SMYD3 is a methyltransferase overexpressed in several human tumours; here methylation of the MAP3K2 kinase by SMYD3 is shown to be critical for Ras-induced tumour development in mouse models and human tumour cells, showing an unexpected role for methylation in a kinase signalling pathway and revealing a candidate therapeutic target. SMYD3 is a largely cytoplasmic lysine methyltransferase that is overexpressed in several human tumours. This paper reports that SMYD3 is required for Ras-induced tumour development in mouse models. The enzyme methylates MAP3K2, which inhibits binding of a phosphatase and potentiates the Ras/Raf signalling pathway. These results reveal an unexpected role for lysine methylation in a kinase signalling pathway and establish SMYD3 as a potential therapeutic target. Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics1. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours2,3,4. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

NUT midline carcinoma (NMC) is a lethal pediatric tumor defined by the presence of BRD-NUT fusion proteins that arrest differentiation. Here we explore the mechanisms underlying the ability of BRD4-NUT to prevent squamous differentiation. In both gain-of and loss-of-expression assays, we find that expression of BRD4-NUT is associated with globally decreased histone acetylation and transcriptional repression. Bulk chromatin acetylation can be restored by treatment of NMC cells with histone deacetylase inhibitors (HDACi), engaging a program of squamous differentiation and arrested growth in vitro that closely mimics the effects of siRNA-mediated attenuation of BRD4-NUT expression. The potential therapeutic utility of HDACi differentiation therapy was established in three different NMC xenograft models, where it produced significant growth inhibition and a survival benefit. Based on these results and translational studies performed with patient-derived primary tumor cells, a child with NMC was treated with the FDA-approved HDAC inhibitor, vorinostat. An objective response was obtained after five weeks of therapy, as determined by positron emission tomography. These findings provide preclinical support for trials of HDACi in patients with NMC. Cancer Res; 71(7); 2686–96. ©2011 AACR.

In a subset of poorly differentiated and highly aggressive carcinoma, a chromosomal translocation, t(15;19)(q13;p13), results in an in‐frame fusion of the double bromodomain protein, BRD4, with a testis‐specific protein of unknown function, NUT (nuclear protein in testis). In this study, we show that, after binding to acetylated chromatin through BRD4 bromodomains, the NUT moiety of the fusion protein strongly interacts with and recruits p300, stimulates its catalytic activity, initiating cycles of BRD4–NUT/p300 recruitment and creating transcriptionally inactive hyperacetylated chromatin domains. Using a patient‐derived cell line, we show that p300 sequestration into the BRD4–NUT foci is the principal oncogenic mechanism leading to p53 inactivation. Knockdown of BRD4–NUT released p300 and restored p53‐dependent regulatory mechanisms leading to cell differentiation and apoptosis. This study demonstrates how the off‐context activity of a testis‐specific factor could markedly alter vital cellular functions and significantly contribute to malignant cell transformation.

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark...