## Replication Study: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth <br> **Abstract:** As part of the [Reproducibility Project: Cancer Biology][1], we published a Registered Report ([Bhargava et al., 2016][2]) 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][3]). 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][4]). 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][5]). 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][6]). 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. ---------- ### Contents **Reports:** Read the [Replication Study][7] or view the [earlier versions][8]. **Note**: In order to successfully run and knit the Replication Study Manuscript R Markdown file, you must install a series of necessary R packages. You can review the necessary packages included in the checkpoint manifest in the markdown file [here][9] or run and knit the following r markdown file and they will be downloaded automatically. To reproduce the Replication Study manuscript text run this in [R Studio][10] (note: downloads [R markdown file][11] directly from osf.io: library(httr) library(rmarkdown) GET("https://osf.io/k2xve/?action=download",write_disk("Replication_Study_8.Rmd", overwrite = T)) render("Replication_Study_8.Rmd", output_format = "word_document") <br> Also, explore the Registered Report and materials related to the Registered Report [here][12] and read the decision letter and author response from the peer review of this paper at *eLife* [here][13]. **Data and Material Availability:** - All other associated data, protocols, analysis scripts, and other digital materials are available within this OSF project. **Experiments replicated**: Reproduce and explore the figures, analyses, data, and methods generated in this replication attempt. <br> - [Assessing cell viability of a panel of cancer cell lines treated with RAF and MEK inhibitors][14] <br> - Replication of Figure 1A of Hatzivassiliou et al., 2010 - [Assessing CRAF and BRAF roles in drug-dependent activation of MEK][15] <br> - Replication of Figure 2B of Hatzivassiliou et al., 2010 - [Biochemical heterodimerization assay with recombinant RAF proteins in the presence or absence of RAF inhibitors][16] <br> - Replication of Figure 4A of Hatzivassiliou et al., 2010 Questions about the project can be directed to [contact+rpcb@cos.io.][17] [1]: https://osf.io/e81xl/wiki/home/ [2]: https://elifesciences.org/articles/09976 [3]: https://doi.org/10.1038/nature08833 [4]: https://doi.org/10.1038/nature08833 [5]: https://doi.org/10.1038/nature08833 [6]: https://doi.org/10.1038/nature08833 [7]: https://www.biorxiv.org/content/10.1101/2021.11.30.470372v2.full [8]: https://osf.io/q4btk/ [9]: https://osf.io/k2xve/ [10]: https://www.rstudio.com [11]: https://osf.io/k2xve/ [12]: https://elifesciences.org/articles/09976 [13]: https://osf.io/zqugv/ [14]: https://osf.io/52zp9/wiki/home/ [15]: https://osf.io/dqr5d/wiki/home/ [16]: https://osf.io/6f3sk/wiki/home/ [17]: mailto:contact+rpcb@cos.io