AT7519, a Potent Multi-Targeted CDK Inhibitor, Is Active in CLL Patient Samples Independent of Stage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3161-3161
Author(s):  
Vicky Lock ◽  
Laurence Cooke ◽  
Murray Yule ◽  
Neil T Thompson ◽  
K. Della Croce ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
B Cell ◽  
Rna Polymerase Ii ◽  
Parp Cleavage ◽  
Regulation Of Transcription ◽  
Cytotoxic Effects ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Cyclin Dependent Kinases

Abstract Cyclin Dependent Kinases (CDKs) play a central role in the eukaryotic cell cycle. The activation of these kinases is modulated by the expression and binding of their regulatory cyclin partners. Their key role in cell cycle progression, coupled to evidence that pathways leading to their activation are deregulated in a number of human cancers makes them attractive therapeutic targets. More recently the role of CDKs 7, 8 and 9 in the regulation of transcription has been explored. CDK9 has been shown to play a role in the regulation of transcription via phosphorylation of RNA polymerase II (RNA pol II). The outcome of transcriptional inhibition via CDK9 exhibits significant variation between cell lines. B-Cell lymphoproliferative disorders, including CLL, rely on the expression of transcripts with a short half-life such as Mcl-1, Bcl-2 and XIAP for survival. In vitro studies have demonstrated that compounds with transcriptional inhibitory effects are effective pro-apoptotic agents in models of this disease. AT7519 is a potent inhibitor of cyclin dependent kinases 1, 2 and 9 and is currently in early phase clinical development. These studies profile the mechanism of action of AT7519 on CLL cells isolated from patients. Primary cell samples were isolated from a total of 15 patients with CLL with various stages of disease (8 Stage 0, 0/I or II and 7 Stage IV) and who were either treatment naïve or had received a variety of prior therapies. Patient samples were characterised for cytogenetic abnormalities (11q, 17p and 13q deletion or trisomy 12) as well IgVH mutation and ZAP70 expression. AT7519 was shown to induce apoptosis (by MTS, morphology and PARP cleavage) in these samples at concentrations of 100–700nM. AT7519 appears equally effective at inhibiting the survival of CLL cells harbouring a variety of mutations including those representative of patients that fall within poorer prognosis treatment groups. The amount of AT7519 required to induce cell death in 50% of the CLL cell population increased as exposure time was decreased but significant cell death was obtained at doses approximating to 1uM following 4–6h of treatment. These doses are equivalent to exposures achieved in ongoing AT7519 clinical studies indicating that cytotoxic doses can be achieved in patients on well tolerated schedules. The mechanism of AT7519 cytotoxic effects was investigated by western blotting for a variety of cell cycle and apoptotic markers following incubation with compound. Short term treatments (4–6h) resulted in inhibition of phosphorylation of the transcriptional marker RNA pol II and the downregulation of the anti-apoptotic protein Mcl-1. Additional antiapoptotic proteins including XIAP and Bcl-2 remained unchanged. The reduction in Mcl-1 protein levels was associated with an increase in the apoptotic marker cleaved PARP. No inhibition of cell cycle markers such as phospho-retinoblastoma protein was observed in the same samples suggesting that the cytotoxic effects of AT7519 in CLL patient samples is due to its transcriptional activity alone. Together the data suggest AT7519 offers a promising treatment strategy for patients with advanced B-cell leukemia and lymphoma.

AT7519, a Potent CDK Inhibitor, Is Active in Leukemia Models and Primary CLL Patient Samples.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3127-3127 ◽  
Author(s):  
Matthew S. Squires ◽  
Ruth E. Feltell ◽  
Victoria Lock ◽  
Donna M. Smith ◽  
Jon E. Lewis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Half Life ◽  
Lymphoproliferative Disorders ◽  
Regulation Of Transcription ◽  
Cure Rate ◽  
Cyclin Dependent Kinases ◽  
Transcriptional Inhibition ◽  
Induction Of Apoptosis

Abstract Cyclin Dependent Kinases (CDKs) play a central role in the eukaryotic cell cycle. The activation of these kinases is modulated by the expression and binding of their regulatory cyclin partners. Their key role in cell cycle progression, coupled to evidence that pathways leading to their activation are deregulated in a number of human cancers makes them attractive therapeutic targets. More recently the role of CDKs 7, 8 and 9 in the regulation of transcription has been explored. CDK9 has been shown to play a role in the regulation of transcription via phosphorylation of RNA polymerase II. The outcome of transcriptional inhibition via CDK9 exhibits significant variation between cell lines. Many leukemic cell lines, which are dependent upon the expression of short half-life transcripts such as Mcl-1 for survival, undergo apoptosis following transcriptional inhibition and the cell cycle effects of such inhibitors are masked. AT7519 is a potent inhibitor of cyclin dependent kinases 1, 2 and 9 and is currently in early phase clinical development. These studies profile the mechanism of action of AT7519 in leukemia cell lines (HL60 and MOLT-4). AT7519 causes rapid induction of apoptosis (within 6 hours of initial exposure) in the absence of cell cycle arrest. This induction of apoptosis occurs in parallel with a reduction in the levels of anti-apoptotic proteins such as Mcl-1. In HL60 xenograft models anti-tumour efficacy is observed following 2 cycles of daily dosing of 15 or 10mg/kg for 5 days followed by 2 days off treatment. We observe a 50% cure rate (4/8 mice) at the 15mg/kg dose and a 30% cure rate at the 10mg/kg dose level 40 days following dosing. Pharmacodynamic biomarker studies demonstrate that a greater extent and duration of Mcl-1 knockdown and apoptosis induction are associated with efficacious doses. B-Cell lymphoproliferative disorders, including CLL, rely on the expression of transcripts with a short half-life such as Mcl-1, Bcl-2 and XIAP for survival. In vitro studies have demonstrated that compounds with transcriptional inhibitory effects are effective pro-apoptotic agents in models of this disease. Therefore, we also characterised the transcriptional effects of AT7519 on CLL cells isolated from patients. AT7519 was shown to induce apoptosis (by MTS, morphology and PARP cleavage) in these samples at concentrations of 100–300nM. These effects were correlated with the cytogenetic background of individual patients and the data supports further clinical investigation of AT7519 in B-Cell lymphoproliferative disorders where survival proteins play a pivotal role.

scholarly journals Cyclin-Dependent Kinases and CTD Phosphatases in Cell Cycle Transcriptional Control: Conservation across Eukaryotic Kingdoms and Uniqueness to Plants

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 279
Author(s):  
Zhi-Liang Zheng
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Large Scale ◽  
Transcriptional Control ◽  
Cell Cycle Control ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pol Ii ◽  
Precise Control ◽  
Cyclin Dependent Kinases ◽  
Entire Cell

Cell cycle control is vital for cell proliferation in all eukaryotic organisms. The entire cell cycle can be conceptually separated into four distinct phases, Gap 1 (G1), DNA synthesis (S), G2, and mitosis (M), which progress sequentially. The precise control of transcription, in particular, at the G1 to S and G2 to M transitions, is crucial for the synthesis of many phase-specific proteins, to ensure orderly progression throughout the cell cycle. This mini-review highlights highly conserved transcriptional regulators that are shared in budding yeast (Saccharomyces cerevisiae), Arabidopsis thaliana model plant, and humans, which have been separated for more than a billion years of evolution. These include structurally and/or functionally conserved regulators cyclin-dependent kinases (CDKs), RNA polymerase II C-terminal domain (CTD) phosphatases, and the classical versus shortcut models of Pol II transcriptional control. A few of CDKs and CTD phosphatases counteract to control the Pol II CTD Ser phosphorylation codes and are considered critical regulators of Pol II transcriptional process from initiation to elongation and termination. The functions of plant-unique CDKs and CTD phosphatases in relation to cell division are also briefly summarized. Future studies towards testing a cooperative transcriptional mechanism, which is proposed here and involves sequence-specific transcription factors and the shortcut model of Pol II CTD code modulation, across the three eukaryotic kingdoms will reveal how individual organisms achieve the most productive, large-scale transcription of phase-specific genes required for orderly progression throughout the entire cell cycle.

scholarly journals Mechanism of RNA polymerase II stalling by DNA alkylation

2017 ◽  
Vol 114 (46) ◽  
pp. 12172-12177 ◽  
Author(s):  
Stefano Malvezzi ◽  
Lucas Farnung ◽  
Claudia M. N. Aloisi ◽  
Todor Angelov ◽  
Patrick Cramer ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Anticancer Agents ◽  
Excision Repair ◽  
Minor Groove ◽  
Dna Alkylation ◽  
Drug Induced ◽  
Rna Pol Ii ◽  
Pol Ii

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

scholarly journals The cryoelectron microscopy structure of the human CDK-activating kinase

2020 ◽  
Vol 117 (37) ◽  
pp. 22849-22857 ◽  
Author(s):  
Basil J. Greber ◽  
Juan M. Perez-Bertoldi ◽  
Kif Lim ◽  
Anthony T. Iavarone ◽  
Daniel B. Toso ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rational Design ◽  
Control Cell ◽  
3D Structure ◽  
Structural Basis ◽  
Pol Ii ◽  
Insight Into ◽  
Cdk Activating Kinase

The human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II (Pol II) subunit RPB1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell cycle progression. Here, we have determined the three-dimensional (3D) structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and to aid in the rational design of therapeutic compounds.

scholarly journals B Lymphocytes Differentially Use the Rel and Nuclear Factor κB1 (NF-κB1) Transcription Factors to Regulate Cell Cycle Progression and Apoptosis in Quiescent and Mitogen-activated Cells

1998 ◽  
Vol 187 (5) ◽  
pp. 663-674 ◽  
Author(s):  
Raelene J. Grumont ◽  
Ian J. Rourke ◽  
Lorraine A. O'Reilly ◽  
Andreas Strasser ◽  
Kensuke Miyake ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Cycle Progression ◽  
Nuclear Factor ◽  
Cycle Progression ◽  
Regulate Cell Cycle Progression

Rel and nuclear factor (NF)-κB1, two members of the Rel/NF-κB transcription factor family, are essential for mitogen-induced B cell proliferation. Using mice with inactivated Rel or NF-κB1 genes, we show that these transcription factors differentially regulate cell cycle progression and apoptosis in B lymphocytes. Consistent with an increased rate of mature B cell turnover in naive nfkb1−/− mice, the level of apoptosis in cultures of quiescent nfkb1−/−, but not c-rel−/−, B cells is higher. The failure of c-rel−/− or nfkb1−/− B cells to proliferate in response to particular mitogens coincides with a cell cycle block early in G1 and elevated cell death. Expression of a bcl-2 transgene prevents apoptosis in resting and activated c-rel−/− and nfkb1−/− B cells, but does not overcome the block in cell cycle progression, suggesting that the impaired proliferation is not simply a consequence of apoptosis and that Rel/NF-κB proteins regulate cell survival and cell cycle control through independent mechanisms. In contrast to certain B lymphoma cell lines in which mitogen-induced cell death can result from Rel/NF-κB–dependent downregulation of c-myc, expression of c-myc is normal in resting and stimulated c-rel−/− B cells, indicating that target gene(s) regulated by Rel that are important for preventing apoptosis may differ in normal and immortalized B cells. Collectively, these results are the first to demonstrate that in normal B cells, NF-κB1 regulates survival of cells in G0, whereas mitogenic activation induced by distinct stimuli requires different Rel/NF-κB factors to control cell cycle progression and prevent apoptosis.

A Phase 1 Trial of SNS-032, a Potent and Specific CDK 2, 7 and 9 Inhibitor, in Chronic Lymphocytic Leukemia and Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3178-3178 ◽  
Author(s):  
William G. Wierda ◽  
R. Chen ◽  
William Plunkett ◽  
Steven Coutre ◽  
Ashraf Z. Badros ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Chronic Lymphocytic Leukemia ◽  
Dose Escalation ◽  
Phase 1 ◽  
Preliminary Evidence ◽  
Lymphocytic Leukemia ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Survival Factors

Abstract SNS-032, formerly BMS-387032, is a highly selective and potent inhibitor of cyclin-dependent kinases (CDK) 2, 7 and 9. CDK2 and CDK7 are involved in cell cycle regulation. CDK7, along with CDK9, regulate RNA polymerase (Pol) II-dependent transcription. Temporary inhibition of RNA Pol II-dependent transcription by SNS-032 has significant effects on short half-life transcripts and proteins, particularly survival factors, cell cycle regulatory proteins, and cytokines that are critical for the survival of malignant B-cells in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). A phase 1 dose-escalation study in patients with MM and CLL is ongoing with separate dose escalations for each indication. The study is designed to evaluate safety, pharmacokinetics (PK) and preliminary evidence of activity of a loading dose (LD) followed by a 6 hour infusion of SNS-032 given weekly for 3 consecutive weeks of each 28-day cycle. Dose and schedule aim to maintain for 6 hours threshold plasma concentrations of 115 ng/mL (in vitro IC90) and higher. The study incorporates an exploratory analysis of potential pharmacodynamic (PD) biomarkers such as decreased phosphorylation of RNA Pol II C-terminal domain to demonstrate inhibition of CDK7 and CDK9, and decreased expression of survival factors to indicate transcriptional inhibition. Methods: Previously treated patients with advanced CLL or MM, measurable disease, and ECOG status 0–1 were eligible. Increasing doses of SNS-032 given as an LD followed by a 6 hr infusion were evaluated. The total starting dose was 15 mg/m2 comprised of a LD of 5 mg/m2 followed by 10 mg/m2 over 6 hr with dose escalation by modified Fibonacci. PD studies of target modulation were performed on peripheral blood mononuclear cells (PBMC) obtained pre- and post-dose. Direct target modulation or downstream effects of target inhibition were evaluated. Results: 35 patients have been treated to date, 18 MM patients and 17 CLL patients. Median age was 61 (range 45–82), with 13 females and 24 males. Median number of prior therapies was 5 (range: 1–11). MM patients have received total doses of 15 – 75 mg/m2. No drug-related dose limiting toxicities (DLTs) or objective responses have been reported thus far in MM. CLL patients have received total doses of 15 –100 mg/m2. No drug-related DLTs were observed through the 50 mg/m2 dose cohort. At 75 mg/m2, concentrations of SNS- 032 exceeded IC90 (mean maximum concentration during the 6 hr infusion was 261 ± 45 ng/mL). Evidence of biochemical tumor lysis syndrome (TLS) was observed in all CLL patients treated at this dose level. One patient experienced a DLT of vascular leak syndrome and failure to receive all 3 cycle 1 doses. One CLL patient has been treated thus far at 100 mg/m2. This patient experienced TLS with a DLT of elevated liver function enzymes for >48 hr and received only 2 of 3 doses in cycle 1. No objective responses have been observed. PD analyses showed evidence of decreased Mcl-1 or XIAP in several patients. Conclusions: The mechanism of action of SNS-032 supports testing this agent in B-cell malignancies such as MM and CLL. A pharmacologically-derived dose regimen that sustains IC90 SNS-032 concentrations or higher for 6 hr is being studied; target levels were achieved and exceeded in cohort 5 (75 mg/m2) for both MM and CLL. No DLTs or objective responses have been observed thus far in MM. AEs and DLTs related to mild to moderate TLS were observed in CLL patients at 75 mg/m2 and higher. No objective responses have been observed. Preliminary evidence of target-specific PD modulation has been demonstrated. Enrollment in this trial is continuing.

The Combination of Romidepsin and Bortezomib Results in Synergistic Induction of Apoptosis in Human B-Lymphoma Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1689-1689 ◽  
Author(s):  
Deshpande S. Deshpande ◽  
Mary Jo Lechowicz ◽  
Rajni Sinha ◽  
Jonathan L. Kaufman ◽  
Lawrence H. Boise ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Mtt Assay ◽  
Research Funding ◽  
Annexin V ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Sequence Specificity ◽  
B Cell Malignancies

Abstract Abstract 1689 Poster Board I-715 Introduction The use of the proteasome inhibitor bortezomib has demonstrated activity in multiple myeloma and lymphomas. The HDAC inhibitor romidepsin is being evaluated in CTCL and PTCL, though its activity in B-cell lymphomas is less clear. We hypothesized that the combination of bortezomib and romidepsin would result in synergistic apoptosis in different B-cell NHL cell lines based upon the observed activity of this combination in more mature B-cell malignancies such as myeloma. Experimental Design Daudi, HT, Ramos and SUDHL-4 cell lines were exposed to different concentrations of bortezomib and romidepsin, separately, concurrently, and sequentially. Cell viability was assessed using MTT-assay, induced apoptosis was evaluated using Annexin V and PI staining from 24-48 hours. Apoptosis was also evaluated using western blot analysis of caspases and PARP cleavage. LC3 and HDAC6 level expressions were performed to determine if the effect of the combination was a result of the aggresome or autophagy pathway. Cell cycle studies were also performed to study if there were any changes after treating cells with the combination. Results The combination of bortezomib and romidepsin resulted in synergistic B-cell apoptosis as measured by MTT-assay with combination indices of < 0.5. This was associated with increased caspases and PARP cleavage as early as 24 hours after exposure. Order of addition experiments demonstrated definite sequence specificity. When romidepsin was added first, and 6 hours later followed by bortezomib, apoptosis was enhanced, compared to both agents being given concurrently or when bortezomib was administered first. Cell cycle analysis studies demonstrated that pretreatment of cells with romidepsin for 6 hours followed by the addition of bortezomib arrested the cells in G2M phase. HDAC6 expression was significantly reduced following combination therapy, and LC3-I was cleaved to LC3-II in treated cells suggesting that the combination affected aggresome formation and autophagy. Conclusion The combination of romidepsin and bortezomib at low nanomolar concentrations suggests that this may be an important clinical combination to test in patients with relapsed or refractory B-cell malignancies. Sequence of administration data is currently being tested to determine if the effect is a result of autophagy inhibition as is seen in myeloma cell lines. Additional mechanistic studies will be presented with the goals of identifying predictors of response that can then be validated in prospective clinical trials. Disclosures Lechowicz: Gloucester: Consultancy. Kaufman:Millennium: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Gloucester: Research Funding; Novartis: Consultancy; BMS: Consultancy; Millennium: Consultancy, Research Funding; Celgene: Consultancy. Flowers:Millennium: Research Funding.

Disruption Of Super Enhancer-Driven Cancer Dependencies In Diffuse Large B-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3021-3021 ◽  
Author(s):  
Bjoern Chapuy ◽  
McKeown Michael ◽  
Charles Y. Lin ◽  
Stefano Monti ◽  
Margaretha GM Roemer ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Advisory Committees ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Dana Farber Cancer Institute ◽  
Super Enhancer ◽  
Equity Ownership

Abstract Diffuse large B-cell lymphoma (DLBCL) exhibits significant biological and transcriptional heterogeneity which is conferred, in part, by pathologic modulation of lineage-specific and growth-associated master regulatory transcription factors (TF). Chromatin associated with TF binding sites is markedly enriched in histone proteins that are post-translationally modified by lysine side-chain acetylation. This mark facilitates the opening of chromatin and recruits a class of co-activators which recognize ε-acetyl lysine through a bromodomain. The sub-family of bromodomain and extra-terminal domain (BET) co-activators (BRD2, BRD3 and BRD4) are appealing, in part, because transgenic expression of BRD2 caused a DLBCL-like neoplasm in mice. We recently developed the first BET inhibitor, JQ1, and now explore the role of BET bromodomains in oncogenic transcription and assess BET family members as therapeutic targets in DLBCL. Nanomolar doses of JQ1 and 3 structurally dissimilar BET bromodomain inhibitors decreased the cellular proliferation of a broad panel of DLBCL cell lines of all transcriptionally defined types whereas the inactive enantiomer, JQ1R, had no effect. BRD2 and BRD4 depletion similarly decreased the proliferation of multiple DLBCL cell lines. We next explored the therapeutic potential of BET inhibition in two independent DLBCL xenotransplantation models, Ly1 and Toledo. In the first xenograft model, JQ1-treated mice had a prolongation of overall survival (p = 0.003). In the second model, JQ1-treated animals had significantly delayed tumor progression and decreased lymphomatous infiltration of spleen and bone marrow. To define the transcriptional pathways regulated by BET bromodomain proteins, we performed transcriptional profiling of multiple vehicle and JQ1-treated DLBCL cell lines. Following JQ1 treatment, we observed downregulation of multiple MYD88/TLR and BCR signaling pathway components and functionally validated MYC and E2F target gene sets. BET inhibition decreased MYC transcripts and protein in the DLBCL cell line panel suggesting that BET bromodomains directly modulate MYC transcription. In contrast, JQ1 treatment did not measurably alter E2F1 transcript or protein abundance suggesting a co-activator role of the BET bromodomains for E2F1. To explore the role of BET bromodomains in oncogenic E2F1 transcriptional signaling, we performed ChIPSeq experiments in Ly1 cells, using a chemical genetic approach. Rank-ordering of all transcriptionally active promoters based on H3K4me3 enrichment and RNA Pol II occupancy identifies pervasive binding and spatial colocalization of BRD4 and E2F1 to active promoter elements. We identified a JQ1-mediated transcriptional elongation defect across E2F1-bound promoters, responsible for the downregulation of E2F1 targets. As oncogenic TFs may signal to RNA Pol II through distal enhancer elements, we also characterized the genome-wide localization of BRD4 to enhancers in the Ly1 DLBCL cell line. Rank-ordering of enhancer regions by H3K27ac enrichment reveals that BRD4 binds to the vast majority of active enhancers in the Ly1 genome. Strikingly, the BRD4 load is asymmetrically distributed throughout the genome at enhancer sites with only a small subset of BRD-loaded “super enhancers (SE)”, 285/18330 (1.6%), accounting for 32% of all BRD4 enhancer binding in the cell. The POU2AF1 locus emerged as the most BRD4-overloaded enhancer in Ly1. BET inhibition reduced RNA Pol II elongation of POU2AF1, with a concomitant increase in promoter-paused RNA Pol II near the transcriptional start site. Accordingly, JQ1 treatment decreased POU2AF1 transcript abundance and protein expression and reduced the expression of a POU2AF1 target gene set. POU2AF1 depletion with independent shRNAs significantly decreased the proliferation of Ly1 and enforced POU2AF1 expression decreased the sensitivity of Ly1 cells to JQ1 treatment. Additional super enhancer-driven genes that were sensitive to JQ1 treatment include ones which promote and maintain the B-cell gene expression program and limit plasma cell differentiation. Our data suggest that BET inhibition limits the growth of DLBCLs by at least two complementary activities: a specific effect on genes that define a given cell type by high BRD4 loading at enhancers and the selective suppression of transcription at E2F- and MYC- driven target genes. + Contributed equally Disclosures: Qi: Patent for JQ1: holds patent for JQ1, holds patent for JQ1 Patents & Royalties. Young:Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Enzon Pharmaceuticals: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Bradner:Tensha Therapeutics: Equity Ownership, Scientific founder of Tensha which is translating drug-like derivatives of the JQ1 chemical probe of BET bromodomains used in this study, as cancer therpeutics. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity. Other; Syros Pharmaceuticals: Equity Ownership, Scientific founder of Syros which is discovering Super Enhancers as a new class of gene control elements. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity., Scientific founder of Syros which is discovering Super Enhancers as a new class of gene control elements. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity. Other.

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