Abstract 1436: Therapeutic targeting of the DNA damage mediators CHK1 and Wee1 in neuroblastoma

Abstract

Abstract Background: High-risk neuroblastoma accounts for only 4% of all pediatric cancer diagnoses, yet is responsible for 12% of pediatric cancer deaths, despite significant intensification of therapy. Through an unbiased RNAi screen, the checkpoint kinase CHK1 was identified as a neuroblastoma therapeutic target (PNAS 2010), and sensitivity to single agent CHK1 inhibition was likely due to myc-induced replication stress - an effect that was synergistically enhanced in combination with conventional chemotherapy. To identify potential mediators of this effect, other members of the pathway were analyzed and Wee1 emerged as an additional target (AACR abstract 4758, 2011). Like CHK1, Wee1 was constitutively phosphorylated in neuroblastoma primary tumors and RNAi depletion in cell lines caused apoptosis. To translate these findings, we have evaluated small molecule inhibitors of these kinases in pre-clinical models of neuroblastoma. Methods: A panel of 10 human and 2 murine neuroblastoma cell lines were selected to screen for Wee1 (MK-1775) & CHK1 (SCH 900776) growth inhibition using the CellTiter-Glo viability assay. The cell lines represented the genomic variation of clinically relevant neuroblastoma. The compounds were tested at ten dose levels to determine the IC50 across a 5-log range. By the method of Chau-Talalay, the compounds were then tested with each other, and in combination with chemotherapy agents to calculate a combination index (CI). At the average IC50, four cell lines were tested for inhibition of downstream CHK1 and Wee1 signaling. Results: Neuroblastoma cell lines (n=10) were sensitive to single agent SCH 900776 (CHK1) and MK-1775 (Wee1) inhibition, with median IC50s of 888 nM and 243 nM, respectively. The murine cell lines were also significantly inhibited by single agent nanomolar CHK1 and Wee1 inhibition and sensitivity correlated with MYCN dosage. SCH 900776 and MK-1775 combined with each other, SN-38 or gemcitabine, demonstrate broad synergistic cytotoxicity, with the combination of the Wee1 and CHK1 inhibitors being the greatest (average CI = 0.3). In four representative neuroblastoma cell lines treated with SCH 900776 or MK-1775, there was decreased phosphorylation of the respective downstream targets p-CHK1 and p-CDC2. However, the induction of DNA damage, as determined by p-γ H2Ax, was apparent only when the two small molecule inhibitors were combined. Conclusion: Neuroblastomas show significant cytotoxicity to the kinase inhibitors SCH 900776 (CHK1) and MK-1775 (Wee1) in vitro as single agents, and this is enhanced in combination with each other or chemotherapy, at doses that are likely to be physiologically feasible. Current efforts are focused on developing the optimal combinatorial strategy in murine models including determining the optimal biomarker for anti-tumor activity prior to translation to an early phase clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1436. doi:1538-7445.AM2012-1436