H3K27me3 Research Articles

Feasibility of probe washing after stereotactic needle biopsy as a novel technique for developing cell lines and xenografts of H3 K27-altered diffuse midline gliomas.

H3 K27-altered diffuse midline gliomas (DMGs) are frequently biopsied to obtain tissue diagnosis, inform clinical decision-making, and determine clinical trial eligibility. Tissue yield from biopsies is typically low, leaving little material available for research. To advance understanding of disease biology and promote preclinical testing of novel therapeutics, collecting viable cellular material from treatment-naive tumors is of paramount importance. Here, the authors report the feasibility of a practicable technique for creating DMG cell lines and patient-derived xenografts (PDXs) without the need for additional biopsy specimens. Tumor cells are obtained by probe washing immediately after completion of biopsy. Wash fluid is collected, and viable cells are expanded in vitro. Cultured cells are used to establish PDX rodent models. A total of 5 patient samples were collected by this technique. Viable tumor cells were obtained from 3 of the 5 samples, and cell lines suitable for experiments were obtained within 6-8 months. Orthotopic implantation and flank engraftment was successful in 1 of the 3 established cell lines. Animals harboring intracranial tumors were euthanized due to disease burden 6-7 months after stereotactic injection. Flank tumors formed within 4-5 months and were serially passaged. Molecular and tissue analyses confirmed retention of H3 K27M expression and loss of H3 K27me3 in all cell lines and PDXs.

Leptomeningeal metastases in glioma revisited: incidence and molecular predictors based on postcontrast fluid-attenuated inversion recovery imaging.

Leptomeningeal metastases (LMs) in glioma have been underestimated given their low incidence and the lack of reliable imaging. Authors of this study aimed to investigate the real-world incidence of LMs using cerebrospinal fluid (CSF)-sensitive imaging, namely postcontrast fluid-attenuated inversion recovery (FLAIR) imaging, and to analyze molecular predictors for LMs in the molecular era. A total of 1405 adult glioma (World Health Organization [WHO] grade 2-4) patients underwent postcontrast FLAIR imaging at initial diagnosis and during treatment monitoring between 2001 and 2021. Collected molecular data included isocitrate dehydrogenase (IDH) mutation, 1p/19q codeletion, H3 K27 alteration, and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status. LM diagnosis was performed with MRI including postcontrast FLAIR sequences. Logistic regression analysis for LM development was performed with molecular, clinical, and imaging data. Overall survival (OS) was compared between patients with and those without LM. LM was identified in 228 patients (16.2%), 110 (7.8%) at the initial diagnosis and 118 (8.4%) at recurrence. Among the molecular diagnostics, IDH-wildtype (OR 3.14, p = 0.001) and MGMT promoter unmethylation (OR 1.43, p = 0.034) were independent predictors of LM. WHO grade 4 (OR 10.52, p < 0.001) and nonlobar location (OR 1.56, p = 0.048) were associated with LM at initial diagnosis, whereas IDH-wildtype (OR 5.04, p < 0.001) and H3 K27 alteration (OR 3.39, p = 0.003) were associated with LM at recurrence. Patients with LM had a worse median OS than those without LM (16.7 vs 32.0 months, p < 0.001, log-rank test), which was confirmed as an independent factor on multivariable Cox analysis (p = 0.004). CSF-sensitive imaging aids the diagnosis of LM, demonstrating a high incidence of LM in adult gliomas. Furthermore, molecular markers are associated with LM development in glioma, and patients with aggressive molecular markers warrant imaging surveillance for LM.

WDR82-Mediated H3K4me3 Is Associated with Tumor Proliferation and Therapeutic Efficacy in Pediatric High-Grade Gliomas.

Pediatric high-grade gliomas (pHGGs) are common malignant brain tumors without effective treatment and poor patient survival. Abnormal posttranslational modification at the histone H3 tail plays critical roles in tumor cell malignancy. We have previously shown that the trimethylation of lysine 4 at histone H3 (H3K4me3) plays a significant role in pediatric ependymoma malignancy and is associated with tumor therapeutic sensitivity. Here, we show that H3K4me3 and its methyltransferase WDR82 are elevated in pHGGs. A reduction in H3K4me3 by downregulating WDR82 decreases H3K4me3 promoter occupancy and the expression of genes associated with stem cell features, cell proliferation, the cell cycle, and DNA damage repair. A reduction in WDR82-mediated H3K4me3 increases the response of pediatric glioma cells to chemotherapy. These findings suggest that WDR82-mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response. This highlights the need for a more thorough understanding of the potential of WDR82 as an epigenetic target to increase therapeutic efficacy and improve the prognosis for children with malignant gliomas.

Protein Arginine Methyltransferase 5 Contributes to Paclitaxel-Induced Neuropathic Pain by Activating Transient Receptor Potential Vanilloid 1 Epigenetic Modification in Dorsal Root Ganglion.

Paclitaxel (PTX), which is a first-line chemotherapy drug used to treat various types of cancers, exhibits peripheral neuropathy as a common side effect that is difficult to treat. Protein arginine methyltransferase 5 (PRMT 5) is a key regulator of the chemotherapy response, as chemotherapy drugs induce PRMT5 expression. However, little is known about the PRMT5-mediated epigenetic mechanisms involved in PTX-induced neuropathic allodynia. Sprague-Dawley rats were intraperitoneally given PTX to induce neuropathic pain. Biochemical analyses were conducted to measure the protein expression levels in the dorsal root ganglion (DRG) of the animals. The von Frey test and hot plate test were used to evaluate nociceptive behaviors. PTX increased the PRMT5 (mean difference [MD]: 0.68, 95% confidence interval [CI], 0.88-0.48; P < .001 for vehicle)-mediated deposition of histone H3R2 dimethyl symmetric (H3R2me2s) at the transient receptor potential vanilloid 1 ( Trpv1 ) promoter in the DRG. PRMT5-induced H3R2me2s recruited WD repeat domain 5 (WDR5) to increase trimethylation of lysine 4 on histone H3 (H3K4me3) at Trpv1 promoters, thus resulting in TRPV1 transcriptional activation (MD: 0.65, 95% CI, 0.82-0.49; P < .001 for vehicle) in DRG in PTX-induced neuropathic pain. Moreover, PTX increased the activity of NADPH oxidase 4 (NOX4) (MD: 0.66, 95% CI, 0.81-0.51; P < .001 for vehicle), PRMT5-induced H3R2me2s, and WDR5-mediated H3K4me3 in the DRG in PTX-induced neuropathic pain. Pharmacological antagonism and the selective knockdown of PRMT5 in DRG neurons completely blocked PRMT5-mediated H3R2me2s, WDR5-mediated H3K4me3, or TRPV1 expression and neuropathic pain development after PTX injection. Remarkably, NOX4 inhibition not only attenuated allodynia behavior and reversed the above-mentioned signaling but also reversed NOX4 upregulation via PTX. Thus, the NOX4/PRMT5-associated epigenetic mechanism in DRG has a dominant function in the transcriptional activation of TRPV1 in PTX-induced neuropathic pain.

Detection of Modified Histones from Oral Mucosa of a Patient with DYT-KMT2B Dystonia

Introduction: DYT-KMT2B is a rare childhood-onset, hereditary movement disorder typically characterized by lower-limb dystonia and subsequently spreads into the craniocervical and laryngeal muscles. Recently, KMT2B-encoding lysine (K)-specific histone methyltransferase 2B was identified as the causative gene for DYT-KMT2B, also known as DYT28. In addition to the fact that many physicians do not have sufficient experience or knowledge of hereditary dystonia, the clinical features of DYT-KMT2B overlap with those of other hereditary dystonia, and limited clinical biomarkers make the diagnosis difficult. Methods: Histone proteins were purified from the oral mucosa of patients with de novo KMT2B mutation causing premature stop codon, and then trimethylated fourth lysine residue of histone H3 (H3K4me3) which was catalyzed by KMT2B was analyzed by immunoblotting with specific antibody. We further analyzed the significance of H3K4me3 in patients with DYT-KMT2B using publicly available datasets. Results: H3K4me3 histone mark was markedly lower in the patient than in the control group. Additionally, a reanalysis of publicly available datasets concerning DNA methylation also demonstrated that KMT2B remained inactive in DYT-KMT2B. Discussion: Although only one case was studied due to the rarity of the disease, the reduction of H3K4me3 in the patient’s biological sample supports the dysfunction of KMT2B in DYT-KMT2B. Together with informatics approaches, our results suggest that KMT2B haploinsufficiency contributes to the DYT-KMT2B pathogenic process.

Correlation between Multiparametric MR Imaging and Molecular Genetics in Pontine Pediatric High-Grade Glioma.

Molecular profiling is a crucial feature in the "integrated diagnosis" of CNS tumors. We aimed to determine whether radiomics could distinguish molecular types of pontine pediatric high-grade gliomas that have similar/overlapping phenotypes on conventional anatomic MR images. Baseline MR images from children with pontine pediatric high-grade gliomas were analyzed. Retrospective imaging studies included standard precontrast and postcontrast sequences and DTI. Imaging analyses included median, mean, mode, skewness, and kurtosis of the ADC histogram of the tumor volume based on T2 FLAIR and enhancement at baseline. Histone H3 mutations were identified through immunohistochemistry and/or Sanger or next-generation DNA sequencing. The log-rank test identified imaging factors prognostic of survival from the time of diagnosis. Wilcoxon rank-sum and Fisher exact tests compared imaging predictors among groups. Eighty-three patients had pretreatment MR imaging and evaluable tissue sampling. The median age was 6 years (range, 0.7-17 years); 50 tumors had a K27M mutation in H3-3A, and 11, in H3C2/3. Seven tumors had histone H3 K27 alteration, but the specific gene was unknown. Fifteen were H3 wild-type. Overall survival was significantly higher in H3C2/3- compared with H3-3A-mutant tumors (P = .003) and in wild-type tumors compared with any histone mutation (P = .001). Lower overall survival was observed in patients with enhancing tumors (P = .02) compared with those without enhancement. H3C2/3-mutant tumors showed higher mean, median, and mode ADC_total values (P < .001) and ADC_enhancement (P < .004), with lower ADC_total skewness and kurtosis (P < .003) relative to H3-3A-mutant tumors. ADC histogram parameters are correlated with histone H3 mutation status in pontine pediatric high-grade glioma.

PGC7 regulates H3K27me3 modification by inhibiting the interaction of YY1 with PRC2.

Primordial germ cell 7 (PGC7)(Dppa3 or Stella) is a small inherently disordered protein that is mainly expressed in oocytes and plays a vital role in the regulation of DNA methylation reprogramming in imprinted loci through interaction with other proteins. Most of PGC7-deficient zygotes are blocked at two-cell stage with an increased tri-methylation at lysine 27 of histone H3 (H3K27me3) level in the nucleus. Our previous work has indicated that PGC7 interacts with yin-yang1 (YY1) that is essential for the recruitment of enhancer of zeste homolog 2 (EZH2)-containing Polycomb repressive complex 2 (PRC2) to H3K27me3 modification sites. Here, we found that the presence of PGC7 weakened the interaction between YY1 and PRC2 without disrupting the assembly of core subunits of the PRC2 complex. In addition, PGC7 promoted AKT to phosphorylate serine 21 of EZH2, resulting in inhibition of EZH2 activity and the dissociation of EZH2 from YY1, thereby decreasing H3K27me3 level. In zygotes, the PGC7-deficient and AKT inhibitor MK2206 both promoted EZH2 to enter the pronuclei but without disturbing the subcellular localization of YY1 and caused an increase in the level of H3K27me3 in the pronuclei, as well as inhibition of the expression of zygote-activating genes regulated by H3K27me3 in two-cell embryos. In summary, PGC7 could affect zygotic genome activation during early embryonic development by regulating the level of H3K27me3 through regulation of PRC2 recruitment, EZH2 activity, and subcellular localization.NEW & NOTEWORTHY PGC7 and YY1 interaction inhibits recruitment of PRC2 by YY1. PGC7 promotes AKT and EZH2 interaction to increase pEZH2-S21 level, which weakens YY1 and EZH2 interaction, thereby decreasing H3K27me3 level. In zygotes, the PGC7-deficient and AKT inhibitor MK2206 promote EZH2 to enter the pronuclei, and increase H3K27me3 level in the pronuclei, as well as inhibition of the expression of zygote-activating genes regulated by H3K27me3 in two-cell embryos, which ultimately affects early embryo development.

Trophoblast PR-SET7 dysfunction induces viral mimicry response and necroptosis associated with recurrent miscarriage

Recurrent miscarriage (RM) is a distressing pregnancy complication. While the etiology of RM remains unclear, growing evidence has indicated the relevance of trophoblast impairment to the pathogenesis of RM. PR-SET7 is the sole enzyme catalyzing monomethylation of H4K20 (H4K20me1) and has been implicated in many pathophysiological processes. However, how PR-SET7 functions in trophoblasts and its relevance to RM remain unknown. Here, we found that trophoblast-specific loss of Pr-set7 in mice led to defective trophoblasts, resulting in early embryonic loss. Mechanistic analysis revealed that PR-SET7 deficiency in trophoblasts derepressed endogenous retroviruses (ERVs), leading to double-stranded RNA stress and subsequent viral mimicry, which drove overwhelming interferon response and necroptosis. Further examination discovered that H4K20me1 and H4K20me3 mediated the inhibition of cell-intrinsic expression of ERVs. Importantly, dysregulation of PR-SET7 expression and the corresponding aberrant epigenetic modifications were observed in the placentas of RM. Collectively, our results demonstrate that PR-SET7 acts as an epigenetic transcriptional modulator essential for repressing ERVs in trophoblasts, ensuring normal pregnancy and fetal survival, which sheds new light on potential epigenetic causes contributing to RM.

DIPG-16. ACUTE VERSUS PROLONGED CONVECTION-ENHANCED DELIVERY OF ALISERTIB INTO H3 K27M-MUTANT MODELS OF DIFFUSE MIDLINE GLIOMA

Abstract Convection-enhanced delivery (CED) is a potentially promising strategy to administer therapeutics directly into the brainstem of children harboring H3 K27-altered diffuse midline glioma (DMG). While early-phase clinical trials have demonstrated the safety of this technique, efficacy has yet to be achieved. Here, we describe how different variations of CED may be exploited to extend survival in a DMG murine patient-derived orthotopic xenograft model. Either 1-day (8 µL/hr) or 7-day (1 µL/hr) continuous CED infusions were performed to deliver an equivalent volume of the aurora kinase inhibitor alisertib to the mouse brainstem. Bioluminescence and magnetic resonance images were acquired to monitor tumor progression, validate CED catheter positioning, and analyze infusion-related imaging changes. Both infusion regimens significantly prolonged survival versus control (median survival benefit of 6.5 and 8 days, p=0.03 and p=0.01, for 1-day and 7-day CED, respectively). Postmortem examination of brains revealed no signs of tissue necrosis, cavitary lesions, or cellular (inflammatory) infiltrate at the site of infusion. However, the tumor parenchyma surrounding the cannula tract was markedly hypocellular in treated animals. Alisertib induced a robust increase in H3 K27 trimethylation along with decreased H3 K27M and Ki-67. Our findings indicate that CED of alisertib is well tolerated and effective, in both the acute and prolonged setting, underscoring the potential of this approach in the management of DMG.

DIPG-24. BRD4 INHIBITION AS A RADIOSENSITIZER THROUGH BLOCKING DNA REPAIR FOR THE TREATMENT OF DIFFUSE MIDLINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) is one of the devastating childhood cancers. Radiation therapy (RT) remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance RT efficacy by combining it with radiosensitizing agents, though none have been successful in doing so. Given this, there is a critical need to identify effective therapeutics to enhance the RT anti-tumor activity in DIPG. Here, we identified BRD4 inhibitors as candidate radiosensitizers from a high throughput drug screening. DIPG cells show increased H3 K27 acetylation (H3K27ac) levels, which bind to BET bromodomain protein 4 (BRD4) and are strongly associated with active transcription. We tested two BRD4 inhibitors (BRD4i: AZD5153 and JQ-1) as well as genetic BRD4 depletion, and observed enhancement of radiation-induced DNA damage, confirming BRD4i as potential radiosensitizers in the treatment of DIPG. We evaluated the effects of BRD4i on gene expression using RNAseq, and observed significant inhibition of DNA-repair proteins such as BRCA1 and RAD51. CUT-RUN qPCR showed decreased H3K27ac at BRCA1 and RAD51 promoters. Combination of BRD4i and RT inhibited cell viability significantly using clonogenic survival assay, apoptosis assay, and also showed cell cycle arrest. Radiation-induced DNA double-strand break (DSB) repair was prolonged with BRD4i with high levels of gH2AX and 53BP1 likely due to inhibition of DNA-repair. In vivo studies revealed increased survival of animals treated with combination therapy of RT and BRD4i in comparison to either monotherapy. Together, these results highlight BRD4i as a potential radiosensitizer and provide a rationale for developing combination therapy with radiation in the treatment of DIPG.

DIPG-15. CONCURRENT GB-13 (IL13.E13K-PE4E) IMPROVES THE EFFICACY OF RADIATION THERAPY IN DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse midline glioma with H3 K27 alterations (DMG) is the most aggressive primary malignant brain tumor in the pediatric population. Radiation therapy (RT) is the standard-of-care; however, it has not demonstrated a significant improvement in overall survival or stalled disease progression, and children usually succumb to disease within 12-15 months of diagnosis. Consequently, finding efficient treatments for DMG remains one of the most critical unmet needs in modern neuro-oncology. Interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is a cell-surface receptor upregulated in ~80% of DMG versus normal brain, posing a potentially promising therapeutic target. Immunotherapies harnessing IL-13Rα2 to selectively delivery cytotoxic payloads, such as pseudomonas exotoxin A (PE), to tumor cells have been previously demonstrated to be safe in DMG patients and efficacious in preclinical models of DMG with high IL-13Rα2 expression using convection-enhanced delivery (CED). Furthermore, there is evidence that PE-based therapies can sensitize cells to RT. In this study, we utilized in vitro and in vivo models of DMG to evaluate clinically-relevant combination treatments of RT and the IL-13/PE immunotoxin GB-13 (IL13.E13K-PE4E). GB-13 improved the effectiveness of low-dose RT in multiple DMG cell lines by promoting caspase activation, which boosted apoptotic cell death. Intratumoral administration of GB-13 via chronic CED decreased tumor burden and prolonged survival in both DMG patient-derived orthotopic xenograft and genetically engineered mouse models. When GB-13 and RT were administered concurrently, this impact became even more prominent, informing future preclinical and clinical investigation of a potentially efficacious therapeutic approach in a subset of DMG.

DIPG-44. DIFFUSE MIDLINE GLIOMA WITH EXTRACRANIAL METASTASIS

Abstract Diffuse midline gliomas (DMGs) are malignant infiltrative gliomas enriched in the pediatric population and characterized by loss of the H3 K27me3 epigenetic marker, most frequently via mutation of the H3F3A gene. Few cases of extracranial DMG metastasis with redemonstrated H3F3A p.K28M (K27M) mutation in metastatic tissue are reported in the literature. Here, we report two such patients, both females (ages 12 and 15), with DMG and extracranial metastasis who died 5 years after initial diagnosis. The first patient with thalamic DMG underwent biopsy, radiation, chemotherapy, and subsequent decompression of spinal metastasis. Initial tumor biopsy demonstrated mutation in BRAF p.V600E, H3F3A K27M, and TERT promoter. The epidural spinal metastasis had mutations in BRAF p.V600E, H3F3A, TERT, and TP53. The second patient with brainstem DMG, diagnosed by radiographic appearance, underwent conventional external beam radiation therapy, chemotherapy including panobinostat and direct delivery of radioimmunotherapy, intra-arterial delivery of bevacizumab, and subsequent decompression of spinal metastasis. The histology and molecular profile for the spinal metastasis was consistent with DMG H3 K27M-mutant, with mutations in BRAF p.G464V, PTPN11 p.R498L, PDGFRA, and KIT. Bone marrow biopsy demonstrated atypical extrinsic cells with strong and diffuse immunolabeling for OLIG2. Autopsy revealed solid organ and osseous metastases. Both patients significantly exceeded the median life expectancy for DMG, raising the possibility that prolonged overall survival permitted progression to a rarely observed disseminated state of disease. There was absence of TP53/p53 modulating pathway mutation seen in classic DMG in the thalamic biopsy of the first patient, as well as the metastatic disease for the second patient, which may contribute to the prolonged survival observed. Molecular analysis of metastatic disease is important, as clinically and prognostically relevant alterations that vary from the primary site of disease may be detected, which shed light on clonal evolution patterns and further our understanding of disease biology.

Targeted Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Regulated by H3K36me3.

Trimethylation of lysine 36 on histone H3 (H3K36me3), an epigenetic mark associated with actively transcribed genes, plays an important role in multiple cellular processes, including transcription elongation, DNA methylation, DNA repair, etc. Aberrant expression and mutations of the main methyltransferase for H3K36me3, i.e., SET domain-containing 2 (SETD2), were shown to be associated with various cancers. Here, we performed targeted profiling of 154 epitranscriptomic reader, writer, and eraser (RWE) proteins using a scheduled liquid chromatography-parallel-reaction monitoring (LC-PRM) method coupled with the use of stable isotope-labeled (SIL) peptides as internal standards to investigate how H3K36me3 modulates the chromatin occupancies of epitranscriptomic RWE proteins. Our results showed consistent changes in chromatin occupancies of RWE proteins upon losses of H3K36me3 and H4K16ac and a role of H3K36me3 in recruiting METTL3 to chromatin following induction of DNA double-strand breaks. In addition, protein-protein interaction network and Kaplan-Meier survival analyses revealed the importance of METTL14 and TRMT11 in kidney cancer. Taken together, our work unveiled cross-talks between histone epigenetic marks (i.e., H3K36me3 and H4K16ac) and epitranscriptomic RWE proteins and uncovered the potential roles of these RWE proteins in H3K36me3-mediated biological processes.

Protein O-GlcNAcylation homeostasis regulates facultative heterochromatin to fine-tune sog-Dpp signaling during Drosophila early embryogenesis

Protein O-GlcNAcylation is a monosaccharide post-translational modification maintained by two evolutionarily conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Mutations in human OGT have recently been associated with neurodevelopmental disorders, although the mechanisms linking O-GlcNAc homeostasis to neurodevelopment are not understood. Here, we investigate the effects of perturbing protein O-GlcNAcylation using transgenic Drosophila lines that overexpress a highly active OGA. We reveal that temporal reduction of protein O-GlcNAcylation in early embryos leads to reduced brain size and olfactory learning in adult Drosophila. Downregulation of O-GlcNAcylation induced by the exogenous OGA activity promotes nuclear foci formation of Polycomb-group protein Polyhomeotic and the accumulation of excess K27 trimethylation of histone H3 (H3K27me3) at the mid-blastula transition. These changes interfere with the zygotic expression of several neurodevelopmental genes, particularly shortgastrulation (sog), a component of an evolutionarily conserved sog-Decapentaplegic (Dpp) signaling system required for neuroectoderm specification. Our findings highlight the importance of early embryonic O-GlcNAcylation homeostasis for the fidelity of facultative heterochromatin redeployment and initial cell fate commitment of neuronal lineages, suggesting a possible mechanism underpinning OGT-associated intellectual disability.

AGAMOUS regulates various target genes via cell cycle-coupled H3K27me3 dilution in floral meristems and stamens.

The MADS domain transcription factor AGAMOUS (AG) regulates floral meristem termination by preventing maintenance of the histone modification lysine 27 of histone H3 (H3K27me3) along the KNUCKLES (KNU) coding sequence. At 2 d after AG binding, cell division has diluted the repressive mark H3K27me3, allowing activation of KNU transcription prior to floral meristem termination. However, how many other downstream genes are temporally regulated by this intrinsic epigenetic timer and what their functions are remain unknown. Here, we identify direct AG targets regulated through cell cycle-coupled H3K27me3 dilution in Arabidopsis thaliana. Expression of the targets KNU, AT HOOK MOTIF NUCLEAR LOCALIZED PROTEIN18 (AHL18), and PLATZ10 occurred later in plants with longer H3K27me3-marked regions. We established a mathematical model to predict timing of gene expression and manipulated temporal gene expression using the H3K27me3-marked del region from the KNU coding sequence. Increasing the number of del copies delayed and reduced KNU expression in a polycomb repressive complex 2- and cell cycle-dependent manner. Furthermore, AHL18 was specifically expressed in stamens and caused developmental defects when misexpressed. Finally, AHL18 bound to genes important for stamen growth. Our results suggest that AG controls the timing of expression of various target genes via cell cycle-coupled dilution of H3K27me3 for proper floral meristem termination and stamen development.

ROS inhibition increases KDM6A-mediated NOX2 transcription and promotes macrophages oxidative stress and M1 polarization.

Reactive oxygen species (ROS) play an essential role in macrophage polarization. However, the adverse effects of ROS reduction by influencing epigenetics are often ignored. In this study, lipopolysaccharide (LPS) was used to stimulate macrophages to increase the ROS in cells, and N-acetylcysteine (NAC) was used to reduce ROS. Inflammatory factors such as interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) were used to evaluate the M1 polarization level of macrophages. Chip was used to detect the tri-methylation at lysine 27 of histone H3 (H3K27me3) level at the promoter site. It was found that the decrease of ROS in macrophages would also cause the increase of the H3K27me3 demethylase KDM6A and lead to the reduction of H3K27me3 in the NOX2 promoter, which would increase the transcription level of NOX2 and the production of ROS and ultimately promote the production of inflammatory factors. Knockout of KDM6A can reduce the transcription of NOX2 and the production of ROS of macrophages, thus preventing the M1 polarization of macrophages. The elimination of ROS in macrophages will affect macrophages by increasing KDM6A and making them produce more ROS, thus inducing oxidative stress. In comparison, direct inhibition of KDM6A can reduce ROS production and inhibit macrophage M1 polarization more effectively.

CRL4B complex-mediated H2AK119 monoubiquitination restrains Th1 and Th2 cell differentiation.

CD4+ T helper (Th) cell differentiation is regulated by lineage-specific expression of transcription factors, which is tightly associated with epigenetic modifications, including histone acetylation and methylation. However, the factors regulating histone modifications involved in Th cell differentiation remain largely unknown. We herein demonstrated a critical role of Cullin 4B (CUL4B) in restricting Th1 and Th2 cell differentiation. CUL4B, which is assembled into the CUL4B-RING E3 ligase (CRL4B) complex, participates in various physiological and developmental processes through epigenetic repression of transcription. Depletion of Cul4b in CD4+ T cells enhanced Th1 and Th2 cell differentiation. In vivo, an aggravated Th2 response caused by the absence of CUL4B was observed in a murine asthma model. Mechanistically, the CRL4B complex promoted monoubiquitination at H2AK119 (H2AK119ub1) and polycomb repressive complex 2 (PRC2)-mediated trimethylation at H3K27 (H3K27me3) at Tbx21 and Maf and consequently repressed their expression during Th cell differentiation. Our study suggests that CRL4B complex-mediated H2AK119ub1 deposition functions to prevent the aberrant expression of Th1 and Th2 lineage-specific genes.

Abstract 2163: Circulating H3K27 nucleosomes to monitor lung cancer patients during treatment, a universal biomarker quantifying the molecular residual disease (MRD) in plasma samples

Abstract Introduction: Treatment options of lung cancer (LC) comprise radiotherapy, and/or combined treatment approaches, including chemotherapy, immunotherapy and targeted therapies based on the tumoral molecular profile. Following curative-intent first-line therapies, clinical surveillance involves serial CT imaging. However, such surveillance can detect only macroscopic disease recurrence and is frequently inconclusive. NGS has been utilized to help identify and monitor treatment plans. Nucleosomes, complexes of DNA and histones proteins, are released during cell death into blood circulation. Trimethylation of lysine 27 on histone H3 (H3K27Me3), catalyzed by enhancer of zeste homolog 2 (EZH2), is a crucial epigenetic process in tumorigenesis. We investigated if H3K27Me3-nucleosome concentration could be a biomarker for molecular residual disease (MRD). Patients and Methods: Plasmas were retrospectively collected from patients with advanced LC during treatment (CIRCAN’s cohort, n= 200) and from healthy donors (n=100). We carried out standard targeted NGS on paired plasmas. Samples were divided in two sub-groups based on genetical results: ctDNA negative (n=120) or positive (n=80) for presence of somatic alterations. Concentration of circulating H3K27Me3-nucleosome was measured using chemiluminescent Nu.Q® immunoassay (Belgian Volition SRL, Belgium). Results: Significantly elevated concentrations of H3K27Me3-nucleosomes were found in LC plasmas during the follow-up of patients compared to healthy donors (median 14.9 ng/ml vs 6.15 ng/ml, respectively, p&amp;lt;0.001). In addition, H3K27Me3 levels is lower in the ctDNA-negative group compared to ctDNA-positive group (median 12.1 vs 24.8 ng/ml, respectively, p&amp;lt;0.001). At a clinical cut-off of 14 ng/ml, 62% of samples were positive for either H3K27Me3 or ctDNA, or for both, suggesting an active disease progression compared to only 40% detection using the NGS assay alone. 38% of the patients have low levels of H3K27Me3 and were ctDNA-negative strongly suggesting a therapeutic response under treatment. Conclusions: High levels of Nu.Q® H3K27Me3 could allow physicians to detect MRD in LC patients following treatment with curative intent. This could be achieved by monitoring testing of patients at defined intervals of treatment and recovery, alongside imaging, to incorporate analyses of evolving molecular landscapes during treatment. In this setting, the H3K27Me3-nucleosome quantification, to complete the molecular exploration of cfDNA is highly encouraging, especially in advanced NSCLC, where re-tissue biopsies are impractical, expensive, and may cause undue harm. H3K27Me3-nucleosome quantification may also be useful in patient identification for specific treatments such as EZH2 inhibitor, but this requires investigation. Citation Format: Emmanuel Grolleau, Julie Candiracci, Arnaud Gauthier, Gaelle Lescuyer, David Barthelemy, Christine Haon, Florence Geiguer, Margaux Raffin, Nathalie Hardat, Julie Balandier, Rémi Rabeuf, Anne-Sophie Wozny, Guillaume Rommelaere, Claire Rodriguez-Lafrasse, Fabien Subtil, Sébastien Couraud, Marielle Herzog, Lea Payen-Gay. Circulating H3K27 nucleosomes to monitor lung cancer patients during treatment, a universal biomarker quantifying the molecular residual disease (MRD) in plasma samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2163.

Abstract 3567: Cancer-epigenetic mouse models for medulloblastoma

Abstract Medulloblastoma (MB) is the most common childhood brain tumors (up to 20% of all childhood brain tumors) and occurs mainly in children (&amp;lt;15 years old. MB is located in the cerebellum in the brain and can spread to the spine or to other parts of brain. Because MB growth is very fast, MB is categorized as a type of high-grade malignant primary tumors. MB development is often associated with the dysregulation of signaling pathways, such as the wingless (WNT) and sonic hedgehog (SHH). Methylated histone H3 lysine 4 (H3K4) is a major methylation mark. Trimethyl H3K4 (H3K4me3) occupies as much as 75% of all human gene promoters. We previously reported that the H3K4 methyltransferase KMT2D (also called MLL4, MLL2, and ALR; a transcriptional coactivator, is required for neuronal differentiation of human neuron-lineage NT2/D1 stem cells. Interestingly, our analysis of several databases has shown that Kmt2d is the most frequently mutated epigenetic modifier (8%‒10%) in medulloblastoma (MB). Recently, we showed that brain-specific knockout of Kmt2d alone induces spontaneous MB in mice. Kmt2d loss highly upregulated several oncogenic signaling programs and downregulated tumor suppressive genes. In this study, we sought to determine whether Kmt2d loss cooperates with another oncogenic event in MB genesis. Particularly, PTCH (also known as PTCH1) is one of the most frequently mutated genes in MB, and KMT2D is known to be a shared driver gene mutation in MB. For these reasons, we examined the effect of Kmt2d loss on of Ptch+/−-driven MB genesis. A heterozygous loss of Kmt2d increased the incidence and progression of Ptch+/−-driven MB genesis. We have shown that Kmt2d loss reduces epigenomic signals for super-enhancers/enhancers and broad H3K4me3 in 4-month-old cerebella. In addition, we determine the effect of heterozygous Kmt2d loss on cell proliferation by comparing the Nes-Cre Kmt2dfl/+ Ptch+/- (KP) cerebella to Ptch+/- cerebella at 2- &amp; 4-month-old time points using IHC staining of Ki-67 (a proliferation marker). MB’s from KP mice showed two different mechanisms by activating oncogenic cellular signaling kinases (e.g., phospho [p]-AKT and p-p38) levels as well as MYCN signaling pathway. Our transcriptomic analysis revealed several tumor suppressive and transcriptional corepressor genes to be downregulated such as Ncor2 and Dnmt3a. We found Ncor2 depletion in Ptch-/+ neurospheres shows upregulation of similar oncogenic genes as in KP mice tumors. Kmt2d loss reduced cerebellar granule cell differentiation. Our analysis for oncogenic kinases may provide a basis for a novel MB treatment plan involving the use of cellular kinase inhibitors. As KMT2D is among the most often mutated genes in human MBs, this mouse model may be useful for future preclinical therapeutic experiments for the treatment of MB patients. KMT2D in medulloblastoma genesis using mouse models will provide epigenetic and mechanistic insights into how heterozygous KMT2D loss promotes medulloblastoma. Citation Format: Shilpa S. Dhar, Ali S. Rizvi, Lauren Reed, Calena Brown, Min Gyu Lee. Cancer-epigenetic mouse models for medulloblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3567.

Abstract 1226: SETD2 loss in renal epithelial cells induces epithelial to mesenchymal transition in a TGFβ-independent manner

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma (RCC) and accounts for majority of the deaths from renal cancer. SETD2, the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is frequently mutated in ccRCC. Studies have shown that SETD2 mutations are often subclonal and the mutation rate dramatically increases from 25% in primary ccRCC to more than 60% in metastatic ccRCC, suggesting SETD2 loss plays an important role in renal cancer metastasis. However, mechanisms underpinning how SETD2 mutation drives invasion and metastasis in ccRCC is largely unknown. Here, by performing RNA-seq and ATAC-seq in a non-tumorigenic renal epithelial cell line isogenic for SETD2 loss-of-function, we found that SETD2 inactivation induces dramatic changes in the transcriptome and drastically enhances chromatin accessibility, which allows binding of a variety of transcription factors. We also discovered that SETD2 loss induces a TGFβ-independent EMT program, which is a major pathway linked to metastasis. Correlation of RNA-seq with ATAC-seq identifies SOX2, OCT2 and PRRX1 as key transcription factors promoting the SETD2 loss-driven EMT program. Furthermore, we found that this distinct EMT program is in part triggered by secreted factors, which suggests that SETD2 mutation promotes a metastasis-permissive tumor microenvironment through paracrine signaling. In summary, our findings reveal that SETD2 is a key regulator of EMT phenotypes that may explain the role of SETD2 mutations in ccRCC metastasis and further uncover potential therapeutic targets for SETD2-mutant ccRCC patients. Citation Format: Tianchu Wang, Ryan Wagner, Ryan Hlady, Sungho Kim, Xiaoyu Pan, Thai Ho, Keith Robertson. SETD2 loss in renal epithelial cells induces epithelial to mesenchymal transition in a TGFβ-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1226.