The goal of this competitive renewal is to interrogate a paradigm-shifting hypothesis that a combination of CB- 839, a glutaminase inhibitor, and 5-FU induces neutrophil extracellular trap (NET) in PIK3CA mutant colorectal cancers (CRCs), which in turn kills these cancer cells. NETs are extracellular web-like structures of cytosolic and granule proteins assembled on de-condensed 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. This proposal is built on the novel findings that we made during the current funding period: 1) PIK3CA mutations render CRCs more dependent on glutamine; 2) CB-839 preferentially inhibits xenograft growth of PIK3CA mutant CRCs; 3) the combination of CB-839 with 5-FU induces xenograft tumor regression of PIK3CA mutant CRC in nude mice; 4) the combination of CB-839 and 5-FU preferentially induces NETs in PIK3CA mutant CRCs; 5) depletion of neutrophils in nude mice significantly attenuates the efficacy of the drug combination; 6) disruption of NETs by DNase I treatment in xenograft tumors also attenuates the efficacy of the drug combination. These exciting observations lead us to hypothesize that NETs induced by the drug combination kill PIK3CA mutant CRCs. Two aims are proposed to test this exceptionally novel hypothesis. Aim 1 will determine if the combination of CB-839 and 5-FU induces NETs in immune-competent mice and human CRC patients. Aim 2 will elucidate the mechanisms by which NETs kill PIK3CA mutant CRCs. Although we focus on colon cancer in this proposal, our proposed studies may have a broader impact beyond colorectal cancer because PIK3CA is mutated in ~ 20% of all human cancer. Moreover, the mechanisms identified in the study may also apply to other NET-related diseases.