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Le rhabdomyosarcome (RMS) est le cancer des tissus mous le plus courant chez l'enfant, et moins de 30 % des patients à haut risque obtiennent une rémission. Par conséquent, il existe un besoin pour une immunothérapie nouvelle et efficace. Les cellules tueuses naturelles (NK), avec leur capacité intrinsèque à tuer les cellules cancéreuses, représentent un outil thérapeutique prometteur. Cependant, leur efficacité clinique est limitée. Ainsi, nous proposons de concevoir ces cellules avec un récepteur antigénique chimérique (CAR) qui permettra aux cellules NK de cibler plus efficacement les cellules de RMS. De plus, nous proposons aussi de concevoir grâce à la technologie CRISPR-Cas9, des NK n’exprimant pas NKG2A, un récepteur impliqué dans l'inhibition des cellules NK par le microenvironnement tumoral. Nous avons développé un vecteur lentiviral codant pour une construction CAR reconnaissant B7-H3 et FGFR4, deux protéines surexprimées à la surface des cellules RMS, associées à une queue intracellulaire optimisée pour l'activation des NK. Les cellules NK primaires expandues ont été transduites et triées en fonction de l'expression du CAR, conduisant à une population de cellules CAR+- NK enrichie. L'efficacité des deux CAR a été évaluée par des tests cytotoxiques et de dégranulation contre les lignées cellulaires de RMS, RH-30 et RD, toutes deux exprimant B7-H3 et FGFR4. Les résultats préliminaires ont montré une augmentation de la cytotoxicité de 20 % par rapport aux NK de type sauvage pour les CAR anti-B7-H3. Les cellules NK ont également été transduites pour éliminer l’expression du gène KLRC1, codant pour NKG2A, en utilisant CRISPR Cas9. Ceci a permis d’augmenter la cytotoxicité des NK de 20% à 25% comparativement aux NK qui expriment NKG2A. Nous avons aussi combiné les deux modifications génétiques, obtenant ainsi des NK qui expriment un CAR contre les cellules du RMS et n’exprimant pas NKG2A. Des résultats préliminaires nous ont permis d’observer que les NK doublement modifiées étaient 60% plus cytotoxiques que les NK non-transduites et 20% plus efficaces que les CAR-NK ou les NK n’exprimant pas NKG2A. Ce projet sera une preuve de principe qu'une thérapie hautement innovante basée sur l'ingénierie des cellules NK est efficace et applicable au cancer solide. / Rhabdomyosarcoma (RMS) is the most common soft tissue cancer in childhood, and less than 30% of high-risk patients achieve remission. Therefore, there is a need for new and efficient immunotherapy. Natural killer (NK) cells, with their intrinsic ability to kill cancer cells, represent a promising therapeutic tool. However, their clinical efficacy is limited. Thus, we propose to engineer these cells with a Chimeric Antigen Receptor (CAR) that will allow NK cells to target RMS cells more efficiently and though the knock-out of NKG2A, a receptor involved in the inhibition of NK cells by the tumor microenvironment. We developed a lentiviral vector coding for a CAR construct recognizing B7-H3 and FGFR4, two proteins overexpressed at the surface of RMS cells, combined to an intracellular tail optimized for NK activation. Expanded primary NK cells were transduced and sorted based on CAR expression, leading to an enriched CAR+ -NK cells population. Efficacy of both CARs was evaluated by cytotoxic and degranulation assays against RH-30 and RD RMS cell lines, both expressing B7-H3 and FGFR4. Preliminary results showed an increase in cytotoxicity of 20% compared to wild type NK for CAR anti-B7-H3. NK cells were also knocked-out for the gene coding for NKG2A, using CRISPR Cas9, thereby increasing cytotoxicity by 20% to 25%. The combination of both genetic modifications should significantly increase the efficacy of NK-cell based therapy in RMS. Indeed, preliminary results allowed us to observe that doubly modified NKs were more than 60% more cytotoxic than non-transduced NKs and 20% more effective than CAR-NKs or NKs not expressing NKG2A. This project will be a proof of principle that a highly innovative therapy based on NK-cell engineering is efficient and applicable to solid cancer.

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Le rhabdomyosarcome de l'adulte est une tumeur rare au pronostic. Le présent travail propose d'étudier les caractéristiques cliniques et moléculaires et la prise en charge des adolescents et adultes atteints de rhabdomyosarcome ainsi que la possibilité de combinaison de thérapie ciblées sur lignées cellulaires in vitro. Nous avons anamysé rétrospectivement 239 patients âgés de 10 ans ou plus, atteints de rhabdomyosarcome au MD Anderson Cancer Center entre 1957 et 2003 et leur statut fusionnel pour PAX-FOXO1 par hybridation in situ en fluorescence. Trois lignées cellulaire de sarcome à petites cellules ont été soumises à des combinaisons de thérapies ciblées avec analyse de la viabilité. Les patients de plus de 50 ans avaient une survie globale à 5 ans de 13 % (médiane de survi à 1.7 ans) en dépit d'une maladie localisée. Approximativement 13 % des patients métastasiques de moins de 50 ans ont eu une survie prolongée de plus de 15 ans. L'utilisation d'une stratégie thérapeutique triple, intégrant chirurgie, chimiothérapie et radiothérapie était signifcativement associée à une survie prolongée. Auniveau molécualire, la présence du transcrit de fusio PAX3/7-FOXO1 était significativement liée à un risque accru de maladie métastatique. L'étude in vitro de thérapies ciblées a permis d'identifier la combinaison du vorinostat plus le 17DMAG associée à la doxorubicine comme ayant une meilleure efficacité. La prise en charge du rhabdomyosarcome de l'adolescent et de l'adulte semble souffrir d'une approche moins agressive comparée au rhabdomyosarcome pédiatrique. De plus, des combianaisons de thérapies ciblées peuvent être intégrées aux protocoles de chimiothérapies standards. / Rhabdomyosarcoma is a rare entity adult patient with unfavourable outcome. This work describes the clinical and molecular specificities of adolescent and adult type of rhabdomyosarcoma and investigates the optimal integration of targetd therapy combinations on small cell sarcoma cell lines in vitro. We retrospectively analyzed 239 patients, 10 years of age and greater, diagonsed withrhabdomyosarcoma at MD Anderson Cancer Center from 1957 trough 2003 and their PAX-FOXO1 fusion gene status by fluorescence in situ hybridization on tissues microarray. Three samll cell sarcoma cell lines were exposed to targetd agent combinations. PAtient with metastatic rhabdomyosarcoma were found to have a 18 % survival rate at 5 years from diagnosis with an 12 %survival past 15 years. This outcome was even poorer for patients over 50 of age, even with localized disease. Younger patients were more likely to receive multidisciplinary therapy than their older counterparts. The presence of PAX-FOXO1 tranlocation was significantly associated with a higher frequency of metastatic disease. The four agents with the exception of abacavir synergized two by two with each other in vitro but the triple combinations did not perform beter than the bitherapies. The dual therapies vorinostat 5HDAC inhibitor) plus 17-DMAG (Hsp90 inhibitor) added with doxorubicin achvied better results than dual or triple therapies. Adult patient with rhabdomyosarcoma present similar molecular and clinical characteristics compared pediatric patients but outcome decrease with age partly du to a less multimodal management. Moreover targeted combinations should be integrated to chemotherapy backbone.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, with two predominant histologic subtypes: embryonal and alveolar. These histologies display distinct clinical courses, and despite refinements in dose and duration of multimodality therapy, the 5-year overall survival of patients diagnosed with metastatic RMS remains <30%. Thus, there is an urgent need to define novel targets for therapeutic intervention. Interrogation of cancer cell signal transduction pathways that regulate the pathogenic behaviours of tumor cells has been successful in defining targets in numerous tumor types. These have ultimately yielded clinically-relevant drugs that have improved the disease-free and overall survival of patients diagnosed with cancer. Work contained in this thesis describes the interrogation of several potential targets for inhibition in RMS. Interruption of RMS cell proliferation, survival and apoptosis is examined through disruption of the protein kinase integrin-linked kinase (ILK) and the nuclear receptor estrogen-receptor β. ILK, in particular, is demonstrated to have dual competing functions through the regulation of c-jun amino-terminal kinase (JNK) signaling: an oncogene in alveolar, and a tumor suppressor in embryonal RMS. These findings are recapitulated in other tumor cell lines, indicating that expression levels of JNK1 correlate with ILK function in a broad spectrum of tumor types. Furthermore, interruption of rhabdomyosarcoma cell migration as a surrogate marker of metastasis is examined through disruption of the stromal-cell derived factor 1α/chemokine (CXC)receptor 4 signaling network, as well as through cooperative interactions between ILK and the mammalian target of rapamycin. Finally, we demonstrate that the insulin-like growth factor pathway is a potential target for therapeutic inhibition, which also distinguishes tumors of embryonal and alveolar histology. These studies provide a rationale for the development of novel agents, as well as the use of established drugs targeting these pathways in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, with two predominant histologic subtypes: embryonal and alveolar. These histologies display distinct clinical courses, and despite refinements in dose and duration of multimodality therapy, the 5-year overall survival of patients diagnosed with metastatic RMS remains <30%. Thus, there is an urgent need to define novel targets for therapeutic intervention. Interrogation of cancer cell signal transduction pathways that regulate the pathogenic behaviours of tumor cells has been successful in defining targets in numerous tumor types. These have ultimately yielded clinically-relevant drugs that have improved the disease-free and overall survival of patients diagnosed with cancer. Work contained in this thesis describes the interrogation of several potential targets for inhibition in RMS. Interruption of RMS cell proliferation, survival and apoptosis is examined through disruption of the protein kinase integrin-linked kinase (ILK) and the nuclear receptor estrogen-receptor β. ILK, in particular, is demonstrated to have dual competing functions through the regulation of c-jun amino-terminal kinase (JNK) signaling: an oncogene in alveolar, and a tumor suppressor in embryonal RMS. These findings are recapitulated in other tumor cell lines, indicating that expression levels of JNK1 correlate with ILK function in a broad spectrum of tumor types. Furthermore, interruption of rhabdomyosarcoma cell migration as a surrogate marker of metastasis is examined through disruption of the stromal-cell derived factor 1α/chemokine (CXC)receptor 4 signaling network, as well as through cooperative interactions between ILK and the mammalian target of rapamycin. Finally, we demonstrate that the insulin-like growth factor pathway is a potential target for therapeutic inhibition, which also distinguishes tumors of embryonal and alveolar histology. These studies provide a rationale for the development of novel agents, as well as the use of established drugs targeting these pathways in rhabdomyosarcoma.

No description available.

No description available.