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Recent studies have shown that G protein-coupled receptors (GPCRs), the largest signal-conveying receptor family, are targets for mutations occurring frequently in different cancer types. GPCR alterations associated with cancer development represent significant challenges for the discovery and the advancement of targeted therapeutics. Among the different molecules that can activate GPCRs, we focused on two molecules that exert their biological actions regulating many typical features of tumorigenesis such as cellular proliferation, survival, and invasion: somatostatin and melatonin. The modulation of signaling pathways, that involves these two molecules, opens an interesting scenario for cancer therapy, with the opportunity to act at different molecular levels. Therefore, the aim of this review is the analysis of the biological activity and the therapeutic potential of somatostatin and melatonin, displaying a high affinity for GPCRs, that interfere with cancer development and maintenance.

Parvovirus B19 (B19V), an ssDNA virus in the family Parvoviridae, is a human pathogenic virus, responsible for a wide range of clinical manifestations, still in need of effective and specific antivirals. DNA structures, including G-quadruplex (G4), have been recognised as relevant functional features in viral genomes, and small-molecule ligands binding to these structures are promising antiviral compounds. Bioinformatic tools predict the presence of potential G4 forming sequences (PQSs) in the genome of B19V, raising interest as targets for antiviral strategies. Predictions locate PQSs in the genomic terminal regions, in proximity to replicative origins. The actual propensity of these PQSs to form G4 structures was investigated by circular dichroism spectroscopic analysis on synthetic oligonucleotides of corresponding sequences. No signature of G4 structures was detected, and the interaction with the G4 ligand BRACO-19 (N,N&prime, (9-{[4-(dimethylamino)phenyl]amino}acridine-3,6-diyl)bis(3-pyrrolidin-1-ylpropanamide) did not appear consistent with the stabilisation of G4 structures. Any potential role of PQSs in the viral lifecycle was then assessed in an in vitro infection model system, by evaluating any variation in replication or expression of B19V in the presence of the G4 ligands BRACO-19 and pyridostatin. Neither showed a significant inhibitory activity on B19V replication or expression. Experimental challenge did not support bioinformatic predictions. The terminal regions of B19V are characterised by relevant sequence and symmetry constraints, which are functional to viral replication. Our experiments suggest that these impose a stringent requirement prevailing over the propensity of forming actual G4 structures.

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level, inducing the degradation of the target mRNA or translational repression. MiRNAs are involved in the control of a multiplicity of biological processes, and their absence or altered expression has been associated with a variety of human diseases, including cancer. Recently, extracellular miRNAs (ECmiRNAs) have been described as mediators of intercellular communication in multiple contexts, including tumor microenvironment. Cancer cells cooperate with stromal cells and elements of the extracellular matrix (ECM) to establish a comfortable niche to grow, to evade the immune system, and to expand. Within the tumor microenvironment, cells release ECmiRNAs and other factors in order to influence and hijack the physiological processes of surrounding cells, fostering tumor progression. Here, we discuss the role of miRNAs in the pathogenesis of multicomplex diseases, such as Alzheimer’s disease, obesity, and cancer, focusing on the contribution of both intracellular miRNAs, and of released ECmiRNAs in the establishment and development of cancer niche. We also review growing evidence suggesting the use of miRNAs as novel targets or potential tools for therapeutic applications.

Extracellular vesicles (EVs) have attracted interest as mediators of intercellular communication following the discovery that EVs contain RNA molecules, including non-coding RNA (ncRNA). Growing evidence for the enrichment of peculiar RNA species in specific EV subtypes has been demonstrated. ncRNAs, transferred from donor cells to recipient cells, confer to EVs the feature to regulate the expression of genes involved in differentiation, proliferation, apoptosis, and other biological processes. These multiple actions require accuracy in the isolation of RNA content from EVs and the methodologies used play a relevant role. In liver, EVs play a crucial role in regulating cell–cell communications and several pathophysiological events in the heterogeneous liver class of cells via horizontal transfer of their cargo. This review aims to discuss the rising role of EVs and their ncRNAs content in regulating specific aspects of hepatocellular carcinoma development, including tumorigenesis, angiogenesis, and tumor metastasis. We analyze the progress in EV-ncRNAs’ potential clinical applications as important diagnostic and prognostic biomarkers for liver conditions.

Parvovirus B19 (B19V), a single-stranded DNA virus in the family Parvoviridae, is a human pathogenic virus responsible for a wide range of clinical manifestations. Currently there is no approved antiviral therapy for parvovirus infection. The acyclic nucleoside phosphonate cidofovir (CDV) has been demonstrated to inhibit replication of B19V in vitro. The aim of the present study was to evaluate whether brincidofovir (BCV), a novel lipid conjugate of CDV, could also inhibit B19V replication. Experiments were carried out in erythroid progenitor cells (EPCs) and UT7/EpoS1 cells, infected with B19V and cultured in the presence of different concentrations of BCV and CDV for comparison. The dynamics of viral replication was evaluated by a qPCR-based assay and the extent of inhibition of viral replication exerted by the compounds determined, along with the effect of the compounds on cell viability and cell proliferation rates. Results confirmed that BCV showed significantly higher antiviral activity against B19V compared to CDV in both cell-based systems. For BCV, the calculated EC50 values were in the range 6.6–14.3 μM in EPCs and 0.22–0.63 μM in UT7/EpoS1 cells. In comparison, the EC50 values for CDV were >300 μM in EPCs and 16.1 μM in UT7/EpoS1 cells. Concurrently, the effects on cell viability were observed at a much higher concentration of BCV, with calculated CC50 values in the range 93.4–102.9 μM in EPCs and 59.9–66.8 μM in UT7/Epos1. The antiviral activity was observed specifically with the metabolically active stereoisomer of BCV suggesting that CDV-diphosphate, the metabolite of both BCV and CDV, was the active antiviral. Our results support a selective role for BCV in the inhibition of B19 viral replication.

Parvovirus B19 (B19V) is a human pathogenic virus associated with a wide range of clinical conditions. Currently, there are no recognized antiviral drugs for B19V treatment, therefore, efforts in the search for compounds inhibiting B19V replication are now being pursued. Coumarins (chromen-2-ones) are considered a privileged structure for designing novel orally bioavailable and non-peptidic antiviral agents. To further contribute to the development of new drugs against B19V, our research was focused on the synthesis, characterization and evaluation of antiviral activity of some new 3-(imidazo[2,1-b]thiazol-6-yl)-2H-chromen-2-one derivatives. The effects of the synthesized compounds on cell viability and viral replication were investigated by employing two relevant cellular systems, the myeloblastoid cell line UT7/EpoS1 and primary erythroid progenitor cells (EPCs). Some of the tested compounds showed inhibitory activity both on cell viability and on viral replication, depending on the cellular system. These results suggest that the mechanism involved in biological activity is sensitive to small structural changes and that it is possible to direct the activity of the 3-(imidazo[2,1-b]thiazol-6-yl)-2H-chromen-2-one core.

We have identified previously undiscovered BRAF in-frame deletions near the αC-helix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers. These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the β3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors dimer formation. Expression of L485-P490–deleted BRAF is able to transform NIH/3T3 cells in a BRAF dimer–dependent manner. BRAF homodimer is confirmed to be the dominant RAF dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions, LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive. Significance: This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer. Cancer Discov; 6(3); 300–15. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 217

Parvovirus B19 (B19V), a human pathogenic ssDNA virus in the Parvoviridae family, is characterized by a strict tropism towards erythroid progenitor cells in bone marrow. Infections commonly induce self-limiting clinical manifestations, as erythema infectiosum in children and arthropathies mainly in adults, but, in subjects with underlying hematological disorders or with immune system deficits, the viral-induced arrest of erythropoiesis can lead to severe clinical manifestations requiring hospitalization and supportive transfusion or passive immunization treatments.

none 5 no Parvovirus B19 (B19V) infection is restricted to erythroid progenitor cells (EPCs) of the human bone marrow, leading to transient arrest of erythropoiesis and severe complications mainly in subjects with underlying hematological disorders or with immune system deficits. Currently, there are no specific antiviral drugs for B19V treatment, but identification of compounds inhibiting B19V replication can be pursued by a drug repositioning strategy. In this frame, the present study investigates the activity of hydroxyurea (HU), the only disease-modifying therapy approved for sickle cell disease (SCD), towards B19V replication in the two relevant cellular systems, the UT7/EpoS1 cell line and EPCs. Results demonstrate that HU inhibits B19V replication with EC50 values of 96.2μM and 147.1μM in UT7/EpoS1 and EPCs, respectively, providing experimental evidence of the antiviral activity of HU towards B19V replication, and confirming the efficacy of a drug discovery process by drug repositioning strategy. The antiviral activity occurs in vitro at concentrations lower than those affecting cellular DNA replication and viability, and at levels measured in plasma samples of SCD patients undergoing HU therapy. HU might determine a dual beneficial effect on SCD patients, not only for the treatment of the disease but also towards a virus responsible for severe complications. Bonvicini, Francesca; Bua, Gloria; Conti, Ilaria; Manaresi, Elisabetta; Gallinella, Giorgio Bonvicini, Francesca; Bua, Gloria; Conti, Ilaria; Manaresi, Elisabetta; Gallinella, Giorgio

Central to genetic studies for Parvovirus B19 (B19V) is the availability of genomic clones that may possess functional competence and ability to generate infectious virus. In our study, we established a new model genetic system for Parvovirus B19. A synthetic approach was followed, by design of a reference genome sequence, by generation of a corresponding artificial construct and its molecular cloning in a complete and functional form, and by setup of an efficient strategy to generate infectious virus, via transfection in UT7/EpoS1 cells and amplification in erythroid progenitor cells. The synthetic genome was able to generate virus with biological properties paralleling those of native virus, its infectious activity being dependent on the preservation of self-complementarity and sequence heterogeneity within the terminal regions. A virus of defined genome sequence, obtained from controlled cell culture conditions, can constitute a reference tool for investigation of the structural and functional characteristics of the virus.

TPS195 Background: Outcomes with first-line (1L) chemotherapy for GC/GEJC are limited and new strategies to increase efficacy without added toxicity are needed. Avelumab, a human anti-PD-L1 IgG1 monoclonal antibody, is approved for metastatic Merkel cell carcinoma (US and EU) and advanced urothelial carcinoma after progression on platinum therapy (US). In phase 1 studies in advanced GC/GEJC, avelumab showed antitumor activity and a manageable safety profile, including in a subgroup of patients (pts) without progression on 1L chemotherapy who received avelumab as maintenance therapy. JAVELIN Gastric 100 (NCT02625610) is a global, randomized, open-label, phase 3 trial comparing maintenance therapy with avelumab vs continuation of 1L chemotherapy in pts with advanced GC/GEJC who have not progressed after 12 weeks of 1L oxaliplatin/fluoropyrimidine chemotherapy. Methods: Eligible pts have histologically confirmed, unresectable, measurable, locally advanced or metastatic GC/GEJC adenocarcinoma, no prior chemotherapy for advanced disease, no prior therapy targeting T-cell coregulatory proteins, and ECOG PS of 0–1. Pts are not preselected based on PD-L1 expression and pts with HER2+ tumors are excluded. Approximately 466 pts who achieve at least stable disease following 12 weeks of 1L oxaliplatin/fluoropyrimidine chemotherapy will be randomized 1:1 to receive maintenance therapy with either avelumab 10 mg/kg by IV infusion Q2W or continuation of 1L chemotherapy. Maintenance therapy is given until disease progression, unacceptable toxicity, or withdrawal. Primary endpoints are overall survival and progression-free survival. Secondary endpoints include best overall response, quality of life measures, safety (NCI-CTCAE v4.03), and tumor biomarkers. Responses are evaluated per RECIST 1.1 and adjudicated by independent review committee. Enrollment is ongoing at sites in North/South America, Asia-Pacific, and Europe. Clinical trial information: NCT02625610.

Nitric oxide (NO*) is a gaseous mediator synthesized by Nitric oxide sinthases. NO* is involved in the modulation of inflammation, but its role in airway inflammation remains controversial. We investigated the role of NO* in the synthesis of the chemok Nes Interleukin-8 and Monocyte Chemotactic Protein-1, and of Intercellular Adhesion Molecule-1 by human airway epithelial cells. normal human bronchial epithelial cells and the bronchial epithelial cell line BEAS-2B were used. Neterleukin-8 (IL-8) and Monocyte Chemotactic Protein-1 (MCP-1) secretion and Intercellular Adhesion Molecule-1 (ICAM-1) expression were measured by ELISA. mRNA was assessed by semiquantitative RTI-PCR. Neterleukin-8 secretion was significantly reduced after 24h incubation with the NO* donor, sodium nitroprusside. The effect was dose-dependent. Similar results were obta Ned with S-Nitroso-N-D,L-penicillam Ne and S-Nitroso-L-glutathione. Inhibition of endogenous NO* with the Nitric oxide synthase inhibitor N-Nitro-L-arg N Ne-methyl-esther caused an increase in IL-8 secretion by lypopolisaccharide- and cytok Ne-stimulated BEAS-2B cells. Sodium nitroprusside also caused a reduction in Monocyte Chemotactic Protein-1 secretion by both cell types. In contrast, Intercellular Adhesion Molecule-1 expression was upregulated by sodium NItroprusside. RTI-PCR results indícate that the modulation of protein levels was paralleled by modification in mRNA levels. NO* has divergent effects on the synthesis of different inflammatory mediators in human bronchial epithelial cells.

The chemokine system has evolved primarily to control the trafficking of leukocytes during immune or inflammatory responses. However, through their expression of chemokine ligands and receptors, cancers have commandeered various aspects of this host defence system in order to enhance their growth. Although engineered over-expression of some tumour-derived chemokines can stimulate host antitumour respones, this is unlikely to be the reason that tumour cells express them. Rather, a growing body of clinical and laboratory evidence indicates that cancer cells may secrete chemokines in order to attract host cells that supply the tumours with growth and angiogenic factors. In addition, chemokine receptor expression by tumour cells may permit them to use the host's pre-existing leukocyte trafficking system to invade target tissues during metastatic spread. Together, these observations suggest that therapies directed against chemokine ligands or receptors may be beneficial in cancer.

10501 Background: Options for patients (pts) with unresectable/metastatic STS are limited. While Dox is standard of care, Dox combinations have yet to improve overall survival (OS) over Dox alone. Olaratumab (IMC-3G3), a fully human monoclonal antibody, selectively binds PDGFRα (a signaling pathway implicated in STS), blocks ligand binding, and enhances Dox activity in preclinical sarcoma models. Methods: We conducted a phase Ib, open-label, randomized phase II study of Dox ± olaratumab in unresectable/metastatic STS (NCT01185964). Pts received Dox (75 mg/m2 Day 1) with (Arm A) or without (Arm B) olaratumab (15 mg/kg Days 1 and 8, every 21 days), a dose and schedule confirmed safe in phase Ib, for up to 8 cycles. In Arm A, olaratumab monotherapy continued after Dox until disease progression; in Arm B, pts could cross over to olaratumab at progression. The primary endpoint was progression-free survival (PFS; target hazard ratio [HR] = 0.67, 80% power, α = 0.2) with OS as a secondary endpoint. Results: Of 1...

Mutations in KRas, NRas, BRaf and NF-1 that activate the Ras and mitogen-activated protein kinase (MAPK) pathway are among the most common oncogenic drivers in many cancers, including melanoma, lung, colorectal, and pancreatic cancer. Two BRaf selective inhibitors, vemurafenib and dabrafenib, have been approved for the treatment of melanoma patients harboring the BRaf V600E/K mutation. However, both compounds have been reported to promote paradoxical MAPK pathway activation in BRaf wild-type cells through induction of active Raf dimers. Therefore, they are believed to be contraindicated for treatment of cancers with BRaf wild type background. In this study, we have identified and characterized a pyrido-pyrimidine derivative inhibitor of all three Raf isoforms. A whole-cell mass spectrum-based analysis revealed that LY3009120 binds to ARaf, BRaf and CRaf isoforms with similar affinity in cells with activating mutations of BRaf or KRas, while vemurafenib or dabrafenib have little or modest CRaf activity. Additionally, LY3009120 induces BRaf-CRaf heterodimerization, but inhibits the phosphorylation of downstream MEK and ERK, indicating that it effectively inhibits the kinase activity of BRaf-CRaf heterodimer. Due to its activity against the three Raf isoforms and dimer, LY3009120 induces minimal paradoxical pathway activation in NRas or KRas mutant cells. These unique pharmacological properties of LY3009120 further distinguish it from selective BRaf inhibitors by its physiologically-relevant activities against tumor cells with NRas or KRas mutations. LY3009120 inhibits MEK phosphorylation and cell proliferation in vitro, and exhibits anti-tumor activity in multiple xenograft models carrying mutations in BRaf, NRas or KRas. LY3009120 is also active against melanoma cells with acquired resistance to vemurafenib or dabrafenib in the setting of MAPK reactivation and cyclin D1 upregulation caused by RTK/Ras activation, BRaf splice variants, or NRas Q61K mutation. Collectively, our findings identify LY3009120 as a potentially best-in-class inhibitor of three Raf isoforms and Raf dimer, with activity against tumor cells with BRaf, NRas or KRas mutations, as well as melanoma cells with acquired resistance to current BRaf therapies. These unique features support investigation of LY3009120 in clinical studies. Citation Format: Sheng-Bin Peng, James Henry, Michael Kaufman, Wei-Ping Lu, Bryan D. Smith, Subha Vogeti, Scott Wise, Youyan Zhang, Robert Van Horn, Xiaoyi Zhang, Tinggui Yin, Vipin Yadav, Lysiane Huber, Lisa Kays, Jennie Walgren, Denis McCann, Phenil Patel, Sean Buchanan, Ilaria Conti, James J. Starling, Daniel L. Flynn. Identification of LY3009120 as a pan inhibitor of Raf isoforms and dimers with minimal paradoxical activation and activities against BRaf or Ras mutant tumor cells. [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 DDT02-02. doi:10.1158/1538-7445.AM2014-DDT02-02

Genetic analyses of cancer in humans indicate that chemokines and their receptors are unlikely to play direct roles in pathogenesis. However, these molecules have pleiotropic effects that impact on cancer pathobiology in animal models, and there is evidence that they may do the same in humans. Given their protean properties, chemokines could have tumor-promoting, tumor-suppressing activities, or either depending on context. An example is found in CCL2, a chemokine that attracts and activates mononuclear cells. In some settings, it stimulates host anti-tumor activities. However, tumor cells themselves secrete CCL2 suggesting that it has growth promoting effects. These have been documented in animal models and clinical epidemiological studies. If CCL2's protumorigenic activities can be validated, then CCL2 and its receptor CCR2 may be therapeutic targets in cancer.

Cell-derived microparticles (MP) are membrane fragments shed by virtually all eukaryotic cells upon activation or during apoptosis that play a significant role in physiologically relevant processes, including coagulation and inflammation. We investigated whether MP derived from monocytes/macrophages have the potential to modulate human airway epithelial cell activation. Monocytes/macrophages were isolated from the buffy coats of blood donors by Ficoll gradient centrifugation, followed by overnight culture of the mononuclear cell fraction. Adherent cells were washed and incubated with the calcium ionophore, A23187, or with histamine. The MP-containing supernatant was incubated with cells of the human bronchial epithelial line BEAS-2B and of the human alveolar line A549. IL-8, MCP-1, and ICAM-1 production was assessed by ELISA and by RT-PCR. In some experiments, monocytes/macrophages were stained with the fluorescent lipid intercalating dye PKH67, and the supernatant was analyzed by FACS. Stimulation of monocytes/macrophages with A23187 caused the release of particles that retain their fluorescent lipid intercalating label, indicating that they are derived from cell membranes. Incubation with A549 and BEAS-2B cells up-regulate IL-8 synthesis. Ultrafiltration and ultracentrifugation of the material abolished the effect, indicating that particulate matter, rather than soluble molecules, is responsible for it. Up-regulation of MCP-1 and ICAM-1 was also demonstrated in A549 cells. Similar results were obtained with histamine. Our data show that human monocytes/macrophages release MP that have the potential to sustain the innate immunity of the airway epithelium, as well as to contribute to the pathogenesis of inflammatory diseases of the lungs through up-regulation of proinflammatory mediators.

278 Background: We report the primary analysis of JAVELIN Gastric 100, which compared avelumab (anti–PD-L1) maintenance after 1L CTx vs continued CTx in patients (pts) with GC/GEJC. Methods: In this global, open-label, phase 3 trial (NCT02625610), eligible pts had previously untreated, unresectable, locally advanced/metastatic (LA/M) HER2− GC/GEJC. Pts without progressive disease (PD) after 12 weeks of 1L oxaliplatin/fluoropyrimidine induction were randomized 1:1 to avelumab 10 mg/kg Q2W switch maintenance or continued CTx, stratified by region (Asia vs non-Asia). Primary endpoint was overall survival (OS) post induction in all randomized or PD-L1+ (≥1% of tumor cells, 73-10 assay) pts. Results: 805 pts received induction CTx and 499 pts were randomized (avelumab, n = 249; CTx, n = 250). At data cutoff (Sep 13, 2019), minimum follow-up was 18 months. In the avelumab and CTx arms, median OS post induction/randomization was 10.4 months (95% CI 9.1-12.0) vs 10.9 months (95% CI 9.6-12.4), hazard ratio (HR) 0.91 (95% CI 0.74-1.11; p = 0.1779); 24-month OS rates were 22.1% (95% CI 16.8-28.0) vs 15.5% (95% CI 10.8-20.9), respectively. The HR for OS in PD-L1+ pts (n = 54) was 1.13 (95% CI 0.57-2.23). No OS trend was seen in Asian pts (n = 114; HR 0.90 [95% CI 0.59-1.36]) or other subgroups, except for a potential benefit with avelumab in pts with no metastatic sites at randomization (n = 60; HR 0.52 [95% CI 0.28-0.98]). Progression-free survival was similar between arms (HR 1.04 [95% CI 0.85-1.28]). In the avelumab vs CTx arms, objective response rates (post randomization only) were 13.3% (95% CI 9.3-18.1) vs 14.4% (95% CI 10.3-19.4), and 12-month rates for duration of response were 62.3% (95% CI 40.9-77.9) vs 28.4% (95% CI 13.2-45.7), respectively. Treatment-related adverse event rates (all grades/grade ≥3) were 61.3%/12.8% with avelumab and 77.3%/32.8% with CTx. Conclusions: Avelumab maintenance showed clinical activity and favorable safety vs continued CTx in pts with LA/M GE/GEJC; however, JAVELIN Gastric 100 did not meet its primary objective of demonstrating superior OS in the randomized or PD-L1+ population. Clinical trial information: NCT02625610.