Decrease In H3K27me3 Research Articles

Activating NSD2 Mutations Drive Oncogenic Reprogramming By Disturbing Epigenetic Landscape in Mantle Cell Lymphoma

Background: NSD2/MMSET/WHSC1, a histone lysine methyltransferase (HMT), is an oncoprotein first characterized by its overexpression in multiple myeloma (MM). NSD2 mutations within the catalytic SET domain are frequently identified in lymphoid malignancies including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL). We previously demonstrated that NSD2 p.E1099K mutation caused an imbalance of H3K36me2/H3K27me3 and drove glucocorticoid resistance in pediatric ALL. In MCL, NSD2 mutations, especially p.E1099K and p.T1150A, are found in 10-15% of cases of MCL and are enriched in MCL patients who relapse from targeted therapies such as ibrutinib, an inhibitor of B cell signaling. However, the activity of NSD2 mutations in MCL remains unexplored. We hypothesize that NSD2 mutations contribute to disease progression and therapy resistance due to aberrant chromatin modification. Aim: To demonstrate the role of NSD2 mutations in MCL progression due to aberrant chromatin modification. Methods: We created isogenic MCL cell lines by knock-in NSD2 p.E1099K and NSD2 p.T1150A mutation into Z138 and Jeko-1 cell lines using CRISPR/Cas9 gene editing. We then determined H3K36me2/H3K27me3/H3K36me3 using immunoblot and the biological activities including cell growth (IncuCyte), apoptosis (Annexin V/PI staining), cell cycle (BrdU incorporation), and in vivo experiments using luciferase-tagged cell lines in NOD-SCID mice. We determined alteration of gene transcriptome using RNA-Seq and histone modification using CUT&RUN (H3K36me2, H3K27me3 and H3K4me3) and ChIP-Seq (H3K27ac) in isogenic Z138 cell lines. Finally, we integrated multi-omics analysis of our MCL cell lines and human patient samples and to comprehensively disclose the epigenetic landscape in MCL with NSD2 mutations. Results: We successfully created isogenic Z138 cell lines with NSD2 p.E1099K mutation or p.T1150A mutation, and isogenic Jeko-1 cell line with NSD2 p.E1099K mutation. Insertion of either NSD2 p.E1099K mutation or p.T1150A mutation by CRISPR/Cas9 gene editing led to a striking increase of H3K36me2, a decrease of H3K27me3 but no change of H3K36me3. NSD2 p.E1099K mutation significantly increased cellular growth and enhanced S phase entry while decreased G0/G1 phase and apoptosis. In NOD-SCID mouse xenografts, NSD2 p.E1099K mutation resulted in a higher tumor burden and shorter survival lifespan in mice. RNA-Seq analysis demonstrated striking changes in gene expression with 1794 genes upregulated and 1492 genes downregulated in NSD2 mutant MCL cells. Among them, anti-apoptotic genes BCL2 and BCL2L2 were upregulated while pro-apoptotic genes BAX, BID, and BIK were downregulated in NSD2 mutant cells, which was consistent with the biological phenotypes. Surprisingly, CCND1, CCNE1, CCNE2, TP53, and CDKN2C ( p21) were downregulated while CDKN2D, CDKL3, and CDKL5 were upregulated in NSD2 mutant cells. Altered gene expression in isogenic Z138 cells was compared with patient expression profiles, with gene ontology revealing significant overlap and enrichment of cell adhesion, neural pathways, and evidence of activated signaling. NSD2 mutation drove these oncogenic reprogramming by disturbing epigenetic landscape as increased levels of H3K36me2, H3K4me3 and H3K27ac but decreased levels of H3K27me3, which contributed to aberrant gene expression patterns. Conclusions: The NSD2 mutation led to increased tumor cell growth but decreased apoptosis due to the dysregulation of epigenetic landscape and gene expression, suggesting an oncogenic reprogramming driven by activated NSD2 mutations in MCL.

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3 (Bromodomain and WD repeat-containing protein 3), a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased H3K4me1 (H3 lysine 4 monomethylation) levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels but also causes a decrease in H3K4me3 (H3 lysine 4 trimethylation) levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific lysine demethylase 5 (KDM5/Lid), an enzyme that removes tri- and dimethyl marks from H3K4. Moreover, analysis of ChIP-seq (chromatin immunoprecipitation sequencing) data revealed that BRWD3 and KDM5 are significantly colocalized throughout the genome and H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

Histone Modification of Osteogenesis Related Genes Triggered by Substrate Topography Promotes Human Mesenchymal Stem Cell Differentiation.

The clinical success of orthopedic implants is closely related to their integration in the bone tissue promoted by rough device surfaces. The biological response of precursor cells to their artificial microenvironments plays a critical role in this process. In this study, we elucidated the relation between cell instructivity and surface microstructure of polycarbonate (PC)-based model substrates. The rough surface structure (hPC) with an average peak spacing (Sm) similar to the trabecular spacing of trabecular bone improved osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), as compared to the smooth surface (sPC) and the surface with a moderate Sm value (mPC). The hPC substrate promoted the cell adhesion and assembling of F-actin and enhanced cell contractile force by upregulating phosphorylated myosin light chain (pMLC) expression. The increased cell contractile force led to YAP nuclear translocation and the elongation of cell nuclei, presenting higher levels of active form of Lamin A/C. The nuclear deformation alternated the histone modification profile, particularly the decrease of H3K27me3 and increase of H3K9ac on the promoter region of osteogenesis related genes (ALPL, RUNX2, and OCN). Mechanism study using inhibitors and siRNAs elucidated the role of YAP, integrin, F-actin, myosin, and nuclear membrane proteins in such a regulatory process of surface topography on stem cell fate. These mechanistical insights on the epigenetic level give a new perspective in understanding of the interaction of substrate and stem cells as well as provide valuable criteria for designing bioinstructive orthopedic implants.

BASIC PENTACYSTEINE2 fine-tunes corm dormancy release in Gladiolus.

Bud dormancy is an important trait in geophytes that largely affects their flowering process and vegetative growth after dormancy release. Compared with seed dormancy, the regulation of bud dormancy is still largely unclear. Abscisic acid (ABA) acts as the predominant hormone that regulates the whole dormancy process. In Gladiolus (Gladiolus hybridus), cold storage promotes corm dormancy release (CDR) by repressing ABA biosynthesis and signaling. However, the mechanisms governing ABA-related processes during CDR via epigenetics are poorly understood. Here, we show that class I BASIC PENTACYSTEINE2, (GhBPC2) directly binds to 9-CIS-EPOXYCAROTENOID DIOXYGENASE (GhNCED) and ABA INSENSITIVE5 (GhABI5) loci and down-regulates their expression to accelerate CDR. During CDR, histone modifications change dramatically at the GhBPC2-binding loci of GhABI5 with an increase in H3K27me3 and a decrease in H3K4me3. GhBPC2 is involved in both H3K27me3 and H3K4me3 and fine-tunes GhABI5 expression by recruiting polycomb repressive complex 2 (PRC2) and the chromatin remodeling factor EARLY BOLTING IN SHORT DAYS (GhEBS). These results show GhBPC2 epigenetically regulates CDR in Gladiolus by mediating GhABI5 expression with PRC2 and GhEBS.

Epigenetic and Metabolic Changes in Diffuse Intrinsic Pontine Glioma.

Diffuse midline glioma (DMG), hitherto known as diffuse intrinsic pontine glioma (DIPG), is a rare and aggressive form of brain cancer that primarily affects children. Although the exact cause of DMG/DIPG is not known, a large proportion of DMG/DIPG tumors harbor mutations in the gene encoding the histone H3 protein, specifically the H3K27M mutation. This mutation decreases the level of H3K27me3, a histone modification that plays a vital role in regulating gene expression through epigenetic regulation. The mutation also alters the function of polycomb repressive complex 2 (PRC2), thereby preventing the repression of genes associated with cancer development. The decrease in H3K27me3 caused by the histone H3 mutation is accompanied by an increase in the level of H3K27ac, a post-translational modification related to active transcription. Dysregulation of histone modification markedly affects gene expression, contributing to cancer development and progression by promoting uncontrolled cell proliferation, tumor growth, and metabolism. DMG/DIPG alters the metabolism of methionine and the tricarboxylic acid cycle, as well as glucose and glutamine uptake. The role of epigenetic and metabolic changes in the development of DMG/DIPG has been studied extensively, and understanding these changes is critical to developing therapies targeting these pathways. Studies are currently underway to identify new therapeutic targets for DMG/DIPG, which may lead to the development of effective treatments for this devastating disease.

Reversion of methionine addiction of osteosarcoma cells to methionine independence results in loss of malignancy, modulation of the epithelial-mesenchymal phenotype and alteration of histone-H3 lysine-methylation.

Methionine addiction, a fundamental and general hallmark of cancer, known as the Hoffman Effect, is due to altered use of methionine for increased and aberrant transmethylation reactions. However, the linkage of methionine addiction and malignancy of cancer cells is incompletely understood. An isogenic pair of methionine-addicted parental osteosarcoma cells and their rare methionine-independent revertant cells enabled us to compare them for malignancy, their epithelial-mesenchymal phenotype, and pattern of histone-H3 lysine-methylation. Methionine-independent revertant 143B osteosarcoma cells (143B-R) were selected from methionine-addicted parental cells (143B-P) by their chronic growth in low-methionine culture medium for 4 passages, which was depleted of methionine by recombinant methioninase (rMETase). Cell-migration capacity was compared with a wound-healing assay and invasion capability was compared with a transwell assay in 143B-P and 143B-R cells in vitro. Tumor growth and metastatic potential were compared after orthotopic cell-injection into the tibia bone of nude mice in vivo. Epithelial-mesenchymal phenotypic expression and the status of H3 lysine-methylation were determined with western immunoblotting. 143B-P cells had an IC50 of 0.20 U/ml and 143B-R cells had an IC50 of 0.68 U/ml for treatment with rMETase, demonstrating that 143B-R cells had regained the ability to grow in low methionine conditions. 143B-R cells had reduced cell migration and invasion capability in vitro, formed much smaller tumors than 143B-P cells and lost metastatic potential in vivo, indicating loss of malignancy in 143B-R cells. 143B-R cells showed gain of the epithelial marker, ZO-1 and loss of mesenchymal markers, vimentin, Snail, and Slug and, an increase of histone H3K9me3 and H3K27me3 methylation and a decrease of H3K4me3, H3K36me3, and H3K79me3 methylation, along with their loss of malignancy. These results suggest that shifting the balance in histone methylases might be a way to decrease the malignant potential of cells. The present results demonstrate the rationale to target methionine addiction for improved sarcoma therapy.

Histone H3 proline 16 hydroxylation regulates mammalian gene expression.

Histone post-translational modifications (PTMs) are important for regulating various DNA-templated processes. Here, we report the existence of a histone PTM in mammalian cells, namely histone H3 with hydroxylation of proline at residue 16 (H3P16oh), which is catalyzed by the proline hydroxylase EGLN2. We show that H3P16oh enhances direct binding of KDM5A to its substrate, histone H3 with trimethylation at the fourth lysine residue (H3K4me3), resulting in enhanced chromatin recruitment of KDM5A and a corresponding decrease of H3K4me3 at target genes. Genome- and transcriptome-wide analyses show that the EGLN2-KDM5A axis regulates target gene expression in mammalian cells. Specifically, our data demonstrate repression of the WNT pathway negative regulator DKK1 through the EGLN2-H3P16oh-KDM5A pathway to promote WNT/β-catenin signaling in triple-negative breast cancer (TNBC). This study characterizes a regulatory mark in the histone code and reveals a role for H3P16oh in regulating mammalian gene expression.

The epigenetic state of EED-Gli3-Gli1 regulatory axis controls embryonic cortical neurogenesis.

SummaryMutations in the embryonic ectoderm development (EED) cause Weaver syndrome, but whether and how EED affects embryonic brain development remains elusive. Here, we generated a mouse model in which Eed was deleted in the forebrain to investigate the role of EED. We found that deletion of Eed decreased the number of upper-layer neurons but not deeper-layer neurons starting at E16.5. Transcriptomic and genomic occupancy analyses revealed that the epigenetic states of a group of cortical neurogenesis-related genes were altered in Eed knockout forebrains, followed by a decrease of H3K27me3 and an increase of H3K27ac marks within the promoter regions. The switching of H3K27me3 to H3K27ac modification promoted the recruitment of RNA-Pol2, thereby enhancing its expression level. The small molecule activator SAG or Ptch1 knockout for activating Hedgehog signaling can partially rescue aberrant cortical neurogenesis. Taken together, we proposed a novel EED-Gli3-Gli1 regulatory axis that is critical for embryonic brain development.

Aging-Associated Changes in Cognition, Expression and Epigenetic Regulation of Chondroitin 6-Sulfotransferase Chst3.

Understanding changes in the expression of genes involved in regulating various components of the neural extracellular matrix (ECM) during aging can provide an insight into aging-associated decline in synaptic and cognitive functions. Hence, in this study, we compared the expression levels of ECM-related genes in the hippocampus of young, aged and very aged mice. ECM gene expression was downregulated, despite the accumulation of ECM proteoglycans during aging. The most robustly downregulated gene was carbohydrate sulfotransferase 3 (Chst3), the enzyme responsible for the chondroitin 6-sulfation (C6S) of proteoglycans. Further analysis of epigenetic mechanisms revealed a decrease in H3K4me3, three methyl groups at the lysine 4 on the histone H3 proteins, associated with the promoter region of the Chst3 gene, resulting in the downregulation of Chst3 expression in non-neuronal cells. Cluster analysis revealed that the expression of lecticans—substrates of CHST3—is tightly co-regulated with this enzyme. These changes in ECM-related genes were accompanied by an age-confounded decline in cognitive performance. Despite the co-directional impairment in cognitive function and average Chst3 expression in the studied age groups, at the individual level we found a negative correlation between mRNA levels of Chst3 and cognitive performance within the very aged group. An analysis of correlations between the expression of ECM-related genes and cognitive performance in novel object versus novel location recognition tasks revealed an apparent trade-off in the positive gene effects in one task at the expense of another. Further analysis revealed that, despite the reduction in the Chst3 mRNA, the expression of CHST3 protein is increased in glial cells but not in neurons, which, however, does not lead to changes in the absolute level of C6S and even results in the decrease in C6S in perineuronal, perisynaptic and periaxonal ECM relative to the elevated expression of its protein carrier versican.

Abstract 1824: EZH2 inhibition induces blast differentiation in acute myeloid leukemia

Abstract Enhancer of zeste homolog 2 (EZH2) is a part of the polycomb repressive complex and catalyzes the trimethylation of lysine 27 on histone H3 (H3K27me3). EZH2 inhibition has a complex role in the pathogenesis of acute myeloid leukemia (AML), in that it has been shown to be either a tumor suppressor or an oncogene depending on the stage of AML development and the genes that EZH2 is regulating during each stage. Unlike follicular and diffuse large B-cell lymphoma where EZH2 mutations result in gain of function, EZH2 mutations are typically loss of function in myeloid diseases. However, we hypothesized that in AML patients without EZH2 mutations, loss of EZH2 function may produce a phenotype that would allow for therapeutic targeting without influencing normal hematopoiesis. We used EPZ011989 (EPZ), an EZH2 inhibitor tool compound, to inhibit H3K27me3 in our studies. We started by treating the MOLM-13 AML cell line with EPZ and confirmed a decrease in H3K27me3. This reduction in H3K27me3 resulted in a slight decrease in metabolic activity via MTS assays as well as decreased colony formation in methocult. These studies were followed up with EPZ inhibition in primary AML samples in vitro. We found that EZH2 inhibition resulted in decreased self-renewal of primary AML samples but not of CD34+ bone marrow cells from normal donors. Furthermore, we found that after 7-day treatment with EPZ, primary AML samples undergo moderate differentiation as suggested by an increase in CD11b surface expression via flow cytometry. These results are further supported by the morphological changes seen after 14-days of EPZ treatment in vitro. Based on these results, we hypothesize that EZH2 inhibition in primary AML samples promotes the differentiation of AML blasts. Furthermore, our preliminary data suggests that daily treatment with 150 mg/kg of EPZ results in a survival advantage and reduced disease burden in the MOLM-13-luciferase murine xenograft model. Despite loss of function EZH2 mutations portending poor outcomes in myeloid malignancies, we demonstrate that pharmacologic EZH2 inhibition reduces AML blast stemness and promotes differentiation into mature myeloid cells. In contrast, no change in normal CD34+ stem cells occurs with EZH2 inhibition, offering the opportunity to selectively target myeloid leukemia. Citation Format: Sydney Fobare, Ola A. Elgamal, Emily H. Stahl, Abeera Mehmood, Jean Truxall, Mariah L. Johnson, Amina Abdul-Aziz, John C. Byrd, Erin Hertlein. EZH2 inhibition induces blast differentiation in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1824.

L-Methionine prevents β-cell damage by modulating the expression of Arx, MafA and regulation of FOXO1 in type 1 diabetic rats

L-Methionine (L-Met) is an essential sulphur-containing amino acid having a vital role in various key cellular processes. Here we investigated the effect of L-Met on streptozotocin-induced β-cell damage model of diabetes mellitus in Sprague Dawley rats. At the end of study biochemical parameters, immunoblotting, qRT-PCR and ChIP-qPCR are performed. L-Met was administered orally (250 and 500 mg/kg/day) to diabetic animals for 8 weeks improved plasma glucose and insulin levels. Pancreas immunohistochemistry showed significant increase in insulin expression, decrease in glucagon and Bax expression. Interestingly, L-Met inhibited the expression of Arx; upregulated MafA and FOXO1 which play a critical role in the maintenance of β-cell identity. Our data also showed a decrease in H3K27me3 and an increase in H3K4me3 ("bivalent domain" alteration) in diabetic rats and these recovered by L-Met. Furthermore, the chromatin-immunoprecipitation assay showed a decreased enrichment of H3K27me3 on the promoter of the FOXO1 gene in diabetic rats and L-Met prevents this decrease. Our results showed the first evidence of the involvement of H3K27me3 in regulating the expression of the FOXO1 gene and the prevention of β-cell injury by L-Met treatment. In conclusion, we report the involvement of L-Met in the modulation of α-cell identity marker (Arx), β-cell identity marker (MafA) and regulation of FOXO1 by histone methylation marks for the first time. We are of the opinion that this employed as a novel therapeutic approach for mitigating diabetes-induced β-cell death.

Loss of Polycomb Repressive Complex 2 Function Alters Digestive Organ Homeostasis and Neuronal Differentiation in Zebrafish.

Polycomb repressive complex 2 (PRC2) mediates histone H3K27me3 methylation and the stable transcriptional repression of a number of gene expression programs involved in the control of cellular identity during development and differentiation. Here, we report on the generation and on the characterization of a zebrafish line harboring a null allele of eed, a gene coding for an essential component of the PRC2. Homozygous eed-deficient mutants present a normal body plan development but display strong defects at the level of the digestive organs, such as reduced size of the pancreas, hepatic steatosis, and a loss of the intestinal structures, to die finally at around 10–12 days post fertilization. In addition, we found that PRC2 loss of function impairs neuronal differentiation in very specific and discrete areas of the brain and increases larval activity in locomotor assays. Our work highlights that zebrafish is a suited model to study human pathologies associated with PRC2 loss of function and H3K27me3 decrease.

Abstract 2117: Immunomodulatory functions of SMYD3 in HPV-negative head and neck squamous cell carcinoma

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) causes approximately 10,000 deaths annually in the United States, with human-papilloma-virus (HPV)-negative patients having a particularly poor prognosis. Pembrolizumab, a programmed-death-1 (PD-1) antibody, was recently approved as a first-line therapy for patients with relapsed disease, however response rates are as low as 19%, underscoring the need for an improved understanding of immunotherapy resistance mechanisms. We have previously shown that HPV-negative HNSCC tumors with higher mRNA levels of SET and MYND Domain Containing 3 (SMYD3), a chromatin modifier known to trimethylated H3K4 (H3K4me3), have significantly lower mRNA levels of CD8 and CD8+ T-cell attracting chemokines, and that SMYD3 depletion in HNSCC cells in vitro leads to upregulation of CD8+ T-cell attracting chemokines. The goal of this work was to elucidate epigenetic mechanisms of immune evasion mediated by SMYD3 in HPV-negative HNSCC. Methods: siRNA- mediated SMYD3 knockdown was performed for 3 days to investigate immuno-modulatory epigenetic mechanisms mediated by SMYD3. RT-qPCR for immune-related genes, Western blotting for histone marks, ChIP-sequencing for H3K4me3, ChIP-qPCRs to determine genomic interactions of SMYD3 and ATAC-sequencing were performed. SMYD3 and CD8 immunohistochemical staining (IHC) of normal squamous, dysplastic epithelium and HPV-negative HNSCC tumors was also performed. Results: siRNA mediated knockdown of SMYD3 resulted in enrichment of inflammatory pathways and upregulation of type 1 interferon response and antigen presentation machinery genes in HNSCC cell lines. Immunoblotting for histone marks showed no reduction in H3K4me3 following SMYD3 depletion for 3 days. ChIP-sequencing for H3K4me3 showed differential distribution pattern with an increase of H3K4me3 peaks on immune-related genes, and a decrease of H3K4me3 on UHRF1, a reader of the repressive histone mark H3K9me3. RNA-seq, RT-PCR and ChIP-qPCR showed that SMYD3 may regulate the transcription of UHRF1 by binding to its promoter. SMYD3 depletion also led to decreased protein levels of UHRF1. ChIP for UHRF1 and H3K9me3 showed that UHRF1 binds directly on the promoters of CXCL9 and CXCL10 which are enriched for H3K9me3. IHC on Normal Squamous Epithelium and Squamous Cell Carcinoma tissues of increasing cancer stages showed increase in SMYD3 levels with the higher stage of cancer, and an inverse correlation with CD8+ T cell infiltration within the tumors. Conclusion: This is the first study reporting that the chromatin modifier SMYD3 may attenuate type I IFN responses in HPV-negative HNSCC by regulating the expression of a major epigenetic regulator, UHRF1, which may silence the expression of immune-related genes through H3K9me3. Citation Format: Nupur Nigam, Benjamin Jonah Bernard, Kyunghee Burkitt, Sohyoung Kim, Yvette Robbins, Richard L. Bennett, Jonathan D. Licht, Vassiliki Saloura. Immunomodulatory functions of SMYD3 in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2117.

The Role of MMSET E1099K Mutation in Gene Dysregulation, Drug Resistance and Clonogenicity in Acute B-Lymphoblastic Leukemia

Background: While outcomes for pediatric patients with newly-diagnosed acute B-lymphoblastic leukemia (ALL) have dramatically improved over the past 20 years, relapse remains a major cause of treatment failure and death. Discovering the underlying biological pathways that lead to drug resistance and relapse is therefore a top priority. MMSET (NSD2 / WHSC1) encodes a histone methyltransferase that dimethylates histone 3 lysine 36 (H3K36me2) with subsequent decrease in histone 3 lysine 27 trimethylation (H3K27me3). Overexpression of MMSET, as in the t(4;14) translocation in multiple myeloma (MM), has been shown to affect proliferation, gene expression, chromatin accessibility and DNA damage repair. Mutations in the SET domain of MMSET resulting in a glutamate to lysine substitution at amino acid 1099 (p.E1099K) have been recognized as one of the most common mutations in relapsed ALL in children. While it has been shown that this leads to increased enzyme activity, the downstream effects of this mutation and the underlying mechanisms of tumor escape are poorly understood. Understanding the biological pathways affected by this mutation is key to discovering new therapies to improve outcomes.Objective: To determine the role of the MMSET p.E1099K mutation in cell proliferation, drug resistance and gene regulation.Design/Method: B-lineage REH and 697 ALL cell lines were transduced with lentiviral vectors overexpressing either wild-type (WT) or mutant (p.E1099K) MMSET or an empty vector (EV) control. Western Blot analysis confirmed overexpression of MMSET as well as increase in H3K36me2 and decrease in H3K27me3 in overexpressed WT and mutant cell lines. The generated cell lines were then plated with and without stroma and evaluated for differences in apoptosis and proliferation in response to chemotherapeutic agents commonly used in the treatment of ALL (e.g. mercaptopurine, thioguanine, methotrexate, prednisone, cytarabine, etoposide, doxorubicin and L-asparaginase). Clonogenic growth in vitro was also assessed using MethoCultTM media. Differences in gene regulation in the REH cell lines were assessed by performing ChIPseq for CCCTC-binding factor (CTCF) and histone marks (H3K9Ac, H3K27Ac, H3K36me2 and H3K27me3) and the results were correlated with RNAseq data.Results: The transduced WT, mutant and EV cell lines did not display differences in proliferation, cell cycle regulation or adhesion. Moreover we were unable to detect any intrinsic drug resistance between the cell lines with respect to apoptosis or proliferation both with and without stroma in vitro . There was, however, a marked increase in clonogenicity in the cell lines overexpressing WT or mutant MMSET compared to the EV control (348% and 285% increased colony number, respectively) when plated in MethoCultTM media.Significant differences in the epigenetic landscape were observed between WT and mutant vs. EV control. As previously described in t(4;14) MM cell lines, over 99% of peaks for the H3K27me3 and H3K36me2 were lost in both the overexpressed WT and mutant cell lines compared to the EV control. However, whereas the decrease in peaks in H3K27me3 represents an overall loss of the mark, the lower peak number for H3K36me2 is actually due to widespread enrichment of the mark throughout the genome.Moreover, RNAseq data revealed significant differences in gene expression between the three cell lines. Notably, the overexpressed WT and mutant cell lines revealed 83 and 90 upregulated genes and 44 and 99 downregulated genes, respectively, compared to EV control. Notably, 70% of upregulated genes and 80% of downregulated genes were shared between cell lines. When compared with previously published data on t(4;14) MM cell lines, the genes upregulated by overexpressed WT or mutant MMSET appear to be unique to leukemia. Interestingly, ETV5, which is involved in transcriptional regulation , is upregulated in both our cell lines and MM cell lines.Conclusion: Catalytically active MMSET rendered by the p.E1099K substitution in ALL leads to an altered chromatin landscape and distinct gene expression signature (distinct from the t(4;14)) but does not result in intrinsic drug resistance in vitro . The increased clonogenic potency may suggest an increased leukemic stem cell population but understanding the precise mechanism for clonal expansion and relapse is the subject of additional in vitro and in vivo experiments. DisclosuresNo relevant conflicts of interest to declare.

The COMPASS-like complex modulates fungal development and pathogenesis by regulating H3K4me3-mediated targeted gene expression in Magnaporthe oryzae.

Histone‐3‐lysine‐4 (H3K4) methylation is catalysed by the multiprotein complex known as the Set1/COMPASS or MLL/COMPASS‐like complex, an element that is highly evolutionarily conserved from yeast to humans. However, the components and mechanisms by which the COMPASS‐like complex targets the H3K4 methylation of plant‐pathogenic genes in fungi remain elusive. Here we present a comprehensive analysis combining biochemical, molecular, and genome‐wide approaches to characterize the roles of the COMPASS‐like family in the rice blast fungus Magnaporthe oryzae, a model plant pathogen. We purified and identified six conserved subunits of COMPASS from M. oryzae: MoBre2 (Cps60/ASH2L), MoSpp1 (Cps40/Cfp1), MoSwd2 (Cps35), MoSdc1 (Cps25/DPY30), MoSet1 (MLL/ALL), and MoRbBP5 (Cps50), using an affinity tag on MoBre2. We determined the sequence repeat in dual‐specificity kinase splA and ryanodine receptors domain of MoBre2 can interact directly with the DPY30 domain of MoSdc1 in vitro. Furthermore, we found that deletion of the genes encoding COMPASS subunits of MoBre2, MoSPP1, and MoSwd2 caused similar defects regarding invasive hyphal development and pathogenicity. Genome‐wide profiling of H3K4me3 revealed that it has remarkable co‐occupancy at the transcription start site regions of target genes. Significantly, these target genes are often involved in spore germination and pathogenesis. Decreased gene expression caused by the deletion of MoBre2, MoSwd2, or MoSpp1 was highly correlated with a decrease in H3K4me3. These results suggest that MoBre2, MoSpp1, and MoSwd2 function as a whole COMPASS complex, contributing to fungal development and pathogenesis by regulating H3K4me3‐targeted genes in M. oryzae.

Identification of a differentiation stall in epithelial mesenchymal transition in histone H3-mutant diffuse midline glioma.

BackgroundDiffuse midline gliomas with histone H3 K27M (H3K27M) mutations occur in early childhood and are marked by an invasive phenotype and global decrease in H3K27me3, an epigenetic mark that regulates differentiation and development. H3K27M mutation timing and effect on early embryonic brain development are not fully characterized.ResultsWe analyzed multiple publicly available RNA sequencing datasets to identify differentially expressed genes between H3K27M and non-K27M pediatric gliomas. We found that genes involved in the epithelial-mesenchymal transition (EMT) were significantly overrepresented among differentially expressed genes. Overall, the expression of pre-EMT genes was increased in the H3K27M tumors as compared to non-K27M tumors, while the expression of post-EMT genes was decreased. We hypothesized that H3K27M may contribute to gliomagenesis by stalling an EMT required for early brain development, and evaluated this hypothesis by using another publicly available dataset of single-cell and bulk RNA sequencing data from developing cerebral organoids. This analysis revealed similarities between H3K27M tumors and pre-EMT normal brain cells. Finally, a previously published single-cell RNA sequencing dataset of H3K27M and non-K27M gliomas revealed subgroups of cells at different stages of EMT. In particular, H3.1K27M tumors resemble a later EMT stage compared to H3.3K27M tumors.ConclusionsOur data analyses indicate that this mutation may be associated with a differentiation stall evident from the failure to proceed through the EMT-like developmental processes, and that H3K27M cells preferentially exist in a pre-EMT cell phenotype. This study demonstrates how novel biological insights could be derived from combined analysis of several previously published datasets, highlighting the importance of making genomic data available to the community in a timely manner.

Low Maternal Dietary Intake of Choline Regulates Toll-Like Receptor 4 Expression Via Histone H3K27me3 in Fetal Mouse Neural Progenitor Cells.

Choline is an essential nutrient and a primary dietary source of methyl groups that are vital for brain development. Low choline (LC) in the maternal diet during pregnancy alters neurogenesis in the fetal brain and leads to low cognitive performance. However, the key signaling pathways that are sensitive to maternal choline supply during neural progenitor cell (NPC) development and the epigenetic mechanisms by which choline availability regulates gene expression are unclear. Timed-pregnant Nestin-CFPnuc transgenic mice are fed either a control diet or LC diet during E11-17. Gene expression changes in sorted E17 NPCs are identified by RNA sequencing. A maternal LC diet significantly increases Tlr4 transcription, causing premature neuronal differentiation and enhanced ethanol-induced NLRP3 inflammasome activation. No changes in DNA methylation at the Tlr4 gene promoter region are detected; however, a 70% decrease in H3K27me3 is observed in the LC-treated NPCs. Inhibition of EZH2 decreases H3K27me3 levels and increases Tlr4 expression. Conversely, the application of catalytically inactive Cas9 with EZH2 to increase H3K27me3 at the Tlr4 promoter causes reduced Tlr4 expression. These data reveal an epigenetic mechanism for the effect of maternal choline availability on brain development, suggesting a likely intervention for neurodevelopmental diseases.

NSD2-E1099K Mutation Leads to Glucocorticoid-Resistant B Cell Lymphocytic Leukemia in Mice

Background: NSD2 (nuclear receptor binding SET domain protein 2) is a histone methyltransferase specific for dimethylation of histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. In pediatric acute lymphoblastic leukemia (ALL), particularly at relapse, a gain of function mutation (E1099K) of NSD2 is found in 10-15% of cases. The NSD2 mutation is found in addition to fusion proteins such as E2A-PBX and ETV6-RUNX1. The mutation can be subclonal at diagnosis and dominant at relapse, suggesting a link to therapeutic resistance. The NSD2-E1099K mutation affects gene expression through an increase in H3K36me2 and a decrease in H3K27me3. Using CRISPR/Cas9-edited isogenic ALL cell lines, we found that NSD2-E1099K mutation drove oncogenic programming by altering chromatin architecture, gene expression and enhancing cell growth, migration and infiltration to the central neural system (CNS). NSD2 mutation caused resistance of ALL cells to glucocorticoids (GC) by blocking genome wide binding of the glucocorticoid receptor (GR, encoded by NR3C1 gene) preventing GC-mediated induction of pro-apoptotic genes. NR3C1 levels were depressed in NSD2-E1099K cells and GC failed to induce autoactivation of NR3C1. While H3K27me3 was globally decreased by NSD2-E1099K, increased H3K27me3 was noted at the promoter of NR3C1, suggesting a novel role of polycomb repressive complex 2 as a therapeutic target for relapsed ALL with NSD2 mutation. While NSD2 is highly expressed in B cells and NSD2 knockout causes defects in B cell development, how the NSD2 mutation affects B cell development and leukemia occurrence in vivo is uncertain. Aims: To determine the role of NSD2 mutation in the pathogenesis of lymphocytic malignancies and GC resistance in a mouse model. Methods: We generated a conditional NSD2-E1099K knock-in mouse model in which the NSD2-E1099K allele was placed in the Rosa26 locus and expressed in B cells under the control of Cd19-Cre (Cd19+/-NSD2E1099K/WT). The resulting phenotype was characterized through peripheral blood counts, cellular morphology and histology of blood smears, bone marrow (BM), spleen and liver, flow cytometric analysis, germinal center B cells (GCB) immunization, BM transplantation, and hematopoiesis analysis in a CD3-/- background. We further established mouse leukemia cell lines with NSD2 mutation for functional analysis. RNA-Seq, real time PCR, immunoblotting, and apoptosis analysis (Annexin V/PI staining) following GC treatment were performed to demonstrate the effects of NSD2 mutation on histone modifications, transcriptome and GC resistance. Results: The NSD2-E1099K mutation increased H3K36me2 and decreased H3K27me3 in isolated B cells from mouse BM and spleen. Mice were aged and did not develop signs of malignancy and RNA-sequencing showed few differences between B cells with or without the NSD2 mutation. However, after immunizing the mice with sheep red blood cells (SRBC), more GCBs were seen in the spleen of NSD2 mutant mice, suggesting mutant NSD2 stimulated germinal center hyperplasia. Transplantation of BM cells from mice expressing NSD2-E1099K into lethally irradiated recipients lead to an expansion of B cells while myeloid and T cells and life span of the recipients impaired. The NSD2 knock-in mouse model was crossed with Cd3-/- mice to create Cd19+/-Cd3-/-NSD2E1099K/WT mice, which within 2 months of birth developed a disease resembling an immature B lymphocytic leukemia (B220+CD19+IgM+IgD-CD5-) with infiltration of the spleen, liver and CNS and a median survival of 4.8 months. These tumors could be transplanted into immunodeficient mice but not immunocompetent mice. RNA seq analysis of these cells revealed 6,815 genes (3,295 upregulated and 3,520 downregulated) differentially expressed in NSD2 mutant B cells compared to normal B cells. The upregulated genes were related to abnormal immunoglobulin level , B cell activation, T-helper 1 physiology, and decreased B cell apoptosis. Importantly, the NSD2 mutant leukemic cells displayed depressed level of NR3C1 gene expression and GC resistance. Conclusions: The NSD2 mutation alters B cell development, particularly in an immunodeficient background and causes B cells to become resistant to glucocorticoids. The inability of the mutation to generate disease on its own except in an immunodeficient background suggests genes that collaborate with NSD2 in ALL may play a role in immune escape. Disclosures No relevant conflicts of interest to declare.

Abstract 462: The mutated chromatin modifier gene, H3F3AK27M, is a druggable target of DNA hypomethylating agents in pediatric high-grade glioma

Abstract Purpose: With the current standard of care for Pediatric high-grade glioma (pHGG) patients the 5-year survival rate remain low at 5-35%. Thus, there is an urgent need to develop improved therapies for pHGG patients. A specific point mutation of histone H3 (H3F3A) has been identified at amino acid 27 (lysine to methionine (K27M)), resulting in inhibition of EZH2 (histone H3K27 tri-methylase), and reduced global levels of H3K27 tri-methylation (H3K27me3). The H3K27M alteration can be found in the majority (~80%) of pHGGs and represents an attractive target for diagnostic and treatment. Since the H3K27me3 mark correlate with gene silencing, mutations resulting in global decrease in H3K27me3 may result in aberrant gene regulation. Majority of demethylated promoters remain silenced after treatment with hypomethylating agent (HMA) due to the presence of repressive H3K27me3. HMAs treatment is also rendered ineffective in case of repression of endogenous retroviruses (ERVs) due to the presence of H3K27me3. Hence, pHGGs harboring the H3K27M mutation with low levels of H3K27me3 represent an attractive therapeutic target for treatment with HMAs. HMAs are approved by FDA for treatment of several forms of human cancers. Methods: We had introduced mutant H3K27M in human pediatric glioma cell (SF188). Pediatric glioma cell lines (wild type and endogenous or endogenous expressed with H3K27M) were treated with DNA hypomethylating agents (HMAs). Cell doubling time was calculated. DNA methylation and gene expression were monitored at different time points. Additional RNA-seq data were obtained from pediatric glioma patient and patient derived cell lines. Results: We demonstrate that pHGG cells carrying H3K27M mutation are more aggressive and showed down-regulation of innate immune response genes (immune deficient or immune cold) and up-regulation of cancer stem like genes as compared to the wild-type pHGG cells. These findings are further confirmed with published data from patient-derived pHGG cells. The prolonged antitumor effects in pHGG cells with H3K27M mutation observed after HMA treatment may be due to the failure of DNA remethylation and upregulated innate immune response pathway genes due to ERV reactivation. Conclusion: Our findings suggest that HMA treatment may be effective against pHGGs with the H3K27M mutation and critical for converting immune deficient cells into immune reactive cells (immune hot). The success of this project will lead to clinical trials for pHGG patients to evaluate HMAs with immune-based therapies in pHGG patients, for whom there are currently no effective treatments. Citation Format: Hemant Gujar, Lian Zhang, Hong-Tao Li, Hitoshi Otani, Daniel J. Weisenberger, Anat Erdreich-Epstein, Gangning Liang. The mutated chromatin modifier gene, H3F3AK27M, is a druggable target of DNA hypomethylating agents in pediatric high-grade glioma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 462.

Low-Phosphate Chromatin Dynamics Predict a Cell Wall Remodeling Network in Rice Shoots.

Phosphorus (P) is an essential plant macronutrient vital to fundamental metabolic processes. Plant-available P is low in most soils, making it a frequent limiter of growth. Declining P reserves for fertilizer production exacerbates this agricultural challenge. Plants modulate complex responses to fluctuating P levels via global transcriptional regulatory networks. Although chromatin structure plays a substantial role in controlling gene expression, the chromatin dynamics involved in regulating P homeostasis have not been determined. Here we define distinct chromatin states across the rice (Oryza sativa) genome by integrating multiple chromatin marks, including the H2A.Z histone variant, H3K4me3 modification, and nucleosome positioning. In response to P starvation, 40% of all protein-coding genes exhibit a transition from one chromatin state to another at their transcription start site. Several of these transitions are enriched in subsets of genes differentially expressed under P deficiency. The most prominent subset supports the presence of a coordinated signaling network that targets cell wall structure and is regulated in part via a decrease of H3K4me3 at transcription start sites. The P starvation-induced chromatin dynamics and correlated genes identified here will aid in enhancing P use efficiency in crop plants, benefitting global agriculture.