Acute Myeloid Leukaemia (AML) is the second most common blood cancer with a 5-year survival rate of ~26%. Hypomethylating agents (HMAs), like Decitabine (DAC) and Azacytidine (AZA), are used to treat elderly AML patients that are unfit for standard cytotoxic chemotherapy. While HMA treatment can increase survival in some patients, response is variable, and relapse is prevalent. HMAs incorporate into DNA during replication and cause global loss of DNA methylation. We hypothesized that relapse may originate from low-cycling leukaemic stem cells that reside in the hypoxic bone marrow, and so aim to compare the efficacy of HMAs in hypoxic and normoxic conditions.
AML cell lines (MOLM-13, MV-4-11, HL-60) were exposed to normoxic (20% O2) and hypoxic (1% O2) conditions for 72hrs before treatment with DAC (100nM) and AZA (500nM) for an additional 72hrs. A trypan exclusion assay was used to monitor growth and viability, while colony-forming assays assessed self-renewal capacity. DNA methylation was then analyzed using bisulphite sequencing.
In normoxia, DAC significantly decreased cell growth in all cell lines, while AZA was only effective on HL-60’s. Alone, hypoxia significantly increased the doubling time of all cell lines by ~3-fold, however HMAs showed no additional effect. Colony counts were reduced to similar extents following HMA treatment in both normoxic and hypoxic conditions. In normoxia, both HMAs significantly decreased DNA methylation across all cell lines, with AZA having the most substantial effect. In hypoxia, the effect of AZA was less pronounced, while DAC maintained its influence on DNA methylation.
In conclusion, despite reduced cell growth in hypoxia, DAC remains equally effective at decreasing methylation. This suggests that either a sub-population of cells proliferates rapidly in hypoxia, or DAC decreases DNA methylation in a replication-independent manner. To explore these possibilities, flow cytometry will be used to confirm the low division rates during hypoxia.