Epigenetic and Transcriptional Regulation of Self Renewal in Acute Myeloid Leukemia

Abstract

Acute myeloid leukemia (AML) is diagnosed in >20,000 people/year in the United States alone and is associated with a poor prognosis. AML arises due to altered transcriptional programs resulting from mutations and chromosomal rearrangements. Frequently, this altered transcription is a consequence of epigenetic deregulation. Indeed, over 70% of AML patients harbor mutated epigenetic modifiers, which regulate chromatin accessibility and gene expression. Aberrant expression of the HOXA gene cluster, which can result from epigenetic deregulation, drives transformation of ~50% of AML, including those associated with poor prognosis. One manner in which the HOXA gene cluster becomes aberrantly expressed is through 11q23 chromosomal translocations involving the Mixed Lineage Leukemia 1 (MLL1) gene. These events result in the formation of fusion genes encoding MLL fusion oncoproteins which transcriptionally activate oncogenes, including the HOXA cluster. Our lab and others have demonstrated that the Polymerase Associated Factor complex (PAF1c), an epigenetic regulator complex, interacts directly with and recruits wildtype MLL1 and MLL-fusion oncoproteins to target loci like HOXA9 and MEIS1. The PAF1c-MLL interaction is required for leukemia cell proliferation, but dispensable for normal hematopoiesis. Mutations and aberrant expression of subunits of the PAF1c are observed in various malignancies, suggesting that the PAF1c must be tightly regulated for proper cellular development. However, the biochemical regulation of the PAF1c that allows for its dynamic regulation of gene expression in AML is not fully understood. To better understand the regulation of the PAF1c, we use a proteomics approach to identify novel interaction partners of the PAF1c in AML cells. This study reveals a novel interaction between the PAF1c and the H3K9 methyltransferase SETDB1. The PAF1c-SETDB1 interaction represses the target genes Hoxa9 and Meis1 in murine MLL-AF9 driven leukemic cells and human AML cell lines. SETDB1 mediated transcriptional repression is correlated with an increase in promoter H3K9 trimethylation (H3K9me3). These data suggest that SETDB1 epigenetically represses pro-leukemic gene expression in AML. Therefore, we next explore the biological impact of SETDB1 expression and H3K9 methylation on AML. We note that expression of SETDB1 in AML patient samples is significantly lower compared to normal hematopoietic cells. Further, higher SETDB1 expression correlates with a significantly better overall survival in AML patients. These data are consistent with SETDB1 negatively regulating pro­leukemic genes and suggests that SETDB1 expression and H3K9 methylation levels may be correlated with AML patient prognosis. We demonstrate that overexpression of SETDB1 significantly delays MLL­AF9 mediated leukemogenesis in vivo by inducing differentiation of leukemic cells. We also explore how chemical inhibition of H3K9 methylation affects AML transformation. Treatment with H3K9 methyltransferase inhibitor UNC0638 is antagonistic to established AML cell growth. In contrast, UNC0638 preserves mouse hematopoietic stem and progenitor cells (HSPCs) in culture and increases the amenability of bone marrow cells to be transformed by the MLL-AF9 oncogene. Transcriptome analyses demonstrate that overexpression of SETDB1 downregulates Hoxa and pluripotency gene programs. ChIP-sequencing and ATAC-sequencing of AML cells show that overexpression of SETDB1 leads to the acquisition of a more compact, epigenetically silenced chromatin state at the promoters of genes that are critical for AML, including Dock1 and MLL-AF9 target genes Hoxa9 and Six1, and others. Together, these data reveal a previously unrecognized role for SETDB1 and H3K9 methylation in suppressing AML by epigenetically silencing pro-leukemic target genes and promoting differentiation.