Histone Methyltransferase KMT2D Research Articles

Deficiency of TET2-mediated KMT2D self-transcription confers a targetable vulnerability in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has become a leading cause of cancer-related mortality worldwide. Conventional therapies tend to exacerbate comorbidities, liver dysfunction, and relapse, rendering an urgent demand for novel strategy for management of HCC. Here, we reported that DNA dioxygenase TET2 collaborates with histone methyltransferase KMT2D to enable transcription of KMT2D and ARID1A in HCC. Mechanistically, KMT2D and ARID1A are the major epigenetic targets of TET2 through RNA-seq analysis. Moreover, KMT2D recruits TET2 to facilitate self-transcription via oxidation of 5-methylcytosine in promoter, thereby maintaining expression of ARID1A. Physiologically, KMT2D was identified as a tumor suppressor and mediates the antitumor effect of vitamin C in HCC. Tumors with depleted KMT2D present growth advantage over control group. Vitamin C is able to impair tumor growth, which is compromised by deficiency of KMT2D. Furthermore, loss of KMT2D sensitizes HCC tumors to cisplatin with reduced tumor weight and high level of DNA damage. Ultimately, TET2-KMT2D axis correlates with prognosis of patients with HCC. Patients with high amounts of TET2 and KMT2D present better outcome. Our findings not only put forth a heretofore unrecognized mechanism underlying cross-talk between TET2 and KMT2D in mediating self-transcription of KMT2D, but also propose a targetable vulnerability for HCC therapy on the basis of TET2-KMT2D axis.

The histone methyltransferase KMT2D is essential for embryo implantation via regulating precise differentiation of endometrial cells

Embryo implantation failures are a major challenge in reproductive medicine, but the underlying mechanism remains poorly understood. Successful implantation requires dynamic remodeling of the endometrium through integrated proliferation and differentiation of endometrial cells including luminal epithelial, glandular epithelial, and stromal cells. Conversely, their disruption causes infertility. Spatiotemporal control of transcription is required for these processes; however, the underlying epigenetic regulation is largely unknown. In this study, we examined expression data from the human endometrium during implantation and discovered that expression of the histone lysine methyltransferase KMT2D was significantly suppressed in patients with recurrent implantation failure. Further study revealed that uterine deletion of Kmt2d in mice caused infertility due to implantation failure. Morphological analysis discovered a reduction in the number of uterine glands and aberrant differentiation of the luminal and glandular epithelium into stratified phenotypes in Kmt2d knockout uteri. Administration of leukemia inhibitory factor protein, which is expressed in uterine glands and is essential for implantation, did not rescue implantation failure in Kmt2d knockout mice, suggesting that infertility was not solely due to uterine gland dysfunction. RNA sequencing analysis revealed that Kmt2d knockout uteri displayed suppressed expression of genes involved in ion homeostasis, which may affect the uterine luminal morphology. Our study suggests that KMT2D plays an essential role in facilitating successful embryo implantation by regulating the coordinated differentiation of endometrial cells, providing valuable insights into unexplained implantation failures in women.

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.

Abstract 1245: The histone methyltransferase KMT2D mediates subtype-specific transcriptional regulation and therapeutic response in prostate cancer

Abstract Primary prostate cancer is characterized by its dependence on androgen receptor (AR) and often has hyperactive PI3K signaling, most frequently through loss of PTEN. PI3K/AKT pathway inhibitors are in late-stage clinical development in combination with anti-AR therapy. Androgen deprivation therapy is a cornerstone of treatment for advanced prostate cancer, but the development of castration-resistant prostate cancer (CRPC) eventually occurs. CRPC remains largely dependent on AR for growth with a subset of CRPC that lose AR dependence. There is an urgent need to study the molecular pathways leading to AR activation and the loss of AR reliance in order to identify therapeutic targets. KMT2D is a histone methyltransferase and an important regulator of gene expression. Here we found that KMT2D establishes the chromatin competence necessary for the recruitment of AR and FOXA1 transcription factors (TFs) to activate AR-dependent transcription in AR-high prostate models. In AR-low models such as the stem cell-like (SCL) subtype, KMT2D controls residual AR-FOXA1 gene expression programs and AP-1 TFs such as FOSL1, a driver of the SCL subtype. In SCL, single cell RNA-seq and single cell chromatin assays confirm a key role for KMT2D in the maintenance of a mixed lineage cell state through the regulation of AP-1 and FOXA1 TFs. Combined suppression of PI3K/AKT and KMT2D reduces cell proliferation in prostate cancer cells and patient derived organoids, providing a rationale for epigenetically informed combination therapies with PI3K/AKT inhibitors in PTEN-deficient prostate cancer. Together, these data establish KMT2D as a major mediator of subtype-specific chromatin accessibility and transcriptomic landscape in CRPC, required for prostate cancer growth and therapeutic response. Citation Format: Srushti Kittane, Erik Ladewig, Taibo Li, Jillian Love, Amaia Arruabarrena-Aristorena, Xinyu Guo, Mirna Sallaku, Liliana Garcia-Martinez, Ryan Blawski, Javier Carmona Sanz, Christopher Simpkins, Wanlu Chen, Bujamin Vokshi, Peihua Zhao, Nachiket Kelkar, Laura Baldino, Ingrid Kalemi, Pau Castel, Emiliano Cocco, Lluis Morey, Charles Sawyers, Hongkai Ji, Maurizio Scaltriti, Alexis Battle, Christina Leslie, Wouter Karthaus, Eneda Toska. The histone methyltransferase KMT2D mediates subtype-specific transcriptional regulation and therapeutic response in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1245.

Comparison of mutation landscapes of pretreatment versus recurrent squamous cell carcinoma of the oral cavity: The possible mechanism of resistance to standard treatment.

A high recurrent rate of oral squamous cell carcinoma (OSCC) is a major concern in head and neck cancer treatment. The study of the genetic mutation landscape in recurrent OSCC may provide information on certain mutations associated with the pathobiology and treatment response of the OSCC. We investigated the mutation landscape of matched pretreatment and recurrent tumors to understand the influence of genetic mutations on the pathobiology and clinical outcomes in OSCC. We sequenced 33 formalin-fixed paraffin-embedded (FFPE) recurrent tumors, primary tumors, and primary tumors before recurrence that matched the recurrent tumors collected from Rajavithi Hospital during 2019-2021. We identified recurrent mutations from these samples by the Oncomine Ion Torrent-based next-generation sequencing on the 517 cancer-associated gene panel. From the results, we found that the most commonly mutated gene in the cohort is a histone methyltransferase KMT2D (54.55%), implicating that aberrance in epigenetic regulation may play a role in oral cancer tumorigenesis. Functional protein association network analysis of the gene frequently mutated in the recurrent tumors showed enrichment of genes that regulate the cancer cell cycle, that is, MRE11A, CDKN2A, and CYLD. This finding was confirmed in the primary-recurring matched pair. We found that recurrent tumors possess a small but recurring group of genes, with presumably the subclonal mutations driving the recurrence of the tumor, suggesting that the recurrent disease originated from a small fraction of the cancer cell that survives standard treatment. These genes were absent in the primary tumor with a good response to the standard treatment. On the other hand, we found an enrichment of DNA repair genes, namely ATR, BRCA1, BRCA2, RAD50, and MUTYH, in nonrecurrent tumors suggesting that the mutations in the DNA repair pathway may at least partially explain the different response to the standard treatment. Our pilot study identified pathways of carcinogenesis in oral cancer and specific gene sets that indicate treatment responses and prognoses in this group of patients.

KMT2D mutations promoted tumor progression in diffuse large B-cell lymphoma through altering tumor-induced regulatory T cell trafficking via FBXW7-NOTCH-MYC/TGF-β1 axis.

Histone methyltransferase KMT2D is one of the most frequently mutated genes in diffuse large B-cell lymphoma (DLBCL) and has been identified as an important pathogenic factor and prognostic marker. However, the biological relevance of KMT2D mutations on tumor microenvironment remains to be determined. KMT2D mutations were assessed by whole-genome/exome sequencing (WGS/WES) in 334 patients and by targeted sequencing in 427 patients with newly diagnosed DLBCL. Among all 761 DLBCL patients, somatic mutations in KMT2D were observed in 143 (18.79%) patients and significantly associated with advanced Ann Arbor stage and MYC expression ≥ 40%, as well as inferior progression-free survival and overall survival. In B-lymphoma cells, the mutation or knockdown of KMT2D inhibited methylation of lysine 4 on histone H3 (H3K4), downregulated FBXW7 expression, activated NOTCH signaling pathway and downstream MYC/TGF-β1, resulting in alterations of tumor-induced regulatory T cell trafficking. In B-lymphoma murine models established with subcutaneous injection of SU-DHL-4 cells, xenografted tumors bearing KMT2D mutation presented lower H3K4 methylation, higher regulatory T cell recruitment, thereby provoking rapid tumor growth compared with wild-type KMT2D via FBXW7-NOTCH-MYC/TGF-β1 axis.

Histone methyltransferase KMT2D inhibits ENKTL carcinogenesis by epigenetically activating SGK1 and SOCS1.

Epigenetic alteration plays an essential role in the occurrence and development of extranodal natural killer/T cell lymphoma (ENKTL). Histone methyltransferase (HMT) KMT2D is an epigenetic regulator that plays different roles in different tumors, but its role and mechanism in ENKTL are still unclear. We performed immunohistochemical staining of 112 ENKTL formalin-fixed paraffin-embedded (FFPE) samples. Then, we constructed KMT2D knockdown cell lines and conducted research on cell biological behavior. Finally, to further investigate KMT2D-mediated downstream genes, ChIP-seq and ChIP -qPCR was performed. The low expression of KMT2D was related to a decreased abundance in histone H3 lysine 4 mono- and trimethylation (H3K4me1/3). In KMT2D knockdown YT and NK-YS cells, cell proliferation was faster (P < 0.05), apoptosis was decreased (P < 0.05), the abundance of S phase cells was increased (P < 0.05), and the level of H3K4me1 was decreased. Notably, ChIP-seq revealed two crucial genes and pathways downregulated by KMT2D. KMT2D is a tumor suppressor gene that mediates H3K4me1 and influences ENKTL proliferation and apoptosis by regulating the cell cycle. Moreover, in ENKTL, serum- and glucocorticoid-inducible kinase-1 (SGK1) and suppressor of cytokine signaling-1 (SOCS1) are downstream genes of KMT2D.

The KDM6A-KMT2D-p300 axis regulates susceptibility to diverse coronaviruses by mediating viral receptor expression.

Identification of host determinants of coronavirus infection informs mechanisms of pathogenesis and may provide novel therapeutic targets. Here, we demonstrate that the histone demethylase KDM6A promotes infection of diverse coronaviruses, including SARS-CoV, SARS-CoV-2, MERS-CoV and mouse hepatitis virus (MHV) in a demethylase activity-independent manner. Mechanistic studies reveal that KDM6A promotes viral entry by regulating expression of multiple coronavirus receptors, including ACE2, DPP4 and Ceacam1. Importantly, the TPR domain of KDM6A is required for recruitment of the histone methyltransferase KMT2D and histone deacetylase p300. Together this KDM6A-KMT2D-p300 complex localizes to the proximal and distal enhancers of ACE2 and regulates receptor expression. Notably, small molecule inhibition of p300 catalytic activity abrogates ACE2 and DPP4 expression and confers resistance to all major SARS-CoV-2 variants and MERS-CoV in primary human airway and intestinal epithelial cells. These data highlight the role for KDM6A-KMT2D-p300 complex activities in conferring diverse coronaviruses susceptibility and reveal a potential pan-coronavirus therapeutic target to combat current and emerging coronaviruses. One Sentence Summary: The KDM6A/KMT2D/EP300 axis promotes expression of multiple viral receptors and represents a potential drug target for diverse coronaviruses.

Histone methyltransferase KMT2D promotes prostate cancer progression through paracrine IL-6 signaling.

Histone methyltransferase KMT2D plays a critical role as a human oncogene in prostate cancer (PCa). Dysregulated inflammatory responses and cytokine signaling are implicated in cancer progression. Furthermore, interleukin 6 (IL-6) is a pleiotropic cytokine that contributes to PCa progression; however, the association between KMT2D and IL-6 in PCa remains unclear. PCa cell proliferative potential, migratory potential, and apoptosis invitro were determined using cell counting kit-8 (CCK-8), EdU incorporation, wound healing, and apoptosis assays. Proliferation and migratory potential were impaired and apoptosis was induced in PCa cells cultured with the conditioned medium from KMT2D-depleted cells. Cytokine array analysis showed that IL-6 was the most affected cytokine in the conditioned media. KMT2D knockdown significantly downregulated the expression of IL-6 in PCa cells. What's more, proliferation and migration were also impaired and apoptosis was also induced by silencing IL-6R expression. Immunohistochemistry (IHC) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were performed to validate the positive correlation between KMT2D and IL-6 in PCa tissue samples. Chromatin immunoprecipitation (ChIP)-PCR demonstrated that KMT2D and H3K4me1 occupied IL-6 enhancer regions and therefore, directly regulated IL-6 expression. The present study revealed that the KMT2D knockdown suppressed prostate cancer progression through the downregulation of paracrine IL-6 signaling. These results suggest that KMT2D could be regarded as a potential new target for PCa therapy.

Loss of Histone Methyltransferase KMT2D Attenuates Angiogenesis in the Ischemic Heart by Inhibiting the Transcriptional Activation of VEGF-A

Angiogenesis occurred after myocardial infarction (MI) protects heart failure (HF). The aim of our study was to explore function of histone methyltransferase KMT2D (MLL4, mixed-lineage leukemia 4) in angiogenesis post-MI. Western blotting showed that KMT2D protein expression was elevated in MI mouse myocardial. Cardiomyocyte-specific Kmt2d-knockout (Kmt2d-cKO) mice were generated, and echocardiography and immunofluorescence staining detected significantly attenuated cardiac function and insufficient angiogenesis following MI in Kmt2d-cKO mice. Cross-talk assay suggested that Kmt2d-KO H9c2-derived conditioned medium attenuates EA.hy926 EC function. ELISA further identified that VEGF-A released from Kmt2d-KO H9c2 was significantly reduced. CUT&Tag and RT-qPCR revealed that KMT2D deficiency reduced Vegf-a mRNA expression and enrichment of H3K4me1 on the Vegf-a promoter. Moreover, KMT2D silencing in ECs also suppressed endothelial function. Our study indicates that KMT2D depletion in both cardiomyocytes and ECs attenuates angiogenesis and that loss of KMT2D exacerbates heart failure after MI in mice.

Pan-Cancer Analysis of Histone Methyltransferase KMT2D with Potential Implications for Prognosis and Immunotherapy in Human Cancer.

Pan-cancer analysis is an efficient tool to obtain a panoramic view of cancer- related genes and identify their oncogenic processes, facilitating the development of new therapeutic targets. Lysine methyltransferase 2D (KMT2D), acting as a major enhancer coactivator for mammalian cells, is one of the most frequently mutated genes across various cancer types and is considered an oncogene and a rationale for epigenetic therapeutic targets. This study was designed to explore the potential role of KMT2D in human cancer through a pan-cancer analysis. The expression of KMT2D was assessed in normal tissues and cell lines, and pancancers from The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), and Genotype-Tissue Expression (GTE) datasets were used to explore its correlation with prognosis, immune cell infiltration, tumor mutation burden, microsatellite instability, and mismatch repair. KMT2D expression was heterogeneous across different cancer types. Increased KMT2D indicated a worse prognosis in adrenocortical carcinoma (ACC), brain lower-grade glioma (LGG), and mesothelioma (MESO), while patients with high KMT2D expression showed better outcomes in renal clear cell carcinoma (KIRC). Moreover, KMT2D expression was positively correlated with immune cell infiltration and negative tumor mutation burden in multiple cancers. In addition, a significant correlation between KMT2D and immune checkpoint-related genes or mismatch repair genes was identified. These findings support the hypothesis that KMT2D is not only a potential biomarker for prognosis and immunotherapy response prediction but also an essential immune regulator in human cancer.

Histone methyltransferase KMT2D contributes to the protection of myocardial ischemic injury.

Histone H3 lysine 4 (H3K4) methyltransferase 2D (KMT2D) plays an important role in cell development in early life. However, the function of KMT2D in adult cells such as cardiomyocytes or neurons has not been reported. In this study, cardiomyocyte-specific KMT2D knockout (KMT2D-cKO) and control (KMT2D-Ctl) mice were exposed to sham or myocardial ischemia (MI) surgery. Depletion of KMT2D aggravated the ischemic area, led to the increased mortality (26.5% in KMT2D-cKO vs 12.5% in KMT2D-Ctl) of the mice, and weakened the left ventricular systolic function. RNA-seq analysis in cardiac tissues identified genes whose expression was changed by MI and KMT2D deletion. Combined with the genome-wide association study (GWAS) analysis, cardiac disease-associated genes Rasd1, Thsd7a, Ednra, and Tns1 were identified. The expression of the Rasd1 was significantly decreased by MI or the loss of KMT2D in vivo. Meanwhile, ChIP assays demonstrated that either MI or loss of KMT2D attenuated monomethylated H3K4 (H3K4me1) enrichment on the enhancer of Rasd1. By generating a KMT2D knockout (H9C2-KO) H9C2 monoclone, we verified that the expression of Rasd1 was controlled by KMT2D, and the expression of Rasd1 was decreased by serum starvation but not low-(O2) treatment in H9C2 cells. KMT2D has a protective effect on ischemic myocardium by regulating cardiac disease-associated genes including Rasd1. KMT2D is required for the H3K4me1 deposition on the enhancer of Rasd1. Our data for the first time suggest that KMT2D-mediated Rasd1 expression may play an important protective effect on adult cells during nutritional deficiency caused by ischemic injury.

Abstract B002: Modeling evolutionary therapy for GI malignancies

Abstract Despite development of personalized treatments GI cancer like pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) remain dismal outcomes. While some patients are potentially curable with surgery and chemotherapy, most unresectable cases are invariably lethal. To advance patient care, we rely on clinical trials to test new therapeutic approaches. Here, we assume that the highest dose of a drug that does not cause severe toxicities is optimal for cancer treatment. However, this treatment strategy, in many cases, may be more harmful for the patient. Adopting principles from the field of evolutionary biology and pest management could offer more effective strategies that can be used for patients with metastatic PDAC and CRC. Resistance mechanisms are assumed to come with an energy cost, making the organism/cell less fit than others in the population. When high doses of treatment are applied, it results in a significant selection gradient by eliminating sensitive populations, freeing resources and facilitating resistant population growth. Such evolutionary dynamics and interactions within the tumor microenvironment can be modeled and exploited, in the form of adaptive strategies, for treatment of CRC and PDAC. It is our central hypothesis that integration of evolutionary dynamics and into PDAC and CRC treatment will delay emergence of resistance, enhance quality of life, and improve clinical outcomes. Our preliminary observations in co-cultured drug sensitive and resistant cell containing spheroids show that a higher proportion of drug sensitive cells inhibits the proliferation of drug resistant cells over time, in the presence of chemotherapeutic agents 5-FU and Oxaliplatin. We also demonstrate a role for the MLL family of histone methyltransferases in the epigenetic regulation of genes involved in chemoresistance. RNA-seq data has shown differential gene expression in metabolic genes regulated by histone methyltransferase KMT2A, KM2TB and KMT2D knockout (KO) cells including Gstt1, CYP26B1 and PYGL. In addition to this, KMT2A, KM2TB and KMT2D KOs show differential sensitivity to chemotherapeutic agents Gemcitabine, SN-38, 5-FU and Oxaliplatin. Further, analysis of KMT2A, KM2TB and KMT2D KO cells will provide mechanistic insight of the epigenetic regulation of metabolic patterns in drug resistant cells. This strategy would provide an approach for reducing exposure to toxic drugs, improving patient quality of life and renewing hope for prolonged control of metastatic PDAC and CRC. This proposal represents a bedside-to-bench-to-bedside project; identifying a shortcoming in how we currently treat PDAC and CRC patients, developing solutions in the lab and ultimately translating such strategies to benefit patients. Citation Format: Glancis Luzeena Raja Arul, Ezequiel Tolosa, Ryan M. Carr, Martin E. Fernandez-Zapico. Modeling evolutionary therapy for GI malignancies [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B002.

Histone methyltransferase KMT2D cooperates with MEF2A to promote the stem-like properties of oral squamous cell carcinoma

BackgroundEpigenetic reprogramming is involved in multiple steps of human cancer evolution and is mediated by a variety of chromatin-modifying enzymes. Specifically, the histone lysine methyltransferase KMT2D is among the most frequently mutated genes in oral squamous cell carcinoma (OSCC). However, the mechanisms by which KMT2D affects the development of OSCC remain unclear.ResultsIn the present study, we found that the expression of KMT2D was elevated in OSCC compared to paracancerous specimens and was correlated with a more advanced tumor grade. More importantly, knockdown of KMT2D impaired their reconstitution in patient-derived organoids and decreased the expression of CD133 and β-catenin in OSCC cells. In in vitro and in vivo models, knockdown of KMT2D reduced the colony formation, migration and invasion abilities of OSCC cells and delayed tumor growth. Mechanistically, the dual-luciferase reporter and co-immunoprecipitation assays in two individual OSCC cell lines indicated that KMT2D may cooperate with MEF2A to promote the transcription activity of CTNNB1, thereby enhancing WNT signaling.ConclusionThe upregulation of KMT2D contributes to stem-like properties in OSCC cells by sustaining the MEF2A-mediated transcriptional activity of CTNNB1.

KMT2D deficiency disturbs the proliferation and cell cycle activity of dental epithelial cell line (LS8) partially via Wnt signaling.

Lysine methyltransferase 2D (KMT2D), as one of the key histone methyltransferases responsible for histone 3 lysine 4 methylation (H3K4me), has been proved to be the main pathogenic gene of Kabuki syndrome disease. Kabuki patients with KMT2D mutation frequently present various dental abnormalities, including abnormal tooth number and crown morphology. However, the exact function of KMT2D in tooth development remains unclear. In this report, we systematically elucidate the expression pattern of KMT2D in early tooth development and outline the molecular mechanism of KMT2D in dental epithelial cell line. KMT2D and H3K4me mainly expressed in enamel organ and Kmt2d knockdown led to the reduction in cell proliferation activity and cell cycling activity in dental epithelial cell line (LS8). RNA-sequencing (RNA-seq) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis screened out several important pathways affected by Kmt2d knockdown including Wnt signaling. Consistently, Top/Fop assay confirmed the reduction in Wnt signaling activity in Kmt2d knockdown cells. Nuclear translocation of β-catenin was significantly reduced by Kmt2d knockdown, while lithium chloride (LiCl) partially reversed this phenomenon. Moreover, LiCl partially reversed the decrease in cell proliferation activity and G1 arrest, and the down-regulation of Wnt-related genes in Kmt2d knockdown cells. In summary, the present study uncovered a pivotal role of histone methyltransferase KMT2D in dental epithelium proliferation and cell cycle homeostasis partially through regulating Wnt/β-catenin signaling. The findings are important for understanding the role of KMT2D and histone methylation in tooth development.

Expression Analysis and Mutational Status of Histone Methyltransferase KMT2D at Different Upper Tract Urothelial Carcinoma Locations.

The gene coding for histone methyltransferase KMT2D is found among the top mutated genes in upper tract urothelial carcinoma (UTUC); however, there is a lack of data regarding its association with clinicopathologic features as well as survival outcomes. Therefore, we aimed to investigate KMT2D expression, mutation patterns, and their utility as prognostic biomarkers in patients with UTUC. A single-center study was conducted on tumor specimens from 51 patients treated with radical nephroureterectomy (RNU). Analysis of KMT2D protein expression was performed using immunohistochemistry (IHC). Customized next-generation sequencing (NGS) was used to assess alterations in KMT2D exons. Cox regression was used to assess the relationship of KMT2D protein expression and mutational status with survival outcomes. KMT2D expression was increased in patients with a previous history of bladder cancer (25% vs. 0%, p = 0.02). The NGS analysis of KMT2D exons in 27 UTUC tumors revealed a significant association between pathogenic KMT2D variants and tumor location (p = 0.02). Pathogenic KMT2D variants were predominantly found in patients with non-pelvic or multifocal tumors (60% vs. 14%), while the majority of patients with a pelvic tumor location (81% vs. 20%) did not harbor pathogenic KMT2D alterations. Both IHC and NGS analyses of KMT2D failed to detect a statistically significant association between KMT2D protein or KMT2D gene alteration status and clinical variables such as stage/grade of the disease or survival outcomes (all p > 0.05). KMT2D alterations and protein expression were associated with UTUC features such as multifocality, ureteral location, and previous bladder cancer. While KMT2D protein expression and KMT2D mutational status do not seem to have prognostic value in UTUC, they appear to add information to improve clinical decision-making regarding the type of therapy.

P111 - Expression analysis and mutational status of histone methyltransferase KMT2D at different upper tract urothelial carcinoma locations

P111 - Expression analysis and mutational status of histone methyltransferase KMT2D at different upper tract urothelial carcinoma locations

Cancer-epigenetic function of the histone methyltransferase KMT2D and therapeutic opportunities for the treatment of KMT2D-deficient tumors.

Epigenetic mechanisms are central to understanding the molecular basis underlying tumorigenesis. Aberrations in epigenetic modifiers alter epigenomic landscapes and play a critical role in tumorigenesis. Notably, the histone lysine methyltransferase KMT2D (a COMPASS/ Set1 family member; also known as MLL4, ALR, and MLL2) is among the most frequently mutated genes in many different types of cancer. Recent studies have demonstrated how KMT2D loss induces abnormal epigenomic reprograming and rewires molecular pathways during tumorigenesis. These findings also have clinical and therapeutic implications for cancer treatment. In this review, we summarize recent advances in understanding the role of KMT2D in regulating tumorigenesis and discuss therapeutic opportunities for the treatment of KMT2D-deficient tumors.

Histone Methyltransferase KMT2D Regulates H3K4 Methylation and is Involved in the Pathogenesis of Ovarian Cancer

To investigate the function of histone-lysine N-methyltransferase 2D (KMT2D) on the methylation of H3 lysine 4 (H3K4) in the progression of Ovarian cancer (OV). KMT2D, ESR1 and H3K4me expressions in surgical resected tumors and tumor adjacent tissues of OV from 198 patients were determined using immunohistochemistry (IHC). Human OV cell lines including SKOV3, HO-8910 cells and normal ovarian epithelial cell line IOSE80 were employed for in vitro experiment, and BALB/C female nude mice were used for in vivo study. qRT-PCR and Western blotting were implemented for measuring the KMT2D, ESR1, PTGS2, STAT3, VEGFR2, H3K4me and ELF3 levels. Chromatin immunoprecipitation (ChIP) analysis was used for studying the binding between ESR1 and H3K4me. Edu staining assay was executed to determine cell viability, and colony formation and cell invasion assay. The immunofluorescence method was utilized for the visualization of protein expression and distribution in cells. In this study, KMT2D, ESR1 and H3K4me were found upregulated in OV progression. Mutated H3K4me could inhibit the proliferation, colony formation and invasion ability of OV cells. Mutated H3K4me could also hinder the ESR1 in SKOV3 expressions and HO-8910 cells, which would further mediate PTGS2/STAT3/VEGF pathway. In vivo studies also demonstrated that mutated H3K4me inhibited OV progression via targeting ESR1. All the ChIP-PCR analysis indicated the moderator effect of H3K4me on ESR1. Our findings indicated that ESR1 played an important role in the OV progression. Besides, H3K4me could promote cell proliferation and inhibit apoptosis of OV cells. Meanwhile, it could also targets the ESR1 production to enhance the migration and invasion of OV cells, which was through the activation of ESR1-ELF3-PTGS2-STAT3-VEGF cascade signaling pathway.