Background: Increased activation of receptor tyrosine kinase (RTK) c-Kit via mutation or overexpression has been associated with many tumours including a sub-group of melanomas. Treatment with imatinib mesylate and other RTK inhibitors has shown early clinical efficacy against certain c-Kit mutant melanomas, but relapse is common and the mechanisms of acquired resistance in melanoma are not yet understood.
Methods: The M230 melanoma cell line carries the common oncogenic c-Kit L576P mutation and is dependent on c-Kit signalling for survival. After prolonged exposure to the RTK inhibitors imatinib mesylate or nilotinib we generated single-cell derived drug resistant clones. These clones were investigated for additional c-Kit mutations, sensitivity to c-Kit loss, and response to RTK inhibitors imatinib, nilotinib, sunitinib and dasatinib.
Results: Ten of twelve expanded clones showed an additional secondary mutation in c-Kit, either T670I (3 imatinib-resistant and 6 nilotinib-resistant clones) or A829P (one imatinib-resistant clone). Both these secondary mutations are associated with RTK inhibitor resistance in cancer. Importantly, these cells remained highly sensitive to targeted c-Kit silencing, indicating a continued dependence on c-Kit signalling. L576P/T670I cells proved refractory to imatinib, nilotinib and dasatinib, but highly sensitive to sunitinib; L576P/A829P cells proved refractory to imatinib and sunitinib, but sensitive to nilotinib and dasatinib. Furthermore, all sub-clones retained parent-like sensitivity to combined MAPK and PI3K inhibition.
The two remaining imatinib-resistant clones showed no additional c-Kit mutation, c-Kit independence, significant downregulation of both MAPK and PI3K signalling and marked morphological changes.
Conclusions: Resistance to RTK inhibitors in melanoma is frequently driven by secondary mutations in c-Kit, and the identification of these mutations can provide rational second-line therapeutic strategies. Alternatively, targeting critical survival pathways downstream of c-Kit may provide a more sustained and potent clinical response.