Lysine Methyltransferase Research Articles

EHMT2 affects microglia polarization and aggravates neuronal damage and inflammatory response via regulating HMOX1.

This research was designed to ascertain the function of euchromatic histone lysine methyltransferase 2 (EHMT2) in ischemic stroke-induced neuronal damage and inflammatory response and its regulatory mechanism. Mouse microglia (BV-2 cells) were induced by oxygen glucose deprivation/reoxygenation (OGD/R) to establish a cellular model, and then co-cultured with HT22 hippocampal neurons. After that, HT22 cell viability and apoptosis were evaluated, followed by the measurement of apoptosis-related factors (B-cell lymphoma-2, Bcl-2 associated X, and cleaved-Caspase 3). Meanwhile, the expression of inducible nitric oxide synthase (M1 microglia polarization marker) and arginase 1 (M2 microglia polarization marker) in BV-2 cells was detected, as well as the levels of inflammatory factors (tumor necrosis factor-α, interleukin [IL]-6, IL-10, IL-1β, and IL-4). Additionally, the expression of EHMT2 and heme oxygenase 1 (HMOX1) in BV-2 cells was assessed by quantitative reverse transcription polymerase chain reaction and western blot, and the binding between EHMT2 and HMOX1 was predicted and verified. OGD/R treatment led to decreased cell viability and increased cell apoptosis in HT22 cells, and aggravated inflammatory response in BV-2 cells. In OGD/R-induced BV-2 cells, EHMT2 and HMOX1 were increasingly expressed, and knockdown of EHMT2 or HMOX1 in BV-2 cells could inhibit neuronal damage and inflammatory response. Moreover, EHMT2 promoted HMOX1 transcription level by histone methylation. Collected evidence showed that down-regulation of EHMT2 relieved neuronal damage and inflammatory response by inhibiting HMOX1 expression.

The Roles of Histone Lysine Methyltransferases in Heart Development and Disease.

Epigenetic marks regulate the transcriptomic landscape by facilitating the structural packing and unwinding of the genome, which is tightly folded inside the nucleus. Lysine-specific histone methylation is one such mark. It plays crucial roles during development, including in cell fate decisions, in tissue patterning, and in regulating cellular metabolic processes. It has also been associated with varying human developmental disorders. Heart disease has been linked to deregulated histone lysine methylation, and lysine-specific methyltransferases (KMTs) are overrepresented, i.e., more numerous than expected by chance, among the genes with variants associated with congenital heart disease. This review outlines the available evidence to support a role for individual KMTs in heart development and/or disease, including genetic associations in patients and supporting cell culture and animal model studies. It concludes with new advances in the field and new opportunities for treatment.

The cytoplasmic fraction of the histone lysine methyltransferase Setdb1 is essential for embryonic stem cells

The major lysine methyltransferase (KMT) Setdb1 is essential for self-renewal and viability of mouse embryonic stem cells (mESCs). Setdb1 was primarily known to methylate the lysine 9 of histone 3 (H3K9) in the nucleus, where it regulates chromatin functions. However, Setdb1 is also massively localized in the cytoplasm, including in mESCs, where its role remains elusive. Here, we show that the cytoplasmic Setdb1 (cSetdb1) is essential for the survival of mESCs. Yeast two-hybrid analysis revealed that cSetdb1 interacts with several regulators of mRNA stability and protein translation machinery, such as the ESCs-specific E3 ubiquitin ligase and mRNA silencer Trim71/Lin41. We found that cSetdb1 is required for the integrity of Trim71 complex(es) involved in mRNA metabolism and translation. cSetdb1 modulates the abundance of mRNAs and the rate of newly synthesized proteins. Altogether, our data uncovered the cytoplasmic post-transcriptional regulation of gene expression mediated by a key epigenetic regulator.

Mettl13 protects against cardiac contractile dysfunction by negatively regulating C-Cbl-mediated ubiquitination of SERCA2a in ischemic heart failure.

Ischemic heart failure (HF) remains a leading cause of morbidity and mortality. Maintaining homeostasis of cardiac function and preventing cardiac remodeling deterioration are critical to halting HF progression. Methyltransferase-like protein 13 (Mettl13) has been shown to regulate protein translation efficiency by acting as a protein lysine methyltransferase, but its role in cardiac pathology remains unexplored. This study aims to characterize the roles and mechanisms of Mettl13 in cardiac contractile function and HF. We found that Mettl13 was downregulated in the failing hearts of mice post-myocardial infarction (MI) and in a cellular model of oxidative stress. Cardiomyocyte-specific overexpression of Mettl13 mediated by AAV9-Mettl13 attenuated cardiac contractile dysfunction and fibrosis in response to MI, while silencing of Mettl13 impaired cardiac function in normal mice. Moreover, Mettl13 overexpression abrogated the reduction in cell shortening, Ca2+ transient amplitude and SERCA2a protein levels in the cardiomyocytes of adult mice with MI. Conversely, knockdown of Mettl13 impaired the contractility of cardiomyocytes, and decreased Ca2+ transient amplitude and SERCA2a protein expression in vivo and in vitro. Mechanistically, Mettl13 impaired the stability of c-Cbl by inducing lysine methylation of c-Cbl, which in turn inhibited ubiquitination-dependent degradation of SERCA2a. Furthermore, the inhibitory effects of knocking down Mettl13 on SERCA2a protein expression and Ca2+ transients were partially rescued by silencing c-Cbl in H2O2-treated cardiomyocytes. In conclusion, our study uncovers a novel mechanism that involves the Mettl13/c-Cbl/SERCA2a axis in regulating cardiac contractile function and remodeling, and identifies Mettl13 as a novel therapeutic target for ischemic HF.

Regulation of Keap1-Nrf2 axis in temporal lobe epilepsy-hippocampal sclerosis patients may limit the seizure outcomes.

Accumulation of reactive oxygen species (ROS) exacerbates neuronal loss during seizure-induced excitotoxicity. Keap1 (Kelch-like ECH-associated protein1)-nuclear factor erythroid 2-related factor 2 (Nrf2) axis is one of the known active antioxidant response mechanisms. Our study focused on finding the factors influencing Keap1-Nrf2 axis regulation in temporal lobe epilepsy (TLE) associated with hippocampal sclerosis (HS) patients. Based on post-surgical follow-up data, patient samples (n = 26) were categorized into class 1 (completely seizure-free) and class 2 (only focal-aware seizures/auras), as suggested by International League Against Epilepsy (ILAE). For molecular analyses, double immunofluorescence assay and Western blot analysis were employed. A significant decrease in expression of Nrf2 (p < 0.005), HO-1; p < 0.02) and NADPH Quinone oxidoreductase1 (NQO1; p < 0.02) was observed in ILAE class 2. Keap1 (p < 0.02) and histone methyltransferases (HMTs) like SetD7 (SET7/9; SET domain-containing 7 histone lysine methyltransferase) (p < 0.009) and enhancer of zeste homolog 2 (EZH2; p < 0.02) and methylated histones viz., H3K4me1 (p < 0.001), H3K9me3 (p < 0.001), and H3K27me3 (p < 0.001) was upregulated in ILAE class 2. Nrf2-interacting proteins viz., p21 (p < 0.001) and heat shock protein 90 (HSP90; p < 0.03) increased in class 1 compared to class 2 patients. Upregulation of HMTs and methylated histones can limit phase II antioxidant enzyme expression. Also, HSP90 and p21 that interfere with Keap1-Nrf2 interaction could contribute to a marginal increase in HO-1 and NQO1 expression despite histone methylation and Keap1. Based on our findings, we conclude that TLE-HS patients prone to seizure recurrence were found to have dysfunctional antioxidant response, in part, owing to Keap1-Nrf2 axis. The significance of Keap1-Nrf2 signaling mechanism in generation of phase II antioxidant response. Keap1-Nrf2 controls antioxidant response through regulation of phase II antioxidant enzymes like HO-1 (heme oxygenase-1), NQO1 (NADPH-Quinone Oxidoreductase1), and glutathione S-transferase (GST). Release of Nrf2 from negative regulation by Keap1 causes its translocation into nucleus, forming a complex with cAMP response-element binding protein (CBP) and small Maf proteins (sMaf). This complex subsequently binds antioxidant response element (ARE) and elicits and antioxidant response involving expression of phase II antioxidant enzymes. Reactive oxygen species (ROS) modify Cysteine 151 residue, p62 (sequsetosome-1), and interacts with Nrf2- binding site in Keap 1. p21 and HSP90 prevent Nrf2 interaction with Keap1. At transcriptional level, histone methyltransferases like EZH2 (enhancer of zeste homologue2), and SetD7 (SET7/9; SET domain-containing 7 histone lysine methyltransferase) and corresponding histone targets viz., H3K27me3, H3K9me3, and H3K4me1 influence Nrf2 and Keap1 expression respectively.

The epigenetic modifier lysine methyltransferase 2C is frequently mutated in gastric remnant carcinoma.

Gastric remnant carcinoma (GRC), which occurs in the stomach after partial gastrectomy, is a rare and aggressive form of gastric adenocarcinoma (GAC). Comprehensive profiling of genomic mutations in GRC could provide the basis for elucidating the origin and characteristics of this cancer. Herein, whole-exome sequencing (WES) was performed on 36 matched tumor-normal samples from patients with GRC and identified recurrent mutations in epigenetic modifiers, notably KMT2C, ARID1A, NSD1, and KMT2D, in 61.11% of cases. Mutational signature analysis revealed a low frequency of microsatellite instability (MSI) in GRC, which was further identified by MSIsensor, MSI-polymerase chain reaction, and immunohistochemistry analysis. Comparative analysis demonstrated that GRC had a distinct mutation spectrum compared to that of GAC in The Cancer Genome Atlas samples, with a significantly higher mutation rate of KMT2C. Targeted deep sequencing (Target-seq) of an additional 25 paired tumor-normal samples verified the high mutation frequency (48%) of KMT2C in GRC. KMT2C mutations correlated with poor overall survival in both WES and Target-seq cohorts and were independent prognosticators in GRC. In addition, KMT2C mutations were positively correlated with favorable outcomes in immune checkpoint inhibitor-treated pan-cancer patients and associated with higher intratumoral CD3+ , CD8+ tumor-infiltrating lymphocyte counts, and PD-L1expression in GRC samples (p = 0.018, 0.092, 0.047, 0.010, and 0.034, respectively). Our dataset provides a platform for information and knowledge mining of the genomic characteristics of GRC and helps to frame new therapeutic approaches for this disease.

Establishment of a t(11;19), KMT2A Rearranged B-ALL Cell Line for Preclinical Evaluation and Novel Therapeutics Development for Refractory Infant Leukemia.

Leukemia, diagnosed in children less than 12 months of age, is a rare condition with an aggressive disease presentation and poor response to conventional chemotherapeutic agents. In addition, the unique vulnerability of the affected population does not always permit the use of markedly intense regimens with higher doses of cytotoxic agents. However, the unique biology of these leukemic cells also provides opportunities for the identification of effective and potentially well-tolerated targeted therapeutic strategies. In this report, we describe the establishment and characterization of a cell line from the blasts of an infant diagnosed with refractory B-cell acute lymphoblastic leukemia (ALL) carrying the characteristic histone lysine methyltransferase 2A (KMT2A) gene rearrangement. This cell line consists of rapidly proliferating clones of cells with chemosensitivity patterns previously described for KMT2A rearranged leukemia cells, including relative resistance to glucocorticoids and sensitivity to cytarabine. We also show effective targetability with menin inhibitors, indicating the activity of abnormal KMT2A-related pathways and the potential utility of this cell line in comprehensive drug library screens. Overall, our findings report the establishment and in vitro validation of a cell line for research into key aspects of infant leukemia biology and targeted therapeutics development.

QSAR study of benzofuran and indole derivatives to predict new compounds as histone lysine methyl transferase inhibitors

Initiation and progression of several diseases by post-translational histone modifications are considered a worldwide problem. Enhancer of Zeste Homologue 2 (EZH2), which belongs to the histone-lysine N-methyl transferase (HKMT) family, has been emphasised as a promising target for cancer therapy. It is a major challenge for the scientific community to find novel approaches to treating this disease. In this study, a series of 51 derivatives of the benzofuran and indole families, previously experimentally evaluated against HKMT, was used to develop the best model with promising anticancer activity. The multiple linear regression (MLR) method, implemented in QSARINS software, was used with a genetic algorithm for variable selection. According to QSARINS, the model with two descriptors (minHBint4 and Wlambdal.unity) was found to be the best and its parameters fit well, and its validation was well established. The applicability domain was also validated for this model. Furthermore, its robustness (R2 = 0.9328), stability (Q2LOO = 0.9212, Q2LMO = 0.9187), and good predictive power (R2ext = 0.929) were also verified. Hence, this model was assumed to have predictive HKMT anticancer activity for designing active compounds. Molecular docking was also performed to identify binding interactions, and new molecules with better predicted biological activity (pIC50) were designed. The binding energy of the three designed compounds demonstrated higher binding activity at the target receptor, followed by complex stability, determined by a 100 ns molecular dynamics simulation and binding free energy calculation. Density functional theory (DFT) and pharmacokinetic analyses also confirmed their drug-like properties. Finally, it can be declared that the proposed tools allow rapid and economical identification of potential anti-HKMT drugs (anticancer drugs) for further development.

SMYD3 drives the proliferation in gastric cancer cells via reducing EMP1 expression in an H4K20me3-dependent manner

Protein lysine methyltransferase SET and MYND domain-containing 3 (SMYD3) is aberrantly expressed in various cancer settings. The mechanisms that SMYD3 activates the expression of critical pro-tumoral genes in an H3K4me3-dependent manner have been well described in previous reports. Besides H3K4me3, H4K20me3 is another catalytic product of SMYD3, however it is a transcriptionally repressive hallmark. Since it is not clear that how SMYD3-elicited transcriptionally repressive program functions in cancer, we used gastric cancer (GC) as a model to investigate the roles of SMYD3-H4K20me3. Herein, online bioinformatics tools, quantitative PCR, western blotting and immunohistochemistry assays demonstrated that SMYD3 expression was markedly increased in GC tissues from our institutional and The Cancer Genome Atlas (TCGA) cohort. Additionally, aberrantly increased SMYD3 expression was closely associated with aggressive clinical characteristics and poor prognosis. Depletion of endogenous SMYD3 expression using shRNAs significantly attenuates the proliferation in GC cells and Akt signaling pathway in vitro and in vivo. Mechanistically, chromatin immunoprecipitation (ChIP) assay showed that SMYD3 epigenetically repressed the expression of epithelial membrane protein 1 (EMP1) in an H4K20me3-dependent manner. Gain-of-function and rescue experiments validated that EMP1 inhibited the propagation of GC cells and reduced p-Akt (S473) level. Based on these data, pharmaceutical inhibition of SMYD3 activity using the small inhibitor BCI-121 deactivated Akt signaling pathway in GC cells and further impaired the cellular viability in vitro and in vivo. Together, these results demonstrate that SMYD3 promotes the proliferation in GC cells and may be a valid target for therapeutic intervention of patients with GC.

Identification of a KDM6A somatic mutation responsible for Kabuki syndrome by excluding a conflicting KMT2D germline variant through episignature analysis

Kabuki syndrome (KS) is a congenital disorder caused by mutations in either KMT2D on chromosome 12 or KDM6A on chromosome X, encoding a lysine methyltransferase and a lysine demethylase, respectively. A 9-year-4-month-old male patient with a normal karyotype presented with KS and autism spectrum disorder. Genetic testing for KS was conducted by Sanger sequencing and episignature analysis using DNA methylation array data. The patient had a mosaic stop-gain variant in KDM6A and a heterozygous missense variant (rs201078160) in KMT2D. The KDM6A variant is expected to be deleterious. The KMT2D variant pathogenicity has been inconsistently reported in the ClinVar database. Using biobanking resources, we identified two heterozygous individuals possessing the rs201078160 variant. In a subsequent episignature analysis, the KS patient showed the KS episignature, but two control individuals with the rs201078160 variant did not. Our results indicate that the mosaic stop-gained variant in KDM6A, but not the rs201078160 variant in KMT2D, is responsible for the KS phenotype in the patient. This study further demonstrated the utility of DNA methylation information in diagnosing rare genetic diseases and emphasized the importance of a reference dataset containing both genotype and DNA methylation information.

Structural basis for recognition and methylation of p97 by METTL21D, a valosin-containing protein lysine methyltransferase

p97 is a human AAA+ (ATPase associated with diverse cellular activities, also known as valosin-containing protein [VCP]) ATPase, which is involved in diverse cellular processes such as membrane fusion and proteolysis. Lysine-specific methyltransferase of p97 (METTL21D) was identified as a class I methyltransferase that catalyzes the trimethylation of Lys315 of p97, a so-called VCP lysine methyltransferase (VCPKMT). Interestingly, VCPKMT disassembles a single hexamer ring consisting of p97-D1 domain and methylates Lys315 residue. Herein, the structures of S-adenosyl-L-methionine-bound VCPKMT and S-adenosyl-L-homocysteine-bound VCPKMT in complex with p97 N/D1 (N21-Q458) were reported at a resolution of 1.8Å and 2.8Å, respectively. The structures revealed the molecular details for the recognition and methylation of monomeric p97 by VCPKMT. Using biochemical analysis, we also investigated whether the methylation of full-length p97 could be sufficiently enhanced through cooperation between VCPKMT and the C terminus of alveolar soft part sarcoma locus (ASPL). Our study provides the groundwork for future structural and mechanistic studies of p97 and inhibitors.

Methyltransferase like 21C interaction with Hsc70 affects chicken myoblast differentiation

Skeletal muscle has an essential role in boiler production, which depends on the muscle fibre development during the embryonic stage. Whereas the posttranslational modifications of both histone and non-histone proteins are known to regulate postnatal muscle development, the biological significance of non-histone methylation is poorly understood. Recently, the methyltransferase like 21 C (METTL21C) is identified as a non-histone lysine methyltransferase whose role in chicken muscle function remains elusive. In this study, METTL21C was found by comparing free-range and caged modes in Lueyang black-boned chicken aged 60 and 120 days. Quantitative real-time PCR (qPCR) analysis demonstrated that METTL21C was mainly enriched in chicken soleus (SOL) and gastrocnemius muscle (GA) (p < .05). Meanwhile, METTL21C had a rapid increase during chicken myoblast differentiation. After METTL21C overexpression, myogenic differentiation (MyoD) and myogenin (MyoG) expression were significantly increased, indicating that METTL21C promotes chicken myoblast differentiation. Using co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometry (LC-MS/MS) analysis, heat shock cognate 71 kDa protein (Hsc70) was confirmed to interact with METTL21C, while the effects of Hsc70 Lys-561 (Hsc70 K561) mutation suggested that Hsc70 was a METTL21C target protein. These results identify the molecular mechanism of METTL21C interaction with Hsc70 associated with chicken myoblast differentiation in the postnatal period. Highlights The mRNA of methyltransferase like 21C (METTL21C) was significantly increased in free-rang mode and during the chicken myoblast differentiation. Overexpression of METTL21C promoted the primary chicken myoblasts proliferation and differentiation. METTL21C could mediate the lysine methylation modification of heat shock cognate 71 kDa protein (Hsc70) to regulate the proliferation of chicken myoblasts.

Lysine Methyltransferase SMYD1 Regulates Myogenesis via skNAC Methylation.

The SMYD family is a unique class of lysine methyltransferases (KMTases) whose catalytic SET domain is split by a MYND domain. Among these, Smyd1 was identified as a heart- and skeletal muscle-specific KMTase and is essential for cardiogenesis and skeletal muscle development. SMYD1 has been characterized as a histone methyltransferase (HMTase). Here we demonstrated that SMYD1 methylates is the Skeletal muscle-specific splice variant of the Nascent polypeptide-Associated Complex (skNAC) transcription factor. SMYD1-mediated methylation of skNAC targets K1975 within the carboxy-terminus region of skNAC. Catalysis requires physical interaction of SMYD1 and skNAC via the conserved MYND domain of SMYD1 and the PXLXP motif of skNAC. Our data indicated that skNAC methylation is required for the direct transcriptional activation of myoglobin (Mb), a heart- and skeletal muscle-specific hemoprotein that facilitates oxygen transport. Our study revealed that the skNAC, as a methylation target of SMYD1, illuminates the molecular mechanism by which SMYD1 cooperates with skNAC to regulate transcriptional activation of genes crucial for muscle functions and implicates the MYND domain of the SMYD-family KMTases as an adaptor to target substrates for methylation.

KMT2A is targeted by miR-361-3p and modulates leukemia cell’s abilities to proliferate, migrate and invade

ABSTRACTObjective:The lives and safety of humans are significantly threatened by acute myeloid leukemia (AML), which is proven to be the most prevalent acute leukemia. This work is therefore intended to investigate and analyze the expressions of miR-361-3p and Histone Lysine Methyltransferase 2A (KMT2A) in tissues and cell lines of AML and identify an advanced and novel target for the therapy of AML.Methods:The qRT-PCR and western blot assays were conducted to find expressions of miR-361-3p/KMT2A in AML PB and cell lines. After then, tests using CCK-8 and EdU were run to see how KMT2A affected the growth of AML cells. Transwell migration and invasion assay was conducted to evaluate KMT2A's contribution to the migration and invasion of AML cells. ENCORI and miRWalk predicted the association between KMT2A and miR-361-3p, and the dual-luciferase reporter experiment verified it. Furthermore, rescue studies were used to ascertain how KMT2A affected the miR-361-3p-regulated AML cells’ abilities to proliferate, migrate, and invade.Results:miR-361-3p was poorly expressed while KMT2A was abundantly expressed. Additionally, KMT2A downregulation prevented AML cells from proliferating. PCNA and Ki-67 protein levels fell when KMT2A was silent. Furthermore, AML cells’ motility, invasion, and metastasis were inhibited by low KMT2A expression. KMT2A was also identified as a direct target of miR-361-3p and negatively correlated with miR-361-3p. Finally, the over-expression of KMT2A partially reversed the inhibitory effects of up-regulation of miR-361-3p.Conclusion:A potential therapeutic candidate target for the treatment of AML may be miR-361-3p/KMT2A.

The Distinctive Features behind the Aggressiveness of Oral and Cutaneous Squamous Cell Carcinomas

Squamous cell carcinomas arise from stratified squamous epithelia. Here, a comparative analysis based on recent studies defining the genetic alterations and composition of the stroma of oral and cutaneous squamous cell carcinomas (OSCC and CSCC, respectively) was performed. Both carcinomas share some but not all histological and genetic features. This review was focused on how mutations in tumor suppressor genes and protooncogenes cooperate to determine the differentiation, aggressiveness, and metastatic potential of OSCC and CSCC. In fact, driver mutations in tumor suppressor genes are more frequently observed in OSCC than CSCC. These include mutations in TP53 (encoding pP53 protein), CDKN2A (encoding cyclin dependent kinase inhibitor 2A), FAT1 (encoding FAT atypical cadherin 1), and KMT2D (encoding lysine methyltransferase 2D), with the exception of NOTCH (encoding Notch receptor 1), whose mutation frequency is lower in OSCC compared to CSCC. Finally, we describe the differential composition of the tumor microenvironment and how this influences the aggressiveness of each tumor type. Although both OSCC and CSCC tumors are highly infiltrated by immune cells, high levels of tumor-infiltrating lymphocytes (TILs) have been more frequently reported as predictors of better outcomes in OSCC than CSCC. In conclusion, OSCC and CSCC partially share genetic alterations and possess different causal factors triggering their development. The tumor microenvironment plays a key role determining the outcome of the disease.

The Histone Methyltransferase SETDB2 Modulates Tissue Inhibitors of Metalloproteinase-Matrix Metalloproteinase Activity During Abdominal Aortic Aneurysm Development.

To determine macrophage-specific alterations in epigenetic enzyme function contributing to the development of abdominal aortic aneurysms (AAAs). AAA is a life-threatening disease, characterized by pathologic vascular remodeling driven by an imbalance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Identifying mechanisms regulating macrophage-mediated extracellular matrix degradation is of critical importance to developing novel therapies. The role of SET Domain Bifurcated Histone Lysine Methyltransferase 2 (SETDB2) in AAA formation was examined in human aortic tissue samples by single-cell RNA sequencing and in a myeloid-specific SETDB2 deficient murine model induced by challenging mice with a combination of a high-fat diet and angiotensin II. Single-cell RNA sequencing of human AAA tissues identified SETDB2 was upregulated in aortic monocyte/macrophages and murine AAA models compared with controls. Mechanistically, interferon-β regulates SETDB2 expression through Janus kinase/signal transducer and activator of transcription signaling, which trimethylates histone 3 lysine 9 on the TIMP1-3 gene promoters thereby suppressing TIMP1-3 transcription and leading to unregulated matrix metalloproteinase activity. Macrophage-specific knockout of SETDB2 ( Setdb2f/fLyz2Cre+ ) protected mice from AAA formation with suppression of vascular inflammation, macrophage infiltration, and elastin fragmentation. Genetic depletion of SETDB2 prevented AAA development due to the removal of the repressive histone 3 lysine 9 trimethylation mark on the TIMP1-3 gene promoter resulting in increased TIMP expression, decreased protease activity, and preserved aortic architecture. Lastly, inhibition of the Janus kinase/signal transducer and activator of the transcription pathway with an FDA-approved inhibitor, Tofacitinib, limited SETDB2 expression in aortic macrophages. These findings identify SETDB2 as a critical regulator of macrophage-mediated protease activity in AAAs and identify SETDB2 as a mechanistic target for the management of AAAs.

Decreased bone resorption in Ezh2 myeloid cell conditional knockout mouse model.

Osteoclasts derived from hematopoietic stem cells control bone resorption. Identifying novel molecules that can epigenetically regulate osteoclastogenesis is important for developing novel treatments for osteoporosis and other disorders associated with bone deterioration and promoting healthy bone formation. The polycomb group (PcG) protein enhancer of zeste homolog 2 (Ezh2), a histone lysine methyltransferase, is associated with epigenetic regulation of numerous cellular processes, but its involvement in bone cell development and homeostasis is not yet clear. Here, LysM-Cre mice were crossed with Ezh2flox/flox mice to delete Ezh2 in myeloid cell lineage mature macrophages. Conditional knockout of Ezh2 (CKO) in myeloid cell line resulted in significant increases in postnatal bone growth in the first 6 months of life for both male and female mice. For female mice, optimal bone mass was seen for mice with Ezh2 deleted in both chromosomes in a pair (f/f Cre+ ; CKO). For male mice, optimal bone mass was found after deletion of Ezh2 from just one chromosome (f/- Cre+ ) with no difference in bone phenotype between f/- Cre+ and CKO male mice. In addition to the gender-specific difference in bone phenotype, Ezh2 CKO mice had significantly less macrophages (CD11b+) present in the bone marrow compared with control mice as well as significantly more mature osteoblasts and bone formation biomarkers present (P1NP, osteocalcin). Inflammatory array for protein lysed from bone tissue revealed deletion of Ezh2 decreased inflammatory milieu in both male and female mice compared with controls. Unexpectedly, myeloid cell deletion of Ezh2 also increased the number of mature osteoblast present in the bone. Deletion of Ezh2 also led to an increase in gene expression of osteoclast-suppressive genes IRF8, MafB, and Arg1 due to a decrease in the presence of the suppressive H3K27me3 epigenetic mark. These findings suggest that manipulation of Ezh2 expression may be a viable strategy to combat bone resorptive disorders such as osteoporosis or arthritis.

Structure-Based Design and Characterization of the Highly Potent and Selective Covalent Inhibitors Targeting the Lysine Methyltransferases G9a/GLP.

Protein lysine methyltransferases G9a and GLP, which catalyze mono- and di-methylation of histone H3K9 and nonhistone proteins, play important roles in diverse cellular processes. Overexpression or dysregulation of G9a and GLP has been identified in various types of cancer. Here, we report the discovery of a highly potent and selective covalent inhibitor 27 of G9a/GLP via the structure-based drug design approach following structure-activity relationship exploration and cellular potency optimization. Mass spectrometry assays and washout experiments confirmed its covalent inhibition mechanism. Compound 27 displayed improved potency in inhibiting the proliferation and colony formation of PANC-1 and MDA-MB-231 cell lines and exhibited enhanced potency in reducing the levels of H3K9me2 in cells compared to noncovalent inhibitor 26. In vivo, 27 showed significant antitumor efficacy in the PANC-1 xenograft model with good safety. These results clearly indicate that 27 is a highly potent and selective covalent inhibitor of G9a/GLP.

Genomic characterization of MSI-H and TMB in gastrointestinal cancer.

e15524 Background: Microsatellite instability (MSI) is a molecular marker of a deficient in mismatch repair (MMR) system. It is one of the main hallmarks in colorectal cancer (CRC) and gastric cancer (GC), occurring in 15% and 18% of CRC and GC respectively. Mutations occur in MMR system genes will impair DNA mismatch recognition and cause accumulation of mutations in DNA microsatellite regions. As a result, microsatellite instability-high (MSI-H) patients often possess an abnormally high number of somatic mutations and would lead to high tumour mutation burden (TMB), which is a prevalent biomarker in cancer immunotherapy. Here we analysed the genomic landscape of 974 gastrointestinal cancer patients to provide new insights on the relationship between MSI, TMB and other genetic mutation characteristics. Methods: Panel sequencing targeting 680 cancer-related genes were performed on patients with colonrectal cancer (n = 569), gastric cancer (n = 375), intestinal cancer (n = 25) and oesophagal cancer (n = 5) on either fresh tumour tissue or formalin-fixed paraffin embedded tumor DNA. Genomic alterations including single nucleotide variations, short insertions and deletions, copy number variations, tumour mutation burden and micro-satellite instability were analysed. Results: Among the 974 patients, 86 patients (8.83%) were classified as MSI-H based on evaluation of 117 MMR-related loci, while rest of the patients (n = 888, 91.2%) were classified as microsatelite stable (MSS). In MSI-H cohort, 68.6% of the patients were classified as TMB-H with a TMB level higher than 10 Muts/Mb, while in MSS cohort, only 3.71% of the patients were classified as TMB-H. The TMB value of MSI-H cohort is also significantly higher than the MSS cohort (p &lt; 0.0001, two-tailed T-test). Meanwhile, we discovered that proportion of patients with mutated lysine methyltransferase family gene KMT2D (54.65% vs 7.17%, p &lt; 0.0001), KMT2B (48.84% vs 3.25%, p &lt; 0.0001) and KMT2C (44.19% vs 7.17%, p &lt; 0.0001) showed significant difference between MSI-H and MSS cohorts (Two-sided Fisher’s exact test). Conclusions: This study demonstrated that MSI-H is highly correlated with TMB-H, providing a new insight of evaluating gastrointestinal cancer treatment on MSI-H patients. The study also revealed novel MSI-H genetic mutation landscape for KMT gene family. Concurrent with recent researches, this study further boosts the potential of KMT genes to be used as a biomarker in both diagnosis and immunotherapy treatment.

Targeting intracellular proteins with cell type-specific functions for cancer immunotherapy

Abstract Immune checkpoint inhibitors (ICIs) use antibodies that block cell surface immune checkpoint proteins with great efficacy in treating immunogenic or “immune hot” tumors such as melanoma, kidney, and lung adenocarcinoma. ICIs have limited response rates to other non-immunogenic cancers. The tumor microenvironment (TME) consists of many cell types that collectively promote tumor progression. Cancer therapeutics are commonly designed to target one molecule in one defined cell type. There is growing evidence that long-term therapeutic responses require the targeting of cancer cells and tumor-promoting populations within the TME. The question remains whether we can identify targetable molecules/pathways that are critical for multiple cell types. Here, we will discuss several molecular targets that may fit a “two or multiple birds, one stone” model, including the B-cell lymphoma-2 (BCL-2) family pro-survival factors, transcriptional factors including signal transducer and activator of transcription 3, the nuclear receptor 4A family (NR4A1, NR4A2, and NR4A3), as well as epigenetic regulators such as bromodomain and extra-terminal (BET) family proteins, histone deacetylase family, SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), and lysine-specific demethylase 1 (LSD1/KDM1A). We will focus on the rationale of these targets in immune modulation, as well as the strategies for targeting these important proteins for cancer therapy.