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Resistance Mechanisms to ALK Tyrosine Kinase Inhibitors (Tkis) in NSCLC Gonzalo Recondo

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Resistance Mechanisms to ALK Tyrosine Kinase Inhibitors (Tkis) in NSCLC Gonzalo Recondo Resistance Mechanisms to ALK Tyrosine Kinase Inhibitors (TKIs) in NSCLC Gonzalo Recondo To cite this version: Gonzalo Recondo. Resistance Mechanisms to ALK Tyrosine Kinase Inhibitors (TKIs) in NSCLC. Cancer. Université Paris Saclay (COmUE); Universidad de Buenos Aires, 2019. English. NNT : 2019SACLS248. tel-02337169 HAL Id: tel-02337169 https://tel.archives-ouvertes.fr/tel-02337169 Submitted on 29 Oct 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Resistance mechanisms to ALK tyrosine kinase inhibitors in NSCLC Thèse de doctorat de l’Université Paris-Saclay 2019SACLS248 préparée à l’Université Paris-Sud : NNT Inserm U981, Gustave Roussy Campus (Villejuif) École Doctorale N°582 : Cancérologie – Biologie – Médecine – Santé Disciplines : Sciences de la vie et de la santé Aspects moléculaires et cellulaires de la biologie Soutenue publiquement le 12 septembre 2019 à Gustave Roussy par Gonzalo Recondo Composition du Jury: Pr Jean-Yves Scoazec Professeur des Universités-Praticien Hospitalier de l'Université Paris-Sud Président Pr. Fabrice Barlesi Professeur des Universités-Praticien Hospitalier, Hôpital Nord, Marseille Rapporteur Pr. Gilles Favre Professeur des Universités-Praticien Hospitalier, l'Université de Toulouse Rapporteur Pr. Benjamin Besse Professeur des Universités-Praticien Hospitalier de l'Université Paris-Sud Examinateur 1 Dr. Luc Friboulet Chef d’Équipe, INSERM U981, Gustave Roussy Campus, Villejuif. Directeur du thèse 2 Acknowledgements En premier lieu, je souhaite remercier la Fondation Nelia et Amadeo Barletta qui m’a soutenu financièrement pendant ma thèse. Je remercie ensuite les membres de mon jury. Merci au professeur Fabrice Barlesi et professeur Gilles Favre d’avoir accepté d’être les rapporteurs de mon travail. Je remercie le professeur Jean-Yves Scoazec de présider mon jury de thèse et mon examinateur, le professeur Benjamin Besse pour avoir bien voulu participer à ce jury. I would like to thank Pr. Fabrice André, director of the INSERM U981 for accepting my candidacy at this prestigious research group. I would like also to thank Pr. Jean-Charles Soria for giving me this opportunity by welcoming me in your team, for your advice and guidance during the time we shared together. My deepest gratitude to my thesis director, Dr. Luc Friboulet. Luc, thank you so much for being my Mentor, it has been one of the greatest learning experiences of my life. Thank you for your kind guidance, your constant support and advice. During these three years, you helped me to forge and develop scientific reasoning, how to organize my work and goals, and to aim for high quality standards. You taught me how to generate hypothesis, design experiments and interpret results. But most of all, you were there for me, helped me develop the skills I needed to push myself forward to overcome countless boundaries, and learn to trust myself in the field of science. Thank you for your many teachings, I am forever grateful. I would like to extend my gratitude to all the members of our team in the lab during my thesis: Ken, Tony, Rosa, Francesco, Mei, Justine, Clement, Natalia, Florien, Daphné, Roman, Nico, Marlene, Clemence, Sophie, Ludo, Dina, Chloé. Thank you so much for teaching me with kindness and for your friendship and support. I would like to thank the Thoracic Oncology Team, Pr. Benjamin Besse, Pr. David Planchard and Dr. Laura Mezquita together with all the great MATCH-R team at Gustave Roussy, for working together in this project. 3 I would like to thank the patients and their families, for trusting in us to search new treatment options through research, and help more patients in the future. A special thanks to the researchers and fellow PhD and Master students at the INSERM U981, for your kindness and warmth. A special thanks to Lau, Ingrid and José, for becoming part of my family in France and making me feel at home. My deepest gratitude to Rosa Frias, my colleague and friend during my first year of PhD. Rosi, thank you for all your help and patience, you continue to inspire me. In hand, I would like to express my gratitude to Dr. Laura Mezquita. Laura, thank you for encouraging and motivating me to pursue my goals and your wonderful friendship. In Argentina, I would like to thank Dr. Maria Ines Vaccaro, my co-director and all the authorities of the Research Department and the University at CEMIC, as well as the Medical Oncology Department and the Molecular Pathology Laboratory. Thank you for encouraging me and supporting me to pursue this dream, for teaching me and guiding me. I would like to extend this to Elisa Bal, Lili and Tefo and the rest of the research team at the Agel H. Roffo Oncology Institute. I would like to thank my Mentor, Dr. Guillermo Del Bosco. Guillermo, most of what I have done so far, has been inspired in your example. Thank you for showing me the way and for being beside me throughout this journey. My deepest gratitude to Pr. Esteban Cvitkovic. Esteban, thank you for this opportunity, for believing and trusting in me. Thanks for all the wisdom, clarity, and vision you have passed on. Thanks for putting me back into my feet, every time, and inspiring me to help many patients back in Argentina. I owe my deepest gratitude to my parents, Isabel and Gonzalo, I am the most grateful and proud son, thank you for your unconditional love and support. Thanks also to my sisters, Marcelo and Rosario for their support. Last but not least, I want to thank my wife Josefina and my kids, Baltazar and Joaquina, this work is dedicated to you. Jose, thank you for encouraging and supporting me with your love throughout these years, and for sharing with joy this adventure. Balta and Joaqui, you make our days the happiest. 4 Abstract The molecular study and classification of lung adenocarcinomas has led to the development of selective targeted therapies aiming to improve disease control and survival in patients. The anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor from the insulin tyrosine kinase receptor family, with a physiologic role in neural development. Gene rearrangements involving the ALK kinase domain occur in ~3-6% of patients with lung adenocarcinoma. The fusion protein dimerizes leading to transactivation of the ALK kinase domain in a ligand-independent and constitutive manner. Lorlatinib is a third generation ALK inhibitor with high potency and selectivity for this kinase in vitro and in vivo, and elevated penetrance in the central nervous system. Lorlatinib can overcome resistance mediated by over 16 secondary kinase domain mutations occurring in 13 residues upon progression to first- and second- generation ALK TKI. In addition, treatment with lorlatinib is effective for patients who have been previously treated with a first and a second generation or a second generation ALK TKI upfront and is currently approved for this indication. The full spectrum of biological mechanisms driving lorlatinib resistance in patients remains to be elucidated. It has been recently reported that the sequential acquisition of two or more mutations in the kinase domain, also referred as compound mutations, is responsible for disease progression in about 35% of patients treated with lorlatinib, mainly by impairing its binding to the ALK kinase domain. However, the effect of these compound mutations on the sensitivity to the repertoire of ALK inhibitors can vary, and other resistance mechanisms occurring in most patients are unknown. My PhD thesis aimed at exploring resistance to lorlatinib in patients with ALK-rearranged lung cancer through spatial and temporal tumor biopsies and development of patient-derived models. Within the institutional MATCH-R study 5 (NCT02517892), we performed high-throughput whole exome, RNA and targeted next-generation sequencing, together with plasma sequencing to identify putative genomic and bypass mechanisms of resistance. We developed patient-derived cell lines and characterized novel mechanisms of resistance and personalized treatment strategies in vitro and in vivo. We characterized three mechanisms of resistance in five patients with paired biopsies. We studied the induction of epithelial-mesenchymal transition (EMT) by SRC activation in two patient-derived cell lines exposed to lorlatinib. Mesenchymal cells were sensitive to combined SRC and ALK co-inhibition, showing that even in the presence of an aggressive and challenging phenotype, combination strategies can overcome ALK resistance. We identified three novel ALK kinase domain compound mutations, F1174L/G1202R, C1156Y/G1269A, L1196M/D1203N occurring in three patients treated with lorlatinib. We developed Ba/F3 cell models harboring single and compound mutations to study the differential effect of these mutations on lorlatinib resistance. Finally, we characterized a novel mechanism of resistance caused by NF2 loss of function at the time of lorlatinib progression through the development of patients derived PDX and cell lines, and in vitro validation of NF2 knock-out with CRISPR/CAS9 gene editing. Downstream activation of mTOR was found to drive lorlatinib resistance by NF2 loss of function and was overcome by providing treatment with mTOR inhibitors. This study shows that mechanisms of resistance to lorlatinib are more diverse and complex than anticipated. Our findings also emphasize how longitudinal studies of tumor dynamics allow deciphering TKI resistance and identifying reversing strategies. 6 Résumé Les analyses moléculaires et la classification des adénocarcinomes bronchiques ont conduit au développement de thérapies ciblées sélectives visant à améliorer le contrôle de la maladie et la survie des patients.
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