RNA binding proteins may be important mediators of the activity-dependent transport of mRNAs to dendritic spines of activated synapses. We used fluorescence microscopy and digital imaging techniques applied to both fixed and live cultured hippocampal neurons to visualize the localization of the mRNA binding protein, zipcode binding protein 1 (ZBP1), and its dynamic movements in response to KCl-induced depolarization at high spatial and temporal resolution. With the use of immunofluorescence, image deconvolution, and three-dimensional reconstruction, ZBP1 was localized in the form of granules that were distributed in dendrites, spines, and subsynaptic sites. KCl depolarization increased the dendritic localization of ZBP1 that was not attributed to an increase in ZBP1 expression. Live cell imaging of single cells before and after perfusion of KCl revealed the rapid and directed efflux of ZBP1 granules from the cell body into dendrites in a proximo-distal gradient. High-speed imaging of enhanced green fluorescence protein-ZBP1 granules revealed rapid anterograde and retrograde movements in dendrites as well as dynamic movements in dendritic spines. A population of ZBP1 granules colocalized with beta-actin mRNA, and their spatial association in dendrites was increased by KCl depolarization. The NMDA receptor antagonist AP-5 impaired the dendritic localization of ZBP1 and beta-actin mRNA and inhibited the KCl-induced transport of ZBP1. The activity-dependent trafficking of ZBP1 and its dynamic movements within dendritic spines provide new evidence to implicate RNA binding proteins as regulators of mRNA transport to activated synapses in response to synaptic activity.

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Growing evidence indicates that both seizure (glutamate) and cocaine (dopamine) treatment modulate synaptic plasticity within the mesolimbic region of the CNS. Activation of glutamatergic neurons depends on the localized translation of neuronal mRNA products involved in modulating synaptic plasticity. In this study, we demonstrate the dendritic localization of HuR and HuD RNA‐binding proteins (RBPs) and their association with neuronal mRNAs following these two paradigms of seizure and cocaine treatment. Both the ubiquitously expressed HuR and neuronal HuD RBPs were detected in different regions as well as within dendrites of the brain and in dissociated neurons. Quantitative analysis revealed an increase in HuR, HuD and p‐glycogen synthase kinase 3β (GSK3β) protein levels as well as neuronal mRNAs encoding Homer, CaMKIIα, vascular early response gene, GAP‐43, neuritin, and neuroligin protein products following either seizure or cocaine treatment. Inhibition of the Akt/GSK3β signaling pathway by acute or chronic LiCl treatment revealed changes in HuR, HuD, pGSK3β, p‐Akt, and β‐catenin protein levels. In addition, a genetically engineered hyperdopaminergic mouse model (dopamine transporter knockout) revealed decreased expression of HuR protein levels, but no significant change was observed in HuD or fragile‐X mental retardation protein RBPs. Finally, our data suggest that HuR and HuD RBPs potentially interact directly with neuronal mRNAs important for differentiation and synaptic plasticity.

Ewing's sarcoma (ES) is the second most common primary tumor of bone in young adults and accounts for 40% of bone tumors in children and adolescents. With modern multimodality management, including chemotherapy, surgery, and radiotherapy, ES patients can expect a 5-year overall survival (OS) of approximately 70% for those with localized disease and 20-30% for those with metastatic disease. The oncogenic drivers behind ES, are the result of a characteristic set of EWS chromosomal translocations, most commonly the t(11;22)(q24;q12) involving the FLI1 locus. Recent studies have demonstrated that both the EWS-FLI and EWS-ERG transcription factors interact with PARP-1 and that PARP inhibitors alter transcription targets of EWS-FLI1/ERG, resulting in significantly reduced cell survival in vitro and reduced tumor growth and formation of lung metastases in animal models. Niraparib (formerly MK-4827) is a potent, orally active PARP inhibitor that is currently being evaluated in Phase III clinical trials for ovarian and BRCA related breast cancer. In an ongoing investigation of cancers with niraparib sensitivity, we investigated niraparib activity as a single agent and in combination with second line chemotherapeutic agents commonly used in Ewing's Sarcoma in patient-derived TumorGraft ES models. Champions TumorGraft models of EWS-FLI1 positive Ewing's sarcoma (CTG-0816 and CTG-0142) utilized in this study were derived directly from primary tumors of heavily pretreated ES patients. Mice bearing established subcutaneous TumorGrafts were treated with regimens of niraparib (p.o., 50-100 mg/kg/day), temozolomide (p.o., 25-50 mg/kg/day) or irinotecan (i.p., 50-100 mg/kg/week) as single agent or doublet combinations. Treatment of tumor bearing mice with single agent niraparib, temozolomide or irinotecan exhibited minimal to modest tumor growth inhibition (0-65%) across models and regimens. When niraparib was used in combination with either high dose temozolomide or irinotecan, synergistic activity was observed resulting in significant tumor regression; however toxicity was evident with pronounced weight loss and myelosuppression. By contrast, complete tumor regression with minimal evidence of toxicity was observed when full dose niraparib, given for 4 weeks, was combined with reduced doses of either chemotherapeutic; for example, niraparib administered with a reduced dose of temozolomide during week 1 and 3 or with irinotecan for three weeks. In addition to its role of inhibiting PARP mediated DNA repair, preliminary gene expression analysis of treated tumors indicates that single agent niraparib reverses components of EWS-FLI1 gene regulation. The ability to both improve tolerability while retaining efficacy when a reduced dose of chemotherapy is combined with full dose niraparib presents important translational opportunities for the clinical investigation of chemosensitization by PARP inhibitors. In conclusion, the current study utilized patient derived tumors to identify well tolerated and efficacious combinations of common second line ES therapies with niraparib. This data supports the clinical investigation of niraparib in combination with temozolomide or irinotecan in patients with Ewing's sarcoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A258. Citation Format: Keith M. Wilcoxen, David G. Brooks, Dhanrajan Tiruchinapalli, Nathan Anderson, Rodney Donaldson, McIver Nivens, Charlie Cook, Tin Khor, Bin Lu, Elizabeth De Oliveira, Erin Hawley, Elizabeth Bruckheimer. The PARP inhibitor niraparib demonstrates synergy with chemotherapy in treatment of patient derived Ewing's sarcoma tumorGraft models. [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 A258.

Cancers are known to be heterogeneous diseases. Tumor cell proliferation and the development of metastatic disease involve the activation of multiple, sometime parallel and often compensatory signal transduction pathways. We have developed a cell-based assay platform that measures specific kinase activity within cells, thus allowing us to conduct pathway-based analyses. This platform can be used for 1) discovering new biomarkers and 2) guiding the design of new therapeutic strategies in personalzed medicine, specifically tailored to the patient's genotype. In the present study, we have used this platform to study the impact of G-protein coupled receptor (GPCR) mediation on IGF-1r (RTK) signal transduction cascades in the presence/absence of oestradiol (ER) using MCF-7 breast cancer cells. The results of these studies indicate that mediating neuroendocrine pathways may be an alternative approach that could enhance the efficacies of specific target therapeutics for the treatment of breast 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 236. doi:1538-7445.AM2012-236

Prostate cancer (PCa)is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in males, with the highest incidence rates recorded in developed countries such as those in North America (Jemal et al., 2011). While the five year-survival rate for PCa patients with localized disease is 100%, it is only 30.6% once the cancer metastasizes (Vishnu et al., 2010). One of the biggest challenges for finding a better treatment for metastatic PCa is the lack of predictable and accurate preclinical models that closely recapitulate different stages of the pathogenesis of human PCa. Champions Oncology focuses on the development of Champions TumorGraft™ models derived from the direct implantation of patient tumors into immunocompromised mice. Compared to traditional cell line-based xenograft models, the patient-derived TumorGraft models maintain stable gene-expression patterns and mutational status and correlate to clinical predictability. To overcome the challenge for PCa, Champions Oncology has utilized its TumorGraft technology to establish and characterize a new androgen-dependent prostate cancer TumorGraft model, CTG-0488. This involved the implantation of a primary human prostate tumor in immunocompromised mice in a manner that preserves the biological properties of the original human tumor. In this preliminary work, the characteristics of Champion TumorGraft™ model CTG-0488 are showcased, including tumor histology, mutational status, and chemosensitivity profiles to several standard of care compounds. Overall the development of an androgen-dependent prostate cancer Champions TumorGraft™ model will significantly aid in the development of oncology compounds focused on androgen-dependent prostate cancer. Citation Format: Tin Oo Khor, Keren Paz, Dhanrajan Tiruchinapalli, David Sidransky, Elizabeth M. Bruckheimer. Development of an androgen-dependent prostate Champions TumorGraft™ cancer model. [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 2793. doi:10.1158/1538-7445.AM2013-2793

A personalized approach towards the therapy of colorectal carcinoma is critical for a successful outcome. In recent years, direct transfer xenograft models (or the “tumor graft”) have proven to be highly predictive and are being adapted in clinical practice. However, direct drug evaluation in tumor graft models for each individual patient is not feasible due to time and cost constraints. Predictive biomarker discovery is an extremely promising application of the tumor graft model, since the discovered biomarker signatures will direct therapy choice in a much broader patient population. In collaboration with the Catholic Health Initiatives Center for Translational Research we at Caliper Life Sciences have established a collection of tumor graft samples of primary human colorectal carcinomas. All samples were collected fresh from consenting patients following an IRB-approved protocol and implanted in vivo on the day of surgical tumor resection. Tumors were grafted either orthotopically or subcutaneously in female NIH-III mice. We present here the results of a comparative study of gene and protein expression profiles for the primary tumor (clinical samples) and the direct transfer xenografts. These data will be correlated with drug sensitivity profiles generated by using the same samples. The resulting data sets will be used to characterize predictive biomarker signatures that will be a valuable tool for selection of the most effective therapy tailored to the individual patient. 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 1319. doi:1538-7445.AM2012-1319

Introduction: Molecularly targeted agents, such as the BRAF inhibitor vemurafenib, may produce short-term responses in some patients; however, most patients are intrinsically resistant, or develop resistance through restructuring of signal transduction pathways. SnapPath™ is a live-cell-processing platform that utilizes ex vivo signal transduction modulation of live tumor samples to produce Functional Signaling Profiles (FSPs). Application of this technology to Champions TumorGraft models may provide novel insights to guide oncology drug development as these models preserve the biological properties of the original human tumor. Methods: Fresh melanoma tumor specimens were collected from patients and implanted into immunodeficient mice. Fine needle aspiration biopsies were performed on each melanoma TumorGraft model and processed on the SnapPath™ platform (BioMarker Strategies) to modulate tumor cell signal transduction networks through brief ex vivo exposure to the vemurafenib tool compound PLX-4720. Cell lysates were then analyzed using a multiplexed immunoassay to assess the inhibition of the downstream MAPK markers pMEK1 and pERK-1/2. FSPs were then created for each TumorGraft model based on baseline and modulated levels of each phosphoprotein. In parallel, the in vivo sensitivity to vemurafenib and BRAF mutation status was evaluated in each Champions TumorGraft model. FSPs were then compared with in vivo efficacy, gene expression and genotype data. Results: Functional profiling stratified the TumorGraft models into two distinct groups upon ex vivo exposure to a BRAF inhibitor: 1) MAPK markers suppressed and 2) MAPK markers not suppressed. As anticipated, TumorGraft models that showed resistance to ex vivo BRAF inhibition demonstrated vemurafenib resistance in vivo and were BRAF wild type. There were other models that displayed MAPK suppression with ex vivo BRAF inhibition and vemurafenib sensitivity in vivo or MAPK suppression with ex vivo BRAF inhibition but demonstrated vemurafenib resistance in vivo. One of these TumorGrafts contained a BRAF V600E mutation, suggesting the activation of an alternate pathway that conferred resistance. The other TumorGraft contained a novel BRAF insertion. The functional profiling suggests that this insertion may activate BRAF and is susceptible to vemurafenib inhibition, but the tumor may contain an alternate pathway that confers resistance. Analysis of gene expression data demonstrated hierarchical clustering of BRAF mutated TumorGraft models. Conclusions: These results demonstrate the capability of the SnapPath™ platform to generate FSPs from FNAs of Champions melanoma TumorGraft models. Overall, the combination of Champions TumorGraft models with functional profiling represents a powerful tool for pharmacodynamic assessment of targeted therapeutics in clinically relevant models and has the potential to guide oncology therapy. Citation Format: Elizabeth M. Bruckheimer, Adam Schayowitz, Kala Barnes, Greg Bertenshaw, Tin Khor, James Cotton, Jay Friedman, Dhanrajan Tiruchinapalli, Douglas P. Clark. Functional profiling of Champions TumorGraft™ models from metastatic melanoma patients. [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 3498. doi:10.1158/1538-7445.AM2013-3498