A combinatorial screen of the CLOUD uncovers a synergy targeting the androgen receptor

Abstract

Approved drugs are invaluable tools to study biochemical pathways, and further characterization of these compounds may lead to repurposing of single drugs or combinations. Here we describe a collection of 308 small molecules representing the diversity of structures and molecular targets of all FDA-approved chemical entities. The CeMM Library of Unique Drugs (CLOUD) covers prodrugs and active forms at pharmacologically relevant concentrations and is ideally suited for combinatorial studies. We screened pairwise combinations of CLOUD drugs for impairment of cancer cell viability and discovered a synergistic interaction between flutamide and phenprocoumon (PPC). The combination of these drugs modulates the stability of the androgen receptor (AR) and resensitizes AR-mutant prostate cancer cells to flutamide. Mechanistically, we show that the AR is a substrate for γ-carboxylation, a post-translational modification inhibited by PPC. Collectively, our data suggest that PPC could be repurposed to tackle resistance to antiandrogens in prostate cancer patients.

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Figure 1: The CLOUD.
Figure 2: A combinatorial HTS of the CLOUD uncovers the synergy between flutamide and PPC.
Figure 3: The combination of antiandrogens and vitamin K antagonists induces apoptosis of LNCaP prostate cancer cells.
Figure 4: The combination of flutamide and PPC induces AR degradation.
Figure 5: The AR is post-translationally modified by γ-glutamyl carboxylation.

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Acknowledgements

We thank M. Iskar (EMBL) and P. Bork (EMBL) for providing us with DIPS scores, R. Schüle (Albert-Ludwigs-University Freiburg, Germany) for LAPC4 cells, and G. Winter (CeMM) and G. Superti-Furga (CeMM) for thoughtful discussions and initializing synergy screenings at CeMM. S.K. acknowledges support by a Marie Curie Career Integration Grant, the Austrian Federal Ministry of Science, Research and Economy and the National Foundation for Research, Technology, and Development and the Austrian Science Fund (FWF): F4701-B20.

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Contributions

P.M., F.K. and S.K. designed and assembled the CLOUD. M.P.L., F.K., C.-H.L. and S.K. designed and performed the screen of the CLOUD. M.P.L., F.K., M.C. and J.M. analyzed the data from the screen. M.P.L. and A.R. designed and performed viability, RT-qPCR, western blotting, immunoprecipitation and immunofluorescence experiments. S.S. performed immunofluorescence experiments. A.R. and E.S. designed and performed gene expression and immunoprecipitation experiments. A.R. and B.B. designed and performed knockdown experiments. A.C.M. and K.L.B. designed and performed proteomics experiments. A.W., R.H. and K.K. performed and analyzed 2D gel electrophoresis experiments. T.P., M.S. and C.B. performed and analyzed RNA-seq experiments. G.D. and J.C. performed DIPS score analyses. Y.F. and P.S. analyzed patient data in PCBaSe. V.I. synthesized, provided and quality controlled chemicals. M.P.L. and S.K. wrote the manuscript.

Corresponding author

Correspondence to Stefan Kubicek.

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Competing interests

M.P.L. and S.K. have filed a patent based on findings described in this manuscript (WO2016170102 A1). V.I. is an employee of Enamine, Ltd and may also own shares in the company.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–21 (PDF 29615 kb)

Supplementary Data Set 1

STEAM and CLOUD drugs. (XLS 338 kb)

Supplementary Data Set 2

Data from CLOUD combinatorial screen. (XLS 4999 kb)

Supplementary Data Set 3

Synergies and antagonisms defined by both Bliss and Loewe scores. (XLS 207 kb)

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Licciardello, M., Ringler, A., Markt, P. et al. A combinatorial screen of the CLOUD uncovers a synergy targeting the androgen receptor. Nat Chem Biol 13, 771–778 (2017). https://doi.org/10.1038/nchembio.2382

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