The Mechanisms of HOXA9-Mediated Oncogenic Transformation

Abstract

Hox genes encode a family of homeodomain-containing transcription factors that are critical for body plan specification and tissue morphogenesis during embryonic development. Hoxa9, in particular, is required for adult hematopoiesis in which it promotes stem cell renewal and expansion. Most importantly, Hoxa9 is commonly dysregulated in various types of acute leukemia, including acute myeloid leukemia (AML), and T- and B-precursor acute lymphoblastic leukemia (B-ALL and T-ALL). Together with its co-factor MEIS1, HOXA9 plays a causal role in driving leukemic transformation. Hoxa9 dysregulation is also linked to various types of solid tumors, and both gain and loss of function have been implicated in tumorigenesis. Despite its central role, the mechanism through which HOXA9 mediates oncogenic transformation remains poorly understood. Previous work in our lab found that in a HOXA9/MEIS1-driven AML cell line, HOXA9 primarily binds to promoter-distal regions of the genome. Its target regions predominately carry the epigenetic signatures indicative of active enhancers. A substantial portion of HOXA9 binding sites are co-occupied by lineage-determining factors, such as C/EBPα and PU.1. However, it remains unknown 1) whether HOXA9 drives the formation of active enhancers and globally alters the enhancer landscape; 2) whether HOXA9 strictly acts downstream of other transcription factors, or it can play a pioneer role and acts upstream of all other transcription factors and chromatin regulators; 3) if its regulatory functions are conserved in other cell lineages. To address these questions, I show that in the myeloid lineage, HOXA9/MEIS1-transformed cells are characterized by significant alterations of the enhancer landscape and exhibit prominent emergence of de novo enhancers. These de novo enhancers are absent of enhancer modifications in any hematopoietic cells, and are associated with activation of a leukemia-specific transcription program. HOXA9 acts as a pioneer factor at these de novo regions and is required for the recruitment of myeloid lineage factor C/EBPα while it is dispensable for the formation of the normal hematopoietic enhancers. Together, these results suggest an active role of HOXA9 in altering enhancer landscapes during leukemic transformation. To explore the mechanisms of HOXA9-mediated enhancer formation, I assessed the role of the histone H3K4 methyltransferase MLL3/MLL4 complex in this alteration of enhancer landscape. Using immunoprecipitation and ChIP-seq analysis, I found physical interaction between HOXA9 and the MLL3/MLL4 complex. In addition, I determined that the MLL3/MLL4 complex is required for formation of de novo enhancers, as well as for in vivo leukemogenesis driven by HOXA9/MEIS1. Collectively, these findings provide strong evidence for an essential role for the MLL3/MLL4 complex in HOXA9-mediated leukemic transformation. I have also collected preliminary data pertaining to HOXA9’s function in other cell lineages. I found that HOXA9 localizes to active enhancer regions in B-lineage leukemia cells and reshape the enhancer landscape; hence, confirming HOXA9’s enhancer binding characteristics. Furthermore, I discovered that HOXA9 efficiently blocks the adipogenic program in pre-adipocytes by preventing the upregulation of the key adipogenesis factor, Pparg. These data highlight a coherent role for HOXA9 in regulating gene expression and modulating cellular differentiation across different lineages. In summary, this dissertation reveals a previously uncharacterized role of HOXA9 in leukemogenesis and cellular transformation, and provides a strong rationale for targeting the HOXA9-collaborating chromatin modulators, as well as the leukemia-specific enhancers, for the therapeutic development of acute leukemia.PHDMolecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138797/1/yuqings_1.pd