Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase

Beck, Halfdan; Nähse, Viola; Larsen, Marie Sofie Yoo; Groth, Petra; Clancy, Trevor; Lees, Michael; Jørgensen, Mette; Helleday, Thomas; Syljuåsen, Randi G.; Sørensen, Claus Storgaard

doi:10.1083/jcb.200905059

Cited by 150 publications

(185 citation statements)

References 24 publications

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“…The gH2AX-positive cells were fairly evenly distributed through S-phase, in contrast to an earlier report by Beck et al in which Wee1 knockdown resulted in the accumulation of gH2AX-labeled cells in late S-phase. 18 Furthermore, unlike the earlier paper, we did not see significant rescue from this effect in any of our 8 Wee1 knockdown clones treated with the general cyclin-dependent kinase (CDK) inhibitor roscovotine (Fig. 1C).…”

Section: Effects Of Wee1 Knockdowncontrasting

confidence: 51%

“…Consistently, however, Wee1 knockdown or pharmacological inhibition results in the increased gH2AX labeling of S-phase cells, in vitro and in vivo, as was seen in the present study. This likely reflects increased DNA damage due the unregulated activity of Cdk2, as previously suggested, 18 and provides a rationale for testing Wee1 inhibitors in combination with S-phase specific agents like gemcitabine. However, our results testing the drug combination in 3 orthotopicallygrown PDX models do not provide strong support for this.…”

Section: Mk-1775 (Now Azd1775mentioning

confidence: 90%

“…This is similar to the earlier report by Beck et al, although there are differences in detail that likely reflect differences in the models and techniques for Wee1 silencing. 18 Interestingly, Beck et al comment that they noticed phosphorylation of H3S10 suggestive of premature mitosis following Wee1 knockdown, 18 which is also noticeable in the dual PH3 vs DNA plot of A2 cells seen in Figure 1C. The effects of MK-1775 on OCIP23 cells were much more dramatic than Wee1 knockdown, with the accumulation of cells in early S-phase showing H3S10 phosphorylation, associated with high levels of Cyclin A2 and Cyclin B1 that are normally highly expressed in late S-phase and G2.…”

Section: Mk-1775 (Now Azd1775mentioning

confidence: 99%

“…Wee1 silencing by small interfering RNA results in the rapid accumulation of DNA damage in S-phase that is substantially reduced when cells are exposed to the general CDK inhibitor roscovotine. 18 Subsequently it was reported that treatment with the clinical Wee1 inhibitor MK-1775 (AZD-1775) likewise had striking cytokinetic effects, including features suggestive of premature entry into mitosis during early S-phase. 19,20 Of the Wee1 inhibitors, MK-1775 is currently the most advanced in clinical development, and it is reported to be highly selective toward Wee1 (21).…”

Section: Introductionmentioning

confidence: 99%

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Cytokinetic effects of Wee1 disruption in pancreatic cancer

et al. 2016

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The Wee1 kinase, which is activated in response to DNA damage, regulates exit from G2 through inhibitory phosphorylation of Cdk1/Cdc2, and is an attractive drug target. However, recent work has highlighted effects of Cdk2 phosphorylation by Wee1 on movement through S-phase, suggesting the potential to sensitize to S-phase specific agents by Wee1 inhibitors. In this paper we applied multiparametric flow cytometry to patient-derived pancreatic cancer xenograft tumor cells to study the cell cycle perturbations of Wee1 disruption via the small molecule inhibitor MK-1775, and genetic knockdown. We find that in vitro treatment with MK-1775, and to a lesser degree, Wee1 RNA transcript knockdown, results in the striking appearance of S-phase cells prematurely entering into mitosis. This effect was not seen in vivo in any of the models tested. Here, although we noted an increase of S-phase cells expressing the damage response marker gH2AX, treatment with MK-1775 did not significantly sensitize cells to the cytidine analog gemcitabine. Treatment with MK-1775 did result in a transient but large increase in cells expressing the mitotic marker phosphorylated H3S10 that reached a peak 4 hours after treatment. This suggests a role for Wee1 regulating the progression of genomically unstable cancer cells through G2 in the absence of extrinsically-applied DNA damage. A single dose of 8Gy ionizing radiation resulted in the time-dependent accumulation of Cyclin A2 positive/phosphorylated H3S10 negative cells at the 4N position, which was abrogated by treatment with MK-1775. Consistent with these findings, a genomescale pooled RNA interference screen revealed that toxic doses of MK-1775 are suppressed by CDK2 or Cyclin A2 knockdown. These findings support G2 exit as the more significant effect of Wee1 inhibition in pancreatic cancers.

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Section: Effects Of Wee1 Knockdowncontrasting

confidence: 51%

Section: Mk-1775 (Now Azd1775mentioning

confidence: 90%

Section: Mk-1775 (Now Azd1775mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cytokinetic effects of Wee1 disruption in pancreatic cancer

et al. 2016

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“…The mechanism that leads to replication stress in DKO cells is still unknown, and it would be interesting to determine whether it involves illegitimate replication origin firing and fork stalling, as shown for replication stress caused by overexpression of Cdt1 33 or by aberrant CDK activity 34 in some cell types. A controlled CDK activity is key to maintain genome integrity during S phase, 35 and it has been reported that most of the DNA replication stress arises as a result of elevated CDK activity. 36 Interestingly, this phenotype can be alleviated by nucleotide addition.…”

Section: E2f-p53 Regulatory Axis In Tissue Homeostasismentioning

confidence: 99%

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

et al. 2015

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Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53 − / − mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy.

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Development of Potent Pyrazolopyrimidinone‐Based WEE1 Inhibitors with Limited Single‐Agent Cytotoxicity for Cancer Therapy

2018

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WEE1 kinase regulates the G /M cell-cycle checkpoint, a critical mechanism for DNA repair in cancer cells that can confer resistance to DNA-damaging agents. We previously reported a series of pyrazolopyrimidinones based on AZD1775, a known WEE1 inhibitor, as an initial investigation into the structural requirements for WEE1 inhibition. Our lead inhibitor demonstrated WEE1 inhibition in the same nanomolar range as AZD1775, and potentiated the effects of cisplatin in medulloblastoma cells, but had reduced single-agent cytotoxicity. These results prompted the development of a more comprehensive series of WEE1 inhibitors. Herein we report a series of pyrazolopyrimidinones and identify a more potent WEE1 inhibitor than AZD1775 and additional compounds that demonstrate that WEE1 inhibition can be achieved with reduced single-agent cytotoxicity. These studies support that WEE1 inhibition can be uncoupled from the potent cytotoxic effects observed with AZD1775, and this may have important ramifications in the clinical setting where WEE1 inhibitors are used as chemosensitizers for DNA-targeted chemotherapy.

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Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase

Abstract: WEE1 and CHK1 jointly regulate Cdk activity to prevent DNA damage during replication.

Cited by 150 publications

References 24 publications

Cytokinetic effects of Wee1 disruption in pancreatic cancer

Cytokinetic effects of Wee1 disruption in pancreatic cancer

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

Development of Potent Pyrazolopyrimidinone‐Based WEE1 Inhibitors with Limited Single‐Agent Cytotoxicity for Cancer Therapy

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