An Integrated Chemical Biology Approach Provides Insight into Cdk2 Functional Redundancy and Inhibitor Sensitivity

Echalier, Aude; Cot, E.; Camasses, Alain; Hodimont, E.; Hoh, François; Jay, Philippe; Sheinerman, Felix B.; Krasińska, Liliana; Fisher, Daniel

doi:10.1016/j.chembiol.2012.06.015

Cited by 39 publications

(40 citation statements)

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“…We found no evidence of CCNE1 copy number change or CDK2 mutation in resistant cell lines derived after extended exposure to PHA-533533. Recent studies in Xenopus show that engineering of compound mutations in the kinase domain of Cdk2 can achieve resistance to CDK inhibitors (25). However, the requirement for multiple residue changes may limit the likelihood of emergence of resistance by mutation in vivo.…”

Section: Discussionmentioning

confidence: 99%

Resistance to CDK2 Inhibitors Is Associated with Selection of Polyploid Cells inCCNE1-Amplified Ovarian Cancer

Etemadmoghadam

Au-Yeung

Wall

et al. 2013

Clinical Cancer Research

100

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Purpose: Amplification of cyclin E1 (CCNE1) is associated with poor outcome in breast, lung, and other solid cancers, and is the most prominent structural variant associated with primary treatment failure in highgrade serous ovarian cancer (HGSC). We have previously shown that CCNE1-amplified tumors show amplicon-dependent sensitivity to CCNE1 suppression. Here, we explore targeting CDK2 as a novel therapeutic strategy in CCNE1-amplified cancers and mechanisms of resistance.Experimental Design: We examined the effect of CDK2 suppression using RNA interference and smallmolecule inhibitors in SK-OV-3, OVCAR-4, and OVCAR-3 ovarian cancer cell lines. To identify mechanisms of resistance, we derived multiple, independent resistant sublines of OVCAR-3 to CDK2 inhibitors. Resistant cells were extensively characterized by gene expression and copy number analysis, fluorescence-activated cell sorting profiling and conventional karyotyping. In addition, we explored the relationship between CCNE1 amplification and polyploidy using data from primary tumors.Results: We validate CDK2 as a therapeutic target in CCNE1-amplified cells by showing selective sensitivity to suppression, either by gene knockdown or using small-molecule inhibitors. In addition, we identified two resistance mechanisms, one involving upregulation of CDK2 and another novel mechanism involving selection of polyploid cells from the pretreatment tumor population. Our analysis of genomic data shows that polyploidy is a feature of cancer genomes with CCNE1 amplification.Conclusions: These findings suggest that cyclinE1/CDK2 is an important therapeutic target in HGSC, but that resistance to CDK2 inhibitors may emerge due to upregulation of CDK2 target protein and through preexisting cellular polyploidy.

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Section: Discussionmentioning

confidence: 99%

Resistance to CDK2 Inhibitors Is Associated with Selection of Polyploid Cells inCCNE1-Amplified Ovarian Cancer

Etemadmoghadam

Au-Yeung

Wall

et al. 2013

Clinical Cancer Research

100

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“…The majority of studies utilizing RO have used doses of 9–10 µM which, when given to interphase cells is sufficient to prevent mitotic entry, suggesting that this dose blocks the majority of Cdk1 activity 8 , 10 , 11 . When mitotic cells are exposed to comparable doses of Cdk1 inhibitors similar to RO, cells undergo a rapid mitotic exit 8 , 12 .…”

Section: Introductionmentioning

confidence: 99%

Partial inhibition of Cdk1 in G₂phase overrides the SAC and decouples mitotic events

et al. 2014

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Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G2 phase onwards. Addition of low doses of RO3306 in G2 phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G2 phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G2 phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A.

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“…Furthermore, depletion or knockdown experiments might not lead to the same conclusions as in situ experiments in which specific enzymes are inhibited in the presence of all regulators at physiological levels (see Krasinska et al, 2008b for explanation). For example, chemical genetics approaches have shown thatwhen present -Cdk2 has a rate-limiting role in cell cycle progression in human cells (Merrick et al, 2011) and Xenopus egg extracts (Echalier et al, 2012), probably because Cdk2 titrates the nuclear S-phase cyclins A and E. Thus, a single cyclin-Cdk complex might be minimally sufficient, but in a normal in vivo situation, when a variety of complexes are present, several of them might become indispensable as a result of the interactions between them.…”

Section: Cell Cycle Control By Cdk1 -The Quantitative Modelmentioning

confidence: 99%

Phosphorylation network dynamics in the control of cell cycle transitions

Fisher

Krasińska

Coudreuse

et al. 2012

Journal of Cell Science

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143

114

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SummaryFifteen years ago, it was proposed that the cell cycle in fission yeast can be driven by quantitative changes in the activity of a single protein kinase complex comprising a cyclin -namely cyclin B -and cyclin dependent kinase 1 (Cdk1). When its activity is low, Cdk1 triggers the onset of S phase; when its activity level exceeds a specific threshold, it promotes entry into mitosis. This model has redefined our understanding of the essential functional inputs that organize cell cycle progression, and its main principles now appear to be applicable to all eukaryotic cells. But how does a change in the activity of one kinase generate ordered progression through the cell cycle in order to separate DNA replication from mitosis? To answer this question, we must consider the biochemical processes that underlie the phosphorylation of Cdk1 substrates. In this Commentary, we discuss recent findings that have shed light on how the threshold levels of Cdk1 activity that are required for progression through each phase are determined, how an increase in Cdk activity generates directionality in the cell cycle, and why cell cycle transitions are abrupt rather than gradual. These considerations lead to a general quantitative model of cell cycle control, in which opposing kinase and phosphatase activities have an essential role in ensuring dynamic transitions.

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An Integrated Chemical Biology Approach Provides Insight into Cdk2 Functional Redundancy and Inhibitor Sensitivity

Cited by 39 publications

References 50 publications

Resistance to CDK2 Inhibitors Is Associated with Selection of Polyploid Cells inCCNE1-Amplified Ovarian Cancer

Resistance to CDK2 Inhibitors Is Associated with Selection of Polyploid Cells inCCNE1-Amplified Ovarian Cancer

Partial inhibition of Cdk1 in G₂phase overrides the SAC and decouples mitotic events

Phosphorylation network dynamics in the control of cell cycle transitions

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An Integrated Chemical Biology Approach Provides Insight into Cdk2 Functional Redundancy and Inhibitor Sensitivity

Cited by 39 publications

References 50 publications

Resistance to CDK2 Inhibitors Is Associated with Selection of Polyploid Cells inCCNE1-Amplified Ovarian Cancer

Resistance to CDK2 Inhibitors Is Associated with Selection of Polyploid Cells inCCNE1-Amplified Ovarian Cancer

Partial inhibition of Cdk1 in G2phase overrides the SAC and decouples mitotic events

Phosphorylation network dynamics in the control of cell cycle transitions

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About

Partial inhibition of Cdk1 in G₂phase overrides the SAC and decouples mitotic events