Hoxa9 influences the phenotype but not the incidence of Mll-AF9 fusion gene leukemia

Abstract

Identification of the targets of mixed lineage leukemia (MLL) fusion genes will assist in understanding the biology of MLL fusion gene leukemias and in development of better therapies. Numerous studies have implicated HOXA9 as one of the possible targets of MLL fusion proteins. To determine if HOXA9 was required for leukemia development by MLL fusion genes, we compared the effects of the Mll-AF9 knock-in mutation in mice in the presence or absence of Hoxa9. Both groups of mice showed myeloid expansion at 8 weeks and then developed myeloid leukemia with a similar incidence and time course. The leukemia in the mice lacking Hoxa9 generally displayed a more immature myeloid phenotype than that in the mice that were wild-type for Hoxa9. Gene expression profiling revealed that expression of Mll-AF9 led to overexpression of Hoxa5, Hoxa6, Hoxa7, Hoxa9, and Hoxa10. Thus, genes of the Hox-a cluster are important in defining the phenotype but not the incidence of Mll-AF9 leukemia. These results demonstrate that the Mll-AF9 fusion gene disrupts the expression of several Hox genes, none of which as a single gene is likely to be necessary for development of leukemia. Instead, we propose that the "Hox code" minimally defined by the Hoxa5-a9 cluster is central to MLL leukemogenesis. ( IntroductionTranslocations involving the mixed lineage leukemia (MLL, ALL-1, HRX) gene are encountered in both myeloid and lymphoid leukemias. These MLL leukemias are often found in infants and also in adults previously treated with chemotherapy for other cancers. 1 The mechanisms by which the translocations cause leukemia remain unknown. Gene expression profile studies demonstrate that lymphoid leukemias with MLL rearrangements exhibit an increase in expression of certain homeobox (HOX) genes compared with phenotype-matched leukemias without MLL rearrangements. [2][3][4][5] The HOXA9 gene may hold an important key to the MLL leukemias because it is the one homeobox gene most frequently overexpressed in these leukemias. 5 Recent evidence also indicates that MLL is part of a multiprotein complex that regulates the transcription of HOXA9 by directly binding to promoter sequences. 6,7 Overexpression of Hoxa9 is also known to transform primary myeloid bone marrow cells. [8][9][10] HOXA9 is directly involved in human leukemia caused by the NUP98-HOXA9 fusion gene 11 and in the BXH-2 mouse model of leukemia. 12 This encouraged us to study the relationship between Hoxa9 and Mll fusion genes and to ask whether Hoxa9 is necessary for the development of Mll leukemia. We were able to test this hypothesis in Mll-AF9 ϩ/Ϫ /Hoxa9 Ϫ/Ϫ mice. Mice expressing Mll-AF9 as a heterozygous knock-in mutation develop myeloid leukemia. 13 The leukemia in these mice occurs with a latency period of about 6 months and is preceded by a preleukemic phase characterized by expansion of myeloid precursors. 14, 15 We compared the Mll-AF9-mediated myeloid expansion and leukemia development in the presence and absence of Hoxa9.To identify other Hox genes that may be involved in leuke...

Epigenetic regulations in hematopoietic Hox code

“…On one hand, Hox genes are orderly activated in normal hematopoietic progenitors and preleukemia cells (Kumar et al, 2004;Yan et al, 2006a), in accordance with the colinear expression pattern that is observed during embryonic development. One the other hand, the expression patterns of paralogous Hox genes seen in embryonic development are not obvious in hematopoietic progenitors and cell lines, but rather whole clusters (or large regions of a cluster) are turned on or off in a lineage-specific manner.…”

“…In accordance with chromatin domain, 5 0 HoxA cluster genes are sequentially activated in MLL-AF9-transduced hematopoietic cells. All 5 0 HoxA cluster genes (Hoxa5-13) are hyperexpressed in MLL-AF9 leukemia cells, whereas only Hoxa5-9 genes are activated in MLL-AF9 preleukemic cells (Kumar et al, 2004;Yokoyama et al, 2005;Yan et al, 2006a). These data suggest that 5 0 HoxA cluster genes are divided into at least two chromatin domains, whereas Hoxa5-9 genes are transcriptionally delimited from Hoxa10-13 by a speculated insulator.…”

“…It is easily concluded that chromosomal translocation results in development of MLL fusion oncogenes and accordingly, mis-expression of Hox genes. However, numerous studies have shown that overexpression of MLL fusion oncoproteins in normal ES and bone marrow cells first promotes nonmalignant expansion of myeloid precursors and Hox deregulation, and then is followed by accumulation of malignant cells (Dobson et al, 1999;Kumar et al, 2004;Yokoyama et al, 2005;Chen et al, 2006). The long latency in vivo and monoclonal nature of the leukemias suggest that secondary genetic transformation is required for the development of leukemia (Dobson et al, 1999;Johnson et al, 2003).…”

Hox genes in hematopoiesis and leukemogenesis

“…Moreover, MLL-ENL immortalization of myeloid progenitor cells could be substituted for by overexpression of HoxA9 and Meis1 (Zeisig et al, 2004), and was dependent on the activity of the oncoprotein c-Myc (Schreiner et al, 2001), a downstream effector of Hoxb4 that is sufficient to induce HSC self-renewal (Satoh et al, 2004). By contrast, analogous studies in a germline mouse model of Mll-AF9-mediated leukemogenesis suggest that Hoxa9 is not necessary for leukemia but does influence the phenotype of the leukemia (Kumar et al, 2004). Gene expression profiling of Mll-AF9-expressing BM cells revealed overexpression of the Hoxa cluster genes, Hoxa5 to Hoxa9, which the authors propose represents the Mll-AF9-triggered 'Hox code' that defines this oncogene.…”

ALL-1/MLL1, a homologue of Drosophila TRITHORAX, modifies chromatin and is directly involved in infant acute leukaemia

“…Further, HOX A9 was found to be indispensable for ALL-1-dependent leukaemogenesis in vivo. Applying a complementary approach, Kumar et al (2004) bred mice to obtain animals transgenic for MLL-AF9 and null for HOX A9 and examined leukaemia development. Surprisingly, the absence of HOX A9 did not affect the incidence and latency of leukaemia, although the malignant cells displayed a more immature myeloid phenotype.…”