Abstract A33: Modeling clinical resistance to RAF inhibitors in an in vivo melanoma model provides insights into prevention of resistance

Abstract

Abstract Small-molecule RAF inhibitors such as vemurafenib have demonstrated proof-of-concept that BRAFV600E is a key driver of proliferation and survival in melanoma, as evidenced by tumor regression and prolonged survival in patients in late stage clinical trials. Unfortunately, in many patients the tumor response can be short lived as resistance to the inhibitor rapidly develops. Understanding the mechanisms that underlie drug resistance will be critical to providing improved treatment options for these patients. Here we model the emergence of resistance to vemurafenib in an early passage primary human melanoma xenograft model, which recapitulates a more physiologically relevant microenvironment, genetic diversity, and selective pressure compared to in vitro models. The selection of resistance in vivo also provides the opportunity to model drug pharmacokinetics which better replicates the selective pressure observed in patients. Long-term continuous treatment of the homozygous BRAFV600E primary melanoma xenografts with RAF inhibitors resulted in the appearance of resistant tumors over the course of 4 to 6 weeks. Pharmacodynamic (PD) analysis of individual tumors indicated that the MAPK pathway is suppressed by the compounds in resistant tumors, although the degree and duration of suppression is less than in sensitive tumors. Furthermore, there is heterogeneity in the kinetics of pathway inhibition and recovery between tumors. Biochemical analyses indicate that BRAFV600E protein levels are upregulated across all resistant tumors and in some cases there is DNA amplification of the mutant BRAF. Taken together with the PD data, this suggests there is a great deal of tumor cell heterogeneity, and that tumors are able to rapidly adapt to the selective pressure being applied. Both in vivo drug withdrawal studies as well as in vitro analyses of tumor derived cell lines indicate that the resistant cells have become dependent on the presence of the inhibitor for their survival. Based on these observations it appears that resistant tumor cells are less fit than sensitive tumor cells in the absence of drug and therefore we tested an intermittent dosing regimen. Intermittent dosing (4 weeks on / 2 weeks off) of RAF inhibitors prevented the evolution of resistance for the duration of the study (4 months). These data could have significant implications in optimizing dose schedules to prevent the emergence of clinical resistance.