Abstract B165: Optimizing selective CDK7 inhibition in MYCN-driven neuroblastoma

Abstract

Abstract Amplification of the MYCN gene defines approximately 50% of high-risk neuroblastomas (NB), and is associated with aggressive disease and a poor clinical outcome. We recently demonstrated that MYCN-amplified NBs are dependent on MYCN-driven global transcriptional amplification, and that they can be selectively targeted using a first-in-class covalent cyclin-dependent kinase (CDK) 7/12 inhibitor, THZ1. CDK7, CDK9 and CDK12 directly phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNAPII) to regulate gene transcription. NB cells expressing high levels of MYCN were 10 times more sensitive to THZ1 than normal cells or NB cells not driven by amplified MYCN. THZ1 caused tumor growth inhibition in murine models of human NB, without general toxicity indicating the clinical potential of this therapeutic strategy. To identify mechanisms of resistance to THZ1, we generated THZ1-resistant NB cells (THZ1-R) by exposing sensitive cells to escalating sub-lethal doses of THZ1. THZ1-R cells exhibited no changes MYCN levels or RNAPII activity, eliminating alterations in CDK7 itself and/or emergence of compensatory pathways to account for the resistance. A survey of common multidrug resistance mechanisms revealed that extended THZ1 exposure exclusively enhanced the transcript levels of ATP-Binding Cassette sub-family member B1 (ABCB1, also MDR1) by 500-fold, which was translated to the protein level as well. The importance of ABCB1 in THZ1-resistance was demonstrated by rescue of THZ1 activity using the selective ABCB1 inhibitor tariquidar and shRNA-mediated gene knockdown. Subsequent screening of CDK inhibitors of the SBI-E class in these ABCB1-overexpressing NB cells afforded the identification of a covalent CDK9/12 inhibitor (SBI-E-9) devoid of efflux-substrate characteristics. Regardless of the expression levels of ABCB1, SBI-E-9 selectively suppressed MYCN expression and MYCN-associated transcriptional activity. In addition, we evaluated the in vitro efficacy of a series of covalent CDK7 inhibitors with distinctive scaffolds from THZ1. Although these compounds do not escape ABCB1-mediated drug efflux, their specific targeting of CDK7 over CDK12 correlated with greater differential selectivity in MYCN-amplified vs. MYCN non-amplified NB cells (30-fold versus 10-fold with THZ1). In conclusion, we report, for the first time, ABCB1-regulated drug efflux as a major mechanism of resistance to the first-generation covalent CDK7/12 inhibitor, THZ1, which can be overcome by a newly identified, covalent CDK9/12 inhibitor, SBI-E-9. Additionally, we have identified second generation covalent CDK7 inhibitors that display a higher selectivity against MYCN-driven NB through augmented CDK7 activity. Hence, these covalent CDK inhibitors represent attractive therapeutic options for MYCN-driven NB, and perhaps other MYC-dependent cancers. Citation Format: Yang Gao, Yanke Liang, Tinghu Zhang, Nathanael S. Gray, Rani E. George. Optimizing selective CDK7 inhibition in MYCN-driven 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 B165.