Cyclin-dependent Kinase 9 Inhibitor Research Articles

Cyclin dependent kinase inhibitor 3 (CDKN3) upregulation is associated with unfavorable prognosis in clear cell renal cell carcinoma and shapes tumor immune microenvironment: A bioinformatics analysis.

Cell cycle regulatory proteins plays a pivotal role in the development and progression of many human malignancies. Identification of their biological functions as well as their prognostic utility presents an active field of research. As a continuation of the ongoing efforts to elucidate the molecular characteristics of clear cell renal cell carcinoma (ccRCC); we present a comprehensive bioinformatics study targeting the prognostic and mechanistic role of cyclin-dependent kinase inhibitor 3 (CDKN3) in ccRCC. The ccRCC cohort from the Cancer Genome Atlas Program was accessed through the UCSC Xena browser to obtain CDKN3 mRNA expression data and their corresponding clinicopathological variables. The independent prognostic signature of CDKN3 was evaluated using univariate and multivariate Cox logistic regression analysis. Gene set enrichment analysis and co-expression gene functional annotations were used to discern CDKN3-related altered molecular pathways. The tumor immune microenvironment was evaluated using TIMER 2.0 and gene expression profiling interactive analysis. CDKN3 upregulation is associated with shortened overall survival (hazard ratio [HR] = 2.325, 95% confident interval [CI]: 1.703-3.173, P < .0001) in the Cancer Genome Atlas Program ccRCC cohort. Univariate (HR: 0.426, 95% CI: 0.316-0.576, P < .001) and multivariate (HR: 0.560, 95% CI: 0.409-0.766, P < .001) Cox logistic regression analyses indicate that CDKN3 is an independent prognostic variable of the overall survival. High CDKN3 expression is associated with enrichment within the following pathways including allograph rejection, epithelial-mesenchymal transition, mitotic spindle, inflammatory response, IL-6/JAK/STAT3 signaling, spermatogenesis, TNF-α signaling via NF-kB pathway, complement activation, KRAS signaling, and INF-γ signaling. CDKN3 is also associated with significant infiltration of a wide spectrum of immune cells and correlates remarkably with immune-related genes. CDKN3 is a poor prognostic biomarker in ccRCC that alters many molecular pathways and impacts the tumor immune microenvironment.

Degradation of Cyclin-Dependent Kinase 9/Cyclin T1 by Optimized Microtubule-Associated Protein 1 Light Chain 3 Beta-Recruiting Coumarin Analogs.

Autophagy is an efficient and attractive protein degradation pathway in addition to the ubiquitin-proteasome system. Herein, systematic optimization of coumarin analogs linked with the CDK9 inhibitor SNS-032 is reported that may bind to cyclin-dependent kinase 9 (CDK9) and microtubule-associated protein 1 light chain 3 beta (LC3B) simultaneously, which leads to the selective autophagic degradation of targeted CDK9/cyclin T1 and is different from the PROTAC degrader THAL-SNS-032. Further mechanism studies revealed an autophagy-lysosome pathway, where the degraders possibly formed a ternary complex with CDK9 and LC3B. In addition, degrader 10 showed antitumor efficacy in vivo. Our work optimized a potent LC3B recruiter and demonstrated the feasibility of autophagy-tethering compounds (ATTECs), which could be applied for the degradation of diverse intracellular pathogenic proteins to treat related diseases.

CDK9 Inhibitor Induces Apoptosis, Autophagy, and Suppression of Tumor Growth in Adult T-Cell Leukemia/Lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is a hematopoietic malignancy with a poor prognosis that develops in approximately 5% of human T-cell leukemia virus type 1 (HTLV-1) carriers. Cyclin-dependent kinase 9 (CDK9), together with Cyclin T, forms a transcription elongation factor, positive transcription elongation factor b (P-TEFb). P-TEFb promotes transcriptional elongation by phosphorylating the second serine (Ser2) of the seven amino acid repeat sequence in the C-terminal domain of RNA polymerase II (RNAP II). CDK9 inhibitors suppress cell proliferation by inducing apoptosis in chronic lymphocytic leukemia and breast cancer but there are no reports on autophagy of CDK9 inhibitors. Here, we investigated the effect of LY2857785, a novel CDK9 selective inhibitor, on cell death in ATL-related cell lines in vitro, freshly isolated cells from ATL patients ex vivo, and on ATL tumor xenografts in NOD/SCID mice in vivo. LY2857785 significantly reduced cell viability and induced apoptosis, as shown by annexin V-positive cells, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase-3, and suppressed the levels of anti-apoptotic protein myeloid cell leukemia-1 (MCL-1). LY2857785 decreased RNAP II Ser2 phosphorylation and downstream c-Myc protein levels. Interestingly, LY2857785 also increased microtubule-associated proteins 1A/1B light chain 3B (LC3)-II binding to autophagosome membranes. Furthermore, LY2857785 decreased the viability of freshly isolated ATL cells and induced apoptosis. Finally, LY2857785 significantly decreased the growth of ATL tumor xenografts. These results suggest that LY2857785 induces cell death of ATL cells by MCL-1-dependent apoptosis and autophagy and has anti-tumor activity.

Abstract P2147: Inhibition Of Cdk7 Attenuates Doxorubicin Cardiotoxicity And Enhances Anticancer Efficacy

Background: The anthracycline family of anticancer agents such as doxorubicin (DOX) can induce apoptotic death of cardiomyocytes and cause cardiotoxicity. We previously reported that DOX-induced apoptosis is accompanied by cardiomyocyte cell cycle-reentry. Cell cycle progression requires cyclin-dependent kinase 7 (CDK7)-mediated activation of downstream cell cycle CDKs. This study aims to determine whether CDK7 can be targeted for cardioprotection during anthracycline chemotherapy. Methods and Results: DOX exposure induced CDK7 activation in mouse heart and isolated cardiomyocytes. Cardiac-specific ablation of Cdk7 attenuated DOX-induced cardiac dysfunction and fibrosis. Treatment with the covalent CDK7 inhibitor THZ1 also protected against DOX-induced cardiomyopathy and apoptosis. DOX induced activation of the proapoptotic CDK2-FOXO1-Bim axis in a CDK7-dependent manner. In response to DOX, endogenous CDK7 directly bound and phosphorylated CDK2 at Thr160 in cardiomyocytes, leading to full CDK2 kinase activation. Importantly, inhibition of CDK7 further suppressed tumor growth when used together with DOX in an immunocompetent mouse model of breast cancer. Conclusions: Activation of CDK7 is necessary for DOX-induced cardiomyocyte apoptosis and cardiomyopathy. Our findings uncover a novel proapoptotic role for CDK7 in cardiomyocytes. Moreover, this study suggests that inhibition of CDK7 attenuates DOX-induced cardiotoxicity, but augments the anticancer efficacy of DOX. Therefore, combined administration of CDK7 inhibitor and DOX may exhibit diminished cardiotoxicity but superior anticancer activity.

Discovery of SHR5428 as a selective and noncovalent inhibitor of CDK7

Cyclin dependent kinase 7 (CDK7) is an attractive target in tumor indications via regulating both cell cycle and transcription. Here, SHR5428 was discovered as a selective and noncovalent CDK7 inhibitor with highly potent CDK7 enzymatic activity and triple negative breast cancer cellular activity on MDA-MB-468 cell. SHR5428 also displayed favorable pharmacokinetic properties in different preclinical species such as mouse, rat and dog, and showed high selectivity over CDK1, CDK2, CDK4, CDK6, CDK9, CDK12 in CDK family. Furthermore, the computational modeling has shed some light on this mechanism. Additionally the in vivo efficacy study in a breast cancer cell line (HCC70 cell) derived xenograft mouse model proved SHR5428 to be orally efficacious with dose-dependent tumor growth inhibition.

Inhibition of CDK1 by RO-3306 Exhibits Anti-Tumorigenic Effects in Ovarian Cancer Cells and a Transgenic Mouse Model of Ovarian Cancer.

Ovarian cancer is the deadliest gynecological malignancy of the reproductive organs in the United States. Cyclin-dependent kinase 1 (CDK1) is an important cell cycle regulatory protein that specifically controls the G2/M phase transition of the cell cycle. RO-3306 is a selective, ATP-competitive, and cell-permeable CDK1 inhibitor that shows potent anti-tumor activity in multiple pre-clinical models. In this study, we investigated the effect of CDK1 expression on the prognosis of patients with ovarian cancer and the anti-tumorigenic effect of RO-3306 in both ovarian cancer cell lines and a genetically engineered mouse model of high-grade serous ovarian cancer (KpB model). In 147 patients with epithelial ovarian cancer, the overexpression of CDK1 was significantly associated with poor prognosis compared with a low expression group. RO-3306 significantly inhibited cellular proliferation, induced apoptosis, caused cellular stress, and reduced cell migration. The treatment of KpB mice with RO-3306 for four weeks showed a significant decrease in tumor weight under obese and lean conditions without obvious side effects. Overall, our results demonstrate that the inhibition of CDK1 activity by RO-3306 effectively reduces cell proliferation and tumor growth, providing biological evidence for future clinical trials of CDK1 inhibitors in ovarian cancer.

Inhibition of CDK1 Overcomes Oxaliplatin Resistance by Regulating ACSL4-mediated Ferroptosis in Colorectal Cancer.

Oxaliplatin is a widely used chemotherapy drug for patients with advanced colorectal cancer (CRC); however, frequent drug resistance limits its therapeutic efficacy in patients. Here, this work identifies cyclin-dependent kinase 1 (CDK1) as a critical contributor to oxaliplatin resistance via in vitro and in vivo CRISPR/Cas9 screening. CDK1 is highly expressed in oxaliplatin-resistant cells and tissues due to the loss of N6-methyladenosine modification. Genetic and pharmacological blockade of CDK1 restore the susceptibility of CRC cells to oxaliplatin in vitro and in cell/patient-derived xenograft models. Mechanistically,CDK1 directly binds to and phosphorylates Acyl-CoA synthetase long-chain family 4 (ACSL4) at S447, followed by recruitment of E3 ubiquitin ligase UBR5 and polyubiquitination of ACSL4 at K388, K498, and K690, which leads to ACSL4 protein degradation. Reduced ACSL4 subsequently blocks the biosynthesis of polyunsaturated fatty acid containing lipids, thereby inhibiting lipid peroxidation and ferroptosis, a unique iron-dependent form of oxidative cell death. Moreover, treatment with a ferroptosisinhibitor nullifies the enhancement of CRC cell sensitivity to oxaliplatin by CDK1 blockade in vitro and in vivo. Collectively, the findings indicate that CDK1 confers oxaliplatin resistance to cells by suppressing ferroptosis. Therefore, administration of a CDK1 inhibitor may be an attractive strategy to treat patients with oxaliplatin-resistant CRC.

N76-1, a novel CDK7 inhibitor, exhibits potent anti-cancer effects in triple negative breast cancer

Emerging evidence suggests that genetically highly specific triple-negative breast cancer (TNBC) possesses a relatively uniform transcriptional program that is abnormally dependent on cyclin-dependent kinase 7 (CDK7). In this study, we obtained an inhibitor of CDK7, N76-1, by attaching the side chain of the covalent CDK7 inhibitor THZ1 to the core of the anaplastic lymphoma kinase inhibitor ceritinib. This study aimed to elucidate the role and underlying mechanism of N76-1 in TNBC and evaluate its potential value as an anti-TNBC drug. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays showed that N76-1 inhibited the viability of TNBC cells. Kinase activity and cellular thermal shift assays showed that N76-1 directly targeted CDK7. Flow cytometry results revealed that N76-1 induced apoptosis and cell cycle arrest in the G2/M phase. N76-1 also effectively inhibited the migration of TNBC cells by high-content detection. The RNA-seq analysis showed that the transcription of genes, especially those related to transcriptional regulation and cell cycle, was suppressed after N76-1 treatment. Moreover, N76-1 markedly inhibited the growth of TNBC xenografts and phosphorylation of RNAPII in tumor tissues. In summary, N76-1 exerts potent anticancer effects in TNBC by inhibiting CDK7 and provides a new strategy and research basis for the development of new drugs for TNBC.

Phase 1 dose-expansion study of oral TP-1287, a cyclin-dependent kinase 9 (CDK9) inhibitor, in patients with Ewing sarcoma (EWS).

TPS11589 Background: Cyclin-dependent kinase 9 (CDK9) blockade inhibits tumor growth and progression by impairing the transcription of key oncogenes, such as myeloid cell leukemia-1 (MCL-1) and c-MYC. CDK9 overexpression has been observed in sarcoma patients (pts), and CDK9 has emerged as a potential therapeutic target in pts with sarcoma. TP-1287 is an investigational orally delivered phosphate prodrug of the CDK9 inhibitor alvocidib. In preclinical studies, TP-1287 has been shown to decrease MCL-1 expression and phosphorylation of RNA polymerase II (RPB1), and inhibit tumor growth in an Ewing sarcoma (EWS) mouse model. Phase 1 dose-escalation in solid tumors has completed and TP-1287 is being investigated in a dose expansion cohort in pts with EWS (NCT03604783). The design of the expansion part of the trial in pts with EWS is herein described. Methods: Up to thirty pts with EWS will be enrolled in this dose expansion cohort. Key eligibility criteria are age ≥18 years (≥12 years, if weight ≥40 kg); histologically confirmed locally advanced or metastatic unresectable EWS; received 1 to 5 prior lines of treatment including an anthracycline; one or more measurable tumors per the RECIST v1.1; ECOG performance status of ≤1; acceptable liver and renal function, and acceptable hematologic and coagulation status; no treatment with surgery, chemotherapy, or investigational therapy within 28 days or 5 half-lives. Eligible pts will be treated with oral TP-1287 monotherapy RP2D established from the dose escalation part (11 mg BID, continuous dosing in a 28-day treatment cycle) and will continue treatment until treatment-related adverse event or disease progression. Assessments will be performed on Day 1 and Day 15 of each cycle. Tumor response assessment will be done after Cycle 2 and at the end of every other cycle thereafter. The primary objectives are objective response rate (ORR) and clinical benefit rate (CBR) ; secondary objectives are median progression-free survival (PFS), PFS rate at 16-weeks and 24-weeks and safety; and exploratory objectives include evaluation of systemic exposure and pharmacodynamics. Safety data will be reviewed on an ongoing basis and a Bayesian approach will be used to assess the efficacy data. Statistical analysis for safety and efficacy parameters will be primarily descriptive in nature. EWS dose expansion cohort is currently recruiting in the United States. Clinical trial information: NCT03604783 .

Inhibition of CDK9 exhibits anticancer activity in hepatocellular carcinoma cells via targeting ribonucleotide reductase

Cyclin-dependent kinase 9 (CDK9) inhibitors are a novel category of anticancer treatment for cancers. However, their effects on hepatocellular carcinoma (HCC) are rarely investigated. Human ribonucleotide reductase (RR, which consists of RRM1 and RRM2 subunits) catalyzes the conversion of ribonucleoside diphosphate into 2′-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools, which play essential roles in DNA synthesis and DNA repair. In this study, we identified that CDK9 protein expression in adjacent non-tumor tissues predicted HCC patients' overall and progression-free survivals. The anticancer activity of a CDK9-selective inhibitor, LDC000067, on HCC cells was positively associated with its ability to inhibit the expression of RRM1 and RRM2. LDC000067 downregulated RRM1 and RRM2 expression through post-transcriptional pathway. Specifically, LDC000067 triggered RRM2 protein degradation via multiple pathways, including proteasome-, lysosome-, and calcium-dependent pathways. Furthermore, CDK9 positively correlates with RRM1 or RRM2 expression in HCC patients, and the expressions of these three genes were associated with the higher infiltration of immune cells in HCC. Taken together, this study identified the prognostic relevance of CDK9 in HCC and the molecular mechanism for the anticancer effect of CDK9 inhibitors on HCC.

Disruption of CDK7 signaling leads to catastrophic chromosomal instability coupled with a loss of condensin-mediated chromatin compaction

Chromatin organization is highly dynamic and modulates DNA replication, transcription, and chromosome segregation. Condensin is essential for chromosome assembly during mitosis and meiosis, as well as maintenance of chromosome structure during interphase. While it is well established that sustained condensin expression is necessary to ensure chromosome stability, the mechanisms that control its expression are not yet known. Herein, we report that disruption of cyclin-dependent kinase 7 (CDK7), the core catalytic subunit of CDK-activating kinase, leads to reduced transcription of several condensin subunits, including structural maintenance of chromosomes 2 (SMC2). Live and static microscopy revealed that inhibiting CDK7 signaling prolongs mitosis and induces chromatin bridge formation, DNA double-strand breaks, and abnormal nuclear features, all of which are indicative of mitotic catastrophe and chromosome instability. Affirming the importance of condensin regulation by CDK7, genetic suppression of the expression of SMC2, a core subunit of this complex, phenocopies CDK7 inhibition. Moreover, analysis of genome-wide chromatin conformation using Hi-C revealed that sustained activity of CDK7 is necessary to maintain chromatin sublooping, a function that is ascribed to condensin. Notably, the regulation of condensin subunit gene expression is independent of superenhancers. Together, these studies reveal a new role for CDK7 in sustaining chromatin configuration by ensuring the expression of condensin genes, including SMC2.

Sirtuin 6 is required for the integrated stress response and resistance to inhibition of transcriptional cyclin-dependent kinases.

Pancreatic ductal adenocarcinoma (PDAC) is classified into two key subtypes, classical and basal, with basal PDAC predicting worse survival. Using in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts (PDXs) of PDAC, we found that basal PDACs were uniquely sensitive to transcriptional inhibition by targeting cyclin-dependent kinase 7 (CDK7) and CDK9, and this sensitivity was recapitulated in the basal subtype of breast cancer. We showed in cell lines, PDXs, and publicly available patient datasets that basal PDAC was characterized by inactivation of the integrated stress response (ISR), which leads to a higher rate of global mRNA translation. Moreover, we identified the histone deacetylase sirtuin 6 (SIRT6) as a critical regulator of a constitutively active ISR. Using expression analysis, polysome sequencing, immunofluorescence, and cycloheximide chase experiments, we found that SIRT6 regulated protein stability by binding activating transcription factor 4 (ATF4) in nuclear speckles and protecting it from proteasomal degradation. In human PDAC cell lines and organoids as well as in murine PDAC genetically engineered mouse models where SIRT6 was deleted or down-regulated, we demonstrated that SIRT6 loss both defined the basal PDAC subtype and led to reduced ATF4 protein stability and a nonfunctional ISR, causing a marked vulnerability to CDK7 and CDK9 inhibitors. Thus, we have uncovered an important mechanism regulating a stress-induced transcriptional program that may be exploited with targeted therapies in particularly aggressive PDAC.

The CDK7 inhibitor CT7001 (Samuraciclib) targets proliferation pathways to inhibit advanced prostate cancer

BackgroundCurrent strategies to inhibit androgen receptor (AR) are circumvented in castration-resistant prostate cancer (CRPC). Cyclin-dependent kinase 7 (CDK7) promotes AR signalling, in addition to established roles in cell cycle and global transcription, providing a rationale for its therapeutic targeting in CRPC.MethodsThe antitumour activity of CT7001, an orally bioavailable CDK7 inhibitor, was investigated across CRPC models in vitro and in xenograft models in vivo. Cell-based assays and transcriptomic analyses of treated xenografts were employed to investigate the mechanisms driving CT7001 activity, alone and in combination with the antiandrogen enzalutamide.ResultsCT7001 selectively engages with CDK7 in prostate cancer cells, causing inhibition of proliferation and cell cycle arrest. Activation of p53, induction of apoptosis, and suppression of transcription mediated by full-length and constitutively active AR splice variants contribute to antitumour efficacy in vitro. Oral administration of CT7001 represses growth of CRPC xenografts and significantly augments growth inhibition achieved by enzalutamide. Transcriptome analyses of treated xenografts indicate cell cycle and AR inhibition as the mode of action of CT7001 in vivo.ConclusionsThis study supports CDK7 inhibition as a strategy to target deregulated cell proliferation and demonstrates CT7001 is a promising CRPC therapeutic, alone or in combination with AR-targeting compounds.

PSMD12 interacts with CDKN3 and facilitates pancreatic cancer progression.

Proteasome 26S subunit, non-ATPase 12 (PSMD12) genes have been implicated in several types of malignancies but the role of PSMD12 in pancreatic cancer (PC) remains elusive. Bioinformatics analysis showed that PSMD12 was highly expressed in PC patients and was associated with shorter overall survival. PSMD12 was also shown to be highly expressed in PC tissues and cell lines. Upregulated PSMD12 showed enhanced cell viability, increased colony formation rate and upregulated levels of PCNA and c-Myc, while the inhibition of PSMD12 abated these levels. PSMD12 knockdown promoted cell apoptosis. The results of xenografts in nude mice confirmed that PSMD12 promoted PC tumor growth in vivo. Protein‒protein interaction network and functional enrichment analyses implied that PSMD12 may have a connection with cyclin-dependent kinase inhibitor 3 (CDKN3). Co‑immunoprecipitation and western blot results confirmed that PSMD12 could interact with and abate the ubiquitination level of CDKN3, thus stabilizing the CDKN3 protein. Rescue assays showed that PSMD12 overexpression caused cell proliferation and that knockdown-induced cell apoptosis could be reversed by CDKN3 regulation. This work reveals the essential roles of PSMD12 in the proliferation and apoptosis of PC development. PSMD12 may regulate CDKN3 expression by interacting with and abating the ubiquitination level of CDKN3, thereby participating in the malignant behavior of PC.

Abstract 1556: CDKN3 inhibition enhances the antitumor effect of paclitaxel by promoting abnormal mitosis of cancer cells

Abstract Background: Paclitaxel (PTX) is one of the key drugs in clinical practice for esophageal squamous cell carcinoma (ESCC), however, therapeutic effects of PTX alone are not sufficient. PTX causes activation of spindle assembly checkpoint resulting in mitotic arrest of cancer cells. Recently, the antitumor effect of PTX has been shown to depend on aberrant chromosome segregation and abnormal spindle formation. Whereas, recent reports showed that cyclin dependent kinase inhibitor 3 (CDKN3) was often overexpressed in various human cancer tissues which was attributed to the higher mitotic activity in cancer cells, and loss of CDKN3 induced abnormal mitosis and centrosome overduplication. Therefore, we hypothesized that the combination of PTX and CDKN3 inhibition may promote mitotic errors in cancer cells. The aim of this study is to examine the combined effect of PTX and CDKN3 inhibition on ESCC. Methods: The expression levels of CDKN3 were compared between ESCC cell lines (TE1, TE5, TE6, TE8, TE10, and TE11) and normal esophageal epithelial cell line (EPC2-hTERT). CDKN3 knockdown ESCC cells using shRNA were created. Cell viability assay (WST-1 and clonogenic assay), immunofluorescence microscopy for measuring the number of centrosomes or spindle microtubules, and time-lapse images for assessing mitosis and cell fate were performed. Results: The expression levels of CDKN3 in ESCC cells were high compared to normal esophageal epithelial cells. Here, TE6 and TE10 ESCC cells were selected for further experiments based on higher CDKN3 expression. WST-1 assays showed an increased sensitivity to PTX after CDKN3 knockdown. Clonogenic assay showed reduced clonogenicity with CDKN3 knockdown, which was further reduced when exposed to 1 nM PTX (no interaction by two-way ANOVA analysis). Quantification of centrosomes revealed an increased frequency of centrosome overduplication in cells with CDKN3 knockdown, and it was further increased when treated with 1 nM PTX. Quantification of microtubules revealed CDKN3 knockdown cells with 1nM PTX had irregular multipolar spindle formation in almost half of the cells in mitosis. In time-lapse images, mitotic errors were more frequent ( &amp;gt; 80% in combination vs 54% in PTX vs 32% in CDKN3 knockdown) and most of the daughter cells after abnormal mitoses resulted in cell death in interphase or did not undergo another cell division. The cumulative cell death was significantly increased in combination treatment with PTX and CDKN3 inhibition compared to PTX treatment alone (Hazard ratio 2.22; 95% CI 1.43 - 3.45, p = 0.0004 ) and CDKN3 inhibition alone (Hazard ratio 3.09; 95% CI 1.92 - 4.98, p &amp;lt; 0.0001). Conclusions: CDKN3 knockdown increased the frequencies of centrosome overduplication and multipolar spindle formation when combined with PTX exposure. These mitotic errors caused multipolar divisions and subsequent cell death. Targeting CDKN3 could be a novel anti-cancer strategy. Citation Format: Shigeki Kataoka, Junichi Matsubara, Trang H. Vu, Tomohiro Kondo, Tomoki Saito, Yosuke Mitani, Osamu Kikuchi, Atsushi Yamada, Shinya Ohashi, Manabu Muto. CDKN3 inhibition enhances the antitumor effect of paclitaxel by promoting abnormal mitosis of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1556.

Abstract 5985: Cyclin dependent kinase 9 (CDK9) inhibition increased efficacy of programmed cell death protein 1 (PD-1) blockade for hepatocellular carcinoma (HCC) through decreasing programmed death-ligand 1 (PD-L1) expression

Abstract Background: PD-1 blockade has been approved as treatment for advanced HCC. We previously demonstrated the efficacy of CDK9 inhibition in reducing the expression of inducible proteins and as a treatment for HCC (Shao et al. Oncology 2022). We thus examined the influence of CDK9 inhibition on the expression of PD-L1 in HCC and the potential of improving the efficacy of PD-1 blockade with the combination of CDK9 inhibitors. Methods: We first examined the influence of specific CDK9 inhibitors, AZD4573 and atuveciclib, on interferon-γ (IFN-γ) induced PD-L1 expression of human HCC cell lines HuH7 and Hep3B. Overexpression of CDK9 and siRNA to CDK9 were used to confirm the findings. Similar experiments were repeated in the mouse HCC cell line BNL. To test the in vivo efficacy, we orthotopically implanted BNL cells in the subcapsular area of BALB/c mice, which were then treated with vehicle, CDK9 inhibitors, anti-PD-L1 antibodies, or their combination. To compare with tumor sizes, all mice were sacrificed after 14 days of treatment. To compare survival, the mice were only sacrificed if they met the criteria of animal euthanasia. Results: Both HuH7 and Hep3B cells had minimal PD-L1 expression, which however could be induced using IFN-γ. AZD4573 and atuveciclib decreased the IFN-γ induced PD-L1 expression of HuH7 and Hep3B cells in a dose dependent manner. The flow cytometry results confirmed that membrane PD-L1 expression was also decreased by CDK9 inhibition. Overexpression of CDK9 decreased the influence of these CDK9 inhibitors on PD-L1 expression. CDK9 knockdown using siRNA reduced the IFN-γ induced PD-L1 expression. AZD4573 and atuveciclib also reduce the PD-L1 expression in BNL cell lines. In the orthotopic animal model, the tumors of the sacrificed mice were significantly smaller in mice treated with atuveciclib and an anti-PD-L1 antibody than mice treatment with either alone. The survival of mice treated with the combination was also longer than mice treated with each therapy alone. Conclusion: We demonstrated that CDK9 inhibition could reduce the IFN-γ induced PD-L1 expression of HCC cells. Combination of CDK9 inhibitors and anti-PD-L1 antibodies was more effective than either therapy alone. Citation Format: Ching-Tso Chen, Yu-Yun Shao, Hung-Wei Hsu, Rita R. Wo, Han-Yu Wang, Ann-Lii Cheng, Chih-Hung Hsu. Cyclin dependent kinase 9 (CDK9) inhibition increased efficacy of programmed cell death protein 1 (PD-1) blockade for hepatocellular carcinoma (HCC) through decreasing programmed death-ligand 1 (PD-L1) expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5985.

Abstract 2688: Inhibition of CDK7 using small molecule inhibitor, TGN-1062, augments the effect of cisplatin treatment in head and neck PDX models

Abstract Purpose: Head and Neck Squamous Cell Carcinoma (HNSCC) is the 6th most prevalent type of cancer in the world with incidence rates projected to increase 30% by 2030. This increase is generally associated with cultural and environmental risk factors such as tobacco consumption, alcohol abuse, airborne pollutants, and exposure to human papilloma virus. Standard of care (SOC) therapy remains multimodal relying primarily on surgical resections followed by chemotherapy and radiation. Screening methods have proven to be ineffective and improvements to treatment over the past decade have only been modest. While there are some successful treatments, like use of cetuximab in patients with EGFR expressing tumors, there is still a major unmet need in this area. Overexpression of Cyclin-Dependent Kinase 7 (CDK7) is highly associated with poor prognosis in HNSCC. CDK7 plays a dual role in tumor progression by regulating the cell cycle via phosphorylation of members of the CDK family and stimulation of transcription via phosphorylation of RNA Polymerase II. CDK7 has also been shown to be associated with super-enhancers which can lead to overexpression of oncogenic driver genes such as BCL2, GFI1, MYC, and pMED1. Previously, we have presented the development of a potent and reversible CDK7 inhibitor, TGN-1062. Here, we have generated data suggesting the potential benefit of CDK7 inhibition in combination with SOC therapy for HNSCC. Methods: HNSCC lines were co-treated with cisplatin and TGN-1062, assessed for viabilities with CellTiter-Glo, and analyzed with the Bliss synergy model. In vivo efficacy studies were conducted at Crown Bioscience. HNSCC PDX tumors were implanted and treated with TGN-1062 with and without Cisplatin. Tumors were measured to determine tumor growth inhibition (TGI) and correlating drug accumulation levels were measured in both tumors and plasma using mass spectrometry. Immunohistochemistry (IHC) and RT-PCR were performed on tumors to determine changes in cleaved Caspase- 3, MYC, and RNA Pol II. Results: TGN-1062 induces a dose dependent change in biomarkers shown via Western blotting. Synergistic effects are observed at certain ratios of TGN-1062 and Cisplatin on HNSCC cells. Co-treatment with cisplatin and TGN-1062 in HN2579 PDX showed the most combination benefit with 64% TGI, an improvement over cisplatin treatment alone at 54% TGI. Improvement to cisplatin treatment was also observed in the HN3540 PDX at end of study with cisplatin and TGN-1062 combination providing 75% TGI compared to cisplatin alone at 68% TGI. Drug accumulation and biomarker analysis from PDX tumors show that combined exposure to TGN-1062 and cisplatin is linked with a better therapeutic response. Conclusion: Targeting CDK7 with TGN-1062 in combination with SOC therapy is a promising approach for treatment of HNSCC. TGN-1062 is a potent CDK7 inhibitor with potential for clinical development. Citation Format: Trason Thode, Alexis Weston, Serina Ng, Shelby Rheinschmidt, Tithi Ghosh Halder, Sydney Adamson, Kate Gutowsky, Brian Durbin, Raffaella Soldi, Mohan Kaadige, Srinivas Kasibhatla, Haiyong Han, Justin Moser, Vincent Chung, Joseph Chao, Victoria Villaflor, Daniel D. Von Hoff, Sunil Sharma. Inhibition of CDK7 using small molecule inhibitor, TGN-1062, augments the effect of cisplatin treatment in head and neck PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2688.

Abstract 5984: CDK9 inhibition as a potential therapeutic strategy in Ewing sarcoma

Abstract Ewing sarcoma (EWS) is the second most common bone and soft tissue sarcoma in children and adolescents and accounts for approximately 2% of all childhood cancer diagnoses. EWS is a highly aggressive cancer: the overall 5-year survival rate for localized and metastatic EWS are 67 and 38%, respectively. Each year, approximately 200 children and adolescents in the United States are diagnosed with EWS. Genetically, nearly all EWS have chromosomal translocations in which a member of the FET gene family is fused with an ETS transcription factor, with 85% of cases being EWSR1-FLI1. Cyclin-dependent kinase 9 (CDK9) is a serine/threonine protein kinase involved in transcription elongation through phosphorylation and activation of RNA polymerase II (RPB1) and increases the levels of key oncogenic genes like myeloid cell leukemia-1 (MCL-1) and c-Myc, which are reported to be involved in chemoresistance. CDK9 may be a viable target in transcriptionally addicted cancers including EWS driven by oncogenic fusion genes like EWSR1-FLI1. TP-1287 is an investigational orally delivered phosphate pro-drug of alvocidib, a potent CDK9 inhibitor. We hypothesized that alvocidib, the pharmaceutically active form of TP-1287, may show activity in EWS cells as a single agent and in combination with standard of care agents. Alvocidib demonstrated the suppression of downstream targets of CDK9 including c-Myc in EWS cell lines after 6-hr treatment. In cell viability assays using CellTiter-Glo, alvocidib resulted in IC50 values of 68 - 125 nM in EWS cell lines after 3-day treatment and 27 - 160 nM in EWS cells from patient derived xenograft (PDX) models, after 6-day treatment. MCL-1 amplified EWS cells were observed significantly more sensitive to alvocidib than MCL-1 diploid EWS cells in the EWS cells from PDX models (p=0.004). Further bioinformatic analysis to predict alvocidib sensitivity is currently underway. Alvocidib demonstrated an additive inhibitory response in combination with topotecan, phosphoramide mustard, a biologically active metabolite of cyclophosphamide, and palifosfamide, a biologically active metabolite of ifosfamide, in EWS cell lines after 3-day treatment. Alvocidib treatment showed reduction of RAD51 which is involved in DNA damage response (DDR), suggesting the activity of alvocidib on DDR may contribute to this additive response. In an in vivo preclinical study using the A-673 model, TP-1287 (2.5 mg/kg, QD) showed tumor growth inhibition (TGI). In vivo combination studies with standard of care agents using EWS xenograft models are currently ongoing. In summary, alvocidib has shown activity inhibiting the growth of EWS cells as a single agent and an additive response in combination with EWS standard of care agents in EWS cell lines. TP-1287 may be a potential therapeutic option for the current regimen in EWS. TP-1287 is being investigated for solid tumors including EWS (clinicaltrials.gov, NCT03604783). Citation Format: Yuta Matsumura, Richard E. Heinz, Curtis A. Allred, Adam Siddiqui, Jason M. Foulks, Steven L. Warner. CDK9 inhibition as a potential therapeutic strategy in Ewing sarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5984.

Evaluation of CDK9 Inhibition by Dinaciclib in Combination with Apoptosis Modulating izTRAIL for the Treatment of Colorectal Cancer.

Treatment options for colorectal cancer (CRC), especially in advanced stages are still insufficient. There, the discovery of Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was a bright spot. However, most cancers show resistance toward apoptotic signals. Cyclin-dependent kinase 9 (CDK9) plays a crucial role in cell cycle progression in most tissues. We recently demonstrated the role of CDK9 in mediating TRAIL resistance. In this work, we investigated the role of CDK9 in colorectal cancer. Immunohistochemical analysis of CDK9 expression in cancer and normal tissues of CRC specimens was performed. The effect of selective CDK9 inhibition in combination with TRAIL on CRC cells was analyzed via cell viability, colony formation, and induction of apoptosis by flow cytometry. The mechanism of action was conducted via western blotting. We now have confirmed overexpression of CDK9 in cancer tissues, with low expression associated with poorer survival in a subset of CRC patients. In-vitro, CDK9 inhibition could strongly promote TRAIL-induced cell death in TRAIL-resistant CRC cells. Mechanistically, CDK9 inhibition induced apoptosis by downregulation of antiapoptotic proteins, myeloid leukemia cell differentiation protein 1 (Mcl-1) and FLICE-inhibitory protein (c-FLIP). Overall, we identified CDK9 as a prognostic marker and combined CDK9 inhibition and TRAIL as a novel and promising therapeutic approaches for colorectal cancer.

Characterization of new highly selective pyrazolo[4,3-d]pyrimidine inhibitor of CDK7

Targeting cyclin-dependent kinase 7 (CDK7) provides an interesting therapeutic option in cancer therapy because this kinase participates in regulating the cell cycle and transcription. Here, we describe a new trisubstituted pyrazolo[4,3-d]pyrimidine derivative, LGR6768, that inhibits CDK7 in the nanomolar range and displays favourable selectivity across the CDK family. We determined the structure of fully active CDK2/cyclin A2 in complex with LGR6768 at 2.6 Å resolution using X-ray crystallography, revealing conserved interactions within the active site. Structural analysis and comparison with LGR6768 docked to CDK7 provides an explanation of the observed biochemical selectivity, which is linked to a conformational difference in the biphenyl moiety. In cellular experiments, LGR6768 affected regulation of the cell cycle and transcription by inhibiting the phosphorylation of cell cycle CDKs and the carboxy-terminal domain of RNA polymerase II, respectively. LGR6768 limited the proliferation of several leukaemia cell lines, triggered significant changes in protein and mRNA levels related to CDK7 inhibition and induced apoptosis in dose- and time-dependent experiments. Our work supports previous findings and provides further information for the development of selective CDK7 inhibitors.