Evolution of botulinum neurotoxin serotype X proteases to induce inflammatory cell death in cancer cells

Triggering protease-activated cell death is a promising strategy for cancer treatment. Here, we used phage-assisted evolution to reprogram botulinum neurotoxin serotype X proteases to cleave and activate procaspase-1 and gasdermin D, key effectors of inflammatory cell death. We also developed an efficient system to broadly characterize the substrate specificity of wild-type and evolved botulinum neurotoxin serotype X protease variants. Evolved proteases triggered robust cell death across multiple cancer cell lines. The gasdermin D-cleaving protease exclusively induced lytic death, whereas the procaspase-1-cleaving variant initiated both lytic and apoptotic cell death. To enable self-delivery into mammalian cells, we reconstitute evolved proteases with a native BoNT translocation domain, selectively killing cultured cancer cells while sparing non-cancerous cells. Expression of the evolved protease targeting caspase-1 reduced tumor growth in a highly drug-resistant tumor mouse model. These findings establish an evolving protease system to modulate inflammatory cell death and highlight the potential of BoNT proteases as programmable tools for targeted cancer therapy.