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Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed in vitro by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an “off-the-shelf” cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors.Significance:These findings demonstrate the feasibility of an “off-the-shelf” vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability.

List of 1087 FSPs suitable for the inclusion in NOUS-209 vaccine.

Upon chronic antigen exposure, CD8(+) T cells become exhausted, acquiring a dysfunctional state correlated with the inability to control infection or tumor progression. In contrast, stem-like CD8(+) T progenitors maintain the ability to promote and sustain effective immunity. Adenovirus (Ad)–vectored vaccines encoding tumor neoantigens have been shown to eradicate large tumors when combined with anti–programmed cell death protein 1 (αPD-1) in murine models; however, the mechanisms and translational potential have not yet been elucidated. Here, we show that gorilla Ad vaccine targeting tumor neoepitopes enhances responses to αPD-1 therapy by improving immunogenicity and antitumor efficacy. Single-cell RNA sequencing demonstrated that the combination of Ad vaccine and αPD-1 increased the number of murine polyfunctional neoantigen-specific CD8(+) T cells over αPD-1 monotherapy, with an accumulation of Tcf1(+) stem-like progenitors in draining lymph nodes and effector CD8(+) T cells in tumors. Combined T cell receptor (TCR) sequencing analysis highlighted a broader spectrum of neoantigen-specific CD8(+) T cells upon vaccination compared to αPD-1 monotherapy. The translational relevance of these data is supported by results obtained in the first 12 patients with metastatic deficient mismatch repair (dMMR) tumors vaccinated with an Ad vaccine encoding shared neoantigens. Expansion and diversification of TCRs were observed in post-treatment biopsies of patients with clinical response, as well as an increase in tumor-infiltrating T cells with an effector memory signature. These findings indicate a promising mechanism to overcome resistance to PD-1 blockade by promoting immunogenicity and broadening the spectrum and magnitude of neoantigen-specific T cells infiltrating tumors.

Oncolytic viruses (OVs) are novel anti-tumor agents with the ability to selectively infect and kill tumor cells while sparing normal tissue. Beyond tumor cytolysis, OVs are capable of priming an anti-tumor immune response via lysis and cross-presentation of locally expressed endogenous tumor antigens, acting as an “endovaccine.” The effectiveness of OVs, similar to other immunotherapies, can be hampered by an immunosuppressive tumor microenvironment. In this study, we modified a previously generated oncolytic herpes simplex virus (oHSV) retargeted to the human HER2 (hHER2) tumor molecule and encoding murine interleukin-12 (mIL-12), by insertion of a second immunomodulatory molecule, murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), to maximize therapeutic efficacy. We assessed the efficacy of this double-armed virus (R-123) compared to singly expressing GM-CSF and IL-12 oHSVs in tumor-bearing mice. While monotherapies were poorly effective, combination with α-PD1 enhanced the anti-tumor response, with the highest efficacy of 100% response rate achieved by the combination of R-123 and α-PD1. Efficacy was T cell-dependent, and the induced immunity was long lasting and able to reject a second contralateral tumor. Importantly, systemic delivery of R-123 combined with α-PD1 was effective in inhibiting the development of tumor metastasis. As such, this approach could have a significant therapeutic impact paving the way for further development of this platform in cancer immunotherapy., Graphical Abstract, Fully virulent tumor retargeted HSV oncolytic viruses (THVs) are novel immunotherapeutic agents with increased specificity, safety, and potency. De Lucia et al. propose the use of a hHER2 THV expressing IL-12 and GM-CSF as a strategy to potentiate anti-tumor efficacy in combination with anti-PD1, opening future perspectives for local and systemic treatment.

Neoantigens are tumor-specific antigens able to induce T-cell responses, generated by mutations in protein-coding regions of expressed genes. Previous studies demonstrated that only a limited subset of mutations generates neoantigens in microsatellite stable tumors. We developed a method, called VENUS (Vaccine-Encoded Neoantigens Unrestricted Selection), to prioritize mutated peptides with high potential to be neoantigens. Our method assigns to each mutation a weighted score that combines the mutation allelic frequency, the abundance of the transcript coding for the mutation, and the likelihood to bind the patient’s class-I major histocompatibility complex alleles. By ranking mutated peptides encoded by mutations detected in nine cancer patients, VENUS was able to select in the top 60 ranked peptides, the 95% of neoantigens experimentally validated including both CD8 and CD4 T cell specificities. VENUS was evaluated in a murine model in the context of vaccination with an adeno vector encoding the top ranked mutations prioritized in the MC38 cell line. Efficacy studies demonstrated anti tumoral activity of the vaccine when used in combination with checkpoint inhibitors. The results obtained highlight the importance of a combined scoring system taking into account multiple features of each tumor mutation to improve the accuracy of neoantigen prediction.

Tumors with microsatellite instability (MSI) are caused by a deficiency in DNA mismatch repair (dMMR) system that leads to the accumulation of mutations within microsatellite regions. These types of mutations can determine a shift of the translational reading frame, generating Frame Shifted Peptides (FSPs). FSPs are tumor-specific neoantigens. Nous-209 is an off-the-shelf vaccine encoding 209 FSPs shared across dMMR/MSI tumors (Leoni et al., Cancer Res, 2020). The combination of the Nous-209 genetic polyvalent cancer vaccine and pembrolizumab has been demonstrated to be safe, highly immunogenic, with promising early signs of clinical efficacy in 12 patients with 7 durable confirmed PRs, 2 durable SDs and 3 PDs observed (Overman et al., SITC Nov 13, 2021 Poster number: 410). Here, we explore immune correlates potentially associated with the clinical outcome with the aim to address the Nous-209 contribution to the activity of pembrolizumab in this patient population. Several immunological analyses were performed on biological samples collected before and after treatment with Nous-209. Analyses were performed on paired blood and tumor biopsy samples. In periphery, vaccine immunogenicity was demonstrated by ex-vivo interferon-gamma ELISpot assay in 67% of subjects in dose level 1 (n=3), and 100% (n=7) of patients in dose level 2 with a mean of ≈1,500 IFN-γ SFCs/million PBMCs at the peak of the immune response. In 3 patients with partial response whose pre/post tumor biopsies were available, the intratumoral TCR repertoire was expanded and diversified post treatment with Nous-209. Moreover, in one out of these three patients it was possible to track vaccine-induced neoantigen specific TCR in the tumor biopsy post Nous-209 treatment. Overall, these results indicate that neoantigen specific CD8+ T cells, induced by Nous-209, expand and diversify upon treatment, and successfully traffic to and infiltrate the tumor microenvironment to exert anti-tumor activity. Citation Format: Anna Morena D'Alise, Guido Leoni, Francesca Langone, Elisa Micarelli, Gabriella Cotugno, Michael James Overman, Marwan Fakih, Dung Le, Théa Faivre, Elisa Scarselli. Characterization of immune correlates of clinical activity for Nous-209, an off-the-shelf immunotherapy, with pembrolizumab for treatment of tumors characterized by microsatellite instability (MSI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB008.

2515 Background: Defective DNA mismatch repair (dMMR) leads to high levels of microsatellite-instability (MSI-H) and insertions or deletions in coding regions, triggering the generation of tumor-specific frameshift peptides (FSPs). We selected 209 shared FSPs among subjects with dMMR/MSI-H cancers, to generate an off-the-shelf vaccine for the treatment of dMMR/MSI-H tumors. Those FSPs were cloned into four proprietary Great Apes Adenoviral (GAd) and four Modified Vaccinia Ankara (MVA) vectors to create a polyvalent viral vectored vaccine named Nous-209 [Leoni, G. et al. Cancer Res, 2020. 80(18): p. 3972-3982.]. Methods: This phase 1 first in human (FIH) study ( NCT04041310 ) evaluates safety and tolerability of two dose levels of the Nous-209 in combination with pembrolizumab, assesses immunogenicity and detects preliminary evidence of anti-tumor activity. Nous-209 is administered intramuscularly, concomitantly with pembrolizumab: one prime (GAd-209-FSP) at the 2nd pembrolizumab infusion and three boosters (MVA-209-FSP) at subsequent infusions each 3 weeks apart. The study is composed of two sequential cohorts i.e. dose escalation and dose expansion. Results: All evaluable subjects with 1st- or 2nd-line metastatic dMMR/MSI-H colorectal (CRC), gastric or gastroesophageal junction (GEJ) cancers enrolled in this phase I (n = 20) were evaluated as of February 03, 2022. Three subjects enrolled in dose level (DL) 1 (2 CRC and 1 GEJ cancer) showed durable confirmed partial responses (PRs). In DL 2 (12 CRC and 5 gastric cancers), 7 subjects had PRs, 6 had stable disease (SD) and 4 had progressive disease (PD) as best response. Most progressors progressed at the 1st CT and none of the responders have progressed. The median follow-up for subjects in DL1 is 24.7 months (22.3-26.7), and 7.6 months (0.9-19.4) in DL 2. The median progression free survival (PFS) and median duration of response (DoR) have not been reached. No dose limiting toxicities (DLTs) were observed. Vaccine immunogenicity was demonstrated in periphery by ex-vivo interferon-gamma ELISpot in 67% of subjects in DL 1, and 82% of subjects with evaluable samples in DL 2. The intratumoral TCR repertoire on pre/post tumor biopsies was analyzed in 3 evaluable subjects with PR and in 1 with PD: expansion and diversification of T cells post treatment with Nous-209 was noted only for the former. Vaccine-reactive TCR clones infiltrating the tumor biopsy post treatment were found in the only subject evaluated to date, with a long-term response. Conclusions: The combination of the Nous-209 and pembrolizumab is safe and well tolerated, and shows signs of clinical efficacy. Nous-209 elicits a neoantigen-specific T cell response expanding within the tumor, possibly contributing to the clinical outcome. Clinical trial information: NCT04041310.

Simple Summary Oncolytic viruses are emerging immunotherapeutics in cancer treatments. The conflicting role of innate immunity in the antitumor activity of oncolytic viruses is still a matter of debate. The STING-dependent DNA sensing axis is considered detrimental for viral replication and cancer cell clearance. Accordingly, we observed that STING loss in tumor cells was associated with improved lytic potential by a herpes-based oncolytic virus. However, STING-knockout cancer cells infected with the oncolytic virus showed impaired immunogenicity, as immunogenic cell death was improperly triggered. In agreement with these observations, STING-knockout tumors raised in a murine syngeneic model were more resistant to a combined treatment of the oncolytic virus with PD-1 blockade. The present study demonstrates the antitumor benefit of antiviral immunity and sheds lights on the mechanisms of immune resistance to oncovirotherapy exerted by STING-loss in tumor cells. Abstract The dichotomic contribution of cancer cell lysis and tumor immunogenicity is considered essential for effective oncovirotherapy, suggesting that the innate antiviral immune response is a hurdle for efficacy of oncolytic viruses. However, emerging evidence is resizing this view. By sensing cytosolic DNA, the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) axis can both counteract viral spread and contribute to the elicitation of adaptive immunity via type I interferon responses. In this paper, we analyzed the tumor-resident function of Sting-mediated DNA sensing in a combined approach of oncovirotherapy and PD-1 immune checkpoint blockade, in an immunocompetent murine model. While supporting increased lytic potential by oncolytic HER2-retargeted HSV-1 in vitro and in vivo, Sting-knockout tumors showed molecular signatures of an immunosuppressive tumor microenvironment. These signatures were correspondingly associated with ineffectiveness of the combination therapy in a model of established tumors. Results suggest that the impairment in antiviral response of Sting-knockout tumors, while favoring viral replication, is not able to elicit an adequate immunotherapeutic effect, due to lack of immunogenic cell death and the inability of Sting-knockout cancer cells to promote anti-tumor adaptive immune responses. Accordingly, we propose that antiviral, tumor-resident Sting provides fundamental contributions to immunotherapeutic efficacy of oncolytic viruses.

Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed in vitro by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an “off-the-shelf” cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors. Significance: These findings demonstrate the feasibility of an “off-the-shelf” vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability.

BackgroundDefective DNA mismatch repair (dMMR) leads to high levels of microsatellite-instability (MSI-H) and insertions or deletions in coding regions, resulting in the generation of tumor-specific frameshift peptides (FSPs). We selected 209 shared FSPs among subjects with first- or second-line metastatic dMMR/MSI-H colorectal (CRC), gastric, and gastroesophageal junction (GEJ) cancers, to develop an off-the-shelf vaccine for the treatment of dMMR/MSI-H tumors. Selected FSPs were cloned into four proprietary Great Apes Adenoviral (GAd) and four Modified Vaccinia Ankara (MVA) vectors to generate a polyvalent viral vectored vaccine called Nous-209 [Leoni G., et al., Cancer Res. 2020]MethodsThis phase 1 first in human (FIH) study (NCT04041310) was designed to evaluate safety and tolerability of two dose levels (one log difference for both GAd and MVA) of Nous-209 genetic polyvalent vaccine in combination with the programmed death receptor-1 (PD-1)-blocking antibody pembrolizumab, to assess immunogenicity of the combination and to detect preliminary evidence of anti-tumor activity. Nous-209 is administered intramuscularly, concomitantly with pembrolizumab (doses and schedule per approved label): one prime (GAd-209-FSP) at the 2nd pembrolizumab infusion and three booster (MVA-209-FSP) injections at subsequent infusions each 3 weeks apart. The study is composed of two sequential cohorts: dose escalation and dose expansionResultsTwelve evaluable subjects with first- or second-line metastatic dMMR/MSI-H cancers were evaluated as of May 28, 2021. Three subjects enrolled in dose level 1 (2 CRC and 1 GEJ cancer) demonstrated durable confirmed partial responses (PRs). In dose level 2 (6 CRC and 3 gastric cancers), 4 subjects had PRs, 2 had stable disease (SD) and 3 had progressive disease (PD). The median follow-up for subjects in dose level 1 is 17.9 months (range 15.3–20 months), and 8 months (range 3.8–12.5 months) for subjects in dose level 2 as of the above cut-off date. No dose limiting toxicities (DLTs) were observed, and the treatment combination was determined to be safe and tolerable. Vaccine immunogenicity was demonstrated by ex-vivo interferon-gamma ELISpot assay in 67% of subjects in dose level 1, and 100% of patients with evaluable samples in dose level 2ConclusionsThe combination of the Nous-209 genetic polyvalent cancer vaccine and pembrolizumab has been demonstrated to be safe, immunogenic, and continues to show early signs of clinical efficacy, which may be attributed to the vaccine contributionReferenceLeoni G, et al. A genetic vaccine encoding shared cancer neoantigens to treat tumors with microsatellite instability. Cancer Res 2020 July 20;80(18):3972–3982.Ethics ApprovalThe study was approved by The University of Texas MD Anderson Cancer Center, IRB number 2019–0651_MOD001. The study was approved by Feinstein Institute for Medical Research Northwell Health, IRB number 19–1086. The study was approved by WCG The Trusted Partner in Ethical Review, IRB number 20200155. The study was approved by Johns Hopkins Medicine, IRB number IRB00220323/CIR00053809. The study was approved by City of Hope, IRB number 19277/176947. The study was approved by Advarra Advancing Better Research. The study was approved by Dana-Faber Cancer Institute. The study was approved by Weill Cornell IRB. The study was approved by Roswell Park IRB.

BackgroundA number of different immune pathways are involved in the effective killing of cancer cells, collectively named as the ‘Cancer Immunity Cycle’. Anti-PD-1 checkpoint blockade (CPB) therapy is active on one of these pathways and reinvigorates anticancer T cell immunity, leading to long-term responses in a limited fraction of patients with cancer. We have previously shown that neoantigens-based adenovirus vectored vaccine in combination with anti-PD-1 further expands pre-existing anticancer immunity and elicits novel neoantigen-specific T cells thereby increasing efficacy to 50% of tumor clearance in mice. Here we added a third component to the CPB plus vaccine combination, which is able to modify the suppressive tumor microenvironment by reducing the number of tumor-infiltrating regulatory T cells (Tregs), as strategy for improving the therapeutic efficacy and overcoming resistance.MethodsThe antitumor efficacy of anti-PD-1, neoantigen vaccine and Treg modulating agents, either Bempegaldesleukin (BEMPEG: NKTR-214) or an anti-CTLA-4 mAb with Treg-depleting activity, was investigated in murine tumor models. We evaluated tumor growth in treated animals, neoantigen-specific T cells in tumors, tumor-infiltrating lymphocytes (TILs) and intratumoral Tregs.ResultsThe addition of BEMPEG or anti-CTLA-4 to the combination of vaccine and anti-PD-1 led to complete eradication of large tumors in nearby 100% of treated animals, in association with expansion and activation of cancer neoantigen-specific T cells and reduction of tumor-infiltrating Tregs.ConclusionThese data support the notion that the integrated regulation of three steps of the cancer immunity cycle, including expansion of neoantigen-specific T cells, reversal of the exhausted T cell phenotype together with the reduction of intratumoral Tregs may represent a novel rationally designed drug combination approach to achieve higher cure rates.

Heterotopic ossification (HO) is a pathological condition characterized by the deposition of mineralized tissue in ectopic locations such as the skeletal muscle. The precise cellular origin and molecular mechanisms underlying HO are still debated. In our study we focus on the differentiation of mesoangioblasts (MABs), a population of multipotent skeletal muscle precursors. High-content screening for small molecules that perturb MAB differentiation decisions identified Idoxuridine (IdU), an antiviral and radiotherapy adjuvant, as a molecule that promotes MAB osteogenic differentiation while inhibiting myogenesis. IdU-dependent osteogenesis does not rely on the canonical BMP-2/SMADs osteogenic pathway. At pro-osteogenic conditions IdU induces a mild DNA Damage Response (DDR) that activates ATM and p38 eventually promoting the phosphorylation of the osteogenesis master regulator RUNX2. By interfering with this pathway IdU-induced osteogenesis is severely impaired. Overall, our study suggests that induction of the DDR promotes osteogenesis in muscle resident MABs thereby offering a new mechanism that may be involved in the ectopic deposition of mineralized tissue in the muscle.

Neoantigens (nAgs) are promising tumor antigens for cancer vaccination with the potential of inducing robust and selective T cell responses. Genetic vaccines based on Adenoviruses derived from non-human Great Apes (GAd) elicit strong and effective T cell-mediated immunity in humans. Here, we investigate for the first time the potency and efficacy of a novel GAd encoding multiple neoantigens. Prophylactic or early therapeutic vaccination with GAd efficiently control tumor growth in mice. In contrast, combination of the vaccine with checkpoint inhibitors is required to eradicate large tumors. Gene expression profile of tumors in regression shows abundance of activated tumor infiltrating T cells with a more diversified TCR repertoire in animals treated with GAd and anti-PD1 compared to anti-PD1. Data suggest that effectiveness of vaccination in the presence of high tumor burden correlates with the breadth of nAgs-specific T cells and requires concomitant reversal of tumor suppression by checkpoint blockade., Vaccination against neo-antigens has resulted in an effective antitumor response in several models. Here, the authors show that delivery of larger sets of neo-antigens using an adenovirus-based vaccination platform, results in much better tumor protection when combined with checkpoint blockade in a mouse model of advanced disease.

Cancer neoantigens (nAgs) have the potential to elicit strong and tumor-specific immune responses and are, therefore, of great interest for cancer immunotherapeutic strategies, including therapeutic vaccines. Here we developed a novel neoantigen cancer vaccine based on adenoviruses derived from non-human Great Apes (GAds). GAds vaccination was highly effective in prophylactic or early therapeutic treatment of mouse tumors, independently of the number of encoded nAgs. In presence of high tumor burden, GAd has no antitumor effect unless combined with anti-PD1 treatment. In this more stringent setting, effectiveness of vaccination required the targeting of many neoantigens. Analysis of gene expression profile of tumors from responder mice showed greater diversification of the T cell repertoire with increased number of clonotypes in combo treated animals compared to anti-PD-1. Data suggest that GAd vaccines encoding a large number of nAgs can synergize with checkpoint inhibitors therapy by increasing the breadth of nAgs-specific T cells. Citation Format: Anna Morena D'Alise, Guido Leoni, Gabriella Cotugno, Fulvia Troise, Francesca Langone, Imma Fichera, Maria De Lucia, Rosa Vitale, Adriano Leuzzi, Veronica Bignone, Elena Di Matteo, Fabio Giovanni Tucci, Lidia Avalle, Valeria Poli, Armin Lahm, Maria Teresa Catanese, Antonella Folgori, Stefano Colloca, Alfredo Nicosia, Elisa Scarselli. A cancer vaccine targeting many neoantigens is required for effective eradication of large tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B105.

SummaryMembers of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a high-density peptide chip technology that allows for probing of the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique, we have experimentally identified thousands of putative SH2-peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2-mediated probabilistic interaction network, which we make available as a community resource in the PepspotDB database. A predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the extracellular signal-regulated kinase activation loop was validated by experiments in living cells.

Assembly of large biochemical networks can be achieved by confronting new cell-specific experimental data with an interaction subspace constrained by prior literature evidence. The SIGnaling Network Open Resource, SIGNOR (available on line at http://signor.uniroma2.it), was developed to support such a strategy by providing a scaffold of prior experimental evidence of causal relationships between biological entities. The core of SIGNOR is a collection of approximately 12 000 manually-annotated causal relationships between over 2800 human proteins participating in signal transduction. Other entities annotated in SIGNOR are complexes, chemicals, phenotypes and stimuli. The information captured in SIGNOR can be represented as a signed directed graph illustrating the activation/inactivation relationships between signalling entities. Each entry is associated to the post-translational modifications that cause the activation/inactivation of the target proteins. More than 4900 modified residues causing a change in protein concentration or activity have been curated and linked to the modifying enzymes (about 351 human kinases and 94 phosphatases). Additional modifications such as ubiquitinations, sumoylations, acetylations and their effect on the modified target proteins are also annotated. This wealth of structured information can support experimental approaches based on multi-parametric analysis of cell systems after physiological or pathological perturbations and to assemble large logic models.

Proline-rich motifs are widely distributed in eukaryotic proteomes and are usually involved in the assembly of functional complexes through interaction with specific binding modules. The tumour - suppressor p53 protein presents a proline-rich region that is crucial for regulating apoptosis by connecting the p53 with a complex protein network. In humans, a common polymorphism determines the identity of residue 72, either proline or arginine, and affects the features of the motifs present in the polyproline domain. The two isoforms have different biochemical properties and markedly influence cancer onset and progression. In this article, we analyse the binding of the p53 proline-rich region with a pool of selected polyproline binding domains (i.e. SH3 and WW), and we present the first demonstration that the purified SH3 domains of the CD2AP/Cin85 protein family are able to directly bind the p53 protein, and to discriminate between the two polymorphic variants P72R.