Id1 Knockdown Research Articles

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.

Identification of ID1 and miR-150 interaction and effects on proliferation and apoptosis in ovine granulosa cells

Granulosa cells (GCs) proliferation and apoptosis play a significantly role in follicular development and atresia. ID1 and miR-150 are involved in cell apoptosis and follicular atresia, but the interaction and function of ID1 and miR-150 in GCs are unclear. This study focuses on ID1 and miR-150 in terms of the interaction and effects on proliferation and apoptosis in ovine granulosa cells. Our findings revealed that ID1 decreased the promoter activity and expression level of oar-miR-150. However, the expression of ID1 was downregulated by miR-150, and ID1 was identified as a target gene of oar-miR-150. miR-150 mimic inhibited proliferation and upregulated apoptosis rate in ovine GCs, while the results of miR-150 inhibitor were opposite. Overexpression of ID1 significantly inhibited ovine GCs proliferation and cell cycle-related genes (CDK1, CDK2, CDK4, CCND2, CDC20, and PCNA) expression, whereas knockdown of ID1 promoted cell proliferation and those genes expression. Overexpression of ID1 significantly downregulated mitochondrial membrane potential and Bcl-2 expression in ovine GCs, and upregulated the expression of pro-apoptosis genes Bax, Caspase-3, and Caspase-9, whereas the results of ID1 knockdown were reversed. Collectively, these findings indicate the interaction and the vital role of ID1 and miR-150 on proliferation and apoptosis in ovine granulosa cells, which may suggest a novel target for ovine follicular development and atresia.

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.

CSIG-09. THERAPEUTIC TARGETING OF PRENATAL PONTINE ID1 SIGNALING IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis. The mechanism behind tumor invasion is currently not well understood. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted. Whole exome, RNA, and ChIP-sequencing were performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Additional in vitro experiments were performed to determine a potential mechanism of action for CBD-mediated effects. Self-reported CBD dosing information was collected from DMG patients. We found that increased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates a similar epigenetically active state at ID1 regulatory regions in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell migration, tumor growth, and to a lesser extent invasion in both murine IUE and multiple patient-derived in vivo DMG models, improving mouse survival. ID1 knockdown significantly decreases the effect of CBD on migration, tumor growth, and invasion. CBD increases reactive oxygen species production, which also affects DMG cell proliferation in a non-ID1 mediated manner. Overall, we find that H3K27M-mediated reactivation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD.

Discovery of a Novel Driver of Radioresistance in Pancreatic Cancer (PC) Using Genome-Wide RNA-Seq

<h3>Purpose/Objective(s)</h3> Discovery of genetic drivers of radioresistance in PC is critical for developing novel therapeutic strategies to combine with radiation in this radioresistant disease. In this study, we used genome-wide RNA-seq to identify genes upregulated in radioresistant (RR) generated PC cell lines and discovered the Inhibitor of DNA Binding 1 (ID1) gene as a promising radiosensitizing target. <h3>Materials/Methods</h3> RR PC cell lines MIA PaCa-2 and PANC-1 were generated by delivering daily ionizing irradiation (IR) (2 Gy/day) <i>in vitro</i> over two weeks (total 20 Gy) followed by standard clonogenic assays to calculate the dose enhancement ratio (DER) following one week from the end of IR. Parental and RR cell lines were submitted for RNA-seq analysis to identify differentially expressed genes. The Limma R package was used to calculate differential expression among genes. Log2 fold change values were calculated for each sample compared to control. Genes with an absolute fold change > 1 were considered significant. Knockdown of target genes via small interfering RNA (siRNA) was confirmed through immunoblotting. Luciferase expressing RR PANC-1 and MIA PaCa-2 with or without shRNA stable knockdown of ID1 were implanted orthotopically in the pancreas of athymic nude mice, and tumors were treated with external beam radiation using a Small Animal Radiation Research Platform to a dose of 20 Gy in 5 daily fractions. Tumor growth was measured weekly via bioluminescence. <h3>Results</h3> Following exposure to two weeks of 2 Gy daily IR <i>in vitro</i>, the two PC cell lines showed significantly greater clonogenic cell survival than parental cell lines (DER 0.4 and 0.5), indicating enhanced RR in these cells. RNA-seq analysis comparing parental and RR cell lines found upregulated seven genes (TNS4, ZDHHC8P1, APLNR, AQP3, SPP1, ID1, ID2) and seven genes downregulated (PTX3, ITGB2, EPS8L1, ALDH1L2, KCNT2, ARHGAP9, IFI16) in both RR cell lines. Western blotting confirmed either increased or decreased expression of these 14 genes in the RR cell lines compared to their parental. siRNA knockdown of each of the seven most significant upregulated genes in the RR MIA PaCa-2 and PANC-1 cells revealed ID1 as the most potent radiosensitizer in the RR cell lines (DER 1.5 and 1.6, respectively). ID1 protein expression was higher in 6 different human PC cell lines than both non-malignant pancreatic and intestinal cell lines. Orthotopic RR tumors with stable knockdown of ID1 showed significantly decreased tumor growth following <i>in vivo IR</i> than the wild-type ID1 tumors (2.5-fold decrease, p<0.001). After the last dose of IR, expression of the DNA damage marker yH2aX in tumors was significantly higher in the ID1 knockdown tumors compared to controls indicating reduced DNA repair in tumors deficient in ID1. <h3>Conclusion</h3> Our analysis indicates a novel role of ID1 in PC radioresistance. Targeting ID1 prior to IR is a promising strategy to overcome the RR of PC, and pre-clinical studies using ID1 inhibitors are ongoing.

Inhibitor of DNA binding 2 knockdown inhibits the growth and liver metastasis of colorectal cancer

BackgroundLiver metastasis of colorectal cancer (CRC) remains high mortality and the mechanism is still unknown. Here we investigated the effects of inhibitor of DNA binding 2 (Id2) on growth and liver metastasis of CRC. MethodsqPCR and western blotting were used to demonstrate mRNA and protein expressions in Id2-knockdown HCT116 cells. Cell growth was observed by cell proliferation assay, colony formation assay and flow cytometry. Cell migration and invasion were observed with wound healing assay and transwell migration and invasion assay. The effects of Id2 knockdown on tumor growth and liver metastasis in vivo were evaluated respectively with subcutaneous tumor model and colorectal liver metastasis model by injecting HCT116 cells into the mesentery triangle of cecum in mice. ResultsId2 overexpression was found in CRC cell lines. Id2 knockdown resulted in a reduction in the proliferation, colony formation, migration and invasion of HCT116 cells. The suppression of cell proliferation was accompanied by the cell cycle arrest in the G0/G1 phase with down-regulation of Cyclin D1, Cyclin E, p-Cdk2/3, Cdk6, p-p27 and up-regulation of p21 and p27. Id2 knockdown reversed epithelial-mesenchymal transition (EMT) through increasing E-Cadherin and inhibiting N-Cadherin, Vimentin, β-catenin, Snail and Slug. Id2 was also found to inhibit CRC metastasis via MMP2, MMP9 and TIMP-1. Furthermore, Id2 knockdown suppressed CRC liver metastasis in vivo. ConclusionId2 promotes CRC growth through activation of the PI3K/AKT signaling pathway, and triggers EMT to enhance CRC migration and invasion.

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

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

ID1 and ID3 are Negative Regulators of TGFβ2-Induced Ocular Hypertension and Compromised Aqueous Humor Outflow Facility in Mice.

PurposeIn POAG, elevated IOP remains the major risk factor in irreversible vision loss. Increased TGFβ2 expression in POAG aqueous humor and in the trabecular meshwork (TM) amplifies extracellular matrix (ECM) deposition and reduces ECM turnover in the TM, leading to a decreased aqueous humor (AH) outflow facility and increased IOP. Inhibitor of DNA binding proteins (ID1 and ID3) inhibit TGFβ2-induced fibronectin and PAI-1 production in TM cells. We examined the effects of ID1 and ID3 gene expression on TGFβ2-induced ocular hypertension and decreased AH outflow facility in living mouse eyes.MethodsIOP and AH outflow facility changes were determined using a mouse model of Ad5-hTGFβ2C226S/C288S-induced ocular hypertension. The physiological function of ID1 and ID3 genes were evaluated using Ad5 viral vectors to enhance or knockdown ID1/ID3 gene expression in the TM of BALB/cJ mice. IOP was measured in conscious mice using a Tonolab impact tonometer. AH outflow facilities were determined by constant flow infusion in live mice.ResultsOver-expressing ID1 and ID3 significantly blocked TGFβ2-induced ocular hypertension (P < 0.0001). Although AH outflow facility was significantly decreased in TGFβ2-transduced eyes (P < 0.04), normal outflow facility was preserved in eyes injected concurrently with ID1 or ID3 along with TGFβ2. Knockdown of ID1 or ID3 expression exacerbated TGFβ2-induced ocular hypertension.ConclusionsIncreased expression of ID1 and ID3 suppressed both TGFβ2-elevated IOP and decreased AH outflow facility. ID1 and/or ID3 proteins thus may show promise as future candidates as IOP-lowering targets in POAG.

BMP9-ID1 signaling promotes EpCAM-positive cancer stem cell properties in hepatocellular carcinoma.

The malignant nature of hepatocellular carcinoma (HCC) is closely related to the presence of cancer stem cells (CSCs). Bone morphologic protein 9 (BMP9), a member of the transforming growth factor‐beta (TGF‐β) superfamily, was recently reported to be involved in liver diseases including cancer. We aimed to elucidate the role of BMP9 signaling in HCC‐CSC properties and to assess the therapeutic effect of BMP receptor inhibitors in HCC. We have identified that high BMP9 expression in tumor tissues or serum from patients with HCC leads to poorer outcome. BMP9 promoted CSC properties in epithelial cell adhesion molecule (EpCAM)‐positive HCC subtype via enhancing inhibitor of DNA‐binding protein 1 (ID1) expression in vitro. Additionally, ID1 knockdown significantly repressed BMP9‐promoted HCC‐CSC properties by suppressing Wnt/β‐catenin signaling. Interestingly, cells treated with BMP receptor inhibitors K02288 and LDN‐212854 blocked HCC‐CSC activation by inhibiting BMP9‐ID1 signaling, in contrast to cells treated with the TGF‐β receptor inhibitor galunisertib. Treatment with LDN‐212854 suppressed HCC tumor growth by repressing ID1 and EpCAM in vivo. Our study demonstrates the pivotal role of BMP9‐ID1 signaling in promoting HCC‐CSC properties and the therapeutic potential of BMP receptor inhibitors in treating EpCAM‐positive HCC. Therefore, targeting BMP9‐ID1 signaling could offer novel therapeutic options for patients with malignant HCC.

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

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

Ursodesoxycholic acid alleviates liver fibrosis via proregeneration by activation of the ID1-WNT2/HGF signaling pathway.

BackgroundThe human liver possesses a remarkable capacity for self‐repair. However, liver fibrosis remains a serious medical concern, potentially progressing to end‐stage liver cirrhosis and even death. Liver fibrosis is characterized by excess accumulation of extracellular matrix in response to chronic injury. Liver regenerative ability, a strong indicator of liver health, is important in resisting fibrosis. In this study, we provide evidence that ursodesoxycholic acid (UDCA) can alleviate liver fibrosis by promoting liver regeneration via activation of the ID1‐WNT2/hepatocyte growth factor (HGF) pathway.MethodsBile duct ligation (BDL) and partial hepatectomy (PH) mouse models were used to verify the effects of UDCA on liver fibrosis, regeneration, and the ID1‐WNT2/HGF pathway. An Id1 knockdown mouse model was also used to assess the role of Id1 in UDCA alleviation of liver fibrosis.ResultsOur results demonstrate that UDCA can alleviate liver fibrosis in the BDL mice and promote liver regeneration via the ID1‐WNT2/HGF pathway in PH mice. In addition, Id1 knockdown abolished the protection afforded by UDCA in BDL mice.ConclusionsWe conclude that UDCA protects against liver fibrosis by proregeneration via activation of the ID1‐WNT2/HGF pathway.

DIPG-59. UPREGULATION OF PRENATAL PONTINE ID1 SIGNALING IN DIPG

BACKGROUNDDiffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors with no curative therapies. Inhibitor of DNA binding (ID) proteins are key regulators of gene differentiation during embryogenesis. Previous work has shown that H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been validated in human DIPG, nor has the regulation and targetability of ID1 been explored in DIPG.RESULTSAnalysis of post-mortem tissue and multiple human datasets showed ID1 to be elevated in DIPG, and to correlate with reduced survival. In a multi-focal autopsy of a DIPG case, we also found ID1 expression to be heterogeneous and to correlate with tumor invasion. Chromatin immunoprecipitation qPCR (ChIP-qPCR) revealed elevated H3K27ac and low H3K27me3 at ID1 regulatory regions (enhancers/promoters) in DIPG tissue compared to normal brain, regardless of H3 or ACVR1 mutation status. Analysis of publicly-available ISH and ChIP-sequencing data of developing murine brains revealed H3K27ac at ID1 enhancers to be elevated in the prenatal hindbrain compared to prenatal forebrain and midbrain, and all postnatal brain regions. ID1 shRNA-mediated knockdown of primary human H3K27M DIPG cells (DIPG007) significantly reduced invasion and migration. We also treated DIPG007 cells with cannabidiol (CBD) and found reduced viability at clinically relevant dosing (IC50=2.4 uM) with dose-dependent reduction in ID1 protein.CONCLUSIONSThese findings indicate that a multifactorial (genetic and regionally-based) epigenetic upregulation of ID1 drives DIPG invasiveness and is targetable with CBD. ID1 knockdown and CBD treatment experiments in murine models of DIPG are ongoing.

TAMI-29. MULTIFACTORIAL UPREGULATION OF ID1 DRIVES DIPG INVASIVENESS AND IS THERAPEUTICALLY TARGETABLE

Abstract Diffuse intrinsic pontine gliomas (DIPGs) are lethal brain tumors with no effective therapies other than radiation. Inhibitor of DNA binding (ID) proteins, key regulators of lineage commitment during embryogenesis, are implicated in tumorigenesis in multiple human cancers. Prior work showed that recurrent H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes. However, this has not been validated in human DIPG. The regulation and targetability of ID1 in DIPG has not been explored either. Exome and transcriptome sequencing analysis of multi-focal DIPG tumors and normal brain tissue from autopsy (n=52) revealed that ID1 expression is significantly elevated in DIPG tissues. Higher ID1 expression correlates with reduced survival in DIPG patients and increased regional invasion in multi-focal autopsy samples. Analyses of developing mouse brain RNA/ChiP-Seq data revealed high ID1 expression and H3K27ac promoter binding in prenatal hind brain compared to all other prenatal and postnatal brain regions. ChIP-qPCR for H3K27ac and H3K27me3 revealed that ID1 gene regulatory regions are epigenetically poised for upregulation in DIPG tissues compared to normal brain, regardless of H3/ACVR1 mutational status. These data support that the developing pons is regionally poised for ID1 activation. Genetic (shRNA) ID1 knockdown in primary human H3.3K27M-DIPG cells (DIPG007) resulted in significantly reduced invasion and migration in vitro. Additionally, DIPG-ID1-KO cells showed improved sensitivity to radiation therapy. Phospho-kinase array analysis of DIPG cells revealed that Akt and WNK1 activity were significantly downregulated upon ID1 knockdown, which was previously shown in lung tumors. Treatment of DIPG007 cells with cannabidiol (CBD) reduced ID1 expression levels and viability/proliferation of DIPG cells in vitro. ID1 knockdown and CBD treatment studies in vivo are ongoing. In summary, our findings indicate that multifactorial (genetic and regional) epigenetic upregulation of ID1 drives DIPG invasiveness and targeting ID1 using CBD may be a potential strategy for the treatment of DIPGs.

BMP4 promotes the metastasis of gastric cancer by inducing epithelial-mesenchymal transition via ID1.

Epithelial-mesenchymal transition (EMT) is a crucial process for cancer cells to acquire metastatic potential, which primarily causes death in gastric cancer (GC) patients. Bone morphogenetic protein 4 (BMP4) is a member of the TGF-β family that plays an indispensable role in human cancers. However, little is known about its roles in GC metastasis. In this study, BMP4 was found to be frequently overexpressed in GC tissues and was correlated with poor patient's prognosis. BMP4 was upregulated in GC cell lines and promoted EMT and metastasis of GC cells both in vitro and in vivo, whereas knockdown of BMP4 significantly inhibited EMT and metastasis of GC cells. Furthermore, the inhibitor of DNA binding 1 (also known as DNA-binding protein inhibitor ID1) was identified as a downstream target of BMP4 using PCR arrays and was upregulated via SMAD1/5/8 phosphorylation. ID1 knockdown attenuated BMP4-induced EMT and invasion in GC cells. Moreover, ID1 overexpression in BMP4 knockdown cells restored the promotion of EMT and cell invasion. In summary, BMP4 induced EMT and promoted GC metastasis by upregulating ID1 expression. Antagonizing BMP4 could be a potential therapeutic strategy for GC metastasis.

Transcription factor ID1 is involved in decidualization of stromal cells: Implications in preeclampsia.

Decidual stromal cells (DSC) from women with preeclampsia (PE) show defective decidualization upon in vitro treatment with cAMP. Decidualization is associated with a multitude of gene expression changes and is a prerequisite for embryo implantation. We reason that the process of decidualization involves a cascade of changes in transcriptional regulators. Our prior studies have found defective decidualization of PE-DSCs as reflected by low prolactin (PRL) levels and other decidualization markers. Transcription factor array analysis identified inhibitor of DNA binding (ID1) and FOXO1 as top differentially expressed genes during decidualization. Unlike ID1, FOXO1 involvement in decidualization has been established. We hypothesized that ID1 plays a major role in regulating stromal cell decidualization. Our data shows basal ID1 mRNA expression is significantly higher in PE DSCs. Cyclic AMP-mediated decidualization significantly upregulates ID1 mRNA expression in DSCs and siRNA-mediated knockdown of ID1 significantly interferes with decidualization as shown by a reduction in PRL and FOXO1 expression, and morphologic criteria. Thus ID1 may serve as a master regulator of stromal cell differentiation and defects in ID1 expression may affect decidualization as seen in PE-DSCs.

Bone morphogenetic protein 2 promotes human trophoblast cell invasion and endothelial-like tube formation through ID1-mediated upregulation of IGF binding protein-3.

Extravillous cytotrophoblasts (EVTs) invade into the uterine wall and remodel spiral arteries for proper placentation. Studies from us and others have demonstrated that the transforming growth factor-β superfamily member bone morphogenetic protein 2 (BMP2) plays important roles in endometrial decidualization and trophoblast cell invasion. However, BMP2 has also been shown to regulate endothelial cell migration and capillary-like tube formation, as has its downstream signaling molecule inhibitor of DNA binding 1 (ID1). Interestingly, insulin-like growth factor binding protein 3 (IGFBP3) also promotes cell migration and angiogenesis in endothelial precursor cell. Moreover, Id1 has a regulatory effect on Igfbp3 expression in rat prostate epithelial cells. However, whether ID1 and IGFBP3 are integrated in BMP2 signaling and involved in the regulation of trophoblast invasive and endovascular differentiation remains unknown. The objective of our study was to examine the effects of BMP2 on ID1 and IGFBP3 expression and their roles in BMP2-regulated human trophoblast invasion and endothelial-like tube formation. Primary and immortalized (HTR8/SVneo) cultures of human trophoblast cells were employed as study models. BMP2 treatment increased ID1 and IGFBP3 mRNA and protein levels in HTR8/SVneo and primary human EVT cells. Intriguingly, ID1 was essential for BMP2-induced IGFBP3 upregulation in both study models, and BMP2-induced trophoblast invasion was attenuated by knockdown of either ID1 or IGFBP3. In addition, BMP2 significantly increased endothelial-like tube formation and knockdown of ID1 and IGFBP3 reduced basal and BMP2-induced tube formation in HTR8/SVneo cells. Similarly, BMP2 increased placenta growth factor (PlGF) production in HTR8/SVneo cells and these effects were attenuated by knockdown of ID1 or IGFBP3. Our results reveal that BMP2 promotes trophoblast cell invasion and endothelial-like tube formation by ID1-mediated IGFBP3 upregulation.

EXTH-47. THERAPEUTIC REVERSAL OF PRENATAL PONTINE ID1 SIGNALING IN DIPG

Abstract Diffuse intrinsic pontine gliomas (DIPGs) are lethal brain tumors with no effective therapies other than radiation. Inhibitor of DNA binding (ID) proteins are key regulators of tissue and lineage-specific gene differentiation during embryogenesis. Previous work has shown that H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes, but this has not been validated in human DIPG, nor has the regulation and targetability of ID1 been explored in DIPG. Analysis of multi-focal post-mortem tumor and normal brain tissue (n=52) as well as multiple human datasets revealed ID1 to be elevated in DIPG. Elevated ID1 was found to correlate with reduced survival in DIPG. In a multi-focal autopsy DIPG case, we found ID1 expression to be heterogeneous and to correlate with tumor invasion. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) was used to quantify H3K27ac and H3K27me3 at ID1 gene regulatory regions (promoters/enhancers) in multi-focal post-mortem tissue. The ID1 loci was found to be epigenetically poised for up-regulation (elevated H3K27ac and low H3K27me3) in DIPG tissue, regardless of H3 or ACVR1 mutation status, compared to normal brain. Analysis of publically-available ISH and ChIP-sequencing data revealed elevated ID1 expression and ID1-enhancer H3K27ac in prenatal mouse hindbrain compared to prenatal forebrain, prenatal midbrain, and all postnatal brain regions. ID1 shRNA-mediated knockdown of primary human H3K27M DIPG cells (DIPG007) resulted in significantly reduced invasion and migration. As cannabidiol (CBD) has successfully been used to therapeutically target ID1 in pre-clinical models of adult human cancers, we treated DIPG007 cells with CBD and found reduced viability at clinically relevant dosing (IC50=2.4 uM) with dose-dependent reduction in ID1 protein. ID1 knockdown and CBD treatment studies in murine models of DIPG are ongoing. These findings indicate that a multifactorial (genetic and regionally-based) epigenetic upregulation of ID1 drives DIPG invasiveness and is targetable with CBD.

Long noncoding RNA ANCR inhibits the differentiation of mesenchymal stem cells toward definitive endoderm by facilitating the association of PTBP1 with ID2

The generation of definitive endoderm (DE) cells in sufficient numbers is a prerequisite for cell-replacement therapy for liver and pancreatic diseases. Previously, we reported that human adipose-derived mesenchymal stem cells (hAMSCs) can be induced to DE lineages and subsequent functional cells. Clarifying the regulatory mechanisms underlying the fate conversion from hAMSCs to DE is helpful for developing new strategies to improve the differentiation efficiency from hAMSCs to DE organs. Long noncoding RNAs (lncRNAs) have been shown to play pivotal roles in developmental processes, including cell fate determination and differentiation. In this study, we profiled the expression changes of lncRNAs and found that antidifferentiation noncoding RNA (ANCR) was downregulated during the differentiation of both hAMSCs and embryonic stem cells (ESCs) to DE cells. ANCR knockdown resulted in the elevated expression of DE markers in hAMSCs, but not in ESCs. ANCR overexpression reduced the efficiency of hAMSCs to differentiate into DE cells. Inhibitor of DNA binding 2 (ID2) was notably downregulated after ANCR knockdown. ID2 knockdown enhanced DE differentiation, whereas overexpression of ID2 impaired this process in hAMSCs. ANCR interacts with RNA-binding polypyrimidine tract-binding protein 1 (PTBP1) to facilitate its association with ID2 mRNA, leading to increased ID2 mRNA stability. Thus, the ANCR/PTBP1/ID2 network restricts the differentiation of hAMSCs toward DE. Our work highlights the inherent discrepancies between hAMSCs and ESCs. Defining hAMSC-specific signaling pathways might be important for designing optimal differentiation protocols for directing hAMSCs toward DE.

Overexpression of ID1 promotes tumor progression in penile squamous cell carcinoma.

Penile squamous cell carcinoma (PSCC) occurs more frequently in developing countries, and is commonly diagnosed at an advanced stage with an unfavorable prognosis. At present, few biomarkers for PSCC have been identified and used in clinical practice. Aberrant expression of inhibitor of DNA binding1 (ID1) has been suggested as a potential regulator of tumor progression in various types cancer. Herein, we evaluated ID1 expression in PSCC and analyzed its association with the clinicopathological parameters of PSCC. Our findings indicated that ID1 overexpression is associated with histological subtype and lymph node metastasis. Kaplan‑Meier survival analysis showed that the overexpression of ID1 is associated with unfavorable cancer‑specific survival. In Cox proportional hazard models, ID1 overexpression was found to be an independent predictor of cancer‑specific survival. Furthermore, we investigated the function of ID1 in PSCC using a PSCC cell line Penl1. Silencing of ID1 expression retarded cell growth, inhibited clonogenesis, and attenuated cell migration and invasion in Penl1 cells. ID1 may regulate key oncogenic and metastasis‑related molecules, as depletion of ID1 expression affected the levels of p‑AKT, p16, PTEN and cleaved caspase‑3, and reduced MMP2/9 secretion in Penl1 cells. Nevertheless, the mRNA expression of p16 and PTEN increased following ID1 knockdown, suggesting that ID1 may repress p16 and PTEN expression in Penl1 cells. Therefore, overexpression of ID1 could serve as a potential prognostic biomarker for the clinical management of PSCC. Strategies targeting ID1‑regulated signaling pathways may have therapeutic benefit in PSCC.

Inhibitor of DNA Binding 2 (ID2) Plays a Key Tumor Suppressor Role in Promoting Oncogenic Transformation in Multiple Myeloma

Dysregulation of transcriptional control is a common phenomenon associated with oncogenesis. Inhibitors of DNA binding (ID) proteins are critical actors in lymphopoiesis, acting as regulators of transcription through a helix-loop-helix (HLH) domain which enables heterodimerization with basic HLH (bHLH) proteins inhibiting their binding to DNA. ID proteins have been implicated in malignant transformation, but their role in multiple myeloma (MM) is unknown. Here, we evaluated the role of ID proteins in biology and transcriptional dysregulation in MM.We first evaluated the expression of the four ID proteins in normal and malignant plasma cells using RNA sequencing data from a cohort of 360 newly diagnosed MM patients and 16 normal plasma cells. We observed significant downregulation of ID2 in primary patient MM cells in comparison to normal plasma cells (p 0.0013).To study ID2 function in MM cells, we next overexpressed ID2 in 2 MM cell lines (MM1S and NCIH929) and observed a significant decrease in proliferation rate, together with G0/G1 phase cell cycle arrest. We performed RNA-sequencing to evaluate the transcriptomic changes following ID2 overexpression. Gene set enrichment analysis (GSEA) revealed significant downregulation of genes involved in E2F pathway and significant changes in pathways related to immune response, regulation of cell death and cell proliferation. In addition, analysis of upstream cis-regulatory motifs of genes significantly dysregulated in both cell lines (>1.5 fold change) showed a highly significant enrichment for bHLH class I transcription factors (E proteins) binding motifs. Conversely, stable ID2 knockdown in 4 MM cell lines (MM1S, NCIH929, RPMI8226 and KMS11) expressing intermediate levels of ID2, showed an increased proliferation rate, assessed by cell counting, H3-thymidine incorporation and ATP production. RNA-sequencing after ID2 knockdown in MM1S and NCIH929 cells showed 600 common genes upregulated in both cell lines (>1.5 fold change). GSEA revealed upregulation of pathways involved in inflammatory response and epithelial-to-mesenchymal transition, while upstream cis regulatory motifs analysis showed a highly significant enrichment for binding motifs of bHLH class I transcription factors E proteins, in particular Tcf3 (p <0.0001).Next, we sought to investigate the mechanisms involved in ID2 downregulation in MM. Since the role of the microenvironment is critical in myelomagenesis, we evaluated the impact of BM microenvironment on ID2 expression in a co-culture system. Using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines, we observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 were able to significantly downregulate ID2 expression at the RNA and protein level. Furthermore, ID2 overexpression in MM cell lines (MM1S and NCIH929) abrogated the impact of BMSC on MM cell proliferation. Next, we evaluated ID2 promoter methylation profile and binding motifs using Sequenom mass array and the assay for transposase-accessible chromatin sequencing (ATAC-seq), respectively. While we didn't observe any increase in methylation of CpG islands located in ID2 promoter in co-culture, explaining ID2 downregulation, we identified several binding motifs corresponding to known driver transcription factors in MM. Especially, we identified SP1 binding motif and we confirmed SP1 binding to ID2 promoter by ChIP-sequencing in MM1S, NCIH929 and U266.These data demonstrate that in MM, ID2 acts as a tumor suppressor by promoting major transcriptomic changes and cell cycle arrest. Bone marrow stromal cells further induce significant downregulation of ID2 in myeloma cells suggesting that ID2/bHLH axis and other ID2 related pathways represent a potential new therapeutic target in myeloma. DisclosuresAnderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy. Munshi:OncoPep: Other: Board of director.