Abstract 2642: KEAP1 mutation in lung cancer cells leads to increased immunosuppressive capability

Abstract

Abstract Considerable advances have been made in lung cancer therapies, but there is still an unmet clinical need to improve survival for lung cancer patients. Immunotherapies have improved survival, although only 20-30% of patients respond to these treatments. Interestingly, cancers with mutations in KEAP1, the negative regulator of the NRF2 cytoprotective pathway, are resistant to immune checkpoint inhibition and correlate with decreased immune cell infiltration. NRF2 is known for promoting an anti-inflammatory phenotype when activated in immune cells, but the study of NRF2 activation in cancer cells has not been adequately assessed. The overall objective of this study is to determine how lung cancer cells with constitutive NRF2 activity interact with the immune microenvironment to promote cancer progression and metastasis. To assess this, we generated CRISPR-edited mouse lung cancer cell lines (Lewis Lung Carcinoma/LL2) with knockouts (KO) of the KEAP1 and NFE2L2 genes. The transcriptomic profiles of 3 KEAP1 KO and 1 NRF2 KO cell lines were compared to the parental wildtype (WT) line using RNA-sequencing. On unsupervised hierarchical clustering of differentially expressed genes, the 3 KEAP1 KO cell lines clustered together and the NRF2 KO clustered with WT cells. Importantly, canonical NRF2 pathway-related genes were significantly increased in KEAP1 KO clones, but the same genes were either decreased or similar to WT in the NRF2 KO clone. We then used flow cytometry to evaluate immunosuppressive surface markers known to suppress T cell function when expressed on cancer cells. Importantly, PD-L1, CD155, CD80, and CD86 were increased in the KEAP1 KO lines compared to WT, but not in the NRF2 KO clone. In addition, the cytokine release profile was differentially regulated in the KEAP1 KO clones in a manner that could alter T cell recruitment and function. These results were also consistent with gene expression in the sequencing dataset. Finally, using an orthotopic allograft model, we completed a preliminary immunophenotyping study of lung tumors for WT, KEAP1 KO, and NRF2 KO cell lines. The activation status of CD8+ T cells and the ratio of CD8+/CD4+CD25+ T cells (a prognostic marker) were decreased in KEAP1 KO tumors. Taken together, these data suggest cancers with KEAP1 mutations may increase tumor growth through suppression of T cells. Citation Format: Christopher John Occhiuto, Karen T. Liby. KEAP1 mutation in lung cancer cells leads to increased immunosuppressive capability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2642.