Fenretinide sensitizes multidrug-resistant human neuroblastoma cells to antibody-independent and ch14.18-mediated NK cell cytotoxicity

Abstract

Neuroblastoma (NB) is an aggressive pediatric malignancy of the sympathetic nervous system responsible for 12% of cancer-associated mortality in children less than 15 years old. Recently, the addition of passive immunotherapy with the anti-GD2 antibody ch14.18 provided a clinical benefit with a 20% improvement in 2-year prevention of relapse for children with high-risk disease. However, still one third of the patients die from their disease emphasizing the need for improved therapy protocols. 4-HPR is a synthetic retinoid known to induce apoptosis in variety of cancer cells mainly via the accumulation of ceramides. 4-HPR is also known to induce the up regulation of death receptors resulting in an enhanced apoptosis via DR pathway. In our work we tested the hypothesis whether treatment with 4-HPR will enhance antibody- independent cellular cytotoxicity (AICC) and ch14.18-mediated anti- neuroblastoma effector functions (CDC, ADCC). We demonstrate that pre- treatment of fenretinide-resistant NB cells with 4-HPR significantly enhanced ch14.18-mediated CDC and ADCC as well as AICC by both human NK cells and PBMCs. Interestingly, we also found an increase of the GD2 binding index and an increase of DRs in all cell lines tested. Our data suggest that the increased overall cytotoxicity towards 4-HPR treated NB cells is based on an up-regulation of GD2 and DRs. Further, we could show that blocking of ganglioside synthesis results in decrease of GD2 expression and nearly complete abrogation of the 4-HPR-mediated increase of ch14.18-dependent cytotoxicity of effector cells and complement, emphasizing a crucial role for GD2 expression as a mechanism for 4-HPR-mediated sensitization of NB towards ch14.18-mediated immunotherapy. Interestingly, blocking of TRAIL-R2 resulted in a significant decrease of AICC, whereas blocking of Fas did not influence the increased AICC, however, the increased production of Granzyme B and perforin by effector cells upon co-culture with 4-HPR-treated NB cells strongly indicates the involvement of other supportive mechanisms, such as a change in expression levels of activating NK cells ligands on target cells. Finally, we could show that tumor xenografts isolated from mice that were treated with 4-HPR exhibit an increased GD2 binding index as well as DR expression compared to tumors isolated from vehicle treated animals. Importantly, this correlates well with a significantly higher ex vivo ADCC and AICC response of human NK cells towards 4-HPR treated tumors than to vehicle treated controls. Moreover, tumors from 4-HPR-treated mice showed a significantly higher percentage of tumor infiltrating NK cells compared to control tumors. In summary, new therapy protocols are needed in order to optimize treatment of children with high-risk NB. We can show here for the first time that 4-HPR treatment sensitizes drug-resistant human NB cells to ADCC and AICC by human NK cells and PBMCs, thereby providing an important baseline for the combination of 4-HPR and passive immunotherapy with ch14.18 in future clinical trials.

Das Neuroblastom ist der häufigste extrakranielle solide Tumor im Kindesalter, der von sympathischen Nerwengewebe ausgehet. Ca. 50% der Patienten gehören zum Zeitpunkt der Diagnosestellung zur Hochrisiko-Gruppe und haben eine 5-Jahres- Überlebensrate von ca. 40% trotz intensiver therapie. Anti GD2-Antikörper hat sich in Kombination- und Monotherapieen als vielversprechend erwiesen. Fenretinid (4-HPR) oder N-(4-hydroxyphenyl)retinamid – ein synthetisches Derivat des Vitamin A. Antitumor Effekt von 4-HPR wurde bereits in Phase I und II Studien bewiesen. 4-HPR induziert Apoptose und Nekrose über: Bildung von reaktiven Sauerstoffspezies (ROS), intrazelluläre Ansammlung von Ceramiden. Außerdem induziert 4-HPR die Heraufregulierung von Todesrezeptoren (DR‘s). In der vorliegenden Arbeit wurde getestet, ob die Vorbehandlung von Multidrug- resistente (MDR) Neuroblastom-Zellen mit 4-HPR die ch14.18-vermittelte Zytotoxizität (ADCC) via erhöhte GD2-Expression und Antikörper-unabhängige Zytotoxizität (AICC) via erhöhte DR-Expression erhöht. Es konnte gezeigt werden, dass 4-HPR erhöht die GD2-Expression auf 4-HPR-resistenten NB Zellen und dadurch ch14.18-vermittelte CDC und ADCC. Die Blockierung von Glucosylceramid-Synthase (GCS), den Srittmacherenzym der Gangliosid-Synthese, senkt GD2-Expression und dadurch ch14.18-vermittelte CDC, ADCC. Ausserdem, 4-HPR steigert auch AICC von 4-HPR-resistenten NB Zellen durch erhöhte Produktion von Effektormolekülen und Expression von Todesrezeptoren. Auch in vivo zeigten die 4-HPR-behandelten Mäusen höhere GD2-Expression auf den Tumorzellen und höhere Sensibilisierung gegenüber NK-Zelllyse. Zusammenfassend, diesen Daten sind eine präklinische Basis für eine Kombinationstherapie mit ch14.18 und 4-HPR zur Behandlung von Hochrisiko- Neuroblastom.