Fig 2: Pooled shRNA library screen identifies PPP6C as a mediator of response to MEKi(A) Schematic of the pooled shRNA MEKi sensitivity screen.(B) Changes in all shRNA hairpins shown as log2(T10/T0) from most depleted to most enriched for each drug condition. Bars representing shRNA hairpins targeting BRAF are shown in blue. All others are shown in gray.(C) Top enriched genes for each drug condition from two replicates of the screen. Colored boxes indicate the genes ranked in the top 50 enriched genes by RIGER for that drug condition not found in the DMSO control condition.(D) Changes in all shRNA hairpins shown as log2(T10/T0) arranged as in (B) but with red bars indicating shRNA hairpins targeting PPP6C. Ranking of each hairpin is shown in Figure S1A.(E) PPP6C+/+ and PPP6C-/- 501mel cells were treated for 72 h with increasing concentrations of trametinib or selumetinib. Cell viability was determined and normalized to vehicle control for each cell line. Dose-response curves and IC50 values for PPP6C+/+ (black) and PPP6C-/- (blue) are shown. The 95% confidence intervals (n = 3) were 8.9–17 nM (PPP6C+/+, trametinib), 65–209 nM (PPP6C-/-, trametinib), 30–66 nM (PPP6C+/+, selumetinib), and 500–1,900 nM (PPP6C-/-, selumetinib). Error bars show SD.(F) PPP6C+/+ and PPP6C-/- 501mel cells were cultured in media containing DMSO or the indicated concentration of trametinib for 2 weeks and stained with crystal violet. Quantification is shown in Figure S2A. n = 3.See also Figures S1B, S2B, and S2C.

Fig 3: PPP6C regulation of ERK signaling is prominent in ERK pathway-driven cancer cells(A) The indicated cell lines were transduced to stably express control (shCTRL) or PPP6C-targeting (shPPP6C-1 or PPP6C-2) shRNAs. Cells were lysed and assessed by immunoblot for levels of phosphorylated and total MEK and ERK. Quantification is shown in Figure S3. n = 2.(B) PPP6C CERES scores for skin cancer cell lines with WT BRAF or hotspot BRAF mutations from the Cancer Dependency Map Project. Cell lines harboring BRAF variants of unknown significance were excluded. Data are represented as mean ± SD. ***p < 0.0005, Welch’s t test.(C) Heatmaps depicting CERES scores of PPP6C, ERK-selective DUSPs, and ERK MAPK cascade components in skin cancer cell lines using data from the Cancer Dependency Map Project. Pearson’s correlation coefficients and p values from linear regression analysis of each gene with PPP6C in the DepMap portal are listed.(D) PPP6C RNA-seq mRNA expression level plotted against MEK1 phosphorylation levels for TCGA tumor samples in cBioPortal. Correlation coefficients and associated p values from linear regression analyses are indicated.(E) PPP6C mRNA levels plotted against ERK2 phosphorylation levels for TCGA tumor samples as in (D).See also Figure S4.