Expression Of NR2F6 Research Articles

Abstract 6531: Anti-tumor immune function of NR2F6, an orphan nuclear receptor, in stromal cells

Abstract Growing evidence points to the importance of the tumor microenvironment (TME) in tumor growth control, progression, and therapy response. Among those is the greater understanding of immune system components within the TME as mediators of tumor growth control. NR2F6 is an orphan nuclear receptor, which was shown to serve as an immune intrinsic checkpoint component and has been suggested to elicit tumor-intrinsic suppression of anti-tumor immunity. Correspondingly, genetic depletion of NR2F6 in CD8 T cells abrogates the immune-activating cytokines (e.g., IL-2, IFNg). In addition, genetic ablation of NR2F6 in melanoma cells activates CD8 T cell-mediated anti-tumor immunity partly by suppressing the expression of tumor-intrinsic immune repressors NACC1 and FKBP10. Notably, combined genetic ablation of tumor cells (NR2F6 KO melanoma) and stroma cells (NR2F6 KO mouse) resulted in a more pronounced inhibition of tumor growth compared with ablation of either tumor cells or TME. The latter provides the foundation for screening NR2F6 inhibitors, which are expected to have a strong systemic (tumor inhibition and immune cell activation) effect on tumor growth. Interestingly, single-cell transcriptomic data from human tumors identified high expression of NR2F6 in stroma cells, cancer-associated fibroblasts (CAFs), and tumor-associated endothelial cells (TAEs). Consistent with these, scRNAseq analysis of mouse melanoma revealed higher expression of NR2F6 in SMA+ immune suppressive myo-CAFs than in immune activating CAFs. Studies that directly reveal the role of stromal NR2F6 in anti-tumor immunity and tumor growth and the state of NR2F6 inhibitors will be discussed. Citation Format: Hyungsoo Kim, Yongmei Feng, Marria Radaeva, Eduard Sergienko, Artem Cherkasov, Ze'ev A. Ronai. Anti-tumor immune function of NR2F6, an orphan nuclear receptor, in stromal cells [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 6531.

The expression and biological effect of NR2F6 in non-small cell lung cancer.

ObjectiveThis study aimed to explore the expression and effect of the nuclear receptor subfamily 2 group F member 6 (NR2F6) gene in non-small cell lung cancer (NSCLC) and provide an experimental basis for the targeted therapy of NSCLC.MethodFirst, the expression of NR2F6 in lung cancer tissues was analyzed using the Gene Expression Omnibus and the Cancer Genome Atlas (TCGA) databases, and the expression of NR2F6 in lung cancer tissues and cells was verified by Western blotting and quantitative polymerase chain reaction. Next, the relationship between NR2F6 expression and the clinicopathological features of lung cancer was analyzed via immunohistochemistry, and the relationship between NR2F6 expression and prognosis was analyzed using the Kaplan–Meier Plotter. The influence of NR2F6 knockdown on the proliferation capacity of lung cancer cells was then verified at cell level. Finally, the expression of heterogeneous nuclear ribonucleoprotein D (HNRNPD) in lung cancer tissue was analyzed using the TCGA database and immunohistochemistry. The impact of HNRNPD knockdown on the proliferation capacity of lung cancer cells was verified at cell level, and the relationship between NR2F6 and HNRNPD was verified by co-immunoprecipitation.ResultsNR2F6 was highly expressed in lung cancer tissues and cells, and its expression was positively correlated with the depth of invasion, lymphatic metastasis, and clinical stage of lung cancer. High expression of NR2F6 in lung cancer was also significantly associated with poor prognosis. At cell level, NR2F6 knockdown was found to inhibit the proliferation of H460 and H358 in lung cancer cells. Furthermore, the TCGA database and immunohistochemical results showed that HNRNPD was highly expressed in lung cancer tissues and was highly consistent with NR2F6 expression in these tissues. Knockdown of HNRNPD also inhibited the proliferation of lung cancer cells. The co-immunoprecipitation experiment verified that NR2F6 interacted with HNRNPD.ConclusionNR2F6 may interact with HNRNPD to jointly regulate the progression of lung cancer, and this conclusion provides a new experimental basis for the study of the molecular targeted therapy of NSCLC.

Functional Screenings Identify Regulatory Variants Associated with Breast Cancer Susceptibility.

Genome-wide association studies (GWAS) have identified more than 2000 single nucleotide polymorphisms (SNPs) associated with breast cancer susceptibility, most of which are located in the non-coding region. However, the causal SNPs functioning as gene regulatory elements still remain largely undisclosed. Here, we applied a Dinucleotide Parallel Reporter sequencing (DiR-seq) assay to evaluate 288 breast cancer risk SNPs in nine different breast cancer cell lines. Further multi-omics analysis with the ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing), DNase-seq (DNase I hypersensitive sites sequencing) and histone modification ChIP-seq (Chromatin Immunoprecipitation sequencing) nominated seven functional SNPs in breast cancer cells. Functional investigations show that rs4808611 affects breast cancer progression by altering the gene expression of NR2F6. For the other site, rs2236007, the alteration promotes the binding of the suppressive transcription factor EGR1 and results in the downregulation of PAX9 expression. The downregulated expression of PAX9 causes cancer malignancies and is associated with the poor prognosis of breast cancer patients. Our findings contribute to defining the functional risk SNPs and the related genes for breast cancer risk prediction.

The role of nuclear receptor transcription factor NR2F6 in tumor

Nuclear receptor subfamily 2, group F, member 6 (NR2F6) is a member of orphan nuclear receptors, which is expressed in major tissues and organs of the human body, and plays an important role in the regulation of various biological functions and gene expressions. Recent studies have shown that the expression of NR2F6 was up-regulated in a variety of malignant tumors and showed significant correlations with cancer progression. These findings triggered the widespread interest in understanding the relationship between NR2F6 and cancer development and progression. In addition, the latest studies have underscored that NR2F6 was involved in enhancing antitumor immune responses that could serve as a potential target for immune regulation. This review summarizes the biological functions of NR2F6 and its role in tumors, with the aim to provide new insights into effective cancer therapies.

Incomplete thermal ablation-induced up-regulation of transcription factor nuclear receptor subfamily 2, group F, member 6 (NR2F6) contributes to the rapid progression of residual liver tumor in hepatoblastoma

ABSTRACT Hepatoblastoma is a kind of extreme malignancy frequently diagnosed in children. Although surgical resection is considered as the first-line treatment for hepatoblastoma, a relatively large population of patients have lost the preferred opportunity for surgery. Administration of locoregional ablation enables local tumor control but with the deficiency of insufficient ablation, residual tumor, and rapid progression. In this study, we integrated 219 hepatoblastoma and 121 non-cancer liver tissues to evaluate the expression of NR2F6, from which a higher NR2F6 level was found in hepatoblastoma compared with non-cancer livers with a standard mean difference (SMD) of 1.04 (95% CI: 0.79, 1.29). The overexpression of NR2F6 also appeared to be an efficient indicator in distinguishing hepatoblastoma tissues from non-cancer liver tissues from the indication of a summarized AUC of 0.90, with a pooled sensitivity of 0.76 and a pooled specificity of 0.89. Interestingly, nude mouse xenografts provided direct evidence that overexpressed NR2F6 was also detected in residual tumor compared to untreated hepatoblastoma. Chromatin immunoprecipitation-binding data in HepG2 cells and transcriptome analysis of HepG2 xenografts were combined to identify target genes regulated by NR2F6. We finally selected 150 novel target genes of NR2F6 in residual tumor of incomplete ablation, and these genes appeared to be associated with the biological regulation of lipid metabolism-related pathway. Accordingly, targeting NR2F6 holds a therapeutic promise in treating residual recurrent hepatoblastoma after incomplete ablation.

MiR-142-3p suppresses the proliferation, migration and invasion through inhibition of NR2F6 in lung adenocarcinoma.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide and lung adenocarcinoma is its main type. MicroRNAs are small, non-coding and single-strand RNAs that regulate gene expression in human cancers. The aim of our study is to investigate the underlying molecular mechanism of miR-142-3p in NSCLC. The expression of miR-142-3p in lung adenocarcinoma tissues and cells was detected by RT-qPCR. Next, cell proliferation, migration, invasion and apoptosis were examined by CCK-8, scratch assay, transwell assay and flow cytometry in A549 and HCC827 cells, respectively. Then, the target of miR-142-3p was predicted by targetscanHuman 7.2 and confirmed using dual-luciferase reporter assay. Additionally, RT-qPCR and western blot were used to detect the expression of NR2F6, MMP2, MMP9 and caspase-3. The results showed that miR-142-3p expression was significantly decreased in tumor tissues and cells. Overexpression of miR-142-3p inhibited the proliferation, migration, invasion and promoted cell apoptosis in vitro, while knockdown of miR-142-3p had reversed function. Furthermore, NR2F6 was identified as a direct target of miR-142-3p, which was negatively correlated with miR-142-3p expression. Finally, miR-142-3p overexpression suppressed the expression of NR2F6, MMP2 and MMP9, but improved caspase-3 expression, while miR-142-3p knockdown got the opposite expression results. Suppressing MMP2 and MMP9 activities inhibited cell invasion. In summary, these findings indicated that miR-142-3p inhibits lung adenocarcinoma cell proliferation, migration and invasion, and enhances cell apoptosis by targeting NR2F6, suggesting that miR-142-3p may be a novel therapeutic target for lung adenocarcinoma treatment.

Nuclear orphan receptor NR2F6 confers cisplatin resistance in epithelial ovarian cancer cells by activating the Notch3 signaling pathway.

The primary challenge facing treatment of epithelial ovarian cancer (EOC) is the high frequency of chemoresistance, which severely impairs the quality of life and survival of patients with EOC. Our study aims to investigate the mechanisms by which upregulation of NR2F6 induces chemoresistance in EOC. The biological roles of NR2F6 in EOC chemoresistance were explored in vitro by Sphere, MTT and AnnexinV/PI assay, and in vivo using an ovarian cancer orthotopic transplantation model. Bioinformatics analysis, luciferase assay, CHIP and IP assays were performed to identify the mechanisms by which NR2F6 promotes chemoresistance in EOC. The expression of NR2F6 was significantly upregulated in chemoresistant EOC tissue, and NR2F6 expression was correlated with poorer overall survival. Moreover, overexpression of NR2F6 promotes the EOC cancer stem cell phenotype; conversely, knockdown of NR2F6 represses the EOC cancer stem cell phenotype and sensitizes EOC to cisplatin in vitro and in vivo. Our results further demonstrate that NR2F6 sustains activated Notch3 signaling, resulting in chemoresistance in EOC cells. Notably, NR2F6 acts as an informative biomarker to identify the population of EOC patients who are likely to experience a favorable objective response to gamma‐secretase inhibitors (GSI), which inhibit Notch signaling. Therefore, concurrent inhibition of NR2F6 and treatment with GSI and cisplatin‐based chemotherapy may be a novel therapeutic approach for NR2F6‐overexpressing EOC. In summary, we have, for the first time, identified an important role for NR2F6 in EOC cisplatin resistance. Our study suggests that GSI may serve as a potential targeted treatment for patients with NR2F6‐overexpressing EOC.

Nuclear orphan receptor NR2F6 as a safeguard against experimental murine colitis

ObjectiveNuclear receptors are known to regulate both immune and barrier functions in the GI tract. The nuclear orphan receptor NR2F6 has been shown to suppress the expression of proinflammatory cytokines...

Abstract 5349: Discovery of retinoic acids as low affinity inhibitors of the leukemia stem cell target NR2F6

Abstract Many human cancers are composed of heterogeneous populations of cells. While most cancer cells have a limited ability to divide, growth of the tumor is carried out by a small population of cancer stem cells, and hence represent the true target of effective anti-cancer therapy. To identify potential therapeutic targets, we previously studied the molecular signature of leukemia stem cells. Our work has led to the discovery of the orphan nuclear receptor NR2F6 (EAR-2) as a potential leukemia stem cell therapeutic target. We have previously shown that NR2F6 is over expressed in patients with acute leukemia and related bone marrow cancers, and that exogenous expression of NR2F6 in mouse bone marrow cells causes acute leukemia in vivo. Furthermore, silencing of NR2F6 expression in human and mouse-leukemia cell lines causes terminal differentiation and death by apoptosis. The discovery of the role of NR2F6 in leukemogenesis and the effects of silencing NR2F6 expression on leukemia cells suggests that this protein is a logical candidate as a therapeutic target in acute leukemia and related disorders for differentiation therapy. This forms the basis of the therapeutic concept that we wished to develop further, by identifying small molecule modulators of NR2F6 function. We hence developed a screening tool that was used to survey the ability of known nuclear receptor ligands to modulate NR2F6 activity. We constructed a hybrid receptor, comprising the NR2F6 ligand binding domain fused to the Gal4 DNA-binding domain. The hybrid receptor was co-transfected into HeLa cells along with a reporter plasmid in which a Gal4 regulatory sequence was placed upstream of a luciferase gene running off a constitutive promoter. The transfected cells were treated with candidate ligands and assayed for luciferase activity by luminometer. Of the 20 nuclear receptor ligands assessed none showed a significant effect at low concentrations (up to 1μM). However, we identifying 9-cis retinoic acid and, to a lesser extent, all-trans retinoic as low affinity ligands for NR2F6 since they were able to show a dose-dependent effect that we began to observe at 20μM. Retinoic acid and its analogues have long been studied in the context of chemoprevention and differentiation therapy based on their anti-proliferative, pro-apoptotic, and ability to induce differentiation. We have now commenced the search for a high affinity small molecular inhibitor of NR2F6 activity by screening commercially available libraries that contain compounds with similar chemical structure to retinoic acid using the luciferase-based assay developed here. Citation Format: Christine V. Ichim, Lap Shu Alan Chan, Dzana Dervovic, David Koos, Richard A. Wells, Thomas Ichim. Discovery of retinoic acids as low affinity inhibitors of the leukemia stem cell target NR2F6. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5349. doi:10.1158/1538-7445.AM2015-5349

A Proteomic Analysis Reveals That Snail Regulates the Expression of the Nuclear Orphan Receptor Nuclear Receptor Subfamily 2 Group F Member 6 (Nr2f6) and Interleukin 17 (IL-17) to Inhibit Adipocyte Differentiation *

Adipogenesis requires a differentiation program driven by multiple transcription factors, where PPARγ and C/EBPα play a central role. Recent findings indicate that Snail inhibits adipocyte differentiation in 3T3-L1 and murine mesenchymal stem cells (mMSC). An in-depth quantitative SILAC analysis of the nuclear fraction of Snail-induced alterations of 3T3-L1 cells was carried out. In total, 2251 overlapping proteins were simultaneously quantified in forward and reverse experiments. We observed 574 proteins deregulated by Snail1 using a fold-change ≥1.5, with 111 up- and 463 down-regulated proteins, respectively. Among other proteins, multiple transcription factors such as Trip4, OsmR, Nr2f6, Cbx6, and Prrx1 were down-regulated. Results were validated in 3T3-L1 cells and mMSC cells by Western blot and quantitative PCR. Knock-down experiments in 3T3-L1 cells demonstrated that only Nr2f6 (and Trip4 at minor extent) was required for adipocyte differentiation. Ectopic expression of Nr2f6 reversed the effects of Snail1 and promoted adipogenesis. Because Nr2f6 inhibits the expression of IL-17, we tested the effect of Snail on IL-17 expression. IL-17 and TNFα were among the most up-regulated pro-inflammatory cytokines in Snail-transfected 3T3-L1 and mMSC cells. Furthermore, the blocking of IL-17 activity in Snail-transfected cells promoted adipocyte differentiation, reverting Snail inhibition. In summary, Snail inhibits adipogenesis through a down-regulation of Nr2f6, which in turn facilitates the expression of IL-17, an anti-adipogenic cytokine. These results would support a novel and important role for Snail and Nr2f6 in obesity control.

NR2F6 (EAR-2) Is a Novel Leukemia Oncogene Whose Cellular Function Is to Regulate Terminal Differentiation of Erythrocytes at the Proerythroblast Stage

The leukemia stem cell model suggests that elucidation of the genes that regulate growth ability within the leukemia cell hierarchy will have important clinical relevance. We showed that the expression of NR2F6 (EAR-2), is greater in clonogenic leukemia single cells than in leukemia cells that do not divide, and that this gene is over-expressed in patients with acute myeloid leukemia and myelodysplastic syndrome. In vivo, overexpression of EAR-2 using a retroviral vector in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome with hypercellular bone marrow, increased blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice, or after secondary-transplantation. Interestingly, animals transplanted with bone marrow that over-expresses EAR-2 develop leukemia that is preceded by expansion of the stem cell compartment in the transplanted mice—suggesting that EAR-2 is an important regulator of hematopoietic stem cell differentiation. Here we report that over-expression of EAR-2 also has a profound effect on the differentiation of erythroid progenitor cells both in vitro and in vivo.Studies of the roles of EAR-2 in normal primary bone marrow cells in vitro showed that overexpression of EAR-2 profoundly impaired differentiation along the erythroid lineage. EAR-2 over-expressing bone marrow cells formed 40% fewer BFU-E colonies, but had greatly extended replating capacity in colony assays. While knockdown of EAR-2 increased the number of cells produced per BFU-E colony 300%.Normal mice transplanted with grafts of purified bone marrow cells that over-expressed EAR-2 developed a rapidly fatal leukemia characterized by pancytopenia, enlargement of the spleen, and infiltration of blasts into the spleen, liver and peripheral blood. Sick animals had profound reduction of peripheral blood cell counts, particularly anemia with a 55% reduction in hemoglobin levels. Anemia was evident even on gross inspection of the blood and the liver in EAR-2 overexpressing animals.Analysis of the leukemic cells revealed an erythroblastic morphology, with the immunophenotype lineageneg, CD71high, TER119med. Hence, we wondered weather EAR-2 caused leukemia by arresting erythroid progenitor cell differentiation. Examination of the bone marrow of pre-leukemic animals showed a four-fold increase in cells with a pro-erythroblastic immunophenotype (CD71highTER119med , region I), and a four-fold decrease in orthochromatophilic erythroblasts (CD71lowTER119high , region IV). We observed no change in the numbers of basophilic erythroblasts (CD71highTER119high , region II) or late basophilic and polychromatophilic erythroblasts (CD71medTER119high, region III). These data suggests that over-expression of EAR-2 blocks erythroid cell differentiation at the pro-erythroblastic stage.Since EAR-2 over-expressing recipients died within 4 week, we wanted to definitively test whether animals had compromised radioprotection. We showed that decreasing the size of the bone marrow graft, reduced survival of the EAR-2 over-expressing cohort by a week, but had no effect on control animals proving that EAR-2 over-expression has a profound effect on erythropoietic reconstitution in vivo.Mechanistically, we show that DNA binding is necessary for EAR-2 function, and that EAR-2 functions in an HDAC-dependent manner, regulating expression of several genes. Pre-leukemic pro-erythroblastic cells (CD71highTER119med) that over-expressed EAR-2 had lower expression of genes involved in erythroid differentiation such as GATA1, EBF1, inhibitor of NFKB (NFKBia), ETV6, CEBP/a, LMO2, and Nfe2, and increased expression of GATA2, GLI1, ID1 and PU.1 than GFP control pro-erythroblasts.These data establish that EAR-2 is a novel oncogene whose cellular function is to regulate terminal differentiation of erythroid cells at the proerythroblastic (CD71highTER119med) stage by deregulating gene expression necessary for erythroid differentiation. DisclosuresIchim:Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding. Koos:Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding.

Expression Profiling Of Nuclear Hormone Receptors In Myeloid Leukemia Reveals Potential Novel Drug Targets For Combination Therapy

Nuclear Hormone Receptors (NHRs) are a large superfamily of ligand dependent transcription factors regulating a plethora of genes involved in metabolism, growth and differentiation. Recent studies identified the overwhelming role played by NHR in determining hematopoiesis as well as HSC function and fate, suggesting that defective proliferation and maturation of progenitors in hematopoietic malignancies could be attributed to altered expression of these transcriptional regulators. NHRs are attractive but relatively unexplored drug targets and there is limited data on their expression profile in hematological malignancies. Our preliminary screening of AML cell lines and primary cells revealed differential expression of PPARg and RXRα among AML cell lines and decreased expression of these targets in resistant cell lines vs. sensitive ones (Fig.1a). We extended this study aiming to identify the expression pattern of all NHRs in myeloid malignancies, compare with ex-vivo chemoresistance and prognostic markers. [Display omitted] Myeloid leukemia cell lines AML(n=12) carrying AML-ETO, Inv-16, PML-RAR fusion transcript, NPM1 or FLT3 mutation and CML(n=8), pooled primary samples harboring only FLT3 ITD mutation and only NPM1 mutation, CD34+ cells and total cellular RNA samples from normal donors were included in the study. Bone marrow samples from AML patients and peripheral blood samples from healthy volunteers were collected after informed consent. PBSC from normal donors (n=7) were enriched for CD34+ cells by magnetic enrichment. RNA extraction followed by purification and cDNA synthesis were done as per manufacturer's recommendation. The expression of 42 NHRs and 42 co-regulators was profiled by qRT PCR using RT2 PCR Profiler array (SABiosciences, Germany). Expression of each gene was normalized to the average of 5 housekeeping genes (GAPDH, b-actin, b2-microglobulin, HGPRT & RPLP0) and differential expression was calculated by normalizing this with normal total RNA samples to determine fold change. Data normalization and differential gene expression were computed using SABiosciences web-based analysis software. Ex-vivo cytotoxicity to cytarabine and daunorubicin was analyzed using MTT assay and IC50 was calculated using Adapt software. Based on Ara-C and Dnr IC50, AML samples were categorized as sensitive or resistant (IC50<6.25µM and >6.25µM for Ara-C;<0.5µM and >0.5µM for Dnr). FLT3-ITD and NPM1 mutations were screened using PCR followed by GeneScan methods.Upon analysis we observed steroid receptors except PPARG, NR5A1, RXRG, NR1D2, glucocorticoid receptors, androgen receptors and their co-regulatory molecules were expressed across all cell lines. In contrast, expression of certain NHRs such as NR2E3, NR1I2, NR1H4, NR0B1, NR0B2, ESRRG, ESRRB, NR1D1 and the co-regulator HDAC7 were less or at undetectable levels, while estrogen receptors and its co-regulatory molecules except PPARGC1B, steroid receptors HNF4A, NR2F1, NR2F2, RARG and NR3C2 were moderately expressed across the cell lines (Fig.1b). Analysis also revealed significantly reduced expression of ESR2, ESRRG, NR2F2, NR2F6 and THRB and increased PPARG in AML compared to CML cell lines. Comparison between CD34+ progenitor population and well differentiated hematopoietic cells demonstrated >6 fold change in NR4A1 expression. Likewise, expression of several differentiation markers NR1H3, NR1D2, RORA, RORB, RARA and VDR were elevated in cells with increased degree of maturation. The receptor inducing terminal differentiation of erythrocytes THRA, had lesser expression (<60 Fold) in erythroleukemic cell lines K562, HEL and LAMA-84 compared to normal (Fig.1c). In Ara-C resistant cell lines, expression of NR1I3, AHR, and HNF4A were up-regulated while PPARG and RXRA were down-regulated. NPM1 positive cell lines and patient samples had increased expression of PPARG, PPARGC1A, NOTCH2, NR1H3 and NR2F2 than the FLT3 mutated group (Fig.1d) and similar expression of PPARG and RXRA was validated in NPM mutated primary AML blasts (Fig.1e).Our comprehensive analysis of NHRs and its co regulatory molecules provides us with insights on NHR expression on differentiation and drug resistance across myeloid leukemia cell lines with various genetic and molecular characteristics. This could be further explored to identify potential novel drug targets to be used as combination therapy in myeloid leukemias to overcome drug resistance. Disclosures:No relevant conflicts of interest to declare.

Abstract 1588: NR2F6, a novel regulator of stem cell maintenance, is necessary and sufficient for initiation of acute leukemia

Abstract The mechanisms that instruct hematopoietic stem cells (HSCs) to choose between self-renewal and differentiation remain elusive. These mechanisms are of particular interest with respect to acute myeloid leukemia (AML) where leukemia stem cell self-renewal is a tantalizing therapeutic target. The orphan nuclear receptor NR2F6 has no previously identified role in hematopoiesis. In a screen of the transcription profiles of single AML cells we found that NR2F6 mRNA expression is 4.6 fold higher in clonogenic vs. non-clonogenic cells. We showed that NR2F6 is expressed highly in long-term HSCs, with expression of NR2F6 generally declining with progenitor cell differentiation. Interestingly, expression of NR2F6 was deregulated in patients with myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia, and AML compared to normal bone marrow (BM). Animals transplanted with retrovirally transduced NR2F6 marked BM were profoundly pancytopenic and rapidly developed AML (median survival of four weeks). Pre-leukemic recipients showed a &amp;gt;10 fold increase in the proportion and absolute number of lineage− Sca1+ cKit+ (LSK) cells. There was also a striking increase (15-18 fold) in the number of CD49b− LSK and CD34− LSK cells, indicating expansion of the long-term HSC population. NR2F6-overexpressing HSCs had increased competitive repopulation ability when transplanted into lethally irradiated hosts. Transplant chimeras reconstituted with a mixture of wild type and NR2F6-overexpressing cells develop key features of MDS including hypercellular BM, increase in blasts, abnormal localization of immature precursors, morphological dysplasia, and eventual progression to AML. In colony forming cell assays, BM from recipients of NR2F6-overexpressing HSCs showed a significant reduction in the number of primary erythroid and myeloid colonies, consistent with the notion that NR2F6 inhibits maturation of normal BM, while augmented serial replating of methylcellulose cultures indicates enhanced in vitro self-renewal ability. Our data suggests that the modulation of NR2F6 function using an antagonistic ligand may be a novel strategy for the targeting of MDS and AML stem cells. As proof of the principle we observed that silencing of NR2F6 using shRNA in two distinct hematopoietic cell lines (U937 human myelomonocytic cells and 32D murine multipotent hematopoietic cells) resulted in spontaneous differentiation and apoptosis. Furthermore, silencing of NR2F6 in murine HSCs rapid depleted LSK cells in ex vivo culture showing that modulation of NR2F6 can force the differentiation of HSCs. Taken together these data suggest that NR2F6 may contribute to the pathogenesis of MDS and AML by affecting HSC homeostasis and progenitor cell differentiation. These data raise the exciting possibility that natural or synthetic antagonist ligands for NR2F6 can be found that may act as anti-leukemic therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1588.

The Orphan Nuclear Receptor NR2F6 Is a Novel Negative Regulator of T-Cell Development.

Abstract 915The orphan nuclear receptor NR2F6 is a mammalian homologue of the Drosophila seven-up gene that plays key roles in decisions of cell fate in neuroblast and retinal cells. We have previously described a novel role for NR2F6 in decisions of cell fate of mammalian haematopoietic cells of the myeloid cell lineage. We have shown that over-expression of NR2F6 in bone marrow cells impairs differentiation and extends the proliferative capacity of myeloid and early progenitor cells eventually leading to acute myeloid leukaemia (AML), while silencing of NR2F6 expression in AML cell lines causes terminal differentiation and apoptosis. A role of NR2F6 in lymphopoiesis has yet to be identified. Here we describe for the first time a role for NR2F6 in the specification of lymphoid cells. NR2F6 expression is heterogeneous throughout the haematopoietic hierarchy, with expression being highest in long-term repopulating HSCs and generally declining with the differentiation of progenitor cells. We report that over-expression of NR2F6 abrogates the developmental program necessary for T-cell lymphopoiesis. We assessed the effects of NR2F6 on lymphopoiesis in vivo by competitive bone marrow transplantation of NR2F6-IRES-GFP or GFP retrovirally transduced grafts (n=43). Competitive repopulation of lethally irradiated murine hosts with GFP transduced bone marrow cells resulted in successful engraftment and T-cell development, with GFP+ T-cells present in the thymus, and periphery at rates comparable to the percent marked cells in the original graft. However over-expression of NR2F6 placed developing T-cells at a dramatic competitive disadvantage. Six weeks post transplant the proportion of CD3+ cells derived from NR2F6 transduced bone marrow cells was greatly diminished relative to control (more than 10 fold), while at 12 weeks post-transplant we observed an abrogation of CD3+ cells derived from NR2F6 transduced T-cells (with the percentage of NR2F6 transduced CD3+ cells being comparable to staining with IgG control) in both the thymus and periphery. This stark competitive disadvantage was observed in all recipients of NR2F6 transduced grafts. We confirmed that this is not a phenomenon specific to the marker CD3 by analysing a portion of the animals for expression of CD4 and CD8, which again showed a lack of mature t-cells. In a second series of bone marrow transplants, cells transduced with NR2F6 or GFP were purified by fluorescence-activated cell sorting and grafts of 100% transduced cells were transferred by tail vein injection into lethally irradiated recipients. Animals transplanted with NR2F6 transduced bone marrow demonstrated a gross decrease in their thymic size and cellularity (∼10 fold decrease, n=17). Furthermore, the thymus of NR2F6 transduced animals contained a larger proportion of non-transduced, GFP negative residual haematopoietic cells than the vector control animals, corroborating the competitive disadvantage that NR2F6 transduced bone marrow cells face in the thymus. As observed in our previous experiments these animals demonstrated a gross reduction in the proportion of CD3+ cells in the thymus, spleen, lymph nodes and peripheral blood. To rule out the possibility that over-expression of NR2F6 is preventing the trafficking of progenitor cells to the thymus we differentiated NR2F6 or GFP transduced haematopoietic stem cells (lin-,c-kit+,sca-1+) into T-cells in vitro on OP9-DL1 cells. We observed a drastic reduction in the number of cells generated from NR2F6 transduced stem/progenitor cells (>50 fold at day 23), suggesting that expression of NR2F6 greatly impairs T-cell development. Mechanistically, others have shown that NR2F6 functions as a transcriptional repressor inhibiting the transactivating ability of genes such as Runx1. We conjecture that in lymphoid progenitors as well NR2F6 functions as a transcriptional repressor preventing the activation of pathways necessary for T-cell survival, proliferation and lymphopoiesis. Taken together, these data establish that the orphan nuclear receptor NR2F6 is a novel negative regulator of T-cell lymphopoiesis, and demonstrate that down-regulation of NR2F6 is important for the survival and proliferation of T-cell progenitors. Disclosures:No relevant conflicts of interest to declare.

NR2F6, the Mammalian Homologue of Drosophila Seven Up, Can Initiate Myelodysplasia and Acute Leukemia.

A central conundrum in the biology of myelodysplastic syndrome (MDS) is the observation that haematopoietic cells derived from the MDS clone have an increased disposition to apoptosis, yet gain clonal dominance in the bone marrow (BM). One explanation for this apparent paradox could be that MDS stem cells attain clonal dominance by acquiring an augmented probability of self-renewal. We therefore sought to identify novel genes associated with self-renewal that are overexpressed in both MDS and AML. By analyzing the growth of individual clonal siblings derived from low passage cultures of OCI-AML4 we found the expression of the orphan nuclear receptor NR2F6 to be four-fold higher in leukaemia cells with high proliferative capacity, compared to those which spontaneously lose proliferative ability. NR2F6 mRNA transcripts were more abundant in patients (n=37) with MDS, CMML, and AML compared to normal BM (n=16; p<0.0003 MDS, <0.03 CMML, <0.0009 AML), validating the clinical relevance of this gene and suggesting that deregulation of NR2F6 might be an early event in disease progression. We studied the effects of NR2F6 on differentiation and proliferation in vitro in the monoblastic cell line U937. Induction of differentiation of U937 cells resulted in a decrease in NR2F6 expression, while forced expression of NR2F6 reduced the doubling time of U937 cells, and inhibited induction of differentiation by retinoic acid. We next studied the effects of NR2F6 on the behaviour of primary haematopoietic cells. Overexpression of NR2F6 in murine bone marrow cells resulted in a significant reduction in the number of BFU-E and CFU-GM and the size of erythroid and myeloid colonies, consistent with the idea that NR2F6 inhibits maturation of normal BM. These results were corroborated by immunophenotyping of BM cultured in suspension, which showed that NR2F6 overexpression resulted in a significant reduction in mature monocytes and granulocytes. In addition, serial replating of haematopoietic colonies revealed greatly extended replating potential in NR2F6-overexpressing BM, consistent with augmented self-renewal capacity. Finally, we assessed the effects of NR2F6 on haematopoiesis in vivo by bone marrow transplantation. Competitive repopulation of lethally irradiated murine hosts with NR2F6-transduced bone marrow cells resulted in successful engraftment and augmented self-renewal as evidenced by increased engraftment in some recipients as well as increased colony formation in serial replating experiments. Recipients of NR2F6-transduced grafts had hypercellular BM, with abnormal localization of immature precursors (ALIP) and an increase in the percentage of blasts. Strikingly, ∼30% of recipients of secondary transplants of NR2F6-transduced bone marrow cells developed acute leukaemia, characterized by infiltration by blasts of bone marrow, peripheral blood, spleen and liver, and by haematopoietic failure. These data establish the importance of NR2F6 in the regulation of haematopoietic cell self-renewal and differentiation. This suggests that deregulated expression of NR2F6 is an important step in the pathogenesis of human MDS and AML and supports the hypothesis that acquisition of augmented self-renewal capacity helps the MDS stem cell gain clonal dominance. Furthermore, the NR2F6 transplant chimera system provides a mouse model of MDS, and the transformation of MDS to AML.

Characterization of a Hierarchy of Proliferative Ability in AML: Identification of a Role for NR2F6 in the Maintenance of the Undifferentiated State.

AML cells are heterogeneous in their capacity to proliferate and form colonies in vitro. Determination of the genes whose expression is necessary for clonogenicity has been hindered by the inability to isolate pure populations of AML cells with defined proliferative abilities. By analyzing the growth of clonal siblings we established that the hierarchical organization of AML can be resolved into distinct strata, permitting the use of clonal siblings as biological reporters of the transcriptional signature of single cells with defined proliferative abilities. We then analyzed low passage cultures of the cell line OCI-AML4. Clones consisting of four cells were micromanipulated so that a single cell was sampled for global RT-PCR while its three clonal siblings served as reporters of clonogenicity. By microarray analysis we found the orphan nuclear receptor NR2F6 to be expressed four-fold lower in leukaemia single cells that spontaneously lose proliferative ability, compared to single cells with greater proliferative capacity. NR2F6 is a poorly characterized member of the COUP transcription factor family. We observed that NR2F6 is overexpressed in AML, CMML and MDS relative to normal bone marrow (BM). We then assessed expression of NR2F6 in U937 leukaemia cells induced to differentiate with a variety of induction agents. All differentiation agents induced significant decreases in NR2F6 expression. To characterize the effect of forced expression of NR2F6 we transduced U937 cells with a retrovirus encoding either NR2F6 or EGFP. Forced expression of NR2F6 reduced the doubling time of these populations, and inhibited retinoic acid induction of U937 cell differentiation. NR2F6 overexpressing cells did not acquire expression of the maturation marker CD11b nor were capable of reducing nitroblue tetrazolium, indicating functional maturity. Assessment of DNA content and analysis of growth kinetics revealed that NR2F6 overexpressing cells did not undergo the proliferative arrest associated with terminally differentiating U937 cells. We then investigated the role of NR2F6 in normal haematopoiesis in the C57/BL6 and Balb/c strains of mice. In both strains we observed that overexpression of NR2F6 resulted in a significant reduction in BFU-E and CFU-GM colony numbers, and colony size. These data are consistent with the notion that NR2F6 inhibits maturation of normal BM. More specifically, analysis of the immunophenotype of BM cultured in suspension for 10 days showed that NR2F6 overexpression resulted in a significant reduction in monocytes, and an increase in mast cells. An excess of mast cells was also observed in cytospin preparations of CFU-GM colonies. Additionally, we have demonstrated that overexpression of NR2F6 augments the in vitro self-renewal ability of BM, using serial replating of semi-solid cultures. Competitive BM transplantation experiments were employed to confirm the role of NR2F6 on stem cell self-renewal and differentiation in vivo. While these investigations are in progress, progenitor assays and analysis of serial-replating in all animals examined have recapitulate our ex vivo observations. Overall, these data establish the importance of NR2F6 in the regulation of normal and leukaemic haematopoietic cell self-renewal and differentiation. Furthermore, our results establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy.