A subset of chromosomal translocations in acute leukemias results in the fusion of the trithorax‐related protein HRX with a variety of heterologous proteins. In particular, leukemias with the t(11;19)(q23;p13.3) translocation express HRX–ENL fusion proteins and display features which suggest the malignant transformation of myeloid and/or lymphoid progenitor(s). To characterize directly the potential transforming effects of HRX–ENL on primitive hematopoietic precursors, the fusion cDNA was transduced by retroviral gene transfer into cell populations enriched in hematopoietic stem cells. The infected cells had a dramatically enhanced potential to generate myeloid colonies with primitive morphology in vitro. Primary colonies could be replated for at least three generations in vitro and established primitive myelomonocytic cell lines upon transfer into suspension cultures supplemented with interleukin‐3 and stem cell factor. Immortalized cells contained structurally intact HRX–ENL proviral DNA and expressed a low‐level of HRX–ENL mRNA. In contrast, wild‐type ENL or a deletion mutant of HRX–ENL lacking the ENL component did not demonstrate in vitro transforming capabilities. Immortalized cells or enriched primary hematopoietic stem cells transduced with HRX–ENL induced myeloid leukemias in syngeneic and SCID recipients. These studies demonstrate a direct role for HRX–ENL in the immortalization and leukemic transformation of a myeloid progenitor and support a gain‐of‐function mechanism for HRX–ENL‐mediated leukemogenesis.

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...

Conditional oncogenes provide a powerful tool for studying early events in transformation. Here we applied this approach to dissect the mechanism of leukemic transformation by MLL fusion proteins. MLL, located at 11q23, is rearranged in a variety of human acute lymphoid and myeloid leukemias. Our goals were to determine if MLL-induced growth transformation is reversible, to characterize the effect of MLL fusion protein expression on cell proliferation, differentiation, and survival in physiologically relevant cells, and finally to identify genes regulated by MLL fusion that are important for transformation.

Mixed lineage leukemia is a very aggressive blood cancer that predominantly occurs in pediatric patients. In contrast to other types of childhood acute leukemias, mixed lineage leukemia presents with a dismal prognosis and despite the availability of advanced treatment methods cure rates have stagnated over the last years. Mixed lineage leukemia is characterized by the presence of MLL fusion proteins that are the result of chromosomal translocations affecting the MLL gene at 11q23. These events juxtapose the amino-terminus of the histone methyltransferase MLL with a variety of different fusion partners that destroy normal histone methyltransferase function of MLL and replace it by heterologous functions contributed by the fusion partner. The resulting chimeras are transcriptional regulators that take control of targets normally controlled by MLL with the clustered HOX homeobox genes as prominent examples. Recent studies suggested that MLL fusion partners activate transcription by two different mechanisms. Some of these proteins are themselves chromatin modifiers that introduce histone acetylation whereas other fusion partners can recruit histone methyltransferases.In particular, histone H3 specific methylation at lysine 79 catalyzed by DOT1L has been recognized as a hallmark of chromatin activated by MLL fusion proteins. Interestingly, several frequent MLL fusion partners seem to coordinate DOT1L activity with a protein complex that stimulates the elongation phase of transcription by phosphorylating the carboxy-terminal repeat domain of RNA polymerase II. The discovery of these novel enzymatic activities that are essentially involved in MLL fusion protein function presents potential new targets for a rational drug development.