Summary • Editorial June 2018 • 30Th

Summary

• Editorial June 2018 • 30th Pezcoller Symposium: Program Abstracts of oral presentations Abstracts of posters • Call for nomination 2019 Pezcoller Foundation-AACR International Award for Extraordinary Achivement in Cancer Research • Call for nomination Scholar-In-Training Awards

News from the Year 28 – No. 50 Pezcoller Foundation Word June 2018 Picture on front page: 2018 Pezcoller Foundation-AACR International Award for Extraordinary Achievement in Cancer Research in the Teatro Sociale of Trento From the left: President Emeritus Gios Bernardi – Pier Paolo Pandolfi Chairman of the Selection Committee – President Enzo Galligioni – Tony Hunter winner –Margaret Foti CEO AACR and Michael Caligiuri Past President AACR June 2018 Editorial Editorial

It’s with a great pleasure that we report that the Dr. Hunter has presented the Pezcoller Lecture recipient of the 2018 Pezcoller Foundation - AACR “Protein phosphorylation: pancreatic cancer and International Award for Extraordinary Achievement new frontiers in histidine phosphorylation ”at the in Cancer Research is Anthony Rex Hunter, Fellow McCormick Place South Convention Center in Chi- of the AACR Academy, American Cancer Society cago during the AACR Annual Meeting on April 15. professor, Renato Dulbecco Chair and professor at On Thursday May 17 dr. Hunter gave his lecture at the Salk Institute for Biological Studies, University the University of Padova Aula Magna “A.Vallisneri” of California, San Diego. while he gave another lecture at the University of The Selection Committee who designated the win- Trento on Friday May 18 at the Centre for Integra- ner met in Philadelphia on December 1st 2017 and tive Biology. was chaired by Pier Paolo Pandolfi. The award was solemnly given on May 19 in the Te- Members of the Committee were Pier Paolo Pan- atro Sociale of Trento introduced by the President dolfi MD PhD ( Chair) Director Cancer Center Beth of the Pezcoller Foundation Enzo Galligioni with Israel Deaconess Medical Center Boston, Carlos the participation of Michael Caligiuri past Presi- L. Arteaga MD FAACR Director Center for Cancer dent of the AACR, Margaret Foti CEO of the AACR Targeted Therapies and Breast Cancer Program and Gios Bernardi President Emeritus. Vanderbilt-Ingram Cancer Center Nashville, Fran- cis R. Balkwill PhD Centre Lead Barts Cancer In- In this issue of the Journal we have the pleasure of stitute London, Michael B. Kastan MD PhD FAACR remembering a few important anniversaries of the Executive Director William & Jane Shingleton Duke Pezcoller Foundation. Cancer Institute Durham NC, Elaine Mardis PhD For the past thirty years the Pezcoller Foundation Co-Director and Professor of Pediatrics Nationwide has been committed to supporting Cancer re- Children’s Hospital Columbus OH, Cornelia Polyak search. MD PhD Department of Medical Oncology Dana Far- In 1988, thirty years ago, in the presence of the ber Cancer Institute Boston, Stefano Piccolo PhD founder prof. Alessio Pezcoller, the First Award Professor of Molecular Biology University of Pad- was given to Vincent de Vita for his studies on the ua, Verda Rotter PhD Professor and Chair Depart- chemotherapy of malignant lymphomas. Also in ment of Molecular and Cellular Biology Weizmann 1988 the first Pezcoller Symposium was organized Institute of Science Rehovot Israel, Charles Swan- in Trento, entitled “ Drug Resistance: Mechanisms ton MD The Francis and Reversal “ Crick Institute and Twenty-five years after his death, in January 1993, UCL Cancer Insti- we remember with gratitude our founder prof. tute London. Alessio Pezcoller Tony Hunter, PhD, We also recall that on April 13, 1987 the agree- is honored for his ment between the Pezcoller Foundation and the significant discov- AACR was stipulated for the assignment of the ery of tyrosine “Pezcoller Foundation-AACR International Award kinases. This pio- for Cancer Research” agreement which has been neering work has renewed this year also with the modification of led to proven suc- the Award title in “Pezcoller Foundation-AACR In- cess in the usage ternational Award for Extraordinary Achievements of cancer chemo- in Cancer Research” therapeutics that target disease-causing tyrosine This year we celebrate the 30th Pezcoller Sym- kinases. Furthermore, Dr. Hunter is credited with posium in Trento on June 25th and 26th entitled: being the first to demonstrate that dysregulated “Overcoming the innate resistance of cancer to tyrosine phosphorylation by activated tyrosine therapy” with the extraordinary Keynote Enrico kinases, such as the viral Src protein, can cause Mihich Lecture by Harold Varmus Nobel Laureate malignant transformation. 1989. Dr. Hunter’s highly regarded work has led to the discovery and development of many effective drugs targeting aberrant kinase signaling in can- Gios Bernardi M.D. cer cells. Editor and President Emeritus

The Pezcoller Foundation Journal - June 2018 3 30th Pezcoller Symposium OVERCOMING THE INNATE RESISTANCE OF CANCER TO THERAPY Trento, Italy • June 25 - 26, 2018

PROGRAM

MONDAY JUNE 25, 2018 TUESDAY JUNE 26, 2018 8.00 Registration Session 4 - Therapeutically resistant Melanoma 8.35 Enzo Galligioni Welcome Chairman: Stefano Piccolo 8.45 David Livingston Focus & Goals 08.30 David Fisher 08.55 The Enrico Mihich Lecture Melanoma: clues to therapeutic efficacy from disease Harold Varmus pathogenesis Pezcoller at 30: Cancer Research, Then and Now 08.55 Ugur Sahin 09.35 Discussion Personalized cancer immunotherapy 09.20 Discussion led by Alberto Mantovani and Mario Colombo Session 1, Pancreas Cancer Chairman: Alberto Bardelli Session 5 - Glioblastoma Chairman: Stefano Piccolo 09.50 David Tuveson Improving pancreatic cancer response to therapy 10.00 Peter Dirks 10.15 Christine Iacobuzio-Donahue Dissecting brain tumour cell fate with functional Evolutionary dynamics of cancer approaches 10.40 Discussion led by Giampaolo Tortora and Davide Melisi 10.25 Ingo Mellinghoff 11.20 Coffee Break Targeting aberrant signaling in primary brain tumors

Session 2, Leukemia 10.50 Discussion led by Felice Giangaspero Chairman: Alberto Bardelli 11.30 Coffee Break

11.30 Brunangelo Falini – Enrico Tiacci Session 6 - Kidney Cancer BRAF inhibition in hairy cell leukemia: biological and Chairman: Massimo Loda clinical aspects 11.55 Pier Paolo Pandolfi 11.40 William Kaelin Modeling resistance identifies vulnerabilities and effective New models and treatments for clear cell renal cell combination therapy in mIDH AML carcinoma 12.20 Discussion led by Pier Paolo Pandolfi 12.05 John Josey 13.00 Lunch Inhibiting the transcriptional factor HIF-2α:treatment of renal cell carcinoma and VHL disease Session 3, Prostate Cancer 12.30 Discussion led by William Kaelin and John Josey Chairman: Pier Paolo Pandolfi 13.10 Lunch

14.00 Charles Sawyers Session 7 - Mesothelioma and Small Cell Lung Cancer Cancer drug resistance Chairman: Alberto Bardelli 14.25 Massimo Loda 14.10 Anton Berns Effect of the microenvironment on prostate cancer biologic Mouse models of thoracic cancers. Phenotypic and clinical behavior consequences of their cell-of-origin 14.50 Discussion led by Francesca Demichelis and Sergio Bracarda 14.35 John Poirier 15.30 Poster Session Molecular mechanisms of acquired chemoresistance in 16.30 Adjourn small cell lung cancer 18.00 visit to Mezzacorona Winery (shuttle transportation provided) 15.00 Discussion led by Alberto Mantovani 20.00 Symposium Dinner at Mezzacorona Winery 15.40 Poster Discussion and Poster Presentation led by Massimo Loda 16.40 David Livingston Concluding Remarks

4 The Pezcoller Foundation Journal - June 2018 30th Pezcoller Symposium OVERCOMING THE INNATE RESISTANCE OF CANCER TO THERAPY Trento, Italy • June 25 - 26, 2018

INVITED PARTECIPANTS

FACULTY

• Bardelli Alberto Candiolo Cancer Institute, University of Torino, Italy • Berns Anton Netherland Cancer Institute, Amsterdam, Holland • Dirks Peter The Hospital for Sick Children, Toronto, Ontario, Canada • Falini Brunangelo Institute of Hematology, University of Perugia, Italy • Fisher David Massachusetts General Hospital, Charlestown, MA • Iacobuzio-Donahue Christine Memorial Sloan Kettering Cancer Center, New York, NY • Josey John Peloton Therapeutics Inc, Dallas, TX • Kaelin G. William Dana-Farber Cancer Institute, Boston, MA • Loda Massimo Dana-Farber Cancer Institute, Boston, MA • Mellinghoff Ingo Memorial Sloan Kettering Cancer Center, New York, NY • Livingston David Dana-Farber Cancer Institute, Boston, MA • Pandolfi Pier Paolo Cancer Center, Beth Israel Deaconness Medical Center, Boston, MA • Piccolo Stefano Molecular Medicine Department, University of Padova, Italy • Poirier John T. Memorial Sloan Kettering Cancer Center, New York, NY • Sahin Ugur BioNTech AG, University of Mainz, Germany • Sawyers Charles Memorial Sloan Kettering Cancer Center, New York, NY • Tiacci Enrico Hematology Department, University of Perugia, Italy • Tuveson David Cancer Center Cold Spring Harbor Lab, Cold Spring, NY

• Varmus Harold Weill Cornell Medicine Meyer Cancer Center, New York, NY

DISCUSSANTS

• Bracarda Sergio Medical Oncology Department, General Hospital, Arezzo, Italy • Colombo Mario Molecular Immunology, Fondazione Istituto Nazionale dei Tumori, Milan, Italy • Demichelis Francesca Centre for Integrative Biology, University of Trento, Italy • Giangaspero Felice Policlinico Umberto I, Università La Sapienza Roma, Italy • Mantovani Alberto Humanitas University, Milan, Italy • Melisi Davide Medical Oncology, University of Verona, Italy • Tortora Giampaolo Medical Oncology, University of Verona, Italy

The Pezcoller Foundation Journal - June 2018 5 30th Pezcoller Symposium Overcoming the innate resistance of cancer to therapy

Abstracts Trento, Italy, June 25-26, 2018

ABSTRACTS OF ORAL PRESENTATIONS

Enrico Mihich Lecture instance, we have recently found that muta- Pezcoller at 30: Cancer Research tions in the KRAS and EGFR proto-oncogenes do not appear in the same lung adenocarcinomas Then and Now. because the combination of these two common mutations is cytotoxic. By attempting to under- Harold Varmus, MD stand how mutations in the EGFR-KRAS signaling pathway affect “downstream” kinases, like ERK, Lewis Thomas University Professor, Weill Cor- and by looking for feedback mechanisms that nell Medicine; regulate such kinases, it may be possible to ge- Senior Associate Member, New York Genome nerate new therapeutic approaches for certain Center cancers. (iii) Cancer genomics teaches us that To commemorate the long history of the Pezcol- mutations In certain genes are more likely to ini- ler meetings, I plan to take a long view. I will tiate tumors in specific cell lineages, providing reflect on the manner in which technical advan- insights into neoplasia that were impossible when ces have enabled the cancer research community carcinogenic events were studied mainly in cul- to approach old questions in new ways, building tured animal fibroblasts. We have, for example, on information acquired with technologies that recently learned to induce pulmonary neuro-en- date back to days before the Pezcoller meetings docrine cells (PNECs), the proposed precursors to and envisioning answers that are emerging from human small cell lung cancers (SCLC), from hu- newer methods to improve our understanding man embryonic stem cells (hESCs) by inhibiting and control of cancer. To illustrate these themes, maturation of NOTCH receptors. We can then I will talk briefly about three projects that are make PNECs oncogenic by simulating the most currently being pursued in my laboratory: common mutations in SCLC: inactivation of two (i) The search for mutant genes that tumor suppressor genes, RB and TP53. I will di- drive carcinogenesis has been made both easier scuss how this approach might be applied in se- and more complex by the application of Next veral situations to improve our understanding of Generation Sequencing (NGS) methods. But early events in human carcinogenesis. some of the implicated mutations, such as those affecting genes that encode parts of the RNA splicing machinery, have been very difficult to understand functionally. I will summarize how Improving Pancreatic Cancer we have attempted (with only limited success) Responses to Therapy to address these issues through work with tran- scriptomes, cancer cell lines, gene editing, and David Tuveson MD, PhD mouse models. (ii) The complex patterns of muta- Cancer Center Cold Spring Harbor Lab, New tions observed in individual cancers with NGS York, NY methods have some unusual features---such as mutual genetic exclusivity---that invite efforts Pancreatic ductal adenocarcinoma (PDAC) is to understand their functional significance. For almost uniformly lethal, and surgical resection

6 The Pezcoller Foundation Journal - June 2018 of localized tumors followed by adjuvant of pancreatic cancer correlate with the gene- chemotherapy is currently the only curati- tic or evolutionary features of the same tumor? ve regimen. Unfortunately, most patients are Our data to be presented relies upon on whole diagnosed with advanced and surgically unre- exome or whole genome sequenced samples of sectable PDAC, due to a lack of early detection primary and metastatic pancreatic cancer tis- methods. Furthermore, such patients oftenti- sues, single cell technologies, and computatio- mes have a rapid disease course due to the nal models. Such questions are of broad interest ineffectiveness of therapies. We developed in cancer biology in general and have a strong mouse and organoid models of PDAC to explo- likelihood to be relevant to understanding me- re the biological aggressiveness of PDAC and chanisms of treatment resistance in other tumor to address these clinical challenges. Accordin- types as well. Abstracts gly, we assembled a large collection of patient derived PDAC organoids, and determined that they accurately represented the primary tu- Braf inhibition in hairy cell leuke- mors from which they were derived. We find that the combination of therapeutic testing mia: biological and clinical aspects and molecular analyses identifies patients who are more prone to respond to available the- Enrico Tiacci and Brunangelo Falini rapeutics. Furthermore, our chemo-sensitivity signatures predict clinical outcomes in PDAC Institute of Hematology and Center for patients who were retrospectively assessed, Hemato-Oncology Research, prompting a further investigation of organoid University and Hospital of Perugia (Italy) profiling as a means to prospectively increase survival in patients. Hairy cell leukemia (HCL) is a rare chronic B-cell neoplasm that is initially sensitive to Tiriac et al, Cancer Discovery 2018, in press chemotherapy with purine analogs. However, up to 50% of patients relapse and become progressively less responsive to these drugs, which Evolutionary dynamics of cancer are also myelotoxic and immunosuppressive. The founding genetic event of HCL is the BRAF- Iacobuzio-Donahue Christine V600E activating kinase mutation, which is present in almost all patients with HCL and in Memorial Sloan Kettering Cancer Center, New almost no patients with other mature B-cell York, NY lymphomas and leukemias (Tiacci et al NEJM, 2011). PDA is the most common neoplasm of the pan- HCL relies heavily (and more than BRAF-mu- creas, and is soon to be the second most com- tated solid tumors) on the V600E mutation for mon cause of cancer deaths in the United Sta- most of its unique features, including the pe- tes. Surgical resection in Stage I/II patients culiar hairy morphology, the distinctive tran- provides the only opportunity for cure, yet >80% scriptional signature and the characteristic of patients will recur and die of their disease immunophenotype, as well as for its viability within 2-3 years. The statistics for Stage III and (Pettirossi et al., Blood 2015). Thus, BRAF- Stage IV PDA are more dismal, having 12 and 6 V600E shapes the hairy cell identity and repre- month median overall survival times, respecti- sents an attractive therapeutic target. vely. Outside of BRCA2 mutations that confer Indeed, the BRAF inhibitor vemurafenib pro- sensitivity to platinum salts or PARP inhibition, ved impressively active in HCL patients heavily or immune checkpoint inhibitors in rare patients pre-treated with (and often refractory to) pu- with mismatch repair deficiency, there are few rine analogues: an oral, brief treatment with actionable targets in the PDA genome. Thus, it this drug produced almost 100% overall reis essential that novel strategies are developed sponses and about 40% of complete remissions to extend survival. To do so it is first imperative (CRs), with no myelotoxicity nor immune-sup- that we develop a deeper understanding of PDA pression (Tiacci et al., NEJM 2015). evolutionary biology in a stage and context de- However, residual leukemic cells persist in the pendent manner. In this presentation we will fo- bone marrow even in complete responders, cus on two questions with clear mechanistic and often showing bypass ERK (re)phosphorylation translational relevance to this goal. First, what downstream of BRAF-V600E, and relapses are are the features of clinically relevant intratumo- common after stopping the drug. To improve ral heterogeneity at the genetic level? Second, response depth and duration, these vemurafe- how do PDA therapies influence evolutionary nib-resistant residual HCL cells should be elimi- trajectories, and can they be more effectively nated, or their emergence prevented altogether, used within the evolutionary context of a tu- ideally by adding to vemurafenib another targe- mor? Second, how to transcriptional subtypes ted, non-myelotoxic drug.

The Pezcoller Foundation Journal - June 2018 7 In this regard, the strategies we are currently in general, independent of cooperating gene- pursuing in the clinic aim at: i) addressing the tic events. On the basis of this data we iden- MEK-ERK dependent mechanisms of resistance tify a powerful APL-like targeted therapy to by concomitantly blocking MEK1/2 (the kinases effectively treat mIDH mouse and human leu- phosphorylated by BRAF) through the addition kemic models. Thus, our findings pave the way to vemurafenib of the MEK1/2 inhibitor cobi- towards the treatment of a sizable fraction metinib; and ii) targeting resistant HCL cells, of human AML through targeted combinatorial whether still depending on MEK-ERK activation therapies. or not, through anti-CD20 immunotherapy in combination with BRAF blockade. Abstracts Lineage Plasticity in Personalized cancer Prostate Cancer Immunotherapy Charles L. Sawyers, M.D.

Ugur Sahin Memorial Sloan Kettering Caner Center, New York, NY BioNTech AG, University of Mainz, Germany The most common mechanism of acquired re- Abstract not received sistance to targeted cancer therapies is alteration of the drug target -- typically through mutation or amplification or through bypass Modelling resistance identifies of the signaling pathway blockade. There is growing evidence of a conceptually distinct vulnerabilities and effective mechanism in which the tumor cells esca- combination therapy in mIDH pe growth inhibition by shifting their lineage AML such that the drug target is no longer essential for the proliferation of that cell type. I Pier Paolo Pandolfi, MD, PhD will present recent work from our laboratory showing how prostate cancers can escape Cancer Research Institute, Beth Israel Deaco- from hormone therapy by undergoing an iden- ness Cancer Center, Department of Medicine tity change from androgen-dependent luminal and Pathology, Beth Israel Deaconess Medical epithelial cells to more basal-like epithelial Center, Harvard Medical School, Boston, Mas- cells. This lineage plasticity occurs in the set- sachusetts, USA ting of combined loss of function mutations in RB and TP53 and is mediated in part by the Although targeted therapies have proven ef- reprogramming transcription factor SOX2. In fective and even curative in human leukemia, addition, we have found that several prostate resistance often ensues. IDH enzymes are mu- cancer driver genes, such as FOXA1, ERF and tated in ~20% of human AML, and currently, ERG, also perturb normal basal-luminal diffe- specific inhibitors of mutant IDH enzymes are rentiation programs. Thus, lineage plasticity actively under clinical investigation as effecti- can have a role in prostate cancer initiation ve treatment for mutant IDH AML. However, as well as in acquired resistance, with impor- resistance to single agent targeted therapies is tant clinical implications for the timing and frequently observed in the clinic. Using mouse context in which hormone therapy should be models, we characterize leukaemia evolution used. from mutant IDH2 (mIDH)-dependence to in- dependence, identifying key vulnerabilities References for both mIDH-dependent and independent Watson PA, Arora VK, Sawyers CL. Emerging leukemias. By applying high-throughput te- mechanisms of resistance to androgen recep- chnologies we identify key vulnerabilities of tor inhibitors in prostate cancer. Nat Rev Can- mIDH leukaemia related to metabolism and si- cer. 2015 15:701-11. PMCID: 4771416. gnalling. Mechanistically, we identify PIN1 and Mu P, Zhang Z, Benelli M, Karthaus WR, Hoover LSD1 to be mediators of mIDH vulnerabilities. E, Chen CC, Wongvipat J, Ku SY, Gao D, Cao Z, We further demonstrate PIN1 to act as both a Shah N, Adams EJ, Abida W, Watson PA, Prandi key regulator of leukaemia maintenance, and D, Huang CH, de Stanchina E, Lowe SW, Ellis L, an inhibitor of differentiation in both sensitive Beltran H, Rubin MA, Goodrich DW, Demichelis (mIDH2 -dependent) and resistant (mIDH2-in- F, Sawyers CL. SOX2 promotes lineage plasti- dependent) disease. city and antiandrogen resistance in TP53- and Importantly, we confirm our findings for leuke- RB1-deficient prostate cancer. Science. 2017 mias harboring both mIDH-1 and -2 mutations 355:84-88. PMCID: 5247742.

8 The Pezcoller Foundation Journal - June 2018 Effect of the microenvironment thelium by 3 month of age, histological chan- on prostate cancer biologic and ges were observed in the stroma. Stromal cells are known to supply Wnt ligands that bind to clinical behavior the Wnt receptors such as Lgr and Frizzled in the epithelium. Hubert Pakula, Svitlana Tyekucheva, We found that there was a progressive incre- Kathryn Penney and Massimo Loda ase in Lgr5 positive cells in the epithelium of T2E and T2E/PTEN-/- adjacent to HGPIN, Departments of Oncologic Pathology and Biosta- while PORCN was progressively up-regulated tistics & Computational Biology, Dana-Farber in the adjacent tumor stroma of these mice.

Cancer Institute; Departments of Biostatistics PORCN acts as a key regulator of the Wnt si- Abstracts and Epidemiology, Harvard T.H. Chan Scho- gnaling pathway by mediating the attachment ol of Public Health; Channing Division of of palmitoleate, a 16-carbon monounsatura- Network Medicine, Department of Medicine, ted fatty acid, to Wnt proteins. Serine pal- Brigham and Women’s Hospital/Harvard Me- mitolylation of WNT proteins is required for dical School efficient binding to frizzled receptors. Taken together, these data suggest that the microe- Most human studies have focused on the muta- nvironment influences prostate cancer initia- tional landscapes in an attempt to predict bio- tion, maintenance, and metastatic progres- logic and clinical behavior of human prostate sion and that Wnt signaling plays a significant cancer. In addition, epigenetic and transcrip- role in this regard. tional epithelial signatures are an important adjunct in predicting aggressive and indolent behavior. While these add additional indepen- Melanoma: clues to therapeutic dent prognostic information, they could be further improved by knowledge of the contri- efficacy from disease pathogenesis bution of stromal elements. Stroma can mor- phologically and functionally change in the David E. Fisher MD, PhD presence of cancer. Compared to normal stroma, there is a swi- Massachusetts General Hospital, Charlestown, tching of the cellular phenotype, remode- MA ling of the extracellular matrix, increases in expression of growth factors and proteases, Cutaneous melanoma is among the few can- increased angiogenesis, and change in the in- cers with a well documented carcinogenic flammatory infiltrate. etiology (UV) yet with unrelenting increases The bidirectional signaling between epithelial in incidence. cells and stromal constituents during normal The melanoma genome typically contains prostate homeostasis is disrupted early in tu- thousands of UV-signature mutations. Many morigenesis. We performed gene expression of these lack oncogenic driver activity, but profiling of laser capture microdissected nor- are recognized to contribute to immune re- mal non-neoplastic prostate epithelial tissue cognition as potential neoantigens. This pre- and compared it to non-transformed and neo- sentation will explore features of cutaneous plastic low and high grade prostate epithelial adaptation to sun/UV, that suggest a powerful tissue from radical prostatectomies, each with set of mechanisms which balance evolutionary its immediately surrounding stroma. Whereas requirements for UV (ie vitamin D) against tis- benign epithelium in prostates with and wi- sue injury inflicted by UV. Carcinogenic fea- thout tumor were similar in gene expression tures of red/blond pigment will be discussed, space, stroma away from tumor was signifi- as well as manipulation of skin pigmentation, cantly different from that in prostates without and behavioral signals triggered by UV expo- cancer. sure. When high to low Gleason grade cases were Despite the high mutational load of melano- compared, upregulation of frizzled receptors ma, the majority of patients fail to achieve was noted. In addition, a stromal gene signa- durable clinical benefit from current immune ture reflecting bone remodeling was seen. In checkpoint therapies. validation data, the signature discriminated A genetically defined murine cancer model cases that developed metastasis from those will be presented, which enables dissection of that did not. the role(s) for UV neoantigens in immunothe- In order to assess mechanistically interactions rapy responses and also permitted the deve- between stroma and transformed epithelium, lopment of novel approaches to “rescue” im- we utilized TMPRSS-ERG (T2E) knock-in mice, munotherapy responsiveness in tumors which and crossed them with PTEN +/- mice. While are deficient in neo-antigens or pre-existing T2E mice did not show alterations in the epi- tumor inflammation.

The Pezcoller Foundation Journal - June 2018 9 The Hospital for Sick Children, sification into subgroups with distinct clini- Toronto, Ontario, Canada cal outcomes, reflected in the revised World Health Organization (WHO) classification of CNS tumors. Unfortunately, advances in our Peter Dirks MD PhD understanding of the molecular pathogenesis of primary brain tumors has not resulted The Hospital for Sick Children, Toronto, On- in better treatment and current therapy still tario, Canada heavily relies on surgery, radiation and chemotherapy. Diffuse tumor growth throughout Brain tumours harbour subpopulations of ne- the brain, uncertain drug delivery, and evo-

Abstracts oplastic stem cells that drive tumourigenesis. lution of the cancer genome during therapy However, the origin of intra-tumoural functio- represent formidable challenges for the deve- nal heterogeneity between diverse tumour lopment of molecularly targeted therapies for cells remains poorly understood. We have stu- brain tumors. died the fate of brain tumour stem cells in There remains an urgent need for a deeper un- mouse medulloblastomas using lineage tracing derstanding of the relationship between brain from Sox2+ cells, revealing that a rare, quie- tumor cells and their microenvironment and scent stem cell is at the root of tumour growth of brain tumor evolution during therapy. and regrowth after conventional and targeted I will discuss our efforts to address these therapy. More recently, we have studied the challenges through sequencing of circulating clonal evolution of barcoded human gliobla- tumor DNA in cerebrospinal fluid, in-situ cha- stoma cells in an unbiased way following serial racterization of the tumor microenvironment, xenotransplantation to define their individual and novel approaches to noninvasive imaging fate behaviours. of brain tumors. Independent of an evolving mutational signature, we show that the growth of GBM clones in vivo is consistent with a remarkably neu- tral process involving a conserved proliferati- New Models and Treatments for ve hierarchy rooted in glioblastoma stem cells Clear Cell Renal Cell Carcinoma (GSCs). Slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capa- William G. Kaelin, Jr., M.D. city, that in turn generates non-proliferative cells. We also identify rare “outlier” clones Howard Hughes Medical Institute, that deviate from these dynamics, and show Dana-Farber Cancer Institute and Brigham that chemotherapy facilitates the expansion and Women’s Hospital, Harvard Medical Scho- of pre-existing drug-resistant GSCs. I will di- ol, Boston, MA 02215 scuss implications of fate mapping approaches to define the patterns of brain tumour growth [email protected] and resistance. Our data support that these tumours reflect Inactivation of the VHL tumor suppressor pro- disturbance of normal developmental hierar- tein, which normally serves to target the al- chies rooted in subpopulations of cells with pha subunit of the HIF transcription factor for stem cell properties. proteasomal degradation, is common in clear cell renal cell carcinoma (ccRCC). In preclinical studies HIF2α, and not HIF1α, Targeting aberrant signaling in appears to drive ccRCC pathogenesis. HIF2α is a basis helix-loop-helix PAS domain protein primary brain tumors with two PAS domains (PAS A and PAS B). Kevin Gardner and Rick Bruick at UTSW identified a Ingo K. Mellinghoff, MD potentially druggable pocket in the HIF2α PAS B domain and further identified chemicals that Memorial Sloan Kettering Cancer Center, can bind to this pocket and induce an alloste- New York ric change that disrupts DNA binding. Medici- nal chemistry efforts at Peloton Therapeutics Primary Brain Tumors comprise a heterogene- produced the tool compound PT2399 and the ous group of diseases with distinct predilection clinical compound PT2385. for certain age groups, locations within the In preclinical models PT2399 is active against central nervous system, growth patterns, and a subset of VHL-/- ccRCC lines and PDXs and response to therapy. Molecular characteriza- PT2385 and has demonstrated activity in a tion of these tumors has uncovered recurrent subset of heavily pretreated ccRCC patien- genetic alterations that allow disease clas- ts. Nonetheless, some ccRCC are insensitive

10 The Pezcoller Foundation Journal - June 2018 to genetic and pharmacological inactivation gonist, PT2977 binds to a hydrophobic pocket of HIF2α, implying a need for both predictive in the Per-Arnt-Sim (PAS)-B domain of HIF-2α, biomarkers and alternative therapies. With re- preventing its dimerization with its obligate spect to the former, our preliminary data indi- partner, HIF-1α, thus inhibiting its transcrip- cate that p53 pathway mutations make ccRCC tional activity. In preclinical models of ccRCC, less dependent on HIF2α. PT2977 selectively antagonizes HIF-2α resul- With respect to alternative therapies, we ting in altered gene expression and potent an- found that high HIF2α levels make ccRCC ti-tumor effects. hyperdependent on EZH1 because of a HIF-dri- The preclinical characterization of PT2977 ven increase in H3K27 demethylases. Finally, and its performance in patients with ccRCC we are using CRISPR/Cas9 to make immuno- will be described. Abstracts competent models of ccRCC. Mouse models of thoracic cancers. Inhibiting the transcription factor Phenotypic consequences of their HIF-2α: treatment of renal cell cell-of-origin carcinoma and VHL disease Anton Berns1, Giustina Ferone1, Ekaterina Se- John A. Josey, Ph.D. menova1, Jitendra Badhai1, Hilde de Vries1, Ji- Ying Song3, Rajith Bhaskaran1, Lorenzo Bom- Peloton Therapeutics, Inc. Dallas, TX bardelli1, Min chul Kwon1, and Ivo Huijbers2

Von Hippel-Lindau (VHL) syndrome is a rare 1The Oncode Institute, Division of Molecular inherited autosomal dominant disorder that Genetics, 2MCCA Transgenic facility, 3Depart- results from mutations in the VHL tumor sup- ment of Pathology, The Netherlands Cancer pressor gene on chromosome 3p25.3. Institute, Amsterdam, The Netherland The syndrome is characterized by the formation of visceral cysts and tumors in numerous Thoracic cancers belong to the most lethal hu- organ systems. Elegant studies have demon- man malignancies. In particular, patients with strated that the von Hippel-Lindau protein small cell lung cancer (SCLC), lung squamous (pVHL) acts as a substrate recognition com- cell carcinoma (LSCC), and malignant me- ponent of an E3 ubiquitin ligase which, under sothelioma (MM) show very poor survival stati- normoxic conditions, tightly regulates the le- stics due to the late detection, disseminated vels of the alpha-subunit of the hypoxia-indu- spread, and chemo-resistance of the tumors. cible factors. We have generated multiple mouse models of VHL patients are prone to develop multifocal these tumors and studied how closely they re- and often bilateral clear cell Renal Cell Carci- semble their human counterpart, how these noma (ccRCC). Both VHL-associated ccRCC and tumors develop over time, from which cells sporadic ccRCC share a common truncal event they originate, what genomic alterations are of loss of pVHL function. recurrently found and how this influence tu- Loss of function of the pVHL tumor suppressor mor characteristics and their response to in- has the immediate downstream consequence terventions. The mouse tumors induced by the of unregulated accumulation of hypoxia-in- lesions most frequently observed in the spe- ducible factors and the pleotropic expression cific human tumor subtypes show remarkable of multiple gene products that contribute to similarity to their human counterpart. tumor angiogenesis, growth, metastasis, and This includes their marker profile, their pri- immune evasion. Specifically, in ccRCC tumor mary site and metastatic spread, their immu- cells with pVHL deficiency, HIF-2α upregulates nophenotype and their response to treatment. the expression of genes that are important to We observe substantial inter- and intra-tumor tumor growth and metastasis, including those heterogeneity and tumor plasticity, this in spi- that encode cyclin D1 (CCND1), vascular en- te of the fact that the mouse tumors are dri- dothelial growth factor A (VEGFA), transfor- ven by the same set of lesions. ming growth factor α, and C-X-C chemokine The cell-of-origin of these tumors can be sur- receptor 4. prisingly diverse with the same set of driver While most transcription factors have histo- mutations giving rise to phenotypically quite rically been considered intractable targets different lesions. Clearly, both the specific set for small molecule modulation, recent effor- of driver lesions as well as the cell-of-origin ts have afforded potent, orally bioavailable of a particular tumor subtype are determining small molecule antagonists of the transcrip- factors for the tumor characteristics and re- tion factor hypoxia-inducible factor-2α (HIF- sponse to treatment. The lessons learned from 2α), a primary driver of ccRCC. One such anta- these models will be discussed.

The Pezcoller Foundation Journal - June 2018 11 Molecular Mechanisms of moresistant cancers. Multiple chemoresistant acquired chemoresistance in small models demonstrated suppression of SLFN11, a factor implicated in sensitivity to a variety cell lung cancer of classes of chemotherapeutic agents. In vivo silencing of SLFN11 was associated with mar- J.T. Poirier, MD ked deposition of H3K27me3, a histone modification placed by EZH2, within the gene body Memorial Sloan Kettering Cancer Center NY of SLFN11, inducing local chromatin condensa- “Small cell lung cancer is initially highly re- tion and gene silencing. sponsive to cisplatin and etoposide but in al- Inclusion of an EZH2 inhibitor with standard

Abstracts most every case becomes rapidly chemoresi- cytotoxic therapies prevented emergence stant, leading to death within one year. We of acquired resistance and augmented che- modeled acquired chemoresistance in vivo motherapeutic efficacy in both chemosensi- using a series of patient-derived xenografts tive and chemoresistant models of small cell to generate paired chemosensitive and che- lung cancer.”

12 The Pezcoller Foundation Journal - June 2018 ABSTRACTS OF POSTERS

boost supports metabolic switch toward glyc- 1. NAMPT is a key element in the Posters olysis or OXPHOS, both strategies exploited by acquisition of resistance to BRAF BRAF-mutated melanomas to adapt to chron- inhibitors becoming an actionable ic exposure to BRAFi. Treatment of melanoma target and a disease marker in cells with NAMPT inhibitors (NAMPTi) depleted NAD, inducing mitochondrial stress, cell cycle metastatic melanoma arrest and ultimately apoptosis. Consistently, NAMPTi were highly effective in the treatment V. Audrito1,2, A. Managò1,2, F. Zamporlini3, F. of melanoma xenografts, inducing complete tu- Gaudino1,2, N. Vitale4, E. Rulli5, S. D’Atri6, G.C. mor regression in the case of M14 cells. Antonini Cappellini7, P. A. Ascierto8, R. Piva4, D. NAMPT up-regulation upon development of re- Massi9, M. Mandalà10, N. Raffaelli3, S. Deaglio1,2 sistance to BRAFi was confirmed in serial bi- opsies from melanoma patients. Furthermore, Affiliations: 1Department of Medical Sciences NAMPT could be dosed in patient plasma where and 4Department of Molecular Biotechnologies it correlated with disease burden, response to and Health Science, University of Turin, Italy; therapy and overall survival. In conclusion, this 2Italian Institute for Genomic Medicine, Turin, work links oncogenic BRAF signaling to meta- Italy; 3Department of Agricultural, Food and bolic reprogramming through NAD biosynthesis Environmental Sciences, Polytechnic Univer- and identifies NAMPT as an actionable target sity of Marche, Ancona, Italy; 5Statistics Unit for melanoma patients with BRAF mutations. Methodology for Clinical research Laborato- ry, Oncology Department, IRCCS, Istituto di Ricerche Farmacologiche Mario Negri, Milan, 2. Maternal immunization against Italy; 6Laboratory of Molecular Oncology and alk hinders tumor progression in 7Department of Oncology and Dermatological Oncology, Istituto Dermopatico dell’Immacola- neuroblastoma-prone offspring ta-IRCCS, Rome, Italy; 8Melanoma, Cancer Im- munotherapy and Innovative Therapies O.U., Giuseppina Barutello1, Federica Riccardo1, Istituto Nazionale Tumori IRCCS Fondazione Claudia Voena2, Elisabetta Bolli1, Elena Qua- “G. Pascale”, Napoli, Italy; 9Division of Patho- glino1, Roberto Chiarle2,3 and Federica Cavallo1 logical Anatomy, Department of Surgery and Translational Medicine, University of Florence, 1Department of Molecular Biotechnology and Italy; 10Unit of Medical Oncology, Department Health Sciences, Molecular Biotechnology Cen- of Oncology and Hematology, Papa Giovanni ter University of Torino, Torino, Italy XXIII Hospital, Bergamo, Italy. 2Department of Molecular Biotechnology and Health Sciences, Center for Experimental Re- e-mail: [email protected] search and Medical Studies, University of Tori- no, Torino, 10126, Italy Although recent clinical trials of BRAF inhibitor 3Department of Pathology, Children’s Hospital (BRAFi) combinations have demonstrated im- Boston, Boston MA, USA proved efficacy in BRAF-mutated melanomas, emergence of acquired resistance limits clini- Introduction and rationale: Neuroblastoma (NB) cal benefit. In resistant cells tumor metabolism is the most common pediatric solid tumor, usu- rewires to accommodate increased energy re- ally diagnosed within the first year of life. One quirements, suggesting that molecules mediat- of the most predisposing genetic alterations ing metabolic adaption may become therapeu- towards NB development is a somatic mutation tic targets overcoming drug resistance. in the anaplastic lymphoma kinase (ALK) gene, Resistance mechanisms are generally linked to resulting in a phenylalanine-to-leucine substi- paradoxical activation of the MAPK signaling tution at codon 1174 (ALKF1174L). This mutation pathway. We found that this oncogenic signa- potentiates the activity of MYCN oncogene, as ture converges on the overexpression of nico- well documented in the ALKF1174L/MYCN pre- tinamide phosphoribosyltransferase (NAMPT), clinical model of spontaneous NB. the main NAD-biosynthetic enzyme, leading Since it has been shown that DNA immuniza- to a marked increase in NAD levels. This NAD tion against ALK oncoantigen is able to induce a

The Pezcoller Foundation Journal - June 2018 13 strong specific immune response against differ- Approximately 20% of patients with pediatric ent mutated form of ALK, impairing the growth acute T-lymphoblastic leukemia (T-ALL) are re- of ALK-positive tumors and enhancing survival in fractory to current glucocorticoid-based ther- mouse models of non-small cell lung cancer and apies. In the present study we investigated the anaplastic large cell lymphoma, ALK targeting possibility to selectively sensitize T-ALL cells to could be exploited against NB. As NB develop- apoptosis by increasing their levels of mitochon- ment occurs very early during life or even during drial reactive oxygen species (mtROS). For this fetal life, we sought to evaluate whether mater- purpose we employed NS1619, a synthetic ben- Posters nal immunization (MI) against ALK could be ap- zimidazolone derivative that increases mtROS plied to cancer immune-prevention in a model of production by opening the large conductance neonatal NB (ALKF1174L/MYCN). Indeed, as a proof Ca2+-activated K+ (BK) channel and dehydroepi- of concept of the effect of MI against an oncoan- androsterone (DHEA), which blunts ROS scaveng- tigen in cencer-prone offspring, we previously ing through inhibition of the pentose phosphate demonstrated the efficacy of MI against Her-2/ pathway. NS1619 and DHEA increased mtROS and neu in hampering tumor progression in a mouse induced death of T-ALL cell lines, patient-derived model of Her-2/neu-driven mammary cancer. xenografts (PDX) and primary cells from T-ALL patients (including cases of refractory T-ALL), Results:Pre-birth immunization against ALK, using but did not induce death of normal human thy- a DNA plasmid coding for the extracellular and mocytes or PBMC. The importance of depowering transmembrane domains of ALK (ALK-ECTM) fol- ROS-scavenging pathways to increase the effica- lowed by electroporation, leads to an extended cy of ROS-producing treatments was underscored tumor-free survival and a lower tumor growth ki- by the finding that NS1619 and DHEA activated F1174L netic in ALK /MYCN offspring born from and NRF2, the master regulator of antioxidant path- fed by ALK-ECTM-vaccinated mothers, as com- ways. NS1619 and DHEA induced cleavage of pared to controls born from control empty vec- OPA1,a mitochondrial protein that controls cris- tor vaccinated mothers. Maternally derived an- tae remodeling and cytochrome c mobilization. ti-ALK antibodies were successfully transferred OPA1 cleavage and cell death were inhibited by from mothers to newborns, as well as immune the ROS scavenger N-acetylcysteine and by siR- IgG-ALK protein complexes. Moreover, MI against NA-mediated knock-down of the mitochondrial ALK induces a decrease in ALK expression in tu- protease OMA1, whose targets include OPA1. Im- mor harbored in ALKF1174L/MYCN offspring born by portantly, NS1619 and DHEA sensitized T-ALL cells ALK-ECTM vaccinated mothers. to death induced by TRAIL or by the glucocorticoid dexamethasone, both of which are known to Conclusions: Overall, these results indicate MI induce mitochondrial outer membrane permea- as a possible intervention to hamper NB devel- bilization (MOMP) through the activation of Bid opment in genetically predestined offspring and could be a valuable treatment for this cancer (TRAIL) or Bim (dexamethasone). type for which efficient preventive options are Taken together, our findings provide the first currently unavailable. evidence for a role of the OMA1-OPA1 axis in sensitizing T-ALL cells to apoptosis, and suggest the feasibility of an integrated pharmacological approach that combines increased mtROS pro- 3. Selective killing of duction, inhibition of scavenging pathways and T-lymphoblastic leukemia (T-ALL) MOMP to treat refractory T-ALL. cells by redox-mediated engagement of the OMA1/OPA1 axis 4. Novel interactions of the Micol Silic-Benussi1, Gloria Scattolin2, Ilaria Ca- von hippel-lindau (pvhl) tumor vallari1, Sonia Minuzzo2, Samuela Francescato3, suppressor with the cdkn1 family Paola del Bianco1, Giuseppe Basso3, Stefano In- draccolo1, Donna M. D’Agostino4 and Vincenzo of cell cycle inhibitors Ciminale1,2* Raissa Bortolotto1,Giovanni Minervini1, Raffaele 1Istituto Oncologico Veneto-IRRCS, via Gattame- Lopreiato1, Antonella Falconieri1, Geppo Sarto- lata 64, 35128 Padova, Italy; 2Department of Sur- ri1, Silvio C.e. Tosatto1,2* gery, Oncology, and Gastroenterology, University of Padova, via Gattamelata 64 35128 Padova, 1Department Of Biomedical Sciences, University Italy; 3Department of Woman and Child Health, Of Padova. Viale G. Colombo 3, 35121, Padova, Haemato-Oncology Division, University of Pado- Italy; 2Cnr Institute Of Neuroscience, Padova, va, via Giustiniani 3, 35128 Padova, Italy; 4De- Viale G. Colombo 3, 35121, Padova, Italy; partment of Biomedical Sciences, University of Padova, via Gattamelata 64, 35128 Padova, Italy *Corresponding Author: [email protected]

14 The Pezcoller Foundation Journal - June 2018 Germline mutations of the von Hippel-Lindau Background (pVHL) tumor suppressor cause several highly vas- Immune checkpoint inhibition (CPI) as a thera- cularized cancers. pVHL, in complex with Elongin peutic principle to boost the immune response C and B, targets the hypoxia-inducible transcrip- to tumors has renewed interest in a phenom- tion factor (HIF-1α) for rapid proteasomal degra- enon that is known as the “abscopal effect” dation, modulating different downstream genes (eliciting a systemic antitumor response using involved in hypoxia response. Prolonged oxygen a local triggering treatment). We present a pa- deprivation is a limiting factor of cell cycle pro- tient case where systemic progression during gression, inducing growth arrest and apoptosis. CPI treatment was followed by a systemic re- Posters However, the exact molecular details leading to sponse after a local radiotherapy treatment. this transition are far from understood. Here, we Material and Methods present a recently published1 novel interaction In August 2016 a seventy-three year old male between pVHL and the cyclin-dependent kinase patient was referred to our Institute for a inhibitor family CDKN1 (p21, p27 and p57). We treatment of bone metastasis from Non Small predicted, dissected and validated their asso- Cell Lung Cancer. His oncological history was ciation through bioinformatics analysis, yeast characterized by previous prostate cancer two-hybrid screening and cell co-immunoprecip- (status post prostatectomy in 2012), urotheli- itation assays. We observe that the N-terminal al cancer (s.p. radical radiotherapy in 2015) tails of CDKN1 proteins share a conserved region and a stage IV squamous cell carcinoma of the mimicking the CODD motif of HIF-1α, necessary lung. After first line chemotherapy with partial for pVHL binding. Interestingly, a site-specific response the patient experienced disease pro- p27 pathological mutation (p.Glu40Lys) alters gression with bilateral lung nodules, a paracar- this novel interaction. Our findings suggest a new diac lesion, mediastinal nodal and bone metas- connection between the pathways regulating hy- tasis. In April 2016 he started immunotherapy poxia and cell cycle progression. with Nivolumab every two weeks. In July 2017 a shoulder CT scan showed a 4.6 cm osteolytic lesion of the left scapula causing severe pain to left shoulder and omolateral upper arm not well controlled by opioid drugs. Due to progression and pain we decided to submit the patient to cytoreductive/pain relieving radiation therapy.

Results The patient underwent a planning CT scan, in supine position, with arms along the body. The target lesion was contoured (GTV-CTV-PTV) including almost all of the left scapula. The patient underwent 3D-Conformal Radiotherapy receiving 30 Gy in ten fractions (3gy/die). At This work was supported by Associazione Ital- the end of the therapy shoulder pain was sig- iana per la Ricerca sul Cancro (AIRC) grants nificantly reduced. The patient was continued MFAG12740 and IG17753 to S.T. on two-weekly Nivolumab and during regular follow up CT scans (February and June 2017) Reference showed a persistent partial response of the 1.Minervini, G., Lopreiato, R., Bortolotto, R. & lesion treated (scapula) concomitantly with a Falconieri, A. Novel interactions of the von Hip- remarkable reduction of lung nodules and the pel-Lindau ( pVHL ) tumor suppressor with the right paracardiac lesion suggesting a systemic CDKN1 family of cell cycle inhibitors. Nat. Publ. response in line with a possible “abscopal ef- Gr. 1–12 (2017). doi:10.1038/srep46562 fect”

Conclusions 5. Cytoreductive Radiotherapy In most clinical situations, radiation therapy concomitant with Nivolumab may be safely delivered concomitantly with immunotherapy. This case report suggests the beyond progression: a local approach synergistic effects of local treatment with new to improve sistemic disease control? drugs being able to enhance the response to systemic therapy probably trough immunologi- Bruni A., D’Angelo E., Meduri B., Vassilieva P., cal pathways (abscopal effect), as also recent- Andolina M., Giacobazzi P, Lohr. F ly suggested by the PEMBRO-RT trial. Further Radiation Oncology Unit, University Hospital studies are needed to confirm these prelimi- “Policlinico of Modena”, Italy nary observations.

The Pezcoller Foundation Journal - June 2018 15 6. Redox homeostasis modulation Gastroenterology, University of Padova, Italy, sensitizes DLBCL cell lines to 3Department of Woman and Child Health, Hae- mato-Oncology Division, University of Padova, venetoclax Italy; 4Department of Biomedical Sciences, University of Padova, Italy. Ciccarese F,1 Vettore L,1 Silic-Benussi M,2 Cimi- nale V1,2 Approximately 20% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients do Posters 1 Department of Surgery, Oncology and Gastro- not respond to standard glucocorticoid-based enterology, University of Padova, Italy. 2Veneto therapies and have a dismal prognosis. Institute of Oncology – IRCCS, Padova, Italy. Hyper-activation of the PI3K/Akt/mTOR oncogenic pathway is a common feature of T-ALL. Presenting author: Ciccarese Francesco, Univer- In addition to controlling cell growth and au- sity of Padova, Department of Surgery, Oncolo- tophagy, mTORC1 influences mitochondrial ac- gy and Gastroenterology, via Gattamelata 64, tivity and oxidative metabolism by controlling Padova, 35128, Italy. the interaction between YY1 and PGC1α. [email protected] In previous studies, we showed that T-ALL cells exhibit high levels of mitochondrial reactive Diffuse Large B-cell Lymphoma (DLBCL), the most oxygen species (ROS). ROS are powerful sig- common type of non-Hodgkin’s lymphoma, is a nalling molecules that can induce apoptosis very heterogeneous malignancy, divided into two through p53 activation, but may also increase molecular subtypes: germinal center B-cell (GCB) cancer cell survival through PTEN oxidation, and activated B-cell (ABC). More than 40% of pa- which results in an increased Akt activity. In the tients (mostly with ABC-DLBCL) are refractory to present study, we tested the possible cross-talk standard chemotherapy (R-CHOP) or will relapse. between mTOR and ROS in T-ALL. As expected, Recent combinatorial regimens with bortezomib, treatment of T-ALL cells with the mTORC1-in- lenalidomide and ibrutinib have proven to be hibitor Everolimus induced LC3 lipidation and more effective against ABC subtype. In addition, p62 degradation, events that suggest the ac- BCL-2 inhibitors promise to be effective both in tivation/induction of autophagy. Importantly, GCB- and in ABC-DLBCLs. Everolimus also increased ROS levels and in- Nevertheless, a significant proportion of DLBCL duced cell death both in T-ALL cell lines and exhibits primary or secondary resistance to these patient-derived T-ALL xenografts (PDX) but compounds. not in primary normal thymocytes. Cell death We investigated a novel pharmacological ap- was reduced by pre-treating cells with the ROS proach aimed at elevating mitochondrial reactive scavenger N-acetyl-cysteine (NAC), indicating oxygen species (ROS) levels, in association with that this effect was ROS-dependent. the BCL-2 inhibitor venetoclax (ABT-199). In vivo experiments carried out in NOD/SCID Our results indicated that elevation of ROS in mice inoculated with glucocorticoid-resistant DLBCL cell lines (N=7 GCB-type, N=7 ABC-type) PDX cells showed that Everolimus significant- strongly increased the pro-apoptotic effect of ly increased the sensitivity of the cells to the ABT-199. Interestingly, the magnitude of this ef- glucocorticoid dexamethasone with resulting fects was higher in cell lines exhibiting an OxPhos increased survival of treated mice. metabolic profile compared to glycolytic cell These studies indicate a connection between lines that were more resistant. mTOR and ROS, and suggest that mTORC1 inhi- Current studies are focused on investigating the bition may prove to be effective to overcome efficacy of our strategy in samples from patients dexamethasone-resistance in refractory T-ALL with resistant/refractory DLBCL. patients.

7. mTORC inhibition increases 8. Generation and reactive oxygen species and death Characterization of TRAIL- in T-lymphoblastic leukemia cells resistant Glioblastoma Cell Lines

Micol Silic-Benussi1,2, Vittoria Raimondi1, Mari- Ahmet Cingoz1, Tunc Morova2, Tugba Bag- on Linseisen2, Loredana Urso2, Ilaria Cavallari1, ci-Onder1,* Sonia Minuzzo1, Paola del Bianco1, Gloria Scat- tolin2, Giuseppe Basso3, Stefano Indraccolo1, 1Koç University School of Medicine, Brain Can- Donna M. D’Agostino4 and Vincenzo Ciminale1,2 cer Research Laboratory, Istanbul, TURKEY 2Koç University School of Medicine, Istanbul, 1Istituto Oncologico Veneto-IRRCS, Padova, TURKEY Italy; 2Department of Surgery, Oncology, and *Correspondence: [email protected]

16 The Pezcoller Foundation Journal - June 2018 Glioblastoma (GBM) is the most common and * = These authors contributed equally to this aggressive primary brain tumor. Tumor ne- work crosis factor–related apoptosis-inducing ligand (TRAIL) causes apoptosis of target cells Breast cancer treatment often includes Dox- and can selectively induce apoptosis in tumor orubicin as adjuvant as well as neoadjuvant cells. However, many cancer cells are resist- chemotherapy. Despite its cytotoxicity cells ant to TRAIL, and mechanisms of resistance can develop drug resistance to Doxorubicin. are not well understood. Uncovering pathways and mechanisms involved In this study, we generated TRAIL-resistant in drug resistance is an urgent and critical aim Posters cell line models, and selected TRAIL-resistant for breast cancer research oriented to improve subpopulations of 4 different GBM cell lines treatment efficacy. (A172, LN18, T98G, and U87MG). Cell viability Here we show that different chemotherapeutic and caspase assays as well as long-term cell drugs, and particularly Doxorubicin, induce the growth assays revealed that the resistant cells expression of ETV7, a transcriptional repressor exhibited sustained TRAIL-resistance. Genome member of the large ETS family of transcription wide RNA sequencing analysis of the cells re- factors. The ETV7 expression led to down-reg- vealed major alterations during the transition ulation of DNAJC15, a co-chaperone protein from TRAIL-sensitive to TRAIL-resistant state whose low expression was previously associat- and identified PI3K pathway to be markedly ed with drug resistance in breast and ovarian changed in all TRAIL-resistant models. In addi- cancer. There was a corresponding reduction tion, our analysis specifically on the apoptosis in Doxorubicin sensitivity of MCF7 and MDA- regulators demonstrated that Bcl-2 and Bcl-XL MB-231 breast cancer cells, also highlighted by expression played a major role in regulating a diminished cell death. the TRAIL response. Furthermore, functional We identified the binding site for ETV7 with- modulation of Bcl-2 and Bcl-XL through over- in the promoter of DNAJC15 and we also expression, shRNA knockdown or BH3 mimetic found that DNA methylation may be a factor applications, changed the apoptotic outcome in ETV-mediated transcriptional repression at of GBM cell lines. To assess whether the se- the DNAJC15 promoter. These findings of an in- lected TRAIL resistant sub-populations also verse correlation between ETV7 and DNAJC15 exhibit resistance to other apoptosis induc- expression in breast cancer cells in terms of ers, we utilized Fas-ligand, Staurosporine and Doxorubicin resistance, correlated well with Temozolomide and observed that there was treatment responses of breast cancer patients heterogeneity for each drugTo then assess the with recurrent disease, based on our analyses possibility of re-sensitizing the resistant cells of reported genome-wide expression arrays. to TRAIL, we applied a proteasome inhibitor, Moreover, we demonstrated that ETV7-medi- Bortezomib, and observed that all resistant ated Doxorubicin resistance involves increased subpopulations can be re-sensitized to TRAIL Doxorubicin efflux via nuclear pumps, which efficiently. could be rescued in part by DNAJC15 up-reg- Our future efforts are directed at understand- ulation. With this study, we propose a novel ing the roles of commonly de-regulated path- role for ETV7 in breast cancer, and we identify ways in resistance mechanisms. Our results DNAJC15 as a new target gene responsible for will provide further understanding of molec- ETV7-mediated Doxorubicin resistance. A bet- ular mechanisms of tumor heterogeneity in ter understanding of the opposing impacts of GBM cells, and help with the novel treatment Doxorubicin could improve the design of com- strategies. binatorial adjuvant regimens with the aim of avoiding resistance and relapse. 9. ETV7 mediates Doxorubicin resistance in breast cancer cells by 10. VEGF removal delays the onset repressing DNAJC15 of acquired resistance to target therapy and increase the efficacy Federica Alessandrini1,*, Laura Pezzè1,*, Daniel Menendez2, Michael A. Resnick2, Yari Ciribilli1 of immune checkpoint inhibitors in BRAF mutated melanoma 1Laboratory of Molecular Cancer Genetics, Centre for Integrative Biology (CIBIO), Uni- Valentina Comunanza, Chiara Gigliotti, versity of Trento, Povo (TN), Italy; Valentina Martin, Gabriella Doronzo, Anna 2Genome Integrity and Structural Biology Lab- Gattuso, Federica di Nicolantonio, Dario San- oratory, National Institute of Environmental giolo, Federico Bussolino Health Sciences (NIHES), NIH, Research Trian- Department of Oncology – University of Turin gle Park, NC Candiolo Cancer Institute FPO-IRCCS

The Pezcoller Foundation Journal - June 2018 17 The introduction of BRAF inhibitors (BRAFi) antitumor effect and we observed total tumor has improved response rate and overall surviv- volume regression in 86% of treated mice. al of metastatic melanoma patients compared Our findings provide biological rationale to to standard chemotherapy. However, acquired explore the association of immunotherapy in drug resistance occurs in nearly all patients. novel combinatorial approaches which could The comprehension of cellular and molecular improve the clinical outcome exerted by on- mechanisms underlying BRAFi resistance could cogene-targeted therapy and further investi- help to identify novel actionable pathways in gation is warranted. Posters the treatment of BRAF dependent tumors. VEGFA is an attractive target for combinatorial cancer therapy and we have recently demon- 11. Detecting cancer biomarkers on strated in melanoma and CRC xenografts that targeting VEGFA enhanced the antitumor ef- polydisperse extracellular vesicles fect of BRAFi by normalizing the tumor vascu- isolated from blood lature, recruiting M1 macrophages and inducing a remodeling of the extracellular matrix Vito G. D’Agostino, Michela Notarangelo, Chi- characterized by a reduction in collagen I and ara Zucal, Francesca Demichelis, Alessandro in cancer-associated fibroblasts (Comunanza Provenzani, Alessandro Quattrone et al EMBO Mol Med 2017). While the previous proof of concept was ob- Centre for Integrative Biology (CIBIO), University tained within in an immunodeficient model, of Trento, Via Sommarive 9, Trento, 38123, Italy here we investigated the therapeutic effect of VEGFA targeting in association with PLX4720 Extracellular vesicles (EVs) are heterogeneous (BRAFi) in a dedicated immune-competent membranous particles intensively studied for model. D4M cells, a BRAFV600E-mutant mela- their potential cargo of diagnostic markers. noma murine cell line, were subcutaneous- We developed a new rapid biochemical meth- ly injected in syngeneic C57BL/6J mice. We od to obtain polydisperse EVs in a physiologi- demonstrated that the association of BRAFi cal pH solution, preserving their morphology, with DC101 (antibody anti VEGFR2) had a weak dispersity, and stability. We challenged the activity while we observed a synergistic anti- reproducibility of this method by isolating EVs tumor effect when combined with B20 (murine from different biological fluids. In plasma of anti-VEGFA neutralizing antibody). Although healthy donors, the abundance of recovered EV targeted inhibition of either BRAF or VEGFA populations, in the range of 1010 per milliliter, delayed the tumor growth, only combined in- positively correlated with the density of blood hibition of both pathways resulted in the re- erythrocytes, platelets, and leukocytes. Quan- gression of initial tumor size, with an evident titative analyses using specific haematopoietic apoptotic effect, and delayed the onset of ac- and epithelial markers demonstrated the un- quired resistance to the BRAF inhibition. biased recovering of polydisperse EV lineages. Since it has been well characterized the im- Individual isolated vesicles were used in new- mune suppressive role of VEGFA in tumors we ly-designed homogeneous assays. We detected further investigated whether contrasting the a picomolar concentration of PSMA on 105 EVs VEGF effect along with simultaneous BRAF isolated from plasma of prostate cancer pa- inhibition can turn into a promotion of both tients and BRAF V600E mutation-carrying mRNA innate and adaptive immunity. Both flow cy- in 103 EVs from plasma of colon cancer pa- tometry and immunofluorescence analysis of tients, reaching unprecedented matching with tumors demonstrated that the combinatorial tissue biopsy results. regimen activated the host immune system, inducing the tumor infiltration by cytotoxic CD8+ lymphocytes, macrophages with tumor 12. A p53/miR-30a/ZEB2 axis suppressive features and NKs. Moreover, the controls triple negative breast association between BRAF targeting and VEG- FA removal reduced the number of circulating cancer aggressiveness CD11b+Ly6ClowLy6G+ polymorphonuclear MDSCs (PMN-MDSCs). Alessandra di Gennaro1#, Valentina Damiano1#, Moreover, we evaluated whether the therapeu- Giulia Brisotto1, Michela Armellin1, Tiziana tic effect obtained by the simultaneous VEGF Perin2, Antonella Zucchetto3, Michela Guardas- blockade and BRAFi could be exploited with cione4, Herman P. Spaink5, Claudio Doglioni6, B. immune-checkpoint modulators and we ob- Ewa Snaar-Jagalska5,Manuela Santarosa1* and served that this synergism is further improved Roberta Maestro1* by the association with checkpoint inhibi- 1Oncogenetics and Functional Oncogenomics tor targeting PD-1. The addition of anti-PD-1 Unit, CRO Aviano National Cancer Institute, blocking antibody significantly enhanced the via F. Gallini 2, Aviano 33081 (PN), Italy. 2Pa-

18 The Pezcoller Foundation Journal - June 2018 thology Unit, CRO Aviano National Cancer In- Center for Thoracic Surgery, University of In- stitute, Aviano (PN), via F. Gallini 2, Aviano subria, Italy; 33081 (PN), Italy. 3Epidemiology and Biosta- 4Scientific and Technological Pole, IRCCS Multi- tistics Unit, CRO Aviano National Cancer In- Medica, Milan, Italy; stitute, Aviano (PN), via F. Gallini 2, Aviano 33081 (PN), Italy. 4Medical Oncology Unit, CRO INTRODUCTION Aviano National Cancer Institute, via F. Gallini Lung cancer is the main cause of cancer-re- 2, Aviano 33081 (PN), Italy. 5Molecular Cell Bi- lated mortality worldwide. Patients with early ology Dept., Institute of Biology, Leiden Uni- stage (I-II) non-small cell lung cancer (NSCLC) Posters versity, Leiden 2333CC, The Netherlands, NL. have a much better prognosis than those diag- 6Ateneo Vita-Salute, Department of Patholo- nosed at late stage. Thus, the development of gy, IRCCS Scientific Institute San Raffaele, Mi- sensitive and non-invasive methods for screen- lan 20132, Italy. ing individuals at high risk for NSCLC is needed. microRNAs (miRNAs) have been suggested as a Inactivation of p53 contributes significantly to novel class of tumor biomarkers; their stability the dismal prognosis of breast tumors, most in biofluids and their change in levels in disease notably triple negative breast cancers (TNBC). suggest their potential application as circulat- How the relief from p53 tumor suppressive ing biomarkers. functions results in tumor cell aggressive behavior is only partially elucidated. In an at- EXPERIMENTAL MODEL tempt to shed light on the implication of mi- Upon a critical review of the literature, we croRNAs in this context, we discovered a new selected 8 miRNAs for a two-step screening of signaling axis involving p53, miR30a and ZEB2. early lung cancer, based on their reported sen- By an in silico approach we identified miR-30a sitivity and specificity and the fact that are not as a putative p53 target and observed that in influenced by hemolysisa. Since smoking habit breast tumors reduced miR-30a expression cor- or inflammatory conditions may influence miR- related with p53 inactivation, lymph node posi- NA levels in serum, we quantified miRNAs of our tivity and poor prognosis. We demonstrate that panels in three groups of controls (non-smok- p53 binds the MIR30a promoter and induces the ers, smokers and COPD patients) and in stage transcription of both miRNA strands, 5p and 3p. I-II NSCLC patients. Droplet digital PCR was ap- Both miR-30a-5p and 3p showed the capacity of plied for quantification of miRNAs. targeting ZEB2, a transcription factor involved in epithelial-mesenchymal transition (EMT), tu- RESULTS mor cell migration and drug resistance. Intrigu- The two-step screening is composed of a pan- ingly, we found that p53 does restrain ZEB2 el of 4 miRNAs endowed with high sensitivity expression via miR-30a. Finally, we provide and a second panel with high specificity. The evidence that the new p53/miR-30a/ZEB2 axis chosen miRNAs should allow to identify true controls tumor cell invasion and distal spread- positive patients, that would undergo CT scan. ing and impinges upon miR-200c expression. For 3 of the 6 miRNAs analyzed there was no Overall, this study highlights the existence of a significant difference among control subgroups novel axis linking p53 to EMT via miR-30a, and (non-smokers, smokers, and subjects affected adds support to the notion that miRNAs rep- by Chronic Obstructive Pulmonary Disease), resent key elements of the complex network whereas miRNA levels were expressed at sig- whereby p53 inactivation affects TNBC clinical nificantly different levels in tumor and control behavior. groups, confirming their possible role as biomarkers previously described in the literature. 13. Serum micrornas as biomarkers CONCLUSIONS The selected miRNAs may help to identify high for early diagnosis of non small cell risk subjects who need further investigation for lung cancer the presence of early NSCLC; in particular, miR- 223 showed a good accuracy in distinguishing Paola D’Antona1, Francesca Moretti2, Emanue- tumor samples from controls. le Finardi2, Marco Barbetta2, Maria Cattoni3, Lorenzo Dominioni3, Douglas M. Noonan4, Albi- REFERENCES no Poli2, Paola Campomenosi1 a Moretti F, D’Antona P, Finardi E, Barbetta M, Dominioni L, Poli A, Gini E, Noonan DM, Im- 1Department of Biotechnology and Life Science, peratori A, Rotolo N, Cattoni M, Campomenosi DBSV, University of Insubria, Varese, Italy; P. Systematic review and critique of circulat- 2Department of Diagnostic and Public Health, ing miRNAs as biomarkers of stage I-II non- University of Verona, Verona, Italy; small cell lung cancer Oncotarget. 2017 Oct 3Department of Medicine and Surgery, DMS, 11;8(55):94980-94996.

The Pezcoller Foundation Journal - June 2018 19 14. Understanding SLC expression aMolecular Immunology Unit, Department of Re- in hematological malignancies search, Fondazione IRCCS Istituto Nazionale dei Tumori, via Amadeo, 42, 20133 Milan, Italy bTumor Immunology Unit, Department of Ruth Eichner1, Enrico Girardi1, Tea Pemovska1, Health Sciences, University of Palermo, Italy Felix Kartnig1, Ulrich Goldmann1, Giulio Su- perti-Furga1,2 Email: [email protected]

Posters CeMM, Research Center for Molecular Medi- Tumor progression is a multifaceted process cine of the Austrian Academy of Sciences, Vi- in which, complex interactions between tu- enna, Austria mor and different types of stromal cells and 2 Center for Physiology and Pharmacology, extracellular matrix components, actively Medical University of Vienna, Vienna, Austria contribute to its phenotypic heterogeneity. Among extracellular matrix proteins, secreted Solute carriers (SLCs) are membrane proteins protein acidic and rich in cysteine (SPARC) has responsible for the exchange of various nutri- been deeply studied since conflicting reports ents, vitamins and ions between cells and their have described its expression to be either in- environment. Their expression pattern depends both on the genetic program of the re- creased or decreased in different cancer set- spective cell type and on the metabolic state tings, also depending on whether it is produced of the cell and its surrounding. To provide by the neoplasm or by the neighboring stroma. enough energy and biomass, various cancers Nevertheless, the different contribution of tu- overexpress a number of SLCs, thus enabling mor- or stromal-derived SPARC in prostate tu- continuous growth and proliferation. Howev- mor microenvironment has not been addressed er, despite their key role in metabolism and yet. Given this evidence, we aimed at provid- growth, not much is known about the drivers of ing new insights into the mechanism by which SLC expression. We therefore aim to systemat- SPARC modulation influences prostate cancer ically investigate both genetic and metabolic development and progression. regulation of a defined set of cancer-relevant We modeled human disease using TRAMP mice, SLCs in hematological malignancies. On the which spontaneously develop autochthonous one side, genome-wide loss-and gain-of-func- prostate tumors following the onset of puberty. tion CRISPR/Cas9 screens will help to identify Crossing TRAMP mice with Sparc-/- mice, we genetic drivers and regulators of SLC expres- found the appearance of focal areas of neu- sion in an unbiased way. roendocrine differentiation within adenocarci- Metabolic perturbations using an established noma. In patients this phenomenon commonly metabolic drug library and a high-content- results after androgen ablation therapy and high-throughput microscopy based readout, correlates with poor prognosis. will in parallel shed more light on the connec- Interestingly, areas of neuroendocrine differ- tion between metabolism and SLC expression. entiation in Sparc-/- TRAMP mice were both The identified regulatory dependencies and positive for cytokeratin 8 and synaptophysin connections will be validated in primary pa- (usually expressed by luminal cells within ad- tient material. enocarcinoma or neuroendocrine cells, respec- Together, these approaches are expected to tively), further suggesting a differentiation of contribute important knowledge on the ex- adenocarcinoma cells to a neuroendocrine-like tensive regulatory network of SLCs in cancer. phenotype. Moreover, immunohistochemis- Together with already available essentiality try showed SPARC positivity not only in scat- data, these insights might lead to the defini- tered tumor cells but also in fibroblasts and tion of new metabolic drug targets and poten- myeloid cells infiltrating TRAMP prostate. Ac- tial synthetic lethal interactions. cordingly, in vitro experiments suggested that stromal-derived SPARC limits neuroendocrine differentiation of prostate cancer cells, while they excluded a role of endogenous SPARC in 15. Adenocarcinoma- this phenomenon. Indeed, prostate cancer cell neuroendocrine transition lines co-cultured in presence of Sparc-deficient of androgen resistant cancer fibroblasts increased or acquired neuroendo- depends on SPARC down- crine features. This likely occurs through the effect of IL-6, a cytokine recently discovered regulation in stromal accessory to induce neuroendocrine differentiation, and cells that we found to be released by Sparc-deficient, but not sufficient, fibroblasts. Claudia Enriqueza, Valeria Cancilab, Data collected so far indicate that stromal Renata Ferria, Ivano Ariolia, Claudio Tripodob, SPARC deficiency skews prostate carcinogen- Mario P. Colomboa , Elena Jachettia esis toward neuroendocrine differentiation. A

20 The Pezcoller Foundation Journal - June 2018 deeper understanding of the molecular mecha- which control the transcriptional activation of nisms governing the balance between prostate oncogenic pathways. Next, we demonstrated adenocarcinoma and neuroendocrine tumors that the enhancer reprogramming occurring according to extracellular matrix composition in vitro is also maintained in in vivo mouse will provide important insights for the develop- TNBC models. In addition, the genes which are ment of new therapeutic strategies. transcriptionally activated by MYC binding on their enhancers, are globally highly expressed in TNBC, in patients with a poor prognosis. Fi- nally, among these genes, we identified MYC Posters 16. MYC-mediated enhancer downstream factors, which are able to reca- reprogramming favors breast pitulate its ability to drive the reprogramming cancers tumorigenesis of mammary epithelial cells and, potentially, tumorigenesis. Luca Fagnocchi1, Vittoria Poli1, Alessandra Fasciani1, Silvio Bicciato2, Silvano Bosari3,4, Conclusion: In conclusion, we demonstrate Matilde Todaro5 and Alessio Zippo1 that the MYC-driven epigenetic reprogramming favors the formation of stem cell-like 1Center for Integrative Biology (CIBIO), Uni- tumor-initiating cells. In addition, we gener- versità di Trento. ate a MYC-dependent oncogenic signature for 2Center for Genome Research, Università di TNBC, identifying downstream targets, which Modena and Reggio Emilia. may represent novel therapeutic targets. 3Fondazione IRCCS Ca’ Granda Ospedale Mag- giore Policlinico, Milano. 4Department of Pathophysiology and Trans- 17. The e3 ubiquitin-protein plantation, Università di Milano. ligase mdm2 is a novel interactor 5 DiBiMIS, Università di Palermo. of the von hippel-lindau Introduction: Breast cancer (BC) is the most tumor suppressor common tumor and the leading cause of death among women cancer patients. Triple negative Antonella Falconieri1#, Giovanni Minervini1#, BC (TNBC) is a particularly aggressive form of Raissa Bortolotto1, Damiano Piovesan1, Raf- BC, which is characterized by the hyper-ac- faele Lopreiato1, Geppo Sartori1, Silvio C.e. tivation of the MYC oncogene. Importantly, Tosatto1,2* cytotoxic chemotherapy represents the only therapeutical option for TNBC. Consequently, 1-Department Of Biomedical Sciences,Univer- the identification of novel biomarkers and tar- sity Of Padova,Viale G. Colombo3,35121,Pado- getable targets in TNBC represents an urgent va,Italy; 2-Cnr Institute Of Neuroscience,Pa- medical need. dova,Viale G. Colombo 3,35121,Padova,Italy; #-Contributed Equally; *-Corresponding Au- Rationale: In this report, we investigated the thor: [email protected] role of MYC during TNBC tumorigenesis, by de- fining its mechanism of action in tumor initia- Mutations of the von Hippel-Lindau tumor sup- tion. In particular, we focused on its activity pressor (pVHL) are causative of familiar pre- on the regulation of distal cis-regulatory ele- disposition to develop different cancers. pVHL ments: the enhancers. Indeed, specific enhanc- is mainly known for its role in regulating hy- er repertoires are critical for cell specification, poxia-inducible factor 1-alpha (HIF-1α) degra- while their decommissioning is a crucial step dation, modulating hypoxia response. Previous towards cell reprogramming. Of importance, studies have suggested that isoform-specific dis-regulation of enhancers could favor tumor- specializations can be associated with human igenesis, by driving the aberrant activation of pVHL. Here, we present a novel interaction oncogenic transcriptional programs. between pVHL and MDM2. Integrating in silico predictions with in vivo assays, we found that Results: Specifically, we show that MYC acts as the accessory acidic tail of pVHL30 is required tumor reprogramming factor by reshaping the for its association with MDM2. Further, we enhancer landscape in mammary epithelial demonstrate that an intrinsically disordered cells. Indeed, it mediates the transcriptional region upstream of the MDM2 tetramerization downregulation of luminal-specific transcrip- domain is responsible for its isoform-specific tion factors, leading to the decommissioning association with pVHL30. This region is mostly of the enhancers which dictated the luminal conserved in higher mammals, including hu- mammary epithelial identity. In addition, it man, similarly to what already proposed for favors the acquisition of a stem cell-like fate the N-terminal tail of pVHL30. Collectively, by driving the activation of de novo enhancers, our data support the idea that the pVHL30

The Pezcoller Foundation Journal - June 2018 21 isoform may play a role in MDM2 regulation, derstood. Here we show that the transcription suggesting a wider interplay between hypoxia factors YAP and TAZ mediate transcription- sensing and cell cycle regulation. al dependencies of transformed cells. YAP/ This work is supported by Associazione Ital- TAZ physically engage the general coactivator iana per la Ricerca sul Cancro (AIRC) grants BRD4, dictating the genome-wide association MFAG12740 and IG17753 to ST. of BRD4 to chromatin. YAP/TAZ flag a large set of enhancers with super-enhancer-like functional properties. Posters YAP/TAZ-bound enhancers mediate recruitment of BRD4 and Pol II at YAP/TAZ-regulated promoters, boosting expression of a host of growth-regulating genes. Treatment with small molecule inhibitors of BRD4 blunts YAP/TAZ pro-tumorigenic activity in several cell/tissue contexts, causes regression of pre-established, YAP/TAZ-addicted neoplastic lesions, and re- verts drug resistance. This work sheds light on essential mediators, mechanisms and genome-wide regulatory elements responsible for transcriptional addiction in cancer and lays the groundwork for a rational use of BET inhibitors according to YAP/TAZ biology.

19. Uncoupling FoxO3A 18. Transcriptional addiction in mitochondrial and nuclear cancer cells is mediated by YAP/ functions in cancer cells TAZ through BRD4 undergoing metabolic stress and Francesca Zanconato*1, Giusy Battilana*1, Mat- chemotherapy tia Forcato2, Letizia Filippi1, Luca Azzolin1, Andrea Manfrin1, Erika Quaranta1, Daniele Di Valentina Celestini1,2, Tugsan Tezil1, Luciana Biagio1, Gianluca Sigismondo3, Vincenza Guz- Russo3, Candida Fasano3, Paola Sanese1, Gio- zardo4, Pascale Lejeune5, Bernard Haendler5, vanna Forte3, Alessia Peserico1, Martina Lep- Jeroen Krijgsveld3, Matteo Fassan4, Silvio Bic- ore Signorile1,4, Giovanna Longo1, Domenico ciato2, Michelangelo Cordenonsi1§# and Stefano De Rasmo5, Anna Signorile6, Raffaella Maria Piccolo1§# Gadaleta7,8, Natasha Scialpi8, Mineko Terao2, Enrico Garattini2, Tiziana Cocco6, Gaetano Vil- 1Department of Molecular Medicine, Universi- lani6, Antonio Moschetta8, Valentina Grossi1 ty of Padua School of Medicine, Viale Colombo and Cristiano Simone1,3 3, 35126 Padua, Italy 2Center for Genome Research, Department of 1Division of Medical Genetics, Department Biomedical Sciences, University of Modena and of Biomedical Sciences and Human Oncology Reggio Emilia, Via G. Campi 287, 41100 Mode- (DIMO), University of Bari Aldo Moro, Bari, na, Italy 70124, Italy 3German Cancer Research Center (DKFZ) and 2Department of Biochemistry and Molecular Heidelberg University, Im Neuenheimer Feld Pharmacology/Laboratory of Molecular Biolo- 581, 69120 Heidelberg, Germany gy; IRCCS - Istituto di Ricerche Farmacologiche 4Department of Medicine (DIMED), Surgical Pa- ‘Mario Negri’, Milano, 20156; Italy thology and Cytopathology Unit, Via Giustini- 3Medical Genetics, National Institute for Gas- ani 2, 35126 Padua, Italy troenterology, IRCCS ‘S. de Bellis’, Castellana 5 Bayer AG, Drug Discovery, Müllerstr. 178, Grotte (Ba), 70013, Italy 13353 Berlin, Germany 4Department of Molecular Medicine, Sapienza University of Rome, 00161 Rome, Italy ABSTRACT 5Institute of Biomembranes and Bioenergetics, Cancer cells rely on dysregulated gene ex- National Research Council (CNR), Bari, 70126, pression. This establishes specific transcrip- Italy tional addictions that may be therapeutically 6Department of Basic Medical Sciences, Neuro- exploited. Yet, the mechanisms ultimately re- sciences and Sense Organs, University of Bari sponsible for these addictions are poorly un- Aldo Moro, Bari, 70124, Italy

22 The Pezcoller Foundation Journal - June 2018 7Division of Digestive Diseases, Department chondrial localization and function and which of Surgery and Cancer, Imperial College Lon- stimuli do they respond to. don, Queen Elizabeth the Queen Mother Wing This will be instrumental to devise personal- (QEQM), London, W2 1NY, UK ized therapeutic strategies-employing molec- 8‘Medicina Interna Universitaria Frugoni’, De- ularly targeted drugs-aimed at manipulating partment of Interdisciplinary Medicine, Uni- cellular metabolism to counteract cancer initi- versity of Bari Aldo Moro, Bari, 70124, Italy ation and progression.

Carcinogenesis is a multistep process by which Posters normal cells evolve to a neoplastic state by 20. Mechanisms of telomere acquiring a succession of cancer hallmarks resulting in the ability to survive and prolif- maintenance in pediatric brain erate in adverse microenvironmental condi- tumors: insights from pre-clinical tions. Cancer cell homeostasis is sustained by models the balance between these newly acquired oncogenic features and pre-existing cellular Aurora I Idilli1, Emilio Cusanelli1, Marialuisa functions. Paradigmatic in this regard is the Cayuela2, Marina Mione1 reprogramming of energy metabolism, where normal cellular processes providing increased 1Centre for Integrative Biology (CIBIO), Univer- energy production, macromolecular biosyn- sity of Trento, Via Sommarive 9, 38123 Trento thesis and maintenance of the redox balance - Italy are ensured by the preservation of key mito- 2 Telomerase, Aging and Cancer Group, Re- chondrial functions. Consistent with this view, search Unit, Department of Surgery, CIBERe- proteins that have been classically considered hd, University Hospital ‘Virgen de la Arrixaca’, as tumor suppressors are sometimes required 30120 Murcia, Spain to be functional for full malignant transformation. This is the case for FoxO3A, which can be The up-regulation of a telomere maintenance both friend and foe to cancer cells depending mechanism (TMM) is a common characteristic of on the cellular context. It is a key determinant cancer cells and represent a hallmark of neopla- of cancer cell homeostasis, playing a dual role sia in 70-80% of cases. in survival/death response to metabolic stress Reliable methods for detecting TMMs in tumor and cancer therapeutics. samples are becoming a pre-requisite in the We recently described a novel mitochondrial clinic, and for the use of target treatments to- arm of the AMPK-FoxO3A axis in normal cells wards telomerase and/or alternative lengthen- upon nutrient shortage. We found that in met- ing of telomeres (ALT) mechanisms. For exam- abolically stressed cancer cells, FoxO3A is re- ple, in pediatric brain tumors ALT is found only cruited to the mitochondria through activation in a subset of malignant brain tumors, whereas of MEK/ERK and AMPK, which phosphorylate pediatric low grade gliomas rarely exhibit an ALT serine 12 and 30, respectively, on FoxO3A phenotype. N-terminal domain. Subsequently, FoxO3A is In our lab, we have developed a juvenile model imported and cleaved to reach mitochondri- of glioma in zebrafish, based on the conditional DNA, where it activates expression of the al expression of human-relevant oncogenes in a mitochondrial genome to support mitochondri- population of brain progenitor cells. al metabolism. Using FoxO3A−/− cancer cells The same model was established also in a back- generated with the CRISPR/Cas9 genome ed- ground of co-overexpression of TERT and TR. We iting system and reconstituted with FoxO3A described how to study TMMs and understand mutants being impaired in their nuclear or mi- related aspects of telomere biology in tumors tochondrial subcellular localization, we show derived from dissected tissues. that mitochondrial FoxO3A promotes survival Each tumor was analyzed by applying techniques in response to metabolic stress. In cancer cells for detecting both tumor-derived telomerase treated with chemotherapeutic agents, accu- and markers of ALT, also if the starting material mulation of FoxO3A into the mitochondria pro- was little. moted survival in a MEK/ERK-dependent man- Using quantitative TRAP assay, we detected tel- ner, while mitochondrial FoxO3A was required omerase activity and correlated it with tert, tr for apoptosis induction by metformin. The in- and atrx RNA levels. Telomere length distribution terplay between the MEK/ERK and the AMPK was evaluated by a non-radioactive TRF assay cascades, which converge on the N-terminal and Q-FISH analysis, in comparison with telomer- domain of FoxO3A, represents the first chap- ic content measured with qPCR. C-circles were ter of the mitochondrial tale of the FoxO3A quantified in genomic tumor DNA. Terra levels code. Further studies are needed to establish were measured by RNA FISH, non-radioactive dot whether other signaling pathways actually tar- - and northen- blot and correlated with telomere get FoxO3A N-terminus to modulate its mito- length. Most of these aspects were studied dur-

The Pezcoller Foundation Journal - June 2018 23 ing the progression from a single cancer initiating ing MCs can interact in vivo with CD40-ex- clone of a few cells to a full tumor. pressing PMN-MDSC, we tested whether Kit- Our data show that this model is ideal to study Wsh-TRAMP mice reconstituted with MCs either how the switch between telomere maintenance sufficient or deficient for CD40L can replace mechanisms occurs in vivo. or not immunosuppression. Only wild type, CD40L competent, MCs could restore PMN-MD- SC suppressive functions, T cell unresponsive- ness and adenocarcinoma development. These

Posters 21. Myeloid Derived Suppressor data unveil an immunoregulatory function of Cells-Mast Cells Cross-Talk Via MCs on PMN-MDSC activity through CD40L- CD40-CD40L Mediates Tumor CD40 interaction favoring immunosuppression Specific Immunosuppression in and tumor onset. In silico analyses provided correlation with clinical data, showing poor Prostate Cancer outcome in prostate cancer patients characterized by high expression of MC/PMN-MDSC 1 2 Elena Jachetti , Valeria Cancila , Alice Rigo- related-genes. ni1, Lucia Bongiovanni2, Barbara Cappetti1, Beatrice Belmonte2, Claudia Enriquez1, Patri- zia Casalini3, Paola Ostano5, Barbara Frossi4, Sabina Sangaletti1, Claudia Chiodoni1, Giovan- 22. Pharmacological activation of na Chiorino5, Carlo E. Pucillo 4, Claudio Tripo- TRPM8 channel overcomes innate do2 and Mario P. Colombo1 resistance to standard-of-care therapies in prostate cancer 1Molecular Immunology Unit, Department of Research, Fondazione IRCCS Istituto Nazion- Alessandro Alaimo1, Marco Lorenzoni1, Arian- ale dei Tumori, Milan, Italy 1 1 2 na Bertossi , Francesco Cambuli , Paolo Am- Tumor Immunology Unit, Department of brosino2, Alice Macchia1,3, Alessandra Bisio1, Health Sciences, University of Palermo, Italy 1 1 3 Sacha Genovesi , Gianluca Petris , Anna Cer- Molecular Targeting Unit, Department of Re- eseto1, Maria Virginia Soldovieri2, Alvaro Vil- search, Fondazione IRCCS Istituto Nazionale larroel4, Mattia Barbareschi5, Marco Durante6, dei Tumori, Milan, Italy Arkaitz Carracedo3, Maurizio Taglialatela7, 4 Department of Medical and Biological Sci- Andrea Lunardi1,§. ence, University of Udine, Italy 5 Fondazione Evo ed Eldo Tempia, Biella, Italy 1Centre for Integrative Biology (CIBIO), Uni- versity of Trento, Trento, Italy. Immunotherapy including checkpoint inhibi- 2Department of Health Sciences, University of tors is currently the most potent therapeutic Molise, Campobasso, Italy. approach for several types of cancers but with 3CIC bioGUNE, Bizkaia Technology Park, 801 limited activity against prostate tumors. Im- building, Derio, Spain. mune suppression instigated by the tumor is 4Instituto Biofisika, Consejo Superior de Inves- thought to be the cause. We indicated mast tigaciones Científicas, CSIC, UPV/EHU, Barrio cells (MCs) as accomplices of adenocarcinoma Sarriena s/n, Leioa, Spain. development within the prostate microenvi- 5Unit of Surgical Pathology, Santa Chiara Hos- ronment, and their immunosuppressive ca- pital, Trento, Italy. pacity prompted our investigation on wheth- 6Trento Institute for Fundamental Physics and er they have part in immunosuppression and Applications (TIFPA), INFN, University of Tren- tolerance to SV40 Large-T antigen, the trans- to, Trento, Italy. forming oncogene in transgenic adenocarcino- 7Department of Neuroscience, University of ma of the mouse prostate (TRAMP) mice. Naples “Federico II”, Naples, Italy. Crossing TRAMP mice with MCs deficient KitWsh §Corresponding Author mice strongly reduced the incidence of ade- Andrea Lunardi, Armenise-Harvard Laboratory nocarcinoma. TRAMP mice are generally tol- of Cancer Biology & Genetics, Centre for In- erant to the SV40 Large T antigen otherwise tegrative Biology, University of Trento, Italy. immunogenic in normal syngeneic B6 mice. E-mail: [email protected] Genetic ablation of MCs restored the capacity of mounting a tumor-specific cytotoxic T Abstract cell response. Notably, in KitWsh-TRAMP mice Discovery of novel ‘druggable’ targets is a pri- the restored T cell immunity correlated with mary goal in cancer translational research. the reduction of polymorphonuclear- myeloid Transient Receptor Potential subfamily M derived suppressor cells (PMN-MDSC) activity, member 8 (TRPM8) is a cation channel almost along with reduced expression of Arg1, Nos2 exclusively expressed by the luminal compart- and Stat3. Having found that CD40L-express- ment of the prostate epithelium in the human

24 The Pezcoller Foundation Journal - June 2018 body. Poorly studied in the context of prostate concurrent with LDR were administered; LDR physiology and homeostasis, the increased ex- was delivered on days 1 and 2, 8 and 9 of each pression of TRPM8 in primary and metastatic CHT cycle, using eight doses of 40 cGy each. prostate cancer lesions suggests a proto-onco- If no progression was observed after CHT-LDR, genic role of the channel. pts received 3 fractions of 8, 10 or 12 Gy (to- Here, by combining a multidisciplinary ap- tal dose 24-36 Gy) of SBRT based on tumor lo- proach to an in vitro genetic platform mod- cation in relation to stomach and duodenum. elling the natural history of prostate tumor- 4D-CT with oral and i.v. contrast was used for igenesis, we demonstrate that potent TRPM8 treatment planning and IGRT-IMRT for delivery. Posters agonists synergize with X-ray treatments to Seven weeks after SBRT tumour re-staging and induce massive apoptotic response in radi- evaluation for surgery was performed. Toxicity oresistant pre-malignant and malignant pro- was scored according to CTCAE v4. Progression totypes of primary prostate lesions. As well, free survival (PFS), freedom from locoregion- TRPM8 activation enhance the efficacy of al progression (FFLRP), freedom from distant docetaxel or enzalutamide in eradicating hor- metastasis (FFDM) and overall survival were mone naïve metastatic PCa cells. calculated using the Kaplan-Meier method. Overall, we propose TRPM8 activation as a Results: Between February 2014 and Decem- valuable clinical strategy for a more effective ber 2017 we enrolled 14 pts. All pts received treatment of low-risk and high-risk organ con- four CHT cycles, except one because of an fined PCa patients. intercurrent myocardial infarction. Two pts developed distant metastases after induction CHT, 12 received SBRT. Total SBRT dose was: 23. Low-dose radiotherapy as a 36 Gy (2 pts), 30 Gy (3 pts) and 24 Gy (7 pts). chemo-enhancer of an induction At present 3/12 pts underwent resection with- chemotherapy regimen with out complications. With an overall median follow-up of 17.6 GemOx and stereotactic body months (range, 3–40), for all patients the radiotherapy for locally advanced locoregional control rate was 57.1% (8/14). pancreatic cancer: preliminary Median PFS, FFLRP, FFDM and OS were 12.2, 19, 14, 18.3 months respectively; estimated results 1-year PFS, FFLRP, FFDM and OS rate were 64.3%, 92.9%, 64.3% and 78% respectively. B.Meduri1*, G. Aluisio1, E.D’Angelo1, C. Tata1, 1 Three pts developed acute G3 or greater he- F. Lohr matologic toxicity (1 anemia and 2 neutrope- nia), 1 pt developed acute G3 gastrointestinal 1Radiation Oncology Unit, Oncology Depart- pain, no further G3 or greater acute nonhema- ment, University Hospital of Modena (IT) tologic toxicity was observed. One patient de- *E-mail: [email protected] - meduri. veloped G2 gastric ulcer and G2 gastric haem- [email protected] orrhage that were medically managed. Late Phone: +39 0594222283 - +39 0594222301 G3 or greater toxicities were not observed. Conclusion: Induction CHT combined with LDR Background and Purpose: Results of published used as a chemo-enhancer and SBRT in three phase II trials have indicated that low-dose fractions resulted in excellent local control radiotherapy (LDR) can enhance the effect of and seems to improve survival outcomes, with chemotherapy and may create an immunolog- a low rate of side effects. Evaluation upon ical microenvironment that enhances immu- enrolment conclusion is awaited to provide fi- nological response against tumors; further- nal results that may provide the rationale to more, phase II studies using stereotactic body further intensify research into modification/ radiotherapy (SBRT) demonstrated excellent modulation of locoregional treatment strate- local control and single radiotherapy doses around 8-10 Gy have also been documented gies for LAPC. to enhance tumor immunogenicity. We report preliminary safety and effectiveness results of an induction chemotherapy (CHT) regimen 24. In silico exploration of combined with LDR used as a chemo-enhancer von hippel-lindau (pvhl) tumor followed by SBRT in locally advanced pancreatic cancer (LAPC) suppressor molecular functions: Methods: Patients (pts) with non-metastatic correlations between disease inoperable LAPC were enrolled on a prospec- mutations, interactors and pathways tive single-institution study (NCT02416609). Four CHT cycles with Gemcitabine and Ox- Giovanni Minervini1#, Federica Quaglia1#, Franc- aliplatin (GEMOX) (day 1-8 of a 21-day cycle) esco Tabaro1, Silvio C.e. Tosatto1,2*

The Pezcoller Foundation Journal - June 2018 25 1-Department Of Biomedical Sciences, University This work is supported by Associazione Ital- Of Padova, Viale G. Colombo 3, 35121, Padova, iana per la Ricerca sul Cancro (AIRC) grants Italy.; 2-Cnr Institute Of Neuroscience, Padova, MFAG12740 and IG17753 to ST. Viale G. Colombo 3, 35121, Padova, Italy.; REFERENCE: 1) TABARO F., MINERVINI G., ET #-Contributed Equally; *-Corresponding Author: AL. VHLDB: A DATABASE OF VON HIPPEL-LINDAU [email protected] PROTEIN INTERACTORS AND MUTATIONS, SCIEN- TIFIC REPORTS, 6:31128, 2016. Posters Familiar cancers represent a privileged point of view for studying the complex cellular events inducing tumor transformation. Von Hippel-Lin- 25. The histone methyl transferase dau syndrome, a familiar predisposition to develop cancer, is a clear example. Here, we DOT1L is a novel epigenetic target present our efforts in deciphering the role of for therapy of endocrine-therapy the von Hippel-Lindau tumor suppressor pro- resistant breast cancer tein (pVHL) in cancer insurgence. We collected high quality information on ca. 1.600 pVHL mu- Giovanni Nassa1, Annamaria Salvati1, Giorgio tations and ca. 160 interactors from VHLdb, a Giurato1,2, Roberta Tarallo1, Francesca Rizzo1, freely accessible database dedicated to pVHL Valerio Gigantino1, Lorenza Mautone1, Tuula A. (1). These were investigated in terms of asso- Nyman3, Alessandro Weisz1 ciation between patient phenotypes, mutated surface and impaired interactions. Our data 1Laboratory of Molecular Medicine and Genom- suggest that different phenotypes correlate ics, Department of Medicine, Surgery and Den- with localized perturbations of the pVHL struc- tistry “Scuola Medica Salernitana”, University ture, with specific cell functions associated of Salerno, Baronissi (SA), Italy, to different protein surfaces. We propose five 2Genomix4Life S.r.l., University of Salerno, different pVHL interfaces to be selectively in- Baronissi (SA), Italy volved in modulating proteins regulating gene 3Proteomics Core Facility, Institute of Clinical expression, protein homeostasis as well as to Medicine, University of Oslo, Oslo, Norway address ECM and ciliogenesis associated functions. Estrogen Receptor alpha (ERα) is a ligand-induc- These data were used to drive molecular dock- ible transcription factor that mediates estrogen ing of pVHL with its interactors and guide Petri signaling in hormone-responsive breast cancer net simulations of the most promising altera- (BC) and is the primary target of specific antican- tions. We predict that disruption of pVHL as- cer therapies. Although ERα blockade with these sociation with certain interactors can trigger drugs is effective, the development of a resist- tumor transformation, inducing metabolism ance to treatment represents the key problem imbalance and ECM remodeling. Collectively in clinical management of patients affected by taken, our findings provide a novel insight into this disease. Understanding the molecular mech- VHL-associated tumorigenesis. This highly inte- anisms underlying ERα action in BC cells may grated in silico approach may help to elucidate help the identification of new therapeutic tar- novel treatment paradigms for VHL disease. gets for more effective pharmacological treat-

26 The Pezcoller Foundation Journal - June 2018 ment of endocrine therapy-resistant tumors. Canada; 6Cancer Research Institute, Beth Israel We recently discovered the epigenetic enzyme Deaconess Cancer Center, Department of Medi- DOT1L (DOT1 Like Histone Lysine Methyltrans- cine and Pathology, Beth Israel Deaconess Medi- ferase) as a novel nuclear partner of ERα in BC cal Center, Harvard Medical School, Boston, MA, cells. To investigate the involvement of DOT1L USA in mediating ERα actions in hormone-responsive and endocrine-resistant BC, physical and func- MicroRNAs (miRs) are small non-coding RNAs that tional interaction between these two molecules act as negative regulators of gene expression on chromatin was mapped by Chromatin Immu- and control tumor progression. We, and others, Posters noprecipitation coupled to Mass Spectrometry demonstrated that miR-214 is upregulated in ma- (ChIP-MS) and Sequencing (ChIP-Seq). Gene si- lignant melanomas and breast tumors. miR-214 lencing and selective pharmacological inhibition coordinates metastasis dissemination via a path- of either protein followed by transcriptome pro- way involving TFAP2C, the adhesion molecule filing (RNA-Seq) were applied and cellular and ALCAM and the anti-metastatic miR-148b. More- functional assays were performed to evaluate over, we showed that the simultaneous inhibition the functional impact of this complex in hor- of miR-214 and overexpression of miR-148b led mone-responsive and antiestrogen-resistant BC to strong reduction of metastasis, thus suggesting cells. ChIP-MS confirmed the co-recruitment of that miR-214 -miR-148b axis can be exploited for the two factors within a chromatin bound multi- combinatorial miR-based therapeutic approach- protein complex. Gene expression profiling and es. In order to study miR-214 function in a model Nascent RNA-Seq before and after DOT1L phar- of endogenous tumors, we generated a locus-spe- macological inhibition highlighted the involve- cific conditional transgenic mouse for miR-214 ment of this enzyme in ERα-mediated transcrip- overexpression and we crossed miR-214 overex- tional regulation of several estrogen responsive pressing animals with a mouse model of breast genes, including ERα itself. Global analysis of cancer progression (MMTV-PyMT mice). While we ERα and DOT1L binding to the genome showed observed a delay in mammary gland tumor onco-recruitment of both proteins on several chro- set in the double transgenic animals, carcinomas matin sites, including regulatory elements of that developed in these mice were more aggres- the ERα gene itself, providing an explanation for sive than controls and an increased number of the inhibition of cell cycle progression and cell lung metastases was observed. Increased aggres- proliferation observed in hormone-responsive siveness can be due, at least in part, to increased and antiestrogen-resistant BC cell models both mesenchymal traits observed in PyMT-miR-214 in vitro and in vivo. The results obtained reveal overexpressing tumors compared to controls. that physical and functional interplay between Moreover, angiogenesis and inflammation result- ERα and DOT1L represent a key molecular event ed to be enhanced in tumors following miR-214 of the estrogen-mediated signaling that control overexpression, thus suggesting a contribution of BC cell functions, suggesting this enzyme as a stromal miR-214 in tumor progression. miR-214 is new potential therapeutic target against endo- highly expressed in stroma cells and its overex- crine-resistant cancers. pression in the stroma compartment is sufficient per se to promote tumor dissemination. Our re- RESEARCH SUPPORTED BY: AIRC (grant IG-17426), sults suggest that when miR-214 is overexpressed CNR Flagship Projects EPIGEN and InterOmics and in an established tumor onset it increases metas- ELIXIRITA HPC@CINECA. tasis formation acting both on tumor and stromal cells. 26. miR-214 overexpressing mice develop more aggressive mammary 27. Diagnosis of Glioma Tumors gland tumors Using Circulating Cell-Free DNA

F. Orso1,2,3, F. Virga1,2,4, D. Baruffaldi1,2, D. Det- Vikrant Palande, Dorith Raviv Shay and Milana tori1, 2, E. Bolli1,2, F. Cavallo1,2,M. Forni1, L. Sal- Frenkel-Morgenstern* mena5, M. Mazzone4, P. P. Pandolfi6, D.Taverna1,2,3 The Azrieli Faculty of Medicine, Bar-Ilan Univer- 1Molecular Biotechnology Center (MBC), 2Dept. sity, 8 Henrietta Szold St, Safed, 1311502, Israel Molecular Biotechnology and Health Sciences Contact us: [email protected], and 3Center for Complex Systems in Molecular [email protected] Biology and Medicine at the University of Torino, Italy; 4Lab of Tumor Inflammation and Angiogen- Introduction esis, Center for Cancer Biology (CCB), VIB, Leu- Gliomas are the most frequent brain tumors, ven, Belgium; 5Princess Margaret Cancer Centre, making up about 30% of all brain and central University Health Network, Toronto, Ontario, nervous system tumors, and 80% of all malignant

The Pezcoller Foundation Journal - June 2018 27 brain tumors. Existing standard diagnostic tech- University of Rome Tor Vergata, Rome, Italy; nique for glioma tumor includes tissue biopsy, 2Fondazione Santa Lucia IRCCS, Rome, Italy; which is a highly invasive and hence a risky tech- 3Department of science medical/chirurgic and nique for the patient’s survival. ‘Liquid biopsy’ is translational medicine, University of Rome “Sa- a new and recently developed non-invasive can- pienza”, Rome, Italy; 4Institute of Human Anato- cer diagnostic technique, which includes use of my and Cell Biology, UniversitàCattolica del Sac- circulating cell-free DNA (cfDNA) fragments for roCuore, Rome, Italy; 5Department of Medical, tracing tumor markers. CfDNA fragments are one Oral & Biotechnological Sciences, G. d’Annunzio Posters of those molecular bits that are released into the University, Chieti, Italy; 6Center of Excellence bloodstream after rapid apoptosis or necrosis of on Aging and Translational Medicine (CeSi-Met), the tumor cells in the cancer patients. Our goal G. d’Annunzio University, Chieti, Italy; 7Depart- is to do comprehensive study between distinct ment of Emergency and Organ Transplantation, types of glioma cancer tumors and cfDNA of the University Aldo Moro , Bari, Italy; 8Department respective patients, to elucidate the scope of cfD- of Surgical and Biomedical Sciences, University NA in liquid biopsy technique for glioma diagnosis. of Perugia, Perugia, Italy;9Department of Experi- mental Medicine and Surgery, University of Rome Methods Tor Vergata, Rome, Italy We collected 8 different glioma patient’s tumor tissue and plasma samples and then isolated tu- Abstract mor DNA from glioma tumor tissue and circulating Non-muscle invasive bladder cancer (NMIBC) is cell-free DNA(cfDNA) from the respective glioma a malignant disease characterized by high het- patient’s plasma. Isolated tumor DNA and cfDNA erogeneity, which corresponds to dysregulated then deeply sequenced on Illumina HiSeq 2500 gene expression and alternative splicing pro- and then NGS data was analyzed to find out single files. Nevertheless, the molecular basis of such nucleotide variants (SNVs) as well as structural aberrant regulation is unknown. Bioinformatics variants on both cfDNA and tumor gDNA. analyses performed by querying public datasets for splicing factors potentially linked to bladder Results cancer progression highlighted a positive correla- We have successfully detected glioma specific tion between the heterogeneous nuclear ribonu- mutations such as IDH1, IDH2, PDGFRA, NOTCH1, cleoprotein I (i.e. PTBP1) mRNA expression and PIK3R1 and TP53, from cfDNA isolated from the NMIBC progression. To validate this observation plasma of glioma patients and could relate this at the protein level, we enrolled a cohort of 152 mutations to the different tumor grades of glio- patients presenting with primary NMIBC (pTa- ma. We are also studying the dynamics of these pT1), for whom detailed follow-up clinical data mutations in response to glioma drug treatment (>2 years)were available. Immunohistochemistry by collecting blood samples at different time in- (IHC) and western blot analyses confirmed that tervals. high PTBP1 expression was associated with worse clinical outcome in terms of incidence of tumor Discussion relapse and survival in NMIBC patients. Next, we This study may help in developing liquid biopsy investigated whether PTBP1 was functionally rel- technique for glioma tumor diagnosis and in its evant for bladder cancer cells. To this end, we prognosis for monitoring the glioma treatment by depleted of PTBP1 expression by transfection of non-invasive approach, and will eventually help PTBP1-specific siRNAs and evaluated its effect on physicians to decide the right treatment on ap- three bladder cancer cell lines. Clonogenic as- propriate time while bypassing the existing ‘wait- says, cytoflourimetric analysis of the cell cycle, and-see’ approach of treatment monitoring. Annexin V and PI assay and immunofluorescence analysis of caspase-3 cleavage demonstrated that PTBP1 favors bladder cancer cell proliferation and survival. Moreover, silencing of PTBP1 28. The splicing factor PTBP1 sensitizes bladder cancer cells to mitomycin C-in- promotes expression of oncogenic duced death, as indicated by double staining with splice variants and predicts poor Annexin V and PI and analysis of caspase 3 cleavage. Mechanistically, we found that PTBP1 mod- prognosis in patients with non- ulates the splicing pattern of bladder cancer-re- muscle invasive bladder cancer lated genes, such as TPM1, FAS, NUMB, MACF1, PKM, CD44, CTNND1, ACTIN1,by directly binding Pamela Bielli1,2, Valentina Panzeri2,3,4, Rossano in proximity of regulated exons and promoting Lattanzio5,6, Simona Mutascio1,2, Marco Pierac- the expression of pro-oncogenic splice variants cioli1,2, Elisabetta Volpe2, Vincenzo Pagliarulo7, of these genes. Notably, pro-oncogenic alterna- Mauro Piantelli5, Antonella Giannantoni8,Savino tive splicing of CD44 correlated with PTBP1 ex- M. Di Stasi9,10 and Claudio Sette2,4,10 pression also in patient’s specimens, suggesting 1Department of Biomedicine and Prevention, the clinical relevance of these results. Thus, our

28 The Pezcoller Foundation Journal - June 2018 study demonstrates an oncogenic role of PTBP1 30. CASP4 gene silencing in in bladder cancer and suggests that its expression epithelial cancer cells leads to and/or its splicing signature can represent novel outcome-predictor markers for NMIBC. impairment of cell migration, cell- matrix adhesion and tissue invasion

29. LMW-PTP targeting to improve Giuliana Papoff1*, Dario Presutti1, Cristiana Lalli1, Giulia Bolasco2, Simonetta Santini1, Can- the effectiveness of chemo- and Posters dida Manelfi1, Valentina Fustaino1, Stefano radiotherapy Alemà1 and Giovina Ruberti1*

*Paolo Paoli, *Erica Pranzini, *Giulia Lori, *Elisa 1 National Research Council, Institute of Cell Pardella, *Anna Caselli, *Paolo Cirri, *Riccardo Biology and Neurobiology - Via E. Ramarini, 32 Marzocchini, Giovanna Caderni, and *Giovanni 00015 - Monterotondo (Rome) – Italy. 2 EMBL Raugei Epigenetic and Neurobiology Unit - Rome – Italy.

*Department of Experimental and Clinical Bio- ABSTRACT medical Sciences “Mario Serio”; University of Inflammatory caspases, including human Florence, Viale Morgani 50, 50134 FIRENZE caspase-4 (CASP4), play key roles in innate im- §NEUROFARBA Department, Section of Pharma- mune and inflammatory responses. The role of cology and Toxicology, University of Florence, 6 inflammatory caspases in cancer cells remains Viale Pieraccini, 50139 Florence, Italy poorly investigated. Here, we explored the consequences of CASP4 expression levels on the mi- The set point of new effective anticancer strat- gratory behavior of epithelial cancer cell lines. egies to overcome cancer resistance is one the By a gene silencing approach and in vitro and major goals of pharmaceutical companies world- in vivo studies we show that down-regulation wide. We demonstrated that Low Molecular of CASP4 leads to impaired cell migration and Weight Protein Tyrosine Phospatase (LMW‐PTP) cell-matrix adhesion. is a new interesting target to revert intrinsic This phenotype is accompanied by an increased resistance of tumors toward chemo- and radio- actin cytoskeleton polymerization, changes in therapy. This enzyme is generally overexpressed the overall organization of adherens junctions in aggressive and therapy-resistant tumors and (AJs) and in the number and size of focal ad- its expression is related to poor prognosis. We hesions. Interestingly, the cell migration deficit demonstrated that LMW-PTP silencing, in many could be reversed by epithelial growth factor different cell lines, allows the reprogramming of treatment, and depletion of calcium ions un- resistant cancer cells toward a phenotype more veiled a role of CASP4 in the novo assembly of sensitive to various anticancer drugs, including AJs, suggesting that the role of CASP4 is not dacarbazine, docetaxel and 5-FU, or radiother- cell-autonomous. Finally, CASP4-silenced A431 apy. Furthermore, LMW-PTP silencing strongly cells exhibited a severe reduction in their ability impairs self-renewal ability of different types to invade lung tissue, when injected into nude of cancer cells, thereby suggesting that this en- mice. Overall, our data support the emerging zyme sustains survival of staminal cancer cells, evidence that inflammatory caspases can reg- promoting relapse. Treating cancer cells with a ulate cell migration through actin remodeling LMW-PTP inhibitors leads to the same effects of and uncover a novel role of CASP4 in cancer cell silencing, improving the effectiveness of chemo- behavior. and radiotherapy. Interestingly, we observed that LMW-PTP inhibitors are not effective on non-can- cerous cells, which express low LMW-PTP levels. 31. Proline dehydrogenase This finding reinforce the hypothesis that expression of LMW-PTP contribute to make cancer cells expression, regulation and function more resistant toward cytotoxic stimuli triggered in non-small cell lung carcinoma by traditional anticancer therapies. Studies conducted on PIRC rats, which spontane- Arianna Parnigoni1, Sarah Grossi1, Paola D’An- ously develop multiple tumours in the colon (a tona1, Elisa Corbetta1, Federica Sanvito1, model for both familial adenomatous polyposis Anna Maria Chiaravalli2, Fausto Sessa2,3, Paola and sporadic colorectal cancer) confirmed that Campomenosi1 treatment with LMW-PTP inhibitors reduces LMW- 1Department of Biotechnology and Life Sciences, PTP expression in rat tumors, increasing sensi- DBSV, University of Insubria, Varese, Italy; 2Pa- tivity of cancer cells toward chemotherapy. In thology Unit, Ospedale di Circolo e Fondazione conclusion, LMW-PTP is a promising target, for Macchi, Varese, Italy; improving the outcome of patients affected by 3Department of Medicine and Surgery, DMS, Uni- cancers refractory to treatments. versity of Insubria, Varese, Italy.

The Pezcoller Foundation Journal - June 2018 29 Introduction. 2 Department of Experimental and Clinical Bi- Non-Small Cell Lung Cancer (NSCLC) is one of omedical Sciences “Mario Serio”, University of the most frequent and deadliest cancers and Florence comprises two main histotypes, adenocarcinoma 3 Department of Surgery and Translational Medi- (ADC) and squamocellular carcinoma (SCC). Iden- cine, University of Florence tification of markers to better define the diagnosis, prognosis and therapeutic options of NSCLC INTRODUCTION is needed. We investigated if proline dehydro- Gastric cancer (GC) is the fifth most common Posters genase (PRODH), a mitochondrial flavoenzyme cancer worldwide and the third leading cause catalyzing the key step in proline degradation, of cancer-related deaths. To date, gastrecto- and involved in the regulation of cell survival, au- my and chemotherapy are the only therapeutic tophagy and apoptosis, may be one such marker. options, but drug resistance is the main cause for treatment failure. Materials and methods. Vasculogenic mimicry (VM) is a new model of We characterized PRODH expression in NSCLC neovascularization in aggressive tumors and by immunohistochemistry and qPCR and test- has been correlated with poor prognosis in GC ed if there was correlation between expression patients. of PRODH and clinical features of the tumors or Our group has developed chemotherapy-resist- expression of other markers. Phenotypic assays ant GC cells using the Caucasian adenocarci- were performed to investigate the cellular influ- noma cell line AGS and three drugs among the enced by PRODH in lung ADC cell lines. We also most used in clinic (5-fluorouracil, cisplatin tested the regulation of the PRODH gene by TTF- and paclitaxel) henceforward denominated 1 by means of transfection experiments, expres- 5FUr, CISr, TAXr. sion analyses and luciferase reporter assays. Our study has highlighted phenotypical differences among chemo-sensitive and chemo-re- Results and discussion. sistant cell lines such as acquisition of stem- We found PRODH immunostaining in the majority like phenotype and increased capacity to form (70%) of lung ADCs. Patients with PRODH positive vessels. tumors had better overall survival than those with negative tumors. Protein staining was paral- MATERIALS AND METHODS leled by high transcript levels. Clonogenic assays Establishment of AGS resistant cell lines expos- showed that PRODH favors survival of ADC cells. ing cells to increasing dilution of drugs for over Moreover, TTF-1, a transcription factor essential 9 months up to dilutions higher than IC50 val- for thyroid and lung development and physiology, ues initially verified on AGS cells through MTT that shows similar expression pattern as PRODH analysis. was found to regulate PRODH expression. This Quantitative RT-PCR, flow cytometry and west- prompted us to investigate if PRODH could be ern blot analysis for stemness and VM markers. a target of TTF-1. Transfection of a TTF-1 ex- Vasculogenic mimicry assay. pression construct into ADC cell lines led to an increase in PRODH transcript and luciferase re- RESULT AND DISCUSSION porter assays showed that a RE was indeed trans- AGS cells acquired chemoresistance as indicat- activated by TTF-1. ed by the increase of IC50 values in drug-treated cells with respect to AGS. Furthermore, MTT Conclusion. assay highlighted that there is not cross-resist- Our data support a possible application of PRODH ance among 5FUr, CISr and TAXr. Supportive immunostaining as a prognostic marker and war- data is that cells are MDR1 negative. rant further research aimed to investigate PRODH Resistant cells showed an upregulation of transactivation by TTF-1 and the role of PRODH in Yamanaka factors either in qPCR and flow cy- the biology of NSCLC. tometer analysis, and particularly interesting is ALDH overexpression in 5FUr. Twist upregulation suggested the investigation 32. Chemotherapy resistance- of VM which resulted particularly enhanced in 5FUr cells which demonstrated their ability to associated epithelial to endothelial form and sustain vessels up to 96 hrs in the transition in gastric cancer tube formation assay. Laminin γ2 and Ephrin A2 showed an increase Sara Peri1,2 & Alessio Biagioni2, Fabio Cianchi3, in resistant cells and especially in 5FUr. Ileana Skalamera3, Fabio Staderini3, Nicola Schi- avone2, Laura Papucci2, Lucia Magnelli2 CONCLUSION One of the most interesting result is that 5FUr 1 Department Of Biotechnology, Chemistry And cells acquire stemness properties and are pos- Pharmacy, University of Siena itive to the tube formation assay suggesting

30 The Pezcoller Foundation Journal - June 2018 that VM might be one mechanisms adopted by mechanism could lead to the identification of cells to avoid drugs exposure. novel breast CSCs vulnerabilities and to the These findings suggest that acquisition of improvement of combinatorial regimens with chemoresistance could cause a relapse of the aim of avoiding resistance and relapse in disease in which tumor cells take advantage breast cancer. of their capability to perform VM in order to self-sustain their growth and that may be cause of poor outcomes. 34. ANP32E as a putative proto- Posters oncogenic factor in MYC over- 33. ETV7 regulates cancer stem expressing cancer cells cells content and resistance to 5-FU Vittoria Poli1, Luca Fagnocchi1, Alessandra Fas- and radiotherapy in breast cancer ciani1,2, Alessio Zippo1,2

Laura Pezzè1, Stefano Pontalti1, Kalina Bad- 1 University of Trento, Center for Integrative owska1, Ira-Ida Skvortsova2, Yari Ciribilli1 Biology (CIBIO) 2 Fondazione Romeo ed Enrica Invernizzi, Isti- 1Laboratory of Molecular Cancer Genetics, CI- tuto Nazionale di Genetica Molecolare (INGM) BIO, University of Trento, Trento, Italy 2Laboratory for Experimental and Translational Breast cancer consists of highly heterogeneous Research on Radiation Oncology (EXTRO-Lab), tumors, whose driver oncogenes result diffi- Department of Therapeutic Radiology and cult to be uniquely defined. We have recently Oncology, Innsbruck Medical University, Inns- reported the central role of MYC in initiating bruck, Austria and sustaining a step-wise cell reprogramming process in differentiated mammary luminal Cancer stem cells (CSCs) are considered the epithelial cells (IMEC) toward a stem cell-like population of cells within a tumor able to drive condition, which favors cell transformation tumorigenesis and known to be highly resistant and tumor initiation. Specifically, over-expres- to conventional chemotherapy as well as radi- sion of MYC induces transcriptional repression otherapy. ETV7 is a poorly studied transcrip- of lineage-specifying transcription factors, tion factor member of the ETS family, known causing decommissioning of luminal-specific to be an interferon-stimulated gene. ETV7 has enhancers. We also provided evidence that been recently found over-expressed in breast the MYC-driven reprogramming favors the cancer (BC), with higher expression levels in formation of tumor initiating cells endowed the more aggressive BC subtypes. In this work, with metastatic capacity, once cells have we investigated the effects of increased ETV7 been challenged with the oncogenic activa- expression on breast CSCs population and re- tion of the PI3K-pathway. This oncogenic set- sistance to chemotherapy and radiotherapy in ting permitted the formation of tumors once BC cells. the IMEC-MYC/PIK3CAH1047R (thereafter named We generated MCF7 and T47D BC-derived cells transformed-IMEC, t-IMEC) have been trans- stably over-expressing ETV7 and we tested sen- planted into the mammary gland of NOD/SCID sitivity of these cells to the chemotherapeutic mice. In addition, we demonstrated that t-IM- drug 5-Flouorouracil (5-FU) and to radiothera- EC retained long-term tumorigenic potential py. By viability assays and apoptosis analyses as xenograft-derived (XD) cells formed tum- we observed that the over-expression of ETV7 ors once re-injected in secondary and tertiary could reduce sensitivity to both 5-FU and ra- recipient mice. Among the different chroma- diotherapy in these cell lines. We could also tin players, we identified the H2A.Z-specific appreciate an increase in ABC transporters and chaperone ANP32E as a putative cofactor that BCL2 anti-apoptotic protein expression follow- may synergize with MYC in driving the ob- ing ETV7 over-expression. These effects were served epigenetic reprogramming. By interro- also accompanied by the observation that al- gating gene expression pattern of chromatin teration of ETV7 expression could significant- regulators, we observed that the histone var- ly affect the population of breast cancer stem iant H2A.Z and its specific chaperone ANP32E cells (CD44+/CD24low cells) in different BC cell was induced in t-IMEC and in XD cells respect lines. We finally associated higher ETV7 expres- to IMEC-MYC. By analyzing the TCGA dataset, sion with altered proliferation rates and in- we found that ANP32E alteration is specifical- creased colony formation potential in soft agar. ly enriched among basal-like breast cancer We propose a novel role for ETV7 in breast can- characterized by MYC deregulation and this cer stem cells plasticity and associated resist- combination correlates with a worst progno- ance to conventional chemotherapy. We finally sis. Altogether, these observations suggested a suggest that an in-depth investigation of this possible cooperative and oncogenic activity of

The Pezcoller Foundation Journal - June 2018 31 ANP32E in the tumor onset and maintenance. cells during tumorigenesis and recent studies To asses this aspect, we targeted XD cells with have highlighted a role for such metabolic re- a lentiviral vector expressing ANP32E and as- programming in adaptation to therapies and sessed effects of ANP32E overexpression on in chemo-resistance development. vitro self-renewing capacity and tumorigenic 5-Fluorouracil (5-FU) is the most commonly activity. Interestingly, ANP32E overexpression used drug in colon cancer therapy. However, in XD cells caused an increment in the capac- despite a promising initial response to the ther- ity to form single cell-derived mammospheres apy, development of drug resistance to 5-FU Posters and to develop cell foci in a semi-solid main human tumor cells is the primary cause of trix. Recent studies reported an ANP32E in- chemotherapy failure. 5-FU exerts its antican- volvement in the DNA damage response (DDR), cer effects through inhibition of thymidylate showing that while H2A.Z incorporation at synthase (TS) and incorporation of its metabo- the damaged chromatin is necessary to allow lites into RNA and DNA. TS catalyzes the meth- a correct recruitment of the repair machin- ylation of dUMP to dTMP using reduced folate ery, its subsequent rapid removal by ANP32E as a methyl donor and its over-expression is is indispensable for the resolution of the widely accepted as a major molecular mecha- damage. In order to assess whether ANP32E nism responsible for 5-FU resistance. overexpression in cancer cells can interfere In our study we established a 5-FU-resistant with the DNA repair machinery, we quantified human colon cancer cell line in order to in- phospho-H2A.X foci in XD cells +/- exogenous vestigate the metabolic adaptation correlated ANP32E and observed that ANP32E overex- to the resistance development, focusing on pressing cells (XD-ANP32E) were characterized one-carbon metabolism and folate cycle. by a strong increment in both phospho-H2A.X Real-time PCR analysis revealed increased foci number and dimension. serine synthesis pathway in resistant cells in A critical component of the DDR is repre- accordance with TS overexpression. Serine sented by ATR, a factor activated by regions supplies carbon to the one-carbon pool, which of single-stranded DNA, which can occur as a is involved in folate metabolism and supports result of oncogene-induced replicative stress. TS activity. Moreover, folate metabolism also Notably, pharmacological treatment with an contributes to the generation of S-adenosyl- ATR inhibitor showed higher sensitivity of XD- methionine, the methyl donor for both DNA ANP32E cells respect to not-transduced cells. and histone methylation, thus influencing epi- This study supports the notion that ANP32E al- genetic control of gene expression. teration in cancer cells subjected to a MYC-re- Radiolabelled metabolic studies and extra- lated replicative stress can promote the de- cellular flux analysis (XF Seahorse analyzer) velopment and maintenance of a tumorigenic showed a significant decrease in glucose uti- phenotype by interfering with the DDR, there- lization together with increased glutamic acid fore favoring the acquisition of oncogenic in- metabolism in resistant cells, suggesting that sults. Of note, the correlation between dereg- this amino acid represents a valid alternative ulated expression of ANP32E and increased cell nutrient to sustain drug resistance. sensitivity to ATR inhibition could establish a Both glutamate and serine support several therapeutic rational for a targeted treatment metabolic processes that are crucial for the of basal-like breast cancers characterized by survival of proliferating cells: they are precur- combined MYC and ANP32E alteration. sors of the nonessential amino acids, are in- corporated into glutathione, a primary cellular antioxidant, and are indispensable for nucleo- 35. Metabolic pathways promoting tides biosynthesis. Together these findings indicate that non-es- colon cancer resistance to sential amino acid metabolism plays an impor- 5-Fluorouracil: the role of non- tant role in 5-FU resistance, suggesting new essential amino acids possible approaches to target resistant cells and overcome colon cancer relapse. Pranzini E.1, Paoli P.1, Leo A.1, Taddei M.L.2, Chiarugi P.1 36. Automated in vivo screen 1Department of Experimental and Clinical Bio- of a zebrafish melanoma model medical Sciences, University of Florence, Flor- identifies FDA-approved drugs for ence, Italy. 2Department of Experimental and Clinical combinatorial treatments Medicine, University of Florence, Florence, Italy Francesca Precazzini1, Viviana Anelli1, Michael Metabolic rearrangements are essential to Pancher2, Valentina Adami2, Pamela Gatto2 satisfy the different requirements of cancer and Maria Caterina Mione1

32 The Pezcoller Foundation Journal - June 2018 1Laboratory of Experimental Cancer Biology, an Brain Research Institute, “Rita Levi-Mon- Centre for Integrative Biology (CIBIO), Univer- talcini” (EBRI), Rome, Italy; 4SYSBIO Center sity of Trento, Via Sommarive 9, 38123 Povo for Systems Biology, Milan, Italy. (TN), Italy * These authors have contributed equally to 2High Throughput Screening (HTS) Facility, this work Centre for Integrative Biology (CIBIO), Univer- sity of Trento, Via Sommarive 9, 38123 Povo Non small cell lung (NSCLC) patients, whose (TN), Italy tumors harbor sensitizing and driving mutations in the epidermal growth factor receptor Posters Kita:GFP-HRAS fish develop cutaneous mela- (EGFR), get a meaningful clinical benefit from noma with high frequency (Santoriello et al., EGFR tyrosine kinase inhibitor treatments. PLoS One, 2010). Already at 3 dpf, transgenic Unfortunately acquired resistance invariably kita-GFP-HRAS larvae show a hyperpigmenta- develops. Increasing evidence points to a key tion phenotype as earliest evidence of abnor- role played by epithelial-mesenchymal transi- mal melanocyte growth. Using this transgenic tion (EMT) in cancer progression and drug re- model we performed a chemical screen with sistance. Importantly, the EMT is not a binary the Prestwick library. The screen is based on process and cancer cells with intermediate or automated detection of a reduction of mel- hybrid epithelial/mesenchymal (E/M) pheno- anocyte number caused by any of 1280 FDA or types characterized by a mixture of epithelial EMA approved drugs of the library. The analysis and mesenchymal traits have been described. showed that 55 molecules were able to reduce The effect of target therapy on the selection of by 60% or more the number of melanocytes intermediated E/M phenotypes in cancer cells per embryo. Hierarchical clustering showed is still poorly investigated. In this study, we that the hits clustered in five major classes: used wet and in silico approaches to investi- anthelmintic, steroids, antisecretory, antifun- gate whether intermediate E/M phenotypes are gal and non steroid anti-inflammatory drugs. associated to resistance to target therapy in a To generate a dose-response curve we further NSCLC model system harboring activating mu- tested two compounds for each class at six dif- tations of the EGFR. The combination of differ- ferent concentrations alone or in combination ent analysis techniques allowed us to describe with a farnesyltransferase inhibitor (Lonafarn- intermediate and complete EMT phenotypes ib), that inhibits an essential post-translation- respectively in HCC827- and HCC4006-derived al modification of HRAS, or two MEK inhibitors drug-resistant human cancer cell lines. Inter- (Trametinib and Selumetinib). Combination of estingly, intermediate E/M phenotypes, a col- Clotrimazole and Lonafarnib showed the most lective cell migration and increased stem-like promising results, allowing to reduce the effec- ability associate to resistance to the EGFR in- tive dosage of both drugs. We are performing hibitor, erlotinib, in HCC827 derived cell lines. validation of these observations in human mel- Moreover, the use of three complementary anoma cultures and investigating the mecha- approaches for gene expression analysis sup- nisms of action of the anti-fungal compound in ported the identification of a small EMT-relat- blocking the proliferation of HRAS-transformed ed gene list, which may have otherwise been melanocytes. overlooked by standard stand-alone methods for gene expression analysis.

37. Characterization of epithelial- 38. Anti-cspg4 dna vaccination mesenchymal transition for the treatment of malignant intermediate/hybrid phenotypes melanoma: a comparative oncology associated to resistance to EGFR trial for translational medicine inhibitors in non-small cell lung cancer cell lines Federica Riccardo1, Giuseppina Barutello1, Lidia Tarone1, Valeria Rolih1, Elisabetta Bol- Presutti D1*, Fustaino V1,2*, Colombo T2, Cardi- li1, Maddalena Arigoni1, Sergio Occhipinti2, nali B1, Papoff G1, Brandi R3, Bertolazzi P4,2, Soldano Ferrone3, Paolo Buracco4, Federica Felici G2, Ruberti G1 Cavallo1

1Institute of Cell Biology and Neurobiology, 1Department of Molecular Biotechnology and National Research Council (IBCN-CNR), Mon- Health Sciences, Molecular Biotechnology terotondo, Rome, Italy; 2Institute for Systems Center, University of Torino, Torino, Italy Analysis and Computer Science “Antonio Ru- 2Center for Experimental Research and Medi- berti” National Research Council, (IASI-CNR), cal Studies (CERMS), Città della Salute e della Rome, Italy; 3Genomics facility of the Europe- Scienza di Torino, Torino, Italy

The Pezcoller Foundation Journal - June 2018 33 3Department of Surgery, Massachusetts Gener- the self antigen in dogs and humans and could al Hospital, Harvard Medical School, Boston, move this approach to standard therapies for MA, USA both veterinary and human clinical application. 4Department of Veterinary Sciences, Universi- ty of Torino, Grugliasco, Italy 39. Identification of DHX30 as Abstract Malignant melanoma (MM) is the sixth most an inhibitor of the translation of Posters frequently diagnosed cancer and after prima- pro-apoptotic mRNAs after p53 ry tumor resection, about 30% of patients will activation by Nutlin experience disease recurrence and metastasis often fatal. In recent years, the development Dario Rizzotto1, Sara Zaccara1, Erik Dassi3, of immune checkpoint inhibitors has changed Matthew D. Galbraith2, Zdenek Andrysik2, Bar- this poor prognosis, though only in a limited tolomeo Bosco1, Annalisa Rossi1, Alessandro subset of MM patients, while most exhibit in- Quattrone3, Joaquin M. Espinosa2, Alberto Inga1 nate resistance and disease progression. There- fore, new therapies are needed. In our studies, 1Laboratory of Transcriptional Networks, Cen- the feasibility and the anti-tumor potential tre for Integrative Biology, CIBIO, University of of targeting the MM-associated oncoantigen Trento, Italy; 2 University of Colorado Anschutz chondroitin sulfate proteoglycan (CSPG)4 with Medical Campus, Aurora, CO, USA; 3Laboratory DNA vaccines delivered by electroporation (EP) of Translational Genomics, Centre for Integra- have been evaluated. tive Biology, CIBIO, University of Trento, Italy. Due to the many similarities with its human (Hu) counterpart, canine (Do) MM offers an Introduction excellent opportunity for translational clinical The transcription factor p53 can be efficiently investigations, being the CSPG4 an attractive activated by the small molecule Nutlin-3 with- immunotherapy target expressed by both hu- out inducing genotoxic stress. Treatment of dif- man and canine MM patients. We have previ- ferent cell lines with this small molecule can ously demonstrated the safety and the clinical result in different phenotypes, ranging from relevance of the intramuscular injection of cell cycle arrest to apoptosis. HCT116 (colon a xenogeneic Hu-CSPG4 plasmid followed by cancer-derived cells) and SJSA1 (osteosarco- EP in dogs with stage II-III surgically resected ma-derived cells) were used to model these CSPG4+ oral MM. Hu-CSPG4 EP caused no side behaviors respectively, by analyzing the tran- effects and resulted in a significantly longer scriptional and translational responses after disease-free (DFI) and overall survival (OS) of Nutlin-3 treatment. vaccinated dogs as compared to controls. How- ever, Hu-CSPG4 vaccine was not effective in Materials and Methods activating T cells from human healthy donors Total and polysomal-bound mRNAs were col- in vitro. To increase the translational power of lected and sequenced after 12h of 10uM Nut- our approach, we employed a hybrid plasmid lin-3 treatment. A bionformatics analysis of coding for a chimeric HuDo-CSPG4 protein, ex- polysome-enriched mRNAs using Weeder led pected to be effective in both veterinary and to identification of a “CG-rich” 3’UTR motif human settings. which is enriched in the tranlationally upreg- We tested the immunogenicity and the anti-tu- ulated mRNAs of SJSA1. The effect of this mo- mor potential of HuDo-CSPG4 intramuscular tif on transalation was evaluated after cloning DNA EP in mice, in surgically resected CSPG4+, its consensus sequence into the 3’UTR of the stage II-IV, oral MM canine patients and in an in b-globin gene, which was put downstream of vitro human setting. Chimeric HuDo-CSPG4 EP the luciferase gene. The activity of the con- is immunogenic in mice. In dogs the procedure struct was evaluated after 12 or 24h of Nut- is safe and induces antibodies (Ab) against both lin-3. The same motif was used for a pull-down Hu- and Do-CSPG4, with a higher affinity and experiment followed by mass spectrometry to anti-tumor potential as compared to Hu-CSPG4 identify interacting proteins. vaccine. From the clinical point of view, the hybrid HuDo-CSPG4 is effective in increasing Results and Discussion both the DFI and the OS of vaccinated canine Our RNA-seq data indicate that HCT116 and MM patients as compared to non-vaccinated SJSA1, although sharing much of the transcrip- controls. Interestingly, data obtained in vitro tional program driven by p53, show little to no with T cells from human healthy donors also overlap at the translational level. SJSA1 pres- suggest HuDo-CSPG4 is more immunogenic than ent different pro-apoptotic translationally-up- Hu-CSPG4 plasmid in a human setting. regulated genes after Nutlin-3, which have one In conclusion, the HuDo-CSPG4 EP is an effec- or more instances of a CG-rich motif in their tive way to break immune tolerance against 3’UTRs. The motif is sufficient to enhance ac-

34 The Pezcoller Foundation Journal - June 2018 tivity of a luciferase reporter when cloned in with non-histone proteins. Thus, the identifica- two copies flanking the 3’UTR of the b-globin tion of novel SMYD3 interactors will be crucial gene, but only in SJSA1. A pull-down experi- for the full comprehension of its role in cancer. ment using the consensus motif as an RNA bait As SMYD3 is involved in cancer growth and cell was used to identify interactors, among which cycle progression, regulating the S/G2 transi- DHX30 was studied further. DHX30 silencing in tion, and considering the impact of cell cycle HCT116 causes: 1) enhanced activity of the regulation on resistance to chemotherapeu- reporter construct after Nutlin treatment; 2) tic drugs (CHTs), we hypothesize that SMYD3 enhanced polysomal association of selected might serve as a molecular target to over- Posters mRNAs containing the motif; 3) enhanced in- come chemoresistance. We focused on the duction of apoptosis as assessed by Annexin-V possibility that BCI-121, which induce S-phase staining. In addition, silencing of DHX30 in arrest, could improve the effects of conven- U2OS cells decreased their survival after Nut- tional chemotherapy by sensitizing cancer lin-3 treatment. cells to S-phase-specific CHTs. In the light of our published data supporting the possibility Conclusions to classify tumor cell types based on SMYD3 We show how the transcriptional program dic- expression levels in order to design an effec- tated by p53 activation can be shaped at a tive SMYD3-targeted epigenetic cancer thera- translational level thanks to the action of a py, we set our experiments in CRC cells with cis-acting “CG-rich” motif, which is enriched high SMYD3 levels. Our results showed that in the 3’UTR of some pro-apoptotic mRNAs. BCI-121-mediated cell cycle arrest decreases The motif can be bound by DHX30, acting as a the time needed for CHTs to exert their growth translational repressor. inhibitory effect, confirming that SMYD3 might The exact mechanism of action and the gen- be used as a molecular target to overcome re- eralization of the model are currently being sistance to CHTs. Through a peptide screening investigated. and an in silico analysis aimed at identifying SMYD3-interacting proteins, we identified a set of new potential partners involved in DNA dam- 40. SMYD3, a novel factor in DNA age and S-phase checkpoint, including BRCA2 damage response, is a molecular and ATM. In vitro and in breast cancer cell line assays confirmed that SMYD3 interacts with a target to overcome cancer drug conserved region of BRCA2, which specifically resistance modulates Homologous Recombination repair on DNA damage. Paola Sanese1, Valentina Grossi1, Valentina Our preliminary data indicate that also ATM Celestini1, Candida Fasano2, Alessia Peserico1, could interact with SMYD3. In an effort to fur- Martina Lepore Signorile1,3, Giuseppina Caret- ther characterize the role of SMYD3 in DNA re- ti4, Alberto Del Rio5 and Cristiano Simone1,2. pair, we found that breast cancer cells exposed to DNA damaging agent showed an increase of 1.Medical Genetics, Department of Biomedical nuclear SMYD3 in cancer cells, which followed Sciences and Human Oncology (DIMO), Univer- the activation of the repair signals, and an sity of Bari Aldo Moro, Bari, 70124, Italy accumulation of unrepaired DNA lesions after 2.Medical Genetics, National Institute for Gas- SMYD3 genetic ablation. troenterology, IRCCS ‘S. de Bellis’, Castellana To exploit the clinical potential of targeting Grotte (Ba), 70013, Italy SMYD3 to sensitize cells to chemotherapy, we 3.Department of Molecular Medicine, Sapienza particularly evaluated the potential of the University of Rome, 00161 Rome, Italy combined treatment with SMYD3 inhibitor and 4.Department of Bioscience, University of Mi- chemotherapy in Triple Negative Breast Cancer lan, Milan, Via Celoria, 26, 20133 MI, Italy (TNBC), which not usually respond to common 5.Institute of Organic Synthesis and Photoreac- therapies. TNBC cell lines expressing high lev- tivity (ISOF), National Research Council (CNR), els of SMYD3 confirmed the efficacy of BCI-121 Bologna, Via P. Gobetti 101, 40129 BO, Italy and S-phase agents combined treatment. Taken together, our data give further strength Human cancers arise from a combination of ge- to the idea that SMYD3 is a key factor in tum- netic and epigenetic changes. Drugs that tar- origenesis, and therefore could be used as a get epigenetic modifiers are a new therapeutic molecular target to counteract cancer devel- challenge, due to the reversibility of epi-modi- opment. Furthermore, unveiling the BRCA2 fications. The SMYD3 histone methyltransferase and/or ATM interaction-mediated involvement has an oncogenic role in various cancer types, of SMYD3 in DNA damage response might help making it a potential target for drug discovery. finding combined pharmacological approaches Recent studies suggest that its oncogenic ac- to take advantage of SMYD3 inhibition to over- tivity might also be mediated by its interaction come chemoresistance.

The Pezcoller Foundation Journal - June 2018 35 41. A screen for drugs that affect in the expression pattern of tumor cells after treatment with the 3 hits, we are investigating brain tumor development and changes in the translatome as a result of drug microglia recruitment in zebrafish treatment.Taken together, these results reinforce the knowledge that zebrafish provides a Valeria Savoca1, Dirk Sieger2 and Marina Mione1 useful instrument to investigate the interactions between tumor and immune cells in GBM. 1Centre for Integrative Biology, University of

Posters Trento, Via Sommarive, 9, Trento 38123, Italy 2Centre for Discovery Brain Sciences, The Uni- 42. Manipulating MAPK/c-Myc versity of Edinburgh, The Chancellor’s Build- ing, 49 Little France Crescent, Edinburgh EH16 axis to overcome chemoresistance 4SB, United Kingdom in CRC

One of the main hallmarks of brain cancer is Martina Lepore Signorile1,2, Valentina Grossi1, the formation of an immune-microenvironment Paola Sanese1, Valentina Celestini1, Candida infiltrated mainly with tumor associated mac- Fasano3, Giovanna Forte3, Vittoria Disciglio3, rophages (TAMs), but also with other types of Cristiano Simone1,3 leukocytes, which contribute to establish a status of chronic inflammation. Thus, the pro- 1 Medical Genetics, Department of Biomedical duction of high amount of immune inhibitory Sciences and Human Oncology (DIMO), Univer- cytokines or inflammatory mediators lead to sity of Bari. increase tumor cells proliferation and invasion. 2 Department of Molecular Medicine, Univer- The most frequent and aggressive form of brain sity of Rome “La Sapienza” 00161 Rome, Italy. tumor, glioblastoma multiforme (GBM), is re- 3 Medical Genetics, National Institute for Gas- sistant to standard of care therapies because troenterology, IRCCS ‘S. de Bellis’, Castellana of its heterogeneity, infiltration properties and Grotte (Ba). immune suppressive microenvironment, therefore innovative therapies are being investigat- Comprehensive genomic profiling is expected ed, among which immunotherapies, whose aim to revolutionize cancer therapy. Recent ad- is to alleviate GBM-associated immune suppres- vances in DNA sequencing technologies provide sion and to boost anti-tumor immune response. unprecedented capacity to comprehensively In this project we have used a zebrafish model identify the alterations, genes and pathways of brain tumor to investigate the immune mi- involved in the tumorigenic process, raising croenvironment of healthy and tumoral brains the hope that targeted therapies against the and the effect of compounds treatment both drivers of cancer can be extended from a few on brain tumor development and immune cells, successful examples to a broader personalized mainly microglia. Leukocytes, including mac- medicine strategy. Colorectal cancer (CRC) rophages and microglia are normally present in poses a formidable challenge in the form of the brain at homeostatic conditions at larval molecular heterogeneity with involvement of and adult stages. In the larval tumor model, several cancer-related pathways and molec- which expresses the oncogene HRAS under the ular changes unique to an individual’s tumor. zic4 enhancer, an increase in the number of Recent discoveries have unveiled an impressive immune cells in the telencephalic region was list of the RAS/RAF/MEK/ERK and PI3K/AKT observed; in the adult model changes were de- pathway inhibitors, offering a new treatment tected more in the cellular morphology of im- paradigm for cancer patients. However, de- mune cells, which dramatically switched to a spite the initial promise, in 2018 only 15.4% of more spherical and less branched phenotype. 610,000 patients with metastatic cancer were The same larval brain tumor model was used to eligible for an FDA-approved, genome-guid- investigate the effects of immune-suppressive ed drug, and just 6.6% of these patients likely compounds (dexamethasone and an inhibitor benefited, also many patients relapse after a of CSF-1R) on the number of microglia cells, couple of years on the drugs, specially in CRC. which were reduced, but these treatments did The occurrence of chemoresistance is respon- not impact on brain size and tumor develop- sible for the limited success of various drugs. ment. Indeed, blocking one pathway, such as RAS, is Given that altered epigenetic landscape is very likely to induce only a cytostatic effect, while common in brain tumors, larvae were screened inhibiting a crosstalk resistance pathway should with a library of compounds targeting epige- induce chemosensitivity and a final cytotoxic netic factors and, as a result, 3 hits were found effect. These considerations highlight the rel- to have an effect in slowing down the develop- evance of molecular profiling and preclinical ment of tumors and in reducing the number of investigation in order to establish new thera- immune cells. In order to characterize changes peutic approaches based on the use of specif-

36 The Pezcoller Foundation Journal - June 2018 ic drugs targeted against the crucial drivers of state that characterize cancer cells resistant cancer-related pathways. Indeed, a paradigm to therapy. Recent studies confirm that acqui- of this view is represented by the p38 MAPK, sition of drug resistance is associated with a which is a central node between the Wnt, ERK, metabolic shift toward respiratory metabolism AMPK and PI3K cascades. In this work we test- in several cancer models. In this scenario, met- ed p38α inhibitors in combination with molec- abolic plasticity acquires a crucial role in the ularly-targeted drugs and chemotherapeutic survival of drug-resistant cells responsible for agents in CRC xenografted nude mice and the tumor relapse. In our model, treatment of co- AOM/DSS colitis-associated carcinoma preclin- lon cancer cells with 5-FU selects a resistant Posters ical model. To this aim, animals were treat- subpopulation with mesenchymal stem-like ed with p38 inhibitor alone or in combination properties that undergo a metabolic repro- with the orally administrable MEK1 inhibitor gramming resulting in addition to OXPHOS. PD0325901, the BRAF inhibitor Sorafenib or the Our results suggest that these mechanisms chemotherapeutic agent Cisplatin. The com- can be activated in response to 5-FU in cells bined use of p38α inhibitor with the indicated that are already resistant to the drug by di- compounds significantly reduced tumor growth rectly modulating miRNAs expression. miRNAs by inducing cytotoxic effect which is mediated are small non-coding RNA molecules that func- by the apoptotic mechanism, the best method tion in post-transcriptional regulation of gene to kill cancer cells. Besides, we show that con- expression and have been described as crucial comitant inhibition of the p38α and MEK/ERK regulators in cancer progression. We identified pathways significantly increases the survival in resistant cells a set of miRNAs different- of APCMin/+ mice in which tumorigenesis is driv- ly expressed in response to the acute treat- en by c-Myc deregulation. Since c-Myc is able ment with respect to untreated resistant cells. to activate cancer stem cells expression pro- Among them, we selected miR210 as a key file, which can be considered responsible for player both in DNA damage repair in response chemoresistance mechanism, it becomes clear to treatment and in metabolic reprogramming. that found a method to neutralized c-Myc can Indeed, downregulation of miR210 in response be the best way to overcome chemoresistance. to 5-FU allows resistant cells to increase DNA Moreover we found that p38α and ERK collab- damage repair and shift cell metabolism to- orate in c-Myc stabilization by inhibiting its ward OXPHOS addiction. proteasomal degradation in CRC cell lines. We These adaptations give resistant cells supply confirmed these results by using Ralimetinib of intermediates for nucleotides synthesis and (p38 inhibitor) and Trametinib (MEK inhibitor), enzyme activity for DNA damage repair. These which are currently in clinical trials for can- results demonstrate a crucial role of miR210 in cer and inflammatory disease. These data vali- acute response of resistant cells to treatment, date that combined inhibition of p38α and ERK suggesting new possible therapeutic approach- pathway or the association with chemotherapy es for drug resistance. could be a promising approach to overcome chemoresistance in CRC. 44. Discoidin Domanin Receptor 1 is a therapeutic target in 43. Role of miRNAs in metabolic radioiodine refractory thyroid plasticity correlated with 5-FU cancer resistance in colon cancer cells Veronica Vella1,2, Maria Luisa Nicolosi3, Patri- Erica Pranzini¶, Paolo Paoli¶, Matteo Ramazzot- zia Cantafio3, Roberta Malaguarnera3, Antoni- ti¶, Paola Chiarugi¶ and Maria Letizia Taddei* no Belfiore2

¶Dipartimento di Scienze Biomediche Sper- 1School of Human and Social Science, University imentali e Cliniche, Università degli Studi di “Kore” of Enna, 94100 Enna, Italy Firenze, Viale Morgagni 50, 50142 Firenze 2Department of Clinical and Sperimental Medi- cine, Endocrinology, University of Catania, Gar- *Dipartimento di Medicina Sperimentale e Cl- ibaldi-Nesima Hospital, 95122 Catania, Italy inica, Università degli Studi di Firenze, Viale 3Department of Health Sciences, Endocrinology, Morgagni 50, 50142 Firenze University of Catanzaro, 88100 Catanzaro, Italy

Highly proliferative cells, including cancer Background: Thyroid cancers unresponsive to cells, respond to their increased energetic and radioiodine (RAI) treatment are indicated as biosynthetic needs primarily through glycoly- RAI-refractory and represent a deadly disease sis. However, this metabolic strategy is not apt with less than 30% survival rate at five years. to sustain the low proliferative and quiescent Overexpression of the insulin receptor isoform

The Pezcoller Foundation Journal - June 2018 37 A (IR-A) and its ligand IGF-2 is a relevant fea- er prognosis when compared to other subtypes ture of these dedifferentiated cancers. Indeed, and lacks targeted therapies.[1] Chemotherapy the IGF-2/IR-A loop has been involved in therapy is the primary established systemic treatment resistance in several malignancies. We recently for early stage and advanced TNBC following identified, in breast cancer, a functional cross- the surgery.[2] However, developing resistance talk between the insulin/IGF axis and discoidin to chemotherapy is a major obstacle in clinics domain receptor 1 (DDR1) a collagen receptor that usually results in relapse and metastasis. with tyrosine kinase activity, which is implicat- Posters ed in cancer progression and metastasis. Both It is known that epigenetic modifications con- IGF-2/IR-A and DDR1 may also play a role in cell tribute to the development and maintenance of stemness. chemotherapy resistant phenotype in different cancers.[3] To date, any comprehensive study Aims: we aimed at investigating whether DDR1 has not been conducted to identify the impor- might affect the biology of RAI-refractory thy- tant epigenetic modifiers regulating chemother- roid cancer partially through its functional apy resistance in TNBC. Therefore, we designed crosstalk with the IGF-2/IR-A loop. Using vari- an Epigenetic Knock-out Library (EPIKOL), which ous RAI-refractory thyroid cancer cells, we then utilizes the CRISPR-Cas9 technology to study the evaluated the effects of DDR1 in cell differenti- functions of wide variety of epigenetic modifiers ation and stem-like phenotype as well as in the modulation of the IGF-2/IR-A axis. on chemotherapy-sensitive and resistant TNBC. EPIKOL is composed of 8000 gRNA (10 gRNA per Results: DDR1 silencing, by specific siRNAs, was gene) targeting the 5’ exons of genes encoding associated with a more differentiated cell phe- chromatin- and DNA-modifying proteins, such notype, characterized by increased thyroid spe- as histone methyltransferases (HMTs), histone cific differentiation markers and concomitant demethylases (HDMs), histone acetyl transferas- decrease of invasiveness ability and stemness es (HATs), histone deacetylases (HDACs), and features. These changes were accompained by DNA methyl transferases (DNMTs). gRNAs target- decreased IR expression with reduced IR-A:IR-B ing the essential genes and non-targeting gRNAs ratio and downregulation of autocrine IGF-2 were also included to serve as positive and neg- production. DDR1 overexpression elicited op- posite effects. In turn, activation of the IGF-2/ ative controls during the screens, respectively. IR-A pathway upregulated DDR1 expression. In- We utilized MDA-MB-231, SUM159 and SUM149 terestingly, DDR1 silencing/inhibition enhanced cell lines to generate chemotherapy-resistant thyroid cancer cells sensitivity to inhibitors of TNBC models. To this end, we exposed these IR/IGF-1R and BRAF in terms of reduced cell vi- cells to escalating doses of doxorubicin and tax- ability and invasion. ol, the most widely used clinical chemothera- peutics, and selected resistant subpopulations. Conclusions: These data suggest that DDR1 is a Comparison of these cell lines will reveal the valuable molecular target for inducing rediffer- differences in the epigenetic landscapes that entiation in RAI-refractory thyroid cancer. result from transitions from a chemosensitive to chemoresistant state. Further screens with our EPIKOL gRNA library 45. Identification of Epigenetic will allow us to identify novel epigenetic regula- Factors That Will Overcome tors of chemotherapy resistance in TNBC. Therapy Resistance in Triple Negative Breast Cancer 1. Foulkes, W.D., I.E. Smith, and J.S. Reis-Fil- ho, Triple-negative breast cancer. N Engl J Med, Yedier Bayram O.1, Kayabolen A.1, Senbabaoglu 2010. 363(20): p. 1938-48. F. 1, Morova T.2, Uyulur F.2, Lack N.2, Onder T.2, 2. Bianchini, G., et al., Triple-negative breast Bagci-Onder T.1,* cancer: challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol, 2016. 1Koç University, School of Medicine, Brain Can- 13(11): p. 674-690. cer Research Laboratory, Istanbul, Turkey 3. Housman, G., et al., Drug resistance in can- 2 Koç University, School of Medicine,Istanbul, cer: an overview. Cancers (Basel), 2014. 6(3): p. Turkey 1769-92. *Correspondence: [email protected]

Triple negative breast cancer (TNBC) is a subtype of breast cancer that does not express receptors for estrogen, progesterone and Her2. It has poor-

38 The Pezcoller Foundation Journal - June 2018 Pezcoller Foundation–AACR international award for extraordinary achievement in Award cancer research

2019 Program guidelines and nomination instructions

AWARD SUMMARY The prestigious Pezcoller Foundation–AACR International Award for Cancer Research was established in 1997 to annually 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; • who continues to be active in cancer research and has a record of recent, noteworthy publications; and • whose ongoing work holds promise for continued substantive contributions to progress in the field of cancer. The award is intended to honor an individual scientist. However, if two candidates are co-nominated to share the Award, curriculum vitae and complete lists of publications for the two candidates must be submitted along with a letter of recommendation that clearly outlines how the work of these individuals is the same or closely related in subject matter and therefore warrants a joint nomination. The award consists of an unrestricted honorarium of €75,000, a commemorative plaque, and full support to the winner and a guest to attend the AACR Annual Meeting. The winner of the 22nd Pezcoller Foundation–AACR International Award for Extraordinary Achievement in Cancer Research will give an award lecture at the AACR Annual Meeting 2019 in Atlanta, GA, USA (March 30-April 3, 2019).

ELIGIBILITY • Eligible candidates are cancer researchers affiliated with institutions in academia, industry, or government that are involved in cancer research, cancer medicine, or cancer-related biomedical science anywhere in the world. • Institutions or organizations are not eligible for the award. • Receipt of other major awards does not preclude a candidate from eligibility for the award. • No regard shall be given to race, gender, nationality, geographic location, or religious or political views.

The Pezcoller Foundation Journal - June 2018 39 NOMINATION PROCESS Nominations may be made by any scientist, whether an AACR member or nonmember, who is now or has been affiliated with any institution involved in cancer research, cancer medicine, or cancer-related biomedical science. Candidates may not nominate themselves. Nominators are asked to maintain the confidentiality of the nomination process and to refrain Award from informing the candidate about the nomination. There is no restriction on the number of candidates that may be nominated by any institution or individual. There is no restriction on the number of candidates that may be nominated by any individual scientist. There is no restriction on the number of nominators that may write nomination let- ters or that may sign a single nomination letter on behalf of a candidate. Nominations must be submitted online at myaacr.aacr.org no later than 4 p.m. U.S. ET on Wednesday, September 12, 2018. Paper nominations cannot be accepted. Full nomination instructions and program guidelines are available through the link below. www.aacr.org/ScientificAwards

NOMINATION MATERIALS 1) Nomination Letter. The letter must: • be addressed to the Selection Committee; be written in English; and not exceed 1,000 words; • specify the AACR Award for which the candidate is being nominated; • contain a concise description of the candidate’s major scientific discovery in basic cancer research or significant contributions to translational cancer research, and the impact of these accomplishments on the field, with publications supporting these accomplishments directly referenced within the letter; • contain a concise description of the candidate’s ongoing work which holds promise for continued substantive contributions to progress in the field of cancer; and • be signed with a handwritten signature by the nominator. If more than one candidate is nominated to share the Award, the nomination letter must clearly outline how the work of the individuals is closely related in subject matter and warrants a joint nomination. 2) Candidate’s CV. The candidate’s curriculum vitae in English, including a complete list of the candidate’s publications. 3) Summary Statement. A statement of no more than 50 words summarizing the candidate’s research accomplishments for which he or she is being nominated.

SELECTION PROCESS Candidates for the award will be considered by a prestigious international Selection Committee of renowned cancer leaders appointed by the president of the AACR in consultation with the Council of the Pezcoller Foundation. The committee will consider all nominations as they have been submitted; the committee may not combine submitted nominations, add a new candidate to a submitted nomination, or otherwise make alterations to the submitted nominations. After careful deliberations by the committee, its recommendations will be forwarded to the Executive Committee of the AACR and the

40 The Pezcoller Foundation Journal - June 2018 Council of the Pezcoller Foundation for final consideration and determination. Selection of the award winner will be made on the basis of the candidate’s scientific accomplishments. No regard will be given to race, gender, nationality, or religious or political view.

RESPONSIBILITIES OF THE AWARD RECIPIENT Award The recipient will also present the Pezcoller Foundation-AACR International Award for Extraordinary Achievement in Cancer Research Lecture, both at the University of Padua and at the University of Trento in Italy, just prior to the official Award ceremony in Trento, in early May 2019. Remarks to be made during the ceremony must be delivered to the Pezcoller Foundation at least four weeks prior to allow sufficient time for translation into Italian. Should the recipient be unable to participate in either event, the award must be forfeited and may be presented instead to the alternate. In the rare event that there are dual winners of the Award, the cash award will be shared equally between them, and the AACR Executive Committee will determine which of the two co-re- cipients will present the Pezcoller-AACR Award Lecture at the AACR Annual Meeting.

CHANGES TO THE NOMINATION Withdrawal of nomination: Please advise the AACR promptly, in writing, should you decide to withdraw your nomination for any reason. Your letter (or e-mail) should include the nominator’s name and institution, the title of the Award and name of the candidate, and the reason for withdrawal.

INQUIRIES Questions regarding eligibility of a candidate may be directed to the AACR Scientific Achie- vement Awards office at [email protected] or by calling (215) 446-7128, prior to submitting an application.

CHANGES TO THE NOMINATION Withdrawal of nomination: Please advise the AACR promptly, in writing, should you decide to withdraw your nomination for any reason. Your letter (or e-mail) should include the nominator’s name and institution, the title of the Award and name of the candidate, and the reason for withdrawal.

The Pezcoller Foundation Journal - June 2018 41 2019 Scholar-In-Training Awards Award

The AACR is proud to offer Scholar-in-Training Awards to enable the participation of meritorious early-career scientists at the Annual Meeting 2018. Since its inception in 1986, the AACR Annual Meeting Scholar-in-Training Award program has provided more than 4,400 grants to young investigators and has received support from more than 50 cancer research foundations, corporations, individuals and other organizations dedicated to the fight against cancer. This year, twenty organizations or individuals generously provided the funding to support this program.

2019 AACR-PEZCOLLER FOUNDATION SCHOLAR-IN-TRAINING AWARDS The Pezcoller Foundation supports these awards to enhance participation in the programs and activities of the AACR by early-career investigators residing in Europe and to provide these outstanding Scholar-in-Training Awardees with an opportunity to share their research findings with the international cancer research community at the AACR Annual Meeting. Selections are made by the criteria from Pezcoller (i.e. European scientists with at least one awardee representing Italy) and based on the meritorious score of the submitted abstract and application. Any questions can be directed to [email protected]

Picture: 2018 Scholar-In-Training Awardees with President Galligioni in Chicago, April 14, 2018

42 The Pezcoller Foundation Journal - June 2018

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

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“The Pezcoller Foundation Journal” year 28, n. 50, Semestrale giugno 2018 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