In vivo CRISPR screening uncovers Folr1-mediated metabolic remodeling that sensitizes colorectal cancer to PD-1 blockade

Introduction: Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide. KRAS mutations are present in approximately 40-50% of CRCs, with G12D being the most prevalent subtype. Accumulating evidence indicates that KRAS G12D CRC responds poorly to immune checkpoint blockade (ICB) therapy, reflecting intrinsic resistance to immunotherapy. To identify key regulators capable of overcoming this resistance, we conducted an in vivo CRISPR/Cas9 genetic screen in a KRAS G12D -driven CRC model. Furthermore, we investigated the underlying mechanisms contributing to immune resistance. Methods: KRAS G12D colorectal cancer cell lines, MC38K and Caco2K were generated. An immune-related sgRNA library was designed using PD-1/PD-L1/MHC-I and ESTIMATE data, and MC38K cells carrying the library were tested under anti-PD1 treatment in vivo. RNA sequencing and metabolic profiling were performed to elucidate immune-related mechanisms, and Seahorse assays were used to validate metabolic alterations. Multiple CRC models were used to evaluate Folr1 inhibition combined with anti-PD1 therapy, and a Folr1-based peptide vaccine was developed to overcome resistance. Results: KRAS G12D overexpression accelerated tumor growth and conferred anti-PD1 resistance in MC38K models. Through the in vivo CRISPR/Cas9 screen, Folr1 emerged as the top depleted gene, ranking first among all candidates associated with immunotherapy resistance. Notably, Folr1 is a clinically actionable target with existing therapeutic antibody currently approved by FDA. RNA-seq analysis of Folr1-knockout MC38K and Caco2K cells revealed profound metabolic remodeling, particularly involving glycolysis. GSEA demonstrated that hallmark pathways including glycolysis, hypoxia, and mTORC1 signaling were downregulated, validated by qRT-PCR, OCR/ECAR assays, and L-lactate measurements. In multiple in vivo models, Folr1 knockout combined with anti-PD1 therapy significantly delayed tumor growth and extended overall survival. Furthermore, the Folr1-based peptide vaccine also markedly suppressed tumor progression. Conclusions: Our study identifies Folr1 as the top-ranked gene mediating immunotherapy resistance in KRAS G12D -driven colorectal cancer. Targeting Folr1 reprograms tumor metabolism, enhances antigen presentation, and restores responsiveness to anti-PD1 therapy. These findings establish Folr1 as a translationally relevant therapeutic target to overcome immune resistance in Kras G12D CRC.