Search

Supplementary Figure 3 - PDF 313K, CRM1 inhibition induces cell cycle arrest and increases cell death when combined with BRAF inhibition

Supplementary Figure 2 - PDF 191K, CRM1 inhibition induces cell cycle arrest and increases cell death when combined with BRAF inhibition

Supplementary Figure 7 - PDF file, TP53 knockdown in A375 reduces the effect of CRM1 inhibition on A375 proliferation but not on caspase-3/7 activity

Supplementary Figure 4 - PDF file 44K, CRM1 inhibition induces a statistically significant increase in caspase-3/7 activity as single therapy or in combination with BRAF inhibition

Supplementary Figure 1 - PDF file 29K, CRM1 and BRAF inhibition decrease melanoma cell proliferation and are synergistic in vitro

Resistance to BRAF inhibitor therapy places priority on developing BRAF inhibitor-based combinations that will overcome de novo resistance and prevent the emergence of acquired mechanisms of resistance. The CRM1 receptor mediates the nuclear export of critical proteins required for melanoma proliferation, survival, and drug resistance. We hypothesize that by inhibiting CRM1-mediated nuclear export, we will alter the function of these proteins resulting in decreased melanoma viability and enhanced BRAF inhibitor antitumoral effects. To test our hypothesis, selective inhibitors of nuclear export (SINE) analogs KPT-185, KPT-251, KPT-276, and KPT-330 were used to induce CRM1 inhibition. Analogs PLX-4720 and PLX-4032 were used as BRAF inhibitors. Compounds were tested in xenograft and in vitro melanoma models. In vitro, we found CRM1 inhibition decreases melanoma cell proliferation independent of BRAF mutation status and synergistically enhances the effects of BRAF inhibition on BRAF-mutant melanoma by promoting cell-cycle arrest and apoptosis. In melanoma xenograft models, CRM1 inhibition reduces tumor growth independent of BRAF or NRAS status and induces complete regression of BRAF V600E tumors when combined with BRAF inhibition. Mechanistic studies show that CRM1 inhibition was associated with p53 stabilization and retinoblastoma protein (pRb) and survivin modulation. Furthermore, we found that BRAF inhibition abrogates extracellular signal–regulated kinase phosphorylation associated with CRM1 inhibition, which may contribute to the synergy of the combination. In conclusion, CRM1 inhibition impairs melanoma survival in both BRAF-mutant and wild-type melanoma. The combination of CRM1 and BRAF inhibition synergizes and induces melanoma regression in BRAF-mutant melanoma. Mol Cancer Ther; 12(7); 1171–9. ©2013 AACR.

Supplementary Figure 5 - PDF file 894K, CRM1 inhibition leads to delayed tumor growth independent of BRAF or NRAS status in melanoma xenograft models

Supplementary Methods and Figure Legends - PDF 133K, Supplementary methods and figure legends

Supplementary Figure 6 - PDF file 57K, CRM1/BRAF inhibition in PTEN null/BRAF mutant melanoma Xenograft model

SINE (Selective Inhibitors of Nuclear Export) block the activity of XPO1/CRM1, 1 of 7 nuclear export proteins in cells, forcing the nuclear retention of key tumor suppressor proteins, leading to selective apoptosis of tumor cells. The purpose of these studies was to evaluate the in vitro activity of SINE against canine tumor cell lines and investigate the biological activity of verdinexor (KPT-335) in dogs with spontaneous cancers as proof of principle for human clinical studies. Several different canine tumor cell lines including those derived from non-Hodgkin Lymphoma (NHL) exhibited growth inhibition and apoptosis in response to nanomolar concentrations of SINE; NHL cells were particularly sensitive with IC50 2-42 nM. A Phase I clinical trial of verdinexor was performed in dogs with cancer with an emphasis on NHL given in vitro activity demonstrated against the tumor cell lines. The MTD was 1.75 mg/kg twice per week although biological activity was observed at 1 mg/kg. Clinical benefit including Partial Response (PR) and Stable Disease (SD) for at least 4 weeks was observed in 9/14 dogs with NHL with a median time to progression of 66 days (range 35-256). A dose expansion study was performed in 6 dogs with NHL given 1.5 mg/kg verdinexor on a Monday/Wednesday/Friday (MWF) regimen; clinical benefit (PR+SD) was observed in 4/6 dogs with a median time to progression of 83 days (range 35-354). Toxicities were primarily gastrointestinal consisting of anorexia, weight loss, vomiting and diarrhea and were manageable with supportive care and dose modulation. A validated health related Quality of Life (QOL) form used to assess dogs during treatment demonstrated that the overall quality of life did not decrease in dogs in this study supporting the notion that clinical toxicities associated with verdinexor are generally well tolerated. Based on these findings, a Phase IIb study was performed in 58 dogs with either newly diagnosed or relapsed NHL. Drug was administered initially at 1.5 mg/kg MWF, but this dosing regimen was changed to 1.25 mg/kg M/Th due to the high rate of anorexia and weight loss on the MWF regimen; dose escalation was permitted to 1.5 mg/kg on the M/Th regimen.. The objective response rate was 29% (1 CR, 16 PR) with an additional 25 dogs experiencing SD for a minimum of 4 weeks, resulting in a of 72% disease control rate. While the median time to progression was approximately 6 weeks, 19 dogs (32%) remained on study drug for more than 8 weeks. Laboratory abnormalities were minimal. Together, these data provide robust evidence that the novel orally bioavailable XPO1 inhibitor vrdinexor exhibits single agent biological activity in a spontaneous large animal model of human NHL. Furthermore, verdinexor was well tolerated even in the absence of supportive care, suggesting that SINE compounds could exhibit good long-term tolerability in people. Citation Format: Cheryl A. London, Luis Feo Bernabe, Sandra Barnard, William Kisseberth, Antonella Borgatti, Michael Henson, Heather Wilson-Robles, Kiersten Jensen, Daisuke Ito, Jaime Modiano, Misty Bear, Michael Pennell, Jean-Richard Saint-Martin, Dilara McCauley, Michael Kauffman, Sharon Shacham. Evaluation of the novel, orally bioavailable selective inhibitor of nuclear export (SINE) KPT-335 (verdinexor) in spontaneous canine cancer: Results of phase I and phase II clinical trials. [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 3809. doi:10.1158/1538-7445.AM2014-3809

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.

The key nuclear export protein CRM1 (chromosomal region maintenance 1, Exportin 1, XPO1) may directly contribute to the pathophysiology of human multiple myeloma (MM). Here, we characterized the role of CRM1 in MM biology and defined the efficacy and molecular mechanisms of novel oral, irreversible, selective inhibitors of nuclear export (SINEs) targeting CRM1 against MM. CRM1 is significantly elevated in patient MM vs. normal plasma cells at transcript and protein levels. CRM1 downregulation by shCRM1 lentiviruses inhibited cell growth and survival of MM cells (p< 0.01). Importantly, SINEs (KPT-185, KPT-251, KPT-276, and KPT-330) blocked proliferation and decreased survival of MM cell lines and patient MM cells (LD50 2-log differences). SINEs potently enhanced nuclear accumulation of multiple CRM1 cargo tumor suppressor proteins, including rapid induction of p53 and IκB (as early as 2h after drug treatment) followed by FOXO1A, FOXO3A, p27, and PP2A in MM cells. Transcripts of p53 and its downstream targets (p21, PUMA, BAX) were induced by KPT-185, thereby inducing strong growth arrest and apoptosis. KPT-185 decreased MM oncogenes (c-myc, c-maf), anti-apoptosis molecules Mcl-1 and Bcl-xL; increased pro-apoptotic protein BAX; as well as inhibited pIκBα. Inhibition of pIκBα further correlated with KPT-185-blocked NFκB p65 DNA-binding activity in MM cells with or without A Proliferation-Inducing Ligand (APRIL) stimulation. KPT-185 further downregulated CRM1 protein, which was blocked by bortezomib; concurrently, KPT-185 (or KPT-330) upregulated CRM1 mRNA in MM cells. Cleavage of caspase 3 and PARP was markedly increased in MM1R cells treated with KPT-185 and bortezomib vs. either drug alone, confirming enhanced cytotoxicity by combination of these agents. Combined dex with KPT-185 (or KPT-276) induced synergistic cytotoxicity against MM cells (combination indices < 1). Moreover, KPT-185 and KPT-330 impaired osteoclastogenesis and bone resorption via blockade of RANKL-induced NFκB activation and NFATc1 in OC precursor cells, without impacting osteoblasts and BMSCs. Finally, SINEs (KPT-251 and KPT-276) suppressed MM cell growth (p< 0.01), diminished MM cell-induced osteolysis, and prolonged survival of SCID mice with diffuse human MM bone lesions. Together, these results identify CRM1 as a promising novel target in MM, strongly supporting clinical development of SINE CRM1 inhibitors to inhibit both MM cell growth and related bone disease. The oral SINE KPT-330 is ongoing in MM and other hematological malignancies. Citation Format: Yu-Tzu Tai, Yosef Landesman, Chirag Acharya, Yolanda Calle, Mike Zhong, Michele Cea, Daniel Tannenbaum, Antonia Cagnetta, Michaela Reagan, Aditya Munshi, William Senapedis, Jean-Richard Saint-Martin, Trinayan Kashyap, Sharon Shacham, Michael Kauffman, Yumei Gu, Lizi Wu, Steve Schey, Irene Ghobrial, Andrew Kung, Nikhil Munshi, Paul Richardson, Kenneth Anderson, Haoqiang Ying. The nuclear export protein CRM1 (XPO1) regulates multiple myeloma cell growth, osteoclastogenesis, and myeloma-induced osteolysis. [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 2142. doi:10.1158/1538-7445.AM2013-2142

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

Inhibition of nuclear export can promote re-activation of tumor suppressor proteins (TSPs) by restoring them to the nucleus. CRM1 (chromosome region maintenance 1, XPO1) is the exclusive exporter of many TSPs. We hypothesized that CRM1 inhibition can be used as a therapeutic target in melanoma. The growth inhibitory and pro-apoptotic effects of KPT-185, KPT-276 and KPT-330, potent, small molecule, selective inhibitors of nuclear export (SINEs) were evaluated in a panel of human metastatic melanoma cell lines using an MTS assay and Annexin V/PI staining, respectively. The weak CRM1 inhibitor, KPT-185 trans-isomer, and DMSO vehicle were controls in all assays. CRM1 protein was expressed in melanoma cell lines regardless of molecular profile (BRAF, NRAS, TP53) as determined by immunoblot. SINEs inhibited cell growth in a concentration-dependent manner and induced apoptosis at nanomolar concentrations (range=139.7nM - 2.41μM). Both BRAF wild type (WT) and BRAF V600E mutant lines were sensitive to apoptosis by SINE, but BRAF V600E lines had an 8.8-fold lower IC50s as compared to BRAF (WT) lines (p=0.018). The cytostatic and pro-apoptotic effects of CRM1 inhibition were associated with nuclear accumulation of p53, and p21 induction in A375 and CHL-1 melanoma cell lines with functional p53 at time points prior to apoptosis, while pERK accumulated in melanoma cells regardless of p53 status. In pharmacokinetic studies in mice, KPT-276 and KPT-330 showed >50% oral bioavailability with Cmax >5μM. Mice bearing either A375 (BRAF V600E) or CHL-1 (BRAF WT) melanoma xenografts were given KPT-276 or KPT-330 SINE by oral gavage. A375 xenografts (p Citation Format: Jennifer Yang, Matthew A. Bill, Gregory S. Young, Yosef Landesman, Sharon Shacham, Michael Kauffman, William Senapedis, Trinayan Kashyap, Jean-Richard Saint-Martin, Gregory Lesinski. Novel small molecule CRM1 inhibitors induce nuclear accumulation of p53, phosphorylated ERK and apoptosis in human melanoma cells. [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 3443. doi:10.1158/1538-7445.AM2013-3443

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

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