CDK9 Activity Research Articles

Discovery of Novel 2-Aminopyridine-Based and 2-Aminopyrimidine-Based Derivatives as Potent CDK/HDAC Dual Inhibitors for the Treatment of Refractory Solid Tumors and Hematological Malignancies.

Co-inhibition of histone deacetylase (HDAC) and cyclin-dependent kinase (CDK) synergizes to produce enhanced antitumor effects and potentially overcomes the drug resistance. In this work, we discovered a series of novel CDK9/HDACs dual inhibitors. Among them, compound 8e was identified to show potent CDK9 and HDAC1 inhibitory activities, with IC50 values at 88.4 and 168.9 nM, respectively, and exhibited antiproliferative capacities against hematological and solid tumor cells. Meanwhile, 8e showed high selectivity for CDK9 and HDAC1, remarkably induced MV-4-11 cell apoptosis and S cell cycle arrests. Furthermore, 8e possessed a significant antitumor potency with a T/C value of 29.98% in the MV-4-11 xenograft model. Interestingly, a potent FLT3/HDAC dual inhibitor 9e was also identified (FLT3/HDAC1/3 IC50 = 30.4/52.4/14.7 nM) and found to possess powerful apoptosis induction ability in MV-4-11 cell and potent antiproliferative capacities against FLT3 mutant-transformed BaF3 cells. Overall, our work provided valuable lead compounds for dual inhibitors with potent anticancer activity.

Abstract 602: Targeting CDK9 with a novel potent and selective inhibitor, YX0798, in CAR-T-relapsed mantle cell lymphoma models

Abstract Introduction: Cyclin-dependent kinase (CDK) family members play critical roles in multiple cellular processes. CDK9 is a central regulator at several stages of PolII transcription, including initiation, elongation, and termination. CDK9 dysfunction resulted in transcriptomic reprogramming and promotion of the development of cancers, including lymphoma and leukemia. We and others reported that transcriptomic reprogramming is associated with disease progression and therapeutic resistance in mantle cell lymphoma (MCL). Targeting CDK9 with its inhibitors AZD4573 and enitociclib is safe and effective for treatment in preclinical MCL models, and they are currently under clinical investigation to assess their efficacy and safety. The data demonstrated that CDK9 is a promising therapeutic target. In this study, we developed a novel CDK9 inhibitor, YX0798, and assessed its potency and safety in preclinical MCL models. Method: Cell viability assays were conducted in a time- and dose-dependent manner to assess the in vitro efficacy of YX0798 as well as AZD4573. Apoptosis assays and western blots were used to determine the mechanism of action in MCL cells. Patient-derived xenograft models from primary MCL patient samples were established in immunodeficient mice and used to determine the therapeutic potential YX0798. Results: Next generation sequencing showed that CDK9 was upregulated and cancer hallmarks MYC-TARGETS-v1 and -v2 were highly enriched in CAR-T-relapsed MCL patients compared to CAR-T-naïve patients. We hypothesized that CDK9 overexpression and activity may greatly contribute to the enrichment of MYC-TARGETS-v1 and -v2 in patients with CAR-T resistance, so targeting CDK9 may overcome that resistance. YX0798 was developed to be a potent and selective CDK9 inhibitor. It markedly inhibited cell viability and induced robust apoptosis in MCL cell lines. As expected, it inhibited CDK9 kinase activity and suppressed the expression of short-lived proteins like c-MYC and MCL-1 that are critical for MCL cell survival and growth. YX0798 at 2 mg/kg for continuous 7 days via pump or at 5 mg/kg daily by oral administration potently inhibited the tumor growth of xenografts derived from a patient with therapeutic relapse to the CAR-T therapy in vivo (P<0.0001). This treatment efficacy was found to be similar at that of AZD4573 but at a higher dosage (15+15 mg/kg, 2 hours split, QW, IP). No adverse effects in mice were observed for any of treatments. Conclusion: These data show that our novel CDK9 inhibitor YX0798 is potent and efficacious in treating aggressive MCL models and overcame CAR-T resistance at a lower dosage than AZD4573. Citation Format: Vivian Jiang, Yu Xue, Hong Kim, Joseph McIntosh, Haiying Chen, Tianci Zhang, Yang Liu, Jia Zhou, Michael Wang. Targeting CDK9 with a novel potent and selective inhibitor, YX0798, in CAR-T-relapsed mantle cell lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 602.

Actin associates with actively elongating genes and binds directly to the Cdk9 subunit of P-TEFb

Nuclear actin has been demonstrated to be essential for optimal transcription, but the molecular mechanisms and direct binding partner for actin in the RNA polymerase complex have remained unknown. By using purified proteins in a variety of biochemical assays, we demonstrate a direct and specific interaction between monomeric actin and Cdk9, the kinase subunit of the positive transcription elongation factor b (P-TEFb) required for RNA polymerase II pause-release. This interaction efficiently prevents actin polymerization, is not dependent on kinase activity of Cdk9 and is not involved with releasing P-TEFb from its inhibitor 7SK snRNP complex. Supporting the specific role for actin in the elongation phase of transcription, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) reveals that actin interacts with genes only upon their active transcription elongation. This study therefore provides novel insights into the mechanisms by which actin facilitates the transcription process.

Synergistic Sensitization of High-Grade Serous Ovarian Cancer Cells Lacking Caspase-8 Expression to Chemotherapeutics Using Combinations of Small-Molecule BRD4 and CDK9 Inhibitors.

Ovarian cancer is one of the most lethal gynecological cancers worldwide, with approximately 70% of cases diagnosed in advanced stages. This late diagnosis results from the absence of early warning symptoms and is associated with an unfavorable prognosis. A standard treatment entails a combination of primary chemotherapy with platinum and taxane agents. Tumor recurrence following first-line chemotherapy with Carboplatin and Paclitaxel is detected in 80% of advanced ovarian cancer patients, with disease relapse occurring within 2 years of initial treatment. Platinum-resistant ovarian cancer is one of the biggest challenges in treating patients. Second-line treatments involve PARP or VEGF inhibitors. Identifying novel biomarkers and resistance mechanisms is critical to overcoming resistance, developing newer treatment strategies, and improving patient survival. In this study, we have determined that low Caspase-8 expression in ovarian cancer patients leads to poor prognosis. High-Grade Serous Ovarian Cancer (HGSOC) cells lacking Caspase-8 expression showed an altered composition of the RNA Polymerase II-containing transcriptional elongation complex leading to increased transcriptional activity. Caspase-8 knockout cells display increased BRD4 expression and CDK9 activity and reduced sensitivities to Carboplatin and Paclitaxel. Based on our work, we are proposing three potential therapeutic approaches to treat advanced ovarian cancer patients who exhibit low Caspase-8 expression and resistance to Carboplatin and/or Paclitaxel-combinations of (1) Carboplatin with small-molecule BRD4 inhibitors; (2) Paclitaxel with small-molecule BRD4 inhibitors, and (3) small-molecule BRD4 and CDK9 inhibitors. In addition, we are also proposing two predictive markers of chemoresistance-BRD4 and pCDK9.

The Selective CDK9 Inhibitor VIP152 Overcame Therapeutic Resistance in Mantle Cell Lymphoma

Introduction Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin lymphoma. Even most advanced therapies are generally followed by resistance and relapse, creating an urgent need for novel drugs to overcome resistance. Our and other studies have shown transcriptome is abnormally reprogrammed in patients with therapeutic resistance. CDK9, as a master transcriptional regulator, is overexpressed in tumors after subsequent relapses to BTK inhibitors (BTKi) and CAR-T therapy. VIP152 is a highly potent and selective CDK9 inhibitor under preclinical and early clinical development. As yet, its potency and safety in patients with MCL, especially those with multiple therapeutic resistances, has not been addressed. Method We performed single-cell RNA sequencing and bulk RNA sequencing to investigate enriched cancer hallmarks in MCL. Cell viability assays were conducted in a time- and dose-dependent manner to assess the in vitro efficacy of VIP152. Apoptosis assays and western blots were used to identify relevant pathways in bypassing resistances. Cell line-derived xenograft and patient-derived xenograft models of MCL created in immunodeficient mice were used to monitor tumor growth and thus determine the in vivo efficacy of VIP152 in overcoming therapeutic resistances. Results Based on single-cell RNA sequencing and bulk RNA sequencing, we found that the cancer hallmark MYC-TARGETS is progressively enriched in MCL patients with subsequent failures to BTKi and CAR-T therapy. CDK9 was found to be overexpressed in CAR-T-relapsed patients compared to CAR-T-naïve but BTKi-refractory/relapsed patients. We hypothesized that CDK9 overexpression and activity may greatly contribute to the transcriptomic reprogramming in patients with therapeutic resistance. Targeting CDK9-mediated transcription with the specific CDK9 inhibitor VIP152 was highly effective in MCL cell lines at low nanomolar range (55-172 nM). VIP152 robustly induced apoptosis in a time- and dose-dependent manner, which was largely dependent on caspase-3 activity. It markedly inhibited CDK9 phosphorylation and down streaming signaling, and suppressed the expression of short-lived proteins like MYC, MCL-1, and cyclin D1 that are critical for MCL cell survival and proliferation. VIP152 effectively inhibited the tumor growth of MCL cell line-derived xenograft models in vivo (P<0.001) with no apparent adverse effects in mice. Moreover, VIP152 markedly inhibited the growth of three patient-derived xenograft tumor models, overcoming BTKi resistance (P<0.001), BTKi-venetoclax dual resistance (P<0.01), and BTKi-CAR-T dual resistance (P<0.001). Conclusion These data showed that CDK9 was a promising target for treating MCL and using VIP152 to specifically target it was efficacious in overcoming a variety of therapeutic resistances in MCL.

Wogonin alleviates BaP-induced DNA damage and oxidative stress in human airway epithelial cells by dual inhibiting CYP1A1 activity and expression.

Benzo[a]pyrene (BaP) is a common air pollutant that has been reported to cause oxidative stress and carcinogenesis. Wogonin, a flavonoid compound extracted from the roots of Scutellaria baicalensis, has been found to possess a variety of pharmacological activities, including anti-inflammatory and anti-cancer effects. The purpose of this study was to examine the ability of wogonin to alleviate the cytotoxicity induced by BaP in human airway epithelial cells and explore the corresponding mechanism. Our study found that wogonin treatment inhibited DNA damage and reactive oxygen species overproduction induced by BaP in human airway epithelial cells. In vitro enzyme assays showed that wogonin significantly inhibited the enzymatic activity of CYP1A1. In addition, wogonin decreased the basal level of CYP1A1 and inhibited the CYP1A1 overexpression induced by BaP, whereas overexpression of CYP1A1 partially reversed the effect of wogonin on BaP-induced DNA damage. Meanwhile, a CYP1A1 inhibitor and CYP1A1 knockdown also showed these same effects. Further studies showed that wogonin regulates CYP1A1 expression by inhibiting CDK7 and CDK9 activity. The use of CDK7 or CDK9 inhibitors decreased BaP-induced cytotoxicity and CYP1A1 expression. Finally, we found that the methoxy group of wogonin was crucial for its inhibitory activity. In conclusion, our data indicated that wogonin could effectively relieve BaP induced cytotoxicity, and its mechanism was related to the dual inhibition of CYP1A1 activity and expression.

Abstract 416: Phosphorylation of CDK9 transduces ALK signaling to promote resistance to PARP inhibitors via RNA Pol II activated homologous recombination repair

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are currently the most promising drugs to induce synthetic lethality in tumor with homologous recombination (HR) deficiency. However, development of resistance to PARPi is an almost universal occurrence in tumor despite initial sensitivity. Additionally, how to identify the right patients for PARPi monotherapy or in combination with other agents remains a substantial clinical challenge. Therefore, understanding the molecular underpinnings of PARPi resistance is critical to the development of rational combinatorial treatment strategies and identification of novel biomarkers which will in turn facilitate optimization of outcomes for patients receiving PARPi. By employing a screening procedure using phospho-RTK antibody array, our study identifies phosphorylated ALK (p-ALK) expression is higher in PARPi/platinum resistant cells relative to sensitive cells. We further demonstrate that p-ALK expression in human ovarian cancer tissue is associated with resistance to PARPi and platinum-based chemotherapy. Notably, FDA approved ALK inhibitors induce an HR-deficient phenotype and sensitize PARPi/platinum-resistant tumors to PARPi in vitro and in vivo. Mechanistically, ALK contributes to PARPi resistance by tyrosine phosphorylating CDK9 at Y19, thereby increasing the protein stability and kinase activity of CDK9 which in turn stabilizes positive transcriptional elongation factor b (P-TEFb) to activate RNA pol II and turn on HR repair genes transcription. Importantly, the phosphorylated counterparts of ALK/CDK9 complex (p-ALK/p-Y19 CDK9) can serve as a biomarker to identify patients with PARPi/platinum-resistant tumors which most likely to respond to dual PARP and ALK inhibition. Together, our findings identify p-ALK/p-Y19-CDK9 mediated stabilization of P-TEFb as a critical mechanism of PARPi resistance and supports a biomarker-guided therapeutic strategy combining ALKi and PARPi to induce synthetic lethality. Citation Format: Yu-Yi Chu, Jianjun Gao, Dihua Yu, Mien-Chie Hung. Phosphorylation of CDK9 transduces ALK signaling to promote resistance to PARP inhibitors via RNA Pol II activated homologous recombination repair [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 416.

Abstract 1555: CDK2 inhibition & disruption of centrosome stoichiometry: a clinically tractable death program for aneuploid cancer cells

Abstract Genomic instability is a cancer hallmark. Aberrant centrosome numbers often occur in cancer and is associated with genomic instability. Mitosis with supernumerary centrosomes leads to abnormal chromosome segregation and aneuploidy in progeny cells. While many cells with abnormal chromosomes are eliminated through apoptosis, some gain a growth advantage and acquire resistance to antineoplastic agents. This aneuploidy is associated with aggressive tumor biology and unfavorable clinical outcomes. Cyclin-dependent kinase 2 (CDK2) plays a major role regulation of cell cycle progression, centrosome duplication and clustering. We previously reported that CDK2 inhibition prevented supernumerary centrosomes clustering, causing multipolar mitosis and anaphase catastrophe in lung and other aneuploid cancers. To elucidate engaged mechanisms focused ion beam scanning electron microscope (FIB-SEM) and immunofluorescent staining were each used to interrogate the ultrastructure of multipolar mitosis after CDK2 inhibition by CYC065-treatment (a CDK2/9 inhibitor). CYC065-treatment induced distinct ultrastructural changes in aneuploid lung cancer cells including formation of chromosome rings and multipolarity. As compared to vehicle controls, CYC065-treatment markedly extended the duration of chromosome rings in lung cancer cells and conferred aberrant mitosis in live cell imagining. Intriguingly, this treatment disrupted centrosome and centriole stoichiometry in cells with chromosome rings or multipolarity. These findings were confirmed using immunofluorescence assays of lung and other cancer cells having supernumerary centrosomes. To elucidate if CDK2 or CDK9 activity directly altered centriole stoichiometry, chromosome rings or multipolarity, immunofluorescent assays were performed in lung cancer cells that independently knocked down CDK2 or CDK9 using siRNAs relative to controls. Centriole stoichiometry abnormalities in cancer cells having chromosome rings or multipolarity were statistically significantly higher in CDK2 knock-down but not in CDK9 knock-down transfectants or controls. Engineered gain of CDK2 but not CDK9 expression rescued CYC065 mediated chromosome rings and multipolarity in aneuploid lung cancer cells. Taken together, this study uncovered that CDK2 inhibition disrupted centrosome stoichiometry, causing apoptotic death of aneuploid cancer cells. This is a clinically-tractable pharmacologic mechanism that conferring cancer cell death. Current work determines if similar mechanisms are engaged in vivo in syngeneic mouse lung cancer models undergoing CDK2 antagonism. Citation Format: Zibo Chen, Xi Liu, Masanori Kawakami, Xiuxia Liu, Allison Baker, Aayush Bhatawadekar, Liliya Tyutyunyk-Massey, Ethan Dmitrovsky, Kedar Narayan. CDK2 inhibition & disruption of centrosome stoichiometry: a clinically tractable death program for aneuploid 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 1555.

Design, synthesis and biological evaluation of 2-((4-sulfamoylphenyl)amino)-pyrrolo[2,3-d]pyrimidine derivatives as CDK inhibitors

To explore the potential use of CDK inhibitors in pancreatic ductal adenocarcinoma (PDAC) therapy, a series of novel 2-((4-sulfamoylphenyl)amino)-pyrrolo[2,3-d]pyrimidine derivatives was designed, synthesised, and investigated for inhibition on both CDK kinase activity and cellular proliferation of pancreatic cancer. Most of new sulphonamide-containing derivatives demonstrated strong inhibitory activity on CDK9 and obvious anti-proliferative activity in cell culture. Moreover, two new compounds suppressed cell proliferation of multiple human pancreatic cancer cell lines. The most potent compound 2g inhibited cancer cell proliferation by blocking Rb phosphorylation and induced apoptosis via downregulation of CDK9 downstream proteins Mcl-1 and c-Myc in MIA PaCa-2 cells. CDK9 knockdown experiment suggests its anti-proliferative activity is mainly mediated by CDK9. Additionally, 2g displayed moderate tumour inhibition effect in AsPC-1 derived xenograft mice model. Altogether, this study provided a new start for further optimisation to develop potential CDK inhibitor candidates for PDAC treatment by alone or combination use.

Biomimetic black phosphorus nanosheets codeliver CDK9 and BRD4 inhibitors for gastric cancer targeted therapy.

Combining the BRD4 and CDK9 inhibitors can trigger the significant down-regulation of the MYC oncogene as well as anti-apoptotic genes and induce tumor cell apoptosis by synergistically impairing RNA synthesis in cancer cells. However, the lack of tumor-targeting capacity and the different pharmacokinetic curves of these two inhibitors may impair the antitumor activity of simultaneous CDK9 and BRD4 inhibition. Herein, CDK9 inhibitor (CI) and BRD4 inhibitor (BI) were codelivered by macrophage membrane-encapsulated black phosphorus nanosheets (M@BP) for the treatment of gastric cancer (GC) via the high expression of BRD4 and CDK9. BP with prominent biocompatibility exhibited a high drug loading efficiency for both CI and BI and could efficiently decrease the expression of the MYC oncogene. More importantly, BP could also serve as a phototherapy agent collaborating with CDK9 and BRD4 inhibition for GC therapy upon near-infrared (NIR) irradiation. Furthermore, the introduction of a macrophage membrane endowed BP with tumor-targeting ability, which could simultaneously deliver CI and BI to tumor tissues. In a murine orthotopic GC model, M@BP could efficiently target and accumulate in the tumor tissues, exhibiting an excellent photothermal effect. The tumor growth monitoring demonstrated that the combination of CI and BI codelivered by M@BP significantly inhibited the tumor progress than the single inhibitors, and the inhibition effect could be further enhanced upon NIR irradiation. Taken together, M@BP with tumor-targeting capacity and high drug loading efficiency for CI and BI could efficiently block the activation of CDK9 and BRD4, exhibiting excellent antitumor activity under NIR irradiation without systemic toxicity in an orthotopic GC model.

547 CDK9 Kinase Activation in Association with AFF1-SEC Initiate Epidermal Progenitor differentiation

Epidermal homeostasis relies on the progenitors to continuously proliferate or initiate differentiation. The molecular mechanisms underlying the earliest events of differentiation initiation remain incompletely understood. Here we show that the kinase activity of CDK9, in association with the Super Elongation Complex (SEC), plays a crucial role in the process. Progenitor-state keratinocytes are associated with inactive CDK9 as part of the SEC. Maintenance of this inactive state of CDK9 relies on the cooperation between HEXIM1 and the SEC-scaffold protein AFF1 but not AFF4. Using a combination of RNAi and RNA-seq, we identified that both HEXIM1 and AFF1 are essential for repressing genes related to “epidermis development” and “keratinization”. Using ChIP-seq, we found that AFF1 and HEXIM1 co-bind near the promoter regions of 2112 genes, including a group of genes with highly enriched Pol II accumulating in the promoter genes (known as “paused” Pol II). CDK9 kinase activation, triggered by SEC disruption or Protein kinase C (PKC) signaling, rapidly upregulates the group of AFF1-HEXIM1 target genes with “paused” Pol II within as short as 1 hour. These rapidly upregulated genes include the transcription factor ATF3, and we found that ATF3 overexpression is sufficient to upregulate other differentiation-activating transcription factors, including OVOL1, PRDM1, ZNF750, and GRHL3. ATF3 overexpression in primary human keratinocytes diminished clonogenicity and led to epidermal tissue hypoplasia. Mechanistically, we found that SEC disruption by peptidomimetic inhibitors, or PKC signaling, disrupts CDK9 from HEXIM1 binding near the promoters, and releases active CDK9 to promote Pol II elongation in the gene bodies. Using co-immunoprecipitation, we further found that the interaction between AFF1 and HEXIM1 is specific to the progenitor state but not in differentiated keratinocytes. Thus, our data suggest that the activity switch of CDK9, in association with AFF1 and HEXIM1, underlies an early step of epidermal progenitor differentiation initiation, in rapid response to cellular signaling such as PKC activation.

The non-apoptotic function of Caspase-8 in negatively regulating the CDK9-mediated Ser2 phosphorylation of RNA polymerase II in cervical cancer

Cervical cancer is the fourth most frequently diagnosed and fatal gynecological cancer. 15–61% of all cases metastasize and develop chemoresistance, reducing the 5-year survival of cervical cancer patients to as low as 17%. Therefore, unraveling the mechanisms contributing to metastasis is critical in developing better-targeted therapies against it. Here, we have identified a novel mechanism where nuclear Caspase-8 directly interacts with and inhibits the activity of CDK9, thereby modulating RNAPII-mediated global transcription, including those of cell-migration- and cell-invasion-associated genes. Crucially, low Caspase-8 expression in cervical cancer patients leads to poor prognosis, higher CDK9 phosphorylation at Thr186, and increased RNAPII activity in cervical cancer cell lines and patient biopsies. Caspase-8 knock-out cells were also more resistant to the small-molecule CDK9 inhibitor BAY1251152 in both 2D- and 3D-culture conditions. Combining BAY1251152 with Cisplatin synergistically overcame chemoresistance of Caspase-8-deficient cervical cancer cells. Therefore, Caspase-8 expression could be a marker in chemoresistant cervical tumors, suggesting CDK9 inhibitor treatment for their sensitization to Cisplatin-based chemotherapy.

CDK9 and PP2A regulate RNA polymerase II transcription termination and coupled RNA maturation

CDK9 is a kinase critical for the productive transcription of protein‐coding genes by RNA polymerase II (pol II). As part of P‐TEFb, CDK9 phosphorylates the carboxyl‐terminal domain (CTD) of pol II and elongation factors, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein‐coding genes. We also confirm the splicing factor SF3B1 as a target of CDK9 and show that SF3B1 in complex with polyadenylation factors is lost from chromatin after CDK9 inhibition. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.

CDK9 activity switch associated with AFF1 and HEXIM1 controls differentiation initiation from epidermal progenitors

Progenitors in epithelial tissues, such as human skin epidermis, continuously make fate decisions between self-renewal and differentiation. Here we show that the Super Elongation Complex (SEC) controls progenitor fate decisions by directly suppressing a group of “rapid response” genes, which feature high enrichment of paused Pol II in the progenitor state and robust Pol II elongation in differentiation. SEC’s repressive role is dependent on the AFF1 scaffold, but not AFF4. In the progenitor state, AFF1-SEC associates with the HEXIM1-containing inactive CDK9 to suppress these rapid-response genes. A key rapid-response SEC target is ATF3, which promotes the upregulation of differentiation-activating transcription factors (GRHL3, OVOL1, PRDM1, ZNF750) to advance terminal differentiation. SEC peptidomimetic inhibitors or PKC signaling activates CDK9 and rapidly induces these transcription factors within hours in keratinocytes. Thus, our data suggest that the activity switch of SEC-associated CDK9 underlies the initial processes bifurcating progenitor fates between self-renewal and differentiation.

460 Rapid activation of epidermal progenitor differentiation via CDK9 activity modulated by AFF1 and HEXIM1

Epidermal barrier function requires differentiation initiation from a sustainable pool of progenitors. The mechanisms underlying the earliest events initiating differentiation remain unclear. Here we show that RNA Polymerase II (Pol II) pause release, controlled by the CDK9 kinase activity, plays a crucial role in the rapid induction of differentiation. Using RNA-seq in combination with Pol II ChIP-seq, we identified a cluster of upregulated genes featuring robust Pol II pause release in keratinocyte differentiation. A subset of these genes showed rapid induction within 3 hours by Peptidomimetic inhibitors targeting the super elongation complex (SEC), in keratinocytes cultured in the undifferentiation condition. A longer-term (24 hour) treatment led to significant changes (fold change >2, p<0.05) of 1286 keratinocyte-differentiation-signature genes. Mechanistically, we found that the SEC scaffold protein AFF1, but not AFF4, is essential for repressing differentiation in the progenitor state. AFF1 knockdown induced epidermal hypoplasia and impaired progenitor regenerative capacity. We further identified that AFF1 represses differentiation in progenitors through sequestering CDK9 in the inactive state via its interaction with HEXIM1. AFF1 and HEXIM1 directly bind near the transcription start sites of 92 rapid-response genes, sustaining Pol II pausing in the progenitor state. These rapid response genes include ATF3, whose overexpression is sufficient to drive the activation of other differentiation-activating transcription factors, including ZNF750, PRDM1, OVOL1, and GRHL3. In addition, we found that the AFF1 and CDK9 activity are both involved in the immediate of response of PKC-signaling activation. Taken together, our findings suggest a model that AFF1 and HEXIM1 mediated CDK9 activity switch underlies the initial steps of epidermal progenitor differentiation, in response to cellular signaling such as PKC activation.

Pyrazole-Curcumin Suppresses Cardiomyocyte Hypertrophy by Disrupting the CDK9/CyclinT1 Complex.

The intrinsic histone acetyltransferase (HAT), p300, has an important role in the development and progression of heart failure. Curcumin (CUR), a natural p300-specific HAT inhibitor, suppresses hypertrophic responses and prevents deterioration of left-ventricular systolic function in heart-failure models. However, few structure–activity relationship studies on cardiomyocyte hypertrophy using CUR have been conducted. To evaluate if prenylated pyrazolo curcumin (PPC) and curcumin pyrazole (PyrC) can suppress cardiomyocyte hypertrophy, cultured cardiomyocytes were treated with CUR, PPC, or PyrC and then stimulated with phenylephrine (PE). PE-induced cardiomyocyte hypertrophy was inhibited by PyrC but not PPC at a lower concentration than CUR. Western blotting showed that PyrC suppressed PE-induced histone acetylation. However, an in vitro HAT assay showed that PyrC did not directly inhibit p300-HAT activity. As Cdk9 phosphorylates both RNA polymerase II and p300 and increases p300-HAT activity, the effects of CUR and PyrC on the kinase activity of Cdk9 were examined. Phosphorylation of p300 by Cdk9 was suppressed by PyrC. Immunoprecipitation-WB showed that PyrC inhibits Cdk9 binding to CyclinT1 in cultured cardiomyocytes. PyrC may prevent cardiomyocyte hypertrophic responses by indirectly suppressing both p300-HAT activity and RNA polymerase II transcription elongation activity via inhibition of Cdk9 kinase activity.

Abstract 2297: Profound anti-tumor efficacy of dual CDK7 and PD1 blockade in novel syngeneic prostate cancer models

Abstract Background: Anti-PD1 immune checkpoint blockade (ICB) has largely failed as a monotherapy for metastatic prostate cancer likely due to inadequate tumor neoantigen load and inadequate T cell filtration (TILs). Strategies to increase TILs that can be tested in novel syngeneic mouse models are needed in order to fulfill this critical medical need. CDK7 is a major regulator of the cell cycle that is under investigation as a novel therapeutic target. CDK7 inhibition can trigger robust anti-tumor immunity by causing genomic instability and DNA damage in lung cancer mouse models. Herein, we characterize our novel syngeneic mouse models and report on the anti-tumor activity of CDK7 alone and in combination with anti-PD1 ICB as an immunotherapeutic strategy. Methods: We generated two models of prostate cancer organoids and syngeneic mouse models. APC and PTEN are tumor suppressor genes commonly lost in metastatic prostate cancer and are emerging as a putative mechanism of resistance to immunotherapy. The MPA and MPP models were established by ex-vivo Lenti-Cas9-sgRNA knock-out of APC or PTEN from the prostate organoids grown in 3D cultures established from the prostate of a MYCT/TP53-/- GEMM. Results: Organoids from the MPA and MPP models form orthotopic high grade prostate adenocarcinoma from 2 to 3 weeks post-implantation and spontaneously develop lung metastases 10-12 weeks after orthotopic implantation. In addition, the MPA model developed immediate castration resistance and neuroendocrine transformation following hormone deprivation in vivo. By contrast, the MPP tumors significantly regressed after in vivo androgen deprivation. Baseline immunoprofiling of the MPA and MPP orthotopic tumors showed increased infiltration from monocytic MDSCs and expression of T cell exhaustion markers. Treatment of the MPA and MPP orthotopic models with YKL-5-124, a CDK7 inhibitor, led to substantial in vivo tumor growth inhibition, which was further augmented by the addition of anti-PD-1 ICB. Immunoprofiling of tumors treated with combined YKL-5-124 and PD-1 blockade showed activation of CD4+ and CD8+ T cells, and a decrease in the Treg, B cell and NK cell population. Conclusions: The MPA and MPP orthotopic models recapitulate the biology and immune suppressive microenvironment of advanced prostate cancer and provide useful tools for immunotherapy drug development. Combination of CDK7 inhibition with anti-PD-1 blockade profoundly inhibited in vivo orthotopic tumor growth in both MPA and MPP model, providing a rationale for exploring CDK7 in combination with PD-1 checkpoint immunotherapy as a novel therapeutical strategy for advanced prostate cancer. Funding Acknowledgements: DRW is funded through 2018YI2295 Prostate Cancer Foundation Young Investigator Award and PC160944 Department of Defense Physician Research Career Development Award. Citation Format: David Wise, Luiza Doro Pereira, Jiansheng Wu, Victor R. Adorno Febles, Jonathan Melamed, Fang-Ming Deng, Hua Zhang, Kwok-Kin Wong. Profound anti-tumor efficacy of dual CDK7 and PD1 blockade in novel syngeneic prostate 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 2297.

Abstract 2641: Examining the potential therapeutic efficacy of the CDK inhibitor alvocidib in a pre-clinical model of myelodysplastic syndrome

Abstract Myelodysplastic Syndrome (MDS) represents a group of hematologic malignancies characterized by pancytopenia and dysplastic morphology. High risk MDS is invariably fatal due to either severe cytopenia, or transformation to acute myeloid leukemia (AML). Inhibitors of transcription offer promising potential for new therapies. Modulation of CDK9 activity, which plays a critical role in controlling gene transcription through phosphorylation of RNA polymerase II, has been proposed as a novel therapeutic approach. In this study, we examined the efficacy of alvocidib, a broad spectrum CDK inhibitor with highest potency against CDK9, which is hypothesized to slow transcription via inhibition of CDK9, leading to stalled RNA Polymerase II and cell death. We employed a previously characterized pre-clinical model of MDS based on the NUP98-HOXD13 (NHD13) fusion gene, which induces MDS that progresses to AML in mice. In addition to alvocidib, we evaluated the in vitro efficacy of 5-Azacytidine (5-Aza) and Decitabine (DAC), which are hypomethylating agents used in treatment of MDS. The in vitro assays demonstrated a potent effect of alvocidib on cell lines that expressed the NHD13 fusion gene. Alvocidib induced a clear cytostatic effect and showed increasing cytotoxic effects with time, peaking at 70% cell death after 72 hours. Annexin-V staining further supported these results, revealing that most cells were apoptotic by 72 hours. Neither 5-Aza nor DAC treatment matched these effects in vitro. The in vivo assays were based on treatment of chimeric mice with both wild type (WT) and MDS hematopoiesis, generated by allogeneic hematopoietic stem cell transplant with bone marrow from WT and NHD13 mice. Four treatment groups (saline, 5-Aza, Alvocidib, and 5-Aza+Alvocidib) were assessed by serial flow cytometry and complete blood counts. In contrast to in vitro studies, in vivo results did not show a clear survival benefit from either group receiving alvocidib, whereas 5-Aza treatment demonstrated statistically significant survival. However, this study revealed several “exceptional responder” mice, defined as mice that demonstrated a ≥75% reduction in WBC count and a ≥50% reduction in NHD13 engraftment levels following transformation to AML; these exceptional responses were observed only in mice receiving alvocidib. This type of dramatic response has not previously been observed in over 20 prior chimeric transplant experiments employing the NHD13 mouse model. Ongoing efforts include follow-up studies to evaluate the frequency of exceptional responders, as well as identification of acquired mutations which collaborate with the initiating mutation (NHD13) to generate AML. Citation Format: Ryan M. Bertoli, Yang Jo Chung, Jason M. Foulks, Anthony S. Wokasch, Peter D. Aplan. Examining the potential therapeutic efficacy of the CDK inhibitor alvocidib in a pre-clinical model of myelodysplastic syndrome [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 2641.

MEDB-28. CDK9 is a druggable mediator sustaining Myc-driven circuitry in medulloblastoma

BACKGROUND: Though long recognized as a master regulator of cell proliferation across a wide range of cancers, Myc has proven elusive to direct therapeutic targeting. The CDK9-containing PTEFb, complexed with either BRD4 or SEC, facilitates Myc-driven transcriptional programs and is necessary for sustaining expression of Myc itself. Advances in development of clinical-grade CDK9 inhibitors creates an opportunity to examine this as a rational therapy for Myc-driven medulloblastoma. METHODS: We used both RNAi depletion and a panel of pharmacologic agents to characterize the mechanistic and functional consequences of CDK9 inhibition in Myc-driven medulloblastoma. We used a combination of clonogenic assays and live cell imaging to assess the cytotoxic effects of CDK9 activity loss. We then performed a combination of CUT&RUN and RNA-seq to evaluate alterations to Myc binding and downstream Myc-driven transcriptional programs. Finally, we employed orthotopic xenograft models of medulloblastoma to assess CNS penetration, tolerability, and anti-tumor efficacy of lead CDK9i candidate compounds. RESULTS: Genetic or pharmacologic inhibition of CDK9 leads to a loss of Myc expression and downregulation of hallmark Myc-driven transcriptional programs. This corresponds to a loss of cell fitness, as measured by decreased proliferation and clonogenic potential. Clinically relevant CDK9 inhibitors show variable efficacy in vivo, but the CNS-penetrant zotiraciclib achieved a significant prolongation in xenograft survival. CONCLUSION: CDK9 catalytic activity represents a druggable vulnerability underpinning Myc-driven transcriptional programs. The development of CNS-penetrant CDK9 inhibitors may open new avenues for rational therapy in these high-risk medulloblastomas.

Discovery of triaromatic flexible agents bearing 1,2,3-Triazole with selective and potent anti-breast cancer activity and CDK9 inhibition supported by molecular dynamics

This study reports an efficient and convenient click synthesis of novel series of chromene scaffold linked to 1,2,3 triazole ring and terminal lipophilic fragments. Structures of all newly synthesized compounds were well characterized by IR, 1H NMR, 13C NMR and elemental analyses. In vitro MTT cytotoxic screening was performed using staurosporine as a reference drug against three different types: aggressive and invasive human breast cancer cell line (MDA-MB 231), Michigan cancer foundation-7 (MCF-7) and human colorectal carcinoma cell line (HCT116). These screening data showed considerable anticancer activity for these newly synthesized compounds compared to reference staurosporine drug with high degree of cell line selectivity and excellent activity with micromolar (µM) half maximal inhibitory concentration (IC50) values against tumor cells. In addition, Cyclin-Dependent Kinase1 and 9 (CDK1 and 9) assays were performed for the most active compounds to get more details about their mechanism of action. In order to assess and explain their binding affinities, molecular docking simulation was studied against CDK9 site. The results obtained from molecular docking study and those obtained from cytotoxic screening and enzyme inhibition were correlated. One of the most prominent analogs is 34 that showed high selective cytotoxicity profile against single breast cancer cell line MCF-7 and MDA-MB 231 with IC50 3.4 µM and 1.4 uM respectively. Molecular dynamics (MD) simulation of the CDK9 complex by Desmond revealed stability during 100 ns with RMSD of CDK9 complex converged at 2.1 Å after 20 ns. During MD, the interaction fractions confirmed multiple interactions of 34 with Cys106, a crucial residue for inhibition of CDK9 activity. A novel 34 triazole compound was introduced for drug design works as potent and selective hit for treatment of breast cancers after processing of optimization steps.