Abstract 1650: CYC065, a novel CDK2/5/9 inhibitor: detailed mechanistic studies, determinants of sensitivity and synergistic combinations

Abstract

Abstract Background: CYC065 is a novel, second generation aminopurine inhibitor highly selective for CDK2, CDK5 and CDK9 over other CDKs and non-CDK enzymes. CYC065 has completed IND-directed drug development and is ready for FIH studies. The unique target profile of CYC065 can be exploited in cyclin E-overexpressing tumors, including a trastuzumab-resistant Her2+ breast cancer model (Scaltriti et al, PNAS 2011; 9:3761). The aim of this study was to gather key dosing information for this compound in advance of FIH studies, and provide understanding of the compound mechanism of action in additional key target indications. CDK9 is a key regulator of mixed lineage leukemia (MLL)-driven leukemogenesis and Mcl-1-dependent survival and therefore represents a potential therapeutic target for AML and MLL leukaemia. Methods: A panel of acute leukemia cell lines with different MLL status (WT, MLL translocations and MLL PTD) was used in this study. Resazurin and flow cytometry-based annexin V assays were used to determine cell viability and apoptosis induction. PI staining was used to examine cell cycle distribution. Protein levels were quantified by Western blotting and quantitative imaging. Combinations were studied using the Chou and Talalay method. The in vivo activity of CYC065 was studied in murine xenograft models. Results: CYC065 rapidly decreased phosphorylation of Ser2 -RNAP II and expression of proteins involved in cell cycle control, survival and leukemogenesis (such as Mcl-1, Cyclin E, PNUTS, Hoxa9 and Meis 1), leading to robust and early induction of apoptosis (within 4 h). Time- and dose-dependent studies indicated that 6 h pulse treatment at 0.5-1 μM CYC065 was sufficient to cause ≥ 90% cell death in sensitive cell lines. Cell lines with MLL rearrangements were highly sensitive to CYC065, whilst sensitivity of AML cell lines with WT MLL correlated with the level of Bcl-2 family proteins. Cell lines with reduced sensitivity to CYC065 could be effectively targeted by exposure to longer, 10 h pulse treatments with CYC065, or in combination with short pulses of Bcl-2 inhibitors. In vivo, CYC065 achieved >90% TGI at well tolerated dose levels in HL60 (WT MLL) and EOL-1 (MLL PTD) xenograft models. Conclusion: CYC065 has therapeutic potential for AML and MLL leukemia. MLL status and the levels of Bcl-2 family proteins might be useful patient stratification markers for predicting sensitivity/resistance. Citation Format: Chiara Saladino, Sheelagh Frame, Susan Davis, David Blake, Daniella Zheleva. CYC065, a novel CDK2/5/9 inhibitor: detailed mechanistic studies, determinants of sensitivity and synergistic combinations. [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 1650. doi:10.1158/1538-7445.AM2015-1650