MLL-Rearranged Leukemias Are Dependent On the Histone H2B Ubiquitin Ligase RNF20

Abstract 649

Histone mono-ubiquitination is a major signaling mechanism that shapes the structure and function of chromatin. Hence, factors that regulate ubiquitin attachment to histones (E2 conjugating enzymes, E3 ligases, and de-ubiquitinating enzymes) often play fundamental roles in epigenetic biological processes, including the pathophysiology of human disease. Here, we explored whether the known regulators of histone ubiquitination/deubiquitination are linked to the pathogenesis of acute myeloid leukemia, a disease often associated with aberrant chromatin signaling. To this end, an shRNA screen was performed where all histone E2s, E3s, and deubiqitinating enzymes were individually suppressed and evaluated for their role in supporting growth of acute myeloid leukemias initiated the MLL-AF9 oncogene. This analysis identified the histone H2B ubiquitin ligase RNF20 (also known as BRE1A) as essential for MLL-AF9 leukemia growth. This finding was verified in a genetically engineered mouse leukemia model (driven by MLL-AF9 together with Nras^G12D) under both in vitro and in vivo conditions. In addition, human leukemia cell lines harboring MLL-rearrangements were found to be uniformly dependent on RNF20 for their proliferation. Upon inhibition of RNF20 and elimination of global H2B mono-ubiquitination, leukemia cells arrest in the G1/G0 stage of the cell-cycle without evidence of myeloid maturation. RNA-SEQ analysis identified that a suite of direct transcriptional targets of MLL-AF9 were downregulated in expression upon RNF20 inhibition, including Hoxa9, Hoxa10, Meis1, and Mef2c. While H2B mono-ubiquitination has been shown previously to promote the catalytic function of H3K4 and H3K79 methyltransferases, we find that in leukemia cells inhibition of RNF20 eradicates global H2B mono-ubiquitination without influencing global H3K4me3 and H3K79me2/me3. However, we find instead that RNF20 is selectively required to maintain local levels of H3K79 methylation in the body of MLL-AF9 target genes, such as Hoxa9. Since MLL-AF9 is known to directly recruit the H3K79 methyltransferase DOT1L to transactivate its target genes, our findings suggest a mechanism whereby targeting RNF20 interferes with the transactivation function of MLL-AF9. Moreover, we found that shRNA-based suppression of RNF20 sensitizes leukemia cells to the growth-arrest induced by the BET bromodomain inhibitor JQ1, without influencing the sensitivity to cytotoxic agents daunorubicin and etoposide. Based on these collective findings, we propose that RNF20 represents a major chromatin-based signaling pathway necessary to maintain the MLL-rearranged subtype of acute myeloid leukemia.