Cyclin-dependent Kinase 9 Inhibitor Research Articles

Abstract 14474: Cdk9-activated Sgk3 Promotes Myocardial Repair and Regeneration After Myocardial Infarction via Gsk-3Å/Å-catenin Pathway

Background: Neonatal heart maintains entire regeneration capacity in a transient regeneration window, but the adult heart loses this function. This process involves numberous core hubs dysregulation of expression and activity that adversely affect the myocardium regeneration ability. Present study screened a functional kinase of serine/threonine-protein kinase 3 (SGK3) in cardiac regeneration after neonatal myocardial infarction (MI) using quantitative phosphoproteomics. Methods: We used quantitative phosphoproteomics data of infarct border zone in newborn heart after MI to identify regeneration related kinases. Gain- and loss-of-function experiments were performed to determine the effect of SGK3 in cardiomyocyte (CM) proliferation and cardiac repair after apical resection (AR) or MI. Pull-down assay and co-immunoprecipitation (Co-IP) experiments were conducted to investigate the direct binding target proteins of SGK3. Results: SGK3 protein expression was highly expressed at postnatal day 1 (P1), reduced at postnatal day 7 (P7) until adult. In vitro, CM proliferation ratio was elevated by SGK3 overexpression, while it was decreased by knockdown of SGK3. In vivo, inhibition of SGK3 shortened the time window of cardiac regeneration after AR in neonatal mice, and overexpression of SGK3 significantly promoted CM proliferation and cardiac repair after MI. Mechanistically, SGK3 could be directly combined with and activated by cyclindependent kinase 9 (CDK9). Inhibition of CDK9 partially abolished the effect of SGK3 on CM proliferation. Moreover, SGK3 could repress GSK-3β activity and increase β-catenin expression. Conclusions: Our study revealed a key role of SGK3 in cardiac regeneration following AR or MI injury, which may reopen a novel therapeutic avenue for MI.

Targeting Transcription Checkpoint Using a Novel CDK9 Inhibitor in Mantle Cell Lymphoma

Introduction Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma that accounts for 5-8% of all non-Hodgkin lymphomas. Despite the Bruton's tyrosine kinase inhibitor ibrutinib and the BH3 mimetic BCL2 inhibitor venetoclax (ABT-199) have proven to be effective therapeutic strategies for MCL, most patients often experience disease progression after treatment. Thus, developing a novel drug to overcome this aggressive relapsed/refractory malignancy is an urgent need. Cyclin-dependent kinase 9 (CDK9) is a serine/threonine kinase belonging to the CDK family which regulates multiple cellular processes, particularly in driving and maintaining cancer cell growth. Unlike classical CDKs, CDK9 is a critical component of the positive transcription elongation factor b (P-TEFb) complex that mediates transcription elongation and mRNA maturation via phosphorylating RNA polymerase II (RNAP2). Previous studies demonstrated that CDK9 inhibition downregulates transcription levels of MCL-1 and MYC, which are crucial in both survival and proliferation of acute myeloid leukemia and diffuse large B-cell lymphoma. We and others found that the MYC signaling pathway was enhanced in MCL, especially in ibrutinib-resistant MCL patients. MYC is a core transcription factor driving lymphomagenesis. It does not possess enzymatic activity and has long been considered to be undruggable. MCL-1 is a key anti-apoptotic protein and is overexpressed in several hematologic malignancies. It was also found to be overexpressed in ibrutinib or venetoclax-resistant MCL cells. Thus, CDK9 is considered as a potential target that may inhibit MYC and MCL-1 pathways. Although recently it was shown that MC180295, a novel selective inhibitor of CDK9, has nanomolar levels anti-cancer potency, whether its beneficial effects extend to relapsed/refractory MCL has not yet been assessed. Methods We use three paired MCL cells sensitive/resistant to ibrutinib or venetoclax to test the efficacy of CDK9 inhibitor MC180295. Cell viability was measured by using Cell Titer Glo (Promega). Cell apoptosis assay and western blot analyses were used to identify affected pathways after MC180295 treatment. Finally, we used patient-derived xenograft (PDX) mouse models to test the therapeutic potential of MC180295 in MCL. Results First, we examined the potential efficacy of a CDK9 inhibitor MC180295 in MCL cells. MC180295 treatment results in growth inhibition of ibrutinib-resistant or venetoclax-resistant MCL cells. By assessing the caspase 3 and PARP activity, we found that MC180295 treatment induces cell death via cell apoptosis in MCL cell lines. Meanwhile, we found that RNAP2 phosphorylation at Ser2, the active form of RNAP2, is downregulated in MC180295 treated MCL cell lines. Consistent to previous studies, MC180295 treatment significantly reduces the protein level of MYC and MCL-1. In addition, we identified several other important proteins, such as cyclin D1 and BCL-XL, were also downregulated upon MCL180295 treatment. MC180295 was able to overcome ibrutinib-venetoclax dual resistance in PDX mouse models without severe side effects. To improve the efficacy of MC180295 as a single agent, we performed in vitro combinational drug screen with a number of FDA-approved or investigational clinical agents and found that MC180295 had a synergistic effect with venetoclax. We are currently investigating the underlying mechanism of action. Conclusion Taken together, our findings showed that targeting CDK9 by its specific inhibitor MC180295 is effective in targeting MCL cells, especially those with ibrutinib or venetoclax resistance and therefore supports the concept that CDK9 is a new target to overcome ibrutinib/venetoclax resistance in MCL. Disclosures Wang: MoreHealth: Consultancy; Dava Oncology: Honoraria; Beijing Medical Award Foundation: Honoraria; OncLive: Honoraria; Molecular Templates: Research Funding; Verastem: Research Funding; Guidepoint Global: Consultancy; Nobel Insights: Consultancy; Oncternal: Consultancy, Research Funding; InnoCare: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Lu Daopei Medical Group: Honoraria; Pulse Biosciences: Consultancy; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding.

Trial in Progress: A Phase I Trial of BTX-A51 in Patients with Relapsed or Refractory AML or High-Risk MDS

Background: For patients with relapsed or refractory (R/R) acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS), low response rates and poor overall survival remain unmet clinical needs. AML cells evade apoptosis through overexpression of antiapoptotic genes and inactivation of p53. The antiapoptotic gene Mcl1 is overexpressed in AML cell lines resistant to venetoclax. Similarly, the sensitivity of AML patients' samples to venetoclax inversely correlates with the presence of a TP53 mutation or low expression of p53. In AML, p53 inactivation more commonly results from overexpression of its negative regulators, Mdmx and Mdm2. BTX-A51 is a novel, oral, direct inhibitor of Casein kinase 1α (CK1α), cyclin dependent kinase 7 (CDK7), and CDK9. CK1α phosphorylates Mdmx and Mdm2 leading to enhanced binding and degradation of p53. CDK7 and CDK9 phosphorylate RNA polymerase II (Pol II) to enable transcriptional initiation and elongation, particularly at large clusters of transcriptional enhancers termed super-enhancers (SE). Preclinical studies have demonstrated that BTX-A51 robustly increased p53 protein levels via CK1a inhibition and Mdm2 downregulation while preferentially decreasing SE transcription of key oncogenes such as Myc andMcl1, enabling selective apoptosis of leukemia cells. The combination of CK1α, CDK7, and CDK9 inhibition was synergistic and prolonged survival in multiple genetic and patient-derived xenograft AML models. Study Design and Methods: This is an open-label, multi-center, first-in-human Phase 1 study evaluating the safety of BTX-A51 in patients with R/R AML or high-risk MDS. The trial will be performed in two phases, a dose escalation (phase 1a) and dose expansion (phase 1b). Phase 1a utilizes a hybrid accelerated titration with single patient cohorts and a Bayesian optimal interval (BOIN) design to assess 8 potential dosing cohorts. The maximum tolerated dose (MTD) will be the dose for which the isotonic estimate of the toxicity rate is closest to the target toxicity rate of 0.3. Up to a maximum of 35 patients will be enrolled in the dose escalation phase of the study at Memorial Sloan Kettering Cancer Center, MD Anderson Cancer Center, and City of Hope Cancer Center. Following determination of the MTD, 15 patients will be enrolled in the dose expansion phase for further evaluation of dose-limiting toxicities (DLTs) and for preliminary evidence of efficacy. BTX-A51 will be dosed 3 weeks on drug, followed by 1 week off drug over a 28-day cycle. For the first cycle, patients will receive tumor lysis syndrome prophylaxis with allopurinol and intravenous fluids and be closely monitored. Key inclusion criteria are age ³ 18 years, R/R AML or R/R high-risk MDS, Eastern Cooperative Oncology Group (ECOG) £ 2 and life expectancy of ³ 6 weeks, and adequate kidney and liver function. Key exclusion criteria are receipt of cancer chemotherapy (other than hydroxyurea) within 2 weeks prior to the start of study drug, transplantation within 3 months prior to screening, active graft-versus-host disease requiring systemic immunosuppressive medications, and a white blood cell count > 20 × 109/L. The primary objective for the Phase 1 study is to determine the MTD and recommended Phase 2 dose (RP2D) of BTX-A51. Secondary objectives include evaluating overall response (complete remission, complete remission with incomplete blood count recovery, and partial remission) according to the European LeukemiaNet (ELN) 2017 criteria (Döhner et al. Blood. 2017), survival (overall survival and event-free survival) and pharmacokinetics. Correlative objectives include determining the changes in SEs and SE-driven expression of antiapoptotic genes by chromatin immunoprecipitation and RNA-sequencing. Recruitment is ongoing and this trial is registered on clinicaltrials.gov: NCT04243785 Disclosures Stein: Amgen: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau. Borthakur:BioLine Rx: Consultancy; Argenx: Consultancy; PTC Therapeutics: Consultancy; FTC Therapeutics: Consultancy; BioTherix: Consultancy; Curio Science LLC: Consultancy; Xbiotech USA: Research Funding; Oncoceutics: Research Funding; Polaris: Research Funding; PTC Therapeutics: Research Funding; Cyclacel: Research Funding; GSK: Research Funding; Jannsen: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding; BioLine Rx: Research Funding; BMS: Research Funding; AstraZeneca: Research Funding; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy. Murray:Salamandra: Current Employment. Kook:Salamandra: Current Employment. Chan:BioTheryx: Current Employment. Stein:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Syros: Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Biotheryx: Consultancy; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Amgen: Consultancy; Celgene Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

CDK9 As a New Therapeutic Vulnerability for Ibrutinib Resistance Mantle Cell Lymphoma (MCL)

MCL is an aggressive B-cell lymphoma with poor prognosis due to emergence of drug resistant populations and lymphoma progression. Ibrutinib is a bruton's tyrosine kinase (BTK) inhibitor that was shown to have high response rates in MCL patients. However, as the use of this drug continues to grow in MCL and other B-cell lymphomas, emergence of resistance and fatal progression are of increasing clinical concern. Currently the mechanisms driving IR are poorly understood and no recurrent driver mutations have been identified in MCL. We have modeled acquired resistance to ibrutinib and implemented chemical proteomics and a cell-based drug screen approaches in IR MCL lines and primary samples, we have shown that MCL cells become resistant to ibrutinib through a kinome-adaptive reprogramming mechanism that lead to constitutive activation of the PI3K/AKT/mTOR pathway with increased levels of Myc and sustained transcription activation. The acquired IR MCL cells have increased rates of growth and augmented adhesion to stromal cells. Collectively, our published and preliminary studies indicate that IR MCL relies on global transcriptome remodeling and subsequent kinome reprogramming, leading to molecularly and clinically aggressive phenotype and resistance to ibrutinib therapy. Rather than there being a single mechanism of acquired IR, kinase networks are rewired in a plethora of ways in MCL cells as they become resistant to ibrutinib treatment. Adaptive remodeling of the kinome creates therapeutic challenges, where even combination targeted kinase inhibitor treatments are unlikely to be successful. Here, using genomic, chemical proteomic and drug screen profiling, we report that enhancer remodeling-mediated transcriptional activation and adaptive signaling changes drive the aggressive phenotypes of IR. Accordingly, IR MCL cells are vulnerable to inhibitors of the transcriptional machinery and especially so to inhibitors of cyclin-dependent kinase 9 (CDK9), the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Further, CDK9 inhibition disables reprogrammed signaling circuits and prevents the emergence of IR in MCL. Finally and importantly, we provided that a novel and facile ex vivo image-based functional drug screening platform could predict clinical therapeutic responses of IR MCL and identified vulnerabilities that can be targeted to disable the evolution of IR. Disclosures Shain: Amgen: Speakers Bureau; GlaxoSmithKline: Speakers Bureau; Adaptive: Consultancy, Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Sanofi/Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; AbbVie: Research Funding; Karyopharm: Research Funding, Speakers Bureau. Shah:Kite/Gilead, Jazz, Incyte: Research Funding; Kite/Gilead, Celgene/Juno/BMS, Novartis, Pfizer, Amgen, Spectrum/Acrotech, Precision Biosciences, Beigene, AstraZeneca, Pharmacyclics/Jansen, Adaptive: Honoraria; NCCN: Vice-Chair, Acute Lymphoblastic Leukemia Working Group: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead, Precision Biosciences, Novartis, AstraZeneca: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Moffitt Cancer Center: Current Employment.

Inhibition of CDK1 Reverses the Resistance of 5-Fu in Colorectal Cancer.

IntroductionAlthough the survival rate of colorectal cancer (CRC) patients can be improved by surgery, radiotherapy, and chemotherapy, the resistance to 5-fluorouracil (5-Fu) affects the effect of chemotherapy and the prognosis of patients. An increasing number of studies showed that 5-Fu resistance was the main reason for the failure of colorectal cancer treatment. The poor prognosis of colorectal cancer greatly harms people’s health. This study aimed to clarify the correlation between cyclin-dependent kinase 1 (CDK1) and 5-Fu-induced tumor resistance.Materials and MethodsCell proliferation and invasion experiments showed that down-regulation of CDK1 inhibited fluorouracil-resistant CRC cell proliferation. The expression level of CDK1 was detected in 5-Fu-resistant CRC cells in vitro. Tumor growth was inhibited by down-regulation of CDK1 in tumor xenograft mouse models.ResultsWe found that CDK1 was highly expressed in tumor tissues, especially in fluorouracil-resistant tissues. We also confirmed that the differential expression of 5-Fu in tumor tissues was related to tumor site, lymph node metastasis and stage. CDK1 promoted migration, invasion and inhibited apoptosis in 5-Fu-resistant CRC cells. Down-regulation of CDK1 inhibited fluorouracil-resistant CRC cell proliferation and tumorigenesis in vivo.ConclusionHigh expression of CDK1 may lead to poor clinical prognosis, and inhibition of CDK1 enhances 5-Fu sensitivity in CRC. Our research suggested that CDK1 may be used to predict 5-Fu efficacy and as a therapeutic target for CRC.

Bromodomain-containing-protein-4 and cyclin-dependent-kinase-9 inhibitors interact synergistically in vitro and combined treatment reduces post-traumatic osteoarthritis severity in mice

Joint injury rapidly induces expression of primary response genes (PRGs), which activate a cascade of secondary genes that destroy joint tissues and initiate post-traumatic osteoarthritis (PTOA). Bromodomain-containing-protein-4 (Brd4) and cyclin-dependent-kinase-9 (CDK9) cooperatively control the rate-limiting step of PRG transactivation, including pro-inflammatory genes. This study investigated whether Brd4 and CDK9 inhibitors suppress inflammation and prevent PTOA development in vitro and in a mouse PTOA model. The effects of Brd4 and CDK9 inhibitors (JQ1 and Flavopiridol) on PRG and associated secondary damage were rigorously tested in different settings. Short-term effects of inflammatory stimuli (IL-1β, IL-6, TNF) on human chondrocyte PRG expression were assessed by RT-PCR and microarray after 5-h. We quantified glycosaminoglycan release from IL-1β-treated bovine cartilage explants after 3-6 days, and osteoarthritic changes in mice after ACL-rupture using RT-PCR (2-24hrs), in vivo imaging of MMP activity (24hrs), AFM-nanoindentation (3-7days), and histology (3days-4wks). Flavopiridol and JQ1 inhibitors act synergistically, and a combination of both almost completely prevented the activation of most IL-1β-induced PRGs in vitro by microarray analysis, and prevented IL-1β-induced glycosaminoglycan release from cartilage explants. Mice given the drug combination showed reduced IL-1β and IL-6 expression, less in vivo MMP activity, and lower synovitis (1.5 vs 4.9) and OARSI scores (2.8 vs 6.0) than untreated mice with ACL-rupture. JQ1 and Flavopiridol work synergistically to reduce injury response after joint trauma, suggesting that targeting Brd4 and/or CDK9 could be a viable strategy for PTOA prevention and treatment of early OA.

Cyclin-Dependent Kinase 9 (CDK9) Inhibitor Atuveciclib Suppresses Intervertebral Disk Degeneration via the Inhibition of the NF-κB Signaling Pathway.

Intervertebral disk degeneration (IVDD) is a spinal disk condition caused by an inflammatory response induced by various proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α. cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator and potential therapeutic target for many diseases, especially in regulating the activation of primary inflammatory response genes. Our study investigated a highly selective CDK9 inhibitor, atuveciclib, which protects nucleus pulposus (NP) cells from proinflammatory stimuli-induced catabolism. The effects of CDK9 inhibition were determined in human and rat NP cells treated with IL-1β in the presence or absence of atuveciclib or small interfering RNA target CDK9. Inhibition of CDK9 led to the attenuation of inflammatory response. In addition, rat intervertebral disk (IVD) explants were used to determine the role of CDK9 inhibition in extracellular matrix degradation. The rat IVDD model also proved that CDK9 inhibition attenuated IVDD, as validated using magnetic resonance imaging and immunohistochemistry. Taken together, CDK9 is a potential therapeutic target to prevent IVDD.

Targeting CDK9: A novel biomarker inthetreatment of endometrial cancer.

Endometrial cancer is one of the three major malignant tumors of the female reproductive system. Although cyclin-dependent kinase 9 (CDK9) has a definitive pathogenic role in various types of cancer, little is known concerning its function in endometrial cancer. Our study was conducted to evaluate the expression and therapeutic potential of CDK9 in endometrial cancer. CDK9 expression was determined by immunohistochemistry in endometrial cancer tissues constructed with paired primary, metastatic, and recurrent tumor tissues from 32 endometrial cancer patients. Small interfering RNA (siRNA) and inhibitors of CDK9 were used to evaluate the effect of CDK9 inhibition on the anti-apoptotic activity and proliferation in endometrial cancer cells. Colony formation assay and wound-healing assays were adopted to assess clonal formation and migratory capacity. The results of the immunohistochemistry demonstrated that CDK9 was highly expressed in the human endometrial cancer cell lines; moreover, it was elevated in metastatic and recurrent endometrial tumor tissue compared when compared with that in patient-matched primary endometrial tumor tissue. Knockdown of CDK9 with siRNA and inhibition of CDK9 activity with the inhibitor suppressed cell proliferation and promoted apoptosis in endometrial cancer. In conclusion, our results provide evidence that CDK9 may be a potential prognostic biomarker and a promising therapeutic target for the treatment of endometrial cancer in the future.

Recent Developments in the Biology and Medicinal Chemistry of CDK9 Inhibitors: An Update.

Cyclin-dependent kinase 9 (CDK9), which regulates transcriptional elongation, is an attractive therapeutic target for many cancers, especially for cancers driven by transcriptional dysregulation. In particular, CDK9 promotes RNA polymerase II pause/release, a rate-limiting step in normal transcriptional regulation that is frequently dysregulated in cancers. Emerging evidence indicates that selective CDK9 inhibition or degradation may provide a therapeutic benefit against certain cancers. Indeed, the development of CDK9 modulators (inhibitors and degraders) has attracted great attention, with several molecules currently under clinical development. This review provides an overview of recent advances in CDK9 modulators in general, with special emphasis on compounds under clinical evaluation and new emerging strategies, such as proteolysis targeting chimeras (PROTACs).

Abstract 5221: Development of TGN-1076, a selective and reversible small molecule inhibitor of CDK7

Abstract Purpose: Tumor genetic heterogeneity can lead to dysregulation of transcriptional programs and cause cancer cells to become highly dependent on certain regulators of gene expression. Super-enhancers (SEs) are a class of regulatory regions that are associated with a strong enrichment of transcriptional coactivators (MED1, BRD4) and chromatin marks (H3K27Ac). It has been shown that tumor cells can acquire SEs at oncogenic driver genes (MYC, TERT, BCL2, GFI1, MED1, OCT4, and SOX2), activate their expression, and induce transcriptional reprogramming. Importantly, the majority of super-enhancers depend on the ability of cyclin-dependent kinase 7 (CDK7) to initiate transcription by phosphorylating the C-terminal domain of RNA Pol II. Recent reports have shown that high level of CDK7 is associated with poor prognosis in breast, gastric, ovarian, pancreatic, SCLC, ATC, and AML cancers. We have embarked on developing a novel therapeutic that specifically targets the CDK7 catalytic ATP pocket. Previously, we reported the discovery of TGN-1062 as a potent inhibitor of CDK7. In this study, we report the development of TGN-1076, an analog of TGN-1062, as a selective and reversible inhibitor of CDK7 due to its ideal candidate criteria. Methods: With structure-guided and iterative medicinal chemistry approaches, the previous lead candidate TGN-1062 was further optimized to improve potency, target selectivity, and desirable drug-like properties. In vitro kinase assays were performed using ADP-GloTM kinase assay kit (Promega). Cell viability assays were performed in a panel of cancer cell lines using CellTiter-Glo assay kit (Promega). RNA Pol II phosphorylation and total MYC protein levels were determined by Western Blotting. Results: TGN-1076 inhibited CDK7 activity with an IC50 of 20 nM and showed 10-500 fold selectivity against the closest members of the CDK family. TGN-1076 blocked growth of multiple cancer cells, among these pancreatic cancer cells were most sensitive with an GI50 of <0.6 µM. More importantly, phosphorylation of RNA Pol II and super-enhancer dependent expression of MYC as well as it's downstream target MCL-1 were significantly reduced in TGN-1076 treated MiaPaca2 pancreatic cancer cells. TGN-1076 showed high micromolar activity (>5 µM) against hERG and multiple P450 isozymes. The results from these studies along with ADME-Tox and pharmacokinetics will be presented. Conclusion: In summary, we have developed a novel selective and reversible inhibitor of CDK7. Our current efforts are focused on further optimization and nomination of a preclinical candidate for ADME-Tox and tumor efficacy studies. Citation Format: Trason Thode, Zhaoliang Li, Alexis Weston, Srikanta Dana, Sherin Daniel Ampanattu, Raffaella Soldi, Mohan Rao Kaadige, Hariprasad Vankayalapti, Vincent Chung, Joseph Chao, Daniel D Von Hoff, Sunil Sharma. Development of TGN-1076, a selective and reversible small molecule inhibitor of CDK7 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5221.

Abstract 4849: YPN-005, an oral CDK7 inhibitor, shows a significant antitumor activity in Myc amplified solid tumors and MCL-1 overexpressed blood cancer

Abstract Background: Cyclin dependent kinase 7 (CDK7) has been recognized as one of potential therapeutic targets in Myc-driven cancers. Previously, we synthesized selective irreversible CDK7 inhibitor and we demonstrated a significant inhibition of the proliferation of TNBC and HCC cells by down-regulation of c-Myc and MCL-1. We further report YPN-005 of the preclinical data with biological characterization. Methods: Antiproliferative activity of YPN-005 was measured in solid tumor cell lines with Myc amplification and in hematologic cancer cells with MCL-1overexpression. To investigate the mechanism of action of YPN-005, the expression levels of phospho-RNA polymerase II, phospho-CDK2 and -CDK4, BClXL, cleaved-PARP, c-Myc and MCL-1 were analyzed by Western Blot analyses. CDK7 occupancy was determined by using a biotinylated small molecule probe following incubating of TNBC, HCC, AML cells and hPBMC with YPN-005. The therapeutic efficacy of YPN005 was evaluated in TNBC and HCC xenograft mouse model. Results: YPN-005 treatment induced apoptosis and cell cycle arrest. Selectivity of YPN-005 on CDK7 was confirmed in the 468 kinases panel assay. YPN-005 significantly inhibited the proliferation of TNBC, ER+ breast cancer, HCC, ovarian cancer, small cell lung cancer (SCLC), pancreas, prostate and various types of blood cancer cells and IC50's were determined in 1-20 nM range. Inhibition of cell proliferation was accompanied by down-regulation of RNA PolII phosphorylation, c-Myc and MCL-1 expression. Washout assay by Western Blot demonstrated that YPN-005 irreversibly blocked RNA PolII phosphorylation. In addition, YPN-005 downregulated the adaptive immune checkpoint PD-L1 by the depletion of c-Myc in TNBCs. CDK7 occupancy over 90% was observed in TNBC, HCC, and AML cells with 100nM of YPN-005. In vivo xenograft models of TNBC and HCC showed that daily oral administration of YPN-005 for 21 days (once, and 10~20mg/kg) efficiently inhibited the growth of tumor. All the mice survived and there was no significant changes in their hematologic profiles. Conclusion: Our findings suggest that YPN-005 is an orally available, potent, and selective CDK7 inhibitor which can be used for the treatment of Myc and MCL-1 overexpressing cancer patients. These data support the rationale for advancing YPN-005 towards IND-enabling studies. Citation Format: Jakyung Yoo, Jun Hee Lee, Mi Jung Lee, Kang Woo Lee, Ji Eun Min, Jinhwan Kim, Ki-Nam Min, Da-Hye Jeon, Dae Seong Lim, Kwang-Ok Lee. YPN-005, an oral CDK7 inhibitor, shows a significant antitumor activity in Myc amplified solid tumors and MCL-1 overexpressed blood cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4849.

Abstract 3564: Anti-tumor effect and HIF1α inhibition by combining CDK4 inhibitor with HSP90 inhibitor in various cancer types including Rb-deficient tumor cells

Abstract As solid tumors outgrow their oxygen supply, regions with abnormal vasculature become poorly oxygenated, known as intratumoral hypoxia. Hypoxia-inducible factor 1α (HIF1α) is the main mediator to hypoxia and promotes angiogenesis, cell survival as well as cancer metastases. Overexpression of HIF1α is correlated with poor prognosis in many malignancies such as colorectal cancers. Targeting HIF1α is a potential strategy for cancer therapy. We have previously shown that cyclin-dependent kinase 1 (CDK1) stabilizes HIF1α through direct phosphorylation of its Ser668 residue in a Von Hippel-Lindau (VHL)-independent manner both under hypoxia and at G2/M under normoxia (Warfel et al., 2013). It has been acknowledged that the heat shock protein 90 (HSP90) is also a VHL-independent HIF1α stabilizer (Isaacs JS et al., 2002). We found that CDK1 may contribute to HSP90-mediated HIF1α stabilization. Combination treatment of HSP90 inhibition and CDK1 knockdown reduces HIF1α expression in hypoxia more robustly than either single treatment. Dual inhibition of CDK1 and HSP90 synergistically decreases cell viability in colorectal cancer cells and suppresses colony formation. We have observed that CDK4 plays a role in HIF1α stabilization. Using the FDA-approved CDK4/6 inhibitors (palbociclib & abemaciclib), we observed that dual inhibition of CDK4 and HSP90 synergistically inhibits cancer viability in colorectal cancer cell lines (eg. HCT116, SW480, DLD1) under both normoxia and hypoxia. Multiple HSP90 inhibitors have been tested, including ganetespib, onalespib, XL888 and TAS116, which indicates such combinational inhibition as a class effect. As expected, the combination treatment of CDK4 knockdown/inhibition and HSP90 inhibition is able to reduce the level of HIF1α and suppress cell viability in various cancer cell lines (eg. colorectal, glioblastoma, breast, and prostate cancers). The combination treatment induces PARP cleavage, indicating activated apoptosis. Interestingly, we found that knockdown of RB does not affect the response of HCT116 or SW480 cells to the combination treatment with abemaciclib and TAS116, although RB deficiency contributes to a relative resistance to CDK4/6 inhibitor monotherapy. Combination of abemaciclib plus TAS116 inhibits cell viability in Rb-deficient cells (eg. H1048 and Saos2). Hypoxia sensitizes the Rb-deficient MDA-MB-468 breast cancer cell line to abemaciclib treatment. Our findings suggest a therapeutic potential for utilizing the combination of CDK4 and HSP90 inhibitors in cancer treatment. Citation Format: Shuai Zhao, Lanlan Zhou, David T. Dicker, Wafik S. El-Deiry. Anti-tumor effect and HIF1α inhibition by combining CDK4 inhibitor with HSP90 inhibitor in various cancer types including Rb-deficient tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3564.

Abstract 5814: Emergent functions of cyclin-dependent kinase 4 regulating aurora b and cenpp transcription

Abstract A better understanding of the precise molecular mechanisms that control cell proliferation is crucial for the identification of novel cancer therapeutic targets. A critical molecule in this process is the Cyclin-Dependent Kinase 4 (CDK4), which regulates key aspects of the G1 to S phase transition through phosphorylation of the retinoblastoma (Rb) family of proteins. Since dysregulation of this cell cycle mechanism is a hallmark of most cancers, drugs have been developed to inhibit the kinase activity of CDK4. However, such drugs have been met with variable degrees of success and, in some cases, little efficacy. Research performed in the last few years has suggested that CDK4 have alternative targets other than the Rb proteins. Consistent with this idea, we found that CDK4 plays additional roles not related to the canonical function in the CDK-Rb axis. Here, we show that CDK4 remains attached to the chromatin fraction of Balb/MK2 keratinocytes upon the extraction of the soluble components (nucleoplasm and cytoplasm). Chromatin-immunoprecipitation (ChIP) analysis shows that CDK4 occupies the regulatory sites of genes associated with chromosomal segregation, such as Aurkb (Aurora B) and Cenpp (Centromere Protein P). Moreover, gain- and loss-of-function experiments showed that the AurkB and Cenpp promoters are regulated through CDK4 binding. In line with these findings, studies in skin carcinogenesis revealed that transgenic expression of CDK4 in mouse epidermis induces chromosome instability (CIN) and centrosome amplification (CA), two events linked to overexpression of Aurora B. Moreover, Aurora-B has a crucial role in chromosome bi-orientation and the spindle-assembly checkpoint, whereas Cenpp is required for proper kinetochore function. Remarkably, a persistent dysregulation of Aurora B has been reported in numerous human cancers. Thus, our results suggest that CDK4 plays a role in the maintenance of chromosomal stability, implying that additional functions could be inhibited by therapeutically targeting CDK4. Our conclusions are supported by previous reports showing that pharmacological inhibition of CDK4 led to delayed progression from G2 to mitosis in Rb-positive and as well as Rb-negative cells. Citation Format: Sung Hyun Lee, Liliana R. Rodriguez, Paula L. Miliani de Marval, Marcelo L. Rodriguez-Puebla. Emergent functions of cyclin-dependent kinase 4 regulating aurora b and cenpp transcription [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5814.

CDK7 inhibitor THZ1 enhances antiPD-1 therapy efficacy via the p38\u03b1/MYC/PD-L1 signaling in non-small cell lung cancer

BackgroundThe cyclin-dependent kinase 7 (CDK7) subunit of TFIIH regulates RNA polymerase-II-based transcription and promotes tumor progression. However, the mechanisms involved in CDK7-mediated immune evasion are unclear in non-small cell lung cancer (NSCLC).MethodsRNA silencing and pharmacologic inhibitors were used to evaluate the functions of CDK7/p38α/MYC/PD-L1 axis in cancer cell proliferation and antiPD-1 therapy resistance. Flow cytometry was performed to detect the status of the immune microenvironment after CDK7 inhibition and antiPD-1 therapy in vivo. CD8 depletion antibodies were used to assess the role of CD8+ T cells in combined CDK7 and PD-1 blockade. The associations among CDK7, p38α, MYC, PD-L1, infiltrating T cells, and survival outcomes were validated in two tissue microarrays and public transcriptomic data of NSCLC.ResultsHigh CDK7 mRNA and protein levels were identified to be associated with poor prognosis in NSCLC. CDK7 silencing and CDK7 inhibitor THZ1 elicited apoptosis and suppressed tumor growth. Moreover, CDK7 ablation specifically suppressed p38α/MYC-associated genes, and THZ1 inhibited MYC transcriptional activity through downregulating p38α. CDK7 inhibition sensitized NSCLC to p38α inhibitor. Further, THZ1 suppressed PD-L1 expression by inhibiting MYC activity. THZ1 boosted antitumor immunity by recruiting infiltrating CD8+ T cells and synergized with antiPD-1 therapy. The CDK7/MYC/PD-L1 signature and infiltrating T cell status collectively stratified NSCLC patients into different risk groups.ConclusionThese data suggest that the combined CDK7 inhibitor THZ1 and antiPD-1 therapy can be an effective treatment in NSCLC.

Targeting complexes of super-enhancers is a promising strategy for cancer therapy.

The hyperactivation and overexpression of critical oncogenes is a common occurrence in multiple types of malignant tumors. Recently, the abnormal activation mechanism of an oncogene by a super-enhancer (SE) has attracted significant attention. A series of changes (insertion, deletion, translocation and rearrangement) in the genome occurring in cancer cells may generate new SEs, leading to the overexpression of SE-driven oncogenes. SEs are composed of typical enhancers densely loaded with mediator complexes, transcription factors, and chromatin regulators, and drive the overexpression of oncogenes associated with cellular identity and disease. Cyclin-dependent kinase 7 (CDK7) and bromodomain protein 4 (BRD4) are critical mediator complexes associated with SE-mediated transcription. Clinical trials have shown that emerging small-molecule inhibitors (CDK7 and BRD4 inhibitor), targeting the SE exert a notable effect on cancer treatment. Increasing evidences has illustrated that the SE and its associated complexes play a critical role in the development of various types of cancer. The present review discusses the composition, function and regulation of SEs and their contribution to oncogenic transcription. In addition, creative therapeutic approaches that target SE, their advantages and disadvantages, as well as the problems with their clinical application are discussed. It was found that targeting SE may be used in conventional treatment and establish more access for patients with cancer.

Transcriptome analysis of neratinib treated HER2 positive cancer model vs untreated cancer unravels the molecular mechanism of action of neratinib

Human estrogen receptor positive cancer cells have mutations and make an excess of the HER2 protein and are far more aggressive than others cancers. Neratinib, an irreversible tyrosine kinase inhibitor is used to treat HER2 positive cancers. Neratinib targets HER2 and blocks its signal transduction resulting in inhibition of cell proliferation and induction of apoptosis without any information about the molecular mechanism involved. To understand the underlying molecular mechanism transcriptome analysis was carried out in normal vs cancer induced SWR/J nude mice. Cancer was induced in SWR/J nude mice with intraperitoneal injection of 5 × 106 SKBR3 cells for 14 days. Histopathology confirmed the induction of cancer in liver and kidney after the tumor size was at least 0.5 cm. Genome wide Mouse U133 Array was used to analyze the effect of neratinib treatment on cancer. Validation of expression was done by qPCR and ELISA. Microscopic examination revealed that neratinib treatment has potential effects on cancerous liver. Transcriptome expression profiling showed 1481 transcripts differentially expressed by neratinib treatment. Transcriptome Analysis Console (TAC) showed that 532 upregulated transcripts were exclusively belonging to cell cycle, inflammation, olfaction, oxidative stress, HER, and EGFR1 while 949 downregulated transcripts were involved in immunology, drug resistance such as histocompatibility, T cell receptors, and immunoglobulins. The differentially expressed genes were considered significant under the criteria of an adjusted p-value < 0.02 and log2 ratios ≥ 1.0 and/or log2 ratios ≤ − 1.0 means two Fold change. qPCR assay and ELISA analysis was used to validate few genes involved in apoptosis and proliferation. This study provides new insights into the neratinib’s mode of action by cyclin-dependent kinase inhibitor-3 and calcium-activated chloride channel 3 as markers for treatment progress.

Multiple myeloma: Combination therapy of BET proteolysis targeting chimeric molecule with CDK9 inhibitor.

Cyclin Dependent Kinase 9 (CDK9) associates with Bromodomain and Extra-Terminal Domain (BET) proteins to promote transcriptional elongation by phosphorylation of serine 2 of RNAP II C-terminal domain. We examined the therapeutic potential of selective CDK9 inhibitors (AZD 4573 and MC180295) against human multiple myeloma cells in vitro. Short-hairpin RNA silencing of CDK9 in Multiple Myeloma (MM) cell lines reduced cell viability compared to control cells showing the dependency of MM cells on CDK9. In order to explore synergy with the CDK9 inhibitor, proteolysis targeting chimeric molecule (PROTAC) ARV 825 was added. This latter drug causes ubiquitination of BET proteins resulting in their rapid and efficient degradation. Combination treatment of MM cells with ARV 825 and AZD 4573 markedly reduced their protein expression of BRD 2, BRD 4, MYC and phosphorylated RNA pol II as compared to each single agent alone. Combination treatment synergistically inhibited multiple myeloma cells both in vitro and in vivo with insignificant weight loss. The combination also resulted in marked increase of apoptotic cells at low dose compared to single agent alone. Taken together, our studies show for the first time that the combination of a BET PROTAC (ARV 825) plus AZD 4573 (CDK9 inhibitor) is effective against MM cells.

Cyclin-Dependent Kinase 9 Inhibition Suppresses Necroptosis and Pyroptosis in the Progress of Endotoxemia.

The host innate immune response stands at the first line of defense against the outburst of pathogen invasion and their byproduct release. The balanced and coordinated expression of genes in normal immune responses is compromised in the progress of endotoxemia with exacerbated inflammation and massive cell death. In the present study, we identified cyclin-dependent kinase 9 (CDK9), the functional subunit of the positive transcription elongation factor b, as a master regulator of inflammatory gene transcription in the process of promoter-proximal pausing to productive elongation. Therapeutic pharmacological inhibition of CDK9 by flavopiridol (FVD) rescued mice from death in experimental models of fatal endotoxemia. In addition to alleviation of the cytokine storm in the circulation system following lethal endotoxin injection, FVD treatment significantly dampened the onset of inflammation in the livers and lungs and reduced the necroptosis and pyroptosis in livers. Moreover, CDK9 inhibition reduced inflammatory cytokine release and decreased cell death in the pro-inflammatory pyroptotic and necroptotic cell death pathway in monocytes in responses to lipopolysaccharide. In conclusion, CDK9 inhibition may affect the progress of endotoxemia by dampening inflammation and cell death including necroptosis and pyroptosis.

Targeting CDK4 (cyclin-dependent kinase) amplification in liposarcoma: A comprehensive review.

Well-differentiated/dedifferentiated (WD/DD) liposarcomas, the most common form of liposarcomas, constitute up to 20 % of all soft tissue sarcomas. Several oncogenes are thought to be involved in liposarcoma pathogenesis, mainly MDM2, CDK4 and HMGA2. While MDM2 inhibitors are now tested in clinical trials, a second actionable and promising target appears to be cyclin-dependent kinase 4 (CDK4), which is amplified in up to 90 % of well differentiated or dedifferentiated (WD/DD) liposarcoma. With the paucity of available therapeutic options, the inhibition of CDK4 represent a potential therapeutic option. In this paper, we review the role of CDK4/6 inhibitors in targeting the commonly identified CDK4 amplification in WD/DD liposarcoma, with an emphasis on the published and currently ongoing trials.

065 CDK7 inhibitor suppresses psoriasis inflammation via inhibiting glycolysis to modulate Th17/Treg balance

Psoriasis is a chronic inflammatory disease characterized by hyper-activated Th17 and suppressive Treg cells, but the mechanism of Th17/Treg cells imbalance is still unclear. Cyclin-dependent kinase 7(CDK7) which is known as a cell cycle regulator has been reported an anti-inflammatory effect in immuno-cells. Here we firstly found CD4+T cells of psoriasis patients expressed higher levels of CDK7 along with an increased glycolysis levels than those in healthy controls. The chemical inhibitor of CDK7 called THZ1 restricted glycolytic metabolism in CD4+T cells of psoriasis patients as well as typical glycolysis related genes. More importantly, THZ1 could suppress Th17 cell differentiation and promote Treg cell differentiation even under Th17 polarizing condition in vitro. Intraperitoneal injection of THZ1 in mice exhibited an alleviated epidermal hyperplasia and alleviated inflammation caused by imiquimod(IMQ) treatment. THZ1-treated IMQ mice had significantly lower ratio of Th17 cells and higher ratio of Treg cells from the splenocytes, in comparison with vehicle-treated IMQ mice. Furthermore, we identified IL-23 as an upstream regulator that stimulated CDK7 expression and glycolysis through p-AKT-Hif-1α signalling pathway. Taken together, our results showed that abnormal CDK7 expression induced by IL-23 in CD4+ T cells in psoriasis patients contributed to the enhanced glycolysis levels which lead to the imbalance of Th17/Treg cells. CDK7 inhibitor THZ1 may serve as an immuno-modulator for psoriasis therapy in the future.