COT/ MAP3K8 drives resistance to RAF inhibition through MAP kinase pathway reactivation

Abstract

Oncogenic mutations in the serine/threonine kinase B-RAF are found in 50–70% of malignant melanomas 1. Pre-clinical studies have demonstrated that the B-RAF ^V600E mutation predicts a dependency on the mitogen activated protein kinase (MAPK) signaling cascade in melanoma 1– 5—an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials 6– 8. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance 9– 11. Identification of resistance mechanisms in a manner that elucidates alternative ‘druggable’ targets may inform effective long-term treatment strategies 12. Here, we expressed ~600 kinase and kinase-related open reading frames (ORFs) in parallel to functionally interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (COT/TPL2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF ^V600E cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signaling. Moreover, COT expression is associated with de novo resistance in B-RAF ^V600E cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibition. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.