Reactivating necroptosis in colorectal cancer potentiates chemotherapy and antitumor immune responses.

Necroptosis, a form of immunogenic cell death (ICD), is frequently impaired in cancer due to the silencing of key regulators, such as RIP3. Despite its potential immunogenic benefits, restoring necroptosis in cancer therapy has been largely underexplored, primarily due to the lack of effective strategies or agents that specifically engage necroptosis without activating apoptosis. In this study, we investigated strategies to restore necroptosis for chemosensitization in RIP3-silenced colorectal cancer (CRC) cells. While reconstituting RIP3 did not impact cell killing by chemotherapy, bypassing RIP3 to induce necroptosis using the natural compound OSW-1 emerged as a promising strategy. OSW-1 displayed strong synergy with 5-fluorouracil (5-FU), effectively eliminating RIP3-silenced CRC cells and tumors through the induction of both necroptosis and apoptosis. Cell death triggered by the OSW-1/5-FU combination was mediated by p53 and CaMKIIδ-driven MLKL phosphorylation, a critical step in the execution of necroptosis. The combination robustly induced ICD hallmarks in CRC cells and immune infiltration in syngeneic tumors. The in vivo antitumor effect of the OSW-1/5-FU combination was largely dependent on antitumor immunity, and abolished by anti-CD8 antibody or immunodeficient hosts. Furthermore, the combination increased immune infiltration in patient-derived primary CRC Air-Liquid Interface (ALI) organoid cultures with autologous immune tumor microenvironment. Collectively, our findings support a novel strategy to activate RIP3-independent necroptosis, providing a compelling rationale to boost the efficacy of chemotherapy via enhanced CRC cell killing and antitumor immunity.