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2019BORD0054.Pdf Année 2019 Thèse n° 84698 THÈSE Pour le DOCTORAT DE L’UNIVERSITE DE BORDEAUX École Doctorale des Sciences de la Vie et de la Santé Spécialité : Génétique Présentée et soutenue publiquement le : 12 Avril 2019 Par Matthieu LEWIS Né le 7 Avril 1990 à Kingston Upon Thames (GBR) Identification de voies de résistance aux inhibiteurs de tyrosine kinase dans la leucémie myéloïde chronique par criblage CRISPR-Cas9 Travail réalisé au sein du laboratoire : « Diversité génétique et réponse au traitement, oncogenèse mammaire et leucémique » INSERM U 1218 ACTION Membres du jury : Pr Martin TEICHMANN – Université de Bordeaux Président Dr Dominique HELMLINGER – Université de Montpellier Rapporteur Dr Franck-Emmanuel NICOLINI – Université de Lyon Rapporteur Pr Anne-Marie MARTINEZ – Université de Montpellier Examinateur Dr Béatrice TURCQ – Université de Bordeaux Invitée Pr François-Xavier MAHON – Université de Bordeaux Directeur de thèse 1 Identification de voies de résistance aux inhibiteurs de tyrosine kinase dans la leucémie myéloïde chronique par criblage CRISPR-Cas9. La caractérisation des tumeurs malignes et la compréhension des mécanismes de résistance aux traitements anticancéreux sont essentielles pour la découverte de nouvelles cibles thérapeutiques. Les criblages génétiques, devenus encore plus puissants avec la technologie d’édition du génome CRISPR-Cas9, le séquençage nouvelle génération et la bioinformatique, sont des outils formidables pour décrypter de nouveaux mécanismes cellulaires, dont la résistance au traitement. La leucémie myéloïde chronique (LMC) est un syndrome myéloprolifératif qui est caractérisé par l’anomalie génétique t(9;22). Cette aberration chromosomique est à l’origine du gène de fusion BCR-ABL1 qui code l’oncogène du même nom responsable de la prolifération anarchique des cellules. L’imatinib mesylate, un inhibiteur de tyrosine kinase, élimine de manière spécifique les cellules leucémiques en ciblant et en bloquant l’activité kinase de cette protéine. Malheureusement, comme pour tout type de thérapie ciblée, une résistance au traitement survient chez certains patients. Afin de repérer des nouvelles voies de résistance à cet inhibiteur de tyrosine kinase, nous avons effectué un criblage génétique avec la librairie « genome-scale CRISPR knock-out » (GeCKO v2) in vitro dans la lignée cellulaire K562. Nous avons découvert plusieurs gènes qui semblent être essentiels pour la réponse au traitement par imatinib, tels que les facteurs pro-apoptotiques BIM et BAX, ou le répresseur de la voie des MAPK, SPRED2. Le rétablissement spécifique de l’apoptose dans les cellules BIM knock-out (KO) par des BH3-mimétiques, ou l’inhibition ciblée de la voie MAPK dans la lignée SPRED2 KO sensibilise de nouveau les lignées résistantes. Dans ce travail, nous avons découvert des mécanismes de résistance déjà connus (l’apoptose, la voie MAPK…) mais nous avons également démontré l’implication de voies peu connues telles que le complexe Mediator, la maturation de ARNm et l’ubiquitinylation de protéines. Spécifiquement cibler ces lésions génétiques avec des thérapies ciblées combinées peut permettre de surmonter les phénotypes de résistance et ouvre la porte à l’utilisation de l’oncologie de précision. Mots clés : inhibiteurs de tyrosine kinase, leucémie myéloïde chronique, criblage CRISPR-Cas9 Genome-wide CRISPR-Cas9 Screening to Identify Pathways Involved in Tyrosine Kinase Inhibitor Resistance in Chronic Myeloid Leukemia. The characterisation of malignant tumour growth and the understanding of resistance mechanisms to treatment in cancer is of utmost importance for the discovery of novel “druggable” targets. Efficient genetic screening, now even more possible with the convergence of CRISPR-Cas9 gene editing technology, next- generation sequencing and bioinformatics, is an important tool for deciphering novel cellular processes, such as resistance to treatment in cancer. Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterised by the t(9;22) genetic abnormality, which encodes the driver of CML, the BCR-ABL1 fusion protein. Imatinib mesylate, a tyrosine kinase inhibitor, specifically eliminates CML cells by targeting and blocking the kinase activity of this protein, yet, as for all targeted therapies in cancer, resistance to treatment exists. In order to discover alternative BCR-ABL1 independent mechanisms of imatinib resistance, we utilized the genome-scale CRISPR knock-out library GeCKO v2 to screen for imatinib sensitising genes in vitro on K562 cells. We revealed genes that seem essential for imatinib induced cell death, such as pro-apoptotic genes (BIM, BAX) or MAPK inhibitor SPRED2. Specifically re-establishing apoptotic capabilities in BIM knock-out (KO) cells with BH3-mimetics, or inhibiting MAP-kinase signalling in SPRED2 KO cells with MEK inhibitors restores sensitivity to imatinib, overcoming resistance phenotypes. In this work, we discovered previously identified pathways (apoptosis, MAP-kinase signalling) and novel pathways that modulate response to imatinib in CML cell lines, such as the implication of the Mediator complex, mRNA processing and protein ubiquitinylation. Targeting these specific genetic lesions with combinational therapy can overcome resistance phenotypes and paves the road for the use of precision oncology. Key words: tyrosine kinase inhibitors, chronic myeloid leukemia, CRISPR-Cas9 screen 2 3 Le laboratoire « Diversité génétique et réponse au traitement, oncogenèse mammaire et leucémique », de l’unité INSERM U1218, dans lequel j’ai effectué ce travail, est depuis plus de 20 ans spécialisé dans la recherche sur une maladie modèle : la leucémie myéloïde chronique (LMC). De nos jours, la LMC est probablement le type de cancer le plus étudié dans le monde pour diverses raisons. Premièrement, elle est un modèle d’étude pour le dévloppement de cancer à plusieurs étapes, la LMC débute classiquement dans une phase chronique puis évolue vers deux phases supplémentaires à mauvais pronostic. L’étude de la progression pathologique dans la LMC pourrait aider à mieux comprendre comment d’autres types de cancer évoluent. Deuxièmement, c’est la première pathologie cancéreuse pour laquelle une caractérisation moléculaire de ses lésions génétiques a eu pour résultat la découverte d’une thérapie anti-cancéreuse ciblée. La démonstration de l’efficacité de la thérapie ciblée contre le cancer dans la LMC a débuté une révolution dans la découverte d’agents anti-cancéreux « intélligents ». Finalement, l’étude du développement de cette pathologie permettrait d’identifier des facteurs génétiques cruciaux à la progression tumorale ou à la résistance au traitement, qui pourraient un jour être de nouvelles cibles thérapeutiques pour la LMC mais aussi pour d’autres types de cancer. Depuis des années, notre équipe cherche à mieux comprendre la résistance au traitement dans la LMC en créant et étudiant des lignées cellulaires résistantes, en analysant des polymorphismes génétiques de patients résistants par séquençage nouvelle génération, en caractérisant l’implication de nouveaux gènes dans la réponse aux thérapies et en élaborant des essais cliniques d’arrêt de traitement pour des patients répondeurs à la thérapie. Depuis 2014, le laboratoire s’est également fortement intéressé à la technologie d’édition du génome Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9. En 2019, une plateforme universitaire autour de cette technique, CRISP’edit, a été créée dans le but d’aider d’autres équipes académiques lors de leurs expériences de manipulations génétiques. 4 Tout premièrement, je remercie le Pr Martin Teichmann pour avoir accepté de présider le jury. J’ai commencé ma carrière scientifique en stage de Master 1 avec toi, et maintenant je vais terminer mes études à tes côtés. Je remercie également le Dr Dominique Helmlinger et le Dr Franck- Emmanuel Nicolini d’avoir accepté d’être rapporteur de ce travail. Je remercie aussi le Pr Marie- Anne Martinez d’avoir accepté de faire partie du jury en tant qu’examinateur. Je souhaite sincèrement remercier François-Xavier Mahon, directeur de thèse et tuteur formidable au cours de ces dernières années. Si, dans ce laboratoire, nous étudions la LMC avec une telle expertise, c’est grâce à toi et aux travaux que t’as effectué pendant ta carrière (y compris tous les Western Blot de ta thèse de science) ! Dès les premiers mois de ma thèse, tu as été nommé directeur de l’institut Bergonié. Ton bureau a changé de localisation et ton emploi du temps s’est intensifié, mais t’étais toujours disponible aux moments critiques de ma thèse pour discuter des résultats scientifiques, pour me pousser à postuler pour participer à des congrès internationaux, et pour m’encourager dans mon travail et dans mes activités de communication et de vulgarisation scientifique. Même si des fois, tu fais des références curieuses à Star Wars, aux Peaky Blinders ou à des vieux acteurs Français que je ne connais pas, je suis très heureux et fier d’avoir partagé cette expérience avec toi. J’ai eu la chance d’avoir, de manière non-officielle, deux chefs dans deux établissements différents : FX à Bergonié et Béatrice Turcq à Carreire. Béa, au début de ma thèse, tu étais mon supérieur hiérarchique qui m’apprenait à extraire des ARN du trizol, et vers la fin de ma thèse, tu es devenue plus qu’une collègue, mais une amie, qui, par contre, m’apprend toujours qu’il faut purifier les amplicons avant le séquençage GATC (oups…). Entre les montagnes de travail (sur ton bureau, littéralement), l’encadrement
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