Methylation of DNMT3B Strongly Associates with the Methylome, Cytogenetic Risk Groups, and Prognosis of Pediatric Acute Myeloid Leukemia

Objective: Epigenetic mechanisms such as DNA methylation are deregulated in cancer. Aberrant DNA methylation is reported to have clinical significance in acute myeloid leukemia (AML) in adults. In this study, we performed a detailed genome-wide screening of DNA methylation to identify the epigenetic signatures that are associated with gene expression and prognosis in pediatric patients with AML.

Methods: Illumina 450K methylation microarray and Affymetrix U133A gene expression microarray profiles were obtained for 151 patients treated on the multicenter clinical trial AML02.  For each gene, canonical correlation analysis (CCA) was performed to summarize its methylation and expression signals into 1 expression score and 1 methylation score.  We then used the statistical tool called projection onto the most interesting statistical evidence  (PROMISE) to test whether the CCA expression and methylation scores were associated with a clinically meaningful pattern of in vitro resistance (IVR) to cytarabine, the presence of detectable minimal residual disease (MRD) after the first course of chemotherapy, and event-free survival (EFS).  In the PROMISE analysis, low IVR, less MRD, and better EFS were defined as a beneficial pattern and the converse was defined as a detrimental pattern for patients.  To address multiplicity, we computed the false discovery rate (FDR) for the results obtained from statistical analysis. 

Results: The DNA methyltransferase gene DNMT3B, which has been implicated in adult AML, was one of thirteen genes with the smallest possible permutation p-value in both CCA and PROMISE analyses.  DNMT3B showed very statistically significant methylation-expression correlation (CCA r = –0.74, P = 7 × 10^–14, FDR = 9.7 × 10^–12).  Also, DNMT3B showed very compelling evidence of a clinically meaningful pattern of association with IVR, MRD, and EFS (Figure 1; PROMISE P < 0.00001, FDR < 0.001).  In particular, a higher CCA-defined methylation score was associated with lower IVR (P = 0.14), less MRD (P < 0.0001), and better EFS (P = 0.00003).  A higher CCA-defined expression score was associated with higher IVR (P = 0.11), greater MRD (P < 0.0001), and poorer EFS (P = 0.00024).  Further, the methylation of DNMT3B was strongly associated with each of the cytogenetic risk groups defined at diagnosis (P = 9.7 × 10^–13) and the genome-wide methylation load (CCA r = 0.80, P < 1 × 10^–16). 

Conclusion:  Taken together, these results suggest that methylation of the DNMT3B locus modulates DNMT3B expression, thereby disrupting epigenetic regulation, altering the methylome, and influencing disease progression in and clinical prognosis of pediatric patients with AML. Our results provide evidence that adding demethylating agents to standard chemotherapy might improve the prognosis of pediatric patients with AML. Additional clinical and basic research is needed to further elucidate the biological mechanisms involved in the epigenetic modifications occurring in AML and to determine the optimal strategy to incorporate demethylating agents into the treatment for AML.  

Figure 1.  (A) Scatterplot of the CCA-defined methylation score and expression score summarizing the signals and correlation of 21 methylation array probe-sets and 1 expression array probe-set annotated to DNMT3B.  Each point represents 1 patient.  The colored barcode panels on the right and top margins of the figure represent bars at the values of the methylation score (right margin panels) and expression score (top margin panels) colored according to whether the patient experienced a pharmacologic or clinical endpoint (IVR, MRD, or EFS) that was statistically indicative of a good (blue bar) or poor (red bar) outcome.  Low-, standard-, and high-risk cytogenetic subgroups are indicated by circles, triangles, and crosses, respectively.  (B) EFS according to the CCA-defined methylation score for DNMT3B. (C) EFS according to the CCA-defined expression score for DNMT3B.