Extended Data Figure 9. Analysis of mutational tolerance of SHOC2 residues based on deep mutational scanning and residue contact points within SHOC2 complex, Impact of SHOC2 variants on growth in low attachment, and Impact of SHOC2 variants on MAPK signaling, and Impact of SHOC2 variants on MAPK signaling in response to MEK inhibition.

a, Violin plot of SHOC2 mean positional viability for surface contacting residues between complex members, PP1C (green, n = 28 positions) and MRAS (maroon, n = 26 positions), compared to core-residues (brown, n = 198 positions) and surface non-contacting residues (yellow, n = 329). Center line represents median, whiskers represent the first and fourth quartiles, box edges represent the second and third quartiles. b, MIA PaCa-2 with knock-out of endogenous SHOC2 and stably re-expressing various SHOC2 gain-of-function (red) and loss-of-function (blue) variants were seeded in ultra-low attachment plates and cultured for 7 days. Viability endpoint via cell titer glow is presented on x-axis along scaled LFC from fitness screen with PaTu-8902. Error bars represent standard deviation of GILA CTG viability (n=6 technical replicates; representative of 3 biological replicates). Line (green) represent simple linear regression model, 95% confidence interval (black dashed lines), R² (goodness of fit), and linear model p-value < 0.0001 (analysis of regression coefficient significantly non-zero) indicated. c, Wild type (WT) and gain- or loss-of-function (GOF/LOF) variants were stably expressed in KRAS mutant cell line MIA PaCa-2 with knock-out of endogenous SHOC2. d, Densitometry quantification of P-S259 RAF1 relative to total RAF1 and normalized to levels from wildtype expressing cells. Center line represents median and whiskers represent interquartile range. ***p<0.001, two-sided t-test between LOF (n = 5 variants) and GOF (n = 6 variants) SHOC2 alleles, representative of 3 biological replicates. e, Wild type (WT) and gain- or loss-of-function (GOF/LOF) variants were stably expressed in KRAS mutant cell line MIA PaCa-2 with knock-out of endogenous SHOC2. Cells were treated with the MEK1/2 inhibitor trametinib (10nM) for 24 hours prior to Western blot. f, Densitometry quantification of P-S259 RAF1 relative to total RAF1 and normalized to levels from wildtype expressing cells. Center line represents median and whiskers represent interquartile range. ***p<0.001, two-sided t-test between LOF (n = 6 variants) and GOF (n = 6 variants) SHOC2 alleles, representative of 3 biological replicates. g, Immunoprecipitation of V5-tagged SHOC2 variants in 293T cells co-transfected with HA-MRAS. h, Densitometry analysis of relative prey including endogenous PP1CB (yellow) and MRAS (maroon) normalized to V5 bait (y-axis) and DMS fitness score (LFC Z-score) (x-axis). Lines represent simple linear regression model, R² (goodness of fit), and linear model p-value < 0.0001 (analysis of regression coefficient significantly non-zero) indicated, representatitve of 3 biological replicates. i, Deep mutational scanning results for N-terminal region of SHOC2 (residues 60-68) depicted via sequence logo plot per amino acid substitution at respective positions (ggseqlogo).