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Cholangiocarcinoma (CCA) is a fatal disease with a 5-year survival of

Targeting BET bromodomain proteins using small molecules is an emerging anticancer strategy with clinical evaluation of at least six inhibitors now underway. Although MYC downregulation was initially proposed as a key mechanistic property of BET inhibitors, recent evidence suggests that additional antitumor activities are important. Using the Eμ-Myc model of B-cell lymphoma, we demonstrate that BET inhibition with JQ1 is a potent inducer of p53-independent apoptosis that occurs in the absence of effects on Myc gene expression. JQ1 skews the expression of proapoptotic (Bim) and antiapoptotic (BCL-2/BCL-xL) BCL-2 family members to directly engage the mitochondrial apoptotic pathway. Consistent with this, Bim knockout or Bcl-2 overexpression inhibited apoptosis induction by JQ1. We identified lymphomas that were either intrinsically resistant to JQ1-mediated death or acquired resistance following in vivo exposure. Strikingly, in both instances BCL-2 was strongly upregulated and was concomitant with activation of RAS pathways. Eμ-Myc lymphomas engineered to express activated Nras upregulated BCL-2 and acquired a JQ1 resistance phenotype. These studies provide important information on mechanisms of apoptosis induction and resistance to BET-inhibition, while providing further rationale for the translation of BET inhibitors in aggressive B-cell lymphomas. Mol Cancer Ther; 15(9); 2030–41. ©2016 AACR.

Recently, treatment with bromodomain and extraterminal protein antagonist (BA) such as JQ1 has been shown to inhibit growth and induce apoptosis of human acute myelogenous leukemia (AML) cells, including those expressing FLT3-ITD. Here, we demonstrate that cotreatment with JQ1 and the FLT3 tyrosine kinase inhibitor (TKI) ponatinib or AC220 synergistically induce apoptosis of cultured and primary CD34+ human AML blast progenitor cells (BPC) expressing FLT3-ITD. Concomitantly, as compared with each agent alone, cotreatment with JQ1 and the FLT3-TKI caused greater attenuation of c-MYC, BCL2, and CDK4/6. Simultaneously, cotreatment with JQ1 and the FLT3-TKI increased the levels of p21, BIM, and cleaved PARP, as well as mediated marked attenuation of p-STAT5, p-AKT, and p-ERK1/2 levels in AML BPCs. Conversely, cotreatment with JQ1 and FLT3-TKI was significantly less active against CD34+ normal bone marrow progenitor cells. Knockdown of BRD4 by short hairpin RNA also sensitized AML cells to FLT3-TKI. JQ1 treatment induced apoptosis of mouse Ba/F3 cells ectopically expressing FLT3-ITD with or without FLT3-TKI–resistant mutations F691L and D835V. Compared with the parental human AML FLT3-ITD–expressing MOLM13, MOLM13-TKIR cells resistant to AC220 were markedly more sensitive to JQ1-induced apoptosis. Furthermore, cotreatment with JQ1 and the pan-histone deacetylase inhibitor (HDI) panobinostat synergistically induced apoptosis of FLT3-TKI–resistant MOLM13-TKIR and MV4-11-TKIR cells. Collectively, these findings support the rationale for determining the in vivo activity of combined therapy with BA and FLT3-TKI against human AML cells expressing FLT3-ITD or with BA and HDI against AML cells resistant to FLT3-TKI. Mol Cancer Ther; 13(10); 2315–27. ©2014 AACR.

The bromodomain and extra-terminal (BET) protein family members, including BRD4, bind to acetylated lysines on histones and regulate the expression of important oncogenes, for example, c-MYC and BCL2. Here, we demonstrate the sensitizing effects of the histone hyperacetylation-inducing pan–histone deacetylase (HDAC) inhibitor panobinostat on human acute myelogenous leukemia (AML) blast progenitor cells (BPC) to the BET protein antagonist JQ1. Treatment with JQ1, but not its inactive enantiomer (R-JQ1), was highly lethal against AML BPCs expressing mutant NPM1c+ with or without coexpression of FLT3-ITD or AML expressing mixed lineage leukemia fusion oncoprotein. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of c-MYC and BCL2 and reduced their levels in the AML cells. Cotreatment with JQ1 and the HDAC inhibitor panobinostat synergistically induced apoptosis of the AML BPCs, but not of normal CD34+ hematopoietic progenitor cells. This was associated with greater attenuation of c-MYC and BCL2, while increasing p21, BIM, and cleaved PARP levels in the AML BPCs. Cotreatment with JQ1 and panobinostat significantly improved the survival of the NOD/SCID mice engrafted with OCI-AML3 or MOLM13 cells ( P < 0.01). These findings highlight cotreatment with a BRD4 antagonist and an HDAC inhibitor as a potentially efficacious therapy of AML. Mol Cancer Ther; 13(5); 1142–54. ©2014 AACR . See related article by Liu et al., [p. 1194][1] [1]: /lookup/volpage/13/1194?iss=5

Introduction: The chemical probe JQ-1 is a thienodiazepine BET-bromodomain inhibitor targeting BRD4 with previously reported efficacy in a patient-derived xenograft mouse model of NMC. TEN-010 is structurally related to JQ1 with superior chemical and biological properties currently under clinical study study solid tumors, including NMC, and hematologic malignancies. NMC is a squamous cell carcinoma commonly observed in midline structures in the lung and mediastinum, and commonly involves a t(15:19) chromosomal translocation encoding a chimeric BRD4-NUT fusion protein. This rare, with estimates of less than 100 people in the United States, disease is typically unresectable, poorly responsive to chemotherapies and clinically aggressive with median survival of ∼ 6 months establishing an unmet need for targeted therapy. Design: A Phase 1 dose escalation “3+3” multi-center study is being conducted in adults with advanced solid tumors. A separate sub-study enrolls patients with NMC at the highest tolerated dose level at the time of patient screening. The NMC patients received subcutaneous daily dosing of TEN-010 for three weeks in a four-week cycle. Patients must have documented NMC by FISH or IHC and cannot not be on chemotherapy at time of treatment. Patients are monitored for safety, pharmacokinetics and pharmacodynamics measured at initial dose and at steady-state, and assessment of anti-tumor activity assessed by RECIST 1.1. (CT) or PET/CT. Pharmacodynamics use a peripheral blood bioassay serially examining systemic integrin expression with TEN-010 dosing. Results: Data are available for the three NMC patients; 1 patient received TEN-010 at 0.1 mg/kg and 2 received 0.45 mg/kg. The 0.1 mg/kg patient had disease progression after 2 weeks. Both patients at the 0.45 mg/kg dose had clinical responses. One patient exhibited a 30% and 50% NMC tumor regression after cycles 1 and 2, respectively. This patient demonstrated rapid symptomatic improvement within two weeks, and remains on therapy into Cycle 3. The other patient exhibited a reduction of ∼50% summed SUV(max) by PET/CT with symptomatic improvement evident after three weeks of therapy during Cycle 1. This patient received two cycles of therapy before having disease progression. Plasma LDH in the 0.45 mg/kg patients, but not the 0.1 mg/kg patient, decreased after one week on treatment; the first 0.45 mg/kg pt had normal LDH through Cycle 2 with continued values in the normal range. On-target pharmacodynamic activity corroborated LDH response. This regimen has been tolerated with grade 1 irritation of the injection site and mild/moderate increases in indirect bilirubin and anorexia. All adverse events have been reversible. Discussion: This is the first documented partial response in NMC using a BET inhibitor. Reduced metabolic activity and clinical responses also are observed Overall the results serve as proof of concept and validate pre-clinical xenograft studies. Further testing and exploration of the optimal dosing regimen are on-going. The results support the promise for TEN-010 as an important new needed treatment for NMC. Citation Format: Geoffrey I. Shapiro, Afshin Dowlati, Patricia M. LoRusso, Joseph P. Eder, Adrienne Anderson, Khanh T. Do, Michael H. Kagey, Cynthia Sirard, James E. Bradner, Steven B. Landau. Clinically efficacy of the BET bromodomain inhibitor TEN-010 in an open-label substudy with patients with documented NUT-midline carcinoma (NMC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A49.

Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is currently lacking. Using H3K27ac and BRD4 ChIP-Seq, coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors responsible for subgroup divergence that validated by ChIP-Seq and implicated candidate cells-of-origin for Group 4. Our integrated analysis of cis-regulatory elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins. Citation Format: Charles Y. Lin, Serap Erkek, Yiai Tong, Linlin Yang, Alexander J. Federation, Marc Zapatka, Parthiv Haldipur, Daisuke Kawauchi, Thomas Risch, Hans-Jörg Warnatz, Barbara Worst, Bensheng Ju, Brent A. Orr, Rhamy Zeid, Donald R. Polaski, Maia Segura-Wang, Sebastian M. Waszak, David TW Jones, Marcel Kool, Volker Hovestadt, Ivo Buchhalter, Laura Sieber, Pascal Johann, Stefan Gröschel, Marina Ryzhova, Andrey Korshunov, Wenbiao Chen, Victor V. Chizhikov, Kathleen J. Millen, Vyacheslav Amstislavskiy, Hans Lehrach, Marie-Laure Yaspo, Roland Eils, Peter Lichter, Jan O. Korbel, Stefan Pfister, James E. Bradner, Paul A. Northcott. Medulloblastoma regulatory circuitries reveal subgroup-specific cellular origins. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B23.

The BET (bromodomain and extraterminal) protein family members including BRD4 bind to acetylated lysines on the histone proteins, help assemble transcriptional regulators at the target gene promoters and enhancers, and regulate the expression of important oncogenes, e.g., Myc and BCL-2. Here we determined the effects of the BET protein inhibitor JQ1 and/or histone deacetylase (HDAC) inhibitor panobinostat (PS) on cultured (JeKo1 and MO2058) and primary human MCL cells harvested from the excised MCL involved lymph nodes. Treatment with JQ1 (100 to 2000 nM), but not its inactive enantiomer (R-JQ1), dose-dependently increased the % of cells in the G1 phase while reducing the % of S phase cells, while concomitantly inducing apoptosis in the cultured (MO2058 > JeKo1) MCL cells. Treatment with JQ1 was also dose-dependently lethal against primary MCL cells. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of MYC, BCL2 and CDK6 promoter in JeKo1 and MO2058 cells. Total RNA from the untreated and JQ1-treated cells was used for the quantitative PCR analysis, which showed depletion of the mRNA of c-MYC, BCL2 and CDK6 genes in JQ1-treated cells. While it had no effect on acetylated histone H3 and BRD4, JQ1 treatment dose-dependently depleted the protein levels of MYC, BCL2, CDK6 and pSer2 RNA POL II, but induced the levels of p21, p27 and cleaved PARP in MCL cells. As compared to each agent alone, co-treatment with JQ1 (but not its inactive enantiomer, R-JQ1) and panobinostat (PS) synergistically induced apoptosis of the cultured and primary MCL cells (combination indices< 1.0 by isobologram analyses), but not of normal CD34+ hematopoietic progenitor cells. This was associated with greater attenuation of MYC, BCL2, MCL1 and cyclin D1, but increase in the levels of p21 and cleaved PARP in cultured MCL cells. Co-treatment with JQ1 and PS was also synergistically lethal against carfilzomib-resistant JeKo1 cells (> 10 fold resistant), which were isolated following selection under a continuous exposure to increasing levels of carfilzomib. Following the tail vein infusion and engraftment of JeKo1 cells (5 million cells/mouse) in the bone marrow and spleen of NOD/SCID mice, co-treatment with JQ1 (50 mg/kg/day, formulated in 10% 2-hydroxypropyl-β-cyclodextrin, administered IP) and PS (5 mg/kg, IP) versus treatment with vehicle control, or JQ1 or PS alone, resulted in significant in vivo attenuation of c-MYC, BCL-2 and cyclin D1 levels in the harvested MCL cells from the mice (p < 0.01). Collectively, these pre-clinical findings demonstrate that the combined treatment with BRD4 antagonist and pan-HDAC inhibitor is a synergistically effective epigenetic therapy targeted against human MCL cells, regardless of their sensitivity to proteasome inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A124. Citation Format: Bhavin Shah, Laxmi Jakkula, Warren Fiskus, Sunil Sharma, Jun Qi, John A. Valenta, Leasha J. Schaub, Melissa Rodriguez, Santhana G.T. Devaraj, James E. Bradner, Kapil N. Bhalla. Co-treatment with BRD4 antagonist and histone deacetylase inhibitor is synergistically lethal against Mantle Cell Lymphoma (MCL) cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A124.

NUT midline carcinoma (NMC) is an aggressive type of squamous cell carcinoma that is defined by the presence of BRD-NUT fusion oncogenes, which encode chimeric proteins that block differentiation and maintain tumor growth. BRD-NUT oncoproteins contain two bromodomains whose binding to acetylated histones is required for the blockade of differentiation in NMC, but the mechanisms by which BRD-NUT act remain uncertain. Here we provide evidence that MYC is a key downstream target of BRD4-NUT. Expression profiling of NMCs show that the set of genes whose expression is maintained by BRD4-NUT is highly enriched for MYC upregulated genes, and MYC and BRD4-NUT protein expression is strongly correlated in primary NMCs. More directly, we find that BRD4-NUT associates with the MYC promoter and is displaced by acetyl histone mimetic BET inhibitor, JQ1, which prevents binding of BET bromodomains to acetylated histones. BRD4-NUT is also required to maintain MYC expression in NMC cell lines, as shown by a dramatic decrease in MYC expression upon JQ1 treatment and knockdown of BRD4-NUT with NUT specific siRNAs. Moreover, both siRNA knockdown of MYC and a dominant-negative form of MYC, omomyc, induce differentiation of NMC cells. Conversely, differentiation of NMC cells induced by knockdown of BRD4-NUT is abrogated by enforced expression of MYC. Together, these findings suggest that MYC is a downstream target of BRD4-NUT that is required for maintenance of NMC cells in an undifferentiated, proliferative state. Our findings support a model in which dysregulation of MYC by BRD-NUT fusion proteins has a central role in the pathogenesis of NMC. Citation Format: Erica M. Walsh, Adlai R. Grayson, Michael J. Cameron, Jernej Godec, Todd Ashworth, Alexandra B. Aserlind, Hongfang Wang, Gerard Evan, Michael J. Kluk, James E. Bradner, Jon C. Aster, Christopher A. French. MYC, a downstream target of BRD-NUT, is necessary and sufficient for the blockade of differentiation in NUT midline carcinoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B39.

An important goal in cancer therapy has been to selectively sensitize cancers to existing drugs that typically work by inducing DNA damage. In particular, many cancers are p53-defective and chemoresistant, thus novel approaches to target p53-defective cancers are needed. One promising strategy to sensitize p53-deficient cells has been to inhibit DNA damage response kinases such as ATM/ATR via ATP-competitive kinase inhibitors. To better understand ATM/ATR activation mechanisms and discover novel probes for the ATM/ATR pathway, we performed a phenotype-based screen of 9,195 small-molecule compounds for inhibitors of hydroxyurea-induced phosphorylation of Chk1, a key ATR substrate. After subsequent biological screens, we selected 4 compounds that inhibited ATR and ATM pathways; 3 were known bioactive agents and 1 was a diversity-oriented-synthetic product. These compounds sensitized p53-deficient cells to diverse DNA-damaging agents. Xenograft experiments were performed on one compound, and it showed synergistic suppression of p53-deficient tumor growth with cisplatin. Importantly, these compounds did not suppress ATR kinase catalytic activity in vitro, unlike typical ATM/ATR kinase inhibitors that are ATP-competitive. To identify molecular targets of one compound, “MARPIN” (ATM and ATR pathway inhibitor), we defined its active site through structure-activity relationship analysis, resulting in synthesis of inactive derivatives of MARPIN. Identification of proteins that specifically bind MARPIN, but not its inactive derivatives, is underway. This phenotype-based chemical genetic screen identified novel ATM/ATR pathway inhibitors that are mechanistically distinct from kinase catalytic inhibitors, and these compounds could serve as probes to identify previously unrecognized druggable targets in DNA damage response pathways. Citation Format: Masaoki Kawasumi, James E. Bradner, Nicola Tolliday, Renee Thibodeau, Heather Sloan, Kay M. Brummond, Paul Nghiem. A chemical genetic screen identifies novel ATM/ATR pathway inhibitors that sensitize p53-deficient cells to DNA-damaging agents without affecting ATR kinase catalytic activity. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B04.