RPA and Rad27 limit templated and inverted insertions at DNA breaks.

Formation of templated insertions at DNA double-strand breaks (DSBs) is very common in cancer cells. The mechanisms and enzymes regulating these events are largely unknown. Here, we investigated templated insertions in yeast at DSBs using amplicon sequencing across a repaired locus. We document very short (most ∼5-34 bp), templated inverted duplications at DSBs. They are generated through a foldback mechanism that utilizes microhomologies adjacent to the DSB. Enzymatic requirements suggest a hybrid mechanism wherein one end requires Polδ-mediated synthesis while the other end is captured by nonhomologous end joining (NHEJ). This process is exacerbated in mutants with low levels or mutated RPA ( rtt105 Δ; rfa1 -t33) or extensive resection mutant ( sgs1 Δ exo1 Δ). Templated insertions from various distant genomic locations also increase in these mutants as well as in rad27 Δ and originate from fragile regions of the genome. Among complex insertions, common events are insertions of two sequences, originating from the same locus and with inverted orientation. We propose that these inversions are also formed by microhomology-mediated template switching. Taken together, we propose that a shortage of RPA typical in cancer cells is one possible factor stimulating the formation of templated insertions.

The therapeutic potential of targeting the CHD protein family in cancer.

Accumulating evidence indicates that epigenetic events undergo deregulation in various cancer types, playing crucial roles in tumor development. Among the epigenetic factors involved in the epigenetic remodeling of chromatin, the chromodomain helicase DNA-binding protein (CHD) family frequently exhibits gain- or loss-of-function mutations in distinct cancer types. Therefore, targeting CHD remodelers holds the potential for antitumor treatment. In this review, we discuss epigenetic regulations of cancer development. We emphasize proteins in the CHD family, delving deeply into the intricate mechanisms governing their functions. Additionally, we provide an overview of current therapeutic strategies targeting CHD family members in preclinical trials. We further discuss the promising approaches that have demonstrated early signs of success in cancer treatment.

Yeast EndoG prevents genome instability by degrading cytoplasmic DNA.

In metazoans release of mitochondrial DNA or retrotransposon cDNA to cytoplasm can cause sterile inflammation and disease 1. Cytoplasmic nucleases degrade these DNA species to limit inflammation 2,3. It remains unknown whether degradation these DNA also prevents nuclear genome instability. To address this question, we decided to identify the nuclease regulating transfer of these cytoplasmic DNA species to the nucleus. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. Nuclear mtDNA (NUMTs) and retrotransposon cDNA insertions increase dramatically in nondividing stationary phase cells. Yeast EndoG (Nuc1) nuclease limits insertions of cDNA and transfer of very long mtDNA (>10 kb) that forms unstable circles or rarely insert in the genome, but it promotes formation of short NUMTs (~45-200 bp). Nuc1 also regulates transfer of cytoplasmic DNA to nucleus in aging or during meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs can originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating cytoplasmic DNA play a role in preserving genome stability.

Transcriptional inhibition after irradiation occurs preferentially at highly expressed genes in a manner dependent on cell cycle progression.

In response to DNA double strand damage, ongoing transcription is inhibited to facilitate accurate DNA repair while transcriptional recovery occurs after DNA repair is complete. However, the mechanisms at play and identity of the transcripts being regulated in this manner are unclear. In contrast to the situation following UV damage, we found that transcriptional recovery after ionizing radiation (IR) occurs in a manner independent of the HIRA histone chaperone. Sequencing of the nascent transcripts identified a programmed transcriptional response, where certain transcripts and pathways are rapidly downregulated after IR, while other transcripts and pathways are upregulated. Specifically, most of the loss of nascent transcripts occurring after IR is due to inhibition of transcriptional initiation of the highly transcribed histone genes and the rDNA. To identify factors responsible for transcriptional inhibition after IR in an unbiased manner, we performed a whole genome gRNA library CRISPR / Cas9 screen. Many of the top hits in our screen were factors required for protein neddylation. However, at short times after inhibition of neddylation, transcriptional inhibition still occurred after IR, even though neddylation was effectively inhibited. Persistent inhibition of neddylation blocked transcriptional inhibition after IR, and it also leads to cell cycle arrest. Indeed, we uncovered that many inhibitors and conditions that lead to cell cycle arrest in G1 or G2 phase also prevent transcriptional inhibition after IR. As such, it appears that transcriptional inhibition after IR occurs preferentially at highly expressed genes in cycling cells.

Heterozygous Kmt2d loss diminishes enhancers to render medulloblastoma cells vulnerable to combinatory inhibition of lysine demethylation and oxidative phosphorylation.

The histone H3 lysine 4 (H3K4) methyltransferase KMT2D (also called MLL4) is one of the most frequently mutated epigenetic modifiers in medulloblastoma (MB) and other types of cancer. Notably, heterozygous loss of KMT2D is prevalent in MB and other cancer types. However, what role heterozygous KMT2D loss plays in tumorigenesis has not been well characterized. Here, we show that heterozygous Kmt2d loss highly promotes MB driven by heterozygous loss of the MB suppressor gene Ptch in mice. Heterozygous Kmt2d loss upregulated tumor-promoting programs, including oxidative phosphorylation and G-protein-coupled receptor signaling, in Ptch-mutant-driven MB genesis. Mechanistically, both downregulation of the transcription-repressive tumor suppressor gene NCOR2 by heterozygous Kmt2d loss and upregulation of the oncogene MycN by heterozygous Ptch loss increased the expression of tumor-promoting genes. Moreover, heterozygous Kmt2d loss extensively diminished enhancer signals (e.g., H3K27ac) and H3K4me3 signature, including those for tumor suppressor genes (e.g., Ncor2). Combinatory pharmacological inhibition of oxidative phosphorylation and the H3K4 demethylase LSD1 drastically reduced tumorigenicity of MB cells bearing heterozygous Kmt2d loss. These findings reveal the mechanistic basis underlying the MB-promoting effect of heterozygous KMT2D loss, provide a rationale for a therapeutic strategy for treatment of KMT2D-deficient MB, and have mechanistic implications for the molecular pathogenesis of other types of cancer bearing heterozygous KMT2D loss.

Low RNA stability signifies strong expression regulatability of tumor suppressors.

RNA expression of a gene is determined by not only transcriptional regulation, but also post-transcriptional regulation of RNA decay. The precise regulation of RNA stability in the cell plays an important role in normal development. Dysregulation of RNA stability can lead to diseases such as cancer. Here we found tumor suppressor RNAs tended to decay fast in normal cell types when compared with other RNAs. Consistent with a negative effect of m6A modification on RNA stability, we observed preferential deposition of m6A on tumor suppressor RNAs. Moreover, abundant m6A and fast decay of tumor suppressor RNAs both tended to be further enhanced in prostate cancer cells relative to normal prostate epithelial cells. Further, knockdown of m6A methyltransferase METTL3 and reader YTHDF2 in prostate cancer cells both posed stronger effect on tumor suppressor RNAs than on other RNAs. These results indicated a strong post transcriptional expression regulatability mediated by abundant m6A modification on tumor suppressor RNAs.

Epigenetic Induction of Smooth Muscle Cell Phenotypic Alterations in Aortic Aneurysms and Dissections.

BACKGROUND: Smooth muscle cell (SMC) phenotypic switching has been increasingly detected in aortic aneurysm and dissection (AAD) tissues. However, the diverse SMC phenotypes in AAD tissues and the mechanisms driving SMC phenotypic alterations remain to be identified. METHODS: We examined the transcriptomic and epigenomic dynamics of aortic SMC phenotypic changes in mice with angiotensin II-induced AAD by using single-cell RNA sequencing and single-cell sequencing assay for transposase-accessible chromatin. SMC phenotypic alteration in aortas from patients with ascending thoracic AAD was examined by using single-cell RNA sequencing analysis. RESULTS: Single-cell RNA sequencing analysis revealed that aortic stress induced the transition of SMCs from a primary contractile phenotype to proliferative, extracellular matrix-producing, and inflammatory phenotypes. Lineage tracing showed the complete transformation of SMCs to fibroblasts and macrophages. Single-cell sequencing assay for transposase-accessible chromatin analysis indicated that these phenotypic alterations were controlled by chromatin remodeling marked by the reduced chromatin accessibility of contractile genes and the induced chromatin accessibility of genes involved in proliferation, extracellular matrix, and inflammation. IRF3 (interferon regulatory factor 3), a proinflammatory transcription factor activated by cytosolic DNA, was identified as a key driver of the transition of aortic SMCs from a contractile phenotype to an inflammatory phenotype. In cultured SMCs, cytosolic DNA signaled through its sensor STING (stimulator of interferon genes)-TBK1 (tank-binding kinase 1) to activate IRF3, which bound and recruited EZH2 (enhancer of zeste homolog 2) to contractile genes to induce repressive H3K27me3 modification and gene suppression. In contrast, double-stranded DNA-STING-IRF3 signaling induced inflammatory gene expression in SMCs. In Sting-/- mice, the aortic stress-induced transition of SMCs into an inflammatory phenotype was prevented, and SMC populations were preserved. Finally, profound SMC phenotypic alterations toward diverse directions were detected in human ascending thoracic AAD tissues. CONCLUSIONS: Our study reveals the dynamic epigenetic induction of SMC phenotypic alterations in AAD. DNA damage and cytosolic leakage drive SMCs from a contractile phenotype to an inflammatory phenotype.

Comprehensive analysis of intercellular communication in the thermogenic adipose niche.

Brown adipose tissue (BAT) is responsible for regulating body temperature through adaptive thermogenesis. The ability of thermogenic adipocytes to dissipate chemical energy as heat counteracts weight gain and has gained considerable attention as a strategy against obesity. BAT undergoes major remodeling in a cold environment. This remodeling results from changes in the number and function of brown adipocytes, expanding the network of blood vessels and sympathetic nerves, and changes in the composition and function of immune cells. Such synergistic adaptation requires extensive crosstalk between individual cells in the tissue to coordinate their responses. To understand the mechanisms of intercellular communication in BAT, we apply the CellChat algorithm to single-cell transcriptomic data of mouse BAT. We construct an integrative network of the ligand-receptor interactome in BAT and identify the major signaling inputs and outputs of each cell type. By comparing the ligand-receptor interactions in BAT of mice housed at different environmental temperatures, we show that cold exposure enhances the intercellular interactions among the major cell types in BAT, including adipocytes, adipocyte progenitors, lymphatic and vascular endothelial cells, myelinated and non-myelinated Schwann cells, and immune cells. These interactions are predicted to regulate the remodeling of the extracellular matrix, the inflammatory response, angiogenesis, and neurite growth. Together, our integrative analysis of intercellular communications in BAT and their dynamic regulation in response to housing temperatures provides a new understanding of the mechanisms underlying BAT thermogenesis. The resources presented in this study offer a valuable platform for future investigations of BAT development and thermogenesis.

Early hyperlipidemia triggers metabolomic reprogramming with increased SAH, increased acetyl-CoA-cholesterol synthesis, and decreased glycolysis.

To identify metabolomic reprogramming in early hyperlipidemia, unbiased metabolome was screened in four tissues from ApoE-/- mice fed with high fat diet (HFD) for 3 weeks. 30, 122, 67, and 97 metabolites in the aorta, heart, liver, and plasma, respectively, were upregulated. 9 upregulated metabolites were uremic toxins, and 13 metabolites, including palmitate, promoted a trained immunity with increased syntheses of acetyl-CoA and cholesterol, increased S-adenosylhomocysteine (SAH) and hypomethylation and decreased glycolysis. The cross-omics analysis found upregulation of 11 metabolite synthetases in ApoE‾/‾ aorta, which promote ROS, cholesterol biosynthesis, and inflammation. Statistical correlation of 12 upregulated metabolites with 37 gene upregulations in ApoE‾/‾ aorta indicated 9 upregulated new metabolites to be proatherogenic. Antioxidant transcription factor NRF2-/- transcriptome analysis indicated that NRF2 suppresses trained immunity-metabolomic reprogramming. Our results have provided novel insights on metabolomic reprogramming in multiple tissues in early hyperlipidemia oriented toward three co-existed new types of trained immunity.

Epigenetic landscape reveals MECOM as an endothelial lineage regulator.

A comprehensive understanding of endothelial cell lineage specification will advance cardiovascular regenerative medicine. Recent studies found that unique epigenetic signatures preferentially regulate cell identity genes. We thus systematically investigate the epigenetic landscape of endothelial cell lineage and identify MECOM to be the leading candidate as an endothelial cell lineage regulator. Single-cell RNA-Seq analysis verifies that MECOM-positive cells are exclusively enriched in the cell cluster of bona fide endothelial cells derived from induced pluripotent stem cells. Our experiments demonstrate that MECOM depletion impairs human endothelial cell differentiation, functions, and Zebrafish angiogenesis. Through integrative analysis of Hi-C, DNase-Seq, ChIP-Seq, and RNA-Seq data, we find MECOM binds enhancers that form chromatin loops to regulate endothelial cell identity genes. Further, we identify and verify the VEGF signaling pathway to be a key target of MECOM. Our work provides important insights into epigenetic regulation of cell identity and uncovered MECOM as an endothelial cell lineage regulator.

Low RNA stability signifies increased post-transcriptional regulation of cell identity genes.

Cell identity genes are distinct from other genes with respect to the epigenetic mechanisms to activate their transcription, e.g. by super-enhancers and broad H3K4me3 domains. However, it remains unclear whether their post-transcriptional regulation is also unique. We performed a systematic analysis of transcriptome-wide RNA stability in nine cell types and found that unstable transcripts were enriched in cell identity-related pathways while stable transcripts were enriched in housekeeping pathways. Joint analyses of RNA stability and chromatin state revealed significant enrichment of super-enhancers and broad H3K4me3 domains at the gene loci of unstable transcripts. Intriguingly, the RNA m6A methyltransferase, METTL3, preferentially binds to chromatin at super-enhancers, broad H3K4me3 domains and their associated genes. METTL3 binding intensity is positively correlated with RNA m6A methylation and negatively correlated with RNA stability of cell identity genes, probably due to co-transcriptional m6A modifications promoting RNA decay. Nanopore direct RNA-sequencing showed that METTL3 knockdown has a stronger effect on RNA m6A and mRNA stability for cell identity genes. Our data suggest a run-and-brake model, where cell identity genes undergo both frequent transcription and fast RNA decay to achieve precise regulation of RNA expression.

Dynamic changes in P300 enhancers and enhancer-promoter contacts control mouse cardiomyocyte maturation.

Cardiomyocyte differentiation continues throughout murine gestation and into the postnatal period, driven by temporally regulated expression changes in the transcriptome. The mechanisms that regulate these developmental changes remain incompletely defined. Here, we used cardiomyocyte-specific ChIP-seq of the activate enhancer marker P300 to identify 54,920 cardiomyocyte enhancers at seven stages of murine heart development. These data were matched to cardiomyocyte gene expression profiles at the same stages and to Hi-C and H3K27ac HiChIP chromatin conformation data at fetal, neonatal, and adult stages. Regions with dynamic P300 occupancy exhibited developmentally regulated enhancer activity, as measured by massively parallel reporter assays in cardiomyocytes in vivo, and identified key transcription factor-binding motifs. These dynamic enhancers interacted with temporal changes of the 3D genome architecture to specify developmentally regulated cardiomyocyte gene expressions. Our work provides a 3D genome-mediated enhancer activity landscape of murine cardiomyocyte development.

CD45 Is Sufficient to Initiate Endothelial-to-Mesenchymal Transition in Human Endothelial Cells-Brief Report.

BACKGROUND: Endothelial-to-mesenchymal transition (EndMT) is a dynamic process in which endothelial cells acquire mesenchymal properties and in turn contribute to tissue remodeling and growth. Previously, we found EndMT associated with mitral valve adaptation after myocardial infarction. Furthermore, mitral valve endothelial cells collected at 6 months post-myocardial infarction expressed the pan-leukocyte marker CD45 and EndMT markers. Additionally, mitral valve endothelial cells induced to undergo EndMT with TGF (transforming growth factor)-ß1 strongly coexpressed CD45 but not CD11b or CD14. Pharmacologic inhibition of the CD45 PTPase (protein tyrosine phosphatase) domain in mitral valve endothelial cells blocked TGFß-induced EndMT. This prompted us to speculate that, downstream of TGFß, CD45 induces EndMT. METHODS: We activated the endogenous CD45 promoter in human endothelial colony forming cells (ECFCs) using CRISPR (cluster regularly interspaced short palindromic repeats)/inactive Cas9 (CRISPR-associated protein 9) transcriptional activation. Bulk RNA sequencing was performed on control ECFCs and CD45-positive ECFCs to identify transcriptomic changes. Three functional assays-cellular migration, collagen gel contraction, and transendothelial electrical resistance-were conducted to assess mesenchymal properties in CD45-positive ECFCs. RESULTS: Activation of the endogenous CD45 promoter in ECFC and 3 additional sources of endothelial cells induced expression of several genes implicated in EndMT. In addition, CD45-positive ECFCs showed increased migration, a hallmark of EndMT, increased collagen gel contraction, a hallmark of mesenchymal cells, and decreased cell-cell barrier integrity, indicating reduced endothelial function. CONCLUSIONS: CD45 is sufficient to incite an EndMT phenotype and acquisition of mesenchymal cell properties in normal human ECFCs. We speculate that CD45, through its C-terminal PTPase domain, initiates signaling events that drive EndMT.

mt-Ty 5'tiRNA regulates skeletal muscle cell proliferation and differentiation.

In this study, we sought to determine the role of tRNA-derived fragments in the regulation of gene expression during skeletal muscle cell proliferation and differentiation. We employed cell culture to examine the function of mt-Ty 5' tiRNAs. Northern blotting, RT-PCR as well as RNA-Seq, were performed to determine the effects of mt-Ty 5' tiRNA loss and gain on gene expression. Standard and transmission electron microscopy (TEM) were used to characterize cell and sub-cellular structures. mt-Ty 5'tiRNAs were found to be enriched in mouse skeletal muscle, showing increased levels in later developmental stages. Gapmer-mediated inhibition of tiRNAs in skeletal muscle C2C12 myoblasts resulted in decreased cell proliferation and myogenic differentiation; consistent with this observation, RNA-Seq, transcriptome analyses, and RT-PCR revealed that skeletal muscle cell differentiation and cell proliferation pathways were also downregulated. Conversely, overexpression of mt-Ty 5'tiRNAs in C2C12 cells led to a reversal of these transcriptional trends. These data reveal that mt-Ty 5'tiRNAs are enriched in skeletal muscle and play an important role in myoblast proliferation and differentiation. Our study also highlights the potential for the development of tiRNAs as novel therapeutic targets for muscle-related diseases.

Targeting Epsins to Inhibit Fibroblast Growth Factor Signaling While Potentiating Transforming Growth Factor-ß Signaling Constrains Endothelial-to-Mesenchymal Transition in Atherosclerosis.

BACKGROUND: Epsin endocytic adaptor proteins are implicated in the progression of atherosclerosis; however, the underlying molecular mechanisms have not yet been fully defined. In this study, we determined how epsins enhance endothelial-to-mesenchymal transition (EndoMT) in atherosclerosis and assessed the efficacy of a therapeutic peptide in a preclinical model of this disease. METHODS: Using single-cell RNA sequencing combined with molecular, cellular, and biochemical analyses, we investigated the role of epsins in stimulating EndoMT using knockout in Apoe-/- and lineage tracing/proprotein convertase subtilisin/kexin type 9 serine protease mutant viral-induced atherosclerotic mouse models. The therapeutic efficacy of a synthetic peptide targeting atherosclerotic plaques was then assessed in Apoe-/- mice. RESULTS: Single-cell RNA sequencing and lineage tracing revealed that epsins 1 and 2 promote EndoMT and that the loss of endothelial epsins inhibits EndoMT marker expression and transforming growth factor-ß signaling in vitro and in atherosclerotic mice, which is associated with smaller lesions in the Apoe-/- mouse model. Mechanistically, the loss of endothelial cell epsins results in increased fibroblast growth factor receptor-1 expression, which inhibits transforming growth factor-ß signaling and EndoMT. Epsins directly bind ubiquitinated fibroblast growth factor receptor-1 through their ubiquitin-interacting motif, which results in endocytosis and degradation of this receptor complex. Consequently, administration of a synthetic ubiquitin-interacting motif-containing peptide atheroma ubiquitin-interacting motif peptide inhibitor significantly attenuates EndoMT and progression of atherosclerosis. CONCLUSIONS: We conclude that epsins potentiate EndoMT during atherogenesis by increasing transforming growth factor-ß signaling through fibroblast growth factor receptor-1 internalization and degradation. Inhibition of EndoMT by reducing epsin-fibroblast growth factor receptor-1 interaction with a therapeutic peptide may represent a novel treatment strategy for atherosclerosis.

Promoting Lymphangiogenesis and Lymphatic Growth and Remodeling to Treat Cardiovascular and Metabolic Diseases.

Lymphatic vessels are low-pressure, blind-ended tubular structures that play a crucial role in the maintenance of tissue fluid homeostasis, immune cell trafficking, and dietary lipid uptake and transport. Emerging research has indicated that the promotion of lymphatic vascular growth, remodeling, and function can reduce inflammation and diminish disease severity in several pathophysiologic conditions. In particular, recent groundbreaking studies have shown that lymphangiogenesis, which describes the formation of new lymphatic vessels from the existing lymphatic vasculature, can be beneficial for the alleviation and resolution of metabolic and cardiovascular diseases. Therefore, promoting lymphangiogenesis represents a promising therapeutic approach. This brief review summarizes the most recent findings related to the modulation of lymphatic function to treat metabolic and cardiovascular diseases such as obesity, myocardial infarction, atherosclerosis, and hypertension. We also discuss experimental and therapeutic approaches to enforce lymphatic growth and remodeling as well as efforts to define the molecular and cellular mechanisms underlying these processes.

Epsin Nanotherapy Regulates Cholesterol Transport to Fortify Atheroma Regression.

BACKGROUND: Excess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, a family of endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In this study, we determined the role of Epsins in macrophage-mediated metabolic regulation. We then developed an innovative method to therapeutically target macrophage Epsins with specially designed S2P-conjugated lipid nanoparticles, which encapsulate small-interfering RNAs to suppress Epsins. METHODS: We used single-cell RNA sequencing with our newly developed algorithm MEBOCOST (Metabolite-mediated Cell Communication Modeling by Single Cell Transcriptome) to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular, and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. We performed this study using myeloid-specific Epsin double knockout (LysM-DKO) mice and mice with a genetic reduction of ABCG1 (ATP-binding cassette subfamily G member 1; LysM-DKO-ABCG1fl/+). The nanoparticles targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis. RESULTS: We revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing lesional macrophage-specific nanoparticle delivery of Epsin small-interfering RNAs, we showed that silencing of macrophage Epsins diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport. In a LysM-DKO-ABCG1fl/+ mouse model, enhanced cholesterol efflux and reverse transport due to Epsin deficiency was suppressed by the reduction of ABCG1. CONCLUSIONS: Our findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis by reducing CD36-mediated lipid uptake and increasing ABCG1-mediated cholesterol efflux.

TNIK regulation of interferon signaling and endothelial cell response to virus infection.

Background: Traf2 and Nck-interacting kinase (TNIK) is known for its regulatory role in various processes within cancer cells. However, its role within endothelial cells (ECs) has remained relatively unexplored. Methods: Leveraging RNA-seq data and Ingenuity Pathway Analysis (IPA), we probed the potential impact of TNIK depletion on ECs. Results: Examination of RNA-seq data uncovered more than 450 Differentially Expressed Genes (DEGs) in TNIK-depleted ECs, displaying a fold change exceeding 2 with a false discovery rate (FDR) below 0.05. IPA analysis unveiled that TNIK depletion leads to the inhibition of the interferon (IFN) pathway [-log (p-value) >11], downregulation of IFN-related genes, and inhibition of Hypercytokinemia/Hyperchemokinemia [-log (p-value) >8]. The validation process encompassed qRT-PCR to evaluate mRNA expression of crucial IFN-related genes, immunoblotting to gauge STAT1 and STAT2 protein levels, and ELISA for the quantification of IFN and cytokine secretion in siTNIK-depleted ECs. These assessments consistently revealed substantial reductions upon TNIK depletion. When transducing HUVECs with replication incompetent E1-E4 deleted adenovirus expressing green fluorescent protein (Ad-GFP), it was demonstrated that TNIK depletion did not affect the uptake of Ad-GFP. Nonetheless, TNIK depletion induced cytopathic effects (CPE) in ECs transduced with wild-type human adenovirus serotype 5 (Ad-WT). Summary: Our findings suggest that TNIK plays a crucial role in regulating the EC response to virus infections through modulation of the IFN pathway.