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Potent and selective eradication of tumor cells by an EpCAM-targeted Ras-degrading enzyme
Valentina Palacio-Castañeda1, Bas van de Crommert1, Elke Verploegen1
1Department of Medical BioSciences, Radboud University Medical Center, Geert Grooteplein 28, 6525 GA Nijmegen, the Netherlands.
Abstract:
Despite decades of efforts, an urgent need remains to develop tumor cell-selective rat sarcoma (Ras)-targeting therapies that can treat patients with Ras-driven tumors. Here we report modular engineered proteins that degrade Ras selectively in tumor cells that overexpress the tumor cell marker epithelial cell adhesion molecule (EpCAM) by fusing the Ras degrader Ras-Rap1-specific endopeptidase with the translocation domain of the Pseudomonas aeruginosa exotoxin A (ETA) or diphtheria toxin (DT). Redirection to EpCAM is achieved by a designed ankyrin repeat protein. In two-dimensional tumor cell cultures, complete degradation of Ras proteins after 24 h was observed with EpCAM-targeted Ras degraders fused to ETA or DT in EpCAM-overexpressing MCF7 and HCT116 cells, with median inhibition concentration values at sub-nanomolar levels. The viability of EpCAM-low non-cancerous fibroblasts remained unaffected. In a three-dimensional (3D) tumor-on-a-chip system that mimics the natural tumor microenvironment, effective Ras degradation and selective toxicity toward tumor cells, particularly with the ETA-fused constructs, was determined on-chip. To conclude, we demonstrate the potential of modular engineered proteins to kill tumor cells highly selectively by simultaneously exploiting EpCAM as a tumor-specific cell surface molecule as well as Ras as an intracellular oncotarget in a 3D system mimicking the natural tumor microenvironment.
Insights
New modular proteins target Ras in EpCAM-overexpressing tumor cells, degrading the oncoprotein and sparing healthy cells. This Ras-targeting therapy shows promise in 3D tumor models.
Area of Science:
- Oncology
- Protein Engineering
- Molecular Biology
Background:
- Ras-driven tumors lack effective, tumor cell-selective therapies.
- Targeting intracellular oncoproteins like Ras requires efficient tumor cell delivery.
Purpose of the Study:
- To develop modular engineered proteins for selective Ras degradation in tumor cells.
- To utilize epithelial cell adhesion molecule (EpCAM) as a tumor-specific targeting marker.
- To evaluate therapeutic efficacy in 2D and 3D tumor models.
Main Methods:
- Fusion of a Ras degrader (Ras-Rap1-specific endopeptidase) with toxin translocation domains (ETA or DT).
- Incorporation of a designed ankyrin repeat protein for EpCAM-mediated targeting.
- Assessment of Ras degradation and cell viability in EpCAM-positive/negative cell lines and a 3D tumor-on-a-chip system.
Main Results:
- EpCAM-targeted Ras degraders achieved complete Ras degradation in EpCAM-overexpressing tumor cells (MCF7, HCT116) at sub-nanomolar concentrations.
- Non-cancerous fibroblasts with low EpCAM expression were unaffected.
- Effective Ras degradation and selective tumor cell toxicity were observed in a 3D tumor-on-a-chip model, particularly with ETA-fused constructs.
Conclusions:
- Modular engineered proteins can selectively degrade Ras within tumor cells.
- Simultaneous targeting of EpCAM and intracellular Ras offers a potent strategy for tumor cell killing.
- This approach demonstrates significant potential for developing novel cancer therapies in a microenvironment-mimicking 3D system.
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