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Background Colorectal cancer (CRC) is a major cause of cancer-related mortality worldwide. 1 While immune checkpoint-blockade (ICB) therapies have shown clinical success in microsatellite instability-high (MSI-H) CRC, the majority of patients, particularly those with microsatellite-stable (MSS) and immune-excluded consensus molecular subtype 2 (CMS2) tumors, remain unresponsive. These ICB-resistant tumors exhibit poor T-cell infiltration and fail to initiate effective cytotoxic immune responses, highlighting an urgent need to uncover tumor-intrinsic mechanisms driving immune evasion and ICB resistance.2 3 This resistance represents a critical barrier to improving patient outcomes, and the tumor-intrinsic factors that enable immune escape and undermine the efficacy of ICB therapies in MSS and CMS2 CRC remain to be determined.Methods A random forest classifier machine-learning model, trained on ICB-treated melanoma datasets was used to identify candidate immune response genes, These candidates were further refined using ElasticNet regression across CRC datasets classified by immune phenotype and molecular subtype.[ http://tiger.canceromics.org, https://portal.gdc.cancer.gov/ ]. Functional validation was performed through targeted deletion in multiple CRC mouse models, including CMS2-like syngeneic CRC models established in humanized PD-1/CTLA-4 double knock-in mice. Immune profiling by flow cytometry and immunohistochemistry, tumor burden, survival, DNA-damage, immune infiltration, and response to pembrolizumab was assessed.Results A prefoldin protein, previously reported as an oncogene implicated in CRC initiation and notably overexpressed in CMS2 tumors, emerged as a top predictive feature in a random forest classifier machine-learning. Its expression correlated strongly with resistance to ICB. Machine-learning models, including ElasticNet and SHAP analysis, confirmed its predictive value (AUC=0.96) and association with immune resistance. Gene set enrichment analysis showed tumors with low expression of this prefoldin had signatures of T cell activation and cytotoxicity. In TCGA CRC data, low prefoldin expression was linked to MSI-H, elevated neoantigen load, and immune-infiltrated microenvironments. Mechanistically, prefoldin knockdown impaired NHEJ, inducing DNA-damage, activating the cGAS-STING-IFN-I axis, and upregulating interferon-stimulated genes. In vivo, prefoldin loss reprogrammed the tumor microenvironment, promoting CD8 + T cell infiltration and inflammation. In human CRC tissues, low prefoldin levels correlated with increased Granzyme B+ cells (p = 0.02). prefoldin inhibition also reduced tumor burden in both genetically engineered mouse models and humanized mice and synergized with anti-PD-1 therapy, resulting in robust tumor regression and extended survival.Conclusions The prefoldin protein emerges as a critical tumor-intrinsic driver of immune evasion in CRC, its inhibition reactivates anti-tumor immunity and converts immune-cold tumors into ICB-responsive ones, offering a powerful therapeutic strategy to overcome resistance in CRC and potentially across other immune-excluded cancers.References Sung H, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–249. https://doi.org:10.3322/caac.21660.Guinney J, et al. The consensus molecular subtypes of colorectal cancer. Nat Med. 2015;21:1350–1356. https://doi.org:10.1038/nm.3967 Lee V, Murphy A, Le DT, Diaz LA, Jr. mismatch repair deficiency and response to immune checkpoint blockade. Oncologist. 2016;21:1200–1211. https://doi.org:10.1634/theoncologist.2016-0046