Targeting PLK1 overcomes T-DM1 resistance via CDK1-dependent phosphorylation and inactivation of Bcl-2/xL in HER2-positive breast cancer

Abstract

Trastuzumab-refractory, HER2 (human epidermal growth factor receptor 2)-positive breast cancer is commonly treated with trastuzumab emtansine (T-DM1), an antibody–drug conjugate of trastuzumab and the microtubule-targeting agent, DM1. However, drug response reduces greatly over time due to acquisition of resistance whose molecular mechanisms are mostly unknown. Here, we uncovered a novel mechanism of resistance against T-DM1 by combining whole transcriptome sequencing (RNA-Seq), proteomics and a targeted small interfering RNA (siRNA) sensitization screen for molecular level analysis of acquired and de novo T-DM1-resistant models of HER2-overexpressing breast cancer. We identified Polo-like kinase 1 (PLK1), a mitotic kinase, as a resistance mediator whose genomic as well as pharmacological inhibition restored drug sensitivity. Both acquired and de novo resistant models exhibited synergistic growth inhibition upon combination of T-DM1 with a selective PLK1 inhibitor, volasertib, at a wide concentration range of the two drugs. Mechanistically, T-DM1 sensitization upon PLK1 inhibition with volasertib was initiated by a spindle assembly checkpoint (SAC)-dependent mitotic arrest, leading to caspase activation, followed by DNA damage through CDK1-dependent phosphorylation and inactivation of Bcl-2/xL. Furthermore, we showed that Ser70 phosphorylation of Bcl-2 directly regulates apoptosis by disrupting the binding to and sequestration of the pro-apoptotic protein Bim. Importantly, T-DM1 resistance signature or PLK1 expression correlated with cell cycle progression and DNA repair, and predicted a lower sensitivity to taxane/trastuzumab combination in HER2-positive breast cancer patients. Finally, volasertib in combination with T-DM1 greatly synergized in models of T-DM1 resistance in terms of growth inhibition both in three dimensional (3D) cell culture and in vivo. Altogether, our results provide promising pre-clinical evidence for potential testing of T-DM1/volasertib combination in T-DM1 refractory HER2-positive breast cancer patients for whom there is currently no treatment available.

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Acknowledgements

This project was supported by TUBITAK-BMBF Bilateral Grant numbers: TUBITAK, 214Z130 (to ÖŞ) and BMBF WTZ, 01DL16003 (to SW). ÖŞ further acknowledges the support from EMBO Installation Grant Number 2791. Special thanks to Genentech, USA Inc. for providing us with T-DM1 under Material Transfer Agreement with MTA number OR-213615. We thank the DKFZ Genomics and Proteomics Core Facility for providing sequencing excellent services. We also thank Deniz Atasoy and Pelin Dilsiz from Istanbul Medipol University for their help with confocal microscopy. ÖK acknowledges support from Baskent University and The Science Academy.

Authors contributions

ÖS designed and performed experiments; acquired, analyzed and interpreted data; and prepared the manuscript. SB, ÖA and SDE performed experiments; acquired and analyzed data; UR contributed to in vivo experimental design and data collection; EE and CA performed the transcriptome data analyses; AA performed the immunohistochemical stainings of xenografts and contributed to data interpretation; ÖK performed the co-immunoprecipitation experiments and contributed to data interpretation; SW designed experiments, interpreted data and critically read and edited the manuscript; ÖŞ designed the study, oversaw experiments and data analysis, and prepared the manuscript. All authors reviewed and commented on the manuscript.

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Correspondence to Özgür Şahin.

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Saatci, Ö., Borgoni, S., Akbulut, Ö. et al. Targeting PLK1 overcomes T-DM1 resistance via CDK1-dependent phosphorylation and inactivation of Bcl-2/xL in HER2-positive breast cancer. Oncogene 37, 2251–2269 (2018). https://doi.org/10.1038/s41388-017-0108-9

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