Extended Data Figure 10: Trametiglue provides durable inhibition of RAS/ERK signaling in models of mutant KRAS and BRAF.
A. (Left) Immunoblot of stable HCT-116 (KRAS G13D) cancer cells including parental, scramble control (shSCR), and KSR1 knockdown (shKSR1). Cells were treated with 10 nM trametinib for the indicated time points and harvested for analysis on the indicated markers. (Right) Quantitative PCR was used to confirm specific knockdown of KSR1 in the shKSR1 cells. KSR1 knockdown slows the rebound of activated RAS-MAPK signaling in the presence of trametinib as measured by recovered phosphorylated-ERK1/2 over time (lanes 1–5 and 6–10 versus 11–15). This data supports that KSR1 plays a positive role in the adaptive resistance of HCT-116 cells to trametinib, suggesting that knockdown or trapping of the KSR-bound MEK complex could mitigate this intrinsic drug resistance mechanism. Experiment was conducted twice with similar results.
B. EC50 values for cell viability assays for the indicated compounds against a series of human cancer cell lines. Mean and standard deviation determined from three independent experiments, each conducted in technical triplicate. Raw data is included in Source Data Extended Data Fig. 10.
C. X-ray crystal structure of trametinib bound to the KSR2:MEK1:AMP-PNP complex. MEK1 and KSR2 are colored pink and green respectively, with several key residues highlighted. Trametinib is shown in stick representation. A Fo-Fc omit electron density map, contoured at 3.0 σ with a 2.0 Å cutoff around ligand, is shown as a blue mesh. Left panel shows the entire inhibitor binding pocket; right panel highlights contacts around the phenyl acetamide group of trametinib.
D. Bar graph plot of mean EC50 values from B.
E. Clonogenic assay of KRAS-mutant and BRAF-mutant cancer cell lines treated with 10 nM trametinib or 10 nM trametiglue, and 10 nM or 50 nM CH5126766 for 10 days. Experiment was conducted twice with similar results.
F. Immunoblot analysis of the indicated cell lines treated for 1 hour with increasing concentrations of trametiglue and trametinib. This data supports that trametiglue, relative to trametinib, is a higher potency inhibitor of RAS-MAPK signaling as measured by phosphorylated ERK1/2 at residues T202 and Y204 (pERK). Experiment was conducted three times with similar results.
G. Immunoblot of KRAS-mutant and BRAF-mutant cancer cell lines treated with 10 nM trametinib or trametiglue for various times. Experiment was conducted twice with similar results.