La leucémie myéloïde chronique (LMC) est une hémopathie maligne causée par l‘apparition du chromosome Philadelphie dans la cellule souche hématopoïétique (CSH), conduisant à l‘expression de la protéine de fusion BCR-ABL1. L‘activité tyrosine kinase dérégulée de cette oncoprotéine provoque l‘activation de plusieurs voies de signalisation critiques dans la leucémogenèse. Si les inhibiteurs de tyrosine kinase (ITK) ciblant BCR-ABL1 représentent des traitements dans l'ensemble très efficaces, plusieurs études montrent que les cellules leucémiques les plus immatures de la moelle osseuse y sont insensibles. Cette thèse propose de compléter les connaissances relatives aux effets de BCR-ABL1 dans la cellule, et plus généralement aux propriétés des CSH de LMC. Notre intérêt s‘est focalisé sur le rôle potentiel des microARN. Dans un premier travail, nous nous sommes intéressés à l‘effet de l‘activité BCR-ABL1 sur le protéome et sur l‘expression des microARN dans la lignée cellulaire K562. Les résultats montrent que BCR-ABL1 régule l'expression d'un microARN fréquemment surexprimé dans les cancers, miR-21. Cet effet dépend du facteur de transcription STAT5, cible bien connue de l'activité kinase de BCR-ABL1. Dans une seconde partie, nous avons montré que dans la moelle osseuse des patients LMC, la fraction cellulaire enrichie en cellules souches (les cellules CD34+CD38low) exprime quatre microARN particuliers: mir-10a, mir-150, miR-155 et miR-146a. Deux de ces microARN (miR-150 et miR-155) sont trouvés spécifiquement dans les cellules des patients, et pas dans celles des individus sains. / In chronic myeloid leukemia (CML), the activity of the constitutively active tyrosine kinase BCR-ABL1 drives the activation of the PI3K/AKT, JAK/STAT, and RAS/RAF/MEK/ERK pathways. Among other consequences, activated or inhibited transcription factors induce important modifications of the CML cells gene expression pattern that could impact cell cycle control, apoptosis and genetic instability, leading to the expansion of the oncogene-transformed cells and to the acquisition of potentially harmful de novo mutations. However, indirect BCR-ABL1-dependant regulations might also occur, for instance through the action of microRNAs (miRNAs). Among the ~2000 miRNAs reported in humans, numerous species are up- or down-regulated in various cancer models. In the context of CML however, there is no clear consensus regarding the role of specific miRNAs, despite several studies. The first aim of this thesis was to study the effects of a clinically relevant concentration of imatinib, a tyrosine-kinase inhibitor (TKI) that blocks BCR-ABL1, on the CML cell line K562: both the microRNA expression profile and the cells proteome were analyzed. Using microarray hybridization, RT-qPCR experiments and a functional assay, we identified miR-21 as one of the most significantly down-regulated microRNA in cells that were treated with imatinib. In parallel, a semi-quantitative proteomic approach identified the tumor suppressor programmed cell death protein 4 (PDCD4) as the most over-expressed protein in imatinib-treated cells. We showed that miR-21 can bind to PDCD4 3'UTR and decrease its expression. The STAT5 - miR-21 - PDCD4 pathway was conserved in CML primary CD34+ cells, and to some extent in acute myeloid leukemia (AML) models as well; the known functions of miR-21 and PDCD4 suggest that their regulation by BCR-ABL1 could participate in the antileukemic response triggered by tyrosine kinase inhibitors. In the second part of this manuscript, we was interested in the immature stem cells population that cannot be eliminated by TKI. The underlying mechanisms of this resistance are not fully understood. The TKI-resistant CML stem cells reside in the CD34+/CD38low subpopulation, that can be sorted from the mononuclear cells fraction using FACS. In this project, we propose to describe the microRNA repertoire of the CML CD34+/CD38low cells to highlight the potential role of microRNA in the resistance mechanisms by identifying some of their targets, using bioinformatic and experimental approaches. This combination of miRNome and functional analysis would allow to increase the knowledge of the biology of the TKI-resistant CML stem cells. Our results have shown that the cellular fraction enriched in stem cells (CD34+CD38low) expressed specifically four microRNA: miR-10a, miR-146, miR-150 and miR-155. It is also interested to notice that only two of them, miR-150 and miR-155, are highly expressed in CML-patient CD34+CD38low cells compared to normal cells.
| Identifer | oai:union.ndltd.org:theses.fr/2016BORD0437 |
| Date | 16 December 2016 |
| Creators | Espadinha, Anne-Sophie |
| Contributors | Bordeaux, Cardinaud, Bruno |
| Source Sets | Dépôt national des thèses électroniques françaises |
| Language | French |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation, Text |
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