Sharon Shechter

Endothelial Akt Signaling Is Rate-Limiting for Rapamycin Inhibition of Mouse Mammary Tumor Progression

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.

Akt Promotes Endocardial-Mesenchyme Transition

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.

RhoB controls coordination of adult angiogenesis and lymphangiogenesis following injury by regulating VEZF1-mediated transcription

Mechanisms governing the distinct temporal dynamics that characterize post-natal angiogenesis and lymphangiogenesis elicited by cutaneous wounds and inflammation remain unclear. RhoB, a stress-induced small GTPase, modulates cellular responses to growth factors, genotoxic stress and neoplastic transformation. Here we show, using RhoB null mice, that loss of RhoB decreases pathological angiogenesis in the ischaemic retina and reduces angiogenesis in response to cutaneous wounding, but enhances lymphangiogenesis following both dermal wounding and inflammatory challenge. We link these unique and opposing roles of RhoB in blood versus lymphatic vasculatures to the RhoB-mediated differential regulation of sprouting and proliferation in primary human blood versus lymphatic endothelial cells. We demonstrate that nuclear RhoB-GTP controls expression of distinct gene sets in each endothelial lineage by regulating VEZF1-mediated transcription. Finally, we identify a small-molecule inhibitor of VEZF1–DNA interaction that recapitulates RhoB loss in ischaemic retinopathy. Our findings establish the first intra-endothelial molecular pathway governing the phased response of angiogenesis and lymphangiogenesis following injury.

RhoB differentially controls Akt function in tumor cells and stromal endothelial cells during breast tumorigenesis

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.

Identification of the PDZ3 Domain of the Adaptor Protein PDZK1 as a Second, Physiologically Functional Binding Site for the C Terminus of the High Density Lipoprotein Receptor Scavenger Receptor Class B Type I

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 (Tyr20 → 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 (Kd = 37.0 μm), albeit with ∼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr253 → Ala substitution. Comparison of the 1.5-Å resolution crystal structure of PDZ3 with its bound target peptide (505QEAKL509) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr20 → Ala (PDZ1) + Tyr253 → Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr253 → 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 [Tyr20 → Ala (PDZ1) + Tyr253 → 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.

Abstract 3775: Pharmacokinetic (PK) / pharmacodynamic (PD) and efficacy relationship of selective inhibitors of nuclear export (KPT-SINE)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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

Abstract 1841: Selective inhibitors of nuclear export (SINE) activate multiple tumor suppressor pathways and kill prostate cancer cells across multiple genotypes in vitro and in vivo

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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/s-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 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

Abstract 2943: KPT-SINE (Selective Inhibitors of Nuclear Export) induce apoptosis in colon cancer cells in-vitro and in-vivo through nuclear localization of Tumor Suppressor Proteins (TSPs)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Chromosome Region Maintenance 1/Exportin-1 (CRM1) is a key nuclear export protein whose inhibition leads to the nuclear accumulation of TSPs and negative regulators of cell proliferation. 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 are developing novel small molecules, KPT-SINE, which irreversibly bind and inhibit CRM1 nuclear export killing a broad range of tumor cell lines and xenografts. We used human colon cancer cells HCT116 to track the molecular and cellular events that follow KPT-SINE treatment and ultimately lead to cancer cell death. Methods: Using our proprietary in silico hierarchical structure-based discovery platform, we designed and tested novel SINE in a cell-based microscopy assay to confirm CRM1-mediated nuclear export inhibition. Cytotoxic IC50s of KPT-SINE were determined on a panel of cancer cell lines. KPT-SINE – KPT-185, -251 and -276 were selected for additional testing based on their potency and pharmacokinetics. These include; 1. Nuclear localization of TSPs, 2. Effects on cell cycle and viability, 3. Effects on TSP mRNA and protein expression. Additionally, we transiently transfected mutant CRM1-C528S into cells and treated with KPT-SINE to show binding to Cysteine 528. We tested the effects of KPT-276 in an in vivo HCT116 xenograft model. Results: KPT-276, a potent inhibitor in the cell-based microscopy assay (EC50 = 130 nM), was a robust inhibitor of HCT116 proliferation (IC50 = 400 nM). 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 CRM1-C528S, confirming KPT-SINE - Cysteine 528 interaction. Washout experiments demonstrated that 4 hrs of KPT-SINE incubation sustained 24 hrs of CRM1 inhibition. We also identified nuclear accumulation of p53, FOXO3a, pRb, APC, IαB, p27, PTEN, and p21, which was followed by cell cycle arrest and cell death. Although KPT-276 treatment had less effect on cell cycle and cytotoxicity assays of p53null HCT116 cells, the p53 wildtype and null HCT116 cells showed similar kill curves after 8 days of treatment. Mouse xenografts treated with 75 mg/kg QDX5 of KPT-276 each week for 4 weeks inhibited tumor growth by more than 70%. Further analysis of the tumor molecular markers from KPT-276 treated animals will be reported. Conclusions: In this study we described the correlative effects of KPT-SINE from the accumulation of nuclear TSPs through to cancer cell death in mouse models. These results suggest that KPT-SINE display potent in vivo efficacy in the xenograft model of human colon cancer. 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 2943. doi:1538-7445.AM2012-2943

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.