Among RAS mutant tumors analyzed we found that
Among RAS mutant tumors analyzed, we found that the efficacy of either the SHP2 inhibitor SHP099, or combined MEK and SHP2 inhibition was best in those expressing RAS mutations at G12 (Figure 7B). Recent studies revealed that cellular RAS(G12C) activity depends on RTK-mediated nucleotide exchange, and binding of an irreversible RAS(G12C) inhibitor promotes dissociation of RAS(G12C) from the GEF SOS (Janes et al., 2018, Lito et al., 2016, Ostrem et al., 2013, Patricelli et al., 2016). In our studies, RAS(G12C) tumor BIBW2992 showed variable degrees of sensitivity to SHP099, which paralleled reported sensitivity of the same cell lines to the RAS(G12C) inhibitors (Lito et al., 2016, Patricelli et al., 2016), consistent with both compounds affecting a common underlying dependence of RAS(G12C) on GEF activity. The combination of MEK and SHP2 inhibition was effective in RAS(G12C) cells, consistent with previously reported data showing low, but detectable, intrinsic GTPase activity retained in such mutants (Hunter et al., 2015). We further found that SHP2 inhibition suppressed ERK activity and that combined MEK and SHP2 inhibition was effective in cells with other RAS(G12X) mutants, such as RAS(G12S) and RAS(G12A). These results suggest that, although these RAS mutants did not show substantial GTPase activity in biochemical assays as purified proteins (Hunter et al., 2015), they may still depend on nucleotide exchange in cells. Alternatively, it is possible that, in certain contexts, pERK activity is regulated by SHP2 via additional mechanisms to GEF (SOS) recruitment. Further biochemical and cell-based studies are warranted to delineate the role of RTK/SHP2 and nucleotide exchange in the regulation of the different RAS mutants in cells. Recent studies reported on the dependency of tumors with mutant RAS on SHP2 (Fedele et al., 2018, Mainardi et al., 2018, Nichols et al., 2018, Ruess et al., 2018). Consistent with those studies, we found RAS(G12X) mutants, but not RAS(Q61X), to be dependent on SHP2. However, although Mainardi et al. (2018) reported that RAS(G13D) signals in a SHP2-dependent manner, we found that RAS(G13D)-driven ERK activity was independent of SHP2 in the cell line models analyzed, and culturing conditions at low serum levels did not confer sensitivity to SHP2 inhibition. RAS(G13D) has been found previously in biochemical assays to retain low, but detectable, intrinsic GTPase activity and, in theory, could require upstream RTK signaling to maintain its active state. However, the same biochemical studies also found that this mutant exhibited an order of magnitude higher rate of nucleotide exchange, compared with wild-type RAS (Hunter et al., 2015, Smith et al., 2013). Thus, the much greater cellular concentration of GTP compared with GDP could result in SOS-independent auto-activation (Figure 7B), consistent with our findings with RAS(G13D) tumor cells. In BRAF(V600E) colorectal and thyroid tumors, we observed upregulation of multiple RTKs in response to ERK signaling inhibition (Figure 7C). By using a combination of pharmacological and knockdown targeting of specific RTKs to dissect the relative contribution of feedback-induced RTKs to RAS activation, we identified a role of EGFR signaling in a subset of colorectal BRAF(V600E) tumor lines, consistent with previous reports (Corcoran et al., 2012, Prahallad et al., 2012). We also identified an example of adaptive resistance to RAF inhibitors driven by another RTK, MET, rather than by the ERBB family. In each case, the tumor cells were also sensitive to combined RAF and SHP2 inhibition, indicating that SHP2 inhibition in combination with RAF and MEK inhibitors may be effective in a broader range of colorectal BRAF(V600E) tumors than combined targeting with EGFR/RAF/MEK, a drug combination recently assessed clinically in this context with modest results (Corcoran et al., 2018). In two BRAF(V600E)-expressing tumor lines, in which both basal and RAF inhibitor-induced p(Y542)SHP2 levels were virtually undetectable (“SHP2-negative”), we identified FGFR signaling driving RAS activation in response to ERK signaling inhibition (Figure 7C). These observations are consistent with previous findings that FGFR is able to signal both dependently or independently of SHP2 in different settings (Hadari et al., 1998, Kouhara et al., 1997). In a third SHP2-negative tumor line, SW1417, selective inhibition of upregulated RTKs detected by RTK array or in our RNA-seq data (not shown) did not affect the pERK rebound after VEM treatment, indicating that another, as-yet-unknown factor mediates feedback-induced RAS activation in those cells. Together these findings raise the possibility that other RTKs, or other RAS-stimulating factors, could signal in an SHP2-independent fashion, depending on cellular context. Identifying which factors and settings drive SHP2-mediated adaptive resistance to ERK signaling inhibitors in various ERK-dependent tumors should enable the development of effective combinatorial pharmacologic strategies tailored for specific tumor contexts.