Functional Characterization of ABCG2 Polymorphisms and Their Influence on Tyrosine Kinase Inhibitor Effects in Chronic Myeloid Leukemia Cells,

The treatment of chronic myeloid leukemia (CML) has seen major improvements since the introduction of the first tyrosine kinase inhibitor (TKI), imatinib. However, follow-up studies show that 30% of CML patients discontinue imatinib mainly due to lack of therapeutic effect and adverse reactions. Second generation TKIs (dasatinib, nilotinib) have been approved for treatment of imatinib-resistant as well as newly diagnosed CML and new candidate drugs are tested (e.g. bosutinib). There is a growing body of evidence that an early-stage response to the drug is important for the long-term therapeutic effect. Therefore, the identification of predictive markers for response that will enable optimized dosing and choice of TKI could significantly improve response rates in CML.

Imatinib undergoes an extensive metabolism, primarily by hepatic CYP3A. We have reported (Gréen et al. 2010) that high CYP3A activity is associated with a better therapeutic response, indicating a clinical significance of imatinib metabolites. All three TKIs in clinical use today (imatinib, dasatinib, nilotinib) are substrates for the efflux transporter ABCG2 which is important for the elimination and resistance to TKIs. Several ABCG2 polymorphisms have been reported and shown to influence imatinib therapeutic outcome.

To investigate the effects of ABCG2 expression and polymorphisms on the in vitro resistance to imatinib, dasatinib, nilotinib, bosutinib and the most abundant CYP3A imatinib metabolite, CGP74588.

ABCG2 polymorphisms were constructed from wild type (wt) human ABCG2 cDNA using site-directed mutagenesis. The polymorphisms (34G>A (rs2231137), 421C>A (rs2231142), 623T>C (rs1061018), 886G>C (rs41282401), 1582G>A (rs45605536) and 1574T>G (rs58818712)) were selected based on frequency and a non-synonymous base-pair exchange. ABCG2 wt or variant cDNA was transferred into a retroviral vector and infected to the CML cell line K562. Successfully infected K562 cells were sorted based on equal levels of the reporter EYFP using FACS Aria. ABCG2 expression on the cell surface was evaluated using anti-human ABCG2 labeling in FACS. In addition, K562/ABCG2/623T>C and K562/ABCG2/1574T>G were permeabilized to detect intracellular ABCG2 protein. The influence of wt and variant ABCG2 on the cytotoxic effect of imatinib, CGP74588, dasatinib, nilotinib and bosutinib was assessed using MTT assays.

Results and discussion: Over-expression of ABCG2 wt in K562 cells resulted in a 2-fold, significant increase in resistance to imatinib when comparing K562/ABCG2 wt and K562 parental IC₅₀ (0.93μM (95% CI: 0.79; 1.08) and 0.45μM (95% CI: 0.41; 0.50), respectively). A similar increase in resistance was seen when cells were exposed to dasatinib or nilotinib while ABCG2 expression did not affect bosutinib cytotoxicity. A 7.8-fold increase in resistance to the imatinib metabolite CGP74588 was seen in K562/ABCG2 wt compared to parental K562 (17.2 μM (95% CI: 16.2; 18.3) and 2.2μM (95% CI: 2.01; 2.45), respectively).

ABCG2 polymorphisms affected the cytotoxicity of imatinib, CGP74588, dasatinib and nilotinib in a similar way and did not seem to alter substrate specificity of the transporter. All polymorphisms, except the 1582G>A for nilotinib showed impaired ABCG2 transport function by significantly lower IC₅₀ compared to K562/ABCG2 wt. Polymorphisms 34G>A, 421C>A and 1582G>A showed intermediate ABCG2 transport function while 623T>C, 886G>C and 1574T>G completely abolished ABCG2 transport. In accordance, FACS analysis revealed that 886G>C, 421C>A and 34G>A variants reduced cell surface ABCG2 expression while 623T>C and 1574T>G seemed to cause a complete failure of the ABCG2 protein to localize to the cell membrane. Despite loss of cell surface ABCG2 in K562/ABCG2/623T>C and 1574T>G, intracellular ABCG2 protein was still detected.

The main imatinib metabolite CGP74588 is more affected by ABCG2 expression than imatinib itself, indicating that variations in ABCG2 activity might affect CGP74588 concentration to a greater extent than imatinib. ABCG2 polymorphisms impaired transport activity and affected the cytotoxicity of imatinib, CGP74588, dasatinib and nilotinib, but not bosutinib. ABCG2/623T>C and 1574T>G prevent ABCG2 incorporation in the cell membrane and trap the protein intracellularly, affecting not only TKI efficacy but most likely all drugs transported by ABCG2.

No relevant conflicts of interest to declare.