Astrocytes reverted to a neural progenitor-like state with transforming growth factor alpha are sensitized to cancerous transformation.

International audienceGliomas, the most frequent primitive central nervous system tumors, have been suggested to originate from astrocytes or from neural progenitors/stem cells. However, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. Transforming growth factor (TGF)-alpha, an epidermal growth factor family member, is frequently overexpressed in the early stages of glioma progression. We previously demonstrated that prolonged exposure of astrocytes to TGF-alpha is sufficient to trigger their reversion to a neural progenitor-like state. To determine whether TGF-alpha dedifferentiating effects are associated with cancerous transforming effects, we grafted intracerebrally dedifferentiated astrocytes. We show that these cells had the same cytogenomic profile as astrocytes, survived in vivo, and did not give birth to tumors. When astrocytes dedifferentiated with TGF-alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. In contrast, irradiation did not modify the lifespan of astrocytes cultivated in serum-free medium. Addition of TGF-alpha after irradiation did not promote their transformation but decreased their lifespan. These results demonstrate that reversion of mature astrocytes to an embryonic state without genomic manipulation is sufficient to sensitize them to oncogenic stress

A biobank of pediatric patient-derived-xenograft models in cancer precision medicine trial MAPPYACTS for relapsed and refractory tumors

Pediatric patients with recurrent and refractory cancers are in most need for new treatments. This study developed patient-derived-xenograft (PDX) models within the European MAPPYACTS cancer precision medicine trial (NCT02613962). To date, 131 PDX models were established following heterotopical and/or orthotopical implantation in immunocompromised mice: 76 sarcomas, 25 other solid tumors, 12 central nervous system tumors, 15 acute leukemias, and 3 lymphomas. PDX establishment rate was 43%. Histology, whole exome and RNA sequencing revealed a high concordance with the primary patient's tumor profile, human leukocyte-antigen characteristics and specific metabolic pathway signatures. A detailed patient molecular characterization, including specific mutations prioritized in the clinical molecular tumor boards are provided. Ninety models were shared with the IMI2 ITCC Pediatric Preclinical Proof-of-concept Platform (IMI2 ITCC-P4) for further exploitation. This PDX biobank of unique recurrent childhood cancers provides an essential support for basic and translational research and treatments development in advanced pediatric malignancies

A biobank of pediatric patient-derived-xenograft models in cancer precision medicine trial MAPPYACTS for relapsed and refractory tumors

Pediatric patients with recurrent and refractory cancers are in most need for new treatments. This study developed patient-derived-xenograft (PDX) models within the European MAPPYACTS cancer precision medicine trial (NCT02613962). To date, 131 PDX models were established following heterotopical and/or orthotopical implantation in immunocompromised mice: 76 sarcomas, 25 other solid tumors, 12 central nervous system tumors, 15 acute leukemias, and 3 lymphomas. PDX establishment rate was 43%. Histology, whole exome and RNA sequencing revealed a high concordance with the primary patient's tumor profile, human leukocyte-antigen characteristics and specific metabolic pathway signatures. A detailed patient molecular characterization, including specific mutations prioritized in the clinical molecular tumor boards are provided. Ninety models were shared with the IMI2 ITCC Pediatric Preclinical Proof-of-concept Platform (IMI2 ITCC-P4) for further exploitation. This PDX biobank of unique recurrent childhood cancers provides an essential support for basic and translational research and treatments development in advanced pediatric malignancies

The Chemokine Receptor CXCR4 Strongly Promotes Neuroblastoma Primary Tumour and Metastatic Growth, but not Invasion

Neuroblastoma (NB) is a heterogeneous, and particularly malignant childhood neoplasm in its higher stages, with a propensity to form metastasis in selected organs, in particular liver and bone marrow, and for which there is still no efficient treatment available beyond surgery. Recent evidence indicates that the CXCR4/CXCL12 chemokine/receptor axis may be involved in promoting NB invasion and metastasis. In this study, we explored the potential role of CXCR4 in the malignant behaviour of NB, using a combination of in vitro functional analyses and in vivo growth and metastasis assessment in an orthotopic NB mouse model. We show here that CXCR4 overexpression in non-metastatic CXCR4-negative NB cells IGR-NB8 and in moderately metastatic, CXCR4 expressing NB cells IGR-N91, strongly increased tumour growth of primary tumours and liver metastases, without altering the frequency or the pattern of metastasis. Moreover shRNA-mediated knock-down experiments confirmed our observations by showing that silencing CXCR4 in NB cells impairs in vitro and almost abrogates in vivo growth. High levels of CXCL12 were detected in the mouse adrenal gland (the primary tumour site), and in the liver suggesting a paracrine effect of host-derived CXCL12 on NB growth. In conclusion, this study reveals a yet unreported NB-specific predominant growth and survival-promoting role of CXCR4, which warrants a critical reconsideration of the role of CXCR4 in the malignant behaviour of NB and other cancers

EXPLORATION OF ANGIOGENESIS INHIBITION IN NEUROBLASTOMA AND CHARACTERIZATION OF ESCAPE MECHANISMS

Adulte ou pédiatrique, les tumeurs solides ont besoin d’oxygène et de nutriments pour se développer et métastaser. Leur apport est assuré par la néo-vascularisation tumorale issue d’un processus multifactoriel appelé l’angiogénèse. Son équilibre est maintenu par une balance entre facteurs pro- et anti-angiogéniques. Elle fait partie des principales cibles pour traiter les cancers et l’inhibition de la voie VEGF en est un facteur clé. Cependant, la réponse aux agents anti-angiogéniques a montré, malgré des résultats encourageants, un effet transitoire associé à l’apparition d’une résistance adaptative de la tumeur.Nous avons étudié l’inhibition de l’angiogénèse et les mécanismes potentiels d’échappement dans les tumeurs pédiatriques solides, et principalement dans le neuroblastome. Le neuroblastome est une tumeur originaire de la crête neurale et atteint généralement l’enfant jeune. Nous avons exploré l’effet anti-tumoral de l’inhibition sélective des récepteurs 1, 2, 3 du VEGF à l’aide de l’inhibiteur à tyrosine kinase axitinib dans divers modèles précliniques de neuroblastome. L’axitinib a montré une activité anti-tumorale modérée associée à une inhibition de la vascularisation. Néanmoins, après un traitement prolongé in vitro, les cellules tumorales IGR-N91-Luc deviennent résistantes à l’axitinib. Elles prolifèrent normalement mais secrètent de «l’ hepatocyte growth factor» (HGF) et activent la voie MAPK. In vivo, le traitement prolongé par axitinib entraîne le développement d’un phénotype plus agressif de la tumeur avec l’augmentation du nombre d’animaux présentant des métastases, associée à une activation de la voie SRC. Ceci nous a conduit à explorer l’effet d’une inhibition ciblant principalement VEGFR2 et MET (récepteur à l’HGF) avec le cabozantinib. Ainsi nous avons contrôlé le développement tumoral en inhibant la néo-vascularisation et l’activation de SRC, et induit la mort cellulaire dans le modèle orthotopique IGR-N91-Luc et inhibé le développement métastatique dans le modèle systémique IMR-32-Luc. Par ailleurs, nous avons étendu notre exploration à d’autres facteurs jouant un rôle dans l’angiogénèse comme FGFR ou PDGFR car ils représentent, comme MET, de puissants oncogènes. Pour cibler simultanément VEGFR et PDGFR, nous avons utilisé l’inhibiteur multi-kinase regorafenib. In vivo, à des doses bien tolérées qui permettent un traitement prolongé, le regorafenib a montré une activité anti-tumorale significative. Cet effet a été associé principalement à une forte inhibition de la vascularisation mais également à l’induction de la mort cellulaire. En élargissant notre étude à d’autres modèles de tumeurs pédiatriques, nous avons observé que son activité est indépendante du type histologique. Compte tenu du caractère oncogénique de PDGFR, nous avons évalué cet inhibiteur dans des modèles présentant une amplification du gène PDGFRA, qui entraine une surexpression et une activation forte du récepteur. Combiné avec des thérapies standards capables d’induire des dommages à l’ADN telles que l’irradiation ou l’irinotecan, l’effet du regorafenib a été potentialisé, notamment dans les modèles amplifiés pour le gène PDGFRA se traduisant par des régressions tumorales. Ces évaluations précliniques soutiennent le développement d’une nouvelle stratégie thérapeutique pour les enfants atteints de tumeurs solides.Solid tumors either adult or pediatric need oxygen and nutrients to grow and metastasize. Their input is provided by tumor neovascularization after a multifactorial process called angiogenesis. Balance is maintained by equilibrium between pro and anti-angiogenic factors. It is one of the main targets for treating cancers and the inhibition of the VEGF pathway is a key factor. However, despite encouraging results, the response to anti-angiogenic agents showed a transient effect associated with the development of an adaptive tumor resistance. We studied the inhibition of angiogenesis and potential escape mechanisms in solid pediatric tumors, mainly in neuroblastoma. Neuroblastoma is a solid tumor derived from the neural crest and it usually affects childhood. We investigated the anti-tumor effect of selective inhibition of VEGF receptors 1, 2, 3 using the tyrosine kinase inhibitor axitinib in various preclinical neuroblastoma l models. Axitinib showed a moderate anti-tumor activity associated with the inhibition of vascularization. However, after prolonged treatment in vitro, tumor cells IGR-N91-Luc become resistant to axitinib. They proliferate normally but secrete the "hepatocyte growth factor" (HGF) and activate the MAPK pathway. In vivo, prolonged treatment by axitinib results in the development of a more aggressive tumor phenotype with an increase in the number of animals exhibiting metastases associated with an activation of SRC signaling. This led us to explore the effect of inhibiting concomitant VEGFR2 and MET (HGF receptor), main cabozantinib targets. So we stabilized tumor growth by inhibiting the neovascularization and activation of SRC, induced cell death in the orthotopic model IGR-N91-Luc and inhibited metastatic development in the IMR-32-Luc systemic model. In addition, we extended our exploration of other factors that play a role in angiogenesis like FGFR or PDGFR because they represent, like MET, powerful oncogenes. To simultaneously target VEGFR and PDGFR, we used the multi-kinase inhibitor regorafenib. In vivo, at well-tolerated doses that allow prolonged treatment, regorafenib showed significant anti-tumor activity. This effect was mainly associated with a strong inhibition of vascularization, but also (with) induction of cell death. By expanding our study to other models of pediatric tumors, we observed that its activity was independent of histologic type. Given the oncogenic character of PDGFR, we evaluated the inhibitor in models which present a PDGFRA gene amplification, which results in a strong activation of the receptor. Combined with standard therapies that can induce DNA damages such as irinotecan or radiation, the effect of regorafenib was potentiated, mainly in PDGFRA gene amplified models, where tumor regressions were obtained. These preclinical evaluations support the development of a new therapeutic strategy for children with solid tumors

Histone deacetylase inhibitor panobinostat induces antitumor activity in epithelioid sarcoma and rhabdoid tumor by growth factor receptor modulation

International audienceBackground: Epithelioid sarcomas and rhabdoid tumors are rare, aggressive malignancies with poor prognosis. Both are characterized by INI1 alterations and deregulation of growth factor receptors albeit their interaction has not been elucidated. Methods: In this study, we investigated the activity of a panel of epigenetic modulators and receptor tyrosine kinase inhibitors in vitro on respective cell lines as well as on primary patient-derived epithelioid sarcoma cells, and in vivo on xenografted mice. Focusing on histone deacetylase (HDAC) inhibitors, we studied the mechanism of action of this class of agents, its effect on growth factor receptor regulation, and changes in epithelial-tomesenchymal transition by using cell-and RT-qPCR-based assays. Results: Pan-HDAC inhibitor panobinostat exhibited potent anti-proliferative activity at low nanomolar concentrations in A204 rhabdoid tumor, and VAESBJ/GRU1 epithelioid sarcoma cell lines, strongly induced apoptosis, and resulted in significant tumor growth inhibition in VAESBJ xenografts. It differentially regulated EGFR, FGFR1 and FGFR2, leading to downregulation of EGFR in epithelioid sarcoma and to mesenchymal-to-epithelial transition whereas in rhabdoid tumor cells, EGFR was strongly upregulated and reinforced the mesenchymal phenotype. All three cell lines were rendered more susceptible towards combination with EGFF inhibitor erlotinib, further enhancing apoptosis. Conclusions: HDAC inhibitors exhibit significant anticancer activity due to their multifaceted actions on cytotoxicity, differentiation and drug sensitization. Our data suggest that the tailored, tissue-specific combination of HDAC inhibitors with therapeutics which target cellular salvage mechanisms might increase their therapeutic relevance

Polo-like Kinase Inhibitor Volasertib Exhibits Antitumor Activity and Synergy with Vincristine in Pediatric Malignancies

Polo-like kinase 1 (PLK1) controls the main cell-cycle checkpoints, suggesting utility of its inhibition for cancer treatment, including of highly proliferative pediatric cancer. This preclinical study explored the selective PLK1 inhibitor volasertib (BI 6727) alone and combined with chemotherapy in pediatric malignancies. Inhibition of proliferation was explored in vitro using dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assay. Mice bearing human xenografts were treated with weekly intravenous injections of volasertib. Volasertib inhibited proliferation in all 40 cell lines tested, with a mean half-maximal growth inhibitory concentration of 313 nmol/l (range: 4-5000 nmol/l). Volasertib was highly active against RMS-1 alveolar rhabdomyosarcoma xenografts, resulting in 100% tumor regression. Activity was associated with complete and prolonged G2/M arrest and subsequent apoptotic cell death. Volasertib showed synergistic activity with vincristine but antagonistic effects with etoposide. These findings support the further exploration of volasertib for pediatric malignancies, particularly alveolar rhabdomyosarcoma, and its combination with mitotic spindle poiso

Induction of glutathione synthesis explains pharmacodynamics of high-dose busulfan in mice and highlights putative mechanisms of drug interaction.

Busulfan is an example of a drug eliminated through glutathione S-transferase (GST)-catalyzed conjugation with reduced glutathione (GSH). We studied the pharmacokinetics and toxicity of busulfan in C57BL6 mice in correlation with liver GST activity and GSH synthesis by accurate determination of precursors, namely, gamma-glutamyl-cysteine and cysteine. A significantly lower incidence of acute toxicity was observed in mice receiving busulfan 16.5 mg/kg twice a day compared with animals receiving 33 mg/kg once a day. In both cases, a total dose of 132 mg/kg was administered over 4 days. The difference in toxicity was explained by pharmacokinetics since a strong induction of clearance was observed only in animals treated twice daily. Induction of metabolism was correlated with an increase in liver cysteine content and enhanced glutathione synthesis rate, whereas GST activity was unchanged. To our knowledge, this is the first time that in vivo flux of GSH synthesis has been shown to be closely related to a drug plasma clearance and toxicity. These results allow hypothesizing that GSH liver synthesis may directly influence busulfan clearance in humans with possible implications in the occurrence of hepatic veno-occlusive disease