502 Spy1 as a Novel Cell Cycle Target Against Medulloblastoma

Abstract

INTRODUCTION: The current treatment strategies for medulloblastoma (MB) are highly aggressive, cytotoxic, and ultimately unsuccessful. One recent therapeutic approach is the use of synthetic cyclin-dependent kinase inhibitors (CKIs). Speedy (Spy1) is a cyclin-like protein that primes its complexes with CDKs insensitive to natural CKIs and promotes uncontrolled cell proliferation by overriding the safety cell cycle checkpoints. Spy1 has been implicated in the expansion of stem-like populations of therapy-resistant tumour initiating cells (TICs) in neural systems. METHODS: We established an in vitro neurosphere platform as well as in vivo high-throughput Zebrafish (ZF) xenograft model using ONS-76 and DAOY MB cell lines derived from three patients. Fluorescent Activated Cell Sorting (FACS) was used to enrich for specific CD133+/- or CD15 +/- populations of MB. Spy1 levels were manipulated using lentivirus to obtain overexpression or knockdown of Spy1 protein levels in the derived TICs as well as heterogenous cell populations. RESULTS: We found that downregulation of Spy1 levels in the employed cell lines resulted in a decrease in neurospheres formation efficiency as well as significantly lower tumour burden in Zebrafish when compared to control (p<0.001). Spy1 overexpression in CD133 or CD15 negative MB populations resulted in the upregulation of Msi1 mRNA levels. Finally, in vivo treatment of ZF xenografts with R-roscovitine, a synthetic CKI, demonstrated declined migration of Spy1 KD MB cells in comparison to control cells (p<0.05). CONCLUSION: This study shows that Spy1 plays a role in the regulation of stemness and aggressive characteristics of TICs in medulloblastoma. The assessed effect on CKI treatment efficacy on the tumours in vivo demonstrated that downregulation of Spy1 may sensitize, at least a subset of MB, to therapy. This work may contribute toward optimizing the design of CKIs and their use in combination therapy for patients with MB.