Supplementary Figure 3: Effects of SHP2 inhibition in RAS pathway-driven cancer cells. | Nature Cell Biology

Supplementary Figure 3: Effects of SHP2 inhibition in RAS pathway-driven cancer cells.

From: RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers

Supplementary Figure 3

(a) Effect of RMC-4550 on RAS-GTP levels in BEAS-2B cells stably transfected with scrambled (shScramble) or NF1 targeting (shNF1) shRNA. Data shows a representative of three independent experiments. (b-d) (b) Cell sensitivities to the knockdowns of the indicated genes were determined using crystal violet staining assays (n = 2). The spectrophotometric intensities of the crystal violet stains were quantified and represented as a bar plot. (c) RAS-GTP levels in cellular lysates are shown by GST-RAF-RBD affinity capture. (d) Individual western blot band intensities were quantified and shown for pERK/ERK, RAS-GTP/RAS (total) level and knock down efficiency, expressed relative to a control scrambled shRNA intensity of 1.0 and the effect of knockdown on pERK. (e-h) (e), (f) Effect of RMC-4550 and the KRASG12C-specific inhibitor ARS-853 on pERK levels in KRASG12C cancer cell lines. Cells were incubated with increasing concentrations of RMC-4550 or ARS-853 for one hour. Cellular lysates were prepared and levels of pERK determined. RMC-4550 produced a concentration-dependent reduction in pERK levels. Geometric mean IC50 values for reduction in pERK by RMC-4550 in NCI-H358 and CALU-1 were 36 nM and 7 nM, respectively. A concentration-dependent reduction in pERK levels was observed for ARS-853 (geometric mean IC50 values of 4700 nM and 430 nM for NCI-H358 and CALU-1 cells, respectively); data represent n = 4 independent experiments, performed in technical duplicate; figures show mean +/− S.D.). (g), (h) Effect of RMC-4550 and ARS-853 on proliferation of two KRASG12C cancer cell lines in 3D culture. NCI-H358 and CALU-1 cells were treated with RMC-4550 or ARS-853 and cell viability measured on Day 7 using Cell Titer Glo (data represent n = 4 independent experiments, performed in duplicate for NCI-H358, and n = 3 independent experiments, performed in duplicate for CALU-1; figures show mean +/− S.D.). RMC-4550 and ARS-853 exhibited geometric mean IC50 values of 43 nM and 1020 nM, respectively, in NCI-H358 cells. Source data is provided in Supplementary Table 9.

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