IntroductionHematopoietic development is controlled by an intricate network of finely tuned transcriptional programs. Consequently, a perturbation of the transcription factors involved can block differentiation. This developmental roadblock cooperates with mutations in pathways that signal growth and/or survival to cause acute leukemias. 1 A prime example for such a mechanism is mixed lineage leukemia. In this disease, the gene for the histone methyltransferase MLL participates in chromosomal translocations that eventually create maturation-blocking and therefore leukemogenic MLL fusion proteins. 2,3 These protein chimeras consist of N-terminal portions of MLL joined to a variety of mostly unrelated fusion partners that replace the original MLL C-terminus, including the methyltransferase domain (http://atlasgeneticsoncology.org/Genes/ MLL.html). MLL fusion proteins are aberrant transcription factors that ectopically activate genes important for hematopoietic development like the abdominal-type Hox genes Hoxa7 and Hoxa9 and their dimerization partner Meis1. [4][5][6][7] Despite intensive study, little is known about the biological function of MLL fusion partners in normal cells, and it is mostly unclear how these proteins activate the oncogenic potential of MLL. In the rare cases where MLL is joined to a cytoplasmatic protein, domains introduced by the partner force a dimerization of the fusion that is crucial for oncogenic activity. 8,9 The overwhelming majority of leukemias with MLL rearrangement, however, involves nuclear proteins as translocation partners. The data available seem to indicate a role of some nuclear MLL partners in transcriptional control and histone modification. Support for this speculation comes from the detection of direct protein-protein interactions of the homologous MLL fusion partners AF4 and AF5q31 that both bind to ENL and the closely related AF9. 10,11 ENL in turn interacts with histone H3. 11 In addition, another MLL fusion partner, AF10, recruits the histone H3 lysine 79-specific methyltransferase DOT1L that introduces a dimethyl mark during transcriptional elongation. 12 The same modification is also a hallmark of genes activated by MLL-ENL. 13 Finally, the proteins CBX8 (chromobox 8) and BCoR (BCL6 corepressor) that are involved in chromatin-dependent gene repression have also been found to associate with ENL and AF9. [14][15][16] In order to learn more about the biological function of a classical MLL fusion partner, we identified proteins associated with the "Eleven Nineteen Leukemia" protein (ENL) originally discovered as an MLL fusion partner in the recurrent translocation t(11;19). Here, we describe the purification and analysis of ENL-associated proteins (EAPs) by tandem immunoprecipitation of ENL. This protein assembly contains several other MLL fusion partners, positive transcription elongation factor b (pTEFb), DOT1L, and polycomb group proteins. The composition of EAP suggests that ENL works in a new unit of transcriptional regulation that coordinates transcriptional elo...

Investigation of the activity of a family of fusion proteins that cause aggressive leukemia suggests transcriptional elongation as a new mechanism for oncogenic transformation.

IntroductionNext to their role in determining the identity of body segments throughout embryogenesis, the clustered HOX homeobox genes also control differentiation and self-renewal of hematopoietic stem and precursor cells. In particular genes of the HOXA cluster and to a lesser extent of the HOXB group are highly transcribed in hematopoietic precursors. During maturation HOX expression is gradually extinguished. 1 An ectopic expression of HOX genes therefore has profound consequences for hematopoiesis. One prominent example is HOXB4, which controls stem cell pool size. As a consequence, artificial introduction of HOXB4 can be used to expand hematopoietic stem cell numbers. 2,3 A relative overexpression of HOX genes also is a hallmark of many hematological malignancies, and a high concentration of HOXA9 in leukemic blasts has been shown to be an adverse prognostic parameter. 4 Proper HOX control is frequently lost in acute myeloid leukemia (AML), where these genes can be erroneously activated by mutations of their upstream regulators CDX2 and CDX4. [5][6][7][8] Also, mixed-lineage leukemia (MLL) fusion proteins in MLL induce aberrant HOX transcription, and the HOX expression pattern is a characteristic diagnostic criterion for this type of leukemia. 9 In several cases of T-cell acute lymphoblastic leukemia (ALL), a recurrent inversion inv(7)(p15q34) places part of the HOXA cluster under control of the promoter normally driving the T-cell receptor. As a consequence, this genetic aberration leads to inappropriate HOX transcription. 10,11 In addition, various HOX genes are directly involved in leukemogenic fusions with nucleoporins. HOXA9, for example, is fused to NUP98 by a translocation t(7;11)(p15;p15) that is frequently detected as sole genetic anomaly in AML. 12,13 In addition to these clinical observations, direct experimental evidence also illustrates the oncogenic capacity of HOX proteins.HOXA9 and HOXA7, as well as their protein dimerization partner MEIS1 (another homeobox gene of the "3-amino-acid-loopextension ϭ TALE" class), frequently were coactivated in retroviral tagging experiments. 14 Likewise, artificial overexpression of HOXA7, HOXA9, or HOXA10 in combination with MEIS1 caused leukemia in animal models. 1 The coexpression of MEIS1 accelerates HOX-driven leukemogenesis by activation of genes that also are present in hematopoietic stem cells. [15][16][17][18] In summary, all available data point to a major role in particular of the "posterior" HOXA genes A7 to A10 as important hematopoietic oncogenes. Despite this HOX dominance in leukemia, a systematic, comparative assessment of the transforming capacity of all HOXA genes has never been undertaken. It is unknown why posterior HOX genes appear to predominate. Moreover, there are conflicting results of whether individual HOX genes are necessary for transformation or whether leukemogenesis is the consequence of a coordinated action involving the combined action of several HOX genes. Especially in the case of MLL fusion proteins that activate expre...

SummaryOncogenic transcription factors such as the leukemic fusion protein RUNX1/ETO, which drives t(8;21) acute myeloid leukemia (AML), constitute cancer-specific but highly challenging therapeutic targets. We used epigenomic profiling data for an RNAi screen to interrogate the transcriptional network maintaining t(8;21) AML. This strategy identified Cyclin D2 (CCND2) as a crucial transmitter of RUNX1/ETO-driven leukemic propagation. RUNX1/ETO cooperates with AP-1 to drive CCND2 expression. Knockdown or pharmacological inhibition of CCND2 by an approved drug significantly impairs leukemic expansion of patient-derived AML cells and engraftment in immunodeficient murine hosts. Our data demonstrate that RUNX1/ETO maintains leukemia by promoting cell cycle progression and identifies G1 CCND-CDK complexes as promising therapeutic targets for treatment of RUNX1/ETO-driven AML.

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