Renal Sympathetic Hyperactivity in Diabetes Is Modulated by 5-HT1D Receptor Activation via NO Pathway.

Renal vasculature, which is highly innervated by sympathetic fibers, contributes to cardiovascular homeostasis. This renal sympathetic outflow is inhibited by 5-HT in normoglycaemic rats. Considering that diabetes induces cardiovascular complications, we aimed to determine whether diabetic state modifies noradrenergic input at renal level and its serotonergic modulation in rats. Alloxan diabetic rats were anaesthetized (pentobarbital; 60 mg/kg i.p.) and prepared for in situ autoperfusion of the left kidney to continuously measure systemic blood pressure (SBP), heart rate (HR), and renal perfusion pressure (RPP). Electrical stimulation of renal sympathetic outflow induces frequency-dependent increases (Δ) in RPP (23.9 ± 2.1, 59.5 ± 1.9, and 80.5 ± 3.5 mm Hg at 2, 4, and 6 Hz, respectively), which were higher than in normoglycaemic rats, without modifying HR or SBP. Intraarterial bolus of 5-HT and 5-CT (5-HT1/5/7 agonist) reduced electrically induced ΔRPP. Only L-694,247 (5-HT1D agonist) reproduced 5-CT inhibition on sympathetic-induced vasoconstrictions, whereas it did not modify exogenous noradrenaline-induced ΔRPP. 5-CT inhibition was exclusively abolished by i.v. bolus of LY310762 (5-HT1D antagonist). An inhibitor of guanylyl cyclase, ODQ (i.v.), completely reversed the L-694,247 inhibitory effect. In conclusion, diabetes induces an enhancement in sympathetic-induced vasopressor responses at the renal level. Prejunctional 5-HT1D receptors, via the nitric oxide pathway, inhibit noradrenergic-induced vasoconstrictions in diabetic rats.

CRISPR-Cas9 Technology as a Tool to Target Gene Drivers in Cancer: Proof of Concept and New Opportunities to Treat Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy produced by a unique oncogenic event involving the constitutively active tyrosine-kinase (TK) BCR/ABL1. TK inhibitors (TKI) changed its prognosis and natural history. Unfortunately, ABL1 remains unaffected by TKIs. Leukemic stem cells (LSCs) remain, and resistant mutations arise during treatment. To address this problem, we have designed a therapeutic CRISPR-Cas9 deletion system targeting BCR/ABL1. The system was efficiently electroporated to cell lines, LSCs from a CML murine model, and LSCs from CML patients at diagnosis, generating a specific ABL1 null mutation at high efficiency and allowing the edited leukemic cells to be detected and tracked. The CRISPR-Cas9 deletion system triggered cell proliferation arrest and apoptosis in murine and human CML cell lines. Patient and murine-derived xenografts with CRISPR-edited LSCs in NOD SCID gamma niches revealed that normal multipotency and repopulation ability of CRISPR edited LSCs were fully restored. Normal hematopoiesis was restored, avoiding myeloid bias. To the best of our knowledge, we show for the first time how a CRISPR-Cas9 deletion system efficiently interrupts BCR/ABL1 oncogene in primary LSCs to bestow a therapeutic benefit. This study is a proof of concept for genome editing in all those diseases, like CML, sustained by a single oncogenic event, opening up new therapeutic opportunities.

A phenolic-rich extract from Ugni molinae berries reduces abnormal protein aggregation in a cellular model of Huntington's disease.

Accumulation of misfolded proteins in the brain is a common hallmark of most age-related neurodegenerative diseases. Previous studies from our group identified the presence of anti-inflammatory and antioxidant compounds in leaves derived from the Chilean berry Ugni molinae (murtilla), in addition to show a potent anti-aggregation activity in models of Alzheimer´s disease. However, possible beneficial effects of berry extracts of murtilla was not investigated. Here we evaluated the efficacy of fruit extracts from different genotypes of Chilean-native U. molinae on reducing protein aggregation using cellular models of Huntington´s disease and assess the correlation with their chemical composition. Berry extraction was performed by exhaustive maceration with increasing-polarity solvents. An unbiased automatic microscopy platform was used for cytotoxicity and protein aggregation studies in HEK293 cells using polyglutamine-EGFP fusion proteins, followed by secondary validation using biochemical assays. Phenolic-rich extracts from murtilla berries of the 19-1 genotype (ETE 19-1) significantly reduced polyglutamine peptide aggregation levels, correlating with the modulation in the expression levels of autophagy-related proteins. Using LC-MS and molecular network analysis we correlated the presence of flavonoids, phenolic acids, and ellagitannins with the protective effects of ETE 19-1 effects on protein aggregation. Overall, our results indicate the presence of bioactive components in ethanolic extracts from U. molinae berries that reduce the load of protein aggregates in living cells.

Biological significance of monoallelic and biallelic BIRC3 loss in del(11q) chronic lymphocytic leukemia progression.

BIRC3 is monoallelically deleted in up to 80% of chronic lymphocytic leukemia (CLL) cases harboring del(11q). In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which has been shown to be a marker for reduced survival in CLL. Nevertheless, the biological mechanisms by which these lesions could contribute to del(11q) CLL pathogenesis and progression are partially unexplored. We implemented the CRISPR/Cas9-editing system to generate isogenic CLL cell lines harboring del(11q) and/or BIRC3 mutations, modeling monoallelic and biallelic BIRC3 loss. Our results reveal that monoallelic BIRC3 deletion in del(11q) cells promotes non-canonical NF-κB signaling activation via RelB-p52 nuclear translocation, being these effects allelic dose-dependent and therefore further enhanced in del(11q) cells with biallelic BIRC3 loss. Moreover, we demonstrate ex vivo in primary cells that del(11q) cases including BIRC3 within their deleted region show evidence of non-canonical NF-κB activation which correlates with high BCL2 levels and enhanced sensitivity to venetoclax. Furthermore, our results show that BIRC3 mutations in del(11q) cells promote clonal advantage in vitro and accelerate leukemic progression in an in vivo xenograft model. Altogether, this work highlights the biological bases underlying disease progression of del(11q) CLL patients harboring BIRC3 deletion and mutation.

Dissecting the role of TP53 alterations in del(11q) chronic lymphocytic leukemia.

BACKGROUND: Several genetic alterations have been identified as driver events in chronic lymphocytic leukemia (CLL) pathogenesis and oncogenic evolution. Concurrent driver alterations usually coexist within the same tumoral clone, but how the cooperation of multiple genomic abnormalities contributes to disease progression remains poorly understood. Specifically, the biological and clinical consequences of concurrent high-risk alterations such as del(11q)/ATM-mutations and del(17p)/TP53-mutations have not been established. METHODS: We integrated next-generation sequencing (NGS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 techniques to characterize the in vitro and in vivo effects of concurrent monoallelic or biallelic ATM and/or TP53 alterations in CLL prognosis, clonal evolution, and therapy response. RESULTS: Targeted sequencing analysis of the co-occurrence of high-risk alterations in 271 CLLs revealed that biallelic inactivation of both ATM and TP53 was mutually exclusive, whereas monoallelic del(11q) and TP53 alterations significantly co-occurred in a subset of CLL patients with a highly adverse clinical outcome. We determined the biological effects of combined del(11q), ATM and/or TP53 mutations in CRISPR/Cas9-edited CLL cell lines. Our results showed that the combination of monoallelic del(11q) and TP53 mutations in CLL cells led to a clonal advantage in vitro and in in vivo clonal competition experiments, whereas CLL cells harboring biallelic ATM and TP53 loss failed to compete in in vivo xenotransplants. Furthermore, we demonstrated that CLL cell lines harboring del(11q) and TP53 mutations show only partial responses to B cell receptor signaling inhibitors, but may potentially benefit from ATR inhibition. CONCLUSIONS: Our work highlights that combined monoallelic del(11q) and TP53 alterations coordinately contribute to clonal advantage and shorter overall survival in CLL.

Changes in the Organosulfur and Polyphenol Compound Profiles of Black and Fresh Onion during Simulated Gastrointestinal Digestion.

This study aims to determine the changes in, and bioaccessibility of, polyphenols and organosulfur compounds (OSCs) during the simulated gastrointestinal digestion of black onion, a novel product derived from fresh onion by a combination of heat and humidity treatment, and to compare it with its fresh counterpart. Fresh and black onions were subjected to in-vitro gastrointestinal digestion, and their polyphenol and OSC profiles were determined by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Although to a lesser extent than in the fresh onion, the phenolic compounds in the black variety remained stable during the digestion process, presenting a higher bioaccessibility index (BI) with recovery corresponding to 41.1%, compared with that of fresh onion (23.5%). As for OSCs, apart from being more stable after the digestion process, with a BI of 83%, significantly higher quantities (21 times higher) were found in black onion than in fresh onion, suggesting that the black onion production process has a positive effect on the OSC content. Gallic acid, quercetin, isorhamnetin, and ɣ-glutamyl-S-(1-propenyl)-L-cysteine sulfoxide were the most bioaccessible compounds in fresh onion, while isorhamnetin, quercetin-diglucoside, ɣ-glutamyl-S-methyl-L-cysteine sulfoxide and methionine sulfoxide were found in black onion. These results indicate that OSCs and polyphenols are more bioaccessible in black onion than in fresh onion, indicating a positive effect of the processing treatment.

Bioaccessibility of Bioactive Compounds of 'Fresh Garlic' and 'Black Garlic' through In Vitro Gastrointestinal Digestion.

Numerous studies have reported health benefits associated with the consumption of fresh and black garlic, which are characterized by the presence of polyphenols and organosulfur compounds (OS). This study aims to analyze the bioaccessibility of the bioactive compounds in fresh and black garlic after in vitro gastrointestinal digestion by monitoring the individual profile of these compounds by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). Polyphenols decreased from the beginning of the digestive process, is mainly affected during intestinal digestion. Regarding the OS, the S-alk(en)yl-L-cysteine (SACs) derivatives were more influenced by the acidic conditions of the gastric digestion, while the γ-glutamyl-S-alk(en)yl-L-cysteine (GSAk) derivatives were more susceptible to intestinal digestion conditions in both the fresh and black garlic samples. In conclusion, after in vitro gastrointestinal digestion, the compounds with the highest bioaccessibility were vanillic acid (69%), caffeic acid (52%), γ-glutamyl-S-methyl-L-cysteine sulfoxide (GSMCS) (77%), and S-allylmercapto-L-cysteine (SAMC) (329%) in fresh garlic. Meanwhile, in black garlic, the main bioaccessible compounds were caffeic acid (65%), GSMCS (89%), methionine sulfoxide (262%), trans-S-(1-propenyl)-L-cysteine (151%), and SAMC (106%). The treatment (heating + humidity) to obtain black garlic exerted a positive effect on the bioaccessibility of OS compounds, 55.3% of them remaining available in black garlic, but only 15% in fresh garlic. Polyphenols showed different behavior regarding bioaccessibility.

Bioavailability of red wine and grape seed proanthocyanidins in rats.

This study explored plasma levels and urinary and fecal excretion of metabolites and microbial-derived catabolites over a 24 h period following the ingestion of red wine (RWP) or grape seed (GSP) proanthocyanidin-rich extracts by rats. In total, 35 structurally-related (epi)catechin metabolites (SREMs) and 5-carbon side chain ring fission metabolites (5C-RFMs) (phenyl-γ-valerolactones and phenylvaleric acids), and 50 phenolic acid and aromatic catabolites were detected after intakes of both extracts. The consumption of the RWP extract, but not the GSP extract, led to the appearance of a ∼200 nmol L-1 peak plasma concentration of SREMs formed from flavan-3-ol monomers. In contrast, ingestion of the GSPs, but not the RWPs, resulted in a substantial increase in microbiota-derived 5-carbon side chain ring fission metabolites (5C-RFMs) in plasma. 5C-RFMs, along with low molecular weight phenolic catabolites were detected in urine after ingestion of both extracts. The GSP and RWP extracts had respective mean degrees of polymerisation 5.9 and 6.5 subunits, and the RWP extract had an upper polymer size of 21 subunits compared to 44 subunits for the GSP extract. The differences in plasma metabolite profiles might, therefore, be a consequence of this polydispersity impacting on the microbiota-mediated rates of cleavage of the proanthocyanidin subunits and their subsequent metabolism and absorption. Urinary excretion of phenolic catabolites indicated that 11% of RWPs and 7% for GSPs were subjected to microbial degradation. In all probability these figures, rather than representing the percentage of proanthocyanidins that are completely degraded, indicate partial cleavage of monomer subunits producing a much higher percentage of shortened proanthocyanidin chains. Obtaining more detailed information on the in vivo fate of proanthocyanidins is challenging because of the difficulties in analysing unabsorbed parent proanthocyanidins and their partially degraded flavan-3-ol subunit chains in feces. Further progress awaits the development of improved purification and analytical techniques for proanthocyanidins and their use in feeding studies, and in vitro fecal and bacterial incubations, with radio and/or stable isotope-labelled substrates.

CRISPR/Cas9-generated models uncover therapeutic vulnerabilities of del(11q) CLL cells to dual BCR and PARP inhibition.

The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.

ETV6/RUNX1 Fusion Gene Abrogation Decreases the Oncogenicity of Tumour Cells in a Preclinical Model of Acute Lymphoblastic Leukaemia.

BACKGROUND: The t(12;21)(p13;q22), which fuses ETV6 and RUNX1 genes, is the most common genetic abnormality in children with B-cell precursor acute lymphoblastic leukaemia. The implication of the fusion protein in leukemogenesis seems to be clear. However, its role in the maintenance of the disease continues to be controversial. METHODS: Generation of an in vitroETV6/RUNX1 knock out model using the CRISPR/Cas9 gene editing system. Functional characterization by RNA sequencing, proliferation assays, apoptosis and pharmacologic studies, and generation of edited-cell xenograft model. RESULTS: The expression of ETV6/RUNX1 fusion gene was completely eliminated, thus generating a powerful model on which to study the role of the fusion gene in leukemic cells. The loss of fusion gene expression led to the deregulation of biological processes affecting survival such as apoptosis resistance and cell proliferation capacity. Tumour cells showed higher levels of apoptosis, lower proliferation rate and a greater sensitivity to PI3K inhibitors in vitro along as a decrease in tumour growth in xenografts models after ETV6/RUNX1 fusion gene abrogation. CONCLUSIONS: ETV6/RUNX1 fusion protein seems to play an important role in the maintenance of the leukemic phenotype and could thus become a potential therapeutic target.

Preclinical Efficacy of Endoglin-Targeting Antibody-Drug Conjugates for the Treatment of Ewing Sarcoma.

PURPOSE: Endoglin (ENG; CD105) is a coreceptor of the TGFß family that is highly expressed in proliferating endothelial cells. Often coopted by cancer cells, ENG can lead to neo-angiogenesis and vasculogenic mimicry in aggressive malignancies. It exists both as a transmembrane cell surface protein, where it primarily interacts with TGFß, and as a soluble matricellular protein (sENG) when cleaved by matrix metalloproteinase 14 (MMP14). High ENG expression has been associated with poor prognosis in Ewing sarcoma, an aggressive bone cancer that primarily occurs in adolescents and young adults. However, the therapeutic value of ENG targeting has not been fully explored in this disease. EXPERIMENTAL DESIGN: We characterized the expression pattern of transmembrane ENG, sENG, and MMP14 in preclinical and clinical samples. Subsequently, the antineoplastic potential of two novel ENG-targeting monoclonal antibody-drug conjugates (ADC), OMTX503 and OMTX703, which differed only by their drug payload (nigrin-b A chain and cytolysin, respectively), was assessed in cell lines and preclinical animal models of Ewing sarcoma. RESULTS: Both ADCs suppressed cell proliferation in proportion to the endogenous levels of ENG observed in vitro. Moreover, the ADCs significantly delayed tumor growth in Ewing sarcoma cell line-derived xenografts and patient-derived xenografts in a dose-dependent manner. CONCLUSIONS: Taken together, these studies demonstrate potent preclinical activity of first-in-class anti-ENG ADCs as a nascent strategy to eradicate Ewing sarcoma.

Sub-hyaloid and sub-internal limiting membrane macular hemorrhage after laser exposure at music festival: a case report.

PURPOSE: To describe the first case of a combined sub-hyaloid and sub-internal limiting membrane macular hemorrhage after recreational laser exposure. METHODS: A 23-year-old patient presented with an acute loss of vision in his right eye (OD) immediately after a brief exposure to a laser beam at a music festival. Color photography, spectral-domain optical coherence tomography (SD-OCT), and multifocal electroretinogram (mfERG) were performed for diagnosis and follow-up. RESULTS: On presentation, visual acuity was 20/400 in the OD and 20/20 in the left eye. Posterior segment examination of his OD revealed a preretinal hemorrhage at the macula. SD-OCT images exposed a combined sub-hyaloid and sub-internal limiting membrane localization. Successful VPP with ILM peeling was carried out. Although sequential mfERG displayed almost complete restoration of the P1 amplitude 6 months posterior to VPP, SD-OCT demonstrated permanent damage to outer retinal layers. Final BCVA on OD was 20/30. CONCLUSION: The expanding and unregulated use of lasers in everyday life has created an increasing amount of cases of laser-induced retinopathy in recent years. Much more attention should be addressed in laser safety and awareness to prevent future ocular injuries. In acute phases of sub-hyaloid hemorrhages blocking direct visualization of the posterior pole, mfERG is a valuable tool to address initial macular pathology.

A critical evaluation of the use of gas chromatography- and high performance liquid chromatography-mass spectrometry techniques for the analysis of microbial metabolites in human urine after consumption of orange juice.

The present study compared and validated two analytical methods, HPLC-HRMS, and GC-MS using MSTFA as derivatization agent, for the analysis of microbiota-derived phenolic acids and aromatic compounds accumulating in urine, collected over a 24 h period after the consumption of 500 mL of orange juice. In addition, purification procedures using SDB-L and HLB solid phase cartridges were compared when HPLC-HRMS technique was used. Both HPLC-HRMS and GC-MS methodologies were successfully validated in terms of specificity, sensitivity, limit of detection and quantification, recovery and matrix effects. HPLC-HRMS, unlike GC-MS, does not require sample derivatization prior to analysis. GC-MS was not suitable for the analysis of phenolic sulfate and glucuronide metabolites because of their lack of volatility. These phase II metabolites could, however, be analysed by HPLC-HRMS which, as a consequence, provided more detailed and complete information on the phenolic compounds derived from microbiota-mediated degradation of orange juice (poly)phenols. Furthermore, the use of SDB-L and HLB cartridges for sample purification prior to HPLC-HRMS analysis is suitable for free phenolics and glucuronide metabolites but not sulfate derivatives. These findings highlight that the use of an inappropriate analytical protocol can adversely affect studies on the bioavailability of dietary (poly)phenols in which microbiota-derived phenolic catabolites play an important role.

Development and validation of an UHPLC-HRMS protocol for the analysis of flavan-3-ol metabolites and catabolites in urine, plasma and feces of rats fed a red wine proanthocyanidin extract.

This study developed, optimized and validated an ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) method to identify and quantify metabolites and microbial-derived catabolites in urine, plasma and feces of rats following ingestion of 50 mg of a red wine proanthocyanidin-rich extract. The method was validated for specificity, linearity, limit of detection (LD) and quantification (LQ), intra-day and inter-day precision, recovery and matrix effects, which were determined for 34 compounds in the three biological matrices. After method validation, three parent flavan-3-ols, four 5-carbon side chain ring fission metabolites, and 27 phenolic acid and aromatic catabolites were quantified in plasma, urine and feces after red wine proanthocyanidin intake. These results establish the value of the UHPLC-HRMS protocol in obtaining a detailed picture of proanthocyanidin metabolites and their microbial-derived catabolites, along with their phase II metabolites, in biological fluids of rat, and potentially in human clinical studies designed to evaluate the bioavailability of dietary flavan-3-ols.

The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia.

CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels. Notably, in a single edited cell-derived clone carrying a frame-shift mutation that prevents p210 oncoprotein expression, the effects were even more drastic, resulting in cell death. These edited cells were injected subcutaneously in immunosuppressed mice and tumor growth was followed for three weeks. BCR/ABL-edited cells developed smaller tumors than those originating from unedited Boff-p210 parental cells. Interestingly, the single edited cell-derived clone was unable to develop tumors, similar to what is observed with the parental Baf/3 cell line.CRISPR/Cas9 genomic editing technology allows the ablation of the BCR/ABL fusion gene, causing an absence of oncoprotein expression, and blocking its tumorigenic effects in vitro and in the in vivo xenograft model of CML. The future application of this approach in in vivo models of CML will allow us to more accurately assess the value of CRISPR/Cas9 technology as a new therapeutic tool that overcomes resistance to the usual treatments for CML patients.

Recent trends in the determination of biogenic amines in fermented beverages - A review.

Biogenic amines (BA) are generally considered as a food hazard, even though there is not a threshold for these biomolecules in the European legislation, except for histamine in fishery products. These compounds are formed during the storage and processing of certain foods through microbiological activity, and when present in high concentrations, could have toxicological effects, causing health problems in consumers, especially to sensitive persons. This fact, in addition to the economical concern involved, makes it necessary to control the amounts of biogenic amines in foods. For all these reasons, literature on biogenic amines in different food products, especially in fermented beverages, is extensive. This review provides an overview of the most recent trends in the determination of biogenic amines in fermented beverages focusing on novelty, improvement and optimization of analytical methods. Hence, the different sample treatment procedures (including derivatization), the most important analytical techniques and the most frequent applications are described and discussed. Although biogenic amines have been determined in wine and other fermented beverages for decades, new advancements and technical possibilities have allowed to increase the accuracy and sensitivity of analytical methods, in order to overcome the challenges posed by the complex matrices and their high intrinsic variability. Thus, the different purposes of BA determination (food safety, production process or food microbiology research) and the most widely employed analytical techniques have been reviewed.

The PARP inhibitor olaparib enhances the sensitivity of Ewing sarcoma to trabectedin.

Recent preclinical evidence has suggested that Ewing Sarcoma (ES) bearing EWSR1-ETS fusions could be particularly sensitive to PARP inhibitors (PARPinh) in combination with DNA damage repair (DDR) agents. Trabectedin is an antitumoral agent that modulates EWSR1-FLI1 transcriptional functions, causing DNA damage. Interestingly, PARP1 is also a transcriptional regulator of EWSR1-FLI1, and PARPinh disrupts the DDR machinery. Thus, given the impact and apparent specificity of both agents with regard to the DNA damage/DDR system and EWSR1-FLI1 activity in ES, we decided to explore the activity of combining PARPinh and Trabectedin in in vitro and in vivo experiments. The combination of Olaparib and Trabectedin was found to be highly synergistic, inhibiting cell proliferation, inducing apoptosis, and the accumulation of G2/M. The drug combination also enhanced γH2AX intranuclear accumulation as a result of DNA damage induction, DNA fragmentation and global DDR deregulation, while EWSR1-FLI1 target expression remained unaffected. The effect of the drug combination was corroborated in a mouse xenograft model of ES and, more importantly, in two ES patient-derived xenograft (PDX) models in which the tumors showed complete regression. In conclusion, the combination of the two agents leads to a biologically significant deregulation of the DDR machinery that elicits relevant antitumor activity in preclinical models and might represent a promising therapeutic tool that should be further explored for translation to the clinical setting.

Trabectedin efficacy in Ewing sarcoma is greatly increased by combination with anti-IGF signaling agents.

PURPOSE: Goal of this study was to identify mechanisms that limit efficacy of trabectedin (ET-743, Yondelis) in Ewing sarcoma (EWS), so as to develop a clinical applicable combination therapy. EXPERIMENTAL DESIGN: By chromatin immunoprecipitation, we analyzed EWS-FLI1 binding to the promoters of several target genes, such as TGFßR2, CD99, insulin-like growth factor receptor 1 (IGF1R), and IGF1, both in vitro and in xenografts treated with trabectedin or doxorubicin. Combined therapy with trabectedin and anti-IGF1R agents (AVE1642 HAb; OSI-906) was tested in vitro and in xenografts. RESULTS: We confirm that both trabectedin and doxorubicin were able to strongly reduce EWS-FLI1 (both type I and type II) binding to two representative target genes (TGFßR2 and CD99), both in vitro and in xenografts. However, trabectedin, but not doxorubicin, was also able to increase the occupancy of EWS-FLI1 to IGF1R promoters, leading to IGF1R upregulation. Inhibition of IGF1R either by the specific AVE1642 human antibody or by the dual IGF1R/insulin receptor inhibitor OSI-906 (Linsitinib) greatly potentiate the efficacy of trabectedin in the 13 EWS cell lines here considered as well as in TC-71 and 6647 xenografts. Combined therapy induced synergistic cytotoxic effects. Trabectedin and OSI-906 deliver complementary messages that likely converge on DNA-damage response and repair pathways. CONCLUSIONS: We showed that trabectedin may not only inhibit but also enhance the binding of EWS-FLI1 to certain target genes, leading to upregulation of IGF1R. We here provide the rationale for combining trabectedin to anti-IGF1R inhibitors.

Characterization of human mesenchymal stem cells from ewing sarcoma patients. Pathogenetic implications.

BACKGROUND: Ewing Sarcoma (EWS) is a mesenchymal-derived tumor that generally arises in bone and soft tissue. Intensive research regarding the pathogenesis of EWS has been insufficient to pinpoint the early events of Ewing sarcomagenesis. However, the Mesenchymal Stem Cell (MSC) is currently accepted as the most probable cell of origin. MATERIALS AND METHODS: In an initial study regarding a deep characterization of MSC obtained specifically from EWS patients (MSC-P), we compared them with MSC derived from healthy donors (MSC-HD) and EWS cell lines. We evaluated the presence of the EWS-FLI1 gene fusion and EWSR1 gene rearrangements in MSC-P. The presence of the EWS transcript was confirmed by q-RT-PCR. In order to determine early events possibly involved in malignant transformation, we used a multiparameter quantitative strategy that included both MSC immunophenotypic negative/positive markers, and EWS intrinsic phenotypical features. Markers CD105, CD90, CD34 and CD45 were confirmed in EWS samples. RESULTS: We determined that MSC-P lack the most prevalent gene fusion, EWSR1-FLI1 as well as EWSR1 gene rearrangements. Our study also revealed that MSC-P are more alike to MSC-HD than to EWS cells. Nonetheless, we also observed that EWS cells had a few overlapping features with MSC. As a relevant example, also MSC showed CD99 expression, hallmark of EWS diagnosis. However, we observed that, in contrast to EWS cells, MSC were not sensitive to the inhibition of CD99. CONCLUSIONS: In conclusion, our results suggest that MSC from EWS patients behave like MSC-HD and are phenotypically different from EWS cells, thus raising important questions regarding MSC role in sarcomagenesis.