L'implication de l'autophagie dans la chimiorésistance du neuroblastome et l'intérêt de son inhibition

Le neuroblastome (NB) est une tumeur fréquente et agressive du jeune enfant. Les tumeurs de haut grade de forme métastatique, généralement développées chez l'enfant de plus de 1 an, sont associées à une importante mortalité malgré un traitement lourd incluant une chimiothérapie à haute dose. La chimiorésistance est donc un problème majeur qui caractérise ces NB de haut grade. Une des hypothèses pour expliquer cette chimiorésistance est celle de l’activation de l’autophagie, un mécanisme auquel recourent les cellules afin de faire face aux situations de stress. D’ailleurs, plusieurs études ont démontré que l'autophagie était activée à la suite des thérapies anticancéreuses. Son inhibition pourrait donc représenter une stratégie thérapeutique très intéressante pour le traitement de cancers. Le but de ce projet de recherche a été de mettre en évidence l'importance de l'autophagie dans les cellules du NB ainsi que l'effet de son inhibition sur la sensibilité des cellules du NB à la chimiothérapie conventionnelle. La présence d'autophagie dans les cellules de NB et sa valeur pronostic ont été évaluées par une étude immunohistochimique et par western blot sur 184 tumeurs patient. Ensuite, dans le but de déterminer l'effet de la chimiothérapie conventionnelle sur le niveau d'autophagie, des études in vitro sur 6 lignées cellulaires ont été effectuées utilisant des tests de mesure d'autophagie (MDC, monodanylcadaverine), de viabilité cellulaire (MTT) et de western blot. Celles ci ont été suivies par des tests d'inhibition de l'autophagie par deux méthodes: l’inactivation du gène ATG5 par un lentivirus contenant un shRNA ciblant ATG5 ou de l'hydroxychloroquine (HCQ), un inhibiteur pharmacologique de l’autophagie. Cette inhibition a été testée seule ou en combinaison avec une chimiothérapie conventionnelle dans le but de déterminer le rôle de l'autophagie dans la sensibilisation des cellules de NB à la chimiothérapie. Ensuite, l’intérêt de l’inhibition de l’autophagie a été évalué sur des modèles murins. Enfin, le niveau d'autophagie a été testé dans des cellules souches de NB. Notre étude a démonté que l'autophagie est présente à un niveau basal dans une majorité des NB mais qu'elle ne représentait pas un facteur pronostic dans ce type de tumeur. Les différentes chimiothérapies testées induisent une augmentation de l'autophagie dans les cellules du NB. Les deux tests d'inhibition ont démontré in vitro que l'autophagie participe à la résistance des cellules aux traitements chimiothérapeutiques classiques du NB. Le blocage de l’autophagie in vivo augmente l’efficacité de la chimiothérapie, cependant certaines données associées au traitement par HCQ devront être complétées. Cette étude démontre que l'inhibition de l'autophagie en combinaison avec la chimiothérapie classique représente une approche thérapeutique prometteuse dans le traitement du NB.Neuroblastoma (NB) is a common tumor in childhood. Despite major advances in treatments, NB still have a poor prognosis with 40% of mortality. Chemoresistance is a major issue that characterizes aggressive NB. This is a consequence of an autophagic mechanism that tumor cells use to overcome stressful situations encountered during treatments. Autophagy has been the subject of several studies showing its activation in response to anticancer therapies. Its inhibition may therefore represent a very interesting therapeutic strategy for cancer treatment. The purpose of this research was to determine the presence of autophagy in NB cells and the effect of autophagy inhibition in sensitizing NB cells to conventional chemotherapy. The presence of autophagy was verified in 184 NB tumors. To determine the effect of conventional chemotherapy on autophagy, the MTT cell viability test and the autophagy measurement test (MDC, monodansylcadaverine) have been used to study 6 NB cell lines in vitro. An immunohistochemical study also allowed the verification of autophagy activation in tumors grown in mouse models. A lentivirus containing a shRNA against ATG5 was used to generate autophagy deficient cells. Using the MTT and MDC tests, we assessed their sensitivity to chemotherapy. In order to determine the effect of HCQ in sensitizing NB cells to chemotherapy, we used HCQ alone or in combination with conventional chemotherapy. This study demonstrated that autophagy is present at a basal level in NB cells. Unlike for LC3, the results showed that Beclin 1 is a factor of poor prognosis. The MTT and MDC tests have shown that vincristine, doxorubicin, cisplatin, temozolomide, rapamycin, and LY294002 induce autophagy in NB cells. Also, immunohistochemical studies showed that cisplatin treatment increased autophagy in vivo in xenograft model of human NB in mice. ATG5 deficient cells showed greater sensitivity to chemotherapy. Furthermore, the use of these cells in mouse models showed an important role of autophagy in tumor progression as well as an increased sensitivity to vincristine. Finally, combination of HCQ with conventional chemotherapy showed an increased sensitivity of NB cells to chemotherapy compared to cells receiving chemotherapy only. This study demonstrates that inhibition of autophagy in combination with conventional chemotherapy is an attractive therapeutic approach for the treatment of NB

Nouvelles approches thérapeutiques pour prévenir les rechutes du neuroblastome : étude préclinique et translationnelle

Le neuroblastome (NB) est la tumeur extra-crânienne la plus fréquente du jeune enfant. Malgré une thérapie multimodale très agressive, 40% des patients atteints de NB à haut risque rechutent. Le traitement de ces patients consiste à éliminer la tumeur par chirurgie, radiothérapie et chimiothérapie, à reconstituer la moelle osseuse par une greffe de cellules souches autologues et enfin à éliminer la maladie résiduelle (MRD) par une immunothérapie visant l’antigène GD2 exprimé par les neuroblastes. Notre étude préclinique a examiné l’efficacité de deux stratégies de traitements qui visent à potentialiser les thérapies actuelles et réduire leur toxicité. La première consiste à réduire la masse tumorale par la radiothérapie ciblée combinée à des radiosensibilisants. La deuxième approche est basée sur l’activation des cellules natural killer (NK) pour potentialiser l’effet de l’immunothérapie anti-GD2 et éliminer la MRD. L’autophagie est un processus catabolique qui élimine les protéines et organelles endommagées par différents stress incluant les irradiations. Par conséquent, inhiber l’autophagie pourrait sensibiliser les neuroblastes aux irradiations. Or, nous avons montré qu’étant très radiosensibles, les neuroblastes ne sont pas davantage éliminés par les irradiations quand ils sont traités avec un inhibiteur de l’autophagie. De plus, l’absence d’un inhibiteur efficace de l’autophagie à usage thérapeutique ne permet pas actuellement d’adopter cette approche. Notre étude a également permis de révéler une nouvelle approche de stimulation des cellules NK par les cellules dendritiques plasmacytoïdes (pDC) activées par un ligand du récepteur Toll-like, capable d’éradiquer la MRD et prévenir les rechutes de NB. Nos résultats ont permis, d’une part, d’élucider les mécanismes impliqués dans la lyse des cellules NK activées par les pDC contre les neuroblastes et, d’une autre part, de démontrer que l’axe pDC-NK chez le patient est fonctionnel, augmente l’efficacité de l’anti-GD2 et élimine efficacement les neuroblastes. Ainsi, l’immunothérapie par les cellules NK est une stratégie très prometteuse pour traiter le NB. Cette étude préclinique servira de base à l’élaboration d’un essai clinique pour traiter les enfants atteints de NB au CHU Sainte Justine.Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Despite aggressive multimodal therapy, 40% of patients with high-risk NB relapse. The current therapy comprises an induction treatment with chemotherapy and surgery, a consolidation treatment including radiotherapy and high-dose chemotherapy followed by bone marrow rescue with autologous hematopoietic stem cell transplantation and finally anti-GD2 immunotherapy targeting the disialoganglioside (GD2) antigen expressed by neuroblasts to treat minimal residual disease (MRD). Our preclinical study proposes two treatment strategies to potentiate current therapies and reduced toxicities. The first aim to reduce tumor mass by targeted radiotherapy combined with radiosensitizers. The second approach is based on the activation of natural killer (NK) cells to potentiate the effect of anti-GD2 therapy and eliminate MRD. Autophagy is a catabolic process that recycle damaged proteins and organelles, induced under various conditions of cellular stress including irradiation. Therefore, inhibiting autophagy could sensitize neuroblasts to irradiation. However, our study showed that neuroblasts were highly sensitive to irradiation and autophagy inhibitor failed to increase neuroblasts sensitization to irradiation. In addition, the absence of a potent autophagy inhibitor for therapeutic use does not allow this approach to be adopted. Our preclinical study demonstrated a novel approach based on NK cell stimulation with Toll-like activated plasmacytoid dendritic cells (pDC) that enhances the efficacy of anti-GD2 immunotherapy and prevent NB relapse. We elucidated the mechanisms involved in pDC-activated NK cells killing of neuroblasts. We further demonstrated that neuroblasts were efficiently killed by patient’s NK cells after stimulation by activated pDC. This is further increased by the addition of anti-GD2 antibody. Altogether, our study demonstrates that NK cell-based immunotherapy has a real potential to enhance anti-GD2 immunotherapy effect and prevent NB relapse. This preclinical study will serve as a basis for the development of a clinical trial to treat children with NB at CHU Sainte Justine

Efficient Killing of High Risk Neuroblastoma Using Natural Killer Cells Activated by Plasmacytoid Dendritic Cells

<div><p>High-risk neuroblastoma (NB) remains a major therapeutic challenge despite the recent advent of disialoganglioside (GD2)-antibody treatment combined with interleukin (IL)-2 and granulocyte monocyte-colony stimulating factor (GM-CSF). Indeed, more than one third of the patients still die from this disease. Here, we developed a novel approach to improve the current anti-GD2 immunotherapy based on NK cell stimulation using toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDCs). We demonstrated that this strategy led to the efficient killing of NB cells. When the expression of GD2 was heterogeneous on NB cells, the combination of pDC-mediated NK-cell activation and anti-GD2 treatment significantly increased the cytotoxicity of NK cells against NB cells. Activation by pDCs led to a unique NK-cell phenotype characterized by increased surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), with increased expression of CD69 on CD56^dim cytotoxic cells, and strong interferon-γ production. Additionally, NB-cell killing was mediated by the TRAIL death-receptor pathway, as well as by the release of cytolytic granules via the DNAX accessory molecule 1 pathway. NK-cell activation and lytic activity against NB was independent of cell contact, depended upon type I IFN produced by TLR-9-activated pDCs, but was not reproduced by IFN-α stimulation alone. Collectively, these results highlighted the therapeutic potential of activated pDCs for patients with high-risk NB.</p></div

NK-cell activation is independent of contact with pDCs, but depends upon type I IFN production by activated pDCs.

<p>A—Analysis of NK-cell cytotoxicity following NK-cell activation with TLR9-stimulated pDCs in contact or in trans-wells. Graphs are representative of three independent experiments. B—Blocking of type I IFN signaling was performed using specific blocking mAbs against IFN-α and type I IFN receptor in NK/pDC co-cultures, followed by cytotoxicity assays performed against SK-N-DZ cells. The graph represents the means of three independent experiments, with error bars representing the standard error of the mean. C—Analysis of NK-cell cytotoxicity following NK-cell activation with TLR9-stimulated pDCs (a-pDC) or IFN-α (1000 IU/mL). The IFN-α concentration was comparable to that estimated under both conditions. Specific lysis of SK-N-DZ, SK-N-AS, and SJNB-7 cells are presented for an E:T ratio of 5:1. Statistical analyses were performed using one-way analysis of variance. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. E:T, effector against target; IFN, interferon; IL, interleukin; mAb, monoclonal antibody; NK, natural killer; pDC, plasmacytoid dendritic cell; TLR, toll-like receptor.</p

Additive effects of anti-GD2 mAb, ADCC, and pDC-activated NK-cell cytotoxicity against NB cells.

<p>A—Cytotoxicity assays were performed against SK-N-DZ, SJNB-7, and SK-N-AS cell lines in the presence or absence of prior incubation of target cells with the anti-GD2 mAb. Effectors were either unstimulated NK cells (NS), NK cells stimulated by overnight incubation with TLR-9-activated pDCs (a-pDC), or NK cells stimulated by low doses of IL-2 alone (IL2/GD2 mAb) or in combination (IL2/GD2 mAb+a-pDC). Graphs present the specific lysis means of three independent experiments, with error bars representing the standard error of the mean. B—NK cell-degranulation assays were performed against SK-N-DZ, SJNB7, and SK-N-AS cells. Unstimulated NK (NS) and stimulated NK cells (a-pDC, IL2/GD2 mAb, or IL2/GD2 mAb+a-pDC) were incubated with target cells at a 1:1 ratio and stained with the anti-CD107a antibody. Graphs represent the means of CD107a MFIs on NK cells (gated on CD56⁺CD3⁻) in three independent experiments. Statistical analyses were performed using one-way analysis of variance. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. ADCC, antibody-dependent cellular cytotoxicity; CD, cluster of differentiation; GD2: disialoganglioside; IL, interleukin; mAb, monoclonal antibody; MFI, mean fluorescence intensity; NB, neuroblastoma; NK, natural killer; pDC, plasmacytoid dendritic cell; TLR, toll-like receptor.</p

Additional file 4: Figure S4. of Autophagy is associated with chemoresistance in neuroblastoma

Effect of ATG5 knockdown xenografts on tumor growth. (11/12) NSG Mice developed xenograft tumor from s.c injection of 5.106 sheGFP cells and only (3/12) from shATG5 cells (Table A). When tumor reached 200 mm3, mice were administered vincristine (0.4 mg/kg/day) or vehicle (saline) for 5 days. Progression of tumor volume was followed in each group. Each data point represents the mean 6 ± SE tumor volume for sheGFP cells and 1 or 2 for shAtg5 cells. At day 6, Tumors size from shATG5 cells were reduced compared with tumors from eGFP (B). Also, tumors from shATG5 cells were more sensitive to vincristine comparing to control cells. Immunohistochemistry was performed on the sections of tumors developed in the mouse model. with LC3 antibody and showed a high expression of LC3 II in control cells and a very low in cells ATG5 knockdown as well as treated (b, d) or not treated (a, c) (C). (PDF 259 kb

Additional file 3: Figure S3. of Autophagy is associated with chemoresistance in neuroblastoma

HCQ sensitizes NB cells to chemotherapy by inhibition of autophagy. A. N91-IGR or NB8-IGR cell viability was measured after vincristine or doxorubicin treatment combined or not to HCQ 30 µM. Results are expressed as percentage of corresponding control and represent mean ± SEM of 4 independent experiments. B. Autophagic activity of N91-IGR or NB8-IGR was measured using MDC agent after increasing concentration of vincristine or doxorubicin treatment combined or not to HCQ 30 µM. Fluorescence was quantified by spectrophotometer. (*: P < 0.05), (**: P < 0.01), (***: P < 0.001). (PDF 215 kb