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Genetics and Gene Transfer in Oncology

ITT Core Research Laboratory
c/o Department of Pharmacology
Viale Pieraccini 6 - 50139 Florence
Tel. 055 4271543 Fax 055 4271280

Principal Investigator: Dr. Rosario Notaro

Staff

The laboratory of "Genetics and Gene Transfer in Oncology" has been established and on March 2007.
It is the first Unit of the Core Research Laboratory of ITT

Main Research Themes

  1. Paroxysmal Nocturnal hemoglobinuria (PNH)
    Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of the hematopoietic stem cell (HSC). PNH is a rare disease, with an estimated population frequency of 1 to 5 cases per million. It is characterized by 3 clinical hallmarks: (1) intravascular hemolysis causing hemoglobinuria, (2) tendency to venous thrombosis, and (3) variable degrees of bone marrow failure. The primary molecular lesion responsible for PNH is a somatic mutation of the X-linked PIG-A gene in HSC that results in complete or partial deficiency of all proteins anchored by the glycosylphosphatidylinositol (GPI) on the membrane of the mutated HSC and of its mature progeny (Figure 1). The deficiency of GPI-anchored molecules on blood cells accounts for most features of PNH, but not for the expansion of PNH (GPI-) clone(s). In fact, very rare GPI– blood cells are present in healthy subjects, but only in PNH patients do the GPI– cells expand and contribute to hematopoiesis to various degrees, side by side with normal (GPI+) hematopoiesis. Clinical observations, in vitro hematopoietic colony studies, and data from PNH mouse models indicate that GPI– HSCs do not have an absolute growth advantage. The close relationship of PNH to idiopathic aplastic anemia (IAA) has suggested that auto-reactive T cells against HSCs believed to be responsible for IAA may also be at work in PNH. Therefore, a plausible model postulates that PNH arises through negative selection against normal HSCs exerted by auto reactive T cells, whereas PNH HSCs escape damage.
    In recent years considerable evidence has accumulated in favor of this autoimmune mechanism for the expansion of the GPI– (PNH) clone: (a) the increased frequency of expanded T-cell clones in both PNH and IAA (Karadimitris et al, Blood 2000); (c) the relatively common association of PNH with the expansion of large granular lymphocytes (LGL) (Karadimitris et al, Br J Haematol 2001); (d) T cells expressing inhibitory receptor superfamily (IRS) molecules in PNH patients, in contrast with healthy controls, belong mainly to activating IRS isoforms (Poggi et al, Blood 2006). We have recently demonstrated how CD8+ CD57+ T cells are oligoclonal in PNH patients and that in the majority of patients this T-cell population bears a set of highly homologous TCR-beta molecules (Gargiulo et al, Blood 2007). Altogether these data strongly support the hypothesis that CD8+CD57+ T cells play a role in PNH pathogenesis and that the expansion of the GPI- blood-cell population in PNH is due to selective damage to normal hematopoiesis, mediated by an autoimmune attack against a single or a restricted range of antigens. The next steps will include formal proof of the responsibility of this T-cell population for the expansion of PNH clone and the identification of their target.

    Figure 1. The molecular basis of PNH. Complex biosynthetic machinery produces the glycosylphosphatidylinositol (GPI) molecule call (see inset) in the endoplasmic reticulum (ER) of a normal cell (left). An early step in this pathway is catalyzed by an acetylglucosaminyl transferase: one of the subunits of this enzyme is encoded by the gene PIG-A, located on the short arm of the X chromosome. A number of cellular proteins become covalently linked to the GPI molecule that serves for conveying and anchoring them on the surface of cell membrane. The PNH cell (right) has a mutation in the PIG-A gene that impairs acetylglucosaminyl transferase activity and causes a total (or partial) block in the synthesis of the GPI molecule. As a result, the proteins requiring a GPI anchor are unable to bind to the membrane and will be lacking from the cell surface. Modified from Karadimitris & Luzzatto. Leukemia, 15:1148-52. 2001.
  2. Pharmacogenetics and chemiotherapy
    The toxicity and efficacy of drugs are extremely variable in the population. The identification of markers associated with such variability could enable an effective tailoring of treatments. A number of factors affect the inter-individual variability of response to drugs. It is likely that most of this variability is associated with inherited variation in the molecules involved in the metabolism and/or in the action of drugs. Topoisomerase-I inhibitor irinotecan is currently used in the therapy of metastatic colorectal cancer but it may have unpredictable severe toxicity: mainly diarrhea and myelosuppression. Its active metabolite, SN38, is inactivated through glucuronidation by uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1). The (TA)7 allele of a common polymorphism in UGT1A1 promoter has been associated with a reduced SN38-glucuronidation rate and with more severe toxicity. However, various studies have reported quite a variable degree of association between UGT1A1 polymorphism and irinotecan toxicity. Since other UGT1A family isoforms, the extra hepatic UGT1A7 and hepatic UGT1A9, are involved in SN38 glucuronidation, we are investigating whether the polymorphic variants of these genes may contribute towards irinotecan toxicity. We have found that in patients with advanced colorectal cancer treated with irinotecan both the low activity alleles of UGT1A1 [(TA)7] and UGT1A7 [622C] are associated with severe toxicity. In addition, we are exploring the possible role of polymorphism of cytochrome P450 family genes (CYP2D6 and CYP19) in the outcome of endocrine therapy of breast cancer.
  3. Gene transfer for the therapy of neoplastic disorders by targeting oncogenetic fusion genes.
    Retroviral vectors (onco-retroviral and lentiviral vectors) are to date the most effective tools for the permanent transfer of genes in eukaryotic cells. Our group has worked on the retrovirally-mediated transfer of genes into hematopoietic stem cells. By using the glucose 6-phosphate dehydrogenase (G6PD) gene as a reporter we have achieved effective retroviral-mediated transfer and expression of G6PD both in vitro (De Angioletti et al, Gene 2000) and in vivo models (Rovira et al, Blood 2000; Shi et al, J Gene Med 2004). In addition we have investigated the safety aspects of retroviral gene transfer (De Angioletti et al, J Virol 2001).
    RNA interference (RNAi) is a highly conserved mechanism of post-transcriptional gene silencing triggered by small interfering RNA (siRNA) that mediate sequence-specific degradation of mRNA. RNAi has been exploited to target specific gene expression in several organisms, including mammals. The specificity of gene targeting by this mechanism makes RNAi potentially useful for the treatment of diseases that result from increased expression of any particular gene. Oncogenic fusion genes are frequent in leukemias and they have recently been identified in solid tumors as well. The transcripts of these fusion genes are, in principle, ideal targets for the therapeutic use of RNAi. Our goal is to test the feasibility of the viral-mediated transfer of specific siRNA in treating neoplasias characterized by the expression of fusion genes (i.e. acute promyelocytic leukemia that is associated with the fusion gene PML-RARα).

Publications

  1. L.Gargiulo, S.Lastraioli, G.Cerruti, M.Serra, F.Loiacono, S.Zupo, L.Luzzatto, R.Notaro. Highly Homologous T-Cell Receptor Beta Sequences Support A Common Target For Auto-Reactive T Cells In Most Patients With Paroxysmal Nocturnal Hemoglobinuria. Blood 109:5036-42. 2007.
  2. G.Balbi, F.Ferrera, M,Rizzi, P.Piccioli, A.Morabito, L.Cardamone, M.Ghio, G.Palmisano, P.Carrara, S.Pedemonte, M.Sessarego, M.De Angioletti, R.Notaro, F.Indiveri, M.P.Pistillo. Association Of -318 C/T And +49 A/G Ctla-4 Gene Polymorphisms With A Clinical Subset Of Italian Patients With Systemic Sclerosis. Clinical And Experimental Immunology 149:40-7. 2007.
  3. Ortolan E, Tibaldi Ev, Ferranti B, Lavagno L, Garbarino G, Notaro R, Luzzatto L, Malavasi F, Funaro A. Cd157 Plays A Pivotal Role In Neutrophil Transendothelial Migration. Blood. 108:4214-22. 2006.
  4. P.Piccioli, M.Serra, V.Gismondi, S.Pedemonte, F.Loiacono, S.Lastraioli, L.Bertario, M.De Angioletti, L.Varesco, R.Notaro. Multiplex Tetra-Primer Amplification Refractory Mutation System Pcr To Detect 6 Common Germline Mutations Of The Mutyh Gene Associated With Polyposis And Colorectal Cancer. Clinical Chemistry. 52:739-43. 2006.
  5. A.Poggi, S.Negrini, M.R.Zocchi, A.M.Massaro, L.Garbarino, S.Lastraioli, L.Gargiulo, L.Luzzatto, R.Notaro. Patients With Paroxysmal Nocturnal Hemoglobinuria Have A High Frequency Of Peripheral-Blood T Cells Expressing Activating Isoforms Of Inhibiting Superfamily Receptors. Blood. 106:2399-408. 2005.
  6. D.J.Araten, D.W.Golde, R.H.Zhang, H.T.Thaler, L.Gargiulo, R.Notaro, L.Luzzatto. A Quantitative Measurement Of The Human Somatic Mutation Rate. Cancer Research. 65:8111-7. 2005.
  7. A.Funaro, E.Ortolan, B.Ferranti, L.Gargiulo, R.Notaro, L.Luzzatto, F.Malavasi. Cd157 Is An Important Mediator Of Neutrophil Adhesion And Migration. Blood. 104:4269-78. 2004.
  8. F.Paglialunga, A.Fico, I.Iaccarino, R.Notaro, L.Luzzatto, G.Martini, S.Filosa. G6pd Is Indispensable For Erythropoiesis After The Embryonic-Adult Hemoglobin Switch. Blood. 104:3148-52. 2004.
  9. P.Shi, M.De Angioletti, R.Donahue, R.Notaro, L.Luzzatto, C.Dunbar. In Vivo Gene Marking Of Rhesus Macaque Long Term Repopulating Hematopoietic Cells Using A Vsv/G Pseudotyped Versus Amphotropic Oncoretroviral Vector. Journal Of Gene Medicine. 6:367-373. 2004.
  10. R.Notaro. Control Of T Lymphocytes: An Alternative Use Of Rituximab. Haematologica. 89:3-4. 2004.
  11. G.Bianchi, A.Banfi, M.Mastrogiacomo, R.Notaro, L.Luzzatto, R.Cancedda, R.Quarto. Ex Vivo Enrichment Of Mesenchymal Cell Progenitors By Fibroblast Growth Factor 2. Experimental Cell Research. 287:98-105. 2003.
  12. A.Karadimitris, D.J.Araten, L.Luzzatto, R.Notaro. Severe Telomere Shortening In Patients With Paroxysmal Nocturnal Hemoglobinuria Affects Both Gpi- And Gpi+ Hematopoiesis. Blood. 102:514-6. 2003.
  13. A.Banfi, G.Bianchi, R.Notaro, L.Luzzatto, R.Cancedda, R.Quarto. Replicative Aging And Gene Expression In Long Term Cultures Of Human Bone Marrow Stromal Cells. Tissue Engineering, 8:901-910. 2002.
  14. D.J., Araten, M.Bessler, S.Mckenzie, H.Castro-Malaspina, B.H.Childs, F.Boulad, A.Karadimitris, R.Notaro, L.Luzzatto. Dynamics Of Hematopoiesis In Paroxysmal Nocturnal Hemoglobinuria (Pnh): No Evidence For Intrinsic Growth Advantage Of Pnh Clones. Leukemia 16:2243-2248. 2002.
  15. L.Longo, O.C.Vanegas, M.Patel, V.Rosti, H.Li, J.Waka, T.Merghoub, P.P.Pandolfi, R.Notaro, K.Manova, L.Luzzatto. Maternally Transmitted Severe Glucose 6-Phosphate Dehydrogenase Deficiency Is An Embryonic Lethal. Embo Journal 21:4229-4239. 2002.
  16. M.Bessler, V.Rosti, Y.Peng, G.Cattoretti, R.Notaro, S.Ohsako, K.B.Elkon, L.Luzzatto. Glycosylphosphatidylinositol-Linked Proteins Are Required For Maintenance Of A Normal Peripheral Lymphoid Compartment But Not For Lymphocyte Development. European Journal Of Immunology 32:2607-2616. 2002.
  17. A.De Renzo, E.Persico, F.De Marino, G.Di Giacomo Russo, R.Notaro, C.Di Grazia, M.Picardi, L.Santoro, R.Torella, B.Rotoli, M.Persico. High Prevalence Of Hepatitis G Virus Infection In Hodgkin's Disease And B-Cell Lymphoproliferative Disorders: Absence Of Correlation With Hepatitis C Virus Infection. Haematologica 87: 714-718, 2002.
  18. R.Notaro, A.De Renzo, G.De Rosa, A.Karadimitris, B.Rotoli. Multiple Myeloma Cured By Conventional Chemotherapy: A Report And A Review. Leukemia And Lymphoma, 43:907-10. 2002.
  19. L.Luzzatto, R.Notaro. Haemoglobin's Chaperone. Nature 417:703-705. 2002.
  20. M.De Angioletti, A.Rovira, M.Sadelain, L.Luzzatto, R.Notaro. Frequency Of Missense Mutations In The Coding Region Of A Eukaryotic Gene Transferred By Retroviral Vectors. Journal Of Virology, 76:1991-1994. 2002.
  21. A.Karadimitris, K.Li, R.Notaro, D.J.Araten, K.Nafa, R.Thertulien, M.Ladanyi, A.E.Stevens, C.S.Rosenfeld, I.A.G.Roberts, L.Luzzatto. Association Of Clonal T-Cell Large Granular Lymphocyte Disease And Paroxysmal Nocturnal Haemoglobinuria (Pnh): Further Evidence For A Pathogenetic Link Between T Cells, Aplastic Anaemia, And Pnh. British Journal Of Haematology, 115:1010-1014. 2001.
  22. D.J.Araten, D.Swirsky, A.Karadimitris, R.Notaro, K.Nafa, M.Bessler, H.T.Thaler, H.Castro-Malaspina, B.H.Childs, F.Boulad, M.Weiss, N.Anagnostopoulos, A.Kutlar, D.G.Savage, R.T.Maziarz, S.Jhanwar L.Luzzatto. Cytogenetic And Morphologic Abnormalities In Paroxysmal Nocturnal Haemoglobinuria. British Journal Of Haematology, 115:360-368. 2001.
  23. L.Luzzatto, R.Notaro. Malaria. Protecting Against Bad Air. Science, 293:442-443. 2001.
  24. M.De Angioletti, A.Rovira, R.Notaro, O.Camacho Vanegas, M.Sadelain, L.Luzzatto. Glucose 6-Phosphate Dehydrogenase Expression Is Less Prone To Variegation When Driven By Its Own Promoter. Gene, 267:221-231. 2001.

Collaborations

  • National Cancer Research Institute of Genoa;
  • "Federico II" University of Naples;
  • University of Turin

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