Profiling Of Signaling Pathways Research Articles

Signaling Pathway Profiling in Multiple Myeloma

Introduction: The pathogenesis of multiple myeloma (MM) involves complex genetic and epigenetic alterations affecting the cellular signaling network, the detailed processes of which remain poorly understood. Recently available genomic data has revealed a diverse mutational landscape with relatively few recurrently mutated genes. Instead, there are clustered mutational hotspots within signaling pathways that have been described to be of pathogenetic relevance in MM. To date, however, only very limited data is available on the activation profile of signaling pathways in MM and most of these studies have been performed in vitro or on sorted patient cells, which may be not representative of primary cells within their native microenvironment.Patient samples and methods: In contrast to in vitro studies, we used an ex vivo immunohistochemistry-based technique to retrospectively analyze the activation status of the five most well-described signaling pathways in MM. Using key activation markers, we interrogated the RAS/RAF/MEK/ERK, JAK-STAT, canonical NF-kB, PI3K-AKT, and c-MYC signaling networks on bone marrow biopsies taken at the time of diagnosis from two independent patient cohorts. The training cohort included 148 newly diagnosed, symptomatic MM patients. The independent validation cohort was comprised of samples from 84 newly diagnosed, well documented MM patients who had been enrolled in the GMMG - HD3/4 clinical multicenter trials. All patients of the validation cohort had undergone upfront high-dose therapy and autologous stem cell transplantation. The activation pattern of each sample was independently scored by two pathologists relative to on-slide positive controls. Activation scores included signal intensity as well as the percentage of positive versus total tumor cells per sample to account for potential clonal heterogeneity. Principal component analysis (PCA) was applied to integrate signaling scores per pathway and sample. Associations of progression-free (PFS) and overall survival (OS) with pathway activation patterns were analyzed using the Cox regression model.Results: We first focused on activation signals that were present in the majority of myeloma cells per sample, potentially representing the major clone of the individual patient’s disease. Several clusters of activation could be distinguished with a NF-κB cluster being the most prominent (77% of cases), followed by samples with activated MEK/ERK signaling (19.6%) partially overlapping with PI3K-AKT activity (9.5%). An additional 5% of cases showed an activation of STAT3 and/or c-MYC in the majority of tumor cells.We next took all activation signals into account, including those present in less than 50% of myeloma cells per sample, potentially representing subclonal populations of tumor cells. Activation of a single pathway was found in 22%, two pathways in 30%, and more than 2 pathways in 47% of cases, while activation of any tested signaling event was absent in 2%. Several clusters of activation could be distinguished with a NF-kB cluster being the most prominent (82% of cases), followed by samples with activated MEK/ERK signaling (49%) partially overlapping with PI3K-AKT activity (41%). 29% of cases showed an activation of the JAK-STAT3 axis while c-MYC expression was detectable in 43% of the samples. Interestingly, the latter two signaling cascades tended to overlap with other activation clusters.A similar distribution of clusters of activation was found in the validation cohort. Importantly, association analyses with clinical data available for this cohort revealed a significantly shorter PFS (HR 4.59; p=0.038) for patients with STAT3 activation and a trend towards a shorter OS. Moreover, patients with c-MYC activation or those with more than one activated pathway had a significantly shorter overall survival (HR 9.54, p=0.019; and HR 3.77, p=0.003, respectively). In contrast, activation of NF-kB was associated with a more favorable outcome (HR 0.20, p=0.034) while MEK/ERK signaling appeared to confer a neutral prognosis.Conclusion: This study is the first comprehensive study of signaling pathways in multiple myeloma. Activation of signaling cascades differs substantially between patients and do not occur randomly but can be distinguished into defined clusters of activation. Most importantly, there appears to be a prognostic hierarchy of these clusters. DisclosuresNo relevant conflicts of interest to declare.

Using an in situ proximity ligation assay to systematically profile endogenous protein-protein interactions in a pathway network.

Signal transduction pathways in the cell require protein-protein interactions (PPIs) to respond to environmental cues. Diverse experimental techniques for detecting PPIs have been developed. However, the huge amount of PPI data accumulated from various sources poses a challenge with respect to data reliability. Herein, we collected ∼ 700 primary antibodies and employed a highly sensitive and specific technique, an in situ proximity ligation assay, to investigate 1204 endogenous PPIs in HeLa cells, and 557 PPIs of them tested positive. To overview the tested PPIs, we mapped them into 13 PPI public databases, which showed 72% of them were annotated in the Human Protein Reference Database (HPRD) and 8 PPIs were new PPIs not in the PubMed database. Moreover, TP53, CTNNB1, AKT1, CDKN1A, and CASP3 were the top 5 proteins prioritized by topology analyses of the 557 PPI network. Integration of the PPI-pathway interaction revealed that 90 PPIs were cross-talk PPIs linking 17 signaling pathways based on Reactome annotations. The top 2 connected cross-talk PPIs are MAPK3-DAPK1 and FAS-PRKCA interactions, which link 9 and 8 pathways, respectively. In summary, we established an open resource for biological modules and signaling pathway profiles, providing a foundation for comprehensive analysis of the human interactome.

Abstract 1608: Profiling c-Met and EGFR kinase inhibitor resistance pathways in non-small lung cancer cells

Abstract Tyrosine kinase inhibitors (TKIs) against c-MET and EGFR have positive therapeutic effects in a subset of Non-Small Cell Lung Cancer (NSCLC) patients; however, their clinical efficacies are limited due to the development of TKI resistance. To determine mechanisms of TKI resistance, two NSCLC model cell lines H2170 and H358 were made resistant to the c-Met/EGFR TKIs SU11274, erlotinib or a combination of both by exposing the cells to progressively increasing concentrations of inhibitor. Initial characterization of these cell lines using phospho-specific antibodies showed that phospho-c-Met was found to be downregulated 4- and 1.5- fold in SU11274-resistant (SR) H2170 and SR H358 cells, respectively. Phospho-EGFR was found to be constitutively autophosphorylated (upregulated 19-fold) in erlotinib-resistant (ER) H2170 cells, but downregulated 6-fold in ER H358 cells. Interestingly, c-Met/EGFR downstream signaling proteins p-mTOR, p-p70S6K and p-ERK were found to be 2-4-fold upregulated, 2-fold upregulated and 2-5-fold upregulated, respectively, in ER H2170 and H358 cells. Phospho-p70S6K was also found to be upregulated 20-fold in SR H2170 cells. These results indicate that alternative signaling pathways may confer TKI resistance in these cell lines. To identify which signaling pathways may be responsible for TKI resistance, H358 parental and resistant cell lines were analyzed by mass spectrometry (MS). Differences in protein expression in cell lines treated with and without EGF and/or erlotinib were measured using higher multiplexing Thermo Scientific™ Tandem Mass Tag™ (TMT™) reagents. More than 1500 protein groups were identified and quantified using a Thermo Scientific™ Orbitrap™ Fusion™ platform with HCD-MS2 and MS3 fragmentation methods. Protein expression in ER H358 cells showed a marked increase in expression of DNA replication proteins, transcription factors, apoptosis inhibitors and some Ras-associated signal transduction pathways. To further investigate the mechanism of TKI resistance, phospho-peptide enrichment was also used in combination with TMT quantitation to profile changes in protein phosphorylation state. Overall, the combination of phospho-peptide enrichment coupled with higher multiplexing TMT and high resolution MS analysis enabled deeper global profiling of TKI resistance signaling pathways compared to previous studies without enrichment. Citation Format: Ryan D. Bomgarden, Ryan Jacobs, Jason Fong, David Moravec, Gregory M. Botting, Michael Blank, Rosa I. Viner, John C. Rogers, Neelu Puri. Profiling c-Met and EGFR kinase inhibitor resistance pathways in non-small lung cancer cells. [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 1608. doi:10.1158/1538-7445.AM2014-1608

Abstract 4517: Evaluation of the TOR kinase inhibitor CC214-2 in a mouse model of hormone-refractory prostate cancer

Abstract Prostate cancer is one of the most common malignancy diagnosed in men. Although androgen deprivation therapy is effective at the beginning of treatment, the majority of patients eventually progress to hormone refractory prostate cancer (HRPC), which leads to a poor outcome. HRPC mouse models have been developed to study transition from hormone dependent (HD) to hormone refractory (HR) disease; however, the exact mechanism behind this phenotypic conversion remains unclear. By using serial in vivo transplantation of LNCap xenograft tumors in castrated male SCID mice, we developed, validated and characterized a mouse model of HRPC, HR-LNCap. In vivo passaged tumor fragments form solid tumors in castrated male SCID mice. By comparison, HR-LNCap tumors grow more aggressively than parental LNCap tumors in SCID mice. A tumor cell line established from HR-LNCap xenograft tumors was able to grow in charcoal stripped serum containing medium, indicating it is androgen independent. HR-LNCap tumors lost sensitivity to androgen receptor antagonist bicalutamide, but remained sensitive to MDV-3100, a second generation AR antagonist approved for treating HRPC. Characterization of this model revealed that HR-LNCap tumors had unique AR signaling pathway profiles, including elevated AR expression and decreased expression of the AR-regulated genes, prostate specific antigen (PSA) and FKBP5, when compared with parental LNCap tumors. RNA sequencing was performed on HR-LNCap tumors to determine expression signature of the model. Interestingly, additional pathway analysis showed that HR-LNCap tumors have significantly higher levels of pAKT in comparison with parental LNCap tumors, indicating activation of AKT/mTOR pathway. Treatment of HR-LNCap tumor bearing mice with a small molecule TOR kinase inhibitor CC214-2 showed significant tumor growth inhibition. Further analysis demonstrated that treatment with CC214-2 inhibited TORC1 and TORC2 pathway markers, pAKT, pS6 and p4EBP1, in HR-LNCap tumors. Taken together, this study describes a mouse model of HRPC with a unique molecular profile, which can be used to evaluate novel therapeutics against HRPC. Citation Format: Wenqing Yang, Jing Jiu, Rama Krishna Narla, Heather K. Raymon. Evaluation of the TOR kinase inhibitor CC214-2 in a mouse model of hormone-refractory prostate cancer. [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 4517. doi:10.1158/1538-7445.AM2014-4517

Gene profiling of human VEGF signaling pathways in human endothelial and retinal pigment epithelial cells after anti VEGF treatment.

BackgroundRanibizumab (Lucentis®) is a Fab-antibody fragment developed from Bevacizumab, a full-length anti-VEGF antibody. Both compounds are used for treating age-related macular degeneration (AMD). The influence of bevacizumab and ranibizumab on genes involved in signal transduction and cell signaling downstream of VEGF were compared in order to detect possible differences in their mode of action, which are not related to their Fab-antibody fragments.MethodsHuman umbilical vein cell lines (EA.hy926) and retinal pigment epithelial cells (ARP-19) were exposed to oxidative stress. The cells were treated with therapeutic concentrations of bevacizumab (0.25 mg/mL) and ranibizumab (125 mg/mL) for 24 hours prior to all experiments, and their effects on gene expressions were determined by RT- PCR.ResultsAfter exposure to bevacizumab, more genes in the endothelial cells were up-regulated (KDR, NFATc2) and down-regulated (Pla2g12a, Rac2, HgdC, PRKCG) compared to non-treated controls. After exposure to ranibizumab, fewer genes were up-regulated (PTGS2) and down-regulated (NOS3) compared to controls. In comparison between drugs, more genes were up-regulated (NFATc2 and KDR) and more were down-regulated (Pla2g12a, Pla2g1b, Ppp3r2, Rac2) by bevacizumab than by ranibizumab. In RPE cells, NOS3 and PGF were up-regulated and Pla2g12b was down-regulated after exposure to ranibizumab, while PIK3CG was up-regulated and FIGF was down-regulated after exposure to bevacizumab, but the differences in gene expression were minor between drugs (PIK3CGand PGF were down-regulated more by ranibizumab than by bevacizumab).ConclusionsThe different gene expressions after exposure to ranibizumab and bevacizumab in endothelial and RPE cells may indicate a somewhat different biological activity of the two compounds.

Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells.

Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer.

Profiling of signaling pathways in live tumor cells to assess drug mechanism of action: A method to predict drug efficacy in a patient.

e11583 Background: This work reports a dynamic real time analysis of live cells to detect drug attenuation of cell signaling related to the MOA of a targeted therapy. The assay employs an impedance...

Activating FLT3 Mutants Show Distinct Gain-of-Function Phenotypes In Vitro and a Characteristic Signaling Pathway Profile Associated with Prognosis in Acute Myeloid Leukemia

About 30% of patients with acute myeloid leukemia (AML) harbour mutations of the receptor tyrosine kinase FLT3, mostly internal tandem duplications (ITD) and point mutations of the second tyrosine kinase domain (TKD). It was the aim of this study to comprehensively analyze clinical and functional properties of various FLT3 mutants.In 672 normal karyotype AML patients FLT3-ITD, but not FLT3-TKD mutations were associated with a worse relapse free and overall survival in multivariate analysis. In paired diagnosis-relapse samples FLT3-ITD showed higher stability (70%) compared to FLT3-TKD (30%). In vitro, FLT3-ITD induced a strong activating phenotype in Ba/F3 cells. In contrast, FLT3-TKD mutations and other point mutations – including two novel mutations – showed a weaker but clear gain-of-function phenotype with gradual increase in proliferation and protection from apoptosis. The pro-proliferative capacity of the investigated FLT3 mutants was associated with cell surface expression and tyrosine 591 phosphorylation of the FLT3 receptor. Western blot experiments revealed STAT5 activation only in FLT3-ITD positive cell lines, in contrast to FLT3-non-ITD mutants, which displayed an enhanced signal of AKT and MAPK activation. Gene expression analysis revealed distinct difference between FLT3-ITD and FLT3-TKD for STAT5 target gene expression as well as deregulation of SOCS2, ENPP2, PRUNE2 and ART3. FLT3-ITD and FLT3 point mutations show a gain-of-function phenotype with distinct signalling properties in vitro. Although poor prognosis in AML is only associated with FLT3-ITD, all activating FLT3 mutations can contribute to leukemogenesis and are thus potential targets for therapeutic interventions.

Reverse Phase Protein Array: A Tool for Signaling Pathway Profiling in the Era of Genome Resequencing

Reverse Phase Protein Array: A Tool for Signaling Pathway Profiling in the Era of Genome Resequencing

Vitamin C Enhances Nanog Expression Via Activation of the JAK/STAT Signaling Pathway

Vitamin C (Vc), also known as ascorbic acid, is involved in many important metabolic and physiological reactions in the body. Here, we report that Vc enhances the expression of Nanog and inhibits retinoic acid-induced differentiation of embryonic stem cells. We investigated Vc regulation of Nanog through Janus kinase/signal transducer and activator of transcription pathway using cell signaling pathway profiling systems, and further confirmed by specific pathway inhibition. Using overexpression and knockdown strategies, we demonstrated that STAT2 is a new positive regulator of Nanog and is activated by phosphorylation following Vc treatment. In addition, site mutation analysis identified that STAT2 physically occupies the Nanog promoter, which was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Taken together, our data suggest a role for Vc in Nanog regulation networks and reveal a novel role for STAT2 in regulating Nanog expression.

Proximity Ligation Assay for High-content Profiling of Cell Signaling Pathways on a Microfluidic Chip

Here, we present the full integration of a proximity ligation assay (PLA) on a microfluidic chip for systematic cell signaling studies. PLA is an in situ technology for the detection of protein interaction, post-translational modification, concentration, and cellular location with single-molecule resolution. Analytical performance advances on chip are achieved, including full automation of the biochemical PLA steps, target multiplexing, and reduction of antibody consumption by 2 orders of magnitude relative to standard procedures. In combination with a microfluidic cell-culturing platform, this technology allows one to gain control over 128 cell culture microenvironments. We demonstrate the use of the combined cell culture and protein analytic assay on chip by characterizing the Akt signaling pathway upon PDGF stimulation. Signal transduction is detected by monitoring the phosphorylation states of Akt, GSK-3β, p70S6K, S6, Erk1/2, and mTOR and the cellular location of FoxO3a in parallel with the PLA. Single-cell PLA results revealed for Akt and direct targets of Akt a maximum activation time of 4 to 8 min upon PDGF stimulation. Activation times for phosphorylation events downward in the Akt signaling pathway including the phosphorylation of S6, p70S6K, and mTOR are delayed by 8 to 10 min or exhibit a response time of at least 1 h. Quantitative confirmation of the Akt phosphorylation signal was determined with the help of a mouse embryonic fibroblast cell line deficient for rictor. In sum, this work with a miniaturized PLA chip establishes a biotechnological tool for general cell signaling studies and their dynamics relevant for a broad range of biological inquiry.

Profiling Inflammatory Responses with Microfluidic Immunoblotting

Rapid profiling of signaling pathways has been a long sought after goal in biological sciences and clinical medicine. To understand these signaling pathways, their protein components must be profiled. The protein components of signaling pathways are typically profiled with protein immunoblotting. Protein immunoblotting is a powerful technique but has several limitations including the large sample requirements, high amounts of antibody, and limitations in assay throughput. To overcome some of these limitations, we have designed a microfluidic protein immunoblotting device to profile multiple signaling pathways simultaneously. We show the utility of this approach by profiling inflammatory signaling pathways (NFκB, JAK-STAT, and MAPK) in cell models and human samples. The microfluidic immunoblotting device can profile proteins and protein modifications with 5380-fold less antibody compared to traditional protein immunoblotting. Additionally, this microfluidic device interfaces with commonly available immunoblotting equipment, has the ability to multiplex, and is compatible with several protein detection methodologies. We anticipate that this microfluidic device will complement existing techniques and is well suited for life science applications.

Abstract 2403: Proteomic profiling of signaling pathways in LKB1 deficient non-small cell lung cancers (NSCLC) identifies novel therapeutic targets including IGF1R pathway.

Abstract Background: LKB1 is a tumor suppressor gene that is lost in ∼35% of non-small cell lung cancers (NSCLC), half of which carry concurrent KRAS mutations. When functioning normally, LKB1 plays a critical role in energy sensing and regulates the PI3K/mTOR pathway. We have previously shown that LKB1 loss is associated with greater resistance to certain targeted drugs. Here, we investigate signaling pathways dysregulated in the setting of LKB1 loss, with a particular focus on potential novel therapeutic targets. Methods: Expression of >140 total and phospho-proteins were measured in 109 NSCLC cell lines by reverse phase protein array (RPPA). Differences in protein expression between LKB1 deficient versus intact cell lines were determined by t-test, with correction for multiple testing. IC50s for targeted therapies were determined by MTS assay in cell lines with and without LKB1. For the drug combination assay, nine NSCLC cell lines with and without LKB1 were treated with the IGFR inhibitor OSI-906 plus serial dilutions of the dual mTOR inhibitor OSI-027. Proliferation was measured after 72 hours. Bliss algorithm was used to calculate synergy and fractional inhibition for each plate independently. Further validation was performed using NSCLC cells stably overexpressing LKB1. Results: Loss of LKB1 via mutation or deletion was strongly correlated with LKB1 protein expression (p<0.0001 by t-test). In agreement with previous studies showing the regulation of mTOR by LKB1, cells with LKB1 loss had significantly greater activation of the mTOR signaling pathway, as indicated by higher levels of phosphorylated (p) mTOR, pS6, and pP70S6K (p<0.014). Other proteins expressed at significantly higher levels in LKB1 deficient NSCLC cell lines included those in the IGF1R pathway (ex., IGF1R and its transcriptional regulator SRC3, p<0.001) and MEK pathway (ex., pERK1/2, pMAPK, p<0.017). Furthermore, we observed increased NRF2 protein levels (0.001) and concomitant downregulation of its suppressor KEAP1 (<0.001) in LKB1 deficient lines, suggesting a potential role in LKB1-mediated regulation of oxidative stress. Inhibition of IGF1R by OSI-906 showed activity in LKB1 deficient lines; however, the combination of IGF1R + mTOR inhibition (OSI-906 + OSI-027) proved to be a more effective therapy, demonstrating synergistic activity at all dose combinations studied. These findings were confirmed in A549 cells stably overexpressing LKB1. Drug combination showed a synergistic effect in A549 control cells compared to A549-LKB1. Conclusions: These studies identify new potential targets in LKB1 deficient NSCLC, which represents a significant portion of lung cancer patients. Specifically, the combination of IGFR and mTOR inhibition demonstrated synergy, supporting further investigation as a therapeutic approach for this molecularly defined subset of NSCLC patients. Citation Format: Lauren Averett Byers, Maria Angelica Cortez, Chao Yang, Jing Wang, Lixia Diao, You Hong Fan, Luc Girard, Adi Gazdar, Ignacio Wistuba, John D. Minna, Matthew O'Connor, Sharon Barr, John V. Heymach. Proteomic profiling of signaling pathways in LKB1 deficient non-small cell lung cancers (NSCLC) identifies novel therapeutic targets including IGF1R pathway. [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 2403. doi:10.1158/1538-7445.AM2013-2403

Abstract 4654: Genomic and proteomic profiling of signaling pathways targeted by a multi-kinase inhibitor, sorafenib.

Abstract Background: Deregulation of intracellular signaling by various genetic alterations is a hallmark of cancer. Therapeutics targeting such activated signaling pathways have achieved significant progress, but the precise pathway(s) targeted by sorafenib has remained unclear. Methods: Using high-density reverse-phase protein arrays, we profiled the phosphorylation status of 180 signaling molecules in the 120 pathways registered on the NCI-Nature curated database in 23 hepatocellular carcinoma (HCC) cell lines showing different sensitivities to sorafenib. We also sequenced the entire coding regions of 518 protein kinase genes in 20 overlapping HCC cell lines. Results: We identified 25 non-synomymous alterations in the 4 mTOR and MAPK pathway genes (RPS6KA1, RPS6KA6, RPS6KB2, and BRAF) in HCC cell lines 1 to 11. We also found that the levels of phosphorylated S6 ribosomal protein and c-Raf were associated with the sensitivity of the HCC cell lines to sorafenib. A mTOR inhibitor, AZD8055, exerted growth-inhibitory activity against sorafenib-resistant HCC cell lines. Discussion: Although a recent phase III clinical trial has demonstrated the effectiveness of sorafenib in patients with unresectable HCC, only a small proportion of the patients achieved the anticipated therapeutic benefits. The present results indicate cross-talk between the mTOR and MAPK signaling pathways in sorafenib-resistant HCC cell lines. Use of a combination of mTOR and MAPK inhibitors may improve the efficacy of molecular targeting therapy against HCC. Citation Format: Mari Masuda, Kazufumi Honda, Tesshi Yamada. Genomic and proteomic profiling of signaling pathways targeted by a multi-kinase inhibitor, sorafenib. [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 4654. doi:10.1158/1538-7445.AM2013-4654

Abstract 3497: Pathway profiling for personalized medicine: Comparison of two immunoassays.

Abstract Cancer therapy is evolving to a detailed molecular analysis of the patient's tumor followed by treatment with selective drug(s) targeting the individual properties of the tumor based upon identification of prognostic and predictive biomarkers. This requires comprehensive, highly sensitive test systems. We therefore sought to develop a sensitive diagnostic test for functionally profiling a spectrum of signaling pathway proteins in tumor samples. Here, we evaluated and compared two assay platforms: CEER (Collaborative Enzyme Enhanced Reactive-Immunoassay; Prometheus Labaratories) and MSD (Meso Scale Discovery). Breast, lung, and prostate cancers as well as glioblastoma and melanoma model cell lines (in all 10 cell lines) with various oncogenic pathway signatures were treated with different concentrations of PI3K inhibitor (BAY 806946) or an inhibitor targeting HER1 and HER2 (Lapatinib). Cells were lysed and the activation status as well as abundance of ten pathway proteins (HER1, HER2, cMET, PI3K, AKT, ERK, MEK, PRAS40, RPS6, and P70S6K) was measured by MSD at Bayer in Berlin and by CEER in a blinded fashion at Prometheus Laboratories. Target-specific inhibition (IC50) and downstream signal modulations were determined and compared. Overall we found a high concordance between the two assays. While target specific inhibitions were observed in relevant cell lines, varying mechanisms of treatment resistance due to redundant pathway activation, feedback loop or pathway cross talks were observed between the PI3K/AKT and RAS/ERK pathways. This study shows that sensitive immunoassays are a suitable tool for the detection and monitoring of biomarkers and provides new insights into the mode of action of targeted agents. Comprehensive profiling of signaling pathways holds promise as an approach to personalize selection of anticancer therapy. Citation Format: Antje Stresemann, Oliver von Ahsen, Oliver Politz, Phillip Kim, Sharat Singh, Khusru Asadullah, Karl Ziegelbauer, Thomas Krahn. Pathway profiling for personalized medicine: Comparison of two immunoassays. [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 3497. doi:10.1158/1538-7445.AM2013-3497

Abstract 2415: Chloroquine disruption of autophagy: A new treatment for DCIS.

Abstract Background: Survival of intraductal, pre-invasive proliferating cells under stress may promote genetic instability and the selection of invasive carcinoma cells. Our analysis of the mechanisms used by DCIS cells to survive in the hypoxic, nutrient deprived intraductal niche has revealed autophagy as a therapeutic target. We hypothesize that the intraductal pre-neoplastic cellular niche will be preferentially sensitive to a short term, anti-autophagy based prevention therapy which targets survival pathways used by pre-invasive carcinomas. Our unique trial design offers a means to functionally screen investigational agents that kill premalignant breast lesions.Methods: DCIS spheroid forming cells have been isolated and propagated from fresh human DCIS lesions. We characterized the DCIS cells by organ culture, xenograft transplantation, molecular cytogenetics (300,000 SNPs) and 59 signal pathway profiling endpoints (Reverse Phase Protein Microarray (RPMA)). Results: Autophagy is a means for intraductal neoplastic cells, accumulating in the duct, to digest intracellular contents and generate energy to survive in the face of severe metabolic, oxidative, and hypoxic stress. DCIS spheroid forming cells were tumorigenic in xenograft models. Autophagy is up-regulated in: human DCIS lesions, DCIS spheroid-forming cells, and the xenograft tumors they generate. Suppression of autophagy by chloroquine kills human DCIS spheroid forming cells in culture and prevents them from forming tumors in xenografts (p<.0003). Based on these preclinical insights our neoadjuvant therapy trial for DCIS patients with any stage/grade DCIS (NCT01023477 PINC Trial), examines the safety and effectiveness of oral chloroquine for 30 days. At the conclusion of treatment, all patients receive standard of care surgical therapy. MRI is performed before and after the treatment period. Endpoints compared before, versus after, therapy are: lesion shrinkage, pathologic regression, disruption of lesion autophagy and proliferation (PCNA), induction of apoptosis, and suppression of xenograft tumorigenicity. Each patient serves as her own control because effectiveness is judged by comparing the patient's DCIS lesion before and after therapy. Conclusions: We have identified the autophagy pathway as a therapeutic target for DCIS. Chloroquine, an inhibitor of autophagy, suppresses the growth and survival of DCIS progenitor, stem-like cells. Autophagy is necessary for survival of intra-ductal DCIS cells and provides justification for this PINC trial. Our initial PINC clinical trial results provide strong evidence that anti-autophagy therapy can generate a measurable reduction in DCIS proliferation index (PCNA) within 4 weeks. In addition to justifying the trial design, these data suggest that at least a subset of DCIS cases may respond to oral chloroquine. Citation Format: Virginia A. Espina, Kirsten Edmiston, Emil Kamar, Geetha Menezes, Hassan Nayer, Lance A. Liotta. Chloroquine disruption of autophagy: A new treatment for DCIS. [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 2415. doi:10.1158/1538-7445.AM2013-2415

Nimesulide and Celecoxib Inhibits Multiple Oncogenic Pathways in Gastric Cancer Cells

Despite being the leading cause of cancer death, targeted therapy for gastric cancer is yet to be established. Wnt/β-catenin signaling is highly deregulated in cancers of gastrointestinal origin including gastric cancers. Stabilization and deregulation of β-catenin occurs at multiple levels and so is being needed to identify a spectrum of Wnt inhibitors to combat deregulated Wnt signaling at the level of various targets and also in different combinations. We developed a luciferase reporter based gastric cancer cellular assay system for Wnt pathway modulator screening and identified nimesulide, a known COX-2 inhibitor as an inhibitor of Wnt/β catenin signaling pathway. Comprehensive signaling pathway profiling revealed that nimesulide could inhibit STAT3, IRF1 and RXR signaling apart from inhibiting Wnt/β-catenin-Myc-E2F signaling cascade. Nimesulide elicits a strong anti-proliferative effect by promoting cell cycle arrest in multiple gastric cancer cell lines. Inhibition of Wnt and STAT3 signaling are found to be COX-2 independent, while the inhibition of RXR and IRF1 pathways are due to the COX-2 inhibiting feature of nimesulide. While nimesulide is capable of activating Notch signaling in gastric cancer cells, celecoxib inhibits Wnt, Myc, E2F, RXR, STAT3, MAPK and Notch signaling pathways in gastric cancer cells. Signaling pathway focused analysis of gastric cancer transcriptome revealed that Wnt, STAT3, IRF1 and RXR signaling pathway are highly deregulated in majority of gastric tumors and indicates the potential of nimesulide and celecoxib class of drugs for targeted gastric cancer therapeutics. The differential inhibition of multiple signaling by nimesulide and celecoxib deserve further investigation.

Evaluation of autophagy as a therapeutic target for DCIS.

189 Background: Survival of intraductal, preinvasive proliferating cells under stress may promote genetic instability and the selection of invasive carcinoma cells. Our analysis of the mechanisms used by DCIS cells to survive in the hypoxic, nutrient deprived intraductal niche has revealed autophagy as a therapeutic target. Methods: DCIS spheroid forming cells have been isolated and propagated from fresh human DCIS lesions. We characterized the DCIS cells by organ culture, xenograft transplantation, molecular cytogenetics (300,000 SNPs) and 59 signal pathway profiling endpoints (Reverse Phase Protein Microarray [RPMA]). Results: DCIS spheroid forming cells were tumorigenic in xenograft models. Autophagy is up-regulated in: human DCIS lesions, DCIS spheroid-forming cells, and the xenograft tumors they generate. Autophagy is a means for intraductal neoplastic cells, accumulating in the duct, to digest intracellular contents and generate energy to survive in the face of severe metabolic, oxidative, and hypoxic stress. Suppression of autophagy by chloroquine kills human DCIS spheroid forming cells in culture and prevents them from forming tumors in xenografts (p < .0003). Based on these preclinical insights we have opened a neoadjuvant therapy trial for DCIS (NCT01023477 PINC Trial) to examine the safety and effectiveness of oral 30-day chloroquine for patients with any stage/grade DCIS. At the conclusion of treatment, all patients receive standard of care surgical therapy. MRI is performed before and after the treatment period. Endpoints compared before, versus after, therapy are lesion shrinkage, pathologic regression, disruption of lesion autophagy and proliferation (Ki67), induction of apoptosis, and suppression of xenograft tumorigenicity. Each patient serves as her own control because effectiveness is judged by comparing the patient’s DCIS lesion before and after oral therapy. Conclusions: We hypothesize that the intraductal pre-neoplastic cellular niche will be preferentially sensitive to a short term, anti-autophagy based prevention therapy which targets survival pathways used by pre-invasive carcinomas. Our unique trial design offers a means to functionally screen investigational agents that kill premalignant breast lesions.

PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways perturbations in non-functioning pituitary adenomas

Non-functioning pituitary adenomas (NFPAs) comprise a heterogeneous group, which are considered the most common pituitary tumor. As no clinically hormone hypersecretion is apparent, non-functioning pituitary adenomas are often diagnosed only when they are large enough to cause tumor mass effects, such as hypopituitarism, visual field defects or headaches. Efficient medical therapy for NFPAs is currently unavailable and surgical treatment of these tumors is not always satisfactory. Characterization of signaling regulatory events in the context of NFPAs may enable the development of new attractive novel strategies. Although data regarding gene expression profiling of signaling pathways in NFPAs have accumulated, studies aimed at fine-classification of NFPAs-specific signaling regulatory mechanisms and feedback loops are scarce.

Abstract 856: Biomarkers of sensitivity and resistance to the BTK inhibitor PCI-32765 in mantle cell lymphoma

Abstract Bruton's tyrosine kinase (Btk) is a central regulator of BCR signaling in B-cell lymphomas. PCI-32765 is a potent and selective inhibitor of Btk which has shown good clinical activity, particularly in mantle cell lymphoma. But its mechanism of action in this disease is poorly understood. In this study, we explored the role of Btk and the activity of PCI-32765 on BCR signaling in PBMC samples from patients, which contain a high percentage of tumor cells, as well as in several MCL lines, including Granta-519, Jeko-1, JVM-2, JVM-13, Maver-1, Mino, NCEB-1, Rec-1 and Z-138. In this work, we present a detailed characterization of signalling pathways inhibited by PCI-32765 downstream of the B-cell receptor in cell lines and tumor cells, studied by gene expression profiling, flow cytometry, and proteomic analyses of signaling pathways and effectors. Correlation with PCI-32765 activity in tumor cells and patients leads to the identification of biomarkers of biomarkers of sensitivity and resistance. Results: Btk and surface IgM protein expression was detected in all MCL lines at variable levels. In a 3-day proliferation assay, JVM-2 & MINO emerged as the most sensitive lines to PCI-32765 (GI50: 1.75-4.4uM). Rec-1 was resistant to PCI-32765 alone (11.3uM) but became much more sensitive (1.45uM) upon BCR stimulation using anti-IgM (10ug/mL). Other lines such as Maver-1, Granta-519 & Jeko-1 all required >10uM of PCI-32765 for inhibition and BCR stimulation did not make much difference. When signaling pathways downstream of BCR activation were studied, intracellular calcium flux following stimulation with IgM was observed in all lines (except JVM-2) and was inhibited at <100nM PCI-32765 in most of them, but no correlation between this and growth inhibition was observed. Constitutive BTK autophosphorylation was observed in all lines and was completely abolished by PCI-32765. BCR stimulation increased p-BTK which was also blocked by PCI-32765 in all lines. Mino and JVM-2 showed constitutive p-ERK activity, which was slightly increased upon BCR stimulation and could be blocked with PCI-32765, whereas the more resistant lines such as Maver-1 and Rec-1 had low endogeneous levels of p-ERK, but which was increased by BCR stimulation and only partially or not reversed by PCI-32765 at 5uM. Little change was observed in levels of p-PLCg1 or p-NF-kB p65. Additionally, in all cell lines stimulation with anti-IgM led to an increased secretion of the chemokines CCL3 and CCL4, which was inhibited by PCI-32765. Blockade of Btk function using PCI-32765 inhibits MCL cell proliferation, BCR signaling and chemokine secretion. These findings highlight the importance of BCR signaling and Btk in MCL, help explain the activity of the Btk inhibitor PCI-32765 in MCL patients, and provide biomarkers that may be of value in the clinic. 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 856. doi:1538-7445.AM2012-856