Inhibitor Of DNA Binding Research Articles

Circ-ATIC Serves as a Sponge of miR-326 to Accelerate Esophageal Squamous Cell Carcinoma Progression by Targeting ID1

In the previous studies, circular RNA (circRNA) has been shown to be closely related to the occurrence and development of various cancers. However, the role and mechanism of circ-ATIC in the progression of esophageal squamous cell carcinoma (ESCC) is not yet clear. Quantitative real-time PCR was used to detect the expression levels of circ-ATIC, microRNA (miR)-326 and inhibitor of DNA binding 1 (ID1) in tissues (n = 50) and cells. Cell counting kit 8 assay, colony formation assay, flow cytometry, wound-healing assay and transwell assay were performed to measure the proliferation, apoptosis, migration, and invasion of cells. In addition, the oxidative stress of cells was evaluated by detecting the productions of superoxide dismutase and malondialdehyde. Animal studies were implied to explore the role of circ-ATIC in ESCC tumor growth. The relationship between circ-ATIC and miR-326 or ID1 was determined by dual-luciferase reporter assay and RNA immunoprecipitation assay. Additionally, the protein expression of ID1 was examined by western blot assay. Circ-ATIC was found to be upregulated in ESCC tissues and cells. Silenced circ-ATIC suppressed the proliferation, migration, invasion, promoted the apoptosis and oxidative stress of ESCC cells. The tumor growth of ESCC also was inhibited by circ-ATIC knockdown. Furthermore, we found that circ-ATIC could sponge miR-326, and miR-326 could target ID1. The rescue experiments revealed that miR-326 inhibitor could reverse the negative regulation of circ-ATIC silencing on ESCC progression, and ID1 overexpression also inverted the inhibitory effect of miR-326 on ESCC progression. In addition, we confirmed that the expression of ID1 was positively regulated by circ-ATIC. Our study showed that circ-ATIC facilitated the progression of ESCC by regulating the miR-326/ID1 axis, indicating that circ-ATIC might be a target for ESCC treatment.

Discovery of the inhibitor of DNA binding 1 as a novel marker for radioresistance in pancreatic cancer using genome-wide RNA-seq.

Purpose/Objective(s): Discovery of genetic drivers of radioresistance is critical for developing novel therapeutic strategies to combine with radiotherapy of radioresistant PDAC. In this study, we used genome-wide RNA-seq to identify genes upregulated in generated radioresistant PDAC cell lines and discovered the Inhibitor of DNA Binding 1 (ID1) gene as a potential regulator of radioresistance in PDAC. Materials/Methods: Radioresistant clones of the PDAC cell lines MIA PaCa-2 and PANC-1 were generated by delivering daily ionizing irradiation (IR) (2 Gy/day) in vitro over two weeks (total 20 Gy) followed by standard clonogenic assays following one week from the end of IR. The generated RR and parental cell lines were submitted for RNA-seq analysis to identify differentially expressed genes. The Limma R package was used to calculate differential expression among genes. Log2 fold change values were calculated for each sample compared to the control. Genes with an absolute fold change > 1 were considered significant. RNA sequencing expression data from the Cancer Genome Atlas (TCGA) database was analyzed through the online databases GEPIA, cBioPortal, and the Human Protein Atlas. Results: Following exposure to two weeks of 2 Gy daily IR in vitro, the two PDAC cell lines showed significantly greater clonogenic cell survival than their parental cell lines, indicating enhanced RR in these cells. RNA-seq analysis comparing parental and RR cell lines found upregulated seven genes (TNS4, ZDHHC8P1, APLNR, AQP3, SPP1, ID1, ID2) and seven genes downregulated (PTX3, ITGB2, EPS8L1, ALDH1L2, KCNT2, ARHGAP9, IFI16) in both RR cell lines. Western blotting confirmed increased expression of the ID1 protein in the RR cell lines compared to their parental cell lines. We found that ID1 mRNA was significantly higher in PDAC tumors compared to matched normal and high ID1 expression correlated with significantly worse disease-free survival (DFS) in PDAC patients (HR = 2.2, log rank P = 0.009). ID1 mRNA expression was also strongly correlated in tumors with TP53 mutation, a known driver of radioresistance. Conclusion: Our analysis indicates a novel role of ID1 in PDAC radioresistance. ID1 expression is higher in tumor tissue compared to normal, and high expression correlates with both worse DFS and association with the TP53 mutation, suggesting that targeting ID1 prior to IR is an attractive strategy for overcoming radioresistance in PDAC.

TAMI-79. THERAPEUTIC REVERSAL OF PRENATAL PONTINE ID1 SIGNALING IN DIPG

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor with rare survival beyond two years. This poor prognosis is largely due to the tumor's highly infiltrative and invasive nature. Nearly 80% of DMGs harbor K27M mutation in the genes encoding histone H3.1 (H3F3A) or H3.3 (HISTIH3B), often with concurrent ACVR1 mutation. Inhibitor of DNA-binding (ID) proteins are key transcriptional regulators of genes involved in lineage commitment and are associated with invasiveness and poor clinical outcomes in multiple human cancers. Introduction of H3K27M and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been confirmed in human tumors or targeted therapeutically. We developed an in-utero electroporation (IUE) murine H3K27M-driven tumor model, which demonstrates increased ID1 expression in H3K27M- and ACVR1-mutated tumor cells. Exome and transcriptome sequencing analysis of multi-focal DMG tumors (n=52) and normal brain tissue revealed that increased ID1 expression is associated with H3K27M/ACVR1-mutation and brainstem location, and correlates with poor survival in patients. ChIP-sequencing for H3K27ac and H3K27me3 in multiple DMG tumors (n=5) revealed that the ID1 gene is epigenetically active, which matches the epigenetic state of murine prenatal hindbrain cells. Higher ID1-expressing astrocyte-like DIPG cells share a similar transcriptional program with ID1+/SPARCL1+ positive oligo/astrocyte-precursor (OAPC) cells from the developing human brain and demonstrate upregulation of gene sets involved in regulation of cell migration. Both genetic and pharmacologic [cannabidiol (CBD)] suppression of ID1 result in decreased DIPG cell invasion/migration in vitro and invasion/tumor growth in multiple in vivo models. Mechanistically, CBD reduces proliferation through production of reactive oxygen species. Further, DIPG patients treated off-trial with CBD (n=15) displayed reduced ID1 tumor expression and improved overall survival. In summary, ID1 is upregulated in DIPG through K27M-mediated epigenetic reactivation of a developmental OAPC-like transcriptional state, and ID1-driven invasiveness of DIPG is therapeutically targetable with CBD.

Transcriptional Deregulation Mediated By ID2-TCF3 Axis Supports MM Cell Growth and Proliferation in the Context of the Bone Marrow Milieu

Multiple myeloma (MM) is a complex and heterogenous disease which is dependent on the surrounding microenvironment for growth and survival. In MM, dysregulation of transcriptional control is a major driver of tumor transformation and progression. To evaluate transcriptional programs activated in MM cells in the context of the bone marrow milieu, we have performed extensive transcriptomic analysis by RNA-seq and ATAC-seq using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines. We observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 cells significantly downregulated expression of Inhibitor of DNA Binding 2 (ID2), while footprint analysis of the open chromatin regions in MM cells upon interaction with BMSC revealed enrichment for binding motifs of the TCF family of transcription factors (E proteins). Inhibitors of DNA binding (ID) proteins control crucial transcriptional programs in B cell maturation via their heterodimerization with E proteins which are members of the basic helix-loop-helix (bHLH) class I family of transcription factors, repressing their DNA binding and therefore transcriptional activity.We found that ID2 expression is significantly lower in primary CD138+ MM cells from patients compared to normal plasma cells (NPCs). Moreover, we have previously implicated the B-cell factor TCF3 as a novel MM dependency. Using MM cell lines and primary samples, we observed elevated enhancer activity at TCF3 locus in primary malignant plasma cells compared to NPCs, which resulted in significant upregulation of TCF3 expression in MM patients. We also showed that TCF3 is regulated by a large proximal enhancer that is bound by MYC and is highly sensitive to chemical perturbation of enhancer co-activators such as BRD4. Genetic perturbation of TCF3 confirmed its critical role on MM cell growth and viability especially in IgH MYC translocated MM cell lines.We here further explored the role of ID2-TCF3 axis and the hypothesis that lower expression of ID2 drives higher TCF3 activity in MM cells, which is further enhanced in presence of the bone marrow microenvironment.Genetic modulation of ID2 significantly affected MM cell viability, with MM cells ectopically expressing ID2 displaying a cell growth arrest even in the presence of the supportive BM milieu. To define the mechanism of the observed oncosuppressive role of ID2 in MM, which is in line with preliminary observations in other hematological malignancy but in contrast with the pro-tumoral role described in solid tumors, we first performed immunoprecipitation of ID2 followed by mass spectrometry in 3 MM cell lines, and identified a very consistent and specific interaction with E proteins TCF3 and TCF12. Next, to explore the transcriptional programs dependent on ID2 we performed RNA-seq of 2 MM cell lines after ID2 overexpression. In line with our in vitro data, gene ontology and gene set enrichment analysis showed a significant downregulation of genes involved in E2F pathway, cell cycle progression and regulation of gene transcription. Interestingly, among the known TCF3 targets in B cells, only XBP-1 was significantly downregulated in MM cells after ID2 overexpression, suggesting the existence of a cell-specific TCF3 dependent transcriptional program in MM. Indeed, ATAC-seq experiments revealed ID2 overexpression led to a significant decrease of TCF3 binding motifs in open chromatin regions, confirming the relevance of ID2 in regulating TCF3 transcriptional activity in MM.In conclusion, while both E and ID proteins have been implicated in malignant transformation, their role in supporting MM transcriptional deregulation and tumor growth in the context of the microenvironment is being defined. Here, we have identified ID2 as a major regulator of the TCF3 dependent transcriptional program in MM, whose downregulation is essential to maintain MM proliferation and to mediate the benefits induced by MM-stroma interaction. DisclosuresAnderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Celgene: Consultancy; Karyopharm: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy.

Bone morphogenetic protein 6–mediated crosstalk between endothelial cells and hepatocytes recapitulates the iron-sensing pathway in vitro

Liver sinusoidal endothelial cell–derived bone morphogenetic protein 6 (BMP6) and the BMP6–small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte–endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.

Screening of host genes regulated by ID1 and ID3 proteins during foot-and-mouth disease virus infection

Foot-and-mouth disease virus (FMDV) is an important pathogen that harms cloven-hoofed animals and has caused serious losses to livestock production since its discovery. Furthermore, inhibitor of DNA binding (ID) proteins have been thoroughly studied in tumorigenesis, differentiation and metastasis, but its role in viral infection is rarely known. In this study, three gene knockout cell lines ID1 KO, ID3 KO, ID1/3 KO were obtained based on BHK-21 cells. We found that ID1 and ID3 genes single or double knockout promote the replication of FMDV. Moreover, compared with negative control cells during virus infection, there were 551 up-regulated genes and 1222 down-regulated genes in the ID1 KO cell line; 916 up-regulated genes and 1845 down-regulated genes in the ID3 KO cell line; 810 up-regulated genes and 1566 down-regulated genes in ID1/3 KO cell line. Further genes expression patterns verification results also showed a good correlation between the data of RT-qRCR and RNA-seq. These findings provide a basis for studying the relevant mechanisms between host genes and ID genes during FMDV infection.

Epstein-Barr virus status of sporadic Burkitt lymphoma is associated with patient age and mutational features.

Sporadic Burkitt lymphoma (BL) is the most frequent tumour of children and adolescents but a rare subtype of lymphomas in adults. To date most molecular data have been obtained from lymphomas arising in the young. Recently, Epstein-Barr virus (EBV) positive and negative BL in young patients was shown to differ in molecular features. In the present study, we present a large age-overarching cohort of sporadic BL (n=162) analysed by immunohistochemistry, translocations of MYC proto-oncogene, basic helix-loop-helix transcription factor (MYC), B-cell leukaemia/lymphoma 2 (BCL2) and B-cell leukaemia/lymphoma 6 (BCL6) and by targeted sequencing. We illustrate an age-associated inter-tumoral molecular heterogeneity in this disease. Mutations affecting inhibitor of DNA binding 3, HLH protein (ID3), transcription factor 3 (TCF3) and cyclin D3 (CCND3), which are highly recurrent in paediatric BL, and expression of sex determining region Y-box transcription factor 11 (SOX11) declined with patient age at diagnosis (P=0·0204 and P=0·0197 respectively). In contrast, EBV was more frequently detected in adult patients (P=0·0262). Irrespective of age, EBV-positive sporadic BL showed significantly less frequent mutations in ID3/TCF3/CCND3 (P=0·0088) but more often mutations of G protein subunit alpha 13 (GNA13; P=0·0368) and forkhead box O1 (FOXO1; P=0·0044) compared to EBV-negative tumours. Our findings suggest that among sporadic BL an EBV-positive subgroup of lymphomas increases with patient age that shows distinct pathogenic features reminiscent of EBV-positive endemic BL.

Discovery of novel ID2 antagonists from pharmacophore-based virtual screening as potential therapeutics for glioma

Glioma, especially the most aggressive type glioblastoma multiforme, is a malignant cancer of the central nervous system with a poor prognosis. Traditional treatments are mainly surgery combined with radiotherapy and chemotherapy, which is still far from satisfactory. Therefore, it is of great clinical significance to find new therapeutic agents. Serving as an inhibitor of differentiation, protein ID2 (inhibitor of DNA binding 2) plays an important role in neurogenesis, neovascularization and malignant development of gliomas. It has been shown that ID2 affects the malignant progression of gliomas through different mechanisms. In this study, a pharmacophore-based virtual screening was carried out and 16 hit compounds were purchased for pharmacological evaluations on their ID2 inhibitory activities. Based on the cytotoxicity of these small-molecule compounds, two compounds were shown to effectively inhibit the viability of glioma cells in the micromolar range. Among them, AK-778-XXMU was chosen for further study due to its better solubility in water. A SPR (Surface Plasma Resonance) assay proved the high affinity between AK-778-XXMU and ID2 protein with the KD value as 129 nM. The plausible binding mode of ID2 was studied by molecular docking and it was found to match AGX51 very well in the same binding site. Subsequently, the cancer-suppressing potency of the compound was characterized both in vitro and in vivo. The data demonstrated that compound AK-778-XXMU is a potent ID2 antagonist which has the potential to be developed as a therapeutic agent against glioma.

Inhibitor of DNA binding 2 (Id2) mediates microtubule polymerization in the brain by regulating \u03b1K40 acetylation of \u03b1-tubulin

Acetylation of α-tubulin lysine 40 (αK40) contributes to microtubule (MT) stability and is essential for neuronal development and function, whereas excessive αK40 deacetylation is observed in neurodegenerative disorders including Alzheimer’s disease (AD). Here we identified inhibitor of DNA binding 2 (Id2) as a novel MT-binding partner that interacts with α-tubulin and enhances αK40 acetylation, leading to MT polymerization in the neurons. Commensurate with our finding that the low levels of Id2 expression along with a reduced αK40 acetylation in the postmortem human AD patient and 5X-FAD, AD model mice brain, Id2 upregulation in the hippocampus of 5X-FAD, which exhibit high levels of Sirt2 expression, increased αK40 acetylation and reconstitutes axon growth. Hence our study suggests that Id2 is critical for maintaining MT stability during neural development and the potential of Id2 to counteract pathogenic Sirt2 activity in AD.

ID1/ID3 mediate the contribution of skin fibroblasts to local nerve regeneration through Itga6 in wound repair.

Cutaneous wound healing requires intricate synchronization of several key processes. Among them, local nerve regeneration is known to be vitally important for proper repair. However, the underlying mechanisms of local nerve regeneration are still unclear. Fibroblasts are one of the key cell types within the skin whose role in local nerve regeneration has not been extensively studied. In our study, we found skin fibroblasts were in tight contact with regenerated nerves during wound healing, while rare interactions were shown under normal circumstances. Moreover, skin fibroblasts surrounding the nerves were shown to be activated and reprogrammed to exhibit neural cell‐like properties by upregulated expressing inhibitor of DNA binding 1 (ID1) and ID3. Furthermore, we identified the regulation of integrin α6 (Itga6) by ID1/ID3 in fibroblasts as the mechanism for axon guidance. Accordingly, transplantation of the ID1/ID3‐overexpressing fibroblasts or topical injection of ID1/ID3 lentivirus significantly promoted local nerve regeneration and wound healing following skin excision or sciatic nerve injury. Therefore, we demonstrated a new role for skin fibroblasts in nerve regeneration following local injury by directly contacting and guiding axon regrowth, which might hold therapeutic potential in peripheral nerve disorders and peripheral neuropathies in relatively chronic refractory wounds.

Targeted Disruption of the Inhibitor of DNA Binding 4 (Id4) Gene Alters Photic Entrainment of the Circadian Clock.

Inhibitor of DNA binding (Id) genes comprise a family of four helix–loop–helix (HLH) transcriptional inhibitors. Our earlier studies revealed a role for ID2 within the circadian system, contributing to input, output, and core clock function through its interaction with CLOCK and BMAL1. Here, we explore the contribution of ID4 to the circadian system using a targeted disruption of the Id4 gene. Attributes of the circadian clock were assessed by monitoring the locomotor activity of Id4−/− mice, and they revealed disturbances in its operation. Id4-mutant mice expressed a shorter circadian period length, attenuated phase shifts in responses to continuous and discrete photic cues, and an advanced phase angle of entrainment under a 12:12 light:dark cycle and under short and long photoperiods. To understand the basis for these properties, suprachiasmatic nucleus (SCN) and retinal structures were examined. Anatomical analysis reveals a smaller Id4−/− SCN in the width dimension, which is a finding consistent with its smaller brain. As a result of this feature, anterograde tracing in Id4−/− mice revealed retinal afferents innovate a disproportionally larger SCN area. The Id4−/− photic entrainment responses are unlikely to be due to an impaired function of the retinal pathways since Id4−/− retinal anatomy and function tested by pupillometry were similar to wild-type mice. Furthermore, these circadian characteristics are opposite to those exhibited by the Id2−/− mouse, suggesting an opposing influence of the ID4 protein within the circadian system; or, the absence of ID4 results in changes in the expression or activity of other members of the Id gene family. Expression analysis of the Id genes within the Id4−/− SCN revealed a time-of-day specific elevated Id1. It is plausible that the increased Id1 and/or absence of ID4 result in changes in interactions with bHLH canonical clock components or with targets upstream and/or downstream of the clock, thereby resulting in abnormal properties of the circadian clock and its entrainment.

Ectopic expression of Id1 or Id3 inhibits transcription of the GATA-4 gene in P19CL6 cells under differentiation condition.

Inhibitor of DNA binding (Id) is a dominant negative form of the E-box binding basic-helix-loop-helix (bHLH) transcription factor since it is devoid of the basic region required for DNA binding and forms an inactive hetero dimer with bHLH proteins. The E-box sequence located in the promoter region of the GATA-binding protein 4 (GATA-4) gene is essential for transcriptional activation in P19CL6 cells. These cells differentiate into cardiomyocytes and start to express GATA-4, which further triggers cardiac-specific gene expression. In this study, expression plasmids for Ids tagged with human influenza hemagglutinin (HA)-FLAG were constructed and introduced into P19CL6 cells. The stable clones expressing the recombinant Id proteins (Id1 or Id3) were isolated. The GATA-4 gene expression in these clones under differentiation condition in the presence of 1% dimethyl sulfoxide (DMSO) was repressed, with concomitant abolishment of the transcription of α-myosin heavy chain (α-MHC), which is a component of cardiac myofibrils. Thus, the increased expression of Id protein could affect GATA-4 gene expression and negatively regulate the differentiation of P19CL6 cells.

The Coffee Diterpene, Kahweol, Ameliorates Pancreatic β-Cell Function in Streptozotocin (STZ)-Treated Rat INS-1 Cells through NF-kB and p-AKT/Bcl-2 Pathways.

Kahweol is a diterpene molecule found in coffee that exhibits a wide range of biological activity, including anti-inflammatory and anticancer properties. However, the impact of kahweol on pancreatic β-cells is not known. Herein, by using clonal rat INS-1 (832/13) cells, we performed several functional experiments including; cell viability, apoptosis analysis, insulin secretion and glucose uptake measurements, reactive oxygen species (ROS) production, as well as western blotting analysis to investigate the potential role of kahweol pre-treatment on damage induced by streptozotocin (STZ) treatment. INS-1 cells pre-incubated with different concentrations of kahweol (2.5 and 5 µM) for 24 h, then exposed to STZ (3 mmol/L) for 3 h reversed the STZ-induced effect on cell viability, apoptosis, insulin content, and secretion in addition to glucose uptake and ROS production. Furthermore, Western blot analysis showed that kahweol downregulated STZ-induced nuclear factor kappa B (NF-κB), and the antioxidant proteins, Heme Oxygenase-1 (HMOX-1), and Inhibitor of DNA binding and cell differentiation (Id) proteins (ID1, ID3) while upregulated protein expression of insulin (INS), p-AKT and B-cell lymphoma 2 (BCL-2). In conclusion, our study suggested that kahweol has anti-diabetic properties on pancreatic β-cells by suppressing STZ induced apoptosis, increasing insulin secretion and glucose uptake. Targeting NF-κB, p-AKT, and BCL-2 in addition to antioxidant proteins ID1, ID3, and HMOX-1 are possible implicated mechanisms.

Id3 Is Essential for Mediating Ezh2-Regulated Persistence of Alloreactive T Cells in Gvhd Target Tissues in Mice

Abstract: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is potentially a curative treatment for hematologic malignancies and non-malignant diseases. However, its application is limited by graft-versus-host disease (GVHD), which is induced by donor T cells that recognize and react to histocompatibilty differences. Despite pharmacological prophylaxis, acute and chronic GVHD still develop in 40-60% of allo-HSCT recipients. Data from our studies and others suggested critical roles of alloantigen-sensitized memory T cells in sustaining host tissue injury during GVHD. However, the molecular pathway(s) that are crucial for the development and maintenance of alloreactive memory T cells mediating persistent GVHD remain largely unknown. Id3 (Inhibitor of DNA Binding 3) is a transcription factor that negatively regulates the DNA-binding activity of E-protein. During response to acute viral infection, Id3 has been shown to regulate the generation of memory T cells, but is dispensable for primary effector response. Whether Id3 is responsible for persistent allo-immunity and tissue damage during GVHD has not been previously examined. Using experimental GVHD models, we demonstrate that Id3 functions as a key molecular gatekeeper for persistent generation of alloreactive T cells mediating tissue damage and GVHD. Infusion of donor T cells from wild-type (WT) B6 mice caused uniformly lethal GVHD in allogeneic BALB/C recipient mice. In contrast, transfer of donor T cells derived from Id3 conditional knockout (Id3-/-) B6 mice did not induce lethal GVHD. Notably, Id3-/- T cell recipients developed similar symptoms of GVHD (e.g. weight loss, hunched back and reduced mobility) as compared to WT T cell recipients within 20 days after allo-HSCT. However, while GVHD continually progressed in WT T cell recipients, which was evidenced by hair loss, severe diarrhea, skin ulceration, weight loss and death, it was gradually diminished in mice receiving allogeneic Id3-/- T-cells without apparent skin inflammation and tissue damage. As a result, all Id3-/- T-cell recipient mice survived over 70 days after allo-HSCT. We found that by 7 days and 14 days after transplantation, there were similar numbers of donor-derived CD4+ and CD8+ T cells in BALB/c recipients (e.g. spleen, liver, skin and gut) of Id3-/- T cells compared with WT T cells. Furthermore, during this acute alloreactive response phase both Id3-/- and WT T cells expressed high levels of activation markers (e.g. CD25, CD44, CD69, CD122) and had high IFN-g production. By 35 days after allo-HSCT, Id3-/- T cell recipient mice showed significantly increased number of donor T cells in the spleen compared to WT T cell recipients, but had 10-fold fewer numbers of alloreactve T cells in GVHD target organs including gut, skin and liver. Analysis of samples from human patients undergoing allo-HSCT revealed that GVHD patients (n=8) had significantly higher frequency of T cells expressing high levels of ID3 compared to time-matched non-aGVHD patients (n=6). These findings suggest that Id3 plays critical roles in mediating the persistence and accumulation of tissue-infiltrating alloreactive T cells, while being dispensable for early activation, proliferation and effector differentiation of alloreactive T cells. Our recent studies have discovered a central role of the epigenetic regulator Ezh2 in controlling allogeneic T cell proliferation, differentiation and function (Blood 2012, Blood 2013 and Blood 2017). Using genome-wide profiling analysis and functional assessment, we found that Ezh2 directly activates Id3 transcription, independent of its canonical role in repressing gene transcription. Retroviral engineering of Ezh2-deficient T cells to express Id3 significantly enhanced the persistence of antigen-activated T cells in vivo, suggesting a critical role of Id3 in mediating Ezh2 regulation of activated T cell survival and maintenance. In summary, our findings identify the importance of Id3 in regulating persistent allo-immunity and tissue damage, and illuminate a novel molecular pathway of Ezh2-Id3 that may be potentially targeted for GVHD therapy. DisclosuresNo relevant conflicts of interest to declare.

Junctional Adhesion Molecule-a, Suppresses Integrin αvβ3 Activation to Control Neovascularization

Introduction: Neovascularization, the formation of new blood vessels from the preexisting vessels, is a complex process. It is strictly regulated in healthy adults. Disruption of endothelial cell-cell junction is critically important in inflammation and pathological angiogenesis. Growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2), two potent angiogenic factors, have been shown to disrupt endothelial cell (EC) junctions. VEGF expression by EC and thus VEGF-dependent neovascularization is normally suppressed in healthy adults. The mechanism of this suppression is not known.Methods: Using Jam-A gene-trap knockout mice, we performed a variety of neovascularization assays such as Matrigel plug, aortic ring sprouting and in vivo tumor growth. Age-matched wild-type (WT) mice were used as controls. Vascular permeability was assessed by Miles assay. Level of VEGF in both the WT and Jam-A null mouse plasma were measured by Elisa assay. Real-time PCR and western blot analyses were used to assess gene expression.Results: Jam-A knockout mice showed significantly enhanced (P<0.004) vascular permeability as assessed by Miles assay. Interestingly, VEGF-, but not FGF-2-dependent neovascularization was significantly augmented (P<0.001) in the absence of Jam-A. ECs isolated from Jam-A null mouse showed significantly enhanced (P<0.05) cell migration in the response to VEGF as compared to WT ECs. We found that the plasma levels of VEGF in the Jam-A null mice were significantly (P<0.000001) and age-dependently increased compared to WT mice. We also found that both mRNA and protein levels of VEGF and its receptor VEGFR2 were significantly increased (P<0.001) in Jam-A null ECs. When tested if the expression of soluble Flt (sFlt), which is a decoy receptor for VEGF that is known to trap VEGF, is downregulated in the absence of Jam-A, we found no significant difference in sFlt mRNA expression in Jam-A null ECs compared to WT. Additionally, augmented vascular permeability observed in Jam-A null mice was completely abrogated upon inhibition of VEGFR2 using a function-blocking antibody DC101, suggesting that the VEGF/VEGFR2 signaling axis is augmented in the absence of Jam-A. In order to determine the mechanism of this upregulation of the VEGF and VEGFR2 in the absence of Jam-A, we tested the expression of hypoxia inducible factor-1α (HIF-1α and inhibitor of DNA binding 1 (Id1), two transcription factors known to upregulate VEGF and VEGFR2 gene expression respectively. Interestingly, mRNA and protein levels of both HIF-1α and Id1 were significantly augmented (P<0.02) in ECs lacking Jam-A compared to WT. Since JAM-A is known to maintain integrin αIIbβ3 in a low affinity state in resting platelets by recruiting CSK to the αIIbβ3-Src complex, we tested if lack of Jam-A will augment integrin αvβ3 signaling in ECs. We found that in fact αvβ3-dependent signaling is augmented in Jam-A null ECs or when Jam-A is inhibited using function-blocking antibody compared to WT. Consistent with this finding, the overexpression of JAM-A in HUVECs attenuated the extent of αvβ3 signaling as well as HIF-1α and Id1 expression, suggesting that Jam-A suppresses αvβ3 signaling to maintain low levels of VEGF and VEGFR2 expression in quiescent ECs.Conclusion: The results presented here suggest that Jam-A suppresses integrin αvβ3 signaling to control VEGF/VEGFR2 expression in adult quiescent ECs thus suppressing neovascularization in adults. During pathological conditions, such as ischemia and inflammation, αvβ3/Jam-A complex is dissociated or Jam-A levels are downregulated allowing αvβ3 signaling, thus supporting pathological angiogenesis. DisclosuresNo relevant conflicts of interest to declare.

EPCT-07. ID1 IS A KEY TRANSCRIPTIONAL REGULATOR OF DIPG INVASION AND IS TARGETABLE WITH CANNABIDIOL

Diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors with no effective therapies beyond radiation. The highly invasive nature of DIPG is key to its aggressive phenotype, but the factors and mechanisms contributing to this aggressive invasion are unknown. Inhibitor of DNA binding (ID) proteins, key regulators of lineage commitment during embryogenesis, are implicated in tumorigenesis in multiple human solid tumors. Prior work showed that recurrent H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes. However, the impact and targetability of ID1 have not been explored in human DIPG. Exome and transcriptome sequencing analyses of multi-focal DIPG tumors and normal brain tissue from autopsy (n=52) revealed that ID1 expression is significantly elevated in DIPG samples. Higher ID1 expression correlates with reduced survival in DIPG patients and increased regional invasion in multi-focal autopsy samples. Analyses of developing mouse brain RNA/ChIP-Seq data revealed high ID1 expression and H3K27ac promoter binding in prenatal hindbrain compared to all other prenatal and postnatal brain regions. ChIP-qPCR for H3K27ac and H3K27me3 revealed that ID1 gene regulatory regions are epigenetically poised for upregulation in DIPG tissues compared to normal brain, regardless of H3/ACVR1 mutational status. These data support that the developing pons is regionally poised for ID1 activation. Genetic (shRNA) ID1 knockdown of primary human H3.3K27M-DIPG cells (DIPG007) resulted in significantly reduced invasion/migration and significantly improved survival of K27M-DIPG mice. Knockdown of ID1 in DIPG cells also resulted in down-regulation of the WNK1-NKCC1 pathway, which regulates tumor cell electrolyte homeostasis and migration. Finally, treatment of DIPG007 cells with cannabidiol (CBD) reduced ID1 levels, viability of DIPG cells and significantly improved survival of K27M-DIPG mice. In summary, our findings indicate that multifactorial (genetic and regional) epigenetic upregulation of ID1 drives DIPG invasiveness; and that targeting ID1 with CBD could potentially be an effective therapy for DIPG.

ERK/Drp1-dependent mitochondrial fission contributes to HMGB1-induced autophagy in pulmonary arterial hypertension.

ObjectivesHigh‐mobility group box‐1 (HMGB1) and aberrant mitochondrial fission mediated by excessive activation of GTPase dynamin‐related protein 1 (Drp1) have been found to be elevated in patients with pulmonary arterial hypertension (PAH) and critically implicated in PAH pathogenesis. However, it remains unknown whether Drp1‐mediated mitochondrial fission and which downstream targets of mitochondrial fission mediate HMGB1‐induced pulmonary arterial smooth muscle cells (PASMCs) proliferation and migration leading to vascular remodelling in PAH. This study aims to address these issues.MethodsPrimary cultured PASMCs were obtained from male Sprague‐Dawley (SD) rats. We detected RNA levels by qRT‐PCR, protein levels by Western blotting, cell proliferation by Cell Counting Kit‐8 (CCK‐8) and EdU incorporation assays, migration by wound healing and transwell assays. SD rats were injected with monocrotaline (MCT) to establish PAH. Hemodynamic parameters were measured by closed‐chest right heart catheterization.ResultsHMGB1 increased Drp1 phosphorylation and Drp1‐dependent mitochondrial fragmentation through extracellular signal‐regulated kinases 1/2 (ERK1/2) signalling activation, and subsequently triggered autophagy activation, which further led to bone morphogenetic protein receptor 2 (BMPR2) lysosomal degradation and inhibitor of DNA binding 1 (Id1) downregulation, and eventually promoted PASMCs proliferation/migration. Inhibition of ERK1/2 cascade, knockdown of Drp1 or suppression of autophagy restored HMGB1‐induced reductions of BMPR2 and Id1, and diminished HMGB1‐induced PASMCs proliferation/migration. In addition, pharmacological inhibition of HMGB1 by glycyrrhizin, suppression of mitochondrial fission by Mdivi‐1 or blockage of autophagy by chloroquine prevented PAH development in MCT‐induced rats PAH model.ConclusionsHMGB1 promotes PASMCs proliferation/migration and pulmonary vascular remodelling by activating ERK1/2/Drp1/Autophagy/BMPR2/Id1 axis, suggesting that this cascade might be a potential novel target for management of PAH.

Altered Responsiveness to TGFβ and BMP and Increased CD45+ Cell Presence in Mitral Valves Are Unique Features of Ischemic Mitral Regurgitation

[Figure: see text].

BONE MORPHOGENETIC PROTEIN 2 PROMOTES HUMAN TROPHOBLAST CELL INVASION AND ENDOTHELIAL-LIKE TUBE FORMATION THROUGH ID1-MEDIATED UP-REGULATION OF IGF BINDING PROTEIN-3

Objective: Extravillous cytotrophoblasts (EVTs) invade into the uterine wall and remodel spiral arteries for proper placentation. Insufficient EVTs invasion and poor spiral arterial remodeling are associated with preeclampsia occurrence. Studies from us and others have demonstrated that the transforming growth factor-beta superfamily member bone morphogenetic protein 2 (BMP2) plays important roles in endometrial decidualization and trophoblast cell invasion. However, BMP2 has also been shown to regulate endothelial cell migration and capillary-like tube formation, as has its downstream signaling molecule inhibitor of DNA binding 1 (ID1). Interestingly, insulin-like growth factor binding protein 3 (IGFBP3) also promotes endothelial precursor cell migration and angiogenesis. Moreover, Id1 shows regulatory effect on Igfbp3 expression in rat prostate epithelial cells. However, whether ID1 and IGFBP3 are integrated in BMP2 signaling and involved in the regulation of trophoblast invasive and endovascular differentiation remains unknown. The objective of our study was to examine the effects of BMP2 on ID1 and IGFBP3 expression and their roles in BMP2-regulated human trophoblast invasion and endothelial-like tube formation. Design and method: Primary and immortalized (HTR8/SVneo) cultures of human trophoblast cells were employed as study models. RT-PCR and Western blot assay were used to study mRNA and protein levels. Small interfering RNA (siRNA) was used to check the roles of key molecules. Matrigel-coated transwell assay and endothelial-like tube formation assay were used to examine cell invasiveness and angiogenesis respectively. Results: BMP2 treatment increased ID1 and IGFBP3 mRNA and protein levels in HTR8/SVneo and primary human EVT cells. Intriguingly, ID1 was essential for BMP2-induced IGFBP3 up-regulation in both study models, and BMP2-induced trophoblast invasion was attenuated by knockdown of either ID1 or IGFBP3. In addition, BMP2 significantly increased endothelial-like tube formation and knockdown of ID1 and IGFBP3 reduced basal and BMP2-induced tube formation in HTR8/SVneo cells. Similarly, BMP2 increased placenta growth factor (PlGF) production in HTR8/SVneo cells and these effects were attenuated by knockdown of ID1 or IGFBP3. Conclusions: Our results reveal that BMP2 promotes trophoblast cell invasion and endothelial-like tube formation by ID1-mediated IGFBP3 up-regulation.

Cannabidiol Treatment Results in a Common Gene Expression Response Across Aggressive Cancer Cells from Various Origins.

Background: We previously reported that cannabidiol (CBD), a cannabinoid with a low toxicity profile, downregulated the expression of the prometastatic gene inhibitor of DNA binding 1 (ID1) in cancer cells, leading to inhibition of tumor progression in vivo. While CBD is broadly used, including in the self-medication of cancer patients, and CBD-based therapies are undergoing clinical evaluation for cancer treatment, its mechanisms of action are still poorly understood. Methods: In this study, using microarray analysis and Western blot analysis for validation, we attempted to identify the full spectrum of genes regulated by CBD across various aggressive cancer cell lines, including the breast, brain, head and neck, and prostate. Results: We confirmed that ID1 was a major target downregulated by CBD and also discovered that CBD inhibited FOXM1 (Forkhead box M1), a transcriptional activator involved in cell proliferation, while simultaneously upregulating GDF15 (growth differentiation factor 15), a cytokine associated with tissue differentiation. Conclusion: Our results suggest that, by modulating expression of shared key cancer-driving genes, CBD could represent a promising nontoxic therapeutic for treating tumors of various origins.