Study of XPO1 abnormalities in B cell lymphomas

L'exportine-1 (ou XPO1) joue un rôle clé dans le transport de nombreux ARN et près de 200 protéines cargos. La mutation « hot-spot » XPO1-E571K est présente chez près de 25% des patients atteints par le lymphome primitif du médiastin (PMBL) et la forme classique du lymphome de hodgkin (cHL), et à plus faible fréquence dans la leucémie lymphoïde chronique (LLC) (3%). Des mutations touchant la voie JAK2/STAT6 sont aussi retrouvées dans le PMBL et le cHL. C’est pourquoi nous avons étudié l’implication de XPO1 dans la voie JAK2/STAT6. Nous avons montré que STAT6 est une protéine cargo de XPO1 par les technique immunofluorescence et PLA (Proximity Ligation Assay). Nous avons montré que le traitement au selinexor induit une accumulation nucléaire de STAT6 sauvage et mutée et que STAT6 peut interagir avec les formes mutée et sauvage de XPO1.Afin de rechercher l’impact fonctionnel de la mutation E571K de XPO1 nous avons comparé plusieurs paramètres physiologiques entre les trois lignées de PMBL. Aucune différence n’a été observée. En effet, la présence de l’amplification de l’allèle sauvage dans la lignée présentant la mutation E571K (MedB1) pourrait masquer les éventuels effets de la mutation. De plus, dans la cohorte de patients que nous avons étudiée la mutation n’est jamais retrouvée à l’état homozygote ou hémizygote indiquant l’importance du dosage génique. Nos expériences de CRISPR-Cas9 sur la lignée U2940 dont le gène XPO1 est sauvage ont confirmé notre hypothèse. De manière intéressante, la présence de la mutation E571K modifie l’affinité de XPO1 pour le selinexor. En effet, le KO de l’allèle muté dans la lignée de cHL UH-01 rend les cellules moins sensibles au selinexor. Nous avons conclu que la balance entre l’allèle sauvage et l’allèle muté est un élément clé pour définir les propriétés oncogéniques de XPO1. Dans une étude préliminaire, une étude protéomique dans les lignées PMBL indique que XPO1-E571K est liée à l’importine-β1 ce qui pourrait modifier la localisation subcellulaire de XPO1.Nous avons montré que la localisation cytoplasmique de cycline D1 contrôle le mécanisme d’invasion et de migration dans cellules de lymphome à cellules du manteau (MCL). Cycline D1 étant une protéine cargo de XPO1 nous avons recherché d’éventuelles anomalies de XPO1 dans les lignées de MCL. Aucune anomalie d’expression de localisation ou de fonction de n’a été observée. Il pourrait être intéressant de déterminer la localisation subcellulaire de cycline D1 au moment du diagnostic afin de proposer une meilleure prise en charge pour les patients. De plus, bien que le selinexor soit toujours en essai clinique, son utilisation pour le traitement du MCL pourrait être envisagée dans les cas les plus agressif où cycline D1 est cytoplasmique.Exportin-1 (or XPO1) plays a key role in the nuclear export of several RNAs and more than 200 proteins. The XPO1-E571K "hot-spot" mutation is present in nearly 25% of patients with primary mediastinal B-cell lymphoma (PMBL), and the classical form of Hodgkin lymphoma (cHL) but at a lower frequency (3%) in chronic lymphocytic leukemia (CLL). Mutations affecting the JAK2/STAT6 pathway are common in PMBL and cHL. We first studied the role of XPO1 in the nuclear export of STAT6 in PMBL cell lines. Using immunofluorescence and proximity ligation assay (PLA) techniques, we showed that STAT6 is a cargo of XPO1. We also showed that a selinexor treatment induced a nuclear accumulation of wild-type and mutant STAT6 whatever the XPO1 status.In order to investigate the functional impact of the XPO1-E571K mutation, we compared several physiological parameters between the three PMBL cell lines bearing or not the mutation. No differences were observed despite the expression of the XPO1E571K allele. However, in the cell line harboring the XPO1 mutation (MedB1), the wild-type (wt) allele was amplified possibly masking the effects of the mutation. In addition, in the cohort of patients we studied, the mutation was never found in the homozygous or hemizygous state indicating the importance of the gene dosage. CRISPR-Cas9 experiments allowing the introduction of the mutation in the U2940 wt cell line confirmed our hypothesis. Interestingly, the presence of the E571K mutation changed the affinity of XPO1 for selinexor. Indeed, the knockout of the mutated allele in the cHL UH-01 line decreased its sensitivity to selinexor. We concluded that the balance between the wt and the mutated alleles is a key element in defining the oncogenic properties of XPO1. In a preliminary study, we conducted a proteomic analysis to identify XPO1E571K partners in the PMBL lines. Our results showed that XPO1E571K interacts with the importin-β1 which could modify the subcellular localization of XPO1.Mantle cell lymphoma (MCL) cells are characterized by the overexpression of cyclin D1. Cyclin D1 being an XPO1 cargo protein, we looked for possible XPO1 abnormalities in several MCL cell lines. No abnormalities in the expression, localization neither function of XPO1 were observed. But, we showed that the cytoplasmic portion of cyclin D1 controlled the invasion and migration of MCL cells. It might be interesting to determine the subcellular localization of cyclin D1 at the time of diagnosis in order to offer a better treatment management for MCL patients. In addition, although selinexor is still in clinical trials, its use for the treatment of MCL could be considered in the most aggressive cases where cyclin D1 is cytoplasmic

STAT6 is a cargo of exportin 1: Biological relevance in primary mediastinal B-cell lymphoma

International audiencePrimary mediastinal B-cell lymphoma (PMBL) is a distinct B-cell lymphoma subtype with unique clinicopathological and molecular features. PMBL cells are characterised by several genetic abnormalities that conduct to the constitutive activation of the Janus kinase 2/signal transducer and activator of transcription 6 (JAK2/STAT6) signalling pathway. Among recurrent genetic changes in PMBL, we previously reported that the XPO1 gene encoding exportin 1 that controls the nuclear export of cargo proteins and RNAs, is mutated (p.E571K) in about 25% of PMBL cases. We therefore hypothesized that STAT6 could be a cargo of XPO1 and that STAT6 cytoplasm/nucleus shuttle could be altered in a subset of PMBL cells. Using immunocytochemistry techniques as well as the proximity ligation assay, we showed that STAT6 bound XPO1 in PBML cell lines and in HEK-293 cells genetically engineered to produce STAT6. Moreover, XPO1-mediated export of STAT6 occurs in cells expressing either a wild-type or the E571K mutated XPO1 protein

Exportin 1 (or XPO1) abnormalities in hematological malignancies: from the gene to targeted therapy

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XPO1 in B cell hematological malignancies: from recurrent somatic mutations to targeted therapy

International audienceMany recent publications highlight the large role of the pivotal eukaryotic nuclear export protein exportin-1 (XPO1) in the oncogenesis of several malignancies, and there is emerging evidence that XPO1 inhibition is a key target against cancer. The clinical validation of the pharmacological inhibition of XPO1 was recently achieved with the development of the selective inhibitor of nuclear export compounds, displaying an interesting anti-tumor activity in patients with massive pre-treated hematological malignancies. Recent reports have shown molecular alterations in the gene encoding XPO1 and showed a mutation hotspot (E571K) in the following two hematological malignancies with similar phenotypes and natural histories: primary mediastinal diffuse large B cell lymphoma and classical Hodgkin's lymphoma. Emerging evidence suggests that the mutant XPO1 E571K plays a role in carcinogenesis, and this variant is quantifiable in tumor and plasma cell-free DNA of patients using highly sensitive molecular biology techniques, such as digital PCR and next-generation sequencing. Therefore, it was proposed that the XPO1 E571K variant may serve as a minimal residual disease tool in this setting. To clarify and summarize the recent findings on the role of XPO1 in B cell hematological malignancies, we conducted a literature search to present the major publications establishing the landscape of XPO1 molecular alterations, their impact on the XPO1 protein, their interest as biomarkers, and investigations into the development of new XPO1-targeted therapies in B cell hematological malignancies

XPO1E571K Mutation Modifies Exportin 1 Localisation and Interactome in B-cell Lymphoma

International audienceSimple Summary: Almost 25% of patients with either primary mediastinal B-cell lymphoma (PMBL) or classical Hodgkin lymphoma (cHL) possess a recurrent mutation of the XPO1 gene encoding the major nuclear export protein. The aim of our study was to assess the molecular function of the mutant XPO1 protein. Using several cell models (including CRISPR-Cas9 edited cells) and high throughput techniques, we determined that the export capacity of the mutant XPO1 was not altered. However, mutant XPO1 accumulated in the cytoplasm due to its binding to importin β1 (or IPO1). The targeting of XPO1 is largely efficient for fighting haemopathies. The inhibition of IPO1 could open new therapeutic perspectives for B-cell lymphomas. Abstract: The XPO1 gene encodes exportin 1 (XPO1) that controls the nuclear export of cargo proteins and RNAs. Almost 25% of primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) cases harboured a recurrent XPO1 point mutation (NM_003400, chr2:g61718472C>T) resulting in the E571K substitution within the hydrophobic groove of the protein, the site of cargo binding. We investigated the impact of the XPO1 E571K mutation using PMBL/cHL cells having various XPO1 statuses and CRISPR-Cas9-edited cells in which the E571K mutation was either introduced or knocked-out. We first confirmed that the mutation was present in both XPO1 mRNA and protein. We observed that the mutation did not modify the export capacity but rather the subcellular localisation of XPO1 itself. In particular, mutant XPO1 bound to importin β1 modified the nuclear export/import dynamics of relevant cargoes

A lowered 26S proteasome activity correlates with mantle lymphoma cell lines resistance to genotoxic stress

International audienceBackground: Mantle cell lymphoma (MCL) is a B-cell hemopathy characterized by the t(11;14) translocation and the aberrant overexpression of cyclin D1. This results in an unrestrained cell proliferation. Other genetic alterations are common in MCL cells such as SOX11 expression, mutations of ATM and/or TP53 genes, activation of the NF-κB signaling pathway and NOTCH receptors. These alterations lead to the deregulation of the apoptotic machinery and resistance to drugs. We observed that among a panel of MCL cell lines, REC1 cells were resistant towards genotoxic stress. We studied the molecular basis of this resistance.Methods: We analyzed the cell response regarding apoptosis, senescence, cell cycle arrest, DNA damage response and finally the 26S proteasome activity following a genotoxic treatment that causes double strand DNA breaks.Results: MCL cell lines displayed various sensitivity/resistance towards genotoxic stress and, in particular, REC1 cells did not enter apoptosis or senescence after an etoposide treatment. Moreover, the G2/M cell cycle checkpoint was deficient in REC1 cells. We observed that three main actors of apoptosis, senescence and cell cycle regulation (cyclin D1, MCL1 and CDC25A) failed to be degraded by the proteasome machinery in REC1 cells. We ruled out a default of the βTrCP E3-ubiquitine ligase but detected a lowered 26S proteasome activity in REC1 cells compared to other cell lines.Conclusion: The resistance of MCL cells to genotoxic stress correlates with a low 26S proteasome activity. This could represent a relevant biomarker for a subtype of MCL patients with a poor response to therapies and a high risk of relapse

Exportin 1‐mediated nuclear/cytoplasmic trafficking controls drug sensitivity of classical Hodgkin lymphoma

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Additional file 3: Figure S2. of A lowered 26S proteasome activity correlates with mantle lymphoma cell lines resistance to genotoxic stress

A. REC1 cells were treated with vehicle or etoposide 4 μg/ml for 2–24 h and harvested. Cells (105 cells per spot) were cytospun on Superfrost glass slides, at 500 g for 3 min, then fixed in 4% paraformaldehyde (PFA) and permeabilized by incubation with 0.5% Triton-X100 (v/v) for 5 min. Slides were then stained with rabbit anti-cyclin D1 primary Ab (sc-718, Santa Cruz Biotech.) and AlexaFuor® 546 goat anti-rabbit IgG (Life Technologies) secondary Ab. DAPI (4′,6-diamidino-2-phenylindole dihydrochloride, Molecular Probes) served for nuclei counterstaining. Slides were mounted, and analyzed with a Fluoview FV 1000 confocal microscope and Fluoview Viewer software (Olympus). B. Cultured JeKo1 and REC1 cells were treated with vehicle (Ctrl) or doxorubicine (Dox 25 nM) for 24 h. Whole cell proteins were purified, separated by SDS-PAGE, and immunoblotted with the indicated antibodies. An anti-β-actin served as a control of charge and transfer. C. Cultured JeKo1 and REC1 cells were treated with vehicle (Ctrl) or bortezomib (10−1-104 nM) or sn38 (10−1-103 nM) for 24 h and then cell viability assessed by a MTS assay as described in the legend of the Fig. 1a. Dose-reponse curves were drawn with the PRISM® software (GraphPad, La Jolla, CA) and the IC50 were deduced from the data. (PPTX 5260 kb

Cytoplasmic cyclin D1 controls the migration and invasiveness of mantle lymphoma cells

Abstract Mantle cell lymphoma (MCL) is a hematologic neoplasm characterised by the t(11;14)(q13;q32) translocation leading to aberrant cyclin D1 expression. The cell functions of cyclin D1 depend on its partners and/or subcellular distribution, resulting in different oncogenic properties. We observed the accumulation of cyclin D1 in the cytoplasm of a subset of MCL cell lines and primary cells. In primary cells, this cytoplasmic distribution was correlated with a more frequent blastoid phenotype. We performed immunoprecipitation assays and mass spectrometry on enriched cytosolic fractions from two cell lines. The cyclin D1 interactome was found to include several factors involved in adhesion, migration and invasion. We found that the accumulation of cyclin D1 in the cytoplasm was associated with higher levels of migration and invasiveness. We also showed that MCL cells with high cytoplasmic levels of cyclin D1 engrafted more rapidly into the bone marrow, spleen, and brain in immunodeficient mice. Both migration and invasion processes, both in vivo and in vitro, were counteracted by the exportin 1 inhibitor KPT-330, which retains cyclin D1 in the nucleus. Our data reveal a role of cytoplasmic cyclin D1 in the control of MCL cell migration and invasion, and as a true operator of MCL pathogenesis