A cellular reporter to evaluate CRM1 nuclear export activity: functional analysis of the cancer-related mutant E571K

García-Santisteban, Iraia; Arregi, Igor; Alonso-Mariño, Marián; Urbaneja, Marı́a A.; García-Vallejo, Juan J.; Bañuelos, Sonia; Rodríguez, José Antonio

doi:10.1007/s00018-016-2292-0

Cited by 26 publications

(53 citation statements)

References 54 publications

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“…In fact, the molecular mechanisms that may be responsible for the pathogenic effect of XPO1 mutations remain to be established. Consistent with the location of the mutational "hotspot" proximal to the NES-binding site [ Figure 2B], it has been reported that the E571K mutation subtly increases the affinity of the receptor for some NESs with a negatively charged carboxy-terminal end [32] . Conceivably, this could lead to altered export of one or more cargos, whose mislocalization might in turn contribute to tumorigenesis.…”

Section: A Recurrent Xpo1 Gene Mutation In Hematological Malignanciessupporting

confidence: 82%

“…This experimental approach cannot be used to demonstrate XPO1-mediated export of proteins that are constitutively located to the nucleus. An alternative approach in this case could be ectopic overexpression of the receptor, which promotes export of NES-containing nuclear cargos to the cytoplasm [32] . Besides LMB-based experiments, the identification, validation and characterization of XPO1 cargos often involve biochemical analyses to demonstrate RanGTP-dependent binding, as well as mutagenesis to map the NES.…”

Section: Xpo1-mediated Protein Nuclear Export: Cargos Mechanisms Andmentioning

confidence: 99%

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Hitting a moving target: inhibition of the nuclear export receptor XPO1/CRM1 as a therapeutic approach in cancer

Sendino¹,

Omaetxebarria²,

Rodríguez³

2018

CDR

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Cellular homeostasis crucially relies on the correct nucleocytoplasmic distribution of a vast number of proteins and RNA molecules, which are shuttled in and out of the nucleus by specialized transport receptors. The nuclear export receptor XPO1, also called CRM1, mediates the translocation of hundreds of proteins and several classes of RNA to the cytoplasm, and thus regulates critical signaling pathways and cellular functions. The normal function of XPO1 appears to be often disrupted in malignant cells due to gene mutations or, most commonly, aberrant overexpression. Due to its important physiological roles and its frequent alteration in human tumors, XPO1 is a promising target for cancer therapy. XPO1 inhibitors have undergone extensive testing as therapeutic agents in preclinical models of cancer, with promising results. One of these inhibitors, Selinexor, is currently being evaluated in multiple clinical trials of different types of solid tumors and hematological malignancies. Here, we review several key aspects of XPO1 function, as well as the mechanisms that may lead to its alteration in cancer, and provide an update on the status of XPO1 inhibitors being developed as drugs for cancer therapy, including the definitive results of the first clinical trials with Selinexor that have been recently published. Figure 1. Receptor-mediated nucleocytoplasmic transport of proteins. A: Illustrative overview of the nuclear import of a cargo protein bearing a "classical" NLS mediated by the Importina/Importinb heterodimer (left) and the nuclear export of a cargo protein bearing a "leucine-rich" NES mediated by XPO1 (right); B: schematic depiction of the nuclear pore complex, illustrating its main structural features; C: examples of nucleocytoplasmic transport signals. The NLSs of SV40 large T antigen (monopartite) and nucleoplasmin (bipartite) are shown, with the basic residues that characterize "classical" NLSs highlighted in red. Below, the NES of PKI and a general consensus sequence of "leucine-rich" NESs are shown. The characteristic hydrophobic residues (represented by f in the consensus) are highlighted in colors and underlined. NLS: nuclear localization signal; NES: nuclear export signal Conception of the work, draft and revision of the work: Sendino M, Omaetxebarria MJ, Rodríguez JA

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Section: A Recurrent Xpo1 Gene Mutation In Hematological Malignanciessupporting

confidence: 82%

Section: Xpo1-mediated Protein Nuclear Export: Cargos Mechanisms Andmentioning

confidence: 99%

Hitting a moving target: inhibition of the nuclear export receptor XPO1/CRM1 as a therapeutic approach in cancer

Sendino¹,

Omaetxebarria²,

Rodríguez³

2018

CDR

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“…Indeed, the change from a negatively charged (glutamate) to a positively charged (lysine) residue could modify the affinity of XPO1 for NESs bearing negative charges [27]. proteins.…”

Section: Discussionmentioning

confidence: 99%

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

Miloudi

Leroy

Jardin

et al. 2018

Cellular Signalling

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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.

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“…We utilized a cellular reporter containing one of the NESs to evaluate the sequencespecific effects of XPO1 hotspot mutations on nuclear export, which were suggested by the structural and proteomic analyses above. We used a modi fied version of a previously described nuclearexport reporter (40) where the first 100 amino acids of the XPO1 substrate survivin (bearing its NES) are fused to a 3× FLAG epitope and two nuclear localization signals ( Supplementary Fig. S9A).…”

Section: Xpo1 E571k Mutations Alter Nes Recognition In a Sequence-spementioning

confidence: 99%

Altered Nuclear Export Signal Recognition as a Driver of Oncogenesis

et al. 2019

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Altered expression of XPO1, the main nuclear export receptor in eukaryotic cells, has been observed in cancer, and XPO1 has been a focus of anticancer drug development. However, mechanistic evidence for cancer-specific alterations in XPO1 function is lacking. Here, genomic analysis of 42,793 cancers identified recurrent and previously unrecognized mutational hotspots in XPO1. XPO1 mutations exhibited striking lineage specificity, with enrichment in a variety of B-cell malignancies, and introduction of single amino acid substitutions in XPO1 initiated clonal, B-cell malignancy in vivo. Proteomic characterization identified that mutant XPO1 altered the nucleocytoplasmic distribution of hundreds of proteins in a sequence-specific manner that promoted oncogenesis. XPO1 mutations preferentially sensitized cells to inhibitors of nuclear export, providing a biomarker of response to this family of drugs. These data reveal a new class of oncogenic alteration based on change-of-function mutations in nuclear export signal recognition and identify therapeutic targets based on altered nucleocytoplasmic trafficking. SIGNIFICANCE: Here, we identify that heterozygous mutations in the main nuclear exporter in eukaryotic cells, XPO1, are positively selected in cancer and promote the initiation of clonal B-cell malignancies. XPO1 mutations alter nuclear export signal recognition in a sequence-specific manner and sensitize cells to compounds in clinical development inhibiting XPO1 function.

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A cellular reporter to evaluate CRM1 nuclear export activity: functional analysis of the cancer-related mutant E571K

Cited by 26 publications

References 54 publications

Hitting a moving target: inhibition of the nuclear export receptor XPO1/CRM1 as a therapeutic approach in cancer

Hitting a moving target: inhibition of the nuclear export receptor XPO1/CRM1 as a therapeutic approach in cancer

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

Altered Nuclear Export Signal Recognition as a Driver of Oncogenesis

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