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 have performed single-cell multi-omic analysis coupled with colony forming assays to characterise the heterogeneous response of AML cells to HMAs.
AML cell lines were stained with CellTrace proliferation marker and treated with HMAs (2000nM azacytidine, AZA; or 100nM decitabine, DAC) for 72h. Single cells were collected by indexed fluorescence activated cell sorting and single-cell nucleosome, methylome and transcriptome sequencing (scNMT-seq) was performed. HMA treatment induced global epigenetic heterogeneity with variable single-cell DNA methylation levels. Heterogeneous loss of DNA methylation was observed in all genomic contexts and did not target specific cellular processes. Genome-wide associations between DNA methylation, chromatin accessibility and gene expression were weakened by HMA treatment. Nonetheless, heterogeneous loss of DNA methylation was correlated to changes in gene expression at several loci of interest.
This analysis also identified rare ‘methylation-retaining’ cells able to evade HMA treatment, and analysis of CellTrace fluorescence demonstrated that these cells proliferated less during treatment. To test whether ’methylation-retaining’ cells had a growth advantage following treatment cessation, we performed colony forming assays. DAC and AZA treated cells formed fewer colonies than untreated cells, and global DNA methylation levels varied between individual colonies. The proportion of colonies with high methylation levels was greater following DAC than AZA treatment, suggesting that ’methylation-retaining’ cells have a growth advantage following DAC. Matched transcriptional profiling of individual colonies identified cholesterol biosynthesis as a potential mechanism by which ‘methylation-retaining’ cells adapt to DAC treatment. Ongoing experiments are assessing the ability of statins to increase DAC efficacy by inhibiting cholesterol biosynthesis.
In conclusion, HMA-induced epigenetic heterogeneity has consequences for AML cell growth. A thorough understanding of the adaptive processes linked to heterogeneous epigenetic states will lead to new co-treatment strategies to prevent or delay AML relapse.