Abstrakta 2018 by Diagnosic Predictive and Experimental ONCOLOGY Days

Abstract book XIV. DIAGNOSTIC, PREDICTIVE AND experimental ONCOLOGY DAYS

Satelli

www.ddpeo.cz

Legionarska 21, 779 00, Olomouc, Czech Republic

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hotel NH Collection Olomouc Congress

hop on Tech s k -t or

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November 19 - 21, 2018

2018

November 20 Olomouc II Hall

ISBN 978-80-270-5084-0

ISSN 2336-8284

CRISPR

Genome Editing

For more information, visit SigmaAldrich.com/CRISPR

The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.

2018

Organizing Institutions MedChemBio Cluster in association 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 Khushboo Agrawal, PhD. Viswanath Das, PhD. Jiří Drábek, PhD. Petr Džubák, MD., PhD. Marián Hajdúch, MD., PhD. Miloš Petřík, PhD. Josef Srovnal, MD., PhD. Marek Svoboda, MD., PhD. Radek Trojanec, PhD.

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

2018

PROGRAM

XIV. DNY DIAGNOSTICKÉ, PREDIKTIVNÍ A EXPERIMENTÁLNÍ ONKOLOGIE / XIV. DIAGNOSTIC, PREDICTIVE AND EXPERIMENTAL ONCOLOGY DAYS PONDĚLÍ / monday - 19. listopadu 2018 / November 19th, 2018 Madrid Hall 13:00 13:15 Ceremonial openning

Human papilomavirus infection in cancer, human reproduction and screening

Chairs: Matejka Rebolj, Marian Hajduch 13:15 14:00 Discordance between human papillomavirus assays in primary cervical cancer screening Matejka Rebolj 14:00 14:15 Human papillomavirus (HPV) and HPV-related diseases Vladimíra Koudelakova 14:15 14:45 Human papillomavirus Infection and fertility alterations Hana Jaworek 14:45 15:00 Proteomic analysis of cervical mucus Tomas Ozdian 15:00 15:30 COFFEE BREAK

Pathophysiology of cancer and molecular targets I

Chairs: Josef Srovnal, Viswanath Das 15:30 16:00 Role of long chain fatty acids in human NK cell cytotoxic activity activity Juan B. De Sanctis 16:00 16:30 The antagonistic functional duality of cyclin-dependent kinase inhibitor p21WAF1/CIP1 in cells derived from multicellular tumor spheroids Viswanath Das 16:30 16:45 Is the inhibition of aldehyde dehydrogenase the way to revert cisplatin resistance in testicular germ cell tumors? Silvia Schmidtova 16:45 17:00 Case-control study of culturable gut microbiota in colorectal cancer Vladislav Raclavsky 17:00 17:15 Targeting the cellular uptake and processing of missfolded proteins: Example of tau aggregates Narendran Annadurai 18:00 19:00 Visit to the IMTM (IF YOU LIKE TO VISIT THE INSTITUTE CONTACT US ON REGISTRATION DESK) Úterý / TUESDAY - 20. listopadu 2018 / November 20th, 2018 Madrid Hall

Pathophysiology of cancer and molecular targets II

Chairs: Martin Mistrik, Petr Muller 9:00 9:30 Mechanisms of HSF1 activation in cancer cells Petr Muller 9:30 10:00 High speed of fork progression induces DNA replication stress and genomic instability Pavel Moudry 10:00 10:15 Genotoxic and proteotoxic stress-response pathways as targetable vulnerabilities of prostate cancer Dusana Majera 10:15 10:30 Urinary bladder tumourigenesis and mesenchymal cells - from reactive cancer stroma to bladder sarcoma Jiri Hatina 10:30 11:00 COFFEE BREAK

PROGRAM

2018

Cancer biomarkers and personalized medicine I

Chairs: Magdalena Ratajska, Jiri Drabek 11:00 11:30 The application of ctDNA based tests in ovarian cancer diagnostic and research Magdalena Ratajska 11:30 11:45 Identification of meningioma patients in high risk of tumor recurrence using microRNA profiling Josef Srovnal 11:45 12:00 ISUP grade groups, tertiary Gleason, periostin and versican in aggressive prostate cancer Gvantsa Kharaishvili 12:00 12:15 IDH1/2 in diffuse gliomas: Retrospective mutation analysis Zuzana Sporikova 12:15 13:30 LUNCH

Cancer therapeutics I

Chairs: Milos Petrik, Martin Mistrik 13:30 14:00 Alcohol abuse drug disulfiram targets cancer via p97 segregate adaptor NPL4 Zdenek Skrott 14:00 14:15 Senolytic cocktail dasatinib+quercetin does not enhance the efficacy of senescence-inducing chemotherapy in liver cancer Kristina Kovacovikova 14:15 14:30 Fluorescent dye labeling nucleolus through interaction with BYSL Zuzana Maceckova 14:30 14:45 In vivo biodistribution study of hydroxyapatite nanoparticles labelled with technecium-99m Zbynek Novy 14:45 15:00 Identification of compounds with cytotoxic activity by cell viability based high - throughput screening Soňa Gurská 15:00 15:15 COFFEE BREAK

Cancer therapeutics II

Chairs: Milan Urban, Jiri Voller 15:15 15:45 Cytotoxic triterpenes and triterpenic conjugates used for studies of mechanism of action Milan Urban 15:45 16:00 6-Substituted purines as ROCK inhibitors with anti-metastatic activity Jiří Voller 16:00 16:15 HTS-likeness and chemical library design Pavlo Polishchuk 16:15 16:30 Betulinic acid derivatives inhibit Hedgehog/Gli-mediated transcription in human glioblastoma cell line Ivo Frydrych 16:30 16:45 Affinity purification as a tool for identifying molecular targets of new triterpenic pyrazine compounds Jana Vaclavkova

Cancer Epigenetics

Chairs: Khushboo Agrawal, Manlio Vinciguerra 16:45 17:15 Chromatin „reader“ machinery as target for overcoming resistance to DNA-demethylating epi-drug decitabine Khushboo Agrawal 17:15 17:30 Gene therapy approaches to increase an efficacy of epigenetic treatment in breast cancer Svetlana Miklikova 17:30 17:45 Loss of histone macroH2A1 in hepatocellular carcinoma triggers paracrine-mediated cancer stemness and promotes adaptive immunity evasion Oriana Lo Re

2018

PROGRAM

17:45 18:00 Search for new epigenetic predictive biomarkers of bevacizumab response in metastatic colorectal cancer patients Rastislav Slavkovský 19:00 22:00 CONFERENCE DINNER

Certified Satellite Workshop on Tech-transfer

Olomouc Hall II - 8:30 - 19:30 - Hosted by Linda Lososova 8:30 - 9:00 REGISTRATION AND COFFEE 9:00 - 9:15

WELCOME SPEECH

9:15 - 10:00 10:00 - 11:00

Innovation for social impact: A hot topic? Monika JAKO – independant senior innovation consultant, Toronto Nenovision: From microscopes to spin-offs Jan NEUMAN, CEO, Nenovision, Brno

11:00 - 11:15 COFFEE BREAK 11:15 - 12:00

DIANA Biotech: Incubation and launching of a biotech spin-off Jaromír ZAHRADKA, CEO, I&I Prague

12:00 - 14:00

NETWORKING LUNCH

14:00 - 15:00 15:00 - 16:00

My UK spin-off failures Pavel KRECMER, CEITEC VUT, Brno Media Venture Start-up Zuzana ZBORILOVA, CEO, OL4you, Olomouc

16:00 - 16:15 COFFEE BREAK

Powerd by:

Projekt: Tým transferu technologií CZ 02.2.69/0.0./0.0/16_014/0000633

16:15 - 17:15 Academic drug development: What we have learned from failures? Marian HAJDUCH, director, Institute of Molecular and Translational Medicine, Olomouc 17:00 - 19:30 Round table discussion on topics presented Keynote speakers: Monica JAKO, Pavel KRECMER, Jan NEUMAN, Jaromir ZAHRADKA, Zuzana ZBORILOVA, Marian HAJDUCH STŘEDA / wednesday - 21. listopadu 2018 / November 21th, 2018 Madrid Hall

Cancer biomarkers and personalized medicine

Chairs: Vladimir Havlicek, Petr Dzubak 9:00 9:30 Urinal siderophores as non-invasive and early biomarkers of infectious underlying diseases in critically ill patients Vladimir Havlicek 9:30 10:00 Non-invasive lung cancer diagnostics using proteomic biomarkers in exhaled breath condensate Petr Dzubak 10:00 10:15 Genetic biomarkers of clinical response to bevacizumab in colorectal cancer patients Karolina Bartakova 10:15 10:45 COFFEE BREAK

PROGRAM

2018

New methods in cancer research and diagnostics

Chairs: Marian Hajduch, Karel Koberna 10:45 11:00 Claire: A system for detection of protein variants from tandem mass spectra Miroslav Hruska 11:00 11:15 Utilization of murine hair follicles for biomarker studies in ionizing radiation response model Hanus Slavik 11:15 11:30 Microsatellite instability testing – Overview of methods and results Alona Rehulkova 11:30 11:45 Quantification of adherent cells using strong enhancers of fluorescence Karel Koberna 11:45 12:00 Closing remarks 12:00 13:15 LUNCH

Workshop Nanostring: From insight to impact Madrid Hall 13:15 - 15:30

The workshop will introduce future users into: 1. Nanostring technology overview 2. Introduction to digital spatial profiling and technology access program 3. PlexSet panels and pathways 4. Panels available for cancer research 5. Practical introduction into the assay - tips and tricks

Postery / Posters 1 Identifying cellular targets of betulinic acid conjugates Jiri Rehulka 2 High-throughput screening system for identification of adenosine receptor ligands Jana Kotulova 3 Mass spectrometry-based proteomic analysis for subtyping of amyloid deposits from FFPE and SFA samples Dusan Holub 4 Circulating biomarkers for pancreatic cancer Martina Jakoubkova 5 Contradictory taxifolin effects on Zeb2 signalling pathway in Hep G2 cells Zdenek Dostal

In-house validation of tumor BRCA1/2 testing Jana Stranska

7 Analysis of molecular mechanisms involved in MSC-mediated chemoresistance in breast cancer Jana Plava 8

Antileishmanial activity of anticancer drugs Ermin Schadich

9 Efficacy of various genotoxic factors in activation of cGAS/STING pathway Tereza Buchtova

QIAsure

IVD

METYLAČNĚ-SPECIFICKÝ REAL-TIME PCR TEST PRO PREDIKCI RIZIKA VÝSKYTU RAKOVINY DĚLOŽNÍHO ČÍPKU

„Výsledky vyšetření na gynekologii prokázaly přítomnost vysoce rizikového kmenu lidského papilomaviru, ale rakoviny děložního čípku se bát nemusíme.“

Pro koho? Test je vhodný jako triáž pro pacientky s lehce abnormálním výsledkem cytologického vyšetření (ASC-US) či pro pacientky s prokázaným vysoce rizikovým typem papilomaviru (high-risk HPV/hrHPV).

Z jakých vzorků? stěry z děložního čípku stěry odebrané do roztoku pro cytologii v tekutém médiu (LBC – liquid-based cytology) vzorky odebrané samoodběrovou sadou

Princip a metodika Na základě vědeckých studií byl prokázán vztah mezi hypermetylací promotorů tumor-supresorových genů FAM19A4 a hsa-mir124-2, a vyšším rizikem výskytu rakoviny děložního čípku. Průkaz hypermetylace tedy poskytuje přesnější informaci o rizikovosti probíhající infekce HPV.

Izolace a kvantifikace DNA

Informace o produktu katalogové č. QIAsure Methylation Test Kit

Multiplex real-time PCR

Automatická analýza a interpretace výsledků

cykler RotorGene Q MDx

software RotorGene Assay Manager

Bisulfitová konverze

616014 72 reakcí

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2018

Human papilomavirus infection in cancer, human reproduction and screening

Chairs: Matejka Rebolj, Marian Hajduch pondělí / 19. listopadu 2018 / Monday / November 19th, 2018 / 13:15 - 15:00 Discordance between human papillomavirus assays in primary cervical cancer screening

Matejka Rebolj King’s College London, London, United Kingdom Introduction Human Papillomavirus (HPV) testing is replacing cytology in primary cervical cancer screening because it is highly sensitive for the detection of high-grade cervical intraepithelial neoplasia (CIN2+). Several HPV assays have been clinically validated, presenting screening laboratories with a challenging choice.

Methods To date, the most well described large study comparing several (4) commercially available, validated, assays is still the Danish Horizon study* (see e.g. https://www.ncbi. nlm.nih.gov/pubmed/24466262). It included samples from ca. 5000 women, of which just below 3000 were undergoing primary cervical screening aged 30-65, the main HPV screening target group. All samples were tested with liquid-based cytology, Hybrid Capture 2, CLART, cobas, and APTIMA assays. Women with HPV+/cyt- samples were followed-up with HPV and cytology at 18 months. Histological outcomes were retrieved from the national pathology database. Results Contrary to expectations, the study showed substantial discordance between the four assays. While in the whole primary screening population 4% of women had abnormal cytology, HPV discordance alone was found in 16% (with an additional 6% who were concordant, i.e. positive on all 4 assays). Subsequent analyses showed that discordant samples were more likely weakly positive,

possibility of vertical transmission and the possible role in human fertility. In our study, impact of HPV infection on men fertility was found. The causal role of high-risk HPV infection was clearly proved in almost all cervical cancers and part of anogenital (vulvar, vaginal, penile and anal) and head and neck (oral, tonsillar, pharyngeal and laryngeal) cancers. Several studies identified Conclusions the HPV DNA presence also in These patterns suggest that the lung and breast cancer cases but calibration of assays to detect HPV the etiological role of HPV in breast infections is the most likely reason and lung carcinogenesis was not for the discordant results. The confirmed to date. Similarly in our discordance may have a relatively study, no HPV was detected in a small effect on the detection of cohort of primary non-small cell lung CIN2+, but a much larger one on cancers. HPV positive women without CIN2+. Considering the availability of HPV vaccination and cervical cancer * The Horizon study was undertaken screening, cervical cancer is at Copenhagen University almost fully preventable disease. Hospital Hvidovre (Jesper Bonde, In the Czech Republic, the main Carsten Rygaard, Sarah Preisler, problem is the low participation of Ditte Ejegod) and University of women in cervical cancer screening Copenhagen (Elsebeth Lynge which is about 55% only and the and Matejka Rebolj). The views low sensitivity of cytology. Recent expressed in this talk will be my own. addressed invitations to cervical cancer screening increased participation in only 8%. Using of Human papillomavirus (HPV) primary HPV screening (instead of and HPV-related diseases cytology) and self-sampling devices Vladimira Koudelakova, Hana in non-attendees could be the solution. Several studies, including Jaworek, Marian Hajduch our study, confirmed the sufficient Institute of Molecular and sensitivity and specificity of HPV Translational Medicine, Faculty of detection in cervicovaginal samples Medicine and Dentistry, Palacky obtained by self-sampling. Primary University Olomouc and University HPV screening was introduced in Hospital Olomouc, Olomouc, several European countries and the Czech Republic current main goal is the selection Results and conclusions of the best triage strategy for HPVHuman papillomavirus (HPV) is a positive women. family of circular double-strand DNA This work was financially supported viruses responsible for about 30% of by NPU LO1304, IGA_LF_2018_005, all infectious agent-related cancers. TE02000058 and EATRIS-CZ. HPV usually infects mucosa and skin, nevertheless, HPV presence was found also in peripheral blood, semen and placenta indicating the cross-reacting to low-risk genotypes, and rarely associated with abnormal cytology or CIN2+. Women with CIN2+ were more likely to test positive on all 4 assays, although even among them the concordance was lower if the CIN2+ was detected after negative cytology. These findings were later confirmed in a systematic review of the literature.

2018

Human papillomavirus infection and fertility alterations

Hana Jaworek1, Vladimira Koudelakova1, Ivana Oborna2,3,4, Blazena Zborilova2, Jana Brezinova4, Dagmar Ruzickova5, Jana Vrbkova1, Pavla Kourilova1, Marian Hajduch1 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Olomouc, Czech Republic. 2 Fertimed Ltd., Olomouc, Czech Republic. 3 Dept. of Obstetrics and Gynaecology, Faculty of Medicine and Dentistry, Olomouc, Czech Republic. 4 SpermBank International Ltd., Olomouc, Czech Republic. 5 Arleta IVF Ltd, Kostelec nad Orlici, Czech Republic 1

and following pregnancy although it was demonstrated that HPV positive embryos exhibit increased apoptosis and delayed early development. Moreover, a higher risk of spontaneous abortion and possible mother-fetus transmission has been also described in HPV-positive women. Unlike other STDs, HPV is not tested obligatorily for gamete donors or infertile couples, although it could significantly affect fertility, efficacy of assisted reproduction treatment, pregnancy or the fetus itself. This work was financially supported by NPU LO1304, IGA_LF_2018_005, TE02000058 and EATRIS-CZ.

Proteomic analysis of cervical mucus

Tomáš Oždian1, Jan Vodička2, Michal Dvořan2, Jiří Dostál2, Veronika Kolářová2, Magda Pešková2, Igor Crha3, Pavel Introduction 3 3 Human papillomaviruses (HPVs) Ventruba , Jana Žáková , Petr 1 2 are agents of a common sexually Džubák , Radovan Pilka , 1 transmitted disease (STDs) that Marián Hajdúch preferentially infects squamous epithelial cells. HPVs are important factors responsible for cancer development in ano-genital and aerodigestive region. The other aspect of HPV infection deserves more attention is its possible association with damage leading to reduced fertility. Results and conclusions HPV was found in female genital tract as well as along the whole male genital tract including semen where it is bound to sperm cells. In our and several other studies, HPV infection was found to be associated with alterations of semen quality like decreased cell count, motility reduction and amplitude of lateral head displacement reduction. Spermatozoa infected by HPV are able to penetrate the oocyte and transfer HPV into hamster eggs. It is not clear if the infected oocyte may lead to a normal early embryonal development and if the infection itself could interfere with the implantation

Institute of molecular and translational medicine, Faculty of Medicine and Dentistry, Palacky Univesity in Olomouc, Olomouc, Czech Republic. 2 Department of Obstetrics and Gynecology, Faculty Hospital and Department of medicine and dentistry, Palacky University in Olomouc, Olomouc, Czech Republic. 3 Department of Obstetrics and Gynecology, Masaryk University in Brno, Brno, Czech Republic For the success of in-vitro fertilization, it is necessary to have synchronized embryo in the blastocyst stage and receptive endometrium. While the embryo is easy to observe, there are currently no markers for endometrial receptivity. For this purpose, cervical mucus, as a non-invasively available specimen of the uterine cervix, is an ideal source of potential biomarkers. The samples of cervical mucus were aspired by neonatal catheters during oocyte aspiration and immediately 1

frozen upon analysis. The samples of cervical mucus were then dissolved using ultrasound needle, digested, purified and analyzed by nanoLC-MS/MS. Mass spectra were evaluated and searched against the human protein database by MaxQuant processing software. The study itself is on the beginning and is currently in the phase of collecting of testing dataset. From already collected samples, 15 samples have been analyzed so far. From this dataset, we have obtained range 282 – 1112 with an average of 596 identified proteins. Those results are much higher than already published cervical mucus proteomic studies and promise higher chance to identify a sensitive biomarker of endometrial receptivity. The study was supported by the Internal Grant of Palacky University (IGA_LF_2018_031); the Czech Ministry of Education, Youth and Sports (LO1304, LM2015064), the Ministry of Health of the Czech Republic (NV18-08-00291).

2018

Pathophysiology of cancer and molecular targets I

Chairs: Josef Srovnal, Viswanath Das pondělí / 19. listopadu 2018 / Monday / November 19th, 2018 / 15:30 - 17:15 Role of long chain fatty acids in human NK cell cytotoxic activity activity

of the aforementioned treatments were incubated with 5000, 10000 or 20000 K562 cells in media with 5 % BSA fatty acid free for 4 hrs. The cells Juan B. De Sanctis were then analyzed by flow cytometry Institute of Molecular and assessing 7ADD as a marker of cell Traslational Medicine. Palacky death. In another set of experiments University, Olomouc, Czech K562 cells were treated with the Republic fatty acids 3 hrs and washed prior the cytotoxicity assay, NK cells were Introduction NK cells are part of innate immune untreated. cells that play a major role in in Results the host-rejection of both tumours Treatment of NK cells by free fatty and virally infected cells. The acids was shown to modulate NK spontaneous cytotoxic activity of NK the expression of KIR activating cells is usually accessed against receptors and not KIR inhibitory K562 cells which do not express MHC receptors. Significant induction was antigens. Tumour cells or any other observed with oleic acid (12.5 ± cells that express MHC class I type 2.8 vs. 53.9 ± 6.6 %, p = 0.001 for C can bind KIR inhibitory receptors at activating and 23.8 ± 7.9 vs. 33.2 ± the surface of NK cells and decrease 8.3 % for inhibitor receptors), and potential cytotoxic response. The eicosapentanoic acid (12.5 ± 2.8 vs. aim of the study was to assess the 59.8 ± 12.3 % p= 0.001 for activating effect of lipids on KIR receptors and and 23.8 ± 7.9 vs. 40.8 ± 14.9 % for cytotoxic response. inhibitor receptors). No major changes were recorded with the other fatty Materials and methods Purified NK cells from 20 normal acids. The expression of Nkp30 and male human normolipemic donors, NKp46 was not altered by fatty acid age 25-40 years, were purified using treatment (expression was above 48 RosetteStep purification kit (Stem % for each case). However, cytotoxic Cell Technologies, Canada). Cells, 2 activity was significantly enhanced in million/ml, were treated for 3 hrs with NK cells treated with saturated fatty 5 % bovine albumin, fatty acid free, acids palmitic acid 27 ± 1.2 litic units, then washed and incubated 3 hrs steric acid 28.2 ± 0.6 litic units, and with 5 % bovine albumin plus different arachidic acid 30.3 ± 2.1 litic units as concentrations (1 pM to 10 μM) of compared to other treatments (mean any of these free fatty acids sodium 22.1 ± 1.1 litic units). If the K562 salts 1) palmitic acid (C16:0), 2) steric cells line were however treated with acid (C18:0), 3) oleic acid (C18:1), 4) these saturated free fatty acids, they arachidic acid (20:0), 5) arachidonic became resistant to NK cytotoxic acid (20:4), 6) eicosapentanoic acid activity suggesting a role in membrane (20:5), and 7) docosahexanoic acid uptake of the fatty acids. K562 cells (C22:6). The cells were than washed were more susceptible to lysis if they and 0.1 million cells were used to were treated with unsaturated fatty assess each of the KIR receptors acids before the cytotoxicity assay by flow cytometry using the pannel was performed, mainly oleic (33.1 of CD158, KIR2DS 3, 4 and 5 ± 1.2 litic unit), arachidonic (33.3 ± (activating receptors), KIR3DL2, and 3.3 litic unit), eicosapentanoic (37.5 KIR3DL4 (inhibitory receptors), and ± 4.8) and docosahexanoic (31.3 ± other cytotoxic receptors Nkp30 and 3.2). Incubation of K562 cells for more NKp46. In parallel, 0.1 million cells than 6 hrs with the fatty acids did not

increase apoptosis or differentiation of the cell line. Conclusions NK cytotoxic response is modulated by incubating the cells with fatty acids. Modulation may affect the expression of activating KIR receptors; however, the expression of the receptors does not necessarily imply an increase in NK cytotoxicity against K562 cells. On the other hand, the target cell line is more susceptible to the unsaturated fatty acid treatment. The saturated fatty acids may enhance NK cytotoxic activity by increasing membrane rigidity. K562 may use these saturated batty acids to survive in nutrition deprived media.

The antagonistic functional duality of cyclin-dependent kinase inhibitor p21WAF1/CIP1 in cells derived from multicellular tumor spheroids

Viswanath Das1,2, Narendran Annadurai1, Dušan Holub1, Marián Hajdúch1,2 Institute of Molecular and Translational Medicine, Olomouc, Czech Republic. Cancer Research Czech Republic, Olomouc, Czech Republic

Introduction The cyclin-dependent kinase inhibitor p21WAF1/CIP1(p21) is a key mediator of p53-dependent cell cycle arrest after DNA damage, in addition to p53-independent mechanisms. Being one of the major transcriptional targets of p53 and due to its anticell proliferative activity, p21 was considered initially as a potent tumor suppressor. However, emerging studies now show the anti-apoptotic and pro-cell proliferative effects of p21, highlighting the oncogenic role of p21 in cancer. In particular, p21 results in genomic instability and

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Průlomové objevy na single-cell úrovni díky ucelenému řetězci řešení

K tomu, abyste učinili vědecký objev, je potřeba postupně překonávát překážky jedním experimentem za druhým... Naším úkolem je Vám pomoci na cestě za tímto průlomovým objevem. Proto jsme pro Vás připravili celistvé řešení zahrnující přístroje, software a reagencie spolu se zkušeným týmem servisních a aplikačních specialistů na světové úrovni, přesně tak, aby Vám nabídl pomocnou ruku v momentě, kdy to potřebujete nejvíce. Zjistěte, proč vědci jako Vy po celém světě spoléhají na naše inovativní technologie.

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2018

the development of aggressive and chemo-resistant traits in a subset of highly proliferating tumor cells through p53-independent pathways. The cellular localization of p21 has been proposed to be critical either in promoting cell survival or in inhibiting cell growth. Materials and Methods Herein, using a combination of gene knockout, cytotoxicity assay, immunofluorescence, immunoprecipitation, and mass spectrometry techniques, we study the consequence of mislocalization of p21 on survival and drug response in cells derived from multicellular spheroids of human colon and cervical cancer cell lines. Results and Conclusions Our study shows that cells derived from multicellular spheroids show an increased expression of p21 despite the reversal from three-dimensional to two-dimensional culture. The p21overexpressing, highly proliferative cells are significantly resistant to a number of standard anti-cancer agents. Further analysis shows that the majority of p21 in this subset of spheroid-derived cells is localized in the cytoplasm and forms a potential ‘anti-apoptosome-like’ complex with mitochondrial apoptosis-associated proteins. These findings add to the shifting paradigm on the role of p21 in tumor cells due to its cellular mislocalization. Acknowledgment: Ministry of Health of the Czech Republic (Gr. Num. LO1304).

Is the inhibition of aldehyde dehydrogenase the way to revert cisplatin resistance in testicular germ cell tumors?

Silvia Schmidtova1, Katarina Kalavska1,2,3, Michal Mego2,3, Katarina Gercakova1, Lucia Kucerova1 Cancer Research Institute, Biomedical Research Center of Slovak Academy of Sciences, Bratislava, Slovakia. Faculty of Medicine, Comenius

strategies. Importantly, ALDH inhibitor disulfiram significantly augmented cisplatin toxicity upon combinatorial treatment. These data suggest novel treatment options Introduction for refractory TGCTs with acquired Testicular germ cell tumors (TGCTs) cisplatin resistance. represent tumors with high curative potential. However, patients with This work was supported by Slovak cisplatin-refractory disease have Research and Development Agency APVV-15-0697, limited treatment options associated (APVV-15-0086, APVV-16-0178), and Scientific Grant with poor prognosis. Development of Agency of The Ministry of Education, new models is needed for unravelling Science, Research and Sport the mechanisms underlying VEGA 1/0043/18. We thank Cancer chemoresistance. Novel agents Research Foundation and League as well as known drugs may prove efficient in enhancing drug sensitivity. against Cancer for financial support. University, Bratislava, Slovakia. Translational Research Unit, National Cancer Institute, Bratislava, Slovakia

Materials and methods Chemoresistant variant of NTERA-2 cell line – NTERA-2 CisR was derived by cultivation in increasing concentrations of cisplatin for 6 months. We determined its chemoresistance by luminescent viability assay, cell migration in 3D spheroid migration assay, expression of aldehyde dehydrogenase (ALDH) by flow cytometry and morphology was visualized by α-Factin staining. Tumorigenicity was determined on immunodeficient mouse model. Upregulated biomarkers were targeted by pharmacological inhibition in 3D tumorosphere assay and in vivo. Results and conclusions NTERA-2 CisR cell line exhibited 30fold higher IC50 values for cisplatin associated with cross-resistance to carboplatin and oxaliplatin. Resistant cells showed different morphology and formed more compact 3D multicellular spheroids. NTERA-2 CisR cells had increased migratory capacity and tumorigenicity. PARP inhibitor veliparib re-sensitized resistant cells to cisplatin in vitro, but it did not work in vivo. More importantly, high expression of ALDH was detected in NTERA-2 CisR cells and its inhibition by disulfiram reverted cisplatin resistance in vitro and in vivo. We derived cisplatin-resistant NTERA-2 CisR cell line as novel clinically relevant model suitable for the evaluation of therapeutic

Case-control study of culturable gut microbiota in colorectal cancer

Lubomír Starý1, Kristýna Mezerová2, Vladislav Raclavský2,1 University Hospital Olomouc, Olomouc, Czech Republic. Palacký University, Olomouc, Czech Republic

Introduction The role of gut microbiota in the development of sporadic colorectal cancer (CRC) has been supported by a number of metagenomic studies recently. However, conclusiveness such studies is limited by small cohort sizes. Also, whole genome sequencing is not available for routine use at a wide scale, and it does not always reach the depth needed for species-level identification. At the same time, MALDI-ToF MS has brought about a revolution in pure culture species identification by reducing labour, time and costs. This brought about a new stimulus to conventional culture techniques. Although many anaerobic bacterial inhabitants of the gut are considered unculturable, this is rather true if culture is performed on a limited number of growth media using simple routine techniques. Actually, culture is performing far better than generally believed. Therefore, we decided to perform a pilot study of the performance of

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extended conventional cultivation followed by MALDI-ToF MS species identification for detection of potential microbial markers associated with newly diagnosed CRC. Rectal swab was used as an easy technique of sampling that can be widely applied in screening studies in future. Material and methods The study was performed in the period 2014-2018. The single centre cohort (n=101) included newly diagnosed colorectal adenoma or cancer. The age-matched controls (n=101) were recruited among patients seeking care for non-adenoma non-CRC diagnosis. Conditions that may affect the composition of gut microbiota profoundly were applied as exclusion criteria, namely any form of inflammatory bowel disease (IBD), antibiotic use within 2 month before sampling, diarrhoea or other symptoms of GIT infection within 2 weeks before sampling, and bowel-clearing within 1 week before sampling Rectal swabs were inoculated on Columbia blood agar (CBA), MacConkey agar (MCA), Brainheart infusion agar with sheep blood (BHI-BA), Schaedler agar (SA), F.A.A. Neomycin agar (FNA), NAS agar, McKay agar and Sabouraud glucose agar (SGA) plates with chloramphenicol (Oxoid, UK). The plates were cultured at 37°C in ambient air supplemented with 5% carbon dioxide (CBA, MCA), 30°C in ambient air (SGA) and at 37°C in the anaerobic gas mixture (80% nitrogen, 10% carbon dioxide, 10% hydrogen; BHI-BA, SA, FNA) and under microaerophilic conditions (the same gas mixture enriched by 1% dioxygen; NAS and McKay agar plates). All colonial morphotypes were identified using MALDI-ToF MS protein profiling following manufacturer’s instructions. Significance of potential associations of particular species with newly diagnosed CRC was evaluated using chi-square test.

Results and conclusions Average age and percentage of males in the CRC group versus control was 67.0 ± 10.52 versus 66.9 ± 10.62, and 60.4 % versus 49.5 %. On average, 9.5 species were identified per sample. Clostridium ramosum, Bacteroides fragilis, Candida albicans and Clostridium perfringens were apparently overrepresented in the CRC group (P = 0.011; 0.024; 0.042 and 0.071; respectively). To our best knowledge, Clostridium ramosum has never been associated with CRC in previous studies. On the contrary, Bacteroides fragilis is widely accepted as a candidate driver of CRC because of its enterotoxin production and positive association with CRC confirmed by several independent studies. Although Candida albicans has not been linked to CRC yet, its role in oral SCC carcinoma is widely accepted and a growing body of evidence supports the view that C. albicans does cause cancer in humans. Interestingly, Clostridium perfringens bacteremia has been associated with subsequent diagnosis of CRC in a recent extensive study. Our results demonstrate that extensive conventional culture is able to detect potential microbial markers of CRC and complement the results of existing metagenomic studies. Economic performance and easy standardization are the main strongpoints of conventional culture, making it suitable for long-term multicenter studies. Candidate status of CRC marker species detected in our study needs to be verified on general population.

Targeting the cellular uptake and processing of missfolded proteins: Example of tau aggregates

Narendran Annadurai, Marian Hajdúch, Viswanath Das Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky

University, Olomouc, Czech Republic Introduction The endosomal-lysosomal and autophagic-lysosomal pathways play a critical role as a quality control system to clear damaged organelles and abnormal and misfolded proteins in order to maintain cellular homeostasis. These defense systems tend to decline during aging, facilitating the manifestation of protein aggregate deposition diseases. Moreover, alterations in degradative pathways are implicated not only in the pathogenies of Alzheimer disease (AD) but also in a host of other diseases including cancer. The misfolded proteins that escape the degradative pathway seed the aggregation of native protein in addition to interfering with the function of other related proteins. This is best characterized by the effect of misfolded tau in AD, huntingtin in Huntington’s disease, α-synuclein in Parkinson’s disease and mutated p53 in cancer. Thus, understanding the mechanisms that result in the endosomal-lysosomal escape of misfolded proteins is essential for understanding the pathogenies of protein aggregationassociated diseases. Materials and Methods Peptides of microtubule-binding repeat 3 (R3) region of tau assemble into fibrils under in vitro conditions. Upon addition to cells, exogenous R3 aggregates seed the aggregation of endogenous tau by unknown mechanisms. Making using of this property of R3 peptides and inhibitors of endocytic pathways, we studied the internalization, endosomallysosomal escape and seeding effect of R3 aggregates in human embryonic kidney HEK-293 cells by live-cell imaging. Results and Conclusions In this study, we used inhibitors of the endosomal-lysosomal pathway to elucidate the steps involved in the internalization and degradation of R3 aggregates, and the resulting effect on the seeding of native tau.

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Our preliminary data show that inhibition of endosomal/lysosomal degradation increases the infectivity of R3 aggregates in HEK-293 cells. The membrane-enclosed R3 aggregates that avoid degradation cause the endosomal membrane to rupture and result in the escape of aggregates into the cytoplasm. The released aggregates then increase

the seeding effect of misfolded R3 peptide in HEK-293 cells expressing aggregation-prone human tau protein. In conclusion, the presented study will help in understanding the mechanism of entry of tau aggregates in cells, and how the alterations or failure in the endosomal-lysosomal pathway define the prion-like seeding and propagation of tau aggregates.

Understanding these mechanisms will provide a foundation for therapeutic approaches targeting the uptake and propagation of misfolded proteins in protein aggregationassociated diseases.

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Pathophysiology of cancer and molecular targets II Chairs: Martin Mistrik, Petr Muller úterý / 20. listopadu 2018 / Tuesday / November 20th, 2018 / 9:00 - 10:30 Mechanisms of HSF1 activation in cancer cells

cell cycle. Replication stress induces fork stalling and fuels genome instability. The mechanistic basis of Petr Muller, Jakub Faktor, replication stress remains Veronika Martinkova, Oliver poorly understood despite its Simoncik, Filip Trcka, Michal emerging role in promoting cancer. Durech, Borivoj Vojtesek Here we show that inhibition of poly(ADP-ribose) polymerase Masaryk Memorial Cancer Institute, (PARP) increases the speed of Brno, Czech Republic fork elongation and does not cause Introduction fork stalling, which is in contrast Activation of stress response to the accepted model in which manifested by increased activity of inhibitors of PARP induce fork chaperones is a natural compensatory stalling and collapse. Aberrant mechanism that accompany acceleration of fork progression malignant transformation. Activation by 40% above the normal velocity of the transcription factor HSF1, leads to DNA damage. Depletion which regulates the expression of of the treslin or MTBP proteins, stress proteins, is then essential for which are involved in origin firing, the survival of tumour cells. Although also increases fork speed above HSF1 and the heat shock response the tolerated threshold, and has been extensively studied, key induces the DNA damage response aspects governing their activation pathway. Mechanistically, we in cancer remain a mystery. Our show that poly(ADP-ribosyl)ation study was therefore aimed to identify (PARylation) and the PCNA proteins that interact with activated interactor p21Cip1 (p21) are crucial HSF1 in response to various stress modulators of fork progression. conditions. High speed of fork progression PARylation and p21 act as induces DNA replication stress suppressors of fork speed in a Materials and methods coordinated regulatory network that For interaction studies, we have and genomic instability is orchestrated by the PARP1 and 1 generated human cell lines Apolinar Maya-Mendoza , expressing SBP tagged HSF1 that Pavel Moudry1,2, Joanna Maria p53 proteins. Moreover, at the fork level, PARylation acts as a sensor exhibit monomeric conformation Merchut-Maya1, MyungHee of replication stress. During PARP under normal conditions. We used 1 1 inhibition, DNA lesions that induce Lee , Robert Strauss , Jiri different stress stimuli including 1,2 fork arrest and are normally heat shock and inhibitors of Hsp90 Bartek resolved or repaired remain or proteasome to find the common 1 Danish Cancer Society Research unrecognized by the replication features of proteins complexes Center, Copenhagen, Denmark. machinery. Conceptually, our associated with activated HSF1. The 2 Institute of Molecular and results show that accelerated samples for pulldown studies were Translational Medicine, Olomouc, replication fork progression collected in several time intervals Czech Republic represents a general mechanism that to monitor the dynamics of HSF1 Accurate replication of DNA requires triggers replication stress and activation, its posttranslational stringent regulation to ensure the DNA damage response. Our modification and assembly of its genome integrity. In human cells, findings contribute to a better protein complexes. thousands of origins of replication understanding of the mechanism of Results and conclusions are coordinately activated during S fork speed control, with implications The quantitative proteomics revealed phase, and the velocity of for genomic (in)stability and several clusters of proteins that replication forks is adjusted to fully rational cancer treatment. replicate DNA in pace with the form distinct protein complexes with activated HSF1 trimers. The most prominent group was represented by proteins assembled into prefoldinlike (R2TP/PFDL) complex and U5 snRNP spliceosome (RUVBL1, RUVBL2, AAR2, EFTUD2, PRPF8 and SNRNP200). Another group of interactors involves AAA+ nuclear proteins such as KIF4A which interaction is associated with HSF1 sumoylation. The last group of interacting proteins was represented by isoforms of Hsp70 and their cochaperones providing negative feedback regulation. These results show the complexity of spaciotemporal changes in protein interactions during stress and reveal important links between HSF1 activation, chromatin remodelling and recruitment of pre-mRNA splicing factors. The work was supported by grant GACR 16-07321S

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Genotoxic and proteotoxic stress-response pathways as targetable vulnerabilities of prostate cancer

Dusana Majera1, Zdenek Skrott1, Jan Bouchal2, Martin Mistrik1, Jiri Bartek1,3,4,5 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. Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Science for Life Laboratory, Karolinska Institute, Stockholm, Sweden. Department of Genome Integrity, Institute of Molecular Genetics of the CAS, v.v.i., Praha, Czech Republic

Introduction Castration-resistant prostate cancer (PCa) represents a serious health and societal challenge, addressed here by model experiments based on mechanistically-supported rationale to explore clinically available inhibitors of PARP1 (PARPi), histone deacetylases (vorinostat), repurposing the alcohol-abuse drug disulfiram (DSF) and survivin inhibitors. Materials and methods PCa cells were examined by colony formation after combined ionizing radiation (IR) and pre-treatment with PARPi or vorinostat (SAHA). Effects of DSF and combined DSF with survivin inhibition was measured by XTT assays. Proteins involved in DNA damage repair, heat-shock and unfolded protein response (UPR) pathways were assessed by immunofluorescence and

immunoblotting. Results and conclusions We observed differential responses of PC3 vs. DU145 cellular models: i) Enhanced sensitivity to PARPi/ IR with lower homologous recombination (HR) repair in PC3; ii) DU145 sensitization to PARPi/IR by vorinostat that decreased mutant p53 and impaired HR, as determined by Rad51 foci. TOPBP1, an important DNA damage response (DDR) factor was decreased after vorinostat treatment. Vorinostat also overcame radio-resistance of stem-cell like DU154-derived cells. All tested PCa models showed sensitivity to DSF combined with copper, and to diethyldithiocarbamate-copper complex (CuET), the anticancer metabolite of DSF. Treatment with DSF/copper or CuET evoked proteotoxic stress, UPR and heat shock responses, effects further enhanced by co-treatment with YM155 (inhibitor of the anti-apoptotic protein survivin), promoting UPRinduced death. CuET and YM155 showed synergistic effects, suggesting an intriguing option for further clinical testing. We propose that targeting genotoxic stress and proteotoxic stress responses by combinations of available drugs could inspire innovative strategies to treat castration-resistant PCa.

Urinary bladder tumourigenesis and mesenchymal cells - from reactive cancer stroma to bladder sarcoma

Jiri Hatina1, Michaela Kripnerová1, Kateřina Houfková1, Pavel Dvořák1, Martin Pešta1, Martina Dolejšová2, Lucie Vištejnová2, Zdeňka Lehečková2, Petra Vohradská3, Milan Hora4, Josef Kopecký5, Jan Gurský6, Kohoutová Michaela7, Kuncová Jitka1, Susanne Jennek8, Karl-Heinz Friedrich8,

Carina Strell9, Wolfgang A. Schulz10 Charles University Medical Faculty in Pilsen, Institut of Biology, Plzen, Czech Republic. 2 Charles University Medical Faculty in Pilsen, Biomedical Center, Plzen, Czech Republic. 3 Teaching Hospital in Pilsen, Institute of Medical Genetics, Plzen, Czech Republic. 4 Teaching Hospital in Pilsen, Department of Urology, Plzen, Czech Republic. 5 Hospital Havířov, Department of Urology, Havířov, Czech Republic. 6 Palacký University, Faculty of Medicine and Dentistry, Institute of Molecular and Translational Medicine, Olomouc, Czech Republic. 7 Charles University Faculty of Medicine in Pilsen, Institute of Physiology, Plzen, Czech Republic. 8 University Hospital Jena, Institute of Biochemistry II, Jena, Germany. 9 Karolinska Institutet, Department of Oncology-Pathology, Stockholm, Sweden. 10 Heinrich Heine University, Department of Urology, Duesseldorf, Germany 1

Urinary tract is by virtue of its constant exposure to urineconcentrated toxins a frequent target of tumourigenesis, with a steadily increasing frequency. Most tumours developping within urinary tract are urothelial carcinomas, originating from urothelial basal or intermediate cells. Urothelial carcinomas represent tumours with a copious stroma, mainly in form of carcinomaassociated fïbroblasts (CAFs). By a fortuitous finding of a primary urothelial carcinoma cell culture (BC44) with a particularly abundant fibroblastic component, we were able to establish urothelial carcinomaassociated fibroblasts (BC44Fibr) as a separate mesenchymal stromal cell line, and we could show that they express typical CAF-marker proteins (vimentin, smooth-muscle actin, fibroblasts activation protein, CD90) as well as connexin-43, a

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typical marker protein of normal suburothelial fibroblasts, hinting at their possible origin. BC44Fibr are distinctly tumour-promoting, and this phenotype derives from a plethora of interconnected activities towards carcinoma cells; we have identified activation of stemness, motility and metabolism, and inhibition of antitumour immune response (1). Tumourigenic transformation of mesenchymal cells on their own is much rarer and results in a continuum of phenotypes, from pseudosarcomatous myofibroblastic proliferation to overt sarcoma. Inflammatory myofibroblastic tumour (IMT) represents a special and still ill-defined diagnosis, whose exact position at the myofibroblastic transformation scale remains a matter of controversy (2). By another serendipity, we were able to establish a cell line (MBT – myofibroblastic bladder tumour) from IMT; to the best of our knowledge the first cell line from this tumour type established worldwide. The cells present

a typical immunophenotype of activated myofibroblasts and a low, but discernible transformation status (motility, anchorage-independent clonogenicity). Interestingly, according to first analyses, MBT cells function interchangeably with BC44Fibr in supporting carcinoma cells. This might be related to episodic observations of synchroneous or metachroneous appearance of particularly aggressive urothelial carcinomas and IMT (3). In conclusion, our new bladder mesenchymal cells lines provide a valuable model for various aspects of bladder tumourigenesis. (1) Hatina J, Kripnerová M, Tuková J, Šrámek J, Dvořák P, Pešta M, Dobrá J, Babuška V, Racek J, Sobol M, Philimonenko A, Hozák P, Czuba Z, Schulz WA, Strell C, Grimm S, Jennek S, Friedrich KH. Tumourstroma interactions in urothelial cancer [in German]. Urologe A 2015 Apr;54(4):516-25.

(2) Cheng L, Foster SR, MacLennan GT, Lopez-Beltran A, Zhang S, Montironi R. Inflammatory myofibroblastic tumors of the genitourinary tract--single entity or continuum? J Urol. 2008 Oct;180(4):1235-40. (3) Montgomery EA, Shuster DD, Burkart AL, Esteban JM, Sgrignoli A, Elwood L, Vaughn DJ, Griffin CA, Epstein JI. Inflammatory myofibroblastic tumors of the urinary tract: a clinicopathologic study of 46 cases, including a malignant example inflammatory fibrosarcoma and a subset associated with highgrade urothelial carcinoma. Am J Surg Pathol. 2006 Dec;30(12):150212. Supported by the Czech Science Foundation project No 17-17636S and by the Charles University Specific Student Research Projects Nr. 260394/2017 and Nr. 260393/2017.

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Cancer biomarkers and personalized medicine I

Chairs: Magdalena Ratajska, Jiri Drabek úterý / 20. listopadu 2018 / Tuesday / November 20th, 2018 / 11:00 - 12:15 The application of ctDNA based tests in ovarian cancer diagnostic and research

Magdalena Ratajska1, Magdalena Koczkowska1, Monika Żuk1,2, Alina Kuźniacka1,2, Bartosz Wasąg1,2 Department of Biology and Medical Genetics, Medical University of Gdansk, Gdansk, Poland. Laboratory of Clinical Genetics, University Clinical Centre, Gdansk, Poland

Introduction Ovarian carcinoma (OC), one of the most common gynecological malignancy, accounts for approximately 239,000 new cases and 152,000 deaths worldwide annually. Because of non-specific symptoms and the lack of effective screening tests, most individuals are diagnosed with an advanced disease (stage III or IV) with the 5-year survival rate below 30%. The overall frequency of germline and somatic variants is estimated at 15-20% and 3-10% respectively, with the majority of mutations being diagnosed in BRCA1/2 genes. Circulating tumor DNA (ctDNA) is a valuable material and an important source of information on the OC molecular pathogenesis and is expected to be a diagnostic and prognostic marker in OC patients. The aim of this study was to establish the prevalence and spectrum of pathogenic and likely pathogenic variants in the 93 cancer-related genes, that may play a role in the development and progression of OC. Material and Methods The study comprises 121 unselected ovarian cancer patients who were referred to the University Hospital in Gdansk and the Red Cross Hospital

in Gdynia between 2012 and 2013. ctDNA was extracted using Cobas cfDNA Sample Preparation Kit, Roche Diagnostics, and mutation screening was performed using the BRCA Tumor MASTR Plus assay (Multiplicom), followed by Human Breast Cancer Panel, Qiagen GmbH. Results In the studied group 24.8% of patients (30/121) were diagnosed with BRCA1/2 pathogenic variants, including 22 and seven individuals with exclusively germline or somatic mutations, respectively and patient with variants of both origin. Within this cohort, seven patients had more than one pathogenic variant. Consequently, patients negative for BRCA1/2 pathogenic variants have been eligible to further screening using the Human Breast Cancer Panel. So far the analysis was successfully completed in 10 patients. Within this group the most representative were pathogenic alterations in the TP53 gene (n=5/10; 50%), followed by pathogenic or likely pathogenic variants in CSMD1, KRAS, NF1, PIK3CA, SMARCA4A. Conclusions Our findings demonstrated that detection of both germline and somatic alterations in ctDNA is feasible and might be helpful as a complementary tool for identification of somatic alterations when the standard diagnostic procedures with using FFPE samples are insufficient.

Identification of meningioma patients in high risk of tumor recurrence using microRNA profiling

Josef Srovnal1, Hanus Slavik1, Vladimir Balik1, Miroslav Vaverka2, Lumir Hrabalek2, Katerina Staffova1, Jana

Vrbkova1, Marian Hajduch1 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic. Department of Neurosurgery, University Hospital Olomouc, Olomouc, Czech Republic

Introduction Meningioma represents one of the most common intracranial tumors. They are generally thought to progress from low to high-grade lesions and relapse. However, the molecular mechanisms underlying their pathogenesis remains to be ellucidated. Identification of meningioma patients with higher risk of recurrence may have significant impact for future clinical management. Methods and patients Screening phase: Formalin-fixed paraffin embedded tumor samples were obtained from 45 meningioma patients (15 recurrent patients, 30 patients without recurrence) and 5 healthy controls (dura mater). Comprehensive clinical-pathological data were mined. There were 15 males and 30 females; median age was 54 years, range 28 – 99 years. Total RNA was purified from FFPE samples after pathological verification using miRNeasyMini kit (QIAGEN). Microarray analysis was performed using the MiRNA 4.0 Array and FlashTagTM Biotin HSR(Affymetrix). Training phase: Pair-matched normalized dataset of 64 meningioma patients with and without recurrence was selected for training phase. The QPCR on LC 480 (Roche) using TaqMan Advanced miRNA Assay (ABI) was performed to obtain miRNA expression data according to the manufactures. The raw data (Cp values triplets) were summarized

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by average and normalized with selected normalization miRNA. Results In the screening phase, we revealed different miRNA profiles in primary meningioma tumors that will relapse in comparison with nonrecurrent tumors. 54 differentially expressed miRNAs were identified in meningioma patients at high risk of recurrence. High-risk patients had significantly higher expression of miR-572 and miR-320c, and lower expression of miR-140 and miR-165p. Twenty-one candidate miRNAs (including miR-107, miR-16-5p, miR320c, miR-371b-5p and miR-15a5p) were chosen for further qPCR validation on independent dataset in training phase. In the training phase 4 candidate miRNAs with minimal differences within the dataset were tested to select the best normalization miRNA. MiR-181 was selected for further analyses. The normalized data confirmed the differences in miRNA expression within recurrent meningioma patients using univariate analysis. Many of tested miRNAs had lower expression in high-risk patients. The multinomial analysis revealed subgroup of seven miRNAs identifying high-risk meningioma patients with high sensitivity and specificity. These seven-MiRNAs model has high positive (79%) and negative (78%) predictive value. Conclusion: We have revealed seven-MiRNAs model (including miR-16, miR-331, miR-146, miR-18, miR130, miR-1271 and miR-15a5p) identifying high-risk meningioma patients with high predictive value. These findings will be further validated on independent dataset. Acknowledgment: This work was financially supported by Ministry of Health of the Czech Republic, grant nr. 15-29021A, IGA UP LF 2018_005, NPU LO1304 and NCMG LM2015091.

ISUP grade groups, tertiary Gleason, periostin and versican in aggressive prostate cancer

Gvantsa Kharaishvili1, Jan Bouchal2, Zdenek Kolar1, Milan Kral3 Department of Clinical and Molecular Pathology, University Hospital,, Olomouc, Czech Republic. Department of Clinical and Molecular Pathology, Palacky University, Olomouc, Czech Republic. Department of Urology, University Hospital, Olomouc, Czech Republic

Introductio A new contemporary prostate cancer grading system was proposed in 2014 for accurate grade stratification (Egevald et al 2016). Tertiary Gleason patterns are reported with increasing frequency in relation with disease recurrence (Epstein et al 2012, Trock et al 2009). We have previously reported association between periostin, Skp2 and Slug in aggressive prostate cancer. We decided to verify association of tertiary Gleason and ISUP contemporary grade groups with expression of these and other selected proteins in our patients. Materials and methods Formalin fixed paraffin embedded tissues of 101 prostate carcinomas were stained immunohistochemically for E-cadherin, beta-catenin, vimentin, Skp2, Slug, Ki67, p53, androgen receptor, PSA, periostin, versican, and scored. Slides were reviewed for the presence of tertiary Gleason (worse than the primary and secondary grade, usually pattern 4 and 5). Carcinomas were classified into localized, advanced and metastatic groups, and ISUP 2014 Gleason grade groups (GG15 [1=GS≤6; 2=GS3+4; 3=GS4+3; 4=GS8, 5=GS9-10], Pierorazio et al 2013 and Epstein et al 2016). Statistical analysis was performed

by SPSS software. Hierarchical cluster analysis (average linkage, within groups) was done to identify subgroups based on protein expression. Kohonen importance network was generated for accurate prediction. Results and conclusions: Tertiary Gleason was recognized in 22% of radical prostatectomy cases, it was more frequent in advanced and metastatic tumors (p Gleason grade groups were in negative association with E-cadherin (Rs -0.203, p=0.045) while with nuclear Skp2 and periostin stromal expressions showed positive association (Rs 0.338 and 0.269, p=0.001 and 0.008, respectively). Periostin stromal positivity correlated with versican stromal expression (Rs 0.368, p Chi-square test based correlation network showed significant relationships between AR, GS groups, stage and clusters (p Kruskal-Wallis Anova test revealed significant differential expressions of stromal periostin (p=0,017), stromal versican (p<0.001), as well as Skp2 (p For the first time, we show significant association of tertiary Gleason and grade groups GG4/5 with periostin, Ki67, Skp2 and beta-catenin. Cluster analysis revealed significant differences of versican, periostin, AR, tertiary Gleason and Grade Groups between patients clusters. Advanced models in larger cohorts are further needed to accurately identify potential aggressive prostate cancer requiring immediate treatment. Acknowledgements Supported by MH CZ – DRO (FNOl, 00098892).

IDH1/2 in diffuse gliomas: Retrospective mutation analysis

Zuzana Šporiková1, Magdalena Megová Houdová1, Rastislav Slavkovský1, Lucie Tučková2, Ondřej Kalita3, Barbora Blumová1, Jiří Ehrmann2,

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Marián Hajdúch1 Institute of molecular and translational medicine, Faculty of medicine and dentistry and Faculty hospital Olomouc, Olomouc, Czech Republic. Department of clinical and molecular pathology, Faculty of medicine and dentistry and Faculty hospital Olomouc, Olomouc, Czech Republic. Department of neurosurgery, Faculty of medicine and dentistry and Faculty hospital Olomouc, Olomouc, Czech Republic

Introduction Gliomas are the most common brain tumours. Despite of the maximal treatment modality are these tumours malignant, highly infiltrating and diffuse with unfavourable outcome. Mutations in isocitrate

dehydrogenase 1 and 2 (IDH1/2) were described for the first time in glioblastomas and low grade gliomas. Mutated IDH1/2 enzymes have de novo aberrant activity leading to consumption of alphaketoglutarate and NADPH and can inhibit degradation of hypoxia inducible factor 1α and increase of oxidative stress in the tumour cells. When the facts are given altogether IDH1/2 mutations are fundamental prognostic biomarker of gliomas and play significant roles in gliomagenesis. Materials and methods Our retrospective study comprise 271 glioma samples obtained from surgical operation within years 2011 to 2017. Using the IHC anti-IDH1 R132H antibody we distinguished IDH1 negative or positive cases. Based on recommendation of the

WHO classification of tumours of the central nervous system (2016) we selected patients under the age of 55 years. For next step of our study we used DNA isolated from FFPE blocks was used for creating a library by Nextera DNA Library Preparation Kit (Illumina). Samples were finally sequenced on next generation sequencing system MiSeq (Illumina). Results and conclusions: Given the fact that IDH1 R132H mutation is present in 90% of IDH1 positive cases, immunohistochemistry is a great tool for distinguishing those patients. For verification these data we suggest to add next generation sequencing for detection of other rare IDH1/2 mutations in IHC IDH1 R132H negative cases.

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2018

Cancer therapeutics I

Chairs: Milos Petrik, Martin Mistrik úterý / 20. listopadu 2018 / Tuesday / November 20th, 2018 / 13:30 - 14:45 Alcohol abuse drug disulfiram targets cancer via p97 segregate adaptor NPL4

Zdenek Skrott1, Martin Mistrik1, Klaus Kaae Andersen2, Soren Friis2, Dusana Majera1, Tomas Ozdian1, Jirina Bartkova2,3, Peter Dzubak1, Jindrich Sedlacek4, Jing Li5, Marian Hajduch1, Boris Cvek4, Raymond J. Deshaies5, Jiri Bartek2,3 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic. Danish Cancer Society Research Center, Copenhagen, Denmark. Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden. Department of Cell Biology & Genetics, Palacky University, Olomouc, Czech Republic. Division of Biology and Biological Engineering, Caltech, Pasadena, USA

Introduction Disulfiram, also known as Antabuse, is an old anti-alcohol drug that has been shown to be effective in various preclinical cancer models. However, the unknown active metabolite, the unclear mechanism of action and unidentified molecular target, all obstruct repurposing disulfiram for use as an anti-cancer agent.

analysed by HR-MRM analysis performed on HPLC-ESI-QTOF system consisting of HPLC chromatograph Thermo UltiMate 3000 with AB Sciex TripleTOF 5600+ mass spectrometer. Results and conclusion: Disulfiram-copper complex was identified as a new active disulfiram metabolite toxic to cancer cells and accumulating in tumours. Moreover, we have found that in the cells, disulfiram-copper complex binds to the NPL4 protein, an adaptor of p97 segregase, which is essential for the degradation of proteins involved in several regulatory and stress responses. Binding of the disulfiram-copper complex to NPL4 induces its conformational change and aggregation. NPL4 aggregates subsequently attract p97 and other stress proteins leading to induction of heat-shock and unfolded-protein responses, impairment of protein degradation, ubiquitin stress, and cell death as a consequence. Collectively, these should encourage further clinical tests, help clinicians to monitor the treatment and identify suitable patients who might benefit from disulfiram. Finally, it should promote eventual repurposing of this old, safe and unexpansive drug to combat cancer worldwide.

Senolytic cocktail dasatinib+quercetin does not enhance the efficacy of senescence-inducing chemotherapy in liver cancer

Kristina Kovacovikova1, Illar Pata2, Martin Mistrik3, Jiri Materials and methods Cell viability was analysed by CFA Bartek3,4, Manlio Vinciguerra1,5 and XTT assays. Cellular proteins were analysed by immunoblotting, immunohistochemistry and immunofluorescence analysis. Disulfiram-copper complex was

International Clinical Research Center (FNUSA-ICRC), Brno, Czech Republic. Tallinn University of Technology, Tallinn, Estonia.

Institute of Molecular and Translational Medicine (IMTM), Palacký University, Olomouc, Czech Republic. Genome Integrity Unit, Danish Cancer Society Research Center , Danish Cancer Society, Copenhagen, Denmark. Institute for Liver and Digestive Health, University College London (UCL), London, United Kingdom

Introduction Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death, which develops in the context of fibrosis and cirrhosis caused by chronic inflammation, in turn due to non-alcoholic fatty liver disease (NAFLD), alcohol consumption and/or hepatitis viral infection. An increased number of senescent cells is associated with age-related tissue degeneration during NAFLD-induced HCC, or during chemotherapeutic treatment. Senolytic agents target selectively senescent cells. A combination of the senolytic drugs dasatinib and quercetin (D+Q) reduced hepatic lipid accumulation and alleviated age-associated physical dysfunction in mice. However, whether D+Q can impact the treatment of HCC, at the endstage of the NAFLD inflammatory spectrum, is unknown. Materials and methods Here, we used two well-established HCC cell lines (HepG2, Huh-7) and xenografted athymic nudemice to investigate the antitumor efficacy of doxorubicin, D+Q, or the combination, against HCC. Results and conclusion We demonstrate that the maximal cytostatic doses for D and/or Q (1 uM+1 uM) lacked efficacy in removing doxorubicin-induced beta-gal-positive senescent cells. Moreover, D+Q did not affect doxorubicin-dependent induction of

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flattened morphology, activation of p16, expression of SASP-associated genes or formation of gH2AX foci. Doxorubicin reduced tumor growth by 30% compared to control mice, while D+Q was ineffective in synergizing with doxorubicin and in clearing doxorubicin-induced HCC senescent cells. Unexpectedly, D+Q alone appeared to have acute protumorigenic effects in control mice. Conclusions: while our data need to be confirmed in animal models that fully recapitulate NAFLD, we demonstrate that these compounds are ineffective, alone or in synergy with senescence-inducing chemotherapy, against experimental HCC.

Fluorescent dye labeling nucleolus through interaction with BYSL

Zuzana Maceckova, Pawel Znojek, Martina Medvedikova, Tomas Ozdian, Petr Dzubak, Marian Hajduch I nstitute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, olomouc, Czech Republic Introduction Our institute has been given a library of fluorescence dyes. After high throughput screening we selected eleven dyes specifically labeling nucleolus. After the following tests, we selected one dye that labeled nucleolus only in green channel and showed covalent biding towards unknown protein. This compound co-localized with ribosomal proteins and with nucleolar protein BYSL. Using three BYSL antibodies we performed an immunoprecipitation detected fluorescent signal. Immunoprecipitated was subjected to MS analysis. Out of this analysis we selected three proteins as the possible target of compound. We suspect that the compound binds specifically to BYSL and we want to prove it in following experiments.

Materials and methods U2OS and K562 cells were fixed with 70% EtOH for 30 min in freezer then incubated with compounds for 10 min in RT. For the purpose of imaging U2OS cells were used. After tagging, the cells were washed with 1X PBS and pictured by youkogawa automated microscope, alternatively, cells were also labeled with immunofluorescence using antibodies specifically against RPL11, RPS26, FLB, NCL and BYSL. K562 cells were used for protein analysis. Cells were lysed with the ripa buffer and then divided on the SDS page. Signal was captured in green chanel using flourecsence scanner. Next, immunoprecipitation using three distinct anti-bysl antibodies was performed. Band emitting signal in green chanel was cut out and submited to MS analysis. Results and conclusions Compound PR312-4 showed best results in image analysis. Furthermore, this compound showed clear colocalisation with BYSL in immunofleorecent detection. Clear approximately 50 kDa band on SDS page in green chanel was visible in six out of eleven compounds. Lysate from cells treated with PR312-4 was submeted to immunoprecipitation using three distinct antibodies againts BYSL, immunuprecipitate using all antibodies showed strong signal. Afterwards band with aproximately 50 kDA was cut out from gel and used for MS analysis. This analysis revield that BYSL and two other proteins are possible targets of PR312-4 compound. Acknowledgment: IGA_LF_2018_005

In vivo biodistribution study of hydroxyapatite nanoparticles labelled with technecium-99m

Zbynek Novy1, Volodymyr Lobaz2, Martin Vlk3, Jan Kozempel3, Martin Hruby2, Marian Hajduch1, Jarmila Drymlova4, Radek Navratil4,

Petrik Milos1 Palacky University Olomouc, Olomouc, Czech Republic. Institute of Macromolecular Chemistry, Prague, Czech Republic. Czech Technical University in Prague, Prague, Czech Republic. University Hospital Olomouc, Olomouc, Czech Republic

Introduction The goal of this study was to image the biodistribution of new type of nanoparticles labelled with 99mTc-HDP. There were four different kinds of hydroxyapatite nanoparticles (coated with different biocompatible polymers) employed in the experiments. Such labelled nanoparticles could serve for imaging of solid tumors based on Enhaced Permeability and Rentention (EPR) Effect in the future. Materials and methods Polymer coated nanoparticles were radiolabelled with 99mTc-HDP and underwent TLC verification of radiochemical purity, then followed by ex vivo biodistribution studies in normal mice in two different time points after administraion (1h, 6h). Biodistribution of labelled nanoparticles was also monitored by µSPECT/CT system 1h, 3h, 6h a 24h post injection and for three different application approaches (in vitro prelabelled nanoparticles, 99m Tc-HDP injected 1 h and 24 h after non-labelled nanoparticles). Final experiment consisted of imaging the biodistribution in vitro prelabelled nanoparticles in tumor mice. The tumor was formed from human colorectal carcinoma cells HT-29 in mouse of SCID strain. Results and conclusions Radiolabelling of selected nanoparticles showed relatively high radiochemical purity, which was in all cases above 90%. Ex vivo biodistribution study revealed predominant accumulation of nanoparticles in liver and spleen. SPECT/CT imaging of mice after administration of prelabelled

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nanoparticles displays mainly liver, spleen and partly bladder, also the heart in early time points post injection. SPECT/CT imaging of tumor mice showed similar results as in case of normal mice, but with no radioactivity detected in tumor site. Biodistribution studies revealed dominant accumulation of nanoparticles in liver and spleen in all monitored time points post injection both in normal and tumor mice. Accumulation of studied nanoparticles was not confirmed in tumor by SPECT/CT. This project was supported by Czech Research Council No. 16-30544A.

Identification of compounds with cytotoxic activity by cell viability based high throughput screening

Soňa Gurská, Petr Džubák, Marián Hajdúch

I nstitute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic High – throughput screening (HTS) has been a significant contributor to the discovery of chemical leads. This technique was developed to evaluate the biological activity of thousands of unique small molecules to identify potential drug candidates in a very short time. This system requires automation, data processing and control software, precise liquid handling devices, and sensitive detectors. One of the methods routinely used in our HTS laboratory is cytotoxicity screening as in vitro cytotoxicity testing has become an essential aspect of drug discovery. In the beginning, the MTS assay as cytotoxicity test was validated. Cytotoxic effects of unique chemical

compounds are currently tested on ten cell lines (8 cancer cell lines and two non-cancer cell lines). Firstly, all compounds are tested at one concentration (50 µM) and the PI (percentage of inhibition) value is calculated. Consequently, compounds which can kill more than 50% of the cell population at tested 50 µM concentration (PI ˃ 50%) are selected for determining the IC50 values. For analysis and calculations the Dotmatics software is used. To quantify the suitability of cytotoxic assay in an HTS the Z-factor is determined for each plate and cell line. Some results obtained in the cytotoxicity testing will be presented and discussed. This study was supported by the Czech Ministry of Education Youth and Sports (LO1304, LM2015063) and Internal grant of Palacky University (IGA_LF_2018_031).

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Cancer therapeutics II

Chairs: Milan Urban, Jiri Voller úterý / 20. listopadu 2018 / Tuesday / November 20th, 2018 / 15:15 - 16:45 using pull-down assays and also Cytotoxic triterpenes and triterpenic conjugates used for with a fluorescent label to visualize studies of mechanism of action them in cells.6 New methodologies

Milan Urban1, Jiří Hodoň1, Lucie Borková2, Jan Pokorný2, Soňa Krajčovičová2, Barbora Lišková1, Miroslav Soural2, Jana Václavková1, Jarmila Staňková1, Dušan Holub1, Petr Džubák1, Marián Hajdúch1, Jan Šarek1, Jiří Řehulka1 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University in Olomouc, Olomouc, Czech Republic. Department of Organic Chemistry, Faculty of Science, Palacky University in Olomouc, Olomouc, Czech Republic

Introduction Triterpenoids are secondary metabolites from plants, fungi, marine invertebrate or other organisms. They often have a variety of biological activities.1 Betulinic acid, for example, has a strong anti-cancer activity and its hemiesters inhibit maturation of HIV.2

for the synthesis of such probes was developed using solid-phase approach.5,6 Conclusion: It was found that the studied compounds easily penetrate to the cells and enter some of the sub-cellular structures. Potential protein targets were found for several active heterocycles. Basic assumptions about SAR, possible pharmacophores and targets will be discussed. References: 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. 6. Krajcovicova, S.; Stankova, J.; Dzubak, P.; Hajduch, M.; Soural, M.; Urban, M. Chem. Eur. J. 2018, 24, 4957.

Results In this work, a number of new semisynthetic triterpenoids of various skeletons (such as lupane, oleanane, ursane, taraxastane etc.) were prepared. Their in vitro cytotoxic activity was measured and it was 6-Substituted purines as ROCK found that the best structures belong inhibitors with anti-metastatic to the analogues of lupane with an activity electronegative substituent at the 1,2 3 position C-23 or with a heterocycle Jiří Voller , Lenka Zahajská , condensed to the A-ring.4 Lucie Plíhalová4, Jana Pharmacological parameters and Jeřábková5, David Burget2, the influence of the best compounds Andreea Csilla Pataki6, Marek on cell cycle and on the DNA/ 4 1 RNA synthesis was then studied. Zatloukal , Vladimír Kryštof , 7 6 The most interesting compounds Jan Brábek , Daniel Rösel , were then equipped with biotin5 to Karel Doležal4, Miroslav identify the potential target proteins Strnad1

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. . Isotope Laboratory, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Prague, Czech Republic. Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, Olomouc, Czech Republic. Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University , Olomouc, Czech Republic. Department of Cell Biology, Faculty of Science, Charles University , Prague, Czech Republic. Department of Cell Biology, Faculty of Science, Charles University , Olomouc, Czech Republic

Introduction Rho-associated serine/threonine kinases (ROCKs) are principal regulators of the actin cytoskeleton. Through its changes, they that regulate the contractility, shape, motility, and invasion of cells. ROCK inhibitors are currently used in therapy of glaucoma and cerebral vasospasm. Because ROCKs are dysregulated in various malignancies, anti-tumor or anti-metastatic potential is being investigated. Materials and methods We explored the relationships between structure and in vitro antiROCK2 activity in a series of novel purine derivatives substituted at the C6 atom by piperidin-1-yl or

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azepan-1-yl groups. The selected derivatives were further tested on 97 protein kinases. Intracellular activity was evaluated in melanoma cell lines by immunodetection of MLC phosphorylation, collagen matrix invasion assay and by analysis of cell morphology (mesenchymal vs. epithelial). Results and conclusions Structure-activity relationship (SAR) analysis shows that anti-ROCK activity is retained, and may be further increased, by substitution of the parent compounds at the C2 atom or by expansion of the C6 side chain. The compounds can reach effective intracellular concentrations, as demonstrated by a decrease in phosphorylation of the ROCK target MLC, and by inhibition of the ROCKdependent invasion of melanoma cells in the collagen matrix. Our study may be useful for further optimization of C6-substituted purine inhibitors of ROCKs and of other sensitive kinases identified by the screening of a broad panel of protein kinases - for example JAK and JNK.

HTS-likeness and chemical library design

Pavlo Polishchuk, Mariia Matveieva Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic

Introduction High-throughput screening (HTS) is one of strategies to identify new hits. Ideally those screening libraries should be small and provide high chances to discover hits. Compounds selected for HTS libraries should satisfy different criteria. Appropriate physicochemical properties estimated by drug-/lead-likeness filters are one of them. There are many developed physicochemical rules commonly used for compound selection. However, almost all of them were derived based on data

sets containing drugs or drug candidates. Therefore they are biased towards drugs rather than to compounds suitable for HTS. Materials and methods We proposed an approach which estimates HTS-likeness of compounds suitable for compound selection. In order to develop a predictive model a large data set of compounds was collected from PubChem database. The data set included 230325 compounds. All of them have been tested in 49 assays. Common physicochemical parameters (number of H-bond donors/ acceptors, lipophilicity, polar surface area, number of rings and rotatable bonds, molecular weight) were calculated for this data set. A custom Random Forest algorithm has been implemented and used to create a model predicting HTSlikeness of compounds based on this features. Models were developed separately for cell-based, biochemical and all assays. Results Another data set of 72670 compounds which have been tested in 45 assays were collected from PubChem and used for validation of the developed model. 28% of compounds were predicted as HTS-like by the model trained on all assays. The median hit rate enrichment was 1.34 that corresponds to 34% increase of a hit rate in these 45 assays in average. Additionally the approach has been validated on the NCI60 data set containing 46982 compounds tested in 68 assays. The model trained on cell-based assays selected 50% of these compounds. The median enrichment was 1.52 whereas the theoretical maximum was 2. Application of different drug-likeness filters to these two data sets did not improve hit rates among selected compounds demonstrating their limited applicability to design of HTS libraries.

Betulinic acid derivatives inhibit Hedgehog/Gli-mediated transcription in human glioblastoma cell line

Ivo Frydrych, Hanus Slavik, Milan Urban, Marian Hajduch Institute of Molecular and Translational Medicine, Olomouc, Czech Republic

Introduction The evolutionary important Hedgehog (Hh)/Gli signaling pathway plays an important role in a variety of processes such as pattern formation, differentiation, proliferation, and organogenesis. Aberrant activation of the Hh/Gli pathway has recently been shown in several forms of solid tumours, such as basal cell carcinoma of the skin, cerebellar medulloblastoma, rhabdomyosarcoma, or cancers of the pancreas, stomach, lung, and prostate. Constitutive activation of Hh/Gli pathway has also been reported in a subset of human gliomas. The Gli family of proteins represent key mediators of the Hh/ Gli pathway. The naturally occurring pentacyclic triterpenoid betulinic acid (BA) has been shown to induce apoptosis and inhibit hedgehog signalling in rhabdomyosarcoma. Inspired by this finding, we screened library of BA derivatives as potential inhibitors of the Hh/Gli pathway. Materials and methods We have developed U87-MG cell line derived luciferase cell-based assay for monitoring Gli1 activation. Cytotoxicity against U87-MG cells has been determined by MTS assay. The effect on cell cycle, DNA, and RNA synthesis has been evaluated by flow cytometry. To examine the molecular mechanism of the phenotypic changes, we performed real-time quantitative (RT-PCR) analysis to monitor the expression changes of critical component of Hh/ Gli pathway and corelated the results with protein expression evaluated by western blot. Results and conclusions We identified three potent

Hh/

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Gli pathway inhibitors with effect even better than Hh/Gli pathway inhibitors cyclopamine or GANT61. These compounds also remarkably decreased U87-MG cells proliferation and induced apoptosis in a dose- and time-dependent manner. The results of RT-PCR analysis showed downregulation of GLI1/2 transcription factors as well as GLI1 targets and the changes in mRNA expression nicely corelated with results at the protein level.

Affinity purification as a tool for identifying molecular targets of new triterpenic pyrazine compounds

Jana Václavková, Jiří Řehulka, Dušan Holub, Jiří Hodoň, Milan Urban, Soňa Gurská, Martina Medvedíková, Petr Džubák, Marián Hajdúch Laboratory of Experimental Medicine, Institute of Molecular a Translational Medicine, Faculty of

Medicine and Dentistry, Palacký University, Olomouc, Czech Republic Introduction Understanding of mechanism of action of newly synthesized compounds is an essential step in the drug development process. In this work, we focused on identifying molecular targets. Our method, affinity purification, seems to be a universal and useful tool to identify molecular targets and will help us in preclinical development of biologically active compounds. Our work is focused on triterpenic pyrazine which showed high selectivity against leukemic cell lines and a drug-resistant leukemic cell lines as well. Materials and methods The pull-down experiments were performed with leukemic CCRFCEM, CCRF-CEM daunorubicine resistant, colorectal carcinoma HCT116 and osteosarcoma U2OS cell lines. Affinity purification was performed using streptavidincoated magnetic beads, which

bind biotinylated analogs of tested compounds, and SILAC labeled cell lysates. We used gel electrophoresis and mass spectrometry (UHPLC/MSESI/LTQ Orbitrap) tools to identify the proteins which are considered as molecular targets of our triterpene. For validation of selected molecular targets we have used isothermal titration calorimetry (ITC). Results and conclusion The identified proteins include oncogenes, parts of mitochondrial electron transport chain, fatty acid metabolism and transporter proteins. The validation steps at the cellular level are at the early beginning and will continue. The candidate proteins could have important biological significance and will be further validated to confirm our findings. The study was supported by the Internal Grant of Palacky University (IGA_LF_2018_031); the Czech Ministry of Education, Youth and Sports (LO1304, LM2015063 and LM2015064).

2018

Cancer epigenetics

Chairs: Khushboo Agrawal, Manlio Vinciguerra úterý / 20. listopadu 2018 / Tuesday / November 20th, 2018 / 16:45 - 18:00 Chromatin “reader” machinery as target for overcoming resistance to DNA-demethylating epi-drug decitabine

Khushboo Agrawal, Petr Vojta, Rastislav Slavkovsky , Ivo Frydrych, Marian Hajduch Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Background: Based on the current understanding of the ‘epigenetic landscape’, cancer methylome is highly disrupted, which makes DNA methylation an excellent target for anti-cancer cures. To date, 5-azacytidine and its congener, 5-aza-2’-deoxycytidine (DAC) are the most successful DNA demethylating epigenetic drugs. However, scientists are yet to find the reversal of clinical resistance to these drugs. Yet another challenge remains to be the decreased efficacy of these promising therapies in the cure of solid tumors.

Materials and methods We used a soild tumor, HCT116 colorectal cancer (CRC) cell line and developed resistance against DAC. DAC-resistant HCT116 cells were used to study the epigenetic crosstalk between DNA methylation and chromatin modifications. Results and conclusions The screening of parental and DAC-resistant CRC cells against inhibitors of epigenetic “writerserasers-readers” unveiled increased sensitivity of resistant cells to BET inhibitor (inhibitor of reader enzymes), (+)-JQ1. BET inhibitor further mediated augmented response on cell cycle phases of resistant cells, increased anti-proliferative effects in xenograft models of resistant cells, and synergystic effects in combination

with DAC in HCT116 parental cells, both in vitroand in vivo. We then sequenced the transcriptome of DACsensitive and -resistant HCT116 cells, and (+)-JQ1 treated-(DAC-resistant) cells, using RNA-seq. The RNA-seq data revealed the overexpression of critical oncogenes, and their binding inactivation of key tumor suppressor genes (TSGs) in resistant cells. The most significant and biologically relevant transcriptional changes were further validated by qRT-PCR, and in addition their methylation status was determined by bisulphite sequencing. We discovered that the expressions of down-regulated TSGs were methylation driven, exposing these tumor-suppressive signatures as biomarkers which might differentiate between DAC-resistance and sensitivity, whereas, overexpression of oncogenes was independent of methylation. Interestingly, the expressions of up-regulated oncogenes which define cell identity, mainly those involved in signaling of inflammatory pathways (including some bromodomain-specific genes) were reversed on treatment with (+)-JQ1. Further, siRNA-mediated genetic inhibition of bromodomains in resistant cells phenocopied therapeutic inhibition by (+)-JQ1. These data unveil the chromatin “reader proteins”, as regulators of dysregulated oncogenic expressions in DAC-resistant cells. The present study provides novel insights into the epigenomic landscape of DACresistant colorectal cancer cells, and put forward, the alternative therapeutic regimen for DACresistant patients. Acknowledgement The study was supported by Internal Grant Agency of Palacky University (LF-2013/016), BIOMEDREG (CZ.l.05/2.1 .00/ 01.0030), and Ministry of Industry and Trade of the Czech Republic (FR-TI4/625).

Gene therapy approaches to increase an efficacy of epigenetic treatment in breast cancer

Verona Buocikova, Martina Poturnajova, Silvia Tyciakova, Katarina Gercakova, Svetlana Miklikova, Bozena Smolkova, Miroslava Matuskova, Lucia Kucerova Cancer Research Institute BMC SAS, Bratislava, Slovakia The last decade has seen a flourishing in the study of the properties of nanoparticles for medical applications. The next generation of nanopharmaceuticals combines a series of advances which enable the creation of multimodal/multifunctional nanodrugs that may contain antitumour agents and targeting ligands. The main goal of Euronanomed II INNOCENT project is to develop a novel nanoparticle-based therapeutical strategy to overcome low efficacy and frequent relapses in breast cancer (BC) treatment. Using the multifunctional nanostructure, we will investigate whether the coencapsulation of multiple therapeutic agents: doxorubicin (DOX) and decitabine (DAC), along with gene therapy, will reduce the proportion of CSCs and inhibit cancer cell growth. The rationale behind epigenetic therapies is the ability of DNA methyltransferase inhibitors to reverse hypermethylation-induced gene silencing of tumour suppressor and other cancer-related genes. The 2’-deoxy-5-azacytidine (DAC), one of the most frequently studied epigenetic agents, is a pro-drug which is metabolized by deoxycitidine kinase (DCK) to its active form. To improve the action of DAC we used the gene therapy approach. The gene coding for DCK was cloned into a DNA expression plasmid under a strong promoter.

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The aim of present work, performed under the scope of INNOCENT project, was to evaluate the efficiency of single (DAC) as well as combined treatment (DAC+DCK) on nonadherent BC cell lines MDA-MB-231 and T-47D. Mutual cooperation between epigenetic treatment and gene therapy will be assessed. The CellTitre-Glo Luminescent Cell Viability assay in 96-well format was used to determine the number of viable cells in culture. Changes in cell biology were evaluated by kinetic live cell imaging (IncuCyte ZOOM™ Kinetic Imaging system). Total RNA was isolated by NucleoSpin RNA kit and reverse transcribed with a RevertAid H minus First Strand cDNA synthesis kit. Gene expression of DCK was evaluated by real-time PCR. Relative expression of target gene DCK, normalized to HPRT, was calculated by the ΔΔCt method. DNA from cell cultures (1-2 x 106 cultured cells) was obtained using a FlexiGene DNA kit. Sodium bisulfite treatment of extracted DNA (1 μg) was performed using an established protocol of the EpiTect Bisulfite Kit. Pyrosequencing was used to evaluate the functional effect of single and combined therapy on DNA methylation. The methylation level of the LINE-1 retrotransposable elements was analyzed with the PyroMark Q24 CpG LINE-1 kit. Gene expression of DCK increased after the transfection. Parental cells were sensitive to the effect of DAC within the concentration of 0.1-10uM. DAC was accompanied by decrease of global DNA methylation. This work was financially supported by the 7FP platform ERA-NET programme EuroNanoMed II Innocent, Scientific Grant Agency (VEGA), contract no. 1/0271/17 and by the Slovak Research and Development Agency project no. APVV 16-0178.

Loss of histone macroH2A1 in hepatocellular carcinoma triggers paracrine-mediated cancer stemness and promotes adaptive immunity evasion

Oriana Lo Re1,2, Matthias Van Haele3, He Feng4, Emmanuel Tsochatzis5, Tommaso Mazza6, Manlio Vinciguerra1,5 International Clinical Research Center (FNUSA-ICRC), Brno, Czech Republic. Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic. Translational Cell & Tissue Research Unit, Department of Imaging & Pathology, Katholieke Universiteit Leuven, Leuven, Belgium. Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, University of California San Diego (UCSD), San Diego, USA. Institute for Liver and Digestive Health, University College London (UCL), London, United Kingdom. Fondazione IRCCS Casa Sollievo della Sofferenza, Laboratory of Bioinformatics, San Giovanni Rotondo , Italy

analyses to phenotype the CSC-like cells. Results and conclusions Tumors from MUP-uPA mice and human non-encapsulated HCC showed low macroH2A1 and high CD44 expression compared to encapsulated tumors. MacroH2A1 knock-down (KD) CSCs exhibited chemoresistance, which could be transferred to control, parental Huh7 HCC cells via macroH2A1 KD conditioned media (CM). This CM reprogrammed the inflammatory and immune transcriptional profiles of parental HCC cells, despite being depleted in several cytokines and chemokines. Finally, the CM activated circulating and liverresident regulatory T cells (Tregs), and reduced the infiltrating pool of hepatic total CD4(+)/CD25(-) T cells. Loss of macroH2A1 in HCC cells drives cancer stem-cell propagation in the tumor microenvironment, and evasion from adaptive immune surveillance by activating Tregs.

Search for new epigenetic predictive biomarkers of bevacizumab response in metastatic colorectal cancer patients

Introduction Cancer stem cells (CSCs) exhibit stem cell-like features and are responsible for tumor relapse and metastasis. In hepatocellular carcinoma (HCC), loss of the histone variant macroH2A1 induces CSC appearance and chemoresistance. How these CSCs interact with neighboring non-stem HCC cells and are cleared by the adaptive immune system is unclear.

Rastislav Slavkovský1, Lucie Kotková1, Karolína Bartáková1, Jana Vrbková1, Hana Študentová 2, Patrik Flodr3, Marie Bartoušková2, Marián Hajdúch1, Jiří Drábek1

Materials and methods We screened tumors from the MUP-uPA mouse model of hepatic tumorigenesis and patients with encapsulated or non-encapsulated HCC for macroH2A1 and CD44 CSC marker expression. We knocked down macroH2A1 in Huh-7 HCC cells, and performed metabolic, transcriptomic and paracrine

The Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University in Olomouc, Olomouc, Czech Republic. Department of Oncology, University Hospital Olomouc, Olomouc, Czech Republic. Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc and the University Hospital Olomouc, Olomouc, Czech Republic

Introduction

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Bevacizumab is an antibody binding VEGF (vascular endothelial growth factor) that contributes to the suppression of angiogenesis, on which the metastatic colorectal carcinoma (mCRC) is often dependent. We assume that methylation pattern changes in tumors may lead to a gene expression change that cause resistance to bevacizumab treatment. Our aim is to identify such methylation change as an epigenetic biomarker. Material and methods Since no reliable predictive biomarker of response to bevacizumab has been found so far, we applied genome-wide screening methods.

From the tumor DNA isolated from the group of responsive and group of non-responsive patients, we enriched the methylated DNA and sequenced it on Illumina HiSeq NGS platform. Differential methylation was determined based on the read counts in the area using MACS2, diffreps, bedtools, and R. Results and conclusion We identified dozens of candidate genome areas, including those in the vicinity of promoters. One of the targets is the gene BAGE5 (B melanoma antigen 5) that is poorly described in tumors, notably in colorectal cancer. 14 candidate genes that can promote angiogenesis

were detected as hypermethylated in responsive patients. We assume that demethylation of the identified angiogenesis-related genes could create alternative evasion angiogenesis pathway during anti-VEGF treatment and thus bring resistance to treatment in non-responsive patients. We plan to validate selected markers of resistance to bevacizumab using alternative methods and a larger group of patients. Grant support acknowledgement This work was supported by the grant from by Ministry of Health of the Czech Republic NV 16-32198A.

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Cancer biomarkers and personalized medicine II

Chairs: Vladimir Havlicek, Petr Dzubak středa / 21. listopadu 2018 / Wednesday / November 21th, 2018 / 9:00 - 10:15 Urinal siderophores as noninvasive and early biomarkers of infectious underlying diseases in critically ill patients

Radim Dobias1, Anton Skriba2, Tomas Pluhacek2, Milos Petrik3, Dominika Luptakova2, Oldrich Benada2, Vladimir Havlicek2 Laboratory of Clinical Mycology, Bacteriology and Mycology, Institute of Public Health in Ostrava, Ostrava, Czech Republic. Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic. Institute of Molecular and Translational Medicine, Palacky University, Olomouc, Czech Republic

Introduction Both early and specific diagnosis of infectious diseases caused by individual pathogens or characterization of mixed infections represent a diagnostic challenge for any microbiology laboratory. Even with good treatment available, a growing incidence of infectious diseases in critically ill patients persists in the Czech Republic.1-3 In patients with malignancies an array of possible nosocomial infections represents a permanent life threatening complication characterized by late implementation of effective antimicrobial therapy. In this talk, we will present an emerging non-invasive diagnostic standard based on microbial secondary metabolites of invading pathogens detected both in animal models as well as in a cohort of 20 human ICU or neutropenic patients with invasive aspergillosis and/or Pseudomonas infection.

siderophores or mycotoxins4 and was originally developed for Aspergillus5 and Pseudomonas6 infections in rat models. Using the first model of experimental aspergillosis in Lewis rats, the fungal siderophores ferricrocin (FC) and triacetylfusarinine C (TAFC) were identified as markers of Aspergillus fumigatus infection7 in high/low dose infection models. Analogously, bacterial pyoverdine E (PyE) was quantified in urine, serum and lung tissues in rats infected with Pseudomonas aeruginosa. Molecular biomarkers were analyzed by matrixassisted laser desorption ionization or electrospray ionization (ESI) using a 12T SolariX Fourier transform ion cyclotron resonance mass spectrometer (MS). Non-invasive diagnoses were performed with animal urine. MS imaging (MSI) experiments on tissues and liquid chromatography (LC)-ESI-MS analyses of rat sera represented the invasive armories. Similar experiments were performed with human urine and sera. In an analogous experiment with Pseudomonas aeruginosa, PyE was quantified in rat urine by LC-FTICR approach by application of PyE non-isobaric analogues. Separation of desferri and ferriforms of these siderophores was achieved on biphenyl LC column.

Results and Discussion Siderophores are low-molecularweight iron-chelating molecules secreted by a diverse set of bacteria and fungi and represent important microbial virulence factors associated with nutrient procurement for microbial growth.4 In high dose rat Aspergillus model, the initial FC signal reflecting the aspergillosis appeared as early as four hours post-infection.8 Results for seven biological replicates showed increasing exponential metabolite profiles in three-day Materials and Methods frame of the experiment. Among nine Our approach is based on mass biological replicates in the low dose spectrometry detection of microbial

model, three animals did not develop any infection up to experiment termination (day 10). One animal experienced an exponential increase of metabolites and died on the day six post-infection. In a different clinical experiment in a set of 20 human ICU patients with probable aspergillosis, siderophore-based approach was even more sensitive than standard galactomannan testing. In parallel, PyE was visualized by MALDI-MSI in infected lung and muscle tissues. Its dereplication was facilitated by our in-house and open software called CycloBranch.9 Lateral distribution of PyE correlated with bacterial bodies visualized by scanning electron microscopy in the multimodal approach. Human clinical data on invasive Pseudomonas infection will also be provided in this talk. Siderophore detection in urine (rat or human) by mass spectrometry represents an early, innovative and non-invasive tool for diagnosing infectious diseases. In theory, it can be applied for detection of any pathogen, either bacterium or fungus, producing siderophores or other low molecular weight virulence factors in the early stages of invasion. Further studies are needed to clarify the effect of antimicrobial prophylaxis to pathogen secondary metabolite profile in human host. We expect that the general application of microbial siderophores in human infectious diagnostics can soon represent the same clinical breakthrough like ribosomal protein typing 10 years ago. Acknowledgements Ministry of Education, Youth and Sports of the Czech Republic (LO1509). References 1. Kocmanová, I.; Lysková, P.; Chrenková, V.; Olišarová, P.; Dobiáš,

2018

R.; Janouškovcová, H.; Soukupová, H.; Mallátová, N.; Svobodová, L.; Hamal, P.; Skružná, M.; Bartoníková, N., Nosocomial candidemia in the Czech Republic in 2012–2015: results of a microbiological multicentre study. Epidemiol. Mikrobiol. Imunol. 2018, 67 (1), 3-10. 2. Dobias, R.; Jaworska, P.; Tomaskova, H.; Kanova, M.; Lyskova, P.; Vrba, Z.; Holub, C.; Svobodova, L.; Hamal, P.; Raska, M., Diagnostic value of serum galactomannan, (1,3)--d-glucan, and Aspergillus fumigatus-specific IgA and IgG assays for invasive pulmonary aspergillosis in non-neutropenic patients. Mycoses 2018, 61 (8), 576586. 3. Herkel, T.; Uvizl, R.; Doubravska, L.; Adamus, M.; Gabrhelik, T.; Htoutou Sedlakova, M.; Kolar, M.; Hanulik, V.; Pudova, V.; Langova, K.; Zazula, R.; Rezac, T.; Moravec, M.; Cermak, P.; Sevcik, P.; Stasek, J.; Malaska, J.; Sevcikova, A.; Hanslianova, M.; Turek, Z.; Cerny, V.; Paterova, P., Epidemiology of hospital-acquired pneumonia: Results of a Central European multicenter, prospective, observational study compared with data from the European region. Biomedical papers of the Medical Faculty of the University Palacky, Olomouc, Czechoslovakia 2016, 160 (3), 448-55. 4. Pluhacek, T.; Lemr, K.; Ghosh, D.; Milde, D.; Novak, J.; Havlicek, V., Characterization of microbial siderophores by mass spectrometry. Mass Spectrom Rev 2016, 35 (1), 3547. 5. Pluhacek, T.; Petrik, M.; Luptakova, D.; Benada, O.; Palyzova, A.; Lemr, K.; Havlicek, V., Aspergillus infection monitored by multimodal imaging in a rat model. Proteomics 2016, 16 (11-12), 178592. 6. Petrik, M.; Umlaufova, E.; Raclavsky, V.; Palyzova, A.; Havlicek, V.; Haas, H.; Novy, Z.; Dolezal, D.; Hajduch, M.; Decristoforo, C., Imaging of Pseudomonas aeruginosa infection with Ga-68 labelled pyoverdine for positron emission

tomography. Scientific Reports 2018, 8 (1), 15698. 7. Luptakova, D.; Pluhacek, T.; Petrik, M.; Novak, J.; Palyzova, A.; Sokolova, L.; Skriba, A.; Sediva, B.; Lemr, K.; Havlicek, V., Noninvasive and invasive diagnoses of aspergillosis in a rat model by mass spectrometry. Scientific reports 2017, 7 (1), 16523. 8. Skriba, A.; Pluhacek, T.; Palyzova, A.; Novy, Z.; Lemr, K.; Hajduch, M.; Petrik, M.; Havlicek, V., Early and Non-invasive Diagnosis of Aspergillosis Revealed by Infection Kinetics Monitored in a Rat Model. Frontiers in Microbiology 2018, 9 (2356). 9. Novák, J.; Škríba, A.; Zápal, J.; Kuzma, M.; Havlíček, V., CycloBranch: An open tool for fine isotope structures in conventional and product ion mass spectra. Journal of Mass Spectrometry 2018, 53 (11), 1097-1103.

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

Petr Dzubak1, Jana Vaclavkova1, Miroslav Hruska1, Jana Vrbkova1, Hanus Slavik1, Josef Srovnal1, Dusan Holub1, Frantisek Kopriva2, Tana Gvozdiakova2, Juraj Kultan3, Petr Jakubec3, Vítězlav Kolek3, Marian Hajduch1 Insititute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic. 2 Pediatric Department, Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech Republic. 3 Department of Respiratory Medicine, Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech Republic Exhaled breath condensate (EBC) can be a unique source of biomarkers 1

such as proteins, lipids, nucleic acids, and small molecules. These biomarkers can provide valuable information about lung-related or systemic diseases as well as. Finding non-invasive and cheap method for early detection of lung cancer would be highly beneficial. Proteomic analysis of EBC has the potential to detect early changes in the status of the respiratory system and has the potential to replace or complement invasive diagnostic methods and provide excellent lung cancer screening technique. Our study aims to identify lung cancer-specific and sensitive protein biomarkers in EBC, assess their diagnostic, prognostic and predictive value for early diagnostics and screening. Exhaled breath condensate proteins were processed by standard tryptic digestion. Samples were purified and diluted for high-resolution HPLC/MS-MS (LTQ Orbitrap Elite – ThermoScientific) analysis. The MS data were analyzed by Proteome Discoverer software and processed by machine learning statistical methods. We have collected and analyzed samples from 163 Non-Small Cell Lung Cancer patients and compared them with 150 healthy controls and samples from patients with other chronic respiratory diseases such as COPD. Using the methods mentioned above the proteins or peptides signatures were identified. Such candidate protein biomarkers will be further studied and validated. This work was supported by the Internal Grant of Palacky University (IGA_LF_2018_031); the Czech Ministry of Education, Youth and Sports (LO1304, LM2015064); Ministry of Health of the Czech Republic (16-32302A, 16-32318A) and Cancer Research Czech Republic.

2018

Genetic biomarkers of clinical response to bevacizumab in colorectal cancer patients

and related 694,000 deaths were reported worldwide. Colorectal cancer is one of the most common cancer diseases in the Czech Republic. Karolína Bartáková1, Although bevacizumab is widely used Jana Vrbková1, Barbora in the treatment of metastatic CRC, Blumová1, Helena Štefanová1, the validated predictive biomarker of the reaction to bevacizumab Veronika Holinková1, treatment is still unknown. In this Rastislav Slavkovský1, project, we try to identify molecular Hana Študentová2, Marie genetics predictive biomarker of Bartoušková2, Patrik Flodr3, bevacizumab. Jiří Drábek1 DNAs from tissues of 48 patients 1 with diagnosed metastatic colorectal Institute of Molecular and carcinoma, treated with bevacizumab Translational Medicine, Olomouc, were obtained from University Czech Republic. 2 Hospital, Olomouc and divided into University Hospital Olomouc, two groups according to progressionOlomouc, Czech Republic. 3 free survival. Copy number analysis Department of Clinical and was performed using the OncoScan Molecular Pathology, Palacký University Olomouc and University FFPE Assay Kit 1.0. The Database for Annotation, Visualization and Hospital Olomouc, Olomouc, Integrated Discovery (DAVID) v6.8 Czech Republic In 2012, nearly 1.4 million of newly was used for gene annotation. diagnosed cases of colorectal cancer DNA isolated from six patient samples

was of poor quality. Raw data (CELL files) of four samples obtained after array scanning did not pass quality control filter during analysis using OncoScan Console 1.3 software. The remaining results from patients designated as responders and non-responders were statistically analyzed and compared. In the both groups, there were identified several genes which can serve after validation as a potential predictive biomarkers of good response to bevacizumab treatment. Acknowledgements This study was supported by Ministry of Health of the Czech Republic, grant number NV16-32198, BBMRICZ CZ.02.1.01/0.0/0.0/16_013/0 001674, and European Regional Development Fund - Project ENOCH CZ.02.1.01/0.0/0.0/16_019/0000868.

2018

New methods in cancer research and diagnostics

Chairs: Marian Hajduch, Karel Koberna středa / 21. listopadu 2018 / Wednesday / November 21th, 2018 / 10:45 - 11:45 Claire: A system for detection of protein variants from tandem mass spectra

information from samples and gives new applications of shotgun proteomics.

Miroslav Hruska1,2, Lakshman Varanasi1, Dusan Holub1, Jana Vaclavkova1, Khushboo Agrawal1, Petr Vojta1, Jiri Voller1, Petr Dzubak1, Marian Hajduch1

Utilization of murine hair follicles for biomarker studies in ionizing radiation response model

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

Introduction Mass spectrometry is the leading technology for analyses of proteome. Standard analyses of protein expression are well-established, but detection of protein variants is challenging and routinely neglected. However, protein variants carry wealth of knowledge and are worth to examine. Materials and methods We have developed Claire, a computational system for detection of variant peptides from tandem mass spectra of proteome. Results and conclusions Claire was validated on data from cancer cell lines, clinical tumor samples and healthy individuals. Detection of peptide variants helped to resolve contamination and mislabeled cell lines within NCI60 proteomes, exomes and SNP chips. Clinical data of colorectal patients showed elevated protein-level tumor heterogeneity in advanced tumor stages. Protein variants also identified individuals against DNA dataset, showing direct use in forensics. In summary, detection of variant peptides extracts more

Hanus Slavik1, Pavlina Duskova1, Martin Mistrik1, Tomas Furst2, Josef Srovnal1, Marian Hajduch1 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic. Faculty of Science, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic

Introduction Hair follicles are relatively accessible as a biological material allowing repeatable and noninvasive sampling. The follicular cells within the follicles can serve as a source of biomarkers of various intrinsic and extrinsic influences. Utility of such a material is promising for example in murine experiments, where repeatable, long-term and harmless sampling is hardly achievable. As a proof of concept, the ionizing irradiation response in murine follicular cells was chosen as a suitable and common external factor for studying cellular senescence, apoptotic pathways, neoplastic transformation, tumor development and cancer therapy. Methods and material Hair follicles were collected from 30 healthy C57Bl/6 mice before X-ray irradiation and 30 min, 3 h, 6 h and 24 h after irradiation. Each group of 10 mice obtained one dose (2, 6 or 10 Gy). RNA was extracted from the

samples using miRNeasy mini kit (Qiagen) and two-tube RT-qPCR was performed for SESN1, p21, MDM2 and HPRT gene expression analysis. Immunofluorescence was done for γ-H2AX and quantitatively evaluated by special software routine. Results and conclusions Total RNA extracted from one pluck was usually less than 35 ng but it had good quality (RIN ≥ 7.5) even after the storage at room temperature. We determined p21 as the best marker for the uniformity and specific dynamic respond for each of the doses. The strongest responds were found after 30 min with 2 Gy (average fold change FCav = 3,24). In case of higher doses, longer remaining p21 overexpression was detected. For instance, FCav = 2,29 even after 24h at dose 10 Gy. Our data were corroborated by immunofluorescence for direct marker of ionizing radiation induced DNA damage γ-H2AX, where 1-3 hair follicles proved to be efficient for microscopic analyses. Strong γ-H2AX signal was detectable within 3h after irradiation followed by rapid decrease within the next 24h. Collected murine hair follicles proved to be a feasible alternative material for molecular analyses which in logistic aspects outreaches phlebotomies or skin biopsies because of repeatable, noninvasive and accurate sampling and non-laborious processing. Dedication This work was financially supported by grants IGA_LF_2018_005, TACR TE02000058 and VG20102014001.

Microsatellite instability testing – overview of methods and results

Alona Rehulkova, Monika Vidlarova, Josef Srovnal I nstitute of Molecular and

2018

Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic Background Microsatellite instability (MSI) is the spontaneous loss or amplification of nucleotides from repetitive DNA regions as a result of genetic hypermutability, which is a consequence of DNA mismatch repair (MMR) breakdown. This phenomenon has been studied in more detail in colorectal cancer and is used for diagnostic phenotyping. However, recent studies have discovered that MSI may be an effective marker for immuno-control therapy in patients with advanced stages of many malignancies because of malignancy associated neoantigens production. Extensive clinical trials have shown improved survival results for patients with MSI-positive tumors who received treatment with inhibitors of programmed cell death 1 ligand (PDL1). Materials and Methods Nowadays, there is a fairly small range of methods for studying the MSI, it is PCR, immunohistochemistry or sequencing. In our work, we use PCR to amplify target microsatellite loci (BAT25, BAT26, D2S123, D5S346, and D17S250, NR-21, NR-24, MONO-27) in germinal and somatic DNA. Further MSI analysis was performed using label-free electrophoresis on Agilent 2100 Bioanalyzer. Results and conclusions MSI is classically associated with colorectal cancer in Lynch syndrome, for which it has well-defined clinical consequences. Nevertheless, we evaluated 68 samples from patients with malignant neoplasms of various origins. We can conclude that this method is accurate and convenient to use, and it is less expensive than sequencing. New smart methods for MSI is coming and will be presented. Acknowledgment: This work was financially supported by Ministry of Health of the Czech Republic,

grant nr. 15-29021A, IGA UP LF 2018_005, NPU LO1304 and NCMG LM2015091.

Quantification of adherent cells using strong enhancers of fluorescence

Anna Ligasová, Karel Koberna I nstitute of Molecular and Translational Medicine, Palacký University Olomouc, Olomouc, Czech Republic Introduction Currently, several methods of cell quantification are available. They are usually based on the time-consuming direct calculation of cells using e.g. a haemocytometer or much easier determination of the relative cell concentrations by methods based on the measurement of the activity of cellular enzymes or on the determination of cellular components by means of specific markers. We have developed a fast, sensitive and cheap method of the quantification of adherent cells. It is based on the incubation of samples with DNA dyes Hoechst 33258, Hoechst 33342 or DAPI, and the elution of the dye from DNA in a solution containing a strong enhancer of the dye’s fluorescence. Materials and Methods Human HeLa, NCI-H2009 and IMR90 cells were cultured in culture flasks or on coverslips (12 mm in diameter) in a Petri dish or in 96well plates at 37°C in a humidified atmosphere containing 5% CO2. The cell quantification was performed by the developed method or, alternatively, by a tetrazolium test (MTT assay), by manual counting or software counting using CellProfiler software. Results and conclusions: The developed method of cell quantification is based on the measurement of DNA content. In contrast to the commonly used methods based on the same strategy, it does not rely on the enhancement of the signal by binding a dye to nucleic

acids. Instead, Hoechst or DAPI dyes bound to DNA are eluted using an elution buffer and enhancement of the signal is performed in the elution buffer. It results in a progressively higher enhancement of the signal compared to DNA-bound dye. In the case of Hoechst 33342, up to a 1,000fold increase of the fluorescence intensity was observed in the elution buffer. As there is no need for cell lysis, the cell cycle analysis using image cytometry can be performed before the elution step. The developed approach is sufficiently sensitive to reveal less than 75 HeLa cells or less than 100 diploid human cells per well in 96-well plates. Depending on the protocol arrangement, around 90 to 120 minutes is required for the procedure completion. The procedure was also successfully tested as a fast substitute of the widely used tetrazolium-based assay for the analysis of cell cytotoxicity. In contrast to the tetrazolium test, the procedure can be interrupted for several months if necessary. Moreover, the signal is stable for at least several weeks at room temperature. As the signal depends on the DNA content of cells, the method is also convenient for the quantification of cells exhibiting low metabolic activity including senescent cells. The approach provides linear data up to the fully confluent well plate. It corresponds to more than 70,000 HeLa cells per well. Funding This research was supported by the Ministry of Education, Youth and Sports, grant number LO1304, the Czech Health Research Council, grant number 15-31604A and the European Regional Development Fund - Project ENOCH, grant number CZ.02.1.01/0.0/0.0/16_019/0000868.

2018

Posterová sekce / Poster section 1

Identifying cellular targets of betulinic acid conjugates

Jiri Rehulka1, Milan Urban2, Miroslav Soural2, Marian Hajduch1, Petr Dzubak1 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic. Department of Organic Chemistry, Institute of Molecular and Translational Medicine, Faculty of Science, Palacky University in Olomouc, Olomouc, Czech Republic

Introduction Betulinic acid has been extensively studied for anticancer, antiinflammatory and antiretroviral activities. Given the complex nature of betulinic acid effects, several models of its action have been proposed. In the present study, we employed affinity purification to identify the drugprotein interactions responsible for the betulinic acid biological activity.

a specific inhibitor of cholesterol biosynthesis BM15.766. Betulinic acid treatment reduced the level of cholesterol and led to an accumulation of its precursor similarly as BM15.766. The study was supported by the Internal Grant of Palacky University (IGA_LF_2018_031); the Czech Ministry of Education, Youth and Sports (LO1304, LM2015063). 2

High-throughput screening system for identification of adenosine receptor ligands

Jana Kotulová, Petr Džubák, Marián Hajdúch Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic

Introduction Adenosine receptors (ARs) are involved in the regulation of a plethora of biological processes, including pathophysiological conditions such as cancer. Anti-cancer properties of ARs were confirmed both, in vitro and in vivo. Given the ubiquitous expression of these receptors and the lack of specificity of compounds targeting them, there is a current need for highly selective and potent agonists and antagonists of ARs. This project aims to develop a highthroughput screening assay for the identification of novel ligands of ARs.

Materials and methods The pull-down assay was based on immobilization of triterpenoid probes and enrichment of their binding partners from whole cell protein lysate. Quantitative mass spectrometry and SILAC metabolic labeling were used to remove unspecifically bound proteins. Mass spectrometric data were processed using MaxQuant Materials and methods and the proteins identified using For high-throughput screening (HTS) SwissProt human database. purposes, double transfected reporter Results and conclusions cell lines expressing mitochondrially Using affinity enrichment, we targeted apoaequorin and one of predicted interactions with several ARs (A1, A2A, A2B or A3) from G proteins involved in the biosynthetic protein-coupled receptor (GPCR) pathway of sterols and fatty acids. family were used in this study. The The inhibition of 7-dehydrocholesterol assay is based on the luminescent reductase by betulinic acid was detection of intracellular calcium flux validated on a cellular level using (AequoScreen platform) caused by

GPCR activation. The screening was performed in 384 well plate format using FLIPR tetra reader, as a part of our automated robotic platform. Obtained data were further analysed in Dotmatics software. Results and conclusions HTS is an essential part of early drug discovery nowadays. We have shown that aequorin-based functional assay is a suitable tool for the identification of new active molecules and could be fully automated resulting in high throughput. In particular, we evaluated each cell line overall performance and optimized conditions accordingly. Furthermore, we tested HTS assay robustness based on various parameters (% CV, Z´ value, Z factor, S/B ratio). Although several ARs ligands are currently in clinical trials for various conditions, including cancer, AR potential is yet to be unrevealed and fully understood. AR ligands are often reproached for their nonselectivity. Hence, emerging new avenues in GPCR pharmacology, such as biased agonism, allosteric modulation, targeting only a single transduction pathway could substantially contribute to the elimination of any potential off-targets. Using AequoScreen platform we can precisely determine which receptors are being activated. Also, we can then specify the mechanism of action in profiling studies. Our HTS method is indeed applicable for other G proteincoupled receptors screening utilizing the same technology and could present guidance on hit identification of other GPCRs in luminescencebased intracellular calcium flux assay step-by-step. The study was supported by the Internal Grant of Palacky University (IGA_LF_2018_031); the Czech Ministry of Education, Youth and Sports (LO1304, LM2015063).

2018

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

Dusan Holub1, Tomas Pika2, Pavla Flodrova3, Patrik Flodr3, Marian Hajduch1, Petr Dzubak1 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University and Faculty Hospital, Olomouc, Czech Republic. Department of Hemato-Oncology, University Hospital, 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 in various organs. Over time, the accumulating amyloid damages the tissue microenvironment and causes organ failure. To date, there are 36 known fibril proteins in human that can cause amyloidosis. Early diagnosis is critical for effective patient management. Immunohistochemistry (IHC) is the preferred method for routine amyloid subtyping. However, it is an antibody-based method with many unspecificities. Therefore we have introduced mass spectrometry-based proteomic analysis for subtyping of amyloid deposits in formalin-fixed paraffin-embedded tissues (FFPE) and abdominal subcutaneous fat aspirates (SFA) samples. Materials and methods So far, we have obtained 293 FFPE and 35 SFA samples for subtyping of amyloid deposits. In FFPE samples, Congo red positive-stained amyloid deposits were dissected using laser microdissection; the proteins were extracted from excised materials and digested using trypsin. In the SFA samples, the proteins were

solubilized and digested directly with trypsin. All samples were subsequently 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 subtyping of different kinds of amyloid proteins (e.g. Ig kappa, Ig lambda, Transthyretin, Fibrinogen, Serum amyloid A, Semenogelin). In all cases with systemic amyloidosis, we usually observed the presence of Serum amyloid P, Apolipoprotein A-IV, Apolipoprotein A-I, Apolipoprotein E, Clusterin, Vimentin and Vitronectin. 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 the Internal Grant of Palacky University (IGA_LF_2018_031); the Czech Ministry of Education, Youth and Sports (LO1304, LM2015091, LM2015064); Ministry of Health of the Czech Republic (16-31156A) and Cancer Research Czech Republic. 4

Circulating biomarkers for pancreatic cancer

Martina Jakoubková, Lakshman Varanasi, Miroslav Hruška, Dušan Holub, Kateřina Grusová, Dalibor Doležal, Jiří Večerka, Viswanath Das, Anna Janošťáková, Veronika Menšíková, Petr Džubák, Marián Hajdúch Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic

Based on the GLOBOCAN 2012, Pancreatic cancer (PC) ranks 7th in cancer-related mortality worldwide. The Czech Republic had the highest incidence of PC in the world in 2012. It is one of the most aggressive types of cancer. Patients are usually diagnosed at the advanced stage due to the asymptomatic course of the disease. Median survival from diagnosis is 4.6 months. Resection remains the most effective treatment of PC. Because only 1520% patients are diagnosed with resectable tumor, early diagnosis is essential to patient survival. Currently there is an effort to identify protein biomarkers in the plasma indicating presence of pathological condition and early detection of various types of disease. To date, the majority of FDA (Food and Drug Administration) approved proteomic tumor markers are glycoproteins. This is because changes in protein glycosylation profiles are typical of tumor cells. Most of the cellular glycosylated proteins are located in the cytoplasmic membrane or secreted from the cell. These proteins are therefore likely to find their way from a neoplasm into the bloodstream. The development of sensitive LC-MS techniques can enable the detection of such proteins, present in human plasma in trace quantities. Candidate biomarkers were identified in serum of mice grafted with human cell lines or patient tumor cells representing various types of cancers of the gastrointestinal tract (GI). Murine serum was collected after the tumor reached a volume of 1 cm3. N-glycopeptides were isolated by solid phase extraction and analyzed by an Orbitrap Fusion (Thermo) mass spectrometer and Ultimate 3000 RSLC Nano liquid chromatography (Thermo) Acquired data were analyzed by software developed in-house and human peptides in murine sera identified. The presence of target peptides in human plasma was verified by a targeted proteomic approach using a QTrap 5500 (AB Sciex) mass spectrometer.

2018

476 human peptides originated in human tumor tissue were detected in murine serum. 128 candidate biomarkers suitable for detection by target proteomic approach were supplemented with 147 peptides from the literature. 64 of these were verified in human plasma from PC patients. The diagnostic and prognostic value of these 64 peptides will be determined in a large cohort of plasma samples from Pancreatic cancer patients and healthy volunteers. 5

Contradictory taxifolin effects on Zeb2 signalling pathway in Hep G2 cells

Zdeněk Dostál1,2, Josef Srovnal2, Kateřina Štaffová2, Lenka Radová3, Jitka Ulrichová1,2, Martin Modrianský1,2 Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic. Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic. Central European Institute of Technology, Masaryk University, Brno, Czech Republic

Introduction Quercetin and taxifolin are abundant in the human diet. Hence, they could be responsible for different health effects due to e.g. regulation of signalling pathways. Our study was focused on modulation of miRNAs, regulators of gene expression, by these compounds and their consequences in experimental models.

chloroform extraction. miRNA expression profiles were assessed via Affymetrix GeneChip™ miRNA 3.0 Array. Electrophoresis and western blot were performed to assess levels of selected proteins. Results and conclusions The results of miRNA arrays revealed deregulation of several miRNAs, which modulate Zeb2 expression, by quercetin (1 µM) and taxifolin (1 µM). Indeed, Zeb2 protein expression was upregulated in a dose dependent manner by taxifolin (1 - 10 µM) but not quercetin (1 - 10 µM). The protein plays an important role in epithelial to mesenchymal transition (EMT), the process related to tumor progression. It results in higher invasiveness and aggressiveness of cancer due to changes in expression of proteins such as E-cadherin or vimentin. Vimentin, marker of EMT, was evaluated by western blot technique which revealed its downregulation. Higher Zeb2 expression should be accompanied by higher vimentin protein levels but our results showed the opposite outcome. This suggests that there is another pathway/impact of taxifolin that reverses the effects of Zeb2 upregulation. Determination of the pathway is a further aim of the study. We can conclude that even though taxifolin can cause upregulation of Zeb2, its overall effect seems to be still positive from the perspective of human health. This work was supported by grants LO1304 and IGA_LF_2018_012. 6

In-house validation of tumor BRCA1/2 testing

Jana Stránská1,2, Rastislav Slavkovský1, Barbora Koblihová1, Barbora Blumová1, Lucie Kotková1,2, Karolína Bartáková1,2, Materials and methods Helena Štefanová1, Gabriela Primary cultures of human 3 4 hepatocytes and Hep G2 cell line Kořínková , Mária Janíková , 1 1 were used as experimental models. Marián Hajdúch , Jiří Drábek Total RNA was isolated by phenol-

Institute of Molecular and

Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic University Hospital Olomouc, Department of Neurology, Olomouc, Czech Republic. University Hospital Olomouc, Department of Clinical and Molecular Pathology, Olomouc, Czech Republic. University Hospital Olomouc, Department of Medical Genetics, Olomouc, Czech Republic

Introduction BReast CAncer type 1/2 susceptibility genes (BRCA) are tumour suppressors, involved in maintenance of genome stability. Testing of somatic BRCA mutations is required prior to the therapy with PARP inhibitors of relapsed platinum-sensitive BRCA mutated ovarian cancer. In this project, we performed analytical verification of the BRCA testing method. Materials and methods We tested BRCA mutations by targeted massively parallel sequencing and/or interpreted results in 3 groups: i) 15 samples with known result from BRCA variant classification run 2 (one mutation per case, result interpretation only, GenQA and EMQN external quality control scheme); ii) 7 samples with known BRCA mutation status (4 cell line samples, 2 Horizon Discovery reference standards, 1 patient sample); iii) 10 FFPE samples of ovarian cancer with unknown BRCA status. Sequencing libraries were prepared with NEBNext Direct® BRCA1/BRCA2 Panel kit, sequenced with MiSeq platform and analyzed with MiSeq Reporter or unique molecular indices (UMI) enabled open source based pipeline containing bwa, fgbio, picard, and vardict tools. Variants were annotated and filtered by VarAFT and Illumina VariantStudio 3.0 software. Results and conclusions In the group i), 15 results from GenQA/EMQN quality scheme were

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classified from level 1 (benign) to level 5 (pathogenic) using 8 publicly available webtools (VarSome, dbSNP, ClinVar, ARUP, BRCA Share, Human Splicing Finder, HGMD, and Cancer Genome Interpreter). Seven variants were clinically interpreted in accordance with a canonical result, 8 variants were mistakenly classified 1 level more pathogenic. Twelve out of 15 recommendations for PARP therapy were correct. In the group ii), all known variants were confirmed by sequencing in several technical replicates. In the group iii), all samples were successfully sequenced and analyzed. We partially suppressed artefacts (large amount of mutations in range of allele frequency 1-5%, position artefacts, etc.) by using modified UMI enabled data processing pipeline. We have identified several pathogenic mutations in 10 tested samples. From wet lab point of view, NEBNextDirect® BRCA1/BRCA2 Panel is a suitable kit for testing of BRCA1/2 variants, fully compatible with our Illumina MiSeq. However, dry lab clinical interpretation of variants is a task where meticulous filtering and a standardized algorithm of classification tools summing up is needed. By now, we are not satisfied with a large number of clinical false positives. Acknowledgements Project was run with a grant support of the European Regional Development Fund - Project ENOCH (No. CZ.02.1.01/0 .0/0.0/16_019/0000868), BBMRI-CZ CZ. 02.1.01/0.0/0.0/16_013/0001674, NPU LO1304, and NCMG LM2015091. 7

Analysis of molecular mechanisms involved in MSCmediated chemoresistance in breast cancer

Jana Plava1,2, Svetlana Miklikova1, Marina Cihova1, Miroslava Matuskova1, Tamara Kukolj3, Drenka

Trivanovic3, Aleksandra Jaukovic3, Diana Bugarski3, Lucia Kucerova1 Laboratory of Molecular Oncology, Cancer Research Institute BMC SAS, , Bratislava, Slovakia. Faculty of Medicine, Comenius University, Bratislava, Slovakia. Institute for Medical Research, University of Belgrade, Belgrade, Serbia

Introduction Chemoresistance is one of the major factors that limit the effectiveness of breast cancer treatment. In the breast, tumour is surrounded mostly by adipose tissue, which is a rich source of mesenchymal stromal cells (MSCs). In our study, we use adipose tissue-derived MSCs isolated from healthy women and breast cancer patients to examine the role of MSCs and breast cancer cells (BCCs) interaction in the modulation of response to chemotherapy. Materials and methods MSCs were isolated from adipose tissue using collagenase digestion followed by their adherence on plastic surface. Effects of chemotherapy (paclitaxel and doxorubicin) on MSCs were determined using Proteome Profiler Cytokine Array Kit (RD Systems) and ELISA (Fine Test). To examine direct effect of isolated MSCs on BCCs, IncuCyte ZoomTM Kinetic Imaging System (Essen BioScience, UK) was used to analyse direct co-culture of these cells. Gap-junctional intercellular communication (GJIC) was evaluated by flow cytometry detection of dye transfer. Tumour cells were stained with DiI and MSCs were labelled with Calcein AM. In addition, we analysed the role of MSCs on BCCs chemoresistance in vivo. Results and conclusions MSCs were shown to be naturally chemoresistant. We have shown that even the morphology of MSCs is unaffected after the chemotherapy pretreatment. On the other hand, we have observed substantial alterations in the MSCs secretion

profile after exposure to doxorubicin and paclitaxel. Pretreated MSCs showed increased secretion of IL6, IL-8 and SerpinE1. Conditioned media from doxorubicin-pretreated MSCs altered the chemosensitivity of BCCs. Even more pronounced effect was observed if they were directly co-cultured. We have detected functional GJIC between MSCs and BCCs by flow cytometric analysis, potentially contributing to altered drug resistance in cancer cells. In the presence of preexposed MSCs the response of the BCCs to the drugs differed substantially and their viability was affected. These effects may involve mechanisms responsible for alterations in apoptosis induction, cell cycle regulation, senescence and cell phenotype. To clarify these molecular mechanisms, the factors secreted by MSCs will be further analysed and the data will be presented. The experimental work cited in this study was supported by the VEGA grants No. 2/0087/15 and 1/0271/17 and by the Slovak Research and Development Agency under the contracts No. APVV-15-0697, APVV16-0010 and APVV-16-0178. 8

Antileishmanial activity of anticancer drugs

Ermin Schadich, Petr Džubák, Marián Hajdúch Institute of Molecular and Transnational Medicine, Palacky University Olomouc, Olomouc, Czech Republic

Introduction Kinetoplastid parasite species of genus Leishmania are associated with leishmaniases, important zoonotic diseases. Our study was focused to determine whether the compounds with known anticancer and other pharmacological properties have in vitro activity against kinetoplastid parasite Leishmania major. Materials and methods We analyzed 1280 compounds

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from Sigma Life Science’s library of pharmacologically active compounds (LOPAC1280) for activity against L. major using Alamar blue assay. Results and conclusions Six compounds had activity against L. major at concentrations relevant to therapeutic measures (IC50 < 10 μM). Three of these compounds have also anti-cancer properties including nitidine chloride, ellipticine and JSK, while three other compounds had other pharmacological activities. The potential of these compounds for future therapeutic measures is highlighted. 9

Efficacy of various genotoxic factors in activation of cGAS/ STING pathway

Tereza Buchtová, Ondřej Šanovec, Martin Mistrík Institute of Molecular and Transnational Medicine, Palacky University Olomouc, Olomouc,

Czech Republic Cyclic GMP-AMP synthase (cGAS) is a cytoplasmic DNA sensor. Once bind to dsDNA it triggers (via STING factor) the expression of inflammatory genes leading to attraction of innate immune system towards the affected cell. Double stranded DNA is usually compartmentalized within nucleus and mitochondria and its presence in the cytosol introduces abnormal situation which may result from endoparasitic attack (bacterial or viral) or excessive genomic instability. The latter case is linked to micronuclei which are DNA particles formed during aberrant mitosis as a result of chromosomal missegregation and/or chromosomal breakage. Thus genomic instability attracts the innate immune system similarly to parasitic infections. Cells particularly prone for genomic instability are cancer cells explaining why tumours often attracts immune T-lymphocytes. At the same time the cancer cells have the ability to block cytotoxic effect of T-cells by activation of so called immune checkpoint.

Novel anticancer therapeutics termed immune checkpoint inhibitors restore the cytotoxic function of T-cells towards the cancer cells and show very promising anticancer effect. Following this rationale, we propose that cancer therapy aimed on increasing the genomic instability of cancer cells combined with the immune checkpoint inhibitors treatment could significantly potentiate such treatment output. In this study, we worked with selected cancer and primary cell lines: U2OS (osteosarcoma), RPE (retinoblastoma), BJ (skin) and mouse fibroblasts. Cells were exposed to various genotoxic factors inducing micronuclei. As a readout for the cGAS/STING pathway activation we measured positivity of the micronuclei for the cGAS signal using quantitative immunofluorescence microscopy. We conclude that fractionated gamma irradiation has the highest ability to form cGAS positive micronuclei. Among other factors, inhibitor of DNA polymerases aphidicolin scores as a potent inducer.

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