Mixed Lineage Kinase MLK4 Is Activated in Colorectal Cancers Where It Synergistically Cooperates with Activated RAS Signaling in Driving Tumorigenesis

Abstract

Colorectal cancers (CRC) are commonly classified into those with microsatellite instability and those that are microsatellite stable (MSS) but chromosomally unstable. The latter are characterized by poor prognosis and remain largely intractable at the metastatic stage. Comprehensive mutational analyses have revealed that the mixed lineage kinase 4 (MLK4) protein kinase is frequently mutated in MSS CRC with approximately 50% of the mutations occurring in KRAS-or BRAF-mutant tumors. This kinase has not been characterized previously and the relevance of MLK4 somatic mutations in oncogenesis has not been established. We report that MLK4-mutated alleles in CRC are constitutively active and increase the transformation and tumorigenic capacity of RAS-mutated cell lines. Gene expression silencing or targeted knockout of MLK4 impairs the oncogenic properties of KRAS-and BRAF-mutant cancer cells both in vitro and in xenograft models. In establishing the role of MLK4 in intracellular signaling, we show it directly phosphorylates MEK1 (MAP2K1) and that MEK/ERK (MAPK1) signaling is impaired in MLK4 knockout cells. These findings suggest that MLK4 inhibitors may be efficacious in KRAS-and BRAF-mutated CRCs and may provide a new opportunity for targeting such recalcitrant tumors. Cancer Res; 73(6); 1912-21. Ó2012 AACR.

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

“…1C and D). Based on a previous study that characterized MLK4 as an oncogene with gain-of-function mutations (H261Y, G291E, R470C and R555*) (24), we performed additional blinded verification of WT and mutant MLK4 activity in an independent laboratory (L.P and T.H), which confirmed the LOF phenotype of MLK4 mutations (Supplementary Fig. S1B).…”

“…To date, there has only been one report evaluating the role of MLK4 in cancer (24). In this study mutations in MLK4 were described as activating, and the authors suggested that MLK4 was oncogenic and promoted tumorigenesis in a mutant KRAS background in CRC (24).…”

“…To date, there has only been one report evaluating the role of MLK4 in cancer (24). In this study mutations in MLK4 were described as activating, and the authors suggested that MLK4 was oncogenic and promoted tumorigenesis in a mutant KRAS background in CRC (24). In contrast, we have characterized the same mutations in MLK4 as loss-of-function and our biochemical, structural, and independent blinded verification unequivocally indicated that these mutations abrogate the activity of the kinase.…”

“…It is important to assess the functional impact of mutations in MLK4 and gain an insight into the role this kinase plays in colorectal tumorigenesis. The functional consequences of MLK4 mutations in CRC have not been determined so far, except for one report, where the authors state that enzymatic activity of mutated MLK4 is increased in KRAS-driven colon cancer (24). Here, we present contradictory findings where we demonstrate that MLK4 mutations in colon cancer are loss-of-function (LOF).…”

76 Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors

“…Consistent with MLK3 regulating the RAF/MEK/ERK pathway, depletion of endogenous MLK3 suppressed activation of the pathway, and mice deficient in both MLK2 and MLK3 have reduced activation of ERK in response to tumour necrosis factor stimulation 2,4 . In addition, various studies suggest that MLKs are oncogenic and mutations in this family of kinases have been observed in several different cancers [5][6][7] . MAP3K9 (MLK1) has been identified as a gene that is frequently mutated in melanoma (12 of 85, or 14%, of melanoma patients evaluated had MLK1 mutations) 8 .…”