Abstract CT233: Multi-modal multi-omic analyses reveal mechanisms of immunotherapy resistance in non-small cell lung cancer (NSCLC)

Abstract Introduction: Overcoming immunotherapy (IO) failure in advanced NSCLC requires distinguishing molecular features of acquired vs primary resistance. Here we used comprehensive multi-omic analyses to define resistance mechanisms. Methods: Analyses were performed on the largest-to-date IO resistance cohort: 1819 biospecimens from 892 patients with NSCLC (371 primary, 521 acquired) from the Phase 2 HUDSON study (NCT03334617) of combination regimens after progression on anti-PD- (L) 1/chemotherapy. Targeted next-generation sequencing of unpaired tumor biopsies pre- (n=226) and post-IO (n=497) was used to profile mutation signatures, clonality, aneuploidy and genomic instability. Post-IO tumor burden was assessed by plasma ctDNA (n=445) ; peripheral T-cell repertoires were analyzed (n=335). Bulk (n=205) and single-cell (sc, n=75) RNA sequencing (RNAseq) of unpaired post-IO tumor biopsies enabled gene set enrichment analysis (GSEA) and high-resolution cell type annotation. Results: Comprehensive analyses revealed acquisition of sub-clonal genomic alterations at the time of acquired resistance. Despite lower systemic ctDNA burden at progression, activating FGF10 and RICTOR and inactivating RBM10 and MSH6 mutations were enriched in acquired vs primary resistance (false discovery rate FDR p0. 05). Post-IO tumors harbored more CDK4, CDK6, and CD22 activating mutations, and inactivating mutations in KDM6A, SMARCA4, and CDKN2A (FDR p0. 05). Genomic instability (increased homologous recombination deficiency signatures, elevated large-scale transitions, telomeric allelic imbalance) was noted in both primary and acquired resistant tumors. Bulk RNAseq GSEA detected upregulation of epithelial-to-mesenchymal transition (EMT), IFNγ response, and inflammatory pathways (FDR p0. 05) in acquired vs primary resistant tumors. Single-cell transcriptomics showed enrichment of tumor-reactive, tissue-resident memory CD8+ T-cell clusters in acquired resistant tumors. Notably, a naïve/stem-like CD8+ T-cell cluster was also enriched in acquired resistant tumors. GSEA in early, central, and tissue-resident memory CD4+ T-cell clusters revealed an upregulation of naïve/stem-like gene sets and a downregulation of antigen processing/presentation gene sets in acquired resistant tumors. scRNAseq and differential expression analysis of epithelial populations highlighted pronounced EMT activation, increased lineage plasticity, and neuroendocrine differentiation gene signatures, implicating cellular reprogramming and phenotypic plasticity as potential contributors to acquired IO resistance. Conclusion: Acquired IO resistance in NSCLC involves dynamic and unique genomic and transcriptomic remodeling, encompassing EMT, lineage plasticity, stem-like programs, and immune reprogramming—highlighting potential avenues for therapeutic intervention. Citation Format: Archana Balan, Sonia Iyer, James Conway, Christopher Cherry, Noushin Niknafs, Mohamed Reda Keddar, Avinash Reddy, Robert McEwen, James White, Grace Kim, Anissa Dallmann, Nima Boluriaan, Sreeharsha Gunda, Mark Awad, Glenwood Goss, Se-Hoon Lee, Keunchil Park, Martin Reck, Michael Thomas, Rachel Karchin, Jane Peters, John F. Kurland, Giuseppe Galletti, Simon T. Barry, Jan Cosaert, J. Carl Barrett, Benjamin Besse, John V. Heymach, Patrick M. Forde, Valsamo Anagnostou. Multi-modal multi-omic analyses reveal mechanisms of immunotherapy resistance in non-small cell lung cancer (NSCLC) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr CT233.