Abstract 2771: CRISPR/Cas9 essentiality screens identify stand-alone and IRF4-linked genetic vulnerabilities in melanoma cells

Melanomas have poor prognosis at the metastatic stage despite impressive progress in targeted- and immuno-therapies. Therefore, identification of novel critical genes involved in melanoma biology with targeting potential is desirable. We and others have previously demonstrated the transcription factor interferon regulatory factor 4 (IRF4) as a dependency factor in various lymphocyte-derived cancers. These findings align with the therapeutic efficacy attained by immunomodulatory drugs which indirectly target IRF4 expression in these cancers. IRF4 also takes part in pigmentation in melanocytes, and its overexpression is prevalent in melanomas. Recently, we showed that high expression of IRF4 is linked to dependency in melanoma cells and mortality in patients. Mechanistically, we demonstrated that IRF4 regulates epigenetic silencing factors involved in DNA and histone H3K27 methylation, and consequently, controls the expression of melanoma-critical tumor suppressor genes and key downstream oncogenic pathways. In order to identify novel genes that melanoma cells rely on for survival or proliferation, we performed CRISPR/Cas9 drop-out screens. One set of screens was aimed at identifying single individual genes necessary for cell fitness. The other set of screens was aimed at uncovering genes, when knocked-out, compromised cell fitness synthetically with IRF4 deficiency. For each set, we screened in replicate two publicly available sub-genomic guide RNA libraries, one targeting genes involved in gene expression and the other targeting genes involved in apoptosis and cancer processes. In order to detect drop-outs between the start and the end of the experiments, we analyzed samples quantitatively via next generation sequencing of guides and the MAGeCK algorithm. Depletion of guides targeting core essential genes verified the screens’ ability to detect dependency genes. We then defined all significant dependency genes in their respective screens. We observed that, in addition to core essential genes, several genes that are deemed druggable show up as essential for melanoma cell fitness, some of which not previously reported. Several additional genes show up only in IRF4-knock out state, suggesting synthetic dependency. We followed up with pathway enrichment analyses to identify the prominent cellular pathways with dependency genes. Here, we observed gene transcription and translation, and cell cycle/mitosis as pathways enriched for melanoma dependency genes, among others. In addition, RNA processing and protein degradation are among the pathways enriched for dependency genes in the IRF4-knockout setting. The dependency genes uncovered in these CRISPR/Cas9 drop-out screens are expected to pave the way for novel targeting approaches in melanoma by themselves or in combination with IRF4. Citation Format: Ulduz Sobhiafshar, N. C. Tolga Emre. CRISPR/Cas9 essentiality screens identify stand-alone and IRF4-linked genetic vulnerabilities in melanoma cells [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 2771.