Aberrant DNA Methylation in CML Is Associated with Disease Progression and Resistance to Imatinib Mesylate.

DNA methylation is important in leukemia pathogenesis and progression. Its role in CML is suggested but incompletely understood. We analyzed 123 blood samples from patients at various CML stages (62 CP, 39 AP, 22 BP) for methylation of promoter-associated CpG islands of 10 genes previously cloned as hypermethylated in K562 leukemic cell line, prostate cancer cell lines, and polycythemia vera cells: ABL, CDH13, DPYS, NOR1 (C1orf102), NPM2, p15^INK4b, PGRA, PGRB (isoforms of progesterone receptor), RIL (PDLIM4), and TFAP2E. Thirty-four patients were untreated, 33 were imatinib naïve (mostly treated by interferon-alpha before the era of imatinib mesylate), and 56 patients were imatinib-resistant or intolerant. Bisulfite treatment of DNA followed by PCR and pyrosequencing were used to quantitatively measure cytosine methylation of CpG sites close to transcription start sites. We used nonparametric tests and the significance level of p<0.05 for all statistical analyses. The highest methylation frequencies over a 15% cutoff level were observed for TFAP2E (72% patients), followed by RIL (57%), DPYS (53%), CDH13 (44%), PGRB (41%), ABL (33%), PGRA (27%), NPM2 (17%), NOR1 (8%), while p15 ^INK4b showed the lowest methylation frequency (5% of all patients). Methylation was generally independent of age and gender. Methylation levels of individual genes significantly correlated with each other. CDH13, NOR1, NPM2 and RIL methylation correlated with the largest number of studied CpG islands (7/10). On the other side of the spectrum, ABL gene methylation correlated only with p15^INK4b and NPM2. Concordant methylation of CpG islands was observed within individual stages of CML as well. We used z-score normalization [z= (value - mean)/standard deviation] to equalize absolute differences in methylation densities between individual genes, and calculated average methylation z-score for all 10 analyzed genes. Average methylation significantly increased with CML stage (z-score increase of 32% in AP and 84% in BP over CP), which was particularly true for the cluster of DPYS, NOR1 and NPM2 genes (z-score increase of 42% in AP and 102% in BP over CP). We compared methylation status separately for each disease stage in groups of imatinib-naïve (19 CP, 11 AP, and 3 BP) versus imatinib-resistant patients (12 CP, 26 AP, and 18 BP). A significant increase in methylation z-scores for NOR1 was observed in imatinib-resistant versus naïve patients in CP (36% increase), AP (59% increase), and BP (96% increase). Imatinib-resistant patients in AP showed significantly higher methylation z-scores also for PGRA, PGRB, and TFAP2E (63–76% increase). On the other hand, ABL gene showed significantly lower methylation z-scores in imatinib-resistant patients: 53% decrease in AP and 103% decrease in BP when compared to imatinib naïve patients. We conclude that aberrant methylation of DNA is associated with progression of CML. NOR1 and several other genes showed more pronounced methylation while ABL was less methylated in patients resistant to imatinib mesylate. Changes in gene silencing by DNA methylation may play a role in developing alternative routes for cells to circumvent the effects of imatinib.