Somatic ASXL1 mutations in chronic myelomonocytic leukemia result in loss of imprinting control and overexpression of DLK1 through deregulation of DNA methylation

Abstract Introduction: Protein truncating somatic ASXL1 mutations in CMML are associated with high-risk features and poor outcomes. We have previously demonstrated that ASXL1-mutant (mt) CMML is characterized by permissive chromatin states and overexpression of leukemia stemness genes (Binder et al. Nat Commun; 2022). DLK1 (delta-like homolog 1) is a pseudo-NOTCH like ligand that is maternally imprinted and paternally expressed and is physiologically not expressed beyond embryogenesis. Aberrant DLK1 expression has been associated with poor outcomes in solid tumors and we have shown differential overexpression in ASXL1mt versus wild-type (wt) CMML. Both the promoter and the last exon of DLK1 contain CpG islands that play a role in regulating DLK1. The CpG island within the fifth exon of DLK1, known as the DLK1-differentially methylated region (DMR) [chr14:100,726,892-100,738,224] is completely unmethylated on the maternal allele and partially methylated on the paternal allele. Previous work (Developmental Cell; 2021) has also identified a DLK1 imprinting control region (ICR) known as the IG-DMR [chr14:100,811,001–100,811,037]. DNA methylation state of the IG-DMR has been shown to impact the expression of MEG3 mRNA (lncRNA) which functions as a cofactor to direct PRC2 to the DLK1 promoter, suppressing DLK1 expression. Since the regulation of DLK1 in ASXL1mt CMML is not well understood, we conducted this multiomic analyses to define epigenetic mechanisms of DLK1 overexpression in ASXL1mt CMML. Methods: After IRB approval, we performed bulk RNA-seq, ChIP-seq (H3K4me1/me3, H3K27ac, H3K27me3) and Illumina MethylationEPIC array on bone marrow (BM) mononuclear cells of 40 CMML patients (19 ASXL1mt, 21 wt). scRNA-seq was conducted on 5 ASXL1mt and 3 wt cases. All libraries were sequenced on an Illumina NovaSeq instrument. We assessed these samples for differential gene expression (RNA-seq) and changes in histone modifications (ChIP-seq) between ASXL1mt and wt CMML, specifically at the DLK1-DMR and IG-DMRregulatory loci. We conducted ddPCR (digital droplet PCR) to determine allelic status of DLK1 in ASXL1mtand wt cases. Global and allele/sequence specific DNA methylation changes were determined using Biomodal's duet EvoC methodology (6 base sequencing, including 5mC and 5hmC) in 5 ASXL1mt and 5 wt CMML cases. Results: We validated the overexpression of DLK1 in 19 ASXL1mt versus 21 wt (log2 fold change +4.85, FDR 4.67e-13) CMML BM samples. scRNA-seq on 5 ASXL1mt versus 3 wt samples demonstrated that DLK1 is only expressed in mutant cases, with the expression being limited to HSPCs. ChIP-seq analysis at the DLK1 regulatory loci validated (a) reduced occupancy of H3K27me3, correlating with ASXL1mt associated disruption of the PRC2-complex, and (b) increased co-occupancy of H3K4me1 and H3K27ac in mt cases, validating a permissive chromatin state in ASXL1mt cases at the DLK1 regulatory regions. Through correlation analysis of RNA-seq and ChIP-seq, we defined the promoter for DLK1, characterized by high Pearson correlation coefficients (R > 0.5) and enrichment of H3K27ac and H3K4me3. Differential methylation analysis identified global hypomethylation around the DLK1-DMR and promoter regions. We observed a mean reduction of ~40% in methylation status at the DLK1 promoter in ASXL1mt versus wt cases, suggesting a loss of epigenetic silencing at this locus. This was corroborated by significant enrichment of 5hmC at the fifth exon of DLK1 indicating active DNA demethylation. Conversely, we observed a gain in methylation at the IG-DMR, suggesting loss of imprinting control through MEG3 silencing and DLK1 overexpression. To further determine the impact of imprinting deregulation on overexpression of DLK1 in ASXL1mt CMML, allele-specific methylation was profiled using the Biomodal whole genome methylation data. This demonstrated that the methylation patterns in ASXL1mt CMML cases were significantly deregulated at the DLK1-DMR, with >30% loss of methylation compared to GRCh38 reference alleles. Furthermore, ddPCR was able to confirm bi-allelic expression of DLK1 in ASXL1mt CMML, suggesting that the observed methylation dysregulation leads to loss of imprinting of the maternal allele. Conclusion: In summary, we demonstrate that ASXL1 mutations in CMML disrupt epigenetic pathways, promoting DNA demethylation, resulting in permissive chromatin states and an aberrant and oncogenic re-expression of maternal DLK1, due to loss of imprinting control.