Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells.

Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.

The Allelic Landscape of Human Blood Cell Trait Variation and Links to Common Complex Disease.

Many common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (<5%) and rare (<1%) variants with a strong impact on blood cell phenotypes. Our data highlight general properties of the allelic architecture of complex traits, including the proportion of the heritable component of each blood trait explained by the polygenic signal across different genome regulatory domains. Finally, through Mendelian randomization, we provide evidence of shared genetic pathways linking blood cell indices with complex pathologies, including autoimmune diseases, schizophrenia, and coronary heart disease and evidence suggesting previously reported population associations between blood cell indices and cardiovascular disease may be non-causal.

Discovery of fluorinated 2­Styryl 4(3H)-quinazolinone as potential therapeutic hit for oral cancer.

Oral squamous cell carcinoma (OSCC) is the most common malignant epithelial neoplasm, affects the mouth and throat, and accounts for 90 % of oral cancers. Considering the associated morbidity with neck dissections and the limitation of existing therapeutic agents, the discovery and development of new anticancer drugs/drug candidates for oral cancer treatment are of the utmost need. In this context, reported here is the identification of fluorinated 2­styryl 4(3H)-quinazolinone as a promising hit for oral cancer. Preliminary studies indicate that the compound blocks the transition of G1 to S phase, thereby leading to arrest in the G1/S phase. Subsequent RNA-seq analysis revealed that the compound induces the activation of molecular pathways involved in apoptosis (such as TNF signalling through NF-κB, p53 pathways) and cell differentiation and suppresses the pathways of cellular growth and development (such as KRAS signaling) in CAL-27 cancer cells. It is noted that identified hit complies with a favorable range of ADME properties as per the computational analysis.

Integrated paired-end enhancer profiling and whole-genome sequencing reveals recurrent CCNE1 and IGF2 enhancer hijacking in primary gastric adenocarcinoma.

OBJECTIVE: Genomic structural variations (SVs) causing rewiring of cis-regulatory elements remain largely unexplored in gastric cancer (GC). To identify SVs affecting enhancer elements in GC (enhancer-based SVs), we integrated epigenomic enhancer profiles revealed by paired-end H3K27ac ChIP-sequencing from primary GCs with tumour whole-genome sequencing (WGS) data (PeNChIP-seq/WGS). DESIGN: We applied PeNChIP-seq to 11 primary GCs and matched normal tissues combined with WGS profiles of >200 GCs. Epigenome profiles were analysed alongside matched RNA-seq data to identify tumour-associated enhancer-based SVs with altered cancer transcription. Functional validation of candidate enhancer-based SVs was performed using CRISPR/Cas9 genome editing, chromosome conformation capture assays (4C-seq, Capture-C) and Hi-C analysis of primary GCs. RESULTS: PeNChIP-seq/WGS revealed ~150 enhancer-based SVs in GC. The majority (63%) of SVs linked to target gene deregulation were associated with increased tumour expression. Enhancer-based SVs targeting CCNE1, a key driver of therapy resistance, occurred in 8% of patients frequently juxtaposing diverse distal enhancers to CCNE1 proximal regions. CCNE1-rearranged GCs were associated with high CCNE1 expression, disrupted CCNE1 topologically associating domain (TAD) boundaries, and novel TAD interactions in CCNE1-rearranged primary tumours. We also observed IGF2 enhancer-based SVs, previously noted in colorectal cancer, highlighting a common non-coding genetic driver alteration in gastric and colorectal malignancies. CONCLUSION: Integrated paired-end NanoChIP-seq and WGS of gastric tumours reveals tumour-associated regulatory SV in regions associated with both simple and complex genomic rearrangements. Genomic rearrangements may thus exploit enhancer-hijacking as a common mechanism to drive oncogene expression in GC.

Increased H3K9 methylation and impaired expression of Protocadherins are associated with the cognitive dysfunctions of the Kleefstra syndrome.

Kleefstra syndrome, a disease with intellectual disability, autism spectrum disorders and other developmental defects is caused in humans by haploinsufficiency of EHMT1. Although EHMT1 and its paralog EHMT2 were shown to be histone methyltransferases responsible for deposition of the di-methylated H3K9 (H3K9me2), the exact nature of epigenetic dysfunctions in Kleefstra syndrome remains unknown. Here, we found that the epigenome of Ehmt1+/- adult mouse brain displays a marked increase of H3K9me2/3 which correlates with impaired expression of protocadherins, master regulators of neuronal diversity. Increased H3K9me3 was present already at birth, indicating that aberrant methylation patterns are established during embryogenesis. Interestingly, we found that Ehmt2+/- mice do not present neither the marked increase of H3K9me2/3 nor the cognitive deficits found in Ehmt1+/- mice, indicating an evolutionary diversification of functions. Our finding of increased H3K9me3 in Ehmt1+/- mice is the first one supporting the notion that EHMT1 can quench the deposition of tri-methylation by other Histone methyltransferases, ultimately leading to impaired neurocognitive functioning. Our insights into the epigenetic pathophysiology of Kleefstra syndrome may offer guidance for future developments of therapeutic strategies for this disease.

ERG and FLI1 binding sites demarcate targets for aberrant epigenetic regulation by AML1-ETO in acute myeloid leukemia.

ERG and FLI1 are closely related members of the ETS family of transcription factors and have been identified as essential factors for the function and maintenance of normal hematopoietic stem cells. Here genome-wide analysis revealed that both ERG and FLI1 occupy similar genomic regions as AML1-ETO in t(8;21) AMLs and identified ERG/FLI1 as proteins that facilitate binding of oncofusion protein complexes. In addition, we demonstrate that ERG and FLI1 bind the RUNX1 promoter and that shRNA-mediated silencing of ERG leads to reduced expression of RUNX1 and AML1-ETO, consistent with a role of ERG in transcriptional activation of these proteins. Finally, we identify H3 acetylation as the epigenetic mark preferentially associated with ETS factor binding. This intimate connection between ERG/FLI1 binding and H3 acetylation implies that one of the molecular strategies of oncofusion proteins, such as AML1-ETO and PML-RAR-α, involves the targeting of histone deacetylase activities to ERG/FLI1 bound hematopoietic regulatory sites. Together, these results highlight the dual importance of ETS factors in t(8;21) leukemogenesis, both as transcriptional regulators of the oncofusion protein itself as well as proteins that facilitate AML1-ETO binding.

Nanotechnological Advancements for the Theranostic Intervention in Anaplastic Thyroid Cancer: Current Perspectives and Future Direction.

Anaplastic thyroid cancer is the rarest, most aggressive, and undifferentiated class of thyroid cancer, accounting for nearly forty percent of all thyroid cancer-related deaths. It is caused by alterations in many cellular pathways like MAPK, PI3K/AKT/mTOR, ALK, Wnt activation, and TP53 inactivation. Although many treatment strategies, such as radiation therapy and chemotherapy, have been proposed to treat anaplastic thyroid carcinoma, they are usually accompanied by concerns such as resistance, which may lead to the lethality of the patient. The emerging nanotechnology-based approaches cater the purposes such as targeted drug delivery and modulation in drug release patterns based on internal or external stimuli, leading to an increase in drug concentration at the site of the action that gives the required therapeutic action as well as modulation in diagnostic intervention with the help of dye property materials. Nanotechnological platforms like liposomes, micelles, dendrimers, exosomes, and various nanoparticles are available and are of high research interest for therapeutic intervention in anaplastic thyroid cancer. The pro gression of the disease can also be traced by using magnetic probes or radio-labeled probes and quantum dots that serve as a diagnostic intervention in anaplastic thyroid cancer.

Rare diseases and pyruvate kinase M2: a promising therapeutic connection.

Pyruvate kinase M2 (PKM2) is a key glycolytic enzyme that regulates proliferating cell metabolism. The role of PKM2 in common diseases has been well established, but its role in rare diseases (RDs) is less understood. Over the past few years, PKM2 has emerged as a crucial player in RDs, including, neoplastic, respiratory, metabolic, and neurological disorders. Herein, we summarize recent findings and developments highlighting PKM2 as an emerging key player in RDs. We also discuss the current status of PKM2 modulation in RDs with particular emphasis on preclinical and clinical studies in addition to current challenges in the field.

Nutritional and microbiota-based therapeutic interventions for alcohol-associated liver disease: From pathogenesis to therapeutic insights.

Alcohol-associated liver disease (ALD) manifests as a consequence of prolonged and excessive alcohol consumption. This disease is closely associated with the interplay between gut health and liver function, which can lead to complex pathophysiological changes in the body. This review offers a comprehensive exploration of ALD's multifaceted nature, with a keen focus on its pathogenesis and the potential of nutritional and microbiota-based therapies. Insights derived from diverse case studies are utilized to shed light on how interventions can rebalance the gut microbiome and enhance liver function in ALD patients. Furthermore, the feasibility of liver transplantation and stem cell therapy as ultimate measures for ALD has been discussed, with acknowledgment of the inherent risks and challenges accompanying them. ALD's complexity underscores the necessity for a thorough understanding of its etiology and progression to devise effective treatments that mitigate its profound impact on an individual's health.

BM-MSC-Loaded Graphene-Collagen Cryogels Ameliorate Neuroinflammation in a Rat Spinal Cord Injury Model.

A major obstacle to axonal regeneration following spinal cord injury (SCI) is neuroinflammation mediated by astrocytes and microglial cells. We previously demonstrated that graphene-based collagen hydrogels alone can decrease neuroinflammation in SCI. Their regenerative potential, however, is poorly understood and incomplete. Furthermore, stem cells have demonstrated both neuroprotective and regenerative properties in spinal cord regeneration, although there are constraints connected with the application of stem cell-based therapy. In this study, we have analyzed the regeneration capability of human bone marrow mesenchymal stem cell (BM-MSC)-loaded graphene-cross-linked collagen cryogels (Gr-Col) in a thoracic (T10-T11) hemisection model of SCI. Our study found that BM-MSC-loaded Gr-Col improves axonal regeneration, reduces neuroinflammation by decreasing astrocyte reactivity, and promotes M2 macrophage polarization. BM-MSC-loaded-Gr-Col demonstrated enhanced regenerative potential compared to Gr-Col and the injury group control. Next-generation sequencing (NGS) analysis revealed that BM-MSC-loaded-Gr-Col modulates the JAK2-STAT3 pathway, thus decreasing the reactive and scar-forming astrocyte phenotype. The decrease in neuroinflammation in the BM-MSC-loaded-Gr-Col group is attributed to the modulation of Notch/Rock and STAT5a/b and STAT6 signaling. Overall, Gene Set Enrichment Analysis suggests the promising role of BM-MSC-loaded-Gr-Col in promoting axonal regeneration after SCI by modulating molecular pathways such as the PI3/Akt pathway, focal adhesion kinase, and various inflammatory pathways.

Isolation and characterization of bioactive alkaloids and GC-MS based identification of volatile oil metabolites from fruits of Glycosmis pentaphylla and evaluation of their cytotoxic activity.

Glycosmis pentaphylla, a member of the Rutaceae family, has been extensively studied for its pharmacological activities, focusing mainly on the cytotoxic properties of its roots and stems. Conversely, limited researched has been done in terms of the phytochemical composition of the fruits. The objective of this study is to isolate and identify the bioactive compounds found in the fruits of G. pentaphylla and then evaluate their potential for anti-cancer activity in oral cancer CAL 27 cell lines. The extraction of bioactive compounds from fruits was done by maceration, and the isolation of alkaloids and volatile oil fractions (F1-F5) was performed by column chromatography. The alkaloids, such as 3-O-methoxyglycocitrine II, noracronycine, 1-hydroxy-3-methoxy-10-methyl-9-acridone and kokusaginine, were first isolated from the fruits of G. pentaphylla. Additionally, GC-MS analysis identified 78 metabolites. The isolated compounds and identified volatile oil fractions were explored for their anti-cancer activity by cell viability assay. Results demonstrated that isolated compounds were found inactive, while the volatile fraction F1 was found active in CAL 27 cell line. Fraction F1 impeded wound healing in CAL 27 cells by scratch assay, and significantly inhibited colony formation in colony formation assay. In cell cycle analysis, treatment with fraction F1 redistributed cells to the S and G2 phases of the cell cycle. α-elemol (2) is the major metabolite identified from the F1 fraction by GC-MS, which could be responsible for the anti-cancer activity. There is potential for future work to further isolate volatile oil metabolites and evaluate their anti-cancer activity through in-vivo techniques.

Targeted suppression of oral squamous cell carcinoma by pyrimidine-tethered quinoxaline derivatives.

Oral cancer (OC) stands as a prominent cause of global mortality. Despite numerous efforts in recent decades, the efficacy of novel therapies to extend the lifespan of OC patients remains disappointingly low. Consequently, the demand for innovative therapeutic agents has become all the more pressing. In this context, we present our work on the design and synthesis of twenty-five novel quinoxaline-tethered imidazopyri(mi)dine derivatives. This was followed by comprehensive investigations into the impact of these molecules on the OC cell line. The in vitro cytotoxicity studies performed in CAL-27 and normal oral epithelial (NOE) cell lines revealed that some of the synthesized molecules like 12d have potent antiproliferative activity specifically towards OC cells with an IC50 of 0.79 µM and show negligible cytotoxicity over NOE cells. Further, 12d arrested cell growth in the S phase of the cell cycle and induced cell death by early apoptosis. The in silico studies validated that 12d binds to the activator binding site on pyruvate kinase M2 (PKM2) overexpressed in OC while the lactate dehydrogenase (LDH)-coupled enzyme assay established 12d as a potent PKM2 activator with an AC50 of 0.6 nM. Hence, this study provides fruitful evidence for the designed compounds as anticancer agents against OC.

Role of epigenetics in OSCC: an understanding above genetics.

Oral cavity cancer is categorized under head and neck cancer that frequently develops from squamous cells hence also known as oral squamous cell carcinoma (OSCC). Although molecular markers for oral cavity cancer are already known, epigenetic signatures for the same haven't been explored much. Epigenetic and genetic alterations were initially thought to be discrete mechanisms driving the tumour but the whole exome sequencing of various cancers has revealed the interdependency of epigenetics and genetic alterations. The reversible nature of these epigenetic changes makes them an alluring target for cancer therapeutics. The primary epigenetic alterations in cancer include DNA methylation and histone modifications. These alterations are useful for patient early detection and prognostication. This review summarizes the epigenetic perspective to understand the etiology, epigenetic biomarkers, and epi-drugs for better predictive diagnosis and treatment of OSCC.

Bugs as Drugs: Understanding the Linkage between Gut Microbiota and Cancer Treatment.

The commensal microbiota is known to regulate host physiology. Dysbiosis or compromised resilience in the microbial ecology is related to the impending risk of cancer. A potential link between cancer and microbiota is indicated by a lot of evidence. The current review explores in detail the various links leading to and /or facilitating oncogenesis, providing sound reasoning or a basis for its utilization as potential therapeutic targets. The present review emphasizes the existing knowledge of the microbiome in cancer and further elaborates on the factors, like genetic modifications, effects of dietary components, and environmental agents, that are considered to assess the direct and indirect effect of microbes in the process of oncogenesis and on the host's health. Strategies modulating the microbiome and novel biotherapeutics are also discussed. Pharmacomicrobiomics is one such niche accounting for the interplay between the microbiome, xenobiotic, and host responses, which is also looked upon. The literature search strategy for this review was conducted by following the methodology of the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA). The method includes the collection of data from different search engines, like PubMed, ScienceDirect, SciFinder, etc., to get coverage of relevant literature for accumulating appropriate information regarding microbiome, cancer, and their linkages. These considerations are made to expand the existing literature on the role of gut microbiota in the host's health, the interaction between host and microbiota, and the reciprocal relationship between the microbiome and modified neoplastic cells. Potential therapeutic implications of cancer microbiomes that are yet unexplored and have rich therapeutic dividends improving human health are discussed in detail in this review.

Chromatin Rewiring by Mismatch Repair Protein MSH2 Alters Cell Adhesion Pathways and Sensitivity to BET Inhibition in Gastric Cancer.

Mutations in the DNA mismatch repair gene MSH2 are causative of microsatellite instability (MSI) in multiple cancers. Here, we discovered that besides its well-established role in DNA repair, MSH2 exerts a novel epigenomic function in gastric cancer. Unbiased CRISPR-based mass spectrometry combined with genome-wide CRISPR functional screening revealed that in early-stage gastric cancer MSH2 genomic binding is not randomly distributed but rather is associated specifically with tumor-associated super-enhancers controlling the expression of cell adhesion genes. At these loci, MSH2 genomic binding was required for chromatin rewiring, de novo enhancer-promoter interactions, maintenance of histone acetylation levels, and regulation of cell adhesion pathway expression. The chromatin function of MSH2 was independent of its DNA repair catalytic activity but required MSH6, another DNA repair gene, and recruitment to gene loci by the SWI/SNF chromatin remodeler SMARCA4/BRG1. Loss of MSH2 in advanced gastric cancers was accompanied by deficient cell adhesion pathway expression, epithelial-mesenchymal transition, and enhanced tumorigenesis in vitro and in vivo. However, MSH2-deficient gastric cancers also displayed addiction to BAZ1B, a bromodomain-containing family member, and consequent synthetic lethality to bromodomain and extraterminal motif (BET) inhibition. Our results reveal a role for MSH2 in gastric cancer epigenomic regulation and identify BET inhibition as a potential therapy in MSH2-deficient gastric malignancies. SIGNIFICANCE: DNA repair protein MSH2 binds and regulates cell adhesion genes by enabling enhancer-promoter interactions, and loss of MSH2 causes deficient cell adhesion and bromodomain and extraterminal motif inhibitor synthetic lethality in gastric cancer.

Overview of oral cavity squamous cell carcinoma: Risk factors, mechanisms, and diagnostics.

Oral cavity squamous cell carcinoma (OCSCC) is the most common malignancy of the oral cavity. The substantial risk factors for OCSCC are the consumption of tobacco products, alcohol, betel quid, areca nut, and genetic alteration. However, technological advancements have occurred in treatment, but the survival decreases with late diagnosis; therefore, new methods are continuously being investigated for treatment. In addition, the rate of secondary tumor formation is 3-7% yearly, which is incomparable to other malignancies and can lead to the disease reoccurrence. Oral cavity cancer (OCC) arises from genetic alterations, and a complete understanding of the molecular mechanism involved in OCC is essential to develop targeted treatments. This review aims to update the researcher on oral cavity cancer, risk factors, genetic alterations, molecular mechanism, classification, diagnostic approaches, and treatment.

CRISPRi enables isoform-specific loss-of-function screens and identification of gastric cancer-specific isoform dependencies.

INTRODUCTION: Genes contain multiple promoters that can drive the expression of various transcript isoforms. Although transcript isoforms from the same gene could have diverse and non-overlapping functions, current loss-of-function methodologies are not able to differentiate between isoform-specific phenotypes. RESULTS: Here, we show that CRISPR interference (CRISPRi) can be adopted for targeting specific promoters within a gene, enabling isoform-specific loss-of-function genetic screens. We use this strategy to test functional dependencies of 820 transcript isoforms that are gained in gastric cancer (GC). We identify a subset of GC-gained transcript isoform dependencies, and of these, we validate CIT kinase as a novel GC dependency. We further show that some genes express isoforms with opposite functions. Specifically, we find that the tumour suppressor ZFHX3 expresses an isoform that has a paradoxical oncogenic role that correlates with poor patient outcome. CONCLUSIONS: Our work finds isoform-specific phenotypes that would not be identified using current loss-of-function approaches that are not designed to target specific transcript isoforms.

Characterization of a genomic region 8 kb downstream of GFI1B associated with myeloproliferative neoplasms.

A genomic locus 8 kb downstream of the transcription factor GFI1B (Growth Factor Independence 1B) predisposes to clonal hematopoiesis and myeloproliferative neoplasms. One of the most significantly associated polymorphisms in this region is rs621940-G. GFI1B auto-represses GFI1B, and altered GFI1B expression contributes to myeloid neoplasms. We studied whether rs621940-G affects GFI1B expression and growth of immature cells. GFI1B ChIP-seq showed clear binding to the rs621940 locus. Preferential binding of various hematopoietic transcription factors to either the rs621940-C or -G allele was observed, but GFI1B showed no preference. In gene reporter assays the rs621940 region inhibited GFI1B promoter activity with the G-allele having less suppressive effects compared to the C-allele. However, CRISPR-Cas9 mediated deletion of the locus in K562 cells did not alter GFI1B expression nor auto-repression. In healthy peripheral blood mononuclear cells GFI1B expression did not differ consistently between the rs621940 alleles. Long range and targeted deep sequencing did not detect consistent effects of rs621940-G on allelic GFI1B expression either. Finally, we observed that myeloid colony formation was not significantly affected by either rs621940 allele in 193 healthy donors. Together, these findings show no evidence that rs621940 or its locus affect GFI1B expression, auto-repression or growth of immature myeloid cells.

CBFß-SMMHC Affects Genome-wide Polycomb Repressive Complex 1 Activity in Acute Myeloid Leukemia.

Mutations and deletions of polycomb repressive complex (PRC) components are increasingly recognized to affect tumor biology in a range of cancers. However, little is known about how genetic alterations of PRC-interacting molecules such as the core binding factor (CBF) complex influence polycomb activity. We report that the acute myeloid leukemia (AML)-associated CBFß-SMMHC fusion oncoprotein physically interacts with the PRC1 complex and that these factors co-localize across the AML genome in an apparently PRC2-independent manner. Depletion of CBFß-SMMHC caused substantial increases in genome-wide PRC1 binding and marked changes in the association between PRC1 and the CBF DNA-binding subunit RUNX1. PRC1 was more likely to be associated with actively transcribed genes in CBFß-SMMHC-expressing cells. CBFß-SMMHC depletion had heterogeneous effects on gene expression, including significant reductions in transcription of ribosomal loci occupied by PRC1. Our results provide evidence that CBFß-SMMHC markedly and diversely affects polycomb recruitment and transcriptional regulation across the AML genome.