Abstract 377: Saturation mutagenesis of KEAP1 to identify pathogenic mutations in lung cancer patients and gain insight into KEAP1 molecular function

Abstract Introduction: KEAP1 and NFE2L2 (NFR2) mutations occur in roughly 20% of non-small cell lung cancer (NSCLC) patients and are associated with resistance to a broad range of therapies including radiotherapy, chemotherapy, targeted therapies, and immunotherapy. In prior work, we demonstrated that not all KEAP1 mutations observed in NSCLC patients are pathogenic. These benign mutations have a wild-type phenotype that does not impart therapy resistance. However, only roughly 100 of ~13, 000 (1%) possible KEAP1 single amino acid substitution mutations have been characterized. Here, we performed saturation mutagenesis of KEAP1 to comprehensively evaluate all possible point mutation phenotypes in vitro. Methods: Saturation mutagenesis was accomplished using the High-throughput Mutagenesis by Integrated TilEs (MITE) method. In brief, the KEAP1 sequence was divided into 21 tiles each containing a 90 bp variable region flanked by a 30 bp constant region. At each codon position, mutations representing the 20 possible natural amino acids and a stop codon were included for a total of 13, 076 KEAP1 variants. The tiles were assembled such that each full length KEAP1 construct contained a single desired mutation. The KEAP1 MITE lentivirus library was transduced into H1299-KEAP1NULL cells that were then treated with vehicle or H2O2 for 24 days. The KEAP1 ORF was amplified from gDNA and mutations were quantified by next generation sequencing. Mutation enrichment analysis was used to derive functional annotations. Results: Oxidative stress resulted in a bimodal distribution of KEAP1 mutations. Cells with KEAP1 nonsense mutations were enriched while cells with silent mutations were depleted. Using a threshold of 95% specificity for silent mutations to distinguish benign from pathogenic mutations, 94% of nonsense mutations and 88% of previously characterized pathogenic mutations were identified as pathogenic. While the majority of KEAP1 mutations were benign (64%), analysis of mutations found in TCGA NSCLC patients showed an enrichment of pathogenic mutations (72%). Overlaying mutation calls onto the KEAP1 structure showed enrichment of pathogenic mutations in the BTB homodimerization interface, hydrophobic core of the oxidative stress sensing IVR domain, and NRF2 binding pocket of the Kelch domain. Conclusions: Saturation mutagenesis of KEAP1 generated a comprehensive list of pathogenic KEAP1 single amino acid substitutions. Assignments strongly agreed with biologic function and prior literature. The assessment of KEAP1 mutation phenotypes is important as benign mutations occur in a significant subset of NSCLC patients and are not expected to impart therapy resistance. Our results will allow identification of benign and pathogenic KEAP1 mutations in clinical studies, which will enable testing of personalized therapeutic strategies for patients with KEAP1 mutations. Citation Format: Noah Kastelowitz, Shashank Shrishrimal, Soyeong Jun, Anni M. Zhang, Rui Wang, Ilayda Ilerten, Maximilian Diehn. Saturation mutagenesis of KEAP1 to identify pathogenic mutations in lung cancer patients and gain insight into KEAP1 molecular function 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 (6Suppl): Abstract nr 377.