## Registered report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation <br> **Abstract:** The [Reproducibility Project: Cancer Biology][1] seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of [citations and Altmetric scores][2] ([Errington et al., 2014][3]). This Registered Report describes the proposed replication plan of key experiments from “COT drives resistance to RAF inhibition through MAPK pathway reactivation” by Johannessen and colleagues, published in *Nature* in 2010 ([Johannessen et al., 2010][4]). The key experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D-E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility Project: Cancer Biology is collaboration between the [Center for Open Science][5] and [Science Exchange][6] and the results of the replications will be published in [*eLife*][7]. ---------- ### Contents This component contains additional materials relating to the Registered Report for Study 12: Johannessen et al., 2010, *Nature*. The Registered Report can be found [here][8]. ---------- **Registered Report Initial Draft**: - This document represents the first draft of the selected experimental protocols. It contains the information that the Core team, with help from scientist volunteers, was able to glean from the original publication and its supplemental materials before the original authors were contacted for their input. - [Study 12 Registered Report Initial Draft][9] **Power Calculations**: - These documents contain details about how power calculations were performed, and include spreadsheets documenting statistical tests as well as any PRISM files, R scripts, or additional materials used. - [Study 12 Power Calculations][10] - [Study 12 Protocol 4 Figure 3I ANOVA.R][11] - [Study 12 Protocol 4 Figure 3I IC50.R][12] - [Study 12 Protocol 5 Figure 4E ANOVA.R][13] - [Figure 4E.csv][14] - [Study 12 Protocol 5 Figure 4E 2% variance ANOVA][15] - [Study 12 Protocol 6 Figure 4F ANOVA.R][16] - [Figure 4F.csv][17] - [Study 12 Protocol 6 Figure 4F 2% variance ANOVA][18] [1]: https://osf.io/e81xl/wiki/home/ [2]: https://osf.io/e81xl/wiki/studies/ [3]: https://doi.org/10.7554/eLife.04333 [4]: https://doi.org/10.1038/nature09627 [5]: http://centerforopenscience.org/ [6]: https://www.scienceexchange.com/ [7]: https://elifesciences.org/ [8]: https://elifesciences.org/articles/11414 [9]: https://osf.io/9awcm/ [10]: https://osf.io/zhnvq/ [11]: https://osf.io/chpkz/ [12]: https://osf.io/mj24h/ [13]: https://osf.io/c2vpt/ [14]: https://osf.io/wfk4r/ [15]: https://osf.io/rm76k/ [16]: https://osf.io/p8yq9/ [17]: https://osf.io/c3jrq/ [18]: https://osf.io/vaq73/