Phosphorylation Of RNA Polymerase Research Articles

Relocalizing transcriptional kinases to activate apoptosis.

Kinases are critical regulators of cellular function that are commonly implicated in the mechanisms underlying disease. Most drugs that target kinases are molecules that inhibit their catalytic activity, but here we used chemically induced proximity to convert kinase inhibitors into activators of therapeutic genes. We synthesized bivalent molecules that link ligands of the transcription factor B cell lymphoma 6 (BCL6) to inhibitors of cyclin-dependent kinases (CDKs). These molecules relocalized CDK9 to BCL6-bound DNA and directed phosphorylation of RNA polymerase II. The resulting expression of pro-apoptotic, BCL6-target genes caused killing of diffuse large B cell lymphoma cells and specific ablation of the BCL6-regulated germinal center response. Genomics and proteomics corroborated a gain-of-function mechanism in which global kinase activity was not inhibited but rather redirected. Thus, kinase inhibitors can be used to context-specifically activate transcription.

Herpes simplex virus 1 inhibits phosphorylation of RNA polymerase II CTD serine-7.

Transcriptional activity of RNA polymerase II (Pol II) is influenced by post-translational modifications of the C-terminal domain (CTD) of the largest Pol II subunit, RPB1. Herpes simplex virus type 1 (HSV-1) usurps the cellular transcriptional machinery during lytic infection to efficiently express viral mRNA and shut down host gene expression. The viral immediate-early protein ICP22 interferes with serine 2 phosphorylation (pS2) by targeting CDK9 and other CDKs, but the full functional implications of this are not well understood. Using Western blotting, we report that HSV-1 also induces a loss of serine 7 phosphorylation (pS7) of the CTD during lytic infection, requiring expression of the two immediate-early proteins ICP22 and ICP27. ICP27 has also been proposed to target RPB1 for degradation, but we show that pS2/S7 loss precedes the drop in total protein levels. Cells with the RPB1 polyubiquitination site mutation K1268R, preventing proteasomal degradation during transcription-coupled DNA repair, displayed loss of pS2/S7 but retained higher overall RPB1 protein levels later in infection, indicating this pathway is not involved in early CTD dysregulation but may mediate bulk protein loss later. Using α-amanitin-resistant CTD mutants, we observed differential requirements for Ser2 and Ser7 for the production of viral proteins, with Ser2 facilitating viral immediate-early genes and Ser7 appearing dispensable. Despite dysregulation of CTD phosphorylation and different requirements for Ser2/7, all CTD modifications tested could be visualized in viral replication compartments with immunofluorescence. These data expand the known means that HSV employs to create pro-viral transcriptional environments at the expense of host responses.IMPORTANCECells rapidly induce changes in the transcription of RNA in response to stress and pathogens. Herpes simplex virus (HSV) disrupts many processes of host mRNA transcription, and it is necessary to separate the actions of viral proteins from cellular responses. Here, we demonstrate that viral proteins inhibit two key phosphorylation patterns on the C-terminal domain (CTD) of cellular RNA polymerase II and that this is separate from the degradation of polymerases later in infection. Furthermore, we show that viral genes do not require the full "CTD code." Together, these data distinguish multiple steps in the remodeling of RNA polymerase during infection and suggest that shared transcriptional phenotypes during stress responses do not revolve around a core disruption of CTD modifications.

Novel meriolin derivatives potently inhibit cell cycle progression and transcription in leukemia and lymphoma cells via inhibition of cyclin-dependent kinases (CDKs)

A key feature of cancer is the disruption of cell cycle regulation, which is characterized by the selective and abnormal activation of cyclin-dependent kinases (CDKs). Consequently, targeting CDKs via meriolins represents an attractive therapeutic approach for cancer therapy. Meriolins represent a semisynthetic compound class derived from meridianins and variolins with a known CDK inhibitory potential. Here, we analyzed the two novel derivatives meriolin 16 and meriolin 36 in comparison to other potent CDK inhibitors and could show that they displayed a high cytotoxic potential in different lymphoma and leukemia cell lines as well as in primary patient-derived lymphoma and leukemia cells. In a kinome screen, we showed that meriolin 16 and 36 prevalently inhibited most of the CDKs (such as CDK1, 2, 3, 5, 7, 8, 9, 12, 13, 16, 17, 18, 19, 20). In drug-to-target modeling studies, we predicted a common binding mode of meriolin 16 and 36 to the ATP-pocket of CDK2 and an additional flipped binding for meriolin 36. We could show that cell cycle progression and proliferation were blocked by abolishing phosphorylation of retinoblastoma protein (a major target of CDK2) at Ser612 and Thr82. Moreover, meriolin 16 prevented the CDK9-mediated phosphorylation of RNA polymerase II at Ser2 which is crucial for transcription initiation. This renders both meriolin derivatives as valuable anticancer drugs as they target three different Achilles’ heels of the tumor: (1) inhibition of cell cycle progression and proliferation, (2) prevention of transcription, and (3) induction of cell death.

Inhibition of neddylation disturbs zygotic genome activation through histone modification change and leads to early development arrest in mouse embryos

Post-translational modification and fine-tuned protein turnover are of great importance in mammalian early embryo development. Apart from the classic protein degradation promoting ubiquitination, new forms of ubiquitination-like modification are yet to be fully understood. Here, we demonstrate the function and potential mechanisms of one ubiquitination-like modification, neddylation, in mouse preimplantation embryo development. Treated with specific inhibitors, zygotes showed a dramatically decreased cleavage rate and almost all failed to enter the 4-cell stage. Transcriptional profiling showed genes were differentially expressed in pathways involving cell fate determination and cell differentiation, including several down-regulated zygotic genome activation (ZGA) marker genes. A decreased level of phosphorylated RNA polymerase II was detected, indicating impaired gene transcription inside the embryo cell nucleus. Proteomic data showed that differentially expressed proteins were enriched in histone modifications. We confirmed the lowered in methyltransferase (KMT2D) expression and a decrease in histone H3K4me3. At the same time, acetyltransferase (CBP/p300) reduced, while deacetylase (HDAC6) increased, resulting in an attenuation in histone H3K27ac. Additionally, we observed the up-regulation in YAP1 and RPL13 activities, indicating potential abnormalities in the downstream response of Hippo signaling pathway. In summary, we found that inhibition of neddylation induced epigenetic changes in early embryos and led to abnormalities in related downstream signaling pathways. This study sheds light upon new forms of ubiquitination regulating mammalian embryonic development and may contribute to further investigation of female infertility pathology.

Discovery of novel CDK9 inhibitor with tridentate ligand: Design, synthesis and biological evaluation

Cyclin-dependent kinase 9 (CDK9) plays a role in transcriptional regulation, which had become an attractive target for discovery of antitumor agent. In this work, beyond traditional CDK9 inhibitor with bidentate ligands in ATP binding domain, a series of novel CDK9 inhibitor with tridentate ligand were designed and synthesized. Surprisingly, this unique tridentate ligand structure endows better CDK9 inhibition selectivity compared to other CDK subtypes, and the lead candidate compound Z4-7a showed effective proliferation inhibition in HCT116 cells with acceptable pharmacokinetic properties. Research on the mechanism indicated that Z4-7a could induce apoptosis in the HCT116 cell line by inhibiting phosphorylation of RNA polymerase II at Ser2, which resulted in the inhibition of apoptosis-related genes and proteins expression. In brief, introduction of tridentate ligand might work as a promising strategy for the development of novel selective CDK9 inhibitor.

TRIM33 plays a critical role in regulating dendritic cell differentiation and homeostasis by modulating Irf8 and Bcl2l11 transcription

The development of distinct dendritic cell (DC) subsets, namely, plasmacytoid DCs (pDCs) and conventional DC subsets (cDC1s and cDC2s), is controlled by specific transcription factors. IRF8 is essential for the fate specification of cDC1s. However, how the expression of Irf8 is regulated is not fully understood. In this study, we identified TRIM33 as a critical regulator of DC differentiation and maintenance. TRIM33 deletion in Trim33fl/fl Cre-ERT2 mice significantly impaired DC differentiation from hematopoietic progenitors at different developmental stages. TRIM33 deficiency downregulated the expression of multiple genes associated with DC differentiation in these progenitors. TRIM33 promoted the transcription of Irf8 to facilitate the differentiation of cDC1s by maintaining adequate CDK9 and Ser2 phosphorylated RNA polymerase II (S2 Pol II) levels at Irf8 gene sites. Moreover, TRIM33 prevented the apoptosis of DCs and progenitors by directly suppressing the PU.1-mediated transcription of Bcl2l11, thereby maintaining DC homeostasis. Taken together, our findings identified TRIM33 as a novel and crucial regulator of DC differentiation and maintenance through the modulation of Irf8 and Bcl2l11 expression. The finding that TRIM33 functions as a critical regulator of both DC differentiation and survival provides potential benefits for devising DC-based immune interventions and therapies.

A novel and ubiquitous miRNA-involved regulatory module ensures precise phosphorylation of RNA polymerase II and proper transcription.

Proper transcription orchestrated by RNA polymerase II (RNPII) is crucial for cellular development, which is rely on the phosphorylation state of RNPII's carboxyl-terminal domain (CTD). Sporangia, developed from mycelia, are essential for the destructive oomycetes Phytophthora, remarkable transcriptional changes are observed during the morphological transition. However, how these changes are rapidly triggered and their relationship with the versatile RNPII-CTD phosphorylation remain enigmatic. Herein, we found that Phytophthora capsici undergone an elevation of Ser5-phosphorylation in its uncanonical heptapeptide repeats of RNPII-CTD during sporangia development, which subsequently changed the chromosomal occupation of RNPII and primarily activated transcription of certain genes. A cyclin-dependent kinase, PcCDK7, was highly induced and phosphorylated RNPII-CTD during this morphological transition. Mechanistically, a novel DCL1-dependent microRNA, pcamiR1, was found to be a feedback modulator for the precise phosphorylation of RNPII-CTD by complexing with PcAGO1 and regulating the accumulation of PcCDK7. Moreover, this study revealed that the pcamiR1-CDK7-RNPII regulatory module is evolutionarily conserved and the impairment of the balance between pcamiR1 and PcCDK7 could efficiently reduce growth and virulence of P. capsici. Collectively, this study uncovers a novel and evolutionary conserved mechanism of transcription regulation which could facilitate correct development and identifies pcamiR1 as a promising target for disease control.

Nanodrug modified with engineered cell membrane targets CDKs to activate aPD-L1 immunotherapy against liver metastasis of immune-desert colon cancer

Immunotherapy based on the PD-1/PD-L1 axis blockade has no benefit for patients diagnosed with colon cancer liver metastasis (CCLM) for the microsatellite stable/proficient mismatch repair (MSS/pMMR)) subtype, which is known as an immune-desert cancer featuring poor immunogenicity and insufficient CD8+ T cell infiltration in the tumor microenvironment. Here, a multifunctional nanodrug carrying a cyclin-dependent kinase (CDK)1/2/5/9 inhibitor and PD-L1 antibody is prepared to boost the immune checkpoint blockade (ICB)-based immunotherapy against MSS/pMMR CCLM via reversing the immunosuppressive tumor microenvironment. To enhance the MSS/pMMR CCLM-targeting efficacy, we modify the nanodrug with PD-L1 knockout cell membrane of this colon cancer subtype. First, CDKs inhibitor delivered by nanodrug down-regulates phosphorylated retinoblastoma and phosphorylated RNA polymerase II and meanwhile arrests the G2/M cell cycle in CCLM to promote immunogenic signal release, stimulate dendritic cell maturation, and enhance CD8+ T cell infiltration. Moreover, CDKi suppresses the secretion of immunosuppressive cytokines in tumor-associated myeloid cells sensitizing ICB therapy in CCLM. Notably, the great efficacy to activate immune responses is demonstrated in the patient-derived xenograft model and the patient-derived organoid model as well, revealing a clinical application potential. Overall, our study represents a promising therapeutic approach for targeting liver metastasis, remolding the tumor immune microenvironment (TIME), and enhancing the response of MSS/pMMR CCLM to boost ICB immunotherapy.

Abstract 5712: Functional dissection of CDK7 in transcription using a highly selective CDK7 inhibitor Q901

Abstract CDK7 is a well-known regulator of both the cell cycle and transcription and plays a crucial role in cell cycle progression by catalyzing T-loop phosphorylation of other CDKs. CDK7 is also an integral subunit of the general transcription factor, TFIIH and regulates transcription through phosphorylation of RNA Polymerase II (Pol II) at CTD Ser5 and CDK9. We used Q901, a highly selective CDK7 inhibitor to further investigate the molecular function of CDK7 in the transcriptional process. Q901 is currently being evaluated in a phase 1 clinical study for selected advanced solid tumors. Compared to previously reported CDK7 inhibitors, Q901 has increased specificity and efficacy, but the impact on transcription have not been studied in detail. In our study, we combined RNA Pol II (pan, Ser5P, Ser2P) ChIP-seq and PRO-seq techniques in the MCF7 breast cancer cell line. The findings demonstrated that the Q901 effectively altered transcription and the RNA Pol II profile in a subset of genes, leading to a significant reduction in Ser5 phosphorylation levels in RNA Pol II. The downregulated genes were associated with critical pathways including downstream of oncogenic genes (MYC and E2F), DNA damage repair, cell cycle checkpoint, and gene expression. To verify whether these genes were the direct targets of CDK7, we performed CDK7 ChIP-seq analysis. CDK7 binding exclusively to the genes that were downregulated by Q901, without any detectable binding to upregulated genes. Despite the universal role of general transcription machinery in transcription, the data demonstrated that the highly selective inhibition of CDK7 in transcription of various oncogenic pathways underlies the potent in vitro and in vivo anti-cancer efficacy of this specific CDK7 inhibitor. Citation Format: Hyerim Jung, Dahun Um, Yoonji Lee, Donghoon Yu, Dayoung Lee, Jiye Ahn, Jeongjun Kim, Seung-Joo Lee, Jaeseung Kim, Kiyean Nam, Tae-Kyung Kim. Functional dissection of CDK7 in transcription using a highly selective CDK7 inhibitor Q901 [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 5712.

Abstract 5966: PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies

Abstract CDK9 is a master regulator of transcription that modulates transcription elongation via phosphorylation of RNA polymerase II. Short-term inhibition of CDK9 depletes short-lived transcripts and labile proteins such as MCL1, BFL1 and MYC to promote cancer cell death. We previously described PRT2527, a novel, potent, highly selective CDK9 inhibitor with anti-leukemic activity in various preclinical models. PRT2527 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT05159518). Here we demonstrate PRT2527 potently inhibits cancer cell growth and induces cell death in various models of Diffuse Large B Cell Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), through depletion of MCL1, BFL1 and MYC. Specifically, we show that brief treatment with PRT2527 (6h) potently induces apoptosis and inhibits proliferation in DLBCL, MCL and CLL cell lines. We also show that the addition of BTK inhibitors (BTKi) or BCL2 inhibitors (BCL2i) potentiates the apoptosis response induced by PRT2527 in vitro and leads to more robust and durable responses including complete tumor regressions in DLBCL CDX and PDX models in vivo. In all studies, the combination of PRT2527 with BTKi or BCL2i is well tolerated. Mechanistically, we show that BTK inhibition upregulates BMF, an endogenous inhibitor of BCL2 and BCL-xL, driving cells towards MCL1 and BFL1 dependency in TMD-8 cells. Concurrent depletion of MCL1 and BFL1 with PRT2527 leads to complete inhibition of Bcl-2- family mediated survival signaling, potently inducing cell death. In similar fashion, Venetoclax-driven BCL2 inhibition also potentiates the anti-tumor activity of PRT2527. Consistent with these findings in vitro, we observe reduced BFL1 and MCL1 as well as increased BMF in tumors of mice treated with BTKi and PRT2527 suggesting complete Bcl-2 family inhibition. We also demonstrate potent induction of apoptosis in peripheral blood mononuclear cells isolated from patients with both treatment-naïve and relapse/refractory CLL following brief treatment with PRT2527 (6h). Co-treatment with BTKi or BCL2i further enhances the apoptotic effect of PRT2527, similarly suggesting combinatorial benefit. Additionally, we characterize the activity of PRT2527 in models of BTKi relapsed/refractory MCL where increased CDK9 dependency has previously been described. In an Ibrutinib-resistant Mino CDX model, we observe increased tumor growth inhibition following treatment with PRT2527 compared to tumors derived from parental Mino cells, suggesting PRT2527 is efficacious in BTKi-resistant MCL. Altogether these data provide a rationale for evaluating PRT2527 in combination with BTK and BCL2 inhibitors for the treatment of patients with relapsed/refractory lymphoid malignancies. Citation Format: Norman Fultang, Ashley M. Schwab, Sophia McAneny-Droz, Diane Heiser, Peggy Scherle, Neha Bhagwat. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, has potent antitumor activity in combination with BTK and BCL2 inhibition in various lymphoid malignancies [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 5966.

Abstract 4605: AZD4573 in combination with CHOP increases combination benefit in preclinical peripheral T-cell lymphoma models

Abstract Peripheral T-cell Lymphoma (pTCL) makes up about 10% of non-Hodgkin's lymphoma in the United States. Patients with relapsed/refractory pTCL have limited treatment options and poor prognosis. Current standard of care for pTCL revolves around the chemotherapy regimens CHOP/E. Here we examine the efficacy of CDK9 inhibition for the treatment of pTCL. CDK9 regulates transcription through phosphorylation of RNA polymerase II. AZD4573 is a highly potent and selective CDK9 inhibitor; acute inhibition with AZD4573 downregulates short-lived proteins such as MCL-1, BFL-1, and c-MYC, which are often overexpressed in hematologic tumors. It has been shown that the expression pattern of the anti-apoptotic BCL-2 family members in pTCL cell line models closely resembled the expression patterns in pTCL patient samples (Koch et al. 2019). Expression of BCL-2 family members is highly heterogeneous in pTCL, with MCL-1 being the most universally expressed. Using MCL-1 inhibitor AZD5991, we showed statistically significant benefit in survival when combined with CHOP in MCL-1 dependent preclinical pTCL PDX models (Koch et al. 2019). In addition, we have previously reported the importance of the anti-apoptotic protein BFL-1 in cell survival in NHL, including a subset of pTCL (Boiko et al 2021). AZD4573 treatment showed a range of activity across the panel of 18 human pTCL cell lines, in a 6-hour caspase-3/7 activation assay. 14 of the cell lines were sensitive, with EC50 values less than 100 nM, and 13 reached a max caspase activation of over 40%. pTCL subtypes that responded to AZD4573 treatment included ALCL, NK-TCL, and PTCL-NOS and CTCL. Acute AZD4573 treatment resulted in decreased pSer2-RNAP2 and reduced protein levels of c-MYC and MCL-1. To determine what was driving the apoptotic phenotype, we used ribonucleoprotein (RNP) mediated CRISPR knock out (KO) of MYC and MCL1 genes. While pTCL cell lines were dependent on c-MYC and MCL-1 for viability after KO, only MCL-1 KO impaired AZD4573 treatment, suggesting that efficacy is mediated through MCL-1 in ALCL pTCL cell lines assessed. To determine if combining AZD4573 with CHOP improved efficacy, we tested 5 pTCL cell lines in a 6x6 combination dosing matrix and measured viability after 24-72 hours using RealTime-Glo after 24hr exposure to CHOP with AZD4573 added for the last 6hrs. Adding AZD4573 to CHOP in vitro increased the efficacy in pTCL ALCL cell lines with MCL-1 dependency, but not in CTCL cell line with BCL-xL dependency and resistance to AZD4573. CHOP treatment in vitro resulted in a decrease in c-MYC levels but not MCL-1, suggesting that combination benefit may be driven through c-MYC. This data suggests that treatment with AZD4573 as a monotherapy or in combination with CHOP regimen would be an effective therapeutic strategy in pTCL. Citation Format: Danielle Sinead Potter, India Ott, Jamal Saeh, Richard Olsson, Stephen Fawell, Lisa Drew, Justine Roderick. AZD4573 in combination with CHOP increases combination benefit in preclinical peripheral T-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 4605.

Abstract 5716: CDK7 as a drug target in advanced stage uveal melanoma

Abstract Uveal melanoma (UM) metastasizes predominantly to the liver, and most metastatic UM are BAP1-deficient. Targeted therapies, such as MEK inhibitors, have failed clinically. There are few FDA-approved therapies for metastatic UM, but these therapies are limited to only a subset of patients and have modest survival benefits. Hence, there is a need to identify novel and effective treatment strategies. Due to the low mutational burden in UM, targeting transcriptional may be an effective strategy. Here, we identified Cyclin Dependent Kinase 7 (CDK7), a key regulator of transcription, as an essential and druggable target in UM. CDK7 directly phosphorylates RNA polymerase II and is expressed in all UMs. siRNA-mediated knockdown of CDK7 decreased the growth of three BAP1-deficient UM cell lines. We tested a clinically relevant, highly specific CDK7 inhibitor, SY-5609, developed by Syros Pharmaceuticals and similarly showed that the cell lines were sensitive to the growth inhibitory effects of SY-5609. Furthermore, we observed synergistic effects on cell growth inhibition when combined with a MEK inhibitor (Trametinib). We performed RNA sequencing and identified several Hallmark Genesets to be positively enriched, including interferon-gamma response, TGF-beta signaling, and NFκB signaling after treatment with SY-5609. These results are validated using a multi-omic approach, utilizing Reverse Phase Protein Array (RPPA) data to validate transcriptome to proteome expression of these enriched pathways. Overall, our studies indicate that targeting CDK7 in uveal melanoma is a potential treatment strategy for BAP1-deficient UM and enhances MEK inhibitor efficacy. Citation Format: Francis J. Waltrich. CDK7 as a drug target in advanced stage uveal melanoma [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 5716.

Abstract 3301: Translational research of CDK12/13 inhibitor, CTX-439, informing clinical trial strategy

Abstract Gene dysregulation is a hallmark of cancer, arising from genetic alterations that invariably lead to dysregulated transcriptional programs. This dysregulation renders cancer cells highly dependent on certain gene expression regulators, leading to a potential for novel therapeutic strategies. Consequently, ongoing research aims to develop small molecules targeting RNA transcriptional processes. Cyclin-dependent kinase 12/13 (CDK12/13) is a member of the cyclin-dependent kinase family of serine/threonine protein kinases. CDK12/13 regulates the RNA transcription elongation by phosphorylating RNA polymerase II and promotes the elongation of transcripts, specifically those involved in DNA damage responses, such as BRCA1/2. The inhibition of CDK12/13 is expected to exert a synergistic effect when combined with PARP inhibitors and chemotherapeutic reagents. Therefore, we developed a novel small molecule inhibitor, CTX-439 (former name: CRD-1835439). Additionally, we demonstrated that CTX-439 exhibits a preclinical efficacy in vitro and in vivo [Cancer Res. (2022) 82 (12_Supplement): 5485]. To identify potential strategies for a clinical trial of CTX-439, in the current study, we conducted translational research to elucidate (1) pharmacodynamics (PD) markers to monitor the suppression of CDK12/13 in humans, (2) patient selection biomarkers to determine sensitivity to CTX-439, and (3) combination strategies with other key medications. Firstly, to identify PD markers of CTX-439, we performed a comprehensive analysis employing RNA-seq, Pol II-ChIP-seq, and Poly(A)-seq. We confirmed that CTX-439 induced the usage of intronic polyadenylation sites by inhibiting CDK12, resulting in the production of shortened mRNA. Of these transcripts, PCF11 emerged as a potential PD marker for clinical trials, which was confirmed using qPCR analysis of human blood. Secondly, to clarify the indication of CTX-439, we performed an in vitro survey involving 400 cell lines and 100 PDX-derived organoids (PDXOs) to identify biomarkers displaying sensitivity and insensitivity to CTX-439. Furthermore, a significant anti-tumor effect of CTX-439 was revealed in PDX models that showed sensitivity in the survey. CTX-439 exhibited a synergistic effect in combination with PARP inhibitors both in vitro and in vivo, highlighting its potential for use in combination strategies. Additionally, CTX-439 showed an anti-tumor effect when combined with other chemotherapy and molecular-targeted drugs. Collectively, the results from these translational research studies provide a foundation for developing a clinical trial by clearly defining PD monitoring, patient stratification biomarkers, and combination strategies. These findings highlight the therapeutic potential of CTX-439 as a single-agent and in combination with inhibitors, such as PARP, for the treatment of cancers characterized by transcriptional addiction. Citation Format: Hiroko Yamakawa, Shunsuke Ebara, Akio Mizutani, Misaki Yoshida, Midori Sugiyama, Koji Yamamoto, Daisuke Komura, Miwako Kakiuchi, Shumpei Ishikawa, Kosuke Yusa, Daisuke Morishita. Translational research of CDK12/13 inhibitor, CTX-439, informing clinical trial strategy [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 3301.

Identification of a Novel Selective CDK9 Inhibitor for the Treatment of CRC: Design, Synthesis, and Biological Activity Evaluation.

Cyclin-dependent kinase 9 (CDK9) is a member of the transcription CDK subfamily. In this work, we preliminarily demonstrated the feasibility of CDK9 as a potent target of treatment for colorectal cancer, and a series of novel CDK9 inhibitors were rationally designed and synthesized based on the structure of AZD5438 (a pan CDKs inhibitor reported by AstraZeneca). A novel selective CDK9 inhibitor named CLZX-205, which possessed significant CDK9 inhibitory activity (IC50 = 2.9 nM) with acceptable pharmacokinetic properties and antitumor efficacy in vitro and in vivo, was developed. Research on the mechanism indicated that CLZX-205 could induce apoptosis in the HCT116 cell line by inhibiting phosphorylation of RNA polymerase II at Ser2, which resulted in the inhibition of apoptosis-related genes and proteins expression, and these results were validated at the cellular and tumor tissue levels. Currently, CLZX-205 is undergoing further research as a promising candidate for CRC treatment.

Endogenous EWSR1 Exists in Two Visual Modalities That Reflect Its Associations with Nucleic Acids and Concentration at Sites of Active Transcription.

EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

Abstract LB_C13: Development of BLX-3030, a potent, selective, orally available CDK9i shows promise in Pancreatic Ductal Adenocarcinoma (PDAC) models

Abstract Background: CDK9 (cyclin-dependent kinase 9), is a Ser/Thr kinase that plays a crucial role in regulating gene expression. It is a component of the P-TEFb complex, which controls transcription elongation by phosphorylating RNA polymerase II. Dysregulated CDK9 has been linked to cancer progression. Pharmacological CDK9 inhibition has recently emerged as a novel and promising approach for cancer therapy, particularly in exocrine pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumors (PNETs). PDAC and PNETs are cancer entities with a dismal prognosis, owing mainly to their resistance to chemo, radiation, and immunotherapies. Dysregulation of CDK9 activity can lead to uncontrolled cell cycle progression and tumor formation. Given the role of CDK9 in promoting PDACs progression, targeting this kinase has emerged as a potential therapeutic strategy for PDACs and PNETs where our late-breaking results of BLX-3030 demonstrated efficacy in pre-clinical tumor models. Materials and Methods: Our proprietary MolecuLernTM fragment-based design platform, in vitro cellular efficacies as a single agent or combination, FACS/apoptosis, cell migration, live-cell imaging, and immunofluorescence assays. Top compounds were evaluated for 7-day tox, PK, selectivity, Nano BRET, ADME-Tox, in vitro safety, hERG, P450, SafetyScreen44 panel, LDH-Glo assay, and KINOMEscan for selectivity. Results: Utilizing a CDK9-refined crystal structure and the MolecuLernTM, we identified novel, reversible, ATP-competitive inhibitors that exhibited potent activity in MYC-expressing pancreatic cancer cells in vitro. BLX-3030, elected from over 90 analogs demonstrated potent activity with an IC50 of 4.2 nM in inhibiting CDK9. CDKs family selectivity and target engagement NanoBRET assay results show the BLX-3030 selectivity and its on-target potency with an IC50 of 24.3 nM. BLX-3030 potently inhibited the growth of pancreatic cancer cells with IC50s ranging from 133 nM to 508 nM in 10 different cell lines. Furthermore, BLX-3030 and its analogs showed excellent PK properties in mice, rats, and dogs with an oral bioavailability of 28%, 38%, and 83%, respectively. BLX-3030 has a low clearance and >3 hours half-life in rodents and canine species. The effect of BLX-3030 in vivo PDAC models including ex vivo results for the levels of CDK9, pCDK9, c-MYC, and cleaved Caspase 3 in tumor tissues will be discussed. Based on these pre-clinical results, BLX-3030 was nominated as a candidate with additional efficacy and safety data supporting IND-enabling studies Conclusion: In summary, BLX-3030 is a potent CDK9i that displayed potent antitumor activity in pancreatic cancer models, with its promising safety, PK, and developable characteristics leading us to select and nominate as clinical candidates. Our late-breaking pre-clinical results from our in vivo study in pancreatic cancer tumor models, including 7-day safety, tolerability, and toxicokineCDK9tic results received are supportive for our planned ND enabling studies and future clinical trials will be presented. Citation Format: Kyle Medley, Zhaoliang Li, Chenyu Lin, Yesenia Barrera-Millan, Lynne Kelley, Haiyong Han, Hariprasad Vankayalapati, David J Bearss. Development of BLX-3030, a potent, selective, orally available CDK9i shows promise in Pancreatic Ductal Adenocarcinoma (PDAC) models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C13.

The suppressive efficacy of THZ1 depends on KRAS mutation subtype and is associated with super-enhancer activity and the PI3K/AKT/mTOR signalling in pancreatic ductal adenocarcinoma: A hypothesis-generating study.

Inhibition of CDK7, a potent transcription regulator, may bring new hope for treating pancreatic ductal adenocarcinoma (PDAC), which is featured by large genetic heterogeneity and abundant KRAS mutations. This investigation aimed at exploring the discrepant efficacies of THZ1, a small-molecule covalent CDK7 inhibitor, on PDACs with different KRAS mutations and the underlying mechanisms. Associations of CDK7 expression with survival by KRAS mutations were first assessed. Effects of THZ1 on PDAC by different KRAS mutations were then investigated in vitro and in vivo. Moreover, the effects of THZ1 on gene transcription and phosphorylation of RNA polymerase II (RNAPOLII) in different KRAS mutant PDACs were assessed, and the effect of THZ1 on super-enhancer activity was evaluated using chromatin immunoprecipitation sequencing. Lastly, the effects of THZ1 on the binding of H3K27ac to PIK3CA and on the PI3K/AKT/mTOR signalling were analysed. High CDK7 expression was significantly linked to worse survival within PDAC patients carrying KRAS-G12V mutation but not in those with KRAS-G12D mutation. The apoptosis-inducing effect of THZ1 was markedly stronger in KRAS-G12V PDAC than KRAS-G12D cancer. THZ1 significantly inhibited the growth of xenograft tumour with KRAS-G12V mutation, and the inhibition was markedly stronger than for KRAS-G12D tumour. In mini-cell-derived xenograft (CDX) models, THZ1 significantly suppressed KRAS-G12V PDAC but not KRAS-G12D cancer. THZ1 significantly suppressed the phosphorylation of RNAPOLII, and this effect was stronger in KRAS-G12V PDAC (especially at ser5). KRAS-G12V PDAC had more H3K27ac-binding super-enhancers, and the inhibition of THZ1 on super-enhancer activity was also stronger in KRAS-G12V PDAC. Furthermore, THZ1 significantly weakened the binding of H3K27ac to PIK3CA in KRAS-G12V PDAC. THZ1 significantly suppressed the PI3K/AKT/mTOR pathway and its downstream markers, and this effect was stronger in KRAS-G12V cells. In this hypothesis-generating study, THZ1 might selectively inhibit certain PDACs with KRAS-G12V mutation more potently compared with some other PDACs with KRAS-G12D mutation, which might be associated with its effect on super-enhancer activity and the PI3K/AKT/mTOR signalling. Our findings might offer novel key clues for the precise management of PDAC and important evidence for future targeted trial design. THZ1 had a stronger effect on PDAC-bearing KRAS-G12V mutation than G12D mutation. Suppressive effect of THZ1 on phosphorylation of RNAPOLII was stronger in KRAS-G12V than KRAS-G12D PDAC. Inhibition of THZ1 on super-enhancer activity and H3K27ac binding to PIK3CA was stronger in KRAS-G12V PDAC. Suppressive effect of THZ1 on PI3K/AKT/mTOR pathway was stronger in KRAS-G12V PDAC.

Transient CDK9 Inhibition with AZD4573 Effectively Induces Apoptosis in Burkitt Lymphoma As a Monotherapy in Pre-Clinical Models

Burkitt Lymphoma (BL) accounts for almost two thirds of all B-cell non-Hodgkin lymphoma cases in children and adolescents. It may affect the jaw, bones of the face, bowel, kidney, ovaries and in some cases spread to the central nervous system. Nearly all types of BL are characterized by c-MYC gene translocation of chromosome 8 with immunoglobulin genes at chromosome 2, 14 or 22 resulting in high levels of the transcription factor. Recently, MCL-1 was shown to be overexpressed in BL and is a critical survival factor in pre-clinical BL models using genetic and chemical approaches. CDK9, a serine/threonine kinase, regulates transcription elongation through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII). AZD4573 is a highly potent and selective CDK9 inhibitor that results in downregulation of short-lived transcripts and labile proteins such as c-MYC and key survival proteins including MCL-1 and BFL-1, resulting in apoptosis in several hematological malignancies (Cidado et al. 2020 & Boiko et al. 2021) We selected several preclinical BL models to assess the rapid apoptogenic potential of AZD4573 in vitro and in vivo. To measure apoptosis, we evaluated cleaved caspase-3 (CC3) induction following acute treatment (6h) using Caspase-Glo 3/7. Using this assay, we found that 3 BL models were sensitive to CDK9 inhibition (EC50 < 100nM; max. CC3 > 50%) and 2 were resistant (EC50 > 100nM; max. CC3 < 50%). Treatment with AZD4573 in these sensitive BL cell line models decreased pSer2-RNAPII, and downregulation of MCL-1 and c-MYC within 6 hours. In AZD4573 resistant lines we observed relatively higher levels of anti-apoptotic proteins BCL2 and BCL-xL, compared to the sensitive cell lines. These increased levels potentially lead to the decreased sensitivity to AZD4573, which does not reduce levels of BCL-2 and BCL-xL due to their longer half-life. Using CRISPR genetic knockouts we demonstrate that BL cell lines are dependent on c-MYC and MCL-1 and that loss of these protein decreases caspase induction after AZD4573 treatment, highlighting the importance of these proteins in driving the efficacy of AZD4573. In vivo AZD4573 dosed on a once weekly schedule resulted in tumor growth inhibition in aggressive BL cell line xenografts Namalwa and Ramos (40-60%) and decreased pSer2-RNAPII, MCL-1 and c-MYC and increased CC3 8 hours after treatment. AZD4573 is currently in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK of AZD4573 in patients with relapsed or refractory Peripheral T-cell lymphomas (pTCL). Our findings demonstrate that targeting CDK9 with AZD4573 can effectively induce apoptosis in pre-clinical BL models and could be an effective therapy for BL patients.

PRT2527, a Novel Highly Selective Cyclin-Dependent Kinase 9 (CDK9) Inhibitor, Has Potent Antitumor Activity in Combination with BTK and BCL2 Inhibition in Various Lymphoid Malignancies

CDK9 is a master regulator of transcription that modulates transcription elongation via phosphorylation of RNA polymerase II. Short-term inhibition of CDK9 depletes short-lived transcripts and labile proteins such as MCL1, BFL1 and MYC to promote cancer cell death. We previously described PRT2527, a novel, potent, highly selective CDK9 inhibitor with anti-leukemic activity in various preclinical models. PRT2527 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT05159518). Here we demonstrate PRT2527 potently inhibits cancer growth and induces cell death in various models of Diffuse Large B Cell Lymphoma (DLBCL), Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL), through depletion of MCL1, BFL1 and MYC. We also show that combination of PRT2527 with BTK inhibitors (BTKi) or BCL2 inhibitors (BCL2i) leads to more robust and durable responses in multiple pre-clinical models. In these studies, the combination of PRT2527 with BTKi or BCL2i was well tolerated. Mechanistically, we show that in cell lines, BTK inhibition upregulates BMF, an endogenous inhibitor of BCL2 and BCL-xL, driving cells towards MCL1 and BFL1 dependency. Concurrent depletion of MCL1 and BFL1 with PRT2527 led to complete inhibition of Bcl-2- family mediated survival signaling, potently inducing cell death. In a similar fashion, Venetoclax-driven BCL2 inhibition also potentiated the anti-tumor activity of PRT2527. Consistent with our findings in vitro, we confirmed potent combinatorial inhibition of tumor growth by PRT2527, administered once weekly, and BTKi or BCL2i, administered daily, in CDX and PDX in vivo models of DLBCL and MCL. We observed reduced BFL1 and MCL1 as well as increased BMF in tumors of mice treated with BTKi and PRT2527 suggesting complete Bcl-2 family inhibition. We also demonstrated enhanced induction of apoptosis by PRT2527 in primary CLL patient samples simultaneously treated with BTKi or BCL2i. Additionally, we demonstrated that PRT2527 potently inhibits growth in Ibrutinib-resistant MCL cell lines. Further work characterizing PRT2527 as monotherapy and in combination with BTKi and BCL2i in in vivo models of BTKi-resistant MCL and CLL is currently ongoing. Altogether these data provide a rationale for evaluating PRT2527 in combination with BTK and BCL2 inhibitors for the treatment of patients with relapsed/refractory hematologic malignancies.

AZD4573 in Combination with CHOP Increases Combination Benefit in Preclinical Peripheral T-Cell Lymphomas Models

Peripheral T-cell Lymphoma (pTCL) make up about 10% of non-Hodgkin's lymphoma in the United States. Patients with relapsed/refractory pTCL have limited treatment options and poor outcomes to standard of care therapy. Current standard of care for pTCL revolves around the chemotherapy regimens CHOP/E and for CD30-postive pTCLs, brentuximab vedotin is approved. CDK9 regulates transcription elongation through phosphorylation of RNA polymerase II. AZD4573 is a highly potent and selective CDK9 inhibitor, acute inhibition with AZD4573 downregulates short-lived proteins such as MCL-1, BFL-1, and c-MYC, which are frequently overexpressed in haematologic tumours. It has been previously shown that the expression pattern of the anti-apoptotic BCL-2 family members in pTCL cell line models closely resembled the expression patterns in pTCL patient samples (Koch et al. 2019). Expression and dependencies of the anti-apoptotic BCL-2 family members is highly heterogeneous in pTCL; however MCL-1 is the most universally expressed BCL-2 family member. Using the MCL-1 inhibitor AZD5991, we have shown statistically significant benefit in survival when combined with CHOP in 2 MCL-1 dependent preclinical pTCL PDX models (Koch et al. 2019). In addition to MCL-1, we have previously reported the importance of the anti-apoptotic protein BFL-1 in mediating cell survival in NHL, including a subset of pTCL (Boiko et al 2021). Consistent with these findings, AZD4573 treatment exhibited a range of activity across the panel of 18 human TCL cell lines, in a 6-hour caspase-3/7 activation assay. 14 of the cell lines were sensitive, having EC 50 values < 100 nM, and 13 reached a max caspase-3/7 activation of greater than 40%. pTCL cell lines that responded to AZD4573 treatment including ALCL, NK-TCL, and PTCL-NOS and CTCL subtypes. Acute AZD4573 treatment of pTCL cell lines resulted in decreased p-SerRNApoll II and reduced levels of c-MYC and MCL-1 protein levels. To determine if MCL-1 or c-MYC was driving the apoptotic phenotype, we used ribonucleoprotein (RNP) mediated CRISPR knock out (KO) of MYC and MCL1 genes. While pTCL cell lines were dependent on c-MYC and MCL-1 after long term KO, only MCL-1 KO impaired AZD4573 treatment, suggesting that AZD4573 efficacy is mediated through MCL-1, in ALCL pTCL cell lines tested. To determine if combining AZD4573 with CHOP improved efficacy, we tested 5 pTCL cell lines in a 6x6 combination dosing matrix and assessed for viability after 24-72 hours using RealTime-Glo after 18hr exposure to CHOP and AZD4573 added for the last 6hrs. Adding AZD4573 treatment to CHOP schedule in vitro increased the combination benefit in pTCL ALCL cell lines that show MCL-1 dependency, but not in CTCL cell line which show a BCL-xL dependency and resistance to AZD4573. CHOP treatment in vitro resulted in a decrease in c-MYC levels at 24-48 hours, but not MCL-1 suggesting that combination benefit may be driven through decreased c-MYC levels, as KO of c-MYC resulted is loss of cell viability in these models. These data suggested that treatment with AD4573 either as a monotherapy or in combination with CHOP, would be an effective therapeutic strategy in pTCL. AZD4573 monotherapy is currently being evaluated in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK in patients with relapsed/refractory pTCL.