Exportin 1-mediated nuclear/cytoplasmic trafficking controls drug sensitivity of classical Hodgkin's lymphoma.

Exportin 1 (XPO1) is the main nuclear export receptor that controls the subcellular trafficking and the functions of major regulatory proteins. XPO1 is overexpressed in various cancers and small inhibitors of nuclear export (SINEs) have been developed to inhibit XPO1. In primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin's lymphoma (cHL), the XPO1 gene may be mutated on one nucleotide and encodes the mutant XPO1E571K . To understand the impact of mutation on protein function, we studied the response of PMBL and cHL cells to selinexor, a SINE, and ibrutinib, an inhibitor of Bruton tyrosine kinase. XPO1 mutation renders lymphoma cells more sensitive to selinexor due to a faster degradation of mutant XPO1 compared to the wild-type. We further showed that a mistrafficking of p65 (RELA) and p52 (NFκB2) transcription factors between the nuclear and cytoplasmic compartments accounts for the response toward ibrutinib. XPO1 mutation may be envisaged as a biomarker of the response of PMBL and cHL cells and other B-cell hemopathies to SINEs and drugs that target even indirectly the NFκB signaling pathway.

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

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 XPO1E571K 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 ß1 modified the nuclear export/import dynamics of relevant cargoes.

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

Primary 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.

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

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.

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

BACKGROUND: 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 ßTrCP 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.

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

Many 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.