br Figure Effect of Pairwise Overexpression of a Kinase
Figure 6. Effect of Pairwise Overexpression of a Kinase and a Phosphatase on Signaling
(A) Workflow of the pairwise overexpression. Two plasmids encoding an FLAG-tagged kinase and a GFP-tagged phosphatase were transfected into HEK293T ASP-1517 successively. Cells were binned into 25 groups according to their FLAG and GFP abundances. The median level of each measured phosphorylation site was computed for each bin.
(B), Kinases MAP2K2, MAPK1, and RPS6KA1 and phosphatases DUSP4, DUSP7, and PTPN2 were selected for the pairwise overexpression, generating nine overexpression combinations in total.
(C) In cells with overexpression of MAP2K2 and DUSP4, median phosphorylation levels of p-MEK1/2 and p-ERK1/2 are plotted for all of the bins over the 1-h EGF stimulation time course.
(D) Signaling trajectories of p-MEK1/2 and p-ERK1/2 plotted as the medians of each individual bin over the 1-h EGF stimulation time course.
(E) Schematic illustrating the modulation of RAF-MEK-ERK cascade signaling states and dynamics upon pairwise overexpression.
(F and G) Analysis of p-ERK1/2 and p-MEK1/2 phosphorylation levels (F) and signaling trajectories (G) on MAPK1-FLAG and DUSP7-GFP abundances.
Figure 7. Kinase Overexpression Induces Resistance to BRAF-MEK Concurrent Inhibition in Melanoma Cells by MEK-Independent ERK Reactivation
(A) Selected kinases were transfected into A375 cells and cultured in the inhibitor- or DMSO-containing media, and signaling states and cell viability were assessed.
(B) Single-cell data from each sample were divided into four bins, depending on the expression level of the GFP-tagged kinase. Signed-BP-R2 analysis was performed to quantify signaling relationships with and without vemurafenib.
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of ERK due to the reduced negative feedback strength. This mechanism may underlie the pro-cancer effects of phosphatase overexpression.
Analysis of the Abundance-Dependent Signaling Relationship Characterizes Drug-Resistance Mechanisms
As protein overexpression has been correlated with drug resis-tance of cancer cells (Johannessen et al., 2010, 2013; Shaffer et al., 2017), we next sought to determine whether our kinome-and phosphatome-wide signaling network profiles could identify kinases or phosphatases that, when overexpressed, induce drug
resistance and could characterize underlying signaling mecha-nisms. In melanoma cells carrying the BRAFV600E mutation, the
overexpression of certain kinases is associated with de novo or acquired resistance to MAPK-ERK inhibition; Johannessen et al. (2010, 2013) have identified resistance-driving candidate genes in viability assays using melanoma A375 cells. Seventeen of these candidates were analyzed in our screen, and the overexpression of 14 caused abundance-dependent signaling modulations to p-ERK1/2 (Thr202/Tyr204) in unstimulated cells (Figure S7A). The 10-min EGF stimulation reduced relationship strengths for each of these kinases (Figure S7B), indicating that these overexpression-related ERK activations were ligand-binding independent (described above with Figure 5C), which has been previously suggested to be a drug-resistance mecha-nism (Guo et al., 2015).
In our kinome- and phosphatome-wide study, we detected 54 POIs that activated ERK in the absence of EGF (Table S7). To determine whether the overexpression of these proteins is
predictive of drug resistance in cells with BRAF mutations, we transfected A375 cells, a melanoma cell line with the BRAFV600E
mutation, using vectors encoding proteins with the strongest abundance-dependent signaling relationship to p-ERK1/2; ABL1, BLK, FES, MAP3K2, MAP3K8, MOS, NTRK2, SRC, and YES1. MEK1DD, a constitutively active kinase, was used as a positive control (Johannessen et al., 2010). Cells were subse-quently treated for 48 h with the BRAFV600E inhibitor vemurafenib or with DMSO (Figure 7A). The strong signaling relationships between these POIs and p-ERK1/2 were observed only in cells treated with vemurafenib, not in control cells treated with DMSO (Figures 7B, S7C, and S7D). This suggests that (1) the constitutive BRAF activation caused by the BRAFV600E mutation leads to strong MAPK-ERK activation that overrides the overex-pression effects, and (2) POI abundance-dependent ERK signaling is independent of RAF activity. To determine whether overexpression of these candidate POIs reduced sensitivity to the BRAFV600E inhibitor vemurafenib, we assigned each single cell into one of four bins based on the abundance of GFP-tagged POI and calculated the percentage of cells in each bin relative to the total cell count (Figure 7B). As expected, the positive control cells that expressed MEK1DD-GFP had significant cell enrich-ment in the fourth bin (i.e., the bin with the highest expression