Epigenetic Regulation Of Transcription Research Articles

LPS-induced neuroinflammation induces changes in the transcriptional profile of members of the CoRest repressive complex in the hippocampus.

The action of Corepressor for Element Silencing Transcription Factor 1 (CoREST) is primarily related to neural fate decisions. However, the molecular mechanisms linking neuroinflammation to the histone modifying complex remain unclear. CoREST is a hub for several cofactors that play important roles in epigenetic remodeling and transcriptional regulation. It allows us to question their functions during the inflammatory response in the Central Nervous System. The impact of LPS-induced neuroinflammation on the transcriptional epigenetic control of members with CoRest repressive complex in the hippocampus was investigated. Characterizing the basal transcriptional profile in the hippocampus of members with CoREST repressive complex showed that the Rcor3 is the most expressed gene, and the Rcor2 is the least expressed one. It was also demonstrated that the levels of Lsd1, CoREST1 (Rcor1), and CoREST2 (Rcor2) transcripts increased in the hippocampus after LPS i.p. administration, while for CoREST3 (Rcor3), no significant difference was observed. A significant increase was noticed in the percentages of the 5-meC mark (Hypermethylation) for the Rcor1 and Lsd1 genes with a positive Pearson correlation between methylation and expression. However, the correlation was directly proportional, ruling out DNA methylation as the main mechanism in transcriptional control.

Epigenomic studies in sorghum reveal differential enrichment of multiple histone marks at clade A PP2C genes in response to drought

Epigenetic regulation is essential for plant development and stress responses, as numerous stress-responsive genes are modulated epigenetically. However, most research has focused on individual histone marks. This study expands on previous work by examining the genome-wide profiles of seven histone marks (H3K9ac, H3K27ac, H3K4me3, H3K36me3, H3K27me3, H2A.Z, and H3K4me2) in sorghum leaves and roots under PEG-induced drought stress. Results revealed that five histone marks (excluding H3K36me3 and H3K27me3) were significantly associated with drought-responsive gene expression. The differential enrichment of these marks may enhance the induction of drought-responsive genes. Specifically, a subset of genes showed multiple histone marks' differential enrichment, with most clade A PP2C genes exhibiting enrichment for four marks after PEG treatment. This suggests a critical role for robust PP2C gene induction in sorghum’s drought response. Additionally, promoter cis-element analysis identified ERF family transcription factors as potential mediators of histone mark enrichment under drought conditions, providing new insights into the interaction between epigenetic modifications and transcriptional regulation in plant stress responses.

H4K20me3-Mediated Repression of Inflammatory Genes is a Characteristic and Targetable Vulnerability of Persister Cancer Cells.

Anti-cancer therapies can induce cellular senescence, which is highly stable, or drug-tolerant persistence, which is efficiently reversed upon therapy termination. While approaches to target senescent cells have been extensively studied, further understanding of the processes regulating persistence is needed to develop treatment strategies to suppress persister cell survival. Here, we used mTOR/PI3K inhibition to develop and characterize a model of persistence-associated arrest in human cancer cells of various origins. Persister and senescent cancer cells shared an expanded lysosomal compartment and hypersensitivity to BCL-XL inhibition. However, persister cells lacked other features of senescence, such as loss of lamin B1, senescence-associated β-galactosidase activity, upregulation of MHC-I, and an inflammatory and secretory phenotype (SASP). Genome-wide CRISPR/Cas9 screening for genes required for the survival of persister cells revealed that they are hypersensitive to the inhibition of one-carbon (1C) metabolism, which was validated by the pharmacological inhibition of SHMT, a key enzyme that feeds methyl groups from serine into 1C metabolism. Connecting 1C metabolism with the epigenetic regulation of transcription, the repressive heterochromatic mark H4K20me3 was enriched at the promoters of SASP and interferon response genes in persister cells, while it was absent in proliferative or senescent cells. Moreover, persister cells overexpressed the H4K20 methyltransferases KMT5B/C, and their downregulation unleashed inflammatory programs and compromised the survival of persister cells. In summary, this study defined distinctive features of persister cancer cells, identified actionable vulnerabilities, and provided mechanistic insight into their low inflammatory activity.

Approaches to anticancer therapy based on modulation of DNA methylation

Background. DNA methylation is a crucial mechanism of epigenetic regulation of transcription. Disturbances in DNA methylation mechanism are associated with various malignancies such as acute myeloid leukaemia, breast cancer, prostate cancer, etc. Influencing the functional status of DNA methyltransferases (DNMTs) enzymes and TET family proteins (TETs), which regulate DNA methylation and demethylation, is the basis of epigenetic anticancer therapy approach. In this review, we have considered the challenges and prospects of nucleoside and non-nucleoside inhibitors of DNMTs as well as TETs inhibitors. The results of clinical trials on the efficacy of DNMTs inhibitors used individually and as part of combination chemotherapy conducted over the last 15 years are also evaluated. Material and Methods. Sources were searched in PubMed, ScienceDirect, Web of Science, eLibrary, CyberLeninka. More than 700 publications were used in the analysis, but the review included mainly works of the last 10 years. A number of articles published earlier than 2015 were used for historical reference. Results. The review provides information on current advances in the development and study of epigenetically active compounds whose action is aimed at the regulation of DNA methylation. Data on the in vitro and in vivo effects of agents considered for use in the therapy of various malignancies are presented. In addition, the data of clinical trials of the most promising epigenetic modulators are presented.

Phenotypes of autism spectrum disorder and schizoaffective disorder associated with SETD1B gene but without intellectual disability and seizures.

The SETD1B gene, located on chromosome 12q24, is one of the chromatin-modifying genes involved in epigenetic regulation of gene transcription. The phenotype of pathogenic variants in the SETD1B gene includes intellectual disability, seizures, and language delay (IDDSELD, OMIM 619000). In this study, we present a family consisting of consanguineous parents who died of cancer and their offspring. This family includes two cases diagnosed with autism spectrum disorder (ASD); six cases diagnosed with schizophrenia, bipolar disorder, or schizoaffective disorder; there cases diagnosed with cancer; and five cases who died of unknown causes in early childhood. Three affected members of this family agreed to genetic testing. We used whole exome sequencing. We report a novel in-frame deletion variant of the SETD1B gene in a family with cases diagnosed with schizoaffective disorder and ASD without seizures and intellectual disability. It was found that the phenotypic features were inherited for at least three generations in the family we presented, and it was shown that the pathogenic variant of the SETD1B gene was transmitted from the affected parent to his affected children. In addition, the father was diagnosed with both schizoaffective disorder and leukemia. We proposed an association between rare variants of SETD1B and phenotypes of ASD and schizoaffective disorder without seizures and intellectual disability. The SETD1B gene is included in both the ASD genetic database of SFARI (https://gene.sfari.org/) and the cancer database of COSMIC (https://cancer.sanger.ac.uk/cosmic). However, there are very few reports of SETD1B gene variants as clinical entities. To our knowledge, the SETD1B gene variant has not been previously reported in an individual diagnosed with both a neuropsychiatric disorder and cancer.

Evaluation of sensomotor development, behavioral reactions and cognitive functions of the second generation of rats with hyperhomocysteinemia

The adverse maternal exposure during pregnancy leads to developmental disorders in the offspring that can be passed on to later generations. Epigenetic regulation of DNA transcription may mediate inherited metabolic diseases. An increase in homocysteine concentration in the blood is associated with epigenetic modifications of the genome, which can alter the fetal brain’s development program and cause cognitive impairment. The aim of our work was to identify changes in sensomotor development, behavioral reactions and cognitive functions of offspring of second generation rats (HcyF2) of hyperhomocysteinemia. Our results indicate that unconditioned reflexes and physical parameters are delayed in HcyF2 rats. In “open field”, HcyF2 rats showed higher levels of anxiety and decreased exploratory and motor activity, while coordination of movements studied in “rotarod” test was not impaired. Decreased limb muscle strength was shown in the “grip strength” test. Additionally, HcyF2 rats demonstrated an impaired learning and longterm memory in the Morris water maze. Biochemical analysis revealed an imbalance in the antioxidant systems, which was attributed to decreased activity of glutathione peroxidases and H2S synthesis enzymes. It was suggested that elevated homocysteine levels during pregnancy may result in epigenetic modifications of the genome, which can impact the metabolism of offspring and be inherited by future generations.

51 Epigenetic regulation of ribosomal RNA transcription and processing by the tumor suppressor SETD2

Abstract Background The methyltransferase SETD2 was originally identified as a tumor suppressor in clear cell renal cell carcinoma (ccRCC), and ccRCC with SETD2 mutations have increased metastatic potential and poor progression free survival. A known epigenetic regulator, SETD2 interacts with actively transcribing RNA polymerase II (RNAPII) and tri-methylates Histone 3 at Lysine 36 (H3K36me3), a modification that correlates with active gene transcription and is recognized by mRNA methylation and splicing factors. Despite these known roles in mRNA transcription, loss of SETD2 does not alter genic transcription, raising the question of whether or how SETD2 regulates RNA biology. Methods Given that SETD2 influences RNA processing, we sought to identify RNA-protein complexes that are regulated by SETD2. To do this in an unbiased and high-throughput manner, we used orthogonal organic phase separation (OOPS) coupled with mass spectrometry to identify changes in RNA binding proteins and pathways between control and SETD2-knockout cells. Results Corroborating previous findings, we identified that RNA binding proteins governing mRNA splicing and cellular metabolism were dysregulated in SETD2 mutant cells. However, the most robust changes in SETD2 knockout cells occurred in pathways regulated ribosome biogenesis (See Figure, orange highlights), resulting in a significant decrease in binding of ribosomal RNA (rRNA) processing factors to RNA. We confirm these alterations lead to defects in rRNA transcription, processing, and ribosome biogenesis. We identify that loss of SETD2 catalytic activity phenocopies these rRNA processing defects, suggesting H3K36me3 is essential for rRNA processing. Disruptions occur to both the large (60S) and small (40S) ribosome subunits, modifying protein translation. Lastly, we identify synthetic lethality between SETD2 deletion or inhibition and small molecules that interfere with rRNA transcription. Conclusions While the majority of research emphasizes the role SETD2 in the regulation of mRNA, our data identify that SETD2 and its catalytic activity play a critical role in the transcription and processing of rRNA. Importantly, rRNA comprises 80-90% of cellular RNA yet is typically overlooked and understudied. Next-generation RNA sequencing or transcriptional assays investigate mRNA or genic transcription while ignoring rRNA, demonstrating why SETD2-mediated rRNA processing has been unexplored. This may also have important implications for prospective clinical trials that utilized RNA-sequencing to identify optimal therapeutic regimens. Our work also identified that SETD2-mutated cells are more sensitive to compounds that inhibit rRNA transcription, potentially uncovering a new therapeutic vulnerability of ccRCC with mono- and bi-allelic loss of SETD2. Alterations in ribosome biogenesis leads to epithelial-to-mesenchymal transition and metastasis in many tumor types, and our future goal will be to investigate the role of ribosomes and rRNA transcription in transformation and metastasis in ccRCC as well as the molecular mechanisms connecting SETD2 to ribosome fidelity. DOD CDMRP Funding: yes

Roles of Lysine Methylation in Glucose and Lipid Metabolism: Functions, Regulatory Mechanisms, and Therapeutic Implications.

Glucose and lipid metabolism are essential energy sources for the body. Dysregulation in these metabolic pathways is a significant risk factor for numerous acute and chronic diseases, including type 2 diabetes (T2DM), Alzheimer's disease (AD), obesity, and cancer. Post-translational modifications (PTMs), which regulate protein structure, localization, function, and activity, play a crucial role in managing cellular glucose and lipid metabolism. Among these PTMs, lysine methylation stands out as a key dynamic modification vital for the epigenetic regulation of gene transcription. Emerging evidence indicates that lysine methylation significantly impacts glucose and lipid metabolism by modifying key enzymes and proteins. This review summarizes the current understanding of lysine methylation's role and regulatory mechanisms in glucose and lipid metabolism. We highlight the involvement of methyltransferases (KMTs) and demethylases (KDMs) in generating abnormal methylation signals affecting these metabolic pathways. Additionally, we discuss the chemical biology and pharmacology of KMT and KDM inhibitors and targeted protein degraders, emphasizing their clinical implications for diseases such as diabetes, obesity, neurodegenerative disorders, and cancers. This review suggests that targeting lysine methylation in glucose and lipid metabolism could be an ideal therapeutic strategy for treating these diseases.

Cytokines drive the formation of memory-like NK cell subsets via epigenetic rewiring and transcriptional regulation.

Activation of natural killer (NK) cells with the cytokines interleukin-12 (IL-12), IL-15, and IL-18 induces their differentiation into memory-like (ML) NK cells; however, the underlying epigenetic and transcriptional mechanisms are unclear. By combining ATAC-seq, CITE-seq, and functional analyses, we discovered that IL-12/15/18 activation results in two main human NK fates: reprogramming into enriched memory-like (eML) NK cells or priming into effector conventional NK (effcNK) cells. eML NK cells had distinct transcriptional and epigenetic profiles and enhanced function, whereas effcNK cells resembled cytokine-primed cNK cells. Two transcriptionally discrete subsets of eML NK cells were also identified, eML-1 and eML-2, primarily arising from CD56bright or CD56dim mature NK cell subsets, respectively. Furthermore, these eML subsets were evident weeks after transfer of IL-12/15/18-activated NK cells into patients with cancer. Our findings demonstrate that NK cell activation with IL-12/15/18 results in previously unappreciated diverse cellular fates and identifies new strategies to enhance NK therapies.

Epigenetic regulation of TERRA transcription and metacyclogenesis by base J in Leishmania major.

The hyper-modified DNA base J helps control termination of Pol II transcription at polycistronic transcription units (PTUs) in T. brucei and L. major , allowing epigenetic control of gene expression. The Telomere Repeat-containing RNA (TERRA) is synthesized in T. brucei by Pol I readthrough transcription of a telomeric PTU. While little is understood regarding TERRA synthesis and function, the hyper-modified DNA base J is highly enriched at telomeres in L. major promastigotes. We now show that TERRA is synthesized by Pol II in L. major and loss of base J leads to increased TERRA. For at least one site, the increased TERRA is by Pol II readthrough transcription from an adjacent PTU. Furthermore, Pol II readthrough defects and increased TERRA correlate with increased differentiation of promastigotes to the infectious metacyclic life stage and decreased cell viability. These results help explain the essential nature of base J in Leishmania and provide insight regarding epigenetic control of coding and non-coding RNA expression and parasite development during the life cycle of L. major .

Unboxing the network among long non-coding RNAs and TGF-β signaling in cancer.

Deeper analysis of molecular mechanisms arising in tumor cells is an unmet need to provide new diagnostic and therapeutic strategies to prevent and treat tumors. The transforming growth factor β (TGF-β) signaling has been steadily featured in tumor biology and linked to poor prognosis of cancer patients. One pro-tumorigenic mechanism induced by TGF-β is the epithelial-to-mesenchymal transition (EMT), which can initiate cancer dissemination, enrich the tumor stem cell population, and increase chemoresistance. TGF-β signals via SMAD proteins, ubiquitin ligases, and protein kinases and modulates the expression of protein-coding and non-coding RNA genes, including those encoding larger than 500 nt transcripts, defined as long non-coding RNAs (lncRNAs). Several reports have shown lncRNAs regulating malignant phenotypes by directly affecting epigenetic processes, transcription, and post-transcriptional regulation. Thus, this review aims to update and summarize the impact of TGF-β signaling on the expression of lncRNAs and the function of such lncRNAs as regulators of TGF-β signaling, and how these networks might impact specific hallmarks of cancer.

Abstract 1721: Epigenetic heterogeneity of osteosarcoma reveals distinct cellular states driven by enhancer regulation

Abstract Osteosarcoma (OS) is characterized by structural rearrangements and copy number changes which drive considerable heterogeneity. What is less clear is how these genetic rearrangements alter are in turn regulated at the epigenetic level to drive gene expression. Defining the epigenetic circuitry of osteosarcoma could help to understand the role of chromatin accessibility in determining cell plasticity. Chromatin accessibility can prime gene expression and define a cellular state. As chromatin accessibility is a major determinant of epigenetic transcriptional regulation, understanding the variability of chromatin accessibility could identify clinically-relevant subtypes and biomarkers of therapy response in Osteosarcoma, a disease that has seen few advances in therapy for over 40 years. Using ATAC-seq, we identified two epigenetic subtypes (EC1 and EC2) of OS with distinct chromatin accessibility. These studies were done in a panel of 11 PDX-derived cell lines and 9 established OS cell lines. We also performed ATACseq on the matching PDX for the 11 PDX-derived lines as well as 8 primary tumor samples. Analysis of transcription factor (TF) motifs and footprints of ATAC-seq peaks identified transcription factors distinctly activated in these two subtypes. For example, EC1 is characterized by high level of activity of developmental transcription factors including RUNX2, MEF2C and homeobox TFs. In contrast, EC2 is characterized by high activity of AP1 transcription factors such as FOSL1 and FOSL2. In order to understand the underlying transcriptional circuitry of these cell states, we performed CUT&RUN assay for the complete panel of PDX-derived cell lines. We found that the subtypes are driven by enhancer regulation (H3K27ac). We defined the specific Core Regulatory Circuitry (CRC) per subtype by defining the superenhancers by ROSE. We overlapped ATAC-seq data in the acetylated regions to demarcate TF binding regions dramatically and reduce the search space for TF cis-regulatory sites. In parallel, we used our PDX-derived cell line panel to screen cluster-specific targeted therapies and found differential responses dependent on cellular states defined by chromatin accessibility. For example, cell lines in the EC2 cluster are marked by upregulation of AP1 transcription factors, have evidence of ERK activation and are highly responsive to ERK inhibitors. In contrast cell lines in the EC1 cluster are sensitive to AURKA and AURKB inhibitors. Single-cell RNAseq analysis indicates that these subtypes may coexist within a single tumor. In summary, we discovered two epigenetically distinct cell states that are controlled by a state-specific collection of transcription factors, creating a gene signature that may aid in distinguishing osteosarcoma subclasses and predicting treatment response. Citation Format: Eunice Lopez Fuentes, Andrew Clugston, Alex Lee, Leanne Sayles, Maria Pons, Alejandro Sweet-Cordero. Epigenetic heterogeneity of osteosarcoma reveals distinct cellular states driven by enhancer regulation [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 1721.

Abstract PR-007: H3.3K27M diffuse midline gliomas are sensitive to SWI/SNF chromatin remodeler degradation

Abstract Diffuse Midline Gliomas (DMG) including Diffuse Intrinsic Pontine Glioma (DIPG) is a lethal pediatric brain tumor. The lysine 27 (K27)-to-methionine (M) point mutation in histone H3.1 or H3.3 (H3.1K27M or H3.3K27M, respectively) affects about 80% of patients establishing H3K27M mutation as a tumor hallmark. Mutated H3 histones inhibit the function of Polycomb repressive complex 2 resulting in global reduction of repressive H3K27me3. In addition, increased acetylation of H3K27 is observed, further contributing to an aberrant chromatin state. Considering the impact of epigenetic alterations in DIPG, we investigated the role of Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodelers in shaping the oncogenic chromatin state in DIPG cells. DIPG cells harboring H3.3K27M mutation were sensitive to combined SMARCA4/SMARCA2/PBRM1 degradation by the proteolysis targeting chimera (PROTAC) AU-15330 degrader. Our premise is based on the SWI/SNF complex altering chromatin states of tumor cells to regulate the tumor immune microenvironment. We hypothesized that the SWI/SNF complex in tumor cells modulates immune responses in DIPG. Cytokine profiler arrays in AU-15330 versus vehicle treated H3.3K27M cells showed a marked decrease of Intercellular Cell Adhesion Molecule-1 (ICAM-1) glycoprotein levels. In addition to its well-known role in leukocyte endothelial transmigration, ICAM-1 contributes to malignant potential of tumor cells. We validated downregulation of ICAM-1 mRNA and protein levels by AU-15330 by immunoblotting, proteomics, and transcriptomics, suggesting an epigenetic regulation of ICAM-1 gene transcription by the SWI/SNF complex. Moreover, silencing of mutant H3.3-encoding gene decreased ICAM-1 protein levels, suggesting ICAM-1 expression is promoted by H3.3K27M mutation-induced SWI/SNF chromatin remodeling in DIPG. In conclusion, our work suggests the H3.3K27M mutation renders DIPG cells dependent on chromatin remodeling by SWI/SNF complexes that can in turn alters the expression of immune system-associated components, such as ICAM-1. Our future work is geared to understanding the role of the SWI/SNF complex in regulating the immune tumor microenvironment in H3K27M DMGs. Citation Format: Mateus Mota, Stefan Sweha, Matt Pun, Siva Kumar Natarajan, Yujie Ding, Chan Chung, Debra Hawes, Fusheng Yang, Alexander Judkins, Susanta Samajdar, Xuhong Cao, Lanbo Xiao, Abhijit Parolia, Arul Chinnaiyan, Sriram Venneti. H3.3K27M diffuse midline gliomas are sensitive to SWI/SNF chromatin remodeler degradation [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-007.

Contributions of transcriptional noise to leukaemia evolution: KAT2A as a case-study.

Transcriptional noise is proposed to participate in cell fate changes, but contributions to mammalian cell differentiation systems, including cancer, remain associative. Cancer evolution is driven by genetic variability, with modulatory or contributory participation of epigenetic variants. Accumulation of epigenetic variants enhances transcriptional noise, which can facilitate cancer cell fate transitions. Acute myeloid leukaemia (AML) is an aggressive cancer with strong epigenetic dependencies, characterized by blocked differentiation. It constitutes an attractive model to probe links between transcriptional noise and malignant cell fate regulation. Gcn5/KAT2A is a classical epigenetic transcriptional noise regulator. Its loss increases transcriptional noise and modifies cell fates in stem and AML cells. By reviewing the analysis of KAT2A-depleted pre-leukaemia and leukaemia models, I discuss that the net result of transcriptional noise is diversification of cell fates secondary to alternative transcriptional programmes. Cellular diversification can enable or hinder AML progression, respectively, by differentiation of cell types responsive to mutations, or by maladaptation of leukaemia stem cells. KAT2A-dependent noise-responsive genes participate in ribosome biogenesis and KAT2A loss destabilizes translational activity. I discuss putative contributions of perturbed translation to AML biology, and propose KAT2A loss as a model for mechanistic integration of transcriptional and translational control of noise and fate decisions. This article is part of a discussion meeting issue 'Causes and consequences of stochastic processes in development and disease'.

Incorporating network diffusion and peak location information for better single-cell ATAC-seq data analysis.

Single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) data provided new insights into the understanding of epigenetic heterogeneity and transcriptional regulation. With the increasing abundance of dataset resources, there is an urgent need to extract more useful information through high-quality data analysis methods specifically designed for scATAC-seq. However, analyzing scATAC-seq data poses challenges due to its near binarization, high sparsity and ultra-high dimensionality properties. Here, we proposed a novel network diffusion-based computational method to comprehensively analyze scATAC-seq data, named Single-Cell ATAC-seq Analysis via Network Refinement with Peaks Location Information (SCARP). SCARP formulates the Network Refinement diffusion method under the graph theory framework to aggregate information from different network orders, effectively compensating for missing signals in the scATAC-seq data. By incorporating distance information between adjacent peaks on the genome, SCARP also contributes to depicting the co-accessibility of peaks. These two innovations empower SCARP to obtain lower-dimensional representations for both cells and peaks more effectively. We have demonstrated through sufficient experiments that SCARP facilitated superior analyses of scATAC-seq data. Specifically, SCARP exhibited outstanding cell clustering performance, enabling better elucidation of cell heterogeneity and the discovery of new biologically significant cell subpopulations. Additionally, SCARP was also instrumental in portraying co-accessibility relationships of accessible regions and providing new insight into transcriptional regulation. Consequently, SCARP identified genes that were involved in key Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to diseases and predicted reliable cis-regulatory interactions. To sum up, our studies suggested that SCARP is a promising tool to comprehensively analyze the scATAC-seq data.

Bromodomain-Containing Protein 9 Regulates Signaling Pathways and Reprograms the Epigenome in Immortalized Human Uterine Fibroid Cells.

Bromodomain-containing proteins (BRDs) are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD dysfunction has been linked to many diseases, including tumorigenesis. However, the role of BRDs in the pathogenesis of uterine fibroids (UFs) is entirely unknown. The present study aimed to determine the expression pattern of BRD9 in UFs and matched myometrium and further assess the impact of a BRD9 inhibitor on UF phenotype and epigenetic/epitranscriptomic changes. Our studies demonstrated that the levels of BRD9 were significantly upregulated in UFs compared to matched myometrium, suggesting that the aberrant BRD expression may contribute to the pathogenesis of UFs. We then evaluated the potential roles of BRD9 using its specific inhibitor, I-BRD9. Targeted inhibition of BRD9 suppressed UF tumorigenesis with increased apoptosis and cell cycle arrest, decreased cell proliferation, and extracellular matrix deposition in UF cells. The latter is the key hallmark of UFs. Unbiased transcriptomic profiling coupled with downstream bioinformatics analysis further and extensively demonstrated that targeted inhibition of BRD9 impacted the cell cycle- and ECM-related biological pathways and reprogrammed the UF cell epigenome and epitranscriptome in UFs. Taken together, our studies support the critical role of BRD9 in UF cells and the strong interconnection between BRD9 and other pathways controlling the UF progression. Targeted inhibition of BRDs might provide a non-hormonal treatment option for this most common benign tumor in women of reproductive age.

Regulation of Angiogenesis by Non-Coding RNAs in Cancer.

Non-coding RNAs, including microRNAs, long non-coding RNAs, and circular RNAs, have been identified as crucial regulators of various biological processes through epigenetic regulation, transcriptional regulation, and post-transcriptional regulation. Growing evidence suggests that dysregulation and activation of non-coding RNAs are closely associated with tumor angiogenesis, a process essential for tumor growth and metastasis and a major contributor to cancer-related mortality. Therefore, understanding the molecular mechanisms underlying tumor angiogenesis is of utmost importance. Numerous studies have documented the involvement of different types of non-coding RNAs in the regulation of angiogenesis. This review provides an overview of how non-coding RNAs regulate tumor angiogenesis. Additionally, we discuss emerging strategies that exploit non-coding RNAs for anti-angiogenic therapy in cancer treatment. Ultimately, this review underscores the crucial role played by non-coding RNAs in tumor angiogenesis and highlights their potential as therapeutic targets for anti-angiogenic interventions against cancer.

The BET inhibitor apabetalone decreases neuroendothelial proinflammatory activation in vitro and in a mouse model of systemic inflammation.

Brain vascular inflammation is characterized by endothelial activation and immune cell recruitment to the blood vessel wall, potentially causing a breach in the blood - brain barrier, brain parenchyma inflammation, and a decline of cognitive function. The clinical-stage small molecule, apabetalone, reduces circulating vascular endothelial inflammation markers and improves cognitive scores in elderly patients by targeting epigenetic regulators of gene transcription, bromodomain and extraterminal proteins. However, the effect of apabetalone on cytokine-activated brain vascular endothelial cells (BMVECs) is unknown. Here, we show that apabetalone treatment of BMVECs reduces hallmarks of in vitro endothelial activation, including monocyte chemoattractant protein-1 (MCP-1) and RANTES chemokine secretion, cell surface expression of endothelial cell adhesion molecule VCAM-1, as well as endothelial capture of THP-1 monocytes in static and shear stress conditions. Apabetalone pretreatment of THP-1 downregulates cell surface expression of chemokine receptors CCR1, CCR2, and CCR5, and of the VCAM-1 cognate receptor, integrin α4. Consequently, apabetalone reduces THP-1 chemoattraction towards soluble CCR ligands MCP-1 and RANTES, and THP-1 adhesion to activated BMVECs. In a mouse model of brain inflammation, apabetalone counters lipopolysaccharide-induced transcription of endothelial and myeloid cell markers, consistent with decreased neuroendothelial inflammation. In conclusion, apabetalone decreases proinflammatory activation of brain endothelial cells and monocytes in vitro and in the mouse brain during systemic inflammation.

SMARCA4 / BRG1 -deficient Uterine Neoplasm With Hybrid Adenosarcoma and Carcinoma Features: Expanding the Molecular-morphologic Spectrum of SMARCA4 -driven Gynecologic Malignancies.

SMARCA4 gene encodes BRG1 , a member of the SWItch/sucrose non-fermentable protein family involved in epigenetic transcriptional regulation of important cellular processes. In the uterine corpus, SMARCA4 / BRG1 deficiency is associated with a novel class of undifferentiated uterine sarcomas, characterized by younger age onset, rhabdoid histology, focal phyllodiform architecture, high-risk pathologic findings, and dismal prognosis. Herein, we report a case of a 34-year-old Asian woman with a SMARCA4 / BRG1 -deficient uterine tumor fulfilling the clinicopathologic features of an undifferentiated uterine sarcoma. However, the tumor exhibited several unique features that have not been previously emphasized, including (1) conspicuous phyllodiform architecture recapitulating conventional adenosarcoma, (2) rhabdoid tumor cells forming cords and keratin-positive cohesive epithelial islands, and (3) cooccurrence with a spatially distinct and discrete endometrial complex atypical hyperplasia from the rest of the proliferation. By immunohistochemistry, the tumor cells were diffusely positive for synaptophysin, whereas BRG1 was lost. Pertinent molecular findings included frameshift mutations in the SMARCA4 gene, mutations in histone modification and chromatin remodeling genes, including KMT2C , ARID1B , KAT6A , and NCOR1 , and mutations in Wnt signaling involving APC and CTNNB1 . Copy number gain in MDM2 and CDK4 were also identified. The tumor mutation burden was intermediate (6.8/MB) and it was microsatellite stable. On balance, our case exhibited morphologic and molecular features that overlap with (1) an undifferentiated uterine sarcoma, (2) an adenosarcoma with sarcomatous overgrowth, and (3) a mixed adenosarcoma and undifferentiated endometrial carcinoma. These hybrid features further expand the molecular-morphologic spectrum of SMARCA4 / BRG1 -deficient uterine neoplasms.

Abstract C109: INTASYL™ PH-894 self-delivering RNAi targeting BRD4 enhances the antigenicity of melanoma cells through MART-1 upregulation

Abstract Background: BRD4 is a member of the family of bromodomain and extra-terminal motif (BET) proteins that act as epigenetic readers and regulators of gene transcription. BRD4 has been shown to be significantly upregulated in cancer including melanoma, with preclinical data supporting BRD4 inhibition as a promising therapeutic target. However, clinical translation of BRD4 inhibitors (BRD4i) has remained elusive. Current small molecule inhibitors of BET proteins are not specific to BRD4 as demonstrated by their inhibition of other members of the BET protein family. Additionally, systemic approaches must overcome a narrow therapeutic window. The INTASYL™ platform is a self-delivering RNAi technology that imparts small molecule-like properties to interfering RNAs, providing efficient delivery to target cells without need for specialized formulations or drug delivery systems. PH-894 is an INTASYL compound that silences BRD4 mRNA. PH-894 has been shown to specifically silence BRD4 mRNA and provide robust direct and abscopal antitumor efficacy in vivo when administered intratumorally (IT). Therefore, IT PH-894 holds promise to realize the therapeutic potential of BRD4 inhibition by avoiding toxicities associated with systemic delivery of small molecule BET inhibitors. Here, we explored the impact of PH-894 mediated BRD4 inhibition on antigenicity of melanoma tumor cells. Specifically, expression of the melanoma tumor-associated antigen MART-1 was studied following PH-894 treatment. Methods: SK-MEL-5 melanoma cells were treated for 72 hours with PH-894 or a non-targeting control (NTC) INTASYL compound. After 72 hours (Day 3), PH-894 was washed out and cells were replated in media containing no INTAYSL compound for the duration of the experiment. Cells were collected for analysis of both BRD4 and MART-1 mRNA and protein from Day 3 through Day 7. Results: PH-894 provided robust silencing of BRD4 mRNA and protein in SK-MEL-5 melanoma cells. Furthermore, PH-894 treatment resulted in a statistically significant upregulation of MART-1 mRNA and protein in these cells. BRD4 silencing and MART-1 upregulation persisted through at least 7 days post-treatment with PH-894. Conclusions: This study demonstrates the ability of PH-894 to efficiently silence BRD4 and upregulate MART-1, thereby increasing the antigenicity of melanoma cells. Local treatment with PH-894 is a potential strategy to decrease BRD4 expression and increase immune response to cancer cells while reducing toxicities associated with systemic BETi. This study supports further development of PH-894 for injectable solid tumor indications such as melanoma. Citation Format: Brianna Rivest, Melissa Maxwell, Andrew Boone, Benjamin Cuiffo, Dingxue Yan, James Cardia. INTASYL™ PH-894 self-delivering RNAi targeting BRD4 enhances the antigenicity of melanoma cells through MART-1 upregulation [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C109.