex issue. MRI has excellent contrast, spatial, and temporal resolution. However, there are many image acquisition variables involved in MRI, which greatly impact image quality, lesion conspicuity, and measurement. Furthermore, the availability of MRI varies globally. As with CT, if an MRI is performed, the technical specifications of the scanning sequences used are optimized for the evaluation of the type and site of disease. Furthermore, as with CT, the modality used at follow-up should be the same as was used at baseline and the lesions should be measured / assessed on the same pulse sequence. For follow-ups, the same type of scanner isused and the image acquisition protocol is followed as closely as possible to prior scans. Body scans are performed with breath-hold scanning techniques, if possible.

[0204] While FDG-PET response assessments need additional study, it is sometimes reasonable to incorporate the use of FDG-PET scanning to complement CT scanning in assessment of progression (particularly possible 'new' disease). New lesions on the basis of FDG-PET imaging are identified according to the following algorithm: (a) Negative FDG- PET at baseline, with a positive FDG-PET at follow-up is a sign of PD based on a new lesion, (b) No FDG-PET at baseline and a positive FDG-PET at follow-up: If the positive FDG-PET at follow-up corresponds to a new site of disease confirmed by CT, this is PD. If the positive FDG-PET at follow-up is not confirmed as a new site of disease on CT, additional follow-up CT scans are needed to determine if there is truly progression occurring at that site (if so, the date of PD will be the date of the initial abnormal FDG-PET scan). If the positive FDG-PET at follow-up corresponds to a pre-existing site of disease on CT that is not progressing on the basis of the anatomic images, this is not PD. (c) FDG-PET may be used to upgrade a response to a CR in a manner similar to a biopsy in cases where a residual radiographic abnormality is thought to represent fibrosis or scarring. The use of FDG-PET in this circumstance is prospectively described in the protocol and supported by disease-specific medical literature for the indication. However, it is acknowledged that both approaches may lead to false positive CR due to limitations of FDG-PET and biopsy resolution / sensitivity.

[0205] A ‘positive’ FDG-PET scan lesion means one which is FDG avid with an uptake greater than twice that of the surrounding tissue on the attenuation corrected image.

[0206] At present, the low dose or attenuation correction CT portion of a combined PET- CT is not always of optimal diagnostic CT quality for use with RECIST measurements. However, if the site can document that the CT performed as part of a PET-CT is of identical diagnostic quality to a diagnostic CT (with IV and oral contrast), then the CT portion of the PET-CT can be used for RECIST measurements and can be used interchangeably with conventional CT in accurately measuring cancer lesions over time. Note, however, that the PET portion of the CT introduces additional data which may bias an investigator if it is not routinely or serially performed.

[0207] Cytology and histology are used to differentiate between partial responses (PR) and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as germcell tumors, where known residual benign tumors can remain). The cytological confirmation of the neoplastic origin of any effusion that appears or worsens during treatment when the measurable tumor has met criteria for response or stable disease is mandatory to differentiate between response or stable disease (an effusion may be a side effect of the treatment) and progressive disease.

[0208] Regarding target lesions, a Complete Response (CR) means the disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. A Partial Response (PR) means at least a 30% decrease in the sum of the diameters of target lesions, using the baseline sum diameters as reference. Progressive Disease (PD) means at least a 20% increase in the sum of the diameters of target lesions, using the smallest sum on study as reference (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. The appearance of one or more new lesions is also considered progression. Stable Disease (SD) means neither sufficient lesion shrinkage to qualify for PR nor sufficient lesion increase to qualify for PD, using the smallest sum diameters while on study as reference.

[0209] Regarding non-target lesions, a Complete Response (CR) means disappearance of all non-target lesions and normalization of tumor marker level. All lymph nodes must be non-pathological in size (<10 mm short axis). If tumor markers are initially above the upper normal limit, they must normalize for a patient to be considered in Complete Response. NonComplete Response / Non-Progressive Disease (Non-CR / Non-PD) means the persistence of one or more non-target lesion(s) and / or maintenance of tumor marker level above the normal limits. Progressive Disease (PD) means the appearance of one or more new lesions and / or unequivocal progression of existing non-target lesions. Unequivocal progression does not trump target lesion status but must be representative of overall disease status change (i.e., not a single lesion increase). Although a clear progression of “non-target” lesions only is exceptional, the opinion of the treating physician prevails in such circumstances, and the progression status is confirmed at a later time by the review panel (or Principal Investigator).

[0210] In the event of the finding of a new lesion, the finding of a new lesion should be unequivocal (i.e., not due to difference in scanning technique, imaging modality, or findings thought to represent something other than tumor). For example, ‘new’ bone lesions may be simply healing or flare of pre-existing lesions. Such lesions are not “new” lesions. If a newlesion is equivocal (e.g., because of small size), a follow-up evaluation clarifies if it represents new disease. If PD is confirmed, progression is declared using the date of the initial scan on which the lesion was discovered.

[0211] The best overall response is the best response recorded from the start of the treatment until disease progression / recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment will depend on the achievement of both measurement and confirmation criteria.

[0212] For participants with measurable disease (i.e., target disease), the study uses the below table summary for evaluating participants’ responses to Compound A22:

[0213] For participants with non-measurable disease (i.e., non-target disease), the study uses the below table summary for evaluating participants’ responses to Compound A22:

[0214] The duration of overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started, or death due to any cause). Participants without events reported are censored at the last disease evaluation.

[0215] The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented, or death due to any cause. Participants without events reported are censored at the last disease evaluation.

[0216] Duration of stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements.

[0217] Overall Survival (OS) is defined as the time from randomization (or registration) to death due to any cause, or censored at date last known alive. Progression-Free Survival (PFS) is defined as the time from registration to the earlier of progression or death due to any cause. Participants alive without disease progression are censored at date of last disease evaluation. Time to Progression (TTP) is defined as the time from randomization (or registration) to progression, or censored at date of last disease evaluation for those without progression reported.

[0218] As a general consideration, the sample size in this study is not justified in terms of statistical hypotheses, but as the equivalent of a phase 1 expansion cohort to ascertain safety and to justify the dosing schedule in this patient population. Of note, two prior dose escalation phase 1 studies have already been completed in different patient populations (acute myeloid leukemia patients in one, and unselected solid tumor patients with no liposarcomas represented in the other) that established a recommended phase 2 dose, which is noted as the starting dose in this study. In most phase I trials, the expansion cohorts usually comprise between 10 to 15 participants to be assured of reasonable safety to proceed into furtherdevelopment. This study has chosen n=12 for safety while also exploring biomarker changes to assess target engagement and early signs of mechanism-based anticancer activities. As a reference, the Objective Response Rate (ORR) will be higher than 20% (respectively, 10%) if more than 5 (respectively, 4) ORRs out of 12 patients are observed, using a one-sided 90%- significance exact binomial confidence interval (90% interval: 21.9%-100% for 20% ORR, 15.4%- 100% for 10%ORR).

[0219] The study follows the below schedule of dose administration and assessments.Schedule of Administration and Assessments

[0220] Schedule notes: a: Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, phosphorus, potassium, total protein, SGOT [AST], SGPT [ALT], sodium, b: Serum pregnancy test (women of childbearing potential). Serum OR urine test is acceptable, c: Off Treatment evaluation: A follow up visit or other contact is required in order to identify SAEs during the 30 days following the end of study drug administration. If the patient is unable to complete an in person visit, a phone call or messaging through the electronic medical record system 1-2 months after completing study drug administration is acceptable for SAE follow up. The lab draws and in person examinations at this time point are to be completed at the time of end of study drug administration (which can be at time of progression and may coincide with the last timepoint of active study participation), d: Follow-Up evaluation. Survival will continue to be captured every 3 months for 1 year from end of drug administration on study or until participant withdrawal, death, or removal from study, e: Off Study evaluation, f: Cycles are 28 days in length, g. For the first cycle, all assessments after C1D1 should occur on the specified day + / -1 day except as otherwise specified, for all later timepoints the assessments can occur + / - 3 days from the specified date unless otherwise specified, h. The on treatment biopsy should occur on C1D22 + / - 8 days. i. Collection of MIC-1 at baseline should be pre-dosing with Compound A22. Collection of MIC-1 on CID 15, C2D1, C3D1 will occur at approximately 2 hours post-dose (e.g approximately + / - 1 hour) on those days. j. BTX-A51 level on C2D1 should be collected pre-dose on that day.Example 6

[0221] A 65 year old female with well differentiated / de-differentiated liposarcoma received 21 mg of Compound A22 on a 3x / week schedule (MWF). Prior to treatment with Compound A22, she was initially diagnosed with a multifocal abdominal / retroperitoneal mass and underwent a debulking surgery, with pathology showing high grade dedifferentiated liposarcoma. Sequencing of her tumor revealed ARID 1 A, ATRX, and CDKN2A mutations and CDK4 and MDM2 amplification. She was treated with liposomal doxorubicin and underwent additional resection. Approximately 18 months later, imaging showed a recurrence and she underwent a third surgery. Post operative imaging again showed enlarging disease and she was started on palbociclib, which she continued for two years with continued slow progression. She then started Compound A22 on a 3 doses per week schedule (MWF) for 28 days per cycle. At baseline, target lesion linear diameter measured 5.5cm.Imaging after 2 cycles of Compound A22 showed reduction in target lesions to 5.2 cm (-6% change).Example 7

[0222] A 67 year old man with well differentiated liposarcoma received 21 mg of Compound A22 on a 3x / week schedule (MWF). Prior to treatment with Compound A22, he was initially diagnosed with a retroperitoneal lipomatous mass that was resected and pathology showed well differentiated liposarcoma measuring 24 cm. Four years later, imaging showed an increasing lipomatous mass that was biopsied and proven to be a of local recurrence of well differentiated liposarcoma which was initially monitored for a year. Next, he was started on palbociclib which was discontinued after 3 months due to continued tumor growth. He then started Compound A22 on a 3 times per week schedule (MWF) for 28 days per cycle. At baseline, target lesion linear diameter measured 5.2cm. Imaging after 1 cycle of 28 days of Compound A22 showed reduction in target lesions to 4.3 cm (-17% change).

[0223] The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the claimed embodiments, and are not intended to limit the scope of what is disclosed herein.Modifications that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to be incorporated herein by reference.

Claims

What is claimed is:

1. A method of treating liposarcoma in a subject, comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of Compound A22,A22, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the liposarcoma is well differentiated.

3. The method of claim 1, wherein the liposarcoma is de-differentiated.

4. The method of any one of claim 1 to 104, wherein the patient has TP53VI .

5. The method of any one of claims 1 to 2, wherein the subject over-expresses MDM2 or has a >4 copy number of the MDM2 gene.

6. The method of any one of claims 1 to 5, wherein the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene.

7. The method of any one of claims 1 to 6, wherein the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene.

8. The method of any one of claims 1 to 7, wherein the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

9. The method of any one of claims 1 to 8, wherein the liposarcoma is refractory.

10. The method of any one of claims 1 to 9, wherein the liposarcoma is metastatic.

11. The method of any one of claims 1 to 10, wherein the liposarcoma is drug-resistant.

12. The method of any one of claims 1 to 11, wherein the subject has failed a prior therapy.

13. The method of any one of claims 1 to 12, wherein the subject is a human.

14. The method of any one of claims 1 to 13, wherein the compound is administered orally.

15. The method of any one of claims 1 to 14, wherein the compound is administered as a tablet or capsule.

16. The method of any one of claims 1 to 15, wherein the therapeutically effective amount ranges from about 0.001 to about 10 mg / kg per day.

17. The method of any one of claims 1 to 16, wherein the therapeutically effective amount ranges from about 15 to about 35 mg per day.

18. The method of any one of claims 1 to 17, wherein the therapeutically effective amount is about 1, about 2, about 3, about 5, about 8, about 10, about 11, about 14, about 15, about 17, about 20, about 21, about 25, about 30, or about 35 mg per day.

19. The method of any one of claims 1 to 18, wherein the compound is administered in one or more cycles.

20. The method of any one of claims 1 to 19, wherein the compound is administered in a 28-cycle.

21. The method of any one of claims 1 to 20, wherein the compound is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days per week.

22. The method of any one of claims 1 to 21, wherein the compound is administered for 3 days or 5 days per week.

23. The method of any one of claims 1 to 22, wherein the compound is administered on Days 1, 2, and 3 in a week.

24. The method of any one of claims 1 to 23, wherein the compound is administered on Days 1, 3, and 5 in a week.

25. The method of any one of claims 1 to 22, wherein the compound is administered on three non-consecutive days in a week.

26. The method of any one of claims 1 to 25, wherein the compound is administered in a 28-day cycle for 3 days per week.

27. The method of claim 26, wherein the compound is administered on three non- consecutive days per week.

28. The method of claim 27, wherein the compound is administered on Days 1, 3, and 5 per week.

29. The method of any one of claims 1 to 22, wherein the compound is administered in a 28-day cycle for 5 days per week.

30. The method of claim 29, wherein the compound is administered in a 28-day cycle on Days 1, 2, 3, 4, and 5 per week.

31. The method of any one of claims 1 to 30, wherein the compound is formulated as a p- toluenesulfonate salt or a di- -toluenesulfonate salt.

32. The method of any one of claims 1 to 31, wherein the therapeutically effective amount is about 21 mg.

33. The method of any one of claims 1 to 31, wherein the therapeutically effective amount is about 30 mg.

34. The method of any one of claims 1 to 31, wherein the therapeutically effective amount is about 35 mg.

35. A method of inhibiting CDK7 in a subject having liposarcoma, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of Compound A22, or a pharmaceutically acceptable salt thereof.

36. The method of claim 35, wherein the liposarcoma is well differentiated.

37. The method of claim 35, wherein the liposarcoma is de-differentiated.

38. The method of any one of claims 35 to 37, wherein the compound is formulated as a -toluenesulfonate salt or a di- -toluenesulfonate salt.

39. The method of any one of claims 35 to 38, wherein the liposarcoma has been resistant to a CDK4 / 6 inhibitor.

40. The method of any one of claims 35 to 39, wherein the subject has TP53VI .

41. The method of any one of claims 35 to 40, wherein the subject over-expresses MDM2 or has a >4 copy number of the MDM2 gene.

42. The method of any one of claims 35 to 41, wherein the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene.

43. The method of any one of claims 35 to 42, wherein the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene.

44. The method of any one of claims 35 to 43, wherein the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

45. The method of any one of claims 35-44, wherein administering comprises administering about 21 mg of Compound A22 three times per week.

46. The method of any one of claims 35-44, wherein administering comprises administering about 30 mg of Compound A22 three times per week.

47. The method of any one of claims 35-44, wherein administering comprises administering about 35 mg of Compound A22 three times per week.

48. A method of treating liposarcoma in a subject having amplified expression of MDM2 or having a >4 copy number of the MDM2 gene, comprising administering to the subject a therapeutically effective amount of Compound A22, or a pharmaceutically acceptable salt thereof.

49. The method of claim 48, wherein the liposarcoma is well differentiated.

50. The method of claim 48, wherein the liposarcoma is de-differentiated.

51. The method of any one of claims 48 to 50, wherein the compound is formulated as a -toluenesulfonate salt or a di- -toluenesulfonate salt.

52. The method of any one of claims 48 to 49, wherein the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene.

53. The method of any one of claims 48 to 52, wherein the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene.

54. The method of any one of claims 48 to 53, wherein the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

55. The method of any one of claims 48-54, wherein administering comprises administering about 21 mg of Compound A22 three times per week.

56. The method of any one of claims 48 to 54, wherein administering comprises administering about 30 mg of Compound A22 three times per week.

57. The method of any one of claims 48 to 54, wherein administering comprises administering about 35 mg of Compound A22 three times per week.

58. A method of inhibiting MDM2 expression in a subject having liposarcoma, comprising administering to the subject a therapeutically effective amount of Compound A22, or a pharmaceutically acceptable salt thereof.

59. The method of claim 58, wherein the liposarcoma is well differentiated.

60. The method of claim 58, wherein the liposarcoma is de-differentiated.

61. The method of any one of claims 58 to 61, wherein the compound is formulated as a -toluenesulfonate salt or a di- -toluenesulfonate salt.

62. The method of any one of claims 58 to 59, wherein the subject over-expresses MDM2 or has a >4 copy number of the MDM2 gene.

63. The method of any one of claims 58 to 62, wherein the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene.

64. The method of any one of claims 58 to 63, wherein the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene.

65. The method of any one of claims 58 to 64, wherein the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

66. The method of any one of claims 58-65, wherein administering comprises administering about 21 mg of Compound A22 three times per week.

67. The method of any one of claims 58-65, wherein administering comprises administering about 30 mg of Compound A22 three times per week.

68. The method of any one of claims 58-65, wherein administering comprises administering about 35 mg of Compound A22 three times per week.

69. A method of inhibiting CDK9 in a subject having liposarcoma, comprising administering to the subject an effective amount of Compound A22, or a pharmaceutically acceptable salt thereof.

70. The method of claim 69, wherein the liposarcoma is well differentiated.

71. The method of claim 69, wherein the liposarcoma is de-differentiated.

72. The method of any one of claims 69 to 71, wherein the compound is formulated as a -toluenesulfonate salt or a di- -toluenesulfonate salt.

73. The method of any one of claims 68 to 70, wherein the liposarcoma has been resistant to a CDK4 / 6 inhibitor.

74. The method of any one of claims 68 to 70, wherein the subject has P53VI .

75. The method of any one of claims 68 to 74, wherein the subject over-expresses MDM2 or has a >4 copy number of the MDM2 gene.

76. The method of any one of claims 68 to 75, wherein the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene.

77. The method of any one of claims 68 to 76, wherein the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene.

78. The method of any one of claims 68 to 77, wherein the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

79. The method of any one of claims 69 to 78, wherein administering comprises administering about 21 mg of Compound A22 three times per week.

80. The method of any one of claims 69 to 78, wherein administering comprises administering about 30 mg of Compound A22 three times per week.

81. The method of any one of claims 69 to 78, wherein administering comprises administering about 35 mg of Compound A22 three times per week.

82. A method of inhibiting CKla in a subject having liposarcoma, comprising administering to the subject an effective amount of Compound A22, or a pharmaceutically acceptable salt thereof.

83. A method of inhibiting CKla and CDK9 in a subject having liposarcoma, comprising administering to the subject an effective amount of Compound A22, or a pharmaceutically acceptable salt thereof.

84. The method of any one of claims 82 to 83, wherein the compound is formulated as a -toluenesulfonate salt or di- -toluenesulfonate salt.

85. The method of any one of claims 82 to 84, wherein the liposarcoma has been resistant to a CDK4 / 6 inhibitor.

86. The method of any one of claims 82 to 84, wherein the subject has P53VI .

87. The method of any one of claims 82 to 86, wherein the subject over-expresses MDM2 or has a >4 copy number of the MDM2 gene.

88. The method of any one of claims 82 to 87, wherein the subject over-expresses MDM2 or has a >6 copy number of the MDM2 gene.

89. The method of any one of claims 82 to 88, wherein the subject over-expresses MDM2 or has a >8 copy number of the MDM2 gene.

90. The method of any one of claims 82 to 89, wherein the subject over-expresses MDM2 or has a >10 copy number of the MDM2 gene.

91. The method of any one of claims 82-90, wherein administering comprises administering about 21 mg of Compound A22 three times per week.

92. The method of any one of claims 82-90, wherein administering comprises administering about 30 mg of Compound A22 three times per week.

93. The method of any one of claims 82-90, wherein administering comprises administering about 35 mg of Compound A22 three times per week.

94. A pharmaceutical composition comprising compound A22,or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, for use in the treatment of a patient diagnosed with liposarcoma.

95. The pharmaceutical composition for use according to claim 94, wherein the patient has P53wt.

96. The pharmaceutical composition for use according to any one of claims 94 to 95, wherein the treatment comprises administration of the pharmaceutical composition to the patient on three non-consecutive days in a week.

97. The pharmaceutical composition for use according to any one of claims 94 to 96, wherein the administration occurs for one or more cycles.

98. The pharmaceutical composition for use according to any one of claims 94 to 97, wherein the cycle is 28 days.

99. The pharmaceutical composition for use according to any one of claims 94 to 98, wherein the pharmaceutical composition is orally administered to the patient.

100. The pharmaceutical composition for use according to any one of claims 94 to 99, wherein the pharmaceutical composition is formulated as an oral unit dosage form.

101. The pharmaceutical composition for use according to any one of claims 94 to 100, wherein the oral unit dosage form comprises about 21 mg of Compound A22.

102. The pharmaceutical composition for use according to any one of claims 94 to 100, wherein the oral unit dosage form comprises about 30 mg of Compound A22.

103. The pharmaceutical composition for use according to any one of claims 94 to 100, wherein the oral unit dosage form comprises about 35 mg of Compound A22.

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