Many anticancer agents induce apoptosis, mitotic catastrophe or senescence. Here, we report the functional characterization of an experimental inducer of TNF-independent necrosis, necrocide-1 (NC1). NC1 (but not its stereoisomer) killed a panel of human cancer cell lines (but not normal cells) at nanomolar concentrations and induced a non-apoptotic, necrotic morphotype, both in vitro and in vivo, in human cancer cells and xenograft models. NC1-induced killing was not inhibited by caspase

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... ors, Bcl-2 overexpression or TNF neutralization, suggesting that NC1 elicits a bona fide necrotic pathway. However, pharmacological or genetic inhibition of necroptosis, pyroptosis and ferroptosis faild to block NC1-mediated regulated necrosis. Instead, NC1 elicited mitochondria ROS production, either elimination of mitochondrial DNA or chemical inhibition of mitochondrial ROS production blocked NC1-induced necrotic cell death. NC1 induced all hallmarks of immunogenic cell death (calreticulin exposure, ATP release and HMGB1 release) in vitro, and NC1-killed mouse cancer cells were able to induce a protective anticancer immune response when injected into histo-compatible, immunocompetent mice. Altogether, we identify a previously uncharacterized signaling cascade leading to necrotic cell death and provided further support to the notion that the induction of programmed necrosis may constitute a future approach for anticancer therapy.