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...
The mixed lineage leukemia (MLL) family of histone methyltransferases has become notorious for the participation of the founding member MLL in fusion proteins that cause acute leukemia. Despite a high overall structural conservation no other MLL homolog has been found involved in leukemogenic fusion proteins. This surprising fact might be either due to a relative genomic stability of the MLL2 locus that prevents chromosomal translocations or MLL2 might be altogether incapable of contributing to oncogenic fusions because of functional constraints. Here we demonstrate the latter to be true for fusion proteins based on MLL2, the closest relative of MLL. A MLL2-ENL protein constructed in analogy to the highly leukemogenic MLL-ENL was incapable of transforming primary hematopoietic cells. Elaborate swap experiments identified the zinc binding “CxxC”- region of MLL2 and an adjacent “post-CxxC” stretch of basic amino acids as the essential determinants for the observed difference between MLL and MLL2. Surprisingly gel shift experiments indicated that the CxxC and post-CxxC domains of MLL and MLL2 conferred almost indistinguishable in vitro DNA binding properties. However, in vivo these motifs guided MLL-ENL and MLL2-ENL to a largely non-overlapping gene repertoire. Divergent nuclear localization, a distinct potency to activate a MLL model promoter in reporter assays, and measurements of homeobox gene levels in primary cells expressing MLL and MLL2 fusion proteins all argued for a separate target site selection of the two fusions. Therefore, the CxxC domain appears to be largely responsible for target finding and as a consequence this domain is a promising object for therapies aimed at MLL fusion proteins without affecting the general function of other MLL family members.
2962 Poster Board II-938 HOX homeobox genes are important regulators of normal and malignant hematopoiesis and abdominal-type HOXA-cluster genes, in particular HOXA7 to HOXA10, are highly leukemogenic. However, little is known about the transforming abilities of anterior HOXA genes HOXA1 to HOXA6 despite a high prevalence of anterior HOX-expression in acute leukemia. Here we performed a comprehensive assessment of the oncogenic potential of every HOXA gene in primary hematopoietic cells. With exception of HOXA2 and HOXA5 all other HOXA genes caused a block or delay of hematopoietic differentiation in serial replating assays and all HOX genes also cooperated with Meis1. No evidence for the alleged tumor-suppressor function of HOXA5 could be found. Whereas all other active HOXA genes induced the outgrowth of mixed granulocytic/monocytic cells, HOXA13 preferentially specified the development of monocytes/macrophages. Albeit more weakly than HOXA9 also the anterior HOXA genes HOXA1, HOXA4, and HOXA6 transformed cells and generated permanent myelomonocytic cell lines arrested at various stages of differentiation. HOX-RNA profiling revealed that each of these lines also transcribed HOXA9. However, kinetic studies with inducible HOX-derivatives showed that HOXA9 expression was not under control by anterior HOXA proteins. Cellular differentiation was induced immediately after loss of anterior HOX activity and HOXA9 down regulation was a secondary event during maturation. This proves that anterior HOX proteins are able to transform hematopoetic cells through an individual contribution independent of HOXA9. In summary our results explain how HOXA9 can predominate in hematologic malignancies while simultaneously it is not necessarily required for transformation. Disclosures: No relevant conflicts of interest to declare.
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