H3K27me3 Research Articles

#1521 EZH2 promotes cyst growth in autosomal dominant polycystic kidney disease

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the leading hereditary kidney disease worldwide. Epigenetic modulations of genes and proteins play important roles in ADPKD pathogenesis. In this study, the role of enhancer of zeste homolog 2 (EZH2), a histone lysine methyltransferase, was investigated in ADPKD. Method To elucidate the role of EZH2 in ADPKD pathogenesis, we employed inducible knockout models, specifically targeting Pkd1 alone (Pkd1fl/fl) or both Pkd1 and EZH2 (Pkd1fl/fl: Ezh2fl/fl) in mouse models. Molecular analyses included assessing EZH2 expression levels and H3K27 trimethylation in Pkd1 knockout cells, kidney tissues from Pkd1 knockout mice, and samples from ADPKD patients. Functional investigations involved the use of EZH2-specific inhibitors, GSK126 and EPZ-6438, to inhibit EZH2 activity. Additionally, Ezh2 knockdown was employed to assess its impact on cystogenesis in MDCK cells. The embryonic kidney cyst model was utilized to evaluate the effects of GSK126 and EPZ-6438 on cyst growth. Results We found that EZH2 expression and the trimethylation of lysine 27 on histone H3 (H3K27) were upregulated in Pkd1 knockout cells and kidney tissues of Pkd1 knockout mice and ADPKD patients. Using EZH2-specific inhibitors GSK126/EPZ-6438 and knockdown of Ezh2 both suppressed cystogenesis in MDCK cells. GSK126 and EPZ-6438 delayed cyst growth in the embryonic kidney cyst model. Double knockout of Pkd1 and Ezh2 significantly decreased the kidney volume, cyst indexes, H3K27 methylation level, and the proliferation of renal cystic epithelial cells compared with Pkd1 knockout mice. Moreover, GSK126 delayed cyst growth and protected renal function in both early- and late-onset Pkd1 conditional knockout mice. EZH2 promoted the proliferation of renal cystic epithelial cells by activating STAT3 through H3K27 methylation. Conclusion Inhibiting EZH2 emerged as a promising strategy to impede cyst growth and attenuate disease progression across various PKD models. These findings establish a molecular foundation for the potential use of EZH2-specific inhibitors in delaying ADPKD cyst growth in the future.

#1409 Enhancer of zeste homolog 2 (EZH2) as a new therapeutic target to prevent indoxyl-sulfate-induced aortic valve calcification

Abstract Background and Aims Calcific aortic valve disease (CAVD) is highly prevalent in chronic kidney disease (CKD) patients and is associated with a poor prognosis. To date, there is no pharmacological treatment to slow down this process. Our group recently reported that indoxyl-sulfate (IS) -induced IL-6 secretion in human interstitial valvular cells (hVICs) promotes their osteogenic transition and calcification in an autocrine manner. Preliminary data suggest that IS also influences macrophage secretion of IL-6. Whether this phenomenon affects the hVICs mineralization remains to be elucidated. In this context, the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2), a key modulator of macrophages’ inflammation, may represent an interesting therapeutic target. Thus, this work aimed to verify whether IS-induced macrophages’ secretion of IL-6 modulates hVICs mineralization, and to determine whether EZH2 is involved in this inflammatory process. Method THP1-derived macrophages were exposed to increasing concentrations of IS (IS normal [Isn]: 0.5 μg/mL, IS uremic [Isu]: 37 μg/mL, IS intermediate [int]: 100 μg/mL or IS maximum [Ismax]: 233 μg/mL) in the presence or absence of an EZH2 inhibitor called GSK-343 (5µM). EZH2 expression and activity (evidenced by the trimethylation of the lysine 27 of the histones H3 (H3K27me3)) were assessed by western blot. Macrophages’ inflammatory potential was checked by RT-qPCR, western blot and Elisa. The impact of macrophages exposed or not to IS and/or GSK-343 on hVICs osteogenic transition was evaluated using conditioned media. HVICs osteogenic transition was assessed following runx2 expression by qRT-PCR and western blot. Mineralization was quantified by the o-cresolphthalein method. Results IS induced macrophages secretion of IL-1β, IL-6, CCL2 and TNF-α. Conditioned media from IS-polarized macrophages promoted a 6-fold increase in hVICs calcification and favored their osteogenic transition as evidenced by increased runx2 expression. IL-6 was the only cytokine able to promote runx2 expression in hVICs, confirming the importance of this specific cytokine in the process. Exposure to IS upregulated macrophages’ expression EZH2, an effect blocked by GSK-343. If GSK-343 did not affect IS-induced macrophages’ secretion of IL-1β, CCL2 and TNF-α, it efficiently blocked the secretion of IL-6 and subsequent induction of runx2 in hVICs. In this model, neither IS, nor GSK-343 modulated macrophages’ H3K27me3, suggesting that EZH2 may act as a transcriptional factor for IL-6 through its non-canonical pathway. In this latter, EZH2 is thought to promote inflammation by binding to p65, rather than through its trimethylation activity. In line with this hypothesis, exposure to IS promoted EZH2 and p65 nuclear translocation in the meantime as well as their co-immunoprecipitation, two phenomena blocked by GSK343. Conclusion This work demonstrates that IS-induced macrophages’ secretion of IL-6 promotes hVICs osteogenic transition and mineralization. Our data suggest that IL-6 secretion in response to IS depends on the EZH2-p65 pathway. The fact that GSK-343 can block this mechanism sheds light on EZH2 as a new therapeutic target to prevent CAVD in CKD patients.

Detection of tumor-derived cell-free DNA in cerebrospinal fluid using a clinically validated targeted sequencing panel for pediatric brain tumors.

Clinical sequencing of tumor DNA is necessary to render an integrated diagnosis and select therapy for children with primary central nervous system (CNS) tumors, but neurosurgical biopsy is not without risk. In this study, we describe cell-free DNA (cfDNA) in blood and cerebrospinal fluid (CSF) as sources for "liquid biopsy" in pediatric brain tumors. CSF samples were collected by lumbar puncture, ventriculostomy, or surgery from pediatric patients with CNS tumors. Following extraction, CSF-derived cfDNA was sequenced using UW-OncoPlex™, a clinically validated next-generation sequencing platform. CSF-derived cfDNA results and paired plasma and tumor samples concordance was also evaluated. Seventeen CSF samples were obtained from 15 pediatric patients with primary CNS tumors. Tumor types included medulloblastoma (n = 7), atypical teratoid/rhabdoid tumor (n = 2), diffuse midline glioma with H3 K27 alteration (n = 4), pilocytic astrocytoma (n = 1), and pleomorphic xanthoastrocytoma (n = 1). CSF-derived cfDNA was detected in 9/17 (53%) of samples, and sufficient for sequencing in 8/10 (80%) of extracted samples. All somatic mutations and copy-number variants were also detected in matched tumor tissue, and tumor-derived cfDNA was absent in plasma samples and controls. Tumor-derived cfDNA alterations were detected in the absence of cytological evidence of malignant cells in as little as 200µl of CSF. Several clinically relevant alterations, including a KIAA1549::BRAF fusion were detected. Clinically relevant genomic alterations are detectable using CSF-derived cfDNA across a range of pediatric brain tumors. Next-generation sequencing platforms are capable of producing a high yield of DNA alterations with 100% concordance rate with tissue analysis.

Regulation of cytokine and chemokine expression by histone lysine methyltransferase MLL1 in rheumatoid arthritis synovial fibroblasts

Histone lysine methylation is thought to play a role in the pathogenesis of rheumatoid arthritis (RA). We previously reported aberrant expression of the gene encoding mixed-lineage leukemia 1 (MLL1), which catalyzes methylation of histone H3 lysine 4 (H3K4), in RA synovial fibroblasts (SFs). The aim of this study was to elucidate the involvement of MLL1 in the activated phenotype of RASFs. SFs were isolated from synovial tissues obtained from patients with RA or osteoarthritis (OA) during total knee joint replacement. MLL1 mRNA and protein levels were determined after stimulation with tumor necrosis factor α (TNFα). We also examined changes in trimethylation of H3K4 (H3K4me3) levels in the promoters of RA-associated genes (matrix-degrading enzymes, cytokines, and chemokines) and the mRNA levels upon small interfering RNA-mediated depletion of MLL1 in RASFs. We then determined the levels of H3K4me3 and mRNAs following treatment with the WD repeat domain 5 (WDR5)/MLL1 inhibitor MM-102. H3K4me3 levels in the gene promoters were also compared between RASFs and OASFs. After TNFα stimulation, MLL1 mRNA and protein levels were higher in RASFs than OASFs. Silencing of MLL1 significantly reduced H3K4me3 levels in the promoters of several cytokine (interleukin-6 [IL-6], IL-15) and chemokine (C–C motif chemokine ligand 2 [CCL2], CCL5, C-X-C motif chemokine ligand 9 [CXCL9], CXCL10, CXCL11, and C-X3-C motif chemokine ligand 1 [CX3CL1]) genes in RASFs. Correspondingly, the mRNA levels of these genes were significantly decreased. MM-102 significantly reduced the promoter H3K4me3 and mRNA levels of the CCL5, CXCL9, CXCL10, and CXCL11 genes in RASFs. In addition, H3K4me3 levels in the promoters of the IL-6, IL-15, CCL2, CCL5, CXCL9, CXCL10, CXCL11, and CX3CL1 genes were significantly higher in RASFs than OASFs. Our findings suggest that MLL1 regulates the expression of particular cytokines and chemokines in RASFs and is associated with the pathogenesis of RA. These results could lead to new therapies for RA.

Epigenetic marks uniquely tune the material properties of HP1α condensates

Biomolecular condensates have emerged as a powerful new paradigm in cell biology with broad implications to human health and disease, particularly in the nucleus where phase separation is thought to underly elements of chromatin organization and regulation. Specifically, it has been recently reported that phase separation of heterochromatin protein 1alpha (HP1α) with DNA contributes to the formation of condensed chromatin states. HP1α localization to heterochromatic regions is mediated by its binding to specific repressive marks on the tail of histone H3, such as trimethylated lysine 9 on histone H3 (H3K9me3). However, whether epigenetic marks play an active role in modulating the material properties of HP1α and dictating emergent functions of its condensates remains to be understood. Here, we leverage a reductionist system, composed of modified and unmodified histone H3 peptides, HP1α, and DNA, to examine the contribution of specific epigenetic marks to phase behavior of HP1α. We show that the presence of histone peptides bearing the repressive H3K9me3 is compatible with HP1α condensates, whereas peptides containing unmodified residues or bearing the transcriptional activation mark H3K4me3 are incompatible with HP1α phase separation. Using fluorescence microscopy and rheological approaches, we further demonstrate that H3K9me3 histone peptides modulate the dynamics and viscoelastic network properties of HP1α condensates in a concentration-dependent manner. Additionally, in cells exposed to uniaxial strain, we find there to be a decreased ratio of nuclear H3K9me3 to HP1α. These data suggest that HP1α-DNA condensates are viscoelastic materials, whose properties may provide an explanation for the dynamic behavior of heterochromatin in cells and in response to mechanostimulation.

Epigenetic regulation of H3K27me3 in laying hens with fatty liver hemorrhagic syndrome induced by high-energy and low-protein diets

BackgroundFatty liver hemorrhagic syndrome (FLHS) in the modern poultry industry is primarily caused by nutrition. Despite encouraging progress on FLHS, the mechanism through which nutrition influences susceptibility to FLHS is still lacking in terms of epigenetics.ResultsIn this study, we analyzed the genome-wide patterns of trimethylated lysine residue 27 of histone H3 (H3K27me3) enrichment by chromatin immunoprecipitation-sequencing (ChIP-seq), and examined its association with transcriptomes in healthy and FLHS hens. The study results indicated that H3K27me3 levels were increased in the FLHS hens on a genome-wide scale. Additionally, H3K27me3 was found to occupy the entire gene and the distant intergenic region, which may function as silencer-like regulatory elements. The analysis of transcription factor (TF) motifs in hypermethylated peaks has demonstrated that 23 TFs are involved in the regulation of liver metabolism and development. Transcriptomic analysis indicated that differentially expressed genes (DEGs) were enriched in fatty acid metabolism, amino acid, and carbohydrate metabolism. The hub gene identified from PPI network is fatty acid synthase (FASN). Combined ChIP-seq and transcriptome analysis revealed that the increased H3K27me3 and down-regulated genes have significant enrichment in the ECM-receptor interaction, tight junction, cell adhesion molecules, adherens junction, and TGF-beta signaling pathways.ConclusionsOverall, the trimethylation modification of H3K27 has been shown to have significant regulatory function in FLHS, mediating the expression of crucial genes associated with the ECM-receptor interaction pathway. This highlights the epigenetic mechanisms of H3K27me3 and provides insights into exploring core regulatory targets and nutritional regulation strategies in FLHS.

EZH2 suppresses ferroptosis in hepatocellular carcinoma and reduces sorafenib sensitivity through epigenetic regulation of TFR2.

Enhancing sensitivity to sorafenib can significantly extend the duration of resistance to it, offering substantial benefits for treating patients with hepatocellular carcinoma (HCC). However, the role of ferroptosis in influencing sorafenib sensitivity within HCC remains pivotal. The enhancer of zeste homolog 2 (EZH2) plays a significant role in promoting malignant progression in HCC, yet the relationship between ferroptosis, sorafenib sensitivity, and EZH2 is not entirely clear. Bioinformatic analysis indicates elevated EZH2 expression in HCC, predicting an unfavorable prognosis. Overexpressing EZH2 can drive HCC cell proliferation while simultaneously reducing ferroptosis. Further analysis reveals that EZH2 amplifies the modification of H3K27 me3, thereby influencing TFR2 expression. This results in decreased RNA polymerase II binding within the TFR2 promoter region, leading to reduced TFR2 expression. Knocking down EZH2 amplifies sorafenib sensitivity in HCC cells. In sorafenib-resistant HepG2(HepG2-SR) cells, the expression of EZH2 is increased. Moreover, combining tazemetostat-an EZH2 inhibitor-with sorafenib demonstrates significant synergistic ferroptosis-promoting effects in HepG2-SR cells. In conclusion, our study illustrates how EZH2 epigenetically regulates TFR2 expression through H3K27 me3, thereby suppressing ferroptosis. The combination of the tazemetostat with sorafenib exhibits superior synergistic effects in anticancer therapy and sensitizes the HepG2-SR cells to sorafenib, shedding new light on delaying and ameliorating sorafenib resistance.

Repressive H3K27me3 drives hyperglycemia-induced oxidative and inflammatory transcriptional programs in human endothelium

BackgroundHistone modifications play a critical role in chromatin remodelling and regulate gene expression in health and disease. Histone methyltransferases EZH1, EZH2, and demethylases UTX, JMJD3, and UTY catalyse trimethylation of lysine 27 on histone H3 (H3K27me3). This study was designed to investigate whether H3K27me3 triggers hyperglycemia-induced oxidative and inflammatory transcriptional programs in the endothelium.MethodsWe studied human aortic endothelial cells exposed to high glucose (HAEC) or isolated from individuals with diabetes (D-HAEC). RT-qPCR, immunoblotting, chromatin immunoprecipitation (ChIP-qPCR), and confocal microscopy were performed to investigate the role of H3K27me3. We determined superoxide anion (O2−) production by ESR spectroscopy, NF-κB binding activity, and monocyte adhesion. Silencing/overexpression and pharmacological inhibition of chromatin modifying enzymes were used to modulate H3K27me3 levels. Furthermore, isometric tension studies and immunohistochemistry were performed in aorta from wild-type and db/db mice.ResultsIncubation of HAEC to high glucose showed that upregulation of EZH2 coupled to reduced demethylase UTX and JMJD3 was responsible for the increased H3K27me3. ChIP-qPCR revealed that repressive H3K27me3 binding to superoxide dismutase and transcription factor JunD promoters is involved in glucose-induced O2− generation. Indeed, loss of JunD transcriptional inhibition favours NOX4 expression. Furthermore, H3K27me3-driven oxidative stress increased NF-κB p65 activity and downstream inflammatory genes. Interestingly, EZH2 inhibitor GSK126 rescued these endothelial derangements by reducing H3K27me3. We also found that H3K27me3 epigenetic signature alters transcriptional programs in D-HAEC and aortas from db/db mice.ConclusionsEZH2-mediated H3K27me3 represents a key epigenetic driver of hyperglycemia-induced endothelial dysfunction. Targeting EZH2 may attenuate oxidative stress and inflammation and, hence, prevent vascular disease in diabetes.Graphical

Morphological Changes Induced by TKS4 Deficiency Can Be Reversed by EZH2 Inhibition in Colorectal Carcinoma Cells.

The scaffold protein tyrosine kinase substrate 4 (TKS4) undergoes tyrosine phosphorylation by the epidermal growth factor receptor (EGFR) pathway via Src kinase. The TKS4 deficiency in humans is responsible for the manifestation of a genetic disorder known as Frank-Ter Haar syndrome (FTHS). Based on our earlier investigation, the absence of TKS4 triggers migration, invasion, and epithelial-mesenchymal transition (EMT)-like phenomena while concurrently suppressing cell proliferation in HCT116 colorectal carcinoma cells. This indicates that TKS4 may play a unique role in the progression of cancer. In this study, we demonstrated that the enhancer of zeste homolog 2 (EZH2) and the histone methyltransferase of polycomb repressive complex 2 (PRC2) are involved in the migration, invasion, and EMT-like changes in TKS4-deficient cells (KO). EZH2 is responsible for the maintenance of the trimethylated lysine 27 on histone H3 (H3K27me3). We performed transcriptome sequencing, chromatin immunoprecipitation, protein and RNA quantitative studies, cell mobility, invasion, and proliferation studies combined with/without the EZH2 activity inhibitor 3-deazanoplanocine (DZNep). We detected an elevation of global H3K27me3 levels in the TKS4 KO cells, which could be reduced with treatment with DZNep, an EZH2 inhibitor. Inhibition of EZH2 activity reversed the phenotypic effects of the knockout of TKS4, reducing the migration speed and wound healing capacity of the cells as well as decreasing the invasion capacity, while the decrease in cell proliferation became stronger. In addition, inhibition of EZH2 activity also reversed most epithelial and mesenchymal markers. We investigated the wider impact of TKS4 deletion on the gene expression profile of colorectal cancer cells using transcriptome sequencing of wild-type and TKS4 knockout cells, particularly before and after treatment with DZNep. Additionally, we observed changes in the expression of several protein-coding genes and long non-coding RNAs that showed a recovery in expression levels following EZH2 inhibition. Our results indicate that the removal of TKS4 causes a notable disruption in the gene expression pattern, leading to the disruption of several signal transduction pathways. Inhibiting the activity of EZH2 can restore most of these transcriptomics and phenotypic effects in colorectal carcinoma cells.

266 Combination Drug Therapy in H3 K27-Altered Diffuse Midline Glioma Enhances Tumor Retention and Treatment Efficacy

INTRODUCTION: Diffuse midline gliomas (DMG) with H3 K27 alterations are uniformly lethal tumors for which no therapy has been found to be effective. One reason for our therapy failure is the blood-brain barrier (BBB), which severely restricts uptake and accumulation of systemically administered drugs in the brain. Convection-enhanced delivery (CED) has emerged as a technique that circumvents the BBB by directly infusing therapeutics into the tumor parenchyma through generation of a mechanical pressure gradient, allowing for homogenous drug distribution at high local concentrations with minimal systemic toxicity. However, efflux transport proteins expressed by the BBB and the tumor, including P-gp and BCRP, rapidly clear the majority of drugs given by CED, severely limiting its therapeutic potential. METHODS: A panel of FDA-approved drugs (ponatinib, topotecan, tretinoin) were evaluated alone and in combination with the known efflux transporter inhibitor everolimus. RESULTS: A high degree of formal synergy was observed in multiple DMG cell lines in vitro, which was attributed to everolimus’ ability to inhibit mTOR signaling and P-gp/BCRP. In vivo, drugs that were expected to penetrate the BBB and be less likely to undergo active efflux persisted at the site of infusion the longest following CED. Concurrent dosing with everolimus further enhanced this effect, which was particularly apparent with the dual P-gp/BCRP substrate ponatinib. CONCLUSIONS: The proclivity of many drugs to be rapidly cleared from the brain by efflux transporters generates a vulnerability that can be exploited for the development of novel therapeutic regimens in DMG, such as with concomitant dosing of P-gp/BCRP-inhibiting drugs when using CED.

Select EZH2 inhibitors enhance viral mimicry effects of DNMT inhibition through a mechanism involving NFAT:AP-1 signaling.

DNA methyltransferase inhibitor (DNMTi) efficacy in solid tumors is limited. Colon cancer cells exposed to DNMTi accumulate lysine-27 trimethylation on histone H3 (H3K27me3). We propose this Enhancer of Zeste Homolog 2 (EZH2)-dependent repressive modification limits DNMTi efficacy. Here, we show that low-dose DNMTi treatment sensitizes colon cancer cells to select EZH2 inhibitors (EZH2is). Integrative epigenomic analysis reveals that DNMTi-induced H3K27me3 accumulates at genomic regions poised with EZH2. Notably, combined EZH2i and DNMTi alters the epigenomic landscape to transcriptionally up-regulate the calcium-induced nuclear factor of activated T cells (NFAT):activating protein 1 (AP-1) signaling pathway. Blocking this pathway limits transcriptional activating effects of these drugs, including transposable element and innate immune response gene expression involved in viral defense. Analysis of primary human colon cancer specimens reveals positive correlations between DNMTi-, innate immune response-, and calcium signaling-associated transcription profiles. Collectively, we show that compensatory EZH2 activity limits DNMTi efficacy in colon cancer and link NFAT:AP-1 signaling to epigenetic therapy-induced viral mimicry.

Abstract 155: Clinico-radiological and histomolecular analyses identify of a new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered

Abstract Diffuse midline gliomas (DMG) H3 K27-altered are incurable grade 4 gliomas and represent a major challenge in neuro-oncology. This tumor type is now classified in four subtypes by the 2021 edition of the WHO Classification of the Central Nervous System (CNS) tumors. However, the H3.3-K27M subgroup still appears clinically and molecularly heterogenous. Recent publications reported that rare patients presenting a co-occurrence of H3.3K27M with BRAF or FGFR1 alterations tended to have a better prognosis. To better study the role of these co-driver alterations, we assembled a large pediatric and adult cohort of 29 tumors H3K27-altered with co-occurring activating mutation in BRAF or FGFR1 as well as 31 previous cases from the literature. We performed a comprehensive histological, radiological, genomic, transcriptomic and DNA methylation analysis. Interestingly, unsupervised t-distributed Stochastic Neighbor Embedding (tSNE) analysis of DNA methylation profiles regrouped BRAFV600E and all but one FGFR1MUT DMG in a unique methylation cluster, distinct from the other DMG subgroups and also from ganglioglioma (GG) or high-grade astrocytoma with piloid features (HGAP). This new DMG subtype harbors atypical radiological and histopathological profiles with calcification and/or a solid tumor component both for BRAFV600E and FGFR1MUT cases. Contrary to other DMG, these tumors occur more frequently in the thalamus (70% for BRAFV600E and 58% for FGFR1MUT) and patients have a longer overall survival with a median above three years. The analyses of a H3.3-K27M BRAFV600E tumor at diagnosis and corresponding in vitro cellular model showed that mutation in H3-3A was the first event in the oncogenesis, indicating a distinct oncogenesis from classical DMG despite an identical founder H3K27 alteration, and possible subclonal evolution.In conclusion, DMG, H3 K27 and BRAF/FGFR1 co-altered represent a new subtype of DMG with distinct genotype-phenotype characteristics, which deserve further attention with respect to trial interpretation and patient management. Our work also lays the foundations for therapeutic development highly-needed against this deadly disease. Citation Format: Lucie Auffret, Yassine Ajlil, Arnault Tauziede-Espariat, Thomas Kergrohen, Chloé Puiseux, Laurent Riffaud, Pascale Blouin, Anne-Isabelle Bertozzi, Pierre Leblond, Klas Blomgren, Sebastien Froelich, Alberto Picca, Mehdi Touat, Marc Sanson, Kevin Beccaria, Thomas Blauwblomme, Volodia Dangouloff-Ros, Nathalie Boddaert, Pascale Varlet, Marie-Anne Debily, Jacques Grill, David Castel. Clinico-radiological and histomolecular analyses identify of a new subtype of diffuse midline glioma, H3 K27 and BRAF/FGFR1 co-altered [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 155.

Abstract 1223: KDM6A orchestrates NK cell response via CD38/48 regulation in multiple myeloma

Abstract Daratumumab (Dara), a first-in-class humanized monoclonal antibody targeting CD38, demonstrates notable efficacy in both newly diagnosed and relapsed/refractory multiple myeloma (MM) when administrated alone or in combination with other agents. However, the development of Dara resistance and subsequent relapse is common.To identify the tumor-intrinsic genes involved in resistance to NK-mediated cytotoxicity in the presence of Dara in MM cells, we conducted an in vitro genome-wide CRISPR-Cas9 knockout (KO) screen and found 433 genes exhibiting a positive correlation with MM cytotoxicity elicited by Dara and primary NK cells. Remarkably, 16 of these genes demonstrated a consistent correlation with cytolytic activity across the majority of the 36 cancer types in the TCGA database.The efficacy of Dara is closely linked to the expression of CD38, with nonresponders exhibiting low CD38 expression. Therefore, we conducted another CRISPR screen to identify the genes whose KO resulted in diminished CD38 expression. The genes that overlapped in these two screens are implicated in regulating both Dara-mediated ADCC and CD38 expression. Our focus turned to KDM6A among the overlapping genes. We validated that KDM6A KO significantly decreased CD38 expression and that reintroducing KDM6A into KDM6A KO cells restored the expression of CD38 and Dara-induced cytotoxicity.KDM6A demethylates lysine 27 of histone H3 (H3K27) to promote gene expression. Our ChIP-seq data unveiled that KDM6A-KO cells exhibited an increased H3K27me3 level at the CD38 promoter area than control cells. Remarkably, this pattern was consistent with Dara-resistant cell lines, which displayed reduced CD38 expression alongside elevated levels of H3K27me3 at the CD38 promoter. Interestingly, we found that CD38 overexpression only partially restored the sensitivity of KDM6A KO cells to Dara treatment, implying the existence of an additional mechanism. We performed RNA-seq in KDM6A KO cells and found CD48, an NK-activating ligand, was significantly repressed. Overexpression of CD48 in KDM6A KO cells resulted in a substantial increase in the secretion of Granzyme B and Perforin by NK cells and restored the activity of NK cells and re-sensitized KDM6A KO cells to Dara treatment. These data suggest that KDM6A mediates Dara-mediated ADCC sensitivity through the dual regulation of CD38 and CD48. KDM6A acts to oppose the EZH2/PRC2 complex by demethylating H3K27me3, and we found that Tazemetostat (Taze), an FDA-approved EZH2 inhibitor, increased the expression of CD38 and CD48, especially in KDM6A-KO cells. Importantly, Taze also restored the sensitivity of KDM6A KO cells to Dara-mediated NK cell cytotoxicity. In summary, our findings illuminate the dual role of KDM6A in enhancing the efficacy of Dara in MM. Imitating KDM6A function using an FDA-approved inhibitor holds promise in overcoming Dara resistance and improving patient outcomes in MM. Citation Format: Jiye Liu, Lijie Xing, Jiang Li, Kenneth Wen, Ning Liu, Yuntong Liu, Annamaria Gulla, Nikhil Munshi, Teru Hideshima, Kenneth Carl Anderson. KDM6A orchestrates NK cell response via CD38/48 regulation in multiple myeloma [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 1223.

Abstract 1729: SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma

Abstract Human Papilloma Virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) poses a significant clinical challenge with limited treatment options. SUV420H1, which encodes for a protein lysine methyltransferase (PMT) that methylates H4K20 (H4K20me3), is recurrently amplified in ~5% of HPV-negative HNSCC tumors and is the second most frequently amplified PMT in this disease, suggesting an oncogenic function in this subset of HPV-negative HNSCC patients. Gene set enrichment analysis using the TCGA database of HPV-negative HNSCC tumors revealed that SUV420H1 overexpressing tumors showed enrichment in the mitotic spindle related pathways, and repression of immune-related pathways. To assess whether SUV420H1 depletion affects the proliferation of HPV-negative HSNCC cells, CCK8 and colony formation assays were pursued after siRNA-mediated knockdown of SUV420H1 in five SUV420H1-overexpressing HPV-negative HNSCC cell lines and revealed a significant reduction in cell proliferation and colony forming capacity. SUV420H1 CRISPR knockout (KO) HPV-negative HNSCC human and mouse cell lines were generated. Proliferation and colony formation assays with the SUV420H1 KO cell lines are ongoing to validate the aforementioned phenotypes. To identify oncogenic mechanism of SUV420H1, genome-wide mapping of H4K20me3 after siRNA-mediated depletion or enzymatic inhibition of SUV420H1 (A-196) was pursued using CUT&RUN assays combined with RNA-seq in a SUV420H1 amplified cell line (SCC-151), and analysis is ongoing. Preliminary RNA-seq analysis revealed upregulation of a number of type I IFN response genes after siRNA-mediated knockdown of SUV420H1. In vivo mouse experiments using SUV420H1 KO mouse cell lines are planned in a syngeneic HPV-negative HNSCC flank mouse model (MOC1) with or without anti-PD-1 treatment with the goal to assess whether SUV420H1 depletion affects tumor growth and/or sensitizes cancer cells to anti-PD-1 therapy. These data provide preliminary support for the function of SUV420H1 as an oncogene in HPV-negative HNSCC. Citation Format: Arfa Moshiri, Sohyoung Kim, Marie Luff, Vassiliki Saloura. SUV420H1 depletion reveals therapeutic potential in HPV-negative head and neck squamous cell carcinoma [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 1729.

Liquid–liquid phase separation of H3K27me3 reader BP1 regulates transcriptional repression

BackgroundBromo-adjacent homology-plant homeodomain domain containing protein 1 (BP1) is a reader of histone post-translational modifications in fungi. BP1 recognizes trimethylation of lysine 27 in histone H3 (H3K27me3), an epigenetic hallmark of gene silencing. However, whether and how BP1 participates in transcriptional repression remains poorly understood.ResultsWe report that BP1 forms phase-separated liquid condensates to modulate its biological function in Fusarium graminearum. Deletion assays reveal that intrinsically disordered region 2 (IDR2) of BP1 mediates its liquid–liquid phase separation. The phase separation of BP1 is indispensable for its interaction with suppressor of Zeste 12, a component of polycomb repressive complex 2. Furthermore, IDR2 deletion abolishes BP1-H3K27me3 binding and alleviates the transcriptional repression of secondary metabolism-related genes, especially deoxynivalenol mycotoxin biosynthesis genes.ConclusionsBP1 maintains transcriptional repression by forming liquid–liquid phase-separated condensates, expanding our understanding of the relationship between post-translational modifications and liquid–liquid phase separation.

Abstract A014: ClpP drives efficacy and mediates acquired resistance with imipridones ONC201 and ONC206 in glioma in vitro

Abstract A014: ClpP drives efficacy and mediates acquired resistance with imipridones ONC201 and ONC206 in glioma <i>in vitro</i>

Epigenetic control on transcription of vernalization genes and whole-genome gene expression profile induced by vernalization in common wheat

Vernalization is necessary for winter wheat to flower. However, it is unclear whether vernalization is also required for spring wheat, which is frequently sown in fall, and what molecular mechanisms underlie the vernalization response in wheat varieties. In this study, we examined the molecular mechanisms that regulate vernalization response in winter and spring wheat varieties. For this purpose, we determined how major vernalization genes (VRN1, VRN2, and VRN3) respond to vernalization in these varieties and whether modifications to histones play a role in changes in gene expression. We also identified genes that are differentially regulated in response to vernalization in winter and spring wheat varieties. We found that in winter wheat, but not in spring wheat, VRN1 expression decreases when returned to warm temperature following vernalization. This finding may be associated with differences between spring and winter wheat in the levels of tri-methylation of lysine 27 on histone H3 (H3K27me3) and tri-methylation of lysine 4 on histone H3 (H3K4me3) at the VRN1 gene. Analysis of winter wheat transcriptomes before and after vernalization revealed that vernalization influences the expression of several genes, including those involved in leucine catabolism, cysteine biosynthesis, and flavonoid biosynthesis. These findings provide new candidates for further study on the mechanism of vernalization regulation in wheat.

Single-genome analysis reveals a heterogeneous association of the herpes simplex virus genome with H3K27me2 and the reader PHF20L1 following infection of human fibroblasts.

Investigating the potential mechanisms of gene silencing for DNA viruses in different cell types is important to understand the differential outcomes of infection, particularly for viruses like herpesviruses that can undergo distinct types of infection in different cell types. In addition, investigating chromatin association with viral genomes informs on the mechanisms of epigenetic regulation of DNA processes. However, there is a growing appreciation for heterogeneity in the outcome of infection at the single cell, and even single viral genome, level. Here we describe a novel assay for quantifying viral genome foci with chromatin proteins and show that a portion of genomes are targeted for silencing by H3K27me2 and associate with the reader protein PHF20L1. This study raises important questions regarding the mechanism of H3K27me2-specific targeting to viral genomes, the contribution of epigenetic heterogeneity to herpesvirus infection, and the role of PHF20L1 in regulating the outcome of DNA virus infection.

AGAMOUS-LIKE24 controls pistil number in Japanese apricot by targeting the KNOTTED1-LIKE gene KNAT2/6-a.

The formation of multi-pistil flowers reduces the yield and quality in Japanese apricot (Prunus mume). However, the molecular mechanism underlying the formation of multi-pistil flowers remains unknown. In the current study, overexpression of PmKNAT2/6-a, a class I KNOTTED1-like homeobox (KNOX) member, in Arabidopsis (Arabidopsis thaliana) resulted in a multi-pistil phenotype. Analysis of the upstream regulators of PmKNAT2/6-a showed that AGAMOUS-like 24 (PmAGL24) could directly bind to the PmKNAT2/6-a promoter and regulate its expression. PmAGL24 also interacted with Like Heterochromatin Protein 1 (PmLHP1) to recruit lysine trimethylation at position 27 on histone H3 (H3K27me3) to regulate PmKNAT2/6-a expression, which is indirectly involved in multiple pistils formation in Japanese apricot flowers. Our study reveals that the PmAGL24 transcription factor, an upstream regulator of PmKNAT2/6-a, regulates PmKNAT2/6-a expression via direct and indirect pathways and is involved in the formation of multiple pistils in Japanese apricot.

Association Between COVID-19 and Neurological Diseases: Evidence from Large-Scale Mendelian Randomization Analysis and Single-Cell RNA Sequencing Analysis

Observational studies have suggested that SARS-CoV-2 infection increases the risk of neurological diseases, but it remains unclear whether the association is causal. The present study aims to evaluate the causal relationships between SARS-CoV-2 infections and neurological diseases and analyzes the potential routes of SARS-CoV-2 entry at the cellular level. We performed Mendelian randomization (MR) analysis with CAUSE method to investigate causal relationship of SARS-CoV-2 infections with neurological diseases. Then, we conducted single-cell RNA sequencing (scRNA-seq) analysis to obtain evidence of potential neuroinvasion routes by measuring SARS-CoV-2 receptor expression in specific cell subtypes. Fast gene set enrichment analysis (fGSEA) was further performed to assess the pathogenesis of related diseases. The results showed that the COVID-19 is causally associated with manic (delta_elpd, − 0.1300, Z-score: − 2.4; P = 0.0082) and epilepsy (delta_elpd: − 2.20, Z-score: − 1.80; P = 0.038). However, no significant effects were observed for COVID-19 on other traits. Moreover, there are 23 cell subtypes identified through the scRNA-seq transcriptomics data of epilepsy, and SARS-CoV-2 receptor TTYH2 was found to be specifically expressed in oligodendrocyte and astrocyte cell subtypes. Furthermore, fGSEA analysis showed that the cell subtypes with receptor-specific expression was related to methylation of lysine 27 on histone H3 (H3K27ME3), neuronal system, aging brain, neurogenesis, and neuron projection. In summary, this study shows causal links between SARS-CoV-2 infections and neurological disorders such as epilepsy and manic, supported by MR and scRNA-seq analysis. These results should be considered in further studies and public health measures on COVID-19 and neurological diseases.