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Abstract

Despite advances in immunotherapy, melanoma remains challenging to treat, particularly in advanced stages where resistance to immune-based therapies frequently develops. Adoptive cell therapy, including chimeric antigen receptor (CAR) T cells, holds promise but has shown limited efficacy in melanoma. In this dissertation, a genome-wide CRISPR/Cas9 screen composed of melanoma cells and CAR-T cells was established to systematically uncover genes that regulate melanoma cell susceptibility to T cell-mediated killing. Using this screening platform, I identified genetic determinants beyond classical immune evasion mechanisms like major histocompatibility complex downregulation or impaired interferon-γ signaling. The co-culture screen demonstrated high reproducibility and robustness, evidenced by consistent changes in sgRNA abundance and the recovery of known tumor-promoting and suppressive targets. I have identified key candidate genes, particularly those involved in oxidative phosphorylation, amino acid transport, NF-κB and mTOR signaling, and the mediator complex, highlighting alternative resistance mechanisms to T cell cytotoxicity. To refine target selection, I have conducted an additional A375 fitness screen to identify essential genes independent of T cell-mediated killing. This allowed for the exclusion of general fitness genes and the prioritization of candidates specifically relevant to immune evasion. I have discovered that CD98 (SLC3A2/SLC7A5) modulates melanoma resistance to T cell-secreted TRAIL, acting via suppression of apoptotic signaling rather than altering T cell activity or TRAIL receptor expression. Additionally, I have performed a meta-analysis across seven screening projects to confirm the technical and functional reliability of the Heidelberg CRISPR sgRNA library. Despite varying experimental contexts, the library maintained consistent sequencing quality and gene essentiality profiles, with most variability stemming from known technical or biological factors. Altogether, this study provides new insight into tumor–immune interactions, uncovers potential therapeutic targets for overcoming TRAIL resistance in melanoma, and reinforces the HD sgRNA library as a robust tool for functional genomics.