Genetics and Gene Transfer in Oncology

ITT Core Research Laboratory (CRL)
c/o Department of Pharmacology

Azienda Ospedaliera Universitaria Careggi (AOU Careggi)
Viale Pieraccini 6, 50139 Florence

Tel. 055 4271543

Fax 055 4271280

Principal Investigator: Rosario Notaro, MD

Team Members

• Maria De Angioletti, PhD
  
• Benedetta Peruzzi, PhD
  
• Chiara Pescucci, PhD
  
• Michela Sica, PhD
  
• Nunzia Passaro, BS
  
• Tommaso Rondelli, BS
  

Introduction
Main Research Themes
References
Main Collaborations
Publications

Introduction

The Laboratory of Genetics and Gene Transfer in Oncology was established in 2007 and it became fully functional in 2008. The laboratory investigates various genetic aspects of clonal diseases with a specific focus on the investigation of individual genetic variability and on the role of somatic mutations.

The main research topics of the laboratory include:

1. Pharmacogenetics and chemotherapy.
2. Study of factors favoring the development and the selective growth of clonal populations of somatic cells.
3. The role of chromosomal rearrangement involving ETS proteins in development and progression of prostate cancer.

Main Research Themes

1. Pharmacogenetics and chemiotherapy
   The response to treatment of individual patients varies considerably both in terms of efficacy and toxicity. A number of factors affect the inter-individual variability of response to treatment. However, it is likely that much of this variability is associated with genetically determined individual differences in how drugs are metabolized. We are exploring the possible role of polymorphisms of a variety of genes (uridine diphosphate glucuronosyl transferase, cytochrome P450, CTLA-4, etc.) in affecting the response to treatments.
   1. Irinotecan toxicity and uridine diphosphate glucuronosyltransferase 1A gene family. Irinotecan, a topoisomerase-I inhibitor used in cancer therapy, may have unpredictable and severe gastrointestinal and hematological toxicity. The active metabolite of irinotecan, SN38, is inactivated through glucuronidation by Uridine diphosphate Glucuronosyltransferase (UGT) 1A1. The association of the UGT1A1 -53(TA)₇ allele with an increased risk of irinotecan toxicity is still controversial. Since other UGT1A family isoforms, the extra hepatic UGT1A7 and hepatic UGT1A9, are involved in SN38 glucuronidation, it is possible that polymorphisms in other members of the UGT1A gene family could help in predicting the risk of irinotecan toxicity. Thus, we are investigating whether the polymorphic variants of these genes may contribute to 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. The identification of genetic markers highly associated with a high risk of toxicity is a good example of how chemotherapy regimens could be more effectively tailored to individual patients.
   2. The role of CTLA-4 polymorphisms in the outcome of allogeneic stem cell transplantation. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene behaves as a negative regulator of T-cell activation. CTLA-4 gene polymorphisms have been found to be associated with susceptibility to autoimmune diseases. Recently, conflicting observations have been reported about the role of CTLA-4 gene polymorphisms in the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have investigated three polymorphisms of the CTLA-4 gene (-318C > T, +49A>G, CT60G>A) in 133 donor/recipient pairs who underwent HLA-matched sibling donor HSCT for hematological malignancies. We found no association of the clinical outcome of the HSCT with either recipient or donor ?318C>T and CT60G>A polymorphisms. At variance, we found a significant association of the donor +49A>G G/G genotype with longer overall survival (OS; P = 0.04), and the number of +49A>G G-alleles in the recipient with longer OS (P < 0.03), longer disease-free survival (DFS; P < 0.04) and reduced relapse rate (P < 0.04). However, multivariate analysis confirmed the independent prognostic significance of only recipient +49A/G genotypes for both OS and DFS. Our data show that the recipient CTLA-4 +49A/G polymorphism, thus far not investigated, appears to be relevant to the clinical outcome of allo-HSCT. This suggests that CTLA-4 expression on leukemic cells and on recipient micro-environment cells might play a role in the post-transplant control of disease (a).
2. Study of inherited and acquired factors favoring the selective growth of abnormal clonal populations of somatic cells
   
   1. Disease model: paroxysmal nocturnal hemoglobinuria. Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired blood disorder characterized by intravascular hemolysis, a tendency for thrombosis, and a variable degrees of bone marrow failure (b). PNH is due to clonal proliferation of a Hematopoietic Stem Cell (HSC) in which a somatic mutation has inactivated the X-linked gene, PIG-A (Figure 1). The resulting deficiency of Glycosylphosphatidylinositol (GPI)-linked proteins on the surface of the progeny of the mutated HSC explains hemolysis and thrombophilia (b). Recently, treatment with a monoclonal antibody that blocks complement protein C5 (eculizumab) almost completely controlled intravascular hemolysis in PNH patients. However, the deficiency of GPIanchored molecules on blood cells does not explain the bone marrow failure and the expansion of the PNH (GPI-negative) clone. Clinical observations, in vitro studies, and data from PNH mouse models indicate that GPI-negative HSC do not have an absolute growth advantage. In addition, very small GPI-negative clones exist in healthy subjects, but only in PNH patients do GPI-negative cells expand and contribute substantially to hematopoiesis. A plausible model that could explain both bone marrow failure and the PNH clone expansion is that normal (GPI-positive) HSC are the target of a selective auto-immune attack to which PNH HSC are resistant. Auto-reactive T-cells against HSCs are responsible for Idiopathic Aplastic Anemia (IAA); the close relationship of PNH with IAA has suggested that they may be present in PNH as well. Indeed, analysis of the TCR repertoire has revealed an increased frequency of expanded T-cell clones in PNH, similar to that observed in IAA. The identity of the putative auto-reactive T-cells and of their targets remains unknown. However, we have found that in PNH patients the expansion of CD8+CD57+ T-cells is relatively common. In addition, by a systematic sequence analysis of the TCR-beta molecules, we have shown that in PNH patients CD8+CD57+ T-cells are oligoclonal and that more than two-thirds of patients share, on this T-cell population, a set of highly homologous TCR-beta molecules (clonotypes) (7). These findings are consistent with the presence in PNH patients of an immune process driven by the same (or similar) antigen(s): probably a non-peptide antigen because patients sharing clonotypes do not all share identical HLA alleles (7). These findings provide strong support to our hypothesis that expansion of the GPI-negative blood-cell population in PNH is due to selective damage to normal hematopoiesis, mediated by an autoimmune attack of CD8+CD57+ T-cells against a nonpeptide antigen(s) that could be the GPI anchor itself. In order to directly test the hypothesis that the GPI anchor itself is the auto-antigen targeted in PNH, the availability of the human GPI anchor is crucial. Mammalian GPI has never been synthesized; however, recently, Cristina Nativi (University of Florence) has succeeded in synthesizing the core structure of the mammalian (human) GPI anchor. The availability of the GPI molecules provides us with a powerful tool to directly investigate the characteristic autoimmune pathogenesis of the clonal expansion observed in PNH. In addition, it is possible that similar autoimmune mechanisms could be responsible for clonal expansion in others acquired clonal cytopenias related to IAA and PNH, such as Myelodysplastic Syndromes (MDS) that are much more frequent than PNH and far more prone to evolve to acute leukemia.

      

      Figure 1 - The molecular basis of PNH. Complex biosynthetic machinery produces the GPI molecule (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 the 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 on the cell surface.
   2. The role of somatic mutation rate in human cancer. Mutations are an inherent risk of cell duplication since they develop even in the absence of any exogenous agent. Thus, the frequency of mutants (f: the fraction of cells harboring a mutation in a given gene) and the mutation rate (μ: the probability of a new mutation occurring in a gene per cell division) are key biological features of any cell population. The accumulation of somatic cells plays a key role in the development of cancer. Since the final common pathway for cancer development is a sequence of somatic mutations, one common risk factor for the development of any tumor must be the rate of somatic mutation (μ). To measure f and μ, a potential sentinel gene is the PIG-A gene that encodes one of the subunits of an enzyme essential in the biosynthesis of GPI (see above, paragraph 2a). Since PIG-A is X-linked, mutational inactivation of the one single copy active in somatic cells results in the absence from the cell surface of all the proteins that require GPI for attachment to the membrane; thus, mutant cells display a GPInegative surface phenotype (see above, Figure 1) that can be easily detected by flow cytometry (Figure 2). The measurement of PIG-A mutants by counting cells with the GPI-negative phenotype has proved to be effective to measure mutant frequency in peripheral blood cells of humans and of others animals (1). Up to now, μ has been exceedingly difficult to measure in human cells (1); however, by using the PIG-A gene as a sentinel in long term culture of B Lymphoblastoid Cell Lines (BLCL), we have a test that makes it possible to measure μ in human cells. By using this approach, we have shown that μ is increased in patients with inherited cancer-prone syndromes, such as Fanconi anemia and ataxia-telangiectasia. Now, we are addressing two important aspects: a) to determine to what extent μ is genetically determined; b) to explore the relationship between the value of μ (and f) and the risk of cancer.
      1. In order to understand whether the normal variation of μ depends more on environmental factors or more on inherited factors, we have measured μ in a small set of clinically normal twins. Intriguingly, the μ variation in the same twin pair was smaller in mono- than in dizygotic twins, supporting the notion that, even within the normal range, μ may have a substantial inherited component. We plan to confirm this notion by testing a large group of pairs of twins.
      2. The determination of μ on BLCLs is still relatively laborious and since it is likely that in terminally differentiated cells the PIG-A mutant frequency (f) will be roughly proportional to the μ value of their progenitor cells, we have resorted to exploring the measurement of the in vivo frequency of mutant cells (f) in peripheral blood granulocytes as a surrogate of μ. We have determined the normal range of f in granulocytes studying a small group of healthy subjects. Now, we intend to compare the f values in granulocytes from a population exposed to environmental carcinogen, namely heavy smokers, and from cancer patients with the distribution of the f values in healthy subjects.
      Our future plans include the identification of the genetic determinants of μ, the investigation of how μ is affected by different environmental exposures (i.e. smoking, etc.) and of how far it correlates with the risk to develop cancer.

      

      Figure 2 - Flow cytometry pattern displaying rare GPI-negative granulocytes from peripheral blood of a normal donor. The mutant (GPI-) cells are clearly resolved from the bulk (GPI+) cells: calculation gives 34 per million
   3. The role of chromosomal rearrangement involving ETS proteins in development and progression of prostate cancer
      Prostate cancer is the most commonly diagnosed cancer in elderly men in Western countries and it is the second leading cause of death in the male population. Recently, chromosomal translocations that juxtaposes the promoter of a gene highly expressed in the prostate to the coding sequence of one member of the ETS gene family (ERG, ETV1, ETV4, ETV5) have been found (c). Since then, a variety of translocations that juxtapose an ETS gene to the promoter of a series of partners that drive its prostate aberrant expression have been identified in prostate cancer. Since these translocations are recurrent in prostate cancer, it is reasonable to hypothesize that the resulting overexpression of an ETS transcription factor plays a direct role in prostate cancer pathogenesis. Evidence for this direct pathogenic role of ERG and ETV1 overexpression in prostate cancer have been reported. We are investigating the pathogenic role of the overexpression of the ETV4 gene that has been documented in a proportion of prostate cancer cases: in some cases it is associated with translocations that juxtapose the ETV4 gene to the promoter of genes highly expressed in the prostate (TMPRSS2, KLK2, DDX5, CANT1), and it is has been found in others without any detectable translocation. We have tested the expression of ETV4 in few human prostate cell lines: ETV4 expression was not detectable in LnCap and V-Cap whereas it was present in PC3 and Du145 cell lines. In order to test the role of ETV4 expression, we have used a doxycycline-inducible expression of short hairpin (sh) RNAs against ETV4: after induction, the growth of DU145 cell line transduced with the shRNA-containing vector was about 50% of that of DU145 transduced with an empty vector. In addition, we have observed that the reduction in ETV4 expression strongly impairs the ability to form colonies in soft agar (? 65% of reduction). ETV4 shRNA interference has produced a similar reduction in cell growth and in the number soft agar colonies in PC3 cell lines. Furthermore, the growth of DU145 xenografts is strongly reduced by the induction of ETV4 shRNA interference. These preliminary experiments show that the expression of ETV4 is functionally important in a cellular model of prostate cancer; thus, they suggest that ETV4 expression may play a direct role in the development and progression of a subset of prostate cancers.

References

1. Piccioli P, Balbi G, Serra M, et al: CTLA-4 +49A>G polymorphism of recipients of HLA-matched sibling allogeneic stem cell transplantation is associated with survival and relapse incidence. Ann Hematol 2010; 89: 613-8.
2. Luzzatto L, Notaro R: Paroxysmal nocturnal hemoglobinuria. In Handin RI, Lux SE, Stossel TP (eds): Blood, principles and practice of hematology (2nd ed.). Philadelphia, PA (USA), Lippincot Williams & Wilkins, 2003; 319-34.
3. Tomlins SA, Rhodes DR, Perner S, et al: Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 2005; 310: 644.

Main Collaborations

With Units within ITT

1. Hematology Unit, AOU Careggi, Firenze
2. Flow Cytometry Unit, AOU Careggi, Firenze
3. Pathology Unit, AOU Careggi, Firenze
4. Laboratory of Mice and Animal Experimentation (L.I.Ge.M.A.), University of Florence
5. Department of Pharmacology, University of Florence
6. Department of Chemistry, University of Florence

With other Italian and Foreign Institutions/Organizations

1. Istituto Nazionale per la Ricerca sul Cancro (IST), Genova
2. "Federico II" University, Napoli
3. University of Turin

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.

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