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Abstract Background HOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies. Results In this study, we found high expression of the HOXD9 gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of HOXD9 in gliomas, we silenced its expression in the glioma cell line U87 using HOXD9-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that HOXD9 contributes to both cell proliferation and/or cell survival. The HOXD9 gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. HOXD9 siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of HOXD9 in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs). Conclusions Our results suggest that HOXD9 may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.

Abstract Background Tumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies. Methods We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas. Results A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide. Conclusions Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.

Neuroblastoma, a rare embryonic tumor arising from neural crest development, is responsible for 15% of pediatric cancer-related deaths. Recently, several single-cell transcriptome studies were performed on neuroblastoma patient samples to investigate the cell of origin and tumor heterogeneity. However, these individual studies involved a small number of tumors and cells, limiting the conclusions that could be drawn. To overcome this limitation, we integrated seven single-cell or single-nucleus datasets into a harmonized cell atlas covering 362,991 cells across 61 patients. We use this atlas to decipher the transcriptional landscape of neuroblastoma at single-cell resolution, revealing associations between transcriptomic profiles and clinical outcomes within the tumor compartment. In addition, we characterize the complex immune-cell landscape and uncover considerable heterogeneity among tumor-associated macrophages. Finally, we showcase the utility of our atlas as a resource by expanding it with additional data and using it as a reference for data-driven cell-type annotation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Transcriptional cofactors of the ETO family are recurrent fusion partners in acute leukemia. We characterized the ETO2 regulome by integrating transcriptomic and chromatin binding analyses in human erythroleukemia xenografts and controlled ETO2 depletion models. We demonstrate that beyond its well-established repressive activity, ETO2 directly activates transcription of MYB, among other genes. The ETO2-activated signature is associated with a poorer prognosis in erythroleukemia but also in other acute myeloid and lymphoid leukemia subtypes. Mechanistically, ETO2 colocalizes with EP300 and MYB at enhancers supporting the existence of an ETO2/MYB feedforward transcription activation loop (e.g., on MYB itself). Both small-molecule and PROTAC-mediated inhibition of EP300 acetyltransferases strongly reduced ETO2 protein, chromatin binding, and ETO2-activated transcripts. Taken together, our data show that ETO2 positively enforces a leukemia maintenance program that is mediated in part by the MYB transcription factor and that relies on acetyltransferase cofactors to stabilize ETO2 scaffolding activity., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). HemaSphere published by John Wiley & Sons Ltd on behalf of European Hematology Association.)

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity., (© 2023. The Author(s).)

Pediatric acute myeloid leukemia expressing the ETO2::GLIS2 fusion oncogene is associated with dismal prognosis. Previous studies have shown that ETO2::GLIS2 can efficiently induce leukemia development associated with strong transcriptional changes but those amenable to pharmacological targeting remained to be identified. By studying an inducible ETO2::GLIS2 cellular model, we uncovered that de novo ETO2::GLIS2 expression in human cells led to increased CASP3 transcription, CASP3 activation, and cell death. Patient-derived ETO2::GLIS2 + leukemic cells expressed both high CASP3 and high BCL2. While BCL2 inhibition partly inhibited ETO2::GLIS2 + leukemic cell proliferation, BH3 profiling revealed that it also sensitized these cells to MCL1 inhibition indicating a functional redundancy between BCL2 and MCL1. We further show that combined inhibition of BCL2 and MCL1 is mandatory to abrogate disease progression using in vivo patient-derived xenograft models. These data reveal that a transcriptional consequence of ETO2::GLIS2 expression includes a positive regulation of the pro-apoptotic CASP3 and associates with a vulnerability to combined targeting of two BCL2 family members providing a novel therapeutic perspective for this aggressive pediatric AML subgroup., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: High-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies., Methods: We combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays., Results: We show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes., Conclusions: Our study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Super Enhancers (SEs) are clusters of regulatory elements associated with cell identity and disease. However, whether these elements are induced by oncogenes and can regulate gene modules cooperating for cancer cell transformation or maintenance remains elusive. To address this question, we conducted a genome-wide CRISPRi-based screening of SEs in ETO2-GLIS2 + acute megakaryoblastic leukemia. This approach revealed SEs essential for leukemic cell growth and survival that are induced by ETO2-GLIS2 expression. In particular, we identified a de novo SE specific of this leukemia subtype and regulating expression of tyrosine kinase-associated receptors KIT and PDGFRA . Combined expression of these two receptors was required for leukemic cell growth, and CRISPRi-mediated inhibition of this SE or treatment with tyrosine kinase inhibitors impaired progression of leukemia in vivo in patient-derived xenografts experiments. Our results show that fusion oncogenes, such as ETO2-GLIS2, can induce activation of SEs regulating essential gene modules synergizing for leukemia progression.

Neuroblastoma, a pediatric cancer of the peripheral sympathetic nervous system, is characterized by an important clinical heterogeneity, and high-risk tumors are associated with a poor overall survival. Neuroblastoma cells may present with diverse morphological and biochemical properties in vitro, and seminal observations suggested that interconversion between two phenotypes called N-type and S-type may occur. In 2017, two main studies provided novel insights into these subtypes through the characterization of the transcriptomic and epigenetic landscapes of a panel of neuroblastoma cell lines. In this review, we focus on the available data that define neuroblastoma cell identity and propose to use the term noradrenergic (NOR) and mesenchymal (MES) to refer to these identities. We also address the question of transdifferentiation between both states and suggest that the plasticity between the NOR identity and the MES identity defines a noradrenergic-to-mesenchymal transition, reminiscent of but different from the well-established epithelial-to-mesenchymal transition.

Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells., (© 2020 by The American Society of Hematology.)

The nuclear receptor binding SET domain protein 1 (NSD1) is recurrently mutated in human cancers including acute leukemia. We show that NSD1 knockdown alters erythroid clonogenic growth of human CD34 + hematopoietic cells. Ablation of Nsd1 in the hematopoietic system of mice induces a transplantable erythroleukemia. In vitro differentiation of Nsd1 -/- erythroblasts is majorly impaired despite abundant expression of GATA1, the transcriptional master regulator of erythropoiesis, and associated with an impaired activation of GATA1-induced targets. Retroviral expression of wildtype NSD1, but not a catalytically-inactive NSD1 N1918Q SET-domain mutant induces terminal maturation of Nsd1 -/- erythroblasts. Despite similar GATA1 protein levels, exogenous NSD1 but not NSD N1918Q significantly increases the occupancy of GATA1 at target genes and their expression. Notably, exogenous NSD1 reduces the association of GATA1 with the co-repressor SKI, and knockdown of SKI induces differentiation of Nsd1 -/- erythroblasts. Collectively, we identify the NSD1 methyltransferase as a regulator of GATA1-controlled erythroid differentiation and leukemogenesis.

Fusion oncogenes are prevalent in several pediatric cancers, yet little is known about the specific associations between age and phenotype. We observed that fusion oncogenes, such as ETO2-GLIS2 , are associated with acute megakaryoblastic or other myeloid leukemia subtypes in an age-dependent manner. Analysis of a novel inducible transgenic mouse model showed that ETO2-GLIS2 expression in fetal hematopoietic stem cells induced rapid megakaryoblastic leukemia whereas expression in adult bone marrow hematopoietic stem cells resulted in a shift toward myeloid transformation with a strikingly delayed in vivo leukemogenic potential. Chromatin accessibility and single-cell transcriptome analyses indicate ontogeny-dependent intrinsic and ETO2-GLIS2 -induced differences in the activities of key transcription factors, including ERG, SPI1, GATA1, and CEBPA. Importantly, switching off the fusion oncogene restored terminal differentiation of the leukemic blasts. Together, these data show that aggressiveness and phenotypes in pediatric acute myeloid leukemia result from an ontogeny-related differential susceptibility to transformation by fusion oncogenes. SIGNIFICANCE: This work demonstrates that the clinical phenotype of pediatric acute myeloid leukemia is determined by ontogeny-dependent susceptibility for transformation by oncogenic fusion genes. The phenotype is maintained by potentially reversible alteration of key transcription factors, indicating that targeting of the fusions may overcome the differentiation blockage and revert the leukemic state. See related commentary by Cruz Hernandez and Vyas, p. 1653 . This article is highlighted in the In This Issue feature, p. 1631 ., (©2019 American Association for Cancer Research.)

Circular RNAs constitute a unique class of RNAs whose precise functions remain to be elucidated. In particular, cancer-associated chromosomal translocations can give rise to fusion circular RNAs that play a role in leukemia progression. However, how and when fusion circular RNAs are formed and whether they are being selected in cancer cells remains unknown. Here, we used CRISPR/Cas9 to generate physiological translocation models of NPM1-ALK fusion gene. We showed that, in addition to generating fusion proteins and activating specific oncogenic pathways, chromosomal translocation induced by CRISPR/Cas9 led to the formation of de novo fusion circular RNAs. Specifically, we could recover different classes of circular RNAs composed of different circularization junctions, mainly back-spliced species. In addition, we identified fusion circular RNAs identical to those found in related patient tumor cells providing evidence that fusion circular RNAs arise early after chromosomal formation and are not just a consequence of the oncogenesis process., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The ETO2-GLIS2 fusion oncoprotein is associated with poor prognosis pediatric acute megakaryoblastic leukemia. Recently, we observed that ETO2-GLIS2 controls enhancers activity at genes regulating haematopoietic progenitor self-renewal and differentiation toward the megakaryocytic lineage. We also showed that targeting ETO2-GLIS2 complex stability inhibits these properties and may represent a novel therapeutic strategy.

Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Glioblastomas, the most common form of primary brain tumors, are the fourth cause of death by cancer in adults. Increasing evidences suggest that glioblastoma resistance to existing radio- and chemotherapies rely on glioblastoma stem cells (GSCs). GSCs are endowed with a unique combination of stem-like properties alike to normal neural stem cells (NSCs), and of tumor initiating properties. The natural polyphenol resveratrol is known to exert opposite actions on neural cells according to their normal or cancerous status. Here, we used resveratrol to explore the molecular mechanisms differing between GSCs and NSCs. We observed a dual action of resveratrol on GSCs: resveratrol blocked GSC proliferation up to 150 μM and induced their necrosis at higher doses. On the opposite, resveratrol had no effect on NSC behavior. To determine the mechanisms underlying resveratrol effects, we focused our attention on the family of NAD-dependent deacetylases sirtuins (SIRT). A member of this family, SIRT1, has been repetitively shown to constitute a preferential resveratrol target, at least in normal cells. Western blot analysis showed that SIRT1 and SIRT3 were expressed by both GSCs and NSCs whereas SIRT2 expression was restricted to GSCs. Pharmacological blockade of SIRT2 activity or down-regulation of SIRT2 expression with siRNAs counteracted the inhibitory effect of resveratrol on cell proliferation. On the contrary, inhibition of SIRT2 activity or expression did not counteract GSC necrosis observed in presence of high doses of resveratrol. Our results highlight SIRT2 as a novel target for altering GSC properties.

Glioblastomas are the most frequent adult primary brain tumors that still remain fatal despite major clinical efforts. As in other solid tumors, populations of glioblastoma stem-like cells (GSCs) endowed with tumor initiating and therapeutic resistance properties have been identified. Glioblastomas are highly vascularized tumors resulting in a rich dialog between GSCs and endothelial cells. In one direction, endothelial cells and their secreted proteins are able to sustain GSC properties while, in turn, GSCs can promote neoangiogenesis, modulate endothelial cell functions and may even transdifferentiate into endothelial cells. Accordingly, targeting tumor vasculature seems a promising issue despite incomplete and transient results obtained from anti-vascular endothelial growth factor therapeutic trials. Recent findings of novel GSC-secreted molecules with pro-angiogenic properties (Semaphorin 3A, hepatoma-derived growth factor) open the path to the design of a concerted attack of glioblastoma vasculature that could overcome the development of resistance to single-targeted therapies while keeping away the toxicity of the treatments., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Presence in glioblastomas of cancer cells with normal neural stem cell (NSC) properties, tumor initiating capacity, and resistance to current therapies suggests that glioblastoma stem-like cells (GSCs) play central roles in glioblastoma development. We cultured human GSCs endowed with all features of tumor stem cells, including tumor initiation after xenograft and radio-chemoresistance. We established proteomes from four GSC cultures and their corresponding whole tumor tissues (TTs) and from human NSCs. Two-dimensional difference gel electrophoresis and tandem mass spectrometry revealed a twofold increase of hepatoma-derived growth factor (HDGF) in GSCs as compared to TTs and NSCs. Western blot analysis confirmed HDGF overexpression in GSCs as well as its presence in GSC-conditioned medium, while, in contrast, no HDGF was detected in NSC secretome. At the functional level, GSC-conditioned medium induced migration of human cerebral endothelial cells that can be blocked by anti-HDGF antibodies. In vivo, GSC-conditioned medium induced neoangiogenesis, whereas HDGF-targeting siRNAs abrogated this effect. Altogether, our results identify a novel candidate, by which GSCs can support neoangiogenesis, a high-grade glioma hallmark. Our strategy illustrates the usefulness of comparative proteomic analysis to decipher molecular pathways, which underlie GSC properties., (Copyright © 2012 AlphaMed Press.)

Zanthoxylum chiloperone var. angustifolium root bark was studied with the aim of finding novel molecules able to overcome cancer stem cell chemoresistance. Purification of a methanol-soluble extract resulted in the isolation of a known pyranocoumarin, trans-avicennol (1). Compound 1 demonstrated antiproliferative activity on glioma-initiating cells, whereas it was inactive on human neural stem cells. trans-Avicennol (1) activated the MAPK/ERK pathway and was also evaluated for its ability to inhibit the enzyme indoleamine-2,3-dioxygenase.

PcG and TrxG proteins mostly with opposite transcriptional activities play key roles in normal and malignant development. In this issue of Cancer Cell, Tan et al. report an unexpected collaboration between CBX8 and MLL-AF9 in leukemia, revealing a far more complicated functional crosstalk between these master epigenetic regulators in oncogenesis., (2011 Elsevier Inc. All rights reserved.)

Gliomas are primary tumors of the human central nervous system with unknown mechanisms of progression. Isocitrate dehydrogenase-1 (IDH1) mutation is frequent in diffuse gliomas such as oligodendrogliomas. To gain insights into the physiopathology of oligodendrogliomas that have a better prognosis than other diffuse gliomas, we combined microdissection, 2-D DIGE and MS/MS focusing on proteome alterations associated with IDH1 mutation. We first compared tumor tissues (TT) and minimally infiltrated parenchymal tissues (MIT) of four IDH1-mutated oligodendrogliomas to verify whether proteins specific to oligodendroglioma tumor cells could be identified from one patient to another. This study resulted in identification of 68 differentially expressed proteins, with functions related to growth of tumor cells in a nervous parenchyma. We then looked for proteins distinctly expressed in TT harboring either mutant (oligodendrogliomas, n=4) or wild-type IDH1 (oligodendroglial component of malignant glio-neuronal tumors, n=4). This second analysis resulted in identification of distinct proteome patterns composed of 42 proteins. Oligodendrogliomas with a mutant IDH1 had noteworthy enhanced expression of enzymes controlling aerobic glycolysis and detoxification, and anti-apoptosis proteins. In addition, the mutant IDH1 migrated differently from the wild-type IDH1 form. Comparative proteomic analysis might thus be suitable to identify proteome alterations associated with a well-defined mutation., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Glioma stem-cells are associated with the brain vasculature. However, the way in which this vascular niche regulates stem-cell renewal and fate remains unclear. Here, we show that factors emanating from brain endothelial cells positively control the expansion of long-term glioblastoma stem-like cells. We find that both pharmacological inhibition of and RNA interference with the mammalian target of rapamycin (mTOR) pathway reduce their spheroid growth. Conversely, the endothelial secretome is sufficient to promote this mTOR-dependent survival. Thus, interfering with endothelial signals might present opportunities to identify treatments that selectively target malignant stem-cell niches.

Background: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs), thought to account for tumorigenesis and therapeutic resistance, have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined., Methodology/principal Findings: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples, regardless of their histopathologies and grades of malignancy (57% of embryonal tumors, 57% of low-grade gliomas and neuro-glial tumors, 70% of ependymomas, 91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (>7 self-renewals), whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities, the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05, chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013, log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers, the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting., Conclusions/significance: In brain tumors affecting adult patients, TSCs have been isolated only from high-grade gliomas. In contrast, our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors.

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. The ASIC3 subunit is largely expressed in the peripheral nervous system, where it contributes to pain perception and to some aspects of mechanosensation. We report here a PDZ-dependent and protein kinase C-modulated association between ASIC3 and the Na+/H+ exchanger regulatory factor-1 (NHERF-1) adaptor protein. We show that NHERF-1 and ASIC3 are co-expressed in dorsal root ganglion neurons. NHERF-1 enhances the ASIC3 peak current in heterologous cells, including F-11 dorsal root ganglion cells, by increasing the amount of channel at the plasma membrane. Perhaps more importantly, we show that the plateau current of ASIC3 can be dramatically increased (10-30-fold) by association with NHERF-1, leading to a significant sustained current at pH 6.6. In the presence of NHERF-1, the ASIC3 subcellular localization is modified, and the channel co-localizes with ezrin, a member of the ezrin-radixin-moesin family of actin-binding proteins, providing the first direct link between ASIC3 and the cortical cytoskeleton. Given the importance of the ASIC3 sustained current in nociceptor excitability, it is likely that NHERF-1 participates in channel functions associated with nociception and mechanosensation.