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Analyse de la régulation des réarrangements précoces du TCRδ dans la lymphopoïèse normale et les anomalies oncogéniques associées dans les leucémies aiguës lymphoblastiques T

Abstract : T cell development is a regulated thymic process during which TCRs δ, γ, β and α rearrangement occurs successively. This process needs somatic recombination V(D)J events which involves RAG1/2 protein, RSS sequences besides V, D and J segments and regulatory elements (enhancers) ensuring a cis-regulation of this process. The control of V(D)J recombination occurs thanks to different mechanisms including epigenetic mechanisms, transcription factor intervention and the RSS conformation/sequence (the 12/23 rule and the B12/23 restriction). T cell acute lymphoblastic leukaemias (T-ALL) are characterized by the proliferation of malignant T lymphoblasts arrested at early stages of maturation. They show common characteristics with T progenitor from which they derived. Most frequent cytogenetics anomalies are chromosomal translocations with TCRα/δ and TCRβ loci. Deregulation, due to chromosomal translocations, of many oncogenesis such as homeodomain gene TLX has been described in T-ALL. Molecular mechanisms of these anomalies, notably the V(D)J machinery role and intimate molecular mechanism of the differentiation blockage observed stay hardly known. TLX1 or TLX3 positive leukaemias are characterised by a maturation blockage at cortical stage. This stage is defined by a complete TCRβ rearrangement, without any TCRα rearrangement or detectable expression of TCRαβ on cell surface. Using a gene reporter assay, we showed a repressive activity of homeodomain TLX1/3 oncoprotein on Enhancer alpha (Eα) which results in a repression of the Eα activity, therefore no TCRα rearrangement. Then, protein interaction between ETS1 and TLX1/3 was demonstrated in vivo in TLX1/3 positive cell lines. Finally, we showed TLX1/3 protein recruitment, via ETS1, on Eα. Interestingly, TLX1direct inactivation using lentiviral strategy (shRNA TLX1) or indirect inactivation (TCRαβ ectopic expression) leads to T cell redifferentiation and a massive cell apoptosis. It is important to note that massive apoptosis was not observed when transduced cells were cultured in a stromal-cell-free standard culture system. In the mean time, TCRδ-TLX1 translocations are detected in the normal thymus. So, TCRδ-TLX1 translocations do not undergo oncogenic selection because of a feed-forward inhibitory effect exerted by TLX1 itself, inducing autorepression of Ea transcriptional activity. Screening for TCRβ and TCRα/δ translocations in 280 T-ALLs allowed identification of four unreported TCR translocated oncogene partner (GNAG, LEF1, NKX2-4 and IL2RB). Molecular mapping of genomic junctions from TCR translocations showed that the majority of oncogenic partner breakpoints are not recombinase mediated and that the regulatory elements predominantly used to drive oncogene expression differ markedly in TCRβ (exclusively enhancer driven) and TCRα/δ translocations, when use of an enhancer-independent cryptic internal promoter is frequent. Our data also imply that oncogene activation takes place at a very immature stage of thymic development whereas the bulk leukemic maturation arrest (DN stage) occurs at a much later (cortical) stage. Moreover, the TCRα/δ translocated cases predominantly demonstrated junctions involving Dδ2 and Dδ3 segments which led us to analyse early stages of human thymopoiesis. Firstly, we show that human TCRδ gene assembly is ordered: Dδ2 to Dδ3 rearrangement followed by a Dδ2-Dδ3 to Jδ1 rearrangement. Dδ2-Dδ3 rearrangement occurs at a specific CD34+ CD7+dim CD1a- early thymic precursor (ETP) stage. Most importantly, we show that RUNX1 is directly involved in this ordered early rearrangement through recruitment to the Dδ2-23RSS and interaction with RAG1. RUNX1 inactivation in CD34+ umbilical cord blood leads to a total absence of human Dδ2-Dδ3 rearrangement. Taken together, these data confirm the role of RUNX1 in ordering early human TCRδ rearrangements and point out for the first time that human tcrd locus is controlled by a B12/23 restriction as opposed to murine tcrd locus regulation. Finally, this results show that the T cell oncogenesis and T cell ontogeny are closely linked. Thus, the physiological T cell maturation process blockage is a central element of the oncogenic process and underlines the potential of T-ALL study for a better understanding of the T ontogeny and show target therapy perspectives.
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Submitted on : Friday, October 12, 2012 - 10:43:29 AM
Last modification on : Monday, October 19, 2020 - 11:02:35 AM
Long-term archiving on: : Saturday, December 17, 2016 - 12:10:00 AM

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Sandrine Le Noir. Analyse de la régulation des réarrangements précoces du TCRδ dans la lymphopoïèse normale et les anomalies oncogéniques associées dans les leucémies aiguës lymphoblastiques T. Biologie cellulaire. Université Paris-Diderot - Paris VII, 2012. Français. ⟨tel-00741229⟩

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