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Background Ferritin receptor (CD71) is an example of a very attractive cancer target, since it is highly expressed in virtually all tumor types, including metastatic loci. However, this target can be considered to be inaccessible to conventional target therapies, due to its presence in many healthy tissues. Here, we describe the preclinical evaluation of a tumor proteases-activatable human ferritin (HFt)-based drug carrier (The-0504) that is able to selectively deliver the wide-spectrum topoisomerase I inhibitor Genz-644282 to CD71-expressing tumors, preventing the limiting toxic effects associated with CD71-targeting therapies. Methods CD71 expression was evaluated using flow cytometry and immunohistochemistry techniques. The-0504 antiproliferative activity towards several cancer cell lines was assessed in vitro. The-0504 antitumor efficacy and survival benefit were evaluated in different human tumors, which had been grown either as xenografts or patient-derived xenografts in mice. The-0504 toxicology profile was investigated in multiple-cycle repeat-dose study in rodents. Results In vitro studies indicate that The-0504 is highly specific for CD71 expressing cells, and that there is a relationship between CD71 levels and The-0504 anticancer activity. In vivo treatments with The-0504 showed a remarkable efficacy, eradicating several human tumors of very diverse and aggressive histotypes, such as pancreas, liver and colorectal carcinomas, and triple-negative breast cancer. Conclusions Durable disease-free survival, persistent antitumor responses after discontinuation of treatment and favorable toxicology profile make The-0504 an ideal candidate for clinical development as a novel, CD71-targeted, low-toxicity alternative to chemotherapy.

Background Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive and lethal disease, lacking effective therapeutic approaches. Available therapies only marginally prolong patient survival and are frequently coupled with severe adverse events. It is therefore pivotal to investigate novel and safe pharmacological approaches. We have recently identified the ABC transporter, ABCC3, whose expression is dependent on mutation of TP53, as a novel target in PDAC. ABCC3-mediated regulation of PDAC cell proliferation and tumour growth in vivo was demonstrated and was shown to be conferred by upregulation of STAT3 signalling and regulation of apoptosis. Methods To verify the potential of ABCC3 as a pharmacological target, a small molecule inhibitor of ABCC3, referred to here as MCI-715, was designed. In vitro assays were performed to assess the effects of ABCC3 inhibition on anchorage-dependent and anchorage-independent PDAC cell growth. The impact of ABCC3 inhibition on specific signalling pathways was verified by Western blotting. The potential of targeting ABCC3 with MCI-715 to counteract PDAC progression was additionally tested in several animal models of PDAC, including xenograft mouse models and transgenic mouse model of PDAC. Results Using both mouse models and human cell lines of PDAC, we show that the pharmacological inhibition of ABCC3 significantly decreased PDAC cell proliferation and clonal expansion in vitro and in vivo, remarkably slowing tumour growth in mice xenografts and patient-derived xenografts and increasing the survival rate in a transgenic mouse model. Furthermore, we show that stromal cells in pancreatic tumours, which actively participate in PDAC progression, are enriched for ABCC3, and that its inhibition may contribute to stroma reprogramming. Conclusions Our results indicate that ABCC3 inhibition with MCI-715 demonstrated strong antitumor activity and is well tolerated, which leads us to conclude that ABCC3 inhibition is a novel and promising therapeutic strategy for a considerable cohort of patients with pancreatic cancer. Electronic supplementary material The online version of this article (10.1186/s13046-019-1308-7) contains supplementary material, which is available to authorized users.

Simple Summary Head and neck cancer (HNC) is a complex and extremely heterogeneous disease that includes a wide variety of cancer subtypes. Despite notable advances in understanding the molecular mechanisms involved in the disease, which allowed the increase of the therapeutic armamentarium, HNC treatment remains very challenging. In fact, to date the average 5-year survival rate for this disease is around 65%; hence, HNC continues to be one of the most aggressive solid tumors. Surgical removal is the first treatment of choice for HCN; however, in addition to this treatment modality, a broad spectrum of new therapies has been developed so far, ranging from multimodal chemotherapy to targeted and immune-therapy, mainly through the use of antibodies. In this work, we systematically reviewed the progress obtained in antibody-drug conjugate (ADC) development for the treatment of HNC. Abstract Background: Antibody-drug conjugates (ADCs) are designed to deliver potent cytotoxic agents into tumor tissues. During the last two decades, a plethora of ADCs have been successfully developed and used for several indications, including hematologic and solid tumors. In this work, we systematically reviewed the progress in ADC development for the treatment of HNC. Methods: This review was registered in PROSPERO database. A comprehensive search was conducted following PRISMA guidelines and using PubMed, Scopus and Web of Science database. Results: In total, 19 studies were included. Due to the significant heterogeneity of the outcome measures, meta-analysis was not performed, and data were summarized in tables. HNC results are poorly represented in the cohorts of completed clinical trials; published data are mostly focused on safety evaluation rather than efficacy of ADCs. Conclusions: Although several novel agents against a wide range of different antigens were investigated, showing promising results at a preclinical level, most of the targets reported in this review are not specific for HNC; hence, the development of ADCs tailored for the HNC phenotype could open up new therapeutic perspectives. Moreover, the results from the present systematic review call attention to how limited is the application of current clinical trials in HNC.

Despite their centrality to rockfall risk management, two issues are frequently overlooked: the role of forests in rockfall dynamic and the fragmentation phenomenon. To investigate the importance of these issues we have developed advanced modelling case studies in two representative sites that have been recently affected by rockfall events in the Aosta Valley Region (Western Italian Alps). In the Saint Oyen case study, about 17,500 m3 of rock detached in March 2019 and reached a service road and the sport center in the lower part of the slope, passing through a mature fir forest. The presence of the forest has significantly influenced the rocks distribution along the slope, increasing the lateral dispersion of trajectories and reducing the mobility. For the design of defensive works, 3D rockfall models of three future potential risk scenarios were therefore performed by using the tree-impact algorithm of the code HY-STONE (Frattini et al., 2012). This algorithm provides the location of impacts on trees, the absorbed energy, and the deviation angle. The input parameters (i.e., the value of diameter at breast height and the forest density) were based on direct measurements of the fir forest. Compared with a traditional simulation without the protective role of forests, the results of 3D numerical modelling with tree-impact algorithm show a decrease in the number of blocks impacting the barriers (91%), no variations in the bouncing heights (for 95th percentile), and an increase in the kinetic energies due to a filter effect by the forest (85% for 95th percentile). In the Roisan case study, about 1,050 m3 of rock toppled in October 2019. While the main body of the rockfall stopped in a relatively flat area close to the failure, two blocks were exceptionally able to reach the foot of the slope causing the interruption of a municipal road. An attempt to back-calibrate this event with HY-STONE showed difficulties to describe the behaviour of these isolated blocks with respects to the main landslide body. A possible explanation for this behaviour is that the detached volume fragmented soon after impacting the slope, giving rise to flying fragments with higher mobility. To test this hypothesis we accounted for fragmentation through a specific algorithm of HY-STONE that fragments the falling blocks when their energy overcomes a certain threshold and simulate the behaviour of the resulting fragments. This approach allowed to accurately replicate the rockfall event. We therefore adopted this approach for defensive-works design, simulating all the unstable volumes overhanging the municipal road. Compared with a traditional simulation, the results of 3D numerical modelling with fragmentation algorithm show an increase in the number of blocks impacting the barriers (86%) and in the bouncing heights (96% for 95th percentile), with a decrease of the kinetic energy due to comminution (39% for 95th percentile). These two case studies demonstrate the importance of accounting for the forest or for fragmentation in the design of cost-effective defensive works. Frattini P, Crosta GB, Agliardi F (2012) Rockfall characterization and modeling. Landslides: types, mechanisms and modelling 22:267-281

The risk management of rainfall-induced landslides requires reliable rainfall thresholds to issue early warning alerts. The practical application of these thresholds often leads to misclassifications, either false negative or false positive, which induce costs for the society. Since missed-alarm (false negative) and false-alarm (false positive) cost may be significantly different, it is necessary to find an optimal threshold that accounts for and minimises such costs, tuning the false-alarm and missed-alarm rates. In this paper, we propose a new methodology to develop cost-sensitive rainfall thresholds, and we also analyse several factors that produce uncertainty, such as the accuracy of rainfall intensity values at landslide location, the time of occurrence, the minimum rainfall amount to define the non-triggering event, and the variability of cost scenarios. Starting from a detailed mapping of landslides that occurred during five large-scale rainfall events in the Italian Central Alps, we first developed rainfall threshold curves with a ROC-based approach by using both rain gauge and bias-adjusted weather radar data. Then, based on a reference cost scenario in which we quantified several cost items for both missed alarms and false alarms, we developed cost-sensitive rainfall threshold curves by using cost-curve approach (Drummond and Holte 2000). Finally, we studied the sensitivity of cost items. The study confirms how important is the information regarding rainfall intensity at the landslide site for the development of rainfall thresholds. Although the use of bias-corrected radar strongly improves these values, a large uncertainty related to the exact time of landslide occurrence still remains, negatively affecting the analysis. Accounting for the different missed-alarm and false-alarm misclassification costs is important because different combinations of these costs make an increase or decrease of the rainfall thresholds convenient. In our reference cost scenario, the most convenient threshold is lower than ROC-based thresholds because it seeks to minimise the number of missed alarms, whereas the missed-alarm costs are almost seven times greater than false-alarm costs. However, for different cost scenarios, threshold may vary significantly, as much as half an order of magnitude.

Gastrointestinal tumors, including pancreatic and colorectal cancers, represent one of the greatest public health issues worldwide, leading to a million global deaths. Recent research demonstrated that the human heavy chain ferritin (HFt) can encapsulate different types of drugs in its cavity and can bind to its receptor, CD71, in several solid and hematological tumors, thus highlighting the potential use of ferritin for tumor-targeting therapies. Here, we describe the development and characterization of a novel nanomedicine based on the HFt that is named The-0504. In particular, this novel system is a nano-assembly comprising an engineered version of HFt that entraps about 80 molecules of a potent, wide-spectrum, non-camptothecin topoisomerase I inhibitor (Genz-644282). The-0504 can be produced by a standardized pre-industrial process as a pure and homogeneously formulated product with favourable lyophilization properties. The preliminary anticancer activity was evaluated in cultured cancer cells and in a mouse model of pancreatic cancer. Overall results reported here make The-0504 a candidate for further preclinical development against CD-71 expressing deadly tumors.

Tumor cell dissemination in cancer patients is associated with a significant reduction in their survival and quality of life. The ubiquitination pathway plays a fundamental role in the maintenance of protein homeostasis both in normal and stressed conditions and its dysregulation has been associated with malignant transformation and invasive potential of tumor cells, thus highlighting its value as a potential therapeutic target. In order to identify novel molecular targets of tumor cell migration and invasion we performed a genetic screen with an shRNA library against ubiquitination pathway-related genes. To this end, we set up a protocol to specifically enrich positive migration regulator candidates. We identified the deubiquitinase USP19 and demonstrated that its silencing reduces the migratory and invasive potential of highly invasive breast cancer cell lines. We extended our investigation in vivo and confirmed that mice injected with USP19 depleted cells display increased tumor-free survival, as well as a delay in the onset of the tumor formation and a significant reduction in the appearance of metastatic foci, indicating that tumor cell invasion and dissemination is impaired. In contrast, overexpression of USP19 increased cell invasiveness both in vitro and in vivo, further validating our findings. More importantly, we demonstrated that USP19 catalytic activity is important for the control of tumor cell migration and invasion, and that its molecular mechanism of action involves LRP6, a Wnt co-receptor. Finally, we showed that USP19 overexpression is a surrogate prognostic marker of distant relapse in patients with early breast cancer. Altogether, these findings demonstrate that USP19 might represent a novel therapeutic target in breast cancer., Facultad de Ciencias Médicas, Centro de Investigaciones Inmunológicas Básicas y Aplicadas

The MYC family of transcription factors is a major driver of human cancer and potential therapeutic target. However, no clinically viable drugs have been yet developed that are able to directly tackle MYC oncoproteins. In our laboratory, we are exploring alternative approaches aiming to disturb signalling downstream of MYC. MYCN is frequently activated in neuroblastoma, a paediatric solid malignancy that, in its metastatic form, has a very poor prognosis. An important pathway regulated by MYC is the CKS1/SKP2/p27kip1 axis. In this study, we have repurposed the anti-psychotic drug Prozac to disrupt CKS1/SKP2/p27Kip1 signalling and assess its potential as an anti-neuroblastoma agent in vitro and in vivo. Using DNA editing technology, we show that stabilisation of p27Kip1 operated by Prozac in MYC-activated cells is essential for the anti-neuroblastoma activity of the drug. Furthermore, dosing mice with a concentration of Prozac equivalent to that used in long-term clinical trials in children with psychiatric disorders caused a significant reduction of metastatic disease in two models of high-risk neuroblastoma. The favourable toxicity profile of Prozac suggests that long-term treatments might be implemented in children with MYC/CKS1high neuroblastomas.

Pancreatic Ductal Adenocarcinoma (PDAC) is a very aggressive disease, lacking effective therapeutic approaches and leaving PDAC patients with a poor prognosis. The life expectancy of PDAC patients has not experienced a significant change in the last few decades with a five-year survival rate of only 8%. To address this unmet need, novel pharmacological targets must be identified for clinical intervention. ATP Binding Cassette (ABC) transporters are frequently overexpressed in different cancer types and represent one of the major mechanisms responsible for chemoresistance. However, a more direct role for ABC transporters in tumorigenesis has not been widely investigated. Here, we show that ABCC3 (ABC Subfamily C Member 3; previously known as MRP3) is overexpressed in PDAC cell lines and also in clinical samples. We demonstrate that ABCC3 expression is regulated by mutant p53 via miR-34 and that the transporter drives PDAC progression via transport of the bioactive lipid lysophosphatidylinositol (LPI). Disruption of ABCC3 function either by genetic knockdown reduces pancreatic cancer cell growth in vitro and in vivo. Mechanistically, we demonstrate that knockdown of ABCC3 reduce cell proliferation by inhibition of STAT3 and HIF1α signalling pathways, previously been shown to be key regulators of PDAC progression. Collectively, our results identify ABCC3 as a novel and promising target in PDAC therapy.

Background Phospholipase Cγ1 (PLCγ1) is highly expressed in human tumours. Our previous studies reported that both stable and inducible PLCγ1 down-regulation can inhibit formation of breast-cancer-derived experimental lung metastasis. Further, high expression of PLCγ1 and its constitutively activated forms (i.e., PLCγ1-pY1253, PLCγ1-pY783) is associated with worse clinical outcome in terms of incidence of distant metastases, but not of local relapse in T1-T2, N0 breast cancer patients. Methods In the present retrospective study, we analysed the prognostic role of PLCγ1 in early breast cancer patients stratified according to the St. Gallen criteria and to their menopausal status. PLCγ1-pY1253 and PLCγ1-pY783 protein expression levels were determined by immunohistochemistry on tissue microarrays, and were correlated with patients’ clinical data, using univariate and multivariate statistical analyses. Results In our series, the prognostic value of PLCγ1 overexpression was restricted to Luminal type tumours. From multivariate analyses, pY1253-PLCγ1High was an independent prognostic factor only in postmenopausal patients with Luminal-B tumours (hazard ratio [HR], 2.4; 95% confidence interval [CI], 1.1–5.3; P = 0.034). Conversely, PLCγ1-pY783High was a remarkably strong risk factor (HR, 20.1; 95% CI, 2.2–178.4; P = 0.003) for pre/perimenopausal patients with Luminal-A tumours. Conclusions PLCγ1 overexpression is a strong predictive surrogate marker of development of metastases in early Luminal-A and -B breast cancer patients, being able to discriminate patients with high and low risk of metastases. Therefore, targeting the PLCγ1 pathway can be considered of potential benefit for prevention of metastatic disease. Electronic supplementary material The online version of this article (10.1186/s12885-019-5949-x) contains supplementary material, which is available to authorized users.

Simple Summary Antibody Drug Conjugates are an emerging class of biopharmaceuticals that have seen an impressive increase of attention in the field of cancer therapy. Here, we describe the therapeutic activity of 1959-sss/DM3, a non-internalizing ADC targeting LGALS3BP, a secreted, extracellular vesicles-associated protein expressed by the majority of human cancers, including neuroblastoma. We show that 1959-sss/DM3 treatment can cure mice with established neuroblastoma tumours in pseudometastatic, orthotopic and Patient Derived Xenograft models. Abstract Neuroblastoma is the most common extra-cranial solid tumor in infants and children, which accounts for approximately 15% of all cancer-related deaths in the pediatric population. New therapeutic modalities are urgently needed. Antibody-Drug Conjugates (ADC)s-based therapy has been proposed as potential strategy to treat this pediatric malignancy. LGALS3BP is a highly glycosylated protein involved in tumor growth and progression. Studies have shown that LGALS3BP is enriched in extracellular vesicles (EV)s derived by most neuroblastoma cells, where it plays a critical role in preparing a favorable tumor microenvironment (TME) through direct cross talk between cancer and stroma cells. Here, we describe the development of a non-internalizing LGALS3BP ADC, named 1959-sss/DM3, which selectively targets LGALS3BP expressing neuroblastoma. 1959-sss/DM3 mediated potent therapeutic activity in different types of neuroblastoma models. Notably, we found that treatments were well tolerated at efficacious doses that were fully curative. These results offer preclinical proof-of-concept for an ADC targeting exosomal LGALS3BP approach for neuroblastomas.

Breast cancer, the most common malignancy among women, is usually detected at an early stage and has a low risk of relapse. Nevertheless, a significant number of patients cannot be cured solely by local treatment. Distinguishing between patients who are of low risk of relapse from those who are of high risk may have important implications to improve treatment outcomes. The PLC-γ1 signaling pathway promotes many physiological processes, including cell migration and invasion. Increasing evidence shows aberrant PLC-γ1 signaling implication in carcinogenesis including breast cancer. In this review, the role of PLC-γ1 in breast cancer and its clinical implications will be discussed, as well as its potential as a prognostic factor and a therapeutic target.

Doxorubicin is employed alone or in combination for the treatment of several hematological and solid malignancies; despite its efficacy, there are associated cardiotoxicity limits both in its application in patients with heart disease risk factors and also in its long-term use. HFt-MP-PAS40 is a genetically engineered human ferritin heavy chain (HFt)-based construct able to efficiently entrap and deliver doxorubicin to cancer cells. HF-MP-PAS contains a short motif sequence (defined as MP) responsive to proteolytic cleavage by tumor matrix metalloproteases (MMPs), located between each HFt subunit and a masking polypeptide sequence rich in proline (P), alanine (A), and serine (S) residues (PAS). This carrier displayed excellent therapeutic efficacy in a xenogenic pancreatic cancer model in vivo, leading to a significant increase in overall animal survival in treated mice. Herein, we describe the HFt-MP-PAS40-Dox efficacy against squamous cell carcinomas of the head and neck (HNSCC) with the goal of validating the application of our nano-drug for the treatment of different solid tumors. In addition, a tolerability study in healthy mice was also performed. The results indicate that HFt-MP-PAS40-Dox produced increased anti-tumor effects both in vitro and in vivo in comparison to the free drug in several HNSCC cell lines. In the acute toxicity studies, the maximum tolerated dose (MTD) of HFt-MP-PAS40-Dox was about 3.5 higher than the free drug: 25 mg/kg versus 7 mg/kg doxorubicin equivalents. Importantly, evaluation of heart tissues provided evidence that doxorubicin is less cardio-toxic when encapsulated inside the ferritin carrier. In conclusion, HFt-MP-PAS40-Dox may be administered safely at higher doses compared with the free drug, resulting in superior efficacy to control HNSCC malignancies.

// Emily Capone 1, 2 , Enza Piccolo 1 , Imma Fichera 1, 6 , Paolo Ciufici 3 , Daniela Barcaroli 3 , Arturo Sala 3, 4 , Vincenzo De Laurenzi 2 , Valentina Iacobelli 1, 5 , Stefano Iacobelli 1, 2, * and Gianluca Sala 1, 2, * 1 MediaPharma s.r.l., 66100, Chieti, Italy 2 Dipartimento di Scienze Mediche, Orali e Biotecnologiche; University “G. d’Annunzio” Chieti-Pescara, Centro Studi sull’Invecchiamento, CESI-MeT, 66100 - Chieti, Italy 3 Dipartimento di Scienze Psicologiche, della Salute e del Territorio, University “G. d’Annunzio” Chieti-Pescara, Centro Studi sull’Invecchiamento, CESI-MeT, 66100 - Chieti, Italy 4 College of Health and Life Sciences, Brunel University London, UK 5 Department of Gynecology and Obstetrics, University of Rome “La Sapienza”, 00100 - Rome, Italy 6 Current address: Nouscom SRL 100 I-00128 Rome, Italy * These authors share senior authorship Correspondence to: Stefano Iacobelli, email: s.iacobelli@mediapharma.it Keywords: endosialin, ADC, sarcoma, duocarmycin, target therapy Received: May 11, 2017 Accepted: June 19, 2017 Published: July 22, 2017 ABSTRACT The endosialin/CD248/TEM1 receptor is expressed on the cell surface of tumor-associated stroma cells as well as in sarcoma and neuroblastoma cells. This receptor is emerging as an attractive molecule in diagnostics and therapeutics because of its expression across the stroma of many human tumors, the low to absent expression in normal tissues and accessibility from the vascular circulation. In this study, we present evidence of the preclinical efficacy of a novel Antibody-Drug Conjugate (ENDOS/ADC). It consists of a humanized endosialin monoclonal antibody, named hMP-E-8.3, conjugated to a potent duocarmycin derivative. In endosialin expressing cancer cell lines, this ENDOS/ADC showed a powerful, specific and target-dependent killing activity. High expression levels of endosialin in cells correlated with efficient internalization and cytotoxic effects in vitro . Efficacy studies demonstrated that ENDOS/ADC treatment led to a long-lasting tumor growth inhibition of a cell line-based model of human osteosarcoma. Taken together, our results demonstrate that endosialin is an attractive target in sarcoma and suggest that ENDOS/ADC has the potential to be developed into a bio-therapeutic agent for these malignancies.

This paper reports the improvement of ModSCA, a simulation model for compressed air systems (CAS). It was conceived to figure out how CAS work and what their weaknesses are, either in a design or in a retrofitting phase, from an energy conversion standpoint. It can be used to evaluate energy savings resulting from retrofitting works, before setting them in place, or to design a new compressed air system from scratches, hence the aim is not only educational, but it is functional to support facility managers. The original ModSCA frame is designed in Simulink in a modular way suitable for further developments, as the ones described in such a work. Special attention is focused on the following topics: (i) further development of the network layout, implementation of new modules (ii) to dynamically assess pressure drops in filters and (iii) to assess the energy role of mass losses. A new control scheme, based on Model Predictive Control (MPC), for variable speed compressors, has been included and some simulations are shown. Besides, an electrical equivalence of the CAS is introduced and its effectiveness shown, as a standalone simulation; this approach shows to be suitable to be embedded in ModSCA, in a further release. The improvements in the behavior of the simulator against real complex system make it keen to be used both by industry and academia.

The TEM-1/Endosialin/CD248 receptor is expressed in the cell surface of tumor-associated stroma cells, as well as in sarcoma and neuroblastoma cells. This receptor is emerging as an attractive molecule in diagnostics and therapeutics because of its expression across the stroma of many human tumors, the low to absent expression in normal tissues and accessibility from the vascular circulation. In this study, we present evidence of the preclinical efficacy of a novel Antibody-Drug Conjugate (ADC). It consists of a humanized TEM-1 monoclonal antibody (E.8-3) conjugated to a highly potent payload (TEM-1-ADC). In TEM-1 expressing cancer cell lines, this TEM-1-ADC demonstrated a powerful, specific and target-dependent killing activity. High expression levels of TEM-1 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro. Efficacy studies demonstrated that TEM-1-ADC treatment leads to a long lasting tumor growth inhibition of cell line-based models of human sarcoma. Taken together, our results demonstrated that TEM-1 is an attractive target in sarcoma and suggest that TEM-1-ADC has the potential to be developed into a biotherapeutic agent in these malignancies. Citation Format: Gianluca Sala, Stefano Iacobelli, Emily Capone, Enza PIccolo, Jean-Fred Sauniere, Vanessa Vannucci Douet. Development of a novel antibody-drug conjugate targeting endosialin/TEM-1: potent antitumor activity in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 40. doi:10.1158/1538-7445.AM2017-40

Over the past decade, we have witnessed a significant increase in the clinical development of targeted therapeutics for the treatment of cancer. Smart drugs have been designed to boost the activity of traditional chemotherapy by selectively blocking signaling pathways on which cancer cells rely. A common feature of all these drugs is that they, directly or indirectly, prevent AKT or ERK activation, i.e. those signaling kinases mainly involved in the control of the critical oncogenic programs, including proliferation, migration, invasion and survival. A large group of these compounds is now routinely used in the clinical setting, including EGFR and HER-2 inhibitors (Gefitinib, Erlotinib and Lapatinib), BRAF inhibitor (Vemurafenib) and MEK1/2 inhibitor (Trametinib) and monoclonal antibodies either naked or in the form of drug-conjugates, targeting HER-2 (Trastuzumab, Pertuzumab and T-DM1), EGFR (Cetuximab and Panitunumab) and VEGF (Bevacizumab). Along these approved compounds, many others are currently under development, including a large number of PI3K and BRAF/MEK inhibitors. Despite promising results at both the preclinical and clinical level, researchers and clinicians very soon had to reduce their enthusiasms once they encountered the problem of acquired drug resistance. Indeed, it is now an accepted paradigm that the prolonged use of these kind of inhibitors, and the resulting block of corresponding signaling pathways often leads to the relief of physiological feedback control mechanisms. This results in the activation of compensatory signaling pathways to maintain the oncogenic signaling and thereby contribute to inhibitor resistance [1]. With this in mind, clinicians and scientists quickly convened that only combination therapies and personalized medicine strategies would be able to overcome drug resistance in cancer therapy. In this respect, studies from different groups have validated the concept that the HER-3 receptor is critically involved in drug resistance in multiple cancers, acting as the main hub for escape mechanisms. HER-3 receptor is the preferred dimerization partner for the HER-2 receptor, and initially considered a poor target for therapy due to its limited intrinsic kinase activity. However, over the past ten years the role of this receptor in tumor development and therapy resistance has become clear and is no longer matter of debate [2]. Recently, the pharmacological inhibition of both PI3K/AKT and BRAF/MEK axes has been shown to relieve the feedback suppression of HER-3 signaling and other receptor tyrosyne kinases (RTK)s, which attenuates the therapeutic response to these agents [3, 4]. Mechanistically, up-regulation of HER-3 pathway mainly occurs at transcriptional level and results in an increased expression of the receptor on the surface of the treated cells, as documented in melanoma, thyroid and breast cancer models. Thus, the upregulation of HER-3 expression and signaling serves as an adaptive pro-survival response to different inhibitors leading to drug resistance. In reciprocal manner, NRG-1, the most potent HER-3 ligand has been found to be released by tumor-associated stromal cells, further promoting the idea that the HER-3 activation is vital in sustaining tumor growth. We have recently extended the evidence in support of a role for the HER-3 pathway in drug resistance by showing that this receptor is upregulated in response to vemurafenib in a model of patient-derived colon cancer stem-like cells (CSC)s with documented BRAFV600E mutation [5]. We found that exogenous NRG-1β is able to stimulate colon CSCs and rescue the anti-proliferative effects of vemurafenib through the activation of HER-3. Importantly, we demonstrated that ligand dependent HER-3 activation is increased in vemurafenib treated cells and that this can be blocked by EV20, a humanized HER-3 antibody developed by our group [6, 7]. This suggests that HER-3 inhibition may be required to prevent escape from vemurafenib treatment. In summary, evidences accumulated by many research groups, including our own, indicate that concurrent inhibition of HER-3 and PI3K/MEK pathways can lead to an optimal inhibition of cancer growth. Several monoclonal antibodies targeting HER-3 are now being developed by major pharmaceutical companies and some of them entered clinical testing. Conclusive results expected from ongoing trials and from other future trials using combinations not yet investigated, will reveal the true potential of HER-3 inhibition in the clinical practice.

Incidence and death rate of Pancreatic Ductal Adenocarcinoma (PDAC) has increased in recent years, therefore the identification of novel targets for treatment is extremely important. Interactions between cancer and stromal cells are critically involved in tumor formation and development of metastasis. We have recently shown (manuscript in press) that PDAC cells secrete BAG3 that binds and activates macrophages trough a specific receptor (IFITM2), inducing their activation and the secretion of PDAC supporting factors. Treatment with a murine monoclonal antibody (AC-2) targeting BAG3 reduced tumor growth and impaired metastasis formation in mouse models. Here we report further characterization of the in vivo activity of the antibody. Moreover, we have now successfully generated humanized variants of the AC-2 antibody and report the in vitro and in vivo activity of the humanized antibodies. Both appear to have the ability to inhibit macrophage activation in vitro and reduce tumor growth in PDAC models to an extent comparable to the murine parental antibody (AC-2). In conclusion, we have identified a novel paracrine loop involved in PDAC growth and metastatic spreading and shown that its pharmacological blockage with an anti-BAG3 antibody has therapeutic potential for PDAC treatment. Citation Format: Vincenzo De Laurenzi, Alessandra Rosati, Gianluca Sala, Maria Caterina Turco. Targeting BAG3-dependent paracrine loop reduces growth and metastatic spreading of Pancreatic adenocarcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A14.

Background: Pancreatic cancer (PC) is one of the most lethal cancers and there is an urgent need to find out new therapeutic approaches. Deregulated signaling through ErbB-1 and ErbB-2, members of the epidermal growth factor receptor family, frequently occurs in PC. However, attempts aiming at inhibiting ErbB-1 and ErbB-2 have revealed only a modest impact on disease outcome. A growing body of evidence suggests that ErbB-3 and its ligand NRG-1β are key players in driving oncogenic signaling and resistance to targeted therapy in PC. We have recently developed a novel humanized ErbB-3 antibody, named EV20, which displayed a potent antitumor activity by promoting receptor downregulation in vitro and in vivo. Here we hypothesized that targeting of ErbB-3 with EV20 would enhance the therapeutic effect of ErbB-2 inhibitors in PC. Materials and Methods: Three different PC cell lines (BxPC-3, HPAF II and SW1990) were chosen based on their metastatic origin, genetic background (KRAS status) and responsiveness to erlotinib. The expression of ErbB-1, ErbB-2 and ErbB-3 was analyzed by FACS and WB. The effects of trastuzumab, cetuximab, EV20 and their combinations on NRG-1β-induced activation of the ErbB3/PI3K/Akt axis, cell proliferation, and in vivo tumor growth were evaluated by standard procedures. Results: ErbB-1 was overexpressed by all the cell lines, whereas the expression levels of ErbB-2 and ErbB-3 were moderate in HPAFII cells and low in BxPC-3 and SW1990 cells. NRG-1β was more effective than EGF (an ErbB-1 ligand) in activating ErbB-3 downstream signaling, which was not inhibited by cetuximab, despite a high ErbB-1 expression in the cells. Conversely, inhibition of ErbB-2 and/or ErbB-3 resulted in a marked suppression of NRG-1β-driven Akt activation. Finally, EV20 in combination with trastuzumab exerted a superior antitumor activity compared to single agent alone in terms of cell proliferation and growth of PC xenografts in nude mice. Conclusions: Dual targeting of ErbB-2 and ErbB-3 could represent a newer therapeutic approach in PC. Note: This abstract was not presented at the meeting. Citation Format: Gianluca Sala, Reza Ghasemi, Emily Capone, Ilario Giovanni Rapposelli, Sara Traini, Cosmo Rossi, Pier Giorgio Natali, Stefano Iacobelli. Dual targeting of ErbB-2 and ErbB-3: A new potential strategy for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5437. doi:10.1158/1538-7445.AM2014-5437

ErbB-3 (HER-3) receptor is involved in tumor progression and resistance to therapy. Development of specific inhibitors impairing the activity of ErbB-3 is an attractive tool for cancer therapeutics. MP-RM-1, a murine monoclonal antibody targeting human ErbB-3, has shown anticancer activity in preclinical models. With the aim to provide novel candidates for clinical use, we have successfully generated a humanized version of MP-RM-1. The humanized antibody, named EV20, abrogates both ligand-dependent and ligand-independent receptor signaling of several tumor cell types, strongly promotes ErbB-3 down-regulation, and efficiently and rapidly internalizes into tumor cells. Furthermore, treatment with EV20 significantly inhibits growth of xenografts originating from prostatic, ovarian, and pancreatic cancers as well as melanoma in nude mice. In conclusion, we provide a novel candidate for ErbB-3-targeted cancer therapy.