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Nuclear export inhibitors avert progression in preclinical models of inflammatory demyelination

Nature Neuroscience volume 18, pages 511–520 (2015)Cite this article

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Abstract

Axonal damage has been associated with aberrant protein trafficking. We examined a newly characterized class of compounds that target nucleo-cytoplasmic shuttling by binding to the catalytic groove of the nuclear export protein XPO1 (also known as CRM1, chromosome region maintenance protein 1). Oral administration of reversible CRM1 inhibitors in preclinical murine models of demyelination significantly attenuated disease progression, even when started after the onset of paralysis. Clinical efficacy was associated with decreased proliferation of immune cells, characterized by nuclear accumulation of cell cycle inhibitors, and preservation of cytoskeletal integrity even in demyelinated axons. Neuroprotection was not limited to models of demyelination, but was also observed in another mouse model of axonal damage (that is, kainic acid injection) and detected in cultured neurons after knockdown of Xpo1, the gene encoding CRM1. A proteomic screen for target molecules revealed that CRM1 inhibitors in neurons prevented nuclear export of molecules associated with axonal damage while retaining transcription factors modulating neuroprotection.

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Figure 1: Expression of CRM1 in MS gray and white matter lesion areas.
Figure 2: KPTs selectively and covalently bind CRM1 and inhibit binding to NES with pharmacokinetic properties that favor blood brain barrier permeability.
Figure 3: Therapeutic treatment of EAE mice with KPT after the onset of hindlimb paralysis is associated with myelinated axon preservation.
Figure 4: Therapeutic treatment of EAE mice with KPT reduces inflammatory burden in the spinal cord and impairs CD4+ T cell proliferative capacity, but does not affect their ability to produce cytokines.
Figure 5: KPT inhibitors provide axonal protection in mouse models that are both dependent and independent of inflammation.
Figure 6: KPT inhibitors prevent locomotion decline and partially restore motor function in a localized EAE mouse model.
Figure 7: Treatment of cultured neurons with KPT-350 or knockdown of CRM1 prevents the induction of focal axonal damage and prevents damage in models independent of inflammation.
Figure 8: Proteomic screen of neurons treated with KPT-350 led to the identification of target molecules explaining the neuroprotective effect.

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Acknowledgements

We thank T. Flagiello and X. Pedre for assistance with EAE animal experiments. The project was supported by US National Institutes of Health grants R01-NS69385 and R37-NS42925, seed funds from Karyopharm Therapeutics to P.C., and the Fast Forward division of the National Multiple Sclerosis Society to P.C. and S.S. T.K. was supported by funds from the Interdisciplinary Centre for Clinical Research in Münster (KuT3/006/11). O.H. is the recipient of the Mount Sinai Helmsley Award. J.D.H. holds a postdoctoral fellowship from the Multiple Sclerosis Society of Canada and the Fonds de la recherche en santé du Québec. Human tissue samples for western blotting were supplied by the UK Multiple Sclerosis Tissue Bank, funded by the MS Society of Great Britain and Northern Ireland, registered charity 207495.

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Authors and Affiliations

  1. Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Jeffery D Haines, Belén de la Hera, Oscar G Vidaurre, Gregory A Moy & Patrizia Casaccia

  2. Division of Clinical Immunology, Department of Medicine, The Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Olivier Herbin & Konstantina Alexandropoulos

  3. Department of Pharmacology, University of Texas Southwestern Medical Center, Texas, USA

    Qingxiang Sun, Ho Yee Joyce Fung & Yuh Min Chook

  4. Institute of Neuropathology, University Hospital Münster, Münster, Germany

    Stefanie Albrecht & Tanja Kuhlmann

  5. Karyopharm Therapeutics, Natick, Massachusetts, USA

    Dilara McCauley & Sharon Shacham

  6. Department of Neurosciences, Cleveland Clinic, Cleveland, Ohio, USA

    Grahame J Kidd

  7. Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Patrizia Casaccia

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  1. Jeffery D Haines
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Contributions

P.C., S.S., J.D.H. and D.M. were responsible for overall analysis and study design. In vivo and in vitro experiments were performed by J.D.H., O.H., B.d.l.H., O.G.V. and G.A.M. Q.S., H.Y.J.F. and Y.M.C. crystallized CRM1 bound to SINE and performed gel shift assays. S.A. and T.K. performed the human brain immunohistochemistry experiments. G.J.K. helped analyze and interpret the three-dimensional electron microscopy results. Immunology experiments were conceived and interpreted by O.H. and K.A. Pharmacokinetic experiments and characterization of drug properties were performed by D.M. and S.S. The paper was written by J.D.H. and P.C.

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Correspondence to Patrizia Casaccia.

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Competing interests

S.S. and D.M. are currently employed at and hold leadership positions at Karyopharm Therapeutics, as well as hold stock in the company.

Integrated supplementary information

Supplementary Figure 1 Comparison of KPT-276, KPT-185 and KPT-251 bound to CRM1.

(a-c) Chemical structure of the KPT-276 compared to the structure of related compounds KPT-185 and KPT-251 inhibitors, (d-f). Views of the NES-binding groove of CRM1, showing interactions between CRM1 and three different KPT inhibitors. Interactions (≤4.0 Å) between KPT-276 (d, magenta), KPT-185 (e, orange) and KPT-251 (f, gray) and CRM1 (pink) are shown as gray dashed lines. (g-i) Views of the complexes generated by 90° rotations about the vertical axes relative to the views in (d-f). Helices H12A in the CRM1 grooves were removed for clarity. The variable arm of KPT-276 binds in a different orientation from those of the previously reported KPT-185 and KPT-251.

Supplementary Figure 2 CRM1 is ubiquitously expressed in cells of the central nervous system and periphery, and KPT inhibitors are not toxic to in vitro cultured cells.

(a,b) Xpo1 (CRM1) mRNA levels in CNS neurons and glia (oligodendrocytes, microglia, astrocytes) and peripheral immune cells (total splenocytes, CD19+ B cells, CD4+ T cells, CD8+ T cells, CD11b+ monocytes/microglia, and CD11c+ dendritic cells) all normalized to Gapdh. MTT assay of (c) spinal cord neurons, (d) cortical neurons, (e) oligodendrocyte progenitors, (f) oligodendrocytes, (g) astrocytes, or (h) splenocytes treated with the indicated doses of KPT-276 and KPT-350 (concentration range 0.1 to 1000 nM) for 24 hr. Bar graphs represent mean ± SEM, n = 8 samples per conditions, from two independent experiments. Statistical differences in all graphs were determined using one-way ANOVA with Dunnett’s correction (***p < 0.001).

Supplementary Figure 3 Therapeutic treatment of EAE mice with KPT compounds does not negatively impact body weight or body condition.

Mice treated after the development of hindlimb paralysis were also monitored for (a) body weight and (b) body condition. Arrows indicate the start and end of the treatment. Mice were supplemented with softened food and a high calorie nutritional supplement (NutricalR) in order to maintain healthy body weight. Body condition 3 = normal; 1 = anorexic; 5 = obese. (c) FluoroMyelin stained longitudinal sections of EAE spinal cords; scale bear = 200 µm. (d) Quantification of FluoroMyelin intensity from EAE animals. The bar graphs represent mean ± SEM. Statistical differences in: (a), (b) were determined by performing linear regression analysis on the data points during the drug treatment period window; (d) were determined using one-way ANOVA with Dunnett’s correction (**p < 0.01, ***p < 0.001 vs. vehicle).

Supplementary Figure 4 KPT inhibitors do not affect transcript levels of oligodendrocyte lineage markers, nor differentiation or oligodendrocyte progenitor proliferation.

(a) Equal weights of spinal cord tissue was used to determine relative transcript levels of stage specific markers of the oligodendrocyte lineage, in EAE mice treated therapeutically with either vehicle, or KPT-350, normalized to 18S rRNA, n=8 per condition, from two independent experiments. (b) Representative images of oligodendrocytes treated with KPT-276 (10 nM) or KPT-350 (10 nM). Cells were grown in the presence of the KPT for 48 hr and the differentiation markers O4 (green) and MBP (red) were determined. Cells were counterstained with DAPI (blue); scale bar represents 50 μm. (c) Quantification of cells in (b). (d) Values represent mean ± SEM of three fields, from three independent experiments. Statistical differences in (a) were determined using independent t-tests with Bonferroni correction; (c), (d) one-way ANOVA with Dunnett’s correction.

Supplementary Figure 5 Therapeutic treatment of EAE mice with KPT inhibitors reduces total splenocytes and maintains relatively equal proportions of splenic immune cell populations.

(a) Representative images of spleens from vehicle, KPT-276 and KPT-350-treated animals, harvested at d28 of EAE, scale bar = 0.5 cm. (b) Quantification of total splenocytes from spleens of vehicle, KPT-276 and KPT-350-treated animals. (c) Pie graphs depicting the relative proportions of splenic immune cell populations in vehicle, KPT-276, or KPT-350-treated animals. Bar graphs represent mean ± SEM. Statistical differences in: (b) were determined using one-way ANOVA with Dunnett’s correction (***p < 0.001).

Supplementary Figure 6 Prophylactic treatment with KPT-276 and KPT-350 at timing of immunization attenuates disease onset and immune cell numbers in the spinal cord and periphery.

(a) Schematic diagram of treatment paradigm for prophylactic EAE. Clinical scores from prophylactic treatment of 8-week old C57/BL6 female mice with MOG35-55-induced EAE and gavaged either with vehicle, KPT-276, or KPT-350 starting at immunization; drug-treatment time window is indicated by the arrow. The graph represents the average mean value of the clinical score ± SEM, n=12 mice per group. (b) Cumulative clinical scores in mice treated from the time of immunization, n=8 animals per group. (c) Mouse body weight and (d) body condition. Arrows indicate the start and end of the treatment. Mice were supplemented with softened food and a high calorie nutritional supplement (NutricalR) in order to maintain healthy body weight. Body condition 3 = normal; 1 = anorexic; 5 = obese. (e) Quantification by flow cytometry of immune cell infiltrates in spinal cords of mice. Microglial cells were identified as CD11b+/ CD45low and monocytes as CD11b+/CD45high (n=4 mice per condition). (f) Quantification of total splenocytes. (g) Quantification by flow cytometry of splenic populations in vehicle, KPT-276 or KPT-350 treated mice. Monocytes were identified as CD11b+/ CD115+, neutrophils as CD11b+/CD115- and dendritic cells (DC) as CD11c+/MHCIIhigh. All bar graphs represent mean ± SEM. Statistical differences in: (b), (e), (f), (g) were determined using Kruskal-Wallis test with Dunn’s correction (*p < 0.05, **p < 0.01, ***p < 0.001 all relative to vehicle); (c), (d) were determined by performing linear regression analysis on the data points during the drug treatment period window.

Supplementary Figure 7 Washout of KPT inhibitors in naive mice restores spleen immune cell populations.

(a) Schematic diagram of method to assess toxicity of drug in spleen. Quantification of total splenocytes in KPT-276, KPT-276 washout, KPT-350, or KPT-350 washout. (b) Quantification by flow cytometry of splenic populations in vehicle, KPT-276 or KPT-350 treated mice. Animals were treated for 14d with drug, or an identical cohort of animals treated with the compounds followed by drug washout for 14d. Monocytes were identified as CD11b+/ CD115+, neutrophils as CD11b+/CD115- and dendritic cells (DC) as CD11c+/MHCIIhigh. Bar graphs represent mean ± SEM. Statistical differences in: (a) were determined using Kruskal-Wallis test with Dunn’s correction (*p < 0.05 vs. vehicle); (b) were determined using independent t-tests with Bonferroni correction (*p < 0.05, **p < 0.01, ***p < 0.001 all relative to the non-washout cell population).

Supplementary Figure 8 Neurofilament heavy chain transcript levels are upregulated in EAE mice treated with KPT-350.

(a) Equal weight of spinal cord tissue was used to determine relative transcript levels of stage specific markers of the oligodendrocyte lineage, and of neurofilament heavy chain (Nefh) in mice treated either with vehicle, or KPT-350, normalized to 18S rRNA, n=8 per condition, from two independent experiments. The bar graph represents mean ± SEM. Statistical difference was determined using an independent t-test (***p < 0.001 vs vehicle).

Supplementary Figure 9 KPT-350 treatment of mouse hippocampal slice cultures prevents the induction of focal axonal damage in models independent of inflammation.

(a) Schematic diagram of kainic-acid model of induced neurotoxicity in mouse hippocampal slice cultures. Representative confocal images of the hippocampal CA3 region from cultures treated with KPT-350 (10 nM) for 1 hr, followed by exposure to 5 µM kainic acid for 18 hr. Slices were stained with NFH (red) to label axons, SMI-32 (green) to label damaged axons, DAPI (blue) was used to counterstain nuclei; scale bar = 100 µm. (b) Quantification of SMI-32 intensity in (a). Values represent mean pixel intensity ± SEM of n= 3 slices per group from three independent experiments. Statistical differences in (b) were determined using one-way ANOVA with Dunnett’s correction (***p < 0.001 vs. kainic acid).

Supplementary Figure 10 KPT-350 prevents the decrease in mitochondrial spare respiratory capacity induced by excitotoxic and inflammatory damage.

(a) Mitochondrial respiratory function was determined by measuring the oxygen consumption rate of neurons treated with DMSO, glut+TNFα in the absence or presence of KPT (10nM of KPT-350) using a SeaHorse Bioanalyzer. (b) Quantification of spare respiratory capacity of neurons treated in (a). Statistical differences in (b) were determined using one-way ANOVA with Dunnett’s correction (*p < 0.05 vs. DMSO).

Supplementary Figure 11 Full length western blots for human data showing increased expression of CRM1 in MS gray matter.

Full length western blots for human data (corresponding to Figure 1e).

Supplementary Figure 12 Full length western blots from rat neurons to validate CRM1 cargos

Full length western blots for human data (corresponding to Figure 8b).

Supplementary Figure 13 Full length western blots from experiments on EAE mice to validate of CRM1 cargoes

Full length western blots for human data (corresponding to Figure 8d).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 and Supplementary Tables 1–3 (PDF 2705 kb)

Supplementary Methods Checklist (PDF 1113 kb)

Video of EAE mice taken at drug start time, on day 16 post-immunization with MOG35-55 when mice exhibited hindlimb paralysis.

Mice were started on treatment at d16 post-immunization, and were oral gavaged with vehicle three times per week until d28. (MOV 3089 kb)

Video of therapeutic EAE mice treated with vehicle.

Video was taken at d28 post-immunization with MOG35-55. Mice were started on treatment at d16 post-immunization, and were oral gavaged with vehicle three times per week until d28. (MP4 1889 kb)

Video of therapeutic EAE mice treated with KPT-276 (75 mg/kg).

Video was taken at d28 post-immunization with MOG35-55. Mice were started on treatment at d16 post-immunization, and were oral gavaged with KPT-276 three times per week until d28. (MP4 5856 kb)

Video of therapeutic EAE mice treated with KPT-350 (7.5 mg/kg).

Mice were started on treatment at d16 post-immunization, and were oral gavaged with KPT-350 three times per week until d28. (MP4 7075 kb)

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Haines, J., Herbin, O., de la Hera, B. et al. Nuclear export inhibitors avert progression in preclinical models of inflammatory demyelination. Nat Neurosci 18, 511–520 (2015). https://doi.org/10.1038/nn.3953

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