ASXL1 Mutations Are Associated with Widespread and Distinct DNA Methylation Alterations

Background:ASXL1 is frequently mutated in a range of myeloid malignancies, including MDS, CMML, and AML, and is strongly associated with a poor prognosis in these myeloid disorders. ASXL1 is known to play a central role in epigenetic regulation, activating or repressing the transcription of genes involved in either differentiation or proliferation through its effects on histone methylation marks. In particular, ASXL1 is thought to be involved in recruiting PRC2 for its role in epigenetic modification. Even though DNA methylation is critically important in regulating gene expression in hematopoiesis, the effect of ASXL1 mutations on cytosine methylation is not well elucidated.

Methods: We have previously studied the effects of the ASXL1 mutation through the use of the KBM5 CML cell line, which carries a homozygous nonsense ASXL1^G710X mutation, resulting in loss of ASXL1 protein expression (Valetta et al, Oncotarget, 2015) . In particular, we saw that the ASXL1 mutation results in failure of PRC2 to recruit the HOXA cluster, resulting in increased expression of HOXA genes and resultant myeloid transformation. We used CRISPR/Cas9 mediated correction of the ASXL1 mutation in the leukemic KBM5 cell line to generate isogenic cell lines for methylome, expression, and chromatin accessibility analysis.

Results: We have previously shown that correction of the ASXL1^G710X mutation in KBM5 cells by CRISPR gene editing restored ASXL1 protein expression, with restored function of PRC2, including HOXA gene down-regulation with resultant reduced cell growth and increased myeloid differentiation, as evidenced by increased expression of CD11b, CD14 and CD15 by flow cytometry. In this current study, RNA-seq performed on the isogenic KBM5 cells confirmed the expression changes and CRISPR mediated correction of the ASXL1 mutation. Methylation DNA immunoprecipitation demonstrated widespread increased cytosine methylation in the KBM5 ASXL1 mutated cell line when compared to the corrected wild type cells. Evaluating this data alongside the RNA sequencing data, a clear difference in expression was noted between the genes that have methylation peaks in the gene promoter versus those that have peaks in the gene bodies. Specifically, increased methylation of the promoter region of genes was associated with decreased gene expression, while increased methylation of the gene body was associated with increased gene expression. Promoter methylation was found to be associated with down-regulation of differentiation associated CD14 . Gene body methylation was seen in widely over-expressed HOXA cluster transcripts and CD15. ATAC-seq demonstrated corresponding changes in chromatin accessibility. Due to the increased methylation in the mutant cells, we next treated the isogenic KBM5 cells with the DNMT inhibitor 5-Azacytidine. Proliferation assays of the ASXL1 mutated and corrected cells revealed significantly decreased cell viability of the KBM5 mutated cells with concordant increase in differentiation. To validate the results in primary samples, we assessed DNA methylation in a cohort of MDS ASXL1 mutant and wild type peripheral blood mononuclear samples. The HELP assay revealed increased methylation in ASXL1 mutant samples particularly in the gene bodies, consistent with the results seen in the isogenic cell line.

Conclusions: It has been hypothesized that the ASXL1 mutation results in hypomethylation with resultant increased expression of genes, as previously seen with increased expression of the HOXA gene cluster in ASXL1 mutated cells. Our current study demonstrates increased methylation of the ASXL1 mutant versus the corrected cell line and reveals a significant amount of gene body methylation in the mutant cells. Specifically, we demonstrate that aberrant methylation in mutant cells provides a therapeutic window for DNMT inhibitors and is associated with site specific associations with chromatin accessibility and gene expression. Our study reinforces that high resolution whole genome methylation studies are needed to dissect the specific effects of mutations in epigenetic regulators such as ASXL1 in MDS and AML.