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.

The existence of leukemia stem cells has been demonstrated in acute myeloid and lymphoblastic leukemias (AML and ALL). The origins of these cells are unknown, but it has been suggested that they result from the transformation of adult hematopoietic stem cells (HSC). To challenge this hypothesis we tested the ability of representative leukemia oncogenes to transform committed myeloid progenitor cells that lack the capacity for self-renewal. Flow-sorted populations of common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP) were transduced with the fusion oncogenes MOZ-TIF2 and BCR-ABL, respectively and their self-renewal and leukemogenic potential were tested in in vitro and in vivo assays. Utilizing the same experimental design we were also able to address the poorly understood question of the contribution of the cell of transformation to the eventual leukaemia phenotype. In contrast to CMP or GMP transduced with BCR-ABL or non-transduced control cells, CMP or GMP that were retrovirally transduced with MOZ-TIF2 could be serially replated in methylcellulose cultures, and continuously propagated in liquid culture media containing IL-3. In further contrast, transplantation of CMP or GMP transduced with MOZ-TIF2 into recipient mice also resulted in an acute myeloid leukemia (AML). This leukaemia could be transplanted to secondary recipients, documenting the long-term self-renewal properties of the leukemic stem cells, yet in limiting dilution experiments did not cause disease below a transplanted cell dose of 1 x104 cells, suggesting that the probability of transferring leukemia to secondary recipients relates to the frequency of self-renewing leukemic stem cells within the total leukemic population. This in turn suggests that our retroviral bone marrow transduction and transplantation models have the same hierarchical organization of self-renewal as has been shown for human AML. The phenotype of the leukemias were virtually indistinguishable regardless of whether the initially transduced cell population was CMP, GMP or the control populations of whole bone marrow mononuclear cells or HSC, suggesting that MOZ-TIF2 may also have a dominant effect upon the eventual leukaemia phenotype. These observations indicate that MOZ-TIF2, but not BCR-ABL, can confer properties of leukemic stem cells to committed myeloid progenitors. Control experiments conducted with with MOZ-TIF2 point mutants that do not cause leukemia in the murine BMT system and with BCR-ABL, a fully active leukemogenic tyrosine kinase, were insufficient to cause in-vitro changes in self-renewal or leukaemia. Together, these data argue strongly that retroviral insertional mutagenesis alone cannot explain these results. However, we cannot exclude the possibility that an active MOZ-TIF2, but not BCR-ABL, can collaborate with mutations induced by retroviral mutagenesis to confer properties of leukemic stem cells to committed progenitors. These findings have important implications regarding the origin of leukemic stem cells, and provide tools for understanding the transcriptional programs that confer properties of self-renewal in malignant and non-malignant cells.

No abstract

Interferons (IFNs) represent an important host defense against viruses. Type I IFNs induce JAK-STAT signaling and expression of IFN-stimulated genes (ISGs), which mediate antiviral activity. Histone deacetylases (HDACs) perform multiple functions in regulating gene expression and some class I HDACs and the class IV HDAC, HDAC11, influence type I IFN signaling. Here, HDAC4, a class II HDAC, is shown to promote type I IFN signaling and coprecipitate with STAT2. Pharmacological inhibition of class II HDAC activity, or knockout of HDAC4 from HEK-293T and HeLa cells, caused a defective response to IFN-α. This defect in HDAC4−/− cells was rescued by reintroduction of HDAC4 or catalytically inactive HDAC4, but not HDAC1 or HDAC5. ChIP analysis showed HDAC4 was recruited to ISG promoters following IFN stimulation and was needed for binding of STAT2 to these promoters. The biological importance of HDAC4 as a virus restriction factor was illustrated by the observations that (i) the replication and spread of vaccinia virus (VACV) and herpes simplex virus type 1 (HSV-1) were enhanced in HDAC4−/− cells and inhibited by overexpression of HDAC4; and (ii) HDAC4 is targeted for proteasomal degradation during VACV infection by VACV protein C6, a multifunctional IFN antagonist that coprecipitates with HDAC4 and is necessary and sufficient for HDAC4 degradation.

Highlights d Loss-of-function mutations of DDX3X are frequent in MYCdriven B cell lymphomas d DDX3X promotes translation of mRNAs encoding the core protein synthesis machinery d Loss of DDX3X buffers MYC-driven global protein synthesis and proteotoxic stress d DDX3X loss is later rescued by ectopic expression of Ychromosome-encoded DDX3Y