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Data Assimilation can improve forecast accuracy of flood inundation models by an optimal combination of uncertain model simulations and observations. Particle Filter (PF) has gained interest in the research community for its ability of dealing with non-linear systems and with any kind of observation and model error distributions. Using PF, one of the most difficult issues to deal with are degeneracy and sample impoverishment. In this study, we have investigated a novel approach, based on a Tempered Particle Filter (TPF), aiming to circumvent these issues, and increase the persistence in time of the assimilation benefits. Flood probabilistic maps derived from Synthetic Aperture Radar (SAR)data are assimilated into a flood forecasting model. The process is iterative and includes a particle mutation in order to keep diversity within the ensemble. Results show an improvement of the model forecast accuracy as a result of the assimilation with respect to the ensemble without any assimilation (Open Loop, OL): on average the Root Mean Square Error decreases by 80% at the assimilation time and by 60% 2 days after the assimilation.A comparison with a standard PF, the Sequential Importance Sampling (SIS), where degeneracy occurred, is carried out. Results are similar at the assimilation time but the increase of performance using the TPF are lasting longer. For instance, TPF-based RMSE are still lower than the OL RMSE 3 days after the assimilation, while the SIS-based RMSE becomes larger than the RMSE-OL 2 days after the assimilation.

Background: CDK4/6 inhibitors have been approved in combination with endocrine therapy for the treatment of estrogen receptor positive (ER+) metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors. Results: p21-activated kinase 1 (PAK1) is a serine/threonine kinase amplified in several human cancer types. Specifically, the survey of TCGA for copy number alterations and/or expression showed PAK1 amplification and/or overexpression in ~23% of ER+ breast cancer. To test the hypothesis that PAK1-amplification is related to CDK4/6 inhibitors resistance, we generated two ER+ breast cancer cell lines T47D (harbouring PAK1 amplification) and MCF7 (non PAK1-amplified) resistant to the combination of fulvestrant (ER downregulator) and abemaciclib (CDK4/6 inhibitor), namely T47DFAR and MCF7FAR. T47DFAR and MCF7FAR were obtained, exposing the parental cells to increasing doses of drugs. Interestingly, only the T47DFAR but not the MCF7FAR showed higher level of PAK1 expression and activation compared to the parental cell line. PAK1 pathway activation was evaluated measuring by immunoblot the levels of phosphorylation of PAK1, MEK, ERK and Beta-Catenin. Moreover, the genomic knockdown of PAK1 through RNAi causes a decrease in cell proliferation of T47DFAR, but not of MCF7FAR and parental cells, suggesting the role of PAK1 as a possible mediator of drug resistance. Conversely, overexpression of PAK1 in MCF7 and T47D parental cells was able to generate resistance to the drug combination. PAK-1 has shown to have a different role in the cytosol as pathways mediator and in the nucleus as transcriptional factor. Our data obtained by subcellular fractionation showed that after treatment with fulvestrant-abemaciclib, PAK1 protein accumulates into the cytosol in T47D-FAR compared to parental T47D, probably mediating an enhancement in cytoplasmatic cell signalling and pathway activation. To univocally demonstrate that PAK1 mediates tumor resistance to fulvestrant-abemaciclib combination, we are generating data from RNA-Seq and whole exome sequencing from T47DFAR and MCF7FAR and the relative parental cell lines. Also, we will assess the efficacy of PAK pharmacological inhibition in overcoming such resistance. Conclusions: In conclusion, our data suggest the role of PAK1 as a novel therapeutic target in ER+/PAK1-amplified breast cancer model resistant to endocrine therapy and CDK4/6 inhibitors. Citation Format: Stefania Belli, Alberto Servetto, Concetta Di Mauro, Daniela Esposito, Ada Pesapane, Fabiana Napolitano, Antonio Santaniello, Pietro De Placido, Priscilla Cascetta, Annachiara Carratù, Eleonora Mozzillo, Roberta Marciano, Roberto Bianco, Luigi Formisano. Pak1 as a novel mediator of resistance to endocrine therapy and CDK4/6 inhibitors in ER+/PAK-1amplified breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-08.

Operational services in the fields of flood monitoring and prevention are benefitting from the large scale and systematic availability of synthetic aperture radar (SAR) data. The main advantages of SAR data are that they provide synoptic views over wide areas, day and night and all-weather condition acquisitions and a reliable data acquisition schedule. Satellite SAR data availability has increased over the past few years due to renewed efforts of several space agencies to put in place new satellite constellations. The latter enable the reduction of the satellite time access to areas of interest and provide enriched information with increased spatial resolution as well as variable polarizations and frequencies. The current situation tells us that there are regions in the world and land cover classes where SAR-derived flood maps are very reliable and accurate, but others where uncertainty is still very high, or where SAR is even unable to provide flood extent information. Therefore, the aim of this paper is to provide an overall picture of SAR-based floodwater mapping algorithms and their suitability for operational applications.

Data assimilation uses observation for updating model variables and improving model output accuracy. In this study, flood extent information derived from Earth Observation data (namely Synthetic Aperture Radar images) are assimilated into a loosely coupled flood inundation forecasting system via a Particle Filter (PF). A previous study based on a synthetic experiment has shown the validity and efficiency of a recently developed PF-based assimilation framework allowing to effectively integrate remote sensing-derived probabilistic flood inundation maps into a coupled hydrologic-hydraulic model. One of the main limitations of this recent framework based on sequential importance sampling is the sample degeneracy and impoverishment, as particles loose diversity and only few of them keep a substantial importance weight in the posterior distribution. In order to circumvent this limitation, a new methodology is adopted and evaluated: a tempered particle filter. The main idea is to update a set of state variables, namely through a smooth transition (iterative and adaptative process). To do so, the likelihood is factorized using small tempering factors. Each iteration includes subsequent resampling and mutation steps using a Monte Carlo Metropolis Hasting algorithm. The mutation step is required to regain diversity between the particles after the resampling. The new methodology is tested using synthetic twin experiments and the results are compared to the one obtained with the previous approach. The new proposed method enables to substantially improve the predictions of streamflow and water levels within the hydraulic domain at the assimilation time step. Moreover, the preliminary results show that these improvements are longer lasting. The proposed tempered particle filter also helps in keeping more diversity within the ensemble.

Coupled hydrologic and hydraulic models represent powerful tools for simulating streamflow and water levels along the riverbed and in the floodplain. However, input data, model parameters, initial conditions and model structure represent sources of uncertainty that affect the reliability and accuracy of flood forecasts. Assimilation of satellite-based Synthetic Aperture Radar observations into a flood forecasting model are generally used to reduce such uncertainties. In this context, we evaluate how sequential assimilation of flood extent derived from synthetic aperture radar data can help in improving flood forecasts. In particular, we carried out twin experiments based on a synthetically generated data-set with controlled uncertainty. To this end, two assimilation methods are explored and compared: the Sequential Importance Sampling (standard method) and its enhanced method where a tempering coefficient is used to inflate the posterior probability (adapted method) and to reduce degeneracy. The experimental results show that the assimilation of SAR probabilistic flood maps significantly improves the predictions of streamflow and water elevation, thereby confirming the effectiveness of the data assimilation framework. In addition, the assimilation method significantly reduces the spatially averaged root mean square error of water levels with respect to the case without assimilation. The critical success index of predicted flood extent maps is significantly increased by the assimilation. While the standard method proves to be more accurate in estimating the water levels and streamflow at the assimilation time step, the adapted method enables a more persistent improvement of the forecasts. However, although the use of a tempering coefficient reduces the degeneracy problem, the accuracy of model simulation is lower at the assimilation time step.

Background: Recently, immune check-point inhibitors have shown efficacy in triple-negative breast cancer (TNBC). Since Hedgehog (Hh) signalling mediates crosstalk between breast cancer cells and tumor-infiltrating immune cells, we investigated the mechanisms by which Hh can modulate PDL1 expression and the tumor-immune microenvironment. Methods: TNBC tumors from untreated patients were subjects to PDL1 and Gli1 expression analysis by IHC. The correlation of Hh pathway activation and PDL1 expression was assessed in TNBC cells treated with the SMO-inhibitor NVP-LDE225. The main aim was to study the PDL1/Gli1 cross-talk. Results: The expression of PDL1 and Gli1, the major indicator for the canonical Hh signaling activation, were assessed by IHC in a tissue microarray (TMA) of TNBC samples from 237 untreated patients. In 203/237 cases we could analyze both PDL1 and Gli1 protein expression. A significant correlation between PDL1 and Gli1 expression was found. Indeed, of 77/203 (38%) PDL1 positive tumors 42/77 (54%) expressed Gli1. In order to explore correlations between other Hh pathway members and PDL1 in TNBCs, we interrogated the open-access database TCGA; PDL1 positive TNBCs showed high levels of SMO and PTCH1, Hh pathway receptors (Q-value 0.018 and 0.010). To better understand the link between Hh pathway and PDL1, we selected a panel of TNBC cell lines; the pharmacological Hh pathway inhibition led to a reduction of PDL1 protein and mRNA. On the other hand, engineered cells harbouring Gli1 overexpression showed higher levels of PDL1. Therefore, we hypothesized that Hh pathway could be involved in the PDL1 transcriptional modulation. To address this issue, we performed a luciferase reporter assay and a ChIP analysis following by PCR amplification of the PDL1 gene promoter. We found that Gli1 binds the PDL1 promoter, indicating that Gli1 transcriptionally enhances PDL1 expression. Gli1 expression was evaluated also in 4T1 cells, a TNBC murine model; furthermore, in 4T1 cells PDL1 protein expression was inhibited by NVP-LDE225. In vivo experiment will be performed in Balb/C mice orthotopically xenografted with 4T1 cells to confirm the role of Hh pathway to modulate the PDL1 expression and the tumor microenviroment in vivo. Conclusions: Our results suggest that Hh pathway has a crucial role in cancer immune evasion trough PDL-1 modulation. Due to their ability to target both tumor cells and the tumor microenvironment, Hh inhibitors could represent promising therapeutics to be clinically investigated in Gli1 overexpressing TNBC patients. Citation Format: Pietro De Placido, Concetta Di Mauro, Daniela Esposito, Ada Pesapane, Stefania Belli, Fabiana Napolitano, Antonio Santaniello, Priscilla Cascetta, Annachiara Carratù, Eleonora Mozzillo, Roberta Marciano, Alberto Servetto, Roberto Bianco, Luigi Formisano. Hedgehog pathway is involved in cancer immune surveillance through PDL1 modulation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-17.

// Vincenza Ciaramella 1 , Carminia Maria Della Corte 1 , Concetta Di Mauro 2 , Stefano Tomassi 3 , Salvatore Di Maro 3 , Teresa Troiani 1 , Erika Martinelli 1 , Roberto Bianco 2 , Sandro Cosconati 3 , Riccardo Pierantoni 4 , Rosaria Meccariello 5 , Rosanna Chianese 4 , Fortunato Ciardiello 1 and Floriana Morgillo 1 1 Oncologia Medica, Dipartimento di Internistica Clinica e Sperimentale “F. Magrassi”, Universita degli Studi della Campania Luigi Vanvitelli, Naples, Italy 2 Oncologia Medica, Dipartimento di Medicina Clinica e Chirurgia, Universita degli Studi di Napoli “Federico II”, Naples, Italy 3 DISTABIF, Universita degli Studi della Campania Luigi Vanvitelli, Caserta, Italy 4 Dipartimento di Medicina Sperimentale sez ‘F. Bottazzi’, Universita degli Studi della Campania Luigi Vanvitelli, Naples, Italy 5 Dipartimento di Scienze Motorie e del Benessere, Universita di Napoli Parthenope, Napoli, Italy Correspondence to: Floriana Morgillo, email: florianamorgillo@yahoo.com Keywords: mesothelioma; KiSS1; biomarker; metastasis; EMT Received: February 03, 2018 Accepted: February 27, 2018 Published: April 10, 2018 ABSTRACT Purpose: Kisspeptin signaling, via its receptors GPR54, could be an essential players in the inhibition of mesothelioma progression, invasion and metastasis formation. The loss of KiSS1 by tumor cells has been associated with a metastatic phenotype but the mechanistic insights of this process are still unknown. Experimental design: The blockade of the metastatic process at early stage is a hot topic in cancer research. We studied the role of KiSS1 on proliferation, invasiveness, migration abilities of mesothelioma cell lines focusing on the effect on epithelial-to-mesenchymal transition (EMT). Results: Treatment with the KiSS1 peptide or with a synthesis peptide with longer half-life, the FTM080, significantly inhibited cell proliferation, migration and invasion of mesothelioma cell lines; the same treatment reduced the activity of MMP-2 and MMP-9 determining consequently a marked reduction in the invasiveness of primary tumors and metastases. Thespecificexpression of EMT markers, as E-caderin, Vimentin, Slug and Snail, suggested the inhibition of EMT after treatment with KiSS1 as well as the preservation of epithelial components. Conclusion: Our results support anti-proliferative effect of KiSS1 in cancer cells and suggest that targeting the KiSS1/GPR54 system may represent a novel therapeutic approach for mesothelioma.

The urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored cell membrane receptor that focuses urokinase (uPA) proteolytic activity on the cell surface. Its expression is increased in many human cancers, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and correlates with a poor prognosis and early invasion and metastasis. uPAR is able to control, through a cross-talk with tyrosine kinase receptors, the shift between tumor dormancy and proliferation, that usually precedes metastasis formation. Therefore, we investigated the role of uPAR expression in RAS mutated NSCLC and CRC cells. In this study we provided evidence, for the first time, that RAS mutational condition is functionally correlated to uPAR overexpression in NSCLC and CRC cancer cell lines and patient-derived tissue samples. Moreover, oncogenic features related to uPAR overexpression in RAS mutated NSCLC and CRC, such as adhesion, migration and metastatic process may be targeted, in vitro and in vivo, by new anti-uPAR small molecules, specific inhibitors of uPAR-vitronectin interaction. Therefore, anti-uPAR drugs could represent an effective pharmacological strategy for NSCLC and CRC patients carrying RAS mutations.

Several evidences suggest a marked angiogenic dependency in triple-negative breast cancer (TNBC) tumorigenesis and a potential sensitivity to anti-angiogenic agents. Herein, the putative role of Hedgehog (Hh) pathway in regulating TNBC-dependent angiogenesis was investigated. Background: Several evidences suggest a marked angiogenic dependency in triple-negative breast cancer (TNBC) tumorigenesis and a potential sensitivity to anti-angiogenic agents. Herein, the putative role of Hedgehog (Hh) pathway in regulating TNBC-dependent angiogenesis was investigated.Methods: Expression and regulation of the Hh pathway transcription factor glioma-associated oncogene homolog1 protein (GLI1) were studied on the endothelial compartment and on TNBC-initiated angiogenesis. To evaluate the translational relevance of our findings, the combination of paclitaxel with the Smo inhibitor NVP-LDE225 was tested in TNBC xenografted mice.Results: Tissue microarray analysis on 200 TNBC patients showed GLI1 overexpression paired with vascular endothelial growth factor receptor 2 (VEGFR2) expression. In vitro, Hh pathway promotes TNBC progression in an autocrine manner, regulating the VEGF/VEGFR2 loop on cancer cell surface, and in a paracrine manner, orchestrating tumour vascularisation. These effects were counteracted by Smo pharmacological inhibition. In TNBC xenografted mice, scheduling NVP-LDE225 rather than bevacizumab provided a better sustained inhibition of TNBC cells proliferation and endothelial cells organisation.Conclusions: This study identifies the Hh pathway as one of the main regulators of tumour angiogenesis in TNBC, thus suggesting Hh inhibition as a potential new anti-angiogenic therapeutic option to be clinically investigated in GLI1 overexpressing TNBC patients.

PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG) conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments.

// Carminia Maria Della Corte 1 , Vincenza Ciaramella 1 , Concetta Di Mauro 2 , Maria Domenica Castellone 3 , Federica Papaccio 1 , Morena Fasano 1 , Ferdinando Carlo Sasso 4 , Erika Martinelli 1 , Teresa Troiani 1 , Ferdinando De Vita 1 , Michele Orditura 1 , Roberto Bianco 2 , Fortunato Ciardiello 1 , Floriana Morgillo 1 1 Oncologia Medica, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale “F. Magrassi e A. Lanzara”, Seconda Universita degli Studi di Napoli, Naples, Italy 2 Oncologia Medica, Dipartimento di Medicina Clinica e Chirurgia, Universita degli studi di Napoli “Federico II”, Naples, Italy 3 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Istituto di Endocrinologia ed Oncologia Sperimentale “G. Salvatore” (IEOS), University of Naples “Federico II”, Naples, Italy 4 Medicina Interna, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale “F. Magrassi e A. Lanzara”, Seconda Universita degli Studi di Napoli, Naples, Italy Correspondence to: Floriana Morgillo, e-mail: florianamorgillo@yahoo.com Keywords: metformin, MEK, selumetinib, pimasertib, NSCLC Received: August 13, 2015 Accepted: November 25, 2015 Published: December 11, 2015 ABSTRACT Purpose: Metformin, widely used as antidiabetic drug, showed antitumoral effects expecially in combination with chemotherapy. Our group recently has demonstrated that metformin and gefitinib are synergistic in LKB1 -wild-type NSCLC cells. In these models, metformin as single agent induced an activation and phosphorylation of mitogen-activated-protein-kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization. Experimental design: Since single agent metformin enhances proliferating signals through the RAS/RAF/MAPK pathway, and several MEK inhibitors (MEK-I) demonstrated clinical efficacy in combination with other agents in NSCLC, we tested the effects of metformin plus MEK-I (selumetinib or pimasertib) on proliferation, invasiveness, migration abilities in vitro and in vivo in LKB1 positive NSCLC models harboring KRAS wild type and mutated gene. Results: The combination of metformin with MEK-I showed a strong anti-proliferative and proapoptotic effect in Calu-3, H1299, H358 and H1975 human NSCLC cell lines, independently from the KRAS mutational status. The combination reduced the metastatic behaviour of NSCLC cells, via a downregulation of GLI1 trascritional activity, thus affecting the transition from an epithelial to a mesenchymal phenotype. Metformin and MEK-Is combinations also decreased the production and activity of MMP-2 and MMP-9 by reducing the NF-jB (p65) binding to MMP-2 and MMP-9 promoters. Conclusions: Metformin potentiates the antitumor activity of MEK-Is in human LKB1 -wild-type NSCLC cell lines, independently from the KRAS mutational status, through GLI1 downregulation and by reducing the NF-jB (p65)-mediated transcription of MMP-2 and MMP-9.

Background The human DKC1 gene is causative of X-linked dyskeratosis congenita (X-DC), a syndrome characterized by mucocutaneous features, bone marrow failure, tumor susceptibility, perturbation of stem cell function, and premature aging. DKC1 is thought to produce a single protein, named dyskerin, which shows strict nucleolar localization and participates in at least two distinct nuclear functional complexes: the H/ACA small nucleolar ribonucleoproteic complex involved in RNA pseudouridylation and the active telomerase complex. Methods By bioinformatics and molecular analyses we identified a DKC1 splice variant able to encode a truncated form of dyskerin, confirmed its active expression in diverse human tissues by RT-PCR, and showed by immunoblotting and immunocytochemistry experiments that it actually encodes a novel protein. Stably transfected clones over-expressing the new isoform were analyzed for growth, morphology and adhesion properties. Results Our results show that DKC1 encodes a new alternatively spliced mRNA able to direct the synthesis of a variant dyskerin with unexpected cytoplasmic localization. Intriguingly, when over-expressed in HeLa cells, the new isoform promotes cell to cell and cell to substratum adhesion, increases the cell proliferation rate and leads to cytokeratin hyper-expression. Conclusions and general significance Our results highlight a novel degree of complexity and regulation of the human DKC1 gene and reveal that it can play a further, unpredicted role in cell adhesion. The identification of a dyskerin cytoplasmic variant reinforces the view that other mechanisms, in addition to telomere instability, can significantly contribute to the pathogenesis of the X-DC, and suggests that DKC1 nucleolar and cytoplasmic functions might cumulatively account for the plethora of manifestations displayed by this syndrome.

Inhibition of the mechanistic target of rapamycin (mTOR) is a promising treatment strategy for several cancer types. Rapamycin derivatives such as everolimus are allosteric mTOR inhibitors acting through interaction with the intracellular immunophilin FKBP12, a prolyl isomerase with different cellular functions. Although mTOR inhibitors have significantly improved survival of different cancer patients, resistance and lack of predictive factors of response remain unsolved issues. To elucidate the mechanisms of resistance to everolimus, we evaluated Met activation in everolimus-sensitive/resistant human cancer cells, in vitro and in vivo. Biochemical and computational analyses were performed. Everolimus-resistant cells were xenografted into mice (10/group) and studied for their response to everolimus and Met inhibitors. The statistical significance of the in vitro results was evaluated by Student's t test. Everolimus reduced Met phosphorylation in everolimus-sensitive cells. This event was mediated by the formation of a Met-FKBP12 complex, which in turn is disrupted by everolimus. Aberrant Met activation in everolimus-resistant cells and overexpression of wild-type/mutant Met caused everolimus resistance. Pharmacological inhibition and RNA silencing of Met are effective in condition of everolimus resistance (P

Resistance to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, often related to Ras or secondary EGFR mutations, is a relevant clinical issue in Non- Small Cell Lung Cancer (NSCLC). Although Src TK has been involved in such resistance, clinical development of its inhibitors has been so far limited. To better define the molecular targets of the Src TKIs saracatinib, dasatinib and bosutinib, we used a variety of in vitro/in vivo studies. Kinase assays supported by docking analysis demonstrated that all the compounds directly inhibit EGFR TK variants. However, in live cells only saracatinib efficiently reduced EGFR activation, while dasatinib was the most effective agent in inhibiting Src TK. Consistently, a pronounced anti-proliferative effect was achieved with saracatinib, in EGFR mutant cells, or with dasatinib, in wt EGFR/Ras mutant cells, poorly dependent on EGFR and erlotinib-resistant. We then identified the most effective drug combinations to overcome resistance to EGFR inhibitors, both in vitro and in nude mice: in T790M EGFR erlotinib-resistant cells, saracatinib with the anti-EGFR mAb cetuximab; in Ras mutant erlotinib-resistant models, dasatinib with the MEK inhibitor selumetinib. Src inhibitors may act with different mechanisms in NSCLCs, depending on EGFR/Ras mutational profile, and may be integrated with EGFR or MEK inhibitors for different cohorts of NSCLCs.