Interferon Regulatory Factor 2 Binding Protein 2 Research Articles

Super-Enhancer-Driven IRF2BP2 is Activated by Master Transcription Factors and Sustains T-ALL Cell Growth and Survival.

Super enhancers (SEs) are large clusters of transcriptional enhancers driving the expression of genes crucial for defining cell identity. In cancer, tumor-specific SEs activate key oncogenes, leading to tumorigenesis. Identifying SE-driven oncogenes in tumors and understanding their functional mechanisms is of significant importance. In this study, a previously unreported SE region is identified in T-cell acute lymphoblastic leukemia (T-ALL) patient samples and cell lines. This SE activates the expression of interferon regulatory factor 2 binding protein 2 (IRF2BP2) and is regulated by T-ALL master transcription factors (TFs) such as ETS transcription factor ERG (ERG), E74 like ETS transcription factor 1 (ELF1), and ETS proto-oncogene 1, transcription factor (ETS1). Hematopoietic system-specific IRF2BP2 conditional knockout mice is generated and showed that IRF2BP2 has minimal impact on normal T cell development. However, in vitro and in vivo experiments demonstrated that IRF2BP2 is crucial for T-ALL cell growth and survival. Loss of IRF2BP2 affects the MYC and E2F pathways in T-ALL cells. Cleavage under targets and tagmentation (CUT&Tag) assays and immunoprecipitation revealed that IRF2BP2 cooperates with the master TFs of T-ALL cells, targeting the enhancer of the T-ALL susceptibility gene recombination activating 1 (RAG1) and modulating its expression. These findings provide new insights into the regulatory network within T-ALL cells, identifying potential new targets for therapeutic intervention.

Transcriptional regulation of adipocyte lipolysis by IRF2BP2.

Adipocyte lipolysis controls systemic energy levels and metabolic homeostasis. Lipolysis is regulated by post-translational modifications of key lipolytic enzymes. However, less is known about the transcriptional mechanisms that regulate lipolysis. Here, we identify the transcriptional factor interferon regulatory factor-2 binding protein 2 (IRF2BP2) as a repressor of adipocyte lipolysis. Deletion of IRF2BP2 in primary human adipocytes increases lipolysis without affecting glucose uptake, whereas IRF2BP2 overexpression decreases lipolysis. RNA-seq and ChIP-seq analyses reveal that IRF2BP2 directly represses several lipolysis-related genes, including LIPE ( HSL , hormone sensitive lipase), which encodes the rate-limiting enzyme in lipolysis. Adipocyte-selective deletion of Irf2bp2 in mice increases Lipe expression and free fatty acid levels, resulting in elevated adipose tissue inflammation and glucose intolerance. Altogether, these findings demonstrate that IRF2BP2 restrains adipocyte lipolysis and opens new avenues to target lipolysis for the treatment of metabolic disease.

Novel hypermorphic variants in IRF2BP2 identified in patients with common variable immunodeficiency and autoimmunity

The interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a transcriptional regulator, functioning a transcriptional corepressor by interacting with the interferon regulatory factor-2. The ubiquitous expression of IRF2BP2 by diverse cell types and tissues suggests its potential involvement in different cell signalling pathways. Variants inIRF2BP2have been recently identified to cause familial common variable immunodeficiency (CVID) characterized by immune dysregulation.This study investigated three rare novel variants inIRF2BP2, identified in patients with primary antibody deficiency and autoimmunity by whole exome-sequencing (WES). Following transient overexpression of EGFP-fused mutants in HEK293 cells and transfection in Jurkat cell lines, we used fluorescence microscopy, real-time PCR and Western blotting to analyze their effects on IRF2BP2 expression, subcellular localization, nuclear translocation of IRF2, and the transcriptional activation of NFκB1(p50).We found altered IRF2BP2 mRNA and protein expression levels in the mutants compared to the wild type after IRF2BP2 overexpression. In confocal fluorescence microscopy, variants in the C-terminal RING finger domain showed an irregular aggregate formation and distribution instead of the expected nuclear localization compared to the variants in the N-terminal zinc finger domain and their wildtype counterpart. Immunoblotting revealed an impaired IRF2 and NFκB1 (p50) nuclear localization in the mutants compared to the IRF2BP2 wildtype counterpart. LPS stimulation reduced IRF2BP2 mRNA expression in the variants compared to the wild type.Our findings significantly contribute to understanding the clinical significance of IRF2BP2 mutations in the pathogenesis of immunodeficiency and immune dysregulation. We observed impairment of the nuclear translocation of IRF2 and NFκB1 (p50) due to the upregulation of IRF2BP2, potentially affecting specific gene expressions involved in immune regulation.

Abstract A10: Targeting MHC-I antigen presentation for cancer immune evasion in acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is a clonal hematopoietic neoplasm, accounting for ~80% of acute leukemia cases in adults. The poor clinical outcome of AML patients (~27% five-year overall survival) is largely due to the inefficiency and toxicity of standard therapies. In the past decade, immunotherapies or cellular therapies (CAR-T/TCR-T cells) have achieved FDA approvals in certain types of solid tumors and leukemia, but currently there is no approved immunotherapy in AML. One potential reason is that AML actively inhibits antigen presentation (AP) to reduce its immunogenicity, thus dampening the efficacies of immunotherapies. This growing consensus is supported by the clinical evidence that AML exhibits low levels of AP machinery at diagnosis and even lower when relapsed from conventional chemotherapies. Besides, better outcomes could be achieved when combining immune checkpoint blockade with neo-antigens-inducing hypomethylating agents, highlighting the importance of AP in AML. We thus hypothesize that targeting tumor-endogenous AP suppressors will enhance AML immunogenicity and benefit immunotherapies. Recent studies have suggested that autophagy or soluble protein PCSK9 can mediate MHC-I degradation or disrupt MHC-I recycling, respectively. Transcription/epigenetic repressors TRAF3 and EZH2, as well as thymidylate synthase, were identified as MHC-I inhibitors. However, their specificity in MHC-I modulation and precise roles in regulating tumor antigenic peptide-MHC-I complexes (pMHC-I) are still unclear. Hence, there is an increasing need to identify AML-specific AP regulation mechanisms. To map such mechanisms, we performed pMHC-I-guided CRISPR screens in both human and mouse AML cell lines. Through these screens, we constructed positive and negative regulatory networks of pMHC-I modulation and compared the role of these novel regulators in the simultaneous modulation of MHC-I expression. This is the first-in-class systematic identification of AP regulators in AML. Among these negative AP regulators, we are particularly interested in the interferon regulatory factor 2 binding protein 2 (IRF2BP2), an AML-specific transcriptional regulator that is highly expressed in AML compared with normal hematopoietic stem and progenitor cells (HSPCs). Notably, IRF2BP2 expression negatively correlates with major histocompatibility complex class I (MHC-I) expression, interferon (IFN) response signatures, and T cell activity in AML patients. Ablation of IRF2BP2 enhanced AP in AML and facilitated T cell-mediated elimination of AML. Moreover, IRF2BP2 depletion synergized with IFN treatment to further boost AP at both transcriptional and protein levels. Our findings reveal a new class of tumor-associated immune-evasion mechanisms that target AP, with potential application as therapeutic targets for next-generation cancer immunotherapies. Citation Format: Xufeng Chen, Qiao Lu, Hua Zhou, Jia Liu, Bettina Nadorp, Audrey Lasry, Zhengxi Sun, Jiangyan Zhang, Michael Cammer, Kun Wang, Zoe Ciantra, Jia You, Qianjin Guo, Hongbing Zhang, Debrup Sengupta, Ahmad Boukhris, Cheng Liu, Peter Cresswell, Patricia L. M. Dahia, Jun Wang, Iannis Aifantis. Targeting MHC-I antigen presentation for cancer immune evasion in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A10.

IRF2BP2 attenuates gestational diabetes mellitus by activating AMPK signaling

Purpose: To investigate the role of interferon regulatory factor 2 binding protein 2 (IRF2BP2) in gestational diabetes mellitus (GDM).
 Methods: Mice were injected intraperitoneally with streptozotocin to establish a model of GDM and then subjected to intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) to determine glucose and insulin tolerances. Lipid metabolism was evaluated by enzyme-linked immunosorbent assay (ELISA). The histomorphology of pancreatic islets was assessed by hematoxylin and eosin staining.
 Results: IRF2BP2 was downregulated in pancreatic tissues of mice with GDM (p < 0.001). Mice in GDM group showed higher blood glucose levels than those in normal pregnancy group. However, overexpression of IRF2BP2 reduced glucose and insulin levels in mice with GDM. Overexpression of IRF2BP2 increased the level of high-density lipoprotein (HDL) and reduced triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL) levels in mice with GDM (p < 0.001). The histopathological changes in the islets of mice with GDM were also ameliorated by overexpression of IRF2BP2. Overexpression of IRF2BP2 reduced IL-6, IL-1β, and TNF-α levels and increased protein expression of p-AMPK in mice with GDM.
 Conclusion: IRF2BP2 ameliorates the outcomes of GDM and suppressed inflammation in mice with GDM through activation of AMPK signaling. Thus, IRF2BP2 is a potential therapeutic strategy for the management of GDM.
 Keywords: IRF2BP2; inflammation; gestational diabetes mellitus; AMPK; mice; insulin tolerance

In This Issue

In This Issue

Autoimmune Cytopenias in Common Variable Immunodeficiency Are a Diagnostic and Therapeutic Conundrum: An Update.

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency (PID). CVID is a heterogenous condition and clinical manifestations may vary from increased susceptibility to infections to autoimmune manifestations, granulomatous disease, polyclonal lymphoproliferation, and increased risk of malignancy. Autoimmune manifestations may, at times, be the first and only clinical presentation of CVID, resulting in diagnostic dilemma for the treating physician.Autoimmune cytopenias (autoimmune haemolytic anaemia and/or thrombocytopenia) are the most common autoimmune complications seen in patients with CVID. Laboratory investigations such as antinuclear antibodies, direct Coomb’s test and anti-platelet antibodies may not be useful in patients with CVID because of lack of specific antibody response. Moreover, presence of autoimmune cytopenias may pose a significant therapeutic challenge as use of immunosuppressive agents can be contentious in these circumstances. It has been suggested that serum immunoglobulins must be checked in all patients presenting with autoimmune cytopenia such as immune thrombocytopenia or autoimmune haemolytic anaemia.It has been observed that patients with CVID and autoimmune cytopenias have a different clinical and immunological profile as compared to patients with CVID who do not have an autoimmune footprint. Monogenic defects have been identified in 10-50% of all patients with CVID depending upon the population studied. Monogenic defects are more likely to be identified in patients with CVID with autoimmune complications. Common genetic defects that may lead to CVID with an autoimmune phenotype include nuclear factor kappa B subunit 1 (NF-kB1), Lipopolysaccharide (LPS)-responsive beige-like anchor protein (LRBA), cytotoxic T lymphocyte antigen 4 (CTLA4), Phosphoinositide 3-kinase (PI3K), inducible T-cell costimulatory (ICOS), IKAROS and interferon regulatory factor-2 binding protein 2 (IRF2BP2). In this review, we update on recent advances in pathophysiology and management of CVID with autoimmune cytopenias.

Exercise ameliorates high-fat diet-induced insulin resistance accompanied by changes in protein levels of hepatic ATF3-related signaling in rats

PurposeExercise is an effective way to alleviate insulin resistance (IR). However, the underlying mechanisms remain to be elucidated. Previous studies demonstrated that cardiolipin synthase 1 (CRLS1)/interferon-regulatory factor-2 binding protein 2 (IRF2bp2)-activating transcription factor 3 (ATF3)-adiponectin receptor 2 (AdipoR2)-adaptor protein containing pH domain, PTB domain and leucine zipper motif 1 (APPL1)-protein kinase B (AKT/PKB)-related signaling was closely associated with obesity-induced IR-related diseases, but the correlation between exercise training alleviating obesity-induced IR and the protein levels of hepatic CRLS1/IRF2bp2-ATF3-AdipoR2-APPL1-AKT-related signaling in rats is unknown. Therefore, We want to investigate the effect of exercise training on IR and the protein levels of hepatic CRLS1/IRF2bp2-ATF3-AdipoR2-APPL1-AKT-related signaling in rat. MethodsThe male healthy Sprague-Dawley rats were divided into four groups: normal control group (NCG, n = 10), diet-induced obesity-sedentary group (DIO-SG, n = 10), diet-induced obesity-chronic exercise group (DIOCEG, n = 10) received chronic swim exercise training and diet-induced obesity-acute exercise group (DIO-AEG, n = 10) received acute swim exercise training. We measured the levels of IR-related indicators and the protein levels of hepatic CRLS1/IRF2bp2-ATF3-AdipoR2-APPL1-AKT-related signaling in NCG, DIO-SG, DIOCEG and DIO-AEG. ResultsWe found that high-fat diet (HFD)-induced obesity decreased insulin sensitivity in rats accompanied by decreased protein levels of hepatic CRLS1, IRF2bp2, AdipoR2, APPL1, p-AKT and increased protein level of hepatic ATF3. The acute exercise and the chronic exercise both increased insulin sensitivity in rats. The chronic exercise decreased hepatic ATF3 protein level and increased CRLS1, IRF2bp2, AdipoR2, APPL1, p-AKT protein levels in HFD-fed rats. The acute exercise decreased hepatic ATF3 protein level and increased hepatic IRF2bp2, APPL1 and p-AKT protein levels in HFD-fed rats. The acute exercise had no significant effect on hepatic CRLS1 and AdipoR2 protein levels in HFD-fed rats. ConclusionOur current findings indicated that exercise alleviated obesity-induced IR accompanied by changes in protein levels of hepatic ATF3-related signaling in rats. Our results are meaningful for exploring the molecular mechanism of exercise alleviating IR symptoms.

Unleashing Cell-Intrinsic Inflammation As a Strategy to Kill AML Blasts

Leukemic blasts are immune cells gone awry. We hypothesized that dysregulation of inflammatory pathways contributes to the maintenance of their leukemic state and can be exploited as cell-intrinsic, self-directed immunotherapy. To test the hypothesis, we analyzed three large, independent data collections from primary acute myeloid leukemia (AML) samples and identified an AML subgroup of approximately 40% of all samples enriched for immune and inflammatory pathways. Moreover, we observed a positive correlation between the enrichment of inflammatory response and a monocytic lineage signature in these primary AML samples. To discover genetic vulnerabilities in AML cells implicated in inflammatory pathways, we integrated data from the Cancer Dependency Map on 789 cancer cell lines with two independent genome-wide screens. We identified the immune modulator interferon regulatory factor 2 binding protein 2 (IRF2BP2) as a selective dependency in AML. We validated AML cell dependency on IRF2BP2 with orthogonal genetic and degradation approaches in vitro and in vivo. Genetic perturbation and degradation of IRF2BP2 translated into a reduction of AML cell line viability and decreased colony formation capacity in vitro and impaired leukemia progression in vivo in AML cell line xenografts. Moreover, inducible knock-out of IRF2BP2 significantly attenuated disease burden in multiple patient-derived xenograft models of AML and prolonged survival. Mechanistically, IRF2BP2 repression induced cell death with hallmarks of apoptosis, evidenced by an increase in annexin V/PI-positive cells and induction of cleaved caspase 3 and 8.To decipher the role of IRF2BP2 in inflammatory signaling, we studied its localization on chromatin and its relationship to histone marks and RNA polymerase II binding in AML cells. We determined that IRF2BP2 binds at both enhancers and promotors. Global gene expression profiling, performed six hours after degradation of IRF2BP2, identified significantly more genes increased than decreased in expression. Most upregulated genes were bound by IRF2BP2 at baseline and gained H3K27ac at enhancers with IRF2BP2 degradation. All told, our data support a role for IRF2BP2 as a transcriptional repressor in AML.Gene set enrichment analysis of the genes directly regulated by IRF2BP2 identified immune response signatures as the top enriched gene sets, with genes regulated by NF-kB in response to TNFa being the most significantly enriched. We thus hypothesized that IRF2BP2 is a repressor of NF-kB mediated TNFa signaling that, when acutely perturbed, leads to leukemia cell death. Indeed, we observed an activation of NF-κB signaling in a luciferase reporter assay, an increase in nuclear NF-kB (RELA) protein levels, and a gain in NF-κB chromatin binding following degradation of IRF2BP2 in AML cells. Moreover, a mutant “super-repressor” allele of IkBa rescued the impaired cell growth upon IRF2BP2 perturbation in AML cells, supporting cell death associated with IRF2BP2 loss being mediated through activation of NF-kB signaling. In addition, we identified IL-1ß as an enhancer of the inflammatory response repressed by IRF2BP2.In summary, we have demonstrated that IRF2BP2 represses IL-1ß/TNFα signaling via NF-κB, and IRF2BP2 perturbation results in an acute inflammatory state leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for regulated cell death, and establish IRF2BP2-mediated transcriptional repression as a mechanism for blast survival. DisclosuresDharia: Genentech: Current Employment. Benajiba: Gilead: Research Funding; Pfizer: Research Funding. Gray: Gatekeeper, Syros, Petra, C4, Allorion, Jengu, B2S, Inception and Soltego: Consultancy, Research Funding. Stegmaier: Auron Therapeutics, Kronos Bio, AstraZeneca, Novartis Institute of Biomedical Research: Consultancy, Research Funding.

IRF2BP2 3′UTR Polymorphism Increases Coronary Artery Calcification in Men

Interferon regulatory factor 2 binding protein 2 (IRF2BP2) suppresses the innate inflammatory response of macrophages. A 9-nucleotide deletion (rs3045215) in the 3′ untranslated region (3′-UTR) of human IRF2BP2 mRNA confers risk of coronary artery disease (CAD) in the Ottawa Heart Genomics Study (OHGS). Here, we sought to identify regulatory mechanisms that may contribute to this risk. We tested how lipopolysaccharides (LPS) affects IRF2BP2 expression in human THP-1 macrophages and primary aortic smooth muscle cells (HAoSMC) genotyped for the deletion allele. Both cell types are implicated in coronary atherosclerosis. We also examined how the deletion affects interaction with RNA binding proteins (RBPs) to regulate IRF2BP2 expression. LPS altered allele-specific binding of RBPs in RNA gel shift assays with the THP-1 macrophage protein extracts. The RBP ELAVL1 suppressed the expression of a luciferase reporter carrying the 3′UTR of IRF2BP2 with the deletion allele. Other RBPs AUF1 or KHSRP did not confer such allele specific regulation. Since it is co-inherited with a risk variant for osteoporosis, a condition tied to arterial calcification, we examined the association of the deletion allele with coronary artery calcification in individuals who had undergone computed tomography angiography in the OHGS. In 323 individuals with a minimal burden of atherosclerosis (<30% coronary stenosis) and 138 CAD cases (>50% stenosis), Mendelian randomization revealed that the rs3045215 deletion allele significantly increased coronary artery calcification in men with minimal coronary stenosis. Thus, not only does the rs3045215 deletion allele predict atherosclerosis, but it also predisposes to early-onset calcification in men.

The Transcriptional Co-factor IRF2BP2: A New Player in Tumor Development and Microenvironment

Interferon regulatory factor 2-binding protein 2 (IRF2BP2) encodes a member of the IRF2BP family of transcriptional regulators, which includes IRF2BP1, IRF2BP2, and IRF2BPL (EAP1). IRF2BP2 was initially identified as a transcriptional corepressor that was dependent on Interferon regulatory factor-2 (IRF-2). The IRF2BP2 protein is found in different organisms and has been described as ubiquitously expressed in normal and tumor cells and tissues, indicating a possible role for this transcriptional cofactor in different cell signaling pathways. Recent data suggest the involvement of IRF2BP2 in the regulation of several cellular functions, such as the cell cycle, cell death, angiogenesis, inflammation and immune response, thereby contributing to physiological cell homeostasis. However, an imbalance in IRF2BP2 function may be related to the pathophysiology of cancer. Some studies have shown the association of IRF2BP2 expression in hematopoietic and solid tumors through mechanisms based on gene fusion and point mutations in gene coding sequences, and although the biological functions of these types of hybrid and mutant proteins are not yet known, they are thought to be involved in an increase in the likelihood of tumor development. In this review, we address the possible involvement of IRF2BP2 in tumorigenesis through its regulation of important pathways involved in tumor development.

IRF2BP2 prevents ox-LDL-induced inflammation and EMT in endothelial cells via regulation of KLF2.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction contributes to the progression of atherosclerosis. Interferon regulatory factor 2-binding protein 2 (IRF2BP2) attenuates macrophage-mediated inflammation and susceptibility to atherosclerosis. However, the effects of IRF2BP2 on vascular endothelial cells in atherosclerosis have not been fully elucidated. In the present study, the effects of IRF2BP2 on cell viability, inflammation and endothelial-to-mesenchymal transition (EMT) of human umbilical vein endothelial cells (HUVECs) were assessed using Cell Counting Kit-8 (CCK-8) assays, ELISA kits and western blot analysis, respectively. In addition, the expression levels of Krüppel-like factor 2 (KLF2) were determined by reverse transcription-quantitative PCR and immunofluorescence assays. A Nitrate/Nitrite assay kit was utilized to detect the production of nitric oxide (NO). The results demonstrated that ox-LDL induced inflammation and EMT of HUVECs, and decreased the NO levels. Furthermore, IRF2BP2 overexpression protected HUVECs against ox-LDL-induced inflammation, EMT and endothelial dysfunction, and resulted in upregulated expression of KLF2. Additionally, IRF2BP2 was shown to bind to KLF2, and KLF2 knockdown reversed the protective effects of IRF2BP2 on ox-LDL-exposed HUVECs. These findings indicated that IRF2BP2 may prevent ox-LDL-induced endothelial damage via upregulating KLF2 expression.

Inhibition of miR-155 potentially protects against lipopolysaccharide-induced acute lung injury through the IRF2BP2-NFAT1 pathway.

Sepsis-induced lung injury is a lethal complication with no effective treatment options, affecting millions of people worldwide. Oroxylin A (OroA) is a natural flavonoid with potent anticancer effects, but its modulating effect on inflammation through microRNAs (miRs) is not apparent. In this report, we investigated the target genes of the miR pathway mediated by OroA and assessed the potential for novel treatments of septic lung injury. An miR array screening and quantitative polymerase chain reaction identified that miR-155-5p could be a candidate regulated by OroA. Bioinformatics analysis indicated that interferon regulatory factor-2-binding protein-2 (IRF2BP2) might be a target of miR-155-5p, and this hypothesis was verified through reporter assays. In addition, an immunoprecipitation assay demonstrated that OroA increased the binding activity of IRF2BP2 to the nuclear factor of activated T-cells 1 (NFAT1), causing inducible nitric oxide synthase to cause an inflammatory reaction. Finally, the direct injection of short hairpin RNA (shRNA)-miR-155-5p into the bone marrow of mice ameliorated LPS-induced acute lung injury and inflammation in mice. Our results provide new mechanistic insights into the role of the OroA-induced miR-155-5p-IRF2BP2-NFAT1 axis in sepsis, demonstrating that direct bone marrow injection of lentivirus containing shRNA-155-5p could prove to be a potential future clinical application in alleviating sepsis-induced acute lung injury.

Interferon Regulatory Factor-2 Binding Protein 2 Ameliorates Sepsis-Induced Cardiomyopathy via AMPK-Mediated Anti-Inflammation and Anti-Apoptosis.

Cardiomyopathy commonly occurs after sepsis and is closely associated with high mortality in clinic. Interferon regulatory factor-2 binding protein 2 (IRF2BP2) has been identified as a negative regulator of inflammation, but its role in septic cardiomyopathy is unknown. The current study aims to illuminate the regulatory function of IRF2BP2 on sepsis-induced cardiomyopathy and to explore the underlying mechanisms. Protein expression of IRF2BP2 in response to sepsis-induced cardiomyopathy was examined in the heart of mice challenged by LPS intraperitoneal injection. AAV9-delivered IRF2BP2 overexpression in the heart was applied to evaluate the regulatory role of IRF2BP2 in sepsis-induced myocardial depression, inflammatory response, and cell death. The molecular mechanisms underlying IRF2BP2-regulated cardiomyopathy were explored using western blot screening assay. Primary cardiomyocytes have been isolated to further confirm the role and mechanism of IRF2BP2 during septic cardiomyopathy. IRF2BP2 expression was dramatically increased in the heart of mice after LPS administration. AAV9-mediated IRF2BP2 overexpression significantly improved sepsis-induced cardiac dysfunction, inhibited inflammatory cell infiltration and cytokine production, and blocked cell death after LPS treatment. Mechanistically, IRF2BP2 activated AMPK signaling in cardiomyocytes, while inhibiting AMPK activation largely reversed IRF2BP2-benefited inflammatory suppression and cell survival. These findings clearly demonstrated that IRF2BP2 is a potent suppressor of sepsis-induced myocardial depression and related heart impairment. Targeting IRF2BP2 represents a promising therapeutic strategy for septic cardiomyopathy.

The Tumor Suppressor Interferon Regulatory Factor 2 Binding Protein 2 Regulates Hippo Pathway in Liver Cancer by a Feedback Loop in Mice.

The conserved Hippo pathway regulates organ size, tissue homeostasis, and tumorigenesis. Interferon regulatory factor 2 binding protein 2 (IRF2BP2) was originally identified as a transcriptional corepressor. However, the association between IRF2BP2 and the Hippo pathway remains largely unknown. In addition, the biological function and regulation mechanism of IRF2BP2 in liver cancer are poorly understood. In this study, we uncovered the clinical significance of IRF2BP2 in suppressing hepatocellular carcinogenesis. We showed that IRF2BP2, a direct target repressed by the Yes-associated protein (YAP)/TEA domain transcription factor 4 (TEAD4) transcriptional complex, inhibited YAP activity through a feedback loop. IRF2BP2 stabilized vestigial-like family member 4 (VGLL4) and further enhanced VGLL4's inhibitory function on YAP. Moreover, liver-specific IRF2BP2 overexpression suppressed tumor formation induced by Hippo pathway inactivation. These results revealed the important role of IRF2BP2 in repressing liver cancer progression and highlighted a feedback loop underlying the Hippo pathway in organ-size control and tumorigenesis.

Hepatic IRF2BP2 Mitigates Nonalcoholic Fatty Liver Disease by Directly Repressing the Transcription of ATF3

Although knowledge regarding the pathogenesis of nonalcoholic fatty liver disease (NAFLD) has profoundly grown in recent decades, the internal restrictive mechanisms remain largely unknown. We have recently reported that the transcription repressor interferon regulatory factor-2 binding protein 2 (IRF2BP2) is enriched in cardiomyocytes and inhibits pathological cardiac hypertrophy in mice. Notably, IRF2BP2 is abundantly expressed in hepatocytes and dramatically down-regulated in steatotic livers, whereas the role of IRF2BP2 in NAFLD is unknown. Herein, using gain-of-function and loss-of-function approaches in mice, we demonstrated that while hepatocyte-specific Irf2bp2 knockout exacerbated high-fat diet-induced hepatic steatosis, insulin resistance and inflammation, hepatic Irf2bp2 overexpression protected mice from these metabolic disorders. Moreover, the inhibitory role of IRF2BP2 on hepatosteatosis is conserved in a human hepatic cell line in vitro. Combinational analysis of digital gene expression and chromatin immunoprecipitation sequencing identified activating transcription factor 3 (ATF3) to be negatively regulated by IRF2BP2 in NAFLD. Chromatin immunoprecipitation and luciferase assay substantiated the fact that IRF2BP2 is a bona fide transcription repressor of ATF3 gene expression via binding to its promoter region. Functional studies revealed that ATF3 knockdown significantly relieved IRF2BP2 knockout-exaggerated hepatosteatosis in vitro. IRF2BP2 is an integrative restrainer in controlling hepatic steatosis, insulin resistance, and inflammation in NAFLD through transcriptionally repressing ATF3 gene expression.

Down-regulation of interferon regulatory factor 2 binding protein 2 suppresses gastric cancer progression by negatively regulating connective tissue growth factor.

Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a transcriptional repressor involved in regulating gene expression and other biological processes, including tumorigenesis. However, the clinical significance and roles of IRF2BP2 in human gastric cancer (GC) remain uncertain. Clinical GC tissues were obtained from GC patients at the First Affiliated Hospital of Nanchang University. Immunohistochemistry (IHC) was conducted to detect the IRF2BP2 protein in clinical paraffin specimens. Cell proliferation, migration and invasion were evaluated by MTT, colony formation assays and transwell assays. Co‐immunoprecipitation was conducted to detect the interaction between TEA domain family members 4 (TEAD4) and vestigial‐like family member 4 (VGLL4) or Yes‐associated protein 1 (YAP1). Dual‐luciferase reporter assay was used to confirm the binding of miR‐101‐3p to the 3′‐UTR. The expression of IRF2BP2 was significantly higher in GC tissues than in normal tissues. Patients with higher IRF2BP2 protein expression had lower survival. IRF2BP2 knockdown inhibited proliferation, migration, invasion and epithelial‐mesenchymal transition in GC cells. IRF2BP2 knockdown decreased the mRNA and protein levels of connective tissue growth factor (CTGF). The interaction between IRF2BP2 and VGLL4 increased the binding of TEAD4 to YAP1, resulting in the transcriptional coactivation of CTGF. In addition, miR‐101‐3p suppressed the expression of CTGF by directly targeting the 3′‐UTR of IRF2BP2. Taken together, these findings provide a model for the role of miR‐101‐3p‐IRF2BP2‐CTGF signalling axis in GC and a novel insight into the mechanism of GC progression and metastasis.

IRF2BP2 modulates the crosstalk between glucocorticoid and TNF signaling

IRF2BP2 (interferon regulatory factor-2 binding protein-2) is an uncharacterized interaction partner of glucocorticoid (GC) receptor (GR), an anti-inflammatory and metabolic transcription factor. Here, we show that GC changes the chromatin binding of IRF2BP2 in natural chromatin milieu. The GC-induced IRF2BP2-binding sites co-occur with GR binding sites and are associated with GC-induced genes. Moreover, the depletion of IRF2BP2 modulates transcription of GC-regulated genes, represses cell proliferation and increases cell movement in HEK293 cells. In A549 cells, the depletion extensively alters the responses to GC and tumor necrosis factor α (TNF), including metabolic and inflammatory pathways. Taken together, our data support the role of IRF2BP2 as a coregulator of both GR and NF-κB, potentially modulating the crosstalk between GC and TNF signaling.

A NOVEL GENE MUTATION ASSOCIATED WITH COMMON VARIABLE IMMUNODEFICIENCY AND PYODERMA

Introduction Common Variable Immunodeficiency (CVID) is a primary immune deficiency characterized by decreased levels of IgG, IgA and/or IgM in the setting of recurrent sino-pulmonary infections and frequently in association with inflammatory or autoimmune disorders. The cause of CVID is known in less than 10% of cases. We describe a novel mutation of the Interferon Regulatory Factor 2 Binding Protein 2 (IRF2BP2) associated with CVID and pyoderma gangrenosum. Case Description The patient is a 63-year old male who had presented with a history of few but severe infections including pneumonia. He then developed facial pyoderma gangrenosum. The patient was found to have with decreased levels of IgG, IgM, and IgA, and no response to pneumococcal immunization. The patient was diagnosed with CVID. He was tried on a myriad of therapies, including rituximab and high-dose intravenous immunoglobulin, with no significant sustained improvement in his facial lesions. He was then placed on anakinra, and though his lesions did not resolve, they improved in both number and severity. Whole exome sequencing identified a heterozygous mutation in IRF2BP2 (c.1180 A>C; p.T394P). Discussion CVID is one of the most common symptomatic primary immunodeficiency syndromes, however, the exact cause of this disease entity is not known in most cases. Several genes have been postulated to be involved in the pathogenesis of CVID including IRF2BP2. A heterozygous IRF2BP2 mutation (c.1652G>A;p. 551N) was recently reported to be associated with CVID and autoimmune disease. We describe a novel mutation of IRF2BP2 associated with CVID and pyoderma gangrenosum. Common Variable Immunodeficiency (CVID) is a primary immune deficiency characterized by decreased levels of IgG, IgA and/or IgM in the setting of recurrent sino-pulmonary infections and frequently in association with inflammatory or autoimmune disorders. The cause of CVID is known in less than 10% of cases. We describe a novel mutation of the Interferon Regulatory Factor 2 Binding Protein 2 (IRF2BP2) associated with CVID and pyoderma gangrenosum. The patient is a 63-year old male who had presented with a history of few but severe infections including pneumonia. He then developed facial pyoderma gangrenosum. The patient was found to have with decreased levels of IgG, IgM, and IgA, and no response to pneumococcal immunization. The patient was diagnosed with CVID. He was tried on a myriad of therapies, including rituximab and high-dose intravenous immunoglobulin, with no significant sustained improvement in his facial lesions. He was then placed on anakinra, and though his lesions did not resolve, they improved in both number and severity. Whole exome sequencing identified a heterozygous mutation in IRF2BP2 (c.1180 A>C; p.T394P). CVID is one of the most common symptomatic primary immunodeficiency syndromes, however, the exact cause of this disease entity is not known in most cases. Several genes have been postulated to be involved in the pathogenesis of CVID including IRF2BP2. A heterozygous IRF2BP2 mutation (c.1652G>A;p. 551N) was recently reported to be associated with CVID and autoimmune disease. We describe a novel mutation of IRF2BP2 associated with CVID and pyoderma gangrenosum.

Abstract A87: Characterization of the involvement of transcriptional repressor IRF2BP2 on T-cell proliferative response

Abstract IRF2BP2 (Interferon Regulatory Factor-2 Binding Protein-2) is a transcriptional repressor involved in the regulation of gene expression in several different biologic contexts. It has been shown that IRF2BP2 expression is induced by the tumor suppressor protein p53 during genotoxic stress in osteosarcoma cell lines, which leads to a prevention of cell death. At least two translocations generating fusion genes involving IRF2BP2 were described distinct types of neoplasia as the IRF2BP2-CDX1 in chondrosarcoma and the IRF2BP2-RARA in acute promyelocytic leukemia. Recently, our group demostrated that IRF2BP2 represses NFAT1 (Nuclear factor of activated T-cells) transcriptional activity in CD4 T lymphocytes. In the present work we investigate the involvement of IRF2BP2 in the biology of CD4 T lymphocytes activation. We showed that ectopic expression of IRF2BP2 repressing CD4 T cell proliferation induced upon activation. We did not observe any differences in cell death of transduced cell cultures overexpressing IRF2BP2; however, we did observe a deleterious role in cell survival in the presence of an apoptotic stimulus by making these cells more prone to die after irradiation. In vivo, transferred IRF2BP2-overexpressing transduced CD4 T cells displayed an impaired expansion capacity compared to controls. Next, we generated a genetic model of conditional transgenic mice that overexpresses IRF2BP2 on T lymphocytes (IRF2BP2fl/flLck-cre+). Analysis by qRT-PCR demonstrated that CD4 T lymphocytes from IRF2BP2fl/flLck-cre+ mice presented an 8- to 10-fold increase of IRF2BP2 mRNA in comparison to wild-type animals (IRF2BP2+/+Lck-cre-). To evaluate the involvement of IRF2BP2 overexpression in T cell homeostasis we characterized the lymphocyte population in the thymus, spleen, and peripheral lymph nodes. In fact, IRF2BP2fl/flLck-cre+ animals showed a decrease in the total T-cell population in lymph nodes and thymus compared with controls. Furthermore, our results showed a reduction on CD3+ cells and a compensatory increase in B220+ compartment in the spleen and lymph nodes. IRF2BP2fl/flLck-cre+ animals also showed fewer counts of CD4+ and CD8+ cells in the thymus, spleen, and lymph nodes. Taken together, our data suggest a role for IRF2BP2 in controlling different parameters of CD4 T-cell function such as proliferation and cell survival, and may act as a regulator of immunologic checkpoint. Financial Support: FAPERJ, CNPq, INCT-Cancer. Citation Format: Barbara L. M. O Vieira, Cristiane S. Secca, Giuliana Mognol, Anjana Rao, João P. B. Viola. Characterization of the involvement of transcriptional repressor IRF2BP2 on T-cell proliferative response [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A87.