Different FLT3 Inhibitors Display Limited, Non-Overlapping Profiles of Specific FLT3-ITD Resistance Mutations in a Cell-Based Screen.

Mutations causing resistance to therapeutic kinase inhibition can be identified in target kinases in various malignant diseases, such as Bcr-Abl in CML, FLT3-ITD in AML, cKit in GIST, EGFR in NSCLC, and FIP1L1-PDGFRalpha in HES/CEL. Thus, mutations in target kinases constitute a general mechanism of resistance to therapeutic kinase inhibiton. It has been shown that sensitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor. We therefore intended to determine, whether different FLT3 inhibitors would produce distinct profiles of secondary, FLT3 resistance mutations. Using a cell-based screening approach, we generated FLT3-ITD expressing cell lines resistant to the FLT3 inhibitors su5614 and PKC412. The frequency of resistant clones per million cells in the presence of inhibitor at 10 times the IC50 was 0.17 for su5614 (1,5?M) and 0 with PKC412 (100nM). When FLT3-ITD transformed cells were mutagenized with ENU (ethylnitrosourea) prior to addition of inhibitors, the frequency of resistant clones increased to 2.33 with su5614, and to 0.34 with PKC412. When we analyzed FLT3-ITD TK1 and TK2 for mutations that may confer inhibitor resistance, we found mutations in 127 out of 179 cell clones (71%) with su5614. Nine different exchanges affecting six positions in TK2 were identified, within or shortly behind the FLT3-ITD A-loop, with exchanges of D835 predominating. We did not detect exchanges affecting TK1. With PKC412, four out of 26 resistant clones (15%) contained mutations. In contrast to su5614, no mutations of TK2 were identified. However, three different amino acid exchanges emerging with PKC412 exclusively affected N676, which is located in TK1 of the FLT3-ITD split kinase domain. This is in line with the observation of a FLT3-ITD N676K exchange identified in a single AML patient with clinical resistance to PKC412 (Heidel et al, 2006). When expressed in cell lines, the identified TK2 A-loop mutations shifted dose-response curves to higher concentrations of su5614, but did not affect response to PKC412, su11248 or sorafenib. However, Y842D caused a strong resistance towards sorafenib, but not to su11248 or PKC412. Recent results also demonstrate a differential response of TK1 N676 exchanges towards different FLT3 inhibitors. We are currently analyzing su11248- and sorafenib- resistant FLT3-ITD clones for specific resistance mutations. Our results demonstrate that cell-based resistance screening is a useful and valid tool for prediction of resistance mutations to kinase inhibitors. In contrast to Bcr-Abl kinase inhibitors such as imatinib, nilotinib and dasatinib, which display highly overlapping resistance profiles, FLT3 kinase inhibitors may generate distinct, non-overlapping profiles. This opens the possibility of using combined FLT3 inhibitor therapy in AML to prevent resistance due to FLT3-ITD kinase domain mutations.