Abstract A079: Directed clonal evolution: Leveraging divergent MAPK inhibitor resistance for convergent hypersensitivity to MAPK hyperactivation

Abstract Targeted therapies inhibiting oncogenic signaling have profoundly improved cancer patient survival. In melanoma, where oncogenic ERK MAPK pathway activation spurred the development of MAPK pathway inhibitors (MAPKi), the combination of BRAF and MEK inhibitors is now the standard of care for targeted therapy for BRAFV600E patients. This has significantly improved response rates and progression-free survival, yet current treatment options, including targeted therapies and immunotherapies, rarely achieve complete responses due to pervasive intrinsic and acquired resistance. Although resistance to MAPK pathway inhibition is predominantly driven by MAPK reactivation, the inherent intra-tumoral heterogeneity of resistance mechanisms complicates efforts to effectively combat this challenge. We propose a novel therapeutic strategy: leveraging MAPK reactivation-mediated MAPKi resistance for collateral sensitivity to non-cross-resistant cytotoxic oncogenic hyperactivation. "Activation lethalities," particularly those mediated by hyperactivation of ERK MAPK signaling, represent an underappreciated therapeutic modality. Here, we establish in a comprehensive panel of melanoma cell lines and in vivo xenograft models that BRAF-mutant melanoma exhibits selective sensitivity to MAPK activation lethality, a susceptibility further heightened by developing MAPKi resistance. Crucially, we discovered that divergent MAPKi resistance mechanisms all lead to a convergent hypersensitivity to MAPK hyperactivation. This renders cells, once evolutionarily guided towards MAPKi resistance, maximally vulnerable, resulting in complete loss of viability in response to MAPK hyperactivation achieved via inducible ERK2 overexpression. Using high-complexity cellular barcode tracing, we observed that combining MAPK pathway inhibition with subsequent hyperactivation synergistically reduced clonal diversity more effectively than either intervention alone. The absence of shared clones between these two conditions further confirmed a lack of cross-resistance, strongly supporting the rationale for a durable, cyclical treatment strategy. Additionally, we demonstrate that a subset of RAS-mutant tumors are also strongly sensitive to MAPK hyperactivation or can be sensitized to MAPK hyperactivation via the MAPKi resistance development. Our findings establish a strong rationale for developing non-cross-resistant combinations of MAPK pathway activators and inhibitors, delivered sequentially or cyclically, offering promising curative potential against MAPK-driven cancers. Citation Format: Eunice Cho, Matthew R. Pacheco, William R. Sellers. Directed clonal evolution: Leveraging divergent MAPK inhibitor resistance for convergent hypersensitivity to MAPK hyperactivation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A079.