MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor efficacy of various DNA-damaging agents, including 5-fluorouracil

Hirai, Hiroshi; Arai, T.; Okada, Megumu; Nishibata, Toshihide; Kobayashi, Makiko; Sakai, Naoko; Imagaki, Kazuhide; Ohtani, Junko; Sakai, Takumi; Yoshizumi, Takashi; Mizuarai, Shinji; Iwasawa, Yoshikazu; Kotani, Hidehito

doi:10.4161/cbt.9.7.11115

Cited by 206 publications

(184 citation statements)

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“…The extent of chromosome fragmentation per cell was analyzed by counting the fragments in 10 randomly chosen metaphases. Significance was determined by the Student's t-test and the P-value is shown by asterisks (***P-valueo0.001) 25,41,42 In this scenario,…”

Section: Discussionmentioning

confidence: 99%

Strong antitumor synergy between DNA crosslinking and HSP90 inhibition causes massive premitotic DNA fragmentation in ovarian cancer cells

et al. 2016

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All current regimens for treating ovarian cancer center around carboplatin as standard first line. The HSP90 inhibitor ganetespib is currently being assessed in advanced clinical oncology trials. Thus, we tested the combined effects of ganetespib and carboplatin on a panel of 15 human ovarian cancer lines. Strikingly, the two drugs strongly synergized in cytotoxicity in tumor cells lacking wild-type p53. Mechanistically, ganetespib and carboplatin in combination, but not individually, induced persistent DNA damage causing massive global chromosome fragmentation. Live-cell microscopy revealed chromosome fragmentation occurring to a dramatic degree when cells condensed their chromatin in preparation for mitosis, followed by cell death in mitosis or upon aberrant exit from mitosis. HSP90 inhibition caused the rapid decay of key components of the Fanconi anemia pathway required for repair of carboplatin-induced interstrand crosslinks (ICLs), as well as of cell cycle checkpoint mediators. Overexpressing FancA rescued the DNA damage induced by the drug combination, indicating that FancA is indeed a key client of Hsp90 that enables cancer cell survival in the presence of ICLs. Conversely, depletion of nuclease DNA2 prevented chromosomal fragmentation, pointing to an imbalance of defective repair in the face of uncontrolled nuclease activity as mechanistic basis for the observed premitotic DNA fragmentation. Importantly, the drug combination induced robust antitumor activity in xenograft models, again accompanied with depletion of FancA. In sum, our findings indicate that ganetespib strongly potentiates the antitumor efficacy of carboplatin by causing combined inhibition of DNA repair and cell cycle control mechanisms, thus triggering global chromosome disruption, aberrant mitosis and cell death. 6 This provides a strong rationale for HSP90 inhibitors in cancer therapy 7 and raises the possibility that HSP90 inhibitors can be used in combination with DNAdamaging chemotherapeutics. 8 Ovarian cancer is among the most common gynecological tumor types and carries the worst prognosis. HSP90 is highly expressed in advanced ovarian carcinoma 9 and thus constitutes a potential therapeutic drug target. 10 In support, HSP90 inhibitors were found effective against ovarian cancer in preclinical models, 11-14 alone or in combination with conventional chemotherapy. 15,16 Carboplatin is the key component of all current first-line therapies for ovarian carcinoma. However, while initially often responding with regression, most tumors relapse and develop resistance within less than a year. 17,18 Like other platinum compounds, carboplatin acts by forming crosslinks within DNA. 17 Platinum compounds form intrastrand DNA lesions but also interstrand crosslinks (ICLs) by covalently linking opposite DNA strands. Such ICLs represent major obstacles to the DNA replication fork 19 and are therefore considered the principal toxic lesion of carboplatin's mode of action. The ICLs can only be removed by a sophisticated DNA repair systemthe Fanconi ...

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Section: Discussionmentioning

confidence: 99%

Strong antitumor synergy between DNA crosslinking and HSP90 inhibition causes massive premitotic DNA fragmentation in ovarian cancer cells

et al. 2016

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“…Accordingly, there is interest in the development and testing of drugs that target G2 or mitotic spindle checkpoints. [5][6][7][8][9] Wee1 is a major regulator of mitotic entry. It phosphorylates and inactivates cyclin-dependent kinase 1 (Cdk1), and its functional role is considered to be in G 2 checkpoint signaling.…”

Section: Introductionmentioning

confidence: 99%

“…11 Preclinical studies have shown that Wee1 inhibition can sensitize cells to mitotic catastrophe following exposure to DNA damaging agents, and several studies are ongoing to test this possibility this in the clinic. 7,[12][13][14][15] Wee1 has an additional role as a Cdk2 kinase, which is relevant to the development of cancer treatment strategies that incorporate Wee1 inhibitors. 16,17 The activation of Cdk2 promotes origin firing during S-phase, and regulates the overall timing of DNA replication.…”

Section: Introductionmentioning

confidence: 99%

“…13 Several clinical trials are underway combining MK-1775 with conventional chemotherapy agents such as gemcitabine, where the effects relieving Weel 0 s restraint of Cdk2 during S-phase might be particularly relevant. 7,15,20,22 There is also a longstanding interest combining checkpoint inhibitors with radiotherapy, since this might differentially improve the radiation response of p53-deficient cancers, where the G1 checkpoint that normally facilitates repair is compromised. Here the anticipated effect of Wee1 inhibition would be to abrogate the G2 checkpoint, resulting in cells entering mitosis with unresolved radiation-induced chromosomal damage, and thereby undergoing death through mitotic catastrophe.…”

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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“…Wee1 is one of the G 2 /M cell cycle checkpoint kinases for which selective chemical inhibitors have been developed as anticancer drugs (3,18). Inhibition of Wee1 potentiates DNA-damaging chemotherapeutics and radiotherapy and has cytotoxic effects as a single agent (19)(20)(21)(22).…”

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confidence: 99%

A haploid genetic screen identifies the G₁/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

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et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The Wee1 cell cycle checkpoint kinase prevents premature mitotic entry by inhibiting cyclin-dependent kinases. Chemical inhibitors of Wee1 are currently being tested clinically as targeted anticancer drugs. Wee1 inhibition is thought to be preferentially cytotoxic in p53-defective cancer cells. However, TP53 mutant cancers do not respond consistently to Wee1 inhibitor treatment, indicating the existence of genetic determinants of Wee1 inhibitor sensitivity other than TP53 status. To optimally facilitate patient selection for Wee1 inhibition and uncover potential resistance mechanisms, identification of these currently unknown genes is necessary. The aim of this study was therefore to identify gene mutations that determine Wee1 inhibitor sensitivity. We performed a genome-wide unbiased functional genetic screen in TP53 mutant near-haploid KBM-7 cells using gene-trap insertional mutagenesis. Insertion site mapping of cells that survived long-term Wee1 inhibition revealed enrichment of G 1 /S regulatory genes, including SKP2, CUL1, and CDK2. Stable depletion of SKP2, CUL1, or CDK2 or chemical Cdk2 inhibition rescued the γ-H2AX induction and abrogation of G 2 phase as induced by Wee1 inhibition in breast and ovarian cancer cell lines. Remarkably, live cell imaging showed that depletion of SKP2, CUL1, or CDK2 did not rescue the Wee1 inhibition-induced karyokinesis and cytokinesis defects. These data indicate that the activity of the DNA replication machinery, beyond TP53 mutation status, determines Wee1 inhibitor sensitivity, and could serve as a selection criterion for Wee1-inhibitor eligible patients. Conversely, loss of the identified S-phase genes could serve as a mechanism of acquired resistance, which goes along with development of severe genomic instability.cell cycle | checkpoint | AZD-1775 | MK-1775 | polyploidy

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MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor efficacy of various DNA-damaging agents, including 5-fluorouracil

Cited by 206 publications

References 23 publications

Strong antitumor synergy between DNA crosslinking and HSP90 inhibition causes massive premitotic DNA fragmentation in ovarian cancer cells

Strong antitumor synergy between DNA crosslinking and HSP90 inhibition causes massive premitotic DNA fragmentation in ovarian cancer cells

Cytokinetic effects of Wee1 disruption in pancreatic cancer

A haploid genetic screen identifies the G₁/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

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MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor efficacy of various DNA-damaging agents, including 5-fluorouracil

Cited by 206 publications

References 23 publications

Strong antitumor synergy between DNA crosslinking and HSP90 inhibition causes massive premitotic DNA fragmentation in ovarian cancer cells

Strong antitumor synergy between DNA crosslinking and HSP90 inhibition causes massive premitotic DNA fragmentation in ovarian cancer cells

Cytokinetic effects of Wee1 disruption in pancreatic cancer

A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

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A haploid genetic screen identifies the G₁/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity