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Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma.

ABSTRACT: Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

SUBMITTER: Bragelmann J

PROVIDER: S-EPMC5622049 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma.

Brägelmann Johannes J Dammert Marcel A MA Dietlein Felix F Heuckmann Johannes M JM Choidas Axel A Böhm Stefanie S Richters André A Basu Debjit D Tischler Verena V Lorenz Carina C Habenberger Peter P Fang Zhizhou Z Ortiz-Cuaran Sandra S Leenders Frauke F Eickhoff Jan J Koch Uwe U Getlik Matthäus M Termathe Martin M Sallouh Muhammad M Greff Zoltán Z Varga Zoltán Z Balke-Want Hyatt H French Christopher A CA Peifer Martin M Reinhardt H Christian HC Örfi László L Kéri György G Ansén Sascha S Heukamp Lukas C LC Büttner Reinhard R Rauh Daniel D Klebl Bert M BM Thomas Roman K RK Sos Martin L ML

Cell reports 20170901 12

Kinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodo ...[more]

PMID: 28930680

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Project description:BackgroundThe 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.MethodsCompound-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.ResultsWe 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.ConclusionsOur results highlight that dual inhibition of cdc7 and CDK9 by PHA-767491 is a potential strategy for targeting TNBC resistant to EGFR-TKIs.

Dataset Information

Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma.

Publications

Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma.

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