Abstract Neutrophil extracellular traps (NETs) are extracellular web-like structures of cytosolic and granule proteins assembled on decondensed chromatin. NETs trap and kill bacteria, fungi, viruses, and parasites. Although some studies implicate NETs in tumor metastasis, whether NETs can kill cancer cells is largely unexplored. We previously showed that PIK3CA mutations render colorectal cancers (CRCs) more dependent on glutamine, and that a combination of CB-839, a glutaminase inhibitor, and 5-FU induced tumor regression in PIK3CA-mutant CRCs. Surprisingly, the combination of CB-839 and 5-FU induced NETs in PIK3CA mutant CRCs in xenograft, syngeneic, and genetically engineered mouse models. Disruption of NETs by DNase I treatment or neutrophil depletion in CRC tumors attenuated the efficacy of the drug combination. Mechanistically, the drug combination preferentially induced IL-8 expression in PIK3CA-mutant CRCs to attract neutrophils into the tumors. Moreover, the drug combination increased reactive oxygen species levels in neutrophils, thereby inducing NET formation. Cathepsin G, a serine protease localized in NETs, enters CRC cells by binding to a cell surface protein RAGE. The internalized cathepsin G cleaves 14-3-3 proteins, releases Bax, and triggers apoptosis in CRC cells (Fig. 1). We have completed a phase II clinical trial of the combination of CB-839 and capecitabine, an oral prodrug of 5-FU, in PIK3CA mutant metastatic CRC patients who were refractory to prior 5-FU treatment. An analysis of tumor biopsies from these patients showed that NETs were significantly up-regulated in post-treatment biopsies compared to their pre-treatment counterparts. Moreover, higher levels of NETs were associated with longer progression-free survival, suggesting that the drug combination-induced NETs inhibit tumor growth in patients. Lastly, we found that a variety of other chemotherapy drugs, including camptothecin/irinotecan, gemcitabine, daunorubicin, epirubicin, oxaliplatin, and regorafenib, also induced NETs, suggesting that NET induction may be a general mechanism by which chemotherapy inhibits tumor growth. Interestingly, the drug-induced NETs formation requires cathepsin G (CTSG), as these drugs failed to induce NETs in CTSG knockout neutrophils. Moreover, the drug treatment caused CTSG-dependent cleavage of histone H3, which is also distinguished from the classical NET formation process. We are currently working to elucidate the molecular mechanism by which histone H3 cleavage triggers NET formation. In summary, our studies illuminated a paradigm-shifting concept: drug-induced NETs kill cancer cells through a previously unrecognized molecular mechanism. Citation Format: Zhenghe John . Wang. Killing cancer cells by suicidal bombing attack with neutrophil extracellular traps abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B074.
Zhenghe Wang (Wed,) studied this question.