Genomic stability at the coding regions of the multidrug transporter gene ABCB1: insights into the development of alternative drug resistance mechanisms in human leukemia cells

Aim:Despite considerable efforts to reverse clinical multidrug resistance(MDR),targeting the predominant multidrug transporter ABCB1/P-glycoprotein(P-gp)using small molecule inhibitors has been unsuccessful,possibly due to the emergence of alternative drug resistance mechanisms.However,the non-specific P-gp inhibitor cyclosporine(CsA)showed significant clinical benefits in patients with acute myeloid leukemia(AML),which likely represents the only proof-of-principle clinical trial using several generations of MDR inhibitors.Nevertheless,the mutational mechanisms that may underlie unsuccessful MDR modulation by CsA are not elucidated because of the absence of CsA-relevant cellular models.In this study,our aims were to establish CsA-resistant leukemia models and to examine the presence or absence of ABCB1 exonic mutations in these models as well as in diverse types of human cancer samples including AMLs.Methods:Drug-resistant lines were established by stepwise drug co-selection and characterized by drug sensitivity assay,rhodamine-123 accumulation,[3H]-labeled drug export,ABCB1 cDNA sequencing,and RNase protection assay.The genomic stability of the ABCB1 coding regions was evaluated by exome sequencing analysis of variant allele frequencies in human populations.Moreover,the mutational spectrum of ABCB1 was further assessed in diverse types of cancer samples including AMLs in the Cancer Genome Atlas(TCGA)at the National Cancer Institute.Results:We report the development of two erythroleukemia variants,RVC and RDC,which were derived by stepwise co-selection of K562/R7 drug-resistant leukemia cells with the etoposide-CsA and doxorubicin-CsA drug combinations,respectively.Interestingly,both RVC and RDC cell lines,which retained P-gp expression,showed altered multidrug-resistant phenotypes that were resistant to CsA modulation.Strikingly,no mutations were found in the ABCB1 coding regions in these variant cells even under long-term stringent drug selection.Genomically,ABCB1 displayed relatively low variant allele frequencies in human populations when compared with several ABC superfamily members.Moreover,ABCB1 also exhibited a very low mutational frequency in AMLs compared with all types of human cancer.In addition,we found that CsA played a role in undermining the selection of highly drug-resistant cells via induction of low-level and unstable drug resistance.Conclusion:Our data indicate that ABCB1 coding regions are genomically stable and relatively resistant to drug-induced mutations.Non-ABCB1 mutational mechanisms are responsible for the drug-resistant phenotypes in both RVC and RDC cell lines,which are also prevalent in clinical AML patients.Accordingly,we propose several relevant models that account for the development of alternative drug resistance mechanisms in the absence of ABCB1 mutations.