Upregulated cholesterol biosynthesis facilitates the survival of methylation-retaining AML cells following decitabine treatment [single_colony_TEM-Seq]
DNA hypomethylating agents (HMAs) are used to treat acute myeloid leukaemia (AML) and myelodysplasia patients who are unsuitable for intensive chemotherapy, but low response rates and therapy-resistant relapse remain significant challenges. To optimise HMA efficacy, we must understand how resistance and relapse arise from cells that survive treatment. Here we combine single-cell multi-omic analysis with parallel colony-forming assays to link HMA-induced molecular heterogeneity with functional consequences in AML cells. HMAs, azacytidine cytidine (azacytidine ) and decitabine (decitabine ), induced global epigenetic heterogeneity associated with upregulation of inflammatory responses and cell death pathways in a subset of hypomethylated cells. Some AML cells maintained high DNA methylation during treatment, and these methylation-retaining cells had increased self-renewal capacity following decitabine , but not azacytidine . Molecular profiling of individual colonies revealed upregulated cholesterol biosynthesis as an adaptation to HMA treatment, and inhibition by rosuvastatin enhanced decitabine  effects in vitro and in vivo. Thus, HMA-induced heterogeneity has important implications for AML cell growth and statins are a candidate co-treatment strategy to delay or prevent HMA-resistant relapse. Overall design: Leukaemia cell lines (HL-60, MOLM-13 and MV-4-11) were treated with hypomethylating agents (HMAs) over 72 hours (doses at 0, 24 and 48 hours) with either Decitabine (DAC), or azacytidine cytidine (AZA) and compared to untreated cells. After 72 hours treatment, cells were seeded into MethoCult and cultured for 14 days. Individual colonies were picked and assessed by parallel TEM-seq and RNA-seq.