SETDB1 Mediated H3K9 Methylation Suppresses MLL-Fusion Target Expression and Leukemic Transformation
Authors: Muntean Andrew G. , Ropa James , SAHA Nirmalya , Hu Hsiang-Yu , Serio Justin , Zhao Lili , Basrur Venkatesha , Nesvizhskii Alexei , Maillard Ivan 
Published: February 2020

Genes encoding epigenetic proteins are mutated in about 70% of Acute Myeloid Leukemia patients underscoring their role in leukemic transformation. Epigenetic deregulation of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia (AML), such as those harboring MLL rearrangements, NPM mutations and others. Indeed, expression of Hoxa9 and its co-factor Meis1 is sufficient to transform bone marrow cells into a lethal AML. We have shown that epigenetic regulation of Hoxa9 and Meis1 is mediated through the Polymerase Associated Factor 1 complex (PAF1c). The PAF1c binds to RNA polymerase II and recruits enzymes that alter epigenetic landscapes and influence gene transcription. We and others have shown that the PAF1c directly binds and recruits MLL and MLL fusion proteins to target genes such as Hoxa9 and Meis1 to deliver H3K4 methylation or H3K79 methylation activity respectively. Importantly, the PAF1c-MLL1 interaction is necessary for the growth of MLL-fusion leukemia cells but appears dispensable for normal hematopoietic stem and progenitor cells (HSPCs) function. This suggests the PAF1c is differentially required in leukemic cells compared to its normal counterparts. Thus, we aimed to 1) define the role of the PAF1c in normal hematopoiesis, and 2) identify mechanisms regulating PAF1c function that may be deregulated in leukemia. To understand the role of the PAF1c in hematopoiesis, we conditionally deleted the PAF1c subunit Cdc73 in HSPCs in hematopoietic cells. Conditional hematopoietic deletion of Cdc73 leads to lethality within a week due to a depletion of c-kit+ cells. Characterization revealed a cell autonomous requirement for Cdc73 to prevent apoptosis in HSPCs. Gene expression profiling on both c-kit+ hematopoietic cells and AML cells revealed that Hoxa9/Meis1 gene programs are differentially responsive to loss of Cdc73. This work revealed an essential role for the PAF1c subunit in maintaining viability of HSPCs and points to AML specific functions for the PAF1c. Thus, we utilized immunoprecipitation followed by mass spectroscopy to identify novel PAF1c interactions in AML cells. We found a novel interaction with several H3K9 methyltransferases including SETDB1. We demonstrate that the PAF1c-SETDB1 interaction reduces expression of Hoxa9 and Meis1 by promoting deposition of H3K9me3. These findings mirrored human AML patient samples that showed SETDB1 expression was inversely correlated with HOXA9 and MEIS1 expression. Further, SETDB1 expression is correlated to disease status and overall survival in AML patients. We recapitulated these findings in mice, where high expression of SETDB1 delayed MLL-AF9 mediated disease progression by promoting differentiation of AML cells. We also explored the biological impact of treating normal and malignant hematopoietic cells with an H3K9 methyltransferase inhibitor, UNC0638. While AML cells demonstrate cytotoxicity to UNC0638 treatment, normal bone marrow cells exhibit an expansion of HSPCs. Consistent with these data, we show that bone marrow treated with UNC0638 is more amenable to transformation by MLL-AF9. Next generation sequencing of AML cells shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with AML, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in AML that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing AML.

Disclosures
Maillard: Genentech: Consultancy; Regeneron: Consultancy.