Fig. 4: Selective degradation of RAS mutants with a VHL–monobody fusion protein.

a The time courses of the KRAS(G12C) and KRAS(WT) levels and the pERK level in RASless MEFs after the induction of the expression of the VHL–12VC1.2 degrader. MLN4924 (1 µM) or MG132 (5 µM) were added after 24 h of degrader expression for an additional 24 h (see the scheme). Total RAS was quantified and normalized relative to t = 0. b Degradation of endogenous KRAS(G12V) by a monobody degrader and its effect on the pERK level in PATU8902. The experimental schematic was identical to that for (a). The graphs show the quantification of the total RAS levels and KRAS levels from immunoblots (biological replicates, n = 2). c The time courses of the RAS levels and pERK level after the expression of a monobody inhibitor (blue) or a degrader (red) in the H23 cells (biological replicates, n = 3, representative results shown). A Flag-tagged mVenus–12VC1 fusion (inhibitor) and a HA-tagged VHL–12VC1.2 fusion (degrader) were expressed upon addition of doxycycline (1 µg/mL). The expression levels of the inhibitor or degrader were quantified using known amount of protein containing both Flag and HA tags (ctrl) as references. After 72 h of induction (black arrow), the media were replaced with serum- and doxycycline-free media to examine the persistent effects of intracellular inhibitors or degraders. d Mouse xenograft experiments with the H23 cell line expressing the VHL–monobody fusion proteins. Mice (5 per group for 12VC1.2 and MB(Neg)+dox, 4 per group for MB(Neg)−dox) were subcutaneously injected at the right and left flanks with H23 cell lines that express either fusion protein under a dox-inducible promotor, with the exception of one mouse in the 12VC1 group that was injected only at the right flank with tumor cells. One group of mice per cell line was given dox-containing feeds when the average tumor size was above 100 mm³ (black arrow) through the end of the experiment. The sizes of the tumors in MB(Neg) group (red squares) and 12VC1.2 group (blue circles) with (solid symbols) and without (empty symbols) dox feeds are plotted (mean ± SEM) as a function of time after cell injection. e Tumors were extracted at the end of experiment and their weights were compared (n = 8 and 10 for tumors for MB(Neg) without and with dox, respectively, and n = 9 tumors for 12VC1.2, two-way ANOVA, 12VC1.2 −dox vs +dox, p-value = 0.004, 12VC1.2 +dox vs MB(Neg) +dox, p-value = 0.01, MB(Neg) +dox vs −dox, p-value > 0.99). The bar centers show the average tumor weights of the population and the error bars show SD. f An image of extracted tumors from each group with a ruler for scale, ranked from large to small in weight.