Abstract 5107: Alterations in cancer-associated fibroblasts are associated with a dysfunctional T cell phenotype and antioxidant response in LKB1/KEAP1-mutant lung adenocarcinoma

Abstract Background: LKB1/STK11 and KEAP1 are the second and third most-commonly mutated tumor suppressor genes in lung adenocarcinoma. STK11 and KEAP1 mutations often co-occur together and with KRAS mutations and have been associated with an immunosuppressive microenvironment, lack of response to anti-PD-(L)1 therapy, and enhanced antioxidant response. Cancer-associated fibroblasts (CAFs) have been associated with tumor progression and worse survival in lung adenocarcinoma. However, the role of this cell population in promoting the phenotype of LKB1/KEAP1-mutant lung adenocarcinoma remains undefined. Methods: We analyzed single-cell RNA sequencing (scRNAseq) data from syngeneic KrasMUT models (LKR10 and LKR13) of lung adenocarcinoma with or without LKB1 or KEAP1 deficiency. Stk11 and Keap1 genes were knocked-out from LKR10K and LKR13K cell lines using CRISPR-Cas9 technology, and mice were injected subcutaneously with KrasMUT (K), KrasMUT/Lkb1MUT (KL), KrasMUT/Keap1MUT (KK), and KrasMUT/Lkb1MUT/Keap1MUT (KLK) tumor cells. CAF populations were analyzed by scRNAseq using R software packages. Results: CAF proportion among non-tumor cells was increased in KL and KK populations compared to K, with the highest percentage observed in the KLK tumors. In the LKR10 model UMAP, KK CAFs clustered distinctly from other samples, showing their different phenotype from other three groups. This was not observed in the LKR13 model where differentially expressed genes (DEGs) from KK-vs-K and KL-vs-K comparisons had considerable overlap, indicating similarities in this population. Twelve genes overlapped in DEGs from LKR10 KL-vs-K and KK-vs-K, of which four were related to MHC class I antigen presentation (B2m, H2-D1, H2-Q7, Psmb8), suggesting a possible role of CAFs in antigen-presentation to CD8 T cells and their exhaustion, a phenomenon previously described in colorectal carcinoma. Npc2, a gene involved in CD1d-mediated lipid antigen presentation to NKT cells, was also upregulated in both groups. In the LKR10 model, classical and non-classical MHC class I genes had the highest expression in KK CAFs, and this was also observed for KL CAFs in the LKR13 model. However, MHC class I upregulation was not replicated in KLK models. Sod3, a secretory extracellular superoxide dismutase that generates an antioxidant response in the microenvironment, was upregulated in both LKR10 KL and KK CAFs. Concurrently, LKR13KLK CAFs also demonstrated increased Sod3 expression compared to LKR13K CAFs. Conclusions: Our analysis demonstrated that CAFs display distinct phenotypes in LKB1 and KEAP1 deficient tumors, indicating that these cells may in part contribute to the immunosuppressive tumor microenvironment and enhanced antioxidant response, hypothetically partly through the upregulation of MHC class I molecules and secretion of antioxidant protein Sod3. Citation Format: Amirali Karimi, Yu Qian, Ana Galan-Cobo, Fuduan Peng, Monique Nilsson, Linghua Wang, Ferdinandos Skoulidis, John V. Heymach. Alterations in cancer-associated fibroblasts are associated with a dysfunctional T cell phenotype and antioxidant response in LKB1/KEAP1-mutant lung adenocarcinoma [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 5107.