NEK10 tyrosine phosphorylates p53 and controls its transcriptional activity

In response to genotoxic stress, multiple kinase signalling cascades are activated, many of them directed towards the tumour suppressor p53 which coordinates the DNA damage response (DDR). Defects in DDR pathways lead to an accumulation of mutations that can promote tumorigenesis. Emerging evidence implicates multiple members of the NimA-related kinase (NEK) family (NEK1, NEK10 and NEK11) in the DDR. Here, we describe a function for NEK10 in the regulation of p53 transcriptional activity through tyrosine phosphorylation. NEK10 loss increases cellular proliferation through modulation of the p53-dependent transcriptional output, by directly phosphorylating p53 on Y327, revealing NEK10’s unexpected substrate specificity. A p53 mutant at this site (Y327F) acts as a hypomorph, causing an attenuated p53-mediated transcriptional response. Consistently, NEK10-deficient cells display heightened sensitivity to DNA damaging agents and low NEK10 expression is an independent predictor of a favorable response to radiation treatment in WT TP53 breast cancer patients.

ABSTRACT 27 28 In response to genotoxic stress, multiple kinase signalling cascades are 29 activated, many of them directed towards the tumour suppressor p53 which coordinates

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Loss of NEK10 leads to increased proliferation and DNA replication 109 To directly investigate the biological function of NEK10, we generated A549 lung 110 adenocarcinoma cell lines with a loss of NEK10 function by CRISPR-Cas9 mediated 111 deletion of exon 24 of the NEK10 gene (A549 NEK10 Δ/Δ cells). The targeted exon 112 contains the "DFG" motif which is required for NEK10 protein kinase activity ( Figure S1). Considering that PI staining of asynchronous cells only provides a "snapshot" of the 120 DNA content of a population of cells, we pulse-labeled cells with 5-ethynyl-2′-121 deoxyuridine (EdU), a nucleoside analog of thymidine that is incorporated into the DNA 122 during active DNA synthesis. Measurement of the proportion of cells with active DNA 123 synthesis (EdU + cells), showed increased DNA synthesis in NEK10 Δ/Δ cells (Figure 1e).

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In addition, staining of cells for phosphorylated Histone H3 (Ser10), a mitotic marker, 125 indicated that a higher proportion of NEK10 Δ/Δ cells were undergoing mitosis, further 126 supporting a function for NEK10 in the control of cellular proliferation (Figure 1f). To explore the mechanism of NEK10-mediated control of cellular proliferation 131 and DNA synthesis, we first sought to determine the activity of the pro-growth signalling 132 PI3K and MAPK pathways and found that there was no measureable difference in the 133 degree of pAKT and pERK levels between the NEK10 +/+ cells and NEK10 Δ/Δ cells 134 (Figure 2a) [43]. We next assessed the cellular levels of p53 and the p53-responsive 135 genes p21 and MDM2 [44][45][46][47]. When compared to parental A549 and NEK10 +/+ cells,     Radiotherapy is often administered as a means to prevent breast cancer 242 recurrence, and is associated with a moderate increase in overall survival (p=0.0057) 243 ( Figure 8c) [57,58]. Interestingly, in the Low NEK10 cohort, there was a 27% increase 244 in 10-year overall survival for patients who had received radiation therapy compared to 245 those that had not (p=0.0003) (Figure 8d, right panel). In contrast, there was no additive 246 benefit of radiation therapy in the High NEK10 patient subset (Figure 8d, left panel). The 247 radio-response of tumours with WT TP53 was highly dependent on NEK10 expression.

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In particular, tumours from the Low NEK10 cohort were the most responsive to 249 radiotherapy, as these patients saw a 35% increase in overall survival (p=0.0001)

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This hypomorphic p53 response elicited by NEK10 loss was manifested by 299 differential reaction to DNA-damaging agents, such as cisplatin, where despite 300 comparable increases in p53 protein and p53 S15 levels in response to this genotoxic characterized changes in p53 S15 phosphorylation and an increase in p53 309 transcriptional activity, Y327F mutation or NEK10 loss did not affect S15 310 phosphorylation. (Figure 6 a-b, d-e). This observation suggests that Y327 311 phosphorylation does not affect S15 phosphorylation and S15-mediated p53 312 stabilization, but likely acts to promote transactivation of p53 target genes.

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The weakened p53 response in NEK10 Δ/Δ cells likely contributed to their 314 sensitivity to olaparib and cisplatin when compared to the NEK10 +/+ cells, presenting as expression represent a cohort that would likely benefit from radiotherapy, as lack of 337 such treatment in this group led to a striking increase in cancer recurrence.

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Taken together, our results demonstrate that NEK10 supports p53 transcriptional 339 activity through Y327 phosphorylation, with loss of NEK10 causing an impaired p53 340 response and a hypomorphic p53 phenotype. Recent studies have identified subsets of 341 breast cancers that are highly sensitive to genotoxic agents and radiation therapy, such 342 as those with DDR defects or genomic instability (i.e. BRCA1/2 deficiency or 343 "BRCAness") [77][78][79][80][81][82][83]. Breast cancers with reduced NEK10 expression may constitute a 344 similar breast cancer subset that is exquisitely sensitive to genotoxic treatments and 345 radiotherapy due to an attenuated p53 response, rather than a direct defect in DNA 346 repair. Thus, low NEK10 expression levels represent a potential prognostic biomarker 347 for the already standard of care chemo/radiotherapeutic treatments for breast cancer.

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Moreover, the yet to be developed NEK10 kinase inhibitors are predicted to act as 349 chemo-and radio-sensitizing agents in breast cancer treatment and improve patient 350 outcomes.

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The following antibodies were purchased from Santa Cruz Biotechnology: