Sharon Tamir

Verdinexor, a Novel Selective Inhibitor of Nuclear Export, Reduces Influenza A Virus Replication In Vitro and In Vivo

ABSTRACT Influenza is a global health concern, causing death, morbidity, and economic losses. Chemotherapeutics that target influenza virus are available; however, rapid emergence of drug-resistant strains is common. Therapeutic targeting of host proteins hijacked by influenza virus to facilitate replication is an antiviral strategy to reduce the development of drug resistance. Nuclear export of influenza virus ribonucleoprotein (vRNP) from infected cells has been shown to be mediated by exportin 1 (XPO1) interaction with viral nuclear export protein tethered to vRNP. RNA interference screening has identified XPO1 as a host proinfluenza factor where XPO1 silencing results in reduced influenza virus replication. The Streptomyces metabolite XPO1 inhibitor leptomycin B (LMB) has been shown to limit influenza virus replication in vitro; however, LMB is toxic in vivo, which makes it unsuitable for therapeutic use. In this study, we tested the anti-influenza virus activity of a new class of orally available small-molecule selective inhibitors of nuclear export, specifically, the XPO1 antagonist KPT-335 (verdinexor). Verdinexor was shown to potently and selectively inhibit vRNP export and effectively inhibited the replication of various influenza virus A and B strains in vitro, including pandemic H1N1 virus, highly pathogenic H5N1 avian influenza virus, and the recently emerged H7N9 strain. In vivo, prophylactic and therapeutic administration of verdinexor protected mice against disease pathology following a challenge with influenza virus A/California/04/09 or A/Philippines/2/82-X79, as well as reduced lung viral loads and proinflammatory cytokine expression, while having minimal toxicity. These studies show that verdinexor acts as a novel anti-influenza virus therapeutic agent. IMPORTANCE Antiviral drugs represent important means of influenza virus control. However, substantial resistance to currently approved influenza therapeutic drugs has developed. New antiviral approaches are required to address drug resistance and reduce the burden of influenza virus-related disease. This study addressed critical preclinical studies for the development of verdinexor (KPT-335) as a novel antiviral drug. Verdinexor blocks progeny influenza virus genome nuclear export, thus effectively inhibiting virus replication. Verdinexor was found to limit the replication of various strains of influenza A and B viruses, including a pandemic H1N1 influenza virus strain, a highly pathogenic H5N1 avian influenza virus strain, and a recently emerging H7N9 influenza virus strain. Importantly, oral verdinexor treatments, given prophylactically or therapeutically, were efficacious in limiting lung virus burdens in influenza virus-infected mice, in addition to limiting lung proinflammatory cytokine expression, pathology, and death. Thus, this study demonstrated that verdinexor is efficacious against influenza virus infection in vitro and in vivo.

A Nuclear Attack on Traumatic Brain Injury: Sequestration of Cell Death in the Nucleus

Exportin 1 (XPO1/CRM1) plays prominent roles in the regulation of nuclear protein export. Selective inhibitors of nuclear export (SINE) are small orally bioavailable molecules that serve as drug‐like inhibitors of XPO1, with potent anti‐cancer properties. Traumatic brain injury (TBI) presents with a secondary cell death characterized by neuroinflammation that is putatively regulated by nuclear receptors.

Human exportin-1 is a target for combined therapy of HIV and AIDS related lymphoma

Infection with HIV ultimately leads to advanced immunodeficiency resulting in an increased incidence of cancer. For example primary effusion lymphoma (PEL) is an aggressive non-Hodgkin lymphoma with very poor prognosis that typically affects HIV infected individuals in advanced stages of immunodeficiency. Here we report on the dual anti-HIV and anti-PEL effect of targeting a single process common in both diseases. Inhibition of the exportin-1 (XPO1) mediated nuclear transport by clinical stage orally bioavailable small molecule inhibitors (SINE) prevented the nuclear export of the late intron-containing HIV RNA species and consequently potently suppressed viral replication. In contrast, in CRISPR-Cas9 genome edited cells expressing mutant C528S XPO1, viral replication was unaffected upon treatment, clearly demonstrating the anti-XPO1 mechanism of action. At the same time, SINE caused the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity in PEL cells resulting in cell cycle arrest and effective apoptosis induction. In vivo, oral administration arrested PEL tumor growth in engrafted mice. Our findings provide strong rationale for inhibiting XPO1 as an innovative strategy for the combined anti-retroviral and anti-neoplastic treatment of HIV and PEL and offer perspectives for the treatment of other AIDS-associated cancers and potentially other virus-related malignancies.

Antiviral Efficacy of Verdinexor In Vivo in Two Animal Models of Influenza A Virus Infection

Influenza A virus (IAV) causes seasonal epidemics of respiratory illness that can cause mild to severe illness and potentially death. Antiviral drugs are an important countermeasure against IAV; however, drug resistance has developed, thus new therapeutic approaches are being sought. Previously, we demonstrated the antiviral activity of a novel nuclear export inhibitor drug, verdinexor, to reduce influenza replication in vitro and pulmonary virus burden in mice. In this study, in vivo efficacy of verdinexor was further evaluated in two animal models or influenza virus infection, mice and ferrets. In mice, verdinexor was efficacious to limit virus shedding, reduce pulmonary pro-inflammatory cytokine expression, and moderate leukocyte infiltration into the bronchoalveolar space. Similarly, verdinexor-treated ferrets had reduced lung pathology, virus burden, and inflammatory cytokine expression in the nasal wash exudate. These findings support the anti-viral efficacy of verdinexor, and warrant its development as a novel antiviral therapeutic for influenza infection.

247 Selective inhibitors of nuclear export (SINE) block the expression of DNA damage repair proteins and sensitize cancer cells to DNA damage therapeutic agents

Conclusions: These studies reveal no evidence for an allosteric trapping mechanism and indicate that all PARP inhibitors examined trap PARP1 via catalytic inhibition. The potency of PARP inhibitors with respect to trapping and catalytic inhibition is linearly correlated in biochemical systems. In cells, trapping potency is related to concentrations required for potentiation in animal models. Detection of PARP trapping in cells requires supraphysiologic conditions that exhaust cellular NAD and PAR, exceed concentrations required to elicit synergism and are not tolerated in vivo. In addition trapping potency appears to be inversely correlated with tolerability. Quantitation of the degree of trapping that is tolerable and is required for therapeutic benefit is under active investigation. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Abstract 3810: Selinexor (KPT-330), a novel selective inhibitor of nuclear export (SINE), shows single agent efficacy against alveolar soft part sarcoma (ASPS) in vivo

Chromosomal Region Maintenance Protein 1/Exportin 1 (CRM1/XPO1) is a key nuclear export protein whose inhibition leads to the nuclear accumulation of Tumor Suppressor Proteins (TSPs) and renders cancer cells susceptible to apoptosis. Selinexor is orally bioavailable and represents a novel class of small molecule compounds with potent activity against a wide variety of cancers. Selinexor is currently in Phase 1 clinical studies in hematological and solid cancer patients (Clinicaltrials.gov NCT01607892 and NCT01607905). We tested the activity of Selinexor in a soft tissue sarcoma – ASPS that is resistant to traditional chemotherapy and irradiation treatment. Here we report in vitro activity of Selinexor against ASPS and in vivo efficacy results in xenograft models. Methods We used MTT, FACS, qPCR, immunofluorescence, immunostaining and immunoblots to measure the in vitro and in vivo effects of KPT-330 on the ASPS-KY cell line and in xenograft models. Results The IC 50 of the ASPS-KY cell line treated with Selinexor for 72 hours was 10 μM. This concentration induced nuclear accumulation of p53, p21, IκB and FOXO3A within 4 hours of treatment, and by 24 hours cells stopped DNA synthesis and were arrested at G1 phase of the cell cycle. By 72 hours, 25% of the cells died. Prior to cell death, the drug reduced the survival protein BCL2 as well as other pro-proliferative proteins such as CDK4, Cyclin D and E2F. In addition, Selinexor induced dephosphorylation of pRb activating its tumor suppressor activity and also induced Caspase 3/7 and PARP cleavage. To assess the in vivo activity of Selinexor in a xenograft model of ASPS, we treated mice with 10 or 20 mg/kg of KPT-330 using a 3 times weekly oral dosing schedule. Following a week of treatment, tumors showed accumulation of TSPs as well as significant reduction of the proliferation marker Ki-67. Following treatment with Selinexor for 40 days, tumor growth was inhibited by 70% at 10 mg/kg and by 80% at 20 mg/kg compared with vehicle treated animals. Analysis of immunoblots from these tumors showed induction of p21, with corresponding reduction of the pro-survival and proliferation markers c-Jun, c-Met, survivin, ERK and HSP70. Histological analysis revealed apoptosis and large areas of fibrosis in treated tumors. These results indicated that Selinexor not only inhibited tumor growth, but also induced ASPS cell death in vivo. Conclusions Our results demonstrated that Selinexor is effective against ASPS in vivo as a single agent and further support the development of SINE-based therapies for alveolar soft part sarcoma that has currently no cure. We will perform further studies to test Selinexor in drug combination studies to identify synergism with other therapies. Citation Format: Marsha L. Crochiere, Trinayan Kashyap, Boris Klebanov, William Senapedis, Diego del Alamo, Sharon Tamir, Erkan Baloglu, Dilara McCauley, Robert Carlson, Michael Kauffman, Sharon Shacham, George Demetri, Andrew Wagner, Ewa Sicinska, Prafulla Gokhale, Nancy Kohl, Amy Saur, Yosef Landesman. Selinexor (KPT-330), a novel selective inhibitor of nuclear export (SINE), shows single agent efficacy against alveolar soft part sarcoma (ASPS) in vivo . [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3810. doi:10.1158/1538-7445.AM2014-3810

In vitro toxicity and efficacy of verdinexor, an exportin 1 inhibitor, on opportunistic viruses affecting immunocompromised individuals

Infection of immunocompromised individuals with normally benign opportunistic viruses is a major health burden globally. Infections with viruses such as Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), Kaposi’s sarcoma virus (KSHV), adenoviruses (AdV), BK virus (BKPyV), John Cunningham virus (JCPyV), and human papillomavirus (HPV) are significant concerns for the immunocompromised, including when these viruses exist as a co-infection with human immunodeficiency virus (HIV). These viral infections are more complicated in patients with a weakened immune system, and often manifest as malignancies resulting in significant morbidity and mortality. Vaccination is not an attractive option for these immune compromised individuals due to defects in their adaptive immune response. Verdinexor is part of a novel class of small molecules known as SINE (Selective Inhibitor of Nuclear Export) compounds. These small molecules demonstrate specificity for the nuclear export protein XPO1, to which they bind and block function, resulting in sequestration of XPO1-dependent proteins in the nucleus of the cell. In antiviral screening, verdinexor demonstrated varying levels of efficacy against all of the aforementioned viruses including previously with HIV. Studies by other labs have discussed likely mechanisms of action for verdinexor (ie. XPO1-dependence) against each virus. GLP toxicology studies suggest that anti-viral activity can be achieved at a tolerable dose range, based on the safety profile of a previous phase 1 clinical trial of verdinexor in healthy human volunteers. Taken together, these results indicate verdinexor has the potential to be a broad spectrum antiviral for immunocompromised subjects for which vaccination is a poor option.

Abstract B198: Selinexor (KPT-330), a novel Selective Inhibitor of Nuclear Export (SINE) shows marked NF-kB inhibition and significant anticancer activity against Non-Small Cell Lung Cancer (NSCLC).

Selinexor (KPT-330) is a novel small molecule inhibitor of nuclear export through covalent binding to Exportin 1 (XPO1/CRM1) leading to forced nuclear retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IκB, resulting in selective death of cancer cells. Preclinical studies have shown that oral Selinexor is well tolerated, even with prolonged (4-8 month) administration. Human Phase 1 clinical studies with Selinexor in hematological and solid cancer patients are ongoing (Clinicaltrials.gov NCT01607892 and NCT01607905). In this study we evaluated the mechanisms involved in Selinexor-induced growth inhibition of NSCLC in vitro and in vivo. Methods: A panel of NSCLC cells with various genetic backgrounds were treated with Selinexor and tested in proliferation (MTT), clonogenic survival, and flow cytometric assays. The cellular localization of TSPs were evaluated by immunofluorescence (IF). Western blotting was used to study levels of cell cycle regulating proteins and quantify markers of cell survival. Functional inhibition of NF-κB was measured using a transcription factor assay kit. A SCID mouse xenograft model of NSCLC, adenocarcinoma A549 (wt p53, NF-κB IC50 of 1.19 uM), was used to evaluate Selinexor effects in vivo and tumors were evaluated by histology and immunohistochemistry (IHC). Results: Selinexor induced cell cycle arrest, inhibited tumor cell growth and clonogenic formation and induced apoptosis in lung cancer cell lines regardless of genetic background (IC50 25-700 nM). Treated normal human dermal fibroblasts entered into cell cycle arrest but resisted apoptosis induction (IC50 4.0 uM). IF of the TSPs p53, p21, IκB, E2F4, and survivin demonstrated strong nuclear localization after 4 to 24 hour treatment with Selinexor followed by apoptosis as determined by cleavage of PARP, caspases 3 and 8 by immunoblot analysis. Functional evaluation of NF-kB in A549 and H1299 revealed transcriptional repression with IC50 values similar to MTT assays. Finally, A549 xenografts treated with Selinexor (10 - 20 mg/kg QoDx3) showed marked tumor suppression when compared to vehicle and cisplatin-treated mice (growth inhibition was 78% at 20 mg/kg, p Conclusions: In NSCLC, Selinexor forces nuclear retention of TSPs, inhibits NF-κB transcriptional activity, inhibits tumor growth and induces cell death regardless of p53 status. Selinexor thus has therapeutic potential for the treatment of NSCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B198. Citation Format: William Senapedis, Marsha Crochiere, Tami Rashal, Trinayan Kashyap, Boris Klebanov, Jean-Richard Saint-Martin, Ori Kalid, Sharon Shechter, Diego del Alamo, Mwanasha Hamuza, Gali Golan, Eran Shacham, Sharon Tamir, Dilara McCauley, Erkan Baloglu, Michael Kauffman, Sharon Shacham, Yosef Landesman. Selinexor (KPT-330), a novel Selective Inhibitor of Nuclear Export (SINE) shows marked NF-kB inhibition and significant anticancer activity against Non-Small Cell Lung Cancer (NSCLC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B198.

Abstract A188: SINE resistant fibrosarcoma cells reveal changes in profile of gene expression, but continue to be sensitive to combination treatment by proteasome inhibition.

SINE (Selective Inhibitors of Nuclear Export) are novel small molecule drugs in phase I clinical trials for advanced cancers. SINEs inhibit nuclear export through covalent binding to Exportin 1 (XPO1/CRM1) leading to forced nuclear retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IkB, resulting in selective death of cancer cells. Resistant cells were created by treating the sensitive fibrosarcoma cell line HT1080 with increasing concentrations of SINE over 10 months. Gene chip analysis of parental-sensitive and drug-resistant cells treated with SINE demonstrated activation of distinct pathways that mediate either cell death or survival. In addition, SINE resistance was overcome by drug combination with proteasome inhibition. Methods: Resistant cells were generated with selection in increasing concentrations of SINE. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. FACS and immunoblots were used to measure effects on cell cycle, protein expression and cell death. RNA from nai[[Unable to Display Character: ]]ve and drug treated sensitive/resistant cells was analyzed by Affymetrix microarrays and qPCR. A drug combination study was performed to evaluate whether resistance to SINE could be overcome with proteasome inhibition. Results: Treatment of SINE-sensitive fibrosarcoma cell line HT1080 (IC50 = 13.6nM) with gradually increasing concentrations of SINE for 10 months resulted in > 100 fold decrease in sensitivity to SINE cytotoxicity (IC50 = 1.7μM). Resistant cells displayed prolonged cell cycle (∼72 vs 24 hrs) compared to parental cells. Resistant cells did not show increased MDR1 and MRP1 activity, suggesting that resistance to SINE was not mediated by induction of the multidrug resistance mechanism. Sequencing of XPO-1 from the SINE resistant cells revealed no mutations in the SINE / cargo binding pocket including the reactive Cys528. Upon exposure to SINE, resistant cells had reduced nuclear accumulation of p53, p21, FOXO1A, IkB, p27, and PP2A proteins compared with SINE-sensitive cells. SINE treatment of both sensitive and resistant cells resulted in pRB de-phosphorylation and induction of p53 and its downstream target p21. In addition, SINE treatment reduced the levels of the anti-apoptotic protein Mcl-1, and induced the apoptotic markers Caspase 3 and PARP cleavage. Microarray analysis revealed changes in cell survival and cell death pathways, which were confirmed by qPCR. In spite of the changes, resistant cells continue to show synergistic death effects induced by SINE in combination with proteasome inhibition. Conclusions: The extensive selection time (10 months) needed to achieve drug resistance suggests that generation of resistance may be difficult and that drug response may be prolonged. However such a resistance would be overcome by drug combination treatment. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A188. Citation Format: Marsha L. Crochiere, Trinayan Kashyap, Jean-Richard Saint-Martin, Sharon Shechter, Ori Kalid, Eran Shacham, William Senapedis, Boris Klebanov, Sharon Tamir, Diego del Alamo, Mwanasha Hamuza, Gali Golan, Erkan Baloglu, Dilara McCauley, Michael Kauffman, Sharon Shacham, Yosef Landesman. SINE resistant fibrosarcoma cells reveal changes in profile of gene expression, but continue to be sensitive to combination treatment by proteasome inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A188.

Abstract 875: Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitors of Nuclear Export (SINE).

Abstract SINE are novel small molecules in human phase I clinical trials for advanced cancers. SINEs inhibit nuclear export through covalent binding to Exportin 1 (XPO1/CRM1) leading to forced nuclear retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, PTEN, pRB and IKB, leading to selective death of cancer cells. Resistant cells were created by treating the sensitive fibrosarcoma cell line HT1080 with increasing concentrations of SINE over 12 months. Gene chip analysis of parental-sensitive and drug-resistant cells that were treated with SINE resulted in activation of distinct pathways that mediate mechanisms of response and drug resistance. Methods Resistant cells were generated by selection in increasing concentrations of SINE. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. FACS, qPCR and immunoblots were used to measure effects on cell cycle, gene expression and cell death. RNA from naive and drug treated sensitive/resistant cells was analyzed by Affymetrix microarrays. Results Treatment of SINE-sensitive fibrosarcoma cell line HT1080 (IC50 = 13.6nM) with gradually increasing concentrations of SINE for ∼1 year resulted with > 100 fold decrease in sensitivity to SINE cytotoxicity (IC50 = 1.7μM). Resistant cells displayed prolonged cell cycle (∼72 vs 24hrs) compared to parental cells. Resistant cells did not show increased PgP activity, suggesting that resistance to SINE was not the result of the induction of the multidrug resistance mechanism. Sequencing of CRM1 from the SINE resistant cells revealed no mutations in the SINE / cargo binding pocket including the reactive Cys528. Upon exposure to SINE, resistant cells had reduced nuclear accumulation of p53, p21, FOXO1A, IKB and p27 compared with SINE-sensitive cells. Interestingly, in SINE-sensitive cells, but not in resistant cells, SINE treatment resulted in potent pRB dephosphorylation (growth suppressive form of pRB), nuclear localization and the activation of p53 as measured by the induction of p53-downstream targets p21 and MIC1 (GDF15). In addition, SINE reduced levels of the anti-apoptotic protein Mcl-1, and induced the apoptotic markers Caspase 3 and PARP cleavage in sensitive but not in SINE resistant cells. Genes involved in cell adhesion, cytoskeleton formation, tight junctions, vesicle transport, cell cycle, and apoptosis were identified as key pathways correlating with drug response and resistance. Conclusions This study identified key pathways and genes that are likely mediating response and resistance to SINE antagonists. The extensive selection time (∼1 year) needed to achieve drug resistance suggests that generation of resistance may be difficult and that drug response may be prolonged. Citation Format: Yosef Landesman, Sharon Shechter, Jean-Richard Saint-Martin, Trinayan Kashyap, Marsha Crochiere, William Senapedis, Paul Ippolitti, Boris Klebanov, Sharon Tamir, Diego del Alamo, Mwanasha Hamuza, Gali Golan, Ori Kalid, Erkan Baloglu, Dilara McCauley, Michael Kauffman, Sharon Shacham. Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitors of Nuclear Export (SINE). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 875. doi:10.1158/1538-7445.AM2013-875