55 Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which confer a poor prognosis and are often refractory to conventional therapies. Recent efforts have begun to unravel the molecular pathogenesis of these malignancies. Several groups have demonstrated that MLL-fusions associate with two macromolecular chromatin complexes; the polymerase associated factor (PAFc) complex, which interacts with the N-terminal domain of MLL, a portion of the protein that is retained in all the described fusions, or the super elongation complex (SEC), via interaction with the C-terminal fusion partner. These complexes play an integral role in regulating transcriptional elongation and this function appears to be aberrantly co-opted by the MLL-fusions to initiate and perpetuate transcriptional programmes that culminate in leukaemia. In this study we used a systematic global proteomic survey incorporating quantitative mass spectrometry to demonstrate that MLL-fusions, as part of SEC and PAFc complexes, are associated with the BET family of acetyl lysine recognition chromatin “adaptor” proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. Targeting the BET proteins to alter aberrant transcriptional elongation has recently been demonstrated to be possible using small molecule inhibitors that selectively bind the tandem bromodomain at the amino-terminus of the ubiquitously expressed BET proteins (BRD2/BRD3/BRD4). We developed a novel class of potent small molecule inhibitors to the BET family, which is chemically distinct to previously published BET-inhibitors. We then used this new compound (I-BET151) to demonstrate its profound and selective efficacy against human MLL-fusion leukaemic cell lines in liquid culture as well as clonogenic assays in methylcellulose. We also establish that primary murine progenitors retrovirally transformed with MLL-ENL and MLL-AF9 are equally susceptible to treatment with I-BET151. We show that the main phenotypic consequence of BET inhibition in MLL fusion leukaemia is a dramatic early induction of cell cycle arrest and apoptosis. Global gene-expression profiling, following I-BET151 treatment in two different human MLL-fusion leukaemia cell lines (expressing MLL-AF4 and MLL-AF9), highlights a common differentially expressed gene signature that accounts for this phenotype. Importantly, chromatin immunoprecipitation analyses at direct MLL target genes including BCL2, C-MYC and CDK6, indicate that I-BET151 selectively inhibits the recruitment of BET family members BRD3/BRD4, and SEC and PAFc components. These events result in the inefficient phosphorylation and release of paused POL-II from the TSS of these genes providing mechanistic insight into the mode of action of I-BET151 in MLL-fusion leukaemia. We subsequently established the therapeutic efficacy of I-BET151 in vivo by demonstrating dramatic disease control in murine models of MLL-AF4 and MLL-AF9 leukaemia. Finally, we also demonstrate that I-BET151 accelerates apoptosis in primary leukaemic cells from a large number of patients with various MLL-fusion leukaemias, by affecting a similar transcription programme to that identified in the human leukaemic cell lines. Importantly, we also demonstrate that I-BET151 significantly reduces the clonogenic potential of isolated primary leukaemic stem cells, suggesting that disease eradication may be possible. These data highlight a new paradigm for drug discovery targeting the protein-protein interactions of chromatin-associated proteins. We demonstrate that small molecules that perturb the interaction of BRD3/4 with chromatin have therapeutic potential in MLL fusion leukaemias and moreover, we provide the molecular mechanism to account for this therapeutic efficacy. Finally, our results emphasize an emerging role for targeting aberrant transcriptional elongation in oncogenesis. Disclosures: Prinjha: GSK: Employment. Chung:GSK: Employment. Lugo:GSK: Employment. Beinke:GSK: Employment. Soden:GSK: Employment. Mirguet:GSK: Employment. Jeffrey:GSK: Employment. Lee:GSK: Employment. Kouzarides:GSK: Consultancy.

In this article (Cancer Cell 7,, the name of one of the authors was listed incorrectly. The author incorrectly listed as "Azam Mohammed" is actually named Mohammad Azam.

No abstract

The hallmark of chronic myeloid leukemia (CML) is theBCR-ABL fusion gene, which is usually formed as a result of the t(9;22) translocation. Patients with CML show considerable heterogeneity both in their presenting clinical features and in the time taken for evolution to blast crisis. In this study, metaphase fluorescence in situ hybridization showed that a substantial minority of patients with CML had large deletions adjacent to the translocation breakpoint on the derivative 9 chromosome, on the additional partner chromosome in variant translocations, or on both. The deletions spanned up to several megabases, had variable breakpoints, and could be detected by microsatellite polymerase chain reaction in unfractionated bone marrow and purified peripheral blood granulocytes. The deletions were likely to occur early and possibly at the time of the Philadelphia (Ph) chromosome translocation: deletions were detected at diagnosis in 11 patients, were found in all Ph-positive metaphases, and were more prevalent in patients with variant Ph chromosomes. Kaplan-Meier analysis showed a median survival time of 36 months in patients with a deletion; patients without a detectable deletion survived > 90 months. The survival-time difference was significant on log-rank analysis (P = .006). Multivariate analysis demonstrated that the prognostic importance of deletion status was independent of age, sex, percentage of peripheral blood blasts, and platelet count. Our data therefore suggest that an apparently simple, balanced translocation may result not only in the generation of a dominantly acting fusion oncogene but also in the loss of one or more genes that influence disease progression.