A gemcitabine sensitivity screen identifies a role for NEK9 in the replication stress response

Abstract

The Replication Stress Response (RSR) is a signaling network that recognizes challenges to DNA replication and coordinates diverse DNA repair and cell-cycle checkpoint pathways. Gemcitabine is a nucleoside analogue that causes cytotoxicity by inducing DNA replication blocks. Using a synthetic lethal screen of a RNAi library of nuclear enzymes to identify genes that when silenced cause gemcitabine sensitization or resistance in human triple-negative breast cancer cells, we identified NIMA (never in mitosis gene A)-related kinase 9 (NEK9) as a key component of the RSR. NEK9 depletion in cells leads to replication stress hypersensitivity, spontaneous accumulation of DNA damage and RPA70 foci, and an impairment in recovery from replication arrest. NEK9 protein levels also increase in response to replication stress. NEK9 complexes with CHK1, and moreover, NEK9 depletion impairs CHK1 autophosphorylation and kinase activity in response to replication stress. Thus, NEK9 is a critical component of the RSR that promotes CHK1 activity, maintaining genome integrity following challenges to DNA replication.

Figure 1.

Synthetic lethal screen identifies gemcitabine sensitivity genes in TNBC. (A) Schematic representation of primary siRNA screen targeting 1006 unique human genes in MDA-MB-231 TNBC cells using a pool of four siRNAs per gene. (B) Summary of results of primary screen. The z-score of the log₂ ratio of gemcitabine treated compared with untreated cell viability relative to a NT siRNA for each gene is shown. The shaded areas indicate positive hit criteria with a z-score <1.5 and >1.5. (C) Volcano plot of primary screen. The gemcitabine treated to untreated cell viability ratio and −log P-value for each gene relative to NT siRNA is shown. The shaded areas indicates positive hit criteria with a gemcitabine treated to untreated ratio <0.7 or >1.3 and a −log P-value >1. (D) Genes causing significant gemcitabine hypersensitivity upon silencing by known gene ontology function.

Figure 2.

NEK9 depletion causes replication stress hypersensitivity. (A) MDA-MB-231 cells were transfected with NT, ATR or NEK9 siRNA, split 1:4 24 h later, and treated 24 h later with or without 5 μM gemcitabine for 72 h prior to assaying for cell viability. Gemcitabine treated to untreated cell viability relative to NT siRNA is shown. (B) Western blot analysis demonstrating efficiency of NEK9 knockdown with indicated siRNAs. (C and D) MDA-MB-231 cells transfected with NT or NEK9 siRNA were seeded for colony formation and treated with indicated concentrations of gemcitabine (C), mitomycin-C (D), HU (E) or camptothecin (F) for 24 h (h). Surviving colonies were counted 8–12 days later. Survival fraction of colonies from treated versus untreated cells is indicated. For (A, C, D), mean and standard deviation from at least two replicas is shown. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

NEK9 depletion leads to accumulation of DNA damage and RPA70 foci. (A) U2OS cells were transfected with NT, CHK1 or NEK9 siRNA and processed 72 h later for γH2AX staining by indirect immunofluorescence. Representative images are shown. Scale bar indicates 10 μm. (B) The percentage (mean and standard deviation) of γH2AX positive cells (≥12 foci per cell) is shown. (C) U2OS cells were transfected with NT, CHK1 or NEK9 siRNA and processed 72 h later for RPA70 staining by indirect immunofluorescence. Representative images are shown. Scale bar indicates 10 μm. (D) The percentage (mean and standard deviation) of RPA70 foci positive cells (≥12 foci per cell) is shown. (E) Western blot analysis demonstrating efficiency of ATR, ATRIP, CHK1 and NEK9 knockdown with indicated siRNAs 72 h after transfection in U2OS cells. For (B and D) *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4.

NEK9 depletion impairs recovery from replication stress. (A) U2OS cells were transfected with NT, ATR, ATRIP or NEK9 siRNA, treated 72 h later with 3 mM HU for 20 h (arrested), washed and released into nocodazole for 10 h (released). DNA content was analyzed by flow cytometry. (B) The percentage (mean and standard deviation) of cells that completed DNA synthesis in three replicate experiments is shown. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5.

NEK9 protein levels increase in response to replication stress. (A) Western blot analysis of lysate from MDA-MB-231 cells treated with 1 mM gemcitabine for the indicated times. (B) Western blot analysis of lysate from HEK 293T cells treated with 3 mM HU for the indicated times. (C) Western blot analysis of lysate from HEK 293T cells treated with 1 mM gemcitabine or 3 uM mitomycin C for 6 h.

Figure 6.

NEK9 complexes with and regulates the activity of CHK1. (A and B) Endogenous NEK9 or CHK1 was immunoprecipitated from cell lysates. Immunocomplexes were washed, separated by SDS-PAGE and immunoblotted with antibodies against NEK9 or CHK1. (C) NEK9 depletion impairs CHK1 autophosphorylation in response to replication stress. HeLa cells were transfected with NT, CHK1 or NEK9 siRNA, and treated with 3 mM HU for 6 h. Cell lysates were separated by SDS-PAGE, and immunoblotted with antibodies against NEK9, P-CHK1 Ser296, CHK1 and GAPDH. (D) NEK9 depletion impairs CHK1 kinase activity. CHK1 was purified from HeLa cells transfected with NT, ATR or NEK9 siRNA, treated with or without 1 mM gemcitabine for 3 h, incubated in an in vitro kinase reaction with ³²P, and processed by autoradiography. The reaction mixtures were separated by SDS-PAGE and immunoblotted with antibodies against ATR, NEK9, CHK1 and GAPDH.