SMYD3 Knockdown Research Articles

SMYD3 associates with the NuRD (MTA1/2) complex to regulate transcription and promote proliferation and invasiveness in hepatocellular carcinoma cells

BackgroundSMYD3, a member of the SET and MYND domain-containing (SMYD) family, is a histone methyltransferase (HMT) and transcription factor that plays an important role in transcriptional regulation in human carcinogenesis.ResultsUsing affinity purification and mass spectrometry assays to identify SMYD3-associated proteins in hepatocellular carcinoma (HCC) cells, we found several previously undiscovered SMYD3-interacting proteins, including the NuRD (MTA1/2) complex, the METTL family, and the CRL4B complex. Transcriptomic analysis of the consequences of knocking down SMYD3, MTA1, or MTA2 in HCC cells showed that SMYD3/NuRD complex targets a cohort of genes, some of which are critically involved in cell growth and migration. qChIP analyses showed that SMYD3 knockdown led to a significant reduction in the binding of MTA1 or MTA2 to the promoters of IGFBP4 and led to a significant decrease in H4K20me3 and a marked increase in H4Ac at the IGFBP4 promoter. In addition, we demonstrated that SMYD3 promotes cell proliferation, invasion, and tumorigenesis in vivo and in vitro and found that its expression is markedly upregulated in human liver cancer. Knockdown of MTA1 or MTA2 had the same effect as knockdown of SMYD3 on proliferation and invasion of hepatocellular carcinoma cells. Catalytic mutant SMYD3 could not rescue the phenotypic effects caused by knockdown of SMYD3. Inhibitors of SMYD3 effectively inhibited the proliferation and invasiveness of HCC cells.ConclusionsThese findings revealed that SMYD3 could transcriptionally repress a cohort of target genes expression by associating with the NuRD (MTA1/2) complex, thereby promoting the proliferation and invasiveness of HCC cells. Our results support the case for pursuing SMYD3 as a practical prognostic marker or therapeutic target against HCC.

Abstract 2968: Interrogating the function of histone methyltransferases in MYC-driven medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor of the cerebellum, with four distinct molecular subgroups, each carrying different clinical prognoses. Group 3 (G3) is the most aggressive subgroup and is associated with amplifications of the MYC gene. MYC expression and function are controlled in part by epigenetic regulation, and interestingly, the few mutations that are found in G3 medulloblastoma are mostly located in genes encoding epigenetic regulators. Surprisingly, many tumors had no mutations raising the question as to whether non-mutated or non-amplified epigenetic regulators may be drivers of G3MB tumorigenesis. We previously found that dysregulation of the H3K27 methyltransferase EZH2 cooperates with MYC in G3MB, indicating that epigenetic regulators may provide a source of potential therapeutic targets. Therefore, to identify novel epigenetic drivers in G3MB, we performed an unbiased shRNA screen of 243 known chromatin regulators. The most intriguing candidate was SMYD3, a multidomain-containing protein with histone H4-lysine 5 (H4K5) methyltransferase activity. Knockdown of SMYD3 suppressed G3MB proliferation in vitro and tumor progression in vivo. SMYD3 protein is nuclear and associated with the MYC promoter. Deletion of SMYD3 results in loss of MYC, G2/M and epithelial-to-mesenchymal target gene expression. Using a multi-omics approach including CUT&RUN sequencing, RNA-seq, DNA methylation, cryo-electron microscopy (cryo-EM) and proteomics we aim to elucidate how SMYD3 controls G3MB tumorigenesis. These results highlight the role of a potentially targetable new epigenetic regulator of tumorigenesis in G3MB. Citation Format: Audrey L. Mercier, Jennifer L. Stripay, Silvija Bilokapic, Justin Williams, Stephanie Nance, Jingjing Liu, Steven Philips, Suresh Kandikonda, Marie Morfouace, Aude Bramoulle, Tamara Advedissian, Jiyang Yu, Junmin Peng, Aseem Ansari, Mario Halic, Adam Durbin, Christopher Vakoc, Martine F. Roussel. Interrogating the function of histone methyltransferases in MYC-driven medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2968.

Abstract 6250: Investigating the immunomodulatory mechanisms of Smyd3 depletion in HPV-negative head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world, with Human Papilloma virus (HPV)-negative patients having a ~50% recurrence rate and poor median overall survival of ~13 months in the recurrent/metastatic setting. Pembrolizumab, a programmed-death-1 (PD-1) checkpoint inhibitor is active but with a response rate as low as 19%. Our preliminary work has identified SET and MYND Domain containing 3 (SMYD3) as a chromatin modifier that is significantly overexpressed in HPV-negative HNSCC tumor tissues compared to normal epithelium and is associated with low CD8+ T-cell infiltration in HPV-negative HNSCC. Furthermore, we have shown that knockdown of human SMYD3 upregulates the expression of multiple type I IFN response and antigen presentation machinery (APM) genes. This study aims to investigate whether Smyd3 depletion potentiates immune-mediated antitumor efficacy in anti-PD-1 resistant syngeneic mouse models of HPV-negative HNSCC (MOC1), as well as relevant mechanisms. SiRNA-mediated knockdown of Smyd3 in mouse HPV-negative MOC1 cells followed by RNA-seq revealed upregulation of multiple type I IFN response and APM genes in vitro. Multicolor flow cytometry of MOC1 tumors treated with control anti-sense oligonucleotides (ASOs) or Smyd3 ASOs showed that Smyd3 depletion in a syngeneic, heterotopic mouse model (C57BL/6) of flank MOC1 tumors induced intratumoral infiltration of CD8+ T-cells, as well as upregulation of H2-Kb (MHC class I) and mouse Pd-l1 in MOC1 cells. Combined treatment of Smyd3 ASOs with anti-PD-1 induced complete regressions of flank MOC1 tumors in 4/8 treated mice and significant tumor growth restriction in 2/8 mice, while 2/8 tumors “escaped” the treatment effect. Single-cell RNA seq of MOC1 tumors treated with control or Smyd3 ASOs is ongoing to evaluate the effects of Smyd3 depletion on MOC1 cancer cells and immune cell subsets of the tumor microenvironment and to identify potential mechanisms of resistance of Smyd3 ASO + anti-PD-1 combination treatment. Genome-wide mapping of Smyd3 and the repressive mark H4K20me3, which is known to be written by Smyd3, in MOC1 cells using CUT&RUN assays is also ongoing and aims to decipher whether Smyd3 directly binds to and regulates the expression of immune-related genes through H4K20me3. Ex-vivo cytotoxicity assays aim to evaluate whether CRISPR Smyd3 KO in MOC1 cells sensitizes cancer cells to TIL-mediated cytotoxicity. Smyd3 depletion induces an inflamed tumor microenvironment and may potentiate the immune-mediated antitumor efficacy of anti-PD-1 in syngeneic mouse models of HPV-negative HNSCC. This work may lay the biological rationale to combine SMYD3 inhibition with T-cell based immunotherapeutic approaches in HPV-negative HNSCC. Citation Format: Daniel E. Tsai, Yvette Robbins, Angel Huynh, Andrew Sinkoe, Cem Sievers, Madhavi Murali, Xiaolin Luo, Clint T. Allen, Vassiliki Saloura. Investigating the immunomodulatory mechanisms of Smyd3 depletion in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6250.

SMYD3 Promotes Cell Cycle Progression by Inducing Cyclin D3 Transcription and Stabilizing the Cyclin D1 Protein in Medulloblastoma.

Simple SummaryMedulloblastoma is the most common malignant pediatric brain tumor and is classified into four molecular subgroups: Wnt, Shh, Group 3, and Group 4. Of these subgroups, patients with Myc+ Group 3 MB have the worst prognosis. Using an RNAi functional genomic screen, we identified the lysine methyltransferase SMYD3 as a crucial epigenetic regulator responsible for promoting Group 3 MB cell growth. We demonstrated that SMYD3 drives MB cell cycle progression by inducing cyclin D3 transcription and preventing cyclin D1 ubiquitination. Using in vitro and ex vivo studies, we showed that SMYD3 suppression by shRNA and BCI-121 significantly impaired proliferation, resulting in the downregulation of cyclin D3, cyclin D1, and pRBSer795. Moreover, we are the first to show that SMYD3 methylates the cyclin D1 protein, indicating that the SMYD3 stabilizes cyclin D1 through post-translational modification. Collectively, our studies position SMYD3 as a promising treatment option for Group 3 Myc+ MB patients.Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Maximum safe resection, postoperative craniospinal irradiation, and chemotherapy are the standard of care for MB patients. MB is classified into four subgroups: Shh, Wnt, Group 3, and Group 4. Of these subgroups, patients with Myc+ Group 3 MB have the worst prognosis, necessitating alternative therapies. There is increasing interest in targeting epigenetic modifiers for treating pediatric cancers, including MB. Using an RNAi functional genomic screen, we identified the lysine methyltransferase SMYD3, as a crucial epigenetic regulator that drives the growth of Group 3 Myc+ MB cells. We demonstrated that SMYD3 directly binds to the cyclin D3 promoter to activate its transcription. Further, SMYD3 depletion significantly reduced MB cell proliferation and led to the downregulation of cyclin D3, cyclin D1, pRBSer795, with concomitant upregulations in RB in vitro. Similar results were obtained following pharmacological inhibition of SMYD3 using BCI-121 ex vivo. SMYD3 knockdown also promoted cyclin D1 ubiquitination, indicating that SMYD3 plays a vital role in stabilizing the cyclin D1 protein. Collectively, our studies demonstrate that SMYD3 drives cell cycle progression in Group 3 Myc+ MB cells and that targeting SMYD3 has the potential to improve clinical outcomes for high-risk patients.

The Lysine Methylase SMYD3 Modulates Mesendodermal Commitment during Development.

SMYD3 (SET and MYND domain containing protein 3) is a methylase over-expressed in cancer cells and involved in oncogenesis. While several studies uncovered key functions for SMYD3 in cancer models, the SMYD3 role in physiological conditions has not been fully elucidated yet. Here, we dissect the role of SMYD3 at early stages of development, employing mouse embryonic stem cells (ESCs) and zebrafish as model systems. We report that SMYD3 depletion promotes the induction of the mesodermal pattern during in vitro differentiation of ESCs and is linked to an upregulation of cardiovascular lineage markers at later stages. In vivo, smyd3 knockdown in zebrafish favors the upregulation of mesendodermal markers during zebrafish gastrulation. Overall, our study reveals that SMYD3 modulates levels of mesendodermal markers, both in development and in embryonic stem cell differentiation.

H3K4 Methyltransferase Smyd3 Mediates Vascular Smooth Muscle Cell Proliferation, Migration, and Neointima Formation.

[Figure: see text].

Abstract 3651: Deciphering immunomodulatory roles of the SMYD3 methyltransferase in HPV-negative head and neck squamous cell carcinoma

Abstract Human Papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) is a cancer with a 50% recurrence rate and poor overall survival. While immunotherapy with programmed-death-1 (PD-1) checkpoint inhibitors was recently approved in the first-line recurrent/metastatic setting, response rates are as low as 20%. A main reason for this is that a majority of these tumors have a low CD8+ T-cell intratumoral infiltrate, rendering primary resistance to T-cell based immunotherapy. We recently found that SET and MYND Domain containing 3 (SMYD3), a chromatin modifier that trimethylates histone 3 lysine 4 (H3K4me3), is overexpressed in HPV-negative HNSCC compared to normal epithelium, and that higher expression of SMYD3 is associated with lower mRNA levels of CD8A and CXCL9, 10 and 11 chemokines, which are key mediators of CD8+ T-cell trafficking, in HPV-negative HNSCC tumors (TCGA). Furthermore, SMYD3 depletion induced upregulation of CXCL9, 10, and 11 in human HNSCC cells in vitro. The purpose of this study is to evaluate whether targeting SMYD3 increases intratumoral CD8+ T-cell infiltration and enhances responses to PD-1 checkpoint inhibition, and to decipher relevant mechanisms. To investigate this hypothesis, a syngeneic, heterotopic mouse model (C57BL/6) using a doxycycline-inducible smyd3 knockdown HPV-negative mouse oral carcinoma cell line (MOC1) was generated. Experiments to assess whether smyd3 knockdown alone and in combination with PD-1 inhibition induce tumor growth restraint are planned. RT-PCR to evaluate the effects of smyd3 depletion on the expression of immune-related (type I IFN response and antigen presentation machinery) genes in the doxycycline-inducible MOC1 cell line, as well as in parental MOC1 cells using siRNA interference is being conducted. Western blotting to assess the effect of smyd3 depletion on global histone methylation marks is also being conducted. Furthermore, to assess whether smyd3 has a cell autonomous effect on the growth of MOC1 cells, cell viability assays (MTT) and cell cycle analysis are being pursued. Preliminary results have shown that smyd3 depletion in parental MOC1 cells induced significant upregulation of mouse cxcl9 and 10. Smyd3 depletion also led to significant cell death in parental MOC1 cells. Experiments to assess the effect of smyd3 depletion on CD8+ T-cell infiltration and tumor growth restraint in vivo are ongoing. This is the first study to investigate the role of SMYD3 as a regulator of antitumor immune response, and may lay the rationale to translate SMYD3 inhibitors in patients with HPV-negative HNSCC with the goal to render them susceptible to T-cell based immunotherapeutic approaches. Citation Format: Benjamin Bernard, Kyunghee Burkitt, Yvette Robbins, Nupur Nigam, Clint Allen, Lyuba Varticovski, Gordon Hager, Carter Van Waes, Vassiliki Saloura. Deciphering immunomodulatory roles of the SMYD3 methyltransferase in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3651.

Histone methyltransferase Smyd3 is a new regulator for vascular senescence.

Endothelial cell senescence is one of the main risk factors contributing to vascular diseases. As increasing number of “epigenetic drugs” entering clinical trials, understanding the mechanism of epigenetic regulation in vascular aging has significant implications in finding targets to cure vascular diseases. However, the epigenetic regulation of endothelial senescence remains unclear. Based on the findings that increased protein level of histone H3 lysine 4 (H3K4) methyltransferase Smyd3 and elevated H3K4me3 modification happened in angiotensin II (Ang II)‐induced senescence in rat endothelial cells, we are curious about whether and how Smyd3 can regulate endothelial senescence. We found that an increase of Smyd3 alone promoted senescence‐associated phenotypes, while knockdown of Smyd3 blocked senescence in endothelial cells. Furthermore, Smyd3‐specific inhibitor reversed vascular senescence‐associated phenotypes at cellular level. Importantly, Ang II‐induced vascular senescence can be greatly alleviated in Smyd3 knockout (KO) mice and those treated with Smyd3 inhibitor. Mechanistically, Smyd3 directly bound to the promoter region of Cdkn1a (coding for p21), then caused its increased H3K4me3 level and elevated gene expression, and ultimately gave rise to senescence‐associated phenotypes. Intriguingly, Smyd3‐mediated p21 upregulated expression also exists in human tissues of vascular disease, indicating it is probably an evolutionarily conserved mechanism in regulating vascular senescence. Thus, Smyd3 can act as a novel factor regulating endothelial senescence through transcriptionally promoting p21 expression. Blocking the Smyd3‐p21 signaling axis may also have potential medical implications in treating diseases related to vascular aging.

SMYD3 promotes implant metastasis of ovarian cancer via H3K4 trimethylation of integrin promoters

Detachment of cancer cells from the primary tumor and formation of spheroids in ascites is required for implantation metastasis in epithelial ovarian cancer (EOC), but the underlying mechanism of this process has not been thoroughly elucidated. To mimic this process, ovarian cancer cells were grown in 3D and 2D culture. Hey and OVCA433 spheroids exhibited decreased cell proliferation and enhanced adhesion and invasion. SMYD3 expression was elevated in ovarian carcinoma spheroids in association with increased H3K4 methylation. Depletion of SMYD3 by transient siRNA, stable shRNA knockdown and the SMYD3 inhibitor BCI-121 all decreased spheroid invasion and adhesion. Gene expression arrays revealed downregulation of integrin family members. Inhibition assays confirmed that invasion and adhesion of spheroids are mediated by ITGB6 and ITGAM. SMYD3-deficient cells regained the ability to invade and adhere after forced overexpression of SMYD3, ITGB6 and ITGAM. However, this biological ability was not restored by forced overexpression of SMYD3 in ITGB6- and/or ITGAM-deficient cancer cells. SMYD3 and H3K4me3 binding at the ITGB6 and ITGAM promoters was increased in spheroids compared to that in monolayer cells, and the binding was decreased when SMYD3 expression was inhibited, consistent with the expression changes in integrins. SMYD3 expression and integrin-mediated adhesion were also activated in an intraperitoneal xenograft model and in EOC patient spheroids. In vivo, SMYD3 knockdown inhibited tumor metastasis and reduced ascites volume in both the intraperitoneal xenograft model and a PDX model. Overall, our results suggest that the SMYD3-H3K4me3-integrin pathway plays a crucial role in ovarian cancer metastasis to the peritoneal surface.

Effect of SMYD3 on biological behavior and H3K4 methylation in bladder cancer.

PurposeOur goal was to investigate the effect of SMYD3 on the biological behavior and histone 3 lysine-4 (H3K4) methylation of bladder cancer (BLAC).Patients and methodsqRT-PCR identified that SMYD3 expression level in BLAC cell lines (T24, 5637, BUI-87 and J-82) and human normal uroepithelial cell line SV-HUC1. We also constructed green fluorescence protein lentiviral vector using the gene short hairpin RNA (shRNA) system. We used Western blot to analyze the SMYD3, H3K4me1, H3K4me2 and H3K4me3 expression levels in shRNA transfection lines. We also performed a colony-forming assay to determine colony-forming ability, cell counting kit-8 for cell proliferation detection, Transwell assay to determine cell migration and invasion and Annexin V-FITC/PI double staining to analyze cell apoptosis.ResultsThe SMYD3 expression level was significantly higher in BLAC cell lines (T24, 5637, BUI-87 and J-82) than in human normal uroepithelial cell line SV-HUC1, and exhibited the highest expression level in T24 cells, among the cell lines tested. qRT-PCR and Western blot analysis results showed that SMYD3 was successfully suppressed in shRNA transfection lines, and identified that SMYD3 suppression resulted inhibited H3K4me2 and H3K4me3 but not H3K4me1. SMYD3 knockdown cells accelerated cell apoptosis and exhibited low cell colony-forming ability, proliferation ability, inhibition of cell migration and invasion compared with normal cells.ConclusionSMYD3 may be activated in BLAC cells to increase H3K4 activity to modulate cell proliferation, migration and invasion ability. The data will be a useful source for future therapy.

Behavioral, circuitry, and molecular aberrations by region-specific deficiency of the high-risk autism gene Cul3.

Cullin 3 (Cul3) gene, which encodes a core component of the E3 ubiquitin ligase complex that mediates proteasomal degradation, has been identified as a true high-risk factor for autism. Here, by combining behavioral, electrophysiological, and proteomic approaches, we have examined how Cul3 deficiency contributes to the etiology of different aspects of autism. Heterozygous mice with forebrain Cul3 deletion displayed autism-like social interaction impairment and sensory-gating deficiency. Region-specific deletion of Cul3 leads to distinct phenotypes, with social deficits linked to the loss of Cul3 in prefrontal cortex (PFC), and stereotypic behaviors linked to the loss of Cul3 in striatum. Correlated with these behavioral alterations, Cul3 deficiency in forebrain or PFC induces NMDA receptor hypofunction, while Cul3 loss in striatum causes a cell type-specific alteration of neuronal excitability in striatal circuits. Large-scale profiling has identified sets of misregulated proteins resulting from Cul3 deficiency in different regions, including Smyd3, a histone methyltransferase involved in gene transcription. Inhibition or knockdown of Smyd3 in forebrain Cul3-deficient mice ameliorates social deficits and restores NMDAR function in PFC. These results have revealed for the first time a potential molecular mechanism underlying the manifestation of different autism-like behavioral deficits by Cul3 deletion in cortico-striatal circuits.

SMYD3 promotes the epithelial-mesenchymal transition in breast cancer.

SMYD3 is a methylase previously linked to cancer cell invasion and migration. Here we show that SMYD3 favors TGFβ-induced epithelial–mesenchymal transition (EMT) in mammary epithelial cells, promoting mesenchymal and EMT transcription factors expression. SMYD3 directly interacts with SMAD3 but it is unnecessary for SMAD2/3 phosphorylation and nuclear translocation. Conversely, SMYD3 is indispensable for SMAD3 direct association to EMT genes regulatory regions. Accordingly, SMYD3 knockdown or its pharmacological blockade with the BCI121 inhibitor dramatically reduce TGFβ-induced SMAD3 association to the chromatin. Remarkably, BCI121 treatment attenuates mesenchymal genes transcription in the mesenchymal-like MDA-MB-231 cell line and reduces their invasive ability in vivo, in a zebrafish xenograft model. In addition, clinical datasets analysis revealed that higher SMYD3 levels are linked to a less favorable prognosis in claudin-low breast cancers and to a reduced metastasis free survival in breast cancer patients. Overall, our data point at SMYD3 as a pivotal SMAD3 cofactor that promotes TGFβ-dependent mesenchymal gene expression and cell migration in breast cancer, and support SMYD3 as a promising pharmacological target for anti-cancer therapy.

Network analyses elucidate the role of SMYD3 in esophageal squamous cell carcinoma.

SMYD3 is a member of the SET and myeloid‐Nervy‐DEAF‐1 (MYND) domain‐containing protein family of methyltransferases, which are known to play critical roles in carcinogenesis. Expression of SMYD3 is elevated in various cancers, including esophageal squamous cell carcinoma (ESCC), and is correlated with the survival time of patients with ESCC. Here, we dissect gene expression data, from a previously described KYSE150 ESCC cell line in which SMYD3 had been knocked down, by integration with the protein–protein interaction (PPI) network, to find the new potential biological roles of SMYD3 and subsequent target genes. By construction of a specific PPI network, differentially expressed genes (DEGs), following SMYD3 knockdown, were identified as interacting with thousands of neighboring proteins. Enrichment analyses from the DAVID Functional Annotation Chart found significant Gene Ontology (GO) terms associated with transcription activities, which were closely related to SMYD3 function. For example, YAP1 and GATA3 might be a target gene for SMYD3 to regulate transcription. Enrichment annotation of the total DEG PPI network by GO ‘Biological Process’ generated a connected functional map and found 532 significant terms, including known and potential biological roles of SMYD3 protein, such as expression regulation, signal transduction, cell cycle, cell metastasis, and invasion. Subcellular localization analyses found that DEGs and their interacting proteins were distributed in multiple layers, which might reflect the intricate biological processes at the spatial level. Our analysis of the PPI network has provided important clues for future detection of the biological roles and mechanisms, as well as the target genes of SMYD3.

Protein lysine methyltransferase SMYD3 is involved in tumorigenesis through regulation of HER2 homodimerization.

HER2 is a receptor tyrosine kinase, which is amplified and overexpressed in a subset of human cancers including breast and gastric cancers, and is indicated in its involvement in progression of cancer. Although its specific ligand(s) has not been detected, HER2 homodimerization, which is critical for its activation, is considered to be dependent on its expression levels. Here, we demonstrate a significant role of HER2 methylation by protein lysine methyltransferase SMYD3 in HER2 homodimerization. We found that SMYD3 trimethylates HER2 protein at lysine 175. HER2 homodimerization was enhanced in the presence of SMYD3, and substitution of lysine 175 of HER2 with alanine (HER2‐K175A) reduced the formation of HER2 homodimers. Furthermore, HER2‐K175A revealed lower level of autophosphorylation than wild‐type HER2. We also identified that knockdown of SMYD3 attenuated this autophosphorylation in breast cancer cells. Our results imply that SMYD3‐mediated methylation of HER2 at Lysine 175 may regulate the formation of HER2 homodimer and subsequent autophosphorylation and suggest that the SMYD3‐mediated methylation pathway seems to be a good target for development of novel anti‐cancer therapy.

SMYD3-mediated lysine methylation in the PH domain is critical for activation of AKT1.

AKT1 is a cytosolic serine/threonine kinase that is overexpressed in various types of cancer and has a central role in human tumorigenesis. Although it is known that AKT1 is post-translationally modified in various ways including phosphorylation and ubiquitination, methylation has not been reported so far. Here we demonstrate that the protein lysine methyltransferase SMYD3 methylates lysine 14 in the PH domain of AKT1 both in vitro and in vivo. Lysine 14-substituted AKT1 shows significantly lower levels of phosphorylation at threonine 308 than wild-type AKT1, and knockdown of SMYD3 as well as treatment with a SMYD3 inhibitor significantly attenuates this phosphorylation in cancer cells. Furthermore, substitution of lysine 14 diminishes the plasma membrane accumulation of AKT1, and cancer cells overexpressing lysine 14-substiuted AKT1 shows lower growth rate than those overexpressing wild-type AKT1. These results imply that SMYD3-mediated methylation of AKT1 at lysine 14 is essential for AKT1 activation and that SMYD3-mediated AKT1 methylation appears to be a good target for development of anti-cancer therapy.

Abstract 4613: SMYD3-mediated lysine methylation is critical for the activation of AKT1 in human cancer

Abstract v-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1), a serine/threonine-protein kinase also known as protein kinase B Alpha (PKB Alpha), is a key mediator of a signaling pathway that governs various cellular processes regulating cell growth, survival, glucose metabolism, genome stability and neovascularization. It is known that dysregulation of AKT1 activity is one of the critical factors for the development and/or progression of a variety of human cancers. Posttranslational modifications of AKT1, including phosphorylation, ubiquitination and glycosylation, are well-analyzed, and it is known that the phosphorylation status of AKT1 is strongly correlated with its oncogenic activity. However, biological functions of methylation on AKT1 have never been analyzed so far. In the present study, we demonstrated that the protein lysine methyltrasnferase SMYD3 methylates lysine 14 in the PH domain of AKT1, which is known to play a critical role in the regulation of AKT1 activity through binding to phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) or phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2), in vitro and in vivo. Lysine 14-substituted AKT1 shows significantly lower levels of phosphorylation at threonine 308, which is the major indicator of AKT1 activity, than wild-type AKT1, and knockdown of SMYD3 as well as treatment with a SMYD3 inhibitor significantly attenuates this phosphorylation in cancer cells. Furthermore, substitution of lysine 14 diminishes the plasma membrane accumulation of AKT1, and cancer cells overexpressing lysine 14-substiuted AKT1 shows lower growth rate than those overexpressing wild-type AKT1. These results imply that SMYD3-mediated methylation of AKT1 at lysine 14 plays a central role in the oncogenic activity of AKT1. Although Phosphatidylinositide 3-kinase (PI3K)-dependent AKT1 phosphorylation by Pyruvate Dehydrogenase Kinase, Isozyme 1 (PDK1) and mTOR Complex 2 (mTORC2) has long been considered to be the primary mechanism accounting for AKT1 activation, these members are not sufficient enough to explain how AKT1 hyperactivation can occur in tumors with normal levels of PI3K/Phosphatase And Tensin Homolog (PTEN) activity. Our findings unveiled the novel mechanism of constitutive hyperphosphorylation on AKT1 in cancer cells, mediated by SMYD3-mediated methylation. The inhibition of this methylation pathway appears to be a promising strategy to develop anti-cancer drugs. Citation Format: Yuichiro Yoshioka, Yusuke Nakamura, Ryuji Hamamoto. SMYD3-mediated lysine methylation is critical for the activation of AKT1 in human cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4613.

Upregulated SMYD3 promotes bladder cancer progression by targeting BCLAF1 and activating autophagy

The recent discovery of a large number of histone methyltransferases reveals important roles of these enzymes in regulating tumor development and progression. SMYD3, a histone methyltransferase, is associated with poor prognosis of patients with prostate and gastric cancer. In the study, we attempted to investigate its putative oncogenic role on bladder cancer. Here, we report that SMYD3 frequently amplified in bladder cancer is correlated with bladder cancer progression and poor prognosis. Overexpression of SMYD3 promotes bladder cancer cell proliferation and invasion, whereas SMYD3 knockdown inhibits cancer cell growth and invasion. Mechanically, SMYD3 positively regulates the expression of BCL2-associated transcription factor 1 (BCLAF1). SMYD3 physically interacts with the promoter of BCLAF1 and upregulates its expression by accumulating di- and trimethylation of H3K4 at the BCLAF1 locus. We further show that SMYD3 overexpression in bladder cancer cells promotes autophagy activation, whereas BCLAF1 depletion inhibits SMYD3-induced autophagy. Finally, we demonstrate that SMYD3 promotes bladder cancer progression, at least in part by increasing BCLAF1 expression and activating autophagy. Our results establish a function for SMYD3 in autophagy activation and bladder cancer progression and suggest its candidacy as a new prognostic biomarker and target for clinical management of bladder cancer.

Abstract C85: Identification of a novel potent selective SMYD3 inhibitor with oral bioavailability

Abstract SMYD3 (Set and Mynd Domain containing 3) is a lysine methyltransferase overexpressed in several cancer types including breast, prostrate, pancreatic, and lung, and this overexpression is associated with poor clinical prognosis. Genetic knockdown of SMYD3 by shRNA has been shown to decrease proliferation in a range of cancer cell lines suggesting that inhibition of SMYD3 may have therapeutic utility. In this presentation we describe the discovery and optimization of a novel series of oxindole sulfonamides and sulfamides with SMYD3 inhibitory activity. One of these compounds, EPZ030456, has a SMYD3 biochemical IC50 of 4 nM and is active in cells with an IC50 of 48 nM in a trimethyl MAP3K2 (MEKK2) in-cell western (ICW) assay. The crystal structure of this compound was solved with SMYD3 and the nucleotide substrate, S-adenosylmethionine and shows the oxindole portion of the molecule extends into the SMYD3 lysine binding channel. EPZ030456 shows < 30% inhibition at a 10 uM screening concentration against 17 histone methyltransferase targets tested, including SMYD2. Further optimization within the series resulted in EPZ031686 which has similar potency to EPZ030456 with a biochemical IC50 of 3 nM and an ICW IC50 of 36 nM and in addition exhibits good bioavailability following oral dosing in mice. Hence, EPZ031686 is a suitable tool to study the role of SMYD3 in cancer and other therapeutic areas, using both in vitro and in vivo models. Citation Format: Lorna H. Mitchell, Paula A. Boriack-Sjodin, Sherri Smith, Michael Thomenius, Nathalie Rioux, Michael Munchhof, James E. Mills, Christine Klaus, Jennifer Totman, Thomas V. Riera, Alejandra Raimondi, Suzanne L. Jacques, Megan Foley, Nigel J. Waters, Kevin W. Kuntz, Tim J. Wigle, Margaret Porter Scott, Robert A. Copeland, Jesse J. Smith, Richard Chesworth. Identification of a novel potent selective SMYD3 inhibitor with oral bioavailability. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C85.

Epigenetic regulation of miR-124 by Hepatitis C Virus core protein promotes migration and invasion of intrahepatic cholangiocarcinoma cells by targeting SMYD3

Hepatitis C Virus core protein (HCVc) plays important roles in the development of intrahepatic cholangiocarcinoma (ICC). MicroRNAs (miRNAs) contribute to tumor progression by interacting with downstream target genes. However, the regulation and role of miRNAs in HCV-related intrahepatic cholangiocarcinoma (HCV-ICC) is poorly understood. In this study, we found that miR-124 was down-regulated in HCV-ICC and the induction of DNMT1 by HCVc mediated the suppression of miR-124. Over-expression of miR-124 suppressed cell migration and invasion in vitro, and reduced the protein levels of SMYD3 and downstream target genes (c-Myc and MMP9). Knockdown of SMYD3 inhibited cell migration and invasion resembling that of miR-124 over-expression. In conclusion, our studies indicate that low miR-124 levels mediated by HCVc via DNMT1 promote ICC cell migration and invasion by targeting SMYD3.

Abstract 3145: Smyd3 catalyzes a novel methylation mark and regulates cancer cell proliferation

Abstract SET and MYND domain-containing protein 3 (Smyd3) is a lysine methyltransferase over-expressed in liver, breast and rectal carcinomas. We have shown that knockdown of Smyd3 has an anti-proliferative effect in cancer cell lines and that this effect can be rescued with wild type but not catalytically inactive Smyd3, directly linking Smyd3 catalytic activity to proliferation. Published reports indicate Smyd3 methylates lysine 4 of histone H3, lysine 20 of histone H4 and VEGFR1. However, activity testing of a peptide library and intact recombinant nucleosomes indicate that a unique methylation mark is catalyzed by Smyd3. LC-MS/MS analysis of in vitro reaction products have identified the location of this novel site of methylation. The existence of this novel mark and its dependence on Smyd3 activity was confirmed in cancer cells and mouse embryonic fibroblasts. We speculate that overexpression of Smyd3 causes aberrant methylation and plays an important role in the deregulation of gene expression leading to uncontrolled cellular proliferation in cancer making Smyd3 a promising target for anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3145. doi:1538-7445.AM2012-3145