XPO1 Inhibitor Selinexor Overcomes Intrinsic Ibrutinib Resistance in Mantle Cell Lymphoma via Nuclear Retention of IκB

Mantle cell lymphoma (MCL) represents approximately 7% of non-Hodgkin lymphoma and remains an incurable disease. Aberrant B-cell receptor (BCR) signaling plays a central role in the pathogenesis of MCL (1). Ibrutinib, a BTK inhibitor, has achieved a remarkable 68% of overall response in MCL (2) and has been approved by the FDA for the treatment of relapsed patients. However, approximately one-third of patients do not respond to ibrutinib up front. In addition, in responding patients, nearly all patients relapse. Median overall survival following ibrutinib cessation is less than 3 months, and 1-year survival is below 20% (3). So far, no treatment options have been established, and none of the existing treatments improve outcome in this patient population (3). Thus, how to prevent and overcome ibrutinib resistance represents one of the unmet clinical challenges.

Selinexor (KPT-330) is a first-in-class selective inhibitor of nuclear export (C₁₇H₁₁F₆N₇O; see ref. 4; for its chemical structure). The drug binds and inhibits exportin XPO-1 that mediates nuclear export of proteins and mRNAs. By doing so, selinexor acts on a cellular process rather than a specific molecular signaling pathway, we thus postulate selinexor may have the potential to act broadly in both ibrutinib-sensitive and -resistant mantle lymphoma cells. Inactivation of XPO-1 leads to intranuclear retention of tumor suppressor proteins thereby suppressing neoplastic cell division and tumor growth. Inactivation of the exportin also decreases the export of mRNAs of several protooncogenes to prevent their translation to proteins in the cytoplasm (5, 6). Selinexor is currently under phase II/IIb clinical investigations for the evaluation of its activity against diffuse large B-cell lymphoma (DLBCL) and a variety of solid tumors (7). In April 2018, selinexor received fast-track designation from the FDA for the treatment of patients with highly refractory multiple myeloma. Because many of the oncogenic and tumor suppressor proteins in MCL also shuttle between nuclei and cytoplasm, we speculate that selinexor may be active in MCL cells regardless of their sensitivity to ibrutinib.

To prevent and overcome ibrutinib resistance, a deep and complete understanding of the resistance mechanisms is required. So far, little is known about the pathways that account for inactivity of ibrutinib in patients with intrinsic resistance to ibrutinib (2). In this study, we aim to compare and contrast cellular response to ibrutinib versus selinexor. We aim to identify molecular mechanisms underlying differential cellular response to these two drugs to obtain a deeper understanding of what underlies tumor cells' susceptibility to the drug intervention.

Human MCL cell lines JeKo-1, Mino, and Granta-519 were purchased from the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, and MAVER-1, JVM-2, REC-1 were purchased from ATCC, respectively in 2015. They were authenticated by the suppliers and tested by PCR to be free of Mycoplasma contamination in November 2017 upon completion of the experiments. All lines were cultured in RPMI1640 medium supplemented with 10% FBS and 100 μg/mL penicillin/streptomycin. Cells were maintained in a humidified 37°C/5% CO₂ incubator. Ibrutinib was purchased from Selleckchem and selinexor (KPT-330) was a gift from Karyopharm Therapeutics. Sources of antibodies are: anti-IκBα from Cell Signaling Technology and antibodies against PARP, P65, P50, MYC, CCND1, Lamin B, and GAPDH from Santa Cruz Biotechnology.

Frozen primary MCL samples were obtained from the archives of the Department of Pathology at University of Chicago (Chicago, IL) with Institutional Review Board review and approval. Samples with greater than 85% tumor cellularity and greater than 80% viability were selected.

MCL lines were treated with various concentrations of ibrutinib and selinexor. The metabolic activities of cells were measured following 72 hours of drug treatment using 3-(4,5-dimethylthiazol-2-l)-2,5-diphenyltetrazolium bromide (MTT) assay as per manufacturer's instructions (Roche Applied Science). IC₅₀ was calculated using the Sigma Plot generated with Prism 6 (GraphPad). Cell viability and cell numbers were determined by Muse Cell Analyzer (Millipore) according to the manufacturer's protocol. Cell growth and viability graphs were generated with Prism 6.

MCL cells were treated with various concentrations of ibrutinib and selinexor for 24–72 hours. Annexin V/PI staining was used for the detection of apoptosis by flow cytometry as described previously (8, 9). For cell-cycle analysis, cells were incubated with 20 μmol/L 5-bromo-2-deoxyuridine (BrdU, BD Biosciences) at 37°C for 2 hours, and stained with PE-conjugated anti-BrdU antibody (BD Biosciences) according to the manufacturer's manual. The percentage of cell-cycle distribution was analyzed using FlowJo (Tree Star Inc.).

The RNA-seq experiments were designed on the basis of published recommendations with biological triplicates (10). JeKo-1 and MAVER-1 were treated with ibrutinib and selinexor for 6 hours. Total RNA was isolated using RNA Mini Kit (QIAGEN) following manufacturer's instructions. All samples were quantified with Qubit RNA Assay (Life technology) and quality was assessed using Agilent Bioanalyzer (Santa Clara). Library preparations were performed using TruSeq Stranded RNA Sample Preparation Kit (Illumina). The quality of the library was assessed using D1K ScreenTapes on Agilent TapeStation and quantity was assessed by TaqMan qPCR assay directed to the adaptors. Each library was single-end sequenced with read length of 50 bp on Illumina Hi-Seq 2500 at a depth of 30 million reads. QC metrics were calculated using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were aligned to the transcriptome using STAR (11) and transcript abundance was estimated using Cufflinks (12). Differentially expressed genes between DMSO and drug-treated conditions were identified using DESeq2 (13). Gene set enrichment analysis (GSEA, Version 2.2.2; ref. 14) was used to identify significant pathways associated with the differentially expressed genes. Gene sets enriched in B-cell malignancies were derived from the gene expression database of the Staudt laboratory (Bethesda, MD; http://lymphochip.nih.gov/signaturedb/index.html) and were uploaded to GSEA (14) for analysis as described previously (15).

Imaging flow cytometry was performed using an Amnis Flowsight imaging cytometer (Amnis, part of EMD Millipore). Debris and doublets were gated out. Bright field (430–480 nm), FITC, and DRAG5 channel were recorded, and ≥1 × 10⁴ single-cell events were collected from each sample. FITC was excited at 488 nm, and its emission was read in a 505–560-nm channel; DRAG5 was excited at 642 nm, and its emission read using a 642–740-nm filter. Flow cytometry and qualitative imaging data were acquired with INSPIRE and analyzed with IDEAS software (Amnis).

Cytoplasmic and nuclear lysates were prepared using the Active Motif Nuclear Extract Kit (Active Motif) following manufacturer's protocol. The protein concentration was determined using the protein assay reagent (Bio-Rad). Immunoblot analyses were performed as described previously (8, 16). NFκB activity was measured in nuclear extracts by the TransAM NFκB P65 and P50 Protein Assays (Active Motif) to specifically detect and quantify the DNA-binding activity of the NFκB P65 and P50 subunit. The assay was performed according to manufacturer's protocol and analyzed using a microplate absorbance reader (Eppendorf PlateReader AF2000).

GraphPad Prism 6 software was used to analyze results and derive IC₅₀ values. Data on in vitro assays were analyzed using t test with a robust variance estimate. Data on primary cell assays were analyzed with the parametric paired Student t test. P ≤ 0.05 was considered significant.

We first determined the sensitivity of six MCL cell lines to ibrutinib using a cell-based MTT assay, which measures cellular metabolic activity. As shown in Fig. 1A, at 72 hours post-ibrutinib treatment, a 50% growth-inhibitory effect (IC₅₀) was observed in JeKo-1 at 0.6 μmol/L. IC₅₀ values for three other cell lines including Mino, REC-1, and JVM-2 ranged from 1.1 to 1.5 μmol/L, and two cell lines MAVER-1 and Granta-519 displayed an IC₅₀ value above 2.6 μmol/L. In vivo clinically achievable concentration of ibrutinib is approximately 0.4 μmol/L (16, 17). Using this concentration as a guideline, we defined JeKo-1 as ibrutinib-sensitive cell line, Mino, REC-1, and JVM-2 as cell lines with intermediate sensitivity, MAVER-1 and Granta-519 as resistant cell lines (Fig. 1A). This sensitivity profile is largely consistent with our previously published results (16) and results by others (18).

We then tested these cell lines for their sensitivities to selinexor. Pharmacokinetics and pharmacodynamics studies showed that at a once-daily dose of 85 mg/m², maximal achievable plasma concentration in patients is 3.78 μmol/L (19). IC₅₀ of selinexor in all six cell lines ranged between 0.04 and 0.21 μmol/L (Fig. 1A), we thus concluded, using the in vivo concentration as a guideline, that all MCL cell lines are sensitive to selinexor. Together, these results show that ibrutinib can elicit antilymphoma activity in some, but not in all MCL cells. In comparison, selinexor exhibits antitumor activity in all tested cell lines.

We next tested the effects of ibrutinib and selinexor on cell growth in JeKo-1, MAVER-1, and Granta which represent ibrutinib-sensitive and -resistant cell lines. The growth rate was reduced by ibrutinib in JeKo-1 in a dose- and time-dependent manner, but unaltered in MAVER-1 and Granta cells (Fig. 1B). In contrast, following selinexor treatment, we observed a significant inhibition of cell growth in all three cell lines in a dose- and time-dependent fashion (Fig. 1C). These results are largely consistent with the cell line sensitivity data measured with the MTT assay (Fig. 1A).

To further investigate the differential sensitivity of MCL cells to ibrutinib and selinexor, we examined the effect of both inhibitors on apoptosis and proliferation. To detect apoptosis, cell lines were treated with different concentrations of ibrutinib and selinexor and Annexin V/PI staining was measured. As shown in Fig. 2A, ibrutinib induced little apoptosis in either sensitive or resistant cell lines. These results are in agreement with our previous findings indicating that ibrutinib targets cell proliferation directly rather than cell viability (16). Compared with ibrutinib, selinexor significantly induced cellular apoptosis in these cell lines in a dose- and time-dependent manner (Fig. 2B). Meanwhile, PARP cleavage was observed with selinexor, but not with ibrutinib treatment (Fig. 2C) confirming that selinexor induced tumor cell death.

We next investigated the effects of selinexor on the cell cycle of MCL cells, in comparison with ibrutinib. As shown in Fig. 2D, the fraction of S-phase was reduced by ibrutinib in JeKo-1 cells, but not much in MAVER-1 and Granta cells (Fig. 2D, top, S-fraction represented by the gray boxes). In comparison, selinexor treatment dramatically inhibited S-phase in both ibrutinib-sensitive and -resistant cells (Fig. 2D, bottom). Furthermore, ibrutinib did not have much effect on cell-cycle–regulatory proteins CCND1 and C-MYC in resistant MAVER-1 and Granta cells (Fig. 2E, left three panels; also see Supplementary Figures for statistical analyses of each cell-cycle phases). In contrast, selinexor reduced CCND1 and C-MYC protein levels in all three cell lines (Fig. 2E, right three panels), which correlates with S-phase reduction. Together, with the MTT data, these results show that selinexor induces cell apoptosis and inhibits cell proliferation in both ibrutinib-sensitive and ibrutinib-resistant cell lines.

We have shown previously that specific reduction of BTK with siRNA slows down cellular growth of ibrutinib-sensitive JeKo-1 cells but had minimal effects on ibrutinib-resistant Granta cells suggesting that ibrutinib acts, at least partially, through inhibition of BTK (16). To evaluate whether the inhibitory effects of selinexor are mediated via XPO-1, we took a similar approach by specifically targeting XPO-1 using RNA interference. As shown in Fig. 3A, siRNA effectively reduced the expression of XPO-1 by 40%–60% in JeKo, Maver-1, and Granta cells (Fig. 3B). As expected, cell growth was slowed down in JeKo and Granta cells (Fig. 3C). However, XPO-1 knockdown produced essentially no effect in MAVER-1, suggesting either the residual XPO-1 in MAVER-1is sufficient to support cell growth or the antitumor effects of selinexor are mediated by XPO-1 in some but not all MCL tumor cell lines.

Using RNA sequencing, we then compared the signaling pathways which are altered by ibrutinib or selinexor treatment. In chronic lymphocytic leukemia (CLL), ibrutinib acts mainly by inhibiting the activity of the BCR and NFκB pathways (15, 20–22). We therefore determined whether ibrutinib achieves its therapeutic effect in MCL through similar mechanisms. To capture early changes in RNA transcription, JeKo-1 and MAVER-1 were treated for 6 hours with or without the inhibitors. Low doses of inhibitors achievable in patients were used, 0.4 μmol/L for ibrutinib and 1.5 μmol/L for selinexor. Cells were harvested and subjected to RNA sequencing and differences in biological pathway perturbation were analyzed using GSEA.

As shown in Fig. 4A (GSEA Enrichment plots) and B (heatmaps showing biological triplicates), genes in the BCR signature, previously defined by Staudt and colleagues (14), were significantly downregulated by ibrutinib treatment in the sensitive JeKo-1 cells (Fig. 4A, left; P = 0.001; FDR = 0.2%; Fig 4B, compare JeKo-1, DMSO vs. IBR). Meanwhile, this downregulation did not occur in the resistant MAVER-1 cells (Fig. 4A, middle; P = 0.162; FDR = 38.3%; Fig. 4B, compare MAVER-1, DMSO vs. IBR). As expected, the BCR pathway in MAVER-1 cells was not affected by selinexor treatment because the compound does not directly target any components of the BCR pathway (Fig. 4A, right; P = 0.123; FDR = 32.8%; Fig. 4B, compare MAVER-1, DMSO vs. SEL).

Similarly, the expression of genes in the NFκB signature, previously defined by Staudt and colleagues (14), was significantly downregulated by ibrutinib in sensitive JeKo-1 (Fig. 4C, left; P = 0.0; FDR = 0%; Fig. 4D, compare JeKo-1, DMSO vs. IBR), but not in resistant MAVER-1 cells (Fig. 4C, middle; P = 0.305; FDR = 56.1%; Fig. 4D, compare MAVER-1, DMSO vs. IBR). In contrast to IBR treatment, selinexor treatment downregulated the NFκB controlled genes in MAVER-1 cells (Fig. 4C, right; P = 0.041; FDR = 11.0%; Fig. 4D, compare MAVER-1, DMSO vs. SEL). Together with the phenotypical data on apoptosis and cell cycle (Fig. 2), our findings revealed that the presence or absence of NFκB downregulation correlates well with sensitivity or resistance of MCL cells to ibrutinib and selinexor (Fig. 3E). The results suggest that ibrutinib may act through BCR–NFκB pathways in MCL and downregulation of NFκB may be essential for the drugs to achieve their cellular therapeutic effects.

We then investigated how selinexor downregulates the NFκB pathway. Under resting conditions, NFκB is kept silent via physical interaction with IκB (inhibitor of NFκB) in the cytoplasm. Stimulation of various upstream signals such as BCR, toll-like receptor, or CD40 ligand activates IKK (IκB kinase) that phosphorylates IκBα, directing it to proteasome degradation. P65 and P50, subunits of NFκB, are then released and translocate to nuclei where they bind DNA and activate NFκB transcriptional program.

Because the main mechanism of action of selinexor is to affect nuclear export, we evaluated the subcellular localization of components of the NFκB pathway in both the sensitive and resistant cell lines. Cells were treated with or without selinexor, and subcellular localization of IκBα and NFκB subunits were examined using FlowSight technology, which combines fluorescence microscopy with flow cytometry. Figure 5A shows representative images of IκB in individual cells, for which nuclei were stained in red (Drag5) and IκB in green (FITC). In DMSO-treated JeKo-1 or MAVER-1 cells (Fig. 5A, left two panels), IκB was distributed in the cytoplasm as the overlay of nuclear and IκB staining showing red nuclei with surrounding green cytoplasm (Fig. 5A, left, column M). In comparison, in selinexor-treated JeKo-1 or MAVER-1 cells, IκB was retained in the nuclei as the overlay of nuclear and IκB staining showing nuclei in yellow (Fig. 5A, right, column M). Quantification of the percentage of nuclear IκBα (of 1 × 10⁴ events) was shown in Fig. 5B. In both JeKo-1 and MAVER-1, there was a significant dose-dependent increase in the nuclear retention of IκB. We then examined the subcellular distribution of P65 and P50. Similar to IκB, selinexor exposure increased nuclear retention of P65 as wells as P50 in both JeKo-1 and MAVER-1 cells (Fig. 5C). This observation was confirmed in ibrutinib-resistant Granta-519 cells as well (Supplementary Fig. S2).

To further validate these findings, we also analyzed the subcellular localization of NFκB proteins using conventional immunoblotting with isolated cytosolic and nuclear extracts. As shown in Fig. 5D, the abundance of cytosolic IκB, P50, and P65 was reduced with increasing concentrations of selinexor treatment (Fig. 5D, lanes 1–3 in both left and right panels). This was accompanied by a simultaneous increase of these three proteins in the nuclei (lanes 4–6 in both left and right panels). Overall, the results are entirely consistent with the FlowSight analyses (Fig. 5A and B).

Both Flowsight and immunoblotting analyses demonstrated that IκBα, P50, and P65 are kept in the nuclei upon selinexor treatment. We postulate that subunits of NFκB (p65 and p50), although present in the nuclei, are trapped in an inhibitory complex with IκB. To demonstrate that the function of nuclear NFκB is impaired, we measured the DNA-binding activity of P65 and P50 in selinexor-treated cells. As shown in Fig. 5E, the DNA-binding activity of both P65 and P50 subunits was reduced upon selinexor treatment in a dose-dependent manner and this happened in both JeKo-1 and MAVER-1 cells (Fig. 5E, left and right). Immunoprecipitation with anti-p65 pulled down both p65 and IκB from the nuclear extracts, further corroborating the notion that NFκB is trapped in an inhibitory complex (Supplementary Fig. S3). Taken together the data from these multiple lines of evidence, we conclude that selinexor kept IκBα in the nuclei, which subsequently inhibited NFκB–DNA binding in both ibrutinib-sensitive and -resistant cells. NFκB inhibition then led to downregulation of the NFκB transcriptional program (Fig. 4).

Next, we validated these findings in primary MCL cells derived from 6 patients. Archived frozen tumor samples were used for this purpose (ibrutinib-naïve) and patient characteristics are shown in Fig. 6A. Shown in Fig. 6B, selinexor induced variable but significant amount of apoptosis in primary MCL tumor cells (Fig. 6B; P = 0.0182). Meanwhile, nuclear localization of IκBα was increased upon selinexor exposure in all cases (Fig. 6C, compare column M of DMSO vs. selinexor) that is quantitatively significant (Fig. 6D, counts of 1 × 10⁴ events, P = 0.0207). Moreover, the degree of cellular apoptosis was linearly correlated with the number of cells with nuclear retention of IκBα (Fig. 6E; r = 0.8267, P = 0.0425). Overall, the results are consistent with our cell line data. These data support the conclusion that selinexor-induced apoptosis is accompanied by IκBα nuclear retention.

In this study, we have made the following findings: (i) selinexor had a broad antitumor activity in both ibrutinib-sensitive and ibrutinib-resistant mantle lymphoma cell lines; (ii) unlike ibrutinib, selinexor induced apoptosis as well as inhibited cell-cycle progression; (iii) antitumor effects of selinexor are mediated by XPO-1 in some but not all MCL tumor cell lines; (iv) downregulation of NFκB was a common feature in cell lines displaying sensitivity to either ibrutinib or selinexor, whereas unchanged NFκB was associated with cell line resistance to ibrutinib; (v) selinexor-induced IκB nuclear retention that was accompanied by decreased NFκB DNA binding; and (vi) IκB nuclear retention occurred in selinexor-treated patient-derived primary tumor cells that was well correlated with their cellular apoptotic response.

We demonstrate here that selinexor has a broad activity in MCL tumor cells including those with intrinsic resistance to ibrutinib. Other studies support our findings. It was reported in CLL that selinexor is effective in reducing the proliferation of leukemia cells and in improving the survival of ibrutinib-refractory Eu-TCL1 mice, a model of aggressive CLL (23). It was reported, in an abstract form, that KPT-276, another SINE compound, inhibits MCL cellular growth and tumor growth in SCID mice (24). These studies together show the potential of using selinexor, alone or in combination with ibrutinib, to prevent or overcome primary ibrutinib resistance in B-cell malignancies.

In CLL, previous studies by us and others show that ibrutinib does not induce apoptosis directly at clinically achievable concentrations (20). Its main mechanisms of action include deceleration of cell cycle and impairment of cell adhesion (25). The latter action displaces tumor cells from the lymph nodes to the periphery (lymphocytosis) and cells die in the periphery as a result of lacking nourishment from its natural microenvironment. Lymphocytosis is also observed in patients with MCL on ibrutinib treatment (3). Because tumor cells are not directly and immediately killed, a window of opportunity is left for the tumor cells to generate mutations and escape the drug suppression. Consistent with this proposition, acquired resistance is observed in nearly all patients with MCL treated with ibrutinib (3). In comparison with ibrutinib, selinexor induced apoptosis as well as cell-cycle arrest; thus, the drug has a better chance to eliminate tumor cells and to prevent or reduce disease relapse.

NFκB activation is an important pathogenic mechanism in MCL. Wiestner and colleagues recently provided the direct in vivo evidence showing canonical NFκB along with BCR pathways are ongoing and active in MCL cells resided in the lymph node microenvironment (1). NFκB may also play an important role in response to drugs. By comparing and contrasting ibrutinib with selinexor, we demonstrated that failure to inhibit NFκB transcriptional signature is associated with cell survival, proliferation, and ibrutinib resistance, while the ability to inhibit is associated with cell death, cell-cycle arrest, and drug sensitivity to either ibrutinib or selinexor. Rahal and colleagues have made similar findings with AFN700, a pharmacologic inhibitor of IKKβ, an activating kinase in the NFκB pathway. Proliferation of both ibrutinib-sensitive and ibrutinib-resistant MCL cell lines are suppressed by AFN700 that are accompanied by the downregulation of NFκB signature in all of these cell lines (18).

Notably, sensitivity to selinexor was even observed in Granta-519 and MAVER-1, the cell lines that are resistant to many other pharmacologic agents (18). MAVER-1, in particular, carries the biallelic loss of TRAF3, which negatively regulates the alternative NFκB pathway (18). Loss of TRAF3 leads to upregulation of NFκB-controlled gene expression independent of the BCR pathway and confers primary ibrutinib resistance in this cell line. In contrast to ibrutinib, selinexor effectively inhibited the NFκB signature in this cell line (Fig. 4). By targeting NFκB, an end effector of BCR signaling, the antitumor action of selinexor bypasses resistance caused by either intrinsic mutations such as TRAF3 or acquired mutations such as BTK. Collectively, these studies highlight the key role of NFκB in MCL pathogenesis and in drug response. Effective inhibition of NFκB downstream of BCR may be essential for a drug, whether it is ibrutinib, selinexor, or AFN700, to work effectively against MCL tumor cells.

To further understand how selinexor inhibits the NFκB pathway, we showed that selinexor retains IκB, P65, and P50 in the nuclei by FlowSight and immunoblot analyses. Although P65 and P50 are kept in the nuclei, we showed that they are bound by IκB and are thus inactive in terms of DNA binding. Taken together, our data suggest that selinexor acts, at least partially, by retaining IκB in the nuclei, decreases DNA binding of NFκB, and reduces NFκB-regulated gene expression. We further validated these findings in patient-derived primary MCL tumors. These observations are largely consistent with recent reports by others showing that selinexor induces IkB accumulation in sarcoma and multiple myeloma cell lines and in myeloma primary tumor cells (26, 27). Despite these observations, given the pleiotropic effects of the drug on many other nuclear proteins, more work will need to be done to demonstrate that inhibition of NFκB activity is a predominant mechanism underlying the antitumor activity of selinexor in MCL.

In conclusion, we demonstrated that inhibition of NFκB transcription by retaining IκB in the nuclei is an important action of selinexor in MCL tumor cells and selinexor is effective in ibrutinib-resistant MCL cell lines and has the potential to help prevent and overcome intrinsic ibrutinib resistance. Whether selinexor would work to overcome acquired ibrutinib resistance remains to be determined. Nonetheless, our study warrants further clinical investigation of this compound in MCL.

S. Shacham is the Founder, President, and Chief Scientific Officer of Karyopharm Therapeutics. Y. Landesman is a vice president (research) at Karyopharm Therapeutics. Y.L. Wang reports receiving a commercial research grant from Karyopharm Therapeutics that was used to support parts of the current study. No potential conflicts of interest were disclosed by other authors.

Conception and design: M. Ming, W. Wu, P. Lu, Y.L. Wang

Development of methodology: M. Ming, W. Wu, S. Sharma, P. Lu

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Ming, W. Wu, M. Sukhanova, W. Wang, S. Sharma

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Ming, W. Wu, B. Xie, M. Sukhanova. W. Wang, S. Kadri, J. Lee, N. Maltsev, P. Lu, Y.L. Wang

Writing, review, and/or revision of the manuscript: M. Ming, B. Xie, M. Sukhanova, J. Lee, P. Lu, Y.L. Wang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Shacham, Y. Landesman, Y.L. Wang

Study supervision: Y.L. Wang

The study is partially funded by a research grant from Karyopharm (to Y.L. Wang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.