Abstrakta 2017 by Diagnosic Predictive and Experimental ONCOLOGY Days

ABSTRACT BOOK XIII. DIAGNOSTIC, PREDICTIVE AND EXPERIMENTAL ONCOLOGY DAYS

2017

2017 November 28 - 30 hotel NH Collection Olomouc Congress Legionarska 21, 779 00, Olomouc, Czech Republic

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2017

Organizing Institutions MedChemBio Cluster in asociation with Institute of Molecular and Translational Medicine & Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University EATRIS - CZ European Infrastructure for Translational Medicine Cancer research Czech republic

Professional Guaranteee Section of Diagnostic and Predictive Oncology, the Czech Oncological Society CMA JEP

President of the Conference Marián Hajdúch, MD., PhD.

The Scientific Committee Jiří Drábek, PhD. Petr Džubák, MD., PhD. Marián Hajdúch, MD., PhD. Ondřej Slabý, PhD. Josef Srovnal, MD., PhD. Marek Svoboda, MD., PhD. Radek Trojanec, PhD.

Organizer Peter Vanek peter.vanek@upol.cz +420 585 632 246

PROGRAM

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XIII. DNY DIAGNOSTICKÉ, PREDIKTIVNÍ A EXPERIMENTÁLNÍ ONKOLOGIE / XIII. DIAGNOSTIC, PREDICTIVE AND EXPERIMENTAL ONCOLOGY DAYS ÚTERÝ / TUESDAY - 28. listopadu 2017 / November 28th, 2017 12:30 - 12:45

WELCOME & INTRODUCTION

Cancer therapeutics I.

Chairs: Petr Dzubak, Marian Hajduch 12:45 - 13:30 13:30 - 14:00 14:00 - 14:15

14:15 - 14:30 14:30 - 14:45 14:45 - 15:15

5-ene-thiazolidinone based design of drug-like molecules Roman Lesyk Challenges and best-practices in academic cancer drug development Marian Hajduch Semisynthetic triterpenoid heterocycles - synthesis, cytotoxicity, and studies of their mechanism of action Milan Urban Design of HTS libraries using novel 3D pharmacophore signatures Pavlo Polishchuk Fluoroquinolones as perspective anti-cancer drugs Natalie Taborska COFFEE BREAK

Cancer therapeutics II.

Chairs: Milos Petrik, Milan Urban 15:15 - 15:45 15:45 - 16:00 16:00 - 16:15 16:15 - 16:35 16:35 - 16:50 16:50 - 17:05

Phytohormones cytokinin ribosides as anticancer agents Jiri Voller 99mTc-labelled hydroxyapatite nanoparticles and their in vitro / in vivo characterisation Zbynek Novy Repurposing of anti-cancer drugs as potential inhibitors of toxic Tau aggregates Narendran Annadurai CRISPR-mediated gene tagging with the bioluminescent HiBiT technology Oleksii Ivanov How we do high content/throughput screening of anti-cancer molecules Pawel Znojek Proteomic profiling reveals DNA damage, nucleolar and ribosomal stress are the main responses to oxaliplatin treatment in cancer cells Tomas Ozdian

STŘEDA / WEDNESDAY - 29. listopadu 2017 / November 29th, 2017

Translational cancer research I. Chairs: Ondrej Slaby, Jiri Drabek 9:00 - 9:45

9:45 - 10:30 10:30 - 11:00 11:00 - 11:15

Unconventional transcripts, fusion genes and neocentromeres: the hidden face of genomic amplifications in cancer. Clelia Tiziana Storlazzi How can we target the CXCL12-CXCR4 axis in oncology patients Stefania Scala Genomic data as the basis for personalized therapeutic planning in pediatric oncology Ondrej Slaby COFFEE BREAK

PROGRAM

2017

Molecular targets & biomarkers I. Chairs: Jiri Sana, Josef Srovnal 11:15 - 11:30 11:30 - 11:45 11:45 - 12:00 12:00 - 12:15 12:15 - 12:30

12:30 - 13:30

Immunotherapy prediction in cancer patients using microsatellite instability Josef Srovnal MiRNA profiling in recurrent and non-recurrent meningeomas Hanus Slavik Cytogenetic analysis of recurrent glioblastomas Magdalena Megova The opioid and cannabinoid receptors expression affects survival of colorectal cancer patients Monika Vidlarova Identification of candidate genes underlying soft tissue sarcoma progression using a progression series of murine fibrosarcoma cell lines Jiri Hatina LUNCH AND POSTER PRESENTATION (Note: the authors are kindly requested to present posters in between 13:00-13:30)

Translational cancer research II.

Chairs: Manlio Vinciguerra, Viswanath Das 13:30 - 14:15 14:15 - 14:45 14:45 - 15:00 15:00 - 15:15 15:15 - 15:30 15:30 - 15:45

Optimized design and analysis of preclinical intervention studies in vivo Andreas Scherer Epigenetic and paracrine control of hepatic cancer stemness Manlio Vinciguerra Life is never flat: The rational use of 3D cell cultures as more predictive preclinical tumor models Viswanath Das MicroRNA analysis in cerebrospinal fluid of glioblastoma patients Alena Kopkova Global microRNA expression analysis of Sox-2 positive and negative glioblastoma cells Jiri Sana COFFEE BREAK

New technologies & bioinformatics Chairs: Petr Dzubak, Martin Mistrik 15:45 - 16:15 16:15 - 16:30 16:30 - 16:45 16:45 - 17:00

17:00 - 17:15

19:00

Strategies for molecular target identification Petr Dzubak Bioinformatic analysis of massive parallel sequencing data for cancer research Petr Vojta Variants in shotgun proteomics Miro Hruska Chronic lymphocytic leukemia cases with stereotyped B-cell receptors: knowledgebase, bioinformatics tools, data management and reporting towards personalised biomedical and clinical applications Tomas Reigl A novelty role of CDK12 in regulation of DNA damage response by altering methylation of miR-152 and targeting downstream regulatory genes Marta Dzimkova CONFERENCE DINNER (NH hotel restaurant, for dinner-registered participants only)

PROGRAM

2017

ČTVRTEK / THURSDAY - 30. listopadu 2017 / November 30th, 2017

Molecular targets & biomarkers II. Chairs: Marian Hajduch, Jiri Drabek 9:00 - 9:45 9:45 - 10:00 10:00 - 10:15

10:15 - 10:30 10:30 - 10:45

New biomarkers – host methylation for management of women with a screen positive test Chris Meijer Pitfalls in HPV68a detection Hana Jaworek Molecular cytogenetics of „double/triple hit“ - high-grade B-cell lymphoma: a new entity of mature B-cell neoplasms Helena Urbankova Human papillomavirus infection in non-small cell lung cancer Vladimira Koudelakova COFFEE BREAK

Molecular targets & biomarkers III. Chairs: Milan Reinis, Pavel Moudry 10:45 - 11:00

11:00 - 11:15

11:15 - 11:30

11:30 - 11: 45

11:45 - 12:00 12:00

Comparative analysis of senescent tumour cells induced by docetaxel or immunomodulatory cytokines Milan Reinis Downregulation of uncharacterized human HEATR1 protein activates p53 and induces ribosomal stress Zsofia Turi Prostate cancer cell lines and patient-derived tissues as a model for therapy decision on inhibition of PARP1 or HDAC in combination with gamma irradiation Dusana Majera Prognostic and predictive value of loss of nuclear RAD51 immunoreactivity in resected nonsmall cell lung cancer patients Marjam Gachechiladze CLOSING CEREMONY LUNCH

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Postery / Posters 1

Steps for improving reproducibility in biomedical research Jiri Drabek

Automatic cytogenetic system Metafer®: optimization and comparison with manual method Oliver Pesat

Gene aberrations in IDH-wild type glioblastoma Zuzana Sporikova

OncoScan analysis of good and bad responders to bevacizumab among mCRC patients Karolína Bartakova

Investigation of biocompatibility and cytotoxic effects of TiO2 nanoparticles with organic surface modifications Zuzana Skubalova

Study on influence of surface modifications of apoferritin on intracellular delivery of ellipticine Barbora Tesarova

Stability of apoferritin modified with ANTI-hNET peptides for active targeting of neuroblastoma cells Marketa Charousova

Trisubstituted purine inhibitors of PDGFRα with high selectivity toward human eosinophilic cell line EOL-1 Veronika Malinkova

Search for new anticancer agents among 4-thiazolidinones Danylo Kaminskyy

Non-invasive lung cancer diagnostics using proteomic biomarkers in exhaled breath condensate. Jana Vaclavkova

Finding molecular patterns relevant for toxicity end-points Mariia Matveieva

Impact of hypoxia on sensitivity of colorectal carcinoma cells towards microtubule-targeting agents Jiri Rehulka

Folic acid conjugates with purine cyclindependent kinase inhibitors Denisa Hendrychova

Mass spectrometry-based proteomic analysis for subtyping of amyloid deposits from FFPE and SFA Dusan Holub

Cytotoxic Triterpenes and Study of their Mechanism of Action Jiri Hodon

GLASS: assisted mutation detection in Sanger data Karol Pal

Antiparasitic activity of anticancer drugs against kinetoplastid parasite Leishmania major Ermin Schadich

Pilotní studie molekulárního testování HPV infekce u nádorů z různých oblastí hlavy a krku Kristyna Glendova

Cytotoxicity profiling of new chemical compounds with chelating activities using highthroughput screening Sona Gurska

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2017

Cancer therapeutics I.

Chairs: Petr Dzubak, Marian Hajduch úterý / 28. listopadu 2017 / Tuesday / November 28, 2017 / 12:45 - 14:45 5-ene-thiazolidinone based design of drug-like molecules

Roman Lesyk, Danylo Kaminskyy, Anna Kryshchyshyn Danylo Halytsky Lviv National Medical University, Lviv, Ukraine Introduction Over the years, 4-thiazolidinones are recognized as privileged heterocycles in medicinal chemistry. Main achievements are related to the Introduction of a series of approved drugs into medical practice, especially hypoglycemic glitazones. Among variety of 4-thiazolidinone subtypes and thiazolidinone-bearing compounds 5-substituted thiazolidinones, namely 5-ene derivatives, are of special interest due to their chemical characteristics and pharmacological profiles. Compounds of mentioned sub-type are high-affinity ligands to a number of biological targets, leadcompounds, and drug-candidates with antidiabetic, antimicrobial, antiviral and anticancer activities. Materials/methods The main directions for 5-enethiazolidinones synthesis and modification, mainly within structurebased approach, can be compiled into the next groups: i) complication of C5 fragment (following the thesis about crucial impact of the C5 moiety); ii) Introduction of the substituents in the N3 position (especially fragments with carboxylic/hydroxy groups); iii) synthesis of isosteric heterocycles; iv) combination with other pharmacologically attractive fragments within hybrid pharmacophore approach; v) annealing in complex heterocyclic systems; vi) utilization of 5-ene-thiazolidinones for the synthesis of other heterocycles. The conjugation of C5 double bound

with C4 carbonyl group of main core allows to assign the 5-ene-4thiazolidinones (especially 5-enerhordanines) as Michael acceptors. Two opposite meanings of this functionality are discussed. 5-Ene-4thiazolidinones are often treated as frequent hitters or PAINS (pan-assay interference compounds), which are useless in modern medicinal chemistry because of compounds interaction with thiolic groups of proteins, and apparently their low selectivity. Michael acceptors currently are considered as “new old tool” for new drug creation, especially anticancer agents (Michael acceptors are one of the most effective activators of Nrf2, efﬁcient covalent inhibitors of set of approved biotargets; structural similarity to endogenous Michael acceptors leads to modulation of ROS-dependent regulatory pathways, etc).

Republic, Demand for drug research and development is continually increasing among academic community. However, researchers need to understand the do’s and don’ts in academic drug research. Based on real life case studies from our internal and collaborative research we will zoom in on key components for success, root causes for failure, and main struggles for ongoing projects. We will share our own experiences in cancer drug discovery and development with an emphasis on novel approaches and emerging technologies.

Semisynthetic triterpenoid heterocycles - synthesis, cytotoxicity, and studies of their mechanism of action

Milan Urban1 ,2, Jiří Hodoň1 , Miroslav Soural2, Jana Results and conclusions Václavková1, Dušan Holub1, One of the possible solutions Petr Džubák1, Jan Šarek1, Jiří within privileged substructure1 1 based diversity oriented synthesis Řehulka , Marián Hajdúch is the ﬁxation of 5-ene-4thiazolidinone fragment in the fused heterocycles. The prime examples are thiopyrano[2,3-d]thiazoles obtained from 5-ene-thiazolidinones. Following the study of biological activities, thiopyrano[2,3-d]thiazole based compounds are considered as cyclic mimetics (with the same pharmacological proﬁles) of pharmacologically active 5-ene-4thiazolidinones but without Michael acceptor functionality.

Challenges and best-practices in academic cancer drug development

Marian Hajduch, Peter Dzubak Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech

IMTM, Faculty of Medicine and Dentistry, UP Olomouc, Olomouc, Czech Republic, Department of Organic Chemistry, Faculty of Science, Palacky University in Olomouc, Olomouc, Czech Republic

Introduction Triterpenoids belong to a large group of natural compounds that may be isolated from plants, fungi, marine invertebrate or other organisms. They often have a variety of biological activities. Betulinic acid, for example, has a strong anti-cancer activity and its hemiesters inhibit maturation of HIV. Materials/methods The aim of this work was to synthesize derivatives of various terpenoid scaffolds (such as lupane, oleanane, ursane, taraxastane etc.)

2017

with a heterocycle condensed to the terpenic skeleton. In addition, derivatives substituted in the position 2 with an electron withdrawing substituent were of interest. Several 2-substituted triterpenes (e.g. 2-ﬂuoro-, or 2-aminoderivatives), and many heterocycle containing triterpenes (e.g. pyrazines, aminothiazoles or pyridines) had cytotoxicity lower than 5 μM on CCRF-CEM cell line and often on multiple other cancer cell lines with the selectivity index of 5-99. Inﬂuence of the best compounds on cell cycle and on the DNA/RNA synthesis was studied. O N N

O N H

H N

O O

O NH

Fig. 1 Conjugate of cytotoxic pyrazine with biotin.5

Results and conclusions To better understand the mechanism of action, solid-phase synthesis of conjugates of active compounds with a ﬂuorescent tag or biotin was developed and currently, we attempt to isolate and identify the target proteins in pull down assays. Basic assumptions about SAR, possible pharmacophores and targets will be discussed. Literature 1 Dzubak, P.; Hajduch, M.; Urban, M.; Sarek, J. et al. Nat. Prod. Rep. 2006, 23, 394. 2 Borkova, L.; Urban, M.; Sarek, J. et al. Eur. J. Med. Chem. 2015, 96, 482. 3 Borkova, L.; Gurska, S.; Dzubak, P.; Burianova, R.; Hajduch, M.; Sarek, J.; Popa, I.; Urban, M. Eur. J. Med. Chem. 2016, 121, 120. 4 Vlk, M.; Urban, M.; Sarek, J. et al. J. Rad. Nucl. Chem. 2016, 308, 733. 5 Soural, M.; Hodon, J.; Dickinson, N. J.; Sidova, V.; Gurska, S.; Dzubak, P.; Hajduch, M.; Sarek, J.; Urban M. Bioconjugate chem. 2015, 26, 2563.

Design of HTS libraries using novel 3D pharmacophore signatures

Pavel Polishchuk1, Timur Madzhidov2 1

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech Republic, Kazan Federal University, Kazan, Russia

Introduction The selection and acquisition of high quality libraries of small molecules for high-throughput screening is an important aspect of drug discovery. The procedure of selection of general purpose HTS library is multi-staged and includes several steps: ﬁltering compounds based on their physicochemical and ADME/Tox properties, removing compounds with undesired functionalities and reactive species, diversity analysis. Structural diversity is usually measured and used based on conventional 2D ﬁngerprints. However, diversity of compounds in terms of their pharmacophore representation is also important and this can help to reduce the number of redundant compounds in the created library. Materials/methods We developed a method of representation of 3D pharmacophores with canonical signatures which can be further hashed to facilitate storage and manipulation. Each compound is represented as a set of calculated conformers. Pharmacophore features are assigned to each conformer resulting in separate 3D pharmacophores. Each pharmacophore is encoded in 3D hashed pharmacophore signature and stored to database. Duplicated signatures for each single compound are removed. Thus, each compound is represented by the set of pharmacophore signatures. Pharmacophore signatures of all compounds represent an accessible pharmacophore space. Compounds from the initial pool are selected in order to evenly cover an available

pharmacophore space. This is done by a greedy solution of a set cover problem. We implement two approaches which prioritize compounds with large and small number of pharmacophores. Finally all compounds are ranked and compounds on top can be considered as more representative in terms of pharmacophore space coverage. Results and conclusions The developed approach was applied in retrospective study based on the library of about 350 000 tested in 95 HTS assays. The results demonstrated that using of the developed procedure can substantially increase hit rate enrichment in a majority of assays in the case of using the approach prioritizing compounds with a small number of pharmacophores. That can be explained by the fact that such compounds are less flexible and have preferable entropy binding term. Despite the fact that the selection of such compounds decreases the overall coverage of a pharmacophore space these compounds can give positive results more often than flexible compounds covering a large number of distinct pharmacophores. Comparison with a conventional selection strategy based on a sphere exclusion algorithm and various 2D fingerprints showed the advantage of our approach. It should be noted that pharmacophore fingerprints showed the best performance from all 2D fingerprints.

Fluoroquinolones as perspective anti-cancer drugs

Natálie Táborská, Gabriela Rylová, Jan Hlaváč, Petr Džubák, Marián Hajdúch Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic Introduction Fluoroquinolones are members of a broad-spectrum bactericidal antibiotics - quinolones - with a

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fluorine atom in their chemical structure. Recently it was published that some of them can influence alternative splicing of a target gene encoding MDM4 (MDMX, Mdm2like P53 binding protein). MDM4 is a P53 inhibitor and thus it supports tumor growth. Two fluoroquinolone antibiotics - ciprofloxacin and enoxacin induce MDM4 exon 6 skipping of pre-miRNA molecule, this process leads to P53 increase. Another quickly emerging and very attractive target in the cancer therapy is PKM (Pyruvate Kinase, Muscle). PKM2 isoform is highly upregulated in human cancer cells and plays an important role in the tumor cell glycolysis. Fluoroquinolones are possible influencing factors for the alternative splicing of tumor cell key genes.

and subsequently treated with target antibiotics - ciprofloxacin, enoxacin and ofloxacin. We also used a newly synthesized compound 05-0777 with quinolone-like chemical structure for the treatment in the same manner. 05-0777 was explored in a set of biological tests before the treatment. Treated cells were collected after 24hour incubation, RNA was isolated and cDNA was obtained in a reverse transcription step. This material served as a template for PCR, resp. real-time PCR. For MDM4 expression examination three primer pairs were used, PCR products were separated in agarose gel and then visualized and quantified. Regarding PKM1 and PKM2 quantification, we used one specific primer pair for each isoform, at the end the relative quantification was carried out.

Materials/methods U-2 OS and CCRF-CEM human cell lines were used as a biological material for ﬂuoroquinolones treatment. Cells were plated in T25 cell culture ﬂasks

Results and conclusions MDM4 mRNA alternative splicing was examined by observation and quantiﬁcation of PCR products ampliﬁed with different primer pairs,

we proved that ciprofloxacin and enoxacin treatment lead to the exon 6 skipping - expression of MDM4 shorter form. This phenomenon was obtained from U-2 OS and CCRFCEM human cell lines, in contrary with untreated or DMSO treated cells which express higher amount of nonskipped MDM4 form that is typically upregulated in tumor cells. Ofloxacin did not have this effect and we also did not show it in 05-0777 treatment. We also observed changes in PKM1 and PKM2 expression after the treatment, but these results will be explained and supported by the future results from proteomic and transcriptomic analysis. Alternative splicing of MDM4 and PKM have a great potential in the tumor growth and prosperity influence, thus we look for new small molecule drugs that can induce the target splicing. Supported by IGA_LF_2017_013

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2017

Cancer therapeutics II.

Chairs: Milos Petrik, Milan Urban úterý / 28. listopadu 2017 / Tuesday / November 28, 2017 / 15:15 - 17:05 Phytohormones cytokinin ribosides as anticancer agents

Jiří Voller1 ,2, Tibor Béres1, Marek Zatloukal1, Pierre Alexandr Kaminski3, Karel Doležal1, Petr Džubák2, Marián Hajdúch2, Miroslav Strnad1 1

Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany ASCR & Palacký University, Olomouc, Czech Republic, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech Republic, The Institut Pasteur, Unit é de Biologie des Bactéries Pathogènes à Gram-Positif, Centre National pour la Recherche Scientiﬁque, Paris, France, BIOLOG Life Science Institute, Bremen, Germany

Introduction Cytokinin ribosides (CR) have demonstrated anticancer activity in various cultured cell lines, several xenografts and even a small clinical trial. Classical anticancer CRs, i.e. N6-isopentenyladenosine, N6benzyladenosine (BAR) and kinetin riboside cause ATP depletion and intrinsic apoptosis. Recently, we discovered that two orthohydroxylated BAR derivatives ortho-topolin riboside (oTR) and 2OH3MeOBAR (N6-(2-hydroxy-3methoxybenzyl)adenosine) - are more active than the classical CRs. Here we report inicial studies related to their mechanism of action. Materials/methods Effects of CRs were studied in leukemia cell line CEM. Following cell deathrelated parameters were evaluated: ATP levels (luciferase assay), casp-3 activation (quantiﬁcation of hydrolysis

of fluorigenic substrate), membrane integrity and anexin translocation (flow-cytometry). Inhibitory activity against recombinant DNPH1 was evaluated in biochemical assay. Metabolization of CR was carried out by capilary electrophoresis and HPLC. Finally we analyzed NCI60 activity profiles. Results and conclusions Although the structures of oTR and 2OH3MeOBAR differ only by the presence of 3-methoxy group, their mechanisms of action are distinct. Whereas the effects of oTR on energy levels and membrane permeability resemble those of classical CRs, 2OH3MeOBAR kills the cells without a marked ATP depletion. Notably, an analysis of NCI60 test data suggests that the activity of neither of them is dependent on the p53 status. Mechanism of action of 2OH3MeOBAR remains unclear, however we speculate that inhibition of the candidate oncogene DNPH1 by its metabolite 5’-monophosphate may be involved.

hydroxyapatite nanoparticles modiﬁed with various biocompatible polymers (HAP-PEG5000, HAPPOX5000, HAP-POX10000 a HAPPHPMA12000). The goal of this work was indirect radiolabeling of newly prepared nanoparticles with radioisotope 99mTc via clinically used radiopharmaceutical 99mTcHDP and subsequently to verify the stability of such labelled nanoparticles in vitro and then to reveal their in vivo biodistribution in normal mice using microSPECT/CT system. All studied nanoparticles were tested in means of their in vitro cytotoxicity on selected cell lines.

Materials/methods Radiolabeling - nanoparticles were incubated for 60 minutes with 99mTc-HPD in room temperature. Subsequently radiochemical purity control was employed using ITLC with two different mobile phases (ammonium acetate and acetone). Stability studies of labelled nanoparticles were performed in two environments - saline and human plasma (2h, 4h and 24h). In 99mTc-labelled hydroxyapatite vivo biodistribution was monitored nanoparticles and their in vitro/ using microSPECT/CT and was in vivo characterisation registered 1h, 3h, 6h a 24 h after 1 1 application of studied compound. Zbynek Novy , Milos Petrik , 1 2 Sona Gurska , Jan Kozempel , The 99mTc-HDP imaging was also performed for comparison with Martin Vlk2, Volodimir Lobaz3, labelled nanoparticles. Cytotoxicity Jan Kucka3, Martin Hruby3, chosen nanoparticles (in unlabeled form) were tested using MTS Jarmila Drymlova4, Marian 1 method in six cell lines (B2, BJ, Hajduch MRC5, HCT116, HCT116p53-/-, 1 Palacky University Olomouc, CCRF-CEM) employing highOlomouc, Czech Republic, throughput instrumentation. IC50s 2 2Czech Technical University in were calculated. The incubation Prague, Praha, Czech Republic, time of nanoparticles on cell lines for 3 Czech Academy of Science, Praha, cytotoxicity studies was 72 hours. Czech Republic, Results and conclusions 4 University Hospital Olomouc, Radiolabeling of all studied Olomouc, Czech Republic nanoparticles with 99mTc-HDP resulted in radiochemical purity Introduction Preclinical screening of selected over 90%. Stability studies of such prepared radiopharmaceutical

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revealed decrease in radiochemical purity of maximum 10.7% in saline and 11.0% in human plasma respectively after 24 hours. SPECT/ CT imaging showed very speciﬁc biodistribution of nanoparticles concentrated in liver and spleen even 24 p.i. for all tested compounds. The accumulation of activity was observed in heart and bladder in earlier time points p.i. (1h and 3h). Cytotoxicity - studied nanoparticles were considered nontoxic according to their IC50s obtained using above mention cell lines (IC50 ˃ 1000 µg/ mL resp. ˃ 2000 µg/mL).Tested nanoparticles show relatively facile radiolabeling with clinically used radiopharmaceutical 99mTc-HDP. Their stability in saline and human plasma is in highly satisfactory level. Biodistribution studies revealed speciﬁc accumulation in liver and spleen, where part of injected dose is excreted via kidneys. In vitro toxicity of the nanoparticles is relatively very low. All these parameters suggest the potential of these compounds for future studies aimed into tumor imaging based on EPR effect in vivo. Acknowledgement to project of Czech Research Council No. 1630544A.

Repurposing of anti-cancer drugs as potential inhibitors of toxic Tau aggregates

Narendran Annadurai1, Alfredo Cagnotto2, Antonio Bastone2, Jakub Malohlava1 ,3, Mario Salmona2, Petr Džubák1, Marián Hajdúch1, Viswanath Das1 1

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, Department of Molecular Biochemistry and Pharmacology, IRCCS Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy, Department of Medical Biophysics, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic

Introduction Tau is a microtubule-stabilizing protein found mainly in neurons of the central nervous system. In tauopathies, genetic mutations and dysfunctions in post-translational modifications result in misfolding of tau and its aggregation into neurofibrillary tangles that contribute to neuronal loss. These misfolded and aggregated Tau strains have the ability to seed and stably propagate the aggregation of endogenous tau in the nearby healthy neuronal cells. Inhibition of Tau aggregation and/ or disaggregation of pre-formed tau fibrils along with targeting extracellular aggregated tau seeds are potential therapeutic strategies for the treatment of tauopathies. Drug repositioning and repurposing is regarded as a reasonable approach to use known drugs for different indications. In this study, we propose the repurposing of clinically used anti-cancer drugs as potential tau aggregation inhibitors.

Results and conclusions We found that some of the anti-cancer compounds, such as paclitaxel, doxorubicin, and daunorubicin were effective inhibitors of Tau-R3 aggregation. An additional screening of 25 anti-cancer compounds resulted in the identification of around 5-10 compounds with potent Tau-R3 aggregation inhibitory effects. Some of these compounds also showed the potential to disaggregate pre-formed Tau-R3 fibrils. These preliminary results were further confirmed by atomic force microscopy. Lastly, the active compounds were tested for the inhibition of Tau-R3 aggregate seeding effect in HEK293 cells expressing wild-type and aggregation-prone mutant tau. The results show that doxorubicin and daunorubicin inhibit Tau-R3-mediated aggregation of wild-type and mutant tau in cells. We believe that these active anti-cancer compounds might be potential inhibitors of Tau aggregation and propagation.

Materials/methods We used the synthetic peptide of the third repeat fragment of tau (Tau-R3) for establishing a Thioﬂavin T-based in vitro assay for screening of small molecules. The Tau-R3 peptide has high self-aggregation potential and induces the aggregation of the microtubule-binding domain in cells. Tau-R3 aggregates were prepared by incubating Tau-R3 peptide with heparin in aggregation inducing buffer at 37°C under shaking conditions (1000 rpm) for 24 hours with and without test compounds and the difference in ﬂuorescence was observed using Perkin Elmer Enspire multi-mode plate reader by measuring excitation at 450 nm and emission at 485 nm. The Tau-R3 aggregates with and without test compounds were used as seeds to induce Tau aggregation in HEK293 cells expressing wild-type and aggregation-prone mutant tau. The effect of compounds on Tau-R3induced aggregation of endogenous tau in HEK-293 cells were monitored by miscroscopy.

CRISPR-mediated gene tagging with the bioluminescent HiBiT technology

Oleksii Ivanov Promega GmbH, Mannheim, Germany Introduction Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant tagged proteins. Nevertheless, it is well known that overexpression can cause artificial effects. Regularly used protein tags include either autofluorescent proteins of high molecular weight or small epitope tags which require very tedious, antibody-based detection procedures. Here we describe a new method based on the combination of a new bioluminescent protein tagging system and CRISPR/Cas9. This new method enables the cloning-

2017

free generation of endogenous protein assays and allows the simple quantification of intra- and/ or extracellular protein levels after experimental manipulation. Materials/methods We developed a new protein tagging and detection method – the HiBiT system – which is based on a split version of NanoLuc luciferase. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation with an 18 kDa subunit derived from NanoLuc (LgBiT). The small tag size of HiBiT and the ability to measure protein expression at endogenous levels makes HiBiT well suited for CRISPR-mediated genome editing. We therefore, established a straightforward, cloning-free and rapid CRISPR/Cas9 protocol for the specific insertion of HiBiT into the genome. The applicability of this newly develop method is shown for hypoxia inducible factor 1A (HIF1α). The importance of low endogenous levels of expression in assay response is demonstrated. Results and conclusions HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes HIF1α and several of its downstream transcriptional targets in response to various stimuli. These results demonstrate the ability to efficiently tag endogenous proteins with HiBiT, allowing sensitive quantitation of the response dynamics in their regulated expression.

How we do high content/ throughput screening of anticancer molecules

The presentation will summarize progress of the biological activity proﬁling by high throughput screening and high content analysis (HCA) at IMTM. The results of few examples of successful implementation of complete robotic assay pipeline and screens of large compound libraries will be presented. HCA is a powerful tool for the identiﬁcation of inhibitors / molecular probes of particular molecular targets, cellular pathways, and processes. Crucial steps in development and validation of such advanced assays as Cell cycle, DNA damage pathway analysis, DNA methylation and another cell-based reporter tools will be discussed in context with data analysis and used software tools. The project is supported by the grants IGA_LF_2017_026, LO1304 and LM2015063.

Proteomic proﬁling reveals DNA damage, nucleolar and ribosomal stress are the main responses to oxaliplatin treatment in cancer cells

Tomáš Oždian1, Dušan Holub1, Zuzana Macečková1, Lakshman Varanasi1, Gabriela Rylová1, Jiří Řehulka1, Jana Václavková1, Hanuš Slavík1, Pavel Moudrý2, Pawel Znojek1, Jarmila Stanková1, Juan Bautista de Sanctis3, Marián Hajdúch1, Petr Džubák1 1

Pawel Znojek, Peter Dzubak, Marian Hajduch 1

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic

Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic, Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University in Olomouc, Olomouc, Czech Republic, Institute of Immunology, National Center of Clinical Immunology and FOCIS Center of Excellence,

Faculty of Medicine, Universidad Central de Venezuela, Caracas, Venezuela Introduction Oxaliplatin is widely used to treat colorectal cancer in both palliative and adjuvant settings. It is also being tested for use in treating hematological, esophageal, biliary tract, and other gastric cancers. Oxaliplatin is routinely used in clinic, however little is known about the responses it induces in cancer cells. The whole-cell proteomics study was conducted to address this issue. Materials/methods Chemosensitive CCRF-CEM cells were treated with oxaliplatin at 29.3 μM (5x IC50) for 240 minutes (halftime to caspase activation)and cell lysates were processed using SILAC based high resolution MS proteomic experiment. Results and conclusions The proteomes of un-/treated cells were then compared by highresolution mass spectrometry, revealing 4049 proteins expressed over 3 biological replicates. Among these proteins, 76 were significantly downregulated and 31 significantly upregulated in whole experiment. In agreement with the DNA-damaging effects of platinum drugs, proteins involved in DNA damage responses were present in both the upregulated and downregulated groups. The downregulated proteins were divided into three subgroups; i) centrosomal proteins, ii) RNA processing and iii) ribosomal proteins, which indicates nucleolar and ribosomal stress. In conclusion, our data supported by further validation experiments indicate the initial cellular response to oxaliplatin is the activation of DNA damage response, which in turn or in parallel triggers nucleolar and ribosomal stress. This effect was further verified on other cancer cell lines.

2017

Translational cancer research I.

Chairs: Ondrej Slaby, Jiri Drabek středa / 29. listopadu 2017 / Wednesday / November 29, 2017 / 9:00 - 11:00 Unconventional transcripts, fusion genes and neocentromeres: the hidden face of genomic ampliﬁcations in cancer.

the overexpression of a circular RNA of the long-non coding RNA PVT1 (circPVT1), with miRNA sponging activity, in leukemias with 8q24 amplification. Therefore, the detection of unconventional transcripts Clelia Tiziana Storlazzi (post-transcriptional chimeras and University of Bari “ Aldo Moro”, Bari, deregulated circRNA) indeed open new scenarios in the understanding of Italy the transcriptomic impact of genomic Introduction amplifications in cancer. Very recently, it was shown that extrachromosomal amplification How can we target the as double minutes (dmin) or ring CXCL12-CXCR4 axis in chromosomes occurs in half of all oncology patients malignancies, making it as one Stefania Scala of the most important types of mutations in carcinogenesis. We Istituto Nazionale per lo Studio e la unveiled new phenomena associated Cura dei Tumori, Fondazione “ G. with the origin, the evolution and Pascale”, Naples, Italy the transcriptomic impact of such esults and conclusions chromosome alterations. Regarding The CXC–chemokine receptor-4 their genesis, our findings are ( CXCR4) pathway plays a role in consistent with a step-wise evolution cancer cell homing and metastasis, of amplicons rather than with a and thus represents a potential target single-step event like chromothripsis, for cancer therapy. Although there as suggested by recent literature. has been great enthusiasm, to date We demonstrated that the episome the promise has yet to be fulfilled. A model might be considered as new class of CXCR4-antagonist cyclic a valid mechanism behind their peptides was recently developed emergence, both in leukemias by us (PCT/IB2011/000120; EP 2 and in solid tumors. In this multi- 528 936 B1; US2013/0079292A1). step evolutionary path, scattered Based on a rational design a small acentric dmin would collapse into ring domain was identified as structural chromosomes with neocentromeres, overlapping motif common to leading to the mitotic stabilization of CXCL12 , the CXCR4-CXCR7 extra-chromosomal amplifications activating ligand, and to vMIP-II, an and providing a selective advantage inhibitory chemokine herpes virus to tumor cells. The occurrence 8 secreted (HHV8). Based on this of neocentromeres with a very motif, secured by a cysteine bond , complex internal structure was also a small library of nineteen peptides detected on ring chromosomes or was designed and evaluated for giant rod chromosomes harbouring the in vitro capability to inhibit the high-copy number amplifications in CXCR4 receptor. From the in vitro liposarcomas. screening three peptides emerged as Concerning the oncogenic role powerful antagonists. These peptides of amplifications, we detected were further evaluated in in vivo chimeric transcripts involving models of B16-hCXCR4 metastasis amplified oncogenes derived in immunocompetent mice and from post-transcriptional events, in human renal cancer xenograft i.e. not generated by genomic models. The compound named rearrangements. Moreover, we found Peptide R was identified as the most

active. With the intent to improve efficacy and biodistribution, stealth liposomes decorated with Peptide R were developed (PL-Peptide R). In vitro PL-Peptide R efficiently inhibited CXCR4- dependent migration and in vivo significantly reduced lung metastases and increased overall survival in B16-CXCR4 injected C57BL/6 mice. CXCR4 is expressed on immune cells including monocytes, B cells and naive T cells in peripheral blood and CXCL12 acts as a chemoattractant for immature and mature hematopoietic cells; it thus plays an important role in inflammation and immune surveillance of tissues. Effective immune cell access to tumor is controlled by CXCL12. CXCL12 repels tumor-specific effector T cells and recruits suppressive cell populations at tumor sites thus impairing effective immune response. Peptide R, inhibits CXCR4 receptor in cancer cells and reverts the Tregs immunosuppressive function. Targeting the CXCR4–CXCL12 axis thus offers the possibility of affecting CXCR4-expressing primary tumor cells, modulating the immune response, or synergizing with other targeted anticancer therapies.

Genomic data as the basis for personalized therapeutic planning in pediatric oncology

Ondrej Slaby1, Hana Noskova1,2, Sona Klusova1, Jaroslav Sterba2 CEITEC, Masaryk University, Brno Department of Pediatric Oncology, University Hospital Brno Despite improvements in the treatment of pediatric malignancies during the last decades there remains an unmet need for new drugs for children with cancer to cure those who presently die from their disease and to reduce the sequelae in survivors. However, drug 1 2

2017

development in children is still based on the adult indication although most of targeted oncology drugs might have a Mechanism of Action applicable to a childhood malignancy. The necessary starting point for

the new therapeutic strategies is a detailed molecular characterization of the tumor. In our study, we have explored the clinical applicability and feasibility of genomic data (germline and somatic whole-exome

sequencing) to drive a biologically based rational targeted therapies on a cohort of pediatric cancer patients with high-risk tumors.

2017

Molecular targets & biomarkers I.

Chairs: Jiri Sana, Josef Srovnal středa / 29. listopadu 2017 / Wednesday / November 29, 2017 / 11:15 - 12:30 Immunotherapy prediction in cancer patients using microsatellite instability

Josef Srovnal1, Monika Vidlarova1, Andrea Prokopova1, Alona Rehulkova1, Pavel Skalicky2, Kamil Vyslouzil2, David Vrana3, Karel Cwiertka3, Marian Hajduch1 1

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic, Department of Surgery, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic, Department of Oncology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic

Introduction Microsatellite instability (MSI) is caused by failure of DNA repair mechanisms (MMRs), which normally repair replication errors. Inactivation of some MMR genes, such as hMLH1, hMSH2, hMSH6 and hPMS2, results in MSI. Originally, MSI was described in the context of germline mutation of MMR genes in families with hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome), but in sporadic colorectal carcinomas, MSI is most often caused by hypermethylation of the hMLH1 promoter and occurs frequently in other carcinomas. MSI occurs in about 10-15% of all colorectal carcinomas, and as it affects the prognosis of the disease, MSI analysis is an important tool in clinical research and molecular diagnosis of cancer. Currently,

FDA approved pembrolizumab for metastatic or unresectable solid tumors with MSI or deficient MMR. Materials/methods MSI was tested in 170 colorectal cancer patients using Bethesda panel of five microsatellites. Total DNA was purified from tumor tissue and peripheral blood by DNeasy kit (Qiagen) according manufacturer’s instructions. The PCR products of amplified microsatellites from both tissues were compared by labelfree electrophoresis on the Agilent 2100 Bioanalyzer with the Agilent DNA 1000 kit. Subsequently, the PCR electrophoregrams of the five microsatellite-specific PCR products from the tumor and bone marrow of the same patient were evaluated. Results and conclusions High MSI (instability in 2 or more microsatellites) was found in 22/170 (12.9%) of colorectal cancer patients. Microsatellite stability was revealed in 103/170 (60.6%) of patients. The rest of patients showed low MSI (1 unstable microsatellite). In this case, further testing of next two or more mononucleotide microsatellites is recommended. Conclusion: MSI or dMMR testing is an emerging issue in cancer patients because of the novel therapeutic approaches available for MSI cancer. Moreover, the testing can identify patients in risk of hereditary cancer syndrome. Acknowledgment: This work was financially supported by NCLG LM2015091, IGA UP LF 2017_013 and NPU LO1304.

MiRNA proﬁling in recurrent and non-recurrent meningeomas

Hanuš Slavík, Josef Srovnal, Tereza Lausová, Jana Vrbková, Vladimír Balik, Marián Hajdúch

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic Introduction Meningeomas represent about 20 % of intracranial tumors and they are usually benign. However, these tumors can relapse after surgical removal, also with progression into higher histopathological grade. Identification of the patients with higher probability of a relapse is our main goal. Materials/methods 65 FFPE tissue samples were used for the analysis (30 samples from non-recurrent patients, 30 pair samples from 15 recurrent patients and 5 negative controls from histologically normal tissue samples of brain envelopes). After total RNA extraction with miRNeasy Mini kit (QIAGEN), Affymetrix miRNA 4.0 Array and FlashTagTM Biotin HSR RNA Labeling Kit were used for potential biomarkers identification. RT-qPCR will be used for data validation. Results and conclusions 54 differentially expressed miRNAs have been found in the samples from recurrent patient before relapse. 16 candidate miRNAs have been chosen for further validation using RT-qPCR and 5 potential molecules for data normalization with the most stable expression in whole data set. Weak differences have been found between first and subsequent tumors in recurrent patients. This project was supported by grants IGA_LF_2017_013 and AZV 1529021A.

2017

Cytogenetic analysis of recurrent glioblastomas

Magdalena Houdova Megova1, Zuzana Sporikova1, Radek Trojanec1, Ondrej Kalita2, Jana Vrbkova1, Jiri Drabek1, Vladimira Koudelakova1, Sona Mlcochova1, Miroslava Rabcanova1, Marian Hajduch1 1

Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and Faculty Hospital Olomouc, Olomouc, Czech Republic, Department of Neurosurgery, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacky University in Olomouc, Olomouc, Czech Republic

Introduction Glioblastoma (GBM) is the most frequent and the most malignant brain tumor of adults. Despite aggressive therapy the overall survival of glioblastoma patients still remains only 12-15 months. The long term survivals typically recur. Currently, there is no standard treatment and lack in knowledge of molecular biology of recurrent GBM. We analyzed molecular and genetic aberrations occurring in primary tumor and after recurrence to elucidate biological characteristics of glioblastoma recurrence.

EGFR amplification (19/24 primary tumors, 16/24 recurrent tumors), CDKN2A loss (18/24 primary tumors, 16/24 recurrent tumors), MDM2 gain (4/24 primary tumors), PDGFRA gain (3/24 recurrent tumors) and GSST1 loss in primary tumors (7/24) and GSTM1 gain in recurrent tumors (7/24). Univariate analysis of overall survival for significant copy number alterations shown significantly worse prognosis for patient with GSTT1 deletion (HR = 5,356, CI = 1,161-24,701, p = 0,031) in primary tumor and trend to better prognosis for patients with CDKN2A deletion and GSTM1 amplification in recurrent tumor. Significance of GSST1 and GSTM1 gene were verified in control group of 104 glioblastomas, where multivariate analysis with adjustment for age and Karnofsky score validate significantly worse prognosis for GBM patient with aberrations of GSTT1 gene. Molecular genetic profiling of 24 recurrent GBM showed association between recurrence and status of glutathione S-transferases genes. The financial support of grants NT13581, TACR TE02000058, IGA UP LF_2017_013 is gratefully acknowledged.

The opioid and cannabinoid receptors expression affects survival of colorectal cancer patients

Monika Vidlarova1, Josef Srovnal1, Emil Berta1 ,2, Petr Prasil3, Andrea Prokopova1, Materials/methods Sona Gurska1, Alona 47 recurrent GBM paired samples 1 1 were obtained. 24 paired samples Rehulkova , Jana Vrbkova , 1 were analyzed by OncoScan assay, Marian Hajduch 23 by fluorescent in situ hybridization for determination of EGFR, p53, RB1, MDM2, CDKN2A, GSTM1, GSTT1 genes and 1p, 19q and 10p chromosomal regions statuses, methylation-specific PCR for MGMT promoter methylation status and CADMA PCR for IDH1/2 mutation status. OncoScan data were processed by GISTIC 2 software. Results and conclusions The most significant aberrations were

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic, Ringerike Hospital, Hønefoss, Norway, Department of Anesthesiology, Vienna, Austria

Introduction Colorectal cancer (CRC) is one of

the leading causes of cancer death in the world. CRC patients are often treated with opioid and cannabinoid analgesia for pain, nausea and side effects of chemotherapy, which can affect their survival. These analgetics acts through opioid and cannabinoid receptors. These receptors and their pathways are involved in tumor progression and metastases. In our study we investigated the opioid receptors µ (OPRM), κ (OPRK) and cannabinoid receptor 2 (CNR2) gene expressions in the CRC patients’ tumor tissue and we analyzed relationship between their expression and patients survival. Materials/methods The OPRM, OPRK and CNR2 gene expression was analyzed in RNA puriﬁed from TRIzol lysates of tumor tissues from 157 CRC patients. Expression of OPRM, OPRK and CNR2 was detected using real-time RT-PCR on LightCycler 1536 from Roche. Β-actin gene expression was used for gene expression normalization. Speciﬁc cut-off values were calculated for each marker using maxstat R software, ver. 3.3.1. Relationship between expression of OPRM, OPRK and CNR2 in tumor tissue and patients survival was analyzed using COX regression and Kaplan-Meier method. Results and conclusions We investigated 157 patients (67 females and 90 males, median age 68 years), stages I-III. In total, 62 (39.5 %) of 157 CRC patients died during a median follow-up period 109.5 months, 30 (21.4 %) of them in connection with current cancer disease and 83.5 % of this patients have survival longer than 5 years. OPRM expression was detected in 37 % of all patients, 62.3 % was bellow and 37.7 % was above the cut-off value. OPRK expression was detected in 28 % of patients, 76.9 % was bellow and 23.1 % was above the cut-off value. CNR2 expression was detected in 53 % of all patients, 74.6 % was bellow and 25.4 % was above the cut-off value. We found out that patients with higher OPRM gene expression

2017

have significantly better overall survival (p=0.012) and patients with higher OPRK and CNR2 gene expression have significantly better disease-free survival (p=0.026; p<0.039, respectively) than patients with low opioid/cannabinoid receptors gene expression. Novel approaches for targeting OPRM, OPRK and CNR2 are promising and requires further validation. Acknowledgement: This work was financially supported by NCLG LM2015091, IGA UP LF 2017_013 and NPU LO1304.

Identiﬁcation of candidate genes underlying soft tissue sarcoma progression using a progression series of murine ﬁbrosarcoma cell lines

Jiří Hatina1, Michaela Kripnerová1, Hamendra Singh Parmar1, Jitka Kuncová2, Martin Leba3, Jiří Šána4, Ondřej Slabý4, Martin Pešta1 1

Charles University Medical Faculty in Pilsen, Institut of Biology, Plzeň, Czech Republic, Charles University Medical Faculty in Pilsen, Institute of Physiology, Plzeň, Czech Republic, University of West Bohemia, Faculty of Applied Sciences, Department of Cybernetics, Plzeň, Czech Republic, Central European Institute of Technology, Molecular Oncology II - Solid Cancer, Brno, Czech Republic

Introduction Sarcomas represent a heterogeneous group of mesenchymal tumours featuring a great variability in their clinical behaviour, ranging from rather indolent lesions with a good

prognosis to rapidly metastasing fatal tumours. While our knowledge of sarcoma initiation advanced rapidly in recent years, relatively little is known about mechanisms of sarcoma progression. Progression series of well characterized sarcoma cell lines can be very instrumental in this respect. We have recently established a series of four mouse fibrosarcoma cell lines, JUN-1, JUN-2, JUN-2fos-3, and JUN-3. JUN-1 and -2 were established from a single tumour initiated in a H2K/v-jun transgenic mouse, JUN3 originates from a different tumour in the same animal, and JUN-2fos-3 results from a targeted in vitro transformation of the JUN-2 cell line. The JUN-sarcoma cell lines present with a range of transformation and metabolic characteristics. With regard to the cell proliferation, motility and invasiveness, the original tumour-derived cell lines show a linear progression scale, from the least transformed JUN-2 to the most transformed JUN-3. In contrast, the JUN-2fos-3 cell line exhibits a unique transformation pattern, with little deregulation of cell growth and proliferation, but pronounced motility and invasiveness (1,2). 1. Hatina J, Hajková L, Peychl J, Rudolf E, Fínek J, Červinka M, et al. (2003) Establishment and characterization of clonal cell lines derived from a fibrosarcoma of the H2-K/V-JUN transgenic mouse. A model of H2-K/V-JUN mediated tumorigenesis. Tumor Biol. 24, 176184. 2. Kripnerová M, Tuková J, Šrámek J, Houdek Z, Pešta M, Grundmanova M, Kuncova J, Hatina J (2015) A progression series of murine fibrosarcoma

separates proliferativeand invasive transformation characteristics. 18th ECCO - 40th ESMO European Cancer Congress, Vienna, Austria 2015 (Abstract: Eur J Cancer 51, Suppl S3, S691). Materials/methods This unique distribution of transformation related-traits made us possible to identify two separate groups of genes tentatively involved in sarcoma progression in a single transcriptomic analysis - on the one hand, proliferation-related genes could be identified by their differential expression in JUN-3 compared to both JUN-2 and JUN-2fos3, and, on the other hand, motility and invasiveness-related genes could be identified by their common expression pattern in JUN-2fos3 and JUN-3 cells compared to JUN-2. Results and conclusions We identified 88 proliferation-related genes and 54 invasiveness-related genes At an initial scrutiny, the Ccl-8 chemokine attracted our attention as it has been independently described as a motility activator for various human carcinomas and it represents a potentially druggable target. Contrary to the finding in human breast carcinoma and melanoma, where CCL-8 is exclusively stromaderived (i.e. paracrine), it seems to acts as an autocrine motility factor in our sarcoma cells. Preliminary experiments suggest that its pharmacological inhibition indeed decreases the migratory ability of the highly motile and invasive JUN-3 cells. We believe that the differentially expressed genes identified in this way could provide promissing candidates to explore clinical sarcoma progression.

2017

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2017

Translational cancer research II.

Chairs: Manilo Vinciguerra, Viswanath Das středa / 29. listopadu 2017 / Wednesday / November 29, 2017 / 13:30 - 15:30 Optimized design and analysis of preclinical intervention studies in vivo

Andreas Scherer1, Matti Poutanen2 ,3, Tero Aittokallio1 ,4 1

Institute for Molecular Medicine Finland FIMM, University of Helsinki, Helsinki, Finland, Turku Center for Disease Modeling, University of Turku, Turku, Finland, Department of Physiology, Institute of Biomedicine, University of Turku, Turku, Finland, Department of Mathematics and Statistics, University of Turku, Turku, Finland

Introduction Poor reproducibility and translatability of preclinical animal studies contribute to both the delays and high costs of therapeutic drug development, and are often caused by poor experimental design. We propose a novel modeling approach to improve statistical power of fully-blinded preclinical intervention studies. Materials/methods We analysed two intervention studies of orthotopic xenografts of VCaP prostate cancer cells in immune deficient mice as disease models for castration-resistant prostate cancer (CRPC). Study 1 analysed the efficacy of two androgen receptor antagonists (ARN-509 and MDV3100) to suppress the growth of castration-resistant VCaP tumors, while the study 2 investigated the effect of surgical and pharmaceutic therapies on orchiectomized mice. Our modeling tool based on animal matching is available as an opensource R-package (http://cran.rproject.org /package=hamlet), and through a web-based graphical user interface (http://rvivo.tcdm.fi/).

previously undetected inverse relationship between animal body weight at baseline and ﬁnal PSA level. The treatment effect of MDV3100 became signiﬁcant only after incorporation of the baseline matching information. As to study 2, and for MDV3100 treatment of study 1, a conventional power level of 0.8 could be reached with much smaller animal numbers in the matched analysis. Through normalization of baseline variability, matching improves statistical inference of treatment responses, and increased statistical power to detect true treatment effects. Implementation of the matching algorithm will lead to improved interpretation of preclinical animal study data, to a decrease in animal numbers used in such studies, and to a more effective clinical translation. We envision a need for a larger community effort toward preclinical reproducibility as it relates to animal studies.

Epigenetic and paracrine control of hepatic cancer stemness

Manlio Vinciguerra1 ,2, Oriana Lo Re1 ,3 1

Center for Translational Medicine, International Clinical Research Center (FNUSA-ICRC(, Brno, Czech Republic, Department of Histology and Anatomy, Faculty of Medicine, Masaryk University, Brno, Czech Republic, Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

Introduction Background and Aims: Hepatocellular cancer (HCC) is an aggressive Results and conclusions disease with a poor outcome. Cancer In study 1, we found that application stem cells (CSCs) are responsible for of the matching strategy uncovered

tumor relapse and chemoresistance. The epigenetic bases of self-renewal of CSCs are not well understood, and what are the paracrine mechanisms by which CSCs influence the behavior of surrounding cancer cells (CC) is unknown. MacroH2A1 is a variant of histone H2A1 that is a robust marker of differentiated HCC (Borghesan M et al, Cancer Res 2016); conversely, absence of macroH2A1 transforms HCC cells in CSCs, characterized by resistance to chemotherapy, enhanced potential to generate bigger and undifferentiated tumors, enhanced glycolytic metabolism and resilience to hypoxia (Lo Re O et al, in revision). The aim of this study was to understand if epigeneticallymodified CSCs may secrete specific factors influencing HCC growth and response to therapy. Materials/methods We used as a model system HCC Huh-7 cells knocked down (KD) for macroH2A1 expression, xenograft nude mice and human HCC biopsies. Metabolomics, immunoblotting, ELISA, gene expression (qPCR), immune assays and immunohistochemistry were used to dissect signaling pathways involves in cancer cell stemness. Results and conclusions Mass spectrometry coupled to ultra-high performance liquid chromatography (UHPLC-MS) was used to profile the metabolism of Huh-7 cells KD for macroH2A1: enhanced glycolytic flux, response to hypoxia and expression of redox carriers was found compared to control cells, consistent with CSCs features. Conditioned medium (CM) of Huh-7 KD for macroH2A1 was strikingly depleted of cytokines IL-6, IL-8 and MIP-1d. Exposure of Huh7 cells to CM from Huh-7 KD cells protected them from chemotherapyinduced senescencein vitro and in vivo in nude mice. CM of KD cells

2017

had no inﬂuence on hepatic stellate cells activation, but failed to activate human T cells, whereas normal cell medium led to both CD4+ and CD8+ cell activation. Immunohistochemistry analyses of human HCC biopsies revealed that undifferentiated parts of tumors expressed less IL-6/ IL-8 levels and CD8+ inﬁltrates compared to differentiated areas. Conclusions: HCC cells devoid of histone macroH2A1 acquire a CSC-like phenotype and secrete a cytokine depleted medium which renders normal HCC resistant to chemotherapy and able to escape immune surveillance. These data uncover a new potential mechanisms by which CSCs tweak the cellular environment to favor cancer growth. This work was supported by the European Social Fund and European Regional Development Fund Project MAGNET (No. CZ.02.1.01/0 .0/0.0/15_003/0000492).

Life is never ﬂat: The rational use of 3D cell cultures as More predictive preclinical tumor models

Viswanath Das Institute of Molecular and Translational Medicine, Olomouc, Czech Republic Introduction In real life, cells are physiologically arranged in a three-dimensional (3D) architecture. Cellular systems that better reproduce the complexities of human systems are expected to signiﬁcantly augment oncology drug discovery and development. A number of predictive models, such as patient-derived primary cells, tumor grafts (PDX), and organoids models now offer relevant predictive insights into clinical outcomes and enable precision medicine approaches. One of the representative predictive models widely used across laboratories is the spheroid cultures of routinely used cancer cell lines. Unlike PDX models, spheroids of cancer cell lines have limited histological resemblance to primary cancer. However, spheroids mimic

many clinically relevant features like hypoxia, altered pH, cell-cell interactions, and multicellular organization, which determine how cells respond to external stimuli. The advantages of spheroids over other predictive models are the relative ease of maintenance and genetic manipulation that make spheroids compatible to high-throughput drug discovery. In this talk, I will provide an overview of our current work on spheroid models to appraise the strengths and current limitations in the field. Materials/methods Monotypic and heterotypic cocultures of spheroids were generated using a modified version of liquidoverlay culture technique established in our laboratory. Screening of small molecules in spheroids and spheroidderived cells were performed using standard cytotoxicity assays, and high-content imagining at our High Throughput Screening Facility (http:// imtm.cz/high-throughput-screeningand-high-content-analysis-core). Detailed microscopic analyses and drug penetration studies in spheroids were carried out by light-sheet fluorescence microscopy. Results and conclusions Recent developments clearly indicate that the transition from 2D to 3D cell cultures is promising for translational research. The data presented in this talk present spheroids as an important tool for bridging the gap that existed between 2D cell cultures and animal studies. As evident from the presented data, cells behave and respond differently to external stimuli in a 3D environment than 2D. Although it is becoming increasingly evident that 3D cell cultures are better predictive models than 2D cultures for drug discovery, are we completely ready to do away with 2D systems? Possibly not due to current challenges associated with 3D cell culture-based assays. However, given the predictive value of 3D cellular models, the balanced approach is to rationally harness the combined strength of 2D and 3D cell culture systems to gain new insights

and solutions to challenging scientiﬁc questions.

Analýza mikroRNA v mozkomíšním moku u pacientů s gliomy

Kopková Alena1, Šána Jiří1,2, Večeřa Marek1, Fadrus Pavel3, Smrčka Martin3, Slabý Ondřej1,2 1

Středoevropský technologický institut (CEITEC), Masarykova univerzita, Brno, Klinika komplexní onkologické péče, Masarykův onkologický ústsav, Brno, Neurochirurgická klinika, Fakultní nemocnice Brno, Brno.

Východiska Gliomy jsou rozdělovány podle histopatologických znaků do dvou hlavních skupin a to na gliomy nižšího (LGG) a vyššího (HGG) stupně malignity. Tyto dvě skupiny se značně liší v prognóze, terapii a průměrné době přežívání pacientů, proto její indikace velmi záleží na diagnóze. Nicméně současné diagnostické přístupy jsou u gliomů v mnoha případech limitovány jednak lokalizací a jednak buněčnou heterogenitou. MikroRNA (miRNA) tvoří skupinu krátkých nekódujících RNA, které jsou zapojeny do patogeneze mnoha nádorových onemocnění. Pozměněné hladiny miRNA asociovanými s některými typy mozkových nádorů byly navíc pozorovány v mozkomíšním moku (CSF). Tyto skutečnosti naznačují, že by analýza miRNA v CSF pacientů postižených gliomem mohla vést k vytvoření nové diagnostické platformy umožňující přesnější stanovení diagnózy. Materiál a metody Ve fázi optimalizace izolace RNA z CSF bylo porovnáváno několik komerčně dostupných kitů s různými modiﬁkacemi v protokolu. Jako nejvhodnější byl vyhodnocen Urine microRNA Puriﬁcation Kit (Norgen). Následná vysokokapacitní analýza miRNA v CSF byla provedena na souboru 36 HGG, 3 LGG a 19 nenádorových vzorků. Všechny

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vzorky byly analyzovány pomocí technologie sekvenování nové generace (NGS) na přístroji NextSeq 500 (Illumina), s využitím CleanTag small RNA Library Preparation Kitu (Trilink, Biotechnologies) pro přípravu knihovny a NextSeq 500/550 High Output Kit v2, 65 cyklů (Illumina) pro vlastní sekvenaci.

Global microRNA expression analysis of Sox-2 positive and negative glioblastoma cells

Výsledky Následná statistická analýza odhalila 13 rozdílně exprimovaných miRNA mezi CSF odebranými od pacientů s GBM a nenádorovými vzorky (přizpůsobená hodnota p < 1x10-6; miR-499a-5p, miR-30b-5p, miR-4454, miR-4284, miR-5100, miR-4791, miR-490-5p, miR-36073p, miR-196a-5p, miR-30c-5, miR10a-5p, miR-199b-3p a miR-7641). Zároveň bylo odhaleno 9 miRNA, které byly statisticky významně rozdílně exprimovány mezi HGG a LGG (přizpůsobená p hodnota < 0,025; miR-126-3p, miR-144-5p, miR-4454, miR-208b-3p, miR-133b, miR-452-5p, miR-511-5p, miR-944 a miR-133a-3p).

Introduction: Glioblastoma multiforme (GBM) is the most frequent intracranial malignity of astrocytic origin in adults. This tumor is characterized by infaust prognosis, which is primarily caused by resistance to the therapy and early relapses relate to the presence of glioblastoma stem cells (GSCs). These cells form neurospheres in vitro and express markers of stemness such as Sox-2, Oct-4, and nestin. Targeting of GSCs could be a novel promising therapeutic approach leading to the overcome of therapy resistance and better prognosis of GBM patients. One of the approaches how to successfully regulate GSC is a targeted regulation of microRNAs (miRNAs). These small non-coding RNA molecules post-transcriptionally regulate an expression of more than 2/3 of all human genes that are also involved in stem cell associated signaling pathways. Moreover, deregulated expression of some miRNAs has been observed in many cancers including GBM. Cell lines and Methods: We have prepared eleven Sox-2 positive and negative paired primary GBM cell lines, which have been cultured under DMEM/F12 medium supplemented with bFGF, EGF, and B12 supplement and DMEM medium supplemented with 10% FBS, respectively. The global miRNA expression analysis was performed

Závěr Na základě výsledků se domníváme, že expresní analýza zmíněných miRNA v CSF by mohla být užitečným diagnostickým nástrojem vedoucím ke zpřesnění diagnózy pacientů s gliomy zejména v případech, kdy v současné době užívanými diagnostickými přístupy dochází k přehlédnutí drobných ložisek s vyšším stupněm malignity, a tedy podhodnocení stupně malignity celého nádoru. Výsledky však ještě musejí být ověřeny na širším a nezávislém souboru vzorků. Tato práce byla podpořena MZ ČR grantem 15-34553A a projektem RVO (MOU, 00209805), a MŠMT ČR projektem CEITEC 2020 (LQ1601).

Sana Jiri, Vecera Marek, Slaby Ondrej Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic,

using GeneChip miRNA 4.0 Array (Affymetrix). Targeted regulation of miRNA levels have been carried out by the transient transfection of speciﬁc anti-miRNAs or miRNA mimics in GSC cell lines NCH 601 acquired from Interdisciplinary Center For Neurosciences (Heidelberg, Germany). The sphere formation assay was analyzed using IncuCyte ZOOM instrument (Essen BioScience). Sox-2 and nestin expressions were analyzed on both protein and transcriptional levels using combination of PAGE with Western blotting and real-time PCR, respectively. Results: Analysis of Sox-2 positive and negative paired GBM cell lines revealed 29 differentially expressed miRNAs, from which miR-93-3p, miR-95-5p, miR-106b-5p, miR-223p, and miR-3195 showed high signiﬁcance (adjust. P value < 0.05) and association with both Sox-2 (Spearman R; p < 0.002) and nestin (Spearman R; p < 0.02) expressions. MiR-22-3p and miR-3195 showed lower expression whereas other miRNAs higher expression in Sox2 positive GBM cells. The in vitro analyses also suggest that miR22-3p and miR-106b-5p affect the sphere formation potential and Sox-2 expression in GSC cells. Conclusion: Taken together, our data suggest that miR-22-3p and miR-106b-5p are probably involved in GSC biology and, thus, should be promising molecular targets to overcome GBM therapeutic resistance. This work was supported by Ministry of Health of the Czech Republic, grant nr. 1533158A, 15-34553A, 15-31627A and 15-34678A. All rights reserved.

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New technologies & bioinformatics

Chairs: Petr Dzubak, Martin Mistrik středa / 29. listopadu 2017 / Wednesday / November 29, 2017 / 15:45 - 17:15 Strategies for molecular target identiﬁcation.

Petr Dzubak, Tomas Ozdian, Dusan Holub, Jana Vaclavkova, Jarka Stankova, Gabriela Rylova, Jiri Rehulka, Vishwanath Das, Marian Hajduch. Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc. In the past, we have identified many of active molecules by phenotypic screens. To support their development into the clinical candidates, the target discovery is an essential step. The strategies that are employed by the “bench” scientist may be genetic, genomic or proteomic and each has its rightful place in the drug-target discovery process. As many bioactive molecules interact with protein targets, the identification by affinitybased proteomic techniques, activitybased protein profiling, or photoaffinity protein labeling are currently used in drug discovery. Such experiments draw upon several disciplines, synthetic chemistry, cell biology, biochemistry and mass spectrometry. These proteomic tools are critical to maintaining the drug-candidate supply, in the broader context of drug discovery. This study was supported by grants IGA_LF_2017_026 and by the National Sustainability Program (LO1304).

Bioinformatic analysis of massive parallel sequencing data for cancer research

Petr Vojta1, Rastislav Slavkovsky1, Marian Hajduch1 1

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University in Olomouc, Olomouc, Czech Republic

Introduction: Nowadays, massive parallel sequencing (MPS) is becoming one of the most dominant method in genetic analysis, in both clinical diagnosis of hereditary malignancies or in experimental identification of new cancer biomarkers. In addition, MPS allows very detailed detection of low-copy somatic mutations including clonal identification in heterogeneous cell subpopulations in case of various neoplasias. Characteristic employment of the MPS methods require specific bioinformatic analysis approaches, due to the versatile usage of MPS. Conversely, during the annotation of DNA variants, we can use a number of common techniques, such as comparison with publically accessible database resources (dbSNP, COSMIC, 1000 genome, ExAC, ClinVar) or focusing on individual subsets of genes based on their phenotypic features or molecular functions described in the ontological relationships.

Faculty of Science, Palacky University, Olomouc, Czech Republic Introduction Shotgun proteomics is the leading technology for discovery-oriented identification of peptides. Routine methods are restricted to reference peptides and their post-translational modifications. Although variants carry wealth of information, they are usually not part of computational analyses. Materials/methods System for identification of variant peptides was developed and further scaled up with fast algorithm for spectral match calculation. Results and conclusions System was validated with internal and external matched sequencing data and preserved high correspondence (> 90%). Variants established genetic relationships within family members, with strong evidence of monozygosity of twin sons. Automatic analyses of PRIDE repository gave insights into sensitivity of deep measurements, reaching hundreds of reliably identified variants (e.g., 262 SNPs in PXD004452). In summary, variant peptides provide insights into genetics and can be part of routine analyses of shotgun proteomics samples.

Aim: The aim of the presented contribution is to demonstrate the evaluation of the DNA variants in common issues such as somatic mutation detection in clinical practice (KRAS, EGFR, NRAS, BRAF), assessing of biomarker identiﬁcation in comparison Chronic lymphocytic leukemia of tumor and normal tissue and a demonstration of the DNA variant cases with stereotyped B-cell receptors: knowledgebase, annotation.

bioinformatics tools, data Variants in shotgun proteomics management and reporting Miroslav Hruska1 ,2, Jiri Voller1, towards personalised biomedical and clinical Lakshman Varanasi1, Petr 1 1 applications Dzubak , Marian Hajduch 1

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic, Computer Science Department,

Tomas Reigl1, Kamila Stranska1 ,2, Vojtech Bystry1, Adam Krejci3, Karol Pal1, Sarka Pospisilova1 ,2, Karla Plevova1 ,2, Nikos Darzentas1 1

CEITEC – Central European

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Institute of Technology, Masaryk University Brno, Brno, Czech Republic, University Hospital Brno, Brno, Czech Republic, RECAMO, Masaryk Memorial Cancer Institute, Brno, Czech Republic, University Hospital SchleswigHolstein, Campus Kiel, Kiel, Germany

Introduction Chronic lymphocytic leukemia (CLL) is generally a heterogeneous disease with varying clinical outcome. At the same time, it features subsets of patients with similar, stereotyped B cell receptors (BcR), involving one in three cases in studied cohorts. A number of such ‘CLL subsets’ have been shown to also share biological and clinical markers, with implications for personalised care. To leverage this, diagnosticians need to be able to manage and annotate BcR sequence data, place their results in context of available information, interpret them, and report them consistently. Materials/methods Different components of our Antigen Receptors Research Tool / ARResT bioinformatics platform (bat.infspire. org/arrest/) are utilised: GLASS enables the initial inspection of sequenograms. ARResT/Interrogate annotates sequences with IMGT/ V-QUEST and assigns them to major CLL subsets with ARResT/ AssignSubsets. ARResT/Interrogate also allows the user to access all stored and new data in more detail at any time, and create informative and consistent reports. Results and conclusions In cooperation with the University Hospital Brno, we have now built a prototype of the ARResT-based bioinformatics platform for their cohort of more than two thousand of CLL cases and for daily use in the clinic. It greatly helps to annotate, store, visualise and compare all CLL patient data, and allows seamless

access to publicly available information about any of the major CLL subsets through ARResT/ Subsets (a.k.a. the Encyclopedia of CLL Subsets) to place the result in context of other stereotyped CLL cases and relevant literature, including published biological and clinical data. This leads towards personalised biomedical and clinical applications. Supported by Ministry of Health of the Czech Republic grant nr. 1634272A.

A novelty role of CDK12 in regulation of DNA ramage response by altering methylation of miR-152 and targeting downstream regulatory genes

Marta Dzimková, Dávid Vrábel, Hana Paculová, Jiri Kohoutek Veterinary Research Institute, Brno, Czech Republic Introduction The DNA-damage-response (DDR) pathway is a cellular mechanism which has evolved to protect cellular integrity by detection and repair of DNA lesions. Previously, our group and others demonstrated that the cyclin-dependent kinase 12 (CDK12) maintains genome stability via regulation of transcription of DDR genes, specifically, BRCA1, RAD51 and others. Importantly, downregulation of the CDK12 caused induction of the 53BP1 and γH2AX foci and accumulation of cells in the G2-M phase of the cell cycle. Since various microRNA (miRNA) are situated within coding genes, we hypothesized that expression of some of them might be also affected by CDK12 depletion. Therefore, we conducted a pilot study focused on identification of candidate miRNAs that might be significantly altered in CDK12 deficient cells. Indeed, downregulation of CDK12 protein level led to aberrant expression of several miRNAs. Among studied

miRNAs, the level of miR-152 was significantly elevated. By using predictive algorithm, several proteins that might be specifically targeted by miR-152 were examined. We confirmed that upregulated expression of miR-152 leads to decreased expression of DNMT1, RICTOR and MET proteins which are often found deregulated in rather wide spectrum of oncogenic diseases. Defects in methylation of miR-152 has been observed in several cancers and studies have proven an on/of loop between expressions of DNMT1 and miR152. We speculate that CDK12 participates in DDR machinery by two distinct mechanisms, either by orchestrating transcription of DDR genes or by stabilization of DNMT1 protein by blocking expression of miR-152 targeting DNMT1. Materials/methods Various methods were used to obtain significant results, among them statistical evaluation of MiRNAs present in different cell lines, cultivation of multiple cell lines under different treatment conditions, and evaluating protein levels vie western blot protocol. Results and conclusions CDK12 regulates transcription of specific protein coding genes, such as genes involved in DNA-damage repair (DDR), by phosphorylation of RNA polymerase II. Since majority of ncRNA, among them miRNAs, are located within protein coding genes, we were intrigued by a simple question, whether depletion of CDK12 will affect transcription/ expression of particular miRNAs? We found out, that some genes are indeed affected and also that CDK12 and miR152 might act concordantly with other regulational mechanisms. In summary, besides its known role in DDR pathway, CDK12 also seems to participate in the transcription/expression of unique miRNAs and thus impacts process of tumorigenesis.

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Molecular targets and biomarkers II.

Chairs: Marian Hajduch, Jiri Drabek čtvrtek / 30. listopadu 2017 / Thursday / November 30, 2017 / 9:00 - 10:30 New biomarkers – host methylation for management of women with a screen positive test

Chris Meijer Vrije Universiteit Amsterdam, Amsterdam, The Netherlands Results and conclusions hrHPV is the causative agent for cervical cancer screening and is more sensitive but less specific compared to cytology in the detection of CIN2+. hrHPV detects in a population the women at risk for cervical cancer and due to the lower specificity a triage test is necessary to detect in the HPV positive risk population the women with clinically relevant disease i.e CIN2+. Presently cytology at baseline and 6 months or HPV 16/18 genotyping are used as triage tests. However cytology is subjective and reporting abnormal cells influenced by prior knowledge of the HPV status and HPV 16/18 genotyping results in a population of non-HPV 16/18 pos women who still have a too high cumulative CIN3+ risk for waiting till the next screening round (3-5years) Even for non-HPV 16/18 pos women with normal cytology their CIN3+ tisk is too high (>2%). Recently it has been shown that detection of hypermethylation of promotor regions of host cell genes involved in cervical carcinogenesis i.eFAM19A4 and miR124-2 is a good tool to detect women at high short term risk for cervical cancer. The Qiasure assay is a quantitative methylation specific PCR which detects hypermethylation of FAM19A4 and miR124-2. The assay detects nearly all (99%) of cervical cancers and advanced CIN lesions defined as associated with an HPV infection >5y and characterized by high number of chromosomal aberrations. This makes the assay an ideal triage tool to detect women with clinically

relevant disease both in HPV pos. women as in women with abnormal cytology (threshold ≥ ASC-US). The assay can be used on both cervical scrapes as on cervico-/vaginal selfcollected material. HPV testing on self-collected cervico /vaginal samples followed by the Qiasure assay opens the way to full molecular screening. Importantly the long term risk for cervical cancer of HPV pos women who test Qiasure negative is at least as good as of HPV pos women who are cytology negative.

Pitfalls in HPV68a detection

Hana Jaworek, Katerina Kubanova, Rastislav Slavkovský, Vladimira Koudelakova, Jiri Drabek, Marian Hajduch Institute of molecular and translation medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic Introduction Human papillomavirus (HPV) infection is a known oncogenic agent associated with about 5% of all cancers in particular cervical cancer. HPV68 is relatively rare genotype classiﬁed by International Agency for Research on Cancer (IARC) working group as probably carcinogenic. Despite the proved oncogenic potential of HPV68, this HPV genotype may be excluded from HPV screening tests or newly developed vaccines due to its rarity in cervical cancer. HPV68 may exist in two subtypes (HPV68a and HPV68b) and primers targeting L1 gene are usually not able to detect HPV68a subtype. The aim of the study was to evaluate the efﬁcacy of routinely used cobas® 4800 HPV Test (targeting L1 gene) in HPV68a detection. Materials/methods Cervical smears (n = 2198) obtained

by physicians and cervicovaginal swabs obtained by self-sampling (n = 217) were analyzed for the presence of HPV. DNA was isolated by cobas x 480 automatic extraction. All samples were tested by cobas® 4800 HPV Test (Roche) and PapilloCheck® HPV-Screening test (Greiner BioOne) and results of both tests were compared. For viral load assessment, HPV68 positive samples were further analyzed by a quantitative multiplex real-time PCR (qPCR) detecting of E2 and E6 HPV68 genes and human GAPDH gene (internal control) using speciﬁc TaqMan® probes. Real-time PCR followed by high resolution melting (HRM) curve analysis was used for HPV68a and HPV68b subtypes differentiation. Results of HRM analysis were veriﬁed by sizing of PCR product using Agilent DNA 1000 kit. The study was approved by the ethical board of the Faculty of Medicine and University hospital. Results and conclusions HPV68 was detected in 39 of 2198 (1.77%) cervical swabs and 4 of 217 (1.84%) cervicovaginal swabs using PapilloCheck® HPV-Screening test. HPV68 single-type infection was detected in 33 of 43 (76.7%) cases. HPV68 coinfection with at least one other hrHPV genotype was detected in 10 of 43 (23.3%) cases. Cobas® 4800 HPV Test gave false negative result in 20 of 33 (60.6%) HPV68 single-type positive cases. The low viral load as a possible case of a false negative result was excluded by qPCR. Median viral load in false negative cases was higher compared to true positive cases (2988 E6 copies/ ng with a range from 18 to 109500 vs. 429 E6 copies/ng with a range of 18 to 57000). HPV68a subtype was detected in all (20/20) false negative cases by HRM analysis. HPV68a and HPV68b subtypes were detected in 5 of 13 (38.5%) and 8 of 13 (61.5%) of true positive cases

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respectively. These results were conﬁrmed by Agilent technology. Though the Cobas® 4800 HPV Test is routinely used HPV screening test for HPV diagnostics, a very high discrepancy in HPV68 detection was found in our study. The false negative result was detected in more than half of HPV68 single type infection cases due to its lower sensitivity for HPV68a. Prevalence of HPV68 genotype could be falsely lower because the cobas® 4800 HPV Test was used as a screening test in the majority of published studies.

Molecular cytogenetics of „double/triple hit” - high-grade B-cell lymphoma: a new entity of mature B-cell neoplasms

Helena Urbankova1, Michaela Vatolikova1, Zuzana Kubova1, Vit Prochazka1, Zuzana Pikalova1, Martin Novak1, Patrik Flodr2, Tomas Papajik1 1

Department of Hemato-Oncology, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomouc, Olomouc, Czech Republic, Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc and University Hospital Olomou, Olomouc, Czech Republic

Introduction According to a new advances in our understanding of biology of mature B-cell neoplasms, a new category of World Health Organization (WHO) classiﬁcation have arisen: Highgrade B-cell lymphoma (HGBL), with MYC and BCL2 and/or BCL6 genes rearrangements, also named „double/triple hit” HGBL (HGBL-DH/ TH). Genetic alterations of MYC and BCL2 and/or BCL6 genes are often a part of complex karyotypes containing multiple numerical and structural chromosomal aberrations. These genetic alterations are independent on the morphology of tumour, that may be diffuse large B-cell lymphoma (DLBCL), blastoid, or with features intermediate between DLBCL and

Burkitt lymphoma (BL).Reported median overal survival of patients with HGBL-DH/TH is lower than 2 years and it is clinically relevant to recognize this entity amongst other aggressive B-cell lymphomas. Materials/methods Routine immunohistochemistry is carried out on tumor tissue biopsies and is complemented by morphological and phenotypic assays. Conventional cytogenetics is limited by availability of fresh tissue infiltrated by lymphoma tumor cells, as the most cases of HGBLDH do not involve peripheral blood or bone marrow. Fluorescence in situ hybridisation (FISH) is recommended to identify MYC and BCL2 and/or BCL6 rearrangements using commercially available locus specific translocation probes. FISH is preferentially performed on suspensions of cultured tumor cells, but if not available it is possible to analyse also formalin-fixed paraffinembedded (FFPE) tissue biopsies (with some technical limitations). Results and conclusions Clinical impact of the MYC and BCL2 and/or BCL6 genes rearrangements seems more variable than suggested in the initial studies but the reasons for this diversity are not yet well known. The presence of MYC rearrangements is the determinant factor, but there are several additional events that seem to modulate its biological significance. Interestingly, there is clinical difference in MYC translocations with immunoglobulin genes (IG) partners and with nonIG partners, where MYC/IG translocations are associated with a higher levels of MYC expression and with worse outcome. The aggressiveness of HGBL-DH can be possibly explained by the synergistic action of altered MYC and BCL2 genes: proliferative activity together with inhibition of apoptosis in the context of a complex karyotype. The role of BCL6 rearrangements however still remains controversial. Approximately 25% of DLBCL cases with high expression of MYC and BCL2 protein, but

without genetic alterations are called „dual-expressors” (DE). The outcome of these cases is not so adverse as DH and they are not included in the HGBL category. The importance of recognizing HGBL-DH cases in current diagnostic practice of aggressive B-cell lymphomas seems to be clear, but there is still no consensus, in what cases is reasonable to use FISH examination due to its relatively high costs. A selection of cases based on morphological and phenotypic criteria seems an acceptable compromise. In summary, the molecular cytogenetics together with morphology, phenotyping and immunohistochemistry plays an irreplaceable role in diagnostics of HGBL-DH/TH in current clinical practice. Recently, new promising technologies of non-invasive genotyping of circulating tumor cell free DNA in peripheral blood emerged. So called liquid biopsies in combination with ultra-deep next generation sequencing could possibly complement molecular diagnostics of agressive B-cell lymphomas and help with monitoring of disease in real time.

Human papillomavirus infection in non-small cell lung cancer

Vladimira Koudelakova1, Hana Jaworek1, Rastislav Slavkovsky1, Jiri Drabek1, Jana Potockova1, Jana Vrbkova1, Marian Hajduch1 1

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic.

Introduction Human papillomavirus (HPV) infection is cause of various diseases and cancer, including anogenital warts, recurrent respiratory papillomatosis and anogenital and oropharyngeal cancers. In several studies, HPV DNA was detected also in lung cancer patients. Nevertheless, the signiﬁcance of

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HPV infection in lung cancer patients remains unclear. The objective of this study was to determine the HPV prevalence in non-small cell lung cancers (NSCLC) in the Czech Republic and its potential clinical significance. Materials and methods: A cohort of 80 NSCLC patients was selected. DNA was isolated from both formalin-fixed paraffinembedded tissue (FFPE) as well as fresh frozen (FF) tissue samples of each patient. All samples were tested for KRAS, EGFR and BRAF mutations using BRAF p.Val600Glu kit (IntellMed), Cobas® EGFR Mutation Test (Roche Diagnostics GmBH) and TheraScreen®: KRAS

Mutation kit (Roche Diagnostics GmBH). The presence of HPV16, 18, 31 and 56 DNA was tested by quantitative multiplex real-time polymerase chain reaction (qPCR). qPCR simultaneously detect E2 and E6 HPV genes and human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene (internal control) using speciﬁc TaqMan® probes. Results and conclusion: The method’s limit of detection (LOD) was determined using dilution series of plasmids HPV DNA with concentration range from 4 to 4x105 HPV copies/reaction. The qPCR method reliably detected HPV DNA in samples containing more

than 4 copies of E2 or E6 HPV genes. Sensitivity and specificity was evaluated using DNA samples isolated from cervical swabs where HPV16, 18, 31 or 56 positivity was detected by cobas® 4800 HPV Test (Roche Diagnostics GmBH) and PapilloCheck® HPV-Screening (Greiner Bio-One). In our study cohort, no HPV positivity was found in FFPE as well as FF NSCLC samples despite the very low LOD of this method. In conclusion, our finding did not confirm any etiologic correlation between HPV16, 18, 31 and 56 and primary NSCLC in the Czech population.

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Molecular targets and biomarkers III.

Chairs: Milan Reinis, Pavel Moudry čtvrtek / 30. listopadu 2017 / Thursday / November 30, 2017 / 10:45 - 11:45 Comparative analysis of senescent tumour cells induced by docetaxel or immunomodulatory cytokines

Milan Reinis, Olena Sapega, Romana Mikyskova, Jana Bieblova, Blanka Mrazkova, Zdenek Hodny Institute of Molecular Genetics of the ASCR, v. v. i., Prague, Czech Republic

cytokines IFNγ and TNFα induced senescence/proliferation arrest in B16 cells only. However, despite of the presence of characteristic features of senescent cells, B16 cells started to proliferate upon the cytokines withdrawal and did not induce bystander senescence. This work was supported by research grant No. 15-24769S provided by the Czech Science Foundation.

Downregulation of

uncharacterized human Introduction Cellular senescence is a process HEATR1 protein activates p53 of permanent proliferative arrest and induces ribosomal stress of cells in response to various Zsoﬁa Turi1, Marketa inducers. It is characterized by typical Senkyrikova1, Martin Mistrik1, morphological changes, as well as 2 ,3 1 by secretion of bioactive molecules Jiri Bartek , Pavel Moudry including proinﬂammatory cytokines 1 Laboratory of Genome Integrity, and chemokines (senescence- Institute of Molecular and associated secretory phenotype, Translational Medicine, Palacky SASP). The phenotypes of senescent University, Olomouc, Czech cells are determined both by the Republic, cellular stress inducing agents and 2 Genome Integrity Unit, Danish Cancer Society Research Center, the cell lineages. Copenhagen, Denmark, Materials/methods 3 Department of Medical To characterize phenotypes of Biochemistry and Biophysics, senescent cancer cells, we employed Division of Genome Biology, two different murine cell lines (TC- Science for Life Laboratory, 1 and B16 cells) and two distinct Karolinska Institute, Stockholm, senescence inductors, cytotoxic agent Sweden docetaxel (DTX) and combination of immunomodulatory cytokines Introduction IFNγ and TNFα. We analyzed cell The nucleolus is a membrane proliferation, markers of senescence, less organelle within the nucleus, SASP, as well as the capability of organized around ribosomal RNA senescent cells to induce bystander encoding DNA repeats. Ribosomal biogenesis which consist of the senescence. transcription of ribosomal DNA Results and conclusions (rDNA) genes, maturation of We have demonstrated that DTX ribosomal RNAs (rRNAs) and induced senescence both in the TC-1 assembly of ribosomal subunits, and B16 tumour cell lines, which was occurs in the nucleoli of the cells. This proved by growth arrest, positive beta- process consumes most of the cells galactosidase staining, increased energy, therefore it needs to be tightly p21 expression, typical SASP controlled and synchronized with the and capacity to induce bystander physiological condition of the cell. senescence. On the other hand, the Stress signals (e. g. genotoxic, treatment with combination of Th1 transcriptional, osmotic, etc.)

and pathways initiated by them, converge in the nucleolus to inhibit rRNA transcription. As a consequence, ribosomal proteins are released to the nucleoplasm, to bind MDM2 E3 ubiquitin ligase. The interaction of ribosomal proteins with MDM2 results in the reduced ubiquitination of p53 protein and p53 stabilization leading to cell cycle arrest, apoptosis or senescence. We performed a siRNA-based high content microscopy screen for knockdowns that activate p53 and focused on human uncharacterized protein HEATR1. Materials/methods siRNAs targeting HEATR1 protein was used to deplete endogenous expression of the protein. Cell proliferation assay, EdU incorporation assay and cell cycle analysis by ﬂow cytometry were performed to examine proliferation and cell cycle proﬁle. Localization of HEATR1 and nucleolar marker proteins were analyzed by immunostaining. Immunoprecipitation was carried out to investigate interaction of ribosomal proteins with MDM2. Protein abundance of HEATR1, p53, p21, RPL5 and MDM2 was analyzed by Western blotting. Finally, EU incorporation assay was used to measure the rate of nascent RNA synthesis. Results and conclusions We validated p53 activation by three independent siRNAs targeting HEATR1. We noted that HEATR1depleted cells have impaired proliferation. Further analysis showed reduced percentage of these cells in the S phase and decreased EdU incorporation. Latter phenotype could be rescued by co-depletion of p53 protein. In addition, we observed HEATR1 localization in the nucleolus and its function in rRNA transcription. Moreover, we found that HEATR1 knockdown cells induce the release

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of nucleophosmin and nucleolin from the nucleoli and increased interaction of MDM2 and RPL5, all of which are markers of ribosomal stress. These novel ﬁndings highlight the importance of tight regulation of the nucleolus and its function, ribosomal biogenesis.

Prostate cancer cell lines and patient-derived tissues as a model for therapy decision on inhibition of PARP1 or HDAC in combination with gamma irradiation

Dusana Majera1, Jan Bouchal2, Martin Mistrik1, Jirina Bartkova3, Jan Gursky1, Jiri Bartek3 1

Laboratory of Genome Integrity, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, Department of Clinical and Molecular Pathology, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, Danish Cancer Society Research Center, Copenhagen, Denmark

Introduction Therapy of castration-resistant prostate cancer still represents a serious problem with urgent need for improved targeting of these heterogeneous tumors. Molecular characterisation of particular tumors is necessary for the implementation of new therapies, such as inhibitors of PARP1 or SAHA-inhibor of histone deacetylases (HDAC). Materials/methods Prostate cancer cells were tested by colony formation assay after combination of gamma irradiation and pre-treatment with inhibitors of PARP1 or HDAC. Cell cycle and apoptosis after SAHA treatment were analyzed by ﬂow cytometry. Selected proteins from DNA repair pathways were detected by immunoﬂuorescence

and immunoblotting. Patient derived tissues were ex-vivo irradiated and analysed by immunohistochemistry. Results and conclusions We observed enhanced sensitivity to PARP1 inhibitor and gamma irradiation and lower level of homologous recombination, as determined by quantiﬁcation of Rad51 foci, in PC3 cell line, when compared to DU145 cell line. Treatment with SAHA sensitized DU145 cells to PARP1 inhibitor or gamma irradiation. Western blot analysis of several proteins involved in homologous recombination, and also mutated p53 protein were downregulated by SAHA in DU145 cells. On the contrary, proteins involved in NHEJ were not downregulated by SAHA pre-treated cells. Rad51 were successfully detected in ex-vivo irradiated prostate patients-derived tissues. Assessment of selected proteins in ex-vivo irradiated tissues may improve personalized prostate cancer therapy.

Prognostic and predictive value of loss of nuclear RAD51 immunoreactivity in resected non-small cell lung cancer patients

Mariam Gachechiladze1, Josef Skarda1, Vítezslav Kolek2, Yvona Grygarkova2, Katerina Langova3, Jan Bouchal1, Zdenek Kolar1, Florent Baty4, Rolf Stahel5, Walter Weder6, Alex Soltermann7, Markus Joerger0 1

Department of Clinical and Molecular Pathology, Insitute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic, Department of Tuberculosis and Respiratory Diseases, Faculty of Medicine and Dentistry, Palack´y University and University Hospital, Olomouc, Czech Republic, Department of Medical Biophysics, Faculty of Medicine and Dentistry, Palacky University, Olomouc,

Czech Republic, Department of Pneumology, Cantonal Hospital, St.Gallen, Switzerland, Clinic of Oncology, University Hospital, Zurich, Switzerland, Department of Thoracic Surgery, University Hospital, Zurich, Switzerland, Department of Pathology and Molecular Pathology, University Hospital, Zurich, Switzerland, Department of Medical Oncology and Hematology, Cantonal Hospital, St.Gallen, Switzerland

Introduction In response to DNA damage, recombination proteins are relocalized into sub-nuclear complexes that are microscopically detected as RAD51containing nuclear foci. We aimed for assessing the prognostic and predictive value of loss of nuclear RAD51 immunoreactivity (‘RAD51 loss’) in 2 independent stage I to III non-small cell lung cancer (NSCLC) patient cohorts undergoing surgical resection and eventual perioperative chemo-/radiotherapy (CT/RT). Materials/methods The discovery set included 69 evaluable patients (19 adenocarcinomas, ADC, 50 squamous cell carcinomas, SCC) from Palacky University Hospital, 45/69 (65.2%) with additional platinum-based CT. The replication set entailed 845 evaluable patients (446 ADC, 399 SCC) from University Hospital Zurich, 308/845 (36.5%) with platinum based CT or RT. RAD51 loss was deﬁned as ≤20% of tumor cell nuclei having any nuclear RAD51 expression. We assessed the prognostic value of RAD51 loss in all patients and its predictive value in patients receiving CT/RT. Results and conclusions RAD51 loss was observed in 40/69 (58.0%) and 439/845 (51.9%) evaluable tumors in the discovery and replication set, respectively (p = 0.34). It was more frequent in ADC compared to SCC (57.2% vs 47.4%, p = 0.003). RAD51 loss

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was signiďŹ cantly associated with worse OS in both the discovery (adjusted HR = 2.39, p = 0.039) and replication set (adjusted HR = 1.31, p = 0.008). The unfavourable prognostic effect of RAD51 loss seen in the overall population was

not observed in patients receiving perioperative CT (adjusted HR = 1.07, p = 0.73) or perioperative RT (adjusted HR = 1.05, p = 0.82). RAD51 loss has an unfavourable prognostic impact in NSCLC patients undergoing curative

surgical resection, but it may have a favourable predictive value in the subgroup of patients receiving perioperative platinum-based CT or RT, most likely as a consequence of deďŹ cient DNA repair.

2017

Posterová sekce / Poster section 1

Steps for improving reproducibility in biomedical research

Jiri Drabek IMTM, LF UP, Olomouc, Czech Republic Introduction Scientific papers used to bear an aura of objectivity and correctness. However, the times they are a-changin´ (Bob Dylan). The number of published papers grows exponentially but not their quality. Ninety three percent from 1576 top scientists, contacted by Nature feels crisis of reproducibility in science. Either we look at biomedicine or at its parts oncology and translational medicine, it is always obvious that their perception is evidence-based and vast amount of money and human potential is wasted (Waste Of Money, Brains, And Time; WOMBAT). Results and conclusions This poster explores the cause of low reproducibility in biomedicinal research. It finds the following culprits: a chance, insufficient methods and instruments, insufficient protocol description, publication bias, falsification, cognitive bias, motivation bias, and a mislead results interpretation. At the same time, it discusses the possibilities of improving the current status. 2

Automatic cytogenetic system Metafer®: optimization and comparison with manual method

Oliver Pešát1, Zuzana Šporiková1, Soňa Mlčochová1, Radek Trojanec1, Miroslava Rabčanová1, Magdalena Megová1, Gabriela Kořínková2, Marián Hajdúch1

Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University in Olomouc, Olomouc, Czech Republic, Department of Pathology and Laboratory of Molecular Pathology, Faculty Hospital Olomouc, Olomouc, Czech Republic

samples. Differences in results can be explained by tumour heterogeneity or improper settings of a system. This examination has a diagnostic and predictive signiﬁcance, therefore optimization of an automatic method has an impact on consistent and reliable results. 3

Gene aberrations in IDH-wild

Introduction type glioblastoma Automatic cytogenetic systems have Zuzana Šporiková1, Ondřej greatly developed during last decade 2 1 along with computational technology. Kalita , Marián Hajdúch , 1 Main purpose of automatic Radek Trojanec , Magdalena cytogenetic system is ability to Megová-Houdová1, Miroslav process samples much faster Vaverka2, Lumír Hrabálek2, than conventional microscopy and Miloslava Zlevorová3, David minimize human error. Optimization 3 1 of sample parameters and used Vrána , Jiří Drábek , Lucie 4 methods is necessary to provide best Tučková , Jiří Ehrmann4, Jana results. Comparison with manual Vrbková1 method helps to determine accuracy 1 Institute of Molecular and of automatic evaluation. Translational Medicine, Faculty of Materials/methods Medicine and Dentistry, Palacky Automatic cytogenetic system University in Olomouc, Olomouc, (Metafer® scanning and imaging Czech Republic, 2 platform, MetaSystems) was used in Dpt of Neurosurgery, University interphase FISH mode to determine Hospital Olomouc, Olomouc, amplification of HER2 gene in breast Czech Republic, 3 cancer tissue. Formalin-fixed paraffin- Dpt of Oncology, University embedded (FFPE) tissue sections of Hospital Olomouc, Olomouc, various thicknesses were used along Czech Republic, 4 with different probe preparation kits Dpt of Pathology and Laboratory of Molecular Pathology, University and ready to use probe. Hospital Olomouc, Olomouc, Results and conclusions Czech Republic Ideal thickness of tissue section was Introduction found out along with ideal probe preparation. Settings of automatic The prognosis of glioblastoma system were optimized mainly to multiforme (GBM) patients is probably find out most effective scanning the worse across all tumours and algorithm designed for determination unusual progression is typical for this of HER2 gene amplification in tissue disease. Overall, the 5-year survival samples. Most effective algorithm rate is <10%, with a final mortality is able to highlight most nuclei in a rate of close to 100% (1,2). The sample and gives operator ability prolonged survival is achieved only to manually check and highlight after maximal therapy of resection, or reject additional nuclei before chemotherapy and radiotherapy (3). spot evaluation. Comparison with Discovery of new gliomagenesis manual method was validated on 20 biomarkers during last few years

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helped to determine predictive and prognostic markers, but the molecular-cytogenetic background is not fully understood to this date. Glioblastoma multiforme is characterized by many genetic aberrations in signalling pathways that are currently being targeted in clinical trials. Genetic biomarkers helps to set a personalized therapy by definition of patient population that may profit from specific therapeutic agent. Given the aggressive and resilient nature of GBM, continued efforts to better understand GBM pathophysiology are required to discover novel targets for future therapy. Materials/methods The data of all glioma patients treated in the University Hospital Olomouc, have been collected prospectively and systematically since 06/2006 to 06/2014. FISH analysis was performed on FFPE tissues according to the manufacturers’ instructions with LSI 1p36.3/1q25.2, LSI 9p21.3/CEP9, LSI 19q13/19p13.42, LSI EGFR/ CEP7, LSI MDM2/12p12.1, LSI PTEN/10p11.1, LSI BCR/22q12.2, LSI CCND1/CEP11 (IntellMed, Ltd., Prague, Czech Republic) and LSI p53/CEP17, LSI RB1/13q12.11 (Vysis, Downers Grove, IL, USA) probes. IDH status: The CADMA PCR principle described by Kristensen et al. (4) was used for the detection of IDH1 R132H and IDH1 R132C mutations. Obtained data were statistically analysed by R Statistical Software, Version 3.2.1 Results and conclusions Results and conclusion: The data of IDH-wild type GBM patients are being statistically calculated, the results and conclusion will be presented in poster. Supported by IGA_ LF_2017_013. 1) Deen DF, Chiarodo A, Grimm EA, Fike JR, Israel MA, Kun L LE, et al. Brain Tumor Working Group Report on the 9th International Conference on Brain Tumor Research and Therapy. Organ System Program, National Cancer Institute.

J Neurooncol. 1993;16:243-72. 2) Kleihues P, Sobin LH. World Health Organization classiﬁcation of tumors. Cancer. 2000;88:2887. 3) Adamson C, Kanu OO, Mehta AI, Di C, Lin N, Mattox AK, et al. Glioblastoma multiforme: a review of where we have been and where we are going. Expert Opin Investig Drugs. 2009;18:1061-1083. 4) Kristensen LS, Andersen GB, Hager H, Hansen LL Competitive ampliﬁcation of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis. Hum Mutat. 2012:33(1):264-71. 4

OncoScan analysis of good and bad responders to bevacizumab among mCRC patients

Karolína Bartáková, Barbora Blumová, Helena Štefanová, Veronika Holinková, Miroslava Rabčanová, Jana Vrbková, Rastislav Slavkovský, Jiří Drábek Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Olomouc, Czech Republic Introduction Colorectal cancer (CRC) is a signiﬁcant cause of tumourrelated morbidity and mortality worldwide, with mortality most often attributable to metastases (mCRC). Improvements of cytotoxic chemotherapy and biologic therapy have prolonged the survival in mCRC. The main determinant of selecting panitumumab or cetuximab (anti-EGFR biological therapy) is wildtype status of KRAS and NRAS gene of the tumour cells while bevacizumab (anti-VEGF biological therapy) is applied to patients with tumours mutated in RAS genes. After more than ten years of bevacizumab approval in Europe, no prospectively validated predictive biomarkers of

response to bevacizumab have emerged. In the current era of personalized medicine, it is desirable to determine such biomarker(s). Materials/methods Formalin-fixed paraffin-embedded tumour tissues were obtained from 30 patients of University Hospital, Olomouc with diagnosed metastatic colorectal carcinoma and treated with FOLFOX (leucovorin, 5-FU, oxaliplatin) and bevacizumab therapy in the first line. They were divided into two groups according to progression-free survival (PFS): good responders (PFS ≥10 months) and bad responders (PFS ≤9 months) without no significant difference between two groups in basic clinical characteristics. Total DNA was purified from FFPE samples after pathological verification using proteinase K treatment followed by Cobas DNA Sample Preparation Kit (Roche). Subsequent microarray analysis was performed according to OncoScan assay manual (Thermo Fisher Scientific). Raw data were obtained using OncoScan Console 1.3 (Thermo Fisher Scientific) in the default manner. Subsequently, the data were analyzed using rCGH bioinformatics pipeline (Bioconductor) and GISTIC (Genomic Identification of Significant Targets in Cancer) algorithm (Broad Institute). Results and conclusions Statistically analyzed data from 15 OncoScan arrays of patients designated as good responders and 15 arrays of patients designated as bad responders were compared. Significant amplifications and deletions in aberrant regions of the genome were identified and compared with published data.¨

2017

Investigation of biocompatibility and cytotoxic effects of TiO2 nanoparticles with organic surface modiﬁcations

Zuzana Skubalova1, Hana Buchtelova1, Simona Dostalova1 ,2, Petr Michalek1 ,2, Sona Krizkova1 ,2, Lukas Richtera1 ,2, Vojtech Adam1 ,2, Zbynek Heger1 ,2 1

Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic, Central European Institute of Technology, Brno University of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno, Czech Republic

Introduction Nanoparticles are used in dermatological preparations, particularly to improve existing properties and also to create new ones. There are several research reports providing contradictory information about toxicity and distribution. Thus, we aimed on testing of cytotoxicity titanium oxide nanoparticles (TiO2 NPs), with surface modified using monochloroacetic acid and dichloroacetic acid. Materials/methods After synthesis, TiO2 NPs were characterized using TEM, FTIR and DLS. Cell lines used for testing of cytotoxicity were: prostate (PNT1A), neuroblastoma (SH-SY5Y) and melanoma (A375). Differential proteome analysis was performed using 2D SDS-PAGE followed by MALDI-TOF. Internalization of TiO2 NPs was studied after their noncovalent labeling with tdTomato expressed in E. coli. Results and conclusions TiO2 had oval-to-spherical morphology with diameter ranging between 1520 nm. Using FT-IR we confirmed successful surface modification. Although studied nanoparticles

exhibited only slight cytotoxic effects, using heterologously expressed tdTomato (λexc = 554 nm, λem = 581 nm) we found that they are able to internalize to intracellular space with accumulation within cytosol. Further proteomic analyses revealed that TiO2 NPs induce differential expression patterns and differentially expressed proteins belonged mostly to molecular pathways affecting response to stimulus, metabolical process, cellular component organization and angiogenesis. Overall, we show that despite in vitro cytotoxic screenings show only slight toxic effects of TiO2 NPs, these NPs affect fundamental molecular pathways and long-term exposure could be potentially a risk for health. The authors gratefully acknowledge ﬁnancial support from the AZV project 15-28334A. 6

Study on inﬂuence of surface modiﬁcations of apoferritin on intracellular delivery of ellipticine

Barbora Tesarova1, Simona Dostalova1 ,2, David Hynek1 ,2, Vojtech Adam1 ,2, Zbynek Heger1 ,2 1

Introduction Nanocarriers can serve as an appropriate platform for targeted chemotherapy. Modiﬁcations of their surface with polyethylene glycol (PEGylation) or peptide sequences rich in PRO, ALA and SER (PASylation) can minimize negative interactions of nanocarriers with biological environment; however they can also hamper internalization in target cells. Materials/methods

As a nanocarrier, we used ubiquitous protein apoferritin with encapsulated cytotoxic drug ellipticine. We studied the formation of protein corona on PEGylated and PASylated nanocarriers by fluorescence labelling of serum proteins and the influence of modifications and protein corona formation on their intracellular delivery of ellipticine. Results and conclusions The preparation of apoferritin nanoparticles with encapsulated ellipticine was confirmed by TEM, DLS and fluorescence analyses. Surface modifications were performed in order to decrease negative interactions with biological environment. Both surface modifications showed not to be hemotoxic for blood according to performed in vitro hemolysis assay. Protein coronas were not observed in fetal bovine serum, either in human plasma according to SDSPAGE. All prepared nanocarriers were smaller than 100 nm, showing their suitability for internalization into malignant cells. While PEGylation of the nanocarrier significantly hampered this internalization, PASylated nanocarrier retained its ability to internalize into cells and deliver the drug to its nucleus. The authors gratefully acknowledge financial support from The Czech Science Foundation (GACR 1712816S). 7

Stability of apoferritin modiﬁed with ANTI-hNET peptides for active targeting of neuroblastoma cells

Marketa Charousova1, Yazan Haddad1, Simona Dostálová1 ,2, Vladislav Strmiska1, David Hynek1 ,2, Vedran Milosavljevic1 ,2, Vojtech Adam1 ,2, Zbynek Heger1 ,2 1

Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00

2017

Brno, Czech Republic, Central European Institute of Technology, Brno University of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno, Czech Republic

Introduction Treatment of neuroblastoma, the most common solid tumor among infants, is based on chemotherapy - efficient, but with many serious side effects. To eliminate them, it is necessary to encapsulate cytotoxic drug inside suitable nanocarrier, like apoferritin. By modifying its surface, it is possible to further increase efficacy by active targeting. Materials/methods To characterize stability of our nanocarrier, colloidal stability test, pH-dependent drug release in various physiological solutions, size and shape were evaluated. MTT toxicity test and receptor expression on neuroblastoma cell lines were performed to verify active targeting to hNET receptor. Results and conclusions TEM showed assembled apoferritin with encapsulated ellipticine and bound anti-hNET peptides A and B. Encapsulation efficiency was calculated using absorbance of ellipticine at 420 nm to 67% for ApoElli with anti-hNET peptideA (ApoElli-A) and 73% for ApoElli with anti-hNET peptideB (ApoElli-B). Size analyzed using dynamic light scattering was 10 nm for ApoElli, 18 nm for ApoElli-A and 43 nm for ApoElli-B. After 3 weeks, the average size of all tested nanoformulations increased to approx.. 330 nm. This sorption of multiple nanocarriers was broken after vortexing, when the size decreased back to <100 nm. Colloidal stability showed first sedimentation of loaded apoferritin after 10 h. Loaded drug molecules were significantly released in endosomal environment, while stably loaded in apoferritin cavity in plasma environment. The active targeting proved efficient in intracellular delivery of ellipticine to neuroblastoma cells. The authors gratefully acknowledge

ﬁnancial support from the The Czech Science Foundation (GACR 1712816S). 8

Trisubstituted purine inhibitors of PDGFRα with high selectivity toward human eosinophilic cell line EOL-1

Veronika Malínková1 ,2, Eva Řezníčková2, Radek Jorda2, Tomáš Gucký1, Vladimír Kryštof2 1

Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký Univerzity, Olomouc, Czech Republic, Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University and Institute of Experimental Botany AS CR, Olomouc, Czech Republic

Introduction Platelet-derived growth factor receptor α (PDGFRA, CD140A) is a type III receptor tyrosine kinase (TK) that regulates cell proliferation, differentiation, adhesion and survival. Constitutively activated forms of PDGFRα have been found in various tumors, arising from various mutations in gastrointestinal stromal tumors and melanoma, missense mutations in childhood acute myeloid leukemia, reciprocal translocations in chronic myeloid leukemia, and deletions that give rise to a fusion protein in chronic eosinophilic leukemia. A typical fusion gene encoding a constitutively activated PDGFRα form is FIP1L1PDGFRA, which originates from an 800-kb cryptic interstitial deletion in chromosome 4q12. The fusion gene encodes a ligand independent and constitutively active TK that confers growth factor-independency to hematopoietic cells and is highly sensitive to the kinase inhibitor imatinib. The FIP1L1-PDGFRA fusion has been found in chronic eosinophilic leukemia, hypereosinophilic syndrome, eosinophilia-associated

myeloproliferative disorders, acute myeloid leukemia, and lymphoblastic T-cell non- Hodgkin lymphoma. Identification of novel PDGFRα inhibitors or thorough selectivity determination for previously described compounds is therefore still a challenge and may provide novel possible drugs. Materials/methods Collection of 23 novel 2,6,9-trisubstituted purines derivatives were synthetized by three step synthesis. All prepared compounds were subjected to biochemical assays to determine their activity against CDK2 and PDGFRα kinases and to cytotoxicity against human cancer cell lines with different expression of oncogenic tyrosine kinases. The cell cycle effects of all compounds after 24h treatment at a concentration corresponding to the compound´s GI50 were analysed by flow cytometry using a 488 nm laser. Expected mechanism of cellular activity was confirmed by immunoblotting of STAT3, ERK1/2, MEK1/2 and their phosphoforms. Results and conclusions Novel 2,6,9-trisubstituted purine derivatives displayed nanomolar inhibitory activities against PDGFRα. The compounds demonstrated strong and selective cytotoxicity in the human eosinophilic leukemia cell line EOL-1, which expresses the oncogenic kinase FIP1L1-PDGFRA. Their cytotoxicity in EOL-1 correlated significantly with PDGFRα inhibition. Moreover, they exhibited dosedependent inhibition of PDGFR autophosphorylation and suppressed its downstream signalling pathways in treated cells (STAT3, ERK1/2 and MEK1/2), confirming the proposed cellular mechanism of action. Our results suggest that substituted purines are versatile platforms for preparing tyrosine kinase inhibitors with specific activity towards eosinophilic leukemia and other cancers expressing constitutively activated PDGFRα mutants such as hepatocellular carcinoma, gastrointestinal stromal tumors or melanoma.

2017

Search for new anticancer agents among 4-thiazolidinones

Danylo Kaminskyy1, Julia Senkiv2, Anna Kryshchyshyn1, Rostyslav Stoika2, Roman Lesyk1 1

Danylo Halytsky Lviv National Medical University, Lviv, Ukraine, Institute of Cell Biology of National Academy of Sciences of Ukraine, Lviv, Ukraine

Introduction The search for new anticancer agents among small molecules is one of the most investigated areas in the modern medicinal chemistry. 4-Thiazolidinones as privileged scaffolds in the drug design are of special interest especially in small research groups. Achievements in anticancer 4-thiazolidinones field are associated both with screening programs’ findings and discovering of new high affinity ligands to a number of validated anticancer targets (JSP-1, TNFα, Bcl-XL-BH3, integrin αvβ3, SHP-2, etc.). Despite the chemical diversity of thiazolidinones (2,4-thiazolidinones, rhodanines, 2-amino(imino)thiazolidinones-4 etc.) 5-ene derivatives are most prominent subtype. The majority of the drug-like compounds belong to mentioned 4-thiazolidinones. This had reflected into the thesis about the crucial role of the presence/nature of the C5-substituent for the biological activity realization. Materials/methods Organic synthesis, NMRspectroscopy, X-ray, MS; in vitro study: cytotoxicity assay, fluorescent microscopy, Western blot analysis, cell cycle analysis Results and conclusions Based on the previous results, when designing new 4-thiazolidinones the next items were prpoused: belonging to the 5-ene-4-thiazolidinones; combination of pyrazoline and thiazolidinone cores, mainly within hybride pharmacophore approach;

Introduction of substituents in N3 position of the main core; molecular geometry of three-ring system; presence of ortho-OH group in phenyl moiety (C5 fragment). Thus, the series of 5-ene-4-thiazolidinone derivatives with pyrazole core linked by enamine group were synthesized and screened towards cancer cell panel. The 5-[5-(2-hydroxyphenyl)3-phenyl-4,5-dihydropyrazol-1ylmethylene]-3-(3-acetoxyphenyl)-2thioxothiazolidin-4-one was selected as the most active and selective agent against leukemia cancer cells (IC50 =118 nM HL-60) with low toxicity towards pseudonormal cells. Study of the molecular mode of action suggests the next ﬁndings: the mitochondria-depended apoptosis as the main mode of action; compound induces the G0/G1 arrest of the treated cells and causes inhibition of cell division; induction of ROS production is one of the compound effects; compound was active towards HL-60/ADR (adriamycinresistant cells with over-expression of P-glycoprotein) most probably via P-gp-independed maner (structure does not correspond to structural requirements of inhibitors of P-gp). Analysis of structure-activity relationships allows to outline the directions for further optimization of identiﬁed lead-compound. 10

Non-invasive lung cancer diagnostics using proteomic biomarkers in exhaled breath condensate.

Jana Václavková1, Miroslav Hruška1, Jana Vrbková1, František Kopřiva3, Vendula Látalová3, Dušan Holub1, Juraj Kultan2, Vítězslav Kolek2, Petr Jakubec2, Marián Hajdúch1, Petr Džubák1 ,3 1

Laboratory of Experimental Medicine, Institute of Molecular a Translational medicine, Faculty of Medicine and Dentistry Palacký University, Olomouc, Czech Republic, Department of Respiratory

Medicine, Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech Republic, Department of Paediatrics, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech Republic

Introduction Exhaled breath condensate (EBC) represents a source of biomarkers such as proteins, arachidonic acid metabolites, vasoactive peptides amines, DNA, RNA, microRNA and small molecules. These biomarkers can provide valuable information about respiratory as well as systemic diseases. Finding non-invasive methods for early detection of lung cancer in the background of underlying lung injury (inflammation, smoking, infections, etc.) would be highly beneficial. Proteomic analysis of EBC has the potential to detect early changes in the status of the respiratory system and possibly other organs. It could also replace or complement some invasive sampling methods in future and provide noninvasive lung cancer screening technique. Together, our studies will advance the development and validation of EBC as a novel tool for the proper diagnosis of lung cancer and monitoring of disease activity, treatment efficacy and prognosis. The aim of our study is to identify lung cancer-specific and sensitive protein biomarkers in EBC, assess their value for early diagnostics and screening of lung cancer and evaluate the diagnostic, prognostic and predictive value of lung cancer protein biomarkers in EBC. Materials/methods Exhaled breath condensate proteins in the sample are reduced by DTT, alkylated with iodoacetamide, digested with trypsin and concentration of peptides is measured. Samples are purified and diluted for HPLC/MS analysis which is performed in three technical replicates using high-resolution LTQ Orbitrap Elite mass spectrometer (Thermo Scientific). The MS data are analyzed by recently developed

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bioinformatics tool (Decryptor, http://decryptor.imtm.cz/), enabling data analysis from tandem massspectrometry of human proteome for the presence of point alterations (non-synonymous single nucleotide polymorphisms, mutations), which are expected to detect cancer cells specifically. Decryptor deduces DNA/ mRNA alterations whenever possible and thus enables revolutionary detection of tumor mutations on the protein level. Results and conclusions We have collected and analyzed samples from patients who have lung cancer and compared them with healthy controls and samples from patients with other respiratory diseases such as COPD, asthma, cystic fibrosis and sleep apnea. Using Decryptor bioinformatics tool we have identified genes which belong to identified individual proteins families. In this study we have identified 4438 genes in case of 10% false results and 1438 genes in case of 1% false results. We have identified some candidate protein biomarkers that will be further studied and validated. Study is supported by the grants of AZV 16-32302A and 16.32318A. 11

Finding molecular patterns relevant for toxicity end-points

Mariia Matveieva, Pavel Polishchuk Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University and University Hospital in Olomouc, Olomouc, Czech Republic Introduction Interpretation of QSAR models is useful for searching molecular features and patterns important for the activity modelled. There are many approaches for interpretation developed. However, none of them takes into account molecular context of analyzed fragments that is essential because the same fragment may have substantially different effect on studied end-point

in different surroundings. Materials/methods For the aim of interpretation we applied structural and physicochemical interpretation (SPCI) approach developed earlier [1, 2], which allows to calculate the contribution of any fragment (derived from the molecules of the data set) to the end-point modelled. In the case of regression models the contribution has the same units as activity, thus it can be a quantitative measure of how the fragment influences the activity of a molecule when present in that molecule. We proposed two strategies to take into account molecular context of fragments: unsupervised and supervised. The unsupervised strategy is based on the analysis of the distribution of fragment contributions by means of Gaussian mixture modelling (GMM). GMM allows to cluster contributions of a given fragment calculated from molecules where it appears. Such clustering can be caused by different molecular contexts of the fragment which can be subsequently detected from each cluster separately. The supervised strategy is based on explicit encoding of a molecular context by taking into account nearest atoms at a distance up to 3 bonds from the considered fragment. Thus each fragment has an associated context that makes it possible to distinguish identical fragments in different surroundings. Results and conclusions The GMM approach was first tested on the data set of 1984 compounds studied on toxicity toward Tetrahymena pyriformis. The results show that the clustering technique correctly identifies known toxicophore patterns. Thus, it could be applied to other data sets and end-points. We applied this methodology to four data sets of molecules tested in MTT assay against four cell lines: HCT116, HCT116p53-, K562, and K562-TAX in our institution. We identified fragments which were more “cytotoxic” in certain molecular surroundings than in other ones.

Explicit context analysis of MTT data helped identify patterns having signiﬁcantly different contributions to HCT116 and HCT116p53- endpoints and to K562 and K562-TAX end-points. 12

Impact of hypoxia on sensitivity of colorectal carcinoma cells towards microtubule-targeting agents

Jiri Rehulka1, Narendran Annadurai1, Ivo Frydrych1, Pawel Znojek1, Petr Dzubak1, Peter Northcote2, John H. Miller3, Marian Hajduch1, Viswanath Das1 1

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech Republic, Schools of Chemical and Physical Sciences, Victoria University of Wellington, Wellington, New Zealand, Biological Sciences, Victoria University of Wellington, Wellington, New Zealand

Introduction Low oxygen supply due to insufﬁcient vasculature is an attribute of numerous solid tumours. Hypoxiainducible factor 1α (HIF-1α) is a major mediator of responses to low oxygen levels. Its level is regulated by rapid proteasomal degradation promoted by the ubiquitin E3 ligase von Hippel Lindau tumour suppressor protein (pVHL). The hypoxiadependent HIF-1α stabilization results in transcriptional activation of target genes including VEGF, TGF-β3, erythropoietin and glucose transporters. Hypoxia also promotes the evolution of more aggressive subclones that are associated with enhanced metastatic potential, poor prognosis and risk of relapse. In addition, hypoxia has been shown to reduce the efﬁcacy of chemotherapy. This study evaluated the effect of hypoxia preconditioning on sensitivity towards microtubule targeting agent peloruside A, paclitaxel and

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vincristine. Materials/methods Cytotoxicity against colorectal carcinoma cells HCT116 and HT-29 was determined using MTT assay. The antimitotic effect was evaluated by ﬂow cytometry and stabilization of microtubules was monitored using intracellular tubulin polymerization assay. Results and conclusions Here we report that microtubuletargeting agent peloruside A is comparably effective against normoxia and hypoxia preconditioned cells. In contrast, hypoxia preconditioning altered the sensitivity of HCT116 to paclitaxel. These data report on the cellular and molecular effects of PLA in hypoxia-conditioned cells for the ﬁrst time and will encourage further exploration of PLA as a promising anti-tumour agent. The biological part of this work was supported by the grant from the Ministry of Education, Youth and Sports of the Czech Republic (L01304, LM2015063), AZV (1531984A) and internal grant of Palacky University IGA_LF_2017_026. The infrastructural part (Institute of Molecular and Translational Medicine) was supported by the grant LO1304 from the National Program of Sustainability II. 13

Folic acid conjugates with purine cyclin-dependent kinase inhibitors

Denisa Hendrychová, Eva Řezníčková, Tomáš Gucký, Soňa Krajčovičová, Miroslav Soural, Vladimír Kryštof Palacký University, Olomouc, Czech Republic Introduction The use of small-molecule delivery systems is a promising approach, which can decrease the systemic toxicity of cytotoxic anticancer drugs. One of the most recent and powerful approaches involves targeting the folate receptor (FOLR1). It is overexpressed in a wide range of

human cancers and folate targeting technology has successfully been applied for the delivery of various chemotherapeutic agents to FOLR1-positive cancers. We therefore designed conjugates of 2,6,9-trisubstituted purine cyclindependent kinase inhibitors with folic acid, prepared them and evaluated their binding to cancer cells. Materials/methods The desired conjugates were constructed stepwise using solidphase synthesis starting from immobilized primary amines. The multistep sequence was optimized to obtain the reaction intermediates and final products in high crude purities. For a proof of concept, the conjugates were tested for its binding to the HeLa cell line overexpressing FOLR1 by flow cytometry using the folate-receptor-targeted fluorescence probe FolateRSense 680 as a competing agent. The HeLa cell line was used because of its overexpression of FOLR1 and significantly greater probe uptake over other cell lines. Results and conclusions Flow cytometric measurements demonstrated the ability of an example conjugate to bind to cancer cells overexpressing the folate receptor. The results confirmed that the presence of conjugated CDK inhibitors markedly decreased the percentage of cells with bound FolateRSense 680. Free folate also reduced the percentage of positive cells and thus confirmed function of the probe. We confirmed that CDK inhibitors conjugated to folate could bind FOLR1-overexpressing cells, suggesting that this concept is a possible route for the development of more specific and efficient anticancer drugs. Detailed cellular experiments will be reported elsewhere. The work was supported by projects 15-15264S (Czech Science Foundation) and IGA-LF-2017-028 and IGA_PrF_2017_014 (Palacky University, Olomouc).

Mass spectrometry-based proteomic analysis for subtyping of amyloid deposits from FFPE and SFA

Dušan Holub1, Tomáš Pika2, Pavla Flodrová3, Petr Džubák1, Marián Hajdúch1 1

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University and Faculty Hospital in Olomouc, Olomouc, Czech Republic, Department of Hemato-Oncology, University Hospital Olomouc, Olomouc, Czech Republic, Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech Republic

Introduction The systemic amyloidosis is a rare disorder characterized by the abnormal deposition of misfolded amyloid protein into various organs. Over time, the accumulating amyloid damages the tissue microenvironment and causes organ failure. To date, there are 31 known fibril proteins in human that can cause amyloidosis. Early diagnosis is critical for effective patient management. IHC method is the preferred method for routine amyloid subtyping. However, it is an antibody-based method with a lot of unspecificities. Therefore we have introduced mass spectrometry-based proteomic analysis for subtyping of amyloid deposits in formalin-fixed paraffin-embedded tissues (FFPE) and in abdominal subcutaneous fat aspirates (SFA). Materials/methods During two years, we have obtained 160 FFPE and 30 SFA samples for subtyping of amyloid deposits. In the FFPE samples, the Congo red positive stained amyloid deposits were dissected using laser microdissection, the proteins were extracted from excised materials and digested using trypsin. In the

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SFA samples, the proteins were solubilized and digested with trypsin. All recovered peptide mixture from FFPE or SFA specimens were successively separated by liquid chromatography and individual peptides were acquired by tandem mass spectrometry. Acquired spectra were identified and quantified using a search engine - MaxQuant. The most abundant amyloid protein determines the amyloid subtype. Results and conclusions The mass spectrometry-based proteomic analysis enables subtyped different kinds of amyloid proteins (Ig kappa, Ig lambda, transthyretin, fibrinogen, serum amyloid A, semenogelin). In all cases with systemic amyloidosis, we observed the presence of serum amyloid P, apolipoprotein A-IV, apolipoprotein A-I and apolipoprotein E proteins. All those proteins are associated with the amyloid formation. Mass spectrometry-based proteomic analysis of FFPE and SFA samples offer a powerful tool for typing of systemic amyloidosis. This work was supported by grants IGA_LF_2017_026, AZV_1531984A, AZV_16-31156A and by the National Sustainability Programme (LO1304). 15

Cytotoxic Triterpenes and Study of their Mechanism of Action

Jiří Hodoň1 ,2, Jana Václavková1, Jiří Řehulka1, Dušan Holub1, Petr Džubák1, Marián Hajdúch1, Milan Urban1 ,2 1

IMTM, Faculty of Medicine and Dentistry, UP Olomouc, Olomouc, Czech Republic, Department of Organic Chemistry, Faculty of Science, Palacky University in Olomouc, Olomouc, Czech Republic

Introduction Triterpenes are natural compounds that may be found in almost all living organisms, most often in plants, fungi

and marine animals. Thousands of triterpenes have been isolated from natural sources and many of them are biologically active. Even though many researchers are studying terpenes quite extensively due to its antitumor and antiviral activities, their mechanisms of action often remain elusive. Materials/methods The aim of this study is investigation of mechanism of action of highly cytotoxic triterpenoid pyrazines and pyridines. The conjugates these triterpenoids with glucose, tetraacetyl glucose, galactose, tetraacetyl galactose and other molecules were prepared using Huisgen cycloaddition in order to increase their solubility and bioavailability. Cytotoxic activities of all prepared compounds were measured. The most active derivates were further examined for their mechanism of action: First, influence of the active compounds on cell cycle was studied, then influence on apoptosis and nucleic acid synthesis using flow cytometry methods. Results and conclusions Active compound with the most promising therapeutic index was selected and equipped by biotin and fluorescent tag in order to be used in pull down assays evaluated by quantitative proteomics and SILAC. A set of proteins was found that bind selectively to the active molecule which may be responsible for the activity. Literature: 1. Hill, R. A.; Connolly, J. D. Nat. Prod. Rep. 2015, 32, 273. 2. Dzubak, P.; Hajduch, M.;Vydra, D.; Hustova, A.; Kvasnica, M.; Biedermann, D.; Markova, L.; Urban, M.; Sarek, J. Nat. Prod. Rep. 2006, 23, 394. 3. Antimonova, A. N.; Petrenko, N. I.; Shakirov, M. M.; Rybalova, T. V.; Frolova, T. S.; Shul‘ts, E. E.; Kukina, T. P.; Sinitsyna, O. I.; Tolstikov G. A. Chem. Nat. Comp. 2013, 49, 657. 4. Ong, S. E.; Li, X.; Schenone, M.; Schreiber, S. L.; Carr, S. A. Chemical Proteomics, 2012, 129.

GLASS: assisted mutation detection in Sanger data

Karol Pal1, Vojtech Bystry1, Tomas Reigl1, Martin Demko1, Adam Krejci1, Boris Tichy1, Sarka Pospisilova1 ,2, Jitka Malcikova2, Nikos Darzentas1 1

CEITEC MU, Brno, Czech Republic, The University Hospital Brno, Brno, Czech Republic

Introduction Despite great advances in Next Generation Sequencing, Sanger sequencing still represents a widely used methodology for mutation detection. The reliability and accuracy of detecting mutations is influenced also by the software analysis of Sanger data. Moreover, the uniform designation of mutations according to the HGVS nomenclature is crucial for reporting and inter-laboratory comparison. Materials/methods GLASS is an easy to use, online software tool for analyzing Sanger data, with advanced functionalities to assist mutation detection, interpretation and reporting, while still following the workflow of “manual inspection” of traces. It automatically recognises the reference gene and orientation of the analyzed sample, and also predicts and annotates possible insertions/deletions and their frequencies – while presenting results on intuitive chromatogrambased visualisations. Results and conclusions GLASS is a bioinformatic implementation of best practices of labs with published know-how in the analysis of TP53 and other clinically relevant genes. It is continuously validated against a growing set of manually analyzed samples with expertly confirmed mutations. Most samples are taken directly from the official certification activities of the European Research Initiative on CLL (ERIC). GLASS is freely available online at http://bat.infspire.org/

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genomepd/glass/. Supported by Ministry of Health of the Czech Republic grant nr. 1634272A. 17

Antiparasitic activity of anticancer drugs against kinetoplastid parasite Leishmania major

Ermin Schadich, Petr Džubák, Marián Hajdúch Institute of Molecular and Translational Medicine, Faculty of Medicine, Palacky University,, Olomouc, Czech Republic Introduction The number of anticancer drugs might have multifaceted pharmacological proﬁles that also include the antiparasitic activity. The aim of this study was to determine whether the compounds with known anticancer and other pharmacological properties have in vitro activity against kinetoplastid parasite Leishmania major. Materials/methods We assayed 1280 compounds from Sigma Life Science’s library of pharmacologically active compounds (LOPAC1280) for activity against L. major promastigotes using Alamar blue assay. Results and conclusions Three compounds with with known anticancer properties, nitidine chloride, ellipticine and JS-K, and three compounds with other pharmacological properties had signiﬁcant activity against L. major promastigotes (IC50 < 10 μM). Their potential for development of novel drug candidates in the therapies against Leishmania parasites is indicated.

A pilot study of molecular testing of HPV infection in tumors from different head and neck areas

Kristyna Glendova1, Radka Mihlova1, Robert Gürlich2, Libor Stanek1,2 Laboratory of molecular detection of pathogens, synlab czech, s.r.o., Prague 2 Department of General Surgery, Third Fauculty of Medicine and Faculty Hospital Královské Vinohrady Head and neck areas include tumors of the oropharynx, hard palate, salivary glands. Tumors in the head and neck region are the fourth most common cancer in men in Europe. The incidence of tumors associated with human papillomaviruses (HPV) is rising sharply. These tumors are different both from a molecular and epidemiological point of view, but also from their clinical behavior. In patients with HPV-positive tumors, decision-making about treatment will probably be different. HPV positive tumors could be treated less intensively with less toxicity. The aim of our pilot study was to compare the findings of HPV infection in squamous cell carcinomas. 10 samples from each area (oropharynx, inner buccal area, hard palate and tooth root area) were analyzed using PCR methods. Significantly different findings of HPV infection in different head and neck tumours’ areas were found. HPV infection in the oropharyngeal region, namely in the root of the tongue and in the buccal area were found in 6 and 2 out of 10 samples, respectively. HPV infection in tumors inside the gingiva was not confirmed. 1

Supported data show a different incident of head and neck tumors according to their proper localization. A testing for a decision-making perspective of the therapy is needed. This work was supported by the Charles University research program PROGRES Q 28 (Oncology). 19

Cytotoxicity proﬁling of new chemical compounds with chelating activities using highthroughput screening

Soňa Gurská1, Petr Džubák1, Robert Kaplánek2 ,3, Tomáš Bříza2 ,3, Martin Havlík2 ,3, Vladimír Král2 ,3, Marián Hajdúch1 1

Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Olomouc, Czech Republic, First Faculty of Medicine, Charles University, Prague, Czech Republic, Department of Analytical Chemistry, University of Chemistry and Technology, Prague, Czech Republic

Introduction Although there are lots of new compounds with anticancer activities identiﬁed in last decades; efﬁcient treatment of cancer is still a serious problem. Usually new active substances have adverse side effects or low selectivity toward cancer cells. Therefore it is still important to search for new active compounds. To identify new anticancer agents, novel hybrid compounds with two or more pharmacophores are developed. The aim of this study was to evaluate the cytotoxic effect of group of hydrazones bearing a benzothiazole

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investigated. As an indicator for reactive oxygen species (ROS) in Materials/methods cells the chloromethyl derivative of In the HTS laboratory the cytotoxicity H2DCFDA was used. of compounds was evaluated by the MTS assay. At ﬁrst the cytotoxic Results and conclusions effect of unique chemical compounds The potent anticancer activity was tested on 10 cell lines (8 cancer of several new derivatives was cell lines and 2 non-cancer cell lines). demonstrated. In most cases the As these compounds have the ability cytotoxic properties of compounds to bind the metal ions, the effect of were not modiﬁed by Fe(II) and Fe(II) and Fe(III) metallocomplexes Fe(III) ions, some compounds lost/ on anticancer activity was also decreased their cytotoxic potential. moiety.

But there was identiﬁed the group of compounds with increased cytotoxic properties due to Fe(II) and Fe(III) ions. The evaluation of intracellular ROS production showed that increased cytotoxicity of some compounds by Fe(III) can be caused by oxidative stress. Obtained results can indicate the possible mechanism of action. This study was supported by grants IGA LF_2017_026 and by the National Sustainability Program (LO1304)

Poznรกmky / Notes:

MedChemBio Cluster & Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc would like to thank all who supported the Conference XIII. Diagnostic, Predictive and Experimental Oncology Days

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