Abstract
The Reproducibility Project: Cancer Biology 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 (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF" by Heidorn and colleagues, published in Cell in 2010 (Heidorn et al., 2010). The experiments to be replicated are those reported in Figures 1A, 1B, 3A, 3B, and 4D. Heidorn and colleagues report that paradoxical activation of the RAF-RAS-MEK-ERK pathway by BRAF inhibitors when applied to BRAF(WT) cells is a result of BRAF/CRAF heterodimer formation upon inactivation of BRAF kinase activity, and occurs only in the context of active RAS. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.
Highlights
The RAS-RAF-MEK-ERK signaling pathway is routinely disregulated in many forms of cancer
Heidorn and colleagues first observed that paradoxical activation occurred only in the context of BRAFWT and activated RAS, an observation confirmed by two other groups (Hatzivassiliou et al, 2010; Poulikakos et al, 2010)
Dissecting the mechanism, they reported that the formation of BRAF/CRAF heterodimers was necessary for pathway activation, and formation of those heterodimers required active RAS signaling
Summary
The RAS-RAF-MEK-ERK signaling pathway is routinely disregulated in many forms of cancer. After confirming that drug binding to BRAF drove BRAF binding to CRAF, Heidorn and colleagues tested a kinase dead version of BRAF (BRAFD594A) (Figure 4D) This version of BRAF still bound to CRAF, indicating that it is not drug binding per se, but inhibition of BRAF activity, that drives BRAF binding to CRAF and paradoxical activation of MEK/ERK. This key experiment will be replicated in Protocol 4. Packer and colleagues extended the work of Heidorn and colleagues to examine if other more broadly targeted tyrosine kinase inhibitors were able to paradoxically activate the RAS-RAF pathway They observed paradoxical pathway activation in D04 cells after treatment with imatinib, nilotinib, dasatinib, and the BRAF inhibitor SB590885. Lidsky and colleagues showed that increased levels of NRAS were key to vemurafenib resistance, they did not observe any activating mutations in their resistant cell lines (Lidsky et al, 2014)
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