Phosphorylation Of RNA Polymerase Research Articles

G1 cyclin-Cdk promotes cell cycle entry through localized phosphorylation of RNA polymerase II.

Cell division is thought to be initiated by cyclin-dependent kinases (Cdks) inactivating key transcriptional inhibitors. In budding yeast, the G1 cyclin Cln3-Cdk1 complex is thought to directly phosphorylate the Whi5 protein, thereby releasing the transcription factor SBF and committing cells to division. We report that Whi5 is a poor substrate of Cln3-Cdk1, which instead phosphorylates the RNA polymerase II subunit Rpb1’s C-terminal domain on S5 of its heptapeptide repeats. Cln3-Cdk1 binds SBF-regulated promoters and Cln3’s function can be performed by the canonical S5 kinase Ccl1-Kin28 when synthetically recruited to SBF. Thus, we propose that Cln3-Cdk1 triggers cell division by phosphorylating Rpb1 at SBF-regulated promoters to promote transcription. Our findings blur the distinction between cell cycle and transcriptional Cdks to highlight the ancient relationship between these two processes.

Inhibition of CDK9 activity compromises global splicing in prostate cancer cells

ABSTRACT Cyclin-dependent kinase 9 (CDK9) phosphorylates RNA polymerase II to promote productive transcription elongation. Here we show that short-term CDK9 inhibition affects the splicing of thousands of mRNAs. CDK9 inhibition impairs global splicing and there is no evidence for a coordinated response between the alternative splicing and the overall transcriptome. Alternative splicing is a feature of aggressive prostate cancer (CRPC) and enables the generation of the anti-androgen resistant version of the ligand-independent androgen receptor, AR-v7. We show that CDK9 inhibition results in the loss of AR and AR-v7 expression due to the defects in splicing, which sensitizes CRPC cells to androgen deprivation. Finally, we demonstrate that CDK9 expression increases as PC cells develop CRPC-phenotype both in vitro and also in patient samples. To conclude, here we show that CDK9 inhibition compromises splicing in PC cells, which can be capitalized on by targeting the PC-specific addiction androgen receptor.

Association between CDK8 gene polymorphisms and dilated cardiomyopathy in a Chinese Han population.

Dilated cardiomyopathy (DCM) is one of the most common types of cardiomyopathies. Various genes have been verified to be related to DCM, but the pathogenesis remains unclear. Cyclin-dependent-kinase 8 (CDK8), encoded by the CDK8 gene, is a transcriptional factor that regulates the phosphorylation of RNA polymerase II. It plays an important role in the transcription process and different signaling pathways. This study aimed to investigate the potential role of CDK8 gene polymorphisms in DCM susceptibility and prognosis in a Chinese Han population. Two single nucleotide polymorphisms (SNPs) of CDK8, rs17083838 (A/G) and rs7992670 (A/G), were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 341 DCM patients and 381 healthy controls. Survival analysis was performed using Kaplan-Meier curves and Cox regression analysis. The frequencies of allele A of both SNPs rs17083838 and rs7992670 were increased in DCM patients compared to healthy controls (P<0.05). Genotypic frequencies of rs17083838 and rs7992670 were associated with the susceptibility to DCM in the codominant, and recessive models (P<0.05), and AA/AG genotypes of rs17083838 were also related to DCM susceptibility in the dominant model. AA/AG genotypes of rs17083838 and the AA genotype of rs7992670 in the dominant and recessive genetic models presented a correlation with the poor prognosis of DCM patients in both univariate (P<0.05) and multivariate analyses (P<0.05) after adjusting for age, gender, left ventricular end-diastolic diameter (LVEDD), and left ventricular ejection fraction (LVEF). This research is the first to reveal that CDK8 gene polymorphisms might be related to DCM susceptibility and prognosis in the Chinese Han population.

Nanomaterial-Facilitated Cyclin-Dependent Kinase 7 Inhibition Suppresses Gallbladder Cancer Progression via Targeting Transcriptional Addiction.

Gallbladder cancer (GBC) is the most aggressive malignancy of the biliary tract cancer, and there is a lack of effective treatment. Here, we developed a nanoparticle platform (8P4 NP) that can deliver THZ1, a cyclin-dependent kinase 7 (CDK7) inhibitor, to treat GBC. Analysis of datasets demonstrated that CDK7 was positively correlated with poor prognosis. CDK7 inhibition suppressed cell proliferation, induced apoptosis, and caused cell cycle block in GBC cells. THZ1 downregulated CDK7-mediated phosphorylation of RNA polymerase II (RNAPII), resulting in a significant downregulation of transcriptional programs, with a preferential repression of oncogenic transcription factors. To improve the tumor targeting efficiency of THZ1, 8P4 NPs were prepared and assembled with THZ1 to form THZ1@8P4 NPs. Compared with free THZ1, THZ1@8P4 NPs showed more advantages in prolonging blood circulation, escaping from lysosomes and increasing cellular uptake. Importantly, THZ1@8P4 NPs demonstrated a more significant inhibition effect on GBC cells than free THZ1 in vitro. In addition, THZ1@8P4 NPs could efficiently deliver THZ1 to tumor sites in a patient-derived xenograft model of early recurrence, leading to tumor regression and transcriptional inhibition with minimal toxicity. In summary, we conclude that THZ1@8P4 NPs provide a potent therapeutic strategy that targets CDK7-mediated transcriptional addiction in GBC.

Abstract 1254: Preclinical studies with TGN-1062, a selective and reversible small molecule inhibitor of CDK7

Abstract Purpose: Cancer causing mutations promote unchecked cell cycle progression and altered gene expression to drive tumor growth. Cyclin-dependent Kinase 7 (CDK7) is a critical player in tumorigenesis not only due to its role in cell cycle regulation through the phosphorylation of other CDKs, but also as part of the TFIIH complex where it stimulates transcription via phosphorylation of RNA Polymerase II. It is also known to be associated with Super Enhancers (SEs). When tumor cells acquire SEs at oncogenic driver genes, such as BCL2, GFI1, and MYC, it can lead to the activation of their expression as well as induce transcriptional reprogramming. When CDK7 is overexpressed, it is associated with poor clinical outcomes (AML, breast, ovarian, pancreatic, SCLC, etc.). Inhibition of CDK7 is a promising strategy for abating uncontrolled expression of these oncogenic genes. We have developed TGN-1062, designed to competitively bind the CDK7 catalytic ATP pocket. TGN-1062 is a potent, selective, and reversible inhibitor of CDK7 with desirable drug-like properties. Here, we have further validated our candidate TGN-1062 as a potent inhibitor of CDK7 with efficacy in multiple murine in vivo models. Methods: In vivo efficacy studies were conducted in nude mice engrafted with A2780, HCT116, or MV4-11 tumor cells and treatment with TGN-1062. Tumors were measured to determine tumor growth inhibition (TGI) and correlating drug accumulation were measured in both tumors and plasma using mass spectrometry. Immunohistochemistry (IHC) was performed on tumors to determine changes in cleaved Caspase, cMYC, Ki67, and RNA Pol II. NanoBRET assay was used to demonstrate inhibition of CDK7 activity in living cells. Changes in MCL-1, MYC, cleaved PARP, and RNA Pol II phosphorylation was demonstrated by Western blotting from TGN-1062 treated A2780 and MV4-11 cellular lysates. Drug combinations with TGN-1062 were performed in multiple cancer cell lines. Results: TGN-1062 inhibits CDK7 in the NanoBRET cellular assay with low nanomolar potency (&amp;lt; 0.508 nM) and induces a dose dependent change in biomarkers shown via Western blotting in ovarian and AML cancer cell lines. TGN-1062 showed a TGI of 60% at the total daily dose of 50 mg/kg PO in an ovarian model (A2780) and 50% in the colorectal model (HCT116). Additionally, TGN-1062 had a TGI of 75% at the total daily dose of 50 mg/kg PO and 80% at the total daily dose of 100 mg/kg PO in the AML (MV4-11) xenograft model. Drug accumulation, biomarker analysis, and IHC from murine tumors, show that TGN-1062 treatment decreases RNA POL II phosphorylation and MYC expression, and increases apoptosis markers. Conclusion: TGN-1062 is a novel, selective, and reversible inhibitor of CDK7 and has significant antitumor activity in vivo. TGN-1062 is a potent CDK7 inhibitor with potential for clinical development. Citation Format: Ryan L. Rodriguez del Villar, Trason G. Thode, Alexis Weston, Serina Ng, Raffaella Soldi, Mohan Kaadige, Srinivas Kasibhatla, Vincent Chung, Joseph Chao, Daniel D. Von Hoff, Sunil Sharma. Preclinical studies with TGN-1062, a selective and reversible small molecule inhibitor of CDK7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1254.

CT7001, a Novel Orally Bio-Available CDK7 Inhibitor, Is Highly Active in in-Vitro and in-Vivo Models of AML

CDK7 is considered an important new target for the treatment of human cancer as it controls the activity of key enzymes involved in cell cycle progression, including other cyclin dependent kinases such as CDK1, CDK2, CDK4 and CDK6. As a master regulator of transcription CDK7 also promotes the expression of key oncogenes such as c-Myc through the phosphorylation of RNA polymerase II. Both transcription and cell cycle regulation are dysregulated in Acute Myeloid Leukaemia (AML).Here, we investigated the therapeutic potential of CT7001 (ICEC0942), a novel orally bio-available ATP competitive CDK7 inhibitor (Hazel et al 2017) in pre-clinical AML models. CT7001 potently inhibited the proliferation of several AML cell lines and patient derived AML cells in vitro, with IC50 values ranging from 0.15-2.6 μM. Consistent with the inhibition of CDK7 in cells, treatment of the FLT3 internal tandem duplication (ITD) positive MV-4-11 cells with CT7001 led to a concentration dependent decrease in the phosphorylation of RNA polymerase II at Ser2, Ser5 and Ser7 as well as the phosphorylation of cell cycle signalling intermediates including CDK1, CDK2 and Rb1. Moreover, CT7001 treatment decreased the expression levels of c-Myc and the pro-survival factor Mcl-1 and induced the accumulation of cleaved PARP as a consequence of apoptosis. Finally, once daily oral administration of CT7001 to mice led to a sustained total growth suppression in a MV-4-11 xenograft model.CT7001 is a potent, selective and orally bioavailable inhibitor of CDK7 that shows promise as a potential new treatment for AML.Reference: Hazel et al. ChemMedChem. 2017 12(5):372-380 Inhibitor Selectivity for Cyclin-Dependent Kinase 7: A Structural, Thermodynamic, and Modelling Study [Display omitted] DisclosuresClark:Carrick Therapeutics: Research Funding. Ainscow:Carrick Therapeutics: Employment, Equity Ownership. Peall:Carrick Therapeutics: Research Funding. Thomson:Carrick Therapeutics: Research Funding. Leishman:Carrick Therapeutics: Employment. Elaine:Carrick Therapeutics: Employment, Equity Ownership. Ali:Carrick Therapeutics: Consultancy, Equity Ownership, Patents & Royalties: Patent Inventor for CT7001. Coombes:Carrick Therapeutics: Consultancy, Equity Ownership, Patents & Royalties: Inventor on CT7001 patent. Barrett:Carrick Therapeutics: Consultancy, Equity Ownership, Patents & Royalties: Inventor on CT7001 patent. Bahl:Carrick Therapeutics: Employment, Equity Ownership.

Global Crosstalk between Two Histone Modifications That Control Transcriptional Elongation in MLL Leukemia

The SET domain containing 2 (SETD2) gene encodes the methyltransferase which is responsible for histone 3 lysine 36 tri-methylation (H3K36me3). Previously, we identified SETD2 loss-of-function (LOF) mutations in about 22% of MLL -rearranged acute leukemia patients (MLL leukemia). Using an Mll-AF9 knock-in (MA9) mouse model, we found that knockdown (KD) of Setd2 could significantly accelerate disease development. All of these findings indicate a strong cooperation between SETD2 inactivation and MLL fusions; however, the detailed mechanism remains unclear. H3K36me3 reduction results from SETD2 LOF has been widely reported. In MLL leukemia, high expression levels of MLL fusion target genes (MLL targets), driven by aberrant high level of histone H3 lysine 79 dimethylation (H3K79me2), have been well-studied. Both H3K79me2 and H3K36me3 are enriched at transcriptionally active gene bodies and are associated with phosphorylated RNA Polymerase II. As two frequently dysregulated elongation marks in leukemia patients, we are interested in the potential crosstalk between H3K79me2 and H3K36me3. We aim to clarify whether H3K79me2 changes could affect H3K36me3 levels in MLL leukemia without SETD2 LOF; whether down-regulation of SETD2-H3K36me3 axis could further affect DOT1L-H3K79me2 axis in MLL leukemia with SETD2 LOF; and how this epigenetic crosstalk contribute to leukemia progression.Firstly, to investigate whether H3K79me2 changes could affect H3K36me3 levels in MLL leukemia when there is no genetic alteration for SETD2, we purified c-Kit-positive (c-Kit+) hematopoiesis stem progenitor cells (HSPCs) from MA9 and C57/BL/6 wild-type (WT) mice. Increased H3K79me2 and H3K36me3 levels were observed in MA9 HSPCs using immuno-blotting. Through integrated ChIP-seq and RNA-seq analysis, we found that most of the genes showed increased H3K79me2 and/or H3K36me3 in MA9. Moreover, H3K79me2 targets significantly overlapped with H3K36me3 targets in MA9, suggesting these two marks were co-enriched in, and may also co-regulate, a large set of genes. Changes in H3K79me2 and H3K36me3 were positively correlated with differences in gene expression. However, at mRNA levels, significantly up-regulated genes were mainly MLL targets but not H3K79me2 and H3K36me3 co-enriched genes. Secondly, to explore whether the H3K36me3 reduction mediated by SETD2 LOF could further affect H3K79me2 levels in MLL leukemia, we mimicked the SETD2 LOF with MLL fusions in patients by performing KD of Setd2 in MA9 HSPCs. Interestingly, an increased H3K79me2 level was observed besides a dramatic reduction of H3K36me3 in Setd2 -KD-MA9 cells through immune-blotting and ChIP-seq analysis. At mRNA levels, there was no further activation of the MLL targets, whereas the previously H3K79me2 and H3K36me3 co-enriched genes were changed. Among the co-enriched genes, a set of AML related tumor suppressor genes (TSGs), such as Asxl1, Asxl2, Bcor were down-regulated; but oncogenes, such as Erg, Stat3, Fndc3a, Nampt, Igf1r, were up-regulated. Next, functional studies of TSG Asxl1 and oncogene Erg were performed. We generated a MA9/ Asxl1 -knockout mouse model (Asxl1-/-/MA9). Enhanced cell proliferation were observed in Asxl1-/-/MA9 cells compared to Asxl1+/+/MA9 cells. Overexpression of ERG was observed in both mouse MA9 and human CD34-MA9 cells by Setd2 / SETD2 KD. Importantly, ERG KD partially rescued the proliferation and self-renewal capacities of SETD2 KD effects in mouse and human MA9 cells. These results support the key role of ASXL1 and ERG in MLL leukemia acceleration as SETD2 targets modulated by H3K79me2 and H3K36me3 changes.In conclusion, in MLL leukemia, both H3K79me2 and H3K36me3 are aberrantly elevated and co-enriched in a group of genes. SETD2 inactivation leads to a global reduction of H3K36me3 and a further elevation of H3K79me2, but does not change the expression of known MLL fusion target genes. Instead, this pattern of histone changes is associated with transcriptional deregulation of a novel set of genes; downregulating TSGs (e.g. ASXL1) and upregulating oncogenes (e.g. ERG). Taken together, our findings reveal a global crosstalk between the oncogenic DOT1L-H3K79me2 axis and the tumor suppressive SETD2-H3K36me3 axis in gene regulation, provide molecular insights into how SETD2 mutations accelerate MLL leukemogenesis through differential regulation of additional TSGs and oncogenes. DisclosuresNo relevant conflicts of interest to declare.

Establishment of a fluorescent reporter of RNA-polymerase II activity to identify dormant cells

Dormancy, a reversible quiescent cellular state characterized by greatly reduced metabolic activity, protects from genetic damage, prolongs survival and is crucial for tissue homeostasis and cellular response to injury or transplantation. Dormant cells have been characterized in many tissues, but their identification, isolation and characterization irrespective of tissue of origin remains elusive. Here, we develop a live cell ratiometric fluorescent Optical Stem Cell Activity Reporter (OSCAR) based on the observation that phosphorylation of RNA Polymerase II (RNApII), a hallmark of active mRNA transcription elongation, is largely absent in dormant stem cells from multiple lineages. Using the small intestinal crypt as a model, OSCAR reveals in real time the dynamics of dormancy induction and cellular differentiation in vitro, and allows the identification and isolation of several populations of transcriptionally diverse OSCARhigh and OSCARlow intestinal epithelial cell states in vivo. In particular, this reporter is able to identify a dormant OSCARhigh cell population in the small intestine. OSCAR therefore provides a tool for a better understanding of dormant stem cell biology.

Structure of TFIIK for phosphorylation of CTD of RNA polymerase II.

During transcription initiation, the general transcription factor TFIIH marks RNA polymerase II by phosphorylating Ser5 of the carboxyl-terminal domain (CTD) of Rpb1, which is followed by extensive modifications coupled to transcription elongation, mRNA processing, and histone dynamics. We have determined a 3.5-Å resolution cryo-electron microscopy (cryo-EM) structure of the TFIIH kinase module (TFIIK in yeast), which is composed of Kin28, Ccl1, and Tfb3, yeast homologs of CDK7, cyclin H, and MAT1, respectively. The carboxyl-terminal region of Tfb3 was lying at the edge of catalytic cleft of Kin28, where a conserved Tfb3 helix served to stabilize the activation loop in its active conformation. By combining the structure of TFIIK with the previous cryo-EM structure of the preinitiation complex, we extend the previously proposed model of the CTD path to the active site of TFIIK.

Bromodomain protein BRD4 is an epigenetic activator of B7-H6 expression in acute myeloid leukemia

ABSTRACT B7-H6, a ligand for the NK activating receptor NKp30, has been identified as a biomarker of poor prognosis in several solid cancers. However, little is known about the role of B7-H6 and the mechanisms that control its expression in acute myeloid leukemia (AML). Epigenome modulation, including epigenomic reader dysregulation, is one of the hallmarks of AML. Bromodomain-containing protein 4 (BRD4), the best-known member of the BET family of epigenetic readers, is overexpressed in AML cells and regulates the transcription of genes involved in the pathogenesis of AML, as MYC oncogene. Here, we analyze the role of BRD4 in regulating B7-H6 in AML cells. Results demonstrated that the specific inhibition of BRD4 drastically reduces the expression of B7-H6 in AML cells. Histone acetylation mediated by CBP30/P300 facilitates the binding of BRD4 to the B7-H6 promoter, which recruits the P-TEFb elongation factor that phosphorylates RNA polymerase II, thereby activating B7-H6 transcription. BRD4 also co-bounded with JMJD6 at the distal enhancer of the B7-H6 gene. Metabolic modulation with metformin modifies the acetylation pattern in the B7-H6 promoter, impairing BRD4 binding, thereby inhibiting B7-H6 expression. B7-H6 knockdown induces the apoptosis in HEL-R cell line. Moreover, a high level of B7-H6 expression in AML patients is related to increased BRD4 levels, myelodysplastic-derived AML, and del5q, the two latter being associated with poor prognosis. Our data show that BRD4 is a positive regulator of the pro-tumorigenic molecule B7-H6 and that the blockage of the B7-H6 is a potential therapeutic target for the treatment of AML.

The poly(ADP-ribosyl)ation of BRD4 mediated by PARP1 promoted pathological cardiac hypertrophy

The bromodomain and extraterminal (BET) family member BRD4 is pivotal in the pathogenesis of cardiac hypertrophy. BRD4 induces hypertrophic gene expression by binding to the acetylated chromatin, facilitating the phosphorylation of RNA polymerases II (Pol II) and leading to transcription elongation. The present study identified a novel post-translational modification of BRD4: poly(ADP-ribosyl)ation (PARylation), that was mediated by poly(ADP-ribose)polymerase-1 (PARP1) in cardiac hypertrophy. BRD4 silencing or BET inhibitors JQ1 and MS417 prevented cardiac hypertrophic responses induced by isoproterenol (ISO), whereas overexpression of BRD4 promoted cardiac hypertrophy, confirming the critical role of BRD4 in pathological cardiac hypertrophy. PARP1 was activated in ISO-induced cardiac hypertrophy and facilitated the development of cardiac hypertrophy. BRD4 was involved in the prohypertrophic effect of PARP1, as implied by the observations that BRD4 inhibition or silencing reversed PARP1-induced hypertrophic responses, and that BRD4 overexpression suppressed the anti-hypertrophic effect of PARP1 inhibitors. Interactions of BRD4 and PARP1 were observed by co-immunoprecipitation and immunofluorescence. PARylation of BRD4 induced by PARP1 was investigated by PARylation assays. In response to hypertrophic stimuli like ISO, PARylation level of BRD4 was elevated, along with enhanced interactions between BRD4 and PARP1. By investigating the PARylation of truncation mutants of BRD4, the C-terminal domain (CTD) was identified as the PARylation modification sites of BRD4. PARylation of BRD4 facilitated its binding to the transcription start sites (TSS) of hypertrophic genes, resulting in enhanced phosphorylation of RNA Pol II and transcription activation of hypertrophic genes. The present findings suggest that strategies targeting inhibition of PARP1-BRD4 might have therapeutic potential for pathological cardiac hypertrophy.

Non-epigenetic induction of HEXIM1 by DNMT1 inhibitors and functional relevance

We have been studying the role of Hexamethylene bisacetamide (HMBA) Induced Protein 1 (HEXIM1) as a tumor suppressor whose expression is decreased in breast and prostate cancer. The anti-cancer actions of HEXIM1 in melanomas and AML have been reported by other groups. Previous studies have shown that 5-Aza-2′deoxycytidine (5-AzadC), a DNMT1 inhibitor, induces re-expression of tumor suppressor genes by removing/erasing methylation marks from their promoters. Our studies highlighted another mechanism wherein 5-AzadC induced DNA damage, which then resulted in enhanced occupancy of NF-ĸB, P-TEFb, and serine 2 phosphorylated RNA Polymerase II on the HEXIM1 gene. As a consequence, 5-AzadC induced HEXIM1 expression in prostate cancer cell lines and triple negative breast cancers. 5-AzadC-induced DNA damage enhanced P-TEFb occupancy via a mechanism that involved activation of ATR and ATM and induction of NF-ĸB recruitment to the HEXIM1 promoter. Downregulation of NF-ĸB attenuated 5-AzadC-induced HEXIM1 expression in prostate and breast cancer cells. The functional relevance of 5-AzadC-induced HEXIM1 expression is revealed by studies showing the HEXIM1 is required for the induction of apoptosis. Collectively, our findings support a non-epigenetic mechanism for 5-AzadC-induced re-expression of HEXIM1 protein, and may contribute to the clinical efficacy of 5-AzadC.

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

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

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

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

BRD4 Prevents R-Loop Formation and Transcription-Replication Conflicts by Ensuring Efficient Transcription Elongation

Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintaining genomic integrity and cell survival. Dysregulation of these systems can lead to conflicts between the transcription and replication machinery, causing DNA damage and cell death. BRD4 allows efficient transcriptional elongation by stimulating phosphorylation of RNA polymerase II (RNAPII). We report that bromodomain and extra-terminal domain (BET) protein loss of function (LOF) causes RNAPII pausing on the chromatin and DNA damage affecting cells in S-phase. This persistent RNAPII-dependent pausing leads to an accumulation of RNA:DNA hybrids (R-loops) at sites of BRD4 occupancy, leading to transcription-replication conflicts (TRCs), DNA damage, and cell death. Finally, our data show that the BRD4 C-terminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET protein LOF.

Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency.

A leading pharmacological strategy toward HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.

Cyclin-dependent kinase 12 (CDK12) in chordoma: prognostic and therapeutic value.

To determine the cyclin-dependent kinase 12 (CDK12) expression in chordoma patient tissues and cell lines, its correlation with oncologic outcomes, and its function in chordoma cell proliferation. A chordoma tissue microarray was constructed from fifty-six patient specimens and examined by immunohistochemistry to measure CDK12 expression and its correlation to patient clinical characteristics and survival. CDK12 expression in chordoma cell lines and patient tissues was evaluated via western blot. CDK12 specific small interfering RNA (siRNA) was applied to determine whether its inhibition attenuated chordoma cell growth and proliferation. CDK12 was expressed in the majority of chordoma specimens, with notably higher expression in patients with recurrent or metastatic disease. High CDK12 expression was an independent prognostic predictor for shorter overall and progression-free survival in chordoma by univariate and multivariate analysis. Western blot analysis revealed that CDK12 was also highly expressed in chordoma cell lines, with CDK12 specific small interfering RNA (siRNA) mediated knockdown decreasing proliferation and inducing apoptosis. Mechanistically, inhibition of CDK12 decreased phosphorylation of RNA polymerase II (RNAP II) and the anti-apoptotic proteins Survivin and Mcl-1. High expression of CDK12 is an independent predictor of poor prognosis in chordoma. Inhibition of CDK12 significantly decreased chordoma cell proliferation and induced apoptosis. Our results support CDK12 as a novel prognostic biomarker and therapeutic target in chordoma.

Abstract B17: MEK inhibition induces myogenic differentiation in RAS-driven rhabdomyosarcoma

Abstract Fusion-negative rhabdomyosarcoma (FN-RMS), which lacks PAX3/7 gene rearrangement, arises from skeletal muscle precursor cells that fail to differentiate despite expression of the myogenic master transcription factor, MYOD1. These tumors frequently harbor mutations in RAS isoforms (NRAS, HRAS, or KRAS), but the role of RAS in blocking myogenic differentiation is incompletely understood. In this study, we used a combination of high-throughput drug screening, transcriptomics, and epigenomics approaches to investigate the role of RAS in FN-RMS differentiation and survival. Oncogenic RAS was required for FN-RMS survival and activated the MAPK pathway to block myoblast differentiation. Consistent with these findings, the MEK inhibitor, trametinib, selectively reduced FN-RMS cell viability; upregulated the prodifferentiation myogenic transcription factor, MYOG; and induced myogenic differentiation. Mechanistically, we found that ERK2, a downstream target of MEK, bound to myogenic differentiation genes, including the promoter of MYOG, where it phosphorylated RNA polymerase II, resulting in RNA polymerase II stalling and transcriptional repression. MEK inhibition resulted in release of ERK2 from the MYOG promoter, facilitating MYOG transcription. Accordingly, trametinib treatment also resulted in MYOG-dependent chromatin remodeling, leading to the establishment of super-enhancers at genes required for late myogenic differentiation (including MYH3) and the loss of RAS-dependent super-enhancers at proliferation genes, such as MYC. In vivo, MEK inhibition induced myogenic differentiation FN-RMS cells to suppress their growth as xenografts. We then performed a combinatorial drug screen and identified combinations that might improve the therapeutic efficacy of trametinib. Excitingly, the most synergistic combination in vitro, trametinib and the multikinase inhibitor, BMS-754807, also induced tumor regression in mouse xenograft models of FN- RMS. Synergy was similarly observed between trametinib and the IGF1R monoclonal antibody, ganitumab, establishing the combination of MEK and IGF1R inhibition as synergistic in FN-RMS. Therefore, in addition to uncovering a mechanism by which RAS signaling suppresses MYOG expression to block MYOG-dependent chromatin remodeling and cellular differentiation in FN-RMS, these findings suggest that patients with FN-RMS may benefit from combination therapy with MEK and IGF1R inhibitors. Citation Format: Marielle E. Yohe, Berkley E. Gryder, Hsien-Chao Chou, Young K. Song, Xiaohu Zhang, Donna Butcher, Kristine A. Isanogle, Christina M. Robinson, Xiaoling Luo, Jin-Qiu Chen, Elijah J. Edmondson, Simone Difilippantionio, Craig J. Thomas, Javed Khan. MEK inhibition induces myogenic differentiation in RAS-driven rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B17.

Thirdhand smoke exposure causes replication stress and impaired transcription in human lung cells.

Thirdhand cigarette smoke (THS) is a newly described toxin that lingers in the indoor environment long after cigarettes have been extinguished. Emerging results from both cellular and animal model studies suggest that THS is a potential human health hazard. DNA damage derived from THS exposure could have genotoxic consequences that would lead to the development of diseases. However, THS exposure-induced interference with fundamental DNA transactions such as replication and transcription, and the role of DNA repair in ameliorating such effects, remain unexplored. Here, we found that THS exposure increased the percentage of cells in S-phase, suggesting impaired S-phase progression. Key DNA damage response proteins including RPA, ATR, ATM, CHK1, and BRCA1 were activated in lung cells exposed to THS, consistent with replication stress. In addition, THS exposure caused increased 53BP1 foci, indicating DNA double-strand break induction. Consistent with these results, we observed increased micronuclei formation, a marker of genomic instability, in THS-exposed cells. Exposure to THS also caused a significant increase in phosphorylated RNA Polymerase II engaged in transcription elongation, suggesting an increase in transcription-blocking lesions. In agreement with this conclusion, ongoing RNA synthesis was very significantly reduced by THS exposure. Loss of nucleotide excision repair exacerbated the reduction in RNA synthesis, suggesting that bulky DNA adducts formed by THS are blocks to transcription. The adverse impact on both replication and transcription supports genotoxic stress as a result of THS exposure, with important implications for both cancer and other diseases.

NME2 Is a Master Suppressor of Apoptosis in Gastric Cancer Cells via Transcriptional Regulation of miR-100 and Other Survival Factors.

Tumorigenesis is a result of uncontrollable cell proliferation which is regulated by a variety of complex factors including miRNAs. The initiation and progression of cancer are always accompanied by the dysregulation of miRNAs. However, the underlying mechanism of miRNA dysregulation in cancers is still largely unknown. Herein we found that miR-100 was inordinately upregulated in the sera of patients with gastric cancer, indicating that miR-100 might emerge as a biomarker for the clinical diagnosis of cancer. The abnormal expression of miR-100 in gastric cancer cells was mediated by a novel transcription factor NME2 (NME/NM23 nucleoside diphosphate kinase 2). Further data revealed that the transcription factor NME2 could promote the transcriptions of antiapoptotic genes including miRNA (i.e., miR-100) and protein-encoding genes (RIPK1, STARD5, and LIMS1) through interacting with RNA polymerase II and RNA polymerase II-associated protein 2 to mediate the phosphorylation of RNA polymerase II C-terminal domain at the 5th serine, leading to the suppression of apoptosis of gastric cancer cells both in vitro and in vivo. In this context, our study revealed that the transcription factor NME2 is a master suppressor for apoptosis of gastric cancer cells. IMPLICATIONS: Our study contributed novel insights into the mechanism involved in the expression regulation of apoptosis-associated genes and provided a potential biomarker of gastric cancer.