Supplementary Figure 2: Expression of SHP2^E76K rescues Class 3 BRAF, NF1^LOF and KRAS^G12C mutant cell lines from RMC-4550.

Stable expression of SHP2-WT and SHP2-E76K was induced upon lentiviral transfection of NCI-H508 (Class 3 BRAF), NCI-H1838 (NF1^LOF), and NCI-H358 (KRAS^G12C) cells (n = 3 biologically independent experiments; figures show mean +/− S.D.) a, c, e) Effect of RMC-4550 on proliferation of NCI-H508 (a), NCI-H1838 cells (c), and NCI-H358 cells (e) in 3D culture. Three days after seeding, cells were treated with RMC-4550 and cell viability measured on Day 8 using CTG (data are representative of n = 2 independent experiments for NCI-H508 and NCI-H1838, and n = 3 observations for H358, in technical duplicate; figures show mean +/− S.D.). RMC-4550 exhibited concentration-dependent inhibition of growth in parental and SHP2^WT-expressing cell lines representing all three genotypes but failed to elicit a reduction in proliferation ≥50% of control signal in SHP2^E76K expressing cells up to the maximal test concentration of 10 μM. b, d, f) Effect of RMC-4550 on pERK levels in NCI-H508 (b), NCI-H1838 cells (d), and NCI-H358 cells (f). Cells were grown in 2D culture and incubated with 1 µM RMC-4550 for 90 minutes. Cellular lysates were prepared and levels of pERK determined by immunoblot. RMC-4550 treatment resulted in reduction of pERK levels in parental and SHP2^WT expressing cells across all genotypes, but failed to suppress pERK in SHP2^E76K expressing cells. Shown is one representative example of three independent experiments. Source data is provided in Supplementary Table 9.