A kinase inhibitor screen identifies a dual cdc7/CDK9 inhibitor to sensitise triple-negative breast cancer to EGFR-targeted therapy
Abstract
Background: The effective management of triple-negative cancer of the breast (TNBC) remains a serious clinical challenge. Despite frequent epidermal growth factor receptor (EGFR) overexpression and reliance upon downstream signalling pathways in TNBC, potential to deal with EGFR-tyrosine kinase inhibitors (TKIs) remains endemic. Therefore, the identification of targeted agents, which synergise with current therapeutic options, is vital.
Methods: Compound-based, high-throughput, proliferation screening was utilized to profile the response of TNBC cell lines to EGFR-TKIs, western blotting and siRNA transfection getting used to look at the result of inhibitors on EGFR-mediated signal transduction and cellular reliance on such pathways, correspondingly. A kinase inhibitor combination screen was utilized to recognize compounds that synergised with EGFR-TKIs in TNBC, utilising sulphorhodamine B (SRB) assay as read-out for proliferation. The outcome of drug combinations on cell cycle arrest, apoptosis and signal transduction was assessed using flow cytometry, automated live-cell imaging and western blotting, correspondingly. RNA sequencing was used to solve transcriptomic changes elicited with this synergistic combination and also to permit identification from the signalling systems most responsive to co-inhibition.
Results: We show a dual cdc7/CDK9 inhibitor, PHA-767491, synergises with multiple EGFR-TKIs (lapatinib, erlotinib and gefitinib) to beat potential to deal with EGFR-targeted therapy in a variety of TNBC cell lines. Combined inhibition of EGFR and cdc7/CDK9 led to reduced cell proliferation, supported by induction of apoptosis, G2-M cell cycle arrest, inhibition of DNA replication and abrogation of CDK9-mediated transcriptional elongation, as opposed to mono-inhibition. Furthermore, high expression of cdc7 and RNA polymerase II Subunit A (POLR2A), the direct target of CDK9, is considerably correlated with poor metastasis-free survival inside a cohort of cancer of the breast patients. RNA sequencing revealed marked downregulation of pathways governing proliferation, transcription and cell survival in TNBC cells given the mixture of the EGFR-TKI along with a dual cdc7/CDK9 inhibitor. Numerous genes filled with these downregulated pathways are connected with poor metastasis-free survival in TNBC.
Conclusions: Our results highlight that dual inhibition of cdc7 and CDK9 by PHA-767491 is really a potential technique for targeting TNBC resistant against PHA-767491 EGFR-TKIs.