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There is substantial interest in mining neoantigens for cancer applications. Non-canonical proteins resulting from frameshift mutations have been identified as neoantigens in cancer. We investigated the landscape of non-canonical proteins in non-small cell lung cancer (NSCLC) and their induced immune response in the form of autoantibodies. A database of cryptoproteins was computationally constructed and comprised all alternate open reading frames (altORFs) and ORFs identified in pseudogenes, noncoding RNAs, and untranslated regions of mRNAs that did not align with known canonical proteins. Proteomic profiles of seventeen lung adenocarcinoma (LUAD) cell lines were searched to evaluate the occurrence of cryptoproteins. To assess the immunogenicity, immunoglobulin (Ig)-bound cryptoproteins in plasmas were profiled by mass spectrometry. The specimen set consisted of plasmas from 30 newly diagnosed NSCLC cases, pre-diagnostic plasmas from 51 NSCLC cases, and 102 control plasmas. An analysis of LUAD cell lines identified 420 cryptoproteins. Plasma Ig-bound analyses revealed 90 cryptoproteins uniquely found in cases and 14 cryptoproteins that had a fold-change >2 compared to controls. In pre-diagnostic samples, 17 Ig-bound cryptoproteins yielded an odds ratio ≥2. Eight Ig-bound cryptoproteins were elevated in both pre-diagnostic and newly diagnosed cases compared to controls. Cryptoproteins represent a class of neoantigens that induce an autoantibody response in NSCLC.

GEN1046 (DuoBody®-PD-L1x4-1BB) is an investigational, potential first-in-class bispecific immunomodulatory antibody designed to elicit an anti-tumor immune response by simultaneous and complementary blockade of PD-L1 on tumor or immune cells and conditional 4-1BB stimulation on T cells and NK cells. Previously, we described encouraging preclinical and early clinical activity of GEN1046 (Muik, et al., 2022, Cancer Discovery). We hypothesized that combining GEN1046 with PD-1 blockade would further potentiate anti-tumor activity through distinct and complementary immune modulatory effects. Addition of an anti-PD-1 agent would free up PD-L1 for binding to GEN1046, thus promoting PD-L1-dependent 4-1BB conditional agonism, while maintaining complete blockade of the PD-1 pathway by inhibiting interactions with both PD-L1 and PD-L2. ​Here we provide preclinical evidence supportive of therapeutic synergy by the combination of GEN1046 and anti-PD-1 and describe the mechanisms of enhanced anti-tumor immunity elicited by the combination. In in vitro studies, combining GEN1046 with an anti-PD-1 agent potentiated cytokine release in mixed lymphocyte reaction assays (using either unstimulated T cells or T cells exhausted by repeated CD3/CD28 co-stimulation) and enhanced T-cell expansion and cytokine secretion in antigen-specific proliferation assays compared to each single agent. In in vivo studies in mice bearing syngeneic subcutaneous MC38 tumors, the combination of an anti-mouse PD-L1x4-1BB bispecific antibody with anti-mouse PD-1 potentiated anti-tumor activity with significant enhancement of survival (P≤0.001) and durable, complete tumor regressions (CR) in 7/10 mice compared to no CR observed with either single agent, suggesting therapeutic synergy with the combination. The combination treatment elicited long-lasting immune memory response, as animals with CR were protected from tumor outgrowth upon rechallenge with MC38 cells. Mechanistically, animals treated with the combination showed a trend for ≥1.5-fold increase in the average density of CD3+ and CD4+ tumor-infiltrating lymphocytes (TILs), as well as proliferating (Ki67+) and cytotoxic (GZMB+) CD8+ TILs relative to each single agent, consistent with an amplified anti-tumor immune response. Together, these preclinical results suggest that combining GEN1046-induced conditional 4-1BB stimulation with complete PD-1 blockade can improve the anti-tumor immune response via distinct and complementary immune modulatory effects. The combination of GEN1046 with pembrolizumab is currently being investigated in ongoing clinical studies in patients with advanced NSCLC, who are treatment-naïve (NCT03917381) or have progressed on prior CPI-containing therapy (NCT05117242). Citation Format: Michela Capello, Angelica Sette, Theo Plantinga, Vanessa Spires, Kristina Nuermberger, Jordan Blum, Alexander Muik, Carol Costa Sa, Omar Jabado, Saskia Burm, Aras Toker, Sina Fellermeier-Kopf, Tahi Ahmadi, Brandon Higgs, Suzana Couto, Özlem Türeci, Mark Fereshteh, Ugur Sahin, Maria Jure-Kunkel, Nora Pencheva. GEN1046 (DuoBody®-PD-L1x4-1BB) in combination with PD-1 blockade potentiates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3283.

PURPOSE The combination chemotherapy of fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) has provided clinically meaningful improvement for pancreatic ductal adenocarcinoma (PDAC). We previously uncovered a role for the serine hydrolase carboxylesterase 2 (CES2) in mediating intratumoral activation of the prodrug irinotecan, a constituent of FOLFIRINOX. We aimed to further test the predictive value of CES2 for response to irinotecan using patient-derived xenograft (PDX) models and to elucidate the determinants of CES2 expression and response to FOLFIRINOX treatment among patients with PDAC. METHODS PDXs were engrafted subcutaneously into nude mice and treated for 4 weeks with either saline control or irinotecan. CES2 and hepatocyte nuclear factor 4 alpha (HNF4A) expression in PDAC tissues was evaluated by immunohistochemical and Western blot analysis. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and hemoglobin A1C (HbA1C) levels in patients who underwent neoadjuvant FOLFIRINOX treatment. RESULTS High CES2 activity in PDAC PDXs was associated with increased sensitivity to irinotecan. Integrated gene expression, proteomic analyses, and in vitro genetic experiments revealed that nuclear receptor HNF4A, which is upregulated in diabetes, is the upstream transcriptional regulator of CES2 expression. Elevated CES2 protein expression in PDAC tissues was positively associated with a history of type 2 diabetes (odds ratio, 4.84; P = .02). High HbA1C levels were associated with longer overall survival in patients who received neoadjuvant FOLFIRINOX treatment ( P = .04). CONCLUSION To our knowledge, we provide, for the first time, evidence that CES2 expression is associated with a history of type 2 diabetes in PDAC and that elevated HbA1C, by predicting tumor CES2 expression, may represent a novel marker for stratifying patients most likely to respond to FOLFIRINOX therapy.

The tumor microenvironment forms a complex pro-tumorigenic milieu constituted by extracellular matrix, surrounding stroma, infiltrating cell populations, and signaling molecules. Proteomic studies have the potential to reveal how individual cell populations within the tumor tissue modulate the microenvironment through protein secretion and consequently alter their protein expression and localization to adapt to this milieu. As a result, proteomic approaches have uncovered how these dynamic components communicate and promote tumor development and progression. The characterization of these mechanisms is relevant for the identification of clinically targetable pathways and for the development of diagnostic tools. Here we describe a method based on the isolation of individual cell compartments and the chromatographic fractionation of intact proteins, followed by enzymatic digestion of individual fractions, and mass-spectrometry analysis, for the profiling of tumor microenvironment cell populations.

Background MYC is an oncogenic driver of development and progression in triple-negative breast cancer (TNBC). Ornithine decarboxylase, the rate-limiting enzyme in polyamine metabolism, is a transcriptional target of MYC. We therefore hypothesized that a plasma polyamine signature may be predictive of TNBC development and progression. Methods Using liquid chromatography mass spectrometry, polyamine levels were determined in plasma samples from newly diagnosed patients with TNBC (n = 87) and cancer-free controls (n = 115). Findings were validated in plasma samples from an independent prospective cohort of 54 TNBC, 55 estrogen receptor negative (ER−) and progesterone receptor negative (PR−) and HER2 positive (HER2+), and 73 ER+ case patients, and 30 cancer-free control subjects. Gene expression data and clinical data for 921 and 2359 breast cancer tumors were obtained from The Cancer Genome Atlas repository and the Oncomine database, respectively. Relationships between plasma diacetylspermine (DAS) and tumor spermine synthase (SMS) mRNA expression with metastasis-free survival and overall survival were determined using Cox proportional hazard models; Fisher exact tests were used to assess risk of distant metastasis in relation to tumor SMS mRNA expression. Results An increase in plasma DAS, a catabolic product of spermine mediated through SMS, was observed in the TNBC subtype of breast cancer. Plasma levels of DAS in TNBC associated with increased risk of metastasis (plasma DAS value ≥ 1.16, hazard ratio = 3.06, 95% confidence interval [CI] = 1.15 to 8.13, two-sided P = .03). SMS mRNA expression in TNBC tumor tissue was also found to be predictive of poor overall survival (top 25th percentile hazard ratio = 2.06, 95% CI = 1.04 to 4.08, one-sided P = .04) and increased risk of distant metastasis in TNBC (comparison of lowest SMS quartile [reference] to highest SMS quartile relative risk = 1.90, 95% CI = 0.97 to 4.06, one-sided Fisher exact test P=.03). Conclusions Metabolomic profiling identified plasma DAS as a predictive marker for TNBC progression and metastasis.

A hallmark of cancer, including pancreatic ductal adenocarcinoma (PDA), is a massive stromal and inflammatory reaction. Many efforts have been made to identify the anti- or protumoral role of cytokines and immune subpopulations within the stroma. Here, we investigated the role of interleukin-17A (IL17A) and its effect on tumor fibroblasts and the tumor microenvironment. We used a spontaneous PDA mouse model (KPC) crossed to IL17A knockout mice to show an extensive desmoplastic reaction, without impaired immune infiltration. Macrophages, especially CD80(+) and T cells, were more abundant at the earlier time point. In T cells, a decrease in FoxP3(+) cells and an increase in CD8(+) T cells were observed in KPC/IL17A(−/−) mice. Fibroblasts isolated from IL17A(+/+) and IL17A(−/−) KPC mice revealed very different messenger RNA (mRNA) and protein profiles. IL17A(−/−) fibroblasts displayed the ability to restrain tumor cell invasion by producing factors involved in extracellular matrix remodeling, increasing T cell recruitment, and producing higher levels of cytokines and chemokines favoring T helper 1 cell recruitment and activation and lower levels of those recruiting myeloid/granulocytic immune cells. Single-cell quantitative PCR on isolated fibroblasts confirmed a very divergent profile of IL17A-proficient and -deficient cells. All these features can be ascribed to increased levels of IL17F observed in the sera of IL17A(−/−) mice, and to the higher expression of its cognate receptor (IL17RC) specifically in IL17A(−/−) cancer-associated fibroblasts (CAFs). In addition to the known effects on neoplastic cell transformation, the IL17 cytokine family uniquely affects fibroblasts, representing a suitable candidate target for combinatorial immune-based therapies in PDA.

Irinotecan-based therapy is a common treatment for pancreatic cancer. To elicit its anticancer activity, the drug requires first the hydrolysis action of the enzyme human carboxylesterase 2 (hCES2). It has been established that pancreatic cancer patients have various levels of hCES2, whereby patients having low levels respond poorer to Irinotecan than patients with higher levels, suggesting that hCES2 can be used to predict response. However, current methods that measure hCES2 activity are inaccurate, complex or lengthy, thus being incompatible for use in a clinical setting. Here, we developed a small molecule ratiometric fluorescent chemosensor that accurately measures hCES2 activity in a single-step within complex mixtures. Our chemosensor is highly selective for hCES2 over hCES1, cell permeable and can measure hCES2 activity in pancreatic cancer patient-derived xenografts. Given the simplicity, accuracy and tissue compatibility of our assay, we anticipate our chemosensor can be used to predict patient response to Irinotecan-based therapy.

Pancreatic Ductal Adenocarcinoma (PDA) is an aggressive malignancy with a very poor outcome. Although chemotherapy (CT) treatment has poor efficacy, it can enhance tumor immunogenicity. Tumor-Associated Antigens (TAA) are self-proteins that are overexpressed in tumors that may induce antibody production and can be PDA theranostic targets. However, the prognostic value of TAA-antibody association as Circulating Immune Complexes (CIC) has not yet been elucidated, mainly due to the lack of techniques that lead to their identification. In this study, we show a novel method to separate IgG, IgM, and IgA CIC from sera to use them as prognostic biomarkers of CT response. The PDA Immune-Complexome (IC) was identified using a LTQ-Orbitrap mass spectrometer followed by computational analysis. The analysis of the IC of 37 PDA patients before and after CT revealed differential associated antigens (DAA) for each immunoglobulin class. Our method identified different PDA-specific CIC in patients that were associated with poor prognosis patients. Finally, CIC levels were significantly modified by CT suggesting that they can be used as effective prognostic biomarkers to follow CT response in PDA patients.

Background We applied a training and testing approach to develop and validate a plasma metabolite panel for the detection of early-stage pancreatic ductal adenocarcinoma (PDAC) alone and in combination with a previously validated protein panel for early-stage PDAC. Methods A comprehensive metabolomics platform was initially applied to plasmas collected from 20 PDAC cases and 80 controls. Candidate markers were filtered based on a second independent cohort that included nine invasive intraductal papillary mucinous neoplasm cases and 51 benign pancreatic cysts. Blinded validation of the resulting metabolite panel was performed in an independent test cohort consisting of 39 resectable PDAC cases and 82 matched healthy controls. The additive value of combining the metabolite panel with a previously validated protein panel was evaluated. Results Five metabolites (acetylspermidine, diacetylspermine, an indole-derivative, and two lysophosphatidylcholines) were selected as a panel based on filtering criteria. A combination rule was developed for distinguishing between PDAC and healthy controls using the Training Set. In the blinded validation study with early-stage PDAC samples and controls, the five metabolites yielded areas under the curve (AUCs) ranging from 0.726 to 0.842, and the combined metabolite model yielded an AUC of 0.892 (95% confidence interval [CI] = 0.828 to 0.956). Performance was further statistically significantly improved by combining the metabolite panel with a previously validated protein marker panel consisting of CA 19–9, LRG1, and TIMP1 (AUC = 0.924, 95% CI = 0.864 to 0.983, comparison DeLong test one-sided P= .02). Conclusions A metabolite panel in combination with CA19-9, TIMP1, and LRG1 exhibited substantially improved performance in the detection of early-stage PDAC compared with a protein panel alone.

Background Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal ‘surfaceome' in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling. Patients and methods Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography–mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis. Results Proteomic analysis of the exosome ‘surfaceome' revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation. Conclusion Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA.

Exosomes and other extracellular vesicles (EVs) have been implicated as mediators of intercellular communication. Their release into the circulation has the potential to inform about tumor status. In-depth proteomic characterization of plasma-derived EVs has been limited by challenges in isolating EVs from protein-abundant biological fluids. We implemented a novel single-step density gradient flotation workflow for efficient and rapid isolation of highly enriched circulating EVs from plasma. Mass-spectrometry analysis of plasma EVs from subjects with lung adenocarcinoma and matched controls resulted in the identification of 640 proteins. A total of 108 proteins exhibited significant (p

The yellow-legged hornetVespavelutinais an invasive alien species in many areas of the world. In Europe, it is considered a species of Union concern and national authorities have to establish surveillance plans, early warning and rapid response systems or control plans. These strategies customarily require the assessment of the areas that could be colonised beyond outbreaks or expanding ranges, so as to establish efficient containment protocols. The hornet is spreading through a mix of natural diffusion and human-mediated transportation. Despite the latter dispersion mode is hardly predictable, natural diffusion could be modelled from nest data of consecutive years. The aim of this work is to develop a procedure to predict the spread of the yellow-legged hornet in the short term in order to increase the efficiency of control plans to restrain the diffusion of this species. We used data on the mean distances of colonial nests between years to evaluate the probability of yellow-legged hornet dispersal around the areas where the species is present. The distribution of nests in Italy was mainly explained by elevation (95% of nests located within 521 m a.s.l.) and distance from source sites (previous years’ colonies; 95% within 1.4–6.2 km). The diffusion models developed with these two variables forecast, with good accuracy, the spread of the species in the short term: 98–100% of nests were found within the predicted area of expansion. A similar approach can be applied in areas invaded by the yellow-legged hornet, in particular beyond new outbreaks and over the border of its expanding range, to implement strategies for its containment. The spatial application of the models allows the establishment of buffer areas where monitoring and control efforts can be allocated on the basis of the likelihood of the species spreading at progressively greater distances.

We investigated the potential of in-depth quantitative plasma proteome analysis to uncover proteins predictive of progression and metastasis in triple negative breast cancer (TNBC). Analysis of samples from 24 pre-menopausal and 24 post-menopausal women with newly diagnosed TNBC who subsequently developed metastasis or remained metastasis free were utilized in the proteomic discovery set, which resulted in 43 proteins associated with tumor progression. These proteins were found to form a hierarchical network with TGFβ. The signature was further confirmed and refined by integrating plasma protein data from a murine TNBC model that encompassed mice with rapid- versus slow-growing tumors. Three genes consisting of CLIC1, MAPRE1, and SERPINA3 in the refined TGFβ signature significantly stratified overall survival (log-rank p = 0.0141) in a larger validation cohort irrespective of menopausal status, tumor stage, grade, and size.

Using a combination of mass-spectrometry and aptamer array-based proteomics, we characterized the protein features of circulating extracellular vesicles (EVs) in the context of lung (LUAD) and pancreatic ductal (PDAC) adenocarcinomas. We profiled EVs isolated from conditioned media of LUAD and PDAC cell lines to identify EV-associated protein cargoes released by these cancer cell types. Analysis of the resulting data identified LUAD and PDAC specific and pan-adenocarcinoma EV protein signatures. Bioinformatic analyses confirmed enrichment of proteins annotated to vesicle-associated processes and intracellular compartments, as well as representation of cancer hallmark functions and processes. Analysis of upstream regulator networks indicated significant enrichment of TP53, MYC, TGFB1 and KRAS-driven network effectors (p = 1.69 &times, 10&minus, 77&ndash, 2.93 &times, 49) manifest in the adenocarcinoma sEV protein cargoes. We extended these findings by profiling the proteome of EVs isolated from lung (N = 15) and pancreatic ductal (N = 6) adenocarcinoma patient plasmas obtained at time of diagnosis, along with EVs derived from matched healthy controls (N = 21). Exploration of these proteomic data revealed abundant protein features in the plasma EVs with capacity to distinguish LUAD and PDAC cases from controls, including features yielding higher performance in the plasma EV isolates relative to unfractionated plasmas.

In the last 5 years, novel knowledge on tumor metabolism has been revealed with the identification of critical factors that fuel tumors. Alpha-enolase (ENO1) is commonly over-expressed in tumors and is a clinically relevant candidate molecular target for immunotherapy. Here, we silenced ENO1 in human cancer cell lines and evaluated its impact through proteomic, biochemical and functional approaches. ENO1 silencing increased reactive oxygen species that were mainly generated through the sorbitol and NADPH oxidase pathways, as well as autophagy and catabolic pathway adaptations, which together affect cancer cell growth and induce senescence. These findings represent the first comprehensive metabolic analysis following ENO1 silencing. Inhibition of ENO1, either alone, or in combination with other pathways which were perturbed by ENO1 silencing, opens novel avenues for future therapeutic approaches.

The repertoire of antigens associated with the development of an autoimmune response in breast cancer has relevance to detection and treatment strategies. We have investigated the occurrence of autoantibodies associated with the development of triple-negative breast cancer (TNBC) in the before diagnosis setting and in samples collected at the time of diagnosis of TNBC. Lysate arrays containing protein fractions from the TNBC MDA-MB-231 cell line were hybridized with TNBC plasmas from the Women's Health Initiative cohort, collected before clinical diagnosis and with plasmas from matched controls. An immune response directed against spliceosome and glycolysis proteins was observed with case plasmas as previously reported in estrogen receptor+ breast cancer. Importantly, autoantibodies directed against networks involving BRCA1, TP53, and cytokeratin proteins associated with a mesenchymal/basal phenotype were distinct to TNBC before diagnosis samples. Concordant autoantibody findings were observed with mouse plasma samples collected before occurrence of palpable tumors from a C3(1)-T triple negative mouse model. Plasma samples collected at the time of diagnosis of stage II TNBC and from matched healthy controls were subjected to proteomic analysis by mass spectrometry to identify Ig-bound proteins yielding a predominance of cytokeratins, including several associated with a mesenchymal/basal phenotype among cases compared with controls. Our data provide evidence indicative of a dynamic repertoire of antigens associated with a humoral immune response reflecting disease pathogenesis in TNBC. Cancer Res; 75(16); 3246–54. ©2015 AACR.

Novel synthetic peptides represent smart molecules for antigen antibody interactions in several bioanalytics applications, from purification to serum screening. Their immobilization onto a solid phase is considered a key point for sensitivity increasing. In this view, we exploited Quartz Crystal Microbalance with simultaneous frequency and dissipation monitoring (QCM-D) with a double aim, specifically, as investigative tool for spacers monolayer assembling and its functional evaluation, as well as high sensitive method for specific immunosorbent assays. The method was applied to pancreatic ductal adenocarcinoma (PDAC) detection by studying the interactions between synthetic phosphorylated and un-phosphorylated alpha-enolase peptides with sera of healthy and PDAC patients. The synthetic peptides were immobilized on the gold surface of the QCM-D sensor via a self-assembled alkanethiol monolayer. The presented experimental results can be applied to the development of surfaces less sensitive to non-specific interactions with the final target to suggest specific protocols for detecting PDAC markers with un-labeled biosensors.

L-lactate dehydrogenase (LDH) converts pyruvate to lactate when oxygen is absent or in short supply, and the enzyme plays a crucial role in cancer metabolism. The functions of many mammalian proteins are modulated by posttranslational modifications (PTMs), and it has been reported that LDH was subjected to several PTMs, including phosphorylation, acetylation, and methylation. In this present work, we characterized the PTMs of LDH from pancreatic ductal adenocarcinoma (PDAC) cells by electrophoresis and mass spectrometry, and identified 13 O-methylated residues from the enzyme. In addition, our qualitative analysis revealed differential methylation of LDH from normal duct cells. The preliminary findings from this study provide important biochemical information toward further understanding of the LDH modifications and their functional significance in pathophysiological processes of pancreatic cancer.

In this present work, we characterized the proteomes of pancreatic ductal adenocarcinoma (PDAC) cells and normal pancreatic duct cells by mass spectrometry using LTQ: Orbitrap, and identified more than 1200 proteins from each sample. On the basis of spectra count label-free quantification approach, we identified a large number of differentially expressed metabolic enzymes and proteins involved in cytoskeleton, cell adhesion, transport, transcription, translation, and cell proliferation as well. The data demonstrated that metabolic pathways were altered in PDAC, consistent with Warburg effect. Our analysis provides a potentially comprehensive picture of metabolism in PDAC, which may serve as the basis of new diagnostic and treatment of PDAC.

α-enolase (ENOA) is a metabolic enzyme involved in the synthesis of pyruvate. It also acts as a plasminogen receptor and thus mediates activation of plasmin and extracellular matrix degradation. In tumor cells, EΝΟΑ is upregulated and supports anaerobic proliferation (Warburg effect), it is expressed at the cell surface, where it promotes cancer invasion, and is subjected to a specific array of post-translational modifications, namely acetylation, methylation and phosphorylation. Both ENOA overexpression and its post-translational modifications could be of diagnostic and prognostic value in cancer. This review will discuss recent information on the biochemical, proteomics and immunological characterization of ENOA, particularly its ability to trigger a specific humoral and cellular immune response. In our opinion, this information can pave the way for effective new therapeutic and diagnostic strategies to counteract the growth of the most aggressive human disease.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and no diagnostic markers have, as of yet, been defined. In PDAC patients, α-enolase (ENOA) is up-regulated and elicits the production of autoantibodies. Here, we analyzed the autoantibody response to post-translational modifications of ENOA in PDAC patients. ENOA isolated from PDAC tissues and cell lines was characterized by two-dimensional electrophoresis (2-DE) Western blot (WB), revealing the expression of six different isoforms (named ENOA1,2,3,4,5,6) whereas only 4 isoforms (ENOA3,4,5,6) were detectable in normal tissues. As assessed by 2-DE WB, 62% of PDAC patients produced autoantibodies to the two more acidic isoforms (ENOA1,2) as opposed to only 4% of controls. Mass spectrometry showed that ENOA1,2 isoforms were phosphorylated on serine 419. ROC analysis demonstrated that autoantibodies to ENOA1,2 usefully complement the diagnostic performance of serum CA19.9 levels, achieving approximately 95% diagnostic accuracy in both advanced and resectable PDAC. Moreover, the presence of autoantibodies against ENOA1,2 correlated with a significantly better clinical outcome in advanced patients treated with standard chemotherapy. In conclusion, our results demonstrate that ENOA phosphorylation is associated with PDAC and induces specific autoantibody production in PDAC patients that may have diagnostic value.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality in both men and women in the United States (1). Alarmingly, the incidence of PDAC-related mortality continues to rise and PDAC is projected to be the second deadliest cancer in the U.S. by 2030 (2).

// Valentina Griggio 1, 2 , Giorgia Mandili 2, 3 , Candida Vitale 1, 2 , Michela Capello 2, 3 , Paolo Macor 4 , Sara Serra 5 , Barbara Castella 1, 2, 3 , Silvia Peola 2, 3 , Myriam Foglietta 1, 2, 3 , Daniela Drandi 1, 2 , Paola Omede 1 , Daniele Sblattero 4 , Paola Cappello 2, 3, 6 , Roberto Chiarle 2, 3, 7 , Silvia Deaglio 5 , Mario Boccadoro 1, 2 , Francesco Novelli 2, 3, 6, 8 , Massimo Massaia 1, 2, 3 , Marta Coscia 1, 2 1 Division of Hematology, University of Torino, AOU Citta della Salute e della Scienza di Torino, Torino, Italy 2 Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy 3 Center for Experimental Research and Medical Studies (CeRMS), AOU Citta della Salute e della Scienza di Torino, Torino, Italy 4 Department of Life Sciences – University of Trieste, Trieste, Italy 5 Department of Medical Sciences, University of Torino and Immunogenetics Unit - Human Genetics Foundation (HuGeF), Torino, Italy 6 Molecular Biotechnology Center, Torino, Italy 7 Department of Pathology, Boston Children’s Hospital, Boston, Massachusetts, USA 8 Service of Immunogenetics and Transplantation, AOU Citta della Salute e della Scienza di Torino, Torino, Italy Correspondence to: Marta Coscia, email: marta.coscia@unito.it Keywords: chronic lymphocytic leukemia, humoral responses, tumor antigens, serological proteomics, alpha-enolase Received: July 13, 2016 Accepted: November 22, 2016 Published: November 30, 2016 ABSTRACT In chronic lymphocytic leukemia (CLL) the occurrence and the impact of antibody responses toward tumor-derived antigens are largely unexplored. Our serological proteomic data show that antibodies toward 47 identified antigens are detectable in 29 out of 35 patients (83%) with untreated CLL. The glycolytic enzyme alpha-enolase (ENO1) is the most frequently recognized antigen (i.e. 54% of CLL sera). We show that ENO1 is upregulated in the proliferating B-cell fraction of CLL lymph nodes. In CLL cells of the peripheral blood, ENO1 is exclusively expressed at the intracellular level, whereas it is exposed on the surface of apoptotic leukemic cells. From the clinical standpoint, patients with progressive CLL show a higher number of antigen recognitions compared to patients with stable disease. Consistently, the anti-ENO1 antibodies are prevalent in sera from patients with progressive disease and their presence is predictive of a shorter time to first treatment. This clinical inefficacy associates with the inability of patients’ sera to trigger complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity against leukemic cells. Together, these results indicate that antibody responses toward tumor-derived antigens are frequently detectable in sera from patients with CLL, but they are expression of a disrupted immune system and unable to hamper disease progression.

Objectives MAP4K5 plays an important role in regulating a range of cellular responses and is involved in Wnt signaling in hematopoietic cells. However, its functions in human malignancies have not been studied. The major objectives of this study are to examine the expression, functions and clinical significance of MAP4K5 in pancreatic ductal adenocarcinoma (PDAC). Materials and Methods The expression levels of MAP4K5, E-cadherin, vimentin, and carboxylesterase 2 (CES2) were examined by immunohistochemistry in 105 PDAC and matched non-neoplastic pancreas samples from our institution. The RNA sequencing data of 112 PDAC patients were downloaded from the TCGA data portal. Immunoblotting and RNA sequencing analysis were used to examine the expression of MAP4K5 and E-cadherin in pancreatic cancer cell lines. The effect of knockdown MAP4K5 using siRNA on the expression of CDH1 and vimentin were examined by Real-time RT-PCR in Panc-1 and AsPC-1 cells. Statistical analyses were performed using IBM SPSS Statistics. Results MAP4K5 protein is expressed at high levels specifically in the pancreatic ductal cells of 100% non-neoplastic pancreas samples, but is decreased or lost in 77.1% (81/105) of PDAC samples. MAP4K5-low correlated with the loss of E-cadherin (P = 0.001) and reduced CES2 expression (P = 0.002) in our patient populations. The expression levels of MAP4K5 mRNA directly correlated with the expression levels of CDH1 mRNA (R = 0.2490, P = 0.008) in the second cohort of 112 PDAC patients from The Cancer Genome Atlas (TCGA) RNA-seq dataset. Similar correlations between the expression of MAP4K5 and E-cadherin were observed both at protein and mRNA levels in multiple pancreatic cancer cell lines. Knockdown MAP4K5 led to decreased CDH1 mRNA expression in Panc-1 and AsPC-1 cells. MAP4K5-low correlated significantly with reduced overall survival and was an independent prognosticator in patients with stage II PDAC. Conclusions MAP4K5 expression is decreased or lost in majority of PDACs. The strong associations between low MAP4K5 expression and loss of E-cadherin, reduced CES2 expression and decreased overall survival may suggest an important role of MAP4K5 in epithelial-to-mesenchymal transition, chemotherapy resistance and tumor progression in pancreatic cancer. Targeting impaired MAP4K5 signaling may represent a new therapeutic strategy for pancreatic cancer.

BACKGROUND Serine hydrolases (SHs) are among the largest classes of enzymes in humans and play crucial role in many pathophysiological processes of cancer. We have undertaken a comprehensive proteomic analysis to assess the differential expression and cellular localization of SHs, which uncovered distinctive expression of Carboxylesterase 2 (CES2), the most efficient carboxyl esterase in activating the prodrug irinotecan into SN-38, in pancreatic ductal adenocarcinoma (PDAC). We therefore assessed the extent of heterogeneity in CES2 expression in PDAC and its potential relevance to irinotecan based therapy. METHODS CES2 expression in PDAC and paired nontumor tissues was evaluated by immunohistochemistry. CES2 activity was assessed by monitoring the hydrolysis of the substrate p-NPA and correlated with irinotecan IC50 values by means of Pearson's correlation. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and CES2 expression in patients who underwent neoadjuvant FOLFIRINOX treatment. All statistical tests were two-sided. RESULTS Statistically significant overexpression of CES2, both at the mRNA and protein levels, was observed in PDAC compared with paired nontumor tissue (P < .001), with 48 of 118 (40.7%) tumors exhibiting high CES2 expression. CES2 activity in 11 PDAC cell lines was inversely correlated with irinotecan IC50 values (R = -0.68, P = .02). High CES2 expression in tumor tissue was associated with longer overall survival in resectable and borderline resectable patients who underwent neoadjuvant FOLFIRINOX treatment (hazard ratio = 0.14, 95% confidence interval = 0.04 to 0.51, P = .02). CONCLUSION Our findings suggest that CES2 expression and activity, by mediating the intratumoral activation of irinotecan, is a contributor to FOLFIRINOX sensitivity in pancreatic cancer and CES2 assessment may define a subset of patients likely to respond to irinotecan based therapy.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth cause of cancer-induced death in the Western World. In PDAC patients, alpha-enolase (ENOA), a glycolytic enzyme that also acts as plasminogen receptor, is up-regulated and elicits the production of autoantibodies. Our previous studies revealed that most PDAC patients specifically produce antibodies to Serine(419)phosphorylated ENOA (Ser(419)P-ENOA) isoforms (ENOA1,2), and that this humoral response correlates with a better clinical outcome. Since autoantibody production can be influenced by HLA polymorphisms, and the ENOA sequence presents multiple peptides predicted to preferentially bind HLA-DR molecules, including the peptide containing Ser(419), we hypothesized that the presence of autoantibodies against ENOA1,2 is associated with specific HLA-DRB1 alleles. Here, we demonstrate that the HLA-DRB1*08 allele is significantly more frequent in PDAC patients with autoantibodies to ENOA1,2 (ENOA1,2(+), 8%) compared to healthy controls (3%, p=0.0112). We observed that a Ser(419)P-ENOA peptide, bioinformatically predicted to bind with high affinity to the HLA-DR8 allele coded by HLA-DRB1*08:01 or *08:04 alleles, was able to activate specific CD4(+) T cell clones derived from a HLA-DRB1*08:01. Thus complexes of the Ser(419)P-ENOA peptide with the HLA that trigger T-cell signaling might be relevant for induction of anti-tumor immune response.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers in Europe and the United States. It has a very low 5 years-survival rate and its diagnosis is often late and imprecise due to the lack of specificity of currently used markers for PDAC. As previously demonstrated PDAC patients’ sera may contain autoantibodies towards phosphorylated a-enolase (ENOA), which in combination with other standard markers can increase specificity in diagnosis of PDAC. In this context we realized a microfluidic platform with integrated EIS biosensors. We achieved a specific antibodies detection by immobilizing onto electrodes peptides corresponding to a portion of ENOA. Phosphorylation of peptides was found to influence the recognition of antibodies in PDAC patients’ sera detected by the developed biochip thus validating the EIS technique as a strong tool for quick, cost-saving and label-free analysis of serum samples. Biochip results are in agreement with those from traditional techniques, such as ELISA and western blot, but measurements are much more sensitive and specific, increasing the possibility of PDAC diagnosis. In addition this approach is faster and more reproducible compared to traditional techniques making the developed biochips ideal for a quick, cost-saving and label-free analysis of serum samples.

Pancreatic ductal adenocarcinoma (PDA), an aggressively invasive, treatment-resistant malignancy, is usually detectable only when already inevitably fatal. Despite advances in genetic screening, mapping and molecular characterization, its pathology remains largely elusive. Renewed interest in longstanding doctrines of tumor metabolism has led to the emergence of aberrant signaling pathways as critical factors modulating central metabolic networks that fuel pancreatic tumors. We have previously described α-enolase (ENO1) as a PDA-associated antigen. It is a moonlighting protein that works both as a key metabolic enzyme and a membrane plasminogen receptor. To better characterize ENO1 metabolic and fuelling role in pancreatic cancer, we have silenced ENO1 in three different human PDA cell lines (CFPAC-1, PT45 and T3M4) and evaluated its impact through proteomic, biochemical and functional approaches. Protein expression alterations following ENO1 knockdown were revealed by LC-MS/MS analysis. On the basis of a spectra count label-free quantitation approach several proteins mainly involved in cell adhesion, metabolism and proliferation were found to be differentially expressed in ENO1-silenced cells compared to the control. Indeed, ENO1-silenced PDA cells displayed a delay in proliferation, decreased survival and colony formation capabilities. The cell-cycle profile analysis revealed a strong increase in the number of PDA cells in G2/M phase, a concomitant decrease in G1 phase and no difference in the proportion of cells in S phase after ENO1 silencing as compared to control cells. Moreover, ENO1-silenced cells showed specific morphological changes that were indicative of cellular senescence, as confirmed by an increase in β-galactosidase staining. Of note, ENO1 knockdown PDA cells grew significantly less compared to control cells when injected sub cute in SCID-beige mice. The growth inhibition was partially due to an increased concentration of intracellular reactive oxygen species (ROS) mainly generated through the sorbitol and NADPH oxidase pathways. ENO1 knockdown increase autophagy, the most important stress response for cells to adapt to nutrient starvation and promotes also catabolic pathway adaptations that restore pyruvate and acetyl-CoA bulk. Furthermore, the increased entry of glutamine into the TCA cycle induce a drop in nucleotide bases synthesis and promote oxidative phosphorylation in PDA cells, switching to the aerobic glycolysis typical of cancer cells. These findings may have implications for future therapeutic approaches: the inhibition of ENO1, in fact, can potentially synergize with therapies targeting autophagy and glutamine pathway. Citation Format: Michela Capello, Sammy Ferri-Borgogno, Moitza Principe, Michelle Samuel Chattaragada, Chiara Riganti, Weidong Zhou, Laura Follia, Lance A. Liotta, Emanuel F. Petricoin, III, Paola Cappello, Francesco Novelli. Alpha-enolase knockdown reprograms metabolism and points out targetable pathways to counteract PDA growth. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B49.

Activation of plasminogen on the cell surface initiates a cascade of protease activity with important implications for several physiological and pathological events. In particular, components of the plasminogen system participate in tumor growth, invasion and metastasis. Plasminogen receptors are in fact expressed on the cell surface of most tumors, and their expression frequently correlates with cancer diagnosis, survival and prognosis. Notably, they can trigger multiple specific immune responses in cancer patients, highlighting their role as tumor-associated antigens. In this review, three of the most characterized plasminogen receptors involved in tumorigenesis, namely Annexin 2 (ANX2), Cytokeratin 8 (CK8) and alpha-Enolase (ENOA), are analyzed to ascertain an overall view of their role in the most common cancers. This analysis emphasizes the possibility of delineating new personalized therapeutic strategies to counteract tumor growth and metastasis by targeting plasminogen receptors, as well as their potential application as cancer predictors.

Pyruvate kinase (PK) is an important glycolytic enzyme that catalyzes the dephosphorylation of phospho-enolpyruvate to pyruvate. Human PK isozyme M2 (PKM2), a splice variant of M1, is overexpressed in many cancer cells, and PKM2 has been investigated as a potential tumor marker for diagnostic assays and as a target for cancer therapy. To facilitate identification and characterization of PK, we studied the enzyme from pancreatic cancer cells and normal pancreatic duct cells by electrophoresis and mass spectrometry, and identified multiple O-methylated residues from PK. These findings advance our knowledge of the biochemical properties of PK and will be important in understanding its biological function in cells.

Background: CA19-9, which is currently in clinical use as a pancreatic ductal adenocarcinoma (PDAC) biomarker, has limited performance in detecting early-stage disease. We and others have identified protein biomarker candidates that have the potential to complement CA19-9. We have carried out sequential validations starting with 17 protein biomarker candidates to determine which markers and marker combination would improve detection of early-stage disease compared with CA19-9 alone. Methods: Candidate biomarkers were subjected to enzyme-linked immunosorbent assay based sequential validation using independent multiple sample cohorts consisting of PDAC cases (n = 187), benign pancreatic disease (n = 93), and healthy controls (n = 169). A biomarker panel for early-stage PDAC was developed based on a logistic regression model. All statistical tests for the results presented below were one-sided. Results: Six out of the 17 biomarker candidates and CA19-9 were validated in a sample set consisting of 75 PDAC patients, 27 healthy subjects, and 19 chronic pancreatitis patients. A second independent set of 73 early-stage PDAC patients, 60 healthy subjects, and 74 benign pancreatic disease patients (combined validation set) yielded a model that consisted of TIMP1, LRG1, and CA19-9. Additional blinded testing of the model was done using an independent set of plasma samples from 39 resectable PDAC patients and 82 matched healthy subjects (test set). The model yielded areas under the curve (AUCs) of 0.949 (95% confidence interval [CI] = 0.917 to 0.981) and 0.887 (95% CI = 0.817 to 0.957) with sensitivities of 0.849 and 0.667 at 95% specificity in discriminating early-stage PDAC vs healthy subjects in the combined validation and test sets, respectively. The performance of the biomarker panel was statistically significantly improved compared with CA19-9 alone (P < .001, combined validation set; P = .008, test set). Conclusion: The addition of TIMP1 and LRG1 immunoassays to CA19-9 statistically significantly improves the detection of early-stage PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. By using a proteome approach, we have found that protein recognized with the highest frequency by autoantibodies in PDAC sera was the alpha-enolase (ENO1), a glycolytic enzyme that also acts as a plasminogen-binding receptor. We have found that either ENO1 silencing or treatment of the PDAC cells with single administration of Adeno-Associated Virus-expressing monoclonal antibody against human ENO1 inhibited lung metastasis in immunosuppressed mice injected with PDAC cells. In addition, PDAC ENO1-silenced cells displayed a down modulated expression of cell surface molecules that regulate the adhesion to the extracellular matrix as confirmed by their reduced binding to laminin, collagen and fibronectin. ENO1 silencing in PDAC cells increased reactive oxygen species mainly generated through the sorbitol and NADPH oxidase pathways, as well as autophagy and catabolic pathway adaptations, which together affect PDAC cell growth and induced senescence. This data indicated that ENO1 plays a critical role in PDAC progression and invasion. ENO1 possesses interesting antigenic properties as it is able to elicit T cell proliferation, activation and cytotoxic specific T lymphocytes (CTL) differentiation both in vitro and in vivo. Tumor infiltrating T lymphocytes specific for ENO1 have been identified in PDAC patients and the number of ENO1-specific T cell clones generated from peripheral blood of PDAC positively correlated with a better survival. Antibody and T cell responses to ENO1 are increase in PDAC patient underwent chemotherapy. DNA vaccination to ENO1 elicits an integrated humoral and cellular immune response that was accompanied by a reduced and regulatory T cells and MDSC that significantly extends survival of Genetically Engineered Mice that spontaneously develop PDAC. Notably, mouse anti-human ENO1 monoclonal antibody inhibits MDSC adhesion to pancreatic endothelial cells and in vitro and in vivo migration and decreases MDSC arginase activity and secretion of IL-6. As whole these data demonstrate that the self-antigen ENO1 is a promising target suitable for therapeutic purposes in PDAC. In particular, the antibodies anti-ENO1 may inhibit PDA cell and myeloid cell invasion and positively modulate T cell response, making the immunotherapy more effective. Citation Format: Francesco Novelli, Michela Capello, Michelle Chattaragada, Sammy Ferri Borgogno, Giorgia Mandili, Moitza Principe, Emanuela Mazza, Sara Bulfamante, Roberta Curto, Daniele Giordano, Paola Cappello. Targeting of alpha-enolase (ENO1) as a novel immunotherapeutical strategy for pancreatic cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A042.

A new class of intercellular communication has recently emerged that involves transfer of exosomes, especially during the interactions between tumor and the immune system. However, the advantage of exosome-mediated cell-cell communication over direct cell to cell communication mechanisms or through soluble factors is unclear. We have performed mass spectrometry based proteomic profiling of exosomes isolated from pancreatic cancer cell lines, for the exosome cell surface and for the protein cargo. The resulting profiles were compared with proteomic profiles of corresponding cell lysates. We uncovered substantial enrichment in protein pairs in exosomes across cell lines but not in cell lysates, suggestive of a mechanism of exosome-based protein sorting and packaging that favor specific protein combinations. Stochastic simulations and analytical analysis provide supportive evidence that exosomes enable recipient cells to rewire gene regulatory networks to induce robust phenotypic transitions among different gene states. Moreover, from the simulation of two-way exosome-mediated communication of two cell types, we show that cells could achieve synchronized decision making through exosomes. Our integrative approach provides new insights into unique features of exosome-mediated cell-cell communication. Citation Format: Mingyang Lu, Michela Capello, Satyendra C. Tripathi, Herbert Levine, Eshel Ben-Jacob, Samir M. Hanash, José Onuchic. The role of exosome-mediated cell-cell communication in inducing phenotypic changes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 779.

Purpose: CA19-9, which is currently in clinical use as a pancreatic ductal adenocarcinoma (PDAC) biomarker, has limited performance in detecting early stage disease. We and others have identified protein biomarker candidates that have the potential to complement CA19.9. We have carried out sequential validations starting with 32 protein biomarker candidates to develop a marker panel to improve detection of early stage disease compared with CA19.9 alone. Patients and Methods: Multiple independent sample sets consisting of PDAC cases, benign pancreatic disease and healthy controls were utilized for immunoassays of candidate markers which after sequential validations yielded a panel of three markers that was further tested in a blinded setting. Results: The 32 biomarker candidates were initially tested in a sample set consisting of 75 PDAC cases, 29 healthy controls, and 19 chronic pancreatitis cases which narrowed the list to seven biomarker candidates. A second independent set of 73 early-stage PDAC cases, 60 healthy controls, and 74 benign pancreatic disease cases (60 chronic pancreatitis cases and 14 benign pancreatic cyst cases) yielded a panel of three markers, including CA19-9, with an AUC (95%CI) of 0.949 (0.917-0.981) and sensitivity of 0.849 at 95% specificity. Additional blinded testing of the model was done using an independent set of plasma samples from 39 resectable PDAC patients and 82 matched healthy controls, yielding an AUC of 0.887. The performance of the biomarker panel was significantly improved compared to CA19-9 alone (P < 0.001) in both plasma sample sets. Conclusion: We validated a protein-based three marker panel with significantly improved performance compared to CA19-9 with the potential to contribute to strategies for detecting early-stage PDAC. Citation Format: Michela Capello, Leonidas E. Bantis, Ghislaine Scelo, Yang Zhao, Dilsher S. Dhillon, Hong Wang, James L. Abbruzzese, Anirban Maitra, Margaret A. Tempero, Randall Brand, Matthew A. Firpo, Sean Mulvihill, Matthew H. Katz, Paul Brennan, Ziding Feng, Samir M. Hanash, Ayumu Taguchi. Sequential validation of blood-based protein biomarker candidates for early-stage pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-134.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. Alpha-enolase (ENOA) is a key glycolytic enzyme that is also expressed on the surface of immune and tumor cells where it acts as a plasminogen receptor. In our laboratory it has been demonstrated that ENOA is expressed on the surface of several PDAC cell lines where is crucial for plasminogen-dependent invasion. A monoclonal antibody against ENOA inhibits this effect both in vitro and in vivo (unpublished data). Aim: We investigated the effect of ENOA silencing in PDAC cell line in order to identify key proteins that are different modulated and clarify its multifunctional role in pancreatic tumorigenesis. Methods: CF-PAC-1 human PDAC cell line was infected with a lentiviral vector delivering a short hairpin RNA (shRNA) targeting ENOA 3’UTR or a scrambled shRNA as a control. After silencing, ENOA mRNA was evaluated by real-time PCR and protein level was assessed by and Western Blot Analysis in each subcellular fraction (cytosolic, membrane and nuclear protein). Proliferation was monitored by cell counting and MTT assays. The cell cycle analysis was performed by cytofluorimetry. A pyruvate assay kit was used to determinate pyruvate production. The tumorigenicity of ENOA silenced cells was assessed by a colony forming assay and their invasive capability was tested by a transwell matrigel invasion assay. Adhesion capability of the cells was evaluated by plate fixing and cell counting. Finally the proteomes of ENOA silenced and control (scrambled or normal CF-PAC-1) cells were compared by LTQ-Orbitrap tandem mass spectrometry analysis and spectra count label-free quantification approach. Results: After lentiviral infection, ENOA was silenced both at the transcript and at the protein level by around 80%, and the silencing was evident in all the cellular fractions. Compared to the control cells, ENOA silenced cells displayed a delay in proliferation and a decreased survival capability, even if the pyruvate production was not affected. This effect was due to a partially block of cell cycle in G2/M phase and not to an apoptotic events. In silenced cells, the capability to form colonies in soft agar was reduced to 40-50%, the invasion in response to plasminogen was abolished, and the adhesive capability decreased to 40%. Finally, more than 1500 proteins were identified from each sample by tandem mass spectrometry analysis and a large number of proteins were differentially expressed in ENOA silenced cells compared to control cells. These proteins were mainly involved in cell adhesion, cell metabolism and cell proliferation and mRNA analysis revealed that the most significantly downregulated proteins (cell-cell and cell-matrix adhesion regulators) were controlled either at the transcriptional or post-translational levels. Conclusions: Together, this data confirm that ENOA in PDAC is not only a glycolytic enzyme, but is a key regulator of a broad spectrum of pathways involved in tumorigenesis. The mechanisms through which ENOA controls cell adhesion, proliferation and metabolism in PDAC are currently under investigation. Citation Format: Moitza Principe, Michela Capello, Michelle Samuel Chattaragada, Weidong Zhou, Lance Liotta, Emanuel Petricoin, Francesco Novelli. Effects of alpha-enolase silencing in adenocarcinoma pancreatic cell line. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A65.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness and resistance to treatments. Biomarkers for its early detection are lacking. In this study we used a proteomic-based approach to identify overexpressed and/or aberrantly modified antigens that could have diagnostic value by inducing an autoantibody response in PDAC. By using SERological Proteome Analysis (SERPA), we have identified nineteen tumor-associated antigens (TAAs) that were specifically recognized by IgG from PDAC patients. These autoantibodies were directed, with high frequency, against a number of metabolic enzymes and cytoskeletal proteins, mostly upregulated in PDAC. The protein most frequently recognized was α-enolase (ENOA), a glycolytic enzyme that can localize on the cell surface as a plasminogen receptor. ENOA was expressed on the surface of PDAC cells and elicited T cell proliferation and activation both in vitro and in vivo. Liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) studies have revealed that in PDAC ENOA is subjected to specific post-translational modifications (PTMs). Further characterization studies of the antibody response to ENOA revealed that PDAC patients specifically produce antibodies to ENOA isoforms that are phosphorylated at Serine-419. By exploiting a genetically engineered mouse model (GEM) spontaneously developing PDAC with a well-defined kinetic, we applied the SERPA approach with mouse sera to validate circulating antibodies to TAAs as early diagnostic tools in PDAC. Eight antigens able to induce the production of autoantibodies in pancreatic intraepithelial neoplasia (PanIN) were identified and five of them induced specific IgG also in PDAC patients. A plasma-membrane cytoskeleton linker molecule showed the highest frequency of autoantibodies in GEMs with early disease and in resectable PDAC patients. ROC analysis demonstrated that the combination of autoantibodies to Ser-419-phosphorylated ENOA in association with antibodies to this cytoskeletal protein and CA19.9 serum levels achieved approximately 97% diagnostic accuracy. In conclusion, our results demonstrate that the simultaneous capture of autoantibodies against a panel of pancreatic cancer-associated antigens might have diagnostic value in this tumor. Citation Format: Michela Capello, Michele Milella, Francesco Novelli, Paola Cappello, Sammy Ferri-Borgogno, Weidong Zhou, Giorgia Mandili, Claudia Fredolini, Isabella Sperduti, Federica Linty, Emanuel Petricoin. Autoantibody signature in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A2.

In this present work, we characterized the proteomes of pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1 and normal pancreatic duct cells by mass spectrometry using LTQ-Orbitrap and identified more than 1700 proteins from each sample. On the basis of the spectra count label-free quantification approach, we identified a large number of differentially expressed metabolic enzymes and proteins involved in cytoskeleton, cell adhesion, transport, transcription, translation, and cell proliferation as well. The data demonstrated that metabolic pathways were altered in PANC-1, consistent with the Warburg effect. In addition, the comparative MS analysis unveiled anomalous metabolism of glutamine, suggesting that glutamine was largely consumed as a nitrogen donor in nucleotide and amino acid biosynthesis in PANC-1. Our analysis provides a potentially comprehensive picture of metabolism in PANC-1, which may serve as the basis of new diagnostics and treatment of PDAC.

Abstract 3878 Introduction: In this study, a serological proteome analysis (SERPA) was applied for the first time to identify novel tumor-associated antigens (Ags) capable of eliciting humoral immune responses in patients with chronic lymphocytic leukemia (CLL). SERPA has been demonstrated to be a valuable method to identify tumor associated Ags in several human solid and hematological malignancies. The identification and characterization of circulating antibodies (Abs) and corresponding Ags in CLL can provide useful information to understand cell transformation, predict clinical outcome, and develop immune-based interventions. Methods: SERPA was performed in 21 untreated patients. Proteins extracted from purified CLL cells were separated by 2-D electrophoresis (2-DE) to obtain proteomic maps which were blotted with corresponding sera by Western Blot to reveal Ab-based reactivity with autologous proteins. To verify the CLL specificity of Abs recognition, 7 out of 21 maps were also probed with sera collected from 7 healthy donors (HD). For identification, Ag spots in WB were aligned with proteins in 2-DE maps. The protein spots corresponding to the assigned Ags were excised from the gel, trypsin digested and analyzed by peptide mass fingerprint by MALDITOF Mass Spectrometry (MS) with the software MASCOT. T cells from 6 CLL patients and 3 HD were stimulated with autologous ENOA-pulsed and control dendritic cells (DC) and evaluated by IFNγ ELISPOT assay. Ags surface expression was analyzed by flow cytometry. Statistical correlations were performed using t-test, Mann-Withney rank sum test and χ2-test. Results: Sixteen out of 21 CLL sera (76%) were immunoreactive and produced a total number of 45 Ag spots, whereas HD sera produced only 3 spots (p Statistical correlation analyses showed that immunoreactive CLL patients are characterized by an early stage of disease. Moreover, ENOA-reactive patients have a better preserved immune system because they have higher numbers of CD3+ (p=.02), CD3+/CD4+ (p=.03) and CD3+/CD8+ (p=.05) cells in the peripheral blood than ENOA-unreactive patients. We also investigated the possibility to induce ENOA-specific T-cell immune responses in 6 CLL patients. ENOA-pulsed DC induced IFNγ production in 4/6 patients (66%). The response was ENOA and CLL specific because: 1) it was not induced by unpulsed DC or DC pulsed with an irrelevant protein; 2) it was not induced when T cells from 3 HD were stimulated with autologous ENOA-pulsed DC. Interestingly, ENOA Abs were detectable by SERPA in 3 out of 4 (75%) patients with ENOA-induced T-cell responses, whereas they were undetectable in patients with unresponsive T cells. Correlations with the IGHV mutational status showed that all patients with ENOA-reactive T cells were M. Conclusions: These results indicate that ENOA is able to elicit specific humoral and cellular immune responses suggesting that this protein can be a promising biomarker and a potential target for immunotherapy in CLL. Disclosures: Massaia: Novartis Farma S.p.A: Honoraria, Research Funding.

In this present work, we characterized the phosphoproteomes of pancreatic ductal adenocarcinoma (PDAC) cells and normal pancreatic duct cells by mass spectrometry using LTQ-Orbitrap. We identified more than 700 phosphoproteins from each sample, and revealed differential phosphorylation of many proteins involved in cell adhesion, cell junction, and cytoskeleton. Since post-translational phosphorylation is a common and important mechanism of acute and reversible regulation of protein function in mammalian cells, an understanding of differential phosphorylation of these proteins and resulting signal transduction changes in PDAC will help in comprehending the complex dynamics of tumor invasion and metastasis in pancreatic cancer.

Abstract 917 In this study we applied the serological proteomics-based approach (SERPA) to identify novel tumor associated antigens (TAA) capable of inducing humoral immune responses in patients with chronic lymphocytic leukemia (CLL). Proteins extracted from the leukemic cells isolated from the peripheral blood of 21 untreated CLL patients were separated by 2-DE electrophoresis and transferred onto membranes by electroblotting to obtain 21 2-DE proteomic maps. Each map was subsequently probed with the corresponding autologous serum collected from the same patient. To verify the CLL-specificity of antibodies (Ab) recognition, 7 out of 21 maps obtained from CLL patients were also probed with sera collected from 7 healthy donors (HD). The Western Blot (WB) performed with sera of CLL patients displayed a total of 45 immunoreactive spots. Only 3 antigen spots were detected in HD sera. For identification, antigen spots in WB were aligned with proteins in 2-DE. The protein spots corresponding to the assigned antigens were excised from the gel, destained and subjected to trypsin digestion. The resulting tryptic fragments were analyzed by peptide mass fingerprint by MALDITOF-MS with MASCOT. All the 45 antigen spots were characterized and consisted of 16 different antigens. Sixteen out of 21 CLL sera (76%) showed immunoreactivity against at least 1 of the 16 identified TAA and 69% of these reactive sera recognized from 2 to 6 different antigens. The IGHV mutational status was available in 20 CLL patients and 12 patients were M, while 8 patients were UM. The reactivity rate and number of WB spots were similar in M and UM patients and did not correlate with other parameters of clinical outcome. Sera from 46% CLL patients exhibited immunoreactivity against a protein which was identified by mass spectrometry as α-Enolase (ENOA). Interestingly, ENOA recognition was CLL specific since none of the sera from HD showed reactivity against this protein. The frequency of ENOA recognition was particularly high in M patients. Indeed, ENOA was recognized from sera of 7 out of 12 M patients (59%), but only from sera of 2 out of 8 UM patients (25%). The ability of ENOA to induce antigen-specific T cell responses was assessed. T cells isolated from the PB of a CLL patient with Ab-based ENOA reactivity were stimulated with autologous monocytes-derived ENOA-pulsed dendritic cells (DC). The results showed that CLL-derived ENOA-pulsed DC stimulated autologous T cells to secrete IFN-gamma. This response was ENOA-specific because it was not induced by unpulsed DC or DC pulsed with an irrelevant protein, and also CLL-specific because IFN-gamma release was not induced when T cells from a HD were stimulated with autologous ENOA-pulsed DC. Altogether, these results indicate that ENOA is capable of eliciting CLL-specific humoral and cellular immune responses. Therefore, ENOA can be considered as an alternative and promising biomarker in CLL, as well as a potential target candidate for immunotherapeutic approaches. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia:Novartis: Honoraria, Research Funding.

In T lymphocytes, the internalization of the R2 chain of the IFN-γ receptor (IFN-γR2) prevents the switching-on of pro-apoptotic and anti-proliferative genes induced by the IFN-γ/STAT1 pathway. In fibroblasts, a critical role of controlling the IFN-γR2 internalization is played by the LI 255–256 intracellular motif. Here we show that, in human malignant T cells, the expression of a mutated IFN-γR2 chain in which the LI 255–256 internalization motif is replaced by two alanines (LI 255–256 AA) induces cell surface accumulation of the receptor and reinstates the cell sensitivity to IFN-γ. In comparison with T cells that expressed wild-type IFN-γR2, cells that expressed the mutated receptor displayed, in response to IFN-γ a sustained activation of STAT1. The activation of this signaling pathway leads to higher induction of MHC class I and FasL expression and triggered apoptosis. Malignant ST4 cells transduced with either wild-type or mutated receptor were able to grow in SCID mice, but only the proliferation of T cells expressing the mutated receptor was inhibited by IFN-γ. Finally, lentiviral-mediated transduction of the mutated receptor in T lymphoblasts from healthy donors reinstated their IFN-γ-dependent apoptosis. As a whole, these data indicate that perturbation of IFN-γR2 internalization by mutating the LI 255–256 motif induces a timely coordinated activation of IFN-γ/STAT1 signaling pathways that leads to the apoptosis of T cells.