Sequential inhibitor therapy in CML: in vitro simulation elucidates the pattern of resistance mutations after second- and third-line treatment.

Purpose: Dasatinib and nilotinib are active in imatinib-resistant chronic myelocytic leukemia (CML) and many patients undergo sequential treatment. We aimed at modeling sequential tyrosine kinase inhibitor (TKI) resistance in vitro to compare the sequences imatinib-nilotinib-dasatinib and imatinib-dasatinib-nilotinib.

Experimental Design: We designed an in vitro model for sequential TKI resistance in CML. Replicates of imatinib-resistant cell lines were treated with dasatinib or nilotinib. Second-line resistant replicates were exposed to third-line treatment.

Results: Growth of all replicates in all three lines of treatment was associated with T315I. However, T315I occurred with low abundance and did not increase during sequential treatment. Nilotinib second-line more often gave rise to sequential resistance compared with dasatinib due to pre-existing P-loop mutations, especially at suboptimal drug concentration. In contrast, mutations predisposing to dasatinib resistance such as F317C/V and V299L did not occur before dasatinib exposure. Nilotinib third-line did not overcome imatinib-dasatinib resistance due to pre-existing T315I or P-loop/V299L or P-loop/F317 exchanges. Dasatinib third-line suppressed imatinib-nilotinib-resistant replicates with residual sensitivity.

Conclusions: Sequential acquisition of BCR-ABL drug resistance mutations in CML might be underestimated. Resistance to sequential TKI monotherapy in vitro more often was associated with stepwise acquisition of drug-specific compound mutations compared with T315I. Pre-existing mutations strongly limited the activity of both third-line treatments, and the activity of nilotinib second-line in vitro critically depended on drug concentration.