Selective CDK9 Inhibitors Research Articles

The synthesis of PROTAC molecule and new target KAT6A identification of CDK9 inhibitor iCDK9

Cyclin-dependent kinases (CDKs) have become potential targets for treating various diseases, especially cancer. Compound iCDK9 is an excellent and selective CDK9 inhibitor, but its major limitation is the potential toxicity and poor understanding of the underlying mechanism. The PROTAC (proteolysis targeting chimera) degraders of bioactive molecules can significantly induce in vitro and in vivo degradation of their target protein with high selectivity and effectively reduce the dose-limiting toxicity of small molecule drugs. Therefore, we designed and synthesized the bifunctional PROTAC molecules of iCDK9, being used for identifying its previously unknown target and revealing the underlying pharmacological mechanism. The PROTAC bifunctional molecule CD-5 could selectively and significantly degrade CDK9 with low cell toxicity. Therefore, we selected CD-5 as a chemical prober in the SILAC quantitative proteomic analysis, which disclosed that CD-5 could enormously lessen the lysine acetyltransferase KAT6A. Furthermore, KAT6A degradation induced by CD-5 repressed the levels of H3K14Ac and H3K23Ac. Lastly, the streptavidin immunoprecipitation (IP) assay confirmed a direct interaction between KAT6A and iCDK9. Collectively, our results uncover that KAT6A is a potential non-kinase target of iCDK9. Notably, this study also demonstrates that the PROTAC-SILAC strategy is an alternative approach for cellular target identification of bioactive molecules.

Abstract 2306: BLU-222, an investigational, potent, and selective CDK2 inhibitor, demonstrated robust antitumor activity in CCNE1-amplified ovarian cancer models

Abstract Background: Cyclin-dependent kinases (CDK) are a class of enzymes that, along with their regulatory cyclin binding partners, drive cell cycle progression. Cell cycle dysregulation is a hallmark of cancer and targeting its genetic drivers can confer therapeutic benefits. Cyclin E1 (CCNE1) gene alterations are common in patients with ovarian cancer and can be found in ~20% of high-grade serous ovarian cancer cases, which tend to be platinum therapy resistant and therefore represent a high medical need. Cyclin E1 is the canonical binding partner of CDK2, which becomes constitutively active when CCNE1 is amplified and overexpressed. Selectively inhibiting CDK2 is an attractive therapeutic option for CCNE1-amplified tumors and may limit off-target CDK-driven toxicities. Here we report preclinical validation studies leading to the development of an orally available CDK2 inhibitor, BLU-222, for the treatment of ovarian cancer harboring a CCNE1 amplification. Methods: BLU-222 selectivity was measured by enzyme assays, cellular target engagement assays (NanoBRET), and proliferation assays in a panel of ovarian cancer cell lines. In vitro cellular potency was assessed by phospho-Rb levels. In vivo antitumor activity of BLU-222 as a single agent or in combination with carboplatin was measured in an OVCAR-3 cell line-derived xenograft (CDX) tumor model harboring a CCNE1 amplification. Results: BLU-222 demonstrated selectivity, both biochemically and in cells, with low nanomolar potency for CDK2 vs other CDK family members (CDK1, -4, -6, -7, and -9). In a panel of ovarian cancer cell lines, those with CCNE1 amplifications were highly sensitive to BLU-222. Consistent with the in vitro profiling, BLU-222 exhibited significant antitumor activity in the OVCAR-3 CDX model. While administration of single-agent carboplatin led to stasis in vivo, the combination of BLU-222 + carboplatin induced durable tumor regression even after treatment cessation. Conclusions: These data provide a strong rationale for advancing BLU-222 towards clinical development in patients with CCNE1-amplified ovarian cancer. Citation Format: Victoria Brown, Phil Ramsden, Nealia House, Richard Vargas, Jian Guo, Ruduan Wang, Riadh Lobbardi, Maxine Chen, Douglas Wilson, Joseph Kim, Neil Bifulco, Michelle Maynard, Emanuele Perola, Dean Zhang, Steve Wenglowsky, Yoon Jong Choi. BLU-222, an investigational, potent, and selective CDK2 inhibitor, demonstrated robust antitumor activity in CCNE1-amplified ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2306.

Abstract 2568: ARTS-021 is a potent and selective CDK2 inhibitor that demonstrates anti-cancer activity in preclinical cancer models with CCNE1 amplification

Abstract Cyclin E1 amplification is prevalent in cancers with high unmet medical need such as high grade serous ovarian cancer, stomach cancer and esophageal cancer. Cancer cell lines with high expression level of cyclin E1 gene exhibited profound sensitivity to CDK2 gene depletion, suggesting CDK2 selective inhibitors have the potential to treat patients harboring Cyclin E1 alterations. Here we report the development and preclinical characterization of ARTS-021, an orally bioavailable small molecule CDK2 selective inhibitor. In enzymatic assay against a panel of different CDKs, ARTS-021 potently inhibits the activity of CDK2/CyclinE1 complex with 50% inhibitory concentration (IC50) in the sub-nanomolar range, displaying superior selectivity against other CDKs (CDK1,4,6,7,9). Very low hit rate (S(10)=0.022, at 1uM) is observed when ARTS-021 is assessed in whole Kinome profiling, further revealing the selectivity of this compound. ARTS-021 also demonstrates potent CDK2 inhibition and selectivity against other CDK family members (CDK1,4,6,9) in a series of cellular assays. Consistent with genetic CDK2 dependence data, cell lines with CCNE1 amplification are particularly sensitive to ARTS-021. Double nanomolar ARTS-021 inhibits Rb phosphorylation and blocks G1/S transition, leading to cell growth arrest specifically in CCNE1 amplified cell lines. This CDK2 dependent anti-tumor activity is further validated in vivo, where daily ARTS-021 administration leads to tumor stasis in CCNE1 amplified but not wild type xenograft models. Taken together, these data demonstrate ARTS-021 as a promising CDK2 selective inhibitor with strong potential towards the development of effective therapies for CCNE1 altered cancer. Citation Format: jiaqi Liang, Dai Cheng, Yali Guo, xiaobin zhang, Xipan Liu, Jia Fu, Qiang Ding, Wen Xiong, Fang Li, Yaoyu Chen, Qing Sheng. ARTS-021 is a potent and selective CDK2 inhibitor that demonstrates anti-cancer activity in preclinical cancer models with CCNE1 amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2568.

Abstract 2574: Q901; a highly selective covalent cdk7 inhibitor inducing substantial anti-tumor effect in a broad spectrum of solid tumor lineages

Abstract As a key regulator of its dual role in transcription and cell cycle progression, CDK7 has been determined to be an attractive therapeutic target in a variety of tumor types driven by cell cycle dysregulation and aberrant control of the transcriptional processes. Q901 is an irreversible small molecule inhibitor with extreme selectivity and potent inhibition activity for CDK7, which forms a covalent bond with C312 of the CDK7 C-terminal domain. Q901 demonstrated selective cytotoxic activities in a broad spectrum of solid tumor lineages. TP53 mutation could be a potential predictive marker for the Q901 response as TP53 wild-type cancer cells demonstrates a significantly increased cytotoxic response compared to TP53 mutant cells in the bioinformatic analysis. In addition, gene expression profiling analysis showed a dose-response increase of POLR2A expression upon Q901 treatment and a correlation with tumor growth inhibition rate, indicating that POLR2A might be a potential PD marker for the Q901-induced response. Taken together, these data support the clinical development of Q901 for solid tumor indications. Q901 is progressing to IND-enabling studies to support the start of Phase 1 oncology clinical trials in early 2022. Citation Format: Donghoon Yu, Yeejin Jeon, Seung-Joo Lee, Jaeseung Kim, Kiyean Nam. Q901; a highly selective covalent cdk7 inhibitor inducing substantial anti-tumor effect in a broad spectrum of solid tumor lineages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2574.

Abstract 1859: VIP152, a selective CDK9 inhibitor, demonstrates sensitivity in gynecologic cell lines that are cisplatin sensitive or resistant and delivers in vivo antitumor efficacy

Abstract Background: The Myc transcription factor has a short half-life of 20-30 minutes (Ramsay et al. 1984) and oncogenic activation and transcriptional addiction leads to sustained levels of Myc, (Gabay et al. 2014). Mechanisms of activation include MYC gene amplification, mutation or rearrangement and have been reported to be prevalent in gynecologic malignancies, such as serous ovarian (39.2%), uterine (28.1%) and endometrial (19.5%) (https://www.cancer.gov/tcga). Since CDK9 phosphorylation of RNA polymerase II is required for transcription of Myc mRNA, we evaluate VIP152, a highly selective CDK9 inhibitor (Lücking et al 2021), to deliver antitumor responses in preclinical models of gynecologic malignancies. Methods: Gynecologic cell lines were treated with 9 dose levels of VIP152 with DMSO and cisplatin as controls. IC50 were calculated using CellTiter-Glo luminescent cell viability assay after 72 hours treatment. A preliminary analysis of 19 cell lines was undertaken to determine whether response to VIP152 is associated with baseline mutation, gene expression, or copy number variation data. Monotherapy efficacy of VIP152 was evaluated in a cisplatin sensitive A2780 in vivo xenograft mouse model. Results: Screening of gynecologic cell lines demonstrates a 3-log range of sensitivity with VIP152 IC50s ranging from 38-593nM. Simultaneous cisplatin screening identified sensitive and resistant cell lines in the same panel. Low VIP152 IC50s were observed in cell lines sensitive or resistant to cisplatin. Cell lines were assigned as sensitive and resistant based on the VIP152 IC50 values. Preliminary analysis suggests a gene signature could predict response to VIP152 in gynecologic malignancies. VIP152 was evaluated in an ovarian cancer A2780 in vivo xenograft model. Monotherapy treatment with 4 doses of VIP152 across 5 dose levels from 5- to 15-mg/kg weekly regimens provided increasing tumor growth inhibition compared with vehicle control. Conclusions: VIP152 demonstrates sensitivity in gynecologic cell lines independent of cisplatin sensitivity. An interim gene signature that is associated with VIP152 sensitivity was defined and we plan to optimize this signature with a larger cell line panel. Dose-dependent tumor growth inhibition in an in vivo xenograft model is demonstrated. VIP152 is currently being evaluated in the clinic (ClinicalTrials.gov Identifiers: NCT02635672 and NCT04978779).

Trial in progress: Phase I study of SY-5609, a potent, selective CDK7 inhibitor, with initial expansion in adults with metastatic pancreatic cancer.

TPS4180 Background: SY-5609 is an oral, selective, potent CDK7 inhibitor that targets two fundamental processes in cancer: transcription and cell cycle control. Early results from the Phase 1 dose escalation portion in patients (pts) with advanced solid tumors reported improved tolerability of the intermittent 7 days on followed by 7 days off (7/7) schedule with ongoing dose escalation beyond the continuous daily dosing maximum tolerated dose. Single-agent clinical activity was demonstrated with durable stable disease, target lesion regressions, and reduction in tumor markers observed in multiple tumor types, notably in pancreatic cancer with a disease control rate (DCR) of 38.5% (Sharma 2021). Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of 11% (ACS Cancer Facts and figures, 2022) with limited treatment options and therefore, is a disease in need of novel effective therapies. Oncogenic KRAS mutations are prevalent in PDAC. Mutant KRAS is a potent stimulator of mitogenic MAPK signaling and downstream transcriptional programs for cell proliferation. Preclinical studies have shown that CDK7 inhibition via SY-5609 inhibits tumor growth in KRAS mutant PDAC xenograft models, in many cases leading to regressions. SY-5609 also potentiates gemcitabine (gem) activity in PDAC cells in vitro and in xenografts in vivo (Henry 2021). Therefore, combining SY-5609 with gem +/- nab-paclitaxel (nab-pac) offers a potential new treatment strategy for metastatic PDAC (mPDAC). The expansion portion of this Phase 1 study will evaluate SY-5609 in combination with gem +/- nab-pac in mPDAC pts. Gem +/- nab-pac will be administered on a biweekly schedule as it has shown better tolerability and similar clinical activity compared to the standard of care (SOC) administration schedule (Rehman 2020). Methods: This is an ongoing Phase 1, multi-center study in select solid tumors, amended to open expansion cohorts for mPDAC and expected to enroll approximately 80 mPDAC pts who have progressed on SOC treatments. Objectives of the expansion cohorts include evaluation of safety and efficacy of SY-5609 in combination with gem +/- nab-pac. Key objectives of the two parallel safety lead-in cohorts 1) SY-5609 + gem and 2) SY-5609 + gem + nab-pac are safety and determination of the recommended combination dose of the doublet and triplet for subsequent cohort expansions using a 3+3 escalation design. Key objectives of expansion cohorts are to describe efficacy, defined by progression-free survival, overall response rate, and DCR. Additional objectives include evaluation of pharmacokinetics and pharmacodynamics of SY-5609 in combination with gem +/- nab-pac. SY-5609 will be administered orally once daily on a 7/7 regimen and gem +/- nab-pac will be administered intravenously, in a 4-week cycle. The expansion portion is now open to enrollment. Clinical trial information: NCT04247126.

Stable H-bond networks are crucial for selective CDK4 inhibition revealed from comprehensive in silico investigation

CDK1 and CDK4 are highly similar isoforms but with apparently diverse cellular functions, which makes it fundamental to discover selective CDK4 inhibitors that could accurately control the process of cell cycle of the specific organization so as to restore normal physiological state. In current research, interaction modes of CDK1 and CDK4 inhibitors were investigated through combined in silico strategies to elucidate the selectivity mechanism against CDK4 over CDK1, revealing that H-bond networks formed with key amino acids such as LYS33 and LEU83 of CDK1 and VAL93 of CDK4 are crucial for CDK4 selective inhibition, which would provide a theoretical basis for the design of selective CDK4 inhibitors.

Identification of potent, non-toxic, selective CDK2 inhibitor through the pharmacophore-based scaffold hopping, molecular dynamics simulation-assisted molecular docking study, Lee Richard contour map analysis, and ADMET properties

Identification of potent, non-toxic, selective CDK2 inhibitor through the pharmacophore-based scaffold hopping, molecular dynamics simulation-assisted molecular docking study, Lee Richard contour map analysis, and ADMET properties

Synthesis of 4-styrylpyrazoles and Evaluation of their Inhibitory Effects on Cyclin-dependent Kinases.

Cycle-regulating and transcriptional cyclin-dependent kinases (CDKs) are attractive targets in cancer drug development. Several CDK inhibitors have already been obtained or are close to regulatory approval for clinical applications. Phenylazopyrazole CAN508 has been described as the first selective CDK9 inhibitor with an IC50 of 350 nM. Since the azo-moiety is not a suitable functionality for drugs due to pharmacological reasons, the preparation of carbo-analogues of CAN508 with similar biological activities is desirable. The present work is focused on the synthesis of carbo-analogues similar to CAN508 and their CDK inhibition activity. Herein, the synthesis of 21 novel carbo analogues of CAN508 and their intermediates is reported. Subsequently, target compounds 8a - 8u were evaluated for protein kinase inhibition (CDK2/cyclin E, CDK4/cyclin D, CDK9/cyclin T) and antiproliferative activities in cell lines (K562, MCF-7, MV4-11). Moreover, the binding mode of derivative 8s in the active site of CDK9 was modelled. Compounds 8a - 8u were obtained from key intermediate 7, which was prepared by linear synthesis involving Vilsmeier-Haack, Knoevenagel, Hunsdiecker, and Suzuki-Miyaura reactions. Styrylpyrazoles 8t and 8u were the most potent CDK9 inhibitors with IC50 values of approximately 1 μM. Molecular modelling suggested binding in the active site of CDK9. The flow cytometric analysis of MV4-11 cells treated with the most active styrylpyrazoles showed a significant G1-arrest. The prepared styrylpyrazoles showed inhibition activity towards CDKs and can provide a novel chemotype of kinase inhibitors.

CYCLONE 2: A phase 2/3, randomized, placebo-controlled study of abiraterone acetate plus prednisone with or without abemaciclib in patients with metastatic castration-resistant prostate cancer.

TPS198 Background: Despite recent advances, nearly all patients (pts) with metastatic castration-resistant prostate cancer (mCRPC) experience disease progression and cancer-specific mortality. Persistent or reactivated androgen receptor (AR) signaling and/or activation of pathways in cross-talk with AR signaling are key drivers of mCRPC progression. Evidence suggests that AR signaling promotes translation of D-type cyclins resulting in cyclin-dependent kinase 4 and 6 (CDK4&6) activation and cell cycle progression. Abemaciclib is an oral selective inhibitor of CDK4&6 dosed on a continuous schedule, that is FDA-approved in combination with endocrine therapy or as monotherapy to treat HR+, HER2- metastatic breast cancer pts. Preclinical studies with prostate cancer cell lines and xenograft models showed that abemaciclib induces cell cycle arrest and tumor growth inhibition. The hypothesis is that addition of abemaciclib to AR targeted therapy may be an effective treatment for mCRPC pts. Methods: CYCLONE 2 (NCT03706365) is a phase 2/3, randomized, double-blind, multicenter, placebo-controlled study to assess the safety and efficacy of abemaciclib in combination with abiraterone acetate plus prednisone (AA+P) in pts with mCRPC. CYCLONE 2 is an adaptive study which is designed in three parts. Part 1 is a 30-patient safety lead-in to determine the recommended phase 2 dose (RP2D; 150 mg or 200 mg, twice daily) of abemaciclib in combination with AA (1000 mg, once daily) + P (5 mg, twice daily). In part 2, 150 pts are randomized 1:1 to AA+P with abemaciclib at the RP2D or placebo. The study expands to enroll an additional 170 pts in Part 3 if prespecified expansion criteria are met at a planned adaptive interim analysis performed by an independent data monitoring committee (IDMC). Pts with mCRPC evidenced by radiographic and/or PSA progression during continuous ADT are eligible. Prior docetaxel for mHSPC is permitted. Systemic anti-cancer therapy for mCRPC and prior novel hormonal agents are exclusionary. The primary objective is radiographic progression free survival (rPFS; per RECIST1.1 for soft tissue and PCWG3 for bone). Secondary objectives include safety, objective response rate, duration of response, time to symptomatic and PSA progression, overall survival, and pharmacokinetics. Status: Enrollment in Part 1 & 2 is completed. Based on the recommendation from the IDMC, Part 3 was opened in June 2021 and enrolls pts from about 112 sites across 12 countries. Clinical trial information: NCT03706365.

Abemaciclib in Combination With Endocrine Therapy for Patients With Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer: A Phase 1b Study.

BackgroundCyclin-dependent kinases (CDK) 4 and 6 regulate G1 to S cell cycle progression and are often altered in cancers. Abemaciclib is a selective inhibitor of CDK4 and CDK6 approved for administration on a continuous dosing schedule as monotherapy or as combination therapy with an aromatase inhibitor or fulvestrant in patients with advanced or metastatic breast cancer. This Phase 1b study evaluated the safety and tolerability, pharmacokinetics, and antitumor activity of abemaciclib in combination with endocrine therapy for metastatic breast cancer (MBC), including aromatase inhibitors (letrozole, anastrozole, or exemestane) or tamoxifen.Patients and MethodsWomen ≥18 years old with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) MBC were eligible for enrollment. Eligibility included measurable disease or non-measurable but evaluable bone disease by Response Evaluation Criteria in Solid Tumours (RECIST) v1.1, Eastern Cooperative Oncology Group performance status 0–1, and no prior chemotherapy for metastatic disease. Adverse events were graded by the National Cancer Institute Common Terminology Criteria for Adverse Events v4.0 and tumor response were assessed by RECIST v1.1.ResultsSixty-seven patients were enrolled and received abemaciclib 200 mg every 12 hours in combination with letrozole (Part A, n=20), anastrozole (Part B, n=16), tamoxifen (Part C, n=16), or exemestane (Part D, n=15). The most common treatment-emergent adverse events (TEAE) were diarrhea, fatigue, nausea, and abdominal pain. Grade 4 TEAEs were reported in five patients (one each with hyperglycemia, hypertension, neutropenia, procedural hemorrhage, and sepsis). There was no effect of abemaciclib or endocrine therapy on the pharmacokinetics of any combination study drug. Across all treated patients, the median progression-free survival was 25.4 months (95% confidence interval: 18.0, 35.8). The objective response rate was 38.9% in 36 patients with measurable disease.ConclusionsAbemaciclib in combination with multiple endocrine therapy options exhibited manageable safety and promising antitumor activity in patients with HR+, HER2- MBC.Clinical Trial Registration https://clinicaltrials.gov/, identifier NCT02057133

Abstract P237: PRT2527 is a potent and selective CDK9 inhibitor that demonstrates anti-cancer activity in preclinical models of hematological malignancies and solid tumors with MYC amplification

Abstract Cyclin-dependent kinase 9 (CDK9) is a master regulator of transcription that controls paused RNA polymerase II (RNAP2) release through phosphorylation of its carboxy-terminal domain, resulting in productive transcription elongation. CDK9 has been extensively studied as a potential target for cancer therapy in “transcriptionally addicted” tumors as transient inhibition of CDK9 primarily depletes proteins with short half-lives, such as the oncogenes MCL1 and MYC, making CDK9 a promising target in cancer. Here we show that PRT2527 is a potent and highly selective CDK9 inhibitor with moderate to high clearance that achieves optimal temporal target engagement and exhibits potent in vitro and in vivo activities. PRT2527 inhibited CDK9 enzymatic activity with an IC50 of 0.98 nM in a biochemical assay and showed high selectivity in a panel of kinases when tested at physiologically relevant 1 mM ATP concentration. In vitro, PRT2527 inhibited phosphorylation of Ser2RNAP2 in NCI-H929 cells with an IC50 of 54 nM, and an IC50 of 198 nM in a plasma assay to adjust for human plasma protein binding. Transient treatment of cells with PRT2527 inhibited pSer2RNAP2, depleted MCL1 and MYC proteins, and activated cleaved caspase-3 (CC3) in a concentration-dependent manner. In a proteomic profiling study, MCL1 was identified as one of the major down-regulated proteins following PRT2527 treatment. In a panel of hematological cancer cell lines representing B- and T-ALL, AML, and non-Hodgkin’s lymphoma (NHL), as well as subsets of sarcoma, prostate, adenoid cystic carcinoma (ACC), and non-small cell lung cancer (NSCLC) cell lines, PRT2527 treatment consistently led to a potent, concentration-dependent inhibition of proliferation. In a pharmacokinetic/pharmacodynamic (PK/PD) study, intravenous (IV) administration of PRT2527 achieved transient target engagement, depletion of MCL1 and MYC proteins, and induction of apoptosis in tumor tissue. This PK/PD correlation was successfully translated into in vivo efficacy in multiple models. Once weekly dosing of PRT2527 was well-tolerated and significantly inhibited tumor growth in various AML CDX models and induced tumor regressions in double-hit and triple-hit diffuse large B-cell lymphoma (DLBCL) CDX and PDX models carrying the MYC translocation. Combining PRT2527 with venetoclax achieved complete tumor regressions in a venetoclax resistant OCI-AML3 model. PRT2527 demonstrated potent ex vivo activity in PDX models of B-ALL and T-ALL, as well as various solid tumor PDX models with high levels of MYC amplification and overexpression, including pancreatic carcinoma, gastric and gastroesophageal carcinomas, NSCLC, and sarcoma. In vivo efficacy studies with once weekly IV administration of PRT2527 confirmed significant tumor growth inhibition in select MYC-amplified solid tumor PDX models. Taken together, this preclinical characterization supports the advancement of PRT2527 into clinical studies for transcriptionally addicted hematological malignancies and solid tumors with MYC amplification and/or dysregulation. Citation Format: Yang W. Zhang, Liang Lu, Min Wang, Dave Rominger, Stefan Ruepp, Kirsten Gallagher, William Gowen-MacDonald, Chaofeng Dai, Miles Cowart, Andrew Combs, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRT2527 is a potent and selective CDK9 inhibitor that demonstrates anti-cancer activity in preclinical models of hematological malignancies and solid tumors with MYC amplification [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P237.

Abstract P051: Identification of an orally bioavailable dual Cyclin K glue degrader - CDK12/13 inhibitor

Abstract CDK12 and CDK13 regulate gene expression through translation and splicing of mRNA. Impaired activity of CDK12 has been shown to reduce the expression of a range of genes involved in DNA homologous repair leading to the induction of a ‘BRCAness’ phenotype in cells. CDK12 inhibitors have the potential in many different indications, including overcoming emergent resistance to PARP inhibition, enhancing the efficacy of PARP inhibition in patients with BRCA1/2 wt status [1] and extending the efficacy of immune checkpoint inhibition through immunogenic cell death [2]. Recently, certain classes of CDK12 inhibitors have also demonstrated ‘glue degrader’ properties [1]: causing proteosomal degradation of the obligate co-factor for CDK12 and 13, Cyclin K. Such glue-degraders have the capability of enhancing cellular potency and specificity over classical kinase inhibitors. Taking as a starting point the selective CDK7 inhibitor samuraciclib, we have taken a rational design approach to develop inhibitors that preferentially inhibit CDK12. By utilising differences in the active site between CDK7 and CDK12 we were able to increase potency against CDK12 in biochemical assays by > 100 fold, with no significant inhibition of kinases outside the CDK class. Cellular potency of these molecules was evaluated in the Ewing’s sarcoma cell line A673, which is known to be sensitive to CDK12 inhibition [4]. Remarkably the potency of the molecules to induce cell death was greater than their biochemical potency against CDK12 by > 10 fold. Analysis of Cyclin K levels in cells demonstrated that the molecules lead to Cyclin K degradation. Furthermore, cellular potency was reduced by the presence of the neddylation inhibitor MLN4924 confirming that the compounds enhanced potency was mediated by glue degradation of Cyclin K. The molecules were found to cause potent inhibition of DNA homologous repair genes including BRCA1, FANCF and ERCC4 and lead to the accumulation of DNA strand breaks as assessed by the marker γ-H2AX. In the BRCA wt ovarian cancer cell line, OV-90, strong synergy was observed in the ability to induce cell death with a range of PARP inhibitors. In A673 tumour bearing mice, dosing with the compounds was found to cause significant reduction in the levels of cyclin K in tumours, which was sustained for 24 hr. Additionally, assessment of CDK12 protein in these tumours also showed a reduction, suggesting greater turnover of CDK12 protein in the absence of Cyclin K. Repeat dose studies at doses that lead to Cyclin K degradation in tumours demonstrated that the compounds were well tolerated, with no weight loss in mice. Two molecules, CT7439 and CT7510, were found to have oral bioavailability in mice that indicate that they would be suitable for once-daily oral administration in humans and are being progressed through regulatory toxicity studies. [1] Johnson, et al, (2016) Cell Rep. 17, 2367 [2] Li, et al, (2020) Cancer Lett. 495, 12 [3] Słabicki, et al, (2020) Nature 585, 293 [4] Iniguez, et al, (2018) Cancer Cell 33, 202 Citation Format: Edward K. Ainscow, Adrian Campbell, Michael Cripps, Robert Workman, Stuart Thomson, Kam Chohan, Damien Crepin, Mahiro Sunose, Jamie Patient, Jane Kendrew, Ash Bahl. Identification of an orally bioavailable dual Cyclin K glue degrader - CDK12/13 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P051.

SY5609, a Potent and Selective CDK7 Inhibitor, Potentiates BTK Inhibitor Activity in Mantle Cell Lymphoma Preclinical Models

Introduction: CDK7 is a key regulator of transcription and cell cycle progression and has been implicated in multiple tumor types driven by aberrant transcriptional (e.g., MYC-, ESR1-activation) and/or aberrant cell cycle control (e.g., loss of RB pathway checkpoint function) mechanisms. SY-5609 is a potent and selective CDK7 inhibitor currently in development in patients with solid tumors (NCT04247126). To assess the potential for SY-5609 development in heme malignancies, we evaluated SY-5609 activity in preclinical models of mantle cell lymphoma (MCL). MCL is an aggressive B cell lymphoma driven by genetic alterations in RB pathway genes (e.g. CCND1, RB1, CDKN2A) and by hyper-activation of B-cell receptor (BCR) signaling leading to activation of NF-kB-dependent transcriptional programs that drive cell proliferation and survival. Our results support evaluation of SY-5609 in MCL patients and highlight the potential for combining SY-5609 with Bruton's Tyrosine Kinase (BTK) inhibitors, inhibitors of BCR signal transduction approved for treatment of MCL.Methods: SY-5609 in vitro antiproliferative activity was assessed in a panel of 7 MCL cell lines by evaluating growth rate inhibition (GR) curve metrics GR 50 (drug concentration at which growth rate is inhibited by 50%) and GR max (maximum depth of response) after 5-days of treatment. SY-5609 pharmacodynamic (PD) responses and combination activity with the BTK inhibitor acalabrutinib were further assessed in MCL cell line Mino-1. PD activity was assessed by measuring transcriptional changes in POLR2A using a NanoString-based PD assay from the SY-5609 solid tumor trial. Combination activity in vitro was assessed by comparing GR curve metrics between acalabrutinib, SY-5609, and acalabrutinib in combination with SY-5609. Expression of CCND1 and E2F1, a key transcriptional regulator of DNA replication commitment and progression, were assayed via western blot. Combination activity in vivo was assessed in mice bearing Mino-1 xenograft tumors by comparing tumor growth rate inhibition (GRI) and body weight change in mice treated with acalabrutinib (15mg/kg, QD) and/or SY-5609 (0.5mg/kg, BID, days 1-7 and 15-21) over 25 days.Results: SY-5609, as a single agent, potently inhibited proliferation of all 7 MCL cell lines tested with a GR 50 geometric-mean of 7 nM (range: 2 to 20 nM) and a GR max geometric-mean of -0.2 (range: -0.6 to 0.2). In MCL cell line Mino-1, SY-5609 induced a dose-dependent increase in POLR2A expression to levels associated with SY-5609 antiproliferative activity in Mino-1 cells in vitro and to levels comparable with those observed in peripheral blood mononuclear cells (PBMCs) collected from SY-5609 solid tumor trial patients. In combination with acalabrutinib, SY-5609 demonstrated synergistic antiproliferative activity in Mino-1 cells in vitro, a dose-dependent decrease in acalabrutinib GR 50 (up to ~2-fold at the highest SY-5609 concentration tested, 5nM), and a dose-dependent increase in GR max (up to ~10-fold). In addition, the combination of SY-5609 and acalabrutinib caused dose-dependent decreases in CCND1 and E2F1 protein expression in vitro, which were not observed with either single agent alone. Finally, in mice bearing Mino-1 xenografts, the combination of SY-5609 and acalabrutinib significantly (p < 0.01) increased tumor GRI relative to each single agent at the doses and regimens tested and was well tolerated (no difference in body weight changes relative to vehicle treated mice).Conclusions: SY-5609 is a potent and selective CDK7 inhibitor that demonstrates antiproliferative activity in MCL cells in vitro, associated with PD changes comparable to those observed in patients enrolled in the SY-5609 solid tumor trial (ENA, 2020). The combination of SY-5609 and acalabrutinib is synergistic in MCL cells in vitro and inhibits expression of key cell cycle regulatory proteins CCND1 and E2F1 at concentrations that are preclinically subtherapeutic for either single agent alone. The combination of SY-5609 and acalabrutinib is also significantly more effective at inhibiting MCL xenograft growth in vivo than either single agent. These results support evaluation of SY-5609, including in combination with BTK inhibitors, in patients with mantle cell lymphoma. DisclosuresJohannessen: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Henry: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sawant: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. D'Ippolito: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Ke: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Lefkowitz: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Eaton: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Dworakowski: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Rosario: Syros Pharmaceuticals: Current Employment. Hodgson: Syros Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company.

VIP152, a Selective CDK9 Inhibitor, Induces Complete Regression in a High-Grade B-Cell Lymphoma (HGBL) Model and Depletion of Short-Lived Oncogenic Driver Transcripts, MYC and MCL1, with a Once Weekly Schedule

VIP152, a Selective CDK9 Inhibitor, Induces Complete Regression in a High-Grade B-Cell Lymphoma (HGBL) Model and Depletion of Short-Lived Oncogenic Driver Transcripts, MYC and MCL1, with a Once Weekly Schedule

Aberrant CDK7 Activity Drives the Cell Cycle and Transcriptional Dysregulation to Support Multiple Myeloma Growth: An Attractive Molecular Vulnerability

Multiple myeloma (MM) cells are characterized by cell cycle dysregulation, epigenetic heterogeneity, and perturbation of the transcriptional landscape. We have previously shown that chemical and genetic perturbation of transcriptional regulator CDK7 significantly and selectively impacted MM cell growth and viability, supporting it as a pharmacologically relevant target for MM. Indeed, selective CDK7 inhibitor YKL-5-124 was active against a large panel of MM cell lines and primary MM cells, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs). The efficacy of YKL-5-124 was confirmed in vivo in several murine models of MM, including disseminated models.Gene expression analysis after CDK7 inhibition in several MM cell lines revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells, supporting CDK7 as a central hub in the oncogenic CDK-pRb-E2F pathway in MM cells, with its expression and activity positively correlated with E2F transcriptional output in patient MM cells. Importantly, dual inhibition with low doses of YKL-5-124 and BRD4 inhibitor JQ1, displayed superior activity against a panel of MM cell lines and primary MM cells compared to single perturbation alone by both converging on a subset of key SE-associated dependencies as well as impacting distinct oncogenic expression programs.To identify synthetically lethal targets and mechanisms of resistance to CDK7 inhibition, we performed a genome-wide CRISPR-Cas9 knockout screen in the MM1S cells treated with YKL-5-124 or DMSO. We found that BCL6, NFKBIA and B, TRAF2, TSC1 and CSNK2A1, a subunit of CK2, were top synthetically lethal hits; whereas deletion of RB1, SF3B3 and the DNA-binding transcriptional activator TADA2A that regulates RNA-pol II transcription, led to resistance to YKL-5-124. Molecular mechanisms of intrinsic and acquired resistance to CDK7 inhibition are now under investigation and will be presented.In conclusion, our study demonstrates CDK7 as an attractive molecular vulnerability in MM that can be exploited therapeutically alone or in combination. DisclosuresShirasaki: FIMECS: Consultancy. Chesi: Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Pfizer: Consultancy; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Mitsiades: Arch Oncology: Research Funding; Karyopharm: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; FIMECS: Consultancy, Honoraria; Nurix: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; BMS: Research Funding; TEVA: Research Funding. Munshi: Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy.

VIP152 Is a Novel CDK9 Inhibitor with Efficacy in Chronic Lymphocytic Leukemia

VIP152 Is a Novel CDK9 Inhibitor with Efficacy in Chronic Lymphocytic Leukemia

Selective Cdk9 inhibition resolves neutrophilic inflammation and enhances cardiac regeneration in larval zebrafish.

ABSTRACTSustained neutrophilic inflammation is detrimental for cardiac repair and associated with adverse outcomes following myocardial infarction (MI). An attractive therapeutic strategy to treat MI is to reduce or remove infiltrating neutrophils to promote downstream reparative mechanisms. CDK9 inhibitor compounds enhance the resolution of neutrophilic inflammation; however, their effects on cardiac repair/regeneration are unknown. We have devised a cardiac injury model to investigate inflammatory and regenerative responses in larval zebrafish using heartbeat-synchronised light-sheet fluorescence microscopy. We used this model to test two clinically approved CDK9 inhibitors, AT7519 and flavopiridol, examining their effects on neutrophils, macrophages and cardiomyocyte regeneration. We found that AT7519 and flavopiridol resolve neutrophil infiltration by inducing reverse migration from the cardiac lesion. Although continuous exposure to AT7519 or flavopiridol caused adverse phenotypes, transient treatment accelerated neutrophil resolution while avoiding these effects. Transient treatment with AT7519, but not flavopiridol, augmented wound-associated macrophage polarisation, which enhanced macrophage-dependent cardiomyocyte number expansion and the rate of myocardial wound closure. Using cdk9−/− knockout mutants, we showed that AT7519 is a selective CDK9 inhibitor, revealing the potential of such treatments to promote cardiac repair/regeneration.

Discovery of a Series of 7-Azaindoles as Potent and Highly Selective CDK9 Inhibitors for Transient Target Engagement.

Optimization of a series of azabenzimidazoles identified from screening hit 2 and the information gained from a co-crystal structure of the azabenzimidazole-based lead 6 bound to CDK9 led to the discovery of azaindoles as highly potent and selective CDK9 inhibitors. With the goal of discovering a highly selective and potent CDK9 inhibitor administrated intravenously that would enable transient target engagement of CDK9 for the treatment of hematological malignancies, further optimization focusing on physicochemical and pharmacokinetic properties led to azaindoles 38 and 39. These compounds are highly potent and selective CDK9 inhibitors having short half-lives in rodents, suitable physical properties for intravenous administration, and the potential to achieve profound but transient inhibition of CDK9 in vivo.

14P Preclinical evaluation of intermittent dosing regimens on antitumor and PD activity of SY-5609, a potent and selective oral CDK7 inhibitor, in ovarian cancer xenografts

Selective CDK7 inhibition has been shown to target two fundamental processes in cancer: transcription and cell cycle control. SY-5609 is a potent and selective CDK7 inhibitor in Ph1 clinical development in patients with advanced solid tumors including ovarian cancer (NCT04247126). Here we report on the impact of intermittent SY-5609 dosing regimens on tumor growth inhibition (TGI), pharmacodynamic (PD) activity, and pharmacokinetics (PK) in a xenograft model of high grade serous ovarian cancer (HGSOC).