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Enzyme-based electrochemical biosensors have been widely deployed for the detection of a range of contaminants in different food products due to their significant advantages over other (bio)sensing techniques. Nevertheless, their performance is greatly affected by the sample matrix itself or by the matrix they are presented with in pretreated samples, both of which can impact the accuracy as well as the sensitivity of the measurements. Therefore, and in order to acquire reliable and accurate measurements, matrix effects and their influence on sensor performance should be taken into consideration. Herein, acetylcholinesterase (AChE)-modified electrochemical sensors were employed for the detection of pesticides in vegetable oils. Sensor interrogation with pretreated oil samples, spiked with carbofuran, revealed the inhibitory potential of the extracted matrix varies between different types of vegetable oil and their fatty acid content. In addition, synergies between the extracted matrix from different types of vegetable oils and the carbamate pesticide, carbofuran, were observed, which led to significant deviations of the sensor’s performance from its anticipated behavior in buffered solution. Taking the aforementioned into consideration, appropriate calibration curves for each type of vegetable oil were drafted, which allowed for the highly reproducible determination of different pesticide concentrations in pretreated real samples. Collectively, a better understanding of AChE inhibition by single or multiple contaminants present in vegetable oils was gained, which can find many applications in numerous fields, ranging from sensor development to the design of new pesticides and medicinal products.

Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition of resistance limits its therapeutic potential and therefore poses the need to identify new therapeutic combinations that could improve treatment outcomes. Despite the human kinome has proved to be an undisputable source of druggable targets, our knowledge remains confined to a limited fraction of it, with a multitude of under-investigated proteins yet to be characterised. Using a kinome-wide RNAi screen, we found that abrogation of pantothenate kinase 4 (PANK4) enhances the antiproliferative effects of TMZ in GBM in vitro. Further validation of our top-hit across various TMZ-resistant GBM cell models, patient-derived GBM cell lines and tissue samples, as well as in vivo studies, corroborated the potential translational significance of our findings. We showed that PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. Using a Tandem Mass Tag (TMT)-based quantitative proteomic approach, a comprehensive global protein dynamics analysis was undertaken to identify key response signatures upon PANK4 knockdown, in the presence or absence of TMZ. We revealed that silencing of PANK4 leads to a marked downregulation of a subset of proteins involved in cellular detoxification. More specifically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a synthetic vulnerability, focal point that can lead to critical cellular damage, accumulation of toxic metabolites, and subsequent cell death. Taken together, we unveil a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM.

Deep sequencing of human tumours has uncovered a previously unappreciated role for epigenetic regulators in tumorigenesis. H3K4 methyltransferase KMT2C/MLL3 is mutated in several solid malignancies, including more than 10% of breast tumours. To study the tumour suppressor role of KMT2C in breast cancer, we generated mouse models of Erbb2/Neu, Myc or PIK3CA-driven tumorigenesis, in which the Kmt2c locus is knocked out specifically in the luminal lineage of mouse mammary glands using the Cre recombinase. Kmt2c knock out mice develop tumours earlier, irrespective of the oncogene, assigning a bona fide tumour suppressor role for KMT2C in mammary tumorigenesis. Loss of Kmt2c induces extensive epigenetic and transcriptional changes, which lead to increased ERK1/2 activity, extracellular matrix re-organization, epithelial-to-mesenchymal transition and mitochondrial dysfunction, the latter associated with increased reactive oxygen species production. Loss of Kmt2c renders the Erbb2/Neu-driven tumours more responsive to lapatinib. Publicly available clinical datasets revealed an association of low Kmt2c gene expression and better long-term outcome. Collectively, our findings solidify the role of KMT2C as a tumour suppressor in breast cancer and identify dependencies that could be therapeutically amenable.

Glioblastoma (GBM) is an aggressive malignant primary brain tumor with limited therapeutic options. We show that the angiotensin II (AngII) type 2 receptor (AT

Bioprinting offers great potential for the fabrication of three-dimensional living tissues by the precise layer-by-layer printing of biological materials, including living cells and cell-laden hydrogels. The laser-induced forward transfer (LIFT) of cell-laden bioinks is one of the most promising laser-printing technologies enabling biofabrication. However, for it to be a viable bioprinting technology, bioink printability must be carefully examined. In this study, we used a time-resolved imaging system to study the cell-laden bioink droplet formation process in terms of the droplet size, velocity, and traveling distance. For this purpose, the bioinks were prepared using breast cancer cells with different cell concentrations to evaluate the effect of the cell concentration on the droplet formation process and the survival of the cells after printing. These bioinks were compared with cell-free bioinks under the same printing conditions to understand the effect of the particle physical properties on the droplet formation procedure. The morphology of the printed droplets indicated that it is possible to print uniform droplets for a wide range of cell concentrations. Overall, it is concluded that the laser fluence and the distance of the donor–receiver substrates play an important role in the printing impingement type; consequently, a careful adjustment of these parameters can lead to high-quality printing.

Glioblastoma (GBM) is an aggressive malignant primary brain tumor with limited therapeutic options. We show that the angiotensin II (AngII) type 2 receptor (AT 2 R) is a therapeutic target for GBM and that AngII, endogenously produced in GBM cells, promotes proliferation through AT 2 R. We repurposed EMA401, an AT 2 R antagonist originally developed as a peripherally restricted analgesic, for GBM and showed that it inhibits the proliferation of AT 2 R-expressing GBM spheroids and blocks their invasiveness and angiogenic capacity. The crystal structure of AT 2 R bound to EMA401 was determined and revealed the receptor to be in an active-like conformation with helix-VIII blocking G-protein or β-arrestin recruitment. The architecture and interactions of EMA401 in AT 2 R differ drastically from complexes of AT 2 R with other relevant compounds. To enhance central nervous system (CNS) penetration of EMA401, we exploited the crystal structure to design an angiopep-2–tethered EMA401 derivative, A3E. A3E exhibited enhanced CNS penetration, leading to reduced tumor volume, inhibition of proliferation, and increased levels of apoptosis in an orthotopic xenograft model of GBM.

RXRs are nuclear receptors acting as transcription regulators that control key cellular processes in all tissues. All type II nuclear receptors require RXRs for transcriptional activity by forming heterodimeric complexes. Recent whole-exome sequencing studies have identified the RXRα S427F hotspot mutation in 5% of the bladder cancer patients, which is always located at the interface of RXRα with its obligatory dimerization partners. Here, we show that mutation of S427 deregulates transcriptional activity of RXRα dimers, albeit with diverse allosteric mechanisms of action depending on its dimeric partner. S427F acts by allosteric mechanisms, which range from inducing the collapse of the binding pocket to allosteric stabilization of active co-activator competent RXRα states. Unexpectedly, RXR S427F heterodimerization leads to either loss- or gain-of-function complexes, in both cases likely compromising its tumor suppressor activity. This is the first report of a cancer-associated single amino acid substitution that affects the function of the mutant protein variably depending on its dimerization partner., A cancer-associated missense mutation in the nuclear receptor RXRα acts by allosteric mechanisms and impacts differently the activity of its dimers, depending on the dimerization partner.

Cancer treatment with chemotherapeutic drugs remains to be challenging to the physician due to limitations associated with lack of efficacy or high toxicities. Typically, chemotherapeutic drugs are administered intravenously, leading to high drug concentrations that drive efficacy but also lead to known side effects. Delivery of drugs through transdermal microneedles (MNs) has become an important alternative treatment approach. Such delivery options are well suited for chemotherapeutic drugs in which sustained levels would be desirable. In the context of developing a novel approach, laser induced forward transfer (LIFT) was applied for bioprinting of gemcitabine (Gem) to coat polymethylmethacrylate MNs. Gem, a chemotherapeutic agent used to treat various types of cancer, is a good candidate for MN-assisted transdermal delivery to improve the pharmacokinetics of Gem while reducing efficiency limitations. LIFT bioprinting of Gem for coating of MNs with different drug amounts and successful transdermal delivery in mice is presented in this study. Our approach produced reproducible, accurate, and uniform coatings of the drug on MN arrays, and on in vivo transdermal application of the coated MNs in mice, dose-proportional concentrations of Gem in the plasma of mice was achieved. The developed approach may be extended to several chemotherapeutics and provide advantages for metronomic drug dosing.

The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me3) and arginine-8 (H3R8me2). These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.

Η παρούσα μελέτη αποσκοπεί στην ανάδειξη των παραγόντων που σχετίζονται με τις παιδαγωγικές,διοικητικές και κοινωνικές διαστάσεις του ρόλου των στελεχών της εκπαίδευσης στον 21ο αιώνα.Θεωρώντας ότι η συμβολή των στελεχών στη διαμόρφωση και την επιτυχία των εκπαιδευτικώνδρώμενων και μεταρρυθμίσεων είναι καθοριστική, ωθούμαστε να ερευνήσουμε το ρόλο αυτό και τιςστάσεις τους σε σχέση με το επάγγελμά τους και κυρίως σε σχέση με την άσκηση διοικητικώνκαθηκόντων και ιεραρχικών δομών μέσα στο εκπαιδευτικό σύστημα.Στη διερεύνηση αυτή, χρησιμοποιήσαμε τόσο ποσοτικές όσο και ποιοτικές μεθόδους έρευνας καιπραγματοποιήθηκε τον Μάρτη του 2014, με τη χρήση ερωτηματολογίων.Αποτελέσματα- Συμπεράσματα της έρευναςΣτην έρευνα αυτή συμμετείχαν συνολικά 108 στελέχη της εκπαίδευσης, από τους 108, οι 58 (54%) ήτανάντρες και οι 50 (46%) ήταν γυναίκες.Το μεγαλύτερο ποσοστό των ερωτηθέντων που συμμετείχαν στην έρευνα ήταν από την Κρήτη και ήτανάντρες, ηλικίας 48 - 56 ετών.Όσον αφορά το επίπεδο μόρφωσης ήταν άπαντες κάτοχοι πτυχίου ΑΕΙ και δεν είχαν μεταπτυχιακότίτλο σπουδών.Όσον αφορά την ερώτηση «Έχετε παρακολουθήσει κάποια επιμόρφωση;» σχεδόν όλοι οισυμμετέχοντες απάντησαν θετικά.Στη συνέχεια στην ερώτηση «Είχατε πριν την εμπειρία του συνδικαλισμού;» οι περισσότεροιερωτώμενοι δεν είχαν κάποια τέτοια εμπειρία.Όσον αφορά το θεσμικό πλαίσιο επιλογής τους, όλοι οι υποψήφιοι επικαλούνται τη σχετικήΝομοθεσία.Στην ερώτηση «Δεχθήκατε κάποιου είδους σεμινάριο/ενημέρωση πριν από την τοποθέτησή σας;»παρατηρείται πως δεν είχαν κάποια ενημέρωση ή σεμινάριο πριν την τοποθέτησή τους.Ως προς την ανάληψη των καθηκόντων τους οι ερωτώμενοι αντιμετώπισαν ελάχιστες δυσκολίες.Οι ερωτώμενοι δεν αντιμετώπισαν καμία πίεση για θέματα που άπτονταν των καθηκόντων τουςσύμφωνα με τις απαντήσεις που δόθηκαν στην ερώτηση.Οι συμμετέχοντες φαίνεται πως διαπίστωσαν διάθεση συνεργασίας σε σχέση με το ρόλο και τακαθήκοντά τους βάση των αποτελεσμάτων.Στην ερώτηση «Έχετε ξεκάθαρη άποψη για τους στόχους της εκπαιδευτικής πολιτικής;», απάντησανπως υπήρξε ξεκάθαρη άποψη.Στην ερώτηση «Χρειάστηκε να συμβουλευτείτε ή να συγκρουστείτε με τις εκπαιδευτικές αρχέςπροκειμένου να επιτύχετε το στόχο σας;», φαίνεται πως δεν χρειάστηκε καθόλου να συμβεί κάτιτέτοιο.Στην ερώτηση «Τα διοικητικά σας καθήκοντα σας επέτρεπαν να έχετε ελευθερία κινήσεων/απόψεωνσε τέτοια ζητήματα;» δήλωσαν πως δεν υπήρχε ελευθερία κινήσεων γενικότερα.Στην ερώτηση «Ποιες δραστηριότητες αναπτύξατε στα πλαίσια του παιδαγωγικού/ συμβουλευτικούσας ρόλου;», οι ερωτώμενοι στοχεύουν στο να συμβουλεύουν, να εμψυχώνουν, και να παροτρύνουνγια επιμόρφωση.Οι συμμετέχοντες δεν έκαναν κάποια πρόταση στο Υπουργείο Παιδείας σχετικά με την εκπαιδευτικήκατάσταση.Στην ερώτηση σχετικά με το τι βαρύτητα πιστεύουν ότι έχουν στο σχεδιασμό της εκπαιδευτικήςπολιτικής, διαπιστώνεται μία πολύ μεγάλη απογοήτευση.Η πλειοψηφία των ερωτηθέντων υποστήριξε πως ναι ο ρόλος τους θα ήταν σημαντικότερος σε κάποιαζητήματα, αν το θεσμικό πλαίσιο ήταν διαφορετικό προσφέρει στους συναδέλφους του και γενικότερα στο εκπαιδευτικό του έργο.Στη ερώτηση «Ποια είναι η εμπειρία σας στην τάξη ως μάχιμος δάσκαλος», αρκετοί εκπαιδευτικοίσημείωσαν την εμπειρία τους σε χρόνια προϋπηρεσίας.Στην έρευνα παρατηρήθηκε ότι υπήρξε καλή σχέση μεταξύ των στελεχών και των Προϊσταμένων τουςκαι αυτό υπήρξε μια θετική επιρροή για τα στελέχη.Είναι σημαντικό επίσης να σημειωθεί πως καμία μεταρρύθμιση δεν έπαιξε ρόλο στην επιλογή τους.Ουσιαστικά το επάγγελμα του εκπαιδευτικού θα πρέπει να χαρακτηρίζεται πιο ορθά ως λειτούργημα.Πάντα προτιμώνται τα δημοκρατικά συμμετοχικά μοντέλα διοίκησης γιατί θεωρούνται πιοαποτελεσματικά

Glioblastoma (GBM) is a primary malignant brain tumor with poor clinical outcomes. Standard of care consists of surgical debulking followed by radiation and temozolomide, but the tumor invariably recurs, and median survival is only ~18 months. Repurposing drugs used for the treatment of other diseases is a promising avenue to identify novel treatments for this highly aggressive form of cancer. One such class of compounds is angiotensin II (AngII) receptor blockers, commonly used to control blood pressure. We show that ~80% of primary human GBM express the angiotensin II type 2 receptor (AT2R). In the presence of AngII, inhibition of AT2R using either PD123319 or EMA401 significantly inhibits GBM proliferation. This effect was lost in GBM cells with CRISPR/Cas9 mediated knockdown of AT2R. EMA401 inhibited invasion, angiogenesis, reduced GBM spheroid growth and induced apoptosis through caspase 3/7 activation. Furthermore, EMA401 induced changes in a number of growth regulatory pathways including apoptosis, DNA replication and focal adhesion. The crystal structure of AT2R bound to EMA401 revealed the receptor to be in an active-like conformation with helix-VIII blocking G protein or β-arrestin recruitment. We demonstrate that the architecture and interaction of EMA401 with AT2R differs drastically from complexes of AT2R with other compounds. Conjugation of EMA401 to angiopep-2 enhanced its blood brain barrier passage and reduced tumor volume in an orthotopic xenograft model of GBM. Targeting AT2R is a novel therapeutic strategy to treat GBM that should be explored in patients.

Liquid biopsies are emerging noninvasive diagnostic tools in cancer. MLL3 histone methyltransferase is a transcriptional regulator encoded by the tumor suppressor KMT2C gene. We evaluated the prognostic significance of KMT2C epigenetic changes in plasma of NSCLC patients. The detection of KMT2C promoter methylation in cfDNA predicts poor outcomes in NSCLC and merits further evaluation as a circulating epigenetic biomarker., MLL3 histone methyltransferase, encoded by the KMT2C gene, is a tumor suppressor that has an essential role in cell‐type‐specific gene expression. We evaluated the prognostic significance of KMT2C promoter methylation as a circulating epigenetic biomarker in plasma cell‐free DNA (cfDNA) in non‐small cell lung cancer (NSCLC). We examined the methylation status of KMT2C promoter using a novel highly specific and sensitive real‐time methylation‐specific PCR (MSP) assay in (a) operable NSCLC: 48 fresh‐frozen NSCLC tissues, their corresponding adjacent non‐neoplastic tissues, and 48 matched plasma samples; (b) metastatic NSCLC: 91 plasma samples; and (c) 60 plasma samples from healthy donors (HD). KMT2C promoter methylation in plasma cfDNA was detected in 7/48 (14.6%) patients with operable and in 18/91 (19.8%) patients with advanced NSCLC but in none (0/60, 0%) of the plasma samples from HD. In operable NSCLC, in corresponding adjacent non‐neoplastic tissue samples, KMT2C promoter methylation was detected in 3/48 (6.3%) cases. Moreover, in operable NSCLC, KMT2C promoter methylation in plasma cfDNA was related to reduced disease‐free survival (ΗR = 0.239; P = 0.001) and worse overall survival (OS; HR = 0.342, P = 0.023). In metastatic NSCLC, KMT2C promoter methylation in plasma cfDNA was related to worse progression‐free survival (PFS; HR = 0.431; P = 0.005) and worse OS (HR = 0.306; P

Carfilzomib (Cfz), an irreversible proteasome inhibitor licensed for relapsed/refractory myeloma, is associated with cardiotoxicity in humans. We sought to establish the optimal protocol of Cfz-induced cardiac dysfunction, to investigate the underlying molecular-signaling and, based on the findings, to evaluate the cardioprotective potency of metformin (Met). Mice were randomized into protocols 1 and 2 (control and Cfz for 1 and 2 consecutive days, respectively); protocols 3 and 4 (control and alternate doses of Cfz for 6 and 14 days, respectively); protocols 5A and 5B (control and Cfz, intermittent doses on days 0, 1 [5A] and 0, 1, 7, and 8 [5B] for 13 days); protocols 6A and 6B (pharmacological intervention; control, Cfz, Cfz+Met and Met for 2 and 6 days, respectively); and protocol 7 (bortezomib). Cfz was administered at 8 mg/kg (IP) and Met at 140 mg/kg (per os). Cfz resulted in significant reduction of proteasomal activity in heart and peripheral blood mononuclear cells in all protocols except protocols 5A and 5B. Echocardiography demonstrated that Cfz led to a significant fractional shortening (FS) depression in protocols 2 and 3, a borderline dysfunction in protocols 1 and 4, and had no detrimental effect on protocols 5A and 5B. Molecular analysis revealed that Cfz inhibited AMPKα/mTORC1 pathways derived from increased PP2A activity in protocol 2, whereas it additionally inhibited phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase pathway in protocol 3. Coadministration of Met prevented Cfz-induced FS reduction and restored AMPKα phosphorylation and autophagic signaling. Conclusively, Cfz decreased left ventricular function through increased PP2A activity and inhibition of AMPKα and its downstream autophagic targets, whereas Met represents a novel promising intervention against Cfz-induced cardiotoxicity.

We directly compared two different approaches used for Circulating Tumor Cell (CTC) isolation, a size-dependent microfluidic system versus an EpCAM-dependent positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in Head and Neck Squamous Cell Carcinoma (HNSCC). A size-dependent microfluidic device (Parsortix, ANGLE) and an EpCAM-dependent positive immune-magnetic isolation procedure were applied in parallel, using 10 mL PB from 50 HNSCC patients and 18 healthy donors. Total RNA was isolated from enriched CTCs and RT-qPCR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M (reference gene). Real time methylation specific PCR (MSP) was used to study the methylation status of RASSF1A and MLL3 genes. In identical blood draws, the label-free size-dependent CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-dependent CTC enrichment, since a significantly higher percentage of identical PB samples was found positive at the gene expression and DNA methylation level, while the specificity was not affected. Our results indicate that future studies focused on the evaluation of clinical utility of CTC molecular characterization in HNSCC should be based on size-dependent enrichment approaches.

The Notch signaling pathway has been implicated in homeostasis and disease, including cancer, in various tissues. Moreover, it has been involved both in stem cell maintenance and differentiation, in a context-dependent manner. Stem/progenitor cells, on the other hand, have long been suspected to be the cells of origin in various malignancies. In order to gain insight in the role of the Notch ligand Dll1 in mouse development, we generated a knock-in line expressing an inducible Cre recombinase. We have employed in vivo approaches in mice to genetically mark rare subpopulations of cells expressing Dll1 in various adult tissues. Moreover, we conditionally expressed a constitutively active Ras oncoprotein in these cells and showed that within days, mice develop squamous neoplasias in the skin, as well as in the stomach.

Bioprinting offers great potential for the fabrication of three-dimensional living tissues by the precise layer-by-layer printing of biological materials, including living cells and cell-laden hydrogels. The laser-induced forward transfer (LIFT) of cell-laden bioinks is one of the most promising laser-printing technologies enabling biofabrication. However, for it to be a viable bioprinting technology, bioink printability must be carefully examined. In this study, we used a time-resolved imaging system to study the cell-laden bioink droplet formation process in terms of the droplet size, velocity, and traveling distance. For this purpose, the bioinks were prepared using breast cancer cells with different cell concentrations to evaluate the effect of the cell concentration on the droplet formation process and the survival of the cells after printing. These bioinks were compared with cell-free bioinks under the same printing conditions to understand the effect of the particle physical properties on the droplet formation procedure. The morphology of the printed droplets indicated that it is possible to print uniform droplets for a wide range of cell concentrations. Overall, it is concluded that the laser fluence and the distance of the donor–receiver substrates play an important role in the printing impingement type, consequently, a careful adjustment of these parameters can lead to high-quality printing.

Using an efficient gene targeting approach, we developed a novel mouse line that expresses the tetracycline-controlled transactivator (tTA) from the constitutively active Eef1a1 locus in a Cre recombinase-inducible manner. The temporally and spatially controlled expression of the EF1-LSL-tTA knockin and activation of tTA-driven responder transgenes was tested using four transgenic lines that express Cre under tissue-specific promoters of the pancreas, mammary gland and other secretory tissues, as well as an interferon-inducible promoter. In all models, the endogenous Eef1a1 promoter facilitated a cell-type-specific activation of target genes at high levels without exogenous enhancer elements. The applicability of the EF1-LSL-tTA strain for biological experiments was tested in two studies related to mammary gland development and tumorigenesis. First, we validated the crucial role of active STAT5 as a survival factor for functionally differentiated epithelial cells by expressing a hyperactive STAT5 mutant in the mammary gland during postlactational remodeling. In a second experiment, we assessed the ability of the EF1-tTA to initiate tumor formation through upregulation of mutant KRAS. The collective results show that the EF1-LSL-tTA knockin line is a versatile genetic tool that can be applied to constitutively express transgenes in specific cell types to examine their biological functions at defined developmental stages.

Background Levosimendan (LEVO) an inodilator indicated for the treatment of heart failure exerts multifaceted cardioprotective effects. Case-studies indicate protection against doxorubicin (DXR)-induced cardiotoxicity, but this effect remains elusive. We have previously shown that LEVO exerts cardioprotection against DXR-induced cardiomyopathy in a rat in vivo model, in a PKA/PKG-dependent manner. Purpose We sought to elucidate the mechanism of LEVO's induced cardioprotection and clarify the contribution of PKG and PKA pathways converging onto phospholamban (PLN). Methods As previously observed, LEVO at a dose of 24μg/kg protects against DXR cardiotoxicity, with protein kinase B (Akt)/ endothelial nitric oxide synthase (eNOS) and protein kinase A and G (PKA/PKG) pathways emerging as the main contributors to cardioprotection. Moreover, phospholamban seems to be the end-target of the signaling cascade. To verify the contribution of phospholamban, phospholamban deficient mice (PLN−/−) were assigned to PLN−/−/DXR (18mg/kg) and PLN−/−/DXR+LEVO (acute) (LEVO bolus, 24 μg/kg) groups for 14 days. Echocardiographic analysis was conducted in all groups and protocols. Furthermore, in order to solidify the mechanism of LEVO-mediated cardioprotection, primary adult ventricular murine cardiomyocytes (AVMCs) were isolated and treated with doxorubicin or/and LEVO as well with L-NAME, DT-2 and H-89 (eNOS, PKG and PKA inhibitors, respectively) and cardiomyocyte-toxicity was assessed. Results In the transgenic PLN−/− mice, LEVO did not exert cardioprotection, whilst the co-administration of doxorubicin and levosimendan led to an impaired Left ventricular function [FS (%): PLN−/−/Control: 39.01±0.42 vs PLN−/−/DXR: 38.12±0.51 in (NS); PLN−/−/DXR+LEVO: 35.38±0.86 (**p Conclusions Single-dose LEVO prevented DXR cardiotoxicity through a cAMP-PKA-phospholamban pathway, highlighting the role of inotropy in DXR cardiotoxicity. These preclinical data can stand as promising grounds for further clinical investigations.

The Notch signaling pathway mediates cell-cell communication regulating cell differentiation and proliferation and cell fate decisions in various tissues. In the urinary bladder, Notch acts as a tumor suppressor in mice, while mutations in Notch pathway components have been identified in human bladder cancer as well. Here we report that the genetic inactivation of Notch in mice leads to downregulation of cell-cell and cell-ECM interaction components, including proteins previously implicated in interstitial cystitis/bladder pain syndrome (IC/BPS), structural defects and mucosal sloughing, inflammation, and leaky urine-blood barrier. Molecular profiling of ailing mouse bladders showed similarities with IC/BPS patient tissue, which also presented low Notch pathway activity as indicated by reduced expression of canonical Notch targets. Urothelial integrity was reconstituted upon exogenous reactivation of the Notch pathway, implying a direct involvement of Notch. Despite damage and inflammation, urothelial cells failed to proliferate, uncovering a possible role for α(4) integrin in urothelial homeostasis. Our data uncover a broad role for Notch in bladder homeostasis involving urothelial cell crosstalk with the microenvironment.

DNA damage response, DNA repair and genomic instability have been under study for their role in tumor initiation and progression for many years now. More recently, next-generation sequencing on cancer tissue from various patient cohorts have revealed mutations and epigenetic silencing of various genes encoding proteins with roles in these processes. These findings, together with the unequivocal role of DNA repair in therapeutic response, have fueled efforts toward the clinical exploitation of research findings. The successful example of PARP1/2 inhibitors has also supported these efforts and led to numerous preclinical and clinical trials with a large number of small molecules targeting various components involved in DNA repair singularly or in combination with other therapies. In this review, we focus on recent considerations related to DNA damage response and new DNA repair inhibition agents. We then discuss how immunotherapy can collaborate with these new drugs and how epigenetic drugs can rewire the activity of repair pathways and sensitize cancer cells to DNA repair inhibition therapies.

Aims Levosimendan (LEVO) a clinically-used inodilator, exerts multifaceted cardioprotective effects. Case-studies indicate protection against doxorubicin (DXR)-induced cardiotoxicity, but this effect remains obscure. We investigated the effect and mechanism of different regimens of levosimendan on sub-chronic and chronic doxorubicin cardiotoxicity. Methods and results Based on preliminary in vivo experiments, rats serving as a sub-chronic model of doxorubicin-cardiotoxicity and were divided into: Control (N/S-0.9%), DXR (18 mg/kg-cumulative), DXR+LEVO (LEVO, 24 μg/kg-cumulative), and DXR+LEVO (acute) (LEVO, 24 μg/kg-bolus) for 14 days. Protein kinase-B (Akt), endothelial nitric oxide synthase (eNOS), and protein kinase-A and G (PKA/PKG) pathways emerged as contributors to the cardioprotection, converging onto phospholamban (PLN). To verify the contribution of PLN, phospholamban knockout (PLN−/−) mice were assigned to PLN−/−/Control (N/S-0.9%), PLN−/−/DXR (18 mg/kg), and PLN−/−/DXR+LEVO (ac) for 14 days. Furthermore, female breast cancer-bearing (BC) mice were divided into: Control (normal saline 0.9%, N/S 0.9%), DXR (18 mg/kg), LEVO, and DXR+LEVO (LEVO, 24 μg/kg-bolus) for 28 days. Echocardiography was performed in all protocols. To elucidate levosimendan’s cardioprotective mechanism, primary cardiomyocytes were treated with doxorubicin or/and levosimendan and with N omega-nitro-L-arginine methyl ester (L-NAME), DT-2, and H-89 (eNOS, PKG, and PKA inhibitors, respectively); cardiomyocyte-toxicity was assessed. Single bolus administration of levosimendan abrogated DXR-induced cardiotoxicity and activated Akt/eNOS and cAMP-PKA/cGMP-PKG/PLN pathways but failed to exert cardioprotection in PLN−/− mice. Levosimendan’s cardioprotection was also evident in the BC model. Finally, in vitro PKA inhibition abrogated levosimendan-mediated cardioprotection, indicating that its cardioprotection is cAMP-PKA dependent, while levosimendan preponderated over milrinone and dobutamine, by ameliorating calcium overload. Conclusion Single dose levosimendan prevented doxorubicin cardiotoxicity through a cAMP-PKA-PLN pathway, highlighting the role of inotropy in doxorubicin cardiotoxicity.

A dicyanomethylene-4H-pyran based tumor-targeting and biothiol activatable fluorescent and colorimetric probe, named Biotin-Yellow, has been developed with unprecedented turn-on near-infrared fluorescence properties. Biotin-Yellow is non-toxic and can effectively recognize and discriminate human lung adenocarcinoma cells and urothelial (bladder) carcinoma cells from embryonic kidney cells and embryonic fibroblasts. Biotin-Yellow can also be employed for real-time bioimaging tracking of upregulated biothiol activity in cancer cells. The efficacy and biothiol responsiveness of Biotin-Yellow was further validated in vivo in zebrafish and mice xenografts.

In this paper, we present the development of a photonic biosensor device for cancer treatment monitoring as a complementary diagnostics tool. The proposed device combines multidisciplinary concepts from the photonic, nano-biochemical, micro-fluidic and reader/packaging platforms aiming to overcome limitations related to detection reliability, sensitivity, specificity, compactness and cost issues. The photonic sensor is based on an array of six asymmetric Mach Zender Interferometer (aMZI) waveguides on silicon nitride substrates and the sensing is performed by measuring the phase shift of the output signal, caused by the binding of the analyte on the functionalized aMZI surface. According to the morphological design of the waveguides, an improved sensitivity is achieved in comparison to the current technologies (

Genome‐wide studies in tumor cells have indicated that chromatin‐modifying proteins are commonly mutated in human cancers. The lysine‐specific methyltransferase 2C (KMT2C/MLL3) is a putative tumor suppressor in several epithelia and in myeloid cells. Here, we show that downregulation of KMT2C in bladder cancer cells leads to extensive changes in the epigenetic status and the expression of DNA damage response and DNA repair genes. More specifically, cells with low KMT2C activity are deficient in homologous recombination‐mediated double‐strand break DNA repair. Consequently, these cells suffer from substantially higher endogenous DNA damage and genomic instability. Finally, these cells seem to rely heavily on PARP1/2 for DNA repair, and treatment with the PARP1/2 inhibitor olaparib leads to synthetic lethality, suggesting that cancer cells with low KMT2C expression are attractive targets for therapies with PARP1/2 inhibitors.

Background HRAS is mutated or overexpressed in various cancers. Although numerous studies have demonstrated that HRAS mutations (HRASmut) promote chemoresistance in HNSCC, the effect of wild type HRAS overexpression (wtHRASov) on chemotherapy has not yet been studied. We sought: i. to investigate the repair capacity of wtHRASov tumors in cisplatin- induced damage and ii. to explore the cisplatin sensitizing effect of HRAS inhibitor tipifarnib. Methods Mutation/RNA and cisplatin IC50 data were retrieved from the Cancer Genome Atlas (TCGA) and COSMIC Cell Lines Project respectively. Modified Z-score was used to define HRAS overexpression. Patient derived xenografts (PDXs) were generated from treatment naive HNSCC patients. Mutational and expression profile of tumor grafts was identified by whole exome sequencing, IHC, qRT-PCR and western blot analysis. Cisplatin or cisplatin combined with tipifarnib was used for PDX’s treatment. Therapy response was monitored by sequential tumor volume measurements over time. Results Our analysis of available RNA and cisplatin IC50 data from HNSCC cell lines demonstrated that wild type HRAS levels are positively correlated with cisplatin resistance. To identify resistance signatures in wtHRASov tumors, we compared the TCGA HNSCC transcriptomes of HRAS-altered (HRASmut or wtHRASov) and HRAS- unaltered tumors. Approximately 6% of HNSCCs were overexpressing HRAS, and there was only partial overlap with the 6% of HRASmut tumors. Moreover, ERCC1 was found highly upregulated in HRAS altered group and especially among wtHRASov subcluster. To further study the resistance of wtHRASov tumors, we generated a cohort of HNSCC PDXs and acquired their mutational, expression and chemosensitivity profile. PDX tumors harboring a profile similar to wtHRASov TCGA cluster exhibited a strong chemoresistant squamous phenotype associated with high ERCC1 levels. Interestingly, these tumors were resensitized to cisplatin when subjected to cisplatin/tipifarnib combinatorial treatment. Conclusions ERCC1 expression is a well-established biomarker for cisplatin resistance. Our study demonstrates that wtHRASov tumours represent a distinctive entity that strongly express ERCC1 and can be resensitized to cisplatin by tipifarnib. Legal entity responsible for the study The authors. Funding Kura Oncology. Disclosure T. Rampias: Research grant / Funding (institution): Kura Oncology Company. All other authors have declared no conflicts of interest.

Herein, we describe a modified protocol for the generation of human induced pluripotent stem cells (hiPS) and expansion under defined, serum free and feeder free conditions. These cells exhibit a high level of plasticity towards various differentiation pathways both in vitro and in vivo. Ultimately, hiPS-derived lines achieved high standards of three dimensional differentiations on biomaterial scaffolds and promoted in vivo regeneration of complex organs, such as Anterior Cruciate Ligament (in swine ACL-rupture models) and other tissues as well.

Therapeutic targeting of tumor cells with drug nanocarriers relies upon successful interaction with membranes and efficient cell internalization. A further consideration is that engineered nanomaterials should not damage healthy tissues upon contact. A critical factor in this process is the external coating of drug delivery nanodevices. Using in silico, in vitro and in vivo studies, we show for the first time that magnetic nanoparticles coated with polyarabic acid have superior imaging, therapeutic, and biocompatibility properties. We demonstrate that polyarabic acid coating allows for efficient penetration of cell membranes and internalization into breast cancer cells. Polyarabic acid also allows reversible loading of the chemotherapeutic drug Doxorubicin, which upon release suppresses tumor growth in vivo in a mouse model of breast cancer. Furthermore, these nanomaterials provide in vivo contrasting properties, which directly compare with commercial gadolinium-based contrasting agents. Finally, we report excellent biocompatibility, as these nanomaterial cause minimal, if any cytotoxicity in vitro and in vivo. We thus propose that magnetic nanodevices coated with polyarabic acid offer a new avenue for theranostics efforts as efficient drug carriers, while providing excellent contrasting properties due to their ferrous magnetic core, which can help the future design of nanomaterials for cancer imaging and therapy.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) initiates IRE1α, ATF6, and PERK cascades, leading to a transcriptional/translational response known as unfolded protein response (UPR). Here we show that VEGF activates UPR mediators through a PLCγ-mediated crosstalk with the mTORC1 complex without accumulation of unfolded proteins in the ER. Activation of ATF6 and PERK contributes to the survival effect of VEGF on endothelial cells (ECs) by positively regulating mTORC2-mediated phosphorylation of AKT on Ser473, which is required for full activity of AKT. Low levels of CHOP allow ECs to evade the proapoptotic effect of this UPR product. Depletion of PLCγ, ATF6, or eIF2α dramatically inhibited VEGF-induced vascularization in mouse Matrigel plugs, suggesting that the ER and the UPR machinery constitute components of the VEGF signaling circuit that regulates EC survival and angiogenesis, extending their role beyond adaptation to ER stress.

IKKα is an NSCLC suppressor and its loss in mouse AT-II lung epithelial cells or in human NSCLC lines increased urethane-induced adenoma growth and xenograft burdens, respectively. IKKα loss can up-regulate HIF-1α, enhancing tumor growth under hypoxia., Through the progressive accumulation of genetic and epigenetic alterations in cellular physiology, non–small-cell lung cancer (NSCLC) evolves in distinct steps involving mutually exclusive oncogenic mutations in K-Ras or EGFR along with inactivating mutations in the p53 tumor suppressor. Herein, we show two independent in vivo lung cancer models in which CHUK/IKK-α acts as a major NSCLC tumor suppressor. In a novel transgenic mouse strain, wherein IKKα ablation is induced by tamoxifen (Tmx) solely in alveolar type II (AT-II) lung epithelial cells, IKKα loss increases the number and size of lung adenomas in response to the chemical carcinogen urethane, whereas IKK-β instead acts as a tumor promoter in this same context. IKKα knockdown in three independent human NSCLC lines (independent of K-Ras or p53 status) enhances their growth as tumor xenografts in immune-compromised mice. Bioinformatics analysis of whole transcriptome profiling followed by quantitative protein and targeted gene expression validation experiments reveals that IKKα loss can result in the up-regulation of activated HIF-1-α protein to enhance NSCLC tumor growth under hypoxic conditions in vivo.

Background: Circulating tumor cells (CTCs) heterogeneity is highly affecting the efficiency of their isolation and thus the reliability of downstream analysis. Especially in Head and Neck Squamous Cell Carcinoma (HNSCC) epithelial mesenchymal transition (EMT) is highly affecting CTC isolation and downstream analysis. We directly compared two different approaches used for CTC isolation, a label-independent size-based microfluidic-based system versus an EpCAM-based positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in HNSCC. Methods: Peripheral blood (PB) in EDTA (20mL) was collected from 50 HNSCC patients and 18 healthy donors (HD). A size-based microfluidic device (Parsortix, ANGLE) and an EpCAM-based positive immune-magnetic isolation procedure were applied in parallel, using 10mL PB in each case. Total RNA was isolated from enriched CTCs and RT-qPCR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M. Real time methylation specific PCR (MSP) was used to study the methylation status of SOX17, RASSF1A and MLL3 genes in DNAs isolated from the same enriched CTCs. Results: In identical blood draws, the label-free size-based CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-based CTC enrichment, since a significantly higher percentage of identical PB samples was found positive for all genes tested both at the gene expression and DNA methylation level, while the specificity was not affected. Conclusions: In HNSCC CTC molecular characterization at the gene expression and DNA methylation level should be based on a label-free size-based isolation system. Citation Format: Martha Zavridou, Sophia Mastoraki, Areti D. Strati, George Koutsodontis, Apostolos Klinakis, Amanda Psyrri, Evi S. Lianidou. Molecular characterization of circulating tumor cells in head and neck squamous cell carcinoma: Direct comparison of a label-independent size-based microfluidic device with EpCAM-based CTC enrichment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2241.

SummaryTumor-propagating cells in acute leukemia maintain a stem/progenitor-like immature phenotype and proliferative capacity. Acute myeloid leukemia (AML) and acute T-lymphoblastic leukemia (T-ALL) originate from different lineages through distinct oncogenic events such as MLL fusions and Notch signaling, respectively. We found that Zfx, a transcription factor that controls hematopoietic stem cell self-renewal, controls the initiation and maintenance of AML caused by MLL-AF9 fusion and of T-ALL caused by Notch1 activation. In both leukemia types, Zfx prevents differentiation and activates gene sets characteristic of immature cells of the respective lineages. In addition, endogenous Zfx contributes to gene induction and transformation by Myc overexpression in myeloid progenitors. Key Zfx target genes include the mitochondrial enzymes Ptpmt1 and Idh2, whose overexpression partially rescues the propagation of Zfx-deficient AML. These results show that distinct leukemia types maintain their undifferentiated phenotype and self-renewal by exploiting a common stem-cell-related genetic regulator.

The DNA damage response (DDR) pathway and ARF function as barriers to cancer development. Although commonly regarded as operating independently of each other, some studies proposed that ARF is positively regulated by the DDR. Contrary to either scenario, we found that in human oncogene-transformed and cancer cells ATM suppressed, in a transcription-independent manner, ARF protein levels and activity. Mechanistically, ATM activated protein phosphatase 1, which antagonized Nek2-dependent phosphorylation of nucleophosmin (NPM), thereby liberating ARF from NPM and rendering it susceptible to degradation by the ULF E3-ubiquitin ligase. In human clinical samples, loss of ATM expression correlated with increased ARF levels and in xenograft and tissue culture models, inhibition of ATM stimulated the tumour-suppressive effects of ARF. These results provide insights into the functional interplay between the DDR and ARF anti-cancer barriers, with implications for tumorigenesis and treatment of advanced tumours.

The urothelium is a specialized epithelium that lines the urinary tract. It consists of three different cell types, namely, basal, intermediate and superficial cells arranged in relatively distinct cell layers. Normally, quiescent, it regenerates fast upon injury, but the regeneration process is not fully understood. Although several reports have indicated the existence of progenitors, their identity and exact topology, as well as their role in key processes such as tissue regeneration and carcinogenesis have not been clarified. Here we show that a minor subpopulation of basal cells, characterized by the expression of keratin 14, possesses self-renewal capacity and also gives rise to all cell types of the urothelium during natural and injury-induced regeneration. Moreover, these cells represent cells of origin of urothelial cancer. Our findings support the hypothesis of basally located progenitors with profound roles in urothelial homoeostasis., It is unclear whether there is a progenitor/stem cell in the basal layer of the urothelium in the bladder. Here, the authors identify Keratin14 positive cells that can regenerate the bladder in both a natural and injury-induced manner, and following neoplastic transformation, can give rise to tumours.

The adult kidney contains a population of low-cycling cells that resides in the papilla. These cells retain for long periods S-phase markers given as a short pulse early in life; i.e., they are label-retaining cells (LRC). In previous studies in adult rat and mice, we found that shortly after acute kidney injury many of the quiescent papillary LRC started proliferating (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315–2327, 2009; Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q. J Clin Invest 114: 795–804, 2004) and, with cell-tracking experiments, we found upward migration of some papillary cells including LRC (Oliver JA, Klinakis A, Cheema FH, Friedlander J, Sampogna RV, Martens TP, Liu C, Efstratiadis A, Al-Awqati Q. J Am Soc Nephrol 20: 2315–2327, 2009). To identify molecular cues involved in the activation (i.e., proliferation and/or migration) of the papillary LRC that follows injury, we isolated these cells from the H2B-GFP mice and found that they migrated and proliferated in response to the cytokine stromal cell-derived factor-1 (SDF-1). Moreover, in a papillary organ culture assay, the cell growth out of the upper papilla was dependent on the interaction of SDF-1 with its receptor Cxcr4. Interestingly, location of these two proteins in the kidney revealed a complementary location, with SDF-1 being preferentially expressed in the medulla and Cxcr4 more abundant in the papilla. Blockade of Cxcr4 in vivo prevented mobilization of papillary LRC after transient kidney ischemic injury and worsened its functional consequences. The data indicate that the SDF-1/Cxcr4 axis is a critical regulator of papillary LRC activation following transient kidney injury and during organ repair.

Blocking Dll4–Notch signaling can reverse established diabetes via Flt3-independent induction of immature thymic DCs that enhance Treg cell generation in mice., Delta-like ligand 4 (Dll4)–Notch signaling is essential for T cell development and alternative thymic lineage decisions. How Dll4–Notch signaling affects pro-T cell fate and thymic dendritic cell (tDC) development is unknown. We found that Dll4 pharmacological blockade induces accumulation of tDCs and CD4+CD25+FoxP3+ regulatory T cells (Treg cells) in the thymic cortex. Both genetic inactivation models and anti-Dll4 antibody (Ab) treatment promote de novo natural Treg cell expansion by a DC-dependent mechanism that requires major histocompatibility complex II expression on DCs. Anti-Dll4 treatment converts CD4−CD8−c-kit+CD44+CD25− (DN1) T cell progenitors to immature DCs that induce ex vivo differentiation of naive CD4+ T cells into Treg cells. Induction of these tolerogenic DN1-derived tDCs and the ensuing expansion of Treg cells are Fms-like tyrosine kinase 3 (Flt3) independent, occur in the context of transcriptional up-regulation of PU.1, Irf-4, Irf-8, and CSF-1, genes critical for DC differentiation, and are abrogated in thymectomized mice. Anti-Dll4 treatment fully prevents type 1 diabetes (T1D) via a Treg cell–mediated mechanism and inhibits CD8+ T cell pancreatic islet infiltration. Furthermore, a single injection of anti-Dll4 Ab reverses established T1D. Disease remission and recurrence are correlated with increased Treg cell numbers in the pancreas-draining lymph nodes. These results identify Dll4–Notch as a novel Flt3-alternative pathway important for regulating tDC-mediated Treg cell homeostasis and autoimmunity.

Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b(+) DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esam(hi) DC subset required lymphotoxin beta receptor signaling, proliferated in situ, and facilitated CD4(+) T cell priming. The Notch-independent Esam(lo) DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b(+)CD103(+) DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4(+) T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b(+) DC subsets in the spleen and intestine.

Notch signalling is a central regulator of differentiation in a variety of organisms and tissue types. Its activity is controlled by the multi-subunit γ-secretase (γSE) complex. Although Notch signalling can play both oncogenic and tumour-suppressor roles in solid tumours, in the haematopoietic system it is exclusively oncogenic, notably in T-cell acute lymphoblastic leukaemia, a disease characterized by Notch1-activating mutations. Here we identify novel somatic-inactivating Notch pathway mutations in a fraction of patients with chronic myelomonocytic leukaemia (CMML). Inactivation of Notch signalling in mouse haematopoietic stem cells (HSCs) results in an aberrant accumulation of granulocyte/monocyte progenitors (GMPs), extramedullary haematopoieisis and the induction of CMML-like disease. Transcriptome analysis revealed that Notch signalling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. Our studies identify a novel role for Notch signalling during early haematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumour-promoting and -suppressive roles within the same tissue.

Introduction: Carfilzomib (Cfz) significantly prolongs progression-free survival in relapsed or refractory multiple myeloma patients, as highlighted in the ENDEAVOR trial. However, Cfz has high incidences of cardiotoxicity and heart failure, leading to treatment cessation. Thus, there is an imperative need for preventive therapies. The study aimed to i) establish an in vivo Cfz cardiotoxicity protocol, ii) investigate the molecular mechanism, identify molecular targets and iii) based on initial results, investigate the potential protective effect and mechanism of Metformin (Met). Methods: Male, C57BL/6 mice, were randomized in groups as following: Acute protocol (6 days): Control (n=7), Cfz (n=8); Sub-chronic protocol (14 days): Control (n=5), Cfz (n=8); Pharmacological intervention protocol (6 days): Control (n=8), Cfz (n=8), Cfz+Met (n=8), Met (n=4). Cfz (8 mg/kg, ip) was administered on alternate days and Met (140 mg/kg, po) daily. Glucose levels were monitored following Met administration. Mice underwent echocardiography on baseline and at the end of treatments. Blood and myocardial tissue samples were obtained for histology, proteasome activity, PP2A activity and signaling pathways focused on PI3K/Akt/eNOS axis, NO homeostasis and AMPKα-mTOR-mediated autophagy. Results: Following acute administration, echocardiography in Cfz group presented a significant reduction in fractional shortening (FS%) vs. Control group (39.87±0.47 vs. 43.03±0.50 respectively, p Signaling pathways were studied in the acute protocol that demonstrated suppressed myocardial contractility. Cfz resulted in significant inhibition of proteasome in both myocardium (55.5% inhibition, p Co-administration of Met prevented FS% reduction (Cfz group: 41.55±0.43 vs. 43.24±0.50 and 43.39±0.56, Control and Cfz+Met respectively, p Conclusions: Cfz exhibits decreased global LV function in vivo, without inducing tissue lesions. The molecular mechanism consists of increased PP2A activity leading to inactivation of AMPKα-mTOR and PI3K/Akt/eNOS pathways - combined with NO homeostasis deregulation. In an intervention approach, Met preserved cardiac function via restoring AMPKα-mediated autophagy. Met administration did not restore NO production and homeostasis; these pathways require further investigation. Met emerges to be a promising preventive therapy for Cfz-induced cardiotoxicity. Disclosures Kastritis: Amgen: Consultancy, Honoraria, Research Funding; Genesis Pahrma: Consultancy, Honoraria; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Honoraria; Prothena: Consultancy, Honoraria. Dimopoulos:Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria. Terpos:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: member of steering committee, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Research Funding; BMS: Consultancy; Genesis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Research Funding; Novartis: Consultancy; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: member of DMC, Research Funding; Amgen Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grant, steering committee member, Research Funding.

Therapeutic inhibition of the MYC oncoprotein action for cancer treatment is a challenging aim and various anti-MYC approaches, including the development of small-molecule candidate drugs, are being actively pursued. Using genetically-modified mouse models of cancer, we have demonstrated that the oncogenicity of mutant KRAS causally depends on unperturbed MYC activity. On the basis of this observation, we performed preclinical studies and treated successfully mouse pancreatic, breast and other cancers, and also xenografts of human pancreatic cancer cell lines, by using Mycro3, an orally-bioavailable small-molecule inhibitor of MYC-MAX dimerization. We observed that, in comparison with vehicle-treated controls, tumor growth in Mycro3-treated mice was drastically attenuated.1 To optimize Mycro3, we have used drug design, organic synthesis, in vitro assays, pharmacokinetic profiling, and in vivo efficacy studies. These efforts led to the identification of novel derivatives, one of which (Amy22) exhibits a vastly improved therapeutic window against MYC (>300-fold over Mycro3). It has also superior physicochemical and pharmacokinetic properties (the peak blood concentration is >6-fold better over Mycro3, while the compound bioavailability is maintained for more than 12 hours). The designed candidate anti-Myc compounds were synthesized by coupling properly substituted pyrazolo[1,5-a]pyrimidines with various amines, and then evaluated in vitro using the TGR-1 (Myc+/+) and H015.19 (Myc-/-) sibling cell lines. The in vivo efficacy of Amy22 has been tested successfully in preclinical studies using advantageous mouse models of MYC overexpressing tumors that we have developed, which enable rapid evaluations. Moreover, patient-derived xenografts (PDXs) are being used, while combinatorial therapeutic regimes using anti-Myc treatment with drugs inhibiting other collaborating tumorigenic pathways have been evaluated.2 ZC has been co-funded by the European Commission under the H2020 Research Infrastructures Contract No. 675121 (project VI-SEEM). 1D. Stellas, M. Szabolcs, S. Koul, Z. Li, A. Polyzos, C. Anagnostopoulos, Z. Cournia, C. Tamvakopoulos, A. Klinakis & A. Efstratiadis (2014) Therapeutic Effects of an anti-Myc Drug on Mouse Pancreatic Cancer, J Natl Cancer Inst, 106(12). pii: dju320 2D. Stellas, Z. Cournia, C. Tamvakopoulos, A. Klinakis & A. Efstratiadis. “Novel Compounds for use in treating or preventing cancerous diseases”. International Patent Application number PCT/EP2016/066340 Citation Format: Veroniki P. Vidali, Zoe Cournia, Adriana Papadimitropoulou, Dimitrios Stellas, Apostolos Klinakis, Elias A. Couladouros, Constantin Tamvakopoulos, Argiris Efstratiadis. Design, synthesis, and efficacy of MYC inhibitors in mouse pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-262.

The kidney papilla contains a population of cells with several characteristics of adult stem cells, including the retention of proliferation markers during long chase periods (i.e., they are label-retaining cells [LRCs]). To determine whether the papillary LRCs generate new cells in the normal adult kidney, we examined cell proliferation throughout the kidney and found that the upper papilla is a site of enhanced cell cycling. Using genetically modified mice that conditionally expressed green fluorescence protein fused to histone 2B, we observed that the LRCs of the papilla proliferated only in its upper part, where they associate with “chains” of cycling cells. The papillary LRCs decreased in number with age, suggesting that the cells migrated to the upper papilla before entering the cell cycle. To test this directly, we marked papillary cells with vital dyes in vivo and found that some cells in the kidney papilla, including LRCs, migrated toward other parts of the kidney. Acute kidney injury enhanced both cell migration and proliferation. These results suggest that during normal homeostasis, LRCs of the kidney papilla (or their immediate progeny) migrate to the upper papilla and form a compartment of rapidly proliferating cells, which may play a role in repair after ischemic injury.

T-cell acute lymphoblastic leukaemia (T-ALL) is a blood malignancy afflicting mainly children and adolescents1. T-ALL patients present at diagnosis with increased white cell counts and hepatosplenomegaly, and are at an increased risk of central nervous system (CNS) relapse2,3. For that reason, T-ALL patients usually receive cranial irradiation in addition to intensified intrathecal chemotherapy. The marked increase in survival is thought to be worth the considerable side-effects associated with this therapy. Such complications include secondary tumours, neurocognitive deficits, endocrine disorders and growth impairment3. Little is known about the mechanism of leukaemic cell infiltration of the CNS, despite its clinical importance4. Here we show, using T-ALL animal modelling and gene-expression profiling, that the chemokine receptor CCR7 (ref. 5) is the essential adhesion signal required for the targeting of leukaemic T-cells into the CNS. Ccr7 gene expression is controlled by the activity of the T-ALL oncogene Notch1 and is expressed in human tumours carrying Notch1-activating mutations. Silencing of either CCR7 or its chemokine ligand CCL19 (ref. 6) in an animal model of T-ALL specifically inhibits CNS infiltration. Furthermore, murine CNS-targeting by human T-ALL cells depends on their ability to express CCR7. These studies identify a single chemokine–receptor interaction as a CNS ‘entry’ signal, and open the way for future pharmacological targeting. Targeted inhibition of CNS involvement in T-ALL could potentially decrease the intensity of CNS-targeted therapy, thus reducing its associated short- and long-term complications.

Considering the strong association between dysregulated insulin-like growth factor (IGF) signaling and various human cancers, we have used an expedient combination of genetic analysis and pharmacological treatment to evaluate the potential of the type 1 IGF receptor (Igf1r) for targeted anticancer therapy in a mouse model of mammary tumorigenesis. In this particular strain of genetically modified animals, histopathologically heterogeneous invasive carcinomas exhibiting up-regulation of the Igf1r gene developed extremely rapidly by mammary gland-specific overexpression of constitutively active oncogenic Kras* (mutant Kras G12D ). Immunophenotyping data and expression profiling analyses showed that, except for a minor luminal component, these mouse tumors resembled basal-like human breast cancers. This is a group of aggressive tumors of poor prognosis for which there is no targeted therapy currently available, and it includes a subtype correlating with KRAS locus amplification. Conditional ablation of Igf1r in the mouse mammary epithelium increased the latency of Kras*-induced tumors very significantly (≈11-fold in comparison with the intact model), whereas treatment of tumor-bearing animals by administration of picropodophyllin (PPP), a specific Igf1r inhibitor, resulted in a dramatic decrease in tumor mass of the main forms of basal-like carcinomas. PPP also was effective against xenografts of the human basal-like cancer cell line MDA-MB-231, which carries a KRAS G13D mutation.

The mammalian insulin-like growth factor 1 (IGF1), which is a member of a major growth-promoting signaling system, is produced by many tissues and functions throughout embryonic and postnatal development in an autocrine/paracrine fashion. In addition to this local action, IGF1 secreted by the liver and circulating in the plasma presumably acts systemically as a classical hormone. However, an endocrine role of IGF1 in growth control was disputed on the basis of the results of a conditional, liver-specific Igf1 gene knockout in mice, which reduced significantly the level of serum IGF1, but did not affect average body weight. Because alternate interpretations of these negative data were tenable, we addressed genetically the question of hormonal IGF1 action by using a positive experimental strategy based on the features of the cre / loxP recombination system. Thus, we generated bitransgenic mice carrying in an Igf1 null background a dormant Igf1 cDNA placed downstream of a transcriptional “stop” DNA sequence flanked by loxP sites (floxed) and also a cre transgene driven by a liver-specific promoter. The Igf1 cDNA, which was inserted by knock-in into the mutated and inactive Igf1 locus itself to ensure proper transcriptional regulation, was conditionally expressed from cognate promoters exclusively in the liver after Cre-mediated excision of the floxed block. Our genetic study demonstrated that the endocrine IGF1 plays a very significant role in mouse growth, as its action contributes approximately30% of the adult body size and sustains postnatal development, including the reproductive functions of both mouse sexes.

The involvement of Myc in the development of human breast cancer is known for quite some time, but an analogous role of Notch signaling is only now emerging from fragmented pieces of information. Recently, a Notch/Myc relationship in oncogenesis was revealed from a mouse model, in which mammary tumors are induced by the intracellular domain of Notch1 (N1(IC)). In fact, a combination of genetic and molecular data demonstrated that Myc is a direct transcriptional target of Notch1 participating as an indispensable downstream effector in N1(IC)-induced tumorigenic action. A medically-relevant correlative observation based on immunophenotyping was the coexpression of Notch1 and Myc in a significant fraction of human breast cancer specimens. A Notch1/Myc oncogenic association was also observed in T-cell acute lymphoblastic leukemia.