Recurrent MLK4 Loss-of-Function Mutations Suppress JNK Signaling to Promote Colon Tumorigenesis

Anna A Marusiak, Natalie L Stephenson, Hayeon Baik, Eleanor W Trotter, Yaoyong Li, Karen Blyth, Susan Mason, Phil Chapman, Lorena A Puto, Jon A Read, Claire Brassington, Hannah K Pollard, Chris Phillips, Isabelle Green, Ross Overman, Matthew Collier, Ewelina Testoni, Crispin J Miller, Tony Hunter, Owen J Sansom, John Brognard

MLK4 is a member of the mixed-lineage family of kinases that regulate the JNK, p38, and ERK kinase signaling pathways. MLK4 mutations have been identified in various human cancers including frequently in colorectal cancer, where their function and pathobiological importance has been uncertain. In this study, we assessed the functional consequences of MLK4 mutations in colon tumorigenesis. Biochemical data indicated that a majority of MLK4 mutations are loss-of-function (LOF) mutations that can exert dominant negative effects. In seeking to understand the abrogated activity of these mutants, we elucidated a new MLK4 catalytic domain structure. To determine whether MLK4 is required to maintain the tumorigenic phenotype, we reconstituted its signaling axis in colon cancer cells harboring MLK4 inactivating mutations. We found that restoring MLK4 activity reduced cell viability, proliferation, and colony formation in vitro and delayed tumor growth in vivo. Mechanistic investigations established that restoring the function of MLK4 selectively induced the JNK pathway and its downstream targets, cJUN, ATF3 and the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. Our work indicates that MLK4 is a novel tumor suppressing kinase harboring frequent LOF mutations that lead to diminished signaling in the JNK pathway and enhanced proliferation in colon cancer.