Theodore Vougiouklakis

The NSD family of protein methyltransferases in human cancer

The NSD family of protein lysine methyltransferases consists of NSD1, NSD2/WHSC1/MMSET and NSD3/WHSC1L1. NSD2 haploinsufficiency causes Wolf-Hirschhorn syndrome, while NSD1 mutations lead to the Sotos syndrome. Recently, a number of studies showed that the NSD methyltransferases were overexpressed, amplified or somatically mutated in multiple types of cancer, suggesting their critical role in cancer. These enzymes methylate specific lysine residues on histone tails and their dysfunction results in epigenomic aberrations which play a fundamental role in oncogenesis. Furthermore, NSD1 was also reported to methylate a nonhistone protein substrate, RELA/p65 subunit of NF-κB, implying its regulatory function through nonhistone methylation pathways. In this review, we summarize the current research regarding the role of the NSD family proteins in cancer and underline their potential as targets for novel cancer therapeutics.

SUV420H1 enhances the phosphorylation and transcription of ERK1 in cancer cells.

The oncogenic protein ERK, a member of the extracellular signal-regulated kinase (ERK) cascade, is a well characterized signaling molecule involved in tumorigenesis. The ERK signaling pathway is activated in a large proportion of cancers and plays a critical role in tumor development. Functional regulation by phosphorylation of kinases in the ERK pathway has been extensively studied, however methylation of the ERK protein has not been reported to date. Here, we demonstrated that the protein lysine methyltransferase SUV420H1 tri-methylated ERK1 at lysines 302 and 361, and that substitution of methylation sites diminished phosphorylation levels of ERK1. Concordantly, knockdown of SUV420H1 reduced phosphorylated ERK1 and total ERK1 proteins, and interestingly suppressed ERK1 at the transcriptional level. Our results indicate that overexpression of SUV420H1 may result in activation of the ERK signaling pathway through enhancement of ERK phosphorylation and transcription, thereby providing new insights in the regulation of the ERK cascade in human cancer.

WHSC1L1 drives cell cycle progression through transcriptional regulation of CDC6 and CDK2 in squamous cell carcinoma of the head and neck.

Wolf-Hisrchhorn Syndrome Candidate 1-Like 1 (WHSC1L1) is a protein lysine methyltransferase that is recurrently amplified (8p11.23) in patients with squamous cell carcinoma of the head and neck (SCCHN). In this study, we investigated the oncogenic role of WHSC1L1 in SCCHN. Using immunohistochemistry on tissue microarrays of patients with locoregionally advanced SCCHN, we found that WHSC1L1 is significantly overexpressed in patients with SCCHN, and is associated with poor grade and heavy smoking history. Knockdown of WHSC1L1 expression resulted in significant growth suppression and reduction of H3K36 dimethylation (H3K36me2) in SCCHN cells. Chromatin immunoprecipitation analysis showed that WHSC1L1 and H3K36me2 are enriched in the gene bodies of the cell cycle-related genes CDC6 and CDK2, implying that WHSC1L1 directly regulates the transcription of these genes. According to the importance of CDC6 and CDK2 for G1 to S transition, WHSC1L1 knockdown induced strong G0/G1 arrest which was rescued by introduction of wild-type WHSC1L1 but not by that of enzyme-inactive WHSC1L1. Our results imply that WHSC1L1 and its product H3K36me2 are essential for the transition from G1 to S phase in SCCHN cells and that WHSC1L1 could serve as a rational target for anticancer drug development for patients with head and neck cancer.

WHSC1L1-mediated EGFR mono-methylation enhances the cytoplasmic and nuclear oncogenic activity of EGFR in head and neck cancer.

While multiple post-translational modifications have been reported to regulate the function of epidermal growth factor receptor (EGFR), the effect of protein methylation on its function has not been well characterized. In this study, we show that WHSC1L1 mono-methylates lysine 721 in the tyrosine kinase domain of EGFR, and that this methylation leads to enhanced activation of its downstream ERK cascade without EGF stimulation. We also show that EGFR K721 mono-methylation not only affects the function of cytoplasmic EGFR, but also that of nuclear EGFR. WHSC1L1-mediated methylation of EGFR in the nucleus enhanced its interaction with PCNA in squamous cell carcinoma of the head and neck (SCCHN) cells and resulted in enhanced DNA synthesis and cell cycle progression. Overall, our study demonstrates the multifaceted oncogenic function of the protein lysine methyltransferase WHSC1L1 in SCCHN, which is mediated through direct non-histone methylation of the EGFR protein with effects both in its cytoplasmic and nuclear functions.

Effects of SMYD2-mediated EML4-ALK methylation on the signaling pathway and growth in non-small-cell lung cancer cells

A specific subtype of non‐small‐cell lung cancer (NSCLC) characterized with an EML4‐ALK fusion gene, which drives constitutive oncogenic activation of anaplastic lymphoma kinase (ALK), shows a good clinical response to ALK inhibitors. We have reported multiple examples implying the biological significance of methylation on non‐histone proteins including oncogenic kinases in human carcinogenesis. Through the process to search substrates for various methyltransferases using an in vitro methyltransferase assay, we found that a lysine methyltransferase, SET and MYND domain‐containing 2 (SMYD2), could methylate lysine residues 1451, 1455, and 1610 in ALK protein. Knockdown of SMYD2 as well as treatment with a SMYD2 inhibitor in two NSCLC cell lines with an EML4‐ALK gene significantly attenuated the phosphorylation levels of the EML4‐ALK protein. Substitutions of each of these three lysine residues to an alanine partially or almost completely diminished in vitro methylation of ALK. In addition, we found that exogenous introduction of EML4‐ALK protein with the substitution of lysine 1610 to an alanine in these two cell lines reduced the phosphorylation levels of AKT, one of the downstream oncogenic molecules in the EML4‐ALK pathway, and suppressed the growth of the two cell lines. We further showed that the combination of a SMYD2 inhibitor and an ALK inhibitor additively suppressed the growth of these two NSCLC cells, compared with single‐agent treatment. Our results shed light on a novel mechanism that modulates the kinase activity of the ALK fused gene product and imply that SMYD2‐mediated ALK methylation might be a promising target for development of a novel class of treatment for tumors with the ALK fused gene.

Characterization of the T-Cell Receptor Repertoire and Immune Microenvironment in Patients with Locoregionally Advanced Squamous Cell Carcinoma of the Head and Neck

Purpose: Squamous cell carcinoma of the head and neck (SCCHN) is a lethal cancer with a suboptimal 5-year overall survival of approximately 50% with surgery and/or definitive chemoradiotherapy. Novel treatments are thus urgently awaited. Immunotherapy with checkpoint blockade has emerged as a promising option for patients with recurrent/metastatic SCCHN; however, it has not been investigated in the curative-intent setting yet. The purpose of this study was to investigate the T-cell receptor repertoire and the tumor microenvironment in tumor tissues of SCCHN patients with locoregionally advanced disease. Experimental Design: We performed T-cell receptor sequencing of tumor tissues from 44 patients with locoregionally advanced SCCHN prior to treatment with definitive chemoradiotherapy and correlated the T-cell clonality and the mRNA expression levels of immune-related genes with clinicopathologic parameters. Results: Clonal expansion of T cells was significantly higher in human papilloma virus (HPV)–negative compared with HPV-positive tumors, signifying more robust antigen presentation in HPV-negative tumors. The latter was supported by the higher percentage of HPV-negative tumors expressing HLA-A protein compared with HPV-positive tumors (P = 0.049). Higher GRZB levels correlated significantly with longer recurrence-free survival (log-rank, P = 0.003) independent of tumor size, nodal stage, and HPV status. Conclusions: Our findings support clonal expansion of T cells in SCCHN patients with locoregionally advanced disease and imply differences in the antigen presentation capacity between HPV-negative and HPV-positive tumors. Elevated GRZB mRNA levels may also serve as a favorable and independent predictor of outcome in SCCHN patients treated with chemoradiotherapy. These data provide rationale for the introduction of immunotherapeutic approaches in the curative-intent setting. Clin Cancer Res; 23(16); 4897–907. ©2017 AACR.

Critical roles of SMYD2-mediated β-catenin methylation for nuclear translocation and activation of Wnt signaling

Accumulation of β-catenin in the nucleus is a hallmark of activation of the Wnt/β-catenin signaling pathway, which drives development of a large proportion of human cancers. However, the mechanism of β-catenin nuclear translocation has not been well investigated. Here we report biological significance of SMYD2-mediated lysine 133 (K133) methylation of β-catenin on its nuclear translocation. Knockdown of SMYD2 attenuates the nuclear localization of β-catenin protein in human cancer cells. Consequently, transcriptional levels of well-known Wnt-signaling molecules, cMYC and CCND1, are significantly reduced. Substitution of lysine 133 to alanine in β-catenin almost completely abolishes its nuclear localization. We also demonstrate the K133 methylation is critical for the interaction of β-catenin with FOXM1. Furthermore, after treatment with a SMYD2 inhibitor, significant reduction of nuclear β-catenin and subsequent induction of cancer cell death are observed. Accordingly, our results imply that β-catenin methylation by SMYD2 promotes its nuclear translocation and activation of Wnt signaling.Accumulation of β-catenin in the nucleus is a hallmark of activation of the Wnt/β-catenin signaling pathway, which drives development of a large proportion of human cancers. However, the mechanism of β-catenin nuclear translocation has not been well investigated. Here we report biological significance of SMYD2-mediated lysine 133 (K133) methylation of β-catenin on its nuclear translocation. Knockdown of SMYD2 attenuates the------ nuclear localization of β-catenin protein in human cancer cells. Consequently, transcriptional levels of well-known Wnt-signaling molecules, cMYC and CCND1, are significantly reduced. Substitution of lysine 133 to alanine in β-catenin almost completely abolishes its nuclear localization. We also demonstrate the K133 methylation is critical for the interaction of β-catenin with FOXM1. Furthermore, after treatment with a SMYD2 inhibitor, significant reduction of nuclear β-catenin and subsequent induction of cancer cell death are observed. Accordingly, our results imply that β-catenin methylation by SMYD2 promotes its nuclear translocation and activation of Wnt signaling.

The role of protein methyltransferases as potential novel therapeutic targets in squamous cell carcinoma of the head and neck

Squamous cell carcinoma of the head and neck is a lethal disease with suboptimal survival outcomes and standard therapies with significant comorbidities. Whole exome sequencing data recently revealed an abundance of genetic and expression alterations in a family of enzymes known as protein methyltransferases in a variety of cancer types, including squamous cell carcinoma of the head and neck. These enzymes are mostly known for their chromatin-modifying functions through methylation of various histone substrates, though evidence supports their function also through methylation of non-histone substrates. This review summarizes the current knowledge on the function of protein methyltransferases in squamous cell carcinoma of the head and neck and highlights their promising potential as the next generation of therapeutic targets in this disease.

Development of novel SUV39H2 inhibitors that exhibit growth suppressive effects in mouse xenograft models and regulate the phosphorylation of H2AX

Protein methyltransferase SUV39H2 was reported to methylate histone H2AX at lysine 134 and enhance the formation of phosphorylated H2AX (γ-H2AX), which causes chemoresistance of cancer cells. We found that a series of imidazo[1,2-a]pyridine compounds that we synthesized could inhibit SUV39H2 methyltransferase activity. One of the potent compounds, OTS193320, was further analyzed in in vitro studies. The compound decreased global histone H3 lysine 9 tri-methylation levels in breast cancer cells and triggered apoptotic cell death. Combination of OTS193320 with doxorubicin (DOX) resulted in reduction of γ-H2AX levels as well as cancer cell viability compared to a single agent OTS193320 or DOX. Further optimization of inhibitors and their in vivo analysis identified a compound, OTS186935, which revealed significant inhibition of tumor growth in mouse xenograft models using MDA-MB-231 breast cancer cells and A549 lung cancer cells without any detectable toxicity. Our results suggest that the SUV39H2 inhibitors sensitize cancer cells to DOX by reduction of γ-H2AX levels in cancer cells, and collectively demonstrate that SUV39H2 inhibition warrants further investigation as a novel anti-cancer therapy.

Protein methyltransferases and demethylases dictate CD8+ T-cell exclusion in squamous cell carcinoma of the head and neck

A subset of patients with recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN) benefit from pembrolizumab and nivolumab, but the majority of patients do not probably due to lack of activated cytotoxic CD8+ T-cells in their tumor tissues. Herein, we aim to investigate whether specific protein methyltransferases (PMTs) and demethylases (PDMTs) could play any roles in the CD8+ T-cell exclusion process in HPV-negative SCCHN. RNA sequencing data from the TCGA database were interrogated for HPV-negative SCCHN patients using a 10-gene chemokine signature that classifies SCCHN tissues into CD8+ T-cell inflamed and non-CD8+ T-cell inflamed phenotypes. Among 53 PMT/PDMT genes examined in the TCGA HPV-negative SCCHN database, expression levels of 15 PMT/PDMT genes were significantly negatively correlated with the chemokine signature score and CD8 mRNA expression levels. The expression level of each of these 15 PMT/PDMT genes showed significantly negative correlations with immune-active chemokines, as well as HLA class I and APM molecules. siRNA-mediated knockdown of a candidate PMT, SMYD3, led to upregulation of CXCL9, CXCL10, CXCL11 and TAP1 at mRNA and protein levels in HPV-negative SCCHN cell lines. These findings demonstrate that overexpression of some PMTs and PDMTs seems to be related with the non-CD8+ T-cell inflamed phenotype and may drive CD8+ T-cell exclusion in HPV-negative SCCHN. This study suggests that chromatin modifiers contribute to CD8+ T-cell exclusion and antigen presentation capacity of HPV-negative SCCHN, supporting that targeting of specific PMTs and/or PDMTs could enhance CD8+ T-cell infiltration and increase the proportion of patients that may benefit from immunotherapy.