Summary • Editorial June 2019 • 31St

Summary

• Editorial June 2019 • 31st Pezcoller Symposium - Program - Abstracts of oral presentations - Abstracts of posters • Call for nomination 2020 Pezcoller Foundation-AACR International Award for Extraordinary Achievement in Cancer Research • Call for nomination Scholar-In-Training Awards

News from the Year 29 – No. 52 Pezcoller Foundation World June 2019 Picture on front page: 2019 Pezcoller Foundation-AACR International Award for Extraordinary Achievement in Cancer Research winner: Prof. Alberto Mantovani June 2019 Editorial

Integrative Biology) of the University of Trento. Editorial In the Award Ceremony, President Galligioni highlighted the main points of Mantovani’s researches and discoveries. “Dr. Mantovani is an eminent physician-scientist, he said, a leader in the field of tumor immunology for decades. Presently Scientific Di- rector at the Istituto Clinico Humanitas and Full Professor of General Pathology at the Humanitas University, Mantovani was for many years Head of the Department of Immunology and Cell Biol- ogy, Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy. Furthermore he holds the Chair of Inflammation and Therapeutic Innova- tion, at the Queen Mary University, London, UK. By identifying macrophages in tumors as corrupted policemen promoting cancer progression, and by discovering relevant genes and functions, Al- berto Mantovani highlighted the role of inflammation and immunity in the tumor microenvi- First of all we are reporting with a great pleas- ronment, a paradigm shift fundamental for the ure that the recipient of 2019 Pezcoller Foun- development of tumor immunology and immu- dation – AACR International Award for Extraor- notherapy. In addition, his researches includ- dinary Achievement in Cancer research is Prof. ed Chemokines (CCL2), IL-1/Toll-like receptors Alberto Mantovani, scientific Director at the (TLR) and Humoral innate immunity (PTX3).” Istituto Clinico Humanitas and President of the “Alberto Mantovani is the most cited Italian sci- Fondazione Humanitas for Research in Milan. entist working in Italy (Scopus, Web of Science, Mantovani was chosen among 21 nominees, by Google scholar) and a bibliometric analysis indi- the Selection Committee chaired by Michael cates that he is one of the 10 most quoted im- B. Kastan MD, PhD, who met in Philadelphia on munologists in the world. November 30 2018. The other members of the Committee were: Mariano Barbacid, PhD, FAACR; He currently serves as president of Internation- Nina Bhardwaj, MD, PhD; Carlos M. Caldas MD; al Union of Immunological Societies (IUIS) and Elisabetta Dejana PhD; Joe W. Gray PhD FAACR; is past president of the Italian Society of Immu- Lorenzo Moretta MD; David A. Tuveson MD, PhD; Jennifer Wargo MD. The Award to Mantovani was formally announced at the AACR annual Meeting in Atlanta, on Sun- day, March 31, 2019, where he delivered his award lecture. The award prize was then offi- cially presented to Mantovani by the President of the Pezcoller Foundation Prof. Enzo Galligio- ni, MD on May 11 in the Teatro Sociale of Tren- to, during the Award Ceremony at the presence of the of Elizabeth M. Jaffee, MD Past Presi- dent of AACR, Margaret Foti PhD, MD, CEO of AACR, Gios Bernardi MD, President Emeritus of the Pezcoller Foundation and large part of the Trento Community. Before the Award Ceremony Mantovani gave a Lectio Magistralis at the at the Department of Molecular Medicine of the University of Padova and a second lecture at the CIBIO (Centre for

The Pezcoller Foundation Journal - June 2019 3 nology and the International Cytokine Society. In addition to the Award, I would like to mention He serves as Editor-in-Chief, Seminars in Immu- that on the next June 17-18, we will hold our 31st nology and senior editor of Cancer Immunology Pezcoller Symposium. In this year’s Symposium, Research the focus will be on the newest insights into the processes that give rise to cancer tissue and its Mantovani is actively involved in the fostering persistence. Cancers are, in essence, products of science and scientific policies in Italy and of organ and tissue development gone wrong. cofounded the association “ Gruppo2003” of In fact, many critical cellular and organ-based Italian highly cited scientists (http://www.grup- operations from genome integrity, the behavior Editorial po2003.it) and the website http://www.scien- of one’s own microbiome, oxygen utilization and zainrete.it. “ cell metabolism, orderly tissue and organ anat- omy, disciplined cell behavior, and the immune During the ceremony as President Emeritus I response may become disordered in the interest made some remarks where I tried to point out of tumor development. Also included will be re- the human and social personality of the winner, cent findings that have the potential to influ- referring to Mantovani’s publications aimed to ence clinical cancer therapeutics in novel ways. young scholars: In the next pages you will find the program and …”Alberto Mantovani want to convey the typical the faculty. sense of adventure of science. He recommends Finally I’m pleased to particularly remember, a passion for the work, the modesty of learning among all other activities of the Pezcoller Foun- from everyone, especially from collaborators dation, the Agreement between the Pezcoller and technicians….. Foundation and the Museum of Science of Tren- Mantovani, although deeply engaged on scientif- to (MUSE), aimed to mutually collaborate to the ic researches, never forget his being a physician diffusion of the scientific awareness among gen- and that the ultimate concern, not only for a eral population. physician but also for any scientific researcher, must be the patient, from the bench to the hos- Gios Bernardi pital bed…” Editor

4 The Pezcoller Foundation Journal - June 2019 31th Pezcoller Symposium CANCER AS A CORRUPTED TISSUE Trento, Italy • June 17 - 18, 2019 PROGRAM

MONDAY JUNE 17, 2019 TUESDAY JUNE 18, 2019 8.00 Registration 8.30 Enzo Galligioni Welcome 8.40 David Livingston Focus & Goals Session 4 - Tissue and Organ Formation Chair: Cathrin Brisken The Enrico Mihich Lecture 08.30 Cedric Blanpain Chair: David Livingston Cancer cell of origin, tumor heterogeneity and EMT 8.50 Sean Morrison transitional state The metabolic regulation of cancer progression 9.35 Discussion 08.55 Discussion 09.10 Stefano Piccolo Session 1 - The Microbiome and Cancer Tumors as wounds that never heals: a YAP/TAZ Chair: Maria Rescigno perspective 09.50 Romina Goldszmid 09.35 Discussion Microbiota as a key modulator of the tumor 09.50 Nikolaus Rajewsky microenvironment Principles of Gene Regulation by Single-Cell RNA 10.15 Discussion Sequencing 10.30 Jennifer Wargo The role of the gut and tumor microbiome in response 10.15 Discussion to cancer therapy 10.30 Coffee Break 10.55 Discussion 11.10 Coffee Break Session 5 - Cancer and Metabolism Chair: Massimo Loda Session 2 - Cancer Metastasis 10.50 William Sellers Chair: Stefano Piccolo The Next Generation Characterization of the Cancer Cell 11.30 Christoph Klein Line Encyclopedia - implications for targeting metabolic From early dissemination to manifest alterations in cancer metastasis:Theoretical and practical challenges of an unsolved problem. 11.15 Discussion 11.55 Discussion 11.30 Matt Vander Heiden 12.10 Mikala Egeblad Metabolic limitations of cancer progression Neutrophil extracellular traps generated 11.55 Discussion during inflammation drive cancer cell proliferation and a pro-metastatic microenvironment 12.10 Lunch 12.35 Discussion Session 6 - Cancer and Immunology 12.50 Lunch Chair: Alberto Bardelli Session 3 - Genome Order and Disorder 13.20 Andrea Ablasser Chair: Fabrizio D’Adda di Fagagna Expanding roles of cGAS in immunity and 14.00 Roger Greenberg inflammation The necessity of noncanonical DNA damage reponses 13.45 Discussion for human cancer 14.00 Catherine Wu 14.25 Discussion Addressing cancer heterogeneity: personalized cancer 14.40 Daniel Durocher vaccines Charting the genetic architecture of the DNA damage 14.25 Discussion response 15.05 Discussion 14.40 Poster Discussion and Poster Presentation (led by 15.20 Simon Boulton Massimo Loda) Maintaining telomeres in ALT cancers 15.40 David Livingston 15.45 Discussion Concluding Remarks 16.00 Fabrizio D’Adda di Fagagna The role of non coding RNA in genome intgrity 16.25 Discussion 16.40 Poster View 17.30 Adjourn

19.30 Symposium Dinner

The Pezcoller Foundation Journal - June 2019 5 INVITED PARTECIPANTS

FACULTY

• Ablasser Andrea Swiss Federal Institute of Technology, Lausanne, CH • Bardelli Alberto Candiolo Cancer Institute, Univerwsity of Torino, IT • Blanpain Cédric Université Libre de Bruxelles, BE • Boulton Simon The Francis Crick Institute, London, UK • Brisken Cathrin Swiss Federal Institute of Technology, Lausanne, CH • D’Adda di Fagagna Fabrizio IFOM – The FIRC Institute of Molcecular Oncology, Milano, IT • Durocher Daniel Lunenfeld-Tanenbaum Research Institute Mount Sinay Hospital, Toronto, Canada • Egeblad Mikala Cold Spring Harbor Laboratory, Cold Spring Harbor, NY • Goldszmid Romina National Cancer Institute, Bethesda, MD • Greenberg Roger Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA • Klein Christoph University of Regensburg, Regensburg, D • Livingston David Dana Farber Cancer Institute, Boston, MA • Loda Massimo Weill Cornell Medicine, New York, NY • Morrison Sean Children Research Institute at UT Southwestern, Dallas, TX • Piccolo Stefano Department of Molecular Medicine, University of Padova, Padova, IT • Rajewsky Nikolaus The Berlin Institute for Medical System Biology, Berlin, D • Rescigno Maria Humanitas University, Milano, IT • Sellers William Broad Institute of MIT, Cambridge, MA • Vander Heiden Matthew Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA • Wargo Jennifer MD Anderson Cancer Center, Houston, TX • Wu Catherine Dana Ferber Cancer Institute, Boston, MA

6 The Pezcoller Foundation Journal - June 2019 31th Pezcoller Symposium Cancer as a corrupted tissue Trento, Italy, June 17-18, 2019 Abstracts ABSTRACTS OF ORAL PRESENTATIONS

The metabolic regulation of melano- that rather than treating cancer with anti-oxidants, which have promoted cancer initiation ma metastasis and progression in large clinical trials, that we should devise pro-oxidant therapies that inhib- Sean J. Morrison it cancer progression by exacerbating oxidative stress. A major focus of my lab is understanding Investigator, Howard Hughes Medical Institute the mechanisms cancer cells use to cope with Professor, Children’s Research Institute oxidative stress, with a view to developing ther- University of Texas Southwestern Medical apies that inhibit these mechanisms. Center A recent discovery is that metabolic differences To study the mechanisms that regulate distant among melanomas, and even among melanoma metastasis by melanoma cells, we developed cells from the same tumor, confer differences a xenograft assay in which small numbers of in metastatic potential by modulating the abil- cells from primary human melanomas engraft in ity to cope with oxidative stress. We observe null NOD/SCID IL2Rγ (NSG) mice and spontaneous- differences in Monocarboxylate Transporter 1 ly metastasize (Nature 456:593). We have now (MCT1) expression among melanomas that cor- banked and characterized the metastatic poten- relate with differences in metastatic potential tial of melanomas from more than 150 patients and differences in survival among patients. The in this assay and observed a correlation between main physiological function of MCT1 is to trans- spontaneous metastasis in NSG mice and metas- port lactate in and out of cells. We found that tasis in patients: melanomas that are destined MCT1 function promotes melanoma metastasis to form distant metastases in patients sponta- by reducing oxidative stress. Efficiently and in- neously form distant metastases in the mice efficiently metastasizing patient-derived xeno- (‘efficient’ metastasizers), while melanomas grafts similarly metabolized isotopically-labeled that do not form distant metastases in patients glucose and glutamine; however, efficient me- metastasize more slowly in mice (‘inefficient’ tastasizers took up more lactate. Efficient me- metastasizers) (Science Translational Medicine tastasizers expressed higher levels of MCT1 and 4:159ra149). This demonstrates there are intrin- MCT1 inhibition reduced lactate uptake in vivo. sic differences among melanomas from differ- MCT1 inhibition had little effect on the growth ent patients in their potential to metastasize. of primary subcutaneous tumors but substantially depleted circulating melanoma cells and re- Using this assay, we discovered that metasta- duced metastatic disease burden. MCT1 inhibi- sizing melanoma cells experience high levels tion suppressed the oxidative pentose phosphate of oxidative stress and that the rare melanoma pathway and increased reactive oxygen species cells that successfully metastasize undergo re- (ROS) levels. Anti-oxidant treatment rescued the versible metabolic changes that increase their effect of MCT1 inhibition on metastasis. MCT1high capacity to survive oxidative stress, including and MCT1-/low cells from the same melanomas increased dependence on the folate pathway had similar capacities to form subcutaneous tu- (Nature 527:186). Treatment of xenografted mors, but MCT1high cells formed more metastases mice with anti-oxidants increases the frequen- after intravenous injection. Metabolic differenc- cy of circulating melanoma cells in the blood es among cancer cells htus confer differences as well as metastatic disease burden, demon- in metastatic potential as metastasizing cells strating that oxidative stress is one factor that depend upon MCT1 to manage oxidative stress. limits distant metastasis. These data suggest MCT1 inhibition is thus a pro-oxidant therapy

The Pezcoller Foundation Journal - June 2019 7 that might impair disease progression in patients Experimental Medicine and Therapy Research, with high risk stage II and III melanomas. University of Regensburg and Fraunhofer Insti- tute of Toxicology and Experimental Medicine, Regensburg, Germany Microbiota as a key modulator of the tumor microenvironment Mutation, selection and adaptation are - by convention - thought to occur primarily within, and to a lesser degree, outside the primary tu- Romina S. Goldszmid mour. However, we previously noted in breast cancer and melanoma that metastatic dissem-

Abstracts Inflammatory Cell Dynamics Section, Cancer ination occurs often early and that advanced and Inflammation Program, CCR, NCI, Bethes- tumour stages seed relatively fewer cells. This da, MD. indicates that metastatic founder cells may lodge and evolve considerable periods of time Cancer has historically been viewed as a disease outside the primary tumour. However, it gen- determined by genetic and environmental fac- erates a plethora of questions: How do can- tors, however, it is now clear that inflammation cer cells survive at distant sites? From which affects all stages of the disease: initiation, pro- disseminated cancer cells descent metastatic gression and metastasis formation. The inflamed colonies? Can genomic evolution occur during tumor microenvironment is in part sustained by dormancy in quiescent cells? Does the environ- infiltrating myeloid cells such as macrophages, ment trigger progression of early-disseminated monocytes, dendritic cells, and neutrophils. In cancer cells or do late-arriving cancer cells cancer, as in infection, these cells can induce take over incipient metastatic colonies? We try adaptive immune responses, but in cancer they to address these questions by analysing single mainly promote the tumor’s immune evasion, disseminated cancer cells isolated at various progression, and metastasis. Moreover, the role time points of disease. Our data suggest that of distinct myeloid cell populations in response available models and endpoint analyses – such to cancer therapy remains unclear. We and oth- as the comparison of primary tumours and me- ers have previously uncovered a role for com- tastases - are insufficient to reflect disease mensal microbes in controlling the response to dynamics in patients. Therefore, we have to cancer immuno- and chemotherapy. In this pres- carefully consider the clinical and evolutionary entation, we will discuss the role of the micro- stage of an individual disease when we try to biota in regulating the composition and function address the underlying mechanisms. of the myeloid cell compartment in the tumor microenvironment and the role of these cells in the response to cancer therapy. Targeting myeloid cells in the tumor microenvironment rep- Neutrophil extracellular traps pro- resents a powerful approach to manipulate the duced during inflammation awakens outcome of cancer therapy; therefore, a clear dormant cancer cells understanding of their regulation and functional organization may lead to rational novel cancer Mikela Egeblad immunotherapeutic approaches. Cold Spring Harbor Laboratory Cold Spring Harbor, NY The Role of the Gut and Tumor Microbiome in Response to Cancer Every year, ~40,000 women in the US who had been successfully treated for primary breast Therapy cancer nonetheless have metastatic recurrence. Metastasis requires four key steps: 1) tumor Wargo Jennifer cells leave the tumor; 2) tumor cells enter a new tissue; 3) disseminated tumor cells (DTCs) MD Anderson Cancer Center, Houston, TX re-initiate proliferation; and 4) an inflammatory ( missing) microenvironment is established to support the growing metastasis. Steps 1-2 are rarely amena- ble to intervention, as they usually occur be- From early dissemination to mani- fore the primary tumor is detected and treated. fest metastasis:Theoretical and prac- However, we may be able to target steps 3-4 to ultimately reduce the occurrence of metastasis tical challenges of an unsolved prob- and its associated mortality. lem. Our research on neutrophil extracellular traps (NETs) has provided novel insights into how in- Christoph A. Klein flammation can cause a) DTCs to re-initiate

8 The Pezcoller Foundation Journal - June 2019 proliferation, and b) result in a metastasis-sup- that use templated DNA synthesis to execute ei- porting inflammatory microenvironment. NETs ther high fidelity restoration of lesions to their are released by neutrophils to the extracellular ground state or inherently error prone mech- space in response to infections and inflamma- anisms that result in loss of genome integrity. tion, and they consist of meshes of genomic DNA This prominence of repair mechanism utilization with ~40 associated proteins, including proteas- is illustrated in the setting of homologous re- es and high mobility group box 1 (HMGB1). We combination deficiency due to mutation within recently reported that lung inflammation, in- a network of genes centered around the breast duced by either tobacco smoke exposure or bac- and ovarian cancer suppressor proteins. This terial lipopolysaccharide (LPS), drove quiescent BRCA network is required for high fidelity DNA

DTCs to re-initiate proliferation. This resulted repair by homologous recombination. While de- Abstracts in metastases that killed mice with experimen- ficiency in this canonical homology directed DNA tal lung inflammation in <4 weeks, while control repair pathway is confers cancer susceptibility, mice survived >8 months without metastasis. it also creates vulnerability to agents that tar- Using intravital imaging, we found that DTCs get orthogonal repair mechanisms. Poly(ADP) Ri- that exit quiescence after lung inflammation bose Polymerase (PARP) inhibitors are approved are surrounded by NETs. We further discovered to treat homologous recombination deficiency that proteases on NETs cleave the basement breast and ovarian cancer, with demonstrated membrane protein laminin, generating a b1-in- improvements in progression free survival in tu- tegrin-activating epitope that caused quiescent mors with BRCA mutations. The efficacy of PARP DTCs to re-initiate proliferation. Thus, NET-as- inhibitors depends on expression of the PARP1 sociated proteases drive step 3 of the metastat- enzyme, which becomes trapped on chromatin ic process. Our new data now show NETs also and necessitates intact BRCA dependent HR for drive a complex, feed-forward-loop between repair in S-phase. In the absence of BRCA1, toxic NETs, macrophages, and fibroblasts leading to use of nohomologous endjoining repair mecha- the creation of a highly inflammatory microen- nisms creates dicentric chromosomes that result vironment. We propose that this NET-induced in a loss of viability upon passage through mito- microenvironment is critical for sustaining the sis. The intersection of these repair pathways in proliferation and survival of the metastasizing PARPi response leads to resistance mechanisms cancer cells. By dissecting the mechanisms by that entail either loss of PARP1 expression or which NETs promote re-initiation of proliferation deficiency in nohomologous endjoining repair. of quiescent DTCs and generation of a sustained These are thought to account, at least in part, inflammatory microenvironment, we have iden- for failure of ~50% of BRCA mutated cancers tified several points of potential intervention to to respond to PARPi. Ideally, additional targets prevent dormant cell awakening and prolong the could be identified that circumvent PARPi resist- survival of cancer patients. ance regardless of mechanism and restore efficacy in BRCA mutated cancers. This presentation will describe our unpublished A novel chromatin directed vulnera- results that implicate the chromatin remodeling protein CHD1L as a novel therapeutic tar- bility in BRCA mutated cancers get in BRCA mutated cancers. Functional do- main directed CRISPR-Cas9 screens were used to Priyanka Verma, Junwei Shi and Roger A Green- identify CHD1L as a vulnerability in BRCA1 and berg BRCA2 mutated cells. Our findings demonstrate that CHD1L loss is synthetic lethal in combina- Department of Cancer Biology, Basser Center tion with mutation to either BRCA1 or BRCA2, for BRCA, Perelman School of Medicine, Univer- while conferring extreme PARPi hypersensitivity sity of Pennsylvania, 421 Curie Boulevard, Phil- regardless of resistance mechanism. Models to adelphia, Pennsylvania 19104, USA. conceptualize these findings will be presented as well as their implications for harnessing DNA The DNA damage response encompasses acute damage responses to enhance therapy in homol- and delayed signaling events that culminate in ogous recombination deficient cancers. repair of genomic lesions and the production of inflammatory cytokines that attract immune responses. This multifaceted DNA damage re- Charting the Genetic Architecture sponse is critical to cancer etiology and response to therapy, particularly in light of the of the DNA Damage Response realization that DNA damaging therapies can synergize with immune checkpoint blockade Durocher Daniel to eradicate tumors. A central aspect of the DNA damage response is the induction of myr- Lunenfeld-Tannenbaum Research Institute iad homology directed DNA repair mechanisms Mount Sinai Hospital, Toronto, Canada

The Pezcoller Foundation Journal - June 2019 9 The orchestration of DNA repair is of fundamen- in the ALT process is highlighted by its inactiva- tal importance to the maintenance of genomic tion in a subset of ALT positive osteosarcomas. integrity and tumor suppression. DNA damage Collectively, our findings uncover an SLX4IP-de- must be detected in the context of the varied pendent regulatory mechanism critical for tel- chromatin landscape, its presence must be com- omere maintenance in ALT cancer cells. municated throughout the cell to alter many ongoing processes, and the machinery that will mend the lesion must be recruited to the The role of non coding RNA in ge- damage site. In my presentation, I will discuss our recent efforts in mapping genome mainte- nome integrity

Abstracts nance pathways using genome-scale CRISPR/ Cas9 screens in human cells. I will highlight Fabrizio d’Adda di Fagagna1,2 how these screens can be used to identify new genome stability factors, characterize drug re- 1IFOM Foundation, Milan, 20139, Italy;; 2Istitu- sponses and provide new insights into the genet- to di Genetica Molecolare, National Research ic architecture of the genome stability network Council, Pavia, 27100, Italy. by identifying potentially actionable synthetic lethal genetic interactions. I will argue that so- The DNA damage response (DDR) is a signaling matic genetic screens in human cells are power- pathway physiologically activated in cancer in- ful tools to study the DNA damage response and itiation (Di Micco et al Nature 2006) and ageing its integration within other cellular pathways. (d’Adda di Fagagna et al. Nature 2003, Fumagal- li et al. Nature Cell Biology 2012). More recently, we reported that DNA double- Maintaining telomeres in ALT can- strand breaks (DSBs) trigger the synthesis of damage-induced long non-coding RNA (diln- cers cRNA) that can be processed into shorter DNA damage response RNAs (DDRNAs) (Francia et al Simon J. Boulton Nature 2012). Such transcripts are essential for full DDR activation and their inhibition by anti- Tha Francis Crick Institute, London, UK sense oligonucleotides (ASO) allows site-specific inhibition of DNA damage signalling and repair In non-malignant somatic cells, telomeres un- (Michelini et al Nature Cell Biology 2017; D’Ales- dergo progressive shortening after DNA repli- sandro et al Nature Communications). cation, which eventually results in replicative We will discuss our progress in understanding senescence and checkpoint-driven cell death. the mechanisms of dilncRNA synthesis by the In contrast, tumour cells achieve replicative different factors associated with the RNA pol II immortality by activating one of two distinct holoenzyme in cells and in a novel reconstitut- telomere maintenance mechanisms; cancers ed in vitro system, challenging the distinction either re-express telomerase or induce Alter- between a DNA lesion and a transcriptional pro- native Lengthening of Telomeres (ALT), which moter.In addition, we will show how sequence- involves recombination between telomeres. specific DDR inhibition can be achieved in vivo ALT is utilised in 10-15% of all tumours and is by targeting dilncRNA and DDRNA and we will associated with poor prognosis due to their discuss its potential therapeutic uses. complex karyotype and lack of targeted therapies. Currently, the mechanisms underpinning ALT induction and maintenance remain poorly Cancer cell of origin, tumor hetero- understood. Here, we show that ALT recombination requires coordinate regulation of the geneity and EMT transitional state SMX resolvasome and BTR complex to ensure the appropriate balance of resolution and dis- Cédric Blanpain solution activities at recombining telomeres. Critical to this control is SLX4IP, which accumu- WELBIO, Laboratory of Stem Cells and Cancer, lates specifically at ALT telomeres and interacts Université Libre de Bruxelles (ULB), 1070 Brux- with SLX4, XPF and BLM. Loss of SLX4IP results elles, Belgium in a hyper-ALT phenotype that is incompatible with cell viability following concomitant loss of Different theories have been proposed to ex- SLX4. Inactivation of BLM is sufficient to res- plain tumour heterogeneity including the cancer cue toxic telomere aggregation and synthetic cell of origin. Here, we developed new genet- lethality in this context, suggesting that SLX4IP ically engineered mouse models allowing line- favours SMX-dependent resolution by antago- age tracing together with oncogenic activation nising promiscuous BLM activity during ALT re- in different cell lineages of the skin epidermis combination. The clinical importance of SLX4IP and the mammary gland and assessed whether

10 The Pezcoller Foundation Journal - June 2019 the cancer cell of origin controls tumour hetero- The Next Generation Characteriza- geneity. I will present evidence that the cancer tion of the Cancer Cell Line Ency- cell of origin controls tumour heterogeneity and the underlying molecular mechanisms by which clopedia implications for targeting the cell of origin control tumor differentiation, metabolic alterations in cancer. stemness, EMT, resistance to therapy and metastasis. These results have important implica- William Sellers tions for our understanding of the mechanisms Broad Institute of MIT, Cambridge, MA controlling tumor heterogeneity and the development of new strategies to block tumor initi- The Cancer Cell Line Encyclopedia, a large-scale ation, progression, metastasis and resistance collection of well annotated cell lines, has pro- Abstracts to therapy. This work is supported by the ERC, vided a rigorous backbone upon which to study WELBIO, FNRS, TELEVIE, and the Fondation Bail- genetic variants, candidate targets, small mol- let-Latour. ecule and biologic therapeutics and to identify new marker driven cancer dependencies. With the goal of more fully understanding molecular features that contribute to cancer phenotypes Tumors As Wounds That Never including drug response we have expanded the Heal: A YAP/TAZ Perspective cell line characterizations to include genetic, RNA splicing, DNA methylation, histone H3 mod- Stefano Piccolo ification, miRNA expression, metabolomic and reverse phase protein-array data for 1,072 cell Dip. Medicina Molecolare Università di Padova lines from various lineages and ethnicities. Inte- grating these data with functional characteriza- Enhanced YAP/TAZ activity is emerging as com- tion including drug sensitivity, shRNA knockdown mon trait of multiple solid tumors in humans. and CRISPR/Cas9 knockout data reveals poten- Strikingly, in mouse models, adult organs lack- tial new cancer drug targets and associated bio- ing YAP/TAZ are unable to develop tumors with- markers. In addition, the unbiased association out overt side effects for normal tissue home- analysis linking cancer metabolome to genetic ostasis, making YAP/TAZ prime candidates for alterations, epigenetic reprogramming, and gene dependency provides a detailed descrip- cancer therapy. That said, YAP/TAZ have phys- tion of metabolic consequences of the cancer iological functions during tissue repair, being es- dysregulated genome. sential for organ regeneration after injury. YAP/ Importantly, in the metabolic space we observed TAZ are typically inactive in normal tissues but distinct patterns of cell autonomous synthesis potently induced by mechanical and physical and secretion of kynurenine, an immune-sup- cues that the cell receives from its microenvi- pressive metabolite. Furthermore, we found ronment, such as extracellular matrix stiffness significant variation in the ability to suppress ky- and topology, and 3D architectural features of nurenine with IDO1 selective inhibitors. Finally, the tissue. I will focus on new discoveries on the by manipulating amino acid availability in large- mechanisms by which YAP/TAZ are controlled by scale screens of >500 barcoded cell lines, we mechanotransduction, and on YAP/TAZ as repro- demonstrated that aberrant ASNS hypermeth- gramming factors in epithelial cells. Indeed, in ylation sensitizes subsets of gastric and hepat- normal tissues YAP/TAZ activation turns more ic cancers to asparaginase therapy. Together, differentiated normal cells into cells endowed this dataset and an accompanying public data with stem-like properties; in tumors, YAP/TAZ portal provide a resource to accelerate cancer convert non-stem tumor cells into cancer-stem research. cells, increasing tumor aggressiveness and fueling metastasis. I will also expand on these References: mechanisms, providing new hints for therapeu- Li H, Ning S, Ghandi M1, Kryukov GV, Gopal S, tic intervention. Deik A, Souza A, Pierce K , Keskul P, Hernandez D, Ann J, Shkoza D, Apfel V, Zou1, Vazquez F, Barretina J, Pagliarini RA, Galli GG, Root D1, Principles of Gene Regulation by Hahn WC, Golub TR , Tsherniak A, Giannakis M1,2, Stuart L. Schreiber S, Clish CB, Garraway Single-Cell RNA Sequencing LA, Sellers WR. The Landscape of Cancer Cell Line Metabolism. Nature Medicine 2019 (in Nikolaus Rajewsky press).

The Berlin Institute for Medical System Biology, Ghandi M, Franklin W. Huang FW, Jané-Val- Berlin, Germany buenav J, Kryukov GV, Lo CC, McDonald III ER, (missing ) Barretina J, Gelfand ET, Bielski CM, Li H, Hu

The Pezcoller Foundation Journal - June 2019 11 K, Andreev-Drakhlin AY, Kim J, Hess JM, Haas Addressing cancer heterogeneity: BJ, Aguet F, Weir BA, Rothberg MV, Paolella BR, Lawrence MS, Akbani R, Lu Y, Tiv HL, Gokhale personalized cancer vaccines PC, deWeck A, Mansour AA, Oh C, Shih J, Hadi K, Rosen Y, Bistline J, Venkatesan K, Reddy A, Catherine J. Wu, M.D. Sonkin D, Liu M, Lehar J, Korn JM, Porter DA, Professor of Medicine Jones MD, Golji J, Caponigro G, Taylor JE, Dun- Chief, Division of Stem Cell Transplantation and ning CM, Creech AL, Warren AC, McFarland JM, Cellular Therapies Zamanighomi M, Kauffmann A, Stransky N, Im- Department of Medical Oncology ielinski M, Maruvka YE, Cherniack AD, Tsherni- 2 Abstracts ak A, Vazquez F, Jaffe JD, Lane AA , Weinstock Dana-Farber Cancer Institute and Harvard Med- DM, Johannessen CM, Morrissey MP, Stegmeier ical School, Boston MA USA F, Schlegel R, Hahn WC, Getz G, Mills GB, Boe- hm JS, Golub TR, Garraway LA, Sellers WR Next The recent successes and challenges of cancer generation characterization of the Cancer Cell immunotherapy have motivated intense inves- Line Encyclopedia. Nature 2019 (in press) tigation of the molecular and cellular determi- nants of therapeutic response. The generation of broad computational and analytic tools to Metabolic limitations of cancer pro- directly probe human samples has led to the emergence of systematic approaches to meet gression this challenge. At the heart of productive anti-tumor immune responses is the interaction of Matthew G. Vander Heiden the T cell and the antigen presenting cell, with recognition of antigen by the T cell receptor Koch Institute for Cancer Research, Massachu- (TCR); these interactions are further impact- setts Institute of Technology, Cambridge MA USA ed by heterogeneous immune cell populations within the tumor microenvironment. While the Complex regulatory mechanisms enable cell search for immunogenic tumor antigens has been metabolism to match physiological state. The the subject of decades-long studies, multiple major pathways cells use to turn nutrients into lines of evidence have convincingly demonstrat- energy and to synthesize macromolecules have ed tumor neoantigens as an important class of been elucidated; however, there remain many immunogenic tumor antigens. Neoantigens arise unanswered questions regarding how metabo- from amino acid changes encoded by somatic lism supports cancer cell proliferation and thus mutations in the tumor cell and have the poten- how best to target metabolism for cancer treat- tial to bind to and be presented by personal HLA ment. Of note, many existing cancer therapies molecules. Using next-generation sequencing target metabolic processes that are thought to approaches, we can now systematically identify be essential in all cells. This raises a key ques- mutations leading to amino acid changes that tion of how drugs which target these processes can be potentially recognized immunological- show differential effectiveness to treat can- ly through the implementation of neoantigen cer as well as a therapeutic window between discovery pipelines. In recent studies, we have cells. We have characterized the factors that demonstrated that neoantigen load is associ- drive sensitivity to disruption of specific meta- ated with clinical outcome to immune-based bolic processes, and find that both cancer cell therapies, and neoantigens can be safely and extrinsic and intrinsic factors dictate metabolic feasibly targeted to generate customized can- vulnerabilities. Further, this leads to different cer vaccines. We have been undertaking pilot cells being more reliant on limiting nutrients clinical trials to develop personal cancer vac- for different metabolic processes, creating a cines in melanoma and glioblastoma that utilize framework to understand how cancers become synthetic long peptides as delivery approach for differentially sensitive to agents which target this therapy. Recent results and new directions otherwise essential pathways. will be discussed.

12 The Pezcoller Foundation Journal - June 2019 ABSTRACTS OF POSTERS Posters In-vivo reprogramming of postmito- Alessandro Alaimo1, Marco Lorenzoni1, Paolo Am- brosino2, Arianna Bertossi1, Alessandra Bisio1, tic neurons induces medulloblastoma Alice Macchia1,3, Eugenio Zoni4, Sacha Genove- si1, Francesco Cambuli1, Veronica Foletto1, Dario Giuseppe Aiello CIBIO De Felice1, Maria Virginia Soldovieri2, France- sco Gandolfi1, Gianluca Petris1, Anna Cereseto1, PhD Student: Giuseppe Aiello Armenise-Harvard La- Maria Caterina Mione1, Marco Durante5, Alvaro boratory of Brain Disorders and Cancer Centre for Villarroel6, Mattia Barbareschi7, Arkaitz Carra- Integrative Biology - CIBIO (University of Trento) cedo3, George Thalmann4, Alessandro Romanel1, Maurizio Taglialatela2,8, Marianna Kruithof-de It is widely accepted that the “cell of origin” Julio4,9 and Andrea Lunardi1 of tumors has to possess a proliferative capacity. Particularly for brain cancer, the transition 1Department of Cellular and Computational In- of neural progenitor to differentiated postmi- tegrative Biology (CIBIO), University of Trento, totic neurons is considered irreversible in phys- Trento, Italy. iological and pathological conditions. Therefore, 2Department of Health Sciences, University of postmitotic neurons have not been considered Molise, Campobasso, Italy. as suitable cell of origin of brain cancer. Here, 3CIC bioGUNE, Bizkaia Technology Park, Derio, we show that neurons reprograming may occur Spain. upon Shh activation and it leads to Medulloblas- 4Urology Research Laboratory, Department of toma (MB) formation in vivo. The Shh MB is a cer- Urology and Department of Clinical Research, ebellar tumor, found in infants and adults that University of Bern, Bern, Switzerland. is thought to originate from cerebellar granule 5Trento Institute for Fundamental Physics and neuron progenitors. More recently, it was discov- Applications (TIFPA), INFN, University of Trento, ered that the two different forms of SHH MB are Trento, Italy. distinguished by different transcriptome/methy- 6Instituto Biofisika, University of the Basque lome levels suggesting that the adult SHH MB may Country (CSIC-UPV/EHU), Leioa, Spain. originate from a different cell of origin. Relying 7Unit of Surgical Pathology, Santa Chiara Hospi- on these data, we use a conditional Cre-Lox re- tal, Trento, Italy. combination system that recapitulate the human 8Department of Neuroscience, University of adult medulloblastoma pathogenesis in mice and Naples “Federico II”, Naples, Italy. demonstrate that the post-migratory mature 9Department of Urology, Leiden University Medi- granule neurons can be reprogrammed in-vivo. cal Centre, Leiden, The Netherlands. This process leads to Shh medulloblastoma and the tumor formation is restricted to the cerebel- Corresponding Author lum. Thus suggesting that cerebellar granule neu- Andrea Lunardi, Armenise-Harvard Laboratory of rons have defined characteristics that allow the Cancer Biology & Genetics, Department of Cel- reprogramming process and cancer formation, lular and Computational Integrative Biology (CI- upon specific mutational hits. Our novel model BIO), University of Trento, Via Sommarive 9, Po- of cancer development could explain the human vo-Trento, 38123, Italy. Phone: 39-0461-285288; SHH medulloblastoma onset in adult individuals E-mail: [email protected] where granule neuron progenitors are no more present. We strongly believe that our model rep- Discovery of novel ‘druggable’ targets is a pri- resents an important starting point to study oth- mary goal in cancer translational research. Tran- er tissues where postmitotic cells might originate sient Receptor Potential subfamily M member 8 cancer and therefore this will open a new field in (TRPM8) is a cation channel almost exclusively cancer and stem cell biology. expressed by the luminal compartment of the prostate epithelium in the human body. Primarily associated with calcium homeostasis, TRPM8 lev- Pharmacological activation of els rise in primary and metastatic prostate can- TRPM8 channel overcomes innate cer (PCa), which makes it a possible oncogenic factor and a suitable target of potential clinical resistance to standard-of-care thera- interest. pies in prostate cancer Herein, we show that increased TRPM8 levels in

The Pezcoller Foundation Journal - June 2019 13 prostate cells favor calcium uptake and activa- Cell-autonomous and cell non-au- tion of pro-survival calcium/calmodulin-depend- tonomous downregulation of the ent kinase II (CaMKII). Nevertheless, by combining a multidisciplinary approach to an in vitro genet- tumor suppressor DAB2IP by mi- ic platform modelling prostate tumorigenesis, we croRNA-149-3p promotes cancer demonstrate that potent TRPM8 agonists syner- progression by microenvironment re- gize with X-ray treatments to induce massive ap- optotic response in radioresistant pre-malignant modeling. Posters and malignant preclinical prototypes of primary prostate lesions. As well, TRPM8 activation en- A. Bellazzo, G. Di Minin, E. Valentino, D. Sicari, hances docetaxel and enzalutamide efficacy in D. Torre, L. Marchionni, M. B. Stadler, G. Zucco- eradicating hormone naïve metastatic PCa cells. lotto, I. M. Montagner, A. Rosato, F. Tonon, C. Overall, our findings identify TRPM8 as a valuable Zennaro, C. Agostinis, R. Bulla, M. Mano, G. Del target for the treatment of PCa and provide a Sal, L.Collavin. solid rationale to pursue the clinical testing of TRPM8 agonists in combination with standard-of- The dynamic crosstalk established between tu- care therapies in PCa patients. mor cells and surrounding stroma cells is crucial in carcinogenesis (Quail and Joyce, 2013). In this context, signaling modulators and adaptors Establishment and Analysis of Pa- that dictate intrinsic cell responses to microen- vironmental cues play a fundamental role, often tient-Specific Group3 Medulloblasto- underestimated. The tumor suppressor DAB2IP ma Mouse Models belongs to this category: it is a cytoplasmic Ras- GAP and adaptor protein that negatively modu- Claudio Ballabio - PhD Student lates multiple oncogenic pathways, including ca- Armenise-Harvard Laboratory of Brain Disorders nonical WNT signaling, VEGF signaling via PI3K/ and Cancer Akt, TNF signaling via NF-κB, and Androgen Re- ceptor (AR) activity (Bellazzo et al., 2017). Not Department of Cellular, Computational and Inte- surprisingly, its expression is frequently reduced grative Biology - CIBIO by gene methylation in several tumors, including University of Trento breast and prostate cancer. In addition, various Trento (TN), Italy post-transcriptional mechanisms of DAB2IP inactivation have been reported; in particular, due to Brain cancer is now the deadliest form of child- the long 3’UTR sequence of its main transcript, hood cancer in the United States. In particular, DAB2IP is a strong candidate for microRNAs (miR- Group3 Medulloblastoma (MB) is the pediatric NAs)-mediated regulation (Bellazzo et al., 2017). brain tumor with highest morbidity and mortal- Performing an high-throughput screening of ity. Patients with Group3 MB currently have the a large collection of human miRNAs, we have worst outcome and nearly 50% are metastatic at identified miR-149-3p as a negative modulator the time of diagnosis. However, the cellular and of DAB2IP (Bellazzo et al., 2018). By efficiently molecular mechanisms underlying Group3 MB are downregulating DAB2IP, miR-149-3p enhances NF- still unknown. What is still lacking in the field is κB signaling activation in prostate cancer cells, the possibility to obtain tumors by direct genet- promoting cell motility, and improving the secre- ic modification of mice and to be able to reca- tion of pro-inflammatory and pro-angiogenic fac- pitulate the growth and metastasis formation of tors. Importantly, we found that the inhibition of Group3 MB. endogenous miR-149-3p restored DAB2IP tumor Exploiting in-vivo transfection of mouse cerebel- suppressive functions, and efficiently reduced lar cells with CRISPR-Cas9 and PiggyBac transpo- tumor growth and dissemination of prostate can- sase systems, we tested different combination cer cells in vivo (Bellazzo et al., 2018). DAB2IP of putative oncosuppressors and putative onco- loss can also affect the behavior of endothelial genes, derived from human Medulloblastoma cells surrounding the tumor: in fact, condition- NGS data, for their ability to induce Group3 MB al DAB2IP knockout in vascular endothelial cells in mice. Surprisingly, concomitant overexpression was shown to potently support formation of a of c-Myc and other transcription factors in mouse pre-metastatic niche, facilitating tumor growth cerebellum is able to induce MB in few months. and dissemination in mouse models of melanoma The newly generated mouse model is able to fully and breast cancer (Ji et al., 2015). We discov- recapitulate human Group 3 MB. ered that cancer cells can reduce DAB2IP levels Using this proposed patient-specific model, we in neighboring endothelial cells and fibroblasts were able to unravel the molecular aspects of by exosome-mediated secretion of miR-149-3p, Group 3 medulloblastoma tumorigenesis and stimulating their proliferation and motility, and identify molecules inhibiting tumor growth in a potentially remodeling the tumor microenviron- targeted manner. ment. Notably, experiments with miR-149-3p in-

14 The Pezcoller Foundation Journal - June 2019 hibitors clearly indicate that additional signals years ago. Ewing sarcoma is an aggressive type released by cancer cells also contribute to DAB2IP of bone tumor representing 1% of all childhood downregulation in nearby endothelial cells and fi- cancers, and its initial oncogenic event is rep- broblasts, and these need to be uncovered. resented by a balanced chromosomal transloca- These results have various implications; they tion originating a chimeric oncoprotein, which contribute to clarify the complex mechanisms in 90% of the patients derives from the fusion that govern the dynamic crosstalk between tu- between EWS and FLI1 genes. Ewing sarcoma’s mor cells and surrounding stroma, and they may treatment is currently based on the use of ge- suggest promising therapeutic strategies in can- neric chemo-agents such as doxorubicin, vincris- Posters cer. Indeed, approaches aimed to upregulate tine, cyclophosphamide, and dactinomycin, and DAB2IP levels would offer the unique therapeutic novel targeted treatments are urgently required. opportunity to act on a single protein that can The identification of novel drugs through preclin- negatively modulate multiple oncogenic signals, ical studies is however heavily influenced by the both in cancer cells and in stromal cells. In this model enrolled in the studies. Faithfull preclin- perspective, factors involved in cell non-autono- ical models able to recapitulate the biological mous downregulation of DAB2IP might be optimal characteristics of the human disease should be targets to develop pharmacologic strategies to employed to improve the efficiency of the pre- limit cancer progression by potentiating DAB2IP clinical tests and to decrease the percentage functions in multiple cellular components of the of subsequently failing clinical trials. Currently, tumor tissue. however, no faithful preclinical model of Ewing Sarcoma is available. Therefore, first objective Bellazzo, A., Di Minin, G., and Collavin, L. of this study will be the development of relia- (2017). Block one, unleash a hundred. Mechani- ble models able to faithfully recapitulate Ewing sms of DAB2IP inactivation in cancer. Cell death sarcomagenesis by inducing the expression of and differentiation 24, 15-25. Ewing oncoproteins in the correct cellular mi- Bellazzo, A., Di Minin, G., Valentino, E., Sicari, croenvironment, which is considered to be the D., Torre, D., Marchionni, L., Serpi, F., Stadler, mesenchymal stem cell. As exclusive hallmark of M. B., Taverna, D., Zuccolotto, G., et al. (2018). tumor cells and driving force of the disease, on- Cell-autonomous and cell non-autonomous cochimeras are ideal targets in medical oncology. downregulation of tumor suppressor DAB2IP by Among them, however, transcription factor onco- microRNA-149-3p promotes aggressiveness of proteins such as Ewing sarcoma oncoproteins are cancer cells. Cell death and differentiation. classified as “undruggable” from a conventional Ji, W., Li, Y., He, Y., Yin, M., Zhou, H. J., Bog- pharmacological point of view, lacking in their gon, T. J., Zhang, H., and Min, W. (2015). AIP1 structure convenient targeting pockets, and even Expression in Tumor Niche Suppresses Tumor though much is known regarding the oncogen- Progression and Metastasis. Cancer research 75, ic functions of different chimeras, the success 3492-3504. rate at which this advanced knowledge has been Quail, D. F., and Joyce, J. A. (2013). Microenvi- translated into effective therapies is pitifully ronmental regulation of tumor progression and low. Second objective of this study will therefore metastasis. Nature medicine 19, 1423-1437. be the enrollment of the generated models in preclinical screenings to identify those molecular mechanisms whose pharmacological tuning will A novel platform to study and target tear down Ewing sarcoma lethality by modulating undruggable Ewing onco-chimeras. oncochimera’s stability. By succeeding, our project will definitely break down the ancient dogma postulating the undrug- Arianna Bertossi, Rubens Begaj, Sacha Genove- gability of oncogenic chimeras belonging to the si, Veronica Foletto, Dario De Felice, Alessandro class of transcription factors, unveil new thera- Alaimo, Marco Lorenzoni, Andrea Lunardi peutic strategies towards the eradication of Ew- ing sarcoma, and, finally, provide a pivotal plat- Università di Trento, CIBIO, via Sommarive 9, form easily exportable to all those tumors driven 38123, Povo (TN), Italy by “undruggable” oncogenic chimeras. Cancer in children is relatively rare, yet it is the leading cause of death by disease in developed countries. Development of efficient therapeutic Contribution of centriole appendages strategies in the past years has drastically re- to PIDDosome activation duced the lethality of several common types of pediatric and juvenile forms of cancer such as Matteo Burigotto1, Alessia Mattivi1, Chiara Va- leukemia and lymphoma, but such a tremendous lentini1, Daniele Migliorati1, Giovanni Magnani1, success does not include sarcoma’s treatment, Alexander Schmidt2, Martin Offterdinger3, An- whose mortality rate remains the same as twenty dreas Villunger4, Luca Fava1

The Pezcoller Foundation Journal - June 2019 15 1 Armenise-Harvard Laboratory of Cell Division, ed in several tumor types. On the contrary, very Centre for Integrative Biology (CIBIO), Universi- few data exist on the activity of TAM and of oth- ty of Trento, Trento, Italy er immune cells in osteosarcoma (OS), the most 2 Proteomics Core Facility, Biozentrum, Univer- common primary bone tumors in young adoles- sity of Basel cents and children. 3 Division of Neurobiochemistry, Biocenter, Medi- We have recently demonstrated in immuno-com- cal University of Innsbruck, Austria petent OS mouse models that trabectedin, a ma- 4 Division of Developmental Immunology, Biocen- rine-derived chemotherapic agent, exerts a po- Posters ter, Medical University of Innsbruck, Austria tent anti-tumor activity that is further enhanced by the combination with anti-PD-1 antibody. Be- Failure in the physical separation of two cells sides directly affecting neoplastic cells, trabec- at the end of cell division (i.e. cyctokinesis) is tedin modifies the tumor immune landscape by one of the most common malfunctions of the cell recruiting T lymphocytes at the tumor site. How- division cycle, predisposing cells to neoplastic ever, contrary to what expected from the litera- transformation. Cytokinesis failure is invariably ture, the treatment did not affect TAM. followed by a reduction in the propensity of cells To better investigate the role of TAM in OS, we to commit to additional cell cycles. Fava et al. performed a “proof-of-concept” co-injection ex- demonstrated that this p53-dependent cell cycle periment with OS cells mixed with macrophages arrest depends on the activation of the PIDDo- differentiated in vitro toward classical M1 or M2 some, a multiprotein complex comprising PIDD1, phenotype, or left undifferentiated (M0). While RAIDD and Caspase-2 (Fava et al, 2017). More- over, centrosome abundance appears crucial for the presence of M1 macrophages inhibited OS, determining the cellular behavior in response to neither M0 nor M2 macrophages affected signif- cytokinesis failure and PIDD1 physically associ- icantly tumor growth. This result indicated that ates with the centrosome. the presence of TAM per se does not influence A genetic screen for centrosomal proteins re- OS growth and suggested that their re-direction vealed the PIDD1 position within the epistatic toward a M1 phenotype could exert therapeutic map of centriole appendage proteins. Moreover, activity. To clarify this issue, mice bearing OS tu- delocalization of PIDD1 from the centrosome re- mors on both flanks were treated locally, only in sults into compromised PIDDosome activation and one lesion, with either liposome-encapsulated p53-dependent cell cycle arrest. Taken together, clodronate to deplete TAM, or with SD101, a syn- our data shed light on the role of the PIDDosome thetic oligonucleotide with immunostimulatory as a centrosome counting entity. CpG motifs. Despite clodronate reduced TAM infiltration, tumor growth inhibition was limited, on Fava LL, Schuler F, Sladky V, Haschka MD, Sora- both flanks; on the other hand, SD101 efficiently troi C, Eiterer L, Demetz E, Weiss G, Geley S, halted the growth of both treated and untreated Nigg EA and Villunger A (2017) The PIDDosome lesions. TAM number was not affected by SD101 activates p53 in response to supernumerary cen- treatment, but they showed a significant reduc- trosomes. Genes Dev 31: 34 – 45 tion in the expression of the M2 marker CD206. Additionally, tumor infiltration by CD8 T cells was enhanced in both treated and untreated tumors Targeting tumor-associated ma- by SD101, but unaffected by clodronate. crophages in osteosarcoma: depletion Overall these preliminary results support the hypothesis that re-directing the phenotype of TAM versus re-direction in OS could be therapeutically more efficient than their direct elimination. Adriana Salvaggio1, Valeria Cancila2, Cristiana Guiducci3, Claudio Tripodo2, Katia Scotlandi4, 1 1 Mario P. Colombo and Claudia Chiodoni The glutamine addiction of multiple 1 Molecular Immunology Unit, Department of Re- myeloma cells shapes the metabolic search, Fondazione IRCCS Istituto Nazionale dei microenvironment of the bone mar- Tumori, Milan, Italy. 2 Tumor Immunology Unit, Department of Health row niche impairing osteoblastic dif- Science, Università di Palermo, Italy. ferentiation 3 Cancer Immunotherapy, Dynavax Technologies, Berkeley, CA Martina Chiu, Denise Toscani, Giuseppe Taurino, 4 CRS Development of Biomolecular Therapies, Emanuela Vicario, Fabrizio Accardi, Nicola Giu- Experimental Oncology Lab, Rizzoli Orthopedic liani and Ovidio Bussolati Institute, Bologna, Italy Department of Medicine and Surgery (DiMeC), University of Parma, Italy The pro-tumorigenic role of tumor-associate macrophages (TAM) has been widely demonstrat- Metabolic alterations of cancer cells are final-

16 The Pezcoller Foundation Journal - June 2019 ized to satisfy the increased nutrient needs due tration from 0.6 mM (the average physiological to uncontrolled proliferation but also impact on BM plasma concentration) to 0.4 mM (the average normal cells of tumor microenvironment. Glu- concentration in the BM plasma of MM patients) tamine addiction of multiple myeloma (MM) cells was sufficient to cause a significant reduction of (Bolzoni, Chiu et al., Blood 2016; 128:667-679) osteoblastic markers as well as of ALP activity leads to decreased glutamine levels in the bone and staining. marrow (BM) plasma of patients. Osteolytic bone These preliminary results suggest that glutamine lesions are a hallmark of active MM, and several addiction of MM contributes to osteoblast impair- mechanisms have been implied in their patho- ment in the bone lesions of the MM BM niche. Posters genesis. However, the role of MM-related alterations of glutamine metabolism in bone lesions has not been investigated yet. Bone lesions in MM are JMJD6-mediated Regulation of characterized by loss and impaired differentiation of osteoblasts. Interestingly, Glutamine Syn- Estrogen Receptor in Breast Cancer thetase (GS), the only enzyme able to catalyze and its role in Tamoxifen response intracellular glutamine synthesis, is down-regulated during osteoblast differentiation. Moreover, B. Cioni, D. Lecis, E. Fontanella and M.P. it has been recently demonstrated that the inhi- Colombo bition of glutamine metabolism decreases bone mass in mice (Yu et al., Cell Metabolism 2019; Department of Molecular Immunology, Via Gia- 29, 966–978). Thus, we hypothesized that the como Venezian, 1, 20133 Milano MI, Istituto Na- low-glutamine microenvironment imposed by MM zionale Tumori plasma cells negatively affects osteoblastogenesis. Introduction Human MM cell lines (HMCLs), immortalized BM Breast cancer (BCa) is one of the most common mesenchymal stem cells (MSC) and osteoblas- cancer types in women worldwide, and estrogen tic cell lines (HOBIT and HOB-01) were grown receptor (ER) is expressed in the vast majority in DMEM supplemented with 2 mM Gln and 10% of these tumors. ER-positive BCa relies on estro- fetal bovine serum. Changes in extracellular Gln gen levels for the development and progression were measured with a commercial kit or with of the disease, as activation of ER signaling reg- mass spectrometry, and amino acid uptake was ulates the expression of critical genes involved in assessed as previously described (Bianchi et al. proliferation and migration of tumor cells. Thus, Neuroscience. 2008; 151:1042-1052). The expres- anti-hormonal treatment, through the inhibition sion of osteoblastic markers (ALP, COL1A1 and of ER action, is the mainstay therapeutic option RUNX2), ALP activity and positivity to ALP stain- for high-risk ER-positive breast cancer patients. ing were then assessed during osteoblast differ- It is well known that modulation of chromatin entiation. conformation via epigenetic modifications regu- As expected from data obtained ex vivo, HMCLs lates transcription of many genes, including hor- consumed large amounts of glutamine (750 ± 50 mone receptors (HRs). The bifunctional arginine nmol/106 cells/day) and exhibited fast initial up- demethylase and lysyl-hydroxylase, JMJD6, is an take of the amino acid. When co-cultured with epigenetic modifier often associated with tumor MSC or osteoblasts, HMCLs accelerated the de- progression in many tumors, including BCa. A cru- pletion of extracellular glutamine (+25%/day, cial role of JMJD6 is to demethylate arginine resi- compared to monocultures of MSC/osteoblasts), dues 3 and 2 of histone 4 (H4R3) and 3 (H3R2), re- promoted GS expression in MSC and hindered via- spectively, thus affecting structural conformation bility of osteoblasts but not of MSC. Consistently, of the chromatin and transcription and function osteoblasts were more sensitive to Gln depletion of HRs.

(EC50 of 240 ±15 μM for HOBIT cells and 300± 5 Hypothesis and aims μM for HOB-01cells) than MSC (EC50 80 ± 20 μM). We hypothesized that modulation of the methyl- The expression and the activity of the concentra- ation status of these histones by JMJD6 is critical tive glutamine transporter SNAT2 (SLC38A2) were for the regulation of ER expression and signaling induced during osteoblastogenesis, while the ex- in BCa, thus possibly affecting proliferation and pression of other transporters, such as ASCT2 and metastatic potential of tumor cells. On the oth- SNAT1, was unchanged. SNAT2 induction was also er hand, we expect that increased ER expression associated with the increased expression of Glu- would improve efficacy of hormone therapy in taminase 1 (KGA, long-transcript form), suggest- BCa patients. This project aims to explore the ing higher glutamine demand and consumption in molecular and functional role of JMJD6 in reg- differentiating osteoblast precursors. In agree- ulating ER signaling and response to Tamoxifen ment with the experimental hypothesis, MSC in BCa cell lines and in suitable mouse models. osteoblastic differentiation was substantially Ultimately, we aim to validate our findings in impaired in the absence of glutamine; moreover, hormone treated and not treated BCa patients the decrease of extracellular glutamine concen- cohorts.

The Pezcoller Foundation Journal - June 2019 17 Results preserve their integrity and original dispersity in Our preliminary data reveled that CRISPR-Cas9 solution. Then, we combined it with ultrasensi- JMJD6 knock-out (KO) ER-positive MCF7 showed tive homogeneous assays, such as amplified lu- a strong increased methylation of H4R3, as well minescent proximity homogeneous assay (alpha) as increased ER expression, and expression of or droplet digital PCR (ddPCR), to detect known ER-responsive genes, RARA and GREB1. In agree- cancer biomarkers from retrospectively analysed ment with this, proliferation of JMJD6 KO cells oncological patients. was strongly increased compared to wild-type By applying alpha-NBI, we detected picomolar Posters (WT) control. As expected, stimulation with es- concentrations of prostate-specific membrane tradiol (E2) further enhanced proliferation of antigen (PSMA) in fractions of EVs isolated from both JMJD6 KO and WT MCF7 cells, however, the plasma of prostate cancer patients, discrimi- Tamoxifen treatment equally suppressed prolif- nating them from control subjects. Directly from eration of both cell types. Furthermore, JMJD6 oil-encapsulated EVs for digital PCR, we identi- KO MCF7 cells showed increased migration when fied somatic BRAF and KRAS mutations from the stimulated with E2 due to loss of E-cadherin ex- plasma of metastatic colorectal cancer (CRC) pa- pression, nevertheless, Tamoxifen treatment suc- tients, matching 100% of concordance with tissue cessfully suppressed cell migration. Importantly, diagnostics and higher sensitivity and specificity increased ER expression and improved response compared with immune-enrichment of tumor-de- to Tamoxifen was also observe in ER-low BT474 rived EVs. We propose NBI-combined approaches BCa cell line that poorly respond to Tamoxifen as a further tool to develop liquid biopsy studies treatment in WT condition, suggesting that inhi- and gain advantages from the possibility of pro- bition of JMJD6 levels can improve response to bing tumor heterogeneity from circulating EVs. hormone therapy by modulating ER levels. Sup- porting this hypothesis, we found that reduced levels of JMJD6 were significantly correlated with New platform for the direct profiling longer overall survival of BCa patients treated with hormone therapy in the METABRIC database of micrornas in biofluids compared to hormone treated patients expressing high levels of JMJD6, suggesting that JMJD6 Detassis Simone could be a predictive marker for hormone therapy response in these patients. Department of Cellular, Computational and Inte- Conclusion grative Biology - CIBIO Our data suggest that inhibition of JMJD6 in BCa University of Trento patients could potentially improve response to hormone therapy by increasing ER expression, Circulating microRNAs have been identified as thus providing a rational for the development of potential biomarkers for early detection, pro- JMJD6 inhibitors. gnosis and prediction of several diseases. Their use in clinical diagnostics has been limited by the lack of suitable detection techniques. Most of the Ultrasensitive detection of cancer current technologies suffer from requiring complex protocols, not yet able to deliver robust and biomarkers from blood circulating cost-effective assays in the field of clinical dia- NBI-isolated vesicles. gnostics. In this work, we report the development of a breakthrough platform for profiling circula- VG. D’Agostino1, M. Notarangelo1, C. Zucal1, L. ting microRNAs. The platform comprises a novel Pasini2, H. Beltran3, F. Demichelis1, A. Provenza- silicon photomultiplier-based reader in conjun- ni1, A. Quattrone1. ction with a chemical-based method for nucleic acid detection. Accurate microRNAs profiling 1 Centre for Integrative Biology (CIBIO), Univer- without extraction, pre-amplification or pre-la- sity of Trento belling of target is now achievable. We designed 2 Istituto Scientifico Romagnolo per lo Studio e la and synthesized a set of reagents that combined Cura dei Tumori (IRST) IRCCS, Meldola the chemical-based method with a chemilumi- 3 Department of Medicine, Weill Cornell Medici- nescent reaction. The signals generated were ne, New York, NY, USA read using a novel, compact silicon photomultiplier-based reader. The platform sensitivity was Extracellular vesicles (EVs) are secreted mem- determined by measuring known concentrations branous particles intensively studied for their po- of hsa-miR-21-5p spike-ins. The limit of detection tential cargo of diagnostic markers. An efficient was calculated as 4.7 pmol/L. The platform was and high-throughput study of EVs in the routine also successfully used to directly detect hsa-miR- clinical practice is needed to understand their 21-5p in eight non-small cell lung cancer plasma clinical utility. We designed the nickel-based iso- samples. Levels of plasma hsa-miR-21-5p expres- lation (NBI) procedure to rapidly isolate EVs to sion were also measured via TaqMan RT-qPCR.

18 The Pezcoller Foundation Journal - June 2019 The successful integration of a unique chemi- role in CRI still remains understudied. Recently cal-based method for nucleic acid detection with our group demonstrated the pro-malignant role of a novel silicon photomultiplier-based reader cre- complement activation in models of mesenchymal ated an innovative product (ODG platform) with carcinogenesis, induced by 3-methycolanthrene diagnostic utility, for the direct qualitative and (3-MCA), and epithelial inflammation-driven skin quantitative analysis of microRNA biomarkers in carcinogenesis, induced by dimethylbenz-α-an- biological fluids. thracene/terephthalic acid treatments (DMBA/ TPA). Our results showed that mice deficient for the central complement component C3 were Posters The SWI/SNF complex is a mecha- protected from tumor development. Further experiments revealed that C3-cleavage products noregulated inhibitor of YAP and were deposited on vessels and tumor cells of TAZ tumor tissues, while they were absent in normal subcutaneous tissues. The C3 deposition on Inactivation of ARID1A and other components of tumor cells was also observed in vitro, both on the nuclear SWI/SNF protein complex occurs at 3-MCA-derived sarcoma and on different murine very high frequencies in a variety of human ma- cancer cell lines. Further, in vivo experiments lignancies, suggesting a widespread role of the suggested that complement activation was main- SWI/SNF complex in tumour suppression1. We ly due to the activation of the classical pathway. show that ARID1A-containing SWI/SNF complex Then, we investigated C3-downstream mecha- (ARID1A-SWI/SNF) operates as an inhibitor of the nism(s) of protection in three different murine pro-oncogenic transcriptional coactivators YAP tumor models. We observed that C3aR- but not and TAZ (also known as WWTR1)2. YAP and TAZ C5aR1- and C5L2-deficient mice were protected are necessary to mediate the effects of the in- from tumor growth in a transplantable model of activation of the SWI/SNF complex, such as cell sarcoma (MN-MCA1), as well as in the 3-MCA-in- proliferation, acquisition of stem cell-like traits duced carcinogenesis model, suggesting that the and liver tumorigenesis2. C3a/C3aR axis was most likely responsible for the phenotype showed by C3-deficient mice in sar- 1 Kadoch & Crabtree. Mammalian SWI/SNF Chro- coma models. However, in the DMBA/TPA mod- matin Remodelling Complexes and Cancer: Me- el we observed that the C5a/C5aR1 dependent chanistic Insights Gained from Human Genomi- signaling was mainly responsible for the pheno- cs. Sci Adv e1500477 (2015) type showed by C3-deficient mice, since C5aR1- 2 This Work: Chang L*, Azzolin L*, Di Biagio D*, /- mice were protected from tumor development, Zanconato F, Battilana G, Lucon Xiccato R, Ara- and that the underlying mechanism involved the gona M, Giulitti S, Panciera T, Gandin A, Sigi- hematopoietic compartment Our results indicate smondo G, Krijgsveld J, Fassan M, Brusatin G, that complement activation occurs in tumor and Cordenonsi M, Piccolo S. The SWI/SNF Complex contributes to tumor development, although the is a Mechanoregulated Inhibitor of YAP and TAZ. mechanism/s implicated could be different in Nature. 2018 Nov;563(7730):265-269. the mouse models.

* co-first authors Targeted NGS in pharmacogenes to identify novel rare variants related to Pro-tumoral role of Complement fluoropyrimidines toxicity activation in murine tumor models Ecca F.1,2, De Mattia E.1, Serra F.3, Roncato R.1,- Elena Magrini1, Sabrina Di Marco1, Chiara Pe- Dreussi E.1, Romanato L.1, Buonadonna A.4, De rucchini1, Fabio Pasqualini1, Andrea Ponzetta1, Paoli A.5, Berretta M.4, Mini E.6, Nobili S.7, Toffo- Maria Luisa Barbagallo1, Colombo Piergiuseppe1, li G.1, Cecchin E.1 Ferdinando Cananzi1, Kevin Berthenet1, Antonio Inforzato 1, 2, Sebastien Jaillon1, 2, Andrea Doni1, 1 Exper and ClinPharmacol, Centro Di Riferimen- Alberto Mantovani1, 2, and Cecilia Garlanda1, 2, to Oncologico- National CancerInstitute, Aviano 2 PhD School, University of Udine 1 IRCCS - Humanitas Clinical and Research Center, 3 INSERM U 1065, C3M,Team: Control of Gene Rozzano, Milan, Italy Expression Nice, France. 2 Humanitas University, Milan, Italy 4 Medical OncologyDpt, Centro Di Riferimento Oncologico- National CancerInstitute, Aviano Cancer related inflammation (CRI) plays a funda- 5 Radiation OncologyDpt, Centro Di Riferimento mental role in fueling tumor appearance and de- Oncologico- National CancerInstitute, Aviano velopment. Although the important contribution 6 Section of Internal Med, Dpt of Experimental of complement activation to inflammation, its and Clin Med, University of Florence

The Pezcoller Foundation Journal - June 2019 19 7 Section of Clinical Pharmacol and OncolDpt of Metabolic re-programming by malat1 Health Sciences, University of Florence depletion in prostate cancer cell lines Fluoropyrimidines (FL) are frequently used in and organotypic slice cultures several solid tumours treatment. Unfortunately, about 26% of patient treated with FL will de- S. Nanni1, A. Aiello2, C. Salis1, A. Re2, C. Cencio- velop unpredictable severe to life-threatening ni2, L. Bacci1, F. Pierconti1, F. Pinto3, D. Pugliese1, toxicity (grade ≥3). Common single nucleotide C. Ripoli1, P. Ostano4, E. Iorio5, P. Bassi 1, 3, C. Posters polymorphisms (SNP) provide partial explana- Grassi 1,3, G. Chiorino4, A. Pontecorvi 1,3, C. Gae- tion to the overall adverse reactions (Toffoli tano6 and A. Farsetti 2 et al, 2015). However, most variants affecting proteins phenotype in humans, including those 1 Università Cattolica del Sacro Cuore Rome, involved in pharmacokinetics, were reported to Italy; be novel and rare (Lek et al, 2016; Kozyra et al, 2 National Research Council-IBCN, Rome, Italy; 2017; Ingelman-Sundberg et al, 2018). 3 Fondazione Policlinico Universitario A. Gemelli We aimed to screen 54 pharmacogenes involved IRCCS, Rome, Italy; in the FL absorption, distribution, metabolism, 4 Fondazione Edo ed Elvo Tempia, Biella, Italy; and excretion (ADME) and folate pathway to 5 Istituto Superiore di Sanità, Rome, Italy; identify novel markers of severe FL-related tox- 6 Istituti Clinici Scientifici Maugeri, Pavia, Italy. icity in a selected group of patients with severe FL-related toxicity phenotype. BACKGROUND: Prostate cancer (PCa) is the One hundred twenty patients treated with a FL- most common malignancy in men worldwide based regimen were retrospectively selected with an increased global burden. The continu- based on grade ≥3 toxicity occurrence and ab- ous improvement of next-generation sequencing sence of acknowledged DPYD risk variants (DPY- methods shed light on the direct involvement D*2A, DPYD*13, DPYD-c2846, and DPYD-HapB3). of multiple non-coding RNA (ncRNA) transcripts PharmGkb resources and literature data were in cell biology and homeostasis, cancer growth used for genes selection. A custom Nimble- and progression as well as in other pathophysi- Gen SeqCap EZ Choice (Roche, Inc.) which covers ological contexts. Contextually, cancer metab- the CDS and UTRs of target genes was designed olism recently emerged at the upfront edge of including the flanked splice junctions regions. An anticancer research with the promise to develop additional region of 3kbp was included to address novel therapeutic approaches. Interestingly, very the promoter genetic variants for 3 genes (DPYD, recent evidences linked long (>200nt) ncRNAs (ln- MTHFR and TYMS). The libraries were sequenced cRNAs) to metabolism opening up to novel ther- with MiSeq instrument (Illumina, Inc.). The re- apeutic strategies based on lncRNAs. Therefore, sulting fastQ files were analyzed using different the study of their effects on metabolism might tools in in-house pipeline. unravel overlooked pathways to target in an anti- By a preliminary analysis, eight singleton vari- cancer perspective. ants on DPYD were identified in eight different HYPOTHESIS & AIM: Although MALAT1 has been patients. Seven of them were missense and one one of the earliest identified lncRNAs and its role was located in the 3’UTR. In addition, three pre- in cancer as promoter of tumour progression and viously reported very rare missense variants were metastasis is well defined, little or no information detected. Variants were analytically validated is available about its direct involvement in mi- by Sanger sequencing and were evaluated with tochondrial metabolism regulation. In this light, several amino acid change and splicing defect our working hypothesis is that MALAT1 might play prediction tools. It is interesting to note that the a pivotal role in PCa as a metabolically active sig- identified missense variants have high damage nal integrator. For this reason, the main goal of score and nine of these may modify the exon- our study is to investigate whether MALAT1 might ic splicing sequences (ESE/ESI). Furthermore, represent a metabolically active target for PCa eighty-five new variants were detected in the inhibition in Organotypic Slice Cultures (OSCs), FL-ADME genes, including a deletion of 43bp in an ex vivo human model suitable for gene expres- the 3’UTR of the TYMS and its overlapped regula- sion, gene targeting, and drug testing analyses. tor ENOSF1. rTS (ENOSF1) RNA may downregulate EXPERIMENTAL DESIGN: Our experimental strat- TS (TYMS) RNA expression via 3’UTR interaction egy is made of a multi-pronged approach aimed (Dolnick, R. et al., 2005). at defining a novel role of MALAT1 in PCa metab- In conclusion we preliminarily demonstrated olism. Specifically, we characterized MALAT1-de- that rare and novel genetic variants in DPYD pendent transcriptome in human PCa cells with can be detected in patients with an extreme aggressive/metastatic phenotype and in OSCs toxicity phenotype and can potentially account and identified putative MALAT1-dependent meta- for a defective FL detoxification. We will try to bolic enzymes and metabolites that were further demonstrate the functional role of these variants investigated by NMR-based metabolomics analy- through specific functional assays. sis before and after ex-vivo gene targeting.

20 The Pezcoller Foundation Journal - June 2019 RESULTS: Transcriptomics and metabolomics per- conditional expression of human RAS oncogene formed in PCa cell lines (PC-3, DU145, and C27IM) in brain progenitor cells (Mayrhofer et al. 2017). pointed out malate dehydrogenase (ME3), pyru- Zebrafish brain tumours have long and irregular vate dehydrogenase kinases (PDK1 and PDK3) as telomeres, undergo sister chromatid exchange well as choline kinase A (CHKA) as important met- and are positive for C-Circles, suggesting that abolic enzymes under MALAT1 transcriptional con- ALT mechanisms are predominant. Following ALT trol. Both transcriptomics and metabolic profiles phenotype, DNA damage at telomeres was signifi- were validated by RT-PCR, western blot and specif- cantly higher than in control cells. We found that ic enzymatic analysis (lactate production, NAD+/ during the progression of tumours from single Posters NADH assays, and assessment of mitochondrial OX- cancer initiating clone to a full tumour, down- PHOS complexes). These findings were confirmed regulation of tert through hypomethylation of its in a large number of human OSCs (n=47) obtained promoter precedes the development of ALT and upon surgery from a selected cohort of patients is linked to an increase of Terra expression. To with diverse stage of the disease. Currently, we study the contributions of telomerase in the de- are exploring whether these enzymes belonging to velopment of ALT, we established the same mod- the tricarboxylic acid (TCA) cycle and phosphati- el in a background of co-overexpressed tert and dylcholine pathway are involved in MALAT1 regu- terc. The tumour generated were similar in loca- latory function and whether a direct modulation tion but less aggressive respect tumours without of TCA cycle enzymes or phosphatidylcholine me- tert/terc overexpression. The analysis of tel- tabolites may reproduce the MALAT1-associated omere and ALT biomarkers showed an ALT rescue metabolic environment in PCa. in the function of tert and terc over-expression. In conclusion, our findings suggest an unprece- We also found a reduction of telomeric DNA dam- dented role of MALAT1 as regulator of cell me- age, downregulation of genes of the pre-replica- tabolism and potentially active target for PCa tion complex and the status of heterochromatin inhibition at single patient level. of telomeres was re-established in telomerase positive brain tumours. We suggest that the activity of telomerase can reduce the replication Mechanisms of telomere maintenan- stress at telomeres by regulation of telomeric heterochromatin. Finally, we characterized tel- ce in zebrafish brain tumors: correla- omere maintenance mechanisms in a cohort of tion with pediatric glioblastoma 20 human brain tumours, where we measured telomeric DNA damage in association with telo- Aurora Irene Idilli1, Emilio Cusanelli1, Francesca meric chromatin. Besides reporting the first in Pagani2, Emanuela Kerschbamer1, Francesco Be- vivo genetic model of ALT+ brain tumour, this rardinelli3, Marialuisa Cayuela4, Pietro Luigi Po- study identifies telomeric maintenance mecha- liani2, Marina Mione1 nisms as significant drivers of the coordination between DNA replication and chromatin status at 1 Centre for Integrative Biology (CIBIO), Universi- telomeres in brain cancers. ty of Trento, Trento – Italy 2 Department of Molecular and Translational Me- dicine, University of Brescia School of Medici- Adenocarcinoma-neuroendocrine ne, Brescia, Italy 3 Department of Science, University of Rome transition of castration resistant pro- “Roma Tre”, Rome, Italy state cancer 4 Department of Surgery, CIBERehd, University depends on SPARC down-regulation Hospital ‘Virgen de la Arrixaca’ and Instituto Murciano de Investigación Biosanitaria (IMIB), in stromal accessory cells 30120 Murcia, Spain. Claudia Enriqueza, Valeria Cancilab, Renata Fer- Alternative lengthening of telomeres (ALT) oc- ria, Roberta Sulsentia, Irene Fischettia, Ivano curs in pediatric brain tumours and may develop Ariolia, Claudio Tripodob, Mario P. Colomboa and as a result of chromosomal instability promoted Elena Jachetti by altered histone H3 modifications in subtelo- meric regions and/or in association with ATRX a Molecular Immunology Unit, Department of Re- mutations/downregulation. However, develop- search, Fondazione IRCCS Istituto Nazionale dei ment of ALT may require additional genetic and Tumori, via Amadeo, 42, 20133 Milan, Italy epigenetic changes, at present largely unknown. b Tumor Immunology Unit, Department of Health Here we have investigated whether ALT devel- Sciences, University of Palermo, Italy ops as a result of the coordination between DNA damage accumulation and chromatin status at Adenocarcinoma-neuroendocrine transition of telomeres in brain cancers. We used a model castration resistant prostate cancer can occur of juvenile zebrafish brain tumour based on the in a relevant subset of patients as a mecha-

The Pezcoller Foundation Journal - June 2019 21 nism to resistance to androgen deprivation and Urology and Department of Clinical Research, androgen-receptor targeted drugs. Tumor cell University of Bern, Bern, Switzerland plasticity toward neuroendocrine differentiation 5 Departement of Clinic and Experimental Medi- (NED) can be sustained by signals provided by the cine, Magna Graecia University of Catanzaro, tumor microenvironment. We previously identi- Italy fied the matricellular protein SPARC as a crucial # Present Address: Herbert Irving Comprehensi- modulator of cancer progression and phenotype, ve Cancer Center, Columbia University Medical exerting different functions depending whether Center, NY, USA Posters expressed by tumor or stroma cells. Therefore, § Corresponding Author: Andrea Lunardi_andrea. we aimed at elucidating the different contribu- [email protected] tion of tumor- or stroma-derived SPARC in prostate cancer, utilizing the TRAMP mouse model. Prostate Cancer (PCa) is the second most com- Crossing TRAMP mice with Sparc-/- mice, we ob- mon cancer in men. While several genomic, ge- served appearance of areas of NED, similarly to netic and molecular alterations characterizing what occurs in TRAMP mice relapsing after sur- human PCa have been functionally associated gical castration. Areas of NED were positive for with tumor onset, progression and resistance to both adenocarcinoma (CK8) and neuroendocrine therapy, the role of many other molecular events (SYP) markers, suggesting trans-differentiation remains still unclear. By combining prostate or- from adenocarcinoma. In TRAMP prostates, we ganoids technology with genetic engineering and observed SPARC positivity in scattered tumor CLICK-chemistry coupled Mass Spectrometry ap- cells and in infiltrating fibroblasts. Moreover, ad- proaches, we identified a panel of secreted pro- enocarcinoma cells injected in Sparc-/- mice ac- teins regulated by ETS-related gene (ERG). Based quired SYP expression. This suggested a role for on the nature of the identified factors, ERG activ- stromal-derived SPARC in limiting NED of pros- ity in pre-cancerous human prostate lesions may tate cancer cells. Accordingly, prostate cancer prime prostate cells and the surrounding stroma cell lines co-cultured in presence of Sparc-defi- to create a tolerant and supportive environment cient fibroblasts acquired neuroendocrine fea- during the initial steps of tumorigenesis. tures. This likely occurs through the effect of IL-6, which is released by SPARC deficient, but not sufficient, fibroblasts. Our data indicate that Cross-talk with lung epithelial cells re- stromal SPARC down-regulation controls prostate cancer transition from adenocarcinoma to neu- gulates Sfrp2 expression enabling dis- roendocrine phenotype. A deeper understanding seminated breast cancer cell latency of the molecular mechanisms governing NED ac- cording to extracellular matrix composition will Marco Montagner1,2, ChrisTape3, Erik Sahai1 provide important insights for the development of new therapeutic strategies in prostate cancer. 1 Tumour Cell Biology Laboratory, Francis Crick Institute, London, UK 2 Department. of Molecular Medicine, University of Padua, Padova, Italy Pro-tumorigenic role of ETS-related 3 gene (ERG) in pre-cancerous prostate University College London, UK lesions The process of metastasis is highly complex. In the case of breast cancer, there are frequently Marco Lorenzoni1, Alessandro Alaimo1, Francesco long timespans between cells leaving the primary Cambuli1,#, Veronica Foletto1, Sacha Genovesi1, tumour and the growth of overt metastases. Dur- Michela Zaffagni1, Dario De Felice1, Arianna Ber- ing this period, cancer cells persist in an indolent tossi1, Alessandro Romanel2, Mattia Barbareschi3, or latent state before transitioning back to an ag- Marianna Kruithof-de Julio4 Marco Gaspari5 and gressive growth. Possible reasons for disease in- Andrea Lunardi1,§ dolence include interplay with myeloid and fibro- blastic cells in the tumour microenvironment and 1 The Armenise-Harvard Laboratory of Cancer ongoing immune surveillance. However, the sig- Biology & Genetics, Department of Cellular, nals causing actively growing cells to enter into Computational and Integrative Biology, Univer- an indolent state, and enabling them to survive sity of Trento, Italy for extended periods of time, are not well under- 2 Laboratory of Bioinformatics and Computatio- stood. In this project, we propose that the be- nal Genomics, Department of Cellular, Compu- havior of indolent breast cancer cells in the lung tational and Integrative Biology, University of is determined by their interactions with alveolar Trento, Italy epithelial cells. In vivo data, as well as a new- 3 Unit of Surgical Pathology, Santa Chiara Hospi- ly developed lung organotypic system, revealed tal, Trento, Italy that lung epithelial cells promote the formation 4 Urology Research Laboratory, Department of of fibronectin (FN) fibrils by indolent cells that

22 The Pezcoller Foundation Journal - June 2019 drive integrin-dependent pro-survival signals. the mechanical properties of cells and tissues, Combined in vivo RNA sequencing and drop-out providing a missing genetic evidence for me- screening identified Secreted frizzled-related chanical properties as potent and specific regu- protein 2 (Sfrp2) as a key mediator of this in- lator of liver organ growth, cell-fate and tissue teraction. Sfrp2 is induced by lung epithelial metabolism in vivo. More generally, it indicates cells and promotes FN fibril formation, integrin for the first time that mechanotransduction has activation and survival, while blockade of Sfrp2 a physiological role in maintaining liver homeo- expression reduces the burden of indolent dis- stasis in mammals. Our genetic system will thus ease. Treatment of mice with an integrin-specific open the possibility for testing, in the future, Posters inhibitor, reduces the survival of disseminated the effective and functional role of mechanos- indolent breast cancer cells in vivo, suggesting transduction in multiple other tissues and in al- FDA-approved integrin-inhibitors as a valuable tered context, such as cancer development. adjuvant therapies to eradicated these cells and prevent metastasis. Histone chaperone ANP32E as a pro- to-oncogenic factor in MYC-driven F-actin dynamics regulates mamma- tumorigenesis lian organ growth and cell fate maintenance Vittoria Poli1, Luca Fagnocchi1, Alessandra Fa- sciani1, Miriam Gaggianesi2, Alice Turdo2, Matilde Pocaterra et al Todaro3, Alessio Zippo1 J Hepatol 2019 1 Laboratory of Chromatin Biology & Epigenetics, Department of Molecular Medicine – University Center for Integrative Biology (CIBIO), Univer- of Padova sity of Trento, 38123, Trento, Italy. 2 Department of Surgical, Oncological and Sto- in vitro, cell behavior can be potently regulat- matological Sciences, University of Palermo, ed by the mechanical properties of cells and Palermo, 90127, Italy. of their microenvironment. Cells sense these 3 DiBiMIS, University of Palermo, Palermo, 90127, features through integrin receptors and focal Italy. adhesions and counteract external forces by developing internal pulling forces via their ac- Breast cancer consists of highly heterogeneous tomyosin cytoskeleton, in turns regulating intra- tumors, whose driver oncogenes result difficult cellular pathways, including the transcriptional to be uniquely defined. We have recently re- coactivators YAP/TAZ. Whether mechanical cues ported the central role of MYC in initiating and are relevant for in vivo regulation of adult organ sustaining a step-wise epigenetic reprogram- homeostasis, and whether this occurs through ming process in mammary luminal epithelial cells YAP/TAZ, remains largely unaddressed. We de- (IMEC) toward a stem cell-like condition, which veloped Capzb conditional knockout mice and favors cell transformation and tumor initiation. obtained primary fibroblasts to characterize the Among the chromatin players that may syner- role of CAPZ in vitro. In vivo, functional analy- gize with MYC, we identified the H2A.Z-specific ses were carried out by inducing Capzb inacti- chaperone ANP32E, whose expression is induced vation in adult hepatocytes. We found that the in MYC-transformed IMEC (t-IMEC). Interestingly, F-actin capping protein CAPZ restrains actomyo- analysis of the TCGA dataset showed that ANP32E sin contractility: Capzb inactivation alters stress alteration is specifically enriched among ba- fiber and focal adhesion dynamics leading to en- sal-like breast cancers characterized by MYC de- hanced myosin activity, increased traction forc- regulation and this combination correlates with es, and increased liver stiffness. in vitro, this a worst prognosis. These observations suggested rescues YAP from inhibition by a small cellular a possible cooperation between MYC and ANP32E geometry; in vivo, it induces YAP activation in in tumor formation and maintenance. Of note, parallel to the Hippo pathway, causing extensive ANP32E overexpression in t-IMEC was associated hepatocyte proliferation and leading to striking with increased tumorigenic potential both in vit- organ overgrowth. Moreover, Capzb is required ro and in vivo. Considering that MYC overexpres- for the maintenance of the differentiated sion is cause of replication stress (RS) and that hepatocyte state, for metabolic zonation, and ANP32E-mediated eviction of H2A.Z is required for gluconeogenesis. In keeping with changes in for DNA-damage repair (DDR), we investigated tissue mechanics, inhibition of the contractility whether their simultaneous deregulation could regulator ROCK, or deletion of the Yap1 mech- further impact on RS and result in DNA damage anotransducer, reverse the phenotypes emerg- accumulation. Of note, t-IMEC overexpressing ing in Capzb-null livers. These results indicate a ANP32E (t-IMEC-ANP32E) are characterized by previously unsuspected role for CAPZ in tuning increment of transcriptional R-Loops and high-

The Pezcoller Foundation Journal - June 2019 23 er number of γH2A.X-marked DNA-damage foci, Bone marrow hematopoietic adap- respect to t-IMEC. A critical component of the tation to distant breast cancer begins DDR is represented by ATR, a factor activated by regions of single-stranded DNA, which can occur early in transformation in association as a result of oncogene-induced RS. Interesting- with deregulated circulating microR- ly, both in vitro and in vivo treatment with an NAs ATR inhibitor showed enhanced sensitivity in association with ANP32E overexpression. This study 1 1

Posters Tiziana Ada Renzi , Claudia Chiodoni , Valeria supports the notion that ANP32E alteration in Cancila2, Milena Perrone1, Andrea M. Tomirotti1, cells undergoing MYC-related RS can promote the Sabina Sangaletti1, Laura Botti1, Matteo Dugo3, development and maintenance of a tumorigen- Matteo Milani1, Lucia Bongiovanni2, Maurizio ic phenotype by interfering with the DNA repair Marrale4, Claudio Tripodo2 and Mario P. Colombo1 machinery. The correlation between ANP32E deregulated expression and increased cell sensitiv- 1 Molecular Immunology Unit, Department of Re- ity to ATR inhibition could establish a therapeu- search, Fondazione IRCCS Istituto Nazionale dei tic rational for targeted treatment of basal-like Tumori, Milan, Italy. breast cancers characterized by combined MYC 2 Tumor Immunology Unit, Department of Health and ANP32E alteration. Sciences, Human Pathology Section, University of Palermo School of Medicine, Palermo, Italy. 3 Platform of Integrated Biology - Bioinformatics, Decoding the role of glutaminolysis Department of Applied Research and Technolo- in developmental and tumor angioge- gy Development, Fondazione IRCCS Istituto Na- zionale dei Tumori Milan, Italy. nesis 4 Department of Physics and Chemistry, Universi- ty of Palermo, Palermo, Italy. Giovanna Pontarin, Roxana Oberkersch, Matteo Astone, Marianna Spizzotin, Massimo M. Santoro During tumorigenesis, newly transformed cells initiate an active cross-talk with bystander cells, Department of Biology, University of Padova, mostly of bone marrow (BM) origin, to establish 35131 Padova, Italy a pro-tumorigenic microenvironment. We hypothesized that signs of this cross-talk can be Metabolism is emerging as an important regulator identified in the BM at the very early phases of in co-determining endothelial cell (EC) behavior cancer development, being finalized to the in- and function. Little is known about a possible struction of a tumor-promoting hematopoiesis. In role of glutamine metabolism during new vessel the MMTV-NeuT model of spontaneous mammary sprouting in normal and pathological conditions. carcinoma we showed that gene expression pro- The key rate-limiting step of glutaminolysis is the filing of the bone marrow along disease progres- conversion of glutamine to glutamate by the mi- sion indicates modifications in the hematopoietic tochondrial glutaminases GLS1 and GLS2. Given compartment already at early disease stages that the very low expression level of GLS2 in ECs, in become more relevant with tumor progression. the present study we investigate the role of GLS1 The transcriptional profile of the adapted he- at cellular level and how it affects angiogenesis matopoiesis revealed the induction of programs in vivo. At first we characterized the effect of related with innate immunity and responses to GLS1 on EC behavior. The absence of glutaminase danger signals, alongside the down-modulation of activity by GLS1 specific blocker CB-839 or ex- adaptive immune response. These transcriptional pression by shRNA alters cell cycle progression reprogramming is paralleled by an expansion of and ultimately impairs proliferation, with no the myeloid populations at the expense of eryth- cytotoxicity. In addition, we found that the ab- roid and B lymphoid fractions. The finding of such sence of GLS1 induces a stress response via ATF4, modifications in the BM microarchitecture even resembling the aminoacid starvation response, at earlier stages of cancer development (high- and affects the total level of VEGF R2 receptor grade dysplasia/in situ cancer stage) provided and its activation after stimulation with VEGF A. the first evidence of the BM acting as a very early To determine whether GLS1 plays a role during sensor of peripheral transformation. Moreover, angiogenesis in vivo, we generated endotheli- we profiled plasmatic microRNAs at late and ear- al conditional GLS1 knockout mice by crossing ly stages of tumor progression and found, already floxed Gls1 mice (Gls1flox/flox) with VE-cadherin at early time points, differentially expressed mi- CreRT2 transgenic mice. In xenograft tumors we croRNAs, which could potentially be used as early found that knock-out of GLS1 in vivo significantly biomarkers of cancerogenesis. In conclusion, our inhibited the tumor growth and vascular vessels data lay a first demonstration that BM hemato- formation. These findings underscore the impor- poietic adaptation to cancer is not confined to a tance of glutamine metabolism in ECs and during general immunosuppressive state associated with angiogenesis in vivo. advanced cancers, rather it represents an early

24 The Pezcoller Foundation Journal - June 2019 process co-evolving with malignant clone expan- vaccinated canine MM patients as compared to sion. controls. Data obtained in vitro with T cells from human healthy donors suggested HuDo-CSPG4 is more immunogenic than Hu-CSPG4. Anti-CSPG4 DNA vaccination re- Moreover, we started to investigate CSPG4 role in both canine and human osteosarcoma (OSA) veals potential therapeutic effects for to eventually propose CSPG4 DNA vaccination as + the treatment of CSPG4 tumors: a an innovative comparative therapy for OSA treat- comparative oncology study ment, too. We found a strong correlation be- Posters tween CSPG4 over-expression and a worse prog- Federica Riccardo1, Giuseppina Barutello1, Lidia nosis in both human and canine OSA patients. The Tarone1, Maddalena Arigoni1, Davide Giacobino2, potentiality of CSPG4 immune-targeting for OSA Selina Iussich2, Sergio Occhipinti3, Angela Peti- treatment was demonstrated by the ability of to1, Soldano Ferrone4, Paolo Buracco2, Federica anti-CSPG4 monoclonal antibodies (mAbs) to sig- Cavallo1 nificantly inhibit both canine and human CSPG4+ OSA in vitro proliferation, migration and oste- 1 Department of Molecular Biotechnology and ospheres generation. In addition, anti- CSPG4 Health Sciences, Molecular Biotechnology Cen- mAbs potentiated the anti-proliferative effect of ter, University of Torino, Torino, Italy doxorubicin. Interestingly, sera derived from ca- 2 Department of Veterinary Sciences, University nine MM patients enrolled in our previous veteri- of Torino, Grugliasco, Italy nary trials, were also able to in vitro inhibit OSA 3 Center for Experimental Research and Medi- cells tumorigenic potential in both adherent and cal Studies (CERMS), Città della Salute e della non-adherent conditions. Scienza di Torino, Torino, Italy Overall, these results provide the rationale to 4 Department of Surgery, Massachusetts General propose HuDo-CSPG4 vaccination for the treat- Hospital, Harvard Medical School, Boston, MA, ment of canine CSPG4+ tumors, to be successfully USA translated in a human setting.

Among the most interesting targets for immunotherapeutic approaches, the Chondroitin Sul- A novel integrative strategy to pre- fate Proteoglycan (CSPG)4 stands out, with low vent colorectal cancer within the expression in healthy tissues, high expression in several solid tumors and a key role in cancer diet-host-microbiota triangle: from progression. Because of the translational power organoids to human in vivo reality of dogs as pre-clinical models for human malig- nancies and the CSPG4 over-expression by both Josep Rubert, Andrea Lunardi human and canine malignant melanoma (MM), we demonstrated the safety and the clinical ef- CIBIO - Department of Cellular, Computational fectiveness of a xenogeneic human (Hu)-CSPG4 and Integrative Biology. University of Trento DNA vaccine in client-owned canine patients with (Italy). Via Sommarive 9, 38123 Povo (Trento) stage II-III surgically resected CSPG4+ MM. Howev- er, Hu-CSPG4 vaccine was barely effective in ac- Colorectal cancer (CRC) is one of the most com- tivating human T cells from healthy donors in vit- mon cancers in the western world. Several hun- ro. Based on these results, we aimed to increase dreds of thousands people are diagnosed annually the translational power of our approach and to with CRC and over half of patients die or have co- extend it for the treatment of other CSPG4+ tu- morbidities. Research has suggested that dietary mors besides MM. patterns, dysbiosis and microbial metabolites As a step forward in this direction, we generated may play a pivotal role in, leading to increasing a hybrid plasmid, derived in part from the Hu- interest among scientists. However, despite the and in part from the dog (Do)-CSPG4 sequences fact that microbial metabolites play a crucial (HuDo-CSPG4). We tested the safety, immuno- role in many biological cases, adequate tools for genicity and anti-tumor potential of HuDo-CSPG4 deciphering the relationship between diet-micro- DNA vaccine in mice, in dogs with stage II-IV sur- biome-host are not yet available. TRIANGLE aims gically resected CSPG4+ MM and in a human set- to provide new insight into the mechanisms by ting in vitro. which microbial metabolites may prevent CRC. Chimeric HuDo-CSPG4 vaccination resulted The first objective is targeted at designing in vit- strongly immunogenic in mice. In canine pa- ro models mimicking human organogenesis and tients, the procedure was safe and induced an- tumorigenesis to evaluate the role of microbial tibodies against both Hu- and Do-CSPG4, with a metabolites. To accomplish this, human colon or- higher affinity and anti-tumor potential as com- ganoids/tumoroids will be established. The sec- pared to Hu-CSPG4. Clinically, HuDo-CSPG4 was ond objective is to identify microbial metabolites effective in increasing the overall survival of that can act as cancer-preventive agents. These

The Pezcoller Foundation Journal - June 2019 25 metabolites will be produced with a gastroin- ment of healthy and tumoral brains and the effect testinal model inoculated with faeces from both of compound treatments both on brain tumor de- healthy and CRC patients and determined by velopment and immune cells, mainly microglia. mass spectrometry. Released microbial metabo- Leukocytes, including macrophages and microglia lites from a food model will then be tested in the are normally present in the brain at homeostatic colon organoids/tumoroids, and metabolite sig- conditions at larval and adult stages. Both in the natures of organoids will be studied. Metabolom- larval and adult tumor model an increase in the ics analysis and 3D cell assays of colon organoids/ number of immune cells in the region expressing Posters tumoroids will provide valuable new insights into the HRAS oncogene, together with an increased the mechanisms by which nutrient-gene interac- percentage of amoeboid-like active microglia tion influences colon stem cell niche and CRC, was observed. and will open up new possibilities for CRC under- Given that an altered epigenetic landscape is standing and prevention. In summary, the results very common in brain tumors, we have screened from TRIANGLE through the integration of micro- zebrafish larvae with a library of compounds tar- bial metabolites (diet), gut microbiota and colon geting epigenetic factors and, as a result, 3 hits organoids (host), will be highly multidisciplinary, were found to have an effect in slowing down the and will undoubtedly set the stage towards the development of brain tumors, but only one, an identification of mechanisms contributing to CRC HDAC inhibitor, led to changes in microglia mor- understanding and will ultimately pave the way phology. to phytochemical use and prevention. In order to characterize gene expression changes in tumor cells after treatment with the HDACi, Acknowledgement we have optimized the TRAP (Translating Ribo- This project has received funding from the Eu- somes Affinity Purification) technique to spe- ropean Union’s Horizon 2020 research and in- cifically pull down translating ribosomes in the novation programme under the Marie Skłod- cytoplasm of oncogene-expressing brain cells. owska-Curie grant agreement N. 794417. The translatome of HDACi treated tumor cells was compared with the transcriptome from FACS sorted zebrafish brain tumor cells; from the anal- A screen for drugs that affect brain ysis of the differentially translated or transcribed genes, the translatome emerged as more precise- tumor development and microglia re- ly reflecting the biological phenotype that had cruitment in zebrafish been observed after HDACi treatment. Pathway analysis of the differentially translated genes re- Valeria Savoca1, Isabella Pesce1, Dirk Sieger2 and vealed that the drug treatment caused the down- Marina Mione1 regulation of many factors involved in purine metabolism, which could explain the decrease in 1 Centre for Integrative Biology, University of brain tumor growth in the zebrafish model treat- Trento, Via Sommarive, 9, Trento 38123, Italy ed with HDACi. 2 Centre for Discovery Brain Sciences, The Uni- Taken together, these results reinforce the knowl- versity of Edinburgh, The Chancellor’s Building, edge that the zebrafish provides a useful model 49 Little France Crescent, Edinburgh EH16 4SB, to investigate the effects of HDACi in brain tum- United Kingdom ors and associated microglia.

One of the hallmarks of brain cancer is the formation of an immune-microenvironment in- miRNAs as potential predictive bio- filtrated with tumor associated macrophages (TAMs), which contribute to establish a status of markers of metastases in thin and thi- chronic inflammation. Thus, the production of ck primary cutaneous melanomas high amount of immune inhibitory cytokines or inflammatory mediators leads to an increase in Virginia Valentini1, Veronica Zelli1, Emanuela tumor cells proliferation and invasion. Gaggiano2, Valentina Silvestri1, Piera Rizzolo1, The most frequent and aggressive form of brain Agostino Bucalo1, Stefano Calvieri2, Pasquale tumor, glioblastoma multiforme (GBM), is resist- Frascione3, Pietro Donati4, Antonio Giovanni ant to standard of care therapies because of its Richetta2 and Laura Ottini1 heterogeneity, infiltration properties and immune suppressive microenvironment, therefore inno- 1 Department of Molecular Medicine, “Sapienza” vative therapies are being investigated, among University of Rome, Rome, Italy; which immunotherapies, whose aim is to allevi- 2 Department of Internal Medicine and Medical ate GBM-associated immune suppression and to Specialties, Unit of Dermatology, “Sapienza” boost anti-tumor immune responses. University of Rome, Rome, Italy; In this project we use a zebrafish model of brain 3 Department of Oncological and Preventative tumor to investigate the immune microenviron- Dermatological, San Gallicano Dermatological

26 The Pezcoller Foundation Journal - June 2019 Institute, IRCCS, Rome, Italy; 2 Candiolo Cancer Institute FPO-IRCCS. Candiolo, 4 Laboratory of Cutaneous Histopathology, San Torino, Italy Gallicano Dermatologic Institute, Rome, Italy 3 Department of Oncology at San Luigi Hospital. University of Torino, Torino, Italy Background: The early detection of primary cutaneous melanomas at high-risk of metastat- Epidemiological data support that obesity, due ic dissemination is essential to improve clinical to a high-fat intake typical of Western diet, management and outcomes of melanoma pa- increases the risk of colon cancer (CRC). Re- tients. The high potential of miRNAs as diagnos- cently it has been demonstrated that, high fat Posters tic, prognostic and therapeutic biomarkers in diet (HFD) and exposure to saturated fatty ac- melanoma is well established. The aim of this ids - such as palmitoleic acid (PA) - augments study was to characterize miRNAs expression the numbers of intestinal progenitors and their profile in relation to metastatic process of cuta- tumorigenicity. Instead, polyunsaturated fatty neous melanoma and correlate miRNAs expres- acids such as docosahexaenoic acid (DHA) are sion with clinical and pathological factors. shown to be protective against CRC in epidemio- Materials and Method: Expression levels of six logical studies; however experimental evidence miRNAs, known to be involved in metastatic supporting this anticancer effect, as well as the process (miR-145-5p, miR-150-5p, miR-182-5p, mechanism, is still lacking. We investigated the miR-203-3p, miR-205-5p and miR-211-5p), were potential anti-CRC activity of polyunsaturated analyzed by quantitative Real-Time PCR in a se- fatty acids in our collections of patient-derived ries of 32 metastatic and non-metastatic primary CRC organoids. cutaneous melanomas, including thin and thick DHA decreased CRC growth in vitro, while PA did melanomas. Eight samples of metastases were not show any effect respect to control. Tumor also examined. Associations between miRNA ex- growth inhibition was mainly caused by cell pro- pression levels and clinical-pathologic charac- liferation rate reduction (determined by ki-67 teristics of primary tumors were also evaluated. staining). Notably, this effect was much more All statistical analyses were performed with the prominent in KRAS-mutated compared to KRAS- R software (www.r-project.org.). wild type tumors. We further analyzed the effect Results: A lower miR-205-5p expression was of DHA or PA on organoids tumor initiating cells observed in metastases when compared with by assessing stem cell markers (by qPCR) and primary metastatic melanomas (p=0.04). Fur- clonogenic capacity. DHA treatment diminished thermore, a progressive downregulation of miR- both Lgr5 and SOX2 RNA levels in KRAS-mutat- 205-5p expression was observed from loco-re- ed organoids while PA treatment did not modify gional metastasis to distant metastasis. expression levels significantly. To assess the col- Significantly lower miR-145-5p and miR-203- ony-forming efficiency we dissociated CRC-orga- 3p expression levels were found in cases with noids to single cells and quantified the number Breslow thickness >1mm (p = 0.002 and p = 0.005, of growing organoids. DHA treatment decreased respectively), high Clark level (p = 0.007 and p colony formation respect to control while PA = 0.005, respectively), ulceration (p = 0.00001 did not. Even DHA-pretreated organoids showed and p = 0.0002, respectively) and mitotic rate decreased colony efficiency compared to those ≥1/mm2 (p = 0.02 and p = 0.001 respectively). that were not pre-treated, supporting a role for Conclusion: Our findings add insights into the DHA in modulating the stem cell number in orga- characterization of miRNAs expression profile of noids. Our results indicate that polyunsaturated thin and thick melanomas, pointing to miR-205- fatty acids affect organoid-initiating capacity 5p as potential marker of distant metastases and tumor growth supporting a potential role for and to miR-145-5p and miR-203-3p as markers prevention and treatment of CRC. of aggressiveness in primary tumors.

Study supported by AIRC-IG21389 to L.O.

Polyunsaturated fatty acids reduces in vitro tumor growth of colorectal cancer patient-derived organoids

Vara-Messler M.1, Di Blasio L.2, Monica V.3, So- male D.2, Chiaverina G.1, Puliafito A.1, Palmiero M.1, Peracino B.3, Primo L.1

1 Department of Oncology at Candiolo IRCCS. University of Torino, Torino, Italy.

The Pezcoller Foundation Journal - June 2019 27 Pezcoller Foundation–AACR International Award for

Award Extraordinary Achievement in Cancer Research

2019 Program guidelines and nomination instructions

NOMINATION DEADLINE August 1, 2019

NOMINATION PROCESS Nominations may be submitted by any invdividual, whether an AACR member or nonmember, who is currently or has previously been affiliated with any institution involved in cancer research, cancer medicine, or cancer-related biomedical science. Self-nominations are prohibited. Nominators must maintain strict confidentiality of their nominations and all nominations must be submitted online to myaacr.aacr.org. Paper nominations will not be accepted. Eligible nominations must include the following nomination materials: • A letter of recommendation written in English (Max: 1,000 words) that comprehensively describes the nominee’s major scientific achievement(s) in basic cancer research and/or their significant contributions to translational cancer research. This letter must also outline the nominee’s current research activity and indicate how this research holds promise for continued substantive contributions to the cancer field. • A brief scientific citation (Max: 50 words) highlighting the major scientific contribution(s) justifying the award candidate’s nomination.

AWARD ELIGIBILITY AND CRITERIA The prestigious Pezcoller Foundation-AACR International Award for Extraordinary Achievement in Can- cer Research was established in 1997 to recognize a scientist of international renown who has made a major scientific discovery in basic cancer research or who has made significant contributions to translational cancer research. Eligible candidates must continue to be active in cancer research, have a record of recent, notewor- thy publications, and be conducting ongoing work that holds promise for continued substantive contributions to progress in the field of cancer. The Pezcoller Foundation-AACR International Award for Extraordinary Achievement in Cancer Resear- ch is intended to honor an individual scientist. However, more than one scientist may be co-nomina- ted and selected to share the award in the event that their investigations are intimately related in subject matter and have resulted in work that is worthy of the award and a joint nomination. Cancer researchers affiliated with institutions in academia, industry, or government involved in cancer research, medicine, or cancer-related biomedical science anywhere in the world are eligible. Institutions and/or organizations are not eligible to receive the award.

AWARD SELECTION PROCESS All eligible nominees will be considered by a prestigious Pezcoller Foundation-AACR International

28 The Pezcoller Foundation Journal - June 2019 Award for Extraordinary Achievement in Cancer Research Selection Committee consisting of an international cohort of renowned cancer leaders appointed by the AACR President in consultation with the Pezcoller Foundation Council. The Pezcoller Foundation-AACR International Award for Extraordinary Achievement in Cancer Resear- ch Selection Committee will consider all nominations as they have been submitted and are restricted from combining submitted nominations, adding new nominees, or otherwise making alterations to any submitted nomination. Award Once chosen, the primary and alternate award recipient selections made by the Pezcoller Founda- tion-AACR International Award for Extraordinary Achievement in Cancer Research Selection Commit- tee shall be sent to the AACR Executive Committee and the Pezcoller Foundation Council for final consideration and ratification. Selection of the award winner will be made on the basis of the candidate’s scientific accomplishmen- ts. No regard shall be given to race, gender, nationality, religion or political preference. Selected award recipients will receive an unrestricted grant of €75,000, a commemorative award, and be invited to present a scientific lecture in conjunction with the AACR Annual Meeting immediately following their selection.

THE AWARD RECIPIENT The winner of the Pezcoller Foundation-AACR International Award for Extraordinary Achievement in Cancer Research will present an Award lecture at the AACR Annual Meeting 2020 in San Diego, CA (April 24-29, 2020). The winner will also present the Twenty-third Annual Pezcoller Foundation-AACR International Award for Cancer Research Lecture, just prior to the official Award ceremony to be held in Trento, Italy in May 2020. Should the recipient be unable to participate in either event, the award must be forfeited and will instead be presented to the selected Award alternate. In the rare event that there are dual winners of the Award, the monetary award will be shared equally between both recipients while the AACR Executive Committee will determine which of the two co-recipients will present the Pezcoller-AACR Award Lecture at the AACR Annual Meeting.

The Pezcoller Foundation Journal - June 2019 29 Award????? 2019 Scholar-In-Training Awardees withPresidentGalligioniin Atlanta, March31,2019 Picture: Any [email protected] representing Italy)andbasedonthemeritoriousscore ofthesubmittedabstractandapplication. Selections are made by the criteria from Pezcoller (i.e. European scientists with at least one awardee national cancerresearchcommunityatthe AACR Annual Meeting. Scholar-in-Trainingding Awardees inter the with findings research their share to opportunity an with of the activities outstan to providethese residing inEuropeand investigators AACR byearly-career The Pezcoller Foundationsupportstheseawardstoenhanceparticipationintheprograms and Scholar-in-TrainingFoundation2020 AACR-Pezcoller Awards at the AACR Annual Meeting. meritorious profferedpapers presenting investigators young outstanding recognize and competitive cancer. Scholar-in-Training against fight highly the are to Awards dedicated organizations other and individuals corporations, foundations, research cancer 55 than more from support received has and Meeting Scholar-in-Training Award program has provided more than 4,580 grants to young investigators at the scientists rious early-career the in 1986, its inception Since 2019. Meeting Annual AACR Annual The 2020 Scholar-In-Training Awards 30 has been proud tooffer Scholar-in-Trainingbeen has AACR Awardsof merito- participation the toenable The Pezcoller Foundation Journal-June 2019 - -

Six-monthly review of the Pezcoller Foundation Via Dordi 8 - 38122 Trento - Italy Tel. (39) 0461 980250 Fax (39) 0461 980350 e-mail: [email protected] www.pezcoller.it

Proprietario/editore: Fondazione Prof. Alessio Pezcoller - Trento n. 36 - Registro delle Persone Giuridiche presso il Commissario del Governo della Provincia di Trento Redazione: Via Dordi 8 - 38122 Trento Direttore Responsabile: Gios Bernardi

“The Pezcoller Foundation Journal” year 29, n. 52, Semestrale giugno 2019 Poste Italiane spa Spedizione in abbonamento postale D.L. 353/2003 (conv. In L. 27/02/2004 n. 46) Art. 1, comma 2, CNS Trento taxe percue / tassa riscossa