Abstract B74: Combined inhibition of ALK and CDKs involved in transcriptional regulation is synergistic in ALK-mutated neuroblastoma

Abstract

Abstract Activating mutations in the anaplastic lymphoma receptor tyrosine kinase (ALK) represent important therapeutic targets in neuroblastoma (NB). One of the more common mutations, ALKF1174L, 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 enhance the cytotoxic effect of crizotinib, we conducted a high throughput small molecule screen for compounds that synergize with crizotinib in NB cells expressing the ALKF1174L mutation. We identified two pan-selective cyclin dependent kinase (CDK) inhibitors, AT7519 and SNS-032, which have overlapping efficacy against the cell cycle-regulating CDK2 and transcriptional elongation-regulating CDK9. Both inhibitors demonstrated synergistic activity with crizotinib, leading to downregulation of pALK and downstream signaling and significantly increased apoptosis over that of either single agent alone. This effect was observed in NB cells expressing not only ALKF1174L, but also in the other commonly observed ALKR1275Q mutation. Synergy was also noted with ceritinib (LDK378), a structurally unrelated ALK inhibitor, in combination with both AT7519 and SNS-032. The combination of crizotinib and SNS-032 led to the inhibition of CDK9-mediated transcriptional elongation as indicated by downregulation of RNA polymerase II phosphorylation at serine 2. Additionally, the combination also induced proteolytic cleavage of elongation-regulating BRD4, suggesting alternative mechanisms contributing to transcriptional inhibition in addition to direct inhibition of CDK9 activity These findings were comparable with results obtained using compounds that were highly selective for CDK7, which has roles in transcriptional initiation as well as in CDK9 activation. Finally, in murine xenogaft models of ALK-mutated NB, the combination led to 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. Combined inhibition of ALK and CDKs involved in transcriptional regulation is synergistic in ALK-mutated neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B74.