Goldstein, M. E.; Chu, B.; Carillo, K. J.; Orban, J.; Toth, E. A.; Fuerst, T. R.

Controlling aberrant RAS signaling has been the subject of intensive efforts aimed at developing specific RAS inhibitors, small molecules that promote RAS degradation, and monobodies that inhibit RAS activity. Direct proteolytic degradation of RAS by site-specific proteases has received considerably less attention. A naturally-occurring protease from Vibrio vulnificus toxin cleaves all RAS isoforms at switch I and attenuates RAS signaling in cell models and patient-derived xenografts, thus demonstrating the potential of this approach. We previously designed a RAS-specific protease, called RASProtease (or RASp), that site-specifically cleaves RAS at switch II. Attacking switch II leverages an order to disorder transition that this region undertakes upon conversion to the active form that predominates in cancer. Switch II participates in an allosteric network that controls KRAS oncogenicity, making it a promising target for proteolytic cleavage that modulates RAS signaling. Preferential targeting of active RAS could be particularly useful for studying RAS signaling networks as well as having potential therapeutic value. Here we examined the effects of RASp cleavage on downstream signaling and cell viability in the MIA PaCa-2 cancer cell model, which harbors homozygous KRAS G12C and is KRAS-dependent for growth and survival. We found that cleavage of KRAS G12C coincided with a decrease in MEK-ERK signaling and resulted in extensive MIA PaCa-2 cell death 24 hours after induction of RASp expression. This level of cell death far exceeded that of control HEK 293T cells under the same conditions, underscoring the vulnerability of this cancer cell model to KRAS G12C elimination.