Integrating Molecular, Epigenetic and Pharmacogenetic Approaches in Managing Imatinib Resistance Among Malaysian Chronic Myeloid Leukemia Patients

The BCR-ABL targeted therapy using ImatinibMesylate (IM) is considered the standard care in chronic myeloid leukemia (CML) management. Despite shown to produce superior results, up to 33% of CML patients on IM therapy, develop resistance which can be either BCR-ABLdependent (gene amplification or point mutations of BCR-ABL gene) or BCR-ABL independent. We undertook molecular, epigenetic and pharmacogenetic approaches to elucidate the heterogeneous array of mechanisms of resistance in Malaysian CML patients undergoing IM therapy. Using denaturing High Performance Liquid Chromatography followed by sequencing 122IM resistant CML patients were screened for BCR-ABL mutations. As part of epigenetic approach, 175 CML patients comprising of 83 good response and 92 BCR-ABL non mutated IM resistant CML patients were subjected to Methylation Specific High Resolution Melt Analysis (MS-HRM) to quantitate the promoter methylation level of HOXA4 and HOXA5 genes. For pharmacogenetic study, 215 CML patients consisting of 107 IM good response and 108 IM resistant CML patients were genotyped for polymorphisms in ABCB1 (1236 T>C, 2677 G>T/A, 3435 C>T), ABCG2 (34 G>A, 421 C>A), ABCC1 (2012 G>T, 2168 C>A), ABCC2 (-24 C>T, 1249 G>A, 3972 C>T), SLC22A1 (480 C>G, 1201 G>A, 1222 G>A, 1239 G>A, 1258-1260 ATG del, TGGTAAGT 8+/8- ins/del), PXR (1792 A>G) and CYP3A4 (878 T>C) genes using polymerase chain reaction restriction fragment length polymorphism technique. BCR-ABL mutations were detected in 30/122 patients (24.6%). Seventeen different types of mutations (T315I, G250E, E255K, E255V, M351T, Y253H, V289F, E355G, F359V, L387M, H396R, E355A, D276G, A397P and E281K) including 2 novel mutations (G251E and N368S) were identified in varying frequencies.Detection of mutations during course of IM therapy may aid in risk stratification as well as in determining therapeutic strategies. MS-HRM analysis showed varying hypermethylation levels of HOXA4 and HOXA5 genes in CML patients studied. IM treated CML patients with higher than 62.5% of HOXA4 methylation were found to be associated with a higher risk for developing IM resistance (OR 4.71; 95% CI: 2.46-9.03; p<0.001) when compared to the optimal response group.While in HOXA5, IM treated CML patients with higher than 62.5% of HOXA5 methylation were found to be associated with a higher risk for developing IM resistance (OR 4.26; 95% CI: 2.22-8.17; p<0.001) when compared to the optimal response group.Promoter hypermethylation of HOXA4 and HOXA5 genes could be considered as one of the BCR-ABL independent mechanisms mediating IM resistance and could be a potential epigenetic biomarker in supplement to the BCR-ABL gene mutation in predicting IM treatment response among CML patients. In pharmacogenetic analysis, the homozygous and heterozygous variant genotypes such as ABCB1 1236 CC (OR 3.39;95% CI:1.375-8.362; p=0.007), SLC22A1 480CG (OR 4.96; 95% CI: 2.006-12.471;p<0.001), PXR 1792AG (OR 3.17; 95% CI:1.22-8.28;p=0.018) emerged as genotypes significantly associated with IM resistance whereas ABCB1 2677TT/AA (OR 0.32;95% CI:0.137-0.741; p=0.007), and ABCG2 421AA (OR 0.24;95% CI: 0.086-0.645; p=0.003) genotypes showed significant association with IM good response. Nevertheless, the pharmacogenetic data to be sufficiently conclusive to translate into individual drug dose adjustment, further studies on larger sample size are warranted. The overall results suggested that resistance to IM in CML patients is not due to a single or simple mechanism, but is a multi-factorial phenomenon. Because the inhibition of only one mechanism is not effective enough to overcome clinical resistance, simultaneous suppression of several proteins may be required to increase the efficacy of IM treatment in CML patients.

(Acknowledgment: Financial support from Fundamental Research Grant Scheme Grant Number: 203/PPSP/6171104, Universiti Sains Malaysia Research University Grant Numbers: 1001/PPSP/812067, 1001/PPSP/812070, 1001/PPSP/812103, and APEX Delivering Excellence 2012 Grant Number: 1002/PPSP/910340).