ABCB1 Overexpression May Predispose Imatinib Treated CML Patients to the Development of Abl Kinase Domain Mutations, and May Be an Important Contributor to Acquired Resistance.

Imatinib has resulted in excellent and sustained clinical response in most patients with CML. For a few patients, a treatment limitation has been the development of imatinib resistance, frequently the result of kinase domain mutations. The cause of increased susceptibility to mutation development remains unknown. ABCB1 is a recognized efflux transporter of imatinib. Previous studies have demonstrated high ABCB1 expression in imatinib resistant cell lines. We hypothesize that ABCB1, by facilitating drug efflux and therefore limiting the intracellular concentration of imatinib, may contribute to resistance and mutation development. Using RQ-PCR for ABCB1 expression relative to the control gene BCR, flow cytometric analysis and radioactive drug intracellular uptake and retention studies (IUR) we have assessed 32 imatinib treated chronic phase CML patients pre therapy. 29/32(90%) patients had expression of ABCB1 mRNA less than 65% of control (median 49% range 21–65%). The three patients with higher expression of ABCB1 (73%, 130% and 105%) all subsequently developed kinase domain mutations and disease progression. Only 1/29 patients with low mRNA (<65%) expression subsequently developed a mutation. While the depth of molecular response to imatinib of the remaining 28 patients varied, disease progression was not observed in any patient, and ABCB1 was not predictive of molecular response(p=0.74). We have also analyzed samples from 4 patients after they developed imatinib resistance not associated with kinase domain mutations. We found significantly higher levels of ABCB1 mRNA(median 308% range 178–408% p<0.001), and protein expression (control: median 0.3% range 0.2–7%, non-mutational resistance: median 7% range 2–15% p=0.005). There was also a significantly lower IUR at 2 hrs (control: median 22ng range 11–36ng, non-mutational resistance: median 13ng range 10–16ng p=0.006). Further, in 2 of the 4 patients we performed imatinib IUR in the presence of the ABCB1 inhibitor PSC833. In both cases inhibition of ABCB1 significantly increased IUR (2hr IUR 11.4 and 11.5 ng: 2hr IUR + PSC833 18.8 and 18.9 ng).This finding was not observed in 25 control samples tested (IUR 25.4ng, IUR +PSC833 - 16.56ng/200,000 cells). We have performed longitudinal studies on one patient who developed initial resistance to imatinib, then to dasatinib. An additional copy of the Ph, was reported at the time of resistance, but no kinase domain mutation. Over the disease course we observed a 7 fold rise in the mRNA expression of ABCB1 and 49 fold increase in ABCB1 protein expression (from 0.3 to 14.7%). This rise in expression of ABCB1 cannot be explained by an increase in percentage of immature myeloid cells.

We conclude that patients with high expression of ABCB1 at diagnosis may be predisposed to mutation development. Furthermore increasing expression of ABCB1 over time may be a valuable contributor to acquired resistance. ABCB1 activation may be an important prognostic marker, and potential target for pharmacological manipulation.