Fig. 4. CHOMP circuit enables conditional activation of Casp3 in Ras-activating cells.

(A) The core circuit (left) links Ras activation by SOS_CA or EGFRvIII to Casp3 activation. The full circuit (right) incorporates an additional TVMVP component to enhance selectivity. New regulatory features introduced in this circuit are explained schematically in the corresponding numbered boxes. Box 1: Input from upstream activators of Ras such as SOS_CA and EGFRvIII (pink) activates Ras (light blue), causing it to bind RBD (dark blue), reconstituting RasTEVP. Box 2: Engineered Casp3 (green) tagged with a membrane localization sequence (mts) can be converted from an inactive to an active state by TEVP cleavage. Box 3: TVMVP cleavage detaches Casp3 from the membrane, reducing its ability to be activated by membrane-localized TEVP. (B) TEVP activates the engineered Casp3, whereas TVMVP inhibits this activation. Cells transfected with the indicated components were analyzed to determine the reduction index (percentage of cell number reduction compared to cells transfected with only a fluorescent marker; see materials and methods and fig. S5B). (C) The core circuit preferentially reduced cell number in the presence of ectopic SOS_CA. The full circuit exhibited improved selectivity. (D) The full circuit (top) and a positive-control circuit incorporating a Gly¹²→Val mutation that makes Ras constitutively active and a Cys¹⁵²→Ala mutation that abolishes TVMVP activity (bottom) were each encoded as a single transcript. (E) In a mixed population, the single-transcript circuit (D, top) conditionally reduced the number of EGFRvIII cells (left) and SOS_CA cells (right) compared with that of cocultured control cells. The positive-control circuit (D, bottom) reduced the number of both fractions. The dashed line indicates the upper limit of the reduction index measured with the positive-control circuit. Dots from the same well are color matched.