Registered report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

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 “COT drives resistance to RAF inhibition through MAPK pathway reactivation” by Johannessen and colleagues, published in Nature in 2010 (Johannessen et al., 2010). 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 and Science Exchange and the results of the replications will be published in eLife.DOI: http://dx.doi.org/10.7554/eLife.11414.001

Transcription factor activities enhance markers of drug response in cancer

“…The importance of ATF2 in melanoma is supported by several lines of evidence; ATF2 is required for melanoma tumor development [66] ; nuclear ATF2 (transcriptionally active) is associated with poor prognosis, metastasis and resistance to genotoxic stress; mitochondrial ATF2 (transcriptionally inactive) is associated with increased apoptosis [67,68] . Moreover, PKCε, the kinase mediating ATF2 transcriptional activity, is among the top 10 kinases associated with BRAF-inhibition resistance, which supports the relationship between ATF2 and Dabrafenib resistance [69] . Finally, this observation is also supported by gene-level essentiality scores from Achilles project, where we found a pancancer tendency between the predicted activity for ATF2 and its essentiality in BRAF V600E mutant cells (R = -0.55, p = 0.062, Pearson correlation; Figure S10A) but not in BRAF wt (R = 0.078, p = 0.38, Pearson correlation; Figure S10B).…”

Targeted Therapy in Melanoma and Mechanisms of Resistance

“…Activating mutations in MEK1 / MEK2 make the blocking of BRAF ineffective, as MEK reactivation means that the MAPK/ERK pathway can still transduce the signal below BRAF, regardless of its inhibition [ 25 , 36 ]. The overexpression of the COT protein, probably due to gene amplification or hitherto unidentified mechanisms, can reactivate MEK in the presence of BRAF inhibition, stimulating ERK signaling and the development of resistance to BRAFis [ 42 , 43 ]. Finally, the reactivation of MAP/ERK pathway-dependent transcription factors may result from downstream ERK activation and the loss of the inhibitory function of the ERK kinase [ 29 ].…”

“…MAP3K8-deficient cells are sensitive to BRAFis and present reduced growth and MEK/ERK activity during BRAFi treatment [ 36 , 42 , 43 ]. Administering BRAF inhibitors in the case of primary MAP3K8 overexpression further increases the expression of this protein, which in turn stimulates the proliferation of melanoma cells [ 42 , 43 ]. Mutations in MAP3K8 are present in about 1.5% of all melanoma patients, frequently in Spitz nevi (in about 33% cases) [ 44 ].…”

Mechanisms of melanoma resistance to treatment with BRAF and MEK inhibitors

“…Increased level of COT protein results in maintenance of proliferation despite BRAF inhibition. In the case of primary over-expression of MAP3K8, administration of BRAF inhibitors leads to even higher COT production and, as a result, to further intensification of proliferation [18,19]. MAP3K8 gene modifications occur in about 1.5% of all patients with melanoma, often (in about 33% of cases) they are present in Spitz nevi [20].…”

“…Increased level of COT protein results in maintenance of proliferation despite BRAF inhibition. [18,19] Loss of the NF1 suppressor gene NF1 is the negative regulator for the RAS signalling pathway. Mutation of NF1 resulting in loss of functional neurofibromin 1 protein (NF1) causes an increase in RAS level, activation of CRAF and consequently activation of MAPK pathway, which results in maintenance of proliferation in the presence of BRAF inhibitors.…”