Targeting the Cell Cycle Checkpoints CHK1 and CHK2 is a Novel Therapeutic Approach for Pediatric Neuroblastoma

Abstract

<b>Abstract ID 23073</b> <b>Poster Board 49</b> Neuroblastoma (NB) is the most common extracranial solid pediatric tumor, and the current treatments are highly toxic and lack effectiveness. In particular, high-risk NB has an extremely low survival rate and presents a major clinical challenge due to the frequent relapse of both metastatic and refractory tumors. The issues with current NB treatments warrant the development of novel therapeutic strategies that are less toxic while being more effective. We have analyzed NB patient datasets and found increased CHEK1 and CHEK2 expression (gene coding for CHK1 and CHK2) which are inversely correlated with overall patient survival. Checkpoint Kinases 1 and 2 (CHK1/CHK2) are essential cell cycle regulators that play vital roles in the progression of cell proliferation and growth. Previous studies indicate that dysregulation of CHK1 and CHK2 promotes tumor growth as well as the progression of multiple cancers including NB. In the present study, we have used a specific small molecule inhibitor of CHK1/2, AZD7762 in NB. In vitro methodologies were used to determine the efficacy of AZD7762 in multiple MYCN amplified (LAN-5, NGP, IMR-32) and MYCN non-amplified (SK-N-AS, SH-SY5Y, CHLA-255) NB cell lines. MTT assays for determining cell proliferation showed significant inhibition of NB cell proliferation in a dose-dependent manner in response to AZD7762 with IC50s of 1-2 μM for non-amplified cell lines and 2-3 μM for amplified cell lines. Clonogenic assays further confirmed the effects of AZD7762 by inhibiting the colony formation capacity (60-85%) of different NB cell lines. Furthermore, AZD7762 significantly induces apoptosis in SH-SY5Y and NGP cell lines in a dose-dependent manner. Inhibition of CHK1/2 by AZD7762 significantly blocked cell cycle progression at the G2/M phase compared to the control treatment in NGP and SH-SY5Y NB cell lines. Additionally, AZD7762 treatments inhibit CHEK1 and CHEK2 gene expression and phosphorylation of CHK1 in contrast to control treatment, as detected by qPCR and immunoblotting respectively. Further, we developed a 3D spheroidal assay model that mimics in&nbsp;vivo tumor growth patterns of NB tumors. AZD7762 treatment significantly and in a dose-dependent manner inhibits overall 3D spheroid growth and proliferation by inhibiting the live cells in the 3D spheroids. Overall, our results show that AZD7762 significantly inhibits NB proliferation and colony formation, induces apoptosis, blocks cell cycle progression, and inhibits 3D spheroid growth. These findings support that the dysregulation of CHK1/2 is vital to NB initiation and growth. Therefore, our study highlights that the inhibition of CHK1 and CHK2 using AZD7762 is an effective novel therapeutic strategy for NB treatment. In our future efforts, we will analyze the effects of AZD7762 in NB mice models and combine these strategies with current chemotherapies to develop effective targeted therapeutic approaches for NB.