MAPK Methylation Potentiates RAS Signaling

Sci. Signal.  17 Jun 2014:
Vol. 7, Issue 330, pp. ec162
DOI: 10.1126/scisignal.2005593

Mutations in RAS are common in many cancers, particularly pancreatic ductal adenocarcinoma (PDAC). However, therapeutically targeting RAS directly is challenging. Aberrant posttranslational modification has emerged as a contributing factor to many cancers, and a potential therapeutic strategy is inhibiting histone lysine methyltransferases (KMTs). Mazur et al. found that inhibiting the activity of the KMT SMYD3 (SET and MYND domain-containing protein 3) suppressed signaling by RAS in PDAC and lung adenocarcinoma. In an expression array of KMT-encoding genes in human PDAC, the expression of SMYD3 was the most increased. In tissue from both humans and mice, SMYD3 abundance increased with the onset and progression of adenocarcinoma in the pancreas and lung. SMYD3 knockdown decreased the proliferation of cultured human lung adenocarcinoma or PDAC cells and decreased the growth of PDAC xenografts. Crossing Smyd3-knockout mice with conditional Kras mutant-knockin mice (KrasG12D:Smyd3KO) showed that loss of SMYD3 in the pancreas prevented the in vivo development of inflammation-induced neoplastic lesions and the ex vivo formation of metaplastic ducts. In the lung, Smyd3 knockout did not affect the development of adenoma but prevented the progression of adenoma to adenocarcinoma. Complementing the KrasG12D:Smyd3KO mice with catalytically inactive SMYD3 did not enable tumor growth and progression like that in the KrasG12D mice. KrasG12D:Smyd3KO mice had decreased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), a downstream mediator of RAS signaling, in both lung and pancreas tissue compared with KrasG12D mice. The majority of SMYD3 in human lung and pancreatic tumors was not in the nucleus, and histone H3 Lys4 was not detected, suggesting that its methyltransferase activity may target a cytoplasmic protein to promote RAS signaling. A protein array identified mitogen-activated protein kinase kinase kinase 2 (MAP3K2) as a candidate substrate of SMYD3. In vitro assays indicated that SMYD3 methylated MAPK3K2 at Lys260, a modification also detected in cells. Methylation of MAP3K2 was decreased in lung tumor biopsies from KrasG12D:Smyd3KO mice. Complementation of SMYD3-knockdown or MAP3K2-knockdown lung and pancreatic cancer cells with wild-type, but not catalytically inactive SMYD3 or K260A mutant MAP3K2, respectively, restored the activation of ERK1/2 in response to growth factor or serum stimulation. SMYD3-mediated Lys260 methylation of MAP3K2 inhibited binding of members of the PP2A phosphatase complex, as detected in a quantitative proteomic screen, and potentiated MAP3K2 activity on several downstream targets detected by Western blotting. Tumors occurred and progressed similarly to KrasG12D mice in KrasG12D:Smyd3KO mice treated with a PP2A inhibitor. These findings indicate that MAP3K2 lysine methylation promotes RAS signaling and establishes SMYD3 as a potential therapeutic target in RAS-mutant cancers.

P. K. Mazur, N. Reynoird, P. Khatri, P. W. T. C. Jansen, A. W. Wilkinson, S. Liu, O. Barbash, G. S. Van Aller, M. Huddleston, D. Dhanak, P. J. Tummino, R. G. Kruger, B. A. Garcia, A. J. Butte, M. Vermeulen, J. Sage, O. Gozani, SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer. Nature 510, 283–287 (2014). [PubMed]