Abstract 6385: Heterogeneity in sensitivity to second-line inhibitors of therapy-resistant melanoma

Inhibition of the mutant BRAFV600E protein in melanoma via targeted therapy causes many cells to die, but resistant populations emerge that are refractory to further treatments. Strategies to target resistance have proven difficult to find, which could be explained by the potential heterogeneity of the population. It is assumed that genetic mutations alone cause resistance, but targeting such mutations has met with limited success in melanoma. Work from our lab and others has now established that non-genetic variability also leads to resistance in vitro and in vivo: we have shown that rare clonal cells, marked by differences in gene expression, survive and form resistant colonies. We recently demonstrated in vitro and in vivo that individual resistant colonies have distinct morphological and transcriptional differences, even with a clonal parent population. These results provide a potential explanation for the difficulty in identifying effective second-line therapies: individual resistant types may have different drug susceptibilities, so an effective second-line drug would have to kill all resistant types at once. These findings raise the possibility that there may be therapeutic vulnerabilities to exploit that are invisible in the conventional view of homogeneous resistance. For instance, drug A could kill half of the resistant population while drug B kills the other half. These drugs alone would not be viable second-line treatments, but together they could be highly effective, greatly expanding the pool of drugs that could combat resistance. However, screens to evaluate all combinations of potential drugs would be prohibitively large. This search space could be simplified by determining the drug sensitivities of individual resistant types and using those drugs in combination to eliminate a larger proportion of the total resistant population. However, such per-type classification has not yet been performed in melanoma. We generated clonal cell lines from individual resistant colonies, screening four and the drug-naive line using a library of ∼2200 drugs and small molecules with known pathways and targets in cancers. These genetically identical resistant lines indeed had distinct drug sensitivity profiles, and therapy-resistant lines as a whole had novel sensitivities and resistances compared to the drug-naive line. Treating drug-naive cells with targeted therapy in combination with drugs that kill resistant lines can reduce or eliminate resistance, even when the candidate second-line drugs alone do not kill drug-naive cells. Using a panel of 24 drugs identified as of interest in our initial screens, we screened 13 resistant lines, including the original four and four resistant to a different targeted therapy. We found potential links between the transcriptional and toxicity profiles of resistant colonies, and are continuing to test pairs of drugs that kill distinct fractions of the resistant population. Citation Format: Gianna T. Busch, Ryan H. Boe, Jingxin Li, Pavithran T. Ravindran, Arjun Raj. Heterogeneity in sensitivity to second-line inhibitors of therapy-resistant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6385.