Journal Article

A kinase inhibitor screen identifies a dual cdc7/CDK9 inhibitor to sensitise triple-negative breast cancer to EGFR-targeted therapy

Zhang, Yinghui, van der Noord, Vera E., Redel, Jevin, Martens, John W. M., Smid, Marcel, McLaughlin, Ronan P., He, Jichao, Foekens, John A., van de Water, Bob

2019

Overview

Background The effective treatment of triple-negative breast cancer (TNBC) remains a profound clinical challenge. Despite frequent epidermal growth factor receptor (EGFR) overexpression and reliance on downstream signalling pathways in TNBC, resistance to EGFR-tyrosine kinase inhibitors (TKIs) remains endemic. Therefore, the identification of targeted agents, which synergise with current therapeutic options, is paramount. Methods Compound-based, high-throughput, proliferation screening was used to profile the response of TNBC cell lines to EGFR-TKIs, western blotting and siRNA transfection being used to examine the effect of inhibitors on EGFR-mediated signal transduction and cellular dependence on such pathways, respectively. A kinase inhibitor combination screen was used to identify compounds that synergised with EGFR-TKIs in TNBC, utilising sulphorhodamine B (SRB) assay as read-out for proliferation. The impact of drug combinations on cell cycle arrest, apoptosis and signal transduction was assessed using flow cytometry, automated live-cell imaging and western blotting, respectively. RNA sequencing was employed to unravel transcriptomic changes elicited by this synergistic combination and to permit identification of the signalling networks most sensitive to co-inhibition. Results We demonstrate that a dual cdc7/CDK9 inhibitor, PHA-767491, synergises with multiple EGFR-TKIs (lapatinib, erlotinib and gefitinib) to overcome resistance to EGFR-targeted therapy in various TNBC cell lines. Combined inhibition of EGFR and cdc7/CDK9 resulted in reduced cell proliferation, accompanied by induction of apoptosis, G2-M cell cycle arrest, inhibition of DNA replication and abrogation of CDK9-mediated transcriptional elongation, in contrast to mono-inhibition. Moreover, high expression of cdc7 and RNA polymerase II Subunit A (POLR2A), the direct target of CDK9, is significantly correlated with poor metastasis-free survival in a cohort of breast cancer patients. RNA sequencing revealed marked downregulation of pathways governing proliferation, transcription and cell survival in TNBC cells treated with the combination of an EGFR-TKI and a dual cdc7/CDK9 inhibitor. A number of genes enriched in these downregulated pathways are associated with poor metastasis-free survival in TNBC. Conclusions Our results highlight that dual inhibition of cdc7 and CDK9 by PHA-767491 is a potential strategy for targeting TNBC resistant to EGFR-TKIs.

• Publisher: BioMed Central,BioMed Central Ltd,BMC
• Subject: Antineoplastic Agents - pharmacology / Antineoplastic Agents - therapeutic use / Apoptosis - drug effects / Biomedical and Life Sciences / Biomedicine / Breast cancer / Cancer metastasis / Cancer Research / CDK9/Cdc7 inhibition / Cell cycle / Cell Cycle - drug effects / Cell Cycle Proteins - antagonists & inhibitors / Cell Line, Tumor / Cell Proliferation - drug effects / Cell Survival - drug effects / Cellular signal transduction / Cetuximab / Combination drug therapy / Cyclin-Dependent Kinase 9 - antagonists & inhibitors / DNA / DNA replication / Drug resistance / Drug Resistance, Neoplasm / Drug therapy / EGFR-targeted therapy / Epidermal growth factor receptors / Epidermal growth factors / ErbB Receptors - antagonists & inhibitors / Erlotinib / Female / Gefitinib / Gene Expression Profiling / Genes / Genetic aspects / Health aspects / Humans / Infection / Kinase inhibitors / Lapatinib / Molecular Targeted Therapy / Oncology / Phenols (Class of compounds) / Prognosis / Protein Kinase Inhibitors - pharmacology / Protein Kinase Inhibitors - therapeutic use / Protein Serine-Threonine Kinases - antagonists & inhibitors / Research Article / RNA / RNA sequencing / Signal Transduction / Surgical Oncology / Transcription (Genetics) / Triple negative breast cancer / Triple Negative Breast Neoplasms - drug therapy / Triple Negative Breast Neoplasms - metabolism / Triple Negative Breast Neoplasms - mortality / Tyrosine