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Data from: Fadraciclib (CYC065), a novel CDK inhibitor, targets key pro-survival and oncogenic pathways in cancer


Zheleva, Daniella et al. (2020), Data from: Fadraciclib (CYC065), a novel CDK inhibitor, targets key pro-survival and oncogenic pathways in cancer, Dryad, Dataset,


Cyclin-dependent kinases (CDKs) contribute to the cancer hallmarks of uncontrolled proliferation and increased survival. As a result, over the last two decades substantial efforts have been directed towards identification and development of pharmaceutical CDK inhibitors. Insights into the biological consequences of CDK inhibition in specific tumor types have led to the successful development of CDK4/6 inhibitors as treatments for certain types of breast cancer. More recently, the development of a new generation of pharmaceutical inhibitors of CDK enzymes that regulate the transcription of key oncogenic and pro-survival proteins, including CDK2 and CDK9, have entered clinical development. Here, we provide the first disclosure of the chemical structure of fadraciclib (CYC065), a CDK inhibitor and clinical candidate designed by further optimization from the aminopurine scaffold of seliciclib. We describe its synthesis and mechanistic characterization. Fadraciclib exhibits improved potency and selectivity for CDK2 and CDK9 compared to seliciclib, and also displays high selectivity across the kinome. We show that the mechanism of action of fadraciclib is consistent with potent inhibition of CDK9-mediated transcription, decreasing levels of RNA polymerase II C-terminal domain serine 2 phosphorylation, the pro-survival protein Myeloid Cell Leukemia 1 (MCL1) and MYC oncoprotein, and inducing rapid apoptosis in cancer cells. This cellular potency and mechanism of action translate to promising anti-cancer activity in human leukemia mouse xenograft models. Studies of leukemia cell line sensitivity identify mixed lineage leukemia (MLL) gene status and the level of B-cell lymphoma 2 (BCL2) family proteins as potential markers for selection of patients with greater sensitivity to CYC605. We show that the combination of fadraciclib with BCL2 inhibitors, including venetoclax, is synergistic in leukemic cell models, as predicted from simultaneous inhibition of MCL1 and BCL2 pro-survival pathways. Fadraciclib preclinical pharmacology data support its therapeutic potential in CDK9- or CDK2-dependent cancers and as a rational combination with BCL2 inhibitors in hematological malignancies. Fadraciclib is currently in Phase 1 clinical studies in patients with advanced solid tumors (NCT02552953) and also in combination with venetoclax in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) (NCT03739554) and relapsed refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (NCT04017546)


Western Blot Analysis

Protein samples for Western blot analysis were prepared as follows. AML cells were seeded at 5 x 105 cells/ml and solid tumor cell lines were seeded at approximately 1 million cells per 10cm dish in the appropriate media and incubated overnight. Cells were treated for up to 24 hours with various concentrations of fadraciclib or DMSO as vehicle control. Cells were collected at various time-points as indicated and centrifuged for 5 min at 478 x g. Medium was removed and cells were washed once in PBS. Cells were pelleted by centrifugation and the pellets were snap-frozen in liquid nitrogen and stored at -20°C until analysis. Pellets were lysed in NP40 cell lysis buffer (Life Technologies, Paisley, UK), PMSF, sodium beta-glycerophosphate and protease inhibitor cocktail (1:1000, Sigma-Aldrich) and sonicated. The soluble protein fraction was obtained after centrifugation at 14,000 x g for 10 min. The protein yield was determined using the BCA protein determination kit (Pierce, Thermo Fisher Scientific, Northumberland, UK) as per the manufacturer’s instructions.

Total protein (25µg) was mixed with 4x gel loading buffer and 1x reducing agent and separated in precast NuPAGE 10%, 4-12% Bis-Tris or 3-8% Tris-Acetate polyacrylamide gels using denaturing electrophoretic conditions (Life Technologies). Proteins were transferred to nitrocellulose membranes using an IBlot transfer unit (Life Technologies). Membranes were blocked in 5% nonfat milk in PBS with 0.05% Tween 20 (PBST) for 2 hours and incubated overnight at 4°C with primary antibody diluted in 5% milk in PBST, or 3% BSA for phospho-specific antibodies. Membranes were washed 3 x 10 minutes in PBST and incubated for 1 hour in 5% milk in PBST containing horseradish peroxidase-conjugated secondary antibody. Membranes were washed as before and incubated with enhanced chemiluminescence solution and exposed to X-ray film (Amersham, VWR, Leicestershire, UK). Protein bands were quantified using ImageJ software. Alternatively, Clarity ECL solution was used and images were captured and analyzed on the Chemidoc (both Biorad, UK). The antibodies and their final concentrations used are listed in Supplementary Table 2.

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