Id1 Levels Research Articles

FHL2 is a novel target in pulmonary arterial hypertension

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): - Longfonds Nederland Background/ Objectives Pulmonary arterial hypertension (PAH) is a fatal disorder characterized by excessive proliferation of endothelial (EC) and smooth muscle cells, and impaired BMP signaling in small lung arteries. The LIM-only protein FHL2 is expressed in the vessel wall in ECs and smooth muscle cells and plays a crucial role in vessel wall homeostasis. However, the molecular mechanisms how FHL2 exerts its effects and its role in pulmonary artery EC function remain unclear. Here, we investigated the effect of FHL2 on EC proliferation and unraveled the underlying mechanisms involved. Methods/Results We found that FHL2 is highly expressed in lungs of PAH rats compared to control rats as evidenced by qPCR and western blot analysis. Human microvascular ECs (MVECs) derived from lungs of PAH patients displayed enhanced expression of FHL2. Ectopic expression of FHL2 in MVECs significantly increased mRNA levels of BMPR2 and Id1. To substantiate these results, luciferase assays were performed using BMP response element reporter construct and found that FHL2 increases Id1 expression. Furthermore, overexpression of FHL2 decreased TNF-α induced expression of pro-inflammatory cytokines. Finally, ectopic expression of FHL2 reduced proliferation of ECs. Conclusion Our data indicate that FHL2 is a novel negative regulator of MVEC proliferation through enhancing BMP signaling and inhibiting inflammation. Modulation of FHL2 may potentially represent a novel therapeutic strategy for the treatment of PAH.

400 Brain AVMs-Related MicroRNAs: Machine Learning Algorithm for Expression Profiles of Target Genes

INTRODUCTION: microRNAs (miRNAs) are a class of non-coding RNAs playing a myriad of important roles in regulating gene expression. Of note, recent work demonstrated a critical role of miRNAs in the genesis and progression of brain arteriovenous malformations (bAVMs). METHODS: A PRISMA-based literature review was performed using PubMed/Medline database with the following search terms: "brain arteriovenous malformations", "cerebral arteriovenous malformations", "microRNA", and "miRNA". All preclinical and clinical studies written in English, regardless of date, were selected. For our bioinformatic analyses, miRWalk and miRTarBase machine learning algorithms were employed; the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was quired for associated pathways/functions. RESULTS: 4 studies were ultimately included in the final analyses. Sequencing data consistently revealed the decreased expression of miR-18a in bAVM-endothelial cells, resulting in increased levels of vascular endodermal growth factor (VEGF), Id-1, matrix metalloproteinase, and growth signals. Our analyses also suggest that the downregulation of miR-137 and miR-195* within vascular smooth muscle cells (VSMCs) may foster the activation of inflammation, aberrant angiogenesis, and phenotypic switching. In the peripheral blood, the overexpression of miR-7-5p, miR-629-5p, miR-199a-5p, miR-200b-3p, and let-7b-5p may contribute to endothelial proliferation and nidus development. The machine learning algorithms employed confirmed associations between miRNA-related target networks, vascular rearrangement, and bAVM progression. CONCLUSIONS: miRNAs expression appears to be critical in managing bAVMs' post-transcriptional signals. Targets of microRNAs regulate canonical vascular proliferation and reshaping. Although additional scientific evidence is needed, the identification of bAVM miRNA signatures may facilitate the development of novel prognostic/diagnostic tools and molecular therapies for bAVMs.

Prognostic impact of inhibitors of DNA binding proteins1 and inhibitors of DNA binding proteins4 genes expression on adult Egyptian patients with acute myeloid leukemia

Background Acute myeloid leukemia (AML) pathogenesis and treatment are currently being better understood at an accelerated rate. Determining genetic and epigenetic changes that can identify patients who are at risk of poor outcomes is therefore desired to optimize treatment options. Many solid tumors have been reported to overexpress Inhibitors of DNA binding proteins (ID1), but few research has looked at the clinical significance of ID1 expression in AML. Additionally, little research has been focused on the direct role of ID4 in myeloid malignancies, as well as its expression and methylation patterns. The aim of the current study was to assess ID1 and ID4 gene expression in bone marrow (BM) aspiration specimens of 91 AML patients, compared with 14 control donors of bone marrow transplantation (BMT), using real-time polymerase chain reaction (RT-PCR). Data were correlated with patients’ clinicopathological features, response to treatment, disease-free survival (DFS), and overall survival (OS) rates. Results ID1 transcript level was significantly increased in AML bone marrow samples compared with normal controls (P = 0.002), while ID4 gene expression showed a nonsignificant difference (P = 0.717). In addition, there was a significant increase in ID1 gene expression in fms-like tyrosine kinase 3 (FLT3) mutant group than fms-like tyrosine kinase 3 wild group (P = 0.010). The total leukocytic count (TLC) was significantly higher in patients with high ID1 expression (P = 0.038) and patients with undetected ID4 expression (P = 0.025). No significant associations were detected between ID1 and ID4 expression levels and patients’ clinicopathological characteristics and OS rates. Conclusion In contrast to ID4, overexpressed ID1 can be adopted as a genetic biomarker for diagnosing AML. ID1 and ID4 expressions did not affect the patients’ OS or DFS.

Abstract 5433: Functional involvement of ribosomal protein L9 to colorectal cancer stemness

Abstract We have previously demonstrated that targeting of ribosomal protein L9 (RPL9) suppresses the growth of colorectal cancer (CRC) via the inactivation of Id-1/NF-κB signaling axis which is known to augment tumor stemness. Thus we aimed in this study to investigate whether the function of RPL9 was associated with CRC stemness properties. It was initially evidenced that inhibition of RPL9 expression reduces the migration and invasion abilities of HT29 parental cell population. We have then sorted out CD133+ cancer stem cells (CSCs) from HT-29 parental cell culture and treated RPL9-specific siRNAs to the isolated CSCs to observe the effect of RPL9 targeting on stemness. As results, knockdown of RPL9 significantly suppressed the proliferation potential and sphere forming capacity of CD133+ HT29 CSCs accompanying with the reduction in CD133 and Id-1 levels. Reflecting these molecular alterations, targeting RPL9 also inhibited the abilities of migration and invasion in CD133+ HT29 CSCs population. Taken together, these findings suggest that RPL9 could be a therapeutic target for both primary CRC treatment and the prevention of metastasis and/or recurrence. Citation Format: Eun-Hye Jeon, Keun Uk Park, Hun-Mo Ryoo, Ilseon Hwang, Yun-Han Lee. Functional involvement of ribosomal protein L9 to colorectal cancer stemness [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 5433.

Abstract 7290: Novel plasmid-based screening method for cervical cancer

Abstract Introduction: Alabama (AL) is tied for third-highest incidence rate and has second-highest death rate of cervical cancer as of 2020. Globally, cervical cancer has the fourth-highest incidence among cancers in women; 90% of deaths from cervical cancer in 2020 occurred in low- and middle-income (LMIC) countries. Inhibitor of differentiation/DNA binding 1 (Id1) expression correlates with tumorigenesis, clinical grade/stage, and aggressiveness of cervical cancer. Thus, Id1 expression could be exploited in novel screening approaches that aim to improve survival rates of cervical cancer in LMIC and clinically underserved settings. As a first step in developing an Id1-based screening approach, these preclinical studies utilized a plasmid that induces expression of a secreted reporter enzyme (secreted embryonic alkaline phosphatase, SEAP), controlled by the Id1 promoter sequence (pId1-SEAP). These studies examined relationships between Id1 expression and kinetics of reporter expression following plasmid transfection to detect human cervical cancer cells. Methods and Results: To confirm the relevance of Id1 as a target in cervical cancer, immunohistochemical staining for Id1 was performed on a human cervical cancer tissue microarray. Western blotting was performed to determine Id1 expression in human cervical cancer cell lines (HeLa, SiHa, CaSki). Optimal transfection conditions with pId1-SEAP were determined experimentally for each line, and then media was sampled through a 48-hour period post-transfection to quantify reporter SEAP expression. Immunohistochemical stain for Id1 showed that early stage and late stage human cervical cancer tissues expressed significantly (p < 0.05) higher levels of Id1 compared to normal tissues, with stain optical density values being 3e5±1e5 for early and late stage compared to 3e4±3e4 for normal tissues (early stage p=0.0001, late stage p=0.0002). Western blot for Id1 in HeLa, SiHa, and CaSki lines showed that all three positively expressed Id1, with Beta-actin-normalized expression values of 0.78±0.12, 1.12±0.05, and 0.76±0.08, respectively. The cell lines transfected with pId1-SEAP expressed SEAP at rates significantly higher than control cells, with the highest rates of production seen during the 36-48-hour post-transfection period. Rates of SEAP production (counts/hour) during this time period were 1e4±3e3 (p=0.0278), 8e2±1e2 (p=0.0834), and 3e2±5e1 (p=0.0683) for transfected HeLa, SiHa, and CaSki cells, respectively. Conclusions: The pId1-SEAP system enabled sensitive detection of cervical cancer cells. These preliminary data support future studies evaluating the in vivo performance of the system in preclinical models of early stage cervical cancer. Further development of this approach could enable sensitive and reliable detection of cervical cancer during early stages when treatment is most effective. Citation Format: Abbigael Eli, Yolanda Hartman, Benjamin B. Kasten, Rebecca Arend, Jason Warram. Novel plasmid-based screening method for cervical 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 7290.

Id1 Ablation Rescues Clonal Hematopoiesis and Delays Disease Progression in Tet2 Null Mice

Hematopoietic malignancies are thought to emerge through the gradual acquisition of genetic mutations within hematopoietic stem and progenitor cells (HSPCs). Some mutations impart a selective proliferative/growth advantage to HSPCs, which expand over time and contribute to a substantial percentage of mature blood cells. This increased proliferation and expansion is termed clonal hematopoiesis (CH) and is a preleukemic phase that is associated with an increased risk of accumulating additional mutations and developing leukemia. These mutations are also associated with increased inflammatory stress and increased cytokine production (“cytokine storm”) in the hematopoietic microenvironment which can contribute to clonal hematopoiesis. Inhibitor of DNA binding 1 (ID1) protein is induced by hematopoietic growth factors and other mediators of inflammatory stress and promotes HSPC proliferation by regulating E proteins and their targets. HSCs that lack Id1 are protected from chronic proliferative stress and exhaustion during serial BMT, and other chronic physiologic stress including genotoxic and inflammatory stress, and aging. ID1 is frequently overexpressed in hematopoietic malignancies and enforced expression of Id1 in HSPCs promotes a myeloproliferative disease in mice suggesting that ID1 may drive clonal expansion and regulate the growth of some hematologic malignancies. Mutations in the Ten-Eleven-Translocation-2 ( Tet2) (Tet methylcytosine dioxygenase 2) gene are frequently observed in patients with CH, and mice that lack Tet2 show clonal expansion, increased mutational loads, genomic instability that results in myeloid and lymphoid malignancies after a long latency. Here, we show that ablation of Id1 rescues clonal hematopoiesis and delays disease progression in Tet2 -/- mice. We found that Tet2 -/- HSPCs express high levels of Id1. Therefore, we genetically ablated Id1 in Tet2 -/- mice and generated Tet2 -/-;Id1 -/- (dKO) mice and performed competitive bone marrow transplantation studies (BMT) to study the intrinsic role of these genes in hematopoietic development. We found that genetic ablation of Id1 in Tet2 -/- HSPCs reduces HSPC proliferation/expansion and myeloid skewing by flow cytometry. dKO mice showed reduced spleen size and weight indicating that reducing ID1 levels in Tet2 -/- mice rescues extramedullary hematopoiesis. We confirmed that reducing ID1 levels in Tet2 -/- mice reduces clonal expansion of Tet2 -/- BMCs using limited number of donor BMCs in competitive BMT. Furthermore, we performed functional assays including colony forming assays (CFU-C) and single cell (SC) assays to assess the proliferation of these cells and found that loss of Id1 in Tet2 -/- mice reduced 1) the number of CFU-C's and the serial replating ability of Tet2 -/- HSPCs, and 2) progenitor expansion in SC assays. Moreover, dKO recipient mice BMCs showed increased apoptosis and senescence compared to Tet2 -/-BMCs, which could contribute to reduced expansion. Surprisingly, Id1 ablation in Tet2 -/- mice also resulted in reduced number of chromosomal aberrations indicating that reducing ID1 levels in Tet2 -/- mice rescues genomic instability. Furthermore, dKO recipient mice showed delayed onset of disease in a model of chronic proliferative stress. Mechanistically, we found that p16 expression was significantly increased in the dKO recipient mice, which could account for reduced expansion and increased apoptosis and senescence. shRNA mediated knockdown of p16 in dKO lineage negative cells reduced apoptosis and senescence in these cells confirming the role of p16 in these phenotypes. We also found that 53BP1 and Rad51 expression was reduced in the dKO recipient mice, which could lead to improved DNA repair and genomic stability. Thus, Id1 may represent a potential therapeutic target to reduce clonal expansion and delay the onset of leukemia.

Abstract 17276: NOTCH3-BMPR Interactions Drive Smooth Muscle Cell Proliferation and Medial Hyperplasia in Pulmonary Arterial Hypertension

Background: One of the characteristics of pulmonary arterial hypertension (PAH) is the proliferation of smooth muscle cells. In heritable PAH, aberrant BMPR2 signaling results in low levels of intracellular ID1. An important pathway involved in vascular smooth muscle cell proliferation and the development of non-heritable PAH is the NOTCH3-HES5 signaling pathway. Previous studies have demonstrated that HES-5 and ID-1 are peptides belonging to the basic helix-loop-helix family, and they act as transcriptional enhancers of cell cycle genes. Transcriptional enhancement or repression requires the heterodimerization of different HES and ID proteins. Hypothesis: We hypothesize that HES-5 binds to and degrades IDs in non-heritable cases of PAH. Methods: The half-life of ID proteins was assessed in human small pulmonary artery smooth muscle cells that either constitutively expressed or minimally expressed HES-5. In order to understand whether this effect was due to direct binding between HES-5 and ID1, co-immunoprecipitation experiments were performed. Immunofluorescent staining was used to detect the expression level of ID1 and HES-5. Proximity ligation assay (PLA) was utilized to examine of endogenous in-situ interaction of ID1-HES5. Results: We found that overexpression of HES-5 significantly diminished the half-life of ID1 and ID3, and had little effect on ID2. All three proteins localized to the nucleus of small pulmonary artery smooth muscle cells. Immunofluorescent staining confirmed elevated levels of HES and reduced levels of ID1 and ID3 in the medial layer of PAH vessels. By PLA assay, we show that HES-5 and ID1 as well as HES-5 and ID-3 physically associate with each other to the level of 40 nanometers. Conclusions: Constitutive NOTCH3 signaling and high intracellular HES-5 levels drive vascular smooth muscle cell proliferation in non-familial PAH by binding to and degrading ID intracellular proteins. Our study demonstrates cross-talk between the NOTCH3-pathway and the BMPR2 pathway in this disease, at the level of their downstream effectors: ID1 and HES-5. Further studies should shed light on how these two pathways control the PAH disease process.

FBXO7 Confers Mesenchymal Properties and Chemoresistance in Glioblastoma by Controlling Rbfox2-Mediated Alternative Splicing.

Mesenchymal glioblastoma (GBM) is highly resistant to radio-and chemotherapy and correlates with worse survival outcomes in GBM patients; however, the underlying mechanism determining the mesenchymal phenotype remains largely unclear. Herein, it is revealed that FBXO7, a substrate-recognition component of the SCF complex implicated in the pathogenesis of Parkinson's disease, confers mesenchymal properties and chemoresistance in GBM by controlling Rbfox2-mediated alternative splicing. Specifically, FBXO7 ubiquitinates Rbfox2 Lys249 through K63-linked ubiquitin chains upon arginine dimethylation at Arg341 and Arg441 by PRMT5, leading to Rbfox2 stabilization. FBXO7 controls Rbfox2-mediated splicing of mesenchymal genes, including FoxM1, Mta1, and Postn. FBXO7-induced exon Va inclusion of FoxM1 promotes FoxM1 phosphorylation by MEK1 and nuclear translocation, thereby upregulates CD44, CD9, and ID1 levels, resulting in GBM stem cell self-renewal and mesenchymal transformation. Moreover, FBXO7 is stabilized by temozolomide, and FBXO7 depletion sensitizes tumor xenografts in mice to chemotherapy. The findings demonstrate that the FBXO7-Rbfox2 axis-mediated splicing contributes to mesenchymal transformation and tumorigenesis, and targeting FBXO7 represents a potential strategy for GBM treatment.

Effect of trametinib and anti-PD-1 combo on tumor growth and survival of KRAS-mutant lung cancer by ID1 downregulation.

e14633 Background: Although anti-PD-1/PD-L1 antibodies represent a major breakthrough in NSCLC patients’ survival, this treatment efficacy is highly dependent on tumor PD-L1 expression. We reported the role of ID1 and the efficacy of a combined blockade of PD-1-ID1 in a KRAS mutant NSCLC mouse model (Baraibar et al; Cancers 2020). Trametinib, which is an oral MEK1/2 inhibitor, acts downstream of KRAS. Here we evaluate trametinib as an ID1 inhibitor able to enhance immunotherapy efficacy through PD-L1 overexpression. Methods: To explore whether MEK1/2 inhibition reduces ID1 expression in vitro and in vivo, experiments in NSCLC cells (human and murine) and in murine models were performed. RNAseq analysis was conducted to elucidate the pathways implicated in ID1 inhibition. Trametinib resistant (TR) cells were generated to analyze the effect of ID1 in the acquired resistance to treatment. Apoptosis was measured by flow cytometry. PD-L1 expression was evaluated by flow cytometry in control, ID1 silenced, ID1-overexpressing and TR cell lines. Treatment of trametinib combined with anti-PD1 was performed in KRAS-mutant LUAD models. Results: In vitro experiments in NSCLC cells showed ID1 inhibition at protein level after MEK1/2 blockade. However, we observe an increase in ID1 mRNA levels following treatment. In vivo experiments performed in C57BL6J and in ID1-deficient mice showed no significant differences in tumor growth between ID1 knock-out and ID1 wild-type mice treated with trametinib. IHC analysis of those tumor samples showed reduced ID1 levels (LLC p<0.001; CMT-167 p<0.0001). RNAseq analysis revealed an enrichment of ubiquitin-proteasome pathway after trametinib exposure. Blockade of proteasome activity with a specific inhibitor, restored ID1 levels. TR cells did not show MEKi induced ID1 inhibition, moreover, ID1 blockade rescued the sensitivity to trametinib in human and murine NSCLC TR cells (p<0.001). Flow cytometry analysis showed increased PD-L1 expression (p<0.001) in human and murine NSCLC cell lines after trametinib administration. In contrast, ID1-overexpressing cells and TR cells did not show PD-L1 upregulation after MEK inhibition. In vivo experiments confirmed the therapeutic efficacy of trametinib and anti-PD-1 combo, reducing tumor growth (p<0.05) and increasing mice survival (p<0.05), compared with trametinib monotherapy or immunotherapy alone. Conclusions: Pharmacological inhibition of MEK1/2 decreased ID1 expression in vitro and in vivo, replicating the effect of a specific shRNA against ID1, through proteasome activation. PD-L1 upregulation induced by ID1 blockade after trametinib treatment sensitized KRAS-mutant NSCLC cells to PD-1/PD-L1 immunotherapy, blocking tumor growth and improving overall survival.

Establishment of a human induced pluripotent stem cell derived alveolar organoid for toxicity assessment

Alveolar epithelial cells (AECs) are vulnerable to injury, which can result in epithelial hyperplasia, apoptosis, and chronic inflammation. In this study, we developed human induced pluripotent stem cell (hiPS) cell-derived AECs (iAECs) and the iAECs based organoids (AOs) for testing AEC toxicity after chemical exposure. HiPS cells were cultured for 14 days with differentiation medium corresponding to each step, and the iAECs-based AOs were maintained for another 14 days. SFTPC and AQP5 were expressed in the AOs, and mRNA levels of SOX9, NKX2.1, GATA6, HOPX, and ID2 were increased. The AOs were exposed for 24 h to nine chemical substances, and IC50 values of the nine chemicals were determined using MTT assay. When the correlations between iAECs 2D culture and AOs 3D culture were calculated using Pearson's correlation coefficient r value, the nine chemicals that caused a significant decrease of cell viability in 3D culture were found to be highly correlated in 2D culture. The cytotoxicity and nitric oxide release in AO cultured with macrophages were then investigated. When AOs with macrophages were exposed to sodium chromate for 24 h, the IC50 value and nitric oxide production were higher than when the AOs were exposed alone. Taken together, the AO-based 3D culture system provides a useful platform for understanding biological characteristics of AECs and modeling chemical exposures.

The role of Smad4/TGFB signaling in tumorigenesis of pancreatic ductal adenocarcinoma (PDAC).

748 Background: Pancreatic cancer ranks 4th among cancers as a cause of death in the United States and 85- 90% of pancreatic cancer are pancreatic ductal adenocarcinoma (PDAC). Previous studies have demonstrated 2 major types of precursors to PDAC including pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN); the mechanisms by which IPMNs give rise to PDAC are incompletely defined. We hypothesize that SMAD4 signaling is specifically involved in IPMN pathway with PanINs and IPMN having partially overlapping signaling pathways and molecular markers. Methods: We performed immunohistochemistry staining on different precursor lesions of mouse pancreatic tissue (PanIN precursor: Ptf1aCre LSL-KrasG12D; IPMN precursor: Ptf1aCreERT LSL- KrasG12D Smad4fl/fl treated with tamoxifen) using markers of different IPMN subtypes and signaling pathways known to be involved in PDAC including, MUC1, MUC2, MUC5AC, SMAD4, ERK1/2, SOX9, and YAP. We treated IPMN cells lines derived from Ptf1aCreERTM LSL-KrasG12D GnasR201C mice with TGF-β, BMP-2, or vehicle control and quantified RNA expression of Smad7, Serpine1, Id2, and normalized to Pmm1 expression. We performed a western blot to detect the protein expression of phospho-SMAD1, phospho-SMAD2, SMAD1, and SMAD2. Results: Both PanIN and IPMN mouse models exhibited strong expression of MUC5AC and MUC1, reflecting the pancreatobiliary subtype. The tumor cells in both models expressed phosphorylated substrates of protein kinase A, phosphorylated ERK1/2, and SOX9. Regional differences were noted for SOX9 signaling with IPMN demonstrating a greater expression around the outer edges of the lesion, compared to the center. PanIN model demonstrated a more prominent nuclear YAP staining. Treatment of BMP2 and TGF-β in pancreatic IPMN cells from Ptf1aCreERTM LSL-KrasG12D GnasR201C cell line significantly increased RNA levels of Serpine1 (2.4 and 199.6, respectively; n = 4; p < 0.05) and Smad7 (2.9 and 6.3, respectively; n = 4; p < 0.05). BMP2 treatment significantly increased RNA levels of Id2 (1.9; n = 4; p < 0.05) while TGF-β significantly decreased Id2 level (0.3; n = 4; p < 0.05). Phosphorylated-SMAD2 was detected only with TGF-β treatment while phosphorylated-SMAD1 was detected with vehicle, TGF-β, and BMP2 treatment with the highest level with BMP2 treatment. Conclusions: Knocking out Smad4, concurrent with activating KrasG12D mutation, induces a specific IPMN progression model that shares similar molecular markers as the PanIN progression model, shown in Kras mutation alone. SMAD4 signaling remains intact in IPMN lesions that arise from activating the KrasG12D GnasR201C mutation, possibly suggesting that Smad4 mutation induces the IPMN-PDAC progression model that differs from Gas-cAMP-PAK-YAP1 cascade found in GNAS signaling.

Brain AVMs-Related microRNAs: Machine Learning Algorithm for Expression Profiles of Target Genes.

microRNAs (miRNAs) are a class of non-coding RNAs playing a myriad of important roles in regulating gene expression. Of note, recent work demonstrated a critical role of miRNAs in the genesis and progression of brain arteriovenous malformations (bAVMs). Accordingly, here we examine miRNA signatures related to bAVMs and associated gene expression. In so doing we expound on the potential prognostic, diagnostic, and therapeutic significance of miRNAs in the clinical management of bAVMs. A PRISMA-based literature review was performed using PubMed/Medline database with the following search terms: "brain arteriovenous malformations", "cerebral arteriovenous malformations", "microRNA", and "miRNA". All preclinical and clinical studies written in English, regardless of date, were selected. For our bioinformatic analyses, miRWalk and miRTarBase machine learning algorithms were employed; the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was quired for associated pathways/functions. four studies were ultimately included in the final analyses. Sequencing data consistently revealed the decreased expression of miR-18a in bAVM-endothelial cells, resulting in increased levels of vascular endodermal growth factor (VEGF), Id-1, matrix metalloproteinase, and growth signals. Our analyses also suggest that the downregulation of miR-137 and miR-195* within vascular smooth muscle cells (VSMCs) may foster the activation of inflammation, aberrant angiogenesis, and phenotypic switching. In the peripheral blood, the overexpression of miR-7-5p, miR-629-5p, miR-199a-5p, miR-200b-3p, and let-7b-5p may contribute to endothelial proliferation and nidus development. The machine learning algorithms employed confirmed associations between miRNA-related target networks, vascular rearrangement, and bAVM progression. miRNAs expression appears to be critical in managing bAVMs' post-transcriptional signals. Targets of microRNAs regulate canonical vascular proliferation and reshaping. Although additional scientific evidence is needed, the identification of bAVM miRNA signatures may facilitate the development of novel prognostic/diagnostic tools and molecular therapies for bAVMs.

RSL3 triggers glioma stem cell differentiation via the Tgm2/AKT/ID1 signaling axis

RSL3 is a synthetic molecule that inactivates glutathione peroxidase 4 to induce ferroptosis. However, its effect on glioma stem cells (GSC) remains unclear. In this study, we found that RSL3 significantly suppressed GSC proliferation and induced their differentiation into astrocytes, which was accompanied by the downregulation of stemness-related markers, including Nestin and Sox2. Combined transcriptome and proteome analyses further revealed that RSL3 promoted GSC differentiation by suppressing transglutaminase 2 (Tgm2), but not by ferroptosis-related pathways. Tgm2 overexpression in CSC2078 cells rescued the changes in stemness-related markers and differentiation caused by RSL3, which was mediated by inhibitor of DNA binding 1 (ID1) activation. Further studies identified ID1 as a downstream signaling target of Tgm2. Blocking the phosphoinositide-3 kinase (PI3K)/Akt pathway with LY294002 suppressed PI3K, p-Akt, and ID1 levels but not Tgm2. Tgm2 overexpression abrogated the changes in PI3K, p-Akt, and ID1 levels caused by LY294002. Taken together, we demonstrate that RSL3 does not induce ferroptosis; instead, it inhibits GSC proliferation and triggers their differentiation by suppressing the Tgm2/Akt/ID1 signaling axis.

ID1 marks the tumorigenesis of pancreatic ductal adenocarcinoma in mouse and human

Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly disease that has an increasing death rate but no effective treatment to now. Although biological and immunological hallmarks of PDAC have been frequently reported recently, early detection and the particularly aggressive biological features are the major challenges remaining unclear. In the current study, we retrieved multiple scRNA-seq datasets and illustrated the genetic programs of PDAC development in genetically modified mouse models. Notably, the transcription levels of Id1 were elevated specifically along with the PDAC development. Pseudotime trajectory analysis revealed that Id1 was closely correlated with the malignancy of PDAC. The gene expression patterns of human PDAC cells were determined by the comparative analysis of the scRNA-seq data on human PDAC and normal pancreas tissues. ID1 levels in human PDAC cancer cells were dramatically increased compared to normal epithelial cells. ID1 deficiency in vitro significantly blunt the invasive tumor-formation related phenotypes. IPA analysis on the differentially expressed genes suggested that EIF2 signaling was the core pathway regulating the development of PDAC. Blocking EFI2 signaling remarkably decreased the expression of ID1 and attenuated the tumor-formation related phenotypes. These observations confirmed that ID1 was regulated by EIF2 signaling and was the critical determinator of PDAC development and progression. This study suggests that ID1 is a potential malignant biomarker of PDAC in both mouse models and human and detecting and targeting ID1 may be a promising strategy to treat or even rescue PDAC.

Inhibitor of DNA binding 2 (ID2) regulates the expression of developmental genes and tumorigenesis in ewing sarcoma.

Sarcomas are difficult to treat and the therapy, even when effective, is associated with long-term and life-threatening side effects. In addition, the treatment regimens for many sarcomas, including Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma, are relatively unchanged over the past two decades, indicating a critical lack of progress. Although differentiation-based therapies are used for the treatment of some cancers, the application of this approach to sarcomas has proven challenging. Here, using a CRISPR-mediated gene knockout approach, we show that Inhibitor of DNA Binding 2 (ID2) is a critical regulator of developmental-related genes and tumor growth in vitro and in vivo in Ewing sarcoma tumors. We also identified that homoharringtonine, which is an inhibitor of protein translation and FDA-approved for the treatment of leukemia, decreases the level of the ID2 protein and significantly reduces tumor growth and prolongs mouse survival in an Ewing sarcoma xenograft model. Furthermore, in addition to targeting ID2, homoharringtonine also reduces the protein levels of ID1 and ID3, which are additional members of the ID family of proteins with well-described roles in tumorigenesis, in multiple types of cancer. Overall, these results provide insight into developmental regulation in Ewing sarcoma tumors and identify a novel, therapeutic approach to target the ID family of proteins using an FDA-approved drug.

ILC2s induce adaptive Th2-type immunity in different stages of tuberculosis through the Notch-GATA3 pathway

The study is to investigate the roles of group 2 innate lymphoid cells (ILC2s) in different courses of tuberculosis (TB). Serum and PBMCs were respectively isolated from the TST negative, LTBI (latent TB infection), ATB (active TB) and RTB (recurrent TB) patients. Flow cytometry was used to detect Th1, Th2 and ILC2s in the peripheral blood. The mRNA and protein levels of GATA3, RORα, CRTH2, Hes1, Notch1, NF-κB, and ID2 were detected by qRT-PCR and Western blotting. ILC2 cells from ATB and RTB patients were stimulated with rJagged2 or DAPT in vitro, and co-cultured with CD4+ T cells from TST negative group. ELISA was used to detect cytokine levels. The results showed that compared with the TST negative or LTBI group, Th2 cells and serum IL-4 in ATB group increased dramatically, accompanied by an increase of Th2/Th1 ratio in ATB patients, especially in RTB group. However, ILC2s in the ATB and RTB group increased significantly, along with increased GATA3, RORα, and CRTH2 levels. After rJagged2 stimulation in vitro, the levels of Hes1, Notch1, NF-κB, RORα, GATA3 and ID2 and those of IL-4, IL-5 and IL-13 were significantly increased. These effects were abrogated by DAPT treatment. Then, ILC2s, especially those from RTB patients, induced Th2-type immune response after co-culturing with CD4+ T cells. In conclusion, our results suggest that ILC2s may promote Th2-type immune response in different stages of TB via the Notch-GATA3 pathway.

DNA methylation regulates the expression of the negative transcriptional regulators ID2 and ID4 during OPC differentiation

The differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes is the prerequisite for remyelination in demyelinated disorders such as multiple sclerosis (MS). Epigenetic mechanisms, such as DNA methylation, have been suggested to control the intricate network of transcription factors involved in OPC differentiation. Yet, the exact mechanism remains undisclosed. Here, we are the first to identify the DNA-binding protein inhibitors, Id2 and Id4, as targets of DNA methylation during OPC differentiation. Using state-of-the-art epigenetic editing via CRISPR/dCas9-DNMT3a, we confirm that targeted methylation of Id2/Id4 drives OPC differentiation. Moreover, we show that in the pathological context of MS, methylation and gene expression levels of both ID2 and ID4 are altered compared to control human brain samples. We conclude that DNA methylation is crucial to suppress ID2 and ID4 during OPC differentiation, a process that appears to be dysregulated during MS. Our data do not only reveal new insights into oligodendrocyte biology, but could also lead to a better understanding of CNS myelin disorders.

Junctional Adhesion Molecule-a, Suppresses Integrin αvβ3 Activation to Control Neovascularization

Introduction: Neovascularization, the formation of new blood vessels from the preexisting vessels, is a complex process. It is strictly regulated in healthy adults. Disruption of endothelial cell-cell junction is critically important in inflammation and pathological angiogenesis. Growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2), two potent angiogenic factors, have been shown to disrupt endothelial cell (EC) junctions. VEGF expression by EC and thus VEGF-dependent neovascularization is normally suppressed in healthy adults. The mechanism of this suppression is not known.Methods: Using Jam-A gene-trap knockout mice, we performed a variety of neovascularization assays such as Matrigel plug, aortic ring sprouting and in vivo tumor growth. Age-matched wild-type (WT) mice were used as controls. Vascular permeability was assessed by Miles assay. Level of VEGF in both the WT and Jam-A null mouse plasma were measured by Elisa assay. Real-time PCR and western blot analyses were used to assess gene expression.Results: Jam-A knockout mice showed significantly enhanced (P<0.004) vascular permeability as assessed by Miles assay. Interestingly, VEGF-, but not FGF-2-dependent neovascularization was significantly augmented (P<0.001) in the absence of Jam-A. ECs isolated from Jam-A null mouse showed significantly enhanced (P<0.05) cell migration in the response to VEGF as compared to WT ECs. We found that the plasma levels of VEGF in the Jam-A null mice were significantly (P<0.000001) and age-dependently increased compared to WT mice. We also found that both mRNA and protein levels of VEGF and its receptor VEGFR2 were significantly increased (P<0.001) in Jam-A null ECs. When tested if the expression of soluble Flt (sFlt), which is a decoy receptor for VEGF that is known to trap VEGF, is downregulated in the absence of Jam-A, we found no significant difference in sFlt mRNA expression in Jam-A null ECs compared to WT. Additionally, augmented vascular permeability observed in Jam-A null mice was completely abrogated upon inhibition of VEGFR2 using a function-blocking antibody DC101, suggesting that the VEGF/VEGFR2 signaling axis is augmented in the absence of Jam-A. In order to determine the mechanism of this upregulation of the VEGF and VEGFR2 in the absence of Jam-A, we tested the expression of hypoxia inducible factor-1α (HIF-1α and inhibitor of DNA binding 1 (Id1), two transcription factors known to upregulate VEGF and VEGFR2 gene expression respectively. Interestingly, mRNA and protein levels of both HIF-1α and Id1 were significantly augmented (P<0.02) in ECs lacking Jam-A compared to WT. Since JAM-A is known to maintain integrin αIIbβ3 in a low affinity state in resting platelets by recruiting CSK to the αIIbβ3-Src complex, we tested if lack of Jam-A will augment integrin αvβ3 signaling in ECs. We found that in fact αvβ3-dependent signaling is augmented in Jam-A null ECs or when Jam-A is inhibited using function-blocking antibody compared to WT. Consistent with this finding, the overexpression of JAM-A in HUVECs attenuated the extent of αvβ3 signaling as well as HIF-1α and Id1 expression, suggesting that Jam-A suppresses αvβ3 signaling to maintain low levels of VEGF and VEGFR2 expression in quiescent ECs.Conclusion: The results presented here suggest that Jam-A suppresses integrin αvβ3 signaling to control VEGF/VEGFR2 expression in adult quiescent ECs thus suppressing neovascularization in adults. During pathological conditions, such as ischemia and inflammation, αvβ3/Jam-A complex is dissociated or Jam-A levels are downregulated allowing αvβ3 signaling, thus supporting pathological angiogenesis. DisclosuresNo relevant conflicts of interest to declare.

Combining Gene Mutation with Gene Expression Data Improves Outcomes Prediction in Acute Promyelocytic Leukemia

INTRODUCTIONIn the context of all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy, heterogeneity of clinical outcomes of patients with acute promyelocytic leukemia (APL) may be higher than expected, mainly outside well-controlled clinical trials. Although recent clinical studies suggest that such heterogeneity could be significantly reduced or even abolished in light of the current arsenic trioxide (ATO)-based therapies, this compound is still not available for most reference centers in low- and middle-income countries (LMIC), and, at least for a near future, alternative strategies for predicting outcomes and improve risk stratification in APL should be tested. Here, we combined recurrent mutations with aberrant expression of genes previously associated with poor prognosis in both APL and acute myeloid leukemia, and proposed an integrative score in APL (ISAPL) for outcomes prediction. METHODS: As a learning set, diagnostic bone marrow samples from 150 adult patients with newly diagnosed APL who were enrolled in the IC-APL study were analyzed. The treatment protocol was identical to that of the LPA2005 trial reported by the Programa Español de Tratamiento en Hematologia/Dutch-Belgian Hemato-Oncology Cooperative Group (PETHEMA/HOVON), except for the replacement of idarubicin by daunorubicin due to its better availability and lower cost in the participating centers. Integer weights for the risk score were derived from Cox proportional hazard model, identical to that reported by Damm et al. (Blood. 2011 Apr 28;117(17):4561-8), using OS as primary endpoint. Variables with P -values lower than 0.05 were included in the model. Variables considered for the model inclusion were the following: FLT3 -ITD status, gene expression profile of ∆Np73/TAp73 ratio and transcript levels of KMT2E, BAALC, ID1, and WT1 genes. Other candidates, such as PIM2, PRAME, ERG, and IDH1 rs11554137 and WT1 rs16754 polymorphisms, were not associated with lower OS and not included in the score. Hazard ratios (HR) for OS were calculated for each variable separately. The HR was converted to integer weights according to the following: variables with HR < 1 were excluded from analyses; variables with HR > 1 and < 1.5 were assigned a weight of 1; variables with HR > 1.5 and < 2.5 were assigned a weight of 2; variables with HR > 2.5 were assigned a weight of 3. The final score was the sum of these integer weights. RESULTS: Complete data for ISAPL modeling were available for 99 of 150 patients (median score: 5, range: 0-16). The median age of the learning set was 35 years (range: 18-65 years) with 44 males (44%). According to PETHEMA/GIMEMA criteria for predicting relapse, 14%, 41%, 27% and 60% of patients assigned to the first, second, third and fourth ISAPL quartiles were deemed high-risk patients (P=0.036). Except for a higher leukocyte count at diagnosis in patients assigned to the fourth-ISAPL quartile (P=0.033), no significant differences in baseline features were found among groups. Early mortality (P=0.005), complete remission (P=0.002), overall survival (P <0.001; Figure 1A), disease-free survival (P=0.017; Figure 1B), event-free survival (P <0.001; Figure 1C), and cumulative incidence of relapse (P=0.03; Figure 1D) rates were significantly different among patients assigned to the first-, second-, third- and fourth-ISAPL quartiles. CONCLUSIONS: In summary, ISAPL resulted in the separation of four distinct groups of patients, with significant differences for hematologic recovery, relapse, and survival. We will next validate these findings in internal and external validation cohorts. [Display omitted] DisclosuresBittencourt:Janssen, Takeda: Honoraria. Pagnano:Roche: Speakers Bureau; Amgen: Consultancy; Bristol-Meirs Squibb: Consultancy, Speakers Bureau; Novartis: Consultancy. Lo Coco:Novartis: Honoraria, Speakers Bureau; Lundbeck: Honoraria, Speakers Bureau; TEVA: Honoraria, Speakers Bureau.

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.