Poster Presentation Australian Epigenetics Alliance Conference 2022

Unravelling epigenetic heterogeneity induced by DNA hypomethylating agents in acute myeloid leukaemia (#101)

Danielle R Bond 1 2 , Kumar Uddipto 1 2 , Kooper V Hunt 1 2 , Sean M Burnard 1 2 , Ellise A Roper 1 2 , Sam Humphries 1 2 , Carlos Riveros 1 , Matthew D Dun 1 2 , Nicole M Verrills 1 2 , Anoop K Enjeti 1 3 4 5 , Heather J Lee 1 2
  1. Hunter Medical Research Institute, New Lambton Heights, NSW, Australia
  2. School of Biomedical Sciences & Pharmacy, The University of Newcastle, Callaghan, NSW, Australia
  3. NSW Health Pathology North, John Hunter Campus, New Lambton Heights, NSW, Australia
  4. School of Medicine and Public Health, The University of Newcastle, Callaghan, NSW, Australia
  5. Calvary Mater Newcastle, Waratah, NSW, Australia

DNA hypomethylating agents (HMAs) are epigenetic therapies used to treat acute myeloid leukaemia (AML) patients who are unfit for cytotoxic chemotherapy. However, acquired resistance and relapse are ongoing challenges with clinical HMA use.

Reasoning that relapse could originate from individual cells able to evade therapy, we performed single-cell multi-omics analysis coupled with colony forming assays to characterise the heterogeneous response of AML cells to HMAs. HL60 cells were treated with HMAs (2000nM azacytidine (AZA) or 100nM decitabine (DAC)) for 72h, and single-cell nucleosome, methylome and transcriptome sequencing (scNMT-seq) was performed on indexed FACS sorted single cells. HMAs induced global epigenetic heterogeneity with variable single-cell DNA methylation levels (17-69%), observed across all genomic contexts, and did not target specific biological pathways. Genome-wide associations between DNA methylation, chromatin accessibility and gene expression were weakened by HMA treatment, proving that global methylation loss is insufficient for gene activation.

A small proportion of cells (5-10%) retained DNA methylation at levels similar to untreated cells, and these “methylation retaining” cells appeared to proliferate less during treatment as evidenced by CellTrace fluorescence levels. Colony forming assays were performed to test whether “methylation retaining” cells had a growth advantage following treatment cessation. DAC and AZA treated cells formed fewer colonies than untreated cells (60-70% decrease), with variable levels of global DNA methylation across individual colonies (17-86%). The proportion of colonies with high DNA methylation (comparable to untreated cells) was much greater for DAC than AZA treated cells (35% vs 2%), suggesting that “methylation retaining” cells may have a growth advantage specifically following DAC treatment. DAC treated “methylation retaining” colonies showed upregulation of cholesterol biosynthesis, and HMAs in combination with statins were able to further decrease colony forming capacity in vitro.

In conclusion, epigenetic heterogeneity induced by HMA therapy may have important clinical implications in AML.