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ABSTRACT: Mixed-lineage leukemia (MLL) fusions are potent oncogenes that initiate aggressive forms of acute leukemia. As aberrant transcriptional regulators, MLL-fusion proteins alter gene expression in hematopoietic cells through interactions with the histone H3 lysine 79 (H3K79) methyltransferase DOT1L. Notably, interference with MLL-fusion cofactors like DOT1L is an emerging therapeutic strategy in this disease. Here, we identify the histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) as an additional chromatin regulator that is necessary for MLL-fusion-mediated leukemogenesis. Suppressing the expression of Rnf20 in diverse models of MLL-rearranged leukemia leads to inhibition of cell proliferation, under tissue culture conditions as well as in vivo. Rnf20 knockdown leads to reduced expression of MLL-fusion target genes, effects resembling Dot1l inhibition. Using ChIP-seq, we found that H2B ubiquitination is enriched in the body of MLL-fusion target genes, correlating with sites of H3K79 methylation and transcription elongation. Furthermore, Rnf20 is required to maintain local levels of H3K79 methylation by Dot1l at Hoxa9 and Meis1. These findings support a model whereby cotranscriptional recruitment of Rnf20 at MLL-fusion target genes leads to amplification of Dot1l-mediated H3K79 methylation, thereby rendering leukemia cells dependent on Rnf20 to maintain their oncogenic transcriptional program.
Proceedings of the National Academy of Sciences 03/2013; 110(10):3901-6. · 9.68 Impact Factor
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Harald Herrmann,
Katharina Blatt, Junwei Shi,
Karoline V Gleixner,
Sabine Cerny-Reiterer,
Leonhard Müllauer,
Christopher R Vakoc,
Wolfgang R Sperr,
Hans-Peter Horny,
James E Bradner,
Johannes Zuber,
Peter Valent
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ABSTRACT: Acute 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.
Oncotarget 11/2012; · 4.78 Impact Factor
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Jake E Delmore,
Ghayas C Issa,
Madeleine E Lemieux,
Peter B Rahl, Junwei Shi,
Hannah M Jacobs,
Efstathios Kastritis,
Timothy Gilpatrick,
Ronald M Paranal,
Jun Qi, [......],
Christopher R Vakoc,
P Leif Bergsagel,
Irene M Ghobrial,
Paul G Richardson,
Richard A Young,
William C Hahn,
Kenneth C Anderson,
Andrew L Kung,
James E Bradner,
Constantine S Mitsiades
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ABSTRACT: MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.
Cell 09/2011; 146(6):904-17. · 32.40 Impact Factor
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Johannes Zuber, Junwei Shi,
Eric Wang,
Amy R Rappaport,
Harald Herrmann,
Edward A Sison,
Daniel Magoon,
Jun Qi,
Katharina Blatt,
Mark Wunderlich,
Meredith J Taylor,
Christopher Johns,
Agustin Chicas,
James C Mulloy,
Scott C Kogan,
Patrick Brown,
Peter Valent,
James E Bradner,
Scott W Lowe,
Christopher R Vakoc
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ABSTRACT: Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.
Nature 08/2011; 478(7370):524-8. · 36.28 Impact Factor
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Johannes Zuber,
Amy R Rappaport,
Weijun Luo,
Eric Wang,
Chong Chen,
Angelina V Vaseva, Junwei Shi,
Susann Weissmueller,
Christof Fellmann,
Christof Fellman,
Meredith J Taylor,
Martina Weissenboeck,
Thomas G Graeber,
Scott C Kogan,
Christopher R Vakoc,
Scott W Lowe
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ABSTRACT: Although human cancers have complex genotypes and are genomically unstable, they often remain dependent on the continued presence of single-driver mutations-a phenomenon dubbed "oncogene addiction." Such dependencies have been demonstrated in mouse models, where conditional expression systems have revealed that oncogenes able to initiate cancer are often required for tumor maintenance and progression, thus validating the pathways they control as therapeutic targets. Here, we implement an integrative approach that combines genetically defined mouse models, transcriptional profiling, and a novel inducible RNAi platform to characterize cellular programs that underlie addiction to MLL-AF9-a fusion oncoprotein involved in aggressive forms of acute myeloid leukemia (AML). We show that MLL-AF9 contributes to leukemia maintenance by enforcing a Myb-coordinated program of aberrant self-renewal involving genes linked to leukemia stem cell potential and poor prognosis in human AML. Accordingly, partial and transient Myb suppression precisely phenocopies MLL-AF9 withdrawal and eradicates aggressive AML in vivo without preventing normal myelopoiesis, indicating that strategies to inhibit Myb-dependent aberrant self-renewal programs hold promise as effective and cancer-specific therapeutics. Together, our results identify Myb as a critical mediator of oncogene addiction in AML, delineate relevant Myb target genes that are amenable to pharmacologic inhibition, and establish a general approach for dissecting oncogene addiction in vivo.
Genes & development 08/2011; 25(15):1628-40. · 12.08 Impact Factor
