Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition

BACKGROUND: Rhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumour in children and adolescents. Aberrant expression of Anaplastic Lymphoma Kinase (ALK) and MET gene has been implicated in the malignant progression of RMS, especially in the alveolar subtype. This observation suggests that crizotinib (PF-02341066), a kinase inhibitor against ALK and MET, may have a therapeutic role in RMS, although its antitumour activity in this malignancy has not yet been studied. METHODS: RH4 and RH30 alveolar RMS (ARMS) cell lines were treated with crizotinib and then assessed by using proliferation, viability, migration and colony formation assays. Multiple approaches, including flow cytometry, immunofluorescence, western blotting and siRNA-based knock-down, were used in order to investigate possible molecular mechanisms linked to crizotinib activity. RESULTS: In vitro treatment with crizotinib inhibited ALK and MET proteins, as well as Insulin-like Growth Factor 1 Receptor (IGF1R), with a concomitant robust dephosphorylation of AKT and ERK, two downstream kinases involved in RMS cell proliferation and survival. Exposure to crizotinib impaired cell growth, and accumulation at G2/M phase was attributed to an altered expression and activation of checkpoint regulators, such as Cyclin B1 and Cdc2. Crizotinib was able to induce apoptosis and autophagy in a dose-dependent manner, as shown by caspase-3 activation/PARP proteolytic cleavage down-regulation and by LC3 activation/p62 down-regulation, respectively. The accumulation of reactive oxygen species (ROS) seemed to contribute to crizotinib effects in RH4 and RH30 cells. Moreover, crizotinib-treated RH4 and RH30 cells exhibited a decreased migratory/invasive capacity and clonogenic potential. CONCLUSIONS: These results provide a further insight into the molecular mechanisms affected by crizotinib in ARMS cells inferring that it could be a useful therapeutic tool in ARMS cancer treatment

ALK inhibition activates LC3B-independent, protective autophagy in EML4-ALK positive lung cancer cells.

ALK inhibitors effectively target EML4-ALK positive non-small cell lung cancer, but their effects are hampered by treatment resistance. In the present study, we asked whether ALK inhibition affects autophagy, and whether this may influence treatment response. Whereas the impact of targeted therapies on autophagic activity previously have been assessed by surrogate marker proteins such as LC3B, we here thoroughly examined effects on functional autophagic activity, i.e. on the sequestration and degradation of autophagic cargo, in addition to autophagic markers. Interestingly, the ALK inhibitor Ceritinib decreased mTOR activity and increased GFP-WIPI1 dot formation in H3122 and H2228 EML4-ALK <sup>+</sup> lung cancer cells, suggesting autophagy activation. Moreover, an mCherry-EGFP-LC3B based assay indicated elevated LC3B carrier flux upon ALK inhibition. In accordance, autophagic cargo sequestration and long-lived protein degradation significantly increased upon ALK inhibition. Intriguingly, autophagic cargo flux was dependent on VPS34 and ULK1, but not LC3B. Co-treating H3122 cells with Ceritinib and a VPS34 inhibitor or Bafilomycin A1 resulted in reduced cell numbers. Moreover, VPS34 inhibition reduced clonogenic recovery of Ceritinib-treated cells. In summary, our results indicate that ALK inhibition triggers LC3B-independent macroautophagic flux in EML4-ALK <sup>+</sup> cells to support cancer cell survival and clonogenic growth

Molecular mechanisms of vascular remodeling in pulmonary arterial hypertension : the implication of tyrosine kinase inhibitors and epigenetic events in the disease

L'hypertension artérielle pulmonaire (HTAP) est une maladie rare caractérisée par une obstruction progressive et un remodelage vasculaire des artères pulmonaires distales, conduisant à une pression artérielle pulmonaire moyenne supérieure à 20mmHg. L'augmentation de la pression aboutit à une dysfonction du ventricule droit et la mort. À l'heure actuelle, il n'existe pas de traitement curatif pour l'HTAP. Il est donc primordial d'identifier de nouvelles cibles thérapeutiques. Comme dans le cancer, les cellules musculaires lisses de l'artère pulmonaire des patients atteints d'HTAP présentent un phénotype hyper-prolifératif et résistant à l'apoptose, entraînant un remodelage vasculaire pulmonaire. Ainsi, plusieurs stratégies thérapeutiques anti cancéreuses pourraient être utiles pour le traitement de l'HTAP. Compte tenu de l'expression altérée des récepteurs tyrosines kinases dans l'HTAP ainsi que dans le cancer, les inhibiteurs de tyrosine-kinases (ITK) ont été mise sur le marché pour le traitement de différents types de cancers et ont été envisagées dans le cadre de l'HTAP. Ainsi, l'ITK, Imatinib, a été capable de régresser l'HTAP induite dans des modèles expérimentaux, tandis que l'administration d'un autre inhibiteur, le Dasatinib, était associée au développement de l'HTAP. Récemment, plusieurs études observationnelles ont démontré le développement de l'HTAP chez des patients atteints d'un cancer du poumon non à petites cellules (CPNPC) présentant des réarrangements de la kinase lymphocytaire anaplasique (ALK) et qui ont reçu des ITK ALK/cMET, notamment Xalkori (R-Crizotinib), Ceritinib, Brigatinib et Lorlatinib. Étant donné que l'hypertension pulmonaire peut être associée à plusieurs maladies, y compris le cancer du poumon, la question demeure de savoir si le développement de l'HTAP chez les patients atteints d'un cancer du poumon recevant l'ITK ALK/c-MET représente un événement indésirable du médicament ou un résultat de la propagation de la maladie. Ainsi, l'objectif principal de chapitre 1 est de déterminer si le R-Crizotinib (connu sous le nom de Xalkori et qui est une première ligne de traitement des patients ayant un CPNPC-ALK positif) exacerbe l'HTAP et/ou prédispose à l'HTAP dans des modèles animaux. In vivo, le traitement par R-Crizotinib a entraîné une élévation marquée de la pression systolique du ventricule droit et de la pression artérielle pulmonaire moyenne associée à une augmentation de l'épaisseur de la paroi médiale des artères pulmonaires distales. De plus, R-Crizotinib, administré avant l'exposition à une faible dose de monocrotaline (MCT), induit une réponse hypertensive pulmonaire exagérée, comme en témoigne une augmentation de la pression systolique du ventricule droit et de la pression artérielle pulmonaire moyenne, de l'épaisseur de la paroi médiale et une diminution du débit cardiaque. In vitro, nous avons démontré que le traitement avec R-Crizotinib réduit la prolifération des cellules endothéliales contrôles de l'artère pulmonaire, effet associé à la formation de cellules multinucléées, ce qui est généralement observée dans les cellules qui meurent d'une une catastrophe mitotique. En conclusion, nous avons démontré que l'agent anticancéreux R-Crizotinib favorise le dysfonctionnement des cellules endothéliales, conduisant à la prédisposition et à l'exacerbation de l'HTAP dans des modèles animaux. Les dernières années de recherche ont approuvé l'importance des marques épigénétiques dans le développement de l'HTAP. En effet, nous nous sommes intéressés, dans le deuxième volet de la thèse, au facteur épigénétique « G9a », qui s'est révélé surexprimé dans différents types de cancers, favorisant la survie et la prolifération des cellules. Compte tenu de l'analogie cancer/HTAP, G9a fut un candidat idéal pour l'étude de son potentiel rôle dans le développement de l'HTAP. Ainsi, l'objectif principal du chapitre 2 est de déterminer si G9a est impliqué dans la progression et la pathogenèse de l'HTAP et de déterminer si son inhibition est bénéfique dans les modèles animaux. Nous avons démontré que G9a est surexprimé dans les artères pulmonaires de patients HTAP et dans les modèles expérimentaux. In vitro, l'inhibition pharmacologique de G9a à l'aide de BIX01294 diminue drastiquement la capacité d'hyper prolifération et la résistance à l'apoptose des cellules musculaires lisses HTAP. Grâce au séquençage d'ARN, nous avons démontré que l'inhibition de G9a s'accompagnait d'une altération du flux d'autophagie et d'une accumulation de lipides. Enfin, le traitement thérapeutique avec BIX01294 a réduit le remodelage vasculaire pulmonaire et la pression artérielle pulmonaire moyenne dans un modèle expérimentale de rat et a également amélioré l'hémodynamique pulmonaire et la fonction ventriculaire droite dans un autre modèle de souris. Ces résultats suggèrent que l'inhibition de G9a pourrait représenter une nouvelle approche thérapeutique dans l'HTAP.Pulmonary arterial hypertension (PAH) is a rare and fatal disease characterized by "a progressive loss and obstructive remodeling of pulmonary arteries (PAs) leading to a mean pulmonary arterial pressure (mPAP) greater than 20mmHg. The persistent elevation of pulmonary pressures leads to right ventricular dysfunction and death. Currently, there is no cure for patients with PAH, which increases the need to develop new and effective therapeutic strategies. Pulmonary artery smooth muscle cells (PASMCs) from PAH patients exhibit a "cancer-like" hyperproliferative and apoptosis-resistant phenotype leading to pulmonary vascular remodeling. Therefore, several anti-cancer therapies could be useful for the treatment of PAH. Given the altered expression of receptor tyrosine kinases and their ligands in PAH as well as in cancer, tyrosine kinase inhibitors (TKIs) have been marketed for the treatment of different types of cancers and have been in the spotlight for anti-PAH drug research. Indeed, the tyrosine kinase inhibitor, Imatinib, was able to regress established PAH in experimental models, while the administration of another inhibitor, Dasatinib, was associated with the development of PAH. Recently, several observational studies have highlighted the development of PAH in patients with non-small cell lung cancer (NSCLC) with anaplastic lymphocyte kinase (ALK) rearrangements who received ALK/cMET TKIs, including Xalkori (R-crizotinib), Ceritinib, Brigatinib, and Lorlatinib. Since pulmonary hypertension can be associated with several diseases, including lung cancer, the question remains whether the development of PAH in lung cancer patients receiving cMET/ALK TKIs represents an adverse drug event or a result of disease spread. Thus, the main objective of Chapter 1 is to determine whether R-Crizotinib (known as Xalkori and which is the first-line treatment for patients with advanced ALK-positive NSCLC) exacerbates PAH and/or predisposes to PAH in experimental animal models. In vivo, R-Crizotinib treatment resulted in a marked elevation of the right ventricular systolic pressure (RVSP) and mPAP which was associated with an increased medial wall thickness of the distal PAs. Additionally, we found that pretreatment of rats with R-Crizotinib, induced an exaggerated pulmonary hypertensive response, as evidenced by the increased RVSP, mPAP, medial wall thickness, and decreased cardiac output. In vitro, we have demonstrated that treatment with R-Crizotinib reduces the proliferation of control pulmonary artery endothelial cells, which was accompanied by the appearance of multinucleated cells, a feature commonly seen in cells dying from mitotic catastrophe. In conclusion, we have demonstrated for the first time that the anticancer agent R-Crizotinib promotes endothelial cell dysfunction, leading to susceptibility and exacerbation of PAH in animal models. Previous studies have demonstrated the importance of epigenetic marks in the development and progression of PAH. Indeed, we were interested in the second part of the thesis, in the epigenetic factor "G9a", which was found to be overexpressed in different types of cancers, promoting cell survival and proliferation. Given the similarities between PAH and cancer, G9a was the ideal candidate to study in PAH. Thus, the main objective of Chapter 2 is to determine if G9a is involved in the progression and pathogenesis of PAH and to determine if its inhibition is beneficial in PAH animal models. We demonstrated that G9a is overexpressed in PAs of PAH patients and in experimental models. In vitro, we found that pharmacological inhibition of G9a using BIX01294 drastically reduces the PAH-PASMC proliferation and survival. Through RNA sequencing analysis, we demonstrated that G9a inhibition is accompanied by an impaired autophagy flux and lipid accumulation. Finally, therapeutic treatment with BIX01294 reduced pulmonary vascular remodeling as well as mPAP in an experimental rat model and also improved pulmonary hemodynamics and right ventricular function in another PAH mouse model. These results suggest that G9a inhibition could represent a new therapeutic approach in PAH

Integrated molecular signaling involving mitochondrial dysfunction and alteration of cell metabolism induced by tyrosine kinase inhibitors in cancer

Cancer cells have unlimited replicative potential, insensitivity to growth-inhibitory signals, evasion of apoptosis, cellular stress, and sustained angiogenesis, invasiveness and metastatic potential. Cancer cells adequately adapt cell metabolism and integrate several intracellular and redox signaling to promote cell survival in an inflammatory and hypoxic microenvironment in order to maintain/expand tumor phenotype. The administration of tyrosine kinase inhibitor (TKI) constitutes the recommended therapeutic strategy in different malignancies at advanced stages. There are important interrelationships between cell stress, redox status, mitochondrial function, metabolism and cellular signaling pathways leading to cell survival/death. The induction of apoptosis and cell cycle arrest widely related to the antitumoral properties of TKIs result from tightly controlled events involving different cellular compartments and signaling pathways. The aim of the present review is to update the most relevant studies dealing with the impact of TKI treatment on cell function. The induction of endoplasmic reticulum (ER) stress and Ca2+ disturbances, leading to alteration of mitochondrial function, redox status and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways that involve cell metabolism reprogramming in cancer cells will be covered. Emphasis will be given to studies that identify key components of the integrated molecular pattern including receptor tyrosine kinase (RTK) downstream signaling, cell death and mitochondria-related events that appear to be involved in the resistance of cancer cells to TKI treatments.This study was funded by Institute of Health Carlos III (ISCiii) (PI16/00090, PI19/00838 and PI19/01266), Spanish Ministry of Economy and Competitiveness (BFU2016-80006-P), Andalusian Ministry of Economy, Innovation, Science and Employment (BIO-216 and CTS-6264), Andalusian Ministry of Equality, Health and Social Policies (PI-0198-2016) and Valencian Ministry of Education, Culture and Sports (PROMETEO/2019/027). P de la C-O was supported by FPU predoctoral fellowship (FPU17/00026) from Spanish Ministry of Education, Culture and Sports. E N-V was supported by the the predoctoral i-PFIS IIS-enterprise contract in science and technologies in health (IFI18/00014) from ISCiii. We thank the Biomedical Research Network Center for Cardiovascular Diseases (CIBERcv), and the Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by the ISCiii and co-financed by European Regional Development Fund (ERDF) "A way to achieve Europe" for their financial support

The role of autophagy in resistance to targeted therapies

Autophagy is a self-degradative cellular process, involved in stress response such as starvation, hypoxia, and oxidative stress. This mechanism balances macro-molecule recycling to regulate cell homeostasis. In cancer, autophagy play a role in the development and progression, while several studies describe it as one of the key processes in drug resistance. In the last years, in addition to standard anti-cancer treatments such as chemotherapies and irradiation, targeted therapy became one of the most adopted strategies in clinical practices, mainly due to high specificity and reduced side effects. However, similar to standard treatments, drug resistance is the main challenge in most patients. Here, we summarize recent studies that investigated the role of autophagy in drug resistance after targeted therapy in different types of cancers. We highlight positive results and limitations of pre-clinical and clinical studies in which autophagy inhibitors are used in combination with targeted therapies. Refereed/Peer-reviewe

Targeting YAP to overcome acquired resistance to ALK inhibitors in ALK-rearranged lung cancer

Clinical benefit of ALK tyrosine kinase inhibitors (ALK-TKIs) in ALK-rearranged lung cancer has been limited by the inevitable development of acquired resistance, and bypass-molecular resistance mechanisms remain poorly understood. We investigated a novel therapeutic target through screening FDA-approved drugs in ALK-TKI-resistant models. Cerivastatin, the rate-limiting enzyme inhibitor of the mevalonate pathway, showed anti-cancer activity against ALK-TKI resistance in vitro/in vivo, accompanied by cytoplasmic retention and subsequent inactivation of transcriptional co-regulator YAP. The marked induction of YAP-targeted oncogenes (EGFR, AXL, CYR61, and TGFβR2) in resistant cells was abolished by cerivastatin. YAP silencing suppressed tumor growth in resistant cells, patient-derived xenografts, and EML4-ALK transgenic mice, whereas YAP overexpression decreased the responsiveness of parental cells to ALK inhibitor. In matched patient samples before/after ALK inhibitor treatment, nuclear accumulation of YAP was mainly detected in post-treatment samples. High expression of YAP in pretreatment samples was correlated with poor response to ALK-TKIs. Our findings highlight a crucial role of YAP in ALK-TKI resistance and provide a rationale for targeting YAP as a potential treatment option for ALK-rearranged patients with acquired resistance to ALK inhibitors.ope

Molecular pathways and therapeutic targets in lung cancer

Lung cancer is still the leading cause of cancer death worldwide. Both histologically and molecularly lung cancer is heterogeneous. This review summarizes the current knowledge of the pathways involved in the various types of lung cancer with an emphasis on the clinical implications of the increasing number of actionable molecular targets. It describes the major pathways and molecular alterations implicated in the development and progression of non-small cell lung cancer (adenocarcinoma and squamous cancer), and of small cell carcinoma, emphasizing the molecular alterations comprising the specific blueprints in each group. The approved and investigational targeted therapies as well as the immune therapies, and clinical trials exploring the variety of targeted approaches to treatment of lung cancer are the main focus of this review

Modulation thérapeutique de l’autophagie dans les lymphomes anaplasiques à grandes cellules ALK positif

Au cours de mon doctorat (Toulouse, 1996-1999, INSERM U326, Dir : Dr B. Payrastre) et de mon premier stage post-doctoral (Bruxelles, 1999-2001, IRIBHM, Dir : Dr C. Erneux), j’ai acquis une expertise dans le domaine de la signalisation cellulaire, en particulier dans la régulation du métabolisme des phosphoinositides, dans deux modèles cellulaires hématopoïétiques (plaquette sanguine humaine et lignée de leucémie myéloïde chronique). Mes travaux dans ces deux laboratoires ont porté sur le rôle des enzymes SHIP1 et SHIP2, deux inositol 5-phosphatases, impliquées dans la dégradation du second messager lipidique PI(3,4,5)P3 en PI(3,4)P2. Mes travaux de thèse ont montré que SHIP1 et SHIP2 participaient activement à la signalisation plaquettaire induite par la thrombine en permettant une nouvelle voie de biosynthèse du PI(3,4)P2. Puis mes travaux de post-doctorat ont démontré les propriétés anti-tumorales de la protéine SHIP2 lors de sa surexpression dans la lignée cellulaire K562, du fait de sa capacité, commune avec le suppresseur de tumeur PTEN, à hydrolyser le PI(3,4,5)P3.Mon intérêt croissant pour les mécanismes d’oncogenèse a alors motivé un second stage post-doctoral (Stanford, 2001-2004, CCSR, Dir : Dr D. Felsher) dans un laboratoire spécialisé dans l’étude des mécanismes d’ « addiction oncogénique », de par l’utilisation de différents modèles murins conditionnels de tumorigenèse. Mes travaux ont mis en évidence : (i) le rôle clé du microenvironnement tumoral, et notamment de l’angiogenèse, dans la survenue de rechutes tumorales après inactivation de l’oncogène MYC dans un modèle de lymphome dépendant de cet oncogène ; (ii) l’efficacité de la combinaison thérapeutique : inactivation de l’oncogène MYC et blocage de l’angiogenèse en terme de prévention de ces rechutes.À mon retour en France (Toulouse, 2004-2008, INSERM U563, Dir : Pr G. Delsol), j’ai mis à profit mon expérience américaine pour développer et caractériser des modèles cellulaires et murins conditionnels pour l’expression de l’oncogène ALK (pour Anaplastic Lymphoma Kinase), ceci afin de compléter la grande spécialisation de mon laboratoire d’accueil sur l’étude des lymphomes anaplasiques à grandes cellules (LAGC) ALK positifs.Depuis mon recrutement à l’INSERM, en 2008, en tant que CR1, j’ai utilisé ces modèles cellulaires et animaux pour démontrer (i) que ces lymphomes ALK-positif présentent une addiction pour l’oncogène ALK ; (ii) que l’angiogenèse, et notamment le VEGF (régulé en partie par le microARN 16), participe au développement tumoral et représente donc une cible thérapeutique potentielle; et enfin (iii), que l’inhibition de l’autophagie cytoprotectrice, activée sous traitement Crizotinib, améliorait l’efficacité des thérapies ciblant l’oncogène ALK.Les projets de recherche que je souhaite développer aujourd’hui s’articulent autour de deux mots-clefs : les lymphomes anaplasiques à grandes cellules ALK-positif et l’autophagie. Ils se subdivisent en trois grands axes de recherche : (i) le rôle, (ii) la régulation et (iii) la modulation thérapeutique de l’autophagie dans ces lymphomes ALK positif. Le premier axe consiste à définir le rôle cytoprotecteur ou cytotoxique de l’autophagie sous diverses thérapies ou combinaisons thérapeutiques actuellement proposées pour améliorer le traitement des patients; le second vise à identifier les mécanismes de régulation post-transcriptionnelle de l’autophagie (notamment via les microARNs) ; et le troisième porte sur le développement d’une nouvelle formulation vaccinale anti- ALK, à base d’autophagosomes, pour compléter les thérapies ciblant l’oncogène ALK, et prévenir, sur le long terme, l’apparition de rechutes tumorales

MECHANISMS UNDERLYING THE SENSITIVITY AND RESISTANCE OF GASTRIC CANCER CELLS TO MET INHIBITORS

MET amplification has been clinically credentialed as a therapeutic target in gastric cancer, but the molecular mechanisms underlying sensitivity and resistance to MET inhibitors are still not well understood. Using whole-genome mRNA expression profiling, we identified autophagy as a top molecular pathway that was activated by the MET inhibitor crizotinib in drug-sensitive human gastric cancer cells, and functional studies confirmed that crizotinib increased autophagy levels in the drug sensitive cells in a concentration-dependent manner. We then used chemical and molecular approaches to inhibit autophagy in order to define its role in cell death. The clinically available inhibitor of autophagy, chloroquine, or RNAi-mediated knockdown of two obligate components of the autophagy pathway (ATG5 and ATG7) blocked cell death induced by crizotinib or RNAi-mediated knockdown of MET, and mechanistic studies localized the effects of autophagy to cytochrome c release from the mitochondria. Overall, the data reveal a novel relationship between autophagy and apoptosis in gastric cancer cells exposed to MET inhibitors. The observations suggest that autophagy inhibitors should not be used to enhance the effects of MET inhibitors in gastric cancer patients