Poster Presentation 35th Lorne Cancer Conference 2023

Treatment-induced DNA methylation heterogeneity and implications for cell growth in Acute Myeloid Leukaemia (#114)

Danielle Bond 1 2 , Sean Burnard 1 2 , Kumar Uddipto 1 2 , Kooper Hunt 1 2 , Charley Lawlor - O'Neill 1 2 , Brooke Harvey 1 2 , Ellise Roper 1 2 , Sam Humphries 1 2 , Carlos Riveros 3 , Heather Lee 1 2
  1. School of Biomedical Sciences & Pharmacy, The University of Newcastle, Callaghan, NSW, Australia
  2. Precision Medicine Program, Hunter Medical Research institute., New Lambton Heights, NSW, Australia
  3. Clinical Research Design and Statistics, Hunter Medical Research institute., New Lambton Heights, NSW, Australia

DNA hypomethylating agents (HMAs) are epigenetic therapies used to treat acute myeloid leukaemia (AML) patients who cannot tolerate 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.

AML cell lines were stained with CellTrace proliferation marker and were treated with HMAs (2000nM azacytidine, AZA; or 100nM decitabine, DAC) for 72h. Single-cell nucleosome, methylome and transcriptome sequencing (scNMT-seq) was performed on cells collected by indexed fluorescence activated cell sorting. HMA treatment induced global epigenetic heterogeneity with variable single-cell DNA methylation levels (17 - 69%). Heterogeneous loss of DNA methylation was observed in all genomic contexts and was related to the number of cell divisions during treatment.

A small proportion (5 - 10%) of HMA-treated cells retained DNA methylation at similar levels to untreated cells, and colony forming assays were performed to test whether these “non-responsive” cells had a growth advantage following treatment cessation. DAC and AZA treated cells formed significantly fewer colonies than untreated cells, and global DNA methylation levels were variable across (25 - 85%). The proportion of colonies with high DNA methylation levels (similar to untreated cells) was greater for DAC than AZA treated cells, suggesting that “non-responsive” cells may have a growth advantage following treatment with DAC, but not AZA. Transcriptional profiling of DAC treated colonies with high levels of global DNA methylation showed upregulation genes involved in cholesterol biosynthesis, and inhibition of this pathway by statins further reduced colony-forming capacity following DAC treatment.

In conclusion, HMA treatment induces global DNA methylation heterogeneity in AML cells with potential implications for relapse. Cells that evade HMA treatment may increase cholesterol biosynthesis to support their continued growth following drug withdrawal. Future studies will test the ability of statins to increase the long-term efficacy of HMA treatment in AML patients.