CONCISE REVIEW: PATHWAY OF THE MONTH

Therapeutic Targeting of Nuclear Export Inhibition in Lung Cancer

Arjun Gupta, MD,a Jessica M. Saltarski, BA,b Michael A. White, PhD,b,c Pier P. Scaglioni, MD,a,b David E. Gerber, MDa,b,d,* aDepartment of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas bHarold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas cPfizer, Inc., New York, New York dDepartment of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, Texas

Received 12 April 2017; revised 3 June 2017; accepted 14 June 2017 Available online - 21 June 2017

ABSTRACT cellular processes. Aberrant nucleocytoplasmic transport of tumor suppressor proteins and regulators— Intracellular compartmentalization and trafficking of mediated by importins and exportins—can result in molecules plays a critical role in complex and essential cellular processes. In lung cancer and other malignancies, tumorigenesis and inactivation of . Several aberrant nucleocytoplasmic transport of tumor suppres- malignancies, including lung cancer, feature over- sor proteins and cell cycle regulators results in tumori- expression of these nuclear transport receptors. Phar- genesis and inactivation of apoptosis. Pharmacologic macologic targeting of this process has demonstrated agents targeting this process, termed selective inhibitors antitumor efficacy. In this review article, we describe of nuclear export (SINE), have demonstrated antitumor the mechanism, function, and therapeutic targeting of efficacy in preclinical models and human clinical trials. nuclear transport, with particular focus on application in Exportin-1 (XPO1), which serves as the sole exporter of lung cancer. several tumor suppressor proteins and cell cycle regula- tors, including retinoblastoma, adenomatous polyposis Nuclear Export Machinery coli, p53, p73, p21, p27, forkhead box O, signal transducer The nuclear envelope, comprising an inner and outer and activator of transcription 3, inhibitor of kB, topo- isomerase II, and protease activated receptor 4—is membrane, prevents the unrestricted diffusion of mole- the principal focus of development of SINE. The most cules larger than 40 kD between the nucleus and the extensively studied of the SINE to date, the exportin-1 cytoplasm. This regulated nuclear-cytolasmic transport inhibitor selinexor (KPT-330 [Karyopharm Therapeutics, of proteins and other molecules plays a key role in 1 Inc., Newton Centre, MA]), has demonstrated single-agent cell functioning. Within the nuclear envelope, nuclear anticancer activity and synergistic effects in combination pore complexes provide an aqueous channel for the regimens against multiple cancer types, with principal active transport of molecules. The karyopherin-B protein toxicities of low-grade cytopenias and gastrointestinal family, which includes both importins and exportins, effects. SINE may have particular relevance in KRAS- facilitates transport across these nuclear pores.2 Cargo driven tumors, for which this treatment strategy demon- proteins destined for nuclear export have specific strates significant synthetic lethality. A multicenter phase 1/2 of selinexor in previously treated advanced KRAS-mutant NSCLC is under way. *Corresponding author. Disclosure: The authors declare no conflict of interest. 2017 International Association for the Study of Lung Address for correspondence: David E. Gerber, MD, Division of Cancer. Published by Elsevier Inc. All rights reserved. Hematology-Oncology, Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Mail Code 8852, Dallas, TX 75390-8852. E-mail: david. Keywords: Adenocarcinoma; Exportin-1; KRAS; Pathway; [email protected] Selinexor; ; XPO1 ª 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. Intracellular compartmentalization and trafficking of ISSN: 1556-0864 http://dx.doi.org/10.1016/j.jtho.2017.06.013 molecules plays a critical role in complex and essential

Journal of Thoracic Oncology Vol. - No. -: --- 2 Gupta et al Journal of Thoracic Oncology Vol. - No. - leucine-rich amino acid sequences known as nuclear II, and protease-activated receptor 4.15 Under physio- export signals, which are recognized by exportin pro- logic conditions, the regulated export of these molecules teins.3 Nuclear-cytoplasmic transport is an active pro- prevents their overactivity in the nucleus in the absence cess requiring energy provided by RanGTP ( of oncogenic stimuli or DNA damage. In multiple cancer guanosine triphosphate). A complex between the cargo types, XPO1 overexpression leads to dysregulated protein, the exportin molecule, and RanGTP is formed export of these tumor suppressor proteins into the and transported across the nuclear pore complex to cytoplasm, where they are unable to exercise their the cytoplasm.4 In the cytoplasm, Ran guanosine effects, thereby resulting in aberrant growth signaling, triphosphate causes hydrolysis of the RanGTP, releasing inactivation of apoptosis, and tumor initiation and the cargo (which remains in the cytoplasm) and exportin growth (Fig. 1B). XPO1 overexpression is also associ- protein (which is recycled back to the nucleus) ated with drug resistance on account of export of drug (Fig. 1A).5 At least seven eukaryotic exportins have been targets such as topoisomerase II and galectin-3.16,17 identified (Table 16–12). Although most of these are 13 responsible for transport, exportin-1 (XPO1) (also Nuclear Export Targeting known as chromosomal region maintenance 1) is a more Given the critical role of nuclear export in cell ubiquitous receptor protein responsible for transporting cycle regulation and tumorigenesis, efforts to inhibit approximately 220 proteins.6,14 XPO1 pharmacologically have been undertaken. First- generation XPO1 inhibitors include natural products Role of Nuclear Export Functions in such as leptomycin B (see Table 1). Leptomycin B Normal Cell Physiology and Cancer irreversibly alkylates an XPO1 residue (cysteine XPO1 is the sole exporter of several tumor sup- 528), preventing XPO1 binding to cargo protein nuclear pressor proteins and cell cycle regulators, including export signals. This in turn leads to inhibition of export retinoblastoma, adenomatous polyposis coli, p53, p73, complex formation, as well as to nuclear retention p21, p27, forkhead box O, signal transducer and acti- of tumor suppressor proteins (Fig. 1C).18 Despite vator of transcription 3, inhibitor of kB, topoisomerase promising preclinical studies, strong dose-limiting

Figure 1. Export through the nuclear pore complex. (A) Physiologic state. Export complexes containing exportin-1 (XPO1), a cargo protein, and Ran guanosine triphosphate (RanGTP) are transported across the nuclear pore because of the RanGTP:Ran guanosine diphosphate gradient. Ran guanosine triphosphatase hydrolyzes RanGTP in the cytoplasm, leading to dissociation of the complex in the cytoplasm. (B) Cancer. Up-regulation of XPO1 results in dysregulated cytoplasmic transport of cell cycle regulators, leading to their accumulation in the cytoplasm and inability to exert their effects. In turn, this state leads to aberrant growth signaling, inactivation of apoptosis, and tumor initiation and growth. (C) Pharmacologic inhibition. Selective inhibitors of nuclear export (SINE) bind to XPO1 and prevent its interaction with cargo proteins, thereby inhibiting nuclear export. Cell cycle regulators are retained in the nucleus, leading to growth inhibition. RB, reginoblastoma; APC, adeno- matous polyposis coli; GTPase, guanosine triphosphatase; IkB, inhibitor of kB. --- 2017 Nuclear Export Inhibition 3

Table 1. Characteristics of Exportin Molecules Exportin protein Alternative Name Chromosome Cargo Role in Cancer Reference XPO1 CRM1 protein 2p15 >200 macromolecules, Up-regulated in multiple Stade et al. (1997)6 including protein and cancer types, RNA (including several associated with tumor suppressor genes tumorigenesis and and cell cycle drug resistance regulators, such as p53, p21, Rb, APC, and FOXO) Exportin-2 CAS protein, chromosome 20q13.13 Importin-a Overexpressed in Holzer et al., (2016)7 segregation 1-like thyroid cancer protein Exportin-3, Karyopherin-b, 12q14.2 tRNA — Leisegag et al. (2012)8 (Exportin-T) tRNA exportin Exportin-4 KIAA1721, FLJ13046, 13q12.11 Broad substrate — Kurisaki et al. (2006)9 specificity, including SMAD3 and eIF5A Exportin-5 Ran-binding protein 21, 6p21.1 Proteins bearing a Inactivating mutations Melo et al. (2010)10 KIAA1291 double-stranded RNA associated with binding domain and microsatellite double-stranded RNAs, instability micro-RNA precursors, tRNA, and eIF1A Exportin-6 Ran-binding protein 20, 16p11.2 Actin — Stuven et al. (2003)11 KIAA0370, FLJ22519 Exportin-7 Ran-binding protein 16, 8p21.3 eIF4A1, ARHGAP1, — Mingot et al. (2004)12 KIAA0745 VPS26A, VPS29, VPS35 and SFN, and p50RhoGAP XPO1, exportin-1; CRM1, chromosome region maintenance 1; Rb, retinoblastoma; APC, adenomatous polyposis coli; FOXO, forkhead box O; CAS, cellular apoptosis susceptibility; tRNA, transfer RNA; SMAD3, mothers against decapentaplegic homolog 3; eIF, eukaryotic translation initiation factor; ARHGAP1, rho GTPase activating protein 1; VPS, vacuolar protein sorting–associated protein; SFN, stratifin. toxicities (anorexia and nausea) and minimal clinical response rate of 4% and a stable disease rate of benefit in early studies limited development of lep- 43%.23 tomycin B.19 Newer pharmacologic agents, termed selective inhibitors of nuclear export (SINE), reversibly bind the XPO1 cysteine 528 residue. To date, the most Preclinical Studies and Clinical Trials in extensively studied of the SINE is selinexor (KPT-330 Lung Cancer [Karyopharm Therapeutics, Inc., Newton Centre, MA]). XPO1 is overexpressed in lung cancer cells, partic- In multiple in vitro and in vivo models, selinexor has ularly those arising in the setting of exposure to demonstrated single-agent anticancer activity and syn- nicotine-derived nitrosamine ketone (a tobacco ergistic effects in combination regimens (Table 2). carcinogen).24 Preclinical studies have demonstrated Globally, selinexor has been administered to more than antitumor activity of SINE in NSCLC cell lines and 2100 patients. Common adverse events include low- xenografts.25,26 SINE have shown efficacy against EGFR grade nausea (62%), fatigue (60%), anorexia (51%), inhibitor–resistant NSCLC cell lines in a time- and thrombocytopenia (42%), and vomiting (37%), which dose- dependent manner.26 Synergism with chemo- have generally been readily managed with standard therapy and radiation therapy has been demonstrated supportive care measures. in the presence of diverse molecular alterations, As monotherapy, selinexor has induced responses including EGFR,p53,RAS,and phosphatidylinositol-4, in hematologic malignancies and yielded disease con- 5-bisphosphate 3-kinase catalytic subunit alpha gene trol in solid tumors.20–22 In one study, 31% of evalu- (PIK3CA) mutations.25,27 able patients had an objective response with use Efficacy against KRAS-mutant lung adenocarcinoma, a of selinexor across a spectrum of non-Hodgkin’slym- disease setting lacking specific targeted therapies to phoma subtypes, with a median duration of response date, appears particularly promising. In a multigenomic exceeding 10 months.22 In another study, among 157 screen of 4700 biological processes in more than 100 evaluable patients with advanced or metastatic solid human NSCLC cell lines, nuclear transport machinery tumors, single-agent selinexor resulted in an objective emerged as the sole process exhibiting synthetic-lethal 4 Gupta et al Journal of Thoracic Oncology Vol. - No. -

Table 2. Inhibitors of Nuclear Export Tested in Humans Molecular Trial Indication for Human ClinicalTrials.gov Drug Parent Company Target Phase Use Identifier(s) First-generation nuclear export inhibitor Leptomycin (also Warner-Lambert/Pfizer, Exportin-1 1 Advanced refractory called elactocin) New York, New York solid cancers Second-generation nuclear export inhibitors SL-801 Stemline Therapeutics, Exportin-1 1 Advanced solid tumors NCT02667873 Inc., New York, New York Selinexor (KPT-330) Karyopharm Therapeutics, Exportin-1 2/ 3 Acute myelogenous (>50 trials in total). Inc., Newton Centre, MA , Lung cancer trials: myelodysplastic NCT02536495, syndrome, T-cell NCT02351505, , B-cell NCT03095612, lymphoma, chronic NCT02250885, lymphocytic leukemia, NCT02213133 , glioblastoma, gynecological cancers, lung cancer, , sarcoma, , , , colon cancer, , gastric cancer, esophageal cancer, and salivary gland tumors Verdinexor (KPT-335) Karyopharm Therapeutics, Exportin-1 1 Antiviral agent (approved NCT02431364 Inc., Newton Centre, MA for canine lymphoma) KPT-8602 Karyopharm Therapeutics, Exportin-1 1/ 2 Relapsed refractory NCT02649790 Inc., Newton Centre, MA multiple myeloma interactions in KRAS-driven cancers.28 In this study, 2. Ullman KS, Powers MA, Forbes DJ. Nuclear export receptors: the primary mechanism of cell kill was intolerance to from importin to exportin. Cell. 1997;90(6):967–970. nuclear accumulation of IkB with consequent inhibition 3. Dong X, Biswas A, Suel KE, et al. Structural basis for k leucine-rich recognition by CRM1. of nuclear factor B transcription activity. Rare cases – < Nature. 2009;458:1136 1141. of intrinsic resistance ( 20%) were associated with 4. Monecke T, Guttler T, Neumann P, Dickmanns A, FSTL5 follistatin-like 5 gene ( ) mutations and attributed Gorlich D, Ficner R. Crystal structure of the nuclear to yes-associated protein 1 (YAP1) activation. With few export receptor CRM1 in complex with Snurportin1 and exceptions, nuclear export inhibition had limited efficacy RanGTP. Science. 2009;324:1087–1091. against KRAS wild-type cell lines. 5. Lui K, Huang Y. RanGTPase: a key regulator of In summary, the broad genomic landscape in lung nucleocytoplasmic trafficking. MolCellPharmacol. – cancer makes it an attractive clinical setting for SINE. 2009;1(3):148 156. 6. Stade K, Ford CS, Guthrie C, Weis K. Exportin 1 To date, selinexor trials in advanced squamous cell (Crm1p) is an essential nuclear export factor. Cell. lung cancer (NCT02536495) and relapsed SCLC 1997;90(6):1041–1050. 23 (NCT02351505) have been initiated. A phase 1/2 trial 7. Holzer K, Drucker E, Oliver S, et al. Cellular apoptosis in previously treated advanced KRAS-mutant NSCLC is susceptibility (CAS) is overexpressed in thyroid car- under way (NCT03095612). cinoma and maintains tumor cell growth: a potential link to the BRAFV600E mutation. Int J Oncol. Acknowledgements 2016;48:1679–1687. This study was Funded in part by a National Cancer 8. Leisegang MS, Martin R, Ramirez AS, Bohnsack MT. Exportin t and exportin 5: tRNA and miRNA biogenesis— Institute Midcareer Investigator Award in Patient- and beyond. Biol Chem. 2012;393:599–604. Oriented Research (K24CA201543-01 to Dr. Gerber). 9. Kurisaki A, Kurisaki K, Kowanetz M, et al. The mechanism of nuclear export of Smad3 involves exportin 4 and Ran. References Mol Cell Biol. 2006;26:1318–1332. 1. Schmidt HB, Gorlich D. Transport selectivity of nuclear 10. Melo SA, Moutinho C, Ropero S, et al. A genetic defect in pores, phase separation, and membraneless organelles. exportin-5 traps precursor microRNAs in the nucleus of Trends Biochem Sci. 2016;41:46–61. cancer cells. Cancer Cell. 2010;18:303–315. --- 2017 Nuclear Export Inhibition 5

11. Stuven T, Hartmann E, Gorlich D. Exportin 6: a novel 21. Gounder MM, Zer A, Tap WD, et al. Phase IB study of nuclear export receptor that is specific for profilin.actin selinexor, a first-in-class inhibitor of nuclear export, in complexes. EMBO J. 2003;22:5928–5940. patients with advanced refractory bone or soft tissue 12. Mingot JM, Bohnsack MT, Jakle U, Gorlich D. Exportin 7 sarcoma. J Clin Oncol. 2016;34:3166–3174. defines a novel general nuclear export pathway. EMBO J. 22. Kuruvilla J, Savona M, Baz R, et al. Selective inhibition 2004;23:3227–3236. of nuclear export with selinexor in patients with non- 13. Guttler T, Gorlich D. Ran-dependent nuclear export Hodgkin’s lymphoma. Blood. 2017;129:3175–3183. mediators: a structural perspective. EMBO J. 23. Abdul Razak AR, Mau-Soerensen M, Gabrail NY, et al. 2011;30(17):3457–3474. first-in-class, first-in-human phase i study of selinexor, a 14. Turner JG, Sullivan DM. CRM1-mediated nuclear export selective inhibitor of nuclear export, in patients with of proteins and drug resistance in cancer. Curr Med advanced solid tumors. J Clin Oncol. 2016;34:4142–4150. Chem. 2008;15(26):2648–2655. 24. Gao W, Lu C, Chen L, Keohavong P. Overexpression of 15. Fukuda M, Asano S, Nakamura T, et al. CRM1 is respon- CRM1: a characteristic feature in a transformed pheno- sible for intracellular transport mediated by the nuclear type of lung carcinogenesis and a molecular target for export signal. Nature. 1997;390(6657):308–311. lung cancer adjuvant therapy. J Thorac Oncol. 16. Takenaka Y, Fukumori T, Yoshii T, et al. Nuclear export of 2015;10:815–825. phosphorylated galectin-3 regulates its antiapoptotic 25. Sun H, Hattori N, Chien W, et al. KPT-330 has antitumour activity in response to chemotherapeutic drugs. Mol Cell activity against non-small cell lung cancer. Br J Cancer. Biol. 2004;24:4395–4406. 2014;111:281–291. 17. Turner JG, Dawson J, Sullivan DM. Nuclear export 26. Wang S, Han X, Wang J, Yao J, Shi Y. Antitumor effects of proteins and drug resistance in cancer. Biochem of a novel chromosome region maintenance 1 (CRM1) Pharmacol. 2012;83:1021–1032. inhibitor on non-small cell lung cancer cells in vitro and 18. Meissner T, Krause E, Vinkemeier U. Ratjadone and in mouse tumor xenografts. PLoS One. 2014;9:e89848. leptomycin B block CRM1-dependent nuclear export by 27. Cromie MM, Gao W. Epigallocatechin-3-gallate en- identical mechanisms. FEBS Lett. 2004;576:27–30. hances the therapeutic effects of leptomycin B on 19. Newlands ES, Rustin GJ, Brampton MH. Phase I trial of human lung cancer a549 cells. Oxid Med Cell Longev. elactocin. Br J Cancer. 1996;74:648–649. 2015;2015:217304. 20. Garzon R, Savona M, Baz R, et al. A phase I clinical trial 28. Kim J, McMillan E, Kim HS, et al. XPO1-dependent nu- of single-agent selinexor in . clear export is a druggable vulnerability in KRAS-mutant Blood. 2017;129:3165–3174. lung cancer. Nature. 2016;538:114–117.