br overexpression of certain kinases is associated
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 Lenvatinib (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
level of the GFP-tagged POI), with vemurafenib treatment compared to the DMSO-treated control (Figure 7C). This indi-cates that MEK1DD overexpression contributes to cell survival. Similarly, all nine examined kinases showed an enrichment of cell abundance in the fourth bin; in six cases, this enrichment was statistically significant (Figure 7C). These results reveal that in melanoma A375 cells, the overexpression of kinases capable of ligand-independent ERK activation reduces cellular dependency on signaling inputs from BRAFV600E.
Three of the tested kinases, MAP3K8, MOS, and SRC, were previously suggested to mediate MAPK-ERK inhibition resis-tance in A375 cells (Johannessen et al., 2013). In analyzing the cell viability data in the same study, we noted that MAP3K8 and MOS only induced resistance to RAF inhibition, whereas SRC overexpression caused resistance to both RAF and MEK individual inhibitions and the concurrent RAF-MEK inhibition (Figure S7E) (Johannessen et al., 2013). We hypothesize that the difference in drug responses are due to the MEK dependency of the POI overexpression-induced ERK activation (i.e., an over-expressed POI directly activating ERK will not be influenced by upstream RAF and/or MEK inhibition). To test this, A375 cells were transfected individually with vectors encoding ABL1,
fenib, MEK inhibitor CI1040, or the combination of both inhibitors for 3 h. As expected, cells overexpressing MEK1DD were not sensitive to vemurafenib, whereas treatment with CI1040 or the combination treatment completely blocked ERK activation (Fig-ures 7D and S7F). Similarly, cells that overexpressed MAP3K8, MAP3K2, or MOS were resistant to vemurafenib but sensitive to CI1040, suggesting that MEK activity is necessary for the MAPK-ERK reactivation induced by these three kinases (Fig-ure 7D, purple boxes). Other POIs, including ABL1, BLK, FES, NTRK2, SRC, and YES1, showed abundance-dependent ERK activation, even with the combination treatment of vemurafenib and CI1040, indicating that these proteins activate ERK in an MEK-independent manner (Figures 7D and S7F).