A High-Throughput Chemical Screen Identifies Synergistic Activity Between Crizotinib And Transcriptional Cdk Inhibitors In Alk-Mutated Neuroblastoma

Activating mutations in the anaplastic lymphoma receptor tyrosine kinase (ALK) represent important therapeutic targets in neuroblastoma (NB). One of the more common mutations, ALK F1174L , is sensitive to the FDA-approved ALK inhibitor, crizotinib, only at high doses and mediates acquired resistance to crizotinib in ALK-rearranged cancers. To identify compounds that would potentiate the effect of crizotinib, we performed a high-throughput compound screen using ALK F1174L -dependent human NB SH-SY5Y cells and compared viability between cells treated with the screen compound alone and in combination with crizotinib. The strongest “hits” among the ∼8,000 compounds screened were inhibitors of cyclin dependent kinases (CDKs). The combination of crizotinib and two candidate pan-selective CDK inhibitors, AT7519 and SNS-032 resulted in synergistic activity with significantly increased apoptosis over that of either single agent alone. This effect was observed in NB cells expressing not only ALK F1174L , but also in those expressing ALK R1275Q , the most common NB-associated ALK mutation. Synergy was also seen with ceritinib (LDK378), a structurally unrelated ALK inhibitor, in combination with both AT7519 and SNS-032. We determined that the synergistic effect was mediated preferentially through the transcriptional, rather than the cell cycle activity of these CDK inhibitors, denoted by absence of cell cycle arrest, stalling of RNA polymerase II at representative gene promoters, and loss of Pol II phosphorylation at the transcription elongation marker serine 2 in treated cells. These findings were comparable with results obtained using agents that were highly selective for CDK9 or CDK7, CDKs with roles in transcription regulation. Finally, in murine xenogaft models of ALK-mutated NB, the combination resulted in inhibition of tumor growth and prolongation of survival compared to single agents alone. Together, these data support further pre-clinical and clinical efforts to explore the therapeutic potential of combining ALK inhibitors with transcriptional CDK inhibitors in ALK-mutated NB. Citation Format: Nathan F. Moore, Edmond Chipumuro, Clark M. Hatheway, Tinghu Zhang, Nathanael S. Gray, Rani E. George. A high-throughput chemical screen identifies synergistic activity between crizotinib and transcriptional CDK inhibitors in ALK-mutated neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2195. doi:10.1158/1538-7445.AM2015-2195