Exploring the Role of the Recurrent Exportin 1 (XPO1/CRM1) Mutations E571G and E571K in Chronic Lymphocytic Leukemia

Background

Exportin 1 (XPO1/CRM1) is a critical component of the nuclear to cytoplasmic export machinery. As such, XPO1 shuttles tumor suppressor proteins (e.g. p53, IkB) out of the nucleus thereby preventing their anti-apoptotic function and contributing to enhanced proliferation. XPO1 also transports a subset of mRNAs (e.g. c-fos, IFN-alpha) by virtue of 3'-UTR AU-rich elements. XPO1 is upregulated in most hematologic malignancies, including CLL, where elevated XPO1 expression correlates with poor prognosis and resistance to therapy. To date, the role of nuclear export in CLL is poorly understood. Interestingly, recurrent mutations at a highly conserved residue (E571G; E571K) in the cargo binding pocket of XPO1 have been described in chronic lymphocytic leukemia (CLL) and are associated with worse overall survival. We hypothesized that the E571G/K mutation confers novel cargo-binding abilities to XPO1 and may contribute to the pathogenesis of CLL.

Methods

To interrogate this question in an unbiased, transcriptome-wide manner we developed CLL cell lines expressing mutant XPO1 and a novel RNA immunoprecipitation procedure using RNase I digestion followed by RNA-seq to capture direct interaction sites or 'footprints'. Reads were mapped to the transcriptome and peaks (regions where RNA footprints are enriched) were identified. We examined footprints for enriched sequence motifs using the MEME suite. Mass-array screening identified a cohort of CLL patients bearing the E571G/K mutation for validation purposes. The XPO1 transgenic mice were generated via pronuclear injection of linearized plasmid containing human cDNA corresponding to XPO1^WT, XPO1^E571G, or XPO1^E571K under the heavy chain promoter/enhancer.

Results

We identified a transcriptome-wide pool of >100 XPO1-interacting RNAs in CLL cells and identified RNA footprints of wild-type XPO1 contributing to the first XPO1 RNA profile of a cell line model of CLL. The majority of bound RNA species associated with XPO1 are ribosomal, as expected; however, we identified multiple additional classes of RNAs such as miRs, mRNA, long-non coding RNAs, and many un-annotated RNAs. These data are consistent with additional preliminary data we have generated indicating that XPO1 may regulate mRNAs important in CLL development. We are currently searching for structural motifs. Validation experiments using patient-derived CLL cells with and without XPO1 mutations are ongoing.

To further assess the independent transforming role of XPO1 dysfunction in B-cells, we generated transgenic mice overexpressing XPO1^WT, XPO1^E571G, or XPO1^E571K under the heavy chain promoter/enhancer in the B cell compartment of C57BL/6 mice. Overexpression of XPO1 at the protein level in B cells was confirmed in multiple transgenic founder lines. Preliminary studies were performed to characterize the XPO1 transgenic animals for defects or alterations in major stages of B cell maturation. We analyzed the pro-B (IgM^-CD43⁺), pre-B (IgM^-CD43^-), and immature (IgM⁺CD43^-) B cell stages of development from the bone marrow of XPO1 transgenic animals and littermate controls. We also examined immature (IgM⁺IgD^-) and mature (IgM⁺IgD⁺) B cells isolated from the spleen of these animals. In this preliminary analysis no significant differences in the B cell compartment were observed between transgenic and non-transgenic animals. Functional studies of these B-cells are ongoing. A cohort of animals from each founder line is being followed for evidence of B-cell expansion and overall survival analysis. In addition, the XPO1 transgenic mice were crossed with the Eµ-TCL1 mice to establish the XPO1^WT/E571K/G/TCL1 double transgenic mouse model whose disease is currently being monitored.

Conclusions

XPO1 is a recurring, high variant allele mutation that occurs in a small subset of CLL patients. XPO1 transports select mRNA and also novel RNA species whose function in CLL have yet to be characterized, but points to a novel role of this oncoprotein in the pathogenesis of this disease.