Role Of Id2 Research Articles

Id2 epigenetically controls CD8+ T-cell exhaustion by disrupting the assembly of the Tcf3-LSD1 complex

CD8+ T-cell exhaustion is a state of dysfunction that promotes tumor progression and is marked by the generation of Slamf6+ progenitor exhausted (Texprog) and Tim-3+ terminally exhausted (Texterm) subpopulations. Inhibitor of DNA binding protein 2 (Id2) has been shown to play important roles in T-cell development and CD8+ T-cell immunity. However, the role of Id2 in CD8+ T-cell exhaustion is unclear. Here, we found that Id2 transcriptionally and epigenetically regulates the generation of Texprog cells and their conversion to Texterm cells. Genetic deletion of Id2 dampens CD8+ T-cell-mediated immune responses and the maintenance of stem-like CD8+ T-cell subpopulations, suppresses PD-1 blockade and increases tumor susceptibility. Mechanistically, through its HLH domain, Id2 binds and disrupts the assembly of the Tcf3-Tal1 transcriptional regulatory complex, and thus modulates chromatin accessibility at the Slamf6 promoter by preventing the interaction of Tcf3 with the histone lysine demethylase LSD1. Therefore, Id2 increases the abundance of the permissive H3K4me2 mark on the Tcf3-occupied E-boxes in the Slamf6 promoter, modulates chromatin accessibility at the Slamf6 promoter and epigenetically regulates the generation of Slamf6+ Texprog cells. An LSD1 inhibitor GSK2879552 can rescue the Id2 knockout phenotype in tumor-bearing mice. Inhibition of LSD1 increases the abundance of Slamf6+Tim-3− Texprog cells in tumors and the expression level of Tcf1 in Id2-deleted CD8+ T cells. This study demonstrates that Id2-mediated transcriptional and epigenetic modification drives hierarchical CD8+ T-cell exhaustion, and the mechanistic insights gained may have implications for therapeutic intervention with tumor immune evasion.

Abstract A55: Role of ID2 in acute myeloid leukemia progression and resistance to chemotherapy

Abstract Acute myeloid leukemia (AML) is thought to be maintained by quiescent leukemia stem cells (LSCs), which are resistant to chemotherapy and responsible for relapse. Recent evidence suggests that a subset of LSCs with higher oxidative phosphorylation and/or increased senescence are responsible for resistance to chemotherapy and relapse. While several genes have been identified that are associated with the maintain LSC maintenance and promote resistance and relapse, the precious molecular mechanism(s) are currently not known. Inhibitor of DNA binding (ID) proteins are a group of Helix-Loop-Helix (HLH) transcription regulators, which function as dominant regulators of other HLH proteins (E proteins) that are required for normal hematopoietic, muscle, neuronal and other cell development. We found that loss of ID2 expression in murine hematopoietic cells results in activation, differentiation, and exhaustion of hematopoietic stem cells (HSCs). ID2 promotes quiescence by stabilizing HIF-1a expression. Analysis of TCGA data sets shows that high levels of ID2 expression is associated with reduced overall survival among AML patients, suggesting that ID2 may function to maintain LSCs. Similar to murine cells, knockdown (KD) of ID2 expression in human cord blood cells resulted in a loss of CD34+CD38− progenitors. Furthermore, knockdown of ID2 expression in the UT-7 AML cell line showed significantly reduced leukemogenic potential in NSG mice, suggesting that ID2 is required for AML cell growth in vivo. UT7-ID2-KD cell line shows significant reduction of colony number when cultured in low oxygen condition in vitro, suggesting a potential role of HIF protein. In comparison, overexpression (OE) of ID2 in the human AML cell line, MO7e, promotes their leukemic potential and burden in NSG mice in vivo, suggesting that ID2 may function to preserve LSC quiescence. Initial studies in primary AML-PDX cells showed reduced leukemic potential upon ID2 knock down. High level of ID2 expression is associated with poor chemotherapy response in patients. We found that MO7e-ID2-OE are resistance to Cytarabine (AraC), while UT7-ID2-KD are susceptible to AraC. Furthermore, we found that AML cell lines that survive AraC treatment show increased ID2 expression, suggesting that ID2 may promote survival of chemotherapy resistant AML cells. It has been reported that AraC resistance of AML cells is associated with increased senescence. We found that surviving UT7-ID2-KD cells after AraC treatment have significantly reduced senescence population compared to controls. Taken together, ID2 promotes normal murine and human HSC quiescence, and may function promote LSC maintenance and resistance to chemotherapy. Citation Format: Tanmoy Sarkar, Jennifer Lombardo, Shweta Singh, Kristbjorn O Gudmundsson, Holly M Morris, Shyam Saran, Jonathan R Keller. Role of ID2 in acute myeloid leukemia progression and resistance to chemotherapy [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 A55.

Abstract 1278: ID3 controlled dynamic Pyk2 localization is important for the mobility of cancer stem cells

Abstract Treatment of brain metastases remains a clinical barrier in breast cancer patients. Secondary breast tumors in the brain are reported to be enriched with breast cancer stem cell (BCSC) markers suggesting that BCSCs infiltrate the brain. Brain infiltration by BCSCs is complex involving detachment from the primary tumor, entering the blood, surviving the microvascular niche, and crossing the blood brain barrier (BBB). It is critical to understand how BCSCs move through the BBB to prevent brain metastases. Nuclear respiratory factor 1 (NRF1) transcription factor, Pyk2 non-receptor tyrosine kinase, and inhibitor of differentiation protein 3 (ID3) have been shown to contribute to the pathogenesis of cancer through inducing CSCs or increasing mobility of these cells. Recently, we showed that NRF1-induced BCSCs crossed the BBB which were guided by ID3 overexpressing endothelial stem cells. In this study, we investigated the NRF1/ID3/Pyk2 axis contributing to BCSCs mobility. NRF1 promoted migration of BCSCs and mesenchymal MDAMB231 cells through brain endothelial cells cultured with astrocytes. ID3 overexpressing endothelial cells promoted adhesion with BCSCs and mobility of BCSCs across the BBB model. In addition, ID3 controlled Pyk2 localization which is important for mobility of stem cells and cancer cells. Endothelial cells were treated with a known risk factors for breast cancer, 17β-estradiol (E2) and environmental pollutant PCB153. Treatment with PCB153 or E2 showed increased growth of BCSCs tumor spheroids and mobility of ESCs. Exosomal ID3 released from endothelial cells also supported the growth of BCSCs and provide the basis for paracrine effects by endothelial stem cells associated with breast tumors. These findings show for the first time a novel role for ID3 and Pyk2 by which endothelial stem cells support breast tumor metastases where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3/Pyk2 modifying agents to prevent the spread of breast cancer to the brain. Citation Format: Jayanta Das, Alok Deoraj, Deodutta Roy, Quentin Felty. ID3 controlled dynamic Pyk2 localization is important for the mobility of cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1278.

Brain infiltration of breast cancer stem cells is facilitated by paracrine signaling by inhibitor of differentiation 3 to nuclear respiratory factor 1.

Treatment options for brain metastatic breast cancer are limited because the molecular mechanism for how breast cancer cells infiltrate the brain is not fully understood. For breast tumors to metastasize to the brain first, cells need to detach from the primary tumor, enter in the blood circulation, survive within the microvascular niche, and then cross the blood-brain barrier (BBB) to colonize into the brain. It is critical to understand how breast cancer cells transmigrate through the BBB to prevent brain metastasis. Nuclear respiratory factor 1 (NRF1) transcription factor has been reported to be highly active in several human cancers and its aberrant expression facilitates in the acquisition of breast cancer stem cells (BCSCs). Inhibitor of differentiation protein 3 (ID3), a transcription regulating protein, induces pluripotent endothelial stem cells (ESCs). Herein, we investigated if NRF1-induced BCSCs could cross a BBB model and guiding of BCSCs by ID3-induced ESCs across the BBB. BCSCs and ESCs were subjected to functional gain/loss experiments to determine if NRF1/ID3 contributed to lineage-specific BCSCs organ entry. First, we tested whether NRF1 promoted migration of breast cancer using a BBB model consisting of BCSCs or MDA-MB231 cells, brain endothelial cell layer, and astrocytes. NRF1 overexpression increased the propensity for BCSCs and NRF1-induced MDA-MB231 cells to adhere to brain endothelial cells and migrate across a human BBB model. Increased adhesion of NRF1-induced BCSCs to ESCsID3 was detected. NRF1-induced BCSCs crossed through the BBB model and this was promoted by ESCsID3. We also showed that environmental relevant exposure to PCBs (PCB153 and PCB77) produced differential effects. Treatment with PCB153 showed increased growth of NRF1-induced BCSCs tumor spheroids and increased in vivo migration of ESCsID3. Exosomal ID3 released from endothelial cells also supported the growth of NRF1-induced BCSCs and provide the basis for paracrine effects by ESCsID3 associated with breast tumors. Xenograft experiments showed that ID3 overexpressing brain ESCs not only supported the growth of BCSC tumor spheroids but guided them to the neural crest in zebrafish. These findings show for the first time a novel role for ID3 and NRF1 by which ESCsID3 help guide BCSCsNRF1 to distant metastatic sites where they most likely facilitate the colonization, survival, and proliferation of BCSCs. This knowledge is important for pre-clinical testing of NRF1/ID3 modifying agents to prevent the spread of breast cancer to the brain.

Lineage Tracing Reveals the Dynamic Contribution of Id2+ Progenitor Cells to Branching Morphogenesis.

Branching morphogenesis is an important process in shaping the arborized structures of several organs. However, the driving force that directs this process from progenitor pools remains incompletely understood. In this lineage tracing study, we investigated the role of Id2+ embryonic progenitor cells in branching organs such as the pancreas, kidney, mammary gland, thyroid gland, and salivary gland. We found that a subset of Id2+ distal progenitor cells in the embryonic pancreas and kidney can give rise to multiple lineages of progeny cells during branching morphogenesis. Id2-labeled cells also supported the postnatal development of the mammary glands. However, Id2+ cells did not contribute to the development of the salivary and thyroid glands. We found the Id2+ cells located in the tip progenitor pools of pancreas and kidney have self-renewal potential and contribute descendants to multiple epithelial cell lineages. Our findings enrich the current model of distal progenitor pools driving branching morphogenesis and provide a new marker to investigate the regularity of branching in these organs.

ID2 Inhibits Bladder Cancer Progression and Metastasis via PI3K/AKT Signaling Pathway.

Background: Inhibitors of DNA-binding (ID) proteins are important regulators of cell proliferation and differentiation. The aim of this study was to evaluated the role of ID proteins in bladder cancer (BCa) and related molecular mechanisms.Methods: The TCGA database was analyzed for the expression and clinical significance of ID proteins. The expression of ID2 was determined by qRT-PCR, immunohistochemical staining and western blot. The role of ID2 was determined by CCK-8, colony formation, wound healing, transwell and xenograft tumor assays, and the potential mechanism of ID2 in BCa was investigated by RNA sequencing.Results: ID2 expression was significantly downregulated in TCGA database and clinical samples, and high ID2 expression was associated with low-grade tumor staging and correlated with better overall survival, disease specific survival (DSS) and progress free interval (PFI). In vivo and in vitro experiments showed that knockdown of ID2 promoted proliferation, migration, invasion and metastasis of BCa cells, while overexpression of ID2 significantly inhibited cell proliferation, migration, invasion and metastasis. Mechanistically, ID2 acts as a tumor suppressor through PI3K/AKT signaling pathway to inhibit the progression and metastasis of BCa.Conclusion: Our results suggest that ID2 exerts tumor suppressive effects in BCa through PI3K/AKT signaling pathway, and altered ID2 expression can be used as a biomarker of BCa progression and metastasis.

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.

Id(entifying) the inhibitor of DNA binding 3 in the brain of Nothobranchius furzeri upon aging.

Inhibitors of DNA (Id) are key transcription factors (TFs) regulating neurogenic processes. They belong to the helix-loop-helix (HLH) TF family and are dominant negative regulators of basic HLH proteins (bHLHs). Specifically, they inhibit cell differentiation and enhance cell proliferation and motility. The Id family includes four members, Id1, Id2, Id3, and Id4, which have been identified in nearly all vertebrates. The transcript catalog of the African turquoise killifish, Nothobranchius furzeri, contains all four TFs and has evolved showing positive selection for Id3. N. furzeri, a teleost, is the short-lived vertebrate and is gaining increasing scientific interest as a new model organism in aging research. It is characterized by embryonic diapause, explosive sexual maturation, and rapid aging. In this study, we investigated both the expression and the role of Id3 in the brain of this model organism. Interestingly, Id3 was upregulated age-dependently along with a distribution pattern resembling that of other vertebrates. Additionally, the gene has undergone positive selection during evolution and shows a high degree of conservation relative to that of other vertebrates. These features make N. furzeri a valid tool for aging studies and a potential model in translational research.

ID2 protects retinal pigment epithelium cells from oxidative damage through p-ERK1/2/ID2/NRF2

Age-related macular degeneration (AMD) is the leading cause of blindness during aging. The degeneration of retinal pigment epithelium (RPE) is the main pathologic characteristic of AMD. ID2 is a member of the Inhibitor of DNA binding proteins (ID) family and is involved in regulation of cell proliferation and differentiation. However, currently the role of ID2 in oxidative injury response in RPE cells remains unknown. Here we showed that oxidative stress increased ID2 expression in RPE cells. Knockdown of ID2 promoted cell apoptosis and increased ROS level in RPE cells that were subjected to oxidative damage. In addition, over-expression of ID2 attenuated the oxidative damage response in RPE cells. Mechanistically, ID2 protected RPE cells from oxidative damage through activating NRF2. Furthermore, phosphorylation of ERK1/2 positively regulated the protective function of ID2. Finally, we confirmed that the oxidative damage increased Id2 expression and over-expression of Id2 elevated Nrf2 expression in primary mouse RPE cells. Therefore, ID2 protects RPE cells from oxidative damage through the p-ERK1/2/ID2/NRF2 pathway. Our study contributes to a better understanding of the mechanisms underlying oxidative stress in AMD and may present a new strategy for AMD treatment.

Abstract 342: Helix-loop-helix Transcription Factor Id3 Promotes Ischemia-induced Angiogenesis

The development of new blood vessels is important during the progression of many diseases including cancer, obesity, and ischemia. While inhibiting blood vessel growth can prevent excess adiposity and tumor growth, it can also exacerbate symptoms of ischemia. Using flow cytometry to quantify total endothelial cells (ECs) in the gastrocnemius of mice who have undergone femoral artery resection (FAR), the number of ECs per gram of muscle increases approximately four-fold relative to uninjured gastrocnemius from the same mouse (442% increase, ± 208%) in a model of hindlimb ischemia. The helix-loop-helix (HLH) transcription factor Inhibitor of differentiation (ID3) promotes angiogenesis and whole tissue blood perfusion in models of cancer and obesity, but the potential role for ID3 in promoting ischemia-induced angiogenesis remains uninvestigated. Id3 mRNA levels in injured gastrocnemius are increased three days post-FAR relative to uninjured gastrocnemius (p < 0.05, n = 6). Furthermore, ID3 global knockout (KO) mice have attenuated perfusion recovery compared to wild type littermate controls at 14 days post-FAR (p < 0.05). Gastrocnemius muscles isolated from ID3 global KO mice 7 days post-FAR express higher levels of the cell cycle inhibitor p21 Cip1 mRNA than WT mice (p = 0.10, n = 3). Finally, flow cytometry analysis of cell subsets in a novel Id3 promoter-GFP reporter mouse line revealed that Id3 promoter activation is greater in CD31 + ECs than many other cell types (n = 10), suggesting ECs may be an important cell type in which ID3 is promoting pro-angiogenic effects. Together, these data support the hypothesis that ID3 promotes ischemia-induced angiogenesis by promoting EC proliferation and inhibiting cell cycle inhibitor p21 Cip1 . To investigate the role of ID3 in ECs during angiogenesis, an EC-specific, tamoxifen-inducible Id3 KO mouse (Id3 fl/fl Cdh5-Cre/ERT2) has been generated.

New role of ID3 in melanoma adaptive drug-resistance.

Adaptive resistance to targeted therapy such as BRAF inhibitors represents in melanoma a major drawback to this otherwise powerful treatment. Some of the underlying molecular mechanisms have recently been described: hyperactivation of the BRAF-MAPK pathway, of the AKT pathway, of the TGFβ/EGFR/PDGFRB pathway, or the low MITF/AXL ratio. Nevertheless, the phenomenon of early resistance is still not clearly understood. In this report, we show that knockdown of neural crest-associated gene ID3 increases the melanoma sensitivity to vemurafenib short-term treatment. In addition, we observe an ID3-mediated regulation of cell migration and of the expression of resistance-associated genes such as SOX10 and MITF. In sum, these data suggest ID3 as a new key actor of melanoma adaptive resistance to vemurafenib and as a potential drug target.

ID3 may protect mice from anti‑GBM glomerulonephritis by regulating the differentiation of Th17 and Treg cells.

Anti‑glomerular basement membrane glomerulonephritis (anti‑GBM GN) is an autoimmune disease that leads to severe and rapidly progressive renal injury. Inhibition of DNA‑binding factor 3 (ID3) serves a key role in autoimmune diseases, such as asthma and Sjögren's syndrome, and in experimental allergic encephalitis models. However, the role of ID3 in the progression of anti‑GBM GN remains unknown. In the present study, ID3 mRNA expression increased between 3‑ and 20‑fold in the renal tissues of anti‑GBM GN mice compared with the Control group, with a peak at day 14 post‑induction. In addition, ID3 protein expression was upregulated from day 7 onwards. The expression of ID3 was also examined in the spleen, and was demonstrated to be increased in the spleen of nephritic mice. T helper 17 (Th17) cells and regulatory T (Treg) cells were present throughout the entire period of observation (from day 7 to day 28) in anti‑GBM GN mice, which may vary at different time points, accompanied with the expression of ID3. Invitro, ID3 expression was increased when CD4+ T cells differentiated into Tregs; however, expression was lower in Th17 cells. Following treatment with ID3 small interfering RNA, RAR‑related orphan receptor γt, but not forkhead box P3, expression increased. Furthermore, increased expression of interleukin‑17A was also observed when ID3 was blocked. In addition, ID3 was able to interact with transcription factor E2A. A significant increase in binding between ID3 and E2A was observed in anti‑GBM GN from day 7 onwards, with a peak at day 14 in both renal tissue and spleen. In conclusion, ID3 may be involved in the differentiation of Th17 and Tregs by downregulating Th17 cells, which is probably associated with binding to E2A. The present results suggested that ID3 may offer protection against anti‑GBM GN in mice.

The role of Id3 in regulating NKT cells of the adipose tissue

Abstract Disorders involving obesity, such as diabetes, heart disease, and metabolic syndrome have become a predominant health issues in the United States and around the world. According to the CDC 36% of adults in the US qualify as obese, leading to a variety of negative health outcomes and billions of dollars in medical costs. One of the hallmarks of obesity is inflammation of the adipose tissue mediated by a combination of molecules such as hormones and cytokines produced by immune cells. Natural killer T (NKT) cells are a heterogeneous population of innate lymphocytes, defined in part, by their ability to quickly release cytokines after activation. NKT cells have been implicated in the maintenance of healthy adipose tissue, and loss of NKT cells in obesity has been correlated with the increased inflammation, glucose and insulin intolerance. As a heterogeneous population, the exact manner by which adipose resident NKT cells modulate the adipose tissue is contentious. We hope to further elucidate NKT cells’ role in adipose tissue, after activation. We also hope to define the molecules responsible for mediating NKT cells’ activity in a healthy adipose environment, with the goal of modulating these cells to improve health outcomes in the disease state. Our first molecule of interest is the inhibitor of DNA binding protein, ID3, which has been previously implicated in NKT cell development in the thymus. Here we use both a GFP reporter and a conditionally deficient line to determine the role that Id3 plays in NKT cell activation in healthy adipose tissue.

Introduction of ID2 Enhances Invasiveness in ID2-null Oral Squamous Cell Carcinoma Cells via the SNAIL Axis.

Inhibitor of DNA-binding (ID) proteins are negative regulators of basic helix-loop-helix transcription factors that generally stimulate cell proliferation and inhibit differentiation. However, the role of ID2 in cancer progression remains ambiguous. Here, we investigated the function of ID2 in ID2-null oral squamous cell carcinoma (OSCC) cells. We introduced an ID2 cDNA construct into ID2-null OSCC cells and compared them with empty-vector-transfected cells in terms of cell proliferation, invasion, and activity and expression of matrix metalloproteinase (MMP). ID2 introduction resulted in enhanced malignant phenotypes. The ID2-expressing cells showed increased N-cadherin, vimentin, and E-cadherin expression and epithelial-mesenchymal transition. In addition, cell invasion drastically increased with increased expression and activity of MMP2. Immunoprecipitation revealed a direct interaction between ID2 and zinc finger transcription factor, snail family transcriptional repressor 1 (SNAIL1). ID2 expression triggered a malignant phenotype, especially of invasive properties, through the ID2-SNAIL axis. Thus, ID2 represents a potential therapeutic target for OSCC.

Silencing of Id2 Alleviates Chronic Neuropathic Pain Following Chronic Constriction Injury.

Inhibitor of DNA binding/differentiation 2 (Id2) belongs to a helix-loop-helix family of proteins. Recent studies have showed that Id2 plays a pivotal role in neuronal survival and neuroprotection. However, under neuropathic pain conditions, the role of Id2 is still unclear. In this study, we investigated the effect of Id2 on neuropathic pain in a rat chronic constriction injury (CCI) model. Our results demonstrated that Id2 was upregulated in the dorsal root ganglion (DRG) in a CCI rat in a time-dependent manner. Intrathecal short-hairpin RNA (shRNA)-Id2 attenuates mechanical allodynia and thermal hyperalgesia in CCI rats, and inhibits the expression of TNF-α and IL-1β in the DRG in CCI rats. Furthermore, knockdown of Id2 reduces the expression of NF-κB p65 in the DRG of CCI rats. Taken together, our findings suggest that knockdown of Id2 may alleviate neuropathic pain by inhibiting the NF-κB activation to inhibit the production of pro-inflammatory mediators. Therefore, Id2 may provide an important target of neuropathic pain treatment.

The Helix-Loop-Helix Protein ID2 Governs NK Cell Fate by Tuning Their Sensitivity to Interleukin-15

The inhibitor of DNA binding 2 (Id2) is essential for natural killer (NK) cell development with its canonical role being to antagonize E-protein function and alternate lineage fate. Here we have identified a key role for Id2 in regulating interleukin-15 (IL-15) receptor signaling and homeostasis of NK cells by repressing multiple E-protein target genes including Socs3. Id2 deletion in mature NK cells was incompatible with their homeostasis due to impaired IL-15 receptor signaling and metabolic function and this could be rescued by strong IL-15 receptor stimulation or genetic ablation of Socs3. During NK cell maturation, we observed an inverse correlation between E-protein target genes and Id2. These results shift the current paradigm on the role of ID2, indicating that it is required not only to antagonize E-proteins during NK cell commitment, but constantly required to titrate E-protein activity to regulate NK cell fitness and responsiveness to IL-15.

Abstract 3002: ID3 expression and oxidative DNA damage are associated with growth of benign and malignant vascular lesions

Abstract Higher levels of Id3 expression have been reported in tumor-infiltrating blood vessels from various tumors, but the exact role of Id3 in vascular lesion formation is poorly understood. This study aims at identifying the status of Id3 expression and distribution in benign vascular lesions and attempts to correlate this information with the progression of vascular lesion. In this study we investigated the expression pattern of Id3 in malignant, benign, and normal human vascular tissue specimens. Expression analysis of Id3 was carried out using immunohistochemical analysis in 48 sporadic samples of a heterogeneous group of benign and malignant vascular tissue. Immunohistochemical analysis confirmed that many of the malignant vascular lesions exhibited strong Id3 expression that correlated positively with increased 8-hydroxydeoxyguanosine (8-OHdG) a marker of oxidative DNA damage. In contrast, in normal human vascular tissue, Id3 expression and 8-OHdG was low. In malignant vascular tissue, Id3 protein displayed a stronger nuclear localization when compared to the cytoplasm. Id3 expression levels in benign vascular lesions while still expressing Id3 were to a lesser extent when compared to malignant vascular tissue. To our knowledge, this is the first study to compare benign and malignant vascular lesions to assess the role of Id3 and 8-OHdG. Our data reveals Id3 expression and oxidative DNA damage to be highly associated in malignant cells and to a lesser extent in their benign counterparts, indicating that the growth of vascular lesions may require oxidative stress signaling converging on Id3. The results also support the involvement of Id3 in tumor angiogenesis a process that critically influences the malignant behavior of cancer cells. Citation Format: Quentin H. Felty, Jayanta K. Das. ID3 expression and oxidative DNA damage are associated with growth of benign and malignant vascular lesions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3002. doi:10.1158/1538-7445.AM2014-3002

Abstract 1402: Regulation of Id2 In the proliferation of glioma-initiating neural progenitor cells

Abstract There are more than 14,000 new cases of malignant high grade glioma diagnosed each year in the US, and although surgical resection, radiation, and cytotoxic therapies are available, these current treatment methods are not curative. Inhibitor of DNA Binding 2 (Id2), a helix-loop-helix (HLH) protein, functions as a dominant negative inhibitor of basic helix-loop-helix (bHLH) transcription factors and is highly expressed in glioma. Recent data from our laboratory defines a critical role for Id2 in the pathogenesis of a subset of glioma known as proneural glioma. Id2 protein function has been characterized in neural progenitor cells (NPCs) where it acts to suppress differentiation and enhance cell cycle progression. These are two key characteristics that are shared with glioma-derived stem cells. We have identified three phosphorylation sites on Id2 in proliferating NPCs using mass spectrometry, and prepared altered Id2 molecules that cannot be phosphorylated at each of these sites. Id2-/- NPCs expressing WT and phosphorylation-site-ablated Id2 have been prepared to examine the role of Id2 phosphorylation in mediating its interactions with binding partners and in modulating the rate of proteasome-dependent degradation. We found an increased level of Id2 in NPCs expressing a mutant form of Id2 that cannot by phosphorylated when compared with NPCs expressing WT Id2. Moreover, the level of WT Id2 protein decreases when NPCs exit the cell cycle; however, unphosphorylated Id2 resists this degradation and exhibits a longer half-life. Stable isotope labeling by amino acids in cell culture (SILAC) experiments revealed that unphosphorylated Id2 molecules bind significantly less than WT Id2 to several anaphase promoting complex (APC) subunits. These findings suggested a possible mechanism for phosphorylation-dependent proteasome-mediated degradation of Id2 in NPCs. Understanding the effect of Id2 phosphorylation in modulating steady state levels of this important protein will inform future studies designed to identify pathways of interest in the transformation of NPCs and the malignant progression of NPC-initiated glioma. This work was supported in part by the Theodora B. Betz Foundation. Citation Format: Jaclyn Sullivan, Matthew Havrda, Brenton Paolella, Arminja Kettenbach, Scott Gerber, Mark A. Israel. Regulation of Id2 In the proliferation of glioma-initiating neural progenitor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1402. doi:10.1158/1538-7445.AM2014-1402

Innate lymphoid cells protect host against intestinal pathogen through Id2 dependent IL-22 production and regulation of microbiota. (MUC4P.855)

Abstract Innate lymphoid cells (ILCs) represent a novel family of immune cells that can regulate immunity, inflammation, and tissue repair in multiple barrier surfaces. While Id2 is known to be required for the development of ILCs progenitor, Id2 remains highly expressed in differentiated ILCs with unknown function. Using newly generated conditional KO mice, we demonstrated a novel role for Id2 in RORγt+ ILC3s not only in controlling the further development and maintenance of ILC3s but also regulating IL-22 production by ILC3s through IL-23R - STAT3 pathway. Id2 deficiency in ILC3s increased morbidity and mortality after Citrobacter rodentium infection. Interestingly, the mice with Id2 deficiency in ILC3s exhibited much higher fecal C. rodentium CFU even within 24 hours post inoculation, suggesting that preexisting Id2-dependent gut microenvironment mediated the early colonization resistance against C. rodentium. Furthermore, the wild type germ free mice colonized with the microbiota from the conditional Id2 deficient mice by gavage of cecal material also had higher fecal C. rodentium CFU, compared with the germ free mice that received microbiota derived from wild type mice. Cohousing partially rescued conditional Id2 deficient mice from lethal infection. Together, our data suggest that continued Id2 expression is required for the development and function of ILC3s, and ILC3s protect host against intestinal pathogen through Id2 dependent regulation of microbiota.

Ablation of the Id2 Gene Results in Altered Circadian Feeding Behavior, and Sex-Specific Enhancement of Insulin Sensitivity and Elevated Glucose Uptake in Skeletal Muscle and Brown Adipose Tissue

Inhibitor of DNA binding 2 (ID2) is a helix-loop-helix transcriptional repressor rhythmically expressed in many adult tissues. Our earlier studies have demonstrated a role for ID2 in the input pathway, core clock function and output pathways of the mouse circadian system. We have also reported that Id2 null (Id2−/−) mice are lean with low gonadal white adipose tissue deposits and lower lipid content in the liver. These results coincided with altered or disrupted circadian expression profiles of liver genes including those involved in lipid metabolism. In the present phenotypic study we intended to decipher, on a sex-specific basis, the role of ID2 in glucose metabolism and in the circadian regulation of activity, important components of energy balance. We find that Id2−/− mice exhibited altered daily and circadian rhythms of feeding and locomotor activity; activity profiles extended further into the late night/dark phase of the 24-hr cycle, despite mice showing reduced total locomotor activity. Also, male Id2−/− mice consumed a greater amount of food relative to body mass, and displayed less weight gain. Id2−/− females had smaller adipocytes, suggesting sexual-dimorphic programing of adipogenesis. We observed increased glucose tolerance and insulin sensitivity in male Id2−/− mice, which was exacerbated in older animals. FDG-PET analysis revealed increased glucose uptake by skeletal muscle and brown adipose tissue of male Id2−/− mice, suggesting increased glucose metabolism and thermogenesis in these tissues. Reductions in intramuscular triacylglycerol and diacylglycerol were detected in male Id2−/− mice, highlighting its possible mechanistic role in enhanced insulin sensitivity in these mice. Our findings indicate a role for ID2 as a regulator of glucose and lipid metabolism, and in the circadian control of feeding/locomotor behavior; and contribute to the understanding of the development of obesity and diabetes, particularly in shift work personnel among whom incidence of such metabolic disorders is elevated.