DUC DODON Madeleine, PhD, DR2 Inserm, 58 year old; Researcher in Laboratory of Human Virology (Ecole Normale Supérieure de Lyon, France); Current research: Role of the regulatory proteins Tax, Rex, and HBZ in leukemic process induced by HTLV-I.
POSITIONS/EMPLOYMENT: 1976-83 Graduate Fellow (Laboratory of Virology U51, France); 1983-84 Postdoc. Fellow (Laboratory of Microbiology, Prof. S. Vogel, Bethesda, MD, USA); 1985-87 Inserm Researcher (UMR 5537 Faculty of Medecine Laennec, Lyon, France); 1988-89 Researcher in Dr. WC Greene's laboratory (Duke University, Durham, USA); 1990-2001 Inserm Researcher (UMR 5537 Faculty of Medecine Laennec, Lyon, France); 1995 Directory of Research in Inserm (DR2); since 2002: Researcher in Laboratory of Human Virology (Lyon, France).
In addition to the function of directory of research in the laboratory in Lyon, since April 2010,I am fullfilling the function of Scientific Advisor, at the Biology and Health department of French Ministery of Research (Directorate General for Research and Innovation) in Paris (two days/week).
GRANTS: from ANRS (1999-2001) and ARC (2003-05). Fondation de France (2007-2009), (2009-2011)
Referee for Blood, Oncogene, Intenational Journal of Cancer, Journal of General Virology and Virologie.
The HTLV-1 human oncoretrovirus has been first identified more than 25 years ago as the etiological agent of Adult T cell Leukemia (ATL) and later as the agent of Tropical Spastic Paraparesis. The viral Tax1 transactivator, a major transcription regulator, is involved in cell transformation. In 2002, a mRNA encoding for a novel open reading frame present in the minus strand of HTLV-1 was discovered and characterized. It encodes HBZ (HTLV-1 bZIP factor), a protein that inhibits Tax-dependent viral transcription, that promotes cell proliferation and which is always expressed, including during the late stages of ATL. Tax and HBZ are therefore the major players in HTLV-1 physiopathology.
Our main objectives are:
(i) to understand the physiopathology of the Human T cell Leukemia Virus Type 1 (HTLV-1) infection and in particular the respective roles of the Tax oncoprotein in the initiation of the leukemic process and of HTLV-1 b-ZIP factor (HBZ) in the maintenance of the leukemic state in vitro and in vivo;
(ii) to elaborate new treatments that could either prevent the occurrence of the diseases or cure the patients after the onset of the leukemia.
I. Role of Tax in the early steps of leukemogenesis
Currently, an outstanding question in HTLV pathogenesis is whether ATL originates from the transformation of a differentiated CD4 T cell or from transformation of a hematopoietic precursor cell.
Our hypothesis is that T cell transformation by the HTLV-1 Tax protein might arise from altered gene products, which converge onto common differentiation pathways, leading to aberrant ab T cell development and subsequent neoplastic transformation. This development occurs in the thymus through a number of well-defined developmental stages, among which the b-selection checkpoint. This checkpoint is dependent on the surface assembly of the pre-TCR comprising the newly rearranged TCRb chain as well as the pre-TCR a invariant chain (pTa) and CD3 subunits. Signals emanating from the pre-TCR promote survival and proliferation of immature thymocytes as well as their differentiation into the CD4+CD8+ double-positive (DP) stage, effectively instructing immature thymocytes to the ab T-cell lineage. Interestingly, the pTa gene (exclusively expressed in immature thymocytes) is regulated in part by the E2A proteins, a subset of bHLH proteins. Inactivation of E2A may play a role in leukomogenesis since E2A deficient mice develop T-cell leukemia that originates in the thymus. We have previously reported that Tax down-regulates the transcription of the pTa gene in human immature thymocytes. This suggests that Tax, by interfering with the surface assembly of the pre-TCR, is perturbing the b-selection checkpoint. As Tax intervenes in the modulation of genes linked to the activation and immortalization of HTLV-1 infected T cells, we hypothesize that Tax might be able to palliate the absence of pre-TCR in immature thymocytes, thus allowing these Tax-expressing thymocytes to survive and to differentiate.
In addition, we now produce routinely HIS (Human Immune System) Rag2-/-gc-/- mice, after intrahepatic injection of human CD34+ progenitors into Rag2-/-gc-/- mice. We specifically assessed the development of human T cells in the thymus of engrafted mice. When a majority of immature cells were present in the thymus (four weeks after engraftment), HIS Rag2-/-gc-/- mice were infected with HTLV-1 by intraperitoneal injection of lethally irradiated virus-producing cells. Four months later, infected HIS mice (confined to the BSL-3 laboratory of PBES of Bioscienses-IFR128 Lyon) are sacrificed and shown to display a high proviral load, whereas T cells from peripheral blood, thymus and spleen are expressing viral structural and regulatory proteins (Gag, Tax, Rex and HBZ). Interestingly, some of these mice develop tumors (thymoma, hepatosplenomegaly, lymph node lymphoma) similar to those observed in ATL patients (manuscript in preparation).
Objectives: These observations emphasize the role of Tax the pre-TCR developmental checkpoint and identify human immature thymocytes as the target cells in which HTLV-1 infection initiates T-cell leukemogenic process[rm1] . Consequently we propose to : (i) evaluate the effects of Tax on the transcription of genes involved in cell proliferation, apoptosis and differentiation in this thymocyte subset; (ii) to clarify the role of Tax by HIS Rag2-/-gc-/- mice with HTLV-1 carrying mutated forms of Tax; (iii) to delineate the role of HBZ in the maintenance of the leukemogenic process by injecting HIS Rag2-/-gc-/- mice with HTLV-1 either deleted of HBZ or carrying mutated forms of this viral protein.
II. Role of HBZ in the late stages of cell transformation
Tax is playing a key role in the initiation of the leukemogenic process while HBZ is thought to pay a major player in the evolution and the maintenance of this process. As above mentioned, HBZ is able to regulate the transcription of hTERT in the presence of an essential cellular partner, the JunD AP1 protein, with which it interacts through the C-ter bZip domain to form active HBZ/JunD heterodimers. However, JunD interacts with the N-ter domain of Menin, encoded by the gene MEN-1 tumor suppressor, is responsible for the inhibition of both the transcriptional activity of JunD and proliferative capacity (Agarwal et al., 1999). Finally, Menin has been described as an inhibitor of telomerase (Lin and Elledge, 2003). We propose to analyze precisely the role of the three partners, HBZ, JunD and Menin, in the regulation of hTERT gene. As the leukemic cells proliferate in an uncontrolled manner, we postulate that HBZ, by antagonizing menin, reveals the transcriptional activity of JunD, resulting in the upregulation of telomerase activity.
Objectives: We plan to demonstrate at the molecular level that telomerase may be identified as a prognostic marker of the leukemogenic process. First, we will evaluate how the HBZ and Menin interaction with JunD is influencing the transcriptional regulation of hTERT. Second, ATL cells are characterized by the strong JunD expression, a fingerprint of the initial activation of Tax. However, no study has been conducted to evaluate the expression of JunD in cells from patients at different stages of the disease or during treatment. As HBZ/JunD heterodimers act as pivotal regulators of the expression of hTERT, we hypothesized that this expression could represent a good marker of the evolution of the leukemic process. To address this hypothesis, we propose to proceed with the evaluation of JunD transcripts, but also those of HBZ and Menin in CD4 + T lymphocytes from ATL patients at different stages of the disease and treated with current therapies. This study will be conducted in collaboration with Dr. O. Hermine (Necker Hospital)
III. Preventing leukemogenesis in HTLV-1 infected HIS Rag2-/-gc-/- mice
Actually, most currently used cytotoxic therapies are not targeting the leukemic cells (Tsukasaki et al., 2009). However, these cells have the unique ability to self-renew mainly due to their high telomerase activity.
Objectives: We propose to evaluate a series of therapeutic approaches to decrease the expression of telomerase, based mainly on the use of anti-reverse transcriptase (AZT) and/or of small interfering RNA directed against HBZ (siRNA/ HBZ). A study in collaboration with Dr. Ali Bazarbachi will be performed to analyze the effect of AZT in combination or not with IFNa and As2O3 in HTLV-1 infected HIS Rag2-/-gc-/- mice. In parallel, we will develop nanoparticles to deliver siRNA targeting HBZ with the advantage to specifically target the leukemic cells, since only the leukemic cells of infected individuals express HBZ protein. These experimental approaches should enable us to establish a correlation between in vivo telomerase activity, tumor development and effectiveness of therapies.