Lysine Methyltransferase Activity Research Articles

Identification of fragments targeting SMYD3 using highly sensitive kinetic and multiplexed biosensor-based screening.

A 1056-membered fragment library has been screened against SMYD3 using a novel multiplexed experimental design implemented in a grating coupled interferometry (GCI)-based biosensor. SMYD3 is a prospective target for anticancer drugs and the focus has initially been on discovery of inhibitors of its lysine methyl transferase activity. However, it has multiple protein interaction partners and several potential roles in carcinogenesis. It therefore remains unclear what mode of action ligands targeting the protein should have. Our goal was therefore to identify new ligands and discriminate hits that interact with the active site and those that interact with other sites. In addition, we were interested in selecting hits based on kinetic features rather than affinity. Screening was done in parallel against SMYD3 alone or SMYD3 with the active site blocked by a tight binding inhibitor. Hit selection was primarily based on dissociation rates. In total, 20 fragments were selected as hits, of which half apparently targeted the active site and half targeted other sites. Twelve of the hits were selected for structural analysis using X-ray crystallography in order to identify binding sites and modes of binding. Four of the hits were successfully identified in crystal structures with SMYD3; the others did not show any electron densities for ligands in the crystals. Although it might be possible to optimize the crystallography approach for a better success rate, it was clear that the sensitivity and time resolution of the biosensor assay was exceptional and enabled kinetic rate constants to be estimated for fragments. Fragments are typically considered to interact too rapidly for such quantification to be possible. This approach consequently represents a paradigm shift. In addition, the multiplexed approach allows ligands targeting different sites to be rationally selected already in the fragment library screening stage.

Selenium nanoparticles modulate histone methylation via lysine methyltransferase activity and S-adenosylhomocysteine depletion

At physiological levels, the trace element selenium plays a key role in redox reactions through the incorporation of selenocysteine in antioxidant enzymes. Selenium has also been evaluated as a potential anti-cancer agent, where selenium nanoparticles have proven effective, and are well tolerated in vivo at doses that are toxic as soluble Se. The use of such nanoparticles, coated with either serum albumin or the naturally occurring alkaline polysaccharide chitosan, also serves to enhance biocompatibility and bioavailability. Here we demonstrate a novel role for selenium in regulating histone methylation in ovarian cancer cell models treated with inorganic selenium nanoparticles coated with serum albumin or chitosan. As well as inducing thioredoxin reductase expression, ROS activity and cancer cell cytotoxicity, coated nanoparticles caused significant increases in histone methylation. Specifically, selenium nanoparticles triggered an increase in the methylation of histone 3 at lysines K9 and K27, histone marks involved in both the activation and repression of gene expression, thus suggesting a fundamental role for selenium in these epigenetic processes. This direct function was confirmed using chemical inhibitors of the histone lysine methyltransferases EZH2 (H3K27) and G9a/EHMT2 (H3K9), both of which blocked the effect of selenium on histone methylation. This novel role for selenium supports a distinct function in histone methylation that occurs due to a decrease in S-adenosylhomocysteine, an endogenous inhibitor of lysine methyltransferases, the metabolic product of methyl-group transfer from S-adenosylmethionine in the one-carbon metabolism pathway. These observations provide important new insights into the action of selenium nanoparticles. It is now important to consider both the classic antioxidant and novel histone methylation effects of this key redox element in its development in cancer therapy and other applications.

Keap1 moderates the transcription of virus induced genes through G9a-GLP and NFκB p50 recruitment.

Cells must control genes that are induced by virus infection to mitigate deleterious consequences of inflammation. We investigated the mechanisms whereby Keap1 moderates the transcription of genes that are induced by Sendai virus infection in mouse embryo fibroblasts (MEFs). Keap1-/- deletions increased the transcription of virus induced genes independently of Nrf2. Keap1 moderated early virus induced gene transcription. Virus infection induced Keap1 to bind Ifnb1, Tnf and Il6, and reduced Keap1 binding at Cdkn1a and Ccng1. Virus infection induced G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition. Keap1-/- deletions eliminated G9a-GLP and NFκB p50 recruitment, and H3K9me2 deposition, but they did not affect NFκB p65, IRF3 or cJun recruitment. G9a-GLP inhibitors (BIX01294, MS012, BRD4770) enhanced virus induced gene transcription in MEFs with intact Keap1, but not in MEFs with Keap1-/- deletions. G9a-GLP inhibitors augmented Keap1 binding to virus induced genes in infected MEFs, and to cell cycle genes in uninfected MEFs. G9a-GLP inhibitors augmented NFκB subunit recruitment in MEFs with intact Keap1. G9a-GLP inhibitors stabilized Keap1 retention in permeabilized MEFs. G9a-GLP lysine methyltransferase activity was required for Keap1 to moderate transcription, and it moderated Keap1 binding to chromatin. The interdependent effects of Keap1 and G9a-GLP on the recruitment of each other and on the moderation of virus induced gene transcription constitute a feedback circuit. Keap1 and the electrophile tBHQ reduced virus induced gene transcription through different mechanisms, and they regulated the recruitment of different NFκB subunits. Characterization of the mechanisms whereby Keap1, G9a-GLP and NFκB p50 moderate virus induced gene transcription can facilitate the development of immunomodulatory agents.

Homocysteine-targeting compounds as a new treatment strategy for diabetic wounds via inhibition of the histone methyltransferase SET7/9

In hypoxia and hyperglycemia, SET7/9 plays an important role in controlling HIF-1α methylation and regulating the transcription of HIF-1α target genes, which are responsible for angiogenesis and wound healing. Here, we report the Ir(III) complex Set7_1a bearing acetonitrile (ACN) ligands as a SET7/9 methyltransferase inhibitor and HIF-1α stabilizer. Interestingly, Set7_1a could engage SET7/9 and strongly inhibit SET7/9 activity, especially after preincubation with homocysteine (Hcy), which is elevated in diabetes. We hypothesize that Set7_1a exchanges ACN subunits for Hcy to disrupt the interaction between SET7/9 and SAM/SAH, which are structurally related to Hcy. Inhibition of SET7/9 methyltransferase activity by Set7_1a led to reduced HIF-1α methylation at the lysine 32 residue, causing increased HIF-1α level and recruitment of HIF-1α target genes that promote angiogenesis, such as VEGF, GLUT1, and EPO, in hypoxia and hyperglycemia. Significantly, Set7_1a improved wound healing in a type 2 diabetic mouse model by activating HIF-1α signaling and downstream proangiogenic factors. To our knowledge, this is the first Hcy-targeting iridium compound shown to be a SET7/9 antagonist that can accelerate diabetic wound healing. More importantly, this study opens a therapeutic avenue for the treatment of diabetic wounds by the inhibition of SET7/9 lysine methyltransferase activity.

Abstract 3274: Targeting NSD family histone methyltransferase activity with irreversible inhibitors

Abstract Nuclear SET domain containing (NSD) family member proteins are histone methyltransferases that mono- and di-methylate histone 3 at lysine 36 (H3K36) to alter gene expression in a context-dependent manner. NSD1 and NSD3 are two members of this family which share greater than 80 percent sequence similarity in the catalytic SET domain. NSD1 is found as a fusion with NUP98 in aggressive cases of pediatric leukemia, and the lysine methyltransferase (KMTase) activity of NUP98-NSD1 drives bone marrow progenitor proliferation. Similarly, high levels of enzymatically active NSD3 mRNA are correlated with reduced disease-free survival in breast cancer and catalytic NSD3 expression is sufficient to rescue tumor growth in a xenograft model of triple negative breast cancer. Selective and potent inhibitors of NSD1 and NSD3 KMTase activity are needed to further investigate the therapeutic potential of targeting this function in leukemia and/or solid cancers. Here, we report the development of irreversible small molecule inhibitors of the NSD1 and NSD3 SET domains. We utilized a NMR-based fragment screen of the NSD1 SET domain to identify the hit BT1, which binds reversibly to the SET domain of NSD1. BT1 was extensively optimized into a series of potent irreversible ligands. Mass spectrometry and structural studies revealed that these compounds react with a cysteine near the autoinhibitory loop, providing a rational for the strong inhibition of KMTase activity we observe in biochemical assays. These inhibitors also bind to the NSD1 and NSD3 SET domains in cells and exhibit potent anti-proliferative effects against leukemia cells containing NUP98-NSD1 and breast cancer cells with high levels of NSD3 expression. In summary, we present novel irreversible inhibitors of NSD1 and NSD3 KMTase activity which represent important chemical probes that can be used to investigate the therapeutic potential of targeting NSD family member catalytic activity in cancers. Citation Format: Christina Howard, Sergei Zari, Shuangjiang Li, Huang Huang, EunGi Kim, Se Ra Park, Trupta Purohit, Hongzhi Miao, Xiaotian Zhang, Caroline Nikolaidis, Hao Li, Juliano Ndoj, HyoJe Cho, Jolanta Grembecka, Tomasz Cierpicki. Targeting NSD family histone methyltransferase activity with irreversible inhibitors [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 3274.

Extracellular DEK Treatment Mimics Hypoxic Blockade of Extra Physiologic Oxygen Stress in Human and Mouse Hematopoietic Cells

Hematopoietic stem cell (HSC) and progenitor cell (HPC) self-renewal, proliferation, survival, and function are regulated by extracellular signals from cytokines and chemokines. DEK, a nuclear regulator of chromatin availability, was recently shown to also function as an extracellular protein that regulates HSC and HPC by inducing signaling through the CXC chemokine receptor 2 (CXCR2), enhancing pools of long-term stem cells while decreasing pools of functional HPC [Capitano et al., JCI, 2019, 129(6):2555-70]. Due to differing effects of extracellular (ec)DEK treatment on HSC growth and function compared to the effects on HPC, we hypothesized that DEK differentially regulates HSC compared to HPC. To explore this, we pulse-treated purified human cord blood (CB) CD34+ cells for 18 hours with recombinant human ecDEK protein. We then isolated by flow cytometry stringently immunophenotypically-defined human HSC, multipotent progenitors (MPP), common myeloid progenitors (CMP), and granulocyte-macrophage progenitors (GMP) and performed mRNA-sequencing to profile ecDEK-dependent transcriptomes of these separate populations of HSC/HPC. Interestingly, there are widely different ranges of effects on the transcriptomes of the different isolated HSC/HPC populations. CMP had the most differentially expressed (DE) genes induced by DEK treatment, followed by HSC and MPP, and GMP had relatively few changes, suggesting DEK treatment may primarily affect HSC and earlier HPC. HSC exhibited the strongest magnitude of changes in DE genes. Fast gene set enrichment analysis (FGSEA) revealed that GMP showed upregulation of gene programs associated with leukocyte migration and downregulation of gene programs regulated by MYC, CMP showed upregulation of gene programs associated with myeloid cell development and downregulation of lysine methyltransferase activity, and MPP and HSC both upregulated cytokine signaling responses in response to DEK treatment, though HSC also exhibited a downregulation of genes associated with cell cycle. Despite the identified unique changes upon DEK treatment in these different HSC/HPC populations, we were most interested to discover that all examined cell types demonstrated upregulation of genes associated with hypoxic responses in cells, and a concordant downregulation of genes that are frequently downregulated by hypoxic exposure following DEK treatment. This suggested to us that DEK may stimulate overlapping pathways related to hypoxia responses and/or response to exposure to extra physiologic oxygen. In fact, the effects on HSC and HPC induced by ecDEK is similar with previous work demonstrating that isolating HSC/HPC at 3% O 2 compared to ambient air conditions (~21% O 2) leads to a preserved pool of functional HSC but a lower number of functional HPC, due to a phenomenon termed extracellular physiologic shock/stress (EPHOSS) [Mantel et al., Cell, 2015, 161(7):1553-65]. To explore whether DEK regulates similar stress response pathways that are associated with EPHOSS, we assessed the effects of ecDEK treatment on HSC/HPC isolated at 3% O 2. Mice were treated with DEK in vivo, then bone marrow cells were isolated at 3% O 2 in a hypoxia chamber. Cells were kept at 3% O 2 or split to ambient air to allow equilibration to a higher oxygen tension and then assessed for HSC and functional HPC numbers. In contrast to untreated HSC/HPC isolated at 3% O 2, ecDEK treated cells isolated at 3% O 2 did not exhibit further increases in LT-HSC numbers or decreases in pools of functional HPC over ecDEK treatment alone identified by colony assays. Isolation at 3% O 2 followed by in vitro treatment with DEK showed similar results. This suggests ecDEK may neutralize the effects of EPHOSS by acting on similar gene programs that are induced by ambient air exposure. We also demonstrate that ecDEK prevents the ex vivo apoptosis of CB HSC/HPC populations, again suggesting that ecDEK is preventing the cellular stress associated with ambient air exposure and ex vivo growth, resulting in a preservation of HSC. Taken together, these data show that ecDEK acts as an extracellular signaling protein that prevents cellular stress by activating gene programs that overlap with hypoxia associated gene programs. This has important implications for the role of ecDEK in normal and diseased hematopoiesis, as well as clinical implications wherein ecDEK may be used to mimic the HSC preserving effects of isolation at 3% O 2. DisclosuresMarkovitz: University of Michigan: Patents & Royalties: H84T BanLec and of the H84T-driven CAR construct.

SMYD2-mediated TRAF2 methylation promotes the NF-κB signaling pathways in inflammatory diseases.

BackgroundThe methylation of lysine residues has been involved in the multiple biological and diseases processes. Recently, some particular non‐histone proteins have been elucidated to be methylated by SMYD2, a SET and MYND domain protein with lysine methyltransferase activity.MethodsSMYD2 was evaluated in synovial tissue and cells derived from rheumatoid arthritis patients. We confirmed TRAF2 could be methylated by SMYD2 using Mass spectrometry, pull‐down, immunoprecipitation, methyltransferase assay, ubiquitination assay, luciferase reporter assays, and western blot analyses. Using loss‐ and gain‐of function studies, we explored the biological functions of SMYD2 in vitro and in vivo. Using acute and chronic inflammation with different mice models to determine the impact of SMYD2.ResultsHere, we first time confirmed that the cytoplasmic protein TRAF2 as the kernel node for NF‐κB signaling pathway could be methylated by SMYD2. SMYD2‐mediated TRAF2 methylation contributed to the durative sensitization of NF‐κB signaling transduction through restraining its own proteolysis and enhancing the activity. In addition, we found knocking down of SMYD2 has different degrees of mitigation in acute and chronic inflammation mice models. Furthermore, as the lysine‐specific demethylase, LSD1 could resist methylation on TRAF2 induced by SMYD2.ConclusionsOur data uncovered an unprecedented cytoplasmic protein network that employed methylation of TRAF2 for the maintenance of NF‐κB activation during inflammatory diseases.

Discovery of an Allosteric Ligand Binding Site in SMYD3 Lysine Methyltransferase.

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug‐like compounds was screened in a biosensor‐based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (K D=42 and 84 μM, respectively). Co‐crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C‐terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3–HSP90 binding was confirmed (K D=13 μM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.

The histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells

The chromatin-binding E3 ubiquitin ligase ubiquitin-like with PHD and RING finger domains 1 (UHRF1) contributes to the maintenance of aberrant DNA methylation patterning in cancer cells through multivalent histone and DNA recognition. The tandem Tudor domain (TTD) of UHRF1 is well-characterized as a reader of lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and, more recently, lysine 126 di- and tri-methylation on DNA ligase 1 (LIG1K126me2/me3). However, the functional significance and selectivity of these interactions remain unclear. In this study, we used protein domain microarrays to search for additional readers of LIG1K126me2, the preferred methyl state bound by the UHRF1 TTD. We show that the UHRF1 TTD binds LIG1K126me2 with high affinity and selectivity compared to other known methyllysine readers. Notably, and unlike H3K9me2/me3, the UHRF1 plant homeodomain (PHD) and its N-terminal linker (L2) do not contribute to multivalent LIG1K126me2 recognition along with the TTD. To test the functional significance of this interaction, we designed a LIG1K126me2 cell-penetrating peptide (CPP). Consistent with LIG1 knockdown, uptake of the CPP had no significant effect on the propagation of DNA methylation patterning across the genomes of bulk populations from high-resolution analysis of several cancer cell lines. Further, we did not detect significant changes in DNA methylation patterning from bulk cell populations after chemical or genetic disruption of lysine methyltransferase activity associated with LIG1K126me2 and H3K9me2. Collectively, these studies identify UHRF1 as a selective reader of LIG1K126me2 in vitro and further implicate the histone and non-histone methyllysine reader activity of the UHRF1 TTD as a dispensable domain function for cancer cell DNA methylation maintenance.

H3 K27M and EZHIP Impede H3K27-Methylation Spreading by Inhibiting Allosterically Stimulated PRC2

Diffuse midline gliomas and posterior fossa type A ependymomas contain the recurrent histone H3 lysine 27 (H3 K27M) mutation and express the H3 K27M-mimic EZHIP (CXorf67), respectively. H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. Invivo, these proteins reduce overall H3 lysine 27 trimethylation (H3K27me3) levels; however, residual peaks of H3K27me3 remain at CpG islands (CGIs) through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with PRC2 that is allosterically activated by H3K27me3 at CGIs and impede its spreading. Moreover, H3 K27M oncohistones reduce H3K27me3 in trans, independent of their incorporation into the chromatin. Although EZHIP is not found outside placental mammals, expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved mechanism. Our results provide mechanistic insights for the retention of residual H3K27me3 in tumors driven by H3 K27M and EZHIP.

ASH2L-Promoted HOXC8 Gene Expression Plays a Role in Mixed Lineage Leukemia-Rearranged Acute Leukemia.

BackgroundMixed lineage leukemia (MLL) fusion protein alone exhibits poor histone lysine methyltransferase (HKMT) activity in catalyzing histone H3 Lys4 trimethylation (H3K4me3) in MLL-rearranged acute leukemia.MethodsTo explore the HKMT effect of another regulatory protein within the complex of proteins associated with Set 1 (COMPASS), we analyzed the H3K4me3 modification of the HOXC8 promoter under the action of ASH2L regulation. Small interfering RNA of ASH2L, chromatin immunoprecipitation, real-time-PCR (RT-PCR), and Western blotting were used to detect the expression of specific regions of the HOXC8 promoter, RBBP5, WDR5, MLL, and BRTF in two MLL-rearranged acute leukemia cell lines (RS4:11 and THP-1 cells).ResultsThe gene and protein expression levels of HOXC8 were significantly downregulated upon treatment with ASH2L-siRNA (as analyzed by targeting specific regions of the HOXC8 promoter located 0 and 3 kb (–3.0 kb) upstream of the transcriptional start site in RSH:11 cells; and –3.0 and –2.0 kb upstream of the transcriptional start site, and +1.4 kb downstream of the transcriptional start site in THP-1 cells). The expression levels of the BRTF, RBBP5, WDR5, and MLL genes were significantly downregulated from the different transcriptional start sites of the HOXC8 promoter in the RSH:11 cell line (P < 0.05). Furthermore, the BPTF and RBBP5 genes were downregulated from the HOXC8 promoter in the THP-1 cell line (P < 0.05).ConclusionBased on these results, we suggest a new concept of histone modification of the ASH2L protein in MLL-rearranged acute leukemia, which cannot carry out methyltransferase activity independently. The protein–protein interactions of ASH2L with other COMPASS members, such as MLL, WDR5, RBBP5, and chromatin remodeling factor BRTF, appear to be essential for its role in the activation of HOXC8 gene transcription.

Genetic mutational status of genes regulating epigenetics: Role of the histone methyltransferase KMT2D in triple negative breast tumors.

PurposeEpigenetic regulating proteins like histone methyltransferases produce variations in several functions, some of them associated with the generation of oncogenic processes. Mutations of genes involved in these functions have been recently associated with cancer, and strategies to modulate their activity are currently in clinical development.MethodsBy using data extracted from the METABRIC study, we searched for mutated genes linked with detrimental outcome in invasive breast carcinoma (n = 772). Then, we used downstream signatures for each mutated gene to associate that signature with clinical prognosis using the online tool “Genotype-2-Outcome” (http://www.g-2-o.com). Next, we performed functional annotation analyses to classify genes by functions, and focused on those associated with the epigenetic machinery.ResultsWe identified KMT2D, SETD1A and SETD2, included in the lysine methyltransferase activity function, as linked with poor prognosis in invasive breast cancer. KMT2D which codes for a histone methyltransferase that acts as a transcriptional regulator was mutated in 6% of triple negative breast tumors and found to be linked to poor survival. Genes regulated by KMT2D included RAC3, KRT23, or KRT14, among others, which are involved in cell communication and signal transduction. Finally, low expression of KMT2D at the transcriptomic level, which mirror what happens when KMT2D is mutated and functionally inactive, confirmed its prognostic value.ConclusionIn the present work, we describe epigenetic modulating genes which are found to be mutated in breast cancer. We identify the histone methyltransferase KMT2D, which is mutated in 6% of triple negative tumors and linked with poor survival.

Effect of EZH2 knockdown on preimplantation development of porcine parthenogenetic embryos

The EZH2 protein endows the polycomb repressive complex 2 (PRC2) with histone lysine methyltransferase activity that is associated with transcriptional repression. Recent investigations have documented crucial roles for EZH2 in mediating X-inactivation, stem cell pluripotency and cancer metastasis. However, there is little evidence demonstrating the maternal effect of EZH2 on porcine preimplantation development. Here, we took parthenogenetic activation embryos to eliminate the confounding paternal influence. We showed that the dynamic expression of EZH2 during early development was accompanied by changes in H3K27me3 levels. Depletion of EZH2 in MII oocytes by small interfering RNA not only impaired embryonic development at the blastocyst stage (P < 0.05), but also disrupted the equilibrium of H3K4me3 and H3K27me3 in the embryo. Interestingly, the expression of TET1, a member of Ten-Eleven Translocation gene family for converting 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5hmC), was decreased after EZH2 knockdown, in contrast to the increase of the other two members, TET2 and TET3 (P < 0.05). These results indicate a correlation between histone methylation and DNA methylation, and between EZH2 and TET1. Along with the downregulation of TET1, the expression of the pluripotency gene NANOG was decreased (P < 0.05), which is consistent with a previous finding in mouse ES cells. Meanwhile, the abundance of OCT4 and SOX2 were also down-regulated. Moreover, EZH2 knockdown reduced the capacity of cells in the blastocysts to resist apoptosis. Taken together, our data suggest that EZH2 is integral to the developmental program of porcine parthenogenetic embryos and exerts its function by regulating pluripotency, differentiation and apoptosis.

Lysine methyltransferase SMYD2 promotes triple negative breast cancer progression

We identified SMYD2, a SMYD (SET and MYND domain) family protein with lysine methyltransferase activity, as a novel breast cancer oncogene. SMYD2 was expressed at significantly higher levels in breast cancer cell lines and in breast tumor tissues. Silencing of SMYD2 by RNAi in triple-negative breast cancer (TNBC) cell lines or inhibition of SMYD2 with its specific inhibitor, AZ505, significantly reduced tumor growth in vivo. SMYD2 executes this activity via methylation and activation of its novel non-histone substrates, including STAT3 and the p65 subunit of NF-κB, leading to increased TNBC cell proliferation and survival. There are cross-talk and synergistic effects among SMYD2, STAT3, and NF-κB in TNBC cells, in that STAT3 can contribute to the modification of NF-κB p65 subunit post-translationally by recruitment of SMYD2, whereas the p65 subunit of NF-κB can also contribute to the modification of STAT3 post-translationally by recruitment of SMYD2, leading to methylation and activation of STAT3 and p65 in these cells. The expression of SMYD2 can be upregulated by IL-6-STAT3 and TNFα-NF-κB signaling, which integrates epigenetic regulation to inflammation in TNBC development. In addition, we have identified a novel SMYD2 transcriptional target gene, PTPN13, which links SMYD2 to other known breast cancer associated signaling pathways, including ERK, mTOR, and Akt signaling via PTPN13 mediated phosphorylation.

Abstract IA19: Targeting the PRC2 complex through EED for anti-cancer therapy

Abstract The Polycomb Repressive Complex-2 (PRC2) plays important roles in regulating gene expression through its histone H3 lysine methyl transferase activity. Dysregulation of PRC2 is observed in multiple cancers. For example, gain of function mutations of the EZH2 catalytic subunit of PRC2 have been reported in B-cell lymphomas, melanoma and parathyroid carcinoma. Components of the complex are also overexpressed in a number of cancers. The PRC2-complex have therefore become a major target for anti-cancer drug discovery. We and others, have reported anti-tumor activity of small molecular weight agents targeting the SAM-binding pocket of EZH2 in preclinical models and a number of EZH2 inhibitors ore showing promising activity in clinical trials. EED and SUZ12 are two additional components of the PRC2 core complex required for regulation of its catalytic function. High resolution structures and other reports have showed that binding of EED to tri-methylated histone H3 Lysine 27 (H3K27Me3) further stimulates the catalytic effect of EZh2. This suggests that small molecular weight compounds binding to and interfering with the EED stimulatory effect on Ezh2 may be able to modulate PRC2 activity. Following this possibility, we launched a comprehensive effort to discover non-catalytic site PRC2 binders. The discovery and characterization of EED226, a highly potent and selective EED-binding inhibitor of PRC2 will be presented. Data will be presented to demonstrate that EED226 inhibits PRC2 function via a distinct allosteric mechanism by binding to the H3K27Me3 binding pocket of EED. Furthermore, EED226 potently inhibits cellular H3K27Me3 levels, modulates target gene expression similar to the SAM competitive Ezh2 inhibitors and inhibits the proliferation of cell lines carrying mutant Ezh2. Oral administration of EED226 to EZH2-mutant tumor bearing mice leads to profound tumor regression. Our data therefore demonstrates that the multi-subunit PRC2 complex can be targeted at non-catalytic binding pockets to achieve anti-tumor activity. Some unique features of the anti-tumor activity achieved through targeting PRC2 will be discussed. Citation Format: Peter W. Atadja. Targeting the PRC2 complex through EED for anti-cancer therapy [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA19.

Abstract 1420: The endonuclease Metnase promotes base excision repair of Clustered abasic DNA lesions

Abstract Metnase, a human SET-transposase fusion protein contains two functional domains: a SET domain and transposase domain. Transposase domain exhibit strand transfer and end joining activity while set domain is responsible for histone lysine methyltransferase activity (KMT). Metnase also increases the efficiency of double strand break repair by non-homologous end joining (NHEJ); however, its role in the excision of clustered DNA damage remains to be investigated. We hypothesize that 5’-3’ endonuclease activity of metnase could possibly substitute for APE1 in BER and serve as an alternative initiator of BER in tumor cells lacking APE1. In the present study, we examined in vitro endonuclease activity of Metnase using abasic site containing artificial DNA, and describe its activity in BER. Its unique 5’ endonuclease activity was selective only for abasic lesions, but not other base modifications such as 8-oxoguanine, uracil, hypoxanthine or xanthenes. Metnase specifically initiates removal of reduced abasic lesions from DNA, and allows completion of short-patch or long-patch BER. APE1 has difficulty cleaving 5’ of clustered abasic lesions. This endonuclease that promotes BER in clustered oxidative DNA damage is not known. Metnase also cleaves multiple abasic lesions (clustered DNA damage) and facilitates the repair of DNA if the multiple DNA damages are 3 or more nucleotides apart on the opposing strands. However, repair of multiple DNA damages remains inefficient if these are in close proximity of each other (&amp;lt; 3nt apart on the opposing strands) and results in DNA double strand break (DSB). These results suggest that Metnase can promote BER of oxidative nucleotides, where APE1 is unable to function. Citation Format: Aruna S. Jaiswal, Elizabeth A. Williamson, Bhavita Patel, Gayathri Srinivasan, Satya Narayan, Robert A. Hromas. The endonuclease Metnase promotes base excision repair of Clustered abasic DNA lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1420. doi:10.1158/1538-7445.AM2017-1420

Lysine methyltransferase SMYD2 promotes cyst growth in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is driven by mutations in PKD1 and PKD2 genes. Recent work suggests that epigenetic modulation of gene expression and protein function may play a role in ADPKD pathogenesis. In this study, we identified SMYD2, a SET and MYND domain protein with lysine methyltransferase activity, as a regulator of renal cyst growth. SMYD2 was upregulated in renal epithelial cells and tissues from Pkd1-knockout mice as well as in ADPKD patients. SMYD2 deficiency delayed renal cyst growth in postnatal kidneys from Pkd1 mutant mice. Pkd1 and Smyd2 double-knockout mice lived longer than Pkd1-knockout mice. Targeting SMYD2 with its specific inhibitor, AZ505, delayed cyst growth in both early- and later-stage Pkd1 conditional knockout mouse models. SMYD2 carried out its function via methylation and activation of STAT3 and the p65 subunit of NF-κB, leading to increased cystic renal epithelial cell proliferation and survival. We further identified two positive feedback loops that integrate epigenetic regulation and renal inflammation in cyst development: SMYD2/IL-6/STAT3/SMYD2 and SMYD2/TNF-α/NF-κB/SMYD2. These pathways provide mechanisms by which SMYD2 might be induced by cyst fluid IL-6 and TNF-α in ADPKD kidneys. The SMYD2 transcriptional target gene Ptpn13 also linked SMYD2 to other PKD-associated signaling pathways, including ERK, mTOR, and Akt signaling, via PTPN13-mediated phosphorylation.

PRC2 Mutations Induce Resistance to Conventional Chemotherapy By Inhibiting Mitochondrial Apoptosis in T-Cell Acute Lymphoblastic Leukemia

Although contemporary combination chemotherapy can cure a substantial fraction of patients with T-cell acute lymphoblastic leukemia (T-ALL), front-line therapy fails in 15-20% of children and 50-70% of adults, and these patients have a poor prognosis. Strikingly, half of treatment failure events in childhood T-ALL are induction failure, suggesting pre-existing resistance to chemotherapeutics with distinct molecular targets. The molecular basis for induction failure remains poorly understood. Recent work has shown that mitochondrial apoptosis resistance is a cellular phenotype that predicts chemotherapy failure in some tumor types. However, the molecular mechanisms responsible for the striking variability in chemotherapy response among different patients with seemingly identical tumors remain largely unknown.Using a technique known as BH3 profiling, we analyzed mitochondrial apoptosis sensitivity or resistance in pre-treatment clinical specimens from a cohort of 47 children and adolescents treated on the COG AALL0434 or DFCI 05001 clinical trials. We found that mitochondrial apoptosis resistance was strongly associated with a poor response to induction chemotherapy (P = 0.008), as well as inferior 5-year event-free survival (65% vs 88%; P = 0.036 by log-rank test). Apoptosis resistance was weakly associated with the early T-cell precursor (ETP) immunophenotype (P = 0.08), but univariate and multivariable Cox regression analysis including both revealed that apoptosis resistance predicts clinical outcome more strongly than ETP status.To identify molecular lesions underlying mitochondrial apoptosis resistance, we applied targeted exome sequencing and array CGH to this cohort. We found that loss-of-function mutations in genes encoding core components of the polycomb repressive complex 2 (PRC2), including EZH2, EED or SUZ12, are associated with resistance to mitochondrial apoptosis (P = 0.015). PRC2 is a chromatin-modifying complex best known for its role in transcriptional repression. The PRC2 complex has been implicated as a tumor suppressor in T-ALL, but whether PRC2 plays a direct role in chemotherapy response is unknown.To test whether PRC2 regulates mitochondrial apoptosis in human T-ALL, we performed shRNA knockdown of the PRC2 core components EZH2, EED or SUZ12 in human T-ALL cell lines. Knockdown of each of these genes significantly induced mitochondrial apoptosis resistance, as assessed by BH3-profiling. This effect was dependent on the lysine methyltransferase activity of the PRC2 complex, because the effect of EZH2 knock-down was rescued by expression of wild-type EZH2, but not a point mutant that is methyltransferase-defective (P < 0.001). PRC2 knockdown also induced significant resistance to apoptosis induction (assessed using caspase 3/7 activation or annexin V/PI staining) in response to various chemotherapeutics with distinct molecular targets, including vincristine, dexamethasone, asparaginase, methotrexate, mercaptopurine, nelarabine, cytarabine and etoposide.To test whether PRC2 regulates mitochondrial apoptosis during normal T-cell development, we took advantage of mice heterozygous for a floxed Ezh2 or Eed allele, and induced deletion of one allele in hematopoietic cells using Mx-Cre activation by pIpC. Controls were Ezh2 and Eed wild-type mice with Mx-Cre activation. BH3 profiling analysis revealed that loss of one Ezh2 or Eed allele is sufficient to induce apoptosis resistance in non-transformed double-negative thymocytes (P = 0.003 for Ezh2 and P = 0.008 for Eed), suggesting that chemotherapy resistance can develop prior to oncogenic transformation.To define the transcriptional consequences of PRC2 inhibition in T-ALL, we performed RNA sequencing of T-ALL cells infected with shRNAs targeting EZH2, EED or SUZ12 (2 independent hairpins for each gene), or two control shRNAs. RNA sequencing analysis revealed a number of candidate transcriptional targets linking PRC2 to the mitochondrial apoptotic machinery, which are currently being investigated using functional genetics and small molecule inhibitors.Collectively, these data implicate polycomb repressive complex 2 function as a key determinant of chemotherapy response in childhood T-ALL. Defining the mechanism linking PRC2 to the mitochondria will provide a rational target for therapeutic intervention. DisclosuresTeachey:Novartis: Research Funding. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding.

The Human Mixed Lineage Leukemia 5 (MLL5), a Sequentially and Structurally Divergent SET Domain-Containing Protein with No Intrinsic Catalytic Activity.

Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any in vitro methyltransferase activity on full-length histones and histone H3 peptides. Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism.

Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability.

G9a is a lysine methyltransferase (KMTase) for histone H3 lysine 9 that plays critical roles in a number of biological processes. Emerging evidence suggests that aberrant expression of G9a contributes to tumor metastasis and maintenance of a malignant phenotype in cancer by inducing epigenetic silencing of tumor suppressor genes. Here, we show that G9a regulates Sox2 protein stability in breast cancer cells. When G9a lysine methyltransferase activity was chemically inhibited in the ER(+) breast cancer cell line MCF7, Sox2 protein levels were decreased. In addition, ectopic overexpression of G9a induced accumulation of Sox2. Changes in cell migration, invasion, and mammosphere formation by MCF7 cells were correlated with the activity or expression level of G9a. Ectopic expression of G9a also increased Sox2 protein levels in another ER(+) breast cancer cell line, ZR-75-1, whereas it did not affect Sox2 expression in MDA-MB-231 cells, an ER(-) breast cancer cell line, or in glioblastoma cell lines. Furthermore, treatment of mouse embryonic stem cells with a KMT inhibitor, BIX-01294, resulted in a rapid reduction in Sox2 protein expression despite increased Sox2 transcript levels. This finding suggests that G9a has a novel function in the regulation of Sox2 protein stability in a cell type-dependent manner.