Abstract 2991: Systematic discovery of regulators of adoptive T-cell therapeutic resistance using <i>in vivo</i> CRISPR screening

Abstract Background: Adoptive T-cell therapy has emerged as a promising approach for the treatment of several malignancies, including melanoma. Despite promising outcomes, therapeutic resistance remains a significant challenge, with limited or transient responses observed in many patients. The molecular mechanisms driving resistance pathways have not been thoroughly studied in vivo. Systematic characterization of the genetic regulators of adoptive T-cell therapeutic resistance is thus critical for designing next-generation of synergistic combination therapies and treatment regimens to enhance efficacy and durability. Methods: We developed an in vivo CRISPR knockout screening system to identify genetic factors mediating resistance to adoptive T-cell therapy in a melanoma mouse model. To explore potential synergistic therapeutic combinations, we opted to target the easily druggable subset of the human genome, encompassing all known kinases. CRISPR-edited B16-GFP-OVA cells transduced with a mouse CRISPR KO library (Brie: comprising 2, 852 sgRNAs targeting 713 mouse kinase genes) were engrafted into C57BL/6J mice. The mice were divided into two groups: a PBS control group (n=6) and a T-cell treatment group (n=14). Tumors were harvested either at an early (Day X) or later time points (Day Y) for genomic DNA extraction and next-generation sequencing (NGS)-based guide RNA enrichment analysis. Deep sequencing data were analyzed using the MAGeCK pipeline. Functional validation of our top candidates was performed by generating single clones of single-gene CRISPR knockouts and conducting functional profiling using orthogonal assays, including flow cytometry, LDH cytotoxicity assay, and pharmacological inhibition studies to assess target engagement and efficacy. Results: The CRISPR screen identified two key regulators of adoptive T-cell resistance, termed NU-ATCTr-101, NU-ATCTr-102. Single-gene knockouts of these targets significantly enhanced T-cell-mediated killing in vitro. Pharmacological inhibition of NU-ATCTr-101 demonstrated a dose-dependent increase in cytotoxicity and enhanced T-cell response. Mechanistic studies revealed alterations in antigen presentation, PD-L1 expression, and T-cell activation pathways. Bulk RNA sequencing and additional pathway analyses are ongoing to further elucidate precise molecular mechanisms underpinning these enhanced therapeutic outcomes. Conclusion: Our findings identify NU-ATCTr-101 and NU-ATCTr-102 as potential therapeutic targets to overcome resistance to adoptive T-cell therapy. Synergistic pharmacological interventions targeting these kinases may serve as promising adjuvants towards the development of effective immunotherapeutic strategies. Future studies will focus on validating these findings across additional human cancer model systems to evaluate and expedite their translational potential. Citation Format: Chenlin Zhao, Sharif Ahmed, Xiyue Hu, Ranjit S. Atwal, Zongjie Wang, Sophie Xiao, Shana O. Kelley. Systematic discovery of regulators of adoptive T-cell therapeutic resistance using in vivo CRISPR screening [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 2991.