ST. ANNA KINDERKREBSFORSCHUNG FORSCHUNGSBERICHT 2015

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1 ST. ANNA KINDERKREBSFORSCHUNG FORSCHUNGSBERICHT 2015

2 EINLEITUNG 7 Im Einsatz für das Leben 8 Einleitung des wissenschaftlichen Direktors 10 Danke an unsere großartige Spenderfamilie! 14 DATEN & FAKTEN 19 Kompetitive Drittmittel 20 Zuweisung der Geldmittel 20 Finanzierung 20 Personelle Zusammensetzung 21 Nationen 21 Forschungsnetzwerke 22 Klinische Forschung 24 Überlebensraten von Kindern und Jugendlichen mit Krebs 25 SCIENCE REPORTS 29 Introduction 30 Novel insights into the mutational landscape and signaling pathways in pediatric B-cell precursor acute lymphoblastic leukemia 32 Understanding altered gene regulation in Ewing sarcoma pathogenesis from studying chromatin and transcriptional dynamics 40 Genomic as well as transcriptomic profiling of disseminated tumor cells 44 Persistence, reactivation and intracellular interactions of human adenoviruses 46 Implications for anti-cancer immune therapy 52 Progress in clinical grade production of virus-specific T-cells 56 CAREER 61 Working at CCRI 63 Scientific Staff 64 FINANZBERICHT 69 Richtlinien zur Spendenverwendung 70 Mittelherkunft 74 Mittelverwendung 75 ANHANG 77 Wissenschaftlicher Beirat 78 International fremdgeförderte Projekte 82 National fremdgeförderte Projekte 84 Danksagung 88 Abgeschlossene Diplomarbeiten und Dissertationen Publikationen Impressum 104

3 RESEARCH IS TO TRANSFORM HYPOTHESES INTO RESULTS AND RESULTS INTO BETTER TREATMENTS OR DIAGNOSTICS. IN OTHER WORDS, RESEARCH TURNS IDEAS INTO HOPE AND HOPE INTO REALITY. Sara Colomer-Lahiguera

4 EINLEITUNG

5 Einleitung IM EINSATZ FÜR DAS LEBEN Diesen Jahresbericht vorzulegen, macht mir besondere Freude war eine Periode, in der vieles ge lungen ist. Der Erfolg wissenschaftlichen Arbei tens ist oft nicht planbar. Trotz inten siver Planungen und Überlegungen, trotz sorgfältiger Analysen stellt sich in der Forschung nicht immer sofort der erhoffte Erfolg ein. Ver gangenes Jahr war das anders. Die St. Anna Kinderkrebsforschung kann auf zahlreiche erfolgreich ver laufene Forschungsprojekte verweisen, deren Resultate durch Veröffentlichungen in renommierten Fach journalen große Beachtung gefunden haben. Bewerbungen für mehrere nationale und inter nationale fremdgeförderte Projekte wurden ebenfalls erfolgreich abgeschlossen und werden über mehrere Jahre hinweg relevante Forschungsprojekte ermöglichen. Die große Genugtuung darüber erklärt sich durch das Thema, dem wir uns seit vielen Jahren widmen. Ein sehr emotionales Terrain, da das Leben und Überleben derer betroffen ist, die uns besonders am Herzen liegen: Kinder und Jugendliche. Unser ganzer Einsatz gilt der pädiatrischen Onkologie mit dem Ziel, unseren jungen Patientinnen und Patienten und ihren Eltern Hoffnung zu geben und die Heilungsrate auf 100 Prozent zu steigern. Dank dem hohen wissenschaftlichen Können meiner Kolleginnen und Kollegen und der fantastischen Einsatzbereitschaft aller Mitarbeiterinnen und Mitarbeiter sind wir seit langer Zeit auf einem guten Weg begannen fünf Forschungsgruppen wissenschaftliche Erkenntnisse auf die klinischen Bedürfnisse abzustimmen, Fragen aus der klinischen Situation ins Labor zu bringen, Ursachen und Mechanismen von Kinderkrebs zu erforschen und neue Therapieansätze zu erkunden. Heute arbeiten in der St. Anna Kinderkrebsforschung zwölf inter disziplinäre Teams auf den Gebieten der Tumor genomik, Molekularbiologie, Immunologie, Zellbiologie, Infektiologie und klinische Forschung. Wegweisende Erkenntnisse aus unserem Institut und die hohe Kompetenz der Institutsmitarbeiterinnen und -mitarbeiter sowie der Ärztinnen und Ärzte des St. Anna Kinderspitals haben dazu geführt, dass die St. Anna Kinderkrebsforschung inter national führende Studienzentrale in einigen Bereichen der pädiatrischen Onkologie wurde. Ein Jahresbericht legt immer Rechenschaft über die Leistungen ab. Dass die Bilanz 2015 positiv ausfällt, freut mich auch für alle, die unserem Bemühen mit persönlicher, tätiger und finanzieller Anteilnahme folgen und es tatkräftig unterstützen: die Mitglieder des Unterstützungskomitees, die Vorstandsmitglieder und alle Förderinnen und Förderer, die uns schon über so viele Jahre treu be - gleiten und den Erfolg erst möglich ge macht haben. Dafür sind wir Ihnen dankbar. Univ.-Prof. Dr. Wolfgang Holter Institutsleiter 8 9

6 Einleitung EINLEITUNG DES WISSENSCHAFTLICHEN DIREKTORS Wir leben in aufregenden Zeiten. Der technologische Fortschritt hat alle gesellschaftlichen Bereiche erfasst, und moderne Informationstechnologien ermöglichen jedem, ob Fachmann oder Laie, den fast uneingeschränkten Zugang zu einer kaum überschaubaren Menge an wissenschaftlichen Daten. Diese neue Dimension der Wissensverbreitung bringt zweifelsohne neue Chancen mit sich, birgt aber auch Risiken. Wissenschaftliches und medizinisches Wissen ist nicht mehr nur ärztliches Privileg. Patientinnen bzw. Patienten und deren Verwandte sind oft auf dem neuesten Stand des Wissens, was die Diagnose und Therapie ihrer Leiden betrifft. Vor allem wenn es um ernste, potentiell lebensbedrohliche Erkrankungen wie Krebs geht, erheben sie den berechtigten Anspruch auf die fortschrittlichsten, aber meist auch sehr teuren, diagnostischen und therapeutischen Verfahren, die sich nicht selten noch in einer sehr frühen, experimentellen Phase der klinischen Entwicklung befinden. Der Zugang zu solchen neuen diagnostischen Leistungen und Behandlungen wird jedoch von steigenden regulatorischen Auflagen einerseits und der abnehmenden Leistungsfähigkeit unserer Gesundheitssysteme andererseits begrenzt. Diese Hürden könnten überwunden werden, wenn es gelingt, Diagnoseverfahren und Therapien treffsicherer und effizienter zu gestalten und damit Neben- und Spätfolgen sowie Anzahl und Dauer von Krankenhaus aufenthalten zu reduzieren. Um dieses ehrgeizige Ziel zu erreichen, konzentrieren sich weltweite Anstrengungen darauf, das Genom erkrankter Gewebe von Patientinnen bzw. Patienten auf individuelle, pharmakologisch verwundbare Veränderungen und deren diagnostische Biomarker zu durchforsten. Dieser Jahresbericht legt dar, dass auch das CCRI erste Schritte in diese Richtung für ausgewählte Krebserkrankungen gesetzt hat. Auf diesem Weg erlaubt der Zugang zu genomischen Datensätzen in offenen Datenbanken, die durch geringe Fallzahlen bedingten Limitierungen bei der Erforschung seltener Erkrankungen wie Krebs bei Kindern zu überwinden. Die neuen Technologien ermöglichen, rasch große Informationsmengen auf mehreren genomischen und epigenomischen Ebenen aus kleinsten Mengen von Chromatin, DNA und RNA selbst aus Körperflüssigkeiten zu gewinnen. Diese müssen dann computergestützt prozessiert und mit großen Datenbanken verglichen werden, um die gewonnene Information interpretieren und vali dieren zu können. Damit rückt die moderne bio medizinische Forschung immer mehr vom Feuchtlabor ins Trockenlabor (den Computer). Diese neuen Methoden identifizierten zum Beispiel ein unerwartetes Ausmaß an Intra-Tumorheterogenität der individuellen genetischen Veränderungen auf Grund der Tumor-/Leukämie-Evolution, was Auswirkungen auf die Tumor Progression und damit auf die Therapie von behandlungsresistenten Rückfällen und disseminierten Erkrankungen mit sich bringt. Doch computergestützte Biologie geht weit über die bloße Datenanalyse und -integration hinaus. Sie setzt mathematische Methoden ein, um auf der Basis von experimentell gewonnenen, quantitativen Daten biologische Prozesse zu modellieren und das Verhalten komplexer Systeme wie genregulatorischer Netzwerke und Signalwege vorherzusagen. Als Teil eines europäischen Systembiologie-Projektes gelang es uns, experimentelle Beschränkungen zu überwinden und mittels mathematischer Modellierungen fehlende physische Evidenz zu ersetzen. Damit leisteten wir Pionierarbeit in der Beschreibung eines neuen Onkogengetriebenen genregulatorischen Moduls in einem pädiatrischen Sarkom. Diese und andere Beispiele wissenschaftlicher Forschung, welche im Jahr 2015 veröffentlicht wurden und die einerseits ein besseres Verständnis von Krebserkrankungen bei Kindern und andererseits die Entwicklung und Optimierung neuer Behandlungskonzepte im Bereich zellulärer Therapien zum Inhalt hatten, präsentiert dieser Jahresbericht in knapper Form. Univ.-Prof. Dr. Heinrich Kovar 10 11

7 DIE HOFFNUNG UND DAS ZUTRAUEN SO VIELER MENSCHEN SIND FÜR MICH EIN STARKER AUFTRAG IN MEINER FORSCHUNGSARBEIT MIT HERZ UND HIRN DAS BESTE ZU GEBEN UND ÜBER MICH HINAUSZUWACHSEN. Manfred Lehner

8 Einleitung DANKE AN UNSERE GROSSARTIGE SPENDERFAMILIE! Das Faszinierende an der Arbeit im Spendenbüro der St. Anna Kinderkrebsforschung sind die Menschen, ihre Hilfsbereitschaft, ihre wunderbaren Ideen und ihr großartiges Spendenengagement. Die Vielfalt der Einfälle, um Spenden zu sammeln, ist so bunt wie ein Regenbogen, der gleichsam wie eine Brücke unsere Spenderfamilie direkt mit dem Forschungserfolg verbindet. Dank der konsequenten Forschung können heute bereits vier von fünf Kindern und Jugendlichen, die vor 40 Jahren noch als unheilbar galten, gerettet werden. Finanziert wird die St. Anna Kinderkrebsforschung, die seit 2002 das Österreichische Spendengütesiegel führt und zum steuerlich begünstigten Empfängerkreis gehört, seit Anfang an hauptsächlich durch Spenden. Der Dank gilt unserer großen Spenderfamilie Sie alle schenken krebskranken Kindern eine Chance auf eine gesunde Zukunft. BUNTE EINFÄLLE SPANNENDE AKTIONEN GROSSZÜGIGE SPENDEN Das Spektrum der Spendenmöglichkeiten ist vielfältig und die Unterstützung damit grenzenlos wie einige Beispiele zeigen: Eis für St. Anna! Seit mehr als zehn Jahren engagieren sich die Mitglieder der Berufsgruppe der Eissalons für die St. Anna Kinderkrebsforschung. Für Silvio Molin-Pradel, dem Obmann und Branchensprecher, ist es immer wieder eine besondere Freude zu Beginn der Eisschlecksaison, gemeinsam mit seinen Kollegen einen namhaften Spendenscheck zu überreichen. Blockbuster Das Leben ist ein Film feierte 2015 Benefizpremiere! Rund 120 Filmbegeisterte arbei teten gemeinsam an einem Low-Budget- Kino spielfilm. Prominente Gesichter wie Manuel Rubey, Reinhard Nowak, Katharina Straßer, Thomas Stipsits, Maddalena Hirschal u.v.m. wollten alle ihren Beitrag für Österreichs erstes Fund raising-filmprojekt zugunsten der St. Anna Kinderkrebsforschung leisten. Regisseur und Autor, Autodidakt Vlado Priborsky, zeigt in seinem Film den eigenen, steinigen Lebensweg, auch auf durchaus humorvolle Weise. Die Unterstützung der Bastelrunde Hirtenberg, unter der Leitung von Marianne Brandtner, hat bereits eine langjährige Tradition. Das ganze Jahr über wird für die St. Anna Kinderkrebsforschung gebastelt, gemalt, gestrickt, genäht usw. Die Erlöse des jährlichen Oster- und Adventmarkts sind jedes Mal beeindruckend. Spannendes Wettpaddeln mit Kultcharakter! Bereits zum 12. Mal war im Sommer 2015 wieder viel Spaß für Jung und Alt beim Benefiz-Sautrogrennen in Scharndorf garantiert. Viele Sautrogkapitäne, Fans und Freunde finden sich alljährlich rund um den Scharndorfer Löschteich ein, um einen spannenden und lustigen Tag zu erleben. Der gesamte Erlös wird gespendet und von Jahr zu Jahr auch noch unglaublich gesteigert. Karl Lamac, Spendenkaiser von Favoriten, unterstützt von Brigitte Aschenbrenner, begleitet das Forschungsinstitut seit Anbeginn und ermöglichte wieder eine beachtliche Spendensumme. LANGE NACHT DER KINDERKREBSFORSCHUNG Forschung auf reizvolle Weise entdecken und Zukunft erleben! Dazu lädt die St. Anna Kinderkrebsforschung alljährlich ein. Im Rahmen der Langen Nacht der Kinderkrebsforschung wird ein spielerisches Kennenlernen des Laboralltages möglich und Wissenschaft zum Anfassen greifbar. Interaktiv, spannend und leicht verständlich wird Wissen über die Bausteine des Lebens und die molekularen Grundlagen der Krebsentstehung vermittelt. Vorführungen in den Labors und spannende wissenschaftliche Vorträge verdeutlichen, was unsere Forscherinnen und Forscher zur Verbesserung der Behandlungs qualität und Erhöhung der Heilungschancen an Krebs erkrankter Kinder und Jugendlicher leisten öffneten wir bereits zum 11. Mal unsere Pforten. Sogar die Science Busters waren zu Gast, leider einer der letzten Auftritte in der Urbesetzung. Für beste Unterhaltung sorgte ebenfalls ein treuer Spender: Comedian Fredi Jirkal. Spannende Fragen, gestellt von Wien Heute Chefredakteur Dr. Paul Tesarek, erwarteten kritische und emotionale Antworten. Bei einer Podiumsdiskussion zum Thema Leben mit Kinderkrebs erläuterten u.a. Vertreter unserer Spenderfamilie ihre Motivation, uns zu unterstützen. ST. ANNA KINDERKREBSFORSCHUNG- UNTERSTÜTZUNGSKOMITEE Schon seit einiger Zeit begleitet die St. Anna Kinderkrebsforschung ein prominent besetztes Ehrenkomitee. Die Mentoren, Personen aus Politik, Wirtschaft und Kultur zeichnen sich durch eine besondere Position aus und sind ehrenamtlich tätig. Durch ihre Präsenz in der Öffentlichkeit können sie viel zugunsten der St. Anna Kinderkrebsforschung bewegen. Seit 2015 unterstützt Eva Angyan, Gattin des Intendanten der Gesellschaft der Musikfreunde in Wien Dr. Thomas Angyan, als Komiteepräsidentin das Forschungsinstitut. Im Oktober 2015 gab es, initiiert von Frau Angyan und großartig unterstützt und gesponsert durch einige Mentorinnen und Mentoren, ein exklusives Benefizkonzert-Highlight zu hören. Mit viel Applaus wurde Elisabeth Leonskaja im Barocksaal des Alten Rathauses gefeiert. Die St. Anna Kinderkrebsforschung konnte sich über einen hervorragenden Spendenbetrag freuen. Die nächsten Benefizkonzerte sind schon in Planung. Mag. Andrea Prantl Leiterin Spendenbüro 14 15

9 Einleitung UNSERE KUSCHELTIERE: KLEINE LEBENSRETTER, DIE FREUDE SCHENKEN Bereits seit 23 Jahren sind die Kuscheltiere der St. Anna Kinderkrebsforschung bei Jung und Alt sehr beliebt und als Sammelobjekte auch heiß begehrt! Jedes Jahr im Oktober heißt die Maskottchenfamilie einen Neuzugang willkommen war es: Kathi, das kecke Kälbchen. Die kleinen Lebensretter freuen sich auf ein nettes Zuhause und jede Menge Spielgefährten. Für 12, schenkt man nicht nur krebskranken Kindern eine Chance, sondern auch sich selber oder seinen Lieben Freude. Bank Austria, Erste Bank und einige Sparkassen sowie die Raiffeisen Zentralbank unterstützen die St. Anna Kinderkrebsforschung beim Vertrieb. Darüber hinaus ist das jeweils aktuelle Maskottchen an den Rezeptionen der renommierten Wiener Innenstadt-Hotels Imperial und Bristol erhältlich. Informieren Sie sich auf unserer Internetseite welche Stofftiere wir neben Kathi anbieten und wie sie am einfachsten zu bestellen sind. JEDE SPENDE HILFT! Es gibt viele Möglichkeiten zu spenden: Onlinespende Barspende Spende mit Zahlschein Spende mit Einziehungs- oder Dauerauftrag Benefizveranstaltungen Sammelaktionen Spenden statt Geburtstags- oder Weihnachtsgeschenken Erwerb unserer Kuscheltiere Kranzablösespenden Legat / Testament etc. Ob mit einer persönlichen Spende oder mit einer gemeinsamen Sammelaktion, ob per Zahlschein, Kreditkarte oder Onlinespende jede finanzielle Zuwendung ermöglicht die Fortsetzung unserer Forschungsarbeit im Kampf gegen Kinderkrebs. Firmen spenden immer häufiger den für Kunden- Weihnachtsgeschenke vorgesehenen Betrag. Statt um Geschenke wird bei Geburtstagen, Jubiläen oder anderen Feiern gerne um Spenden gebeten. Auch der Verzicht auf Blumen und Kränze bei Begräbnissen, um stattdessen zu spenden, hilft krebskranken Kindern. Ein Testament oder ein Legat zugunsten der St. Anna Kinderkrebsforschung schenkt ebenfalls langfristig eine gesunde Zukunft. WOLLEN SIE INFORMATIONEN, UNTERLAGEN ODER HABEN SIE FRAGEN? Das Spendenteam und ich freuen uns auf Ihre Kontaktaufnahme: +43 (0) spende@kinderkrebsforschung.at Bank Austria IBAN: AT BIC: BKAUATWW Erste Bank IBAN: AT BIC: GIBAATWW Das Team der St. Anna Kinderkrebsforschung ist dankbar für die langjährige spendenfreudige Unterstützung und ich persönlich für die Begegnung mit so vielen großzügigen, warm herzigen und hilfs bereiten Menschen. Herzlichen Dank! Mag. Andrea Prantl Leiterin Spendenbüro 16 17

10 DATEN & FAKTEN

11 Daten & Fakten KOMPETITIVE DRITTMITTEL (Forschungsförderungen) im Jahr 2015 USA FINNLAND NIEDERLANDE 56,18 % Europäische Union POLEN RUSSLAND 0,83 % Sonstige Drittmittelgeber 4,18 % Österreichische Nationalbank DEUTSCHLAND SLOWAKEI 17,23 % Österreichische Forschungsförderungsgesellschaft (FFG) 21,59 % Fonds zur Förderung der wissenschaftlichen Forschung (FWF) ÖSTERREICH FRANKREICH SPANIEN NATIONEN MitarbeiterInnen der St. Anna Kinderkrebsforschung im Jahr 2015 UNGARN ZUWEISUNG DER GELDMITTEL im Jahr 2015 FINANZIERUNG der Infrastruktur des Forschungsinstitutes und der Forschungsprojekte im Jahr 2015 PORTUGAL ITALIEN NEUSEELAND KROATIEN SERBIEN PERSONELLE ZUSAMMENSETZUNG der MitarbeiterInnen im Jahr 2015 GRIECHENLAND IRAN 92,03 % Forschung 5,02 % Spendenwerbung 2,91 % Verwaltungsaufwand 0,02 % Sonstiger Aufwand 78,86 % Spenden 21,14 % Kompetitive Drittmittel (Forschungsförderung) 69 % Frauen 31 % Männer 20 21

12 Daten & Fakten FORSCHUNGSNETZWERKE National und international N N IE IE AN TIN B L N A EN GE ALI R AR ST AU G I E N L EN BE ILI EN AS R RI B GA L BU ILE CH A IN RK CH MA D NE Ä L AN D CH S T U DE ND NL A H FIN EIC NKR ND L FRA N A CHE E I NIEN GR I TA N R B S S GRO ND IRL A D ISL AN L E ISRA N IE IT A L N A JA P K R O AT IE N LE T TL AN D LUX EM BUR G NEUSEE L AND NIEDERL ANDE NOR WEG EN ÖS TE RR EIC H PO LE N P O R TU G A L RUM ÄN IE N RUSSL AND SCHW EDEN SCHW EIZ SERB IEN SIN GAP UR SLO WA K EI SLO WE NIE S PA N NIE N TSC HE CH TÜ IEN RK EI UK RA IN UN E GA RN UR UG UA US Y A WE IS SR US SL AN D 22 23

13 Daten & Fakten KLINISCHE FORSCHUNG ÜBERLEBENSRATEN VON KINDERN UND JUGENDLICHEN MIT KREBS Die St. Anna Kinderkrebsforschung fungiert im Bereich der Langerhans-Zell-Histiozytose, dem Neuroblastom und der Stammzellentransplantation, als internationales Koordinierungszentrum für die hier abgebildeten Studien. Das Studienmanagement wurde von unserem Koordinierungszentrum, der Abteilung S 2 IRP, durchgeführt. PATIENTENAUFKOMMEN IN S 2 IRP ALS KOORDINIERUNGSZENTRUM FÜR KLINISCHE STUDIEN GesamtpatientInnenanzahl ANSTIEG DER 2-JAHRES-ÜBERLEBENSRATEN VON KREBSKRANKEN KINDERN UND JUGENDLICHEN 100 % 80 % 60 % 40 % 20 % Morbus Hodgkin Maligne Keimzellentumoren Wilms-Tumor Akute lymphoblastische Leukämie Non-Hodgkin Lymphom Neuroblastom und Ganglioneuroblastom Osteosarkom Rhabdomyosarkom Hirntumoren Ewing-Sarkom Akute myeloische Leukämie Quelle: GPOH Internationale PatientInnen PatientInnen in Österreich ANSTIEG DER 5-JAHRES-ÜBERLEBENSRATEN VON KREBSKRANKEN KINDERN UND JUGENDLICHEN 100 % 80 % 60 % 40 % 20 % Morbus Hodgkin Wilms-Tumor Akute lymphoblastische Leukämie Non-Hodgkin Lymphom Rhabdomyosarkom Hirntumoren Neuroblastom und Ganglioneuroblastom Ewing-Sarkom & Osteosarkom Akute myeloische Leukämie Modifiziert nach Pui C.-H. et al. (2011) Nat. Rev. Clin. Oncol. 8,

14 MY SCIENTIFIC MOTIVATION IS TO FIND ANSWERS. MY PERSONAL MOTIVATION IS TO CONTRIBUTE ANSWERS TO IMPROVE THE SURVIVAL CHANCES FOR CHILDREN WITH CANCER! Inge Ambros

15 SCIENCE REPORTS

16 Science Reports INTRODUCTION We are living in very exciting times. Technological progress affects all areas of society, and modern information technology enables everyone, experts and laymen alike, to access vast amounts of scientific data. This new way of disseminating knowledge entails a number of un precedented opportunities and challenges. Scientific and medical knowledge is no longer a privilege of researchers and doctors only. Patients and their relatives are frequently very well informed about the state-of-the-art in diagnosis and treatment of their maladies. So when it comes to serious, potentially life-threatening diseases such as cancer, these patients often request the most advanced, but frequently very expensive, diagnostic tests and treatments, which, however, may still be at a very early, sometimes still experimental stage of clinical development. On the other hand, access to novel diagnostic services and therapies is often limited by the increasing regulatory burden and decreasing capacities of our health care system. These limitations may be overcome if diagnosis and treatment can be tailored to be more precise and efficient, thus not only reducing side and late effects, but also number and periods of hospitalization. To achieve this ambitious goal, worldwide efforts in biomedical research currently concentrate on the mining of patients genetic information for individual pharmacologically targetable disease vulnerabilities and their diagnostic biomarkers. As this annual report demonstrates, the CCRI has, for certain pediatric cancers, made first steps into this new direction. In this quest, deposition of data sets in publicly accessible data bases allows to partly overcome limitations associated with the study of rare diseases such as childhood cancers. The novel technologies enable rapid generation of large amounts of multilayered genomic and epi genomic information from small quantities of chromatin, DNA, or RNA retrieved from different tissues and even body fluids. These need to be computationally processed and compared to large data bases in order to facilitate interpretation and validation of results. Thus, more and more, the scientific work in biomedical research moves from the wet to the dry lab (the computer). These new approaches identified, for example, an unexpected intra-tumor/leukemia heterogeneity as a result of disease evolution, with far-reaching consequences for tumor progression and treatment of therapy-refractory, relapsed and disseminated disease. But computational biology goes far beyond bioinformatical data analysis and integration. It applies mathematics to model biological processes based on experimentally generated quantitative results to predict the behavior of complex systems such as gene regulatory networks and signaling pathways. In the context of a European systems biology project, we managed to overcome experimental limitations by replacing the missing physical evidence with mathematical modelling, pioneering the description of a novel oncogene driven gene regulatory module in a pediatric sarcoma. These and other exciting examples of CCRI research published in 2015 and focusing on a better understanding of pediatric cancers but also on development and optimization of novel treatment approaches based on cellular therapies are briefly presented in this annual report. Univ.-Prof. Dr. Heinrich Kovar 30 31

17 Science Reports NOVEL INSIGHTS INTO THE MUTATIONAL LANDSCAPE AND SIGNALING PATHWAYS IN PEDIATRIC B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood and adolescence. ALL comprises a number of disease entities characterized by chromosomal rearrangements, aneuploidy, structural variants, and sequence mutations that generally perturb cellular pathways including lymphoid development, tumor suppression, cell cycle control, epigenetic regulation as well as kinase and cytokine receptor signaling. Although current treatment protocols ensure that up to 90% of the patients remain in long-term remission, certain subgroups have a high incidence of relapse despite intensive therapy or are a priori refractory to current therapeutic approaches. Equally disconcerting are the risks associated with current curative therapies, whose toxicities and potential late side effects are a major concern. The goal of the basic and translational research of the CCRI's leukemia research groups is therefore to explore the landscape of genetic lesions and the biology of specific ALL subtypes and to pinpoint novel treatment targets and strategies that may eventually improve the outcome and quality of life of the affected individuals. KRAS AND CREBBP MUTATIONS: A RELAPSE-LINKED MALICIOUS LIAISON High hyperdiploidy denotes the largest B-cell precursor ALL (BCP-ALL) subgroup in children and adolescents. Affected patients usually present with low-risk features and respond well to treatment. The relative proportion of relapses (up to 15%) is small but nevertheless constitutes the largest genetically homogeneous fraction in the BCP-ALL cohort. High hyperdiploid (HD) ALL has a modal number of nonrandomly gained chromosomes and is probably caused by the maldistribution of chromosomes during a single abnormal nondisjunction event already in utero (Panzer-Grümayer et al. 2002). To promote disease development, subsequent additional hits are required. However, none of the identified gene deletions or mutations does qualify as an indisputable indicator of an elevated relapse risk. We therefore investigated the mutational landscape in a genome-wide, unbiased and quantitative manner and compared the clonal composition of diagnostic and relapse samples by next generation sequencing in a large cohort of relapsing and norelapsing HD ALL cases. RTK/Ras signaling activating mutations are highly heterogeneous Our analyses revealed that receptor tyrosine kinase (RTK)/Ras pathway mutations predominate in these leukemias, affecting 75% of cases at initial diagnosis and virtually all cases at relapse. While these mutations were primarily subclonal or even oligoclonal at initial diagnosis, they were generally present in the dominant clone at relapse. Furthermore, a substantial proportion of initial mutations was lost at relapse and replaced by other mutations in the same or another gene in the pathway (Figure 1A). CREBBP mutations are generally conserved and accumulate at relapse The second most frequently affected gene was CREBBP, which was present in nearly a fifth of all cases at initial diagnosis, increased to one third of all cases at relapse, and was usually conserved (Figure 1B). This suggests that genetic alterations in this gene are involved in the relapse development and may mediate drug resistance, as suggested previously (Inthal et al. 2012). Figure 1 A AF (%) Rel AF (%) Dx AF (%) Rel AF (%) Dx B PJ FB RS KD20493 RD17412 MB2 792 NH KD Y B 399 PJ13414 A Y KA D HS MJ ZA16211 ST13892 C A G C KE Figure 1. Frequencies and patterns of RTK/Ras pathway (A) and CREBBP (B) mutations. Inter- and intragenic heterogeneity of mutations at diagnosis (Dx; top) and relapse (Rel; bottom) in cases with matched diagnosis and relapse samples. UPN of cases indicated at the x axis; Allelic frequency (AF) of mutations at the y axis; Colors mark specific mutations; black filled circles in bars indicate the conservation of the respective mutation at relapse G K 32 33

18 Science Reports Co-occurrence of CREBBP and KRAS mutations at relapse Our most prominent finding was the coexistence of CREBBP and KRAS mutations in HD ALL relapse clones, an insight that derived from the quantitative overlap of these two mutations. It originally became evident in our whole exome sequencing (WES) data and was subsequently confirmed in the analysis of the targeted sequencing results of the entire cohort, including additional 65 relapse samples (P<0.001). Although KRAS and NRAS mutations at relapse were similarly frequent (23% and 19%, respectively), only the KRAS ones concurred with the CREBBP mutations. This observation implies that these two mutations exert an interdependent function and cooperate in a RAS isoform-specific and synergistic manner under the selective pressure of the applied chemotherapy. The relapse-associated co-occurrence of CREBBP and KRAS mutations also allowed us to determine from which clones they originally derived and, thereby, to reconstruct the respective clonal development in nine suitable cases. The three ensuing patterns of relapse evolution are depicted in Figure 2. They clearly highlight the specific selective advantage of CREBBP- and KRAS-double mutant clones, which eventually culminates in the emergence of a predominant clone at relapse. The unique alliance of these two mutations is further corroborated by the fact that, initially, seven of these nine cases also had clones with other RTK/Ras signaling gene mutations, none of which, however, persisted. CREBBP and KRAS mutations are associated with unfavorable clinical features at relapse Finally, we also checked the clinical consequences of mutations, in particular how they might influence time to relapse, the molecular response to relapse treatment and outcome. Overall, RTK/Ras pathway and CREBBP mutant relapses showed only a slight prevalence of occurring early while KRAS mutant relapses did in fact always develop early; in line, relapses with concurrent CREBBP and KRAS mutations appeared earlier than those in the remaining cases with only one or no mutation in either gene (P = 0.012). PTPN11-mutant relapses, on the other hand, occurred late, and NRAS- and FLT3-mutated ones did not differ from cases without such mutations. Minimal residual disease standard risk (SR) and high-risk (HR) relapses were similarly distributed in RTK/Ras pathway mutated and wild type cases. By contrast, both KRAS- and CREBBPmutated cases fell mainly into the high-risk group, while PTPN11-mutated ones were found in the standard risk group. HD ALL is generally perceived as a disease with a low recurrence risk and an excellent prognosis. Owing to the fact that relapses arise from highly selected cell populations that survived first-line treatment, the ensuing relapse clones have a more restricted repertoire of mutations. Although clonal heterogeneity is a typical characteristic of all malignancies and of childhood ALL in particular, the exceptional position of RTK/Ras pathway mutations in this group is a remarkable and noteworthy, albeit not entirely unprecedented, phenomenon. Despite the previously suggested apparently mutually exclusive occurrence of RTK/Ras pathway mutations, targeted sequencing uncovered an extraordinary inter- and intragenic heterogeneity in the vast majority of cases, particularly at diagnosis. DIAGNOSIS Dx-specific CREBBP KRAS CREBBP RELAPSE KRAS Figure 2. Model for the evolution of relapses with concurring CREBBP and KRAS mutations. Shown are the three most frequently observed scenarios represen ting the diverse composition of initial leukemia populations defined by mutations in CREBBP, KRAS or a combination thereof and their presence at relapse. Note that only mutations in KRAS, but not in the other RTK/Ras pathway genes, are depicted. The clone size was estimated from the means of the allelic frequencies of respective mutations present at initial diagnosis (left) and relapse (right) in corresponding cases. The first and most frequent scenario indicates the initial coexistence of various clones with two major ones carrying either a CREBBP or KRAS mutation in addition to a minor clone that harbors distinct mutations in both genes. At relapse, this double-mutated population constitutes the entire leukemic clone (upper part). The two other scenarios highlight the initial presence of a KRAS- and CREBBP-mutated major or minor clone, in which a minute population of cells also carries a CREBBP (middle) or KRAS (bottom) mutation, respectively. Again, only clones harboring both mutations prevail at relapse. Color code of mutations at the bottom of the graph

19 Science Reports Moreover, two distinct Ras pathway mutations and double-mutated RAS genes were found, albeit at a low frequency. Taken together, these findings rank the activation of the Ras pathway as the currently most important disease-relevant functional change in HD ALL. It comes as a surprise, therefore, that, in contrast to the overall less common CREBBP mutations, which are definitely involved in relapse development, RTK/Ras pathway mutations should have only little or no impact at all on the clinical behavior of the disease. At diagnosis, RTK/Ras pathway mutations are as frequent in cases that eventually experience a relapse as they are in those that stay in long-term remission and they also share a similar distribution. CREBBP mutations are, in contrast, far less common in non-relapsing cases. Moreover, at relapse, RTK/ Ras pathway mutations remain as frequent as at diagnosis, whereas the frequency of CREBBP mutations increases. Of high interest in this context is that of all RTK/Ras pathway mutations, only those affecting KRAS pair with CREBBP mutations. The high concordance rate of KRAS and CREBBP mutations at relapse suggests a first circumstantial, but nevertheless important, clue that these two alterations are functionally linked. The simultaneous deregulation of both proteins may have a synergistic effect, render the affected clone more resistant to therapy and thereby endow it with a considerable proliferative and selective advantage. This notion is further supported by our observation in four cases that already carried two distinct CREBBP-mutated clones at diagnosis. As all these alterations affect the HAT domain and presumably attenuate the acetylation of proteins to a similar extent, it is safe to assume that they are functionally equivalent. Nevertheless, only those that also acquired KRAS mutations eventually evolved into predominant relapse clones. CREBBP acetylates both histone and non-histone proteins, such as H3K18, p53 and BCL6, and missense mutations in the HAT domain generally attenuate or destroy the acetylating and coactivator function of the protein. As CREBBP is a cofactor for more than 400 transcription factors and most likely acetylates even many more proteins than have yet been identified, it is virtually impossible to predict which targets may be affected and be of specific relevance in HD ALL. The functional impact and consequences of different types of RAS mutations in this specific genetic and cellular context are similarly complex and unresolved. In transformed cells, oncogenic RAS operates mainly via the RAF-MEK-ERK and PI3K-AKT-mTOR pathways, which prominently control many functions that are essential for cancer. Whether and to which extent the deregulation of these particular pathways also contributes to leukemia development in HD ALL is, so far, not known. Whereas the balanced frequency of KRAS and NRAS mutations at diagnosis implies that they could exert similar functions, only KRAS seems to profit from attenuated CREBBP and thus cooccurs at relapse. This strongly biased selection provides another good example of the fact that individual RAS isoforms can indeed have distinctive and unique properties, which however might only become relevant and obvious under very specific circumstances. It is likely that CREBBP modulates or alters not only the function of RAS effectors but also of specific RAS proteins as well. In conclusion, KRAS- and CREBBP-double-mutated relapse cases are among those with the highest risk and are therefore prime candidates for novel therapeutic approaches. They could, for instance, combine histone deacetylase inhibitors with Ras pathway inhibitors or drugs that inhibit the connected PI3K-AKT-mTOR pathway, as proposed previously for hypodiploid and HD leukemias with activated Ras signaling. Finally, as somatic CREBBP mutations are also present in a small number of non-relapsing HD ALL cases, they generally fail to be useful predictive or prognostic markers. Malinowska-Ozdowy K, Frech C, Schonegger A, Eckert C, Cazzaniga G, Stanulla M, zur Stadt U, Mecklenbrauker A, Schuster M, Kneidinger D, von Stackelberg A, Locatelli F, Schrappe M, Horstmann MA, Attarbaschi A, Bock C, Mann G, Haas OA, Panzer-Grumayer R (2015) KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia. Leukemia 29: PAX5-JAK2 A JANUS-FACED TRANSCRIPTION FACTOR In B-cell precursor ALL PAX5, a transcription factor essential for B-cell commitment and maintenance, is frequently affected by genetic alterations such as deletions, point mutations, internal amplifications, and chromosomal rearrangements. The latter occur in 2-3% of the cases and result in the expression fusion genes encoding potentially oncogenic chimeric proteins. PAX5 fusion partners comprise a remarkably diverse group of proteins, which play distinct roles in transcription, chromatin remodeling, cell structuring and different signaling pathways (Nebral et al. 2007, 2009; Denk et al. 2014). Generally it is assumed that PAX5 chimeric proteins antagonize wild-type PAX5 activity; however, there is evidence that the partner protein modulates the function of distinct PAX5 fusions (Fortschegger et al. 2014). In this regard, the PAX5-JAK2 chimera is of particular interest, because it fuses the N-terminal DNA-binding domain of PAX5 to the kinase domain of JAK2, which as already shown for other JAK2 fusion proteins may result in the constitutive activation of the JAK-STAT signaling pathway. On the other hand, the PAX5-JAK2 fusion protein may interfere with the B-cell-specific transcription program regulated by wild-type PAX5. Since constitutive activation of the JAK-STAT pathway plays a critical role in malignant transformation and disease maintenance, a number of small molecule inhibitors is currently under clinical development. Therefore, we have assessed the biochemical properties and functional consequences of PAX5-JAK2 and determined whether PAX5-JAK2-positive leukemia might be targeted with JAK2 kinase inhibitors. Our data show that PAX5-JAK2 shares features with both PAX5 and JAK2 chimeric proteins but has also highly distinctive characteristics. While like all PAX5 fusion proteins (Fortschegger et al. 2014) PAX5-JAK2 is a nuclear protein and via the retained PAX5 DNA-binding paired domain is capable of binding to PAX5 target sequences, gene expression profiling of primary leukemia cells showed that the expression signature of PAX5-JAK2-positive leukemia is clearly distinct from leukemias harboring other PAX5 chimeras, such as PAX5-ETV6 and PAX5-C20orf112. Hence, as previously hypothesized (Fortschegger et al. 2014), we have now for the first time demonstrated that on a genome-wide level leukemias with different PAX5 fusions have distinctive gene expression profiles, strongly indicating their differential impact on gene regulation

20 Science Reports Figure 4 A Furthermore, in line with the anticipated dual function of PAX5-JAK2, gene expression profiling showed that typical JAK-STAT as well as PAX5 targets are among the deregulated genes in PAX5-JAK2-positive compared to PAX5 wild-type leukemia. Together, our data illustrate that PAX5- JAK2 deregulates at least a subset of genes of the PAX5 downstream transcriptional network and simultaneously activates JAK-STAT-signaling-specific genes, and thus, by interfering with these two important pathways, may promote leukemogenesis. Expression of PAX5-JAK2 also resulted in a high degree of tyrosine phosphorylation of PAX5-JAK2 itself (Figure 3) and the constitutive phosphorylation and activation of downstream signal transducers and activators of transcription (STAT) proteins. Furthermore, the chimeric protein also enabled the cytokine-independent growth of murine Ba/F3 cells. Hence, PAX5-JAK2 is a constitutively active kinase with transforming potential and triggers JAK-STAT signaling. Intriguingly, however, although main signaling functions of wild-type JAK2 are linked to its cytoplasmatic localization, PAX5-JAK2 localizes exclusively to the nucleus (Figure 3). All other JAK2 fusion proteins described to date are also cytoplasmatic proteins and their majority possesses an oligomerization domain provided by the fusion partner, which facilitates trans-autophosphorylation of the kinase and the constitutive activation of the JAK-STAT signaling cascade; PAX5-JAK2, however, functions as a nuclear, monomeric protein. Even though the Figure 3. PAX5-JAK2 is a nuclear protein. Immunofluorescence of HeLa cells transfected with HA-tagged PAX5-JAK2 using an HA (green) and py1007/8-jak2 (red) antibody and DAPI (blue) for DNA counterstaining, illustrating the exclusive nuclear localization of phosphorylated PAX5-JAK2. Left panel: DAPI counterstain of nuclei; middle panels: green signals, PAX5-JAK2; red signals phosphorylated PAX5-JAK2; right panel: merged images. underlying mechanism of how PAX5-JAK2 becomes phosphorylated remains to be determined, PAX5- JAK2 apparently activates the JAK-STAT pathway in a manner that is independent from classical cytoplasmatic interactions. Consequently, it is conceivable that PAX5-JAK2 phosphorylates STATs in the nucleus, where they are usually present also in an unphosphorylated state. Together, PAX5-JAK2 functions as nuclear catalytically active kinase that autophosphorylates and in turn phosphorylates and activates downstream STATs in a non-canonical mode. Despite the nuclear localization of PAX5-JAK2 and its non-canonical mode of activating JAK-STAT signaling, JAK2 inhibitors efficiently block constitutive hyperactivation of the kinase, resulting in dephosphorylation of its downstream target STAT5, B transcriptional down-regulation of JAK-STAT target genes, and rapid cell death (Figure 4). Hence, our study provides further important insights into the function of a highly intriguing kinase-activating chimeric protein and substantiates the notion that PAX5-JAK2 may represent a druggable target for therapeutic intervention. Figure 4. JAK2 inhibitors block PAX5-JAK2 kinase activity. (A) Cytokine-independent murine Ba/F3 cells expressing PAX5-JAK2 were treated with different concentrations of the indicated inhibitors and intracellular STAT5 phosphorylation (pstat5) was measured by flow cytometry. While treatment of the cells with the Src- family tyrosine kinase inhibitor dasatinib had no effect, application of the JAK2 inhibitors ruxolitinib (left panel) and fedratinib (right panel) resulted in a reduction of pstat5 levels. (B) After 24 and 48 hours of inhibitor treatment, staining for apoptotic and dead cells with Annexin-V-allophycocyanin and DAPI, respectively, showed a rapid induction of cell death. Untreated and DMSO, no drug, negative controls. Schinnerl D, Fortschegger K, Kauer M, Marchante JR, Kofler R, Den Boer ML, Strehl S (2015) The role of the Janus-faced transcription factor PAX5-JAK2 in acute lymphoblastic leukemia. Blood 125: Anderl S, Konig M, Attarbaschi A, Strehl S (2015) PAX5-KIAA1549L: a novel fusion gene in a case of pediatric B-cell precursor acute lymphoblastic leukemia. Mol Cytogenet 8:

21 Science Reports UNDERSTANDING ALTERED GENE REGULATION IN EWING SARCOMA PATHOGENESIS FROM STUDYING CHROMATIN AND TRANSCRIPTIONAL DYNAMICS EWING SARCOMA AN EPIGENETIC DISEASE Ewing sarcoma is a highly aggressive cancer affecting bone and soft tissue in children and adolescents. The majority of tumors are characterized by only few mutations with one recurrent genetic aberration, a gene rearrangement between EWS and an ETS transcription factor gene, most frequently FLI1. The EWS-FLI1 fusion protein binds to chromatin at canonical ETS recognition motifs and at GGAA-microsatellites, and changes the expression of hundreds of genes. There is ample in vitro and in vivo experimental evidence that disrupting EWS-FLI1 function blocks Ewing sarcoma growth, hence, EWS-FLI1 has been proposed the perfect therapeutic target in Ewing sarcoma. However, transcription factors are notoriously difficult to target pharmacologically. Thus, novel alternative therapeutic options may arise from understanding the mechanisms behind EWS-FLI1 mediated widespread transcriptional dysregulation. Within the cell nucleus, the DNA is tightly packed with histones and non-histone proteins into chromatin. Chemical modifications including methylation and acetylation, the so called epigenetic code, influence chromatin compaction and thus accessibility of the DNA for interaction with the transcriptional machinery. For a transcription factor to get access to its target genes, it has either to bind to sites of already open chromatin or attract proteins that open-up chromatin de novo. In order to better understand the modes and mechanisms by which EWS-FLI1 causes transcriptional perturbation, we studied dynamic changes in seven histone marks, open chromatin, DNA methylation, and gene expression before and after experimental modulation of EWS-FLI1 by inducible RNA interference in a Ewing sarcoma model cell line. We showed that EWS-FLI1 induces widespread epigenetic rewiring in proximal and distal enhancers and a genome-wide increase in histone acetylation. Figure 2: Comprehensive epigenetic and RNA expression profiling of a Ewing sarcoma cell line in EWS-FLI1 high and low states establishes the first Ewing sarcoma reference epigenome and reveals distinct modes of promoter regulation and widespread enhancer reprogramming. Figure 1: Like in the ancient African game, in which stones are repeatedly redistributed in the holes of a wooden board, the fusion oncogene EWS-FLI1 redistributes epigenetic marks thus leading to epigenetic reprogramming of Ewing sarcoma. Of all measured marks, histone H3K27 acetylation patterns were the most highly dynamic and strongly associated with EWS-FLI1 binding, suggesting that the oncogenic fusion protein imposes a characteristic H3K27 acetylation program on Ewing sarcoma cells. We discovered that EWS-FLI1 binding hyper-activates promoters of mainly proliferation driving genes and reprogrammes the genome-wide enhancer landscape of Ewing sarcoma. Comparison of this first Ewing sarcoma reference epigenome to those of 72 different human cell types revealed a striking specificity of the EWS-FLI1 imposed histone H3K27 acetylation signature for Ewing sarcoma. To facilitate data access and reuse by other researchers, we developed an EWS-FLI1 epigenome website for online exploration and download ( Our dataset can be used to visualize and investigate epigenetic changes in EWS-FLI1 dependent cells one gene at a time, or to study patterns of epigenetic and transcriptional deregulation on a more global level, for example annotating known and novel non-coding transcripts and alternative promoters that are EWS-FLI1 dependent. Finally, clustering of epigenetic promoter signatures defined classes of EWS-FLI1 regulated genes that responded differently to low-dose treatment with histone deacetylase inhibitors

22 Science Reports BAYESIAN MODEL SELECTION EWS -FLI1 RAD 51 EWS -FLI1 x RAD 51 Taken together, this study provided first insights into Ewing sarcoma as a disease of the epigenome and characterized chromatin changes associated with addiction to the EWS-FLI1 oncogene. These data provide a rationale for the potential use of epigenetic drugs in Ewing sarcoma treatment (Tomazou et al., 2015). DECIPHERING MECHANISMS OF TRANSCRIPTIONAL DYSREGULATION BY SYSTEMS BIOLOGY Experimental modulation of EWS-FLI1 in Ewing sarcoma cells results in decreased expression of genes associated with cellular proliferation. Consistent with many of these genes being regulated by the E2F transcription factor family, we previously identified co-localization of EWS-FLI1 with E2F3 on gene regulatory elements of the vast majority of E2F3 regulated genes. Interestingly, upon knockdown of EWS-FLI1 and chromatin immuno- precipitation at different time points we observed a gradual replacement of transcription activating E2F3 by gene repressive E2F4 on EWS-FLI1/E2F co-regulated genes. We therefore hypothesized that, in Ewing sarcoma, EWS-FLI1 hijacks and activates proliferation driving genes by displacing E2F4 and recruiting E2F3 to its target genes. Importantly, we found this E2F exchange mechanism to also directly control expression of E2F3 itself. Tightly time-resolved RNA expression analysis following inducible EWS-FLI1 knockdown revealed immediate response of E2F3 regulation while other E2F3 target genes lagged behind, suggesting a feed-forward loop in EWS-FLI1 driven regulation of E2F3 target genes. This hypothesis was strengthened by mutation analysis of transcription factor binding motifs in the promoters of a series of EWS-FLI1/E2F3 controlled genes. Destruction of the ETS (EWS-FLI1) binding motif prohibited recruitment of E2F3 and drastically reduced gene expression as monitored by reporter gene assays. These data suggested a requirement of EWS-FLI1 binding for either the displacement of E2F4 or the recruitment of E2F3, which might be best explained by a direct interaction between EWS-FLI1 and at least one of these two opposing players. However, we failed to obtain experimental proof for a physical interaction, presumably due to its likely transitory nature. We therefore decided to adopt a mathematical modelling approach to replace this missing piece of experimental evidence. Four alternative mechanistic models were formulated and optimized as systems of differential equations predicting dynamic gene expression behaviors of EWS-FLI1, E2F3 and its target genes over time. Figure 3: Mathematical modeling of four alternative models explaining cooperative regulation of EWS-FLI1 target genes (i.e. RAD51) by EWS-FLI1 and E2F3 and Bayesian model selection identify model B (complex formation between the two transcription factors) as the most probable explanation of the experimentally observed gene expression dynamics. 1.46% A 87.83% EWS -FLI % E2F3 E2F3 Based on highly time- resolved gene expression measurements and Bayesian model selection, the model proposing complex formation between EWS-FLI1 and E2F3 (model B in the figure) turned out to be the most probable (87.83%) explanation for the observed experimental data. Thus, this study in Ewing sarcoma gives an example of the value of systems biology and mathematical modelling to resolve difficult to experimentally address pathogenic mechanisms (Schwentner et al., 2015). Tomazou EM, Sheffield NC, Schmidl C, Schuster M, Schonegger A, Datlinger P, Kubicek S, Bock C, Kovar H (2015) Epigenome mapping reveals distinct modes of gene regulation and widespread enhancer reprogramming by the oncogenic fusion protein EWS-FLI1. Cell Rep 10: RAD 51 C 0.12% EWS -FLI1 E2F3 E2F3 RAD 51 B D Schwentner R, Papamarkou T, Kauer MO, Stathopoulos V, Yang F, Bilke S, Meltzer PS, Girolami M, Kovar H (2015) EWS-FLI1 employs an E2F switch to drive target gene expression. Nucleic Acids Res 43:

23 Science Reports GENOMIC AS WELL AS TRANSCRIPTOMIC PROFILING OF DISSEMINATED TUMOR CELLS Neuroblastoma, the most common extra-cranial solid tumor in childhood, is characterized by the presence of disseminated tumor cells (DTCs) in the bone marrow (BM) in the majority of patients with metastatic disease (stage M). Tumor relapse is the most frequent reason for therapy failure in those patients, in most instances originating in the BM. Thus, one of the most burning questions concerns the early identification and characterization of the relapse clone in the BM to find a more adequate therapy for this aggressive tumor. However, as DTCs are often the minor cell fraction in the bone marrow, we typically only diagnose a few single cells or a few hundred cells per one million normal bone marrow cells (mononuclear cells). To overcome the technical challenge of analyzing these rare DTCs, we adopted enrichment techniques, ultimately allowing a detailed genomic as well as transcriptomic profiling of these cells. As a result we were able to show that DTCs, even when present at very low frequencies, could be augmented by magnetic beads-based enrichment, thus facilitating full genome analyses by applying high resolution SNP array or sequencing approaches (Abbasi et al. 2015). Additionally, also high quality RNA could be obtained from enriched DTCs, allowing RNA sequencing (Rifatbegovic 2015). These technical improvements now make it possible to fully characterize the relapse clone by SNP array, DNA- and RNA-NGS (next generation sequencing) approaches. METRONOMIC TREATMENT LEADS TO TUMOR CELL SENESCENCE IN MYCN AMPLIFIED NEUROBLASTOMA CELLS Another feature of stage M neuroblastoma is the presence of chromosomal aberrations and, importantly, the amplification of the MYCN oncogene seen in approximately 40% of patients. Especially the MYCN amplification turns the neuroblastic cells into a very aggressive and frequently therapy refractory state. MYCN amplified neuroblastoma cells show a biphasic morphology in cell culture. We were able to show that the large, fibroblast-like cells co-occurring with the typical neuronal cell type represent tumor cells with senescent features (Ambros et al. 1997). Importantly, the number of senescent cells can be increased considerably by adding low dose cytotoxic drugs, such as hydroxyurea or topotecan. We demonstrated that the use of low doses of those drugs resulted in a permanent stop of cell growth of MYCN amplified neuroblastoma cells in vitro and that metronomic topotecan treatment prolonged disease-free survival in an in vivo mouse model leading to long-term cure in 40% of the animals (Narath et al. 2007, Taschner-Mandl et al. 2015). This new mode-of-action of metronomic treatment may be clinically relevant, as metronomic regimens are considered a feasible maintenance treatment option with moderate adverse event profiles in a number of clinical studies. Abbasi MR, Rifatbegovic F, Brunner C, Ladenstein R, Ambros IM, Ambros PF (2015) Bone marrows from neuroblastoma patients: an excellent source for tumor genome analyses. Mol Oncol 9: Figure 1: Enrichment of GD2 pos disseminated tumor cells (DTCs) by a magnetic beadsbased technique. DNA and RNA can be extracted from the negative and positive fractions for further analysis e.g. SNP array or next generation sequencing methods. Figure 2: MYCN amplified, aggressive neuroblastoma cell lines become senescent when treated for 3 5 weeks with low doses of cytotoxic drugs, such as hydroxyurea or topotecan. Long-term topotecan treated cells stop cell growth, change their morphology (flat, enlarged nucleus and cytoplasm) and stain blue for the senescence marker senescence-associatedbeta-galactosidase. Rifatbegovic F, Abbasi MR, Taschner-Mandl S, Kauer M, Weinhausel A, Handgretinger R, Ambros PF (2015) Enriched bone marrow derived disseminated neuroblastoma cells can be a reliable source for gene expression studies a validation study. PLoS ONE 10: e Taschner-Mandl S, Schwarz M, Blaha J, Kauer M, Kromp F, Frank N, Rifatbegovic F, Weiss T, Ladenstein R, Hohenegger M, Ambros IM, Ambros PF (epub 2015) Metronomic topotecan impedes tumor growth of MYCN-amplified neuroblastoma cells in vitro and in vivo by therapy induced senescence. Oncotarget 7:

24 Science Reports PERSISTENCE, REACTIVATION AND INTRACELLULAR INTERACTIONS OF HUMAN ADENOVIRUSES patients (%) Human adenoviruses (HAdVs) represent a large family of genetically diverse pathogens displaying broad tissue tropism and causing a variety of clinical manifestations ranging from mild to severe diseases even in immunocompetent individuals (Lion T 2014). HAdVs are divided into seven species (A-G) currently comprising 70 types (Lion T 2014). Following primary infection, most commonly affecting young children, HAdVs can persist in a latent state in different tissues and may be reactivated in the presence of immunosuppression. The best- documented sites of HAdV-persistence include tonsillar and adenoidal T-lymphocytes, which appear to represent a sanctuary for adeno viral latency. More recently, adenoviral DNA was also found in tumor- infiltrating lymphocytes, in T-cells isolated from the colon, but also in lymphoma cells and various other tissues (Kosulin K et al. 2013; Kosulin K et al. 2014). Reactivation of the viruses in immunocompromised individuals can result in invasive infections which can be associated with high mortality rates, particularly in children undergoing allogeneic hematopoietic stem cell transplantation (allo-hsct) (Lion et al. 2003, Lion et al. 2010). Our earlier findings demonstrated that the onset of invasive HAdV-infection in pediatric transplant recipients is almost invariably preceded by the appearance and proliferation of the virus in the gastrointestinal (GI) tract. Rapidly rising HAdV copy numbers in serial stool specimens, exceeding the threshold of one million virus copies per gram, were shown to correlate with a high risk of invasive infection (Lion et al. 2010). This critical threshold was therefore integrated into an algorithm for diagnostic monitoring and treatment of impending HAdVmediated complications (Lion T 2014). Identification of the actual sources of invasive HAdV-infections in the immunocompromised host may be regarded as a prerequisite for improved risk-assessment and the implementation of strategies preventing severe systemic manifestations. The occasional observation of extremely high viral loads in serial stool specimens of patients with HAdV-reactivation posttransplant, sometimes exceeding 1011 particles per gram, implies the existence of a hitherto unknown compartment in the GI-tract harboring persistent HAdV-infection and providing a site of origin for invasive infections in the immunocompromised host. We have recently extended the HAdV screening assay used in our routine diagnostics (Ebner- Kosulin K et al. 2005) to permit reliable detection of all 70 currently known types of the virus. We have performed systematic screening in children for the presence of persistent adenoviruses in the GI-tract, and patients displaying reactivation of the virus after allo-hsct were investigated by in-situ hybridization techniques to determine the sites of latent infection and viral expansion. PERSISTENCE OF ADENOVIRUSES IN THE GASTROINTESTINAL TRACT We have prospectively screened biopsies from 143 patients undergoing elective gastrointestinal endoscopy for a variety of clinical indications. Overall, 44/143 (31%) of the children and 61/576 (11%) of the biopsies investigated revealed the presence of HAdV-DNA. The relative frequencies of viral DNA detection in biopsies from different sections of the GI-tract are displayed in Figure 1, indicating the highest prevalence of HAdV-persistence in the terminal ileum. Among children who tested positive for HAdV in any section of the GI-tract, the virus was detectable in biopsies from the ileum in virtually all instances and the frequency of virus persistence appeared to decline with increasing distance from the ileum (Figure 1). In the majority of children with documented viral persistence (27/44; 61%), HAdV was detectable in biopsies taken at a single site, in most instances in the ileum. In 17/44 (39%) of children, the virus was detected at two or three different sites of the GI-tract, generally including the ileum. In-situ hybridization for HAdV-DNA in tissue sections from selected patients with PCR-documented virus persistence revealed specific signals primarily within lymphoid cells in the lamina propria, while scattered signals were rarely detected in individual epithelial cells. The analysis of viral protein expression by immunohistochemistry showed positive signals within distinct T-cells of the lamina propria in ileum biopsies of some patients (Kosulin T et al. 2015) oesophagus stomach duodenum jejunum ileum colon rectum Figure 1. Persistence of HAdV in different sections of the GI-tract. Biopsies from 143 patients were analyzed. In 90% of patients who tested positive for HAdV, presence of the virus was detected in the ileum. Prevalence of the virus in other sections was significantly lower

25 Science Reports ADENOVIRUSES IN THE GASTROINTESTINAL TRACT IN HSCT-RECIPIENTS An earlier study performed in pediatric transplant recipients at our center revealed the presence of intestinal HAdV infection in 51/138 (37%) of cases (Lion T et al. 2010). In the most recent analysis performed (Kosulin K et al. 2015), screening of serial stool specimens by PCR during the post-transplant period in 148 children revealed HAdV-positivity in a rather high proportion of instances (84/148; 57%). The occurrence of invasive HAdV-infection, defined by the onset of viremia during the first 100 days after transplantation, was observed in 16/148 (11%) children and occurred exclusively in patients with prior detection of the virus in stool. Among patients who tested HAdV-positive in stool, 16/84 (19%) revealed viremia post-transplant, while no cases of viremia were observed in patients without presence of the virus in stool (0/64; p<0.01). The high rate of HAdV-positivity in stool specimens within the recently investigated cohort of 148 transplant recipients might represent the cumulative result of reactivations and de novo infections. Overall, our findings in nearly 300 pediatric transplant recipients over ten years indicate that HAdV-reactivations or new infections occur in more than one third of cases. Screening for HAdV in stool specimens shortly before allogeneic stem cell transplantation revealed presence of the virus in 21/148 (14%) of cases, indicating that shedding into the stool occurs prior to the onset of severe immunosuppression in some instances. These patients continued testing positive for the virus during post-transplant monitoring of Figure 2. Detection of HAdV in intestinal epithelium of HSCT patients. Left picture: in situ hybridization (dark violet signals). Right picture: immunohistochemical analysis (brown signals). stool specimens in nearly all instances (20/21; 95%), and the rate of progression to invasive infection was significantly higher than in individuals who tested negative for the virus before transplantation (33% versus 7%; p<0.001). Detection of HAdV in stool at any time point investigated within the first 100 days after transplantation showed a significant correlation with later occurrence of viremia by multivariate analysis (p<0.01). In children who tested HAdV-positive in stool already before HSCT, a highly significant correlation with viremia during the post-transplant course was observed, and the difference to patients who revealed HAdVpositivity only after transplantation, without previous detection of intestinal virus shedding, was significant (p=0.020). Investigation of intestinal biopsy specimens by in-situ hybridization was performed in transplant recipients with relatively high HAdV copy numbers in serial stool samples ranging from 1x10 7 to 10 9 virus copies/g stool, and a high density of HAdV-specific hybridization signals was observed in epithelial cells (Figure 2). Moreover, immunohistochemical analysis of viral protein expression also showed strong signals in epithelial cells (Kosulin K et al. 2015). IMPLICATIONS OF HADV PERSISTENCE IN THE GASTROINTESTINAL TRACT AND VIRUS SHEDDING Persistence of HAdV in the gastrointestinal tract was identified in 31% of children, with the highest prevalence in the terminal ileum. Since tonsillar and adenoidal lymphocytes are the best documented sites of HAdV-latency, the greatest prevalence of persisting HAdV in the ileum may be related to the highest density of lymphoid tissue in this section of the GI-tract. The most prevalent HAdV-representatives identified in intestinal biopsies representing persistent infection included species C followed by A, and the same prevalence was also observed in the cohort of HSCT-patients investigated. In-situ hybridization and immunohistochemistry identified HAdV-persistence in lymphoid cells of the lamina propria, whereas biopsies from transplant recipients revealed high numbers of replicating HAdV in intestinal epithelial cells. The prevalence of HAdV species, the frequencies of persistence in the GI-tract and reactivation post-transplant indicate that detection of intestinal HAdV-shedding pre-transplant correlated with high risk of invasive infection. HAdV-persistence in the gastrointestinal tract is a likely origin of infectious complications in immunocompromised children. Intestinal lymphocytes represent a reservoir for HAdV-persistence and reactivation, while the intestinal epithelium is the main site of viral proliferation preceding dissemination. The finding of rare epithelial cells displaying a low number of HAdV-DNA signals in carriers of persistent virus is in accordance with the observation of intestinal shedding of viral particles even in healthy individuals. However, detection of HAdV-shedding into the stool prior to allogeneic HSCT appears to indicate a significantly elevated risk of invasive viral infection, as indicated by clinical observations in the patient cohort investigated. Immunohistochemical analysis of viral protein expression suggested a low level of virus production in lymphoid cells which may primarily serve as sites of long-term persistence, with leakage of viral particles into the environment. This notion is in line with the carrier state described for other viruses. In individuals with adequate immune response, small amounts of infectious virus may be eliminated, thus permitting limited virus proliferation in epithelial cells and shedding into the stool. By contrast, severe immunosuppression may facilitate extensive infection of intestinal epithelial cells which provide an environment optimally conducive to virus proliferation. Intestinal expansion of adenoviruses was shown to precede invasive infection conferring a high risk of life-threatening disease in HSCT-recipients (Lion T et al. Leukemia 2010), and early onset of pre-emptive therapy was shown to be important for clinical outcome. Our findings have important implications for assessing the risk of invasive HAdV-infections. Molecular HAdV-monitoring of stool specimens prior to HSCT can identify patients at high risk of invasive viral infection. As recently suggested for respiratory virus infections commonly associated with post-transplant complications, postponing elective HSCT may be considered. Delaying the transplantation and anti-adenoviral treatment in patients with intestinal HAdV-shedding might be a clinically feasible strategy, depending on the individual risk situation. Early pre-emptive therapy with novel anti-adenoviral agents displaying more favorable toxicity profiles, such as Brincidofovir, or virus-specific T-cells, might help controlling intestinal proliferation and prevent life-threatening invasive infection (Kosulin K et al. 2015)

26 Science Reports REGULATION OF CELLULAR GENE EXPRESSION BY HUMAN ADENOVIRUSES Over the past few years, a number of micrornas (mirnas) have been identified, and their ability to post-transcriptionally regulate the expression of human genes was characterized. The presence of mirna-encoding genes is not restricted to eukaryotes, their presence has also been documented in the genomes of different viruses. Depending on the type, HAdVs encode one or two so-called virus-associated RNAs (VA RNAs) that can be processed into functional mirnas. Members of HAdV species C encode two well-structured VA RNAs, VA RNAI and VA RNAII. These RNAs are approximately 160 nucleotides (nt) long and are generated by RNA polymerase III. They are expressed throughout the infection cycle but reach their highest concentration during the late phase of infection. Whereas VA RNAII is not vital for virus replication, the predominantly produced VA RNAI is essential for efficient translation of mrnas during the late infection phase. A well-documented function of VA RNAI is the inhibition of interferon-inducible protein kinase R (PKR), which is activated in infected cells by viral double-stranded RNA (dsrna), and thus constitutes a component of the antiviral response. Furthermore, VA RNAs I and II were shown to be processed by the Dicer enzyme into mirna-like small RNAs (mivarnas). Since large amounts of VA RNAs are processed by Dicer, and some of the processing products are incorporated into the RNA-induced silencing complex (RISC), VA RNAs and mivarnas were suggested to inhibit the cellular RNA interference (RNAi) pathway. RNAs derived from both VA RNAI and VA RNAII were shown to be produced by both lytically and persistently infected cells. GENE SILENCING BY MIVARNAS Our studies focused particularly on one of the most abundant mivarna single strands derived from the 3 -arm of VA RNAI [3 mivarnai(g)-138] encoded by HAdV-C5. Mimics of the major mivarna isoforms including 5 mivarnai(a)-137, 3 mivarnai(a)-137, and 3 mivarnaii-138 were investigated. To differentiate between targeting by the 5 -arm- and 3 -arm-derived single strands of mivarnai(a), a mimic of 5 mivarnai(g)-137 was also included. The capacity of these mimics to silence genes was evaluated using dual-luciferase assays. The mivarnas derived from the 3 -arm of VA RNAI silenced genes more potently than mivarnas derived from the 5 -arm, in line with earlier data demonstrating that mivarna strands derived from the 3 -arm were generally incorporated into RISCs more efficiently. To co-isolate RISC-associated mirnas and their target mrnas, we immunoprecipitated RISCs from lysates of HAdV-C5-infected cells. Moreover, changes in gene expression upon infection of cells with HadV-C5 or transfection of individual mivarna mimics were studied. Microarray analysis allowed us to identify a set of genes downregulated after mivarna transfection or HAdV-C5 infection. In this study, we determined direct targets of both single strands of mivarnai(a)-137 analyzing mrna isolated from RISC-immunoprecipitates. We focused on this particular mivarna because sequencing of small RNA libraries prepared from RISC-immunoprecipitates indicated that both single strands are abundant in RISCs. Moreover, the single strand derived from the 3 arm of VA RNAI (3 mivarnai-137) was more abundant in RISC than 3 mivarnai(a)-138, for which one direct target had been reported previously. The majority of targets identified in this study were those of 3 mivarnai-137. Examples of biological processes affected included genes involved in apoptosis (BOK), mitochondrial processes (BRP44), membrane-associated processes (SMAGP, TMEM222), or cell growth (LY6K). The greatest reduction of transcripts was observed for BOK, LY6K, and TMEM222. Transcripts of the genes PRR3, SMAGP, and SUPT7L contained predicted target sites for mivarna-137 single strands derived from both strands of VA RNAI, but the vast majority of validated targets represented those of 3 mivarnai-137, i.e. BOK, BRP44, ISOC1, LY6K, PRR3, SMAGP, SUPT7L, TMEM222, all residing in the 3 -UTRs of their genes. By contrast, genes with target sites located exclusively in the 5 -UTR showed no or poor downregulation of expression. Targeting of several genes identified can conceivably support virus propagation, and our goal was to identify HAdV-encoded mivarnas that are likely to contribute most effectively to virus-mediated posttranscriptional downregulation of cellular gene expression. The data generated extend our understanding of cellular genes targeted by adenoviral mirnas which can ultimately serve as a basis for further studies aiming at the inhibition of virus propagation (Bellutti F et al. 2015). Kosulin K, Geiger E, Vecsei A, Huber WD, Rauch M, Brenner E, Wrba F, Hammer K, Innerhofer A, Potschger U, Lawitschka A, Matthes-Leodolter S, Fritsch G, Lion T (epub 2015) Persistence and reactivation of human adenoviruses in the gastrointestinal tract. Clin Microbiol Infect 22: 381 e Bellutti F, Kauer M, Kneidinger D, Lion T, Klein R (2015) Identification of RISC-associated adenoviral micrornas, a subset of their direct targets, and global changes in the targetome upon lytic adenovirus 5 infection. J Virol 89:

27 Science Reports IMPLICATIONS FOR ANTI-CANCER IMMUNE THERAPY BREAKING IMMUNE TOLERANCE THROUGH INTERFERING WITH MAPK OR PI3K SIGNALING IN MYELOID CELLS Myeloid antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages, represent key players in linking innate with adaptive immunity by stimulating immunogenic or tolerogenic T cells. In this context, co-stimulatory or inhibitory molecules expressed by APCs define the nature of T cell responses that either reject or tolerate infected, foreign or mutated tissues like tumors. The Laboratory of Tumor Immunology at the St. Anna Kinderkrebsforschung aims at the characterization of factors that feature exaggerated T cell reactivity leading to allograft responses that causes graftversus host disease (GvHD) after hematopoietic stem cell transplantation (HSCT) and tumor rejection in pediatric cancer patients. On the one hand interference with immunogenic or tolerogenic pathways may help increase tumor immunogenicity and on the other it may reduce the risk of GvHD after adoptive cell transfer of in vitro modulated DCs or T cells. Our observations, published in 2015, highlight immune modulating functions of mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K) signaling molecules in dendritic cells in the context of pathogen-induced auto-immunity. Upon knock down of p38 MAPK-activated kinase 2 (MK2) in DCs, we observed a severe clinical course of experimental autoimmune encephalitis (EAE) (Soukup et al., 2015). In contrast, loss of the PI3K antagonist phosphatase and tensin homolog (PTEN) renders myeloid APCs immune inhibitory leading to reduced PI3K-p38 signaling and weakened EAE severity (Sahin et al., 2015). Based on our observation we propose MK2 and PI3K-mediated signal transduction as dominant mechanisms of immune inhibitory myeloid cell functions. Due to the putative pro-tumor activity of tumor-resident myeloid cells, the particular function of MK2 and PI3K expressed in myeloid cell infiltrates of the tumor microenvironment (TME) is currently under investigation in immunocompetent murine tumor models. First evidence attributes immune inhibitory functions to MK2 in the TME of melanoma and glioblastoma. Hence, pharmacological inhibition of MK2 combined with an immune therapeutic regimen, such as irradiation, might be a promising approach for cancer treatment. Along these lines, tumor irradiation is considered as a potent immune therapeutic intervention which increases tumor immunogenicity upon massive antigen release into the TME, as reviewed by Soukup and Wang, MK2 ATTENUATES DC-MEDIATED TH1 IMMUNITY AND CYTOTOXICITY In the study of Soukup et al. we revealed unexpected immune inhibitory functions of MK2 when expressed in DCs. Under MK2-deficient conditions, we observed a shift towards a Th1-promoting phenotype in pathogen-activated DCs, differentiated from murine bone marrow and human monocytes, or isolated directly from murine spleens. These findings were underlined by our observations in a murine model of multiple sclerosis (EAE) (Figure 1). In mice lacking MK2 specifically in CD11c+ DCs (MK2ΔDC), clinical symptoms, brain damage and increased inflammation correlated strongly with the established classical Th1-mediated autoimmune phenotype and could be abrogated upon administration of an IFN-γ-blocking antibody. Along these lines, down-regulated MK2 expression in DCs was shown to contribute to onset and development of de-regulated autoimmune reactions in the EAE model. Severe clinical symptoms become obvious Figure 1: Lack of MK2 in DCs induces Th1-driven EAE. Clinical course of CD11c-specific MK2 ko (MK2DDC, n = 5) and WT littermate control (n = 5) mice after induction of EAE upon infusion of myelin oligodendrocyte glycoprotein (MOG) and complete freund s adjuvant (CFA) enriched with mycobacterium tuberculosis, followed by a 2 nd boost immunization (day 24) (UPPER LEFT GRAPH) and following additional treatment with an IFN-γ blocking or isotype control antibody (n = 5 or 6 animals for each group) (UPPER RIGHT GRAPH). Mean +/- SEM is shown. Furthermore, immunopathology of spinal cords from EAE mice analyzed at peak of disease (day 33) is shown (LOWER LEFT GRAPH). Areas of myelin loss are visualized in Kluver-Barrera (KLB)-stained sections (ARROWS), degree of inflammation in EAE mice in hematoxylin & eosin (H&E) stainings (ARROWS). Based on our observation in Soukup et al. we propose an MK2-mediated tolerogenic DC phenotype (low IL-12, high IL-10) upon repeated antigen challenge, which dampens Th1 effector mechanisms (low IFN-γ) and cytotoxicity. MK2 blockade, using e.g. the small molecule inhibitor MK2-I3, promotes p38-mediated IL-12 release and cytotoxicity in the context of antigen-specific immune reactions (lower right illustration). upon repeated antigen challenge (2 nd immunization) in MK2ΔDC mice. This observation along with antigen unresponsiveness upon boost immunization in WT littermate controls led us to the conclusion of an essential MK2 function in tolerizing the immune system against foreign or mutated antigens. Of note, pathogens and inflammatory factors strongly induce MK2, leading to negative feedback signaling on p38, which is critical for both pro- and anti-inflammatory immune responses (Figure 1, lower right illustration). For that reason MK2 inhibition results in a complex DC phenotype, primarily displayed by excess IL-12 supply for effective Th1 immunity and cytotoxicity. Therefore, MK2-driven anti-inflammatory functions should be considered in autoimmune, tumor, or transplant models with regard to clinical intervention. Especially in the context of inflammatory diseases such as rheumatoid arthritis, for the treatment of which MK2 inhibitors are being tested, monitoring of potential immune dysregulation will be of utmost importance

28 Science Reports 2 Tumor microenvironment Bone marrow Dendritic cell MYELOID CELL-SPECIFIC LOSS OF PTEN RESULTS IN PI3K-MEDIATED IMMUNE SUPPRESSION AND ATTENUATED AUTO-IMMUNITY In a similar study published by Sahin et al. we showed that tight regulation of PI3K signaling by PTEN in APCs is necessary for the development of Th17-dependent autoimmune pathology. APCs deficient for PTEN exhibit reduced activation of p38 MAPK and reduced expression of T cell polarizing cytokines. Furthermore, PTEN deficiency led to upregulation of markers for alternative activation, such as arginase 1, with concomitant downregulation of inducible NO synthase in APCs in vitro and in vivo. As a result, T cell polarization was dysfunctional in PTEN deficient APCs, in particular affecting the Th17 cell subset. Intriguingly, mice with cell type specific deletion of PTEN-targeting APCs were protected from EAE, which was accompanied by a pronounced reduction of IL-17 and IL-22 producing autoreactive T cells and reduced central nervous system (CNS) influx of classically activated monocytes/macrophages. In conclusion, the regulatory phenotype observed in PTEN-deficient DCs in vitro and the beneficial effects on EAE disease progression in mice lacking PTEN provides genetic evidence that sustained PI3K activation in APCs abrogates pathogenic Th17 cell polarization in autoimmunity. This underscores a critical role for PTEN in disease pathogenesis and warrants further possibilities for therapeutic intervention. In contrast, targeting PI3K signaling in the TME may support tumor rejecting effector T cell functions. CONSEQUENCE OF IMMUNE INHIBITORY FUNCTIONS OF MK2 AND PI3K ON THE TUMOR MICRO ENVIRONMENT The role of tumor-resident immune cells with respect to tumor protection is crucial and unresolved (Figure 2). In particular DCs, like myeloid-derived suppressor cells (MDSCs) or M2-polarized tumor-associated macrophages (TAMs), can acquire immune suppressive phenotypes, which prevent cytotoxic T cells from tumor killing. In addition, DCs are inhibited for proper antigen presentation and caught in the TME due to aberrant chemokine receptor expression. In this context we observed strong upregulation of MK2 in MDSCs and tumor-resident DCs in melanoma and glioblastoma. Interestingly, MK2 deficiency allows DCs to leave the TME and induces physiological immune reactions against the tumors. Upon MK2 knock down this observation is associated with differential regulation of chemokine receptor and ligands, which reportedly drive DCs migration into lymphatic tissues. We therefore propose an important immune inhibitory function, hence pro- tumor activity of MK2 in melanoma and glioblastoma. Together with our published immune inhibitory functions of the MK2 and PI3K pathway in the auto-immune context, we attribute an important role to MK2 and PI3K with regard to protecting tumors from cytotoxic immune responses. Pharmacological inhibition of MK2 or PI3K in combination with immune therapeutic intervention may help to induce strong and effective cytotoxic T cell immunity in tumors Lymph node monocyte Deviated myeloid differentiation in the tumor microenvironment Impaired migration of dendritic cells towards lymph nodes No efficient priming of T cells by dendritic cells in lymph nodes Soukup K, Halfmann A, Le Bras M, Sahin E, Vittori S, Poyer F, Schuh C, Luger R, Niederreiter B, Haider T, Stoiber D, Bluml S, Schabbauer G, Kotlyarov A, Gaestel M, Felzmann T, Dohnal AM (2015) The MAPK-activated kinase mk2 attenuates dendritic cell-mediated th1 differentiation and autoimmune encephalomyelitis. J Immunol 195: Sahin E, Brunner JS, Kral JB, Kuttke M, Hanzl L, Datler H, Paar H, Neuwinger N, Saferding V, Zinser E, Halfmann A, Soukup K, Hainzl E, Lohmeyer T, Niederreiter B, Haider T, Dohnal AM, Kronke G, Bluml S, Schabbauer G (2015) Loss of phosphatase and tensin homolog in APCs impedes Th17-mediated autoimmune encephalomyelitis. J Immunol 195: Soukup K, Wang X (2015) Radiation meets immunotherapy a perfect match in the era of combination therapy? Int J Radiat Biol 91: T cells 1 M2 TAM MDSC M1 TAM Adapted from Adams et al (2015) Nat Rev Drug Discov Figure 2: A simplified view of tumor immune escape. Effective anti-tumor immunity is triggered by professional APCs such as DCs, which present tumor antigens to T cells in lymphatic tissues. Following antigen-specific T cell priming and activation, tumor-specific T cells, in principle, are capable of lysing tumor cells. Tumors overcome these cytotoxic events through interfering with myeloid cell differentiation and migration, which leads to the accumulation of tumorpro moting myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) showing a so called M2 phenotype. DCs impaired in antigen presentation and migration also contribute to an immune suppressive tumor microenvironment (TME). Targeting immune inhibitory molecules, like immune checkpoints or our recently described factors (Soukup et al. and Sahin et al.), in the TME potentially raises anti-tumor immune reactions

29 Science Reports PROGRESS IN CLINICAL GRADE PRODUCTION OF VIRUS-SPECIFIC T-CELLS Figure 1 A Leukocytes B Donor INTRODUCTION For several haematological diseases, allogeneic hematopoietic stem cell transplantation (HSCT) is the only treatment option. Despite substantial progress in this field, human adenovirus (HAdVs), cytomegalovirus (CMV) and Epstein-Barr Virus (EBV) cause serious and potentially life-threatening post HSCT infections in immunosuppressed patients and especially HAdV and CMV infections are associated with mortality rates of up to 30%-60%. Treatment with anti-viral drugs, however, is associated with substantial nephro- and myelo-toxicity and is of limited effectiveness. Evidence indicating that recovery of virus-specific T-cells correlates with viral clearance has prompted several attempts to use donor-derived HAdV- and/or CMV-specific T-cells for adoptive immunotherapy. Regrettably, current therapies are mostly based on cost- and time-intensive and technically demanding immunotherapeutic protocols, a fact that limits their broader clinical application. Therefore, while showing impressive clinical results concerning reduction of viral load without inducing GvHD, only few clinical phase I/II trials have been published to date. Other clinical trials treated patients with virus-specific T-cell lines manufactured over periods of 14 weeks, which is often too long for timely administration of an anti-viral immunotherapy PREPARATORY WORK TO BRING SHORT-TERM EXPANDED VIRUS-SPECIFIC T-CELLS INTO THE CLINIC To avoid time-intensive approaches, we developed a faster short-term method that uses synthetic polypeptides and the cytokine Interleukin-15 to expand virus-specific T-cells within 12 days. This protocol was then adapted to good manufacturing practice (GMP) conditions (but at that time without manufacturing approval) thus enabling first-in-man treatment of two patients on a named patient use basis. As a next step, we aimed to initiate a clinical phase I/II trial analysing feasibility and safety of our newly developed short-term expanded virus-specific T-cell lines in patients after HSCT (funded by the ZIT Life Science Call). To achieve this aim, we had to establish and re-validate the manufacturing procedure including quality control assays according to EU-Pharmacopeia regulations. In May 2015 we received the manufacturing approval for the pro duction of short-term expanded virus-specific T-cells, which was a prerequisite for the planned submission of the clinical trial to the responsible authorities (AGES) in February ADDITIONAL PROTOCOL TO SELECT HADV-SPECIFIC T-CELLS VIA STREPTAMER TECHNOLOGY Another fast approach is the magnetic isolation of CD8+ virus-specific T-cells via removable good manufacturing practise (GMP) peptide-major histocompatibility complex class I (MHC I) streptamers. For this procedure, leukocytes were directly labelled with streptamers (including magnetic beads) to select CMV-specific T-cells. The complete removal of streptamers from bound T-cells after adding D-biotin (see Figure 1A) allows the isolation of so called minimally manipulated T-cells, Figure 1: Schematic drawings of the selection process (A) Leukocytes are first incubated with MHC I streptamers (labeled with magnetic beads), then placed into a strong magnetic field and, finally, magnetically isolated. After isolation, streptamers are dissociated by adding D-biotin resulting in highly pure HAdV-specific T-cells. (B) Depiction of the largescale and small-scale strategy to magnetically select HAdV-specific T-cells via streptamers. The largest part of the donor-derived leukapheresis product is used to isolate PWLs. Simultaneously, the minor part was used to generate sehadv-tcls from PBMCs. Both cell products are then enriched using magnetic isolation as described (A). Dissocation of Strep-Tactin Purified HAdV-specific T-cells ADV-specific T-cell White blood cell Bead Strep-Tactin Strep-Tag / MHC1 Magnet D-Biotin Incubation with strepamers Magnetic isolation of Strepramer-bound T-cells Dissocation of MHC I Strep-tag Streptamer Leukapheresis product Plasma-wash PWLs for large-scale Ficoll-gradient PBMCs sehdv-tcls for small-scale 56 57

30 Science Reports which are considered a less advanced therapy medicinal product (ATMP), thereby avoiding high regulatory barriers. Generally, this method is strictly dependent on the previous definition of immunodominant virus-specific epitopes, which are bound to MHC complexes. The identification and successful testing of a novel HAdV-specific epitope for the frequent HLA-type A*02, which was performed together with the group of Prof. Stevanovic in Tübingen, prompted us to apply for an EU-Grant (EUROSTARS) together with the company IBA GmbH in Upon approval of this EU-Grant, we were able to develop HAdV-streptamers for this novel A*02- as well as four other HAdV-specific epitopes, thus increasing the probability to detect and isolate HAdV-specific T-cells to about 95% of the Caucasian population. Assuming that the generally very low frequency of HAdV-specific T-cells in peripheral blood might negatively influence the purity of magnetically selected HAdV-T-cells, we applied two different strategies to select streptamer+ HAdV-specific T-cells. The first strategy large-scale was based on direct ex vivo magnetic selection of HAdV, alone or in combination with EBV-specific T-cells, starting from high numbers (1-6x109) of plasma-depleted leukocytes (PWLs) (Fig. 1B). The second strategy small-scale started with small amounts (25x106) of short-term expanded HAdV-specific T-cell lines (sehadv-tcls) in order to increase the frequency of virus-specific T-cells in the starting material (Figure 1B). Whereas the purity after selection of HAdV- specific T-cells alone was low, with max. 6.7%, the combination with EBV-specific T-cells increased the purity to max. 50,7%. A further increase to values of up to 98% was achieved by small-scale selected HAdV-specific T-cells. Further analyses revealed high functional activity of streptamer-selected T-cells (Freimüller et al. 2015). CONCLUSION We extended the streptamer technology isolating HAdV-specific T-cells under clinical-scale conditions. Especially the high purities achieved after small-scale selection might pave the way to establish T-cell banks comprising highly specific HAdV-streptamer+ T-cells off-the-shelf. Freimuller C, Stemberger J, Artwohl M, Germeroth L, Witt V, Fischer G, Tischer S, Eiz-Vesper B, Knippertz I, Dorrie J, Schaft N, Lion T, Fritsch G, Geyeregger R (2015) Selection of adenovirusspecific and Epstein-Barr virus-specific T cells with major histocompatibility class I streptamers under Good Manufacturing Practice (GMP)-compliant conditions. Cytotherapy 17:

31 CAREER

32 Career IF YOU WANT TO APPLY FOR A POSITION IF YOU WANT TO APPLY FOR A POSITION please proceed according to the details given on our web site. please proceed according to the details given on our web site. WORKING AT CCRI A SHARED MISSION MAKES A DIFFERENCE IN RESEARCH! The Children s Cancer Research Institute (CCRI) is an internationally renowned biomedical research institute, dedicated to the advancement of diagnosis, prognosis and treatment of childhood cancer. Science at the CCRI addresses a broad range of topics, covered by 12 research groups whose members work closely t ogether and in c ooperation with clinicians from St. Anna Children s Hospital. We are at the forefront of international pediatric cancer research and scientists at CCRI are striving to make the most innovative and effective therapies available to young patients. The CCRI offers an intellectually stimulating environ ment with lots of opportunities for s cientific exchange and discussions in regular seminars, retreats, and national and international conference attendances. The quality of our work is p eriodically reviewed by a panel of top international experts guaranteeing consistently high standards of CCRI research. PhD students are accompanied by PhD-committees to guide them through their thesis and postdocs have the option to exercise their ability to teach by mentoring and training younger colleagues. At CCRI, we are an international team with employees from all over the world, offering students and postdocs an ideal combination of freedom and guidance to foster their personal and i ntellectual development while at the same time introducing them to the world of science and academic competition

33 Career SCIENTIFIC STAFF GROUP LEADERS Peter Ambros Martin Distel Alexander Dohnal Michael Dworzak Gerhard Fritsch Oskar Haas Wolfgang Holter Heinrich Kovar Ruth Ladenstein Thomas Lion Renate Panzer-Grümayer Sabine Strehl AFFILIATED CLINICIANS Andishe Attarbaschi Heidrun Boztug Christofer Diakos Bernhard Fahrner Caroline Hutter Leo Kager Anita Lawitschka Georg Mann Susanne Matthes- Leodolter Milen Minkov Christina Peters Herbert Pichler Christian Posch Andreas Vécsei Volker Witt POSTDOCTORAL FELLOWS & STAFF SCIENTISTS Sarah Ahmadi-Erber Ingeborg Ambros Dave Aryee Jozef Ban Konstantin Byrgazov Stefan Czurda Filomena De Almeida Nogueira Markus Diem Klaus Fortschegger Christian Frech René Geyeregger Dávid Héja Zvenyslava Husak Maximilian Kauer Stefanie Kirchberger Florian Kleber Karin Kosulin Manfred Lehner Chantal Lucini Kamilla Malinowska- Ozdowy Helena Okulski Dagmar Schinnerl Raphaela Schwentner Caterina Sturtzel Sabine Taschner-Mandl Eleni Tomazou-Bock Cornelia Vesely CLINICAL RESEARCH Alisa Alspach Saelde Baumgartner Evgenia Glogova Corinne Grafl Ingeborg Hirsch Dasa Janousek Susanne Karlhuber Monica Kiesewetter Barbara Kristufek Zsuzsanna Lehner Nora Mühlegger Marek Nykiel Ulrike Pötschger Ingrid Pribill Marion Sebek Eva Sorz Elfriede Thiem PHD STUDENTS Reza Abbasi Stefanie Anderl Stephan Bastelberger Dominik Bogen Sara Colomer-Lahiguera Barbara Dillinger Friedrich Erhart Anna-Maria Husa Anna Katschnig Florian Kromp Cornelia Mutz Leonel Pereira Katarzyna Pietrzykowska Christine Portsmouth Julia Proff Fikret Rifatbegovic Benjamin Salzer Klara Soukup Tamara Weiss Martin Zeppetzauer DIPLOMA STUDENTS Clemens Brunner Bernadette Blauensteiner Charlotte Brey Cathrien Czurda Helena Dodig Teresa Gerber Bastien Huber Sophia Hütter Fiona Kraler Katharina Martin Vanessa Mayr Maria Strobl Veronika Triebel TECHNICIANS IN RESEARCH & DIAGNOSTICS Bettina Berkowitsch Maria Berneder Bettina Brunner- Herglotz Helga Daxberger Gudrun Divoky Jasmin Dmytrus Ulrike Engel Susanna Fischer Michaela Fortschegger Nelli Frank Christine Freimüller Brigitte Grimm Angela Halfmann Sabrina Haslinger Andrea Inthal Gunhild Jug Dragana Jugovic Margit König Susanna Koskela Astrid Mecklenbräuker Karin Mühlbacher Bettina Nocker Susana Pascoal Maya-Marisol Plank Michaela Pregesbauer Sandra Preuner-Stix Dieter Printz Christina Satke Daniela Scharner Angela Schumich Sabrina Smelik Manuela Stadler Julia Stemberger Susanne Suhendra Dijana Trbojevic Diana Walder Christian Walterskirchen Eva Winkler Sven Wohlmacher Andrea Ziegler Elke Zipperer RESEARCH SUPPORT OFFICE Nuno-Miguel Andrade Gomes Barbara Brunmair Zoltán Dobai 64 65

34 VERANTWORTUNG MITTRAGEN, UM FORSCHUNG SICHER ZUM PATIENTEN ZU BRINGEN. René Geyeregger

35 FINANZBERICHT

36 Finanzbericht RICHTLINIEN ZUR SPENDENVERWENDUNG Die St. Anna Kinderkrebsforschung wird zum über wiegenden Teil durch private Spenden finanziert. Für den Betrieb des Forschungsinstitutes werden jährlich mehr als sieben Millionen Euro benötigt, der Verein verfügt jedoch über keine Basisfinanzierung durch die öffentliche Hand. Zusätzliche Mittel werden im Rahmen von kompetitiv ausgeschriebenen Projektförderungen von anerkannten nationalen und internationalen Stellen akquiriert. Wir fühlen uns unseren Spenderinnen und Spendern gegenüber zu einer sparsamen und effizienten Verwendung der uns anvertrauten Gelder ver - pflichtet. Aus diesem Grund verwenden wir weniger als 10 % für die Verwaltung und das Fundraising, das bedeutet: mehr als 90 % der Spenden fließen direkt in die Forschung. SPENDENGÜTESIEGEL UND STEUERLICHE ABSETZBARKEIT Seit dem Jahr 2002 trägt die St. Anna Kinderkrebsforschung als eine der ersten Organisationen Öster reichs das Spendengütesiegel der Kammer der Wirtschaftstreuhänder. Für die jährliche Neuverleihung führt ein Wirtschaftsprüfer zusätzlich eine Prüfung der transparenten und ordnungs gemäßen Verwendung der Mittel gemäß den strengen Richt linien des Spendengütesiegels durch. Auf Grundlage eines von der Finanzlandesdirek tion für Wien erlassenen Bescheides zählt die St. Anna Kinderkrebsforschung zum begünstigten Empfänger kreis, sodass Spenden sowohl von der Lohnsteuer als Sonderausgabe, als auch von der Einkommensteuer als Betriebsausgabe steuerlich absetzbar sind. QUALITÄTSSICHERUNG DER WISSENSCHAFTLICHEN ARBEIT Das Forschungsinstitut verfügt über ein Scientific Advisory Board ein Gremium aus externen Experten mit der Aufgabe der laufenden Evalu ierung der wissenschaftlichen Arbeiten und Beratung der Institutsleitung. Darüber hinaus werden regelmäßig neue wissenschaftliche Projekte bei renommierten forschungsfördernden nationalen und internationalen Stellen eingereicht und Forschungsergebnisse in international anerkannten, wissenschaftlichen Journalen publiziert. In regelmäßigen Abständen findet zusätzlich eine objektive Beurteilung der wissenschaftlichen Leistung durch ausgewiesene externe Fachleute auf dem Gebiet statt. Der Jahresabschluss wird gemäß den Bestimmungen des Vereinsgesetzes für große Vereine erstellt, wobei die gleichen Richtlinien wie für Kapitalgesellschaften gelten. Die Finanzgebarung und der Jahresabschluss des Vereins werden jährlich von einem beeideten Wirtschaftsprüfer kontrolliert und mit einem uneingeschränkten Bestätigungsvermerk versehen. Damit wird der sach- und zweckgemäße Umgang mit den erhaltenen Spenden sichergestellt und bestätigt

37 WIR MENSCHEN TENDIEREN DAZU, KOMPLEXE DINGE ZU VEREINFACHEN, UM SIE VERSTEHEN UND FASSEN ZU KÖNNEN. ABER DIE NATUR KANN NICHT VEREINFACHT WERDEN. FORSCHUNG LEHRT EHRFURCHT UND SUCHT DIE HERAUSFORDERUNG. Cornelia Mutz

38 Finanzbericht MITTELHERKUNFT MITTELVERWENDUNG I. Spenden a) ungewidmete 0,00 0,00 b) gewidmete , ,51 II. Mitgliedsbeiträge 0,00 0,00 III. Betriebliche Einnahmen a) betriebliche Einnahmen aus öffentlichen Mitteln , ,78 b) sonstige betriebliche Einnahmen , ,54 IV. Subventionen und Zuschüsse der öffentlichen Hand 0,00 0,00 V. Sonstige Einnahmen a) Vermögensverwaltung 0,00 0,00 b) sonstige andere Einnahmen sofern nicht in Punkt I bis IV festgehalten , ,44 VI. Auflösung von Passivposten für noch nicht widmungsgemäß verwendete Spenden bzw. Subventionen 0,00 0,00 VII. Auflösung von Rücklagen 0,00 0,00 VIII. Jahresverlust 0,00 0, I. Leistungen für die statutarisch festgelegten Zwecke , ,20 II. Spendenwerbung , ,96 III. Verwaltungsaufwand , ,69 IV. Sonstiger Aufwand sofern nicht unter Punkt I bis III festgehalten V. Zuführung zu Passivposten für noch nicht widmungsgemäß verwendete Spenden bzw. Subventionen , , , ,29 VI. Zuführung zu Rücklagen 0,00 0,00 VII. Jahresüberschuss 0,00 0,00 TOTAL , ,27 JAHRESERGEBNIS 0,00 0,00 TOTAL , ,

39 ANHANG

40 Anhang WISSENSCHAFTLICHER BEIRAT PROF. DR. KLAUS-MICHAEL DEBATIN Universitätsklinik für Kinder- und Jugendmedizin Ulm, Germany PROF. JAMES R. DOWNING, MD Scientific Director St. Jude Children s Research Hospital Memphis, TN , USA PROF. LEE J. HELMAN, MD Scientific Director for Clinical Research Center for Cancer Research National Cancer Institute, National Institutes of Health Bethesda, MD , USA PROF. CRYSTAL L. MACKALL, MD Associate Director Stanford Cancer Institute Stanford, CA , USA GREGORY H. REAMAN, MD Office of Hematology and Oncology Products, OND Center for Drug Evaluation and Research U.S. Food and Drug Administration Silver Spring, MD 20993, USA PROF. MEGAN SYKES, MD Columbia University Medical Center New York, NY 10032, USA PROF. STEPHAN LADISCH, MD Bosworth Chair for Cancer Biology Center for Cancer and Immunology Research Children s National Medical Center Washington, DC 20010, USA 78 79

41 I WANT TO MAKE A DIFFERENCE IN OUR PATIENTS LIVES BY TRANSFORMING LARGE, COMPLEX GENOMIC DATA SETS INTO CLINICALLY USEFUL INFORMATION. Christian Frech 80 81

42 Anhang INTERNATIONAL FREMDGEFÖRDERTE PROJEKTE Optimized diagnostics for improved treatment stratification in invasive fungal diseases (FUNGITECT) Coordinator: Thomas Lion (CCRI/Labdia) European Commission Grant FP7 Cooperation Project N : Duration: 01/02/2014 to 31/01/2018 Automation of flow cytometric analysis for quality-assured follow-up assessment to guide curative therapy for acute lymphoblastic leukaemia in children (AutoFLOW) Labdia/CCRI partner: Michael Dworzak Coordinator: Martin Kampel (Technical University of Vienna, Austria) European Commission Grant FP7 Industry- Academia Partnerships and Pathways (Marie Curie Actions) N : Duration: 01/02/2014 to 31/01/2018 EURO EWING Consortium International clinical trials to improve survival from Ewing sarcoma (EEC) CCRI partner: Heinrich Kovar Coordinator: Jeremy Whelan (University College London, UK) European Commission Grant FP7 Cooperation Project N : Duration: 01/09/2013 to 31/08/2018 Molecular mechanisms of human fungal pathogen host interaction (ImResFun) CCRI partner: Thomas Lion Coordinator: Karl Kuchler (Medical University of Vienna, Austria) European Commission Grant FP7 Initial Training Network (Marie Curie Actions) N : Duration: 01/10/2013 to 30/09/2017 Expert paediatric oncology reference network for diagnostics and treatment (ExPO-r-Net) Coordinator: Ruth Ladenstein (CCRI) Grant from the European Consumers, Health, Agriculture and Food Executive Agency (CHAFEA) N : Duration: 01/03/2014 to 31/08/2017 Targeted modulation of immune-system responses in cell therapies (MODICELL) Coordinator: Originally Andreas Heitger ( 5 May 2014), presently Wolfgang Holter European Commission Grant FP7 Industry- Academia Partnerships and Pathways (Marie Curie Actions) N : Duration: 01/01/2013 to 31/12/2016 International study for treatment of childhood relapsed ALL 2010 (IntReALL) CCRI partners: Georg Mann, Andishe Attarbaschi and Ruth Ladenstein Coordinator: Jeremy Whelan (University College London, UK) European Commission Grant FP7 Cooperation Project N : Duration: 01/10/2011 to 31/09/2016 Analysing and Striking the Sensitivities of Embryonal Tumours (ASSET) CCRI partner: Heinrich Kovar Coordinator: Walter Koch (University College Dublin, Ireland) European Commission Grant FP7 Cooperation Project N : Duration: 01/11/2010 to 30/04/2016 PanCare childhood and adolescent cancer survivor care and follow-up studies (PanCareSurFup) CCRI/St Anna Spital partners: Eva Frey Coordinator: Lars Hjorth (Lund University, Sweden) European Commission Grant FP7 Cooperation Project N : Duration: 01/02/2011 to 31/01/2016 European network for cancer research in children and adolescents (ENCCA) CCRI partners: Ruth Ladenstein, Peter Ambros, Michael Dworzak and Heinrich Kovar Coordinator: Ruth Ladenstein (CCRI) European Commission Grant FP7 Network of Excellence N : Duration: 01/01/2011 to 31/12/

43 Einleitung NATIONAL FREMDGEFÖRDERTE PROJEKTE TRANSCALL (Translational research in childhood acute lymphoblastic leukemia) CCRI responsible principal investigator: Renate Panzer-Grümayer Grant from the Austrian Science Fund (FWF), ERA-Net Transcan N : I226-B19 Duration: 01/07/2013 to 30/06/2017 The role of micrornas in the EWS-FLI1 gene regulatory network of Ewing sarcoma CCRI responsible principal investigator: Heinrich Kovar Grant from the Austrian Science Fund (FWF), Stand-Alone program N : P24708-B21 Duration: 01/08/2012 to 31/07/2015 Directed in vitro differentiation of induced pluripotent stem cells towards the B lymphoid lineage (B-different) CCRI coordinator: Klaus Fortschegger Grant from the Austrian Research Promotion Agency (FFG), Call Bridge (Brückenschlagprogramm) N : Duration: 01/06/2014 to 31/05/2017 Permanent consequences in childhood Langerhans cell histiocytosis CCRI responsible principal investigator: Milan Minkov Grant from the Austrian National Bank (OeNB), Jubiläumsfonds N : Duration: 01/09/2015 to 31/08/2017 PROVABES (prospective validation of biomarkers in Ewing sarcoma for personalized treatment) CCRI responsible principal investigator: Heinrich Kovar Grant from the Austrian Science Fund (FWF), ERA-Net Transcan N : I225-B19 Duration: 01/04/2013 to 31/03/2017 Myeloproliferative neoplasms CCRI partner: Thomas Lion Coordinator: Peter Valent (Medical University of Vienna, Austria) Grant from the Austrian Science Fund (FWF), Special Research Programme (SFB) N : F4705-B20 Duration: 01/03/2013 to 28/02/2017 Immune responses in humanized mice with porcine mixed xenogeneic chimerism: Evaluation of T-cell function, tolerance and pathogen clearance CCRI responsible principal investigator: Andreas Heitger Grant from the Austrian Science Fund (FWF), Erwin-Schrödinger Fellowship Duration: 01/01/2013 to 31/12/2015 MK2/3 in immune regulation by dendritic cells CCRI responsible principal investigator: Alexander Dohnal Grant from the Austrian Science Fund (FWF), Stand-Alone program N : P23271-B11 Duration: 01/04/2011 to 30/09/2015 Single molecule array platform for sensitive diagnostics (SmardScout) CCRI partner: Thomas Lion Coordinator: Jan Hesse (Center for Advanced Bioanalysis GmbH, Linz, Austria) Grant from the Austrian Research Promotion Agency (FFG), Research Studios Austria, 4th Call N : Duration: 01/09/2014 to 31/08/2018 Verfahren zur hochautomatisierten Bewertung und Klassifikation von Zellen in Gewebeschnitten anhand räumlicher Markerprofile (TisQuant) LABDIA responsible principal investigator: Peter Ambros Grant from the Austrian Research Promotion Agency (FFG), ERA-SME N : Duration: 01/06/2014 to 31/05/2017 Integrating entertainment and reaction assessment into child cancer therapy (INTERACCT) CCRI/St Anna Spital partner: Anita Lawitschka Coordinator: Helmut Hlavacs (University of Vienna, Austria) Grant from the Austrian Research Promotion Agency (FFG), Call Bridge (Brückenschlagprogramm) N : Duration: 01/05/2013 to 30/04/2016 Prä-klinische Entwicklung einer Off-the-Shelf individualisierten Krebsimmuntherapie (IN SITU DC-CIT) CCRI partner: Alexander Dohnal Coordinator: Wolfgang Schöfberger (University of Linz, Austria) Grant from the Austrian Research Promotion Agency (FFG), Call Bridge (Brückenschlagprogramm) N : Duration: 01/10/2012 to 30/09/2016 Virus-specific immunotherapy (VISIT) Labdia/CCRI: coordinator René Geyeregger (Labdia), partner: Matthes-Leodolter (CCRI) Grant from the Wirtschaftsagentur Wien, Call Life Sciences 2014 N : Duration: 01/04/2015 to 31/03/2018 Development of an epigenetic biomarker for risk stratification in localized Ewing sarcoma CCRI responsible principal investigator: Eleni Tomazou Grant from the Austrian National Bank (OeNB), Jubiläumsfonds N : Duration: 01/01/2014 to 31/12/2015 MicroRNA profiling of tumour tissue as a predictive marker for the clinical outcome of patients treated with a novel dendritic cell-based active immunotherapy (MiRNAs & DC THERAPY) CCRI responsible principal investigator: Friedrich Erhart Grant from the Austrian Society for Haemato- Oncology (OEGHO), ASHO Research Grant 2014 Duration: 01/01/2014 to 30/06/2015 Analysis of the secretory profile and immunogenicity of drug-induced senescent tumor cells in aggressive neuroblastoma CCRI responsible principal investigator: Peter Ambros Grant from the Herzfeldersche Familienstiftung Duration: 01/07/2012 to 31/07/2015 Automated MRD-assessment in AML (flowcluster) Labdia coordinator: Michael Dworzak Grant from the Wirtschaftsagentur Wien, Call Life Sciences 2014 N : Duration: 01/03/2015 to 28/02/

44 DER UNSTILLBARE FORSCHUNGSDRANG NACH DEM WARUM? WIESO? WESHALB? DAS VERBINDET MEINE ARBEIT MIT DEN KINDERN, FÜR DIE ICH FORSCHE. Raphaela Schwentner

45 Anhang DANKSAGUNG Wir möchten uns an dieser Stelle gerne bei unseren zahlreichen privaten Spenderinnen und Spendern bedanken, die uns seit vielen Jahren treu unterstützen. Des Weiteren bedanken wir uns bei den folgenden nationalen und internationalen Fördergebern und Organisationen: 7. Forschungsrahmenprogramm der Europäischen Kommission (FP7) AG Wissenschaft und Forschung der Österreichischen Gesellschaft für Kinder- und Jugendheilkunde Bundesministerium für Wissenschaft, Forschung und Wirtschaft (BMWFW) Dachverband der Österreichischen Kinder-Krebs-Hilfe EMBT (Europäische Gruppe für Blut- und Knochenmarktransplantation) Fonds zur Förderung der wissenschaftlichen Forschung (FWF) Herzfelder sche Familienstiftung Kapsch AG Liddy Shriver Sarcoma Initiative Medac Gesellschaft für klinische Spezialpräparate mbh, Deutschland ÖGKJ im Bereich der hämatoonkologischen Forschung Arbeitsgruppe für Wissenschaft und Forschung der Österreichischen Gesellschaft für Kinder- und Jugendheilkunde, Sektion: Hämatologisch-onkologische Forschung Österreichische Akademie der Wissenschaften Österreichische Forschungsförderungsgesellschaft (FFG) Österreichische Nationalbank (OeNB) SIOPEN Association (Internationale Gesellschaft des Europäischen pädiatrisch-onkologischen Neuroblastom-Forschungsnetzwerks) Wiener Wissenschafts-, Forschungs- und Technologiefonds (WWTF) Wilhelm Sander Stiftung, München Wirtschaftsagentur Wien 88 89

46 Anhang ABGESCHLOSSENE DIPLOMARBEITEN UND DISSERTATIONEN 2015 SARAH AHMADI Costimulation blockade as a means to generate allo-specific tolerant T cells in a murine HSCT/GvHD model Supervised by Assoc. Prof. Andreas Heitger, MD PhD Thesis STEFAN BASTELBERGER Genomic aberrations and deregulation of genes in ETV6/RUNX1-positive childhood leukemia Supervised by Prof. Renate Panzer-Grümayer, MD PhD Thesis DOMINIK BOGEN Introducing a novel treatment approach and deciphering tumor heterogeneity in MYCN-amplified neuroblastoma Supervised by Assoc. Prof. Peter Ambros, PhD, and Javed Khan, MD, (NIH) PhD Thesis CHARLOTTE BREY Exploration of a bispecific antibody approach for T cell based immunotherapy of human cytomegalovirus infection Supervised by Ao. Prof. Florian Rüker (BOKU) and Assoc. Prof. Manfred Lehner, PhD MSc Thesis SARA COLOMER-LAHIGUERA The role of CDKN1B-p27kip1 deregulation in the pathogenesis of pediatric T-cell acute lymphoblastic leukemia Supervised by Sabine Strehl, PhD PhD Thesis URSULA DAUM Vergleich zweier durchflusszytometrischer Methoden zum Nachweis intrazellulärer Signaltransduktion in leukämischen Zellen Supervised by Angela Schumich BSc Thesis TERESA GERBER Investigation of the spatial and functional relationship between extrachromosomal MYCN amplicons and nuclear lamins in topotecan-induced senescent neuroblastoma cell lines Supervised by Sabine Taschner-Mandl, PhD, and Assoc. Prof. Peter Ambros, PhD MSc Thesis FIONA KRALER A cellular model system to investigate the impact of ETV6/RUNX1 in acute lymphoblastic leukemias Supervised by Prof. Renate Panzer-Grümayer, MD MSc Thesis VIKTORIA MARTINKOWITSCH Torque Teno Viren bei stammzelltransplantierten Patienten Supervised by Karin Kosulin, PhD, and Prof. Thomas Lion, MD, PhD BSc Thesis SABINE MÜLLER Alternative lengthening of telomeres (ALT) in neuroblastoma Supervised by Andrea Ziegler BSc Thesis JULIA PROFF Einfluss von Immunevasionsmechanismen des Zytomegalievirus auf die Effektivität von chimären Antigenrezeptoren für die adoptive Immuntherapie Supervised by Prof. Wolfgang Holter, MD, and Assoc. Prof. Manfred Lehner, PhD PhD Thesis VERONIKA TRIEBEL Impact of ETV6-RUNX1 expression in leukemia Supervised by Prof. Renate Panzer-Grümayer, MD MSc Thesis 90 91

47 RESEARCH MAKES MY MIND FLY. MAY IT BRING HOPE TO ME AND TO CHILDREN WITH CANCER! Reza Abbasi

48 Anhang PUBLIKATIONEN 2015 Aalbers AM, van den Heuvel-Eibrink MM, Baumann I, Dworzak M, Hasle H, Locatelli F, De Moerloose B, Schmugge M, Mejstrikova E, Novakova M, Zecca M, Zwaan CM, Te Marvelde JG, Langerak AW, van Dongen JJ, Pieters R, Niemeyer CM, van der Velden VH (2015) Bone marrow immunophenotyping by flow cytometry in refractory cytopenia of childhood. Haematologica 100: Abbasi MR, Rifatbegovic F, Brunner C, Ladenstein R, Ambros IM, Ambros PF (2015) Bone marrows from neuroblastoma patients: an excellent source for tumor genome analyses. Mol Oncol 9: Anderl S, Konig M, Attarbaschi A, Strehl S (2015) PAX5-KIAA1549L: a novel fusion gene in a case of pediatric B-cell precursor acute lymphoblastic leukemia. Mol Cytogenet 8: 48 Arico M, Astigarraga I, Braier J, Donadieu J, Gadner H, Glogova E, Grois N, Henter JI, Janka G, McClain KL, Ladisch S, Potschger U, Rosso D, Thiem E, Weitzman S, Windebank K, Minkov M, Histiocyte S (2015) Lack of bone lesions at diagnosis is associated with inferior outcome in multisystem langerhans cell histiocytosis of childhood. Br J Haematol 169: Bader P, Kreyenberg H, von Stackelberg A, Eckert C, Salzmann-Manrique E, Meisel R, Poetschger U, Stachel D, Schrappe M, Alten J, Schrauder A, Schulz A, Lang P, Muller I, Albert MH, Willasch AM, Klingebiel TE, Peters C (2015) Monitoring of minimal residual disease after allogeneic stem-cell transplantation in relapsed childhood acute lymphoblastic leukemia allows for the identification of impending relapse: results of the ALL-BFM-SCT 2003 trial. J Clin Oncol 33: Bellutti F, Kauer M, Kneidinger D, Lion T, Klein R (2015) Identification of RISC-associated adenoviral micrornas, a subset of their direct targets, and global changes in the targetome upon lytic adenovirus 5 infection. J Virol 89: Bogen D, Wei JS, Azorsa DO, Ormanoglu P, Buehler E, Guha R, Keller JM, Mathews Griner LA, Ferrer M, Song YK, Liao H, Mendoza A, Gryder BE, Sindri S, He J, Wen X, Zhang S, Shern JF, Yohe ME, Taschner-Mandl S, Shohet JM, Thomas CJ, Martin SE, Ambros PF, Khan J (2015) Aurora B kinase is a potent and selective target in MYCNdriven neuroblastoma. Oncotarget 6: Bolling T, Braun-Munzinger G, Burdach S, Calaminus G, Craft A, Delattre O, Deley MC, Dirksen U, Dockhorn-Dworniczak B, Dunst J, Engel S, Faldum A, Frohlich B, Gadner H, Gobel U, Gosheger G, Hardes J, Hawkins DS, Hjorth L, Hoffmann C, Kovar H, Kruseova J, Ladenstein R, Leuschner I, Lewis IJ, Oberlin O, Paulussen M, Potratz J, Ranft A, Rossig C, Rube C, Sauer R, Schober O, Schuck A, Timmermann B, Tirode F, van den Berg H, van Valen F, Vieth V, Willich N, Winkelmann W, Whelan J, Womer RB (2015) Development of curative therapies for Ewing sarcomas by interdisciplinary cooperative groups in Europe. Klin Padiatr 227: Boztug H, Zecca M, Sykora KW, Veys P, Lankester A, Slatter M, Skinner R, Wachowiak J, Potschger U, Glogova E, Peters C, party Epdw (2015) Treosulfan-based conditioning regimens for allogeneic HSCT in children with acute lymphoblastic leukaemia. Ann Hematol 94: Bugarin C, Sarno J, Palmi C, Savino AM, te Kronnie G, Dworzak M, Shumich A, Buldini B, Maglia O, Sala S, Bronzini I, Bourquin JP, Mejstrikova E, Hrusak O, Luria D, Basso G, Izraeli S, Biondi A, Cazzaniga G, Gaipa G, group IBs (2015) Fine tuning of surface CRLF2 expression and its associated signaling profile in childhood B-cell precursor acute lymphoblastic leukemia. Haematologica 100: e Butko E, Distel M, Pouget C, Weijts B, Kobayashi I, Ng K, Mosimann C, Poulain FE, McPherson A, Ni CW, Stachura DL, Del Cid N, Espin-Palazon R, Lawson ND, Dorsky R, Clements WK, Traver D (2015) Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo. Development 142: Creutzig U, Dworzak M, Zimmermann M, Bourquin JP, Gruhn B, Fleischhack G, Graf N, Klingebiel T, Kremens B, Lehrnbecher T, von Neuhoff C, von Stackelberg A, Stray J, Reinhardt D (2015) Randomised introduction of 2-CDA as intensification during consolidation for children with high-risk AML--results from study AML-BFM Klin Padiatr 227: Cross NC, White HE, Colomer D, Ehrencrona H, Foroni L, Gottardi E, Lange T, Lion T, Machova Polakova K, Dulucq S, Martinelli G, Oppliger Leibundgut E, Pallisgaard N, Barbany G, Sacha T, Talmaci R, Izzo B, Saglio G, Pane F, Muller MC, Hochhaus A (2015) Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia. Leukemia 29:

49 Anhang Cseh A, Niemeyer CM, Yoshimi A, Dworzak M, Hasle H, van den Heuvel-Eibrink MM, Locatelli F, Masetti R, Schmugge M, Gross-Wieltsch U, Candas A, Kulozik AE, Olcay L, Suttorp M, Furlan I, Strahm B, Flotho C (2015) Bridging to transplant with azacitidine in juvenile myelomonocytic leukemia: a retrospective analysis of the EWOG- MDS study group. Blood 125: Dantonello TM, Stark M, Timmermann B, Fuchs J, Selle B, Linderkamp C, Handgretinger R, Hagen R, Feuchtgruber S, Kube S, Kosztyla D, Kazanowska B, Ladenstein R, Niggli F, Ljungman G, Bielack SS, Klingebiel T, Koscielniak E, Cooperative Weichteilsarkom S (2015) Tumour volume reduction after neoadjuvant chemotherapy impacts outcome in localised embryonal rhabdomyosarcoma. Pediatr Blood Cancer 62: Defferrari R, Mazzocco K, Ambros IM, Ambros PF, Bedwell C, Beiske K, Benard J, Berbegall AP, Bown N, Combaret V, Couturier J, Erminio G, Gambini C, Garaventa A, Gross N, Haupt R, Kohler J, Jeison M, Lunec J, Marques B, Martinsson T, Noguera R, Parodi S, Schleiermacher G, Tweddle DA, Valent A, Van Roy N, Vicha A, Villamon E, Tonini GP (2015) Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification. Br J Cancer 112: Eckert C, Hagedorn N, Sramkova L, Mann G, Panzer-Grumayer R, Peters C, Bourquin JP, Klingebiel T, Borkhardt A, Cario G, Alten J, Escherich G, Astrahantseff K, Seeger K, Henze G, von Stackelberg A (2015) Monitoring minimal residual disease in children with high-risk relapses of acute lymphoblastic leukemia: prognostic relevance of early and late assessment. Leukemia 29: Fischer U, Forster M, Rinaldi A, Risch T, Sungalee S, Warnatz HJ, Bornhauser B, Gombert M, Kratsch C, Stutz AM, Sultan M, Tchinda J, Worth CL, Amstislavskiy V, Badarinarayan N, Baruchel A, Bartram T, Basso G, Canpolat C, Cario G, Cave H, Dakaj D, Delorenzi M, Dobay MP, Eckert C, Ellinghaus E, Eugster S, Frismantas V, Ginzel S, Haas OA, Heidenreich O, Hemmrich-Stanisak G, Hezaveh K, Holl JI, Hornhardt S, Husemann P, Kachroo P, Kratz CP, Kronnie GT, Marovca B, Niggli F, McHardy AC, Moorman AV, Panzer- Grumayer R, Petersen BS, Raeder B, Ralser M, Rosenstiel P, Schafer D, Schrappe M, Schreiber S, Schutte M, Stade B, Thiele R, Weid N, Vora A, Zaliova M, Zhang L, Zichner T, Zimmermann M, Lehrach H, Borkhardt A, Bourquin JP, Franke A, Korbel JO, Stanulla M, Yaspo ML (2015) Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options. Nat Genet 47: Freimuller C, Stemberger J, Artwohl M, Germeroth L, Witt V, Fischer G, Tischer S, Eiz-Vesper B, Knippertz I, Dorrie J, Schaft N, Lion T, Fritsch G, Geyeregger R (2015) Selection of adenovirus-specific and Epstein-Barr virus-specific T cells with major histocompatibility class I streptamers under Good Manufacturing Practice (GMP)-compliant conditions. Cytotherapy 17: Gaspar N, Hawkins DS, Dirksen U, Lewis IJ, Ferrari S, Le Deley MC, Kovar H, Grimer R, Whelan J, Claude L, Delattre O, Paulussen M, Picci P, Sundby Hall K, van den Berg H, Ladenstein R, Michon J, Hjorth L, Judson I, Luksch R, Bernstein ML, Marec-Berard P, Brennan B, Craft AW, Womer RB, Juergens H, Oberlin O (2015) Ewing sarcoma: current management and future approaches through Collaboration. J Clin Oncol 33: Heitzeneder S, Zeitlhofer P, Potschger U, Nowak E, Seidel MG, Holzl M, Lawitschka A, Forster-Waldl E, Matthes-Martin S, Heja D, Haas OA, Heitger A (2015) Mannan-binding lectin deficiency attenuates acute GvHD in pediatric hematopoietic stem cell transplantation. Bone Marrow Transplant 50: Hochedlinger N, Nitzlnader M, Falgenhauer M, Welte S, Hayn D, Koumakis L, Potamias G, Tsiknakis M, Saraceno D, Rinaldi E, Ladenstein R, Schreier G (2015) Standardized data sharing in a paediatric oncology research network--a proof-ofconcept study. Stud Health Technol Inform 212: Inaba H, Zhou Y, Abla O, Adachi S, Auvrignon A, Beverloo HB, de Bont E, Chang TT, Creutzig U, Dworzak M, Elitzur S, Fynn A, Forestier E, Hasle H, Liang DC, Lee V, Locatelli F, Masetti R, De Moerloose B, Reinhardt D, Rodriguez L, Van Roy N, Shen S, Taga T, Tomizawa D, Yeoh AE, Zimmermann M, Raimondi SC (2015) Heterogeneous cytogenetic subgroups and outcomes in childhood acute megakaryoblastic leukemia: a retrospective international study. Blood 126: Karawajew L, Dworzak M, Ratei R, Rhein P, Gaipa G, Buldini B, Basso G, Hrusak O, Ludwig WD, Henze G, Seeger K, von Stackelberg A, Mejstrikova E, Eckert C (2015) Minimal residual disease analysis by eight-color flow cytometry in relapsed childhood acute lymphoblastic leukemia. Haematologica 100: Klein K, Kaspers G, Harrison CJ, Beverloo HB, Reedijk A, Bongers M, Cloos J, Pession A, Reinhardt D, Zimmerman M, Creutzig U, Dworzak M, Alonzo T, Johnston D, Hirsch B, Zapotocky M, De Moerloose B, Fynn A, Lee V, Taga T, Tawa A, Auvrignon A, Zeller B, Forestier E, Salgado C, Balwierz W, Popa A, Rubnitz J, Raimondi S, Gibson B (2015) Clinical impact of additional cytogenetic aberrations, ckit and RAS mutations, and treatment elements in pediatric t(8;21)-aml: Results from an international retrospective study by the international berlin-frankfurt-munster study group. J Clin Oncol 33:

50 MIR IST DIE AUFGABE MEINER ARBEIT BEWUSST. ES GEHT UM DIE WICHTIGE ENTSCHEIDUNG BEZÜGLICH OPTIMALER THERAPIE. DAS HÄLT MICH WACH. Angela Schumich