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Evaluation of Caspase Activation to Assess Innate Immune Cell Death
Published on: January 20, 2023
Evolution of botulinum neurotoxin serotype X proteases to induce inflammatory cell death in cancer cells
Julia McCreary1,2,3,4, Colin F Hemez1,2,3,4, Michael H Raymond4,5
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
Abstract:
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.
Insights
Researchers reprogrammed botulinum neurotoxin proteases to trigger cancer cell death. This engineered protease system shows potential for targeted cancer therapy by selectively inducing cell death in tumors.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Protease-activated cell death is a key strategy in cancer treatment.
- Botulinum neurotoxin (BoNT) proteases offer potential for targeted therapeutic applications.
Purpose of the Study:
- To reprogram BoNT serotype X proteases to cleave and activate procaspase-1 and gasdermin D.
- To develop a system for characterizing protease substrate specificity.
- To evaluate the efficacy of engineered proteases in cancer cell death induction and in vivo tumor growth inhibition.
Main Methods:
- Phage-assisted evolution was used to engineer BoNT serotype X proteases.
- Substrate specificity of wild-type and evolved proteases was characterized.
- Engineered proteases were reconstituted with a BoNT translocation domain for cell delivery.
- In vivo studies utilized a drug-resistant tumor mouse model.
Main Results:
- Evolved proteases effectively induced cell death in multiple cancer cell lines.
- Gasdermin D-cleaving protease induced lytic cell death; procaspase-1-cleaving variant induced both lytic and apoptotic cell death.
- Engineered proteases selectively killed cancer cells in culture.
- Expression of the caspase-1 targeting protease reduced tumor growth in a resistant mouse model.
Conclusions:
- An evolving protease system was established to modulate inflammatory cell death.
- Reprogrammed BoNT proteases demonstrate potential as programmable tools for targeted cancer therapy.
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