Projects 2017 /18
List of Projects 2017/18
Title: Genomic profiling of myeloid malignancies and development of precision medicine strategies
Supervisor(s): Mario Cazzola, Elisa Rumi
phd student: Ilaria Carola Casetti
Rare cancers are identified as those with an incidence of less than 6 per 100,000 persons per year: most myeloid malignancies belong to this category, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and myelodysplastic/ myeloproliferative neoplasms (MDS/MPN). All tumors are caused by somatic mutation, that is, the occurrence of abnormalities in DNA sequence of genes, and the identification of recurrent genetic events represents one of the most direct approaches to understand cancer biology. Precision medicine is a medical model that combines already established clinical-pathological parameters with advanced genomic profiling in order to create innovative diagnostic, prognostic and therapeutic strategies. In hematology, the paradigmatic example is chronic myeloid leukemia, in which the discovery of the BCR-ABL1 fusion gene and its oncogenic properties has translated into targeted therapy. We are currently combining genomic profiling with clinical studies to develop innovative precision medicine strategies for patients with MDS, MPN or MDS/MPN. State-of-art genomic profiling include mutational characterization of both germline and acquired gene mutations. We preferentially study patients enrolled into observational or interventional clinical trials. We analyze the interactions among driver mutations, gene expression, clinical variables and patient outcome to model natural history of disease and impact of treatment. We aim to identify genomic changes that can be used for classification, risk assignment, enrollment into clinical trials, and prediction of response. Phase I/II as well as innovative clinical trials, aimed to test safety and efficacy of new or repurposed drugs in molecularly characterized patients, are conducted in collaboration with pharmaceutical companies.
1. Rumi E, Pietra D, Ferretti V, Klampfl T, Harutyunyan AS, Milosevic JD, Them NC, Berg T, Elena C, Casetti IC, Milanesi C, Sant’antonio E, Bellini M, Fugazza E, Renna MC, Boveri E, Astori C, Pascutto C, Kralovics R, Cazzola M; Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative Investigators. JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes. Blood 2014;123:1544-1551.
2. Elena C, Gallì A, Such E, Meggendorfer M, Germing U, Rizzo E, Cervera J, Molteni E, Fasan A, Schuler E, Ambaglio I, Lopez-Pavia M, Zibellini S, Kuendgen A, Travaglino E, Sancho-Tello R, Catricalà S, Vicente AI, Haferlach T, Haferlach C, Sanz GF, Malcovati L, Cazzola M. Integrating clinical features and genetic lesions in the risk assessment of patients with chronic myelomonocytic leukemia. Blood 2016;128:1408-1417.
3. Della Porta MG, Gallì A, Bacigalupo A, Zibellini S, Bernardi M, Rizzo E, Allione B, van Lint MT, Pioltelli P, Marenco P, Bosi A, Voso MT, Sica S, Cuzzola M, Angelucci E, Rossi M, Ubezio M, Malovini A, Limongelli I, Ferretti VV, Spinelli O, Tresoldi C, Pozzi S, Luchetti S, Pezzetti L, Catricalà S, Milanesi C, Riva A, Bruno B, Ciceri F, Bonifazi F, Bellazzi R, Papaemmanuil E, Santoro A, Alessandrino EP, Rambaldi A, Cazzola M. Clinical effects of driver somatic mutations on the outcomes of patients with myelodysplastic syndromes treated with allogeneic hematopoietic stem-cell transplantation. J Clin Oncol 2016;34:3627-3637.
Title: Pompe disease: genetic analysis of “extreme phenotypes”
Supervisor(s): Cesare Danesino, Paola De Filippi
phd student: Ilaria Palmieri
Pompe disease (PD) (OMIM 232300) is a rare autosomal recessive disorder; GAA is the only disease causing gene. Late-onset phenotypes show a wide clinical variability for age of onset, rate of disease progression, pattern of muscle involvement. This extensive clinical variability, present both within and among families, suggest that some family members of patients with PD are carriers of two pathogenic mutations, show a very mild clinical phenotype and are not yet diagnosed as affected with PD. The efficacy of Enzyme Replacement Therapy (ERT) is well documented, but several reports show that some patients behave as “poor responders”. It is thus possible to identify “extreme phenotypes” (very mild or very severe) for most of the clinical features of the disease. The clinical variability of PD as well as the unpredictable response to ERT, clearly indicate that factors, other than GAA deficiency, may influence the mechanisms of muscle damage/repair, the severity of other clinical features and the response to ERT. The project is aimed to identify genetic factors, additional to GAA mutations, relevant for modulating the clinical presentation of the disease. We expect, within 1 year, thanks to ongoing nationwide collaborations, to collect 30 patients representative for “extreme phenotype” related to age of onset, muscle damage, respiratory impairment, and response to therapy. These patients will be studied by exome sequencing and genetic variants identified will be selected based on their frequency, possible pathogenic significance, belonging to pathways related to previously listed clinical features and validated. The project has been already funded
1. De Filippi P, Saeidi K, Ravaglia S, Dardis A, Angelini C, Mongini T, Morandi L, Moggio M, Di Muzio A, Filosto M, Bembi B, Giannini F, Marrosu G, Rigoldi M, Tonin P, Servidei S, Siciliano G, Carlucci A, Scotti C, Comelli M, Toscano A, Danesino C. Genotype-phenotype correlation in Pompe disease, a step forward. Orphanet J Rare Dis 2014;9:102.
2. Parenti G), Fecarotta S, la Marca G, Rossi B, Ascione S, Donati MA, Morandi LO, Ravaglia S, Pichiecchio A, Ombrone D, Sacchini M, Pasanisi MB, De Filippi P, Danesino C, Della Casa R, Romano A, Mollica C, Rosa M, Agovino T, Nusco E, Porto C, Andria G. A chaperone enhances blood α-glucosidase activity in Pompe disease patients treated with enzyme replacement therapy. Mol Ther 2014;22:2004-2012.
3. Musumeci O, la Marca G, Spada M, Mondello S, Danesino C, Comi GP, Pegoraro E, Antonini G, Marrosu G, Liguori R, Morandi L, Moggio M, Massa R, Ravaglia S, Di Muzio A, Filosto M, Tonin P, Di Iorio G, Servidei S, Siciliano G, Angelini C, Mongini T, Toscano A; Italian GSD II group. LOPED study: looking for an early diagnosis in a late-onset Pompe disease high-risk population. J Neurol Neurosurg Psychiatry 2016;87:5-11.
Title: Identifying pathogenic, prognostic and theragnostic factors in cancer-associated gastrointestinal inflammation
Supervisor(s): Antonio Di Sabatino
phd student: Paolo Giuffrida
A higher risk for developing small bowel carcinomas (SBC) has been reported in celiac disease (CeD) and Crohn’s disease (CrD). However, most information concerning CeD or CrD-associated-SBC (CeD-SBC) is limited to few, retrospective series or case reports. It has been suggested that CeD-SBC show a survival advantage compared with sporadic SBC. Apart from SBC, CeD may predispose to enteropathy associated T-cell lymphoma (EATL), a rare but aggressive complication. Poor adherence to a gluten-free diet, HLA-DQ2 homozygosity, and late diagnosis of CD are recognized as risk factors for malignant evolution of CD. This tumor derives from the neoplastic transformation of aberrant intraepithelial T lymphocytes emerging in CeD patients unresponsive to a gluten-free diet. In a retrospective multicenter study we will assess more-in depth the features of EATL in CeD and CeD- or CrD-associated SBC. Aim of the project is to describe the spectrum of lymphoproliferations (of B-cell and T/NK cells) arising in CD by a histopathologic review and to assess the potential pathogenetic and prognostic role of the underlying gastrointestinal disorder. Molecular analysis is evaluated by microdissection technique the clonality of lesions as well as the clonal relationship between the inflammatory infiltrate, at different times of the disease evolution to overt lymphoma and, for B-cell lymphomas, the mutational status of immunoglobulin heavy chain genes. In the second part of the project we aim to investigate the immunophenotypic and molecular features of SBC associated with Crohn’s disease and celiac disease and explore possible clinical or molecular biomarkers of cancer evolution.
1. Vanoli A, Di Sabatino A, Biancone L, Martino M, et al. Small bowel Epstein-Barr virus-positive lympho-epithelioma-like carcinoma in Crohn’s disease. Histopathology 2017;70:837-839.
2. Vanoli A, Di Sabatino A, Furlan D, et al. Small Bowel Carcinomas in Coeliac or Crohn’s Disease: Clinico-pathological, Molecular and Prognostic Features. A Study from the Small Bowel Cancer Italian Consortium. J Crohns Colitis 2017 Feb 24. [Epub ahead of print] 3. Vanoli A, Di Sabatino A, Martino M, et al. Small bowel carcinomas in celiac or Crohn’s disease: distintictive histophenotypic, molecular and histogenetic patterns. Mod Pathol, in press
Title: H2O2 membrane crossing by aquaporins may control oxidative eustress and distress
Supervisor(s): Umberto Laforenza
phd student: Giorgia Pellavio
In all cells, H2O2, the most abundant and stable reactive oxygen species (ROS), can have a different effect depending on concentration. Recently, the diffusion of H2O2 from producing cells across plasma membranes to the extracellular fluid has been considered an important ROS scavenging possibility, mediated by some water channel protein – aquaporins (AQPs), isoforms. The proposed project has a dual purpose: – to further understand the mechanisms of AQPs as peroxiporins in mediating H2O2 diffusion through cellular and subcellular compartments. The study will investigate the contribution of different AQPs expressed in HeLa cells by selective silencing of single isoforms and functional experiments of water and H2O2 permeability. Moreover, the gating of AQPs in presence/absence of oxidative stress will be analyzed by testing known antioxidant and novel plant extract compounds. – to investigate whether AQPs expression and functioning is affected by HPV infection in human sperm cells, in which four AQPs were recently identified, involved in volume regulation and ROS elimination. A relationship between sperm number/motility and AQPs functioning has been also demonstrated. HPV infection is closely related to decrease in sperm motility and morphology. Therefore, ejaculated sperm cells from normospermic and sub-fertile subjects screened for HPV infection will be investigated for AQPs expression and functioning. In particular, water and H2O2 permeability as well as the ROS basal quantification in sperm cells from health and HPV infected subjects, both normal and sub-fertile will be also investigated. The results could indicate a correlation between AQPs, HPV infection and male fertility.
1. Medraño-Fernandez I, Bestetti S, Bertolotti M, Bienert GP, Bottino C, Laforenza U, Rubartelli A, Sitia R. Stress Regulates Aquaporin-8 Permeability to Impact Cell Growth and Survival. Antioxid Redox Signal 2016;24:1031-1044.
2. Laforenza U, Pellavio G, Marchetti AL, Omes C, Todaro F, Gastaldi G. Aquaporin-Mediated Water and Hydrogen Peroxide Transport Is Involved in Normal Human Spermatozoa Functioning. Int J Mol Sci 2016;18:E66.
3. Bienert GP, Chaumont F. Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide. Biochim Biophys Acta 2014;1840:1596-1604.
Title: Asparaginase-based Antibody-Drug conjugates
Supervisor(s): Claudia Scotti
phd student: Greta Pessino
E. coli Asparaginase (ASNase) is an enzyme able to catalyse cleaveage of asparagine and glutamine. It has been a key element of the multi-drug approach to treat pediatric Acute Lymphoblastic Leukemia (ALL) for more that 40 years, thanks to the sensitivity of these cells to the deprivation of these two amino acids. Thanks to this therapy, more than 90% cases of ALL are successfully cured. However, several limitations remain, especially for the high-risk forms of LLA and for the side effects ASNase generates. The latter mainly depend on its sistemic effects and on the production of anti-ASNase antibodies by the host. With our research, we are dissecting this enzyme in order to clarify both its mechanism of action and the basis of its problematic features, and to generate improved versions of the molecule. Recent evidence both from our group and others suggest that targeting of ASNase onto cancer cells could be a solution to the systemic effects of the enzyme. In this project, we propose to build on our previous knowledge of the enzyme and on our experience in antibody engineering in order to generate targeted forms of ASNase able to specifically reach cancer cells. scFv-ASNase fusions will be generated selecting specific cell-surface receptors, like CD19 and CD20, present on cancer cells, as targeting antigens. The students will be guided to determine enzymatic and structural features of each fusion and its cytotoxicity on selected leukaemia cell lines.
1. Parmentier JH, Maggi M, Tarasco E, Scotti C, Avramis VI, Mittelman SD. Glutaminase activity determines cytotoxicity of L-asparaginases on most leukemia cell lines. Leuk Res 2015;39:757-762.
2. Maggi M, Chiarelli LR, Valentini G, Scotti C. Engineering of Helicobacter pylori L-asparaginase: Characterization of two functionally distinct groups of mutants. PloS one 2015;10:e0117025.
3. Covini D, Tardito S, Bussolati O, Chiarelli LR, Pasquetto MV, Digilio R, Valentini G, Scotti C. Expanding targets for a metabolic therapy of cancer: L-asparaginase, Recent patents on anti-cancer drug discovery. 2012;7:4-13.
Title: Identification of novel genes and novel pathogenetic mechanisms in congenital ataxias
Supervisor(s): Enza Maria Valente
phd student: Valentina Serpieri
Congenital ataxias (CAs) are heterogeneous disorders of brain development, due to malformations of the cerebellum and often of the brainstem and other brain areas. CAs are characterized by neonatal or paediatric onset of a spectrum of neurological features including hypotonia, cerebellar signs (ataxia, dysarthria), oculomotor anomalies, psychomotor delay and intellectual impairment. While for some CAs the diagnosis is suggested by the presence of peculiar clinical or neuroimaging signs, in other forms the presentation remains unspecific and does not permit to discriminate among distinct disorders with similar onset ages. Recent progresses in genetic technologies have led to an impressive acceleration in the discovery of causative genes yet many patients still remain undiagnosed. Aim of this PhD project is to apply Next Generation Sequencing (NGS) techniques to identify novel CA genes and improve gene-phenotype correlates in over 200 uncharacterized CA patients. This will be pursued through the following tasks: i) optimization of NGS-based panels for simultaneous sequencing of known CA genes; ii) whole-exome sequencing in selected patients negative to panel analysis and functional studies in cellular models (mainly patients’ fibroblasts) to confirm the pathogenicity of identified variants; iii) deep integration of clinical, imaging and genetic data. The PhD student will become confident with a range of NGS strategies, tools for bioinformatic data analysis as well as a range of cell biology and biochemistry techniques. The project is supported by a grant from the Italian Ministry of Health and will be carried on in collaboration with several research groups in Italy and abroad.
1. Roosing S, Romani M, Isrie M, Rosti RO, Micalizzi A, Musaev D, Mazza T, Al-gazali L, Altunoglu U, Boltshauser E, D’Arrigo S, De Keersmaecker B, Kayserili H, Brandenberger S, Kraoua I, Mark PR, McKanna T, Van Keirsbilck J, Moerman P, Poretti A, Puri R, Van Esch H, Gleeson JG, Valente EM. Mutations in CEP120 cause Joubert syndrome as well as complex ciliopathy phenotypes. J Med Genet 2016;53:608-15.
2. Micalizzi A, Moroni I, Ginevrino M, Biagini T, Mazza T, Romani M, Valente EM. Very mild features of dysequilibrium syndrome associated with a novel VLDLR missense mutation. Neurogenetics. 2016 Jul;17(3):191-5.
3. Lee JE, Silhavy JL, Zaki MS, Schroth J, Bielas SL, Marsh SE, Olvera J, Brancati F, Iannicelli M, Ikegami K, Schlossman AM, Merriman B, Attié-Bitach T, Logan CV, Glass IA, Cluckey A, Louie CM, Lee JH, Raynes HR, Rapin I, Castroviejo IP, Setou M, Barbot C, Boltshauser E, Nelson SF, Hildebrandt F, Johnson CA, Doherty DA, Valente EM, Gleeson JG. CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium. Nat Genet 2012;44:193-199.
Project 7 (not funded)
Title: Nanotechnology applications for innovative therapeutic approaches
Supervisor(s): Livia Visai
phd student: Martina Oriano
This study aims to investigate by using biochemical/molecular and biological methods, the applications of nanotechnology for innovative therapeutic approaches as follows: a. gold nanoparticles conjugated with drugs for treatment of breast cancer; b. nanoparticles/nano-antibodies for treatment of bacterial infections; c. nanoparticles as a countermeasure for treatment of osteoporosis.
1. Risi G, Bloise N, Merli D, Icaro-Cornaglia A, Profumo A, Fagnoni M, Quartarone E, Imbriani M, Visai L. In vitro study of multiwall carbon nanotubes (MWCNTs) with adsorbed mitoxantrone (MTO) as a drug delivery system to treat breast cancer. RSC Adv 2014;4:18683.
2. Frasnelli M, Cristofaro F, Sglavo VM, Dirè S, Callone E, Ceccato R, Bruni G, Cornaglia AI, Visai L. Synthesis and characterization of strontium-substituted hydroxyapatite nanoparticles for bone regeneration. Mater Sci Eng C Mater Biol Appl 2017;71:653-662.
3. Liang X, Garcia BL, Visai L, Prabhakaran S, Meenan NA, Potts JR, Humphries MJ, Höök M. Allosteric Regulation of Fibronectin/α5β1 Interaction by Fibronectin-Binding MSCRAMMs. PLoS One 2016;11:e0159118.
Project 8 (supported by dedicated, external funds)
Title: Determinants of ventricular fibrillation during myocardial infarction
Supervisor: Gaetano De Ferrari
phd student: Maria Luce Caputo
Sudden cardiac death is one of the leading causes of death and is caused in most cases by the occurrence of a malignant ventricular arrhythmia. This can occur due to the presence of a predisposing condition such as an arrhythmogenic disorder or an altered myocardial substrate, such as a previous myocardial infarction or a cardiomyopathy. Very often, the malignant arrhythmia is triggered by acute myocardial ischemia in the setting of an acute myocardial infarction. As a matter of fact, sudden cardiac death is often the first manifestation of coronary artery disease. Goal of the present research project will be to search the epidemiologic, clinical and genetic determinants of sudden cardiac death and aborted sudden cardiac death, looking specifically at patients with acute myocardial infarction complicated by an episode of ventricular fibrillation and comparing them with a cohort of patients without ventricular fibrillation.
1. Rocchetti M, Sala L, Dreizehnter L, Crotti L, Sinnecker D, Mura M, Pane LS, Altomare C, Torre E, Mostacciuolo G, Severi S, Porta A, De Ferrari GM, George AL Jr, Schwartz PJ, Gnecchi M, Moretti A, Zaza A. Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes. Cardiovasc Res 2017;113:531-541.
2. Crotti L, Hu D, Barajas-Martinez H, De Ferrari GM, Oliva A, Insolia R, Pollevick GD, Dagradi F, Guerchicoff A, Greco F, Schwartz PJ, Viskin S, Antzelevitch C. Torsades de pointes following acute myocardial infarction: evidence for a deadly link with a common genetic variant. Heart Rhythm 2012;9:1104-1112.
3. Shivkumar K, Ajijola OA, Anand I, Armour JA, Chen PS, Esler M, De Ferrari GM, Fishbein MC, Goldberger JJ, Harper RM, Joyner MJ, Khalsa SS, Kumar R, Lane R, Mahajan A, Po S, Schwartz PJ, Somers VK, Valderrabano M, Vaseghi M, Zipes DP. Clinical neurocardiology defining the value of neuroscience-based cardiovascular therapeutics. J Physiol 2016;594:3911-3954.