Recent studies reported on the

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 Levofloxacin 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.