Necrocide 1 Mediates a Non-Apoptotic Necrotic Cell Death and Immunogenic Response in Human Cancer Cells

Abstract 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 inhibitors, 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.