Therefore, the predictive changes in P-S6 can be rapidly and quantitatively assessed by microscopic imaging in tumors sampled by minimally invasive FNA biopsies. P-S6 can predict responsiveness to RAF inhibition in melanoma patients To establish the feasibility of real-time P-S6 and P-ERK assessment in ideals calculated by Student’s test (unequal variances) are shown relative to before treatment FNA. taken before and 2 weeks after vemurafenib treatment have demonstrated that considerable (typically 80%) inhibition of extracellular signalCregulated kinase (ERK) phosphorylation was required to induce a tumor response (15). Consistent with these findings, we found that cell lines in which vemurafenib or selumetinib failed to substantially decrease the amount of phosphorylated ERK1 and ERK2 (P-ERK) (for example, WM1158 and MM608) were less sensitive to vemurafenib Farampator (Figs. 1B and fig. S2 and S3). Open in a separate windowpane Fig. 1 Reduction of TORC1 activity by RAF or MEK inhibition in sensitive ideals in (C) and (D) were determined with two-tailed Student’s test. However, we also observed a lack of level of sensitivity to vemurafenib or selumetinib in several cell lines (for example, IGR1 and A2058) despite powerful P-ERK inhibition that was comparable to that accomplished in sensitive cell lines (for example, WM164 and 451Lu) (Fig. 1B and figs. S2 and S3). These findings suggest that, although inhibition of P-ERK is clearly necessary, it alone is not Farampator sufficient to forecast level of sensitivity to MAPK inhibition, and some melanoma cell lines may consequently possess ERK-independent survival signals. RAF or MEK inhibition reduces TORC1 activity in drug-sensitive cell lines Analysis of additional signaling changes after RAF or MEK inhibition exposed that a decrease in phosphorylated ribosomal protein S6 (P-S6) levels after vemurafenib or selumetinib treatment correlated well with level of sensitivity to these providers (Fig. 1, B to D). With this cell collection panel, P-S6 suppression was a more effective predictor of level of sensitivity than several other candidate biomarkers previously reported to forecast level of sensitivity in = 0.03 (for VEM) and = 0.001 (for SEL) by two-tailed Student’s test. (B) WM164 or IGR1 cells were treated with or without 3 M vemurafenib (+VEM) in the presence (8055) or absence (con) of 300 nM AZD8055. Cells were lysed for Western blots after 24 hours and were analyzed for apoptosis after 72 hours of treatment. **= 0.001 (versus VEM) and 0.0001 (versus 8055); N.S., not significant, by one-way analysis of variance (ANOVA) with Bonferroni posttest. (C) Induction of apoptosis was measured by annexin V staining in WM164 and IGR1 cells treated in triplicate for 72 hours in the presence or absence of 3 M vemurafenib without (CON) or with 300 nM AZD8055, 1 M GDC0941, or 500 nM BEZ235. ** 0.0001 for combination relative to each single agent alone by one-way ANOVA with Bonferroni posttest. (D) Cells were treated in triplicate for 72 hours with 3 M vemurafenib, 1 M ABT-263, or both Farampator inhibitors in combination and were assessed for apoptosis, as with (C). ** 0.0001 by one-way ANOVA with Bonferroni posttest for combination treatment relative to each single agent alone. Error bars symbolize SD for those experiments. We also observed that inhibition of the prolonged TORC1 signaling in resistant cells restored an apoptotic response to vemurafenib. Inside a resistant mutant melanoma xenografts(A) Tumor xenografts generated from WM164 and IGR1 cells were treated with vehicle (CON) or vemurafenib (VEM) (75 mg/kg) twice daily (individual tumor measurements demonstrated in fig. S10). Error bars symbolize SEM. value determined by two-tailed test for vehicle versus vemurafenib treatment. (B) P-ERK and P-S6 Farampator (s240/244) staining by immunohistochemistry in xenografts harvested CDC25B before or after 48 hours of treatment with vemurafenib, as with (A). Scale pub, 100 M. (C) Serial FNAs were performed on xenograft tumors before treatment and after 24 and 48 hours of vemurafenib treatment and were processed, stained, and analyzed as explained in Materials and Methods. Images of representative cells in the indicated percentiles of P-S6 (s240/244) staining intensity are demonstrated. Green, P-S6; reddish, melanoma markers (HMB45/MART1/NG2); blue, 4,6-diamidino-2-phenylindole (DAPI) nuclear stain. For quantification of P-S6 staining by automated fluorescence microscopy, each open circle represents an individual tumor cell. Histograms showing the HMB45/MART1/NG2 staining intensities of tumor cells used in the analysis are demonstrated above each quantitation. A minimum of 960 cells was analyzed per condition (range, 960 to.