Recurrent XPO1 mutations alter pathogenesis of chronic lymphocytic leukemia.

BACKGROUND: Exportin 1 (XPO1/CRM1) is a key mediator of nuclear export with relevance to multiple cancers, including chronic lymphocytic leukemia (CLL). Whole exome sequencing has identified hot-spot somatic XPO1 point mutations which we found to disrupt highly conserved biophysical interactions in the NES-binding groove, conferring novel cargo-binding abilities and forcing cellular mis-localization of critical regulators. However, the pathogenic role played by change-in-function XPO1 mutations in CLL is not fully understood. METHODS: We performed a large, multi-center retrospective analysis of CLL cases (N = 1286) to correlate nonsynonymous mutations in XPO1 (predominantly E571K or E571G; n = 72) with genetic and epigenetic features contributing to the overall outcomes in these patients. We then established a mouse model with over-expression of wildtype (wt) or mutant (E571K or E571G) XPO1 restricted to the B cell compartment (Eµ-XPO1). Eµ-XPO1 mice were then crossed with the Eµ-TCL1 CLL mouse model. Lastly, we determined crystal structures of XPO1 (wt or E571K) bound to several selective inhibitors of nuclear export (SINE) molecules (KPT-185, KPT-330/Selinexor, and KPT-8602/Eltanexor). RESULTS: We report that nonsynonymous mutations in XPO1 associate with high risk genetic and epigenetic features and accelerated CLL progression. Using the newly-generated Eµ-XPO1 mouse model, we found that constitutive B-cell over-expression of wt or mutant XPO1 could affect development of a CLL-like disease in aged mice. Furthermore, concurrent B-cell expression of XPO1 with E571K or E571G mutations and TCL1 accelerated the rate of leukemogenesis relative to that of Eµ-TCL1 mice. Lastly, crystal structures of E571 or E571K-XPO1 bound to SINEs, including Selinexor, are highly similar, suggesting that the activity of this class of compounds will not be affected by XPO1 mutations at E571 in patients with CLL. CONCLUSIONS: These findings indicate that mutations in XPO1 at E571 can drive leukemogenesis by priming the pre-neoplastic lymphocytes for acquisition of additional genetic and epigenetic abnormalities that collectively result in neoplastic transformation.

Recognition of nuclear export signals by CRM1 carrying the oncogenic E571K mutation.

The E571K mutation of CRM1 is highly prevalent in some cancers, but its mechanism of tumorigenesis is unclear. Glu571 of CRM1 is located in its nuclear export signal (NES)-binding groove, suggesting that binding of select NESs may be altered. We generated HEK 293 cells with either monoallelic CRM1WT/E571K or biallelic CRM1E571K/E571K using CRISPR/Cas9. We also combined analysis of binding affinities and structures of 27 diverse NESs for wild-type and E571K CRM1 with structure-based bioinformatics. While most NESs bind the two CRM1 similarly, NESs from Mek1, eIF4E-transporter, and RPS2 showed >10-fold affinity differences. These NESs have multiple charged side chains binding close to CRM1 position 571, but this feature alone was not sufficient to predict different binding to CRM1(E571K). Consistent with eIF4E-transporter NES binding weaker to CRM1(E571K), eIF4E-transporter was mislocalized in tumor cells carrying CRM1(E571K). This serves as proof of concept that understanding how CRM1(E571K) affects NES binding provides a platform for identifying cargoes that are mislocalized in cancer upon CRM1 mutation. Finally, we showed that large affinity changes seen with some NES peptides (of Mek1 and RPS2) do not always translate to the full-length cargoes, suggesting limitations with current NES prediction methods. Therefore, comprehensive studies like ours are imperative to identify CRM1 cargoes with real pathogenic potential.

pCRM1exportome: database of predicted CRM1-dependent Nuclear Export Signal (NES) motifs in cancer-related genes.

MOTIVATION: The consensus pattern of Nuclear Export Signal (NES) is a short sequence motif that is commonly identified in protein sequences, whether the motif acts as an NES (true positive) or not (false positive). Finding more plausible NES functioning regions among the vast array of consensus-matching segments would provide an interesting resource for further experimental validation. Better defined NES should also allow meaningful mapping of cancer-related mutation positions, leading to plausible explanations for the relationship between nuclear export and disease. RESULTS: Possible NES candidate regions are extracted from the cancer-related human reference proteome. Extracted NES are scored for reliability by combining sequence-based and structure-based approaches. The confidently identified NES candidate motifs were checked for overlap with cancer-related mutation positions annotated in the COSMIC database. Among the ∼700 cancer-related sequences in the COSMIC Cancer Gene Census, 178 sequences are predicted to have possible NES motifs containing cancer-related mutations at their key positions. These lists are organized into our database (pCRM1exportome), and other protein sequences in the human reference proteome can also be retrieved by their UniProt IDs. AVAILABILITY AND IMPLEMENTATION: The database is freely available at http://prodata.swmed.edu/pCRM1exportome. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Structural prerequisites for CRM1-dependent nuclear export signaling peptides: accessibility, adapting conformation, and the stability at the binding site.

Nuclear export signal (NES) motifs function as essential regulators of the subcellular location of proteins by interacting with the major nuclear exporter protein, CRM1. Prediction of NES is of great interest in many aspects of research including cancer, but currently available methods, which are mostly based on the sequence-based approaches, have been suffered from high false positive rates since the NES consensus patterns are quite commonly observed in protein sequences. Therefore, finding a feature that can distinguish real NES motifs from false positives is desired to improve the prediction power, but it is quite challenging when only using the sequence. Here, we provide a comprehensive table for the validated cargo proteins, containing the location of the NES consensus patterns with the disordered propensity plots, known protein domain information, and the predicted secondary structures. It could be useful for determining the most plausible NES region in the context of the whole protein sequence and suggests possibilities for some non-binders of the annotated regions. In addition, using the currently available crystal structures of CRM1 bound to various classes of NES peptides, we adopted, for the first time, the structure-based prediction of the NES motifs bound to the CRM1's binding groove. Combining sequence-based and structure-based predictions, we suggest a novel and more straight-forward approach to identify CRM1-binding NES sequences by analysis of their structural prerequisites and energetic evaluation of the stability at the CRM1's binding site.

Correlation of CRM1-NES affinity with nuclear export activity.

CRM1 (Exportin1/XPO1) exports hundreds of broadly functioning protein cargoes out of the cell nucleus by binding to their classical nuclear export signals (NESs). The 8- to 15-amino-acid-long NESs contain four to five hydrophobic residues and are highly diverse in both sequence and CRM1-bound structure. Here we examine the relationship between nuclear export activities of 24 different NES peptides in cells and their CRM1-NES affinities. We found that binding affinity and nuclear export activity are linearly correlated for NESs with dissociation constants ( Kds) between tens of nanomolar to tens of micromolar. NESs with Kds outside this range have significantly reduced nuclear export activities. These include two unusually tight-binding peptides, one from the nonstructural protein 2 of murine minute virus (MVM NS2) and the other a mutant of the protein kinase A inhibitor (PKI) NES. The crystal structure of CRM1-bound MVM NS2NES suggests that extraordinarily tight CRM1 binding arises from intramolecular contacts within the NES that likely stabilizes the CRM1-bound conformation in free peptides. This mechanistic understanding led to the design of two novel peptide inhibitors that bind CRM1 with picomolar affinity.

Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites.

Liquid-liquid phase separation (LLPS) is believed to underlie formation of biomolecular condensates, cellular compartments that concentrate macromolecules without surrounding membranes. Physical mechanisms that control condensate formation/dissolution are poorly understood. The RNA-binding protein fused in sarcoma (FUS) undergoes LLPS in vitro and associates with condensates in cells. We show that the importin karyopherin-ß2/transportin-1 inhibits LLPS of FUS. This activity depends on tight binding of karyopherin-ß2 to the C-terminal proline-tyrosine nuclear localization signal (PY-NLS) of FUS. Nuclear magnetic resonance (NMR) analyses reveal weak interactions of karyopherin-ß2 with sequence elements and structural domains distributed throughout the entirety of FUS. Biochemical analyses demonstrate that most of these same regions also contribute to LLPS of FUS. The data lead to a model where high-affinity binding of karyopherin-ß2 to the FUS PY-NLS tethers the proteins together, allowing multiple, distributed weak intermolecular contacts to disrupt FUS self-association, blocking LLPS. Karyopherin-ß2 may act analogously to control condensates in diverse cellular contexts.