The aetiology of pathogenic mutations in acute myeloid leukaemia

Ministry of Health Malaysia ; Relapse in acute myeloid leukaemia (AML) is driven by additional cooperating somatic mutations that were acquired (or selected for) after chemotherapy, where the relapsing clone evolves from a cell carrying leukaemia-initiating genetic lesions, including gene fusions such as RUNX1/ETO. Some fusion genes are thought to confer a mutator phenotype that predisposes cells to the acquisition of cooperating mutations. As such, it is important to understand how chemotherapy and fusion gene expression contribute to mutagenesis in relapsing AML. The effect of chemotherapeutic agents on the mutation frequency was determined at the thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyltransferase (HPRT) loci in a fully controlled cell model system using TK6 human lymphoblastoid cell lines. Both daunorubicin and cytarabine were mutagenic to DNA at the TK and HPRT loci. Comparison of the daunorubicin-treated and vehicle-treated mutational spectra at the HPRT coding region revealed a significant increase in large deletions in daunorubicin-treated cells relative to vehicletreated cells, demonstrating daunorubicin as a powerful mutagen, inducing almost exclusively gene deletions presumably via strand break induction. The effect of RUNX1/ETO fusion gene expression on mutation frequency was also determined both spontaneously and after chemotherapy treatment using a cell line transduced with the full-length RUNX1/ETO fusion gene. RUNX1/ETO significantly increases spontaneous mutation frequency at both TK and HPRT loci; however, there is no strong evidence that RUNX1/ETO fusion gene expression sensitises cells to chemotherapy-induced mutation. Interrogation of the spontaneous HPRT base substitution spectrum revealed that RUNX1/ETO significantly increases T:A > G:C transversions in vitro, and particularly at the central base position of 5’ApTpA3’ / 5’TpApT3’ sequences, although this observation was not evident in primary t(8;21) AML. A flow cytometric method was established to evaluate the ...