Abstract Immune checkpoint blockade (ICB) has revolutionized cancer treatment, but resistance limits its clinical effectiveness. Therefore, understanding immune resistance mechanisms and identifying predictive markers are essential. Our transcriptomic analysis of PD-1 blockade-treated patients and non-responsive tumor models identifies WEE1 as a key resistance factor, promoting cancer stem cell (CSC)-like traits and immune resistance phenotypes including low T cell infiltrated into tumor and anti-apoptotic properties to cytotoxic T cells (CTLs). WEE1 drives AKT hyperactivation, enhancing expression of resistance factors such as CYCLIN A which contributes to CSC-like properties, and the anti-apoptotic molecule MCL-1, while also reducing T cell infiltration via CXCL10 downregulation. This signaling axis is conserved across various cancers. Notably, WEE1 inhibition sensitizes tumors to ICB by disrupting these refractory properties and reinvigorating antitumor immunity. These findings underscore the novel role of WEE1 in driving immune resistance and CSC-like characteristics, supporting the use of WEE1 inhibitors as a strategy to overcome resistance to anti-PD-1 therapy. Citation Format: Hyo-Jung Lee, Suyeon Kim, Eunho Cho, Tae Woo Kim. WEE1 confers resistance to PD-1 blockade via hyperactivation of the AKT signaling pathway [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B036.