Abstract LB048: Functional characterization of genetic regulators driving melanoma drug resistance via Single-Cell high-content CRISPR screening

Abstract Melanoma, a highly aggressive form of skin cancer, presents a significant clinical challenge. The identification of BRAFV600E mutations as a driver in roughly half of all melanoma cases has led to the development of targeted therapies, exemplified by Vemurafenib (Vem), which inhibits overactivated MAPK signaling. However, frequent relapses remain a major obstacle. To investigate the complex mechanisms driving drug resistance in melanoma, we developed PerturbFate, a scalable single-cell combinatorial-indexing strategy that integrates multi-modal profiling with CRISPR perturbations. This approach simultaneously captures newly synthesized and pre-existing transcriptomes, chromatin accessibility, and CRISPR sgRNA identity from individual cells. Using this platform, we profiled BRAFV600E melanoma cells (A375) after knocking down each of 143 potential drug resistance candidate genes, both with and without Vem treatment, in a pooled manner. We identified extensive phenotypic state transitions in melanoma cells under genetic and chemical perturbations, revealing that an undifferentiated state is associated with enhanced drug resistance, while a more benign, Vem-responsive melanocytic state exists at the opposite end of the phenotypic spectrum. Temporal gene regulatory network analyses revealed that AP-1 transcription factors (TFs), particularly FOSL1, pioneer other diverse TFs to drive the melanoma dedifferentiation. In contrast, SOX10 is the key determinant of the melanocytic state. We identified MAPK pathway, which primarily promotes proliferation, and Hippo/YAP signaling, which predominantly drives the undifferentiated state transition, as convergent key pathways mediating drug resistance. Their synergistic interplay, executed through the collaborative activation of downstream TFs (e.g., FOSL1, RREB1, SMADs, KLF5)-mediated gene expression, is a critical driver of Vem resistance. To elucidate the mechanisms underlying the observation that perturbation of genes encoding the Mediator Complex components (e.g., MED12, CCNC, MED13, MED19, MED15, MED24) induced autonomous dedifferentiation and/or resistance to Vem in melanoma, we established linkages between their distinct biochemical properties and heterogeneous perturbation responses. We also identified the convergence of dysregulated inflammation, potentially mediated by VEGFC and interleukins (ILs), with Hippo/YAP-mediated transcriptional responses in driving melanoma dedifferentiation and Vem resistance following perturbation of mediator complex genes. Altogether, our study introduces a novel single-cell multi-modal CRISPR perturbation platform, which we applied to comprehensively chart the responses of melanoma cells to the perturbation of all potential Vem resistance candidate genes. These findings pave the way for the identification of precise targets for combination therapies with enhanced efficacy against melanoma drug resistance. Citation Format: Zihan Xu, Ziyu Lu, Aileen Ugurbil, Abdulraouf Abdulraouf, Jianxiang Zhang, Wei Zhou, Junyue Cao. Functional characterization of genetic regulators driving melanoma drug resistance via Single-Cell high-content CRISPR screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB048.