Long-term analysis of the RiBVD phase II trial reveals the unfavorable impact of TP53 mutations and hypoalbuminemia in older adults with mantle cell lymphoma; for the LYSA group.

Between 2011 and 2012, a phase II trial evaluated the use of the RiBVD (Rituximab, Bendamustine, Velcade and Dexamethasone) combination as first-line treatment for mantle cell lymphoma (MCL) patients aged over 65. We have now re-examined the classic prognostic factors, adding an assessment of the mutation status of TP53. Patients (n=74; median age 73 years) were treated with the RiBVD combination. Median Progression Free Survival (mPFS) was 79 months, and median Overall Survival (mOS) was 111 months. TP53 mutation status was available for 54/74 (73%) patients. TP53 mutations (TP53mt) were found in 12 patients (22.2%). In multivariate analysis, among the prognostic factors (PF) evaluated, only TP53mt and an albumin level below 3.6 g/dL (Alb<3.6 g/dL) were independently associated with a shorter mPFS. A hazard ratio (HR) of 3.16 (1.3-9.9, p=0.014) was obtained for TP53mt versus TP53wt, and 3.6 (1.39-9.5, p=0.009) for Alb<3.6 g/dL vs Alb≥3.6 g/dL. In terms of mOS, multivariate analysis identified three PFs: TP53mt (HR: 5.9 (1.77-19.5, p=0.004)), Alb<3.6 g/dL (HR: 5.2 (1.46-18.5, p=0.011)), and ECOG=2 (HR: 3.7 (1.31-10.6, p=0.014)). Finally, a score combining TP53 status and albumin level distinguished three populations based on the presence of 0, 1, or 2 PF. For these populations, mPFS was 7.8 years, 28 months and 2.5 months, respectively. Our prolonged follow-up confirmed the efficacy of the RiBVD regimen, comparing it favorably to other regimens. TP53mt and hypoalbuminemia emerge as strong PF that can be easily integrated into prognostic scores for older adult patients with MCL.

Enantioselective Approach for Expanding the Three-Dimensional Space of Tetrahydroquinoline to Develop BET Bromodomain Inhibitors.

The pharmaceutical industry has a pervasive need for chiral specific molecules with optimal affinity for their biological targets. However, the mass production of such compounds is currently limited by conventional chemical routes, that are costly and have an environmental impact. Here, we propose an easy access to obtain new tetrahydroquinolines, a motif found in many bioactive compounds, that is rapid and cost effective. Starting from simple raw materials, the procedure uses a proline-catalyzed Mannich reaction followed by the addition of BF3 ⋅ OEt2 , which generates a highly electrophilic aza-ortho-quinone methide intermediate capable of reacting with different nucleophiles to form the diversely functionalized tetrahydroquinoline. Moreover, this enantioselective one-pot process provides access for the first time to tetrahydroquinolines with a cis-2,3 and trans-3,4 configuration. As proof of concept, we demonstrate that a three-step reaction sequence, from simple and inexpensive starting compounds and catalysts, can generate a BD2-selective BET bromodomain inhibitor with anti-inflammatory effect.

CYCLON and NPM1 Cooperate within an Oncogenic Network Predictive of R-CHOP Response in DLBCL.

R-CHOP immuno-chemotherapy significantly improved clinical management of diffuse large B-cell lymphoma (DLBCL). However, 30-40% of DLBCL patients still present a refractory disease or relapse. Most of the prognostic markers identified to date fail to accurately stratify high-risk DLBCL patients. We have previously shown that the nuclear protein CYCLON is associated with DLBCL disease progression and resistance to anti-CD20 immunotherapy in preclinical models. We also recently reported that it also represents a potent predictor of refractory disease and relapse in a retrospective DLBCL cohort. However, only sparse data are available to predict the potential biological role of CYCLON and how it might exert its adverse effects on lymphoma cells. Here, we characterized the protein interaction network of CYCLON, connecting this protein to the nucleolus, RNA processing, MYC signaling and cell cycle progression. Among this network, NPM1, a nucleolar multi-functional protein frequently deregulated in cancer, emerged as another potential target related to treatment resistance in DLBCL. Immunohistochemistry evaluation of CYCLON and NPM1 revealed that their co-expression is strongly related to inferior prognosis in DLBCL. More specifically, alternative sub-cellular localizations of the proteins (extra-nucleolar CYCLON and pan-cellular NPM1) represent independent predictive factors specifically associated to R-CHOP refractory DLBCL patients, which could allow them to be orientated towards risk-adapted or novel targeted therapies.

c-MYC and p53 expression highlight starry-sky pattern as a favourable prognostic feature in R-CHOP-treated diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogeneous entity, in which the first-line treatment currently consists of an immuno-chemotherapy regimen (R-CHOP). However, around 30% of patients will not respond or will relapse. Overexpression of c-MYC or p53 is frequently found in DLBCL, but an association with prognosis remains controversial, as for other biomarkers previously linked with DLBCL aggressivity (CD5, CD23, or BCL2). The aim of this study was to explore the expression of these biomarkers and their correlation with outcome, clinical, or pathological features in a DLBCL cohort. Immunohistochemical (c-MYC, p53, BCL2, CD5, and CD23), morphological ('starry-sky' pattern [SSP]), targeted gene panel sequencing by next-generation sequencing (NGS), and fluorescence in situ hybridisation analyses were performed on tissue microarray blocks for a retrospective cohort of 94 R-CHOP-treated de novo DLBCL. In univariate analyses, p53 overexpression (p53high ) was associated with unfavourable outcome (p = 0.04) and with c-MYC overexpression (p = 0.01), whereas c-MYC overexpression was linked with an SSP (p = 0.004), but only tended towards an inferior prognosis (p = 0.06). Presence of a starry-sky morphology was found to be correlated with better survival in p53high DLBCL (p = 0.03) and/or c-MYC-positive DLBCL (p = 0.002). Furthermore, NGS data revealed that these three variables were associated with somatic mutations (PIM1, TNFRSF14, FOXO1, and B2M) involved in B-cell proliferation, survival, metabolism, and immune signalling. Taken together, these results show that the SSP pattern seems to be a protective factor in high-risk DLBCL subgroups and highlight cell death as a built-in failsafe mechanism to control tumour growth.

Adverse outcome in follicular lymphoma is associated with MYC rearrangements but not MYC extra copies.

Follicular lymphomas (FLs) with MYC rearrangements (MYC-R) and extra copies of MYC (MYC-EC) are rare and the prognosis impact is uncertain. We conducted a retrospective study including 321 FL patients, among whom 259 (81%) had no 8q24 alterations and 62 (19%) were assigned to 8qAlt. Forty-five cases were classified as MYC-EC and six as MYC-R. MYC-R patients were significantly older (P = 0·008), had higher follicular lymphoma international prognostic index (FLIPI) stage (P = 0·05) and ß2-microglobulin (ß2m; P = 0·05). Among patients treated with immuno-chemotherapy, four presented a MYC-R and 25 a MYC-EC. Univariate analysis showed the absence of significant difference between MYC-EC and normal MYC (MYC-NL) regarding progression-free survival (PFS; HR1·3; 95% CI [0·4-1·6]) and specific overall survival (SOS; HR 1·6; 95% CI [0·4-5·7]). Those results were compared to data from the PRIMA trial. This confirmed that MYC-EC had no impact on PFS (P = 0·86) or SOS (P = 0·9). Conversely, MYC-R was associated with a trend to inferior outcome regarding PFS (HR : 6·1; 95% CI [2·2-17·1]; P = 0·00026), lymphoma-related death (SOS; HR 13·6; 95% CI [2·9-65]; P = 0·00014) and risk of transformation (transformation-free survival (TFS); HR 82·7; 95% CI [14·8-463·4]; P < 0·0001). In conclusion, MYC-EC has no prognostic impact in FL but MYC-R FL tended to be associated with an increased risk of transformation and poorer outcome.

High Activation of the AKT Pathway in Human Multicystic Renal Dysplasia.

Multicystic renal dysplasia is a congenital cystic anomaly of the kidney caused by abnormal metanephric differentiation with immature tubules. It is surrounded by mesenchymal collars and islands of immature mesenchyma present between the cysts. The PI3K-AKT-mTOR signaling pathway is a key regulator involved in cell growth, proliferation, motility, survival, and apoptosis. Activation of the PI3K-AKT-mTOR pathway results in the survival and proliferation of tumor cells in many cancers. The aim of this study is to analyze the topographic expression of phospho-AKT, phospho-mTOR, and phospho-70S6K in renal development and in the multicystic dysplastic kidney (MCDK). A total of 17 fetal kidneys of development age from the first to the third trimester and 13 cases of pathological kidneys with MCDK were analyzed by immunohistochemistry in order to evaluate the expression of phospho-AKT (S473), phospho-mTOR, and phospho-70S6K. Phospho-AKT and phospho-mTOR were expressed early in renal development and in an identical manner for every structure derived from the ureteric bud, such as collecting ducts and urothelium. Phospho-p70S6K was expressed early in the urothelium and in glomerular mesangial cells. Later, their expressions differed according to the needs of cell proliferation and differentiation over time by becoming more selective. In MCDK, phospho-AKT, phospho-mTOR, and phospho-70S6K have the same profile: a high cytoplasmic expression in cystic epithelium, loose mesenchyma, and primitive tubes. This study demonstrates the essential and specific role of the PI3K-AKT-mTOR pathway in the formation of cysts in multicystic renal dysplasia.

The sVEGFR1-i13 splice variant regulates a ß1 integrin/VEGFR autocrine loop involved in the progression and the response to anti-angiogenic therapies of squamous cell lung carcinoma.

BACKGROUND: While lung adenocarcinoma patients can somewhat benefit from anti-angiogenic therapies, patients with squamous cell lung carcinoma (SQLC) cannot. The reasons for this discrepancy remain largely unknown. Soluble VEGF receptor-1, namely sVEGFR1-i13, is a truncated splice variant of the cell membrane-spanning VEGFR1 that has no transmembrane or tyrosine kinase domain. sVEGFR1-i13 is mainly viewed as an anti-angiogenic factor which counteracts VEGF-A/VEGFR signalling in endothelial cells. However, its role in tumour cells is poorly known. METHODS: mRNA and protein status were analysed by Real-Time qPCR, western blotting, ELISA assay, proximity ligation assay or immunohistochemistry in human tumour cell lines, murine tumourgrafts and non small cell lung carcinoma patients samples. RESULTS: We show that anti-angiogenic therapies specifically increase the levels of sVEGFR1-i13 in SQLC cell lines and chemically induced SQLC murine tumourgrafts. At the molecular level, we characterise a sVEGFR1-i13/ß1 integrin/VEGFR autocrine loop which determines whether SQLC cells proliferate or go into apoptosis, in response to anti-angiogenic therapies. Furthermore, we show that high levels of both sVEGFR1-i13 and ß1 integrin mRNAs and proteins are associated with advanced stages in SQLC patients and with a poor clinical outcome in patients with early stage SQLC. CONCLUSIONS: Overall, these results reveal an unexpected pro-tumoural function of sVEGFR1-i13 in SQLC tumour cells, which contributes to their progression and escape from anti-angiogenic therapies. These data might help to understand why some SQLC patients do not respond to anti-angiogenic therapies.

BET Family Protein BRD4: An Emerging Actor in NFκB Signaling in Inflammation and Cancer.

NFκB (Nuclear Factor-κ-light-chain-enhancer of activated B cells) signaling elicits global transcriptional changes by activating cognate promoters and through genome-wide remodeling of cognate regulatory elements called "super enhancers". BET (Bromodomain and Extra-Terminal domain) protein family inhibitor studies have implicated BET protein member BRD4 and possibly other BET proteins in NFκB-dependent promoter and super-enhancer modulation. Members of the BET protein family are known to bind acetylated chromatin to facilitate access by transcriptional regulators to chromatin, as well as to assist the activity of transcription elongation complexes via CDK9/pTEFb. BET family member BRD4 has been shown to bind non-histone proteins and modulate their activity. One such protein is RELA, the NFκB co-activator. Specifically, BRD4 binds acetylated RELA, which increases its transcriptional transactivation activity and stability in the nucleus. In aggregate, this establishes an intimate link between NFκB and BET signaling, at least via BRD4. The present review provides a brief overview of the structure and function of BET family proteins and then examines the connections between NFκB and BRD4 signaling, using the inflammatory response and cancer cell signaling as study models. We also discuss the potential of BET inhibitors for relief of aberrant NFκB signaling in cancer, focusing on non-histone, acetyl-lysine binding functions.

hEIDI: An Intuitive Application Tool To Organize and Treat Large-Scale Proteomics Data.

Advances in high-throughput proteomics have led to a rapid increase in the number, size, and complexity of the associated data sets. Managing and extracting reliable information from such large series of data sets require the use of dedicated software organized in a consistent pipeline to reduce, validate, exploit, and ultimately export data. The compilation of multiple mass-spectrometry-based identification and quantification results obtained in the context of a large-scale project represents a real challenge for developers of bioinformatics solutions. In response to this challenge, we developed a dedicated software suite called hEIDI to manage and combine both identifications and semiquantitative data related to multiple LC-MS/MS analyses. This paper describes how, through a user-friendly interface, hEIDI can be used to compile analyses and retrieve lists of nonredundant protein groups. Moreover, hEIDI allows direct comparison of series of analyses, on the basis of protein groups, while ensuring consistent protein inference and also computing spectral counts. hEIDI ensures that validated results are compliant with MIAPE guidelines as all information related to samples and results is stored in appropriate databases. Thanks to the database structure, validated results generated within hEIDI can be easily exported in the PRIDE XML format for subsequent publication. hEIDI can be downloaded from http://biodev.extra.cea.fr/docs/heidi .

Roles of paxillin family members in adhesion and ECM degradation coupling at invadosomes.

Invadosomes are acto-adhesive structures able to both bind the extracellular matrix (ECM) and digest it. Paxillin family members-paxillin, Hic-5, and leupaxin-are implicated in mechanosensing and turnover of adhesion sites, but the contribution of each paxillin family protein to invadosome activities is unclear. We use genetic approaches to show that paxillin and Hic-5 have both redundant and distinctive functions in invadosome formation. The essential function of paxillin-like activity is based on the coordinated activity of LD motifs and LIM domains, which support invadosome assembly and morphology, respectively. However, paxillin preferentially regulates invadosome assembly, whereas Hic-5 regulates the coupling between ECM degradation and acto-adhesive functions. Mass spectrometry analysis revealed new partners that are important for paxillin and Hic-5 specificities: paxillin regulates the acto-adhesive machinery through janus kinase 1 (JAK1), whereas Hic-5 controls ECM degradation via IQGAP1. Integrating the redundancy and specificities of paxillin and Hic-5 in a functional complex provides insights into the coupling between the acto-adhesive and ECM-degradative machineries in invadosomes.

Haploinsufficiency for NR3C1, the gene encoding the glucocorticoid receptor, in blastic plasmacytoid dendritic cell neoplasms.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive leukemia for which knowledge on disease mechanisms and effective therapies are currently lacking. Only a handful of recurring genetic mutations have been identified and none is specific to BPDCN. In this study, through molecular cloning in an index case that presented a balanced t(3;5)(q21;q31) and molecular cytogenetic analyses in a further 46 cases, we identify monoallelic deletion of NR3C1 (5q31), encoding the glucocorticoid receptor (GCR), in 13 of 47 (28%) BPDCN patients. Targeted deep sequencing in 36 BPDCN cases, including 10 with NR3C1 deletion, did not reveal NR3C1 point mutations or indels. Haploinsufficiency for NR3C1 defined a subset of BPDCN with lowered GCR expression and extremely poor overall survival (P = .0006). Consistent with a role for GCR in tumor suppression, functional analyses coupled with gene expression profiling identified corticoresistance and loss-of-EZH2 function as major downstream consequences of NR3C1 deletion in BPDCN. Subsequently, more detailed analyses of the t(3;5)(q21;q31) revealed fusion of NR3C1 to a long noncoding RNA (lncRNA) gene (lincRNA-3q) that encodes a novel, nuclear, noncoding RNA involved in the regulation of leukemia stem cell programs and G1/S transition, via E2F. Overexpression of lincRNA-3q was a consistent feature of malignant cells and could be abrogated by bromodomain and extraterminal domain (BET) protein inhibition. Taken together, this work points to NR3C1 as a haploinsufficient tumor suppressor in a subset of BPDCN and identifies BET inhibition, acting at least partially via lncRNA blockade, as a novel treatment option in BPDCN.

Atad2 is a generalist facilitator of chromatin dynamics in embryonic stem cells.

Although the conserved AAA ATPase and bromodomain factor, ATAD2, has been described as a transcriptional co-activator upregulated in many cancers, its function remains poorly understood. Here, using a combination of ChIP-seq, ChIP-proteomics, and RNA-seq experiments in embryonic stem cells where Atad2 is normally highly expressed, we found that Atad2 is an abundant nucleosome-bound protein present on active genes, associated with chromatin remodelling, DNA replication, and DNA repair factors. A structural analysis of its bromodomain and subsequent investigations demonstrate that histone acetylation guides ATAD2 to chromatin, resulting in an overall increase of chromatin accessibility and histone dynamics, which is required for the proper activity of the highly expressed gene fraction of the genome. While in exponentially growing cells Atad2 appears dispensable for cell growth, in differentiating ES cells Atad2 becomes critical in sustaining specific gene expression programmes, controlling proliferation and differentiation. Altogether, this work defines Atad2 as a facilitator of general chromatin-templated activities such as transcription.

A six gene expression signature defines aggressive subtypes and predicts outcome in childhood and adult acute lymphoblastic leukemia.

Abnormal gene expression in cancer represents an under-explored source of cancer markers and therapeutic targets. In order to identify gene expression signatures associated with survival in acute lymphoblastic leukemia (ALL), a strategy was designed to search for aberrant gene activity, which consists of applying several filters to transcriptomic datasets from two pediatric ALL studies. Six genes whose expression in leukemic blasts was associated with prognosis were identified:three genes predicting poor prognosis (AK022211, FASTKD1 and STARD4) and three genes associated with a favorable outcome (CAMSAP1, PCGF6 and SH3RF3). Combining the expression of these 6 genes could successfully predict prognosis not only in the two discovery pediatric ALL studies, but also in two independent validation cohorts of adult patients, one from a publicly available study and one consisting of 62 newly recruited Chinese patients. Moreover, our data demonstrate that our six gene based test is particularly efficient in stratifying MLL or BCR.ABL negative patients. Finally, common biological traits characterizing aggressive forms of ALL in both children and adults were found, including features of dormant hematopoietic stem cells, suggesting new therapeutic strategies.

Identification of a novel BET bromodomain inhibitor-sensitive, gene regulatory circuit that controls Rituximab response and tumour growth in aggressive lymphoid cancers.

Immuno-chemotherapy elicit high response rates in B-cell non-Hodgkin lymphoma but heterogeneity in response duration is observed, with some patients achieving cure and others showing refractory disease or relapse. Using a transcriptome-powered targeted proteomics screen, we discovered a gene regulatory circuit involving the nuclear factor CYCLON which characterizes aggressive disease and resistance to the anti-CD20 monoclonal antibody, Rituximab, in high-risk B-cell lymphoma. CYCLON knockdown was found to inhibit the aggressivity of MYC-overexpressing tumours in mice and to modulate gene expression programs of biological relevance to lymphoma. Furthermore, CYCLON knockdown increased the sensitivity of human lymphoma B cells to Rituximab in vitro and in vivo. Strikingly, this effect could be mimicked by in vitro treatment of lymphoma B cells with a small molecule inhibitor for BET bromodomain proteins (JQ1). In summary, this work has identified CYCLON as a new MYC cooperating factor that autonomously drives aggressive tumour growth and Rituximab resistance in lymphoma. This resistance mechanism is amenable to next-generation epigenetic therapy by BET bromodomain inhibition, thereby providing a new combination therapy rationale for high-risk lymphoma.

Induced malignant genome reprogramming in somatic cells by testis-specific factors.

Germline cell differentiation is controlled by a specific set of genes whose expression is tightly locked into the repressed state in somatic cells. Large-scale epigenome alterations, now evidenced in nearly all cancers, lead to aberrant activation of these normally silenced genes, as attested by the many reports describing the expression of testis-specific factors, known as cancer-testis genes, in various cancer cells. Here, based on the literature, we argue that off-context activity of some of the testis-specific epigenome regulators can reprogram the somatic cell epigenome toward a malignant state by favoring self-renewal and sustaining cell proliferation under stressful conditions, thereby constituting a major oncogenic mechanism.

[Quantitative proteomics by SILAC: practicalities and perspectives for an evolving approach]. / La protéomique quantitative par la méthode SILAC : technique et perspectives.

Mass spectrometry-based quantitative proteomics strategies are ideally adapted to the detection of global protein changes between different biological samples. Among these, SILAC (stable isotope labelling by amino acids in cell culture) has demonstrated a great potential. This method is extremely accurate and relatively easy to apply for the quantification of proteins extracted from cultured cells. SILAC involves cell culture either in regular culture media ("light" protein synthesis) or in media where amino acids have been replaced by their isotopically labelled counterparts ("heavy" protein synthesis). Cell populations to be compared can be mixed and treated as a single sample, which allows downstream sample preparation without the risk of introducing quantification errors. During mass spectrometry analysis, the relative protein abundance between biological samples can be calculated from the intensities of heavy and light peptides. As shown by numerous applications in biological and clinical studies, SILAC represents a promising method for the elucidation of cellular and physio-pathological mechanisms and for the identification of disease biomarkers. The restriction of SILAC to the quantification of proteins from cultured cells has just been overcome with the description in a recent paper of a SILAC mouse, which will allow this technology to be applied to the differential study of -tissues and biological fluids from model animals.

Discovering new bioactive neuropeptides in the striatum secretome using in vivo microdialysis and versatile proteomics.

The striatum, a major component of the brain basal nuclei, is central for planning and executing voluntary movements and undergoes lesions in neurodegenerative disorders such as Huntington disease. To perform highly integrated tasks, the striatum relies on a complex network of communication within and between brain regions with a key role devoted to secreted molecules. To characterize the rat striatum secretome, we combined in vivo microdialysis together with proteomics analysis of trypsin digests and peptidomics studies of native fragments. This versatile approach, carried out using different microdialysis probes and mass spectrometer devices, allowed evidencing with high confidence the expression of 88 proteins and 100 processed peptides. Their secretory pathways were predicted by in silico analysis. Whereas high molecular weight proteins were mainly secreted by the classical mode (94%), low molecular weight proteins equally used classical and non-classical modes (53 and 47%, respectively). In addition, our results suggested alternative secretion mechanisms not predicted by bioinformatics tools. Based on spectrum counting, we performed a relative quantification of secreted proteins and peptides in both basal and neuronal depolarization conditions. This allowed detecting a series of neuropeptide precursors and a 6-fold increase for neurosecretory protein VGF and proenkephalin (PENK) levels. A focused investigation and a long peptide experiment led to the identification of new secreted non-opioid PENK peptides, referred to as PENK 114-133, PENK 239-260, and PENK 143-185. Moreover we showed that injecting synthetic PENK 114-133 and PENK 239-260 into the striatum robustly increased glutamate release in this region. Thus, the combination of microdialysis and versatile proteomics methods shed new light on the secreted protein repertoire and evidenced novel neuropeptide transmitters.

Cellular and molecular mechanisms of abnormal calcification following ischemia-reperfusion injury in human liver transplantation.

Recent studies suggest a possible link between calcification and ischemia-reperfusion injury following liver transplantation. Histological staining, immunolabeling, and biochemical and electron microscopy analyses were applied to assess the possible mechanism(s) of calcification in liver tissue. Although light microscopy studies did not reveal the presence of large necrotic or apoptotic areas, electron microscopy showed the presence of membrane-bound vacuolar structures in hepatocytes, indicative of cell damage. Myofibroblasts were abundant in regions surrounding and within calcification. In these precalcified and calcified areas, myofibroblasts expressed bone-specific matrix proteins, such as osteopontin, type 1 collagen and bone sialoprotein. In addition, transforming growth factor beta (TGFbeta)-1 and BMP2, two growth factors implicated in osteoblast differentiation, and Runx2 and Msx2, two transcription factors targets of TGFbeta-1 and BMP2, were also expressed in these myofibroblasts. These data suggest that liver calcification following transplantation may be a consequence of precipitation of hydroxylapatite emanating from necrotic or apoptotic hepatocytes associated with proliferation of myofibroblasts expressing bone-specific matrix proteins.

Proteomic analysis of tyrosine phosphorylation during human liver transplantation.

BACKGROUND: Ischemia-reperfusion (I/R) causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. RESULTS: Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. CONCLUSION: Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.