Paediatric acute myeloid leukaemia (pAML) is the second most common form of leukaemia in children however, is responsible for the most leukaemia-associated deaths. The majority of children achieve an initial chemotherapeutic response however, 20% of patients will relapse. Receptor tyrosine kinase FLT3 is mutated in 20% of cases and is associated with increased likelihood of forming drug resistance. Histone lysine methyltransferase MLL is mutated in 10% of cases and is commonly co-mutated with FLT3. Cytarabine in combination with anthracycline based drugs are the long-standing treatment for pAML. However, multiple new therapies targeting FLT3 in relapsed pAML have been trialled but unfortunately have not increased overall survival. To identify more effective treatment targets, the mechanisms underpinning the development of resistance to cytarabine and anthracycline based chemotherapies requires characterisation.
We hypothesised, that analysis of protein epigenetic modifications, coupled with signalling pathways analysis would reveal mechanisms underpinning FLT3/MLL mutant pAML survival in cytotoxic conditions. A drug resistant FLT3/MLL mutant MV411 subline was created by culture in increasing concentrations of cytarabine and daunorubicin. Unbiased global analysis of the proteome of parental and drug resistant MV411 lines via label-free Data Dependent Acquisition (DDA) LC-MS/MS identified significantly increased activity of known and novel proteins associated with leukaemogenesis and therapy resistance such as IDH1,2 and 3A. The resistant cells also demonstrated significant changes in histone posttranslational modifications particularly methylation and acetylation, suggesting that altered epigenetic regulation is underpinning protein expression changes driving resistance.
Furthermore, ingenuity pathway analysis of differentially expressed proteins demonstrates the changes in cellular metabolism that occur in therapy-resistant AML cells. To validate these findings western blotting was used.
Our preliminary data, provides important new information on the cooperative mechanisms that underpin resistance to standard of care chemotherapies, providing us with a framework to investigate novel therapeutics to improve the treatment of children with AML.