Neuroblastoma (NB) is a childhood cancer with a highly complex nature. High-risk NB patients undergo intensive treatment regimens that are often followed by long-term side effects. This, in addition to the emergence of resistant cancer cells, highlights a need for novel therapeutic targets and treatment strategies to improve outcome in NB. P21 is a cyclin-dependent kinase inhibitor considered to play a role in tumor resistance and aggressiveness due to its involvement in cell cycle and/or apoptosis. This project aimed to explore the expression of endogenous p21 in high-risk NB cell lines and whether p21 could be a therapeutic target for high-risk NB. Endogenous p21 levels were investigated using RT-qPCR and quantitative immunocytochemistry in eight high-risk NB cell lines. A small molecular inhibitor of p21, UC2288, was used in these cell lines to investigate tumour cell viability following p21 inhibition. In addition, combination treatment with UC2288 and the chemotherapy drug cisplatin was performed on resistant NB cell lines. Our results show variable expression of p21, where cell lines with high endogenous p21 expression showed sensitivity to single agent treatment with cisplatin or UC2288. Moreover, resistant NB cell lines showed lower endogenous p21 expression, however, combination treatment with UC2288 and cisplatin showed reduced viability, indicating sensitivity to combination treatment. This project highlights the potential of using p21 as a therapeutic target as well as a predictive biomarker in high-risk NB.

Le facteur de transcription T-bet joue un rôle central dans la régulation de la différenciation des lymphocytes T. La protéine tyrosine kinase, la glycogène synthase kinase 3 (GSK-3), inhibe l'activation des lymphocytes T et contrôle l'expression de leurs récepteurs inhibiteurs PD-1 et LAG- 3. Bien que l'inhibition de GSK-3 puisse augmenter l'expression de T-bet, l'interrelation entre T-bet et GSK-3 dans l'immunité tumorale est inconnue. Dans cette étude, nous montrons que les souris knock-out T-bet (Tbet - / -) sont compromises dans leur capacité à contrôler la croissance des cellules tumorales du mélanome B16. Cependant, l'injection d'une petite molécule inhibitrice (SMI) de GSK-3 inverse cette condition compromise entraînant le contrôle de la croissance tumorale similaire à celle observée chez les souris de type sauvage. Un examen de Tbet - / - a montré une perte de cellules dendritiques (DC) et de cellules leucocytes polymorphonucléaires (PMN) potentiellement suppressives et de lymphocytes tumoraux T (TILs) CD4 + accompagnée d'une augmentation de cellules T CD8 +. L'analyse viSNE (avancé tSNE) a en outre montré une réduction de la population effectrice expérimentée à l'antigène dans les TILs CD8 + chez Tbet -/-. Cette population est marquée par la réduction de CD44. L'inhibition de GSK-3 n'a montré aucun effet sur la perte de DC, TILs CD4 +, PMN et les TILs CD8 + ainsi que l’expression de Granzyme B (GZMB) sur les cellules T CD8 +. La seule exception était une augmentation mineure néanmoins statistiquement significative du facteur de transcription Eomesdermin (Eomes) dans les TILs CD8 +. L'étude démontre un effet compensatoire inattendu de l'inhibition de GSK-3 sur la perte de T-bet. Il reste à élucider la nature complète du parcours de cette compensation. / The transcription factor T-bet plays a central role in regulating T-cell differentiation, while the protein tyrosine kinase, glycogen synthase kinase 3 (GSK-3) inhibits T-cell activation and controls the expression of inhibitory receptors PD-1 and LAG-3 on T-cells. Although GSK-3 inhibition can increase T-bet expression, the inter-relationship between T-bet and GSK-3 in tumor immunity is unknown. In this study, we show that T-bet knock-out (Tbet-/-) mice are compromised in their ability to control the growth of the B16 melanoma tumor cells. However, the injection of a small molecule inhibitor (SMI) of GSK-3 reverses this compromised condition resulting in the control of tumor growth similar to that seen in wild type mice. An examination of Tbet-/- showed a loss of dendritic cells (DC) and potentially suppressive polymorphonuclear leucocytes (PMN) and CD4+ cell tumor infiltrating lymphocytes (TILs) accompanied by an increase in CD8+ cells. viSNE analysis (advanced tSNE- t-Distributed Stochastic Neighbor Embedding) further showed a reduction of antigen experienced effector marker CD44 in CD8+ TILs in Tbet-/-. GSK-3 inhibition showed no effect on the loss of DCs, CD4+ TILs or the presence of PMNs or CD8+ T-cells or the loss of Granzyme B (GZMB) on CD8+ cells. The one exception was a minor but statistically significant increase in the transcription factor Eomesodermin (Eomes) in CD8+ TILs. The study demonstrates an unexpected compensatory effect of GSK-3 inhibition on the loss of T-bet. The full nature of the pathway that accounts for this compensation remains to be elucidated.