Dot1l Reverses the Abnormal Self-Renewal Conferred on HSPCs through Cohesin Haploinsufficiency By Diminishing Expression of Hoxa7 and Hoxa9

Abstract

Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy with a poor prognosis. Developing novel treatments for AML has been difficult due to the heterogeneity of genetic mutations observed across patients. Recent genomic sequencing of AML patients have identified recurrent somatic mutations in cohesin complex genes in a subset of patients (10-20%). The canonical function of the cohesin complex is to maintain sister chromatid cohesion permitting proper segregation of genomic information to each daughter cell in mitosis. Complete loss of cohesin function causes an early embryonic lethal phenotype in knock-out mice, implying that some level of cohesin function is absolutely required for normal development. Interestingly, somatic cohesin mutations in AML are uniformly heterozygous and not associated with dramatic changes in chromosomal number, implying that cohesin mutations promote AML through a function independent of their role during mitosis.Recent investigations have demonstrated that cohesin haploinsufficiency leads to enhanced self-renewal in Hematopoietic Stem and Progenitor Cells (HSPCs). Previous studies from our lab identified significant increases in the HSPC self-renewal factors Hoxa7 and Hoxa9 in Rad21 -depleted HSPCs concomitant with decreased levels of the repressive histone mark trimethylated lysine 27 on histone H3 (H3K27me3) at the Hoxa7 and Hoxa9 promoters, indicating that Rad21 plays a role in proper PRC2-mediated silencing of these genes. Immunoprecipitation and ChIP experiments revealed that cohesin and PRC2 directly interact and are bound in close proximity to Hoxa7 and Hoxa9 . Interestingly genome-wide levels of PRC2's repressive histone mark H3K27me3 were reduced. Importantly, H3K27me3 levels were significantly decreased at the Hoxa7 / Hoxa9 locus. Knockdown of either Hoxa7 or Hoxa9 in Rad21 -depleted HSPCs suppressed their self-renewal, implying both are critical downstream effectors of reduced cohesin levels.In the present study we sought to delineate additional molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal and evaluate possible therapeutic interventions to reverse the phenotype conferred on HSPCs by cohesin haploinsufficiency. We find that reduction of the core cohesin subunit Rad21 using RNAi increases levels of dimethylated lysine 79 on histone H3 (H3K79me2) genome-wide in contrast to the genome-wide reduction of H3K27me3 levels. H3K79me2 is deposited by the Dot1l chromatin modifying complex and is correlated with active gene expression. Given the established role of Dot1l in regulating Hoxa9 expression, we sought to evaluate whether inhibiting Dot1l methyltransferase activity would reverse the phenotype caused by cohesin haploinsufficiency. As anticipated, we observed that inhibition of Dot1l function, using either of two commercially available inhibitors or RNAi mediated knockdown, reverses the enhanced self-renewal capacity of cohesin haploinsufficient HSPCs. Consistent with our proposed mechanism, this reduced self-renewal occurs with a concomitant reduction in Hoxa7 and Hoxa9 expression. We observe a similar trend using HSPCs from Smc3 conditional knockout mice, a more elegant genetic model. RNA-sequencing and ChIP-sequencing studies are ongoing to evaluate the transcriptome and epigenomic changes imparted by cohesin haploinsufficiency and how these changes are affected by Dot1l inhibition. Our preliminary studies are intriguing because cohesin haploinsufficiency appears to have contrasting effects on genome-wide levels H3K27me3 and H3K79me2, two opposing epigenetic marks. In conclusion, our data demonstrate that Dot1l inhibition negatively regulates self-renewal of cohesin haploinsufficient HSPCs, but not control HSPCs, by abbrogating the abberant expression of Hoxa7 and Hoxa9 . This study establishes feasibility for the use of Dot1l inhibition as a targeted therapeutic treatment for AML patients harboring cohesin mutations. DisclosuresLevine:Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Celgene: Research Funding.