Background Through negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies. Methodology/Principal Findings In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133+/CD15+ tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1+/- p53-/-), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo. Conclusions/Significance Despite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.

Background: In spite of leukemia therapy improvements obtained over the last decades, therapy is not yet effective in all cases. Current approaches in Acute Lymphoblastic Leukemia (ALL) research focus on identifying new molecular targets to improve outcome for patients with a dismal prognosis. In this light phosphoproteomics seems to hold great promise for the identification of proteins suitable for targeted therapy.Methodology/Principal Findings: We employed Reverse Phase Protein Microarrays to identify aberrantly activated proteins in 118 pediatric B-cell precursor (BCP)-ALL patients. Signal transduction pathways were assayed for activation/expression status of 92 key signalling proteins. We observed an increased activation/expression of several pathways involved in cell proliferation in poor clinical prognosis patients. MLL-rearranged tumours revealed BCL-2 hyperphosphorylation through AMPK activation, which indicates that AMPK could provide a functional role in inhibiting apoptosis in MLL-rearranged patients, and could be considered as a new potential therapeutic target. Second, in patients with poor clinical response to prednisone we observed the up-modulation of LCK activity with respect to patients with good response. This tyrosine-kinase can be down-modulated with clinically used inhibitors, thus modulating LCK activity could be considered for further studies as a new additional therapy for prednisone-resistant patients. Further we also found an association between high levels of CYCLIN E and relapse incidence. Moreover, CYCLIN E is more expressed in early relapsed patients, who usually show an unfavourable prognosis.Conclusions/Significance: We conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies.

Instability of tissue protein biomarkers is a critical issue for molecular profiling. Pre-analytical variables during tissue procurement, such as time delays during which the tissue remains stored at room temperature, can cause significant variability and bias in downstream molecular analysis. Living tissue, ex vivo, goes through a defined stage of reactive changes that begin with oxidative, hypoxic and metabolic stress, and culminate in apoptosis. Depending on the delay time ex vivo, and reactive stage, protein biomarkers, such as signal pathway phosphoproteins will be elevated or suppressed in a manner which does not represent the biomarker levels at the time of excision. Proteomic data documenting reactive tissue protein changes post collection indicate the need to recognize and address tissue stability, preservation of post-translational modifications, and preservation of morphologic features for molecular analysis. Based on the analysis of phosphoproteins, one of the most labile tissue protein biomarkers, we set forth tissue procurement guidelines for clinical research. We propose technical solutions for (i) assessing the state of protein analyte preservation and specimen quality via identification of a panel of natural proteins (surrogate stability markers), and (ii) using multi-purpose fixative solution designed to stabilize, preserve and maintain proteins, nucleic acids, and tissue architecture.

Abstract Abstract 2605 Poster Board II-581 Background. Annexin II (ANXA2) is a member of a peripheral membrane-binding protein family acting in a calcium-dependent manner, is involved in many cellular mechanisms, as cell proliferation and membrane physiology and is related to cancer progression. The aim of this study was to assess the ANXA2 expression in B cell precursor acute lymphoblastic leukemia (Bcp-ALL), in the attempt to finally evaluate it as a new potential therapeutic target. Materials and Methods. The ANXA2 expression was tested in 77 newly diagnosed pediatric Bcp-ALL diagnosed and treated in our centers, according with LLA-2000 protocol of Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP). Diagnostic samples and 3 B-ALL cell lines (REH, SEM, 697) were studied by reverse phase protein array (RPPA), western blot and real-time PCR (RQ-PCR) analyses. Furthermore, immunofluorescence on bone marrow smears and cytofluorimetric studies were performed, in order to visualize the protein subcellular location. The associations between the ANXA2 expression, molecular features and prognosis were evaluated. For statistical purpose, multivariate analyses with Wilcoxon test and t-Test with conservative Bonferroni corrections and Kaplan-Mayer analysis were performed. Pearson correlation was used to compare mRNA and protein levels. Results. Our analyses demonstrated a positive correlation between mRNA and protein ANXA2 expression (Pearson correlation or index 0.6). Comparing ANX2 expression and molecular features, we found a statistically significant difference between patients with unfavourable [t(9;22), t(4;11)] and favourable translocations [t(12;21)], showing a higher level of ANXA2 in the former group (p-value <0.05). Additionally ANXA2 resulted upregulated at both mRNA and protein levels in 24 out of 77 patients included in the study, and in the group presenting with high ANXA2 expression, 8 (33%) patients relapsed; in contrast, in the group with low ANXA2 expression only 8 out of 53 cases (15%) suffered from a relapse. Interestingly, 5 patients (21%) with high ANXA2 expression died of progressive disease, while with only one case (2%) in the group with low ANXA2 expression. A multivariate analysis also showed that ANXA2 is an independent predictor of disease's aggressiveness. Due to the heterogeneity of response to treatment among our patients, which imply a stratification based on detection of minimal residual disease (MRD), the correlation between high expression level of ANXA2 with prognosis resulted not statistically significant (Kaplan-Mayer p-value >0.05). However, our data strongly suggested a correlation with a worst prognosis in those cases with high ANXA2 expression. Furthermore, immunofluorescence and cytofluorimetric analyses performed on SEM and 697 cell lines showed that ANXA2 is localized on the cellular membrane's surface, where the protein is usually involved in many cell functions. Conclusions. To date, our study reports on ANXA2 expression and location in pediatric ALL. Our findings suggest that ANXA2 expression represents a marker of aggressiveness in Bcp-ALL, confirmed by the correlation with unfavourable molecular rearrangement such as MLL/AF4. Although the prognostic impact of ANXA2 expression needs to be evaluated with a further retrospective study including a larger and selected population, our data already strongly suggest that ANXA2 expression could be considered as a new potential therapeutic target in pediatric Bcp-ALL. Disclosures: No relevant conflicts of interest to declare.

Little is known about the preanalytical fluctuations of phosphoproteins during tissue procurement for molecular profiling. This information is crucial to establish guidelines for the reliable measurement of these analytes. To develop phosphoprotein profiles of tissue subjected to the trauma of excision, we measured the fidelity of 53 signal pathway phosphoproteins over time in tissue specimens procured in a community clinical practice. This information provides strategies for potential surrogate markers of stability and the design of phosphoprotein preservative/fixation solutions. Eleven different specimen collection time course experiments revealed augmentation (+/-20% from the time 0 sample) of signal pathway phosphoprotein levels as well as decreases over time independent of tissue type, post-translational modification, and protein subcellular location (tissues included breast, colon, lung, ovary, and uterus (endometrium/myometrium) and metastatic melanoma). Comparison across tissue specimens showed an >20% decrease of protein kinase B (AKT) Ser-473 (p < 0.002) and myristoylated alanine-rich C-kinase substrate protein Ser-152/156 (p < 0.0001) within the first 90-min postexcision. Proteins in apoptotic (cleaved caspase-3 Asp-175 (p < 0.001)), proliferation/survival/hypoxia (IRS-1 Ser-612 (p < 0.0003), AMP-activated protein kinase beta Ser-108 (p < 0.005), ERK Thr-202/Tyr-204 (p < 0.003), and GSK3alphabeta Ser-21/9 (p < 0.01)), and transcription factor pathways (STAT1 Tyr-701 (p < 0.005) and cAMP response element-binding protein Ser-133 (p < 0.01)) showed >20% increases within 90-min postprocurement. Endothelial nitric-oxide synthase Ser-1177 did not change over the time period evaluated with breast or leiomyoma tissue. Treatment with phosphatase or kinase inhibitors alone revealed that tissue kinase pathways are active ex vivo. Combinations of kinase and phosphatase inhibitors appeared to stabilize proteins that exhibited increases in the presence of phosphatase inhibitors alone (ATF-2 Thr-71, SAPK/JNK Thr-183/Tyr-185, STAT1 Tyr-701, JAK1 Tyr-1022/1023, and PAK1/PAK2 Ser-199/204/192/197). This time course study 1) establishes the dynamic nature of specific phosphoproteins in excised tissue, 2) demonstrates augmented phosphorylation in the presence of phosphatase inhibitors, 3) shows that kinase inhibitors block the upsurge in phosphorylation of phosphoproteins, 4) provides a rational strategy for room temperature preservation of proteins, and 5) constitutes a foundation for developing evidence-based tissue procurement guidelines.

Background. ALL is the most frequent cancer in childhood. Although current chemotherapy protocols reach an event free survival (EFS) greater than 70%, the remaining cases relapse and ALL is still the first cause of death in children with cancer. Several efforts have been made to better understand the underlying mechanism involved in ALL. Current chemotherapy protocols have reached a plateau of effectiveness. The detection of minimal residual disease (MRD) during the course of treatment will definitively allows us to stratify children with ALL in standard (SR), intermediate (MR) and high (HR) risk, respectively. In the latter group we included patients with a prednisone poor response, with t(9;22) and t(4;11) (HR by default). Despite of this better definition of therapeutic response, the majority of relapses are included in the MR group, suggesting that we are treating different forms of ALL in this subset. For this reason new biomarkers and molecular targets are highly mandatory. The objective of functional proteome analyses of ALL is to bridge clinical applications with new biomarker discoveries that will be useful for determining prognosis and response to therapy and to identify potential new more selective treatments. Materials and Methods. We analyzed 81 diagnostic samples (peripheral blood and/or bone marrow) with B precursor ALL, that were diagnosed and treated at our centers (Catania and Padova) and enrolled in the AIEOP-LLA 2000 protocol. Reverse phase proteomic assay (RPPA) was used to interrogate the expression of 81 phosphorylated or native protein endpoints in our patients’ samples. We evaluated Pro- and Anti-Apoptotic, Protein Kinase, and Growth factor receptor cell signaling pathways. For statistical purposes, we considered the following characteristics: age at diagnosis, gender, immunophenotype, karyotype, white blood cell count and percentage of blast at diagnosis, response to prednisone, level of MRD at day 33 (TP1) and day 78 (TP2) during the induction phase. We distributed patients in 4 groups, based on risk (SR 21, MR 31, HR 10) and occurrence of relapse (19). We also studied patient specimens both at diagnosis and relapse only in those samples with a blast rate greater than 50% (10 cases out of 19). All findings obtained with RPPA were confirmed by Western blot analyses. We applied the Wilcoxon Test and the t-Test, using the ‘R’ software. Both univariate and multivariate analyses were performed. Results. Comparing subgroups for gender, WBC count (higher vs less than 50.000/ml), immunophenotype (Common vs others), karyotype (t(12;21) positive vs negative), prednisone good (PGR) vs poor (PPR) responders, analysis of MRD (SR vs MR, MR vs HR, SR vs HR, MR vs MR relapsed) and diagnoses vs relapses, we did not find any statistically significant data. Conversely, we found a statistically significant higher expression of FosB and Annexin II in the group of children with WBC >50.000/ml (p value Conclusions. Our preliminary data showed that RPPA in association with Western blot analysis, is a reliable strategy for the identification of biomarkers and/or molecules for targeted therapy. Application of this methodology on specific subgroups of children with ALL will allow us to identify new therapeutic strategies.