Regulated Hox Gene Expression Reveals a Novel Role for Hox Genes in Myeloid Cell Proliferation and Survival

Abstract

The Hox family of homeodomain transcription factors are essential for the regulation of hematopoiesis and deregulated expression of some Hox gene is associated with the development of myeloproliferative disorders and leukaemia. In mammals, 39 Hox genes are organized into four clusters (A, B, C or D). The expression of these genes is tightly regulated at particular differentiation points in haematopoiesis. Importantly, the over-expression of Hox genes, HoxB4, HoxA9 and HoxA10 is frequent in acute myeloid leukaemia, and may arise as a result of MLL rearrangements or from translocations fusing Hox genes to the nucleoporin Nup98. Overexpression of murine HoxB8 together with IL-3 results in myeloid leukaemia in mice. Primary myeloid progenitor cells can be immortalised using retroviral expression of Homeobox genes HoxB8 or HoxA9 in the presence of exogenous growth factors Interleukin-3 (IL-3) or GM-CSF. We have exploited this observation to generate IL-3 dependent cell lines from gene-deleted mice to identify which members of the Bcl-2 family of apoptosis regulators are required for apoptosis provoked by IL-3 deprivation (Blood, 2006 108:1461-8). Using a unique lentiviral expression system we have now generated IL-3 dependent myeloid progenitor cell lines in which we can regulate the expression of wild-type (untagged) HoxB8 or HoxA9 using 4-hydroxy tamoxifen (4HT), to determine how these genes immortalise myeloid cells. The mechanisms of action of Hox proteins in leukaemiagenesis remain to be determined but are thought, in part at least, to result from a block in myeloid differentiation. Conditional (growth-factor dependent) immortalisation of myeloid progenitors was possible only in the presence of induced Hox gene expression and surprisingly, withdrawal of HoxB8 expression did not result in terminal differentiation of all cells. Instead, loss of Hox expression, even in the presence of IL-3, induced Go/G1 cell cycle arrest and caspase-dependent cell death. This death was substantially slower that that induced by IL-3 deprivation, indicating that for some time at least, survival signals transduced by IL-3 remained intact. Thus whilst the IL-3 survival signal persisted, the proliferative signal was inhibited. We also show that HoxB8 regulates expression of the pro-apoptotic Bcl-2 family member Bim, since loss of HoxB8 resulted in substantially increased Bim expression and the cell death induced by loss of HoxB8 expression was inhibited in Bim-deficient cells. Importantly, re-addition of 4HT to cell cultures after various periods of no HoxB8 expression restored HoxB8 expression and resulted in an increase in cell viability, cell proliferation and decrease of Bim expression, indicating that at least some cells without HoxB8 expression have not terminally differentiated and retain the ability to proliferate. Our results suggest that overexpression of Hox genes such as HoxB8 (or HoxA9) contribute to myeloid transformation by coupling a growth factor signal to proliferation and also regulate the apoptotic machinery. Using this system will be able to provide proof of principal that leukemia-associated Hox genes are valid therapeutic targets.