Prognostic relevance of epigenetic regulation of drug pharmacology and myeloid genes in pediatric AML

Abstract Introduction: The dysregulation of DNA methylation in acute myeloid leukemia (AML) significantly changes the regulation and expression levels of genes involved in AML chemotherapy pharmacokinetics and pharmacodynamics and a panel of myeloid genes known to be associated with AML progression. The primary objective of this study was to evaluate the impact of the methylation levels of specific genes and their association with treatment outcomes among pediatric AML patients. Methods: The discovery cohort included 924 patients from TARGET database with methylation data obtained from the gene expression omnibus and expression data from genomic data commons. Cox proportional hazard analysis was performed to determine the association of DNA methylation of CpGs in the key myeloid panel genes (n= 107) and genes of relevance to cytarabine, daunorubicin and etoposide pharmacology genes (N= 65) with event-free survival (EFS) and overall survival (OS). Additionally, a Chi-square test was applied to assess the association between CpG methylation M value and status of Minimal Residual Disease after induction I (MRD1). A Bonferroni corrected p value of < 2.17×10-5was used as a significant association threshold. Significant results were validated in an independent patient cohort treated on AML02 trial (n=159). Results: Of the CpGs tested in the pertinent genes, 23 CpGs in drug pathway genes were associated with at least one endpoint at p <2x10−5. It included multiple CpGs (N=7) in ABCA3, were associated with clinical outcome endpoints. Of these for the most significant CpG (cg01278797) in ABCA3 greater methylation showed a consistent association with poor outcome in discovery (OS, HR= 1.43, p<0.0001, EFS HR= 1.35, p <0.0001, and a greater MRD1 positivity, p <0.001) and validation cohort (OS, HR= 1.81, p<0.0001, EFS HR= 1.61, p <0.0001, and a greater MRD1 positivity, p <0.01). For ABCA3, gene expression levels, higher expression was associated with worse OS (HR= 1.62, p <0.0001) and EFS (HR= 1.42, p=0.0002). Further, the methylation and expression of ABCA3 showed a positive correlation (p= 0.046), indicating possible epigenetic regulation. An etoposide metabolic pathway is mediated by myeloperoxidase (MPO), where hypermethylation of eight CpGs was associated with adverse clinical outcome. cg09421562 was the top significant CpG with higher methylation associated with poor outcome in discovery OS, HR= 1.22, 95% CI 1.15-1.29, p <10−11; EFS, HR= 1.15, 95% CI 1.10-1.20, p <10−9; MRD, p<10−7and validation cohort (OS, HR= 1.22, 95% CI 1.15-1.29, p <10−11; EFS, HR= 1.15, 95% CI 1.10-1.20, p <10−9; MRD, p<10−7. Additionally, increased MPO expression were related to better clinical outcomes in discovery (OS, HR=0.87, p <0.0001; EFS, HR= 0.89, p <0.0001) and validation cohorts (OS, HR=0.84, p =0.014; EFS, HR= 0.846, p =0.007). These CpGs and expression stayed significantly associate without come in multivariable analysis after adjusting for age, race, risk group ad WBC count. Other drug pathway related genes with methylation predictive of outcome included ABCC1 and NOS3. Among AML-relevant genes, 43 CpGs were associated with at least one outcome endpoint at Bonferroni corrected p<2.17×10-5. This included KIT, a receptor kinase and MPL, a myeloproliferative leukemia virus oncogene where hypermethylation of CpGs in these genes and the lower gene expression were associated with worse outcomes in both discovery and validation cohorts (OS, and EFS, p<0.001 for both). Methylation and gene expression for KIT and MPL showed significant inverse correlation as expected. Other genes with significant association of methylation and expression with outcome includedDNMT3B, CALR, DNMT3A, NOTCH1, PRPF8, RUNX1, GATA2, VWF. Conclusion: The study demonstrated that interpatient differences in DNA methylation levels of key genes related to AML drugs or disease pathogenesis affect clinical outcomes. This creates opportunities for outcome prediction and the strategic use of hypomethylating agents to enhance patient clinical outcomes.