Person: Bradner, James E
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Bradner
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James E
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Bradner, James E
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Publication Repression of BIM mediates survival signaling by MYC and AKT in high-risk T-cell acute lymphoblastic leukemia(2014) Reynolds, Christine; Roderick, Justine E.; LaBelle, James L.; Bird, Gregory; Mathieu, Ronald; Bodaar, Kimberly; Colon, Diana; Pyati, Ujwal; Stevenson, Kristen E.; Qi, Jun; Harris, Marian; Silverman, Lewis; Sallan, Stephen; Bradner, James E; Neuberg, Donna; Look, A.; Walensky, Loren; Kelliher, Michelle A.; Gutierrez, AlejandroTreatment resistance in T-cell acute lymphoblastic leukemia (T-ALL) is associated with PTEN deletions and resultant PI3K-AKT pathway activation, as well as MYC overexpression, and these pathways repress mitochondrial apoptosis in established T-lymphoblasts through poorly defined mechanisms. Normal T-cell progenitors are hypersensitive to mitochondrial apoptosis, a phenotype that is dependent on expression of proapoptotic BIM. In a conditional zebrafish model, MYC downregulation induced BIM expression in T-lymphoblasts, an effect that was blunted by expression of constitutively active AKT. In human T-ALL cell lines and treatment- resistant patient samples, treatment with MYC or PI3K-AKT pathway inhibitors each induced BIM upregulation and apoptosis, indicating that BIM is repressed downstream of MYC and PI3K-AKT in high-risk T-ALL. Restoring BIM function in human T-ALL cells using a stapled peptide mimetic of the BIM BH3 domain had therapeutic activity, indicating that BIM repression is required for T-ALL viability. In the zebrafish model, where MYC downregulation induces T- ALL regression via mitochondrial apoptosis, T-ALL persisted despite MYC downregulation in 10% of bim wild-type zebrafish, 18% of bim heterozygotes, and in 33% of bim homozygous mutants (P = 0.017). We conclude that downregulation of BIM represents a key survival signal downstream of oncogenic MYC and PI3K-AKT signaling in treatment-resistant T-ALL.Publication Biased Multicomponent Reactions to Develop Novel Bromodomain Inhibitors(American Chemical Society, 2014) McKeown, Michael R; Shaw, Daniel L; Fu, Harry; Liu, Shuai; Xu, Xiang; Marineau, Jason J; Huang, Yibo; Zhang, Xiaofeng; Buckley, Dennis L; Kadam, Asha; Zhang, Zijuan; Blacklow, Stephen; Qi, Jun; Zhang, Wei; Bradner, James EBET bromodomain inhibition has contributed new insights into gene regulation and emerged as a promising therapeutic strategy in cancer. Structural analogy of early methyl-triazolo BET inhibitors has prompted a need for structurally dissimilar ligands as probes of bromodomain function. Using fluorous-tagged multicomponent reactions, we developed a focused chemical library of bromodomain inhibitors around a 3,5-dimethylisoxazole biasing element with micromolar biochemical IC50. Iterative synthesis and biochemical assessment allowed optimization of novel BET bromodomain inhibitors based on an imidazo[1,2-a]pyrazine scaffold. Lead compound 32 (UMB-32) binds BRD4 with a Kd of 550 nM and 724 nM cellular potency in BRD4-dependent lines. Additionally, compound 32 shows potency against TAF1, a bromodomain-containing transcription factor previously unapproached by discovery chemistry. Compound 32 was cocrystallized with BRD4, yielding a 1.56 Å resolution crystal structure. This research showcases new applications of fluorous and multicomponent chemical synthesis for the development of novel epigenetic inhibitors.Publication ATAD2 is an epigenetic reader of newly synthesized histone marks during DNA replication(Impact Journals LLC, 2016) Koo, Seong Joo; Fernández-Montalván, Amaury E.; Badock, Volker; Ott, Christopher; Holton, Simon J.; von Ahsen, Oliver; Toedling, Joern; Vittori, Sarah; Bradner, James E; Gorjánácz, MátyásATAD2 (ATPase family AAA domain-containing protein 2) is a chromatin regulator harboring an AAA+ ATPase domain and a bromodomain, previously proposed to function as an oncogenic transcription co-factor. Here we suggest that ATAD2 is also required for DNA replication. ATAD2 is co-expressed with genes involved in DNA replication in various cancer types and predominantly expressed in S phase cells where it localized on nascent chromatin (replication sites). Our extensive biochemical and cellular analyses revealed that ATAD2 is recruited to replication sites through a direct interaction with di-acetylated histone H4 at K5 and K12, indicative of newly synthesized histones during replication-coupled chromatin reassembly. Similar to ATAD2-depletion, ectopic expression of ATAD2 mutants that are deficient in binding to these di-acetylation marks resulted in reduced DNA replication and impaired loading of PCNA onto chromatin, suggesting relevance of ATAD2 in DNA replication. Taken together, our data show a novel function of ATAD2 in cancer and for the first time identify a reader of newly synthesized histone di-acetylation-marks during replication.Publication MELK is not necessary for the proliferation of basal-like breast cancer cells(eLife Sciences Publications, Ltd, 2017) Huang, Hai-Tsang; Seo, Hyuk-Soo; Zhang, Tinghu; Wang, Yubao; Jiang, Baishan; Li, Qing; Buckley, Dennis L; Nabet, Behnam; Roberts, Justin M; Paulk, Joshiawa; Dastjerdi, Shiva; Winter, Georg E; McLauchlan, Hilary; Moran, Jennifer; Bradner, James E; Eck, Michael; Dhe-Paganon, Sirano; Zhao, Jean; Gray, NathanaelThorough preclinical target validation is essential for the success of drug discovery efforts. In this study, we combined chemical and genetic perturbants, including the development of a novel selective maternal embryonic leucine zipper kinase (MELK) inhibitor HTH-01-091, CRISPR/Cas9-mediated MELK knockout, a novel chemical-induced protein degradation strategy, RNA interference and CRISPR interference to validate MELK as a therapeutic target in basal-like breast cancers (BBC). In common culture conditions, we found that small molecule inhibition, genetic deletion, or acute depletion of MELK did not significantly affect cellular growth. This discrepancy to previous findings illuminated selectivity issues of the widely used MELK inhibitor OTSSP167, and potential off-target effects of MELK-targeting short hairpins. The different genetic and chemical tools developed here allow for the identification and validation of any causal roles MELK may play in cancer biology, which will be required to guide future MELK drug discovery efforts. Furthermore, our study provides a general framework for preclinical target validation.Publication Epigenetic Reprogramming of Lineage-Committed Human Mammary Epithelial Cells Requires DNMT3A and Loss of DOT1L(Elsevier, 2017) Breindel, Jerrica L.; Skibinski, Adam; Sedic, Maja; Wronski-Campos, Ania; Zhou, Wenhui; Keller, Patricia J.; Mills, Joslyn; Bradner, James E; Onder, Tamer; Kuperwasser, CharlotteSummary Organogenesis and tissue development occur through sequential stepwise processes leading to increased lineage restriction and loss of pluripotency. An exception to this appears in the adult human breast, where rare variant epithelial cells exhibit pluripotency and multilineage differentiation potential when removed from the signals of their native microenvironment. This phenomenon provides a unique opportunity to study mechanisms that lead to cellular reprogramming and lineage plasticity in real time. Here, we show that primary human mammary epithelial cells (HMECs) lose expression of differentiated mammary epithelial markers in a manner dependent on paracrine factors and epigenetic regulation. Furthermore, we demonstrate that HMEC reprogramming is dependent on gene silencing by the DNA methyltransferase DNMT3A and loss of histone transcriptional marks following downregulation of the methyltransferase DOT1L. These results demonstrate that lineage commitment in adult tissues is context dependent and highlight the plasticity of somatic cells when removed from their native tissue microenvironment.Publication Transcription control by the ENL YEATS domain in acute leukemia(2017) Erb, Michael A.; Scott, Thomas G.; Li, Bin; Xie, Huafeng; Paulk, Joshiawa; Seo, Hyuk-Soo; Souza, Amanda; Roberts, Justin M.; Dastjerdi, Shiva; Buckley, Dennis L.; Sanjana, Neville E.; Shalem, Ophir; Nabet, Behnam; Zeid, Rhamy; Offei-Addo, Nana K.; Dhe-Paganon, Sirano; Zhang, Feng; Orkin, Stuart; Winter, Georg E.; Bradner, James ERecurrent chromosomal translocations involving the mixed lineage leukemia gene (MLL) give rise to a highly aggressive acute leukemia associated with poor clinical outcome1. The preferential involvement of chromatin-associated factors in MLL rearrangement belies a dependency on transcription control2. Despite recent progress made in targeting chromatin regulators in cancer3, available therapies for this well-characterized disease remain inadequate, prompting the present effort to qualify new targets for therapeutic intervention. Using unbiased, emerging CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in MLL-AF4-positive acute leukemia, we identified ENL (eleven-nineteen leukemia) as an unrecognized dependency particularly indispensable for proliferation in vitro and in vivo. To explain the mechanistic role for ENL in leukemia pathogenesis and dynamic transcription control, we pursued a chemical genetic strategy utilizing targeted protein degradation. Acute ENL loss suppresses transcription initiation and elongation genome-wide, with pronounced effects at genes featuring disproportionate ENL load. Importantly, ENL-dependent leukemic growth was contingent upon an intact YEATS chromatin reader domain. These findings reveal a novel dependency in acute leukemia and a first mechanistic rational for disrupting the YEATS domain in disease.Publication ENL links histone acetylation to oncogenic gene expression in AML(2017) Wan, Liling; Wen, Hong; Li, Yuanyuan; Lyu, Jie; Xi, Yuanxin; Hoshii, Takayuki; Joseph, Julia; Wang, Xiaolu; Loh, Yong-Hwee E.; Erb, Michael A.; Souza, Amanda L.; Bradner, James E; Shen, Li; Li, Wei; Li, Haitao; Allis, C. David; Armstrong, Scott; Shi, XiaobingCancer cells are characterized by aberrant epigenetic landscapes and often exploit chromatin machinery to activate oncogenic gene expression programs1. Recognition of modified histones by “reader” proteins constitutes a key mechanism underlying these processes; therefore, targeting such pathways holds clinical promise, as exemplified by the development of BET bromodomain inhibitors2, 3. We recently identified the YEATS domain as a novel acetyllysine-binding module4, yet its functional importance in human cancer remains unknown. Here we show that the YEATS domain-containing protein ENL, but not its paralog AF9, is required for disease maintenance in acute myeloid leukaemia (AML). CRISPR-Cas9 mediated depletion of ENL led to anti-leukemic effects, including increased terminal myeloid differentiation and suppression of leukaemia growth in vitro and in vivo. Biochemical and crystal structural studies and ChIP-seq analyses revealed that ENL binds to acetylated histone H3, and colocalizes with H3K27ac and H3K9ac on the promoters of actively transcribed genes that are essential for leukaemias. Disrupting the interaction between the YEATS domain and histone acetylation via structure-based mutagenesis reduced RNA polymerase II recruitment to ENL target genes, leading to suppression of oncogenic gene expression programs. Importantly, disruption of ENL’s functionality further sensitized leukaemia cells to BET inhibitors. Together, our study identifies ENL as a histone acetylation reader that regulates oncogenic transcriptional programs in AML and suggests that displacement of ENL from chromatin may be a promising epigenetic therapy alone or in combination with BET inhibitors for AML.Publication Prostate cancer-associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4(2017) Dai, Xiangpeng; Gan, Wenjian; Li, Xiaoning; Wang, Shangqian; Zhang, Wei; Huang, Ling; Liu, Shengwu; Zhong, Qing; Guo, Jianping; Zhang, Jinfang; Chen, Ting; Shimizu, Kouhei; Beca, Francisco; Blattner, Mirjam; Vasudevan, Divya; Buckley, Dennis L.; Qi, Jun; Buser, Lorenz; Liu, Pengda; Inuzuka, Hiroyuki; Beck, Andrew; Wang, Liewei; Wild, Peter J.; Garraway, Levi; Rubin, Mark A.; Barbieri, Christopher E.; Wong, Kwok-Kin; Muthuswamy, Senthil; Huang, Jiaoti; Chen, Yu; Bradner, James E; Wei, WenyiThe bromodomain and extra-terminal (BET) family of proteins, comprised of four members including BRD2, BRD3, BRD4 and the testis-specific isoform BRDT, largely function as transcriptional co-activators 1–3 and play critical roles in various cellular processes, including cell cycle, apoptosis, migration and invasion 4,5. As such, BET proteins enhance the oncogenic functions of major cancer drivers by either elevating their expression such as c-Myc in leukemia 6,7 or by promoting transcriptional activities of oncogenic factors such as AR and ERG in the prostate cancer setting 8. Pathologically, BET proteins are frequently overexpressed and clinically linked to various types of human cancers 5,9,10, therefore pursued as attractive therapeutic targets for selective inhibition in patients. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed 11,12 and shown promising outcomes in early clinical trials. Despite resistance to BET inhibitor has been documented in pre-clinical models 13–15 the molecular mechanisms underlying acquired resistance are largely unknown. Here, we report that Cullin 3SPOP earmarks BET proteins including BRD2, BRD3 and BRD4 for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of BET proteins, leading to their elevated abundance in SPOP-deficient prostate cancer. As a result, prostate cancer cells and prostate cancer patient-derived organoids harboring SPOP mutations are more resistant to BET inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor suppressor role of SPOP in prostate cancer by negatively controlling BET protein stability, and also provide a molecular mechanism for BET inhibitor resistance in prostate cancer patients bearing SPOP mutations.Publication Regulation of MYC Expression and Differential JQ1 Sensitivity in Cancer Cells(Public Library of Science, 2014) Fowler, Trent; Ghatak, Payel; Price, David H.; Conaway, Ronald; Conaway, Joan; Chiang, Cheng-Ming; Bradner, James E; Shilatifard, Ali; Roy, Ananda L.High level MYC expression is associated with almost all human cancers. JQ1, a chemical compound that inhibits MYC expression is therapeutically effective in preclinical animal models in midline carcinoma, and Burkitt’s lymphoma (BL). Here we show that JQ1 does not inhibit MYC expression to a similar extent in all tumor cells. The BL cells showed a ∼90% decrease in MYC transcription upon treatment with JQ1, however, no corresponding reduction was seen in several non-BL cells. Molecularly, these differences appear due to requirements of Brd4, the most active version of the Positive Transcription Elongation Factor B (P-TEFb) within the Super Elongation Complex (SEC), and transcription factors such as Gdown1, and MED26 and also other unknown cell specific factors. Our study demonstrates that the regulation of high levels of MYC expression in different cancer cells is driven by unique regulatory mechanisms and that such exclusive regulatory signatures in each cancer cells could be employed for targeted therapeutics.Publication Small-molecule inhibition of BRD4 as a new potent approach to eliminate leukemic stem- and progenitor cells in acute myeloid leukemia (AML)(Impact Journals LLC, 2012) Herrmann, Harald; Blatt, Katharina; Shi, Junwei; Gleixner, Karoline V.; Cerny-Reiterer, Sabine; Müllauer, Leonhard; Vakoc, Christopher R.; Sperr, Wolfgang R.; Horny, Hans-Peter; Bradner, James E; Zuber, Johannes; Valent, PeterAcute myeloid leukemia (AML) is a life-threatening stem cell disease characterized by uncontrolled proliferation and accumulation of myeloblasts. Using an advanced RNAi screen-approach in an AML mouse model we have recently identified the epigenetic ‘reader’ BRD4 as a promising target in AML. In the current study, we asked whether inhibition of BRD4 by a small-molecule inhibitor, JQ1, leads to growth-inhibition and apoptosis in primary human AML stem- and progenitor cells. Primary cell samples were obtained from 37 patients with freshly diagnosed AML (n=23) or refractory AML (n=14). BRD4 was found to be expressed at the mRNA and protein level in unfractionated AML cells as well as in highly enriched CD34+/CD38− and CD34+/CD38+ stem- and progenitor cells in all patients examined. In unfractionated leukemic cells, submicromolar concentrations of JQ1 induced major growth-inhibitory effects (IC50 0.05-0.5 μM) in most samples, including cells derived from relapsed or refractory patients. In addition, JQ1 was found to induce apoptosis in CD34+/CD38− and CD34+/CD38+ stem- and progenitor cells in all donors examined as evidenced by combined surface/Annexin-V staining. Moreover, we were able to show that JQ1 synergizes with ARA-C in inducing growth inhibition in AML cells. Together, the BRD4-targeting drug JQ1 exerts major anti-leukemic effects in a broad range of human AML subtypes, including relapsed and refractory patients and all relevant stem- and progenitor cell compartments, including CD34+/CD38− and CD34+/CD38+ AML cells. These results characterize BRD4-inhibition as a promising new therapeutic approach in AML which should be further investigated in clinical trials.