Histone Methyltransferase Research Articles

Prospects for breast cancer immunotherapy using microRNAs and transposable elements as objects.

One of the directions in treatment of chemoresistant breast cancer (BC) may include new methods of activating the immune response against tumor cells. Clinically used checkpoint inhibition using antibodies to PD-1 and PD-L1 works in some patients, but the lack of biomarkers means number of respondents is low. The possibility of combining this method with chemotherapy is limited by an increased risk of toxic liver damage, development of immune-related pneumonitis, and thyroid dysfunction. This article includes introduction into the clinic of new methods of immunotherapy for BC, among which epigenetic activation of retroelements, double-stranded transcripts of which stimulate the interferon response against the tumor, is promising. For this purpose, inhibitors of DNA methyltransferase*, histone deacetylase* and histone methyltransferase* are used (* subtitles in the main text). Their antitumor effect is also mediated by removal of repressive epigenetic marks from tumor suppressor genes. However, numerous studies have proven the role of retroelements in the carcinogenesis of various malignant neoplasms, including BC. Moreover, endogenous retroviruses HERV-K and LINE1 retrotransposons are planned to be used as diagnostic biomarkers for BC. Therefore, a rational approach to using viral mimicry in antitumor therapy of BC may be the simultaneous suppression of specific retrotransposons (drivers for carcinogenesis) using reverse transcriptase inhibitors and silencing of specific transposons involved in carcinogenesis using complementary microRNAs. To determine possible pathways of influence in this direction, 35 specific transposon-derived microRNAs* changes in BC were identified, which can become guides for targeted therapy of BC.

Knockdown of SMYD3 by RNA Interference Regulates the Expression of Autophagy-Related Proteins and Inhibits Bone Formation in Fluoride-Exposed Osteoblasts.

This study aimed to explore the role of histone methyltransferase SET and MYND domain containing 3 (SMYD3) in bone metabolism of osteoblasts exposed to fluoride. The levels of urine fluoride, BALP, and OC and the mRNA expression of SMYD3 were determined in patients with skeletal fluorosis and non-fluoride-exposed people on informed consent. The expression of SMYD3 protein, OC contents, and BALP activities were detected in human osteoblast-like MG63 cells and rat primary osteoblasts treated with sodium fluoride (NaF) for 48 h. The autophagosomes were observed by transmission electron microscopy. Then, we knocked down SMYD3 to confirm whether it was involved in the regulation of bone formation and related to autophagy and Wnt/β-catenin pathway. We observed that OC and BALP levels in patients with skeletal fluorosis significantly increased, while the mRNA expression of SMYD3 significantly decreased in the skeletal fluorosis groups. In vitro, the OC contents, BALP activities, and expression of SMYD3 significantly increased, and many autophagosomes were observed in NaF treated osteoblasts. The downregulation of SMYD3 significantly inhibited OC contents, BALP activities, and expression of autophagy-related proteins, but with no significant changes in the Wnt/β-catenin pathway. Our results demonstrated that fluoride exposure with coal-burning pollution caused orthopedic injuries and abnormalities in the levels of OC and BALP and hindered normal bone metabolism. Silencing the SMYD3 gene could significantly reduce OC and BALP levels via inhibiting the increase in autophagy induced by fluoride.

16 Linking VHL and SETD2 in a common oncogenic pathway that converges on the mitoticspindle

Abstract Background Loss of chromosome 3p is a landmark event in clear cell renal cell carcinoma (ccRCC) that results in mono-allelic loss of VHL (von Hippel Lindau) and SETD2 (Set-domain containing 2) (and other tumor suppressors co-located on 3p). Second hits in VHL inactivate this key tumor suppressor initiating tumor progression. SETD2, a histone methyltransferase, was previously shown to have a dual function in methylating both histones and microtubules, thereby contributing to both the histone and tubulin codes. Methylation by SETD2 on microtubules occurs at the mitotic spindle and is essential for normal mitosis and cytokinesis, with loss of SETD2 acting as a strong driver of apoptosis. This raises a conundrum of how cancer cells survive early mono-allelic loss of SETD2, escaping cell death. Methods Using biochemical kinase assays and mass spectrometry we have identified SETD2 as a substrate for AURKA. In addition, we have used immunoblotting and immunofluorescence assays to probe phosphorylation on SETD2 and the impact of phosphorylation of SETD2 both on its chromatin and cytoskeleton targets. Results We have identified the mitotic kinase, Aurora kinase A (AURKA), as a regulator of SETD2. Our data uncover SETD2 as a unique substrate for phosphorylation by AURKA, with mass spectrometry identifying serine 2080 (S2080) as the site of phosphorylation on SETD2. We found phosphorylation of SETD2 by AURKA at S2080 contributes to its methyltransferase (i.e. enzymatic) activity on microtubules but does not impact chromatin methylation on H3K36 which remains unaltered. We show that VHL regulates SETD2 via AURKA, and loss of phosphorylation on SETD2 results in mitotic defects and genomic instability. Importantly, we demonstrate that inhibition of AURKA is synthetic lethal in the setting of VHL and SETD2 deficiency. Conclusions AURKA expression levels are high in VHL-null cells resulting from an inability of VHL to target AURKA for degradation and our data now highlight a direct link between VHL and SETD2, two tumor suppressors believed to independently drive RCC pathogenesis. In summary, our data reveal a tumor-specific vulnerability linked to mitotic fragility that can be precisely targeted to ultimately drive mitotic catastrophe. DOD CDMRP Funding: yes

Interplay between DOT1L and HDAC1 regulates Leishmania donovani infection in human THP-1 cells

Leishmania donovani, a protozoan parasite, causes visceral leishmaniasis. The parasite modifies the global gene expressions of the host genome, facilitating its survival within the host. Thus, the host epigenetic modulators play important roles in host-pathogen interaction and host epigenetic modification in response to infection. Previously, we had reported that the host epigenetic modulator, histone deacetylase 1 (HDAC1) expression was upregulated on Leishmania donovani infection. This upregulation led to the repression of host defensin genes in response to the infection. In this paper, we have investigated the interplay between the host DOT1L, a histone methyltransferase, and HDAC1 in response to Leishmania donovani infection. We show that the expression of DOT1L is upregulated both at transcript and protein level following infection leading to increase in H3K79me, H3K79me2, and H3K79me3 levels. ChIP experiments showed that DOT1L regulated the expression of HDAC1. Downregulation of DOT1L using siRNA resulted in decreased expression of HDAC1 and increased transcription of defensin genes and thereby, lower parasite load. In turn, HDAC1 regulates the expression of DOT1L on Leishmania donovani infection as downregulation of HDAC1 using siRNA led to reduced expression of DOT1L. Thus, during Leishmania donovani infection, an interplay between DOT1L and HDAC1 regulates the expression of these two histone modifiers leading to downregulation of defensin gene expression.

PTIP epigenetically regulates DNA damage-induced cell cycle arrest by upregulating PRDM1

The genome is constantly exposed to DNA damage from endogenous and exogenous sources. Fine modulation of DNA repair, chromatin remodeling, and transcription factors is necessary for protecting genome integrity, but the precise mechanisms are still largely unclear. We found that after ionizing radiation (IR), global trimethylation of histone H3 at lysine 4 (H3K4me3) was decreased at an early (5 min) post-IR phase but increased at an intermediate (180 min) post-IR phase in both human and mouse hematopoietic cells. We demonstrated that PTIP, a component of the MLL histone methyltransferase complex, is required for H3K4me3 upregulation in the intermediate post-IR phase and promotes cell cycle arrest by epigenetically inducing a cell cycle inhibitor, PRDM1. In addition, we found that PTIP expression is specifically downregulated in acute myeloid leukemia patients. These findings collectively suggest that the PTIP-PRDM1 axis plays an essential role in proper DNA damage response and its deregulation contributes to leukemogenesis.

Abstract Mo130: Epigenetic Mechanisms Regulate Sex Differences in Cardiac Reparative Functions of Bone Marrow Progenitor Cells

Historically, lower incidence of CVD and related deaths in women as compared with men of the same age has been attributed to female sex hormones, particularly estrogen and its receptors. Autologous Bone marrow stem cell (BMSC) clinical trials for cardiac cell therapy overwhelmingly included male patients; however, meta-analysis data on these trials suggest a better functional outcome in women as compared with men. A direct comparison of the mechanisms governing sex-specific cardiac reparative activity in bone marrow-derived mesenchymal stem cells (BMSCs), with and without the influence of sex hormones, remains unexplored. This study was designed to identify mechanisms that regulate superior reparative properties of female endothelial progenitor cells (EPCs) post-MI, particularly epigenetic mechanisms. Male (M), female (F), and ovariectomized female (OVX) mice-derived EPCs were subjected to a series of molecular and epigenetic analyses followed by in vivo functional assessments of cardiac repair. RNA sequencing and other quantitative assays showed a similar genetic profile between F-EPCs and OVX-EPCs, distinct from M-EPCs that displayed significant up-regulation of inflammation-related genes. F-EPCs and OVX EPCs secrete higher levels of proangiogenic factors, lower levels of proinflammatory cytokines and show better cardiac reparative activity after intra-cardiac injections in a male mouse model of myocardial infarction (MI). Epigenetic sequencing revealed a marked difference in the occupancy of the gene repressive H3K9me3 mark, particularly at transcription start sites of key angiogenic and proinflammatory genes in male EPCs compared with female and ovariectomized EPCs. Our study unveiled that functional sex differences in EPCs is, in part, mediated by differential epigenetic regulation of the proinflammatory and anti-angiogenic gene CCL3, orchestrated by the control of H3K9me3 by histone methyltransferase, G9a/Ehmt2. Our research highlights the importance of considering sex of donor cells for progenitor-based tissue repair.

H3K36 methyltransferase GhKMT3;1a and GhKMT3;2a promote flowering in upland cotton

BackgroundThe SET domain group (SDG) genes encode histone lysine methyltransferases, which regulate gene transcription by altering chromatin structure and play pivotal roles in plant flowering determination. However, few studies have investigated their role in the regulation of flowering in upland cotton.ResultsA total of 86 SDG genes were identified through genome-wide analysis in upland cotton (Gossypium hirsutum). These genes were unevenly distributed across 25 chromosomes. Cluster analysis revealed that the 86 GhSDGs were divided into seven main branches. RNA-seq data and qRT‒PCR analysis revealed that lysine methyltransferase 3 (KMT3) genes were expressed at high levels in stamens, pistils and other floral organs. Using virus-induced gene silencing (VIGS), functional characterization of GhKMT3;1a and GhKMT3;2a revealed that, compared with those of the controls, the GhKMT3;1a- and GhKMT3;2a-silenced plants exhibited later budding and flowering and lower plant heightwere shorter. In addition, the expression of flowering-related genes (GhAP1, GhSOC1 and GhFT) significantly decreased and the expression level of GhSVP significantly increased in the GhKMT3;1a- and GhKMT3;2a-silenced plants compared with the control plants.ConclusionA total of 86 SDG genes were identified in upland cotton, among which GhKMT3;1a and GhKMT3;2a might regulate flowering by affecting the expression of GhAP1, GhSOC1, GhFT and GhSVP. These findings will provide genetic resources for advanced molecular breeding in the future.

Abstract We033: Heart-Specific Histone Methyltransferase SMYD1 Promotes Cardiac Maturation in the Direct Conversion of Human Fibroblasts into Cardiomyocytes.

Introduction: Direct cardiac reprogramming emerges as a promising strategy for in situ conversion of fibroblasts into induced cardiomyocyte-like cells (iCMs). While the conversion of mouse fibroblasts into functional, beating iCMs has been achieved efficiently, the reprogramming of human iCMs remains challenging, particularly their maturation with existing transcription factor combinations. Given the pivotal role of epigenetic regulation in this process, our study sought to leverage heart-specific epigenetic factors alongside cardiac-enriched transcription factors to enhance cardiac maturation, thereby yielding more reliable human iCMs. Result: Through analyzing the proteomic data across various human tissues, we found that while many epigenetic factors are broadly expressed, SMYD1 is uniquely heart-specific. To determine its involvement in reprogramming, we overexpressed SMYD1 and evaluated the reprogramming efficiency and iCM quality via flow cytometry, immunofluorescence staining, and transcriptome analysis. Notably, we observed an increase in iCMs expressing sarcomere markers, such as α-myosin heavy chain and α-actinin, as well as the ventricular specific myosin-light-chain 2v in SMYD1-treated group. Gene set enrichment analysis of RNA-sequencing data further indicated that SMYD1-induced iCMs exhibit ventricular gene signatures. Additionally, we discovered moderate activation of SMYD1 in mature iCMs generated with MEF2C, GATA4, TBX5, and TBX20. TBX20 is the key factor that leads to the co-binding of MEF2C, TBX5, and TBX20 on the SMYD1 promoter region and increases active histone marks, H3K27ac and H3K4me1. Rescue assay and transcriptomic comparison confirmed SMYD1 as the primary downstream target of TBX20 in promoting iCM functional maturation. Mechanistically, SMYD1 directly interacts with MEF2C and GATA4 to modify histone marks on cardiac regulatory regions. Conclusion: Our study provides comprehensive phenotypic and molecular evidence of SMYD1’s role as a cardiac-specific regulator during direct cardiac reprogramming. By interacting with key reprogramming factors and rewriting histone marks, SMYD1 significantly advances the maturation of human iCMs towards a ventricular subtype identity.

DPPA4 increases aggressiveness of pituitary neuroendocrine tumors by enhancing cell stemness.

Pituitary neuroendocrine tumors, PitNETs, are often aggressive and precipitate in distant metastases that are refractory to current therapies. However, the molecular mechanism in PitNETs' aggressiveness is not well understood. Developmental pluripotency-associated 4 (DPPA4) is known as a stem cell regulatory gene and overexpressed in certain cancers, but its function in the context of PitNETs' aggressiveness is not known. We employed both rat and human models of PitNETs. In the rat pituitary tumor model (RPT), we used prenatal-alcohol-exposed (PAE) female Fischer rats which developed aggressive PitNETs following estrogen treatment, while in the human pituitary tumor (HPT) model, we used aggressively proliferative cells from pituitary tumors of patients undergone surgery. Various molecular, cellular, and epigenetic techniques were used to determine the role of DPPA4 in PitNETs' aggressiveness. We show that DPPA4 is overexpressed in association with increased cell stemness factors in aggressive PitNETs of PAE rats and of human patients. Gene-editing experiments demonstrate that DPPA4 increases the expression of cell stemness and tumor aggressiveness genes and promotes proliferation, colonization, migration, and tumorigenic potential of PitNET cells. ChIP assays and receptor antagonism studies reveal that DPPA4 binds to canonical WINTs promoters and increases directly or indirectly the Wnt/β-catenin control of cell stemness, tumor growth, and aggressiveness of PitNETs. Epigenetic studies show involvement of histone methyltransferase in alcohol activation of DPPA4. These findings support a role of DPPA4 in tumor stemness and aggressiveness and provide a preclinical rationale for modulating this stemness regulator for the treatment of PitNETs.

Dual-target EZH2 inhibitor: latest advances in medicinal chemistry.

Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, plays a crucial role in tumor progression by regulating gene expression. EZH2 inhibitors have emerged as promising anti-tumor agents due to their potential in cancer treatment strategies. However, single-target inhibitors often face limitations such as drug resistance and side effects. Dual-target inhibitors, exemplified by EZH1/2 inhibitor HH-2853(28), offer enhanced efficacy and reduced adverse effects. This review highlights recent advancements in dual inhibitors targeting EZH2 and other proteins like BRD4, PARP1, and EHMT2, emphasizing rational design, structure-activity relationships, and safety profiles, suggesting their potential in clinical applications.

Subcellular one carbon metabolism in cancer, aging and epigenetics.

The crosstalk between metabolism and epigenetics is an emerging field that is gaining importance in different areas such as cancer and aging, where changes in metabolism significantly impacts the cellular epigenome, in turn dictating changes in chromatin as an adaptive mechanism to bring back metabolic homeostasis. A key metabolic pathway influencing an organism's epigenetic state is one-carbon metabolism (OCM), which includes the folate and methionine cycles. Together, these cycles generate S-adenosylmethionine (SAM), the universal methyl donor essential for DNA and histone methylation. SAM serves as the sole methyl group donor for DNA and histone methyltransferases, making it a crucial metabolite for chromatin modifications. In this review, we will discuss how SAM and its byproduct, S-adenosylhomocysteine (SAH), along with the enzymes and cofactors involved in OCM, may function in the different cellular compartments, particularly in the nucleus, to directly regulate the epigenome in aging and cancer.

Insights into the mechanisms driven by H3K4 KMTs in pancreatic cancer.

Pancreatic cancer is a malignancy arising from the endocrine or exocrine compartment of this organ. Tumors from exocrine origin comprise over 90% of all pancreatic cancers diagnosed. Of these, pancreatic ductal adenocarcinoma (PDAC) is the most common histological subtype. The five-year survival rate for PDAC ranged between 5 and 9% for over four decades, and only recently saw a modest increase to ∼12-13%, making this a severe and lethal disease. Like other cancers, PDAC initiation stems from genetic changes. However, therapeutic targeting of PDAC genetic drivers has remained relatively unsuccessful, thus the focus in recent years has expanded to the non-genetic factors underlying the disease pathogenesis. Specifically, it has been proposed that dynamic changes in the epigenetic landscape promote tumor growth and metastasis. Emphasis has been given to the re-organization of enhancers, essential regulatory elements controlling oncogenic gene expression, commonly marked my histone 3 lysine 4 monomethylation (H3K4me1). H3K4me1 is typically deposited by histone lysine methyltransferases (KMTs). While well characterized as oncogenes in other cancer types, recent work has expanded the role of KMTs as tumor suppressor in pancreatic cancer. Here, we review the role and translational significance for PDAC development and therapeutics of KMTs.

Changes in Histone Code Regulation during the Initiation of Paraptosis-Like Death of HEp-2 Tumor Cells by Oxidized Disulfiram Derivatives

Disulfiram (DSF) and its oxidized derivatives (DSFoxy) are currently being investigated as possible anticancer agents. We previously found that DSFoxy initiate paraptosis-like death of tumor cells, which is of potential interest for the treatment of tumors resistant to the initiation of apoptosis. Based on bioinformatics analysis of mass spectrometric data on protein ubiquitination, we formulated a conception about the important role of disruption of the retrograde transport of damaged proteins from the endoplasmic reticulum to the cytosol in the mechanism of initiation of paraptosis-like cell death. In the present work, it was found that DSFoxy, in the process of initiating paraptosis-like death of human adenocarcinoma HEp-2 cells, also enhances the ubiquitination of histones and histone code enzymes. In particular, this applies to the ubiquitination of histone H2BC12, histone methyltransferases responsible for transcription and repair of damaged DNA, as well as acetylating and ubiquitin-conjugating proteins. Bioinformatics analysis of changes in ubiquitination of cell nuclear proteins using the STRING database revealed during this process an increase in the occurrence of ubiquitinated proteins (functional enrichment) of cell cycle regulation, cell response to DNA damage and DNA repair, the regulation of which also depends on the histone code. This directly indicates damage to the cell nucleus and is consistent with confocal microscopy data. These results indicate that when paraptosis-like death is initiated by DSFoxy, along with impairment of retrograde transport and ER stress, there is also a change in the regulation of the histone code, which points to a pleotropic mechanism of cell death induction.

Lysine Methyltransferase 2D Regulates Immune Response and Metastasis in Head and Neck Cancer.

The most frequently altered epigenetic modifier in head and neck squamous carcinoma (HNSC) is the histone methyltransferase KMT2D. KMT2D catalyzes methylation of histone H3K4 resulting in open chromatin and the activation of target genes. Tumor-associated macrophages (TAMs) promote cancer growth by causing T lymphocyte exhaustion. C-C motif chemokine ligand 2 (CCL2) is a potent TAM chemotactic factor. In HNSC, TAMs have been associated with unfavorable patient outcomes and metastasis. The aim of this study was to determine the role of KMT2D in HNSC using genetically engineered in vivo models. KMT2D protein expression was correlated with lymph node metastasis in human HNSC using immunohistochemistry. Genetically engineered KMT2D and CCL2 knockout models of HNSC were created in vivo. HNSC was characterized using qRT-PCR, histopathology, and immunohistochemistry/immunofluorescence microscopy. We also analyzed the effects of KMT2D expression on the proliferation and migration of human HNSC lines. The regulation of the CCL2 gene by KMT2D was characterized using chromatin immunoprecipitation-sequencing assay of transposase accessible chromatin-sequencing, and chromatin conformation capture-sequencing. Human HNSC cases with high KMT2D expression exhibited significantly increased lymph node metastasis. Reduced KMT2D expression in our genetically engineered model correlated with reduced lymph node metastasis, longer latency, and slow tumor growth. CCL2 expression was decreased in KMT2D deficient HNSC, which correlated with a reduced TAM gene expression signature. Genomic experiments demonstrated that KMT2D directly targeted the CCL2 gene. A new genetically engineered in vivo model of CCL2-null HNSC was created, recapitulating the KMT2D deficient phenotype and showing a decreased T lymphocyte exhaustion signature. KMT2D regulates CCL2-mediated immune response and metastasis in HNSC.

The histone methyltransferase KMT2D maintains cellular glucocorticoid responsiveness by shielding the glucocorticoid receptor from degradation

Because of their ability to induce lymphocyte apoptosis, glucocorticoids (GC) are widely used to treat hematological malignancies such as lymphomas and multiple myeloma. Their effectiveness is often limited, however, due to the development of glucocorticoid resistance by a variety of molecular mechanisms. Here we performed an unbiased genome-wide CRISPR screen with the human T cell leukemia cell line Jurkat to find previously unidentified genes required for GC-induced apoptosis. One such gene was KMT2D (also known as MLL2 or MLL4), which encodes a histone lysine methyltransferase whose mutations are associated with a variety of cancers, blood malignancies in particular, and are considered markers of poor prognosis. Knockout of KMT2D by CRISPR/Cas9 gene editing in Jurkat and several multiple myeloma cell lines downregulated GR protein expression. Surprisingly, this was not due to a reduction in GR transcripts, but rather to a decrease in the protein's half-life, primarily due to proteasomal degradation. Reconstitution of KMT2D expression restored GR levels. In contrast to the known ability of KMT2D to control gene transcription through covalent histone methylation, KMT2D-mediated upregulation of GR levels did not require its methyltransferase activity. Co-immunoprecipitation and proximity ligation assays found constitutive binding of KMT2D to the GR, which was enhanced in the presence of GC. These observations reveal KMT2D to be essential for stabilization of cellular GR levels, and suggest a possible mechanism by which KMT2D mutations may lead to GC resistance in some malignancies.

Targeting Smurf1 to block PDK1-Akt signaling in KRAS-mutated colorectal cancer.

The phosphoinositide 3-kinase (PI3K)-Akt axis is one of the most frequently activated pathways and is demonstrated as a therapeutic target in Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated colorectal cancer (CRC). Targeting the PI3K-Akt pathway has been a challenging undertaking through the decades. Here we unveiled an essential role of E3 ligase SMAD ubiquitylation regulatory factor 1 (Smurf1)-mediated phosphoinositide-dependent protein kinase 1 (PDK1) neddylation in PI3K-Akt signaling and tumorigenesis. Upon growth factor stimulation, Smurf1 immediately triggers PDK1 neddylation and the poly-neural precursor cell expressed developmentally downregulated protein 8 (poly-Nedd8) chains recruit methyltransferase SET domain bifurcated histone lysine methyltransferase 1 (SETDB1). The cytoplasmic complex of PDK1 assembled with Smurf1 and SETDB1 (cCOMPASS) consisting of PDK1, Smurf1 and SETDB1 directs Akt membrane attachment and T308 phosphorylation. Smurf1 deficiency dramatically reduces CRC tumorigenesis in a genetic mouse model. Furthermore, we developed a highly selective Smurf1 degrader, Smurf1-antagonizing repressor of tumor 1, which exhibits efficient PDK1-Akt blockade and potent tumor suppression alone or combined with PDK1 inhibitor in KRAS-mutated CRC. The findings presented here unveil previously unrecognized roles of PDK1 neddylation and offer a potential strategy for targeting the PI3K-Akt pathway and KRAS mutant cancer therapy.

Pan‑cancer analysis on the role of KMT2C expression in tumor progression and immunotherapy.

Histone lysine N-methyltransferase 2C (KMT2C) is involved in transcriptional regulation and DNA damage repair. Mutations in KMT2C have been implicated in the progression, metastasis, and drug resistance of multiple cancer types. However, the roles of KMT2C in the regulation of tumor prognosis, immune cell infiltration and the immune microenvironment in these multiple cancer types remain unclear. Therefore, in the present study, data from The Cancer Genome Atlas and Genotype-Tissue Expression databases were used for KMT2C expression analyses. Kaplan-Meier and univariate Cox regression analyses were also performed to investigate the prognostic role of KMT2C. In addition, Gene Set Enrichment Analysis (GSEA) was conducted to study the KMT2C-related signaling pathways. Tumor immune estimation resource 2 and single-sample GSEA were conducted to investigate the correlation between KMT2C expression and immune cell infiltrations, and Spearman's analysis was conducted to study the correlations among KMT2C, tumor mutational burden, microsatellite instability, immune regulators, chemokines and immune receptors. Immunohistochemistry of patient kidney tumor samples was performed to verify the correlation between KMT2C and programmed death-ligand 1 (PD-L1) expression. Finally, RNA interference, wound healing and colony formation assays were conducted to evaluate the effects of KMT2C expression on cell proliferation and metastasis. The results of the present study demonstrated that KMT2C was highly expressed in multiple cancer types, was a protective factor in kidney renal clear cell carcinoma and ovarian serous cystadenocarcinoma, and a risk factor for lung squamous cell carcinoma and uveal melanoma. In addition, KMT2C levels were negatively correlated with immune-activated pathways and the infiltration of immune cells, and positively correlated with inhibitory immune factors and tumor angiogenesis. Patients with low KMT2C expression had higher objective response rates to immunotherapy, and drug sensitivity analysis indicated that topoisomerase, histone deacetylase, DOT1-like histone H3K79 methyltransferase and G9A nuclear histone lysine methyltransferase inhibitors could potentially be used to treat tumors with high KMT2C expression levels. Finally, the KMT2C and PD-L1 expression levels were shown to be positively correlated, and KMT2C knockdown markedly promoted the proliferation and invasion capacities of A549 cells. In conclusion, the present study revealed that low KMT2C expression may be a promising biomarker for predicting the response of patients with cancer to immunotherapy. Conversely, high KMT2C expression was shown to promote tumor angiogenesis, which may contribute to the formation of the immunosuppressive tumor microenvironment.

CCN6 Suppresses Metaplastic Breast Carcinoma by Antagonizing Wnt/β-Catenin Signaling to Inhibit EZH2-Driven EMT.

Metaplastic breast carcinomas (mBrCA) are a highly aggressive subtype of triple-negative breast cancer with histologic evidence of epithelial-to-mesenchymal transition and aberrant differentiation. Inactivation of the tumor suppressor gene cellular communication network factor 6 (CCN6; also known as Wnt1-induced secreted protein 3) is a feature of mBrCAs, and mice with conditional inactivation of Ccn6 in mammary epithelium (Ccn6-KO) develop spindle mBrCAs with epithelial-to-mesenchymal transition. Elucidation of the precise mechanistic details of how CCN6 acts as a tumor suppressor in mBrCA could help identify improved treatment strategies. In this study, we showed that CCN6 interacts with the Wnt receptor FZD8 and coreceptor LRP6 on mBrCA cells to antagonize Wnt-induced activation of β-catenin/TCF-mediated transcription. The histone methyltransferase EZH2 was identified as a β-catenin/TCF transcriptional target in Ccn6-KO mBrCA cells. Inhibiting Wnt/β-catenin/TCF signaling in Ccn6-KO mBrCA cells led to reduced EZH2 expression, decreased histone H3 lysine 27 trimethylation, and deregulation of specific target genes. Pharmacologic inhibition of EZH2 reduced growth and metastasis of Ccn6-KO mBrCA mammary tumors in vivo. Low CCN6 is significantly associated with activated β-catenin and high EZH2 in human spindle mBrCAs compared with other subtypes. Collectively, these findings establish CCN6 as a key negative regulator of a β-catenin/TCF/EZH2 axis and highlight the inhibition of β-catenin or EZH2 as a potential therapeutic approach for patients with spindle mBrCAs. Significance: CCN6 deficiency drives metaplastic breast carcinoma growth and metastasis by increasing Wnt/β-catenin activation to upregulate EZH2, identifying EZH2 inhibition as a mechanistically guided treatment strategy for this deadly form of breast cancer.

The Drosophila histone methyltransferase SET1 coordinates multiple signaling pathways in regulating male germline stem cell maintenance and differentiation.

Many tissue-specific adult stem cell lineages maintain a balance between proliferation and differentiation. Here, we study how the H3K4me3 methyltransferase Set1 regulates early-stage male germ cells in Drosophila. Early-stage germline-specific knockdown of Set1 results in temporally progressive defects, arising as germ cell loss and developing into overpopulated early-stage germ cells. These germline defects also impact the niche architecture and cyst stem cell lineage non-cell-autonomously. Additionally, wild-type Set1, but not the catalytically inactive Set1, rescues the Set1 knockdown phenotypes, highlighting the functional importance of the methyltransferase activity of Set1. Further, RNA-sequencing experiments reveal key signaling pathway components, such as the JAK-STAT pathway gene Stat92E and the BMP pathway gene Mad, which are upregulated upon Set1 knockdown. Genetic interaction assays support the functional relationships between Set1 and JAK-STAT or BMP pathways, as both Stat92E and Mad mutations suppress the Set1 knockdown phenotypes. These findings enhance our understanding of the balance between proliferation and differentiation in an adult stem cell lineage. The phenotype of germ cell loss followed by over-proliferation when inhibiting a histone methyltransferase also raises concerns about using their inhibitors in cancer therapy.

The G9a histone methyltransferase represses osteoclastogenesis and bone resorption by regulating NFATc1 function.

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For invivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.