Inhibitor Id3 Research Articles

Antiproliferative effect of selexipag active metabolite MRE-269 on pulmonary arterial smooth muscle cells from patients with chronic thromboembolic pulmonary hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a group 4 pulmonary hypertension (PH) characterized by nonresolving thromboembolism in the central pulmonary artery and vascular occlusion in the proximal and distal pulmonary artery. Medical therapy is chosen for patients who are ineligible for pulmonary endarterectomy or balloon pulmonary angioplasty or who have symptomatic residual PH after surgery or intervention. Selexipag, an oral prostacyclin receptor agonist and potent vasodilator, was approved for CTEPH in Japan in 2021. To evaluate the pharmacological effect of selexipag on vascular occlusion in CTEPH, we examined how its active metabolite MRE-269 affects platelet-derived growth factor-stimulated pulmonary arterial smooth muscle cells (PASMCs) from CTEPH patients. MRE-269 showed a more potent antiproliferative effect on PASMCs from CTEPH patients than on those from normal subjects. DNA-binding protein inhibitor (ID) genes ID1 and ID3 were found by RNA sequencing and real-time quantitative polymerase chain reaction to be expressed at lower levels in PASMCs from CTEPH patients than in those from normal subjects and were upregulated by MRE-269 treatment. ID1 and ID3 upregulation by MRE-269 was blocked by co-incubation with a prostacyclin receptor antagonist, and ID1 knockdown by small interfering RNA transfection attenuated the antiproliferative effect of MRE-269. ID signaling may be involved in the antiproliferative effect of MRE-269 on PASMCs. This is the first study to demonstrate the pharmacological effects on PASMCs from CTEPH patients of a drug approved for the treatment of CTEPH. Both the vasodilatory and the antiproliferative effect of MRE-269 may contribute to the efficacy of selexipag in CTEPH.

Extracellular vesicles derived from GMSCs stimulated with TNF-α and IFN-α promote M2 macrophage polarization via enhanced CD73 and CD5L expression

Immunoregulatory properties of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are promising. Gingival tissue-derived MSCs (GMSCs) have unique immunoregulatory capacity and secrete large amounts of EVs. Recent findings suggest that priming MSCs with inflammatory stimuli is an effective strategy for cell-free therapy. However, the precise mechanism by which the contents of EVs are customized has not been fully elucidated. Here, we show that EVs derived from GMSCs primed with a combination of two pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interferon-α (IFN-α), synergistically promote anti-inflammatory M2 macrophage polarization by increasing the expression of cluster of differentiation 73 (CD73) and CD5 molecule-like (CD5L). Expression of CD73 by TNF-α/IFN-α stimulation was transcriptionally upregulated by the activation of mammalian target of rapamycin signaling and nuclear translocation of hypoxia-inducible factor 1α in GMSCs. TNF-α/IFN-α treatment also significantly increased the expression of CD5L mRNA via the transcription factor DNA-binding protein inhibitor ID3 and liver X receptor. Interestingly, exosomal CD5L is a prerequisite for the synergistic effect of EVs-mediated M2 macrophage polarization. These results indicate that combined pre-licensing with TNF-α and IFN-α in GMSCs is ideal for enhancing the anti-inflammatory function of EVs, which contributes to the establishment of a therapeutic tool.

Up-regulated DNA-binding inhibitor Id3 promotes differentiation of regulatory T cell to influence antiviral immunity in chronic hepatitis B virus infection

AimElevated Treg is relevant to persistent HBV infection, and the regulatory mechanism of Treg levels remains unclear. E proteins are important transcriptional regulators and could be antagonized by inhibitors of DNA-binding (Id) 1–4. We aim to clarify the role of Ids during HBV infection. Main methodsChanges of Ids and their relationship with Treg were investigated in both HBV transfection model and hepatitis B patients. Significance of Ids was studied by in vitro Treg differentiation induction with Id inhibited or over-expressed. The role of inflammatory cytokines for Id was studied by co-culture. RNA-Seq was conducted to explore the differentially expressed genes in Id-overexpressed CD4 T cells upon Treg differentiation induction conditions. Key findingsId-overexpressed mice attenuated virus clearance in HBV transfection model. In the HBV transfection mouse model, Tregs were up-regulated, with Id3 increased in Treg as well. Clinically, circulating Tregs in chronic hepatitis B (CHB) patients were elevated, and elevated Id3 transcriptional levels were positively correlated with Tregs. IL-1β could up-regulate Id3 in Treg cells induced in vitro. RNA-Seq revealed that increased Id could cause a series of signaling pathway changes during Treg differentiation. SignificanceId3 is elevated during HBV infection to ease Treg differentiation, and the antiviral immunity is influenced that make the infection to develop into chronic state.

Prognostication and Treatment of Burkitt Lymphoma in the Modern Era

Introduction Burkitt lymphoma (BL) is an ultra-aggressive B-cell lymphoma which represents ~1% of adult and ~30% of pediatric lymphomas.1 Apart from the sporadic variant predominant in the United States (US), BL may occur as immunodeficiency-associated or endemic variants. BL is curable in most patients with short-course, dose-intense immunochemotherapy. Molecular underpinning of BL has been well understood, but not yet translated into targeted therapy. About 90% show MYC rearrangement with an immunoglobulin gene (the classical IGH-MYC t(8;14), IGK, or IGL) but otherwise BL has fewer karyotypic abnormalities than other high-grade lymphomas. Commonly mutated genes include the centroblastic transcription factor TCF3 (25%, activating mutations) or its inhibitor ID3 (60%, inactivating mutations), cell cycle regulators CCND3 (30%) and CDKN2A/B (10%), as well as TP53 (35%).2 Of note, tonic PI3K-AKT-mTOR signaling contributes to BL sustained cell survival. The prognostic impact of specific molecular features remains undefined.

Correlation of the levels of DNA-binding inhibitor Id3 and regulatory T cells with SLE disease severity

E proteins, a subset of basic helix-loop-helix (bHLH) proteins, are transcription activators and their functions are inhibited by DNA-binding inhibitor (Id) 1–4. Studies have shown that Treg levels are decreased in Id3 knockout mice. Mice over-expressing Id1 in CD4 T cells possessed a greater number of regulatory T cells (Treg) and exhibited attenuated experimental autoimmune encephalomyelitis (EAE). The significance of Id proteins in human systemic lupus erythematosus (SLE) remains unclear. In this study, we systematically analyzed Id transcription in naïve, memory CD4 cells and regulatory T cells in peripheral blood mononuclear cells (PBMCs) in patients with active or inactive SLE. In parallel, Treg subsets in PBMCs were analyzed using different strategies. Id expression levels were correlated with Treg numbers as well as clinical indicators. We found that Id genes expressed in human peripheral CD4 cells were mainly Id2 and Id3. Id3 levels were significantly elevated in CD4+CD25hi T cells of patients with active SLE. Likewise, Id3 levels were positively correlated with increased CD4+FoxP3+ and CD4+Helios+FoxP3+ Treg cells in these patients. Id3 levels were found to be positively correlated with erythrocyte sedimentation rate (ESR), lupus anticoagulant (LAC), ribosomal antibody and SLE Disease Activity Index (SLEDAI) in patients with active SLE. Mice overexpressing Id1 in CD4+ T cells possessed significantly higher Treg levels in spleen and lower autoantibody concentrations in serum. Our results suggest that during the pathogenesis of SLE, up-regulation of Id3 can promote Treg differentiation to play an inhibitory effect on autoimmune responses.

D3 and D1 receptors: The Yin and Yang in the treatment of restless legs syndrome with dopaminergics.

Dopaminergic treatments targeting the D3 receptor subtype to reduce the symptoms of RLS show substantial initial clinical benefits but fail to maintain their efficacy over time. Sensorimotor circuits in the spinal cord are the gateway for the sensory processing of the symptoms and critical for the associated leg movements that relieve the symptoms and the periodic limb movements that often develop during sleep. There is a high preponderance of the inhibitory D3 receptor in the sensory-processing areas of the spinal cord (dorsal horn), whereas the motor areas in the ventral horn more strongly express the excitatory D1 receptor subtype. D3 and D1 receptors can form functional heteromeric ensembles that influence each other. In the spinal cord, long-term treatment with D3 receptor agonists is associated with the upregulation of the D1 receptor subtype and block of D1 receptor function at this stage can restore the D3 receptor effect. Alternate scenarios for a role of dopamine involve a role for the D5 receptor in regulating motor excitability and for the D4 receptor subtype in controlling D3-like effects. A model emerges that proposes that the behavioral changes in RLS, while responsive to D3 receptor agonists, may be ultimately be the result of unmasked increased D1-like receptor activities.

Transcriptomic Analysis Reveals Erythroferrone Modulates BMP6 Signaling Pathways Involved in Iron Homeostasis and Metabolism

Introduction: Erythroferrone (ERFE), the recently identified erythroid regulator of iron absorption, is a member of the C1q/TNF-related protein (CTRP) family. It is produced in erythroblasts in response to an increased erythropoietic drive, downregulates hepcidin expression and thereby promotes intestinal iron absorption and mobilization. The levels of Erfe are inappropriately high under conditions of ineffective erythropoiesis in inherited anemias such as thalassemia and congenital dyserythropoietic anemias. However, the mechanism of Erfe mediated Hepcidin inhibition remains unknown. Here, we use transcriptomic analysis of the human hepatoma cell line, Huh7 to identify genes and pathways involved in hepatocyte response to Erfe.Methods: Huh7 cells were seeded in a 6-well cell culture plate. Twenty four hours later, the cells were washed with PBS, and treated with a recombinant murine ERFE monomeric Fc (mEFRE-FC, 10µg/ml) protein or a control IgG (10 ug/ml) for 1h, 6h or 24h in growth media. RNA was isolated, followed by RNA quantification and quality assessment using a 2100 Agilent Bioanalyzer. ERFE is known to regulate Hepcidin antimicrobial peptide (HAMP) transcription and a treatment effect on HAMP expression was demonstrated by qPCR prior to sequencing. A total of 27 mRNA sequencing libraries were constructed from 1ug of human total RNA with the Illumina TruSeq Stranded mRNA Sample Prep protocol and single-end 75 bp reads were generated on an Illumina NextSeq 500. DESeq2 statistical package was used for differential expression testing. Multiple comparisons were adjusted for using a false discovery rate of 5%. Additionally, results were filtered to consider only those genes that demonstrated a fold-change point estimate >2 in either direction. Pathway analysis was performed using the Biological Process gene sets from the Gene Ontology annotation.Results: Compared to control treated Huh7 cells, 3 transcripts were differentially regulated following a one hour treatment with mERFE-Fc, ID1 (Log2 fold-change -1.55, q-value 4.99E-09), BPIFB2 (Log2 fold-change -1.88, q-value 0.036) and ANO1 (Log2 fold-change -1.43, q-value 0.019). A larger number of genes were differentially regulated with longer treatments, 32 genes at 6h and 828 genes at 24h. Among selected genes differentially expressed at 24h, between control treated and Erfe treated cells, we observed a significant reduction in expression of genes known to be directly upregulated by bone morphogenetic proteins (BMPs), including DNA binding protein inhibitor ID1(ID1), DNA binding protein inhibitor ID2 (ID2), DNA binding protein inhibitor ID3 (ID3) and HAMP. BMP6 has been demonstrated to regulate several biological processes such as iron metabolism in the liver, adipogenesis, and insulin sensitization. Interestingly, we also observed upregulation of SLC27A1 (or FATP1, fatty acid transporter, Log2 fold-change 2.27, q-value 0.003), GDNF (Log2 fold-change 2.57, q-value 2.4E-05), GIPR (Log2 fold-change 2.53, q-value 7.9E-07) upon treatment with Erfe at 24h. Previous work has demonstrated that all of these genes are downregulated by BMP6 treatment 1-3. Pathway analysis indicated a number of genes differentially regulated in the GOBP (gene ontology biological processes) iron ion homeostasis pathway, BMP signaling or TGF-β receptor signaling pathway. This assessment further indicated downregulation of cellular hormone levels and metabolic processes and ion homeostasis as biological processes impacted by erythroferrone.Conclusions: Significant differences in gene expression occur in hepatocytes upon interaction with erythroferrone. Many genes in the iron homeostasis, BMP6 signaling metabolic processes were also differentially regulated. This further supports the mechanism of ERFE on iron homeostasis. DisclosuresJasuja:Pfizer: Employment. Sawant:Pfizer: Employment. Pittman:Pfizer: Employment. Quan:Pfizer: Employment.

Differential Dopamine D1 and D3 Receptor Modulation and Expression in the Spinal Cord of Two Mouse Models of Restless Legs Syndrome.

Restless Legs Syndrome (RLS) is often and successfully treated with dopamine receptor agonists that target the inhibitory D3 receptor subtype, however there is no clinical evidence of a D3 receptor dysfunction in RLS patients. In contrast, genome-wide association studies in RLS patients have established that a mutation of the MEIS1 gene is associated with an increased risk in developing RLS, but the effect of MEIS1 dysfunction on sensorimotor function remain unknown. Mouse models for a dysfunctional D3 receptor (D3KO) and Meis1 (Meis1KO) were developed independently, and each animal expresses some features associated with RLS in the clinic, but they have not been compared in their responsiveness to treatment options used in the clinic. We here confirm that D3KO and Meis1KO animals show increased locomotor activities, but that only D3KO show an increased sensory excitability to thermal stimuli. Next we compared the effects of dopaminergics and opioids in both animal models, and we assessed D1 and D3 dopamine receptor expression in the spinal cord, the gateway for sensorimotor processing. We found that Meis1KO share most of the tested behavioral properties with their wild type (WT) controls, including the modulation of the thermal pain withdrawal reflex by morphine, L-DOPA and D3 receptor (D3R) agonists and antagonists. However, Meis1KO and D3KO were behaviorally more similar to each other than to WT when tested with D1 receptor (D1R) agonists and antagonists. Subsequent Western blot analyses of D1R and D3R protein expression in the spinal cord revealed a significant increase in D1R but not D3R expression in Meis1KO and D3KO over WT controls. As the D3R is mostly present in the dorsal spinal cord where it has been shown to modulate sensory pathways, while activation of the D1Rs can activate motoneurons in the ventral spinal cord, we speculate that D3KO and Meis1KO represent two complementary animal models for RLS, in which the mechanisms of sensory (D3R-mediated) and motor (D1R-mediated) dysfunctions can be differentially explored.

The role of genes involved in stress, neural plasticity, and brain circuitry in depressive phenotypes: Convergent findings in a mouse model of neglect

Early life neglect increases risk for the development of psychopathologies during childhood and adulthood, including depression and anxiety disorders. We recently reported epigenetic changes in DNA derived from saliva in three genes predicted depression in a cohort of maltreated children: DNA-binding protein inhibitor ID-3 (ID3), Glutamate NMDA Receptor (GRIN1), and Tubulin Polymerization Promoting Protein (TPPP). To validate the role of these genes in depression risk, secondary analyses were conducted of gene expression data obtained from medial prefrontal cortex (mPFC) tissue of mice subjected to a model of maternal neglect which included maternal separation and early weaning (MSEW). Anxiety and depression-like phenotype data derived using the elevated plus maze (EPM) and forced swimming test (FST), respectively, were also available for secondary analyses. Behavioral tests were conducted in MSEW and control adult male mice when they were between 65 and 80days old. ID3, GRIN1 and TPPP gene expression in the mPFC were found to significantly predict behavioral differences in the EPM and FST. These results further support the role of these genes in the etiology of depressive and anxiety phenotypes following early life stress.

Opposing aging-related shift of excitatory dopamine D1 and inhibitory D3 receptor protein expression in striatum and spinal cord.

Normal aging is associated with a decrease in motor function, a concomitant increase in muscle stiffness and tone, and a decrease in dopamine (DA) levels in the spinal cord. The striatum plays a critical role in the control of motor function, and it receives strong DA innervation from the substantia nigra. However, locomotor activity also requires the activation of motoneurons in the lumbar spinal cord, which in the mouse express all five DA receptor subtypes (D1-D5). Of these, the D3 receptor (D3R) expresses the highest affinity to DA and mediates inhibitory actions, while activation of the lower-affinity D1 receptor (D1R) system promotes excitatory effects. To test whether the aging-related decrease in DA levels is associated with corresponding changes in DA receptor protein expression levels, we probed with Western blot and immunohistochemical techniques for D1R and D3R protein expression levels over the normal life span of the mouse. We found that with age D1R expression levels increased in both striatum and spinal cord, while D3R expression levels remained stable in the striatum or slightly decreased in the spinal cord. The resulting D1-to-D3 ratio indicates a strong upregulation of D1R-mediated pathways in old animals, which is particularly pronounced in the lumbar spinal cord. These data suggest that aging may be associated with a shift in DA-mediated pathways in striatum and spinal cord, which in turn could be an underlying factor in the emergence of aging- and DA-related motor dysfunctions such as Parkinson's disease or Restless Legs Syndrome (RLS).

Induced Developmental Arrest of Early Hematopoietic Progenitors Leads to the Generation of Leukocyte Stem Cells

SummarySelf-renewal potential and multipotency are hallmarks of a stem cell. It is generally accepted that acquisition of such stemness requires rejuvenation of somatic cells through reprogramming of their genetic and epigenetic status. We show here that a simple block of cell differentiation is sufficient to induce and maintain stem cells. By overexpression of the transcriptional inhibitor ID3 in murine hematopoietic progenitor cells and cultivation under B cell induction conditions, the cells undergo developmental arrest and enter a self-renewal cycle. These cells can be maintained in vitro almost indefinitely, and the long-term cultured cells exhibit robust multi-lineage reconstitution when transferred into irradiated mice. These cells can be cloned and re-expanded with 50% plating efficiency, indicating that virtually all cells are self-renewing. Equivalent progenitors were produced from human cord blood stem cells, and these will ultimately be useful as a source of cells for immune cell therapy.

The DNA-binding inhibitor Id3 regulates IL-9 production in CD4(+) T cells.

The molecular mechanisms by which TGF-β and interleukin 4 (IL-4) signaling control the differentiation of IL-9-producing CD4+ T (TH9) cells remain incompletely understood. We show here that the DNA-binding inhibitor Id3 regulated Th9 cell differentiation, as deletion of Id3 increased IL-9 production from CD4+ T cells. Mechanistically, TGF-β1 and IL-4 down-regulated Id3 expression and this process required the kinase TAK1. Reduction of Id3 expression enhanced the binding of the transcription factors E2A and GATA-3 in the Il9 promoter region, which promoted Il9 gene transcription. Importantly, Id3 control of TH9 cells differentiation regulated anti-tumor immunity in an experimental melanoma-bearing model in vivo, and also in human CD4+ T cells in vitro. Thus, this study reveals a previously unrecognized TAK1-Id3-E2A-GATA-3 pathway that regulates TH9 cell differentiation.

Abstract A13: Mechanisms of E47 induced quiescence and acinar cell differentiation in human pancreatic cancer cells

Abstract Pancreatic ductal adenocarcinoma (PDA) initiates from quiescent acinar cells that attain a Kras mutation, undergo acinar-ductal metaplasia and rapidly acquire increased growth potential. During this process several transcription factors from the basic helix-loop-helix (bHLH) family are downregulated while expression of their inhibitor Id3 is induced. Previously we showed that Id3 knockdown with siRNA resulted in growth arrest in PDA cells. Here we queried whether aggressive PDA cells can be reprogrammed to revert to their original quiescent acinar cell phenotype by shifting bHLH transcription programs. In order to mitigate Id3 and upregulate bHLH protein expression we engineered human PDA cells to stably express an inducible form of the bHLH factor E47. Remarkably, we found that E47 triggers both cell cycle arrest and re-activation of the acinar cell differentiation program and in PDA cells. E47 acted as a tumor suppressor of human pancreatic cancer cells in vitro and in vivo. In 4 human PDA cell lines E47 led to G0/G1 arrest within 48 hours and induction of E47 reduced tumor formation in vivo. Notably, even temporary induction of E47 in vitro prior to transplantation resulted in small tumors relative to controls, suggesting that E47 induces stable changes in cell growth. Mechanistically, we found that E47 induced arrest requires upregulation of the cyclin-dependent kinase inhibitor (cdki) p21. Simultaneous with cell cycle exit E47 expressing PDA cells exhibited restored acinar cell features. GSEA enrichment analysis of microarray data revealed significant induction of the exocrine gene signature by E47. These findings were confirmed by qPCR demonstrating up to 70,000 fold increases in expression of acinar digestive enzymes. Further, E47 induced feed forward expression of MIST1, the bHLH factor required for acinar maturation, which is profoundly downregulated in PDA. To determine whether induced MIST1 protein was functional in these cells, we examined expression of the MIST1 target genes Foxp2, Copz2, and Rab3d finding that they were significantly increased within 48 hours of inducing E47 activity. Further, PDA cells acquired MIST1-dependent Cx32 gap junctions, a hallmark of differentiated acinar cells. Collectively the data show that PDA cells retain a remarkable degree of plasticity which can be exploited to return them to a quiescent acinar-like state. The discovery that bHLH activity is the critical node regulating PDA cell fate offers new opportunities to manipulate transcription pathways for therapeutic advances in pancreatic cancer. Citation Format: Sangwun Kim, Challeng Yang, Chelsea Riha, Reyhaneh Lamy, Brad L. Jakubison, Stephen F. Konieczny, Pamela Itkin-Ansari. Mechanisms of E47 induced quiescence and acinar cell differentiation in human pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A13.

Child Abuse, Depression, and Methylation in Genes Involved With Stress, Neural Plasticity, and Brain Circuitry

To determine whether epigenetic markers predict dimensional ratings of depression in maltreated children. A genome-wide methylation study was completed using the Illumina 450K BeadChip array in 94 maltreated and 96 healthy nontraumatized children with saliva-derived DNA. The 450K BeadChip does not include any methylation sites in the exact location as sites in candidate genes previously examined in the literature, soatestfor replication of prior research findings was not feasible. Methylation in 3 genes emerged as genome-wide-significant predictors of depression: DNA-Binding Protein Inhibitor ID-3 (ID3); Glutamate Receptor, Ionotropic N-methyl-D-aspartate (NMDA) 1 (GRIN1); and Tubulin Polymerization Promoting Protein (TPPP) (p< 5.0× 10(-7), all analyses). These genes are all biologically relevant with ID3 involved in the stress response, GRIN1 involved in neural plasticity, and TPPP involved in neural circuitry development. Methylation in CpG sites in candidate genes were not predictors of depression at significance levels corrected for whole genome testing, but maltreated and control children did have significantly different β values after Bonferroni correction at multiple methylation sites in these candidate genes (e.g., BDNF, NR3C1, FKBP5). This study suggests that epigenetic changes in ID3, GRIN1, and TPPP genes, in combination with experiences of maltreatment, may confer risk for depression in children. The study adds to a growing body of literature supporting a role forepigenetic mechanisms in the pathophysiology of stress-related psychiatric disorders. Although epigenetic changes are frequently long lasting, they are not necessarily permanent. Consequently, interventions to reverse the negative biological and behavioral sequelae associated with child maltreatment are briefly discussed.

Chronic Active B-Cell Receptor Signaling in Lymphoma

Abstract SCI-26We have developed loss-of-function, RNA interference-based screens to reveal genes essential for cancer cell proliferation and survival. In parallel, we are using high-throughput RNA resequencing (RNA-seq) to identify somatic mutations and other structural abnormalities in cancer. The intersection of these two data sets has helped us to discover novel pathogenetic pathways in lymphoma that are amenable to therapeutic attack. The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) has constitutive activation of the NF-κB pathway, which we traced to the signaling adapter CARD11. In some ABC DLBCL biopsies (∼10%), somatic mutations produce CARD11 isoforms that spontaneously activate NF-κB signaling. In ABC DLBCL tumors with wild-type CARD11, we defined a “chronic active” form of B-cell receptor (BCR) signaling that activates NF-κB. Such ABC DLBCLs are killed by knockdown of BCR signaling components, such as Bruton’s tyrosine kinase (BTK), or components of the BCR itself. Over one-fifth of ABC DLBCLs have mutations in the CD79B or CD79A subunits of the BCR. In 18 percent of cases, mutations occur in a single tyrosine residue in the critical “ITAM” signaling motif, generating BCRs that avoid negative autoregulation by the LYN tyrosine kinase. Based on these findings, we are conducting clinical trials of ibrutinib in relapsed/refractory DLBCL. Ibrutinib is an irreversible and highly selective small-molecule inhibitor of BTK. Thus far, ibrutinib monotherapy has induced many complete and partial responses in patients with ABC DLBCL, including those with “primary refractory” tumors that had never responded to any prior therapy. One patient has been in a sustained complete response for over 19 months, taking ibrutinib daily with no discernible side effects. Of note, ABC DLBCL tumors with and without CD79B mutations have responded, suggesting that BCR pathway addiction may be a prevalent feature in this lymphoma subtype. More recently, we have uncovered a “tonic” form of BCR signaling in Burkitt lymphoma that engages the prosurvival PI(3) kinase pathway. Two-thirds of Burkitt lymphoma cell lines die upon knockdown of BCR subunits or the proximal kinase SYK, due to loss of PI(3) kinase signaling. Moreover, a gene expression signature of PI(3) kinase activity is more highly expressed in Burkitt lymphoma biopsies than in biopsies of other aggressive lymphomas. Tonic BCR signaling in Burkitt lymphoma is mechanistically distinct from chronic active BCR signaling in ABC DLBCL, since it does not engage BTK, CARD11, or NF-κB. RNA-seq revealed that 70 percent of sporadic Burkitt lymphoma cases harbor somatic mutations that potentiate the action of the transcription factor TCF3 by preventing its inhibitory heterodimerization with the DNA-binding inhibitor ID3. TCF3 promotes tonic BCR signaling and PI(3) kinase activity in Burkitt lymphoma by transactivating the immunoglobulin heavy- and light-chain genes, thereby increasing surface BCR expression, and by repressing the phosphatase SHP-1, a potent negative regulator of BCR signaling. Hence, inhibitors of proximal BCR signaling and the PI(3) kinase pathway should be evaluated in Burkitt lymphoma, especially in patients for whom high-dose chemotherapy is infeasible, such as older individuals and those with the endemic form of this lymphoma. Disclosures:Off Label Use: I will be discussing clinical trials of ibrutinib (PCI-32765) in lymphoma. Ibrutinib is an investigational drug that has not yet received FDA approval for any indication.

Repression of the DNA-binding inhibitor Id3 by Blimp-1 limits the formation of memory CD8+ T cells

Blimp-1 is a transcriptional repressor that promotes the differentiation of CD8+ T cells into short-lived KLRG-1+ effector cells (SLEC), but how it operates remains poorly defined. Here we show that Blimp-1 binds and represses the Id3 promoter in SLEC. Repression of Id3 by Blimp-1 was dispensable for SLEC development but limited their capacity to persist as memory cells. Enforced expression of Id3 was sufficient to rescue SLEC survival and enhanced recall responses. Id3 function was mediated in part through inhibition of E2a transcriptional activity and induction of genes regulating genome stability. These findings identify a Blimp-1-Id3-E2a axis as a key molecular switch that determines whether effector CD8+ T cells are programmed to die or enter the memory pool.

Control of the differentiation of regulatory T cells and TH17 cells by the DNA-binding inhibitor Id3

The molecular mechanisms directing Foxp3 gene transcription in CD4+ T cells remain ill defined. We show that deletion of the inhibitory helix-loop-helix (HLH) protein Id3 results in defective Foxp3+ Treg cell generation. We identified two transforming grothw factor-β1 (TGF-β1)-dependent mechanisms that are vital for activation of Foxp3 gene transcription, and are defective in Id3−/− CD4+ T cells. Enhanced binding of the HLH protein E2A to the Foxp3 promoter promoted Foxp3 gene transcription. Id3 was required to relieve inhibition by GATA-3 at the Foxp3 promoter. Further, Id3−/− T cells increased differentiation of Th17 cells in vitro and in a mouse asthma model. A network of factors therefore act in a TGF-β-dependent manner to control Foxp3 expression and inhibit Th17 cell development.

Developmental expression patterns and localization of DNA-binding protein inhibitor (Id3) in the mouse retina

Id3 (inhibitor of DNA binding/differentiation), a member of the Id helix-loop-helix protein family, has long been studied as a positive regulator of proliferation and a negative regulator of differentiation. In this study, we examined the expression pattern and cellular phenotypes of Id3 in postnatal and adult mouse retina. Id3 was mainly expressed in the early postnatal inner retina. From the late postnatal development towards adulthood, Id3 expression was confined to the ganglion cell layer and the inner nuclear layer. Colocalization analysis showed that Id3 positive cells were identified as retinal ganglion cells and amacrine cells. The differential expression profiles of Id3 provide the groundwork for the elucidation of its possible role in retinal development.

Gene expression by the anterior pituitary gland: effects of age and caloric restriction

Biological aging is associated with functional deficits at the cellular, organ, and system levels. The pituitary gland, the central organ of the neuroendocrine system, has been shown to play an important role in the aging process. To gain a better understanding of its functional changes with aging, we compared the gene expression profiles of the anterior pituitary of young and old Brown Norway rats, focusing on the major pituitary hormone genes. We also explored the effects of caloric restriction, an intervention shown to delay or inhibit age-associated pathologic and biologic changes in a number of systems and organisms, on the expression of these genes. Of the total of 1176 genes arrayed on each of the six membranes per group that we used, 542 (46%) were detectable in the anterior pituitary of young and old rats. Significance analysis of microarrays (SAM) of these 542 detectable genes revealed 28 genes that changed significantly with age, among which 24 decreased and 4 increased. Among the five major hormone genes on the membrane, growth hormone (GH) and prolactin decreased with age, the glycoprotein hormone common α subunit gene increased, and follicle-stimulating hormone-β subunit (FSH-β) and thyrotropin-β (TSH-β) subunit did not change. Among these genes, the three found to change by array analysis were confirmed to do so by Northern blot analysis. For the two genes among the five that were not selected (i.e. did not change) by array analysis, TSH-β also showed no significant change by Northern blot; but the other, FSH-β, showed significant increase. Thus, of the five genes checked by Northern blot analysis, the results were consistent with the array data in four cases. Short-term caloric restriction (5 weeks) of young adult animals resulted in 19 genes being significantly down-regulated, while no significantly up-regulated genes were identified. Among the genes that were down-regulated were GH, gonadotropin releasing hormone receptor (GnRH-R), three cytochrome c oxidase subunits and two heat shock proteins. With long-term (21 month) caloric restriction, about 30% of the genes that changed with aging (8/28) were prevented from doing so, and none of the age-related changes was enhanced with long-term caloric restriction. The genes that showed most significant rescue were neuropeptide Y, GnRH-R, DNA-binding protein inhibitor Id-3, and nerve growth factor-induced protein I-B. These results indicate that long-term caloric restriction can partially prevent some of the age-related changes in gene expression in the anterior pituitary of Brown Norway rats, suggesting a benefit of this regimen to be the slowing of the aging process. The fact that fewer than 30% genes derived benefit also suggests that the effect of caloric restriction is rather limit, which is consistent with the thesis that caloric restriction may slow, but not prevent, the aging process.

Thymocyte development in early growth response gene 1-deficient mice.

Early growth response gene 1 (Egr1) codes for a transcriptional regulator that contains a zinc-finger DNA binding domain. Egr1 expression is induced by a variety of extracellular stimuli including TCR-ligand interactions. Its pattern of expression in the thymus and dependence on ERK activation have led to speculation that it has a role in T cell development, but the exact nature of this role has been undefined. To more clearly define the role of Egr1 in thymocyte development, we have analyzed thymocytes from Egr1-deficient mice. We find that thymuses from Egr1-deficient mice contain twice as many cells as age-matched controls, and the increase in thymocyte number is apparent at the early CD4/CD8 double negative stage of development. Subsequent maturation to the CD4/CD8 double positive stage and survival of the double positive cells both appear normal in Egr1-deficient animals. We also find that Egr1 promotes positive selection of both CD4 and CD8 single positive cells without playing a major role in negative selection. Egr1 influences positive selection by enhancing expression of the helix-loop-helix inhibitor Id3 and the anti-apoptosis molecule bcl-2. Thus, Egr1 translates developmental signals into appropriate changes in gene expression at multiple stages of thymocyte development.