Inhibitor Of DNA Binding Proteins Research Articles

Abstract 290: The role of tumor stromal topography-regulated genes in modulating the cellular landscape of tumor stroma in breast cancer

Abstract The tumor remodels the surrounding extracellular matrix fibers to become topographically aligned at the tumor front, further facilitating cancer cell migration. Our recent studies show that aligned tumor stromal topography promotes the detachment of breast cancer cells from the main tumor, then, in the following cell migration, upregulating genes such as genes encoding the cytochrome P450 family 1 subfamily A member 1 (CYP1) and inhibitor of DNA binding (ID) proteins. In this study, we further explored the role of topography-regulated genes in modulating the cellular landscape of tumor stroma. By analyzing the correlation of topography-regulated genes with stromal cell infiltration in bioinformatics databases, we found significant positive correlations between the RNA expression levels of most topography-regulated genes and the infiltration levels of cancer-associated fibroblasts and endothelial cells. Notably, the strongest correlation was between the expression level of ID3 and the infiltration levels of cancer-associated fibroblasts (r = 0.509) and endothelial cells (r = 0.506). Additionally, ID3 remained highly expressed in circulating tumor cells but exhibited low expression at distant metastatic sites. These findings suggest that genes regulated by tumor stromal topography, especially ID3, may play a role in neovascularization and fibrosis of the tumor microenvironment and the survival adaptation of cancer cells in circulation, thereby promoting metastasis. Citation Format: Chia-Yi Su. The role of tumor stromal topography-regulated genes in modulating the cellular landscape of tumor stroma in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 290.

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.

BMP9-ID1 Pathway Attenuates N6-Methyladenosine Levels of CyclinD1 to Promote Cell Proliferation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly lethal malignant neoplasm, and the involvement of bone morphogenetic protein 9 (BMP9) has been implicated in the pathogenesis of liver diseases and HCC. Our goal was to investigate the role of BMP9 signaling in regulating N6-methyladenosine (m6A) methylation and cell cycle progression, and evaluate the therapeutic potential of BMP receptor inhibitors for HCC treatment. We observed that elevated levels of BMP9 expression in tumor tissues or serum samples from HCC patients were associated with a poorer prognosis. Through in vitro experiments utilizing the m6A dot blotting assay, we ascertained that BMP9 reduced the global RNA m6A methylation level in Huh7 and Hep3B cells, thereby facilitating their cell cycle progression. This effect was mediated by an increase in the expression of the inhibitor of DNA-binding protein 1 (ID1). Additionally, using methylated RNA immunoprecipitation qPCR(MeRIP-qPCR), we showed that the BMP9-ID1 pathway promoted CyclinD1 expression by decreasing the m6A methylation level in the 5' UTR of mRNA. This occurred through the upregulation of the fat mass and obesity-associated protein (FTO) in Huh7 and Hep3B cells. In our in vivo mouse xenograft models, we demonstrated that blocking the BMP receptor with LDN-212854 effectively suppressed HCC growth and induced global RNA m6A methylation. Overall, our findings indicate that the BMP9-ID1 pathway promotes HCC cell proliferation by down-regulating the m6A methylation level in the 5' UTR of CyclinD1 mRNA. Targeting the BMP9-ID1 pathway holds promise as a potential therapeutic strategy for treating HCC.

ID2 Promotes Leukemogenesis and Chemoresistance of Acute Myeloid Leukemia By Modulating Cellular Oxidative Metabolism

Acute myeloid leukemia (AML) relies on quiescent leukemia stem cells (LSCs) to evade chemotherapy and drive disease relapse. Recent research indicates that a specific subset of LSCs, characterized by elevated oxidative phosphorylation and/or increased senescence, is responsible for chemotherapy resistance and relapse. While numerous genes associated with LSC maintenance and resistance have been identified, the underlying molecular mechanisms remain elusive. Inhibitor of DNA binding (ID) proteins, a group of Helix-Loop-Helix (HLH) transcription regulators, play a dominant role in regulating other HLH proteins (E proteins) required for the normal development of hematopoietic, muscle, neuronal, and other cells. Our previous investigation revealed that ID2 protects quiescent hematopoietic stem cells (HSCs) activation and exhaustion. Analysis of TCGA data sets demonstrated that high levels of ID2 expression are linked to reduced overall survival in AML patients, suggesting ID2 may be required to maintain some LSCs. Consistent with murine cell findings, we found that knocking down (KD) ID2 expression in human cord blood cells resulted in a loss of CD34+CD38- progenitors. Similarly, ID2-KD in AML cell lines (UT-7, MB-02, and M-07e) significantly reduced their leukemogenic potential in NSG mice, indicating that ID2 is essential for AML cell growth in vivo. Conversely, overexpressing ID2 in the human AML cell line M-07e enhanced their leukemic potential and burden in NSG mice, supporting the notion that ID2 may preserve LSC quiescence. Experiments with primary AML-Patient Derived Xenograft (AML-PDX) cells, which are more clinically relevant, also showed reduced leukemic potential in transplanted NSG mice upon ID2 knockdown. Additionally, ID2-KD cells displayed significantly lower levels of reactive oxygen species (ROS) and mitochondrial activity compared to control cells in both AML cell lines and PDX cells. As ROS-low cells are more susceptible to cytarabine (AraC), we investigated whether ID2-KD renders AML cells more susceptible to AraC. Indeed, MB-02-ID2-KD and UT7-ID2-KD cells showed increased sensitivity to AraC, while M-07e cells overexpressing ID2 exhibited resistance to AraC. Furthermore, AraC effectively eliminated ROS-low cells, but ROS-high cells remained resistant, supporting previous findings. To further investigate the effect of ID2-KD in AraC resistance in vivo, UT7-ID-KD and MB-02-ID2-KD transplanted NSG mice were treated with AraC and monitored for their overall survival, and it was observed that ID2-KD transplanted mice have significantly higher overall survival compared to the control cell transplanted mice. In conclusion, it can be said that ID2 plays a critical role in maintaining normal murine and human HSCs, and it likely contributes to leukemia development and chemotherapy resistance. Therefore, targeting ID2 may offer a promising therapeutic approach to selectively tackle LSCs, followed by chemotherapy and bone marrow transplantation to achieve complete remission.

Reconstruction of the regulatory hypermethylation network controlling hepatocellular carcinoma development during hepatitis C viral infection.

Hepatocellular carcinoma (HCC) has been associated with hepatitis C viral (HCV) infection as a potential risk factor. Nonetheless, the precise genetic regulatory mechanisms triggered by the virus, leading to virus-induced hepatocarcinogenesis, remain unclear. We hypothesized that HCV proteins might modulate the activity of aberrantly methylated HCC genes through regulatory pathways. Virus-host regulatory pathways, interactions between proteins, gene expression, transport, and stability regulation, were reconstructed using the ANDSystem. Gene expression regulation was statistically significant. Gene network analysis identified four out of 70 HCC marker genes whose expression regulation by viral proteins may be associated with HCC: DNA-binding protein inhibitor ID - 1 (ID1), flap endonuclease 1 (FEN1), cyclin-dependent kinase inhibitor 2A (CDKN2A), and telomerase reverse transcriptase (TERT). It suggested the following viral protein effects in HCV/human protein heterocomplexes: HCV NS3(p70) protein activates human STAT3 and NOTC1; NS2-3(p23), NS5B(p68), NS1(E2), and core(p21) activate SETD2; NS5A inhibits SMYD3; and NS3 inhibits CCN2. Interestingly, NS3 and E1(gp32) activate c-Jun when it positively regulates CDKN2A and inhibit it when it represses TERT. The discovered regulatory mechanisms might be key areas of focus for creating medications and preventative therapies to decrease the likelihood of HCC development during HCV infection.

G protein-coupled receptor 17 is regulated by WNT pathway during oligodendrocyte precursor cell differentiation

G protein-coupled receptor 17 (GPR17) and the WNT pathway are critical players of oligodendrocyte (OL) differentiation acting as essential timers in developing brain to achieve fully-myelinating cells. However, whether and how these two systems are related to each other is still unknown. Of interest, both factors are dysregulated in developing and adult brain diseases, including white matter injury and cancer, making the understanding of their reciprocal interactions of potential importance for identifying new targets and strategies for myelin repair. Here, by a combined pharmacological and biotechnological approach, we examined regulatory mechanisms linking WNT signaling to GPR17 expression in OLs. We first analyzed the relative expression of mRNAs encoding for GPR17 and the T cell factor/Lymphoid enhancer-binding factor-1 (TCF/LEF) transcription factors of the canonical WNT/β-CATENIN pathway, in PDGFRα+ and O4+ OLs during mouse post-natal development. In O4+ cells, Gpr17 mRNA level peaked at post-natal day 14 and then decreased concomitantly to the physiological uprise of WNT tone, as shown by increased Lef1 mRNA level. The link between WNT signaling and GPR17 expression was further reinforced in vitro in primary PDGFRα+ cells and in Oli-neu cells. High WNT tone impaired OL differentiation and drastically reduced GPR17 mRNA and protein levels. In Oli-neu cells, WNT/β-CATENIN activation repressed Gpr17 promoter activity through both putative WNT response elements (WRE) and upregulation of the inhibitor of DNA-binding protein 2 (Id2). We conclude that the WNT pathway influences OL maturation by repressing GPR17, which could have implications in pathologies characterized by dysregulations of the OL lineage including multiple sclerosis and oligodendroglioma.

Inhibitor of DNA Binding Protein 3 (ID3) and Nuclear Respiratory Factor 1 (NRF1) Mediated Transcriptional Gene Signatures are Associated with the Severity of Cerebral Amyloid Angiopathy.

Cerebral amyloid angiopathy (CAA) is a degenerative vasculopathy. We have previously shown that transcription regulating proteins- inhibitor of DNA binding protein 3 (ID3) and the nuclear respiratory factor 1 (NRF1) contribute to vascular dysregulation. In this study, we have identified sex specific ID3 and NRF1-mediated gene networks in CAA patients diagnosed with Alzheimer's Disease (AD). High expression of ID3 mRNA coupled with low NRF1 mRNA levels was observed in the temporal cortex of men and women CAA patients. Low NRF1 mRNA expression in the temporal cortex was found in men with severe CAA. High ID3 expression was found in women with the genetic risk factor APOE4. Low NRF1 expression was also associated with APOE4 in women with CAA. Genome wide transcriptional activity of both ID3 and NRF1 paralleled their mRNA expression levels. Sex specific differences in transcriptional gene signatures of both ID3 and NRF1 were observed. These findings were further corroborated by Bayesian machine learning and the GeNIe simulation models. Dynamic machine learning using a Monte Carlo Markov Chain (MCMC) gene ordering approach revealed that ID3 was associated with disease severity in women. NRF1 was associated with CAA and severity of this disease in men. These findings suggest that aberrant ID3 and NRF1 activity presumably plays a major role in the pathogenesis and severity of CAA. Further analyses of ID3- and NRF1-regulated molecular drivers of CAA may provide new targets for personalized medicine and/or prevention strategies against CAA.

Potential anti-obesity/inflammatory flavonoid-derived biomolecules against Obesity to prevent WAT differentiation by targeting a DNA-binding protein inhibitor, ID-1.

A concoction of unhealthy eating, inactivity, and the adverse effects of specific drugs brings on obesity. The primary cause of Obesity is the storage of too much energy and triglycerides in adipocytes, particularly white adipose tissue (WAT). In addition to modifying one's lifestyle, anti-obesity medicines are increasingly used as adjuvant therapy. Flavonoids are the major class of compounds having significant biological impacts and health-improving properties. To find novel flavonoid compounds that fight obesity using computational drug design techniques. This work targets 1DI protein to predict new flavonoid compounds that fight obesity. The study uses computational approaches to anticipate potential anti-obesity/inflammatory flavonoid compounds against obesity to prevent WAT differentiation by targeting ID-1, a DNA-binding protein inhibitor. Our study led to the identification of the protein target inhibitor lead CID: 5280443, which was found to be a potent inhibitor of the receptor. According to the findings of this study, this bio-active molecule may be used as a lead for the development of drugs that preferentially fight obesity without interfering with the functions of the human proteasome. The scientific community will benefit from these discoveries, which could aid in the creation of new medications that treat obesity more successfully.

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.

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.

ID3 mediates BMP2-induced downregulation of ICAM1 expression in human endometiral stromal cells and decidual cells.

Recurrent pregnancy loss (RPL) remains an unsolved problem in obstetrics and gynecology, and up to 50% of RPL cases are unexplained. Unexplained RPL (uRPL) is widely considered to be related to an aberrant endometrial microenvironment. BMP2 is an important factor involved in endometrial decidualization and embryo implantation, and intercellular adhesion molecule 1 (ICAM1) is a critical inflammatory regulator in the endometrium. In this study, we found that endometrial samples obtained from Unexplained RPL patients have significantly lower BMP2 and higher ICAM1 levels than fertile controls. For further research on the relationship between BMP2 and ICAM1 and the potential molecular mechanisms in Unexplained RPL, immortalized human endometrial stromal cells (HESCs) and primary human decidual stromal cells (HDSCs) were used as study models. Our results showed that BMP2 significantly decreased ICAM1 expression by upregulating DNA-binding protein inhibitor 3 (ID3) in both HESCs and HDSCs. Using kinase receptor inhibitors (dorsomorphin homolog 1 (DMH-1) and dorsomorphin) and siRNA transfection, it has been found that the upregulation of ID3 and the following downregulation of ICAM1 induced by BMP2 is regulated through the ALK3-SMAD4 signaling pathway. This research gives a hint of a novel mechanism by which BMP2 regulates ICAM1 in the human endometrium, which provides insights into potential therapeutics for unexplained RPL.

Insulin Elevates ID2 Expression in Trophoblasts and Aggravates Preeclampsia in Obese ASB4-Null Mice.

Obesity is a risk factor for preeclampsia. We investigated how obesity influences preeclampsia in mice lacking ankyrin-repeat-and-SOCS-box-containing-protein 4 (ASB4), which promotes trophoblast differentiation via degrading the inhibitor of DNA-binding protein 2 (ID2). Asb4-/- mice on normal chow (NC) develop mild preeclampsia-like phenotypes during pregnancy, including hypertension, proteinuria, and reduced litter size. Wild-type (WT) and Asb4-/- females were placed on a high-fat diet (HFD) starting at weaning. At the age of 8-9 weeks, they were mated with WT or Asb4-/- males, and preeclamptic phenotypes were assessed. HFD-WT dams had no obvious adverse outcomes of pregnancy. In contrast, HFD-Asb4-/- dams had significantly more severe preeclampsia-like phenotypes compared to NC-Asb4-/- dams. The HFD increased white fat weights and plasma leptin and insulin levels in Asb4-/- females. In the HFD-Asb4-/- placenta, ID2 amounts doubled without changing the transcript levels, indicating that insulin likely increases ID2 at a level of post-transcription. In human first-trimester trophoblast HTR8/SVneo cells, exposure to insulin, but not to leptin, led to a significant increase in ID2. HFD-induced obesity markedly worsens the preeclampsia-like phenotypes in the absence of ASB4. Our data indicate that hyperinsulinemia perturbs the timely removal of ID2 and interferes with proper trophoblast differentiation, contributing to enhanced preeclampsia.

Exploring pradimicin-IRD antineoplastic mechanisms and related DNA repair pathways

DNA-targeting agents have a significant clinical use, although toxicity remains an issue that plays against their widespread application. Understanding the mechanism of action and DNA damage response elicited by such compounds might contribute to the improvement of their use in anticancer chemotherapy. In a previous study, our research group characterized a new DNA-targeting agent – pradimicin-IRD. Since DNA-targeting agents and DNA repair are close-related subjects, the present study used in silico-modelling and a transcriptomic approach seeking to characterize the DNA repair pathways activated in HCT 116 cells following pradimicin-IRD treatment. Molecular docking analysis showed pradimicin-IRD as a DNA intercalating agent and a potential inhibitor of DNA-binding proteins. Furthermore, the transcriptomic study highlighted DNA repair functions related to genes modulated by pradimicin-IRD, such as nucleotide excision repair, telomeres maintenance and double-strand break repair. When validating these functions, PCNA protein levels decreased after exposure to pradimicin. Furthermore, molecular docking analysis suggested DNA-pradimicin-PCNA interaction. In addition, hTERT and POLH showed reduced mRNA levels after 6 h of treatment with pradimicin-IRD. Moreover, POLH-deficient cells displayed higher resistance to pradimicin-IRD than POLH-proficient cells and the compound prevented formation of the POLH/DNA complex (molecular docking). Since the modulation of DNA repair genes by pradimicin-IRD is TP53-independent, unlike doxorubicin, dissimilarities between the mechanism of action and the DNA damage response of pradimicin-IRD and doxorubicin open new insights for further studies of pradimicin-IRD as a new antineoplastic compound.

Chemoproteomic profiling to identify activity changes and functional inhibitors of DNA-binding proteins

DNA-binding proteins are promising therapeutic targets but are notoriously difficult to drug. Here, we evaluate a chemoproteomic DNA interaction platform as a complementary strategy for parallelized compound profiling. To enable this approach, we determined the proteomic binding landscape of 92 immobilized DNA sequences. Perturbation-induced activity changes of captured transcription factors in disease-relevant settings demonstrated functional relevance of the enriched subproteome. Chemoproteomic profiling of >300 cysteine-directed compounds against a coverage optimized bead mixture, which specifically captures >150 DNA binders, revealed competition of several DNA-binding proteins, including the transcription factors ELF1 and ELF2. We also discovered the first compound that displaces the DNA-repair complex MSH2-MSH3 from DNA. Compound binding to cysteine 252 on MSH3 was confirmed using chemoproteomic reactive cysteine profiling. Overall, these results suggested that chemoproteomic DNA bead pull-downs enable the specific readout of transcription factor activity and can identify functional "hotspots" on DNA binders toward expanding the druggable proteome.

Inhibitor of DNA binding proteins revealed as orchestrators of steady state, stress and malignant hematopoiesis.

Adult mammalian hematopoiesis is a dynamic cellular process that provides a continuous supply of myeloid, lymphoid, erythroid/megakaryocyte cells for host survival. This process is sustained by regulating hematopoietic stem cells (HSCs) quiescence, proliferation and activation under homeostasis and stress, and regulating the proliferation and differentiation of downstream multipotent progenitor (MPP) and more committed progenitor cells. Inhibitor of DNA binding (ID) proteins are small helix-loop-helix (HLH) proteins that lack a basic (b) DNA binding domain present in other family members, and function as dominant-negative regulators of other bHLH proteins (E proteins) by inhibiting their transcriptional activity. ID proteins are required for normal T cell, B cell, NK and innate lymphoid cells, dendritic cell, and myeloid cell differentiation and development. However, recent evidence suggests that ID proteins are important regulators of normal and leukemic hematopoietic stem and progenitor cells (HSPCs). This chapter will review our current understanding of the function of ID proteins in HSPC development and highlight future areas of scientific investigation.

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.

Signaling networks controlling ID and E protein activity in T cell differentiation and function.

E and inhibitor of DNA binding (ID) proteins are involved in various cellular developmental processes and effector activities in T cells. Recent findings indicate that E and ID proteins are not only responsible for regulating thymic T cell development but also modulate the differentiation, function, and fate of peripheral T cells in multiple immune compartments. Based on the well-established E and ID protein axis (E-ID axis), it has been recognized that ID proteins interfere with the dimerization of E proteins, thus restricting their transcriptional activities. Given this close molecular relationship, the extent of expression or stability of these two protein families can dynamically affect the expression of specific target genes involved in multiple aspects of T cell biology. Therefore, it is essential to understand the endogenous proteins or extrinsic signaling pathways that can influence the dynamics of the E-ID axis in a cell-specific and context-dependent manner. Here, we provide an overview of E and ID proteins and the functional outcomes of the E-ID axis in the activation and function of multiple peripheral T cell subsets, including effector and memory T cell populations. Further, we review the mechanisms by which endogenous proteins and signaling pathways alter the E-ID axis in various T cell subsets influencing T cell function and fate at steady-state and in pathological settings. A comprehensive understanding of the functions of E and ID proteins in T cell biology can be instrumental in T cell-specific targeting of the E-ID axis to develop novel therapeutic modalities in the context of autoimmunity and cancer.

GDF-11 promotes human trophoblast cell invasion by increasing ID2-mediated MMP2 expression

BackgroundGrowth differentiation factor-11 (GDF-11), also known as bone morphogenetic protein-11, belongs to the transforming growth factor-beta superfamily. GDF-11 was first identified as an important regulator during embryonic development. Increasing evidence has demonstrated that GDF-11 regulates the development of various organs and its aberrant expressions are associated with the risk of cardiovascular diseases and cancers. Extravillous trophoblast (EVT) cells invasion is a critical event for placenta development and needs to be finely regulated. However, to date, the biological function of GDF-11 in the human EVT cells remains unknown.MethodsHTR-8/SVneo, a human EVT cell line, and primary cultures of human EVT cells were used to examine the effect of GDF-11 on matrix metalloproteinase 2 (MMP2) expression. Matrigel-coated transwell invasion assay was used to examine cell invasiveness. A series of in vitro experiments were applied to explore the underlying mechanisms that mediate the effect of GDF-11 on MMP2 expression and cell invasion.ResultsTreatment with GDF-11 stimulates MMP2 expression, in the HTR-8/SVneo and primary human EVT cells. Using a pharmacological inhibitor and siRNA-mediated knockdown approaches, our results demonstrated that the stimulatory effect of GDF-11 on MMP2 expression was mediated by the ALK4/5-SMAD2/3 signaling pathways. In addition, the expression of inhibitor of DNA-binding protein 2 (ID2) was upregulated by GDF-11 and that was required for the GDF-11-stimulated MMP2 expression and EVT cell invasion.ConclusionsThese findings discover a new biological function and underlying molecular mechanisms of GDF-11 in the regulation of human EVT cell invasion.Ede8tEoyGya1QPPVVUV7JYVideo

Expression Profiles of ID and E2A in Ovarian Cancer and Suppression of Ovarian Cancer by the E2A Isoform E47.

Simple SummaryOvarian cancer is one of the most fatal gynecological malignancies in women. Even though primary treatment might result in complete remission, approximately 60–80% patients with advanced-stage ovarian cancer experience a relapse. To improve their survival outcomes, several novel agents have been proposed; however, the majority of these have demonstrated limited efficacy in the treatment of ovarian cancer. Interestingly, an imbalance in the basic helix-loop-helix inhibitor of DNA binding (bHLH/ID) protein networks has been associated with oncogenesis, but the exact expression patterns of bHLH/ID in ovarian cancer are not yet known. We hypothesized that such an imbalance in bHLH/ID activity may be integral to ovarian cancer pathogenesis, and modulating the bHLH/ID balance might be a promising approach to treat ovarian cancer.The E2A and inhibitor of DNA binding (ID) proteins are transcription factors involved in cell cycle regulation and cellular differentiation. Imbalance of ID/E2A activity is associated with oncogenesis in various tumors, but their expression patterns and prognostic values are still unknown. We evaluated ID and E2A expression in ovarian cancer cells, and assessed the possibility of reprogramming ovarian cellular homeostasis by restoring the ID/E2A axis. We analyzed copy number alterations, mutations, methylations, and mRNA expressions of ID 1–4 and E2A using The Cancer Genome Atlas data of 570 ovarian serous cystadenocarcinoma patients. Incidentally, 97.2% cases exhibited gain of ID 1–4 or loss of E2A. Predominantly, ID 1–4 were hypomethylated, while E2A was hypermethylated. Immunohistochemical analysis revealed that ID-3 and ID-4 expressions were high while E2A expression was low in cancerous ovarian tissues. Correlation analysis of ID and E2A levels with survival outcomes of ovarian cancer patients indicated that patients with high ID-3 levels had poor overall survival. We also determined the effect of E2A induction on ovarian cancer cell growth in vitro and in vivo using SKOV-3/Luc cells transduced with tamoxifen-inducible E47, a splice variant of E2A. Interestingly, E47 induced SKOV-3 cell death in vitro and inhibited tumor growth in SKOV-3 implanted mice. Therefore, restoring ID/E2A balance is a promising approach for treating ovarian cancer.

A novel antiproliferative PKCα-Ras-ERK signaling axis in intestinal epithelial cells

We have previously shown that the serine/threonine kinase PKCα triggers MAPK/ERK kinase (MEK)-dependent G1→S cell cycle arrest in intestinal epithelial cells, characterized by downregulation of cyclin D1 and inhibitor of DNA-binding protein 1 (Id1) and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Here, we use pharmacological inhibitors, genetic approaches, siRNA-mediated knockdown, and immunoprecipitation to further characterize antiproliferative ERK signaling in intestinal cells. We show that PKCα signaling intersects the Ras-Raf-MEK-ERK kinase cascade at the level of Ras small GTPases and that antiproliferative effects of PKCα require active Ras, Raf, MEK, and ERK, core ERK pathway components that are also essential for pro-proliferative ERK signaling induced by epidermal growth factor (EGF). However, PKCα-induced antiproliferative signaling differs from EGF signaling in that it is independent of the Ras guanine nucleotide exchange factors (Ras-GEFs), SOS1/2, and involves prolonged rather than transient ERK activation. PKCα forms complexes with A-Raf, B-Raf, and C-Raf that dissociate upon pathway activation, and all three Raf isoforms can mediate PKCα-induced antiproliferative effects. At least two PKCα–ERK pathways that collaborate to promote growth arrest were identified: one pathway requiring the Ras-GEF, RasGRP3, and H-Ras, leads to p21Cip1 upregulation, while additional pathway(s) mediate PKCα-induced cyclin D1 and Id1 downregulation. PKCα also induces ERK-dependent SOS1 phosphorylation, indicating possible negative crosstalk between antiproliferative and growth-promoting ERK signaling. Importantly, the spatiotemporal activation of PKCα and ERK in the intestinal epithelium in vivo supports the physiological relevance of these pathways and highlights the importance of antiproliferative ERK signaling to tissue homeostasis in the intestine.