Modelling of TKI Resistance In CML Cell Lines: Kinase Domain Mutations Usually Arise In the Setting of BCR-ABL Overexpression.

Abstract 3383

There are three currently identified secondary resistance mechanisms observed in chronic myeloid leukemia (CML) patients receiving tyrosine kinase inhibitors (TKIs). These include overexpression of drug-efflux proteins (ABCB1 and ABCG2), increased BCR-ABL expression, and mutations in the kinase domain (KD) of BCR-ABL. We investigated the interplay between these three modes of resistance in vitro, as well as looking for other mechanisms. Three CML blast crisis cell lines (K562, its ABCB1-overexpressing variant, K562 Dox, and KU812) were cultured in gradually increasing concentrations of imatinib (IM) to 2μ M, or dasatinib (DAS) to 200nM. Two IM-resistant K562 lines were established, both with increased IC50s for IM (from 13.7μ M in naïve cells, to ~50μ M), as well as increased IC50s for DAS and nilotinib (NIL). No cell-surface expression of ABCB1 or ABCG2 was observed, nor were KD mutations present. However, BCR-ABL expression was seen to steadily increase in both lines from 178% in naïve cells, to ~380% and 1200% in the resistant lines, suggesting this was the major mode of resistance. However, when a DAS-resistant K562 culture was generated the T315I mutation emerged. Studies of the intermediate stages of resistance revealed that BCR-ABL overexpression occurred in a step-wise fashion, peaking at 1915% in the 3.5nM intermediate, but then dropping significantly to ~1000% in the 5nM intermediate (P=0.0003). BCR-ABL expression then stabilised at this level, and the T315I mutation was subsequently detected. Thus, it appears that BCR-ABL overexpression was the first mechanism of resistance detectable, but was followed by the emergence of a KD mutation which had a clear selective advantage. This sequential selection was observed a further four times: in a DAS-resistant K562 Dox culture, and in three IM-resistant KU812 cultures. BCR-ABL expression in the DAS-resistant K562 Dox culture increased from 186% in naïve cells to 540% in the final culture. Studies of intermediate cultures revealed that BCR-ABL expression peaked at 850% in the 55nM intermediate, but then dropped significantly to ~500% in the 75nM intermediate (P=0.004). This drop in BCR-ABL expression coincided with the appearance of the V299L mutation. Interestingly, the K562 Dox DAS-resistant line also displayed resistance to NIL and IM, likely conferred by BCR-ABL overexpression as the 55nM intermediate (with the highest BCR-ABL expression levels) had the highest IC50s for NIL and IM, while the 75nM intermediate (with the V299L mutation) had increased IC50^DAS but lower NIL and IM IC50s. Thus, BCR-ABL overexpression was the primary event, followed by the KD mutation. Likewise in three IM-resistant KU812 cultures, BCR-ABL expression levels rose from 443% in naïve cells, to peak levels of 6210%, 10,448% and 990% respectively, followed by drops in expression which coincided with the appearance of compound KD mutations, and the F359C mutation respectively (Table). In contrast, three IM-resistant K562 Dox cells were not found to have any KD mutations, nor BCR-ABL overexpression. Instead, the primary cause of resistance in these lines appears to be a further increase in ABCB1 expression. All three lines had increased IC50s for IM (from 12μ M in naïve cells, to ~27μ M), NIL (from 400nM to ~1000nM) and DAS (from 100nM to >625nM). The addition of PSC833 (an ABCB1 inhibitor) decreased the IM, NIL, and DAS IC50s for all three resistant lines to the level of the naïve control (~3μ M, ~250nM and ~10nM respectively), indicating that ABCB1 expression, facilitating active efflux of the drugs, is the primary mechanism of resistance in these lines. We have demonstrated that KD emergence is a stochastic event, as the same mutation did not always occur twice, however BCR-ABL and ABCB1 overexpression were more likely to arise recurrently in predisposed lines. Notably, different TKIs elicited different resistant mechanisms, but all were BCR-ABL dependent. Furthermore, all resistant lines showed cross-resistance to the three TKIs tested (IM, DAS and NIL), suggesting that currently available TKIs share the same susceptibilities to drug resistance.