Abstract 3803: CRISPR screening identifies methionine synthase as a potential therapeutic target in KRAS-driven NSCLC

Abstract One-carbon metabolism, which integrates the folate and methionine cycles, is crucial for cancer growth. Methionine synthase (MTR), a key enzyme in this pathway, converts 5-methyltetrahydrofolate (5-CH3-THF) to active tetrahydrofolate (THF), a cofactor required for nucleotide synthesis. Concurrently, MTR catalyzes the conversion of homocysteine to methionine, effectively linking the folate and methionine cycles. Given its central role, MTR has gained attention as a potential therapeutic target in cancer research, though its function in KRAS-driven lung cancer remains poorly understood. Recently, we performed an in vitro CRISPR screening using a metabolism-focused sgRNA library in mouse KrasG12D/+;p53-/- (KP) lung tumor derived cell line to identify potential metabolic therapeutic targets, where MTR emerged as one of top hits. Functional validation showed that MTR knockout (MtrKO) impaired cell proliferation, reduced colony formation, and inhibited allograft tumor growth. To further evaluate MTR's role in vivo, we used genetically engineered mouse models (GEMMs) of KRAS-driven non-small cell lung cancer (NSCLC). Lentiviral delivery of sgRNAs targeting Mtr, along with Cre recombinase, into KrasLSL_G12D/+;Lkb1flox/flox;Rosa26-LSL-Cas9-EGFP (KL_Cas9) and KrasLSL_G12D/+;p53flox/flox;Lkb1flox/flox;Rosa26-LSL-Cas9-EGFP (KPL_Cas9) mice resulted in conditional MTR knockout in lung tumors, significantly extending survival in mice with KL or KPL lung tumors compared to mice bearing wild-type (MtrWT) KL or KPL lung tumors. Furthermore, MtrKO cells showed increased sensitivity to folate and methionine deprivation compared to MtrWT cells in vitro. Folic acid supplementation did not affect the growth of MtrWT;KP allografts but rescued the growth of MtrKO tumors, confirming that MTR is essential for maintaining THF levels in vivo to support metabolic processes critical for KP tumor growth. Further in vivo isotope tracing revealed that purine salvage pathway was upregulated in MtrKO tumors. Targeting purine salvage pathway, but not the pyrimidine salvage pathway, synergistically suppressed MtrKO KP allograft tumor growth. Moreover, MTR knockout induced S-phase arrest, elevated γH2AX levels (indicative of DNA damage), and enhanced KP cell sensitivity to cisplatin. These findings highlight MTR as a critical metabolic vulnerability and promising therapeutic target in KRAS-driven lung cancers. Citation Format: Wenping Wang, Samuel Wang, Ioana Dobrescu, Hyungsin Kim, Kyle Nunn, Joel Lee, Joshua Martorelli, Fuqian Shi, Eduardo Lopes, Victoria Silva, Hua Zhong, Zhixian Hu, Daniel Herranz, Chang Chan, Eileen White, Jessie Yanxiang Guo. CRISPR screening identifies methionine synthase as a potential therapeutic target in KRAS-driven NSCLC [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 3803.