Replication Study: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Bhargava et al., 2016) that described how we intended to replicate selected experiments from the paper "RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth" (Hatzivassiliou et al., 2010). Here we report the results. We found two unrelated RAF inhibitors, PLX4720 or GDC-0879, selectively inhibited BRAF(V600E) cell proliferation, while the MEK inhibitor, PD0325901, inhibited BRAF(V600E), wild-type RAF/RAS, and mutant RAS cancer cell proliferation, similar to the original study (Figure 1A; Hatzivassiliou et al., 2010). We found knockdown of CRAF, but not BRAF, in mutant RAS cells attenuated the phospho-MEK induction observed after PLX4720 treatment, similar to the original study (Figure 2B; Hatzivassiliou et al., 2010). The original study reported analogous results with GDC-0879, which was not observed in this replication, although unexpected control results confound the interpretation. We also attempted a replication of an assay with recombinant proteins to test the differential effect of RAF inhibitors on BRAF-CRAF heterodimerization (Figure 4A; Hatzivassiliou et al., 2010). Although we were unable to conduct the experiment as planned, we observed differential binding of BRAF by RAF inhibitors; however, it was between BRAF and beads, independent of CRAF. While these data were unable to address whether, under the conditions of the original study, the same observations could be observed, we discuss key differences between the original study and this replication that are important to consider for further experiments. Finally, where possible, we report meta-analyses for each result. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/470372v2_fig1.gif" ALT="Figure 1"> View larger version (7K): org.highwire.dtl.DTLVardef@1f77d0eorg.highwire.dtl.DTLVardef@1333494org.highwire.dtl.DTLVardef@c10824org.highwire.dtl.DTLVardef@fda30f_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Cellular dose response curves for RAF and MEK inhibitors in BRAF(V600E), mutant RAS, and wild-type RAF/RAS cell lines Cell viability assays were performed for RAF inhibitors (PLX4720 and GDC-0879) and MEK inhibitor (PD0325901) against A375, (BRAF(V600E)), HCT116 (mutant RAS), and MeWo (wild-type RAF/RAS) cells. Absolute half-maximum effective concentration (EC50) values ({micro}M) were determined for each biological repeat. EC50 values unable to be accurately estimated are reported as either >20 {micro}M or <0.078 {micro}M, which are the largest and smallest doses tested, respectively. Absolute EC50 values for each biological repeat for A375, HCT116, and MeWo cells treated with (A) PLX4720, (B) GDC-0879, or (C) PD0325901 for this replication attempt are plotted with the EC50 value reported in Hatzivassiliou et al. (2010) displayed as a single point (red triangle) for comparison. Where possible the mean and 95% confidence interval of the replication data are shown. Additional details for this experiment can be found at https://osf.io/52zp9/. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/470372v2_fig2.gif" ALT="Figure 2"> View larger version (34K): org.highwire.dtl.DTLVardef@1dc3058org.highwire.dtl.DTLVardef@e9f5d9org.highwire.dtl.DTLVardef@baf5eorg.highwire.dtl.DTLVardef@126717a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Cellular dose response curves for each biological repeat This is the same experiment as in Figure 1. The dose response curve of each biological repeat [n=4] for A375, HCT116, and MeWo cells treated with (A) PLX4720, (B) GDC-0879, or (C) PD0325901 for this replication attempt are plotted. Additional details for this experiment can be found at https://osf.io/52zp9/. C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/470372v2_fig4.gif" ALT="Figure 4"> View larger version (33K): org.highwire.dtl.DTLVardef@e4357eorg.highwire.dtl.DTLVardef@73e9ceorg.highwire.dtl.DTLVardef@1fb01c6org.highwire.dtl.DTLVardef@31a799_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 4.C_FLOATNO Testing of BRAF and CRAF antibodies used in original study The BRAF and CRAF antibodies reported in the original study (Hatzivassiliou et al., 2010) were tested for suitability by Western blot. (A) Representative Western blots and Ponceau-stained membrane (experiment performed independently 3 times) of 0.5 {micro}g of purified recombinant BRAF kinase domain (KD). Membranes were probed with the BRAF antibody used in the original study (RRID:AB_1840330) or with a different BRAF antibody used in this replication attempt (RRID:AB_2721113). (B) Representative Western blots and Ponceau-stained membrane (experiment performed independently 3 times) of 0.5 {micro}g of purified recombinant GST tagged CRAF KD. Membranes were probed with the CRAF antibody used in the original study (RRID:AB_397552) or with a different CRAF antibody used in this replication attempt (RRID:AB_10989977). (C) Representative Western blots (experiment performed once) of 16 {micro}g of whole cell lysates from the indicated human cell lines. Membranes were probed with the BRAF (RRID:AB_1840330) or CRAF (RRID:AB_397552) antibodies used in the original study. (D) Screenshot from the Constraint-based Multiple Alignment Tool (COBALT) (Papadopoulos and Agarwala, 2007) showing the alignment of full-length BRAF (NCBI Reference Sequence: NP_004324.2), the region of BRAF used as the immunogenic peptide used to generate the BRAF antibody used in the original study (RRID:AB_1840330), the BRAF KD used in this replication attempt, and the BRAF KD used in the original study. (E) Screenshot from COBALT showing the alignment of full-length CRAF (NCBI Reference Sequence: NP_002871.1), the region of CRAF used as the immunogenic peptide used to generate the CRAF antibody used in the original study (RRID:AB_397552), and the CRAF KD used in the original study and this replication attempt. Additional details for this experiment can be found at https://osf.io/6f3sk/. C_FIG