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PDF file - 219K, Effects of RhoB deletion on MT-myc induced mammary tumorigenesis in virgin female mice

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PDF file - 21K, RhoB is downregulated in MDA-MB-231 cells using lentivirus shRNA

PDF file - 54K, RhoB is downregulated in MCF7 cells by antisense oligonucleotides

PDF file - 34K, Level of expression of mRNA of different genes in RhoB+/+ and RhoB-/- mouse tumor cells

PDF file - 52K, Level of silencing of Akt in RhoB-/- and RhoB+/+ mouse cells

PDF file - 26K, Level of EGFR on the surface of human MDA-MB-231 cells

The protein STING (stimulator of interferon genes) is a central regulator of the innate immune system and plays an important role in antitumor immunity by inducing the production of cytokines such as type I interferon (IFN). Activation of STING stems from the selective recognition of endogenous cyclic dinucleotides (CDNs) by the large, polar, and flexible binding site, thus posing challenges to the design of small molecule agonists with drug-like physicochemical properties. In this work we present the design of SNX281, a small molecule STING agonist that functions through a unique self-dimerizing mechanism in the STING binding site, where the ligand dimer approximates the size and shape of a cyclic dinucleotide while maintaining drug-like small molecule properties. SNX281 exhibits systemic exposure, STING-mediated cytokine release, strong induction of type I IFN, potent in vivo antitumor activity, durable immune memory, and single-dose tumor elimination in mouse models via a Cmax-driven pharmacologic response. Bespoke computational methods – a combination of quantum mechanics, molecular dynamics, binding free energy simulations, and artificial intelligence – were developed during the course of the project to design SNX281 by explicitly accounting for the unique self-dimerization mechanism and the large-scale conformational change of the STING protein upon activation. Over the course of the project, we explored millions of virtual molecules while synthesizing and testing only 208 molecules in the lab. This work highlights the value of a multifaceted computationally-driven approach anchored by methods tailored to address target-specific problems encountered along the project progression from initial hit to the clinic.

Targeted protein degradation (TPD) has emerged as a powerful approach for removing (rather than inhibiting) proteins implicated in diseases. A key step in TPD is the formation of an induced proximity complex where a degrader molecule recruits an E3 ligase to the protein of interest (POI), facilitating the transfer of ubiquitin to the POI and initiating the proteasomal degradation process. Here, we address three critical aspects of the TPD process using atomistic simulations: 1) formation of the ternary complex induced by a degrader molecule, 2) conformational heterogeneity of the ternary complex, and 3) degradation efficiency via the full Cullin Ring Ligase (CRL) macromolecular assembly. The novel approach described here combines experimental biophysical data with molecular dynamics (MD) simulations to accurately predict ternary complex structures at atomic resolution. We integrate hydrogen-deuterium exchange mass spectrometry (HDX-MS, which measures the solvent exposure of protein residues) with MD to improve the efficiency and accuracy of the ternary structure predictions of the bromodomain of the cancer target SMARCA2 with the E3 ligase VHL, as mediated by three different degrader molecules. The simulations accurately reproduce X-ray crystal structures -- including a new structure that we determined in this work (PDB ID: 7S4E) -- with root mean square deviations (RMSD) of 1.1 to 1.6 A. The simulations also reveal a structural ensemble of low-energy conformations of the ternary complex. Snapshots from these simulations are used as seeds for additional simulations, where we perform 7.1 milliseconds of aggregate simulation time using Folding@home. The detailed free energy surface captures the crystal structure conformation within a low-energy basin and is consistent with solution-phase experimental data (HDX-MS and SAXS). Finally, we graft a structural ensemble of the ternary complexes onto the full CRL and perform enhanced sampling simulations, which suggest that differences in degradation efficiency may be related to the proximity distribution of lysine residues on the POI relative to the E2-loaded ubiquitin.

Therapeutics are currently unavailable for Venezuelan equine encephalitis virus (VEEV), which elicits flu-like symptoms and encephalitis in humans, with an estimated 14% of cases resulting in neurological disease. Here we identify anti-VEEV agents using in silico structure-based-drug-design (SBDD) for the first time, characterising inhibitors that block recognition of VEEV capsid protein (C) by the host importin (IMP) α/β1 nuclear transport proteins. From an initial screen of 1.5 million compounds, followed by in silico refinement and screening for biological activity in vitro, we identified 21 hit compounds which inhibited IMPα/β1:C binding with IC50s as low as 5 µM. Four compounds were found to inhibit nuclear import of C in transfected cells, with one able to reduce VEEV replication at µM concentration, concomitant with reduced C nuclear accumulation in infected cells. Further, this compound was inactive against a mutant VEEV that lacks high affinity IMPα/β1:C interaction, supporting the mode of its antiviral action to be through inhibiting C nuclear localization. This successful application of SBDD paves the way for lead optimization for VEEV antivirals, and is an exciting prospect to identify inhibitors for the many other viral pathogens of significance that require IMPα/β1 in their infectious cycle.

STING (Stimulator of Interferon Genes) is a master regulator of type I interferons and a key mediator of innate immunity. Activation of STING provides two critical anti-tumor responses – (1) The “spark” for initiating a robust innate immune response and (2) Priming and activation of a potent T cell mediated anti-tumor adaptive response. By serving as a bridge between the two arms of the immune system, activation of STING has the potential to transform the tumor microenvironment from immunologically “cold” to “hot” (inflamed), making otherwise resistant tumors respond to checkpoint blockade as well as other T cell targeting immunotherapies. Many preclinical and clinical drug discovery programs targeting STING activation are based on modifications of the naturally occurring cyclic dinucleotide (CDN) agonists. However, the CDN molecules suffer from poor drug-like properties and consequently cannot be delivered systemically. Thus, the benefits of STING activation by these first generation CDNs will be limited to only a subset of tumors that are accessible to intratumoral injection. The large, polar and charged binding site as well as the mechanism of activation of STING present unique drug discovery challenges to designing a small molecule that can induce the optimal, active conformation of the STING dimer while maintaining the physical-chemical properties required for systemic exposure. Using our proprietary physics-driven discovery engine with our integrative chemistry/biophysics/biology approach we have developed novel small molecule agonists of STING that exhibit selective and potent activity across all known isoforms of the human protein. Our small molecule agonist rapidly activates the STING/TBK1/IRF3 pathway (phosphorylation and translocation) and induces the expression of type I IFN in cells in a STING-dependent manner. Treatment of primary immune cells from healthy human donors results in the activation of dendritic cells and upregulation of costimulatory markers. Drug characteristics and STING pharmacology allow for unique dosing paradigm in vivo with robust induction of type I IFN after intravenous (i.v.) administration. A single i.v. dose produces potent and durable anti-tumor immunity and complete tumor regression in mice bearing syngeneic tumors. Activation of tumor directed T cell response provides long term protection against tumor re-challenge and effective systemic immunity. Our work highlights the tremendous potential of a small molecule STING agonist as a well-tolerated, systemically delivered cancer immunotherapeutic. Citation Format: Meghana M Kulkarni, Timothy Dwight, Kristen Marino, Bryce K Allen, Mohammed Taimi, Cecilia Bastos, Sujen Lai, Cheryl Koh, Ramya Samant, Bethany Tesar, Huang Huang, Brian Chamberlain, James M Rice, Dazhi Tan, Zhixiong Lin, Sharon Shechter, Stella Li, Sam Sparks, Holly Soutter, Steven Swann, Woody Sherman, Christopher Winter. True small molecule STING agonist generates systemic and potent anti-tumor immunity required for effective cancer immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A20. doi:10.1158/1535-7163.TARG-19-LB-A20

There are currently no therapeutics to treat infection with the alphavirus Venezuelan equine encephalitis virus (VEEV), which causes flu-like symptoms leading to neurological symptoms in up to 14% of cases. Large outbreaks of VEEV can result in 10,000 s of human cases and mass equine death. We previously showed that mifepristone (RU486) has anti-VEEV activity (EC50 = 20 μM) and only limited cytotoxicity (CC50 > 100 μM), but a limitation in its use is its abortifacient activity resulting from its ability to antagonize the progesterone receptor (PR). Here we generate a suite of new mifepristone analogues with enhanced antiviral properties, succeeding in achieving >11-fold improvement in anti-VEEV activity with no detectable increase in toxicity. Importantly, we were able to derive a lead compound with an EC50 of 7.2 µM and no detectable PR antagonism activity. Finally, based on our SAR analysis we propose avenues for the further development of these analogues as safe and effective anti-VEEV agents.

Although the alphavirus Venezuelan equine encephalitis virus (VEEV) has been the cause of multiple outbreaks resulting in extensive human and equine mortality and morbidity, there are currently no anti-VEEV therapeutics available. VEEV pathogenicity is largely dependent on targeting of the viral capsid protein (CP) to the host cell nucleus through the nuclear transporting importin (Imp) α/β1 heterodimer. Here we perform a high-throughput screen, combined with nested counterscreens to identify small molecules able to inhibit the Impα/β1:CP interaction for the first time. Several compounds were able to significantly reduce viral replication in infected cells. Compound G281-1564 in particular could inhibit VEEV replication at low μM concentration, while showing minimal toxicity, with steady state and dynamic quantitative microscopic measurements confirming its ability to inhibit CP nuclear import. This study establishes the principle that inhibitors of CP nucleocytoplasmic trafficking can have potent antiviral activity against VEEV, and represents a platform for future development of safe anti-VEEV compounds with high efficacy and specificity.

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology. Cancer Res; 73(1); 50–61. ©2012 AACR.

The nuclear export protein XPO1 is overexpressed in cancer, leading to the cytoplasmic mislocalization of multiple tumor suppressor proteins. Existing XPO1-targeting agents lack selectivity and have been associated with significant toxicity. Small molecule selective inhibitors of nuclear export (SINEs) were designed that specifically inhibit XPO1. Genetic experiments and X-ray structures demonstrate that SINE covalently bind to a cysteine residue in the cargo-binding groove of XPO1, thereby inhibiting nuclear export of cargo proteins. The clinical relevance of SINEs was explored in chronic lymphocytic leukemia (CLL), a disease associated with recurrent XPO1 mutations. Evidence is presented that SINEs can restore normal regulation to the majority of the dysregulated pathways in CLL both in vitro and in vivo and induce apoptosis of CLL cells with a favorable therapeutic index, with enhanced killing of genomically high-risk CLL cells that are typically unresponsive to traditional therapies. More importantly, SINE slows disease progression, and improves overall survival in the Eμ-TCL1-SCID mouse model of CLL with minimal weight loss or other toxicities. Together, these findings demonstrate that XPO1 is a valid target in CLL with minimal effects on normal cells and provide a basis for the development of SINEs in CLL and related hematologic malignancies. ispartof: Blood vol:120 issue:23 pages:4621-4634 ispartof: location:United States status: published

The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1-PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI ("target peptide") binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr(20) → Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the target peptide (K(d) = 37.0 μm), albeit with ∼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr(253) → Ala substitution. Comparison of the 1.5-Å resolution crystal structure of PDZ3 with its bound target peptide ((505)QEAKL(509)) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr(20) → Ala (PDZ1) + Tyr(253) → Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr(253) → Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr(20) → Ala (PDZ1) + Tyr(253) → Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.

Chronic activation of Akt signaling in the endothelium recapitulates the salient features of a tumor vasculature and can be inhibited by rapamycin, an inhibitor of mammalian target of rapamycin. This led to the hypothesis that the antitumor efficacy of rapamycin may be partially dependent on its ability to inhibit endothelial Akt signaling, making rapamycin an antiangiogenic agent and endothelial Akt pathway inhibitor. Dose-response studies with rapamycin showed that primary human endothelial cells and fibroblasts had a bimodal Akt response with effective reductions in phosphorylated Akt (pAkt) achieved at 10 ng/mL. In contrast, rapamycin increased pAkt levels in tumor cell lines. When tumor-bearing mice were treated with rapamycin doses comparable to those used clinically in transplant patients, we observed strong inhibition of mammary tumor growth. To test whether Akt activation in the endothelium was rate-limiting for this antitumor response, we engineered mouse mammary tumor virus–polyoma virus middle T antigen mice with endothelial cell–specific expression of constitutively activated Akt. We observed that the antitumor efficacy of rapamycin was reduced in the presence of elevated endothelial Akt activation. Just as we observed in MCF7 cells in vitro, rapamycin doses that were antiangiogenic resulted in increased pAkt levels in total mouse mammary tumor virus–polyoma virus middle T antigen tumor lysates, suggesting that tumor cells had an opposite Akt response following mammalian target of rapamycin inhibition compared with tumor endothelial cells. Together, these data support the hypothesis that endothelial Akt signaling in the tumor vasculature is an important target of the novel anticancer drug rapamycin. [Cancer Res 2007;67(11):5070–5]

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 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 naïve 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

Abstract Chromosomal 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) such as p53, FOXO, PTEN, pRB and I-κB. SINE are a novel class of compounds currently in clinical development for a variety of cancers. SINE irreversibly block CRM1-dependent nuclear export and force the nuclear retention of TSPs, inducing apoptosis in cancer cells. Here we report in vitro activity of SINE on a broad range of cancer cell lines and in vivo efficacy results in NCI-N87 gastric and HCT-116 colorectal carcinoma murine tumor xenograft models. Methods MTT cytotoxicity assays were used to determine IC50s of KPT-SINE on various cell lines. The effects of SINE on the cell cycle and gene expression were investigated by FACS and quantitative RT/PCR respectively. Inhibition of CRM1 nuclear export was determined by immunofluorescence of CRM1 cargos. The effects of SINE treatment on tumor growth in vivo were assessed by measuring tumor volumes and by imaging with FluoroThymidine Positron Emission Tomography (FLT-PET). In tumors, gene expression was analyzed and immunohistochemistry performed to detect TSP, proliferation and apoptosis markers. Results SINE showed potent cytotoxicity on the majority of the cell lines tested with IC50 values 80%. Treated tumor samples showed very low levels of Ki67 and increased TUNEL staining consistent with induction of cell cycle arrest and apoptosis in SINE-treated groups. Also, marked upregulation of the TSPs p53 and p21 was observed, indicating that SINE induced nuclear localization of these proteins in situ. In the NCI-N87 gastric carcinoma xenograft model, oral SINE treatment inhibited tumor growth >75% compared with vehicle- treated animals. Immunofluorescence analysis with biomarkers of proliferation, along with FLT-PET results, indicated significant inhibition of proliferation in tumors. Histological analysis of the tumors confirmed in vitro observations of cytotoxic effects on tumors, induction of apoptosis and the replacement of cancer cells with fibrotic tissue. Conclusions These studies demonstrate potent in vitro and in vivo efficacy of single agent SINE CRM1 inhibitors against NCI-N87 and HCT-116 xenografts. These data further support the development of SINE-based therapies for gastric and colorectal cancers. Citation Format: Dilara McCauley, Trinayan Kashyap, Mwanasha Hamuza, Yosef Landesman, Paul Ippolitti, Sharon Shechter, Jean Richard Saint-Martin, Marsha Crochiere, William Senapedis, Boris Klebanov, Sharon Tamir, Diego Del Alamo, Erkan Baloglu, Giulio Draetta, Michael Kauffman, Sharon Shacham. Selective inhibitors of nuclear export (SINE) display single agent efficacy against gastric (NCI-N87) and colon (HCT-116) xenografts. [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 4336. doi:10.1158/1538-7445.AM2013-4336

Abstract 2452 CRM1 (chromosomal regional maintenance 1) or XPO1 (exportin 1) is the major protein that mediates nuclear export. XPO1 has been shown to be over-expressed in cancer cells leading to ineffective cytoplasmic localization of multiple tumor suppressor genes such us p53, FOX03a, and Iκ-β. Chronic Lymphocytic Leukemia (CLL) is characterized by disrupted apoptosis caused both by microenviromental stimuli and aberrant activation of survival pathways including PI3K/Akt, NF-kB, and p53. We previously showed that first generations Selective Inhibitor of Nuclear Export (SINE), KPT-185 and KPT-251, specifically bind to and block XPO1 function resulting in re-activation of tumor suppressor pathways and apoptosis in CLL. Furthermore, we showed that KPT-251 at 75mg/Kg, 3 times per week (QODX3) slowed disease progression, and improved overall survival in the Eμ-TCL1-SCID mouse model of CLL (median OS = 130 and 72 days for KPT-330 and vehicle respectively; p=0.01) with minimal weight loss or other toxicities. The goal of this study was to determine the efficacy of KPT-330, a new generation SINE currently in Phase 1 human studies, with improved pharmacokinetic and oral bioavailability, in the Eμ-TCL1-SCID mouse model of CLL. We first compared the in-vitro activity of KPT-330 to KPT-251. KPT-330 induced improved levels of cytotoxicity on CLL cells compared to KPT-251 with retained selective cytotoxicity against tumor cells when compared to peripheral blood mononuclear cells and isolated B, and T cells. Similar to KPT-251, KPT-330 showed enhanced cytotoxic potency on CLL cells under coculture conditions with stromal cells compared to CLL alone (p10 mg/kg QODX3 but was fully reversed by the end of the study and did not appear to adversely affect the animals. Moreover, KPT-330 at doses of >10 mg/kg BIW and QODX3 significantly prevented an increase in circulating CLL cells compared to vehicle. To further validate KPT-330 in mice with leukemic phase (ie, very high tumor burdens), 20 additional mice were engrafted with CD19+ TCL1 leukemia cells and treatment with vehicle or 15 mg/kg KPT-330 (QoDx3/wk) was initiated 70 days post engraftment. Mice treated with KPT-330 had a significant survival advantage (p=0.0008, median OS = undefined; 99 days, for KPT-330 and vehicle respectively). KPT-330 was strikingly active in prolonging survival of TCL1-SCID mice. Using CLL as a disease model we validate the clinical effectiveness of targeting XPO1 with use of SINE, with favorable therapeutic index in-vivo. These data strongly support the ongoing Phase 1 clinical studies of KPT-330 in patients with advanced hematologic malignancies including CLL. Disclosures: Sandanayaka: Karyopharm Therapeutics: Employment. Shechter:Karyopharm Therapeutics: Employment. McCauley:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment.

Chromosome region maintenance 1/Exportin-1 (CRM1) is a key nuclear export protein whose inhibition leads to nuclear accumulation of tumor suppressor (TSP) and growth regulatory proteins (GRP). Through CRM1 inhibition, nuclear localization of these proteins restores cell cycle checkpoints and genome surveying functions culminating in apoptosis of tumor cells. Conversely, CRM1 inhibition of normal cells induces reversible cell cycle arrest. We developed a structural model of CRM1 suited for binding diverse small molecules and used it to guide the discovery and development of KPT-SINE (selective inhibitors of nuclear export). The model predicted that a switching motion of Met545 and Met583 in CRM1 was required for inhibitor accommodation. We found that published CRM1 inhibitors display efficient mimicry of cargo Nuclear Export Signals (NES) and a thiol reactive warhead proximal to Cys528 in CRM1. This was further confirmed by Yuh Min Chook who solved the X-ray structures of CRM1. In silico screening yielded 402 hits (from a library of ˜250,000 drug-like compounds) with 19 molecules active in vitro (IC50 Methods: CRM1 Model: The CRM1 model was developed using the novel consensus Induced Fit Docking approach (cIFD). In vitro/vivo evaluation: Compounds were tested in a cell-based microscopy assay to confirm CRM1-mediated nuclear export inhibition. Cells transfected with mutant CRM1-Cys528Ser were treated with KPT-SINE in order to determine covalent binding to Cys528. Cytotoxic IC50s of KPT-SINE were determined on a panel of cancer cell lines. Multiple KPT-SINE derivatives were tested for: 1) Nuclear localization of TSPs, 2) Effects on cell cycle and viability, 3) Effects on TSP protein expression, and 4) Mouse xenograft model efficacy. Results: KPT-SINE, potent inhibitors in the cell-based microscopy assay (EC50=20-130nM), were robust inhibitors of cancer cell proliferation and inducers of apoptosis. KPT-SINE inhibition of CRM1 started within 30 minutes and reached a maximum in 8 hrs. Additionally, KPT-SINE effects were blocked by transient transfection of mutant CRM1-Cys528Ser, supporting the role of Cys528. We also identified nuclear accumulation of p53, FOXO1a, FOXO3a, APC, IκB, p27, PP2Aα, and p21, which was followed by cell cycle arrest and death of cancer cells while normal cells undergo a reversible block. Both solid (MDA-MB-468) and hematological (Z-138) mouse xenografts treated with KPT-SINE at 25 mg/kg QoDx3 PO weekly for 4 weeks inhibited tumor growth by more than 70%. Immunohistochemical analysis of the tumor molecular markers from KPT-SINE treated animals exhibited nuclear localization of TSPs, a decrease in proliferation (Ki-67), and an increase in apoptosis (TUNEL). Conclusions: We described the correlative effects of KPT-SINE from the accumulation of nuclear TSPs at the molecular level through to potent in vivo efficacy in xenograft models. KPT-SINE, which were discovered and optimized using a structure-based approach, are due for Phase I clinical trials Mid-2012. This proffered talk is also presented as Poster A2. Citation Format: William T. Senapedis, Jr., Erkan Baloglu, Doriana Froim, Dilara McCauley, Yuh Min Chook, Michael Kauffman, Sharon Shacham, Yosef Landesman, Ori Kalid, Sharon Shechter, Jean Richard St Martin, Trinayan Kashyap, Boris Klebanov, Louis Plamondon, Vincent Sandanayaka. In silico designed covalent peptidomimetic inhibitors (KPT-SINE) of CRM1 modulate tumor suppressor protein nuclear export and induce apoptosis in cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr PR1.

e15200 Background: CRM1 (XPO1) is the sole exportin mediating transport of multiple tumor suppressor proteins (TSP) including p53, pRB, FOXO, APC and NPM1 out of the nucleus, abrograting their function. Many tumor types show overexpression of CRM1, thus extinguishing TSP function. CRM1 inhibition forces nuclear accumulation of TSPs inducing apoptosis in cancer cells. Methods: MSKCC PrCa gene database was used for mRNA analyses. Inhibition of CRM1 mediated NE was determined by IHC. MTT assays were used to determine cytotoxicity across divergent PrCa lines: LAPC-4 (p53wt, AR+, low Akt/mTOR); LnCaP (p53wt, AR+, high Akt/mTOR); LnCaP-C81 and LnCaP-C4-2B (p53wt, AR+, androgen independent, high Akt/mTOR); 22rv1 (p53wt, AR+, androgen independent, low Akt/mTOR); PC3 (p53-del, AR–, high Akt/mTOR), DU145 (p53mut, AR–, low Akt/mTOR). BPH1 and prostatic epithelial line EPN were used as non-neoplastic controls. 22rv1 xenografts were initiated in the flanks of SCID mice and treatment begun when tumors reached ~150mm3. Results: Analyses of nuclear pore complex (NPC)-related mRNA levels shows coordinate regulation of transcripts and allow PrCa to be segregated into high and low NPC expression. Low NPC expressing tumors tended to have reduced AR expression; APC, NPM1, and RB1 mRNA levels were also low (p5-20µM). SINE cytotoxic effects are independent all tested genetic alterations. In the aggressive, androgen independent 22rv1 xenograft, oral SINE KPT-251 showed dose-dependent inhibition of tumor growth and synergy with cisplatin. Conclusions: These data show that PrCa shows coordinate NPC regulation, and that selective blockade of CRM1 dependent NE represents a completely novel, neoplasia-selective and well-tolerated target for use as single agent or in combination with chemotherapy for PrCa.

Chromosomal 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) such as p53, FOXO, PTEN, pRB and I-κB. Small molecule, KPT-SINE that block CRM1-dependent nuclear export can force the nuclear retention of TSPs, thus render cancer cells more susceptible to apoptosis and more responsive to other chemotherapies. KPT-SINE display significant in vitro and in vivo activity over a broad range of tumor cell lines and xenografts. To optimize the design of future clinical trials, we developed a pharmacodynamics assay and tested it in cell lines, leukocytes in vitro and leukocytes from animal models. Methods: We used quantitative PCR and immunoblotting to study patterns of gene expression of CRM1, Macrophage Inhibitory Cytokine 1 (MIC1), p53 and p21 in human, mouse, rat and monkey leukocytes. The selection and the validation of the four PD markers included 1. In vitro studies where expression of PD markers was determined in tumor cell lines. 2. In vitro studies where expression of PD markers was determined in purified leukocytes. 3. In vivo studies where expression of PD markers was determined in leukocytes isolated from mice, rats and monkeys that were treated with KPT-SINE in pharmacokinetic studies. Drug levels were determined in the plasma of all animals. Results: KPT-SINE treatment at 75 mg/kg QDX5 each week for 4 weeks inhibited tumor growth by more than 80% in Molt-4, Z-138, Hep3b and U87 xenograft models. In separate PK and TK studies, expression levels of the four PD markers were investigated and were found to correlate well with KPT-SINE exposure and duration. In addition, we found that the full induction of those markers was p53-dependent. However, we also observed partial p53-independent induction of CRM1 and MIC1. PK analysis in treated rats and monkeys indicated that KPT-SINE plasma concentrations above 250 nM were sufficient to induce significant changes in the mRNA expression levels of the PD markers. Those concentrations were higher than the individual cytotoxicity indexes (EC50), of each of the above cancer cell lines, that we measured in vitro by cell proliferation assay. Conclusions: The results from our studies suggest that KPT-SINE display potent in vivo efficacy in various xenograft models. We were able to identify PD markers that correlated well with in vitro and in vivo levels of KPT-SINE. Further studies investigating CRM1, p53, p21 and MIC1 as biomarkers of target inhibition and response to KPT-SINE treatment are on-going. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3775. doi:1538-7445.AM2012-3775

CRM1 (Xpo1) is a key nuclear export protein which controls the nuclear export of multiple tumor suppressor proteins (TSP) and cell proliferation pathways including p53, p21, FOXO, PI3K/AKT, Wnt/ß-catenin and NF-κB. Mislocalization of TSP can abrogate their functions as well as render chemotherapies ineffective. Induction of nuclear expression of TSP and chemotherapy targets, by CRM1 inhibition can restore their tumor suppressor functions and increase drug sensitivity. We have developed orally active, small molecule SINE that irreversibly and potently inhibit CRM1 mediated nuclear export of multiple TSP and other cargoes (IC50 < 100 nM). Here, we describe in vitro results using SINE compounds, KPT-185 in vitro and KPT-251 in vitro and in vivo on seven prostate cancer (PrCa) cell lines representing distinct differentiation/progression states of disease and genotypes: LAPC-4 (Androgen receptor [AR] positive, androgen dependent with low Akt/mTOR activities, p53 wt); LnCaP (AR positive, androgen dependent with high Akt/mTOR activities, p53 wt); LnCaP-C81 and LnCaP-C4-2B (AR positive, androgen independent with high Akt/mTOR activities, p53 wt); 22rv1 (AR positive, androgen independent with low Akt/mTOR activities, p53 wt); PC3 (AR negative, with high Akt/mTOR activites and no p53 function (p53 del) and DU145 (AR negative, with low Akt/mTOR activites and mutant p53). Benign prostatic hyperplasia line (BPH1) and Prostatic epithelial line (EPN) were used as non-neoplastic controls. We show that SINE block CRM1 mediated nuclear export of FOXO and p53 with a IC50 values 5-20 μM). SINE cytotoxic effects are independent of p53 status, and induce caspase-3 activation. SINE display synergistic effects in combination with cisplatin and docetaxel in vitro with combination indices between 0.3 and 0.8. In vivo results indicate that KPT-251 show a dose-dependent inhibition of tumor growth. KPT251 was also synergistic when administered at 30mg/kg/5 days/week per os with docetaxel and cisplatin by using the aggressive p53 wt 22rv1 xenografts. Taken together, CRM1 inhibition represents a completely novel, neoplasia-selective and well- tolerated target for use as single agent or in combination with chemotherapy for PrCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1841. doi:1538-7445.AM2012-1841

Transport of macromolecules across nuclear membrane is fundamental to the proper functioning of living cells. Nuclear localization of intact tumor suppressor proteins (TSPs) and other growth regulatory proteins (GRPs) such as p53, FOXO, pRB, p21, p27 and IκB are critical for their “policing” function. Conversely, mislocalization of a nuclear protein to the cytoplasm can render it ineffective as a TSP/GRP. Cancer cells appear to acquire intracellular mechanisms to export tumor-supressing nuclear proteins as they evolve and spread. In many cases, nuclear export is followed by proteasome dependent degradation. Most known TSP/GRP utilize the nuclear export protein CRM1 (chromosomal region maintenance 1; also called exportin 1 [XPO1]), to exit the nucleus, and overexpression of CRM1 has been reported as a poor prognostic factor in a variety of neoplasms. Therefore, inhibition of CRM1 can force the nuclear localization of key TSPs/GRPs, activating their pathways. This leads to cell cycle arrest followed by induction of cell death in cancer cells. Our SINE are novel, potent and selective, drug-like nuclear export inhibitors (IC50 ∼40-100nM) that derive their activity through direct covalent modification and inhibition of CRM1. SINE exert selective and prolonged inhibition of CRM1-mediated forkhead (FOXO), p53, p21 and IκB nuclear export in a variety of normal and transformed cell lines. Cell cycle analysis following incubation with the SINE compound KPT-0127 revealed that it blocks at both the G1/S and G2/M phases, consistent with arrest at multiple cell cycle checkpoints. SINE-induced apoptosis is observed in many cancer cell lines (e.g. A375, Jurkat, BL-40, HCT-116), but not in normal proliferating cells (e.g. 3T3, mouse embryo fibroblasts). In cytotoxicity assays, SINE compounds showed highly potent cytotoxicity in both hematologic and solid tumor cell lines (IC50s 2000nM). SINE show no significant effect on 37 proteins including several cysteine proteases, minimal CYP inhibition (> 10 μM for all major CYPs) and no hERG inhibition. In single dose mouse toxicology studies, KPT-0127 was generally well tolerated (p.o. or s.c.) up to 560 mg/kg, and no deaths were observed. The pharmacokinetics of KPT-0127 is adequate for s.c. and i.v. dosing in vivo. Tumor xenograft studies have been undertaken in colon cancer HCT-116 and myeloma MM.1S tumor bearing mice. SINE induce potent, dose dependent anticancer activity against both small (∼140mm3) and large (∼1400mm3) tumors. Together, these data demonstrate that SINE represent a novel mechanism of action with robust anti-tumor activity both in vitro and in vivo and promising applications as single agent and for combination therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2580. doi:10.1158/1538-7445.AM2011-2580

430 Background: CRM1 (XPO1) is a key nuclear export protein which controls the location of multiple tumor suppressor (TSP) and growth regulatory (GRP) proteins including p53, PI3K/AKT, Wnt/ß-catenin and NF-kB. Forced nuclear expression of TSP and GRP by CRM1 inhibition can lead to apoptosis in cancer cells while sparing normal cells. Methods: Novel small-moleculeCRM1 inhibitors were synthesized and nuclear distribution studies were performed in cells transfected with HIV-rev GFP proteins. Cell proliferation studies were performed in 16 CRC cell lines: LS-123, SW-626, Colo-201, Colo-205, Colo-320DM, Colo-320HSR, Lovo, DLD-1, HCT-15, WiDi, LS-174T, LS-180, SW-620, C2BBe1, HCT-8, HCT-116, and in human peripheral leukocytes (PBMC). Cellular distribution and apoptosis assays were performed on HCT-116. Antitumor activity is assessed in human HCT-116 xenografts in scid-mice. Results: The lead CRM1 inhibitor, KPT-0127, blocks CRM1 mediated nuclear export of HIV-Rev-GFP, FOXO, and p53 with an IC50 of ∼300 nM. KPT-0127 is cytotoxic to various CRC cell lines with EC50s of 0.07-1.1 μM; in 9 CRC lines EC50s were < 0.3 mM. In contrast, normal cell lines and PBMCs had EC50 > 5-20 μM. In HCT- 116 cells, KPT-0127 induces cell cycle arrest at both G1/S and G2/M checkpoints and dose dependently increases nuclear p53, followed by an increase in caspase 3. KTP-0127 10μM shows no significant effect on 37 proteins including several cysteine proteases. In mice, KPT-0127 given by SC injection of 30-100 mg/kg leads to serum levels exceeding the effective CRM1 inhibitory concentration for at least 4 hours and is well tolerated. KPT-0127 given SC to mice bearing HCT-116 colon xenografts results in dose-dependent antitumor activity. Conclusions: The novel small- molecule CRM1 inhibitor KTP-0127 kills CRC lines with multiple TSP, GRP, and oncogenic abnormalities, including p53 mutations/deletions and PTEN deficiency/AKT activation, while sparing normal cells. This likely reflects the ability of CRM1 inhibition to affect multiple critical and non-redundant regulatory pathways. These results support the development of CRM1 inhibitors for the treatment of CRC. IND-enabling CMC and toxicology work are in preparation. [Table: see text]

Abstract 3012 Background: CRM1 (XPO1) is a key nuclear export protein which controls multiple tumor suppressor proteins (TSP) and cell proliferation pathways including p53, PI3K/AKT, Wnt/ß-catenin and NF-kB. Further, mislocalization of proteins can abrogate TSP functions and render chemotherapies ineffective. For example, multiple myeloma (MM) cells that are grown at high densities (to mimic the in vivo situation) become resistant to topoisomerase 2 (topo2) inhibitors (e.g., doxorubicin) simply because topo2α is exported from the nucleus. Forcing the nuclear expression of chemotherapy targets, TSP and growth regulatory proteins by CRM1 inhibition can restore drug sensitivity and restore checkpoint control/genome surveying functions. These events lead to apoptosis or autophagy in cancer cells while sparing normal cells. Methods: CRM1 inhibitors were synthesized and nuclear distribution studies were performed in U2OS cells transfected with HIV-rev GFP proteins. Cell proliferation studies were performed in multiple myeloma (MM), leukemia and lymphoma cell lines and in human peripheral blood mononuclear cells (PBMCs). Toxicology studies were performed in several mouse strains. Antitumor activity is assessed in a xenograft model of human MM.1 cells growing in scid-mice. Results: The lead CRM1 inhibitor, KPT-0127, blocks CRM1 mediated nuclear export of HIV-Rev-GFP, FOXO, and p53 with an IC50 of ~300 nM. KPT-0127 is selectively cytotoxic to various hematological cell lines with EC50s in the 0.02–1.0 μM range, and shows limited cytotoxicity in similar studies in normal cell lines (NIH-3T3, MRC5, HUVECs) and PBMCs (EC50 >5-20 μM). KPT-0127 increases the nuclear localization of IkB in HUT-78 leukemia cells and human PBMCs, and inhibits TNF-α secretion in LPS stimulated U937 macrophage derived cells, likely through inhibition of NF-kB signaling. In MM cells grown at high densities which actively export topo2 to the cytoplasm via CRM1, blocking CRM1-dependent transport strongly enhanced topo2 nuclear localization and augmented the apoptotic effects of doxorubicin in Conclusions: The sensitivity of tumors with multiple TSP and oncogenic abnormalities, including p53 mutations/deletions and PTEN deficiency/AKT activation, to killing with KPT-0127 likely reflects the ability to affect multiple critical and non-redundant regulatory pathways. CRM1 inhibition also forces topo 2 to remain in the nucleus, and increases levels of nuclear IkB antagonizing NF-kB function, thereby reducing the likelihood of resistance development. These results support the development of small molecule, drug-like CRM1 inhibitors for the treatment of MM and other hematological cancers. IND-enabling CMC and toxicology work are expected to begin in early 2011. Disclosures: Shacham: Karyopharm: Employment, Equity Ownership, Patents & Royalties. Nir:Karyopharm: Consultancy. Draetta:Karyopharm: Consultancy. Sandanayaka:Karyopharm: Employment. Shechter:Karyopharm: Employment. Kauffman:Karyopharm: Consultancy, Equity Ownership, Patents & Royalties.

CRM1 (Xpo1) is the major export factor for proteins from the nucleus to the cytoplasm, including tumor suppressors (TSPs) and other modulators of proliferative responses such as p53, FOXO, c-Abl, pRB and IκB. Leptomycin B (LMB), a non-drug like natural product is a potent inhibitor of CRM1-mediated nucleocytoplasmic transport. LMB and related analogs trap TSPs and other proteins in the nucleus, forcing neoplastic cells into apoptosis while normal cells undergo reversible cell cycle arrest. However, LMB has limited efficacy in vivo due to its severe gastrointestinal toxicity. Here, we describe our lead compound KPT-0127 a novel small molecule, water soluble, drug-like, selective, irreversible CRM1 antagonist. Like LMB, KPT-0127 forms a covalent bond with Cys528 in the CRM1 cargo-binding pocket, abrogating most, but not all, export functions of CRM1. KPT-0127 exerts a potent (EC50 300-400nM) and prolonged inhibition of CRM1- mediated HIV-1 Rev, forkhead (FOXO), and p53 nuclear export in a variety of normal and transformed cell lines. In cytotoxicity assays, KPT-0127 showed high potency in most hematologic cancer cell lines (EC50 5-10µM). Studies in the HCT-116 colon cancer cell line suggested that KPT-0127 dose dependently increases the nuclear levels of p53 and appears to induce cell cycle arrest at both the G1/S and G2/M checkpoints, prior to inducing apoptosis. In normal peripheral blood mononuclear cells (PBMCs) and in Hut78 leukemia cells, KPT-0127 potently increased the nuclear levels of IκB. However, KPT-0127 induced cell death of Hut78 cells with no effect on normal PBMCs. In drug combination studies, KPT-0127 showed additive or synergistic cytotoxicity activity with either 5-FU, carboplatin, or doxorubicin. Mechanism of action studies demonstrated that the Cys528 residue in the cargo-binding pocket of CRM1 is essential for the inhibitory effect of KPT-0127. Mutagenesis of Cys528 to a Ser completely abrogated KPT-0127 inhibition. KPT-0128, the transisomer of KPT-0127, shows little effect on both HIV-Rev nuclear export and in cytotoxicity assays (EC50s > 10μM), supporting the specificity of KPT-0127 for CRM1. Moreover, the selectivity of KTP-0127 was demonstrated across a panel of 37 proteins including several cysteine proteases. In single dose mouse toxicology studies, KPT-0127 was generally well tolerated (oral or SC) up to 560 mg/kg, and no deaths were observed. SC dosing daily for 5 days up to 100mg/kg (the highest dose tested) showed no behavioral, clinical chemistry, or hematogical effects in mice; Pharmacokinetics of KPT-0127 is adequate and in vivo efficacy studies are currently being performed; results will be presented. All together, these data demonstrate that KPT-0127 represents a novel, tumor selective, and well-tolerated irreversible Crm1 inhibitor which may be suitable for clinical development both as a single agent and in combination with standard therapies. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A19.

Background Exportin 1 (XPO1) is a well-characterized nuclear export protein whose expression is up-regulated in many types of cancers and functions to transport key tumor suppressor proteins (TSPs) from the nucleus. Karyopharm Therapeutics has developed a series of small-molecule Selective Inhibitor of Nuclear Export (SINE) compounds, which have been shown to block XPO1 function both in vitro and in vivo. The drug candidate, selinexor (KPT-330), is currently in Phase-II/IIb clinical trials for treatment of both hematologic and solid tumors. The present study sought to decipher the mechanisms that render cells either sensitive or resistant to treatment with SINE compounds, represented by KPT-185, an early analogue of KPT-330. Methods Using the human fibrosarcoma HT1080 cell line, resistance to SINE was acquired over a period of 10 months of constant incubation with increasing concentration of KPT-185. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. Fluorescence activated cell sorting (FACS), quantitative polymerase chain reaction (qPCR), and immunoblots were used to measure effects on cell cycle, gene expression, and cell death. RNA from naïve and drug treated parental and resistant cells was analyzed by Affymetrix microarrays. Results Treatment of HT1080 cells with gradually increasing concentrations of SINE resulted in > 100 fold decrease in sensitivity to SINE cytotoxicity. Resistant cells displayed prolonged cell cycle, reduced nuclear accumulation of TSPs, and similar changes in protein expression compared to parental cells, however the magnitude of the protein expression changes were more significant in parental cells. Microarray analyses comparing parental to resistant cells indicate that a number of key signaling pathways were altered in resistant cells including expression changes in genes involved in adhesion, apoptosis, and inflammation. While the patterns of changes in transcription following drug treatment are similar in parental and resistant cells, the extent of response was more robust in the parental cells. Conclusions These results suggest that SINE resistance is conferred by alterations in signaling pathways downstream of XPO1 inhibition. Modulation of these pathways could potentially overcome the resistance to nuclear export inhibitors. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1790-z) contains supplementary material, which is available to authorized users.

Endothelial to mesenchyme transition (EndMT) can be observed during the formation of endocardial cushions from the endocardium, the endothelial lining of the atrioventricular canal (AVC), of the developing heart at embryonic day 9.5 (E9.5). Many regulators of the process have been identified; however, the mechanisms driving the initial commitment decision of endothelial cells to EndMT have been difficult to separate from processes required for mesenchymal proliferation and migration. We have several lines of evidence that suggest a central role for Akt signaling in committing endothelial cells to enter EndMT. Akt1 mRNA was restricted to the endocardium of endocardial cushions while they were forming. The PI3K/Akt signaling pathway is necessary for mesenchyme outgrowth, as sprouting was inhibited in AVC explant cultures treated with the PI3K inhibitor LY294002. Furthermore, endothelial marker, VE-cadherin, was downregulated and mesenchyme markers, N-cadherin and Snail, were induced in response to expression of a constitutively active form of Akt1 (myrAkt1) in endothelial cells. Finally, we isolated the function of Akt1 signaling in the commitment to the transition using a transgenic model where myrAkt1 was pulsed only in endocardial cells and turned off after EndMT initiation. In this way, we determined that increased Akt signaling in the endocardium drives EndMT and discounted its other functions in cushion mesenchymal cells.