Abstract 6418: Non-genetic drug adaptation to MAPK pathway inhibitors in melanoma

Abstract Over 50% of melanomas are driven by BRAFV600 mutations. Clinically, targeted inhibitors of mutant BRAF and MEK are the primary treatment for more than 50% of melanoma patients that are refractory to standard-of-care immunotherapy. While targeted therapies are initially clinically effective, a subset of residual cancer cells persist and inevitably drive tumor recurrence and drug resistance. While some cells persist due to pre-existing mechanisms (e.g. genetic mutations), others can non-genetically adapt to tolerate drug by rewiring their internal signaling cascades. Importantly, a key feature of these non-genetically adapted persister cells is that their drug-tolerant phenotype is acquired in response to drug pressure, making them extremely challenging to identify and characterize. Using cutting-edge long-term time-lapse microscopy and an automated single-cell tracking pipeline, we studied the behaviors of persister cells in drug-treated BRAFV600E melanoma populations. Notably, we found significant heterogeneity within persister populations. While all persister cells initially enter a state of drug-induced quiescence, a subset of these cells—termed “escapees”—can escape drug action and resume proliferation. By computationally reconstructing single-cell lineages from long-term time-lapse imaging experiments, we monitored key signaling pathways before, during, and after escape from drug. Using this approach, we identified a significant increase in mTorC1 signaling activity in persisters that would go on to escape drug. Additionally, mTorC1 signaling caused increases in protein translation and cell growth rates during the quiescent periods of these cells. The abundance of Cyclin D1, a key driver of proliferation, is extremely sensitive to cellular translation rates due to its short half-life. In order to determine if the observed increase in mTorC1-mediated growth and translation is driving the upregulation of Cyclin D1 levels past a threshold necessary for escape, we used CRISPR to tag Cyclin D1 at the endogenous locus. Importantly, we found that escapees significantly upregulate Cyclin D1 levels in the hours prior to their escape, compared to non-escapees. These findings have important clinical implications because they suggest that Cyclin D1 protein levels are a key bottleneck for cell-cycle re-entry under MAPK inhibition. Additionally, these findings implicate Cyclin D1 as one of the first markers that can distinguish future escapees from non-escapees in the quiescence period before these distinct cell-cycle fates emerge. Citation Format: Varuna Nangia, Humza Ashraf, Nasreen Marikar, Victor Passanisi, Christopher Ryland Ill, Timothy Hoffman, Sabrina Leigh Spencer. Non-genetic drug adaptation to MAPK pathway inhibitors in 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 6418.