Plk1 Inhibition Causes Post-Mitotic DNA Damage and Senescence in a Range of Human Tumor Cell Lines

<div><p>Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the <i>in vitro</i> and <i>in vivo</i> consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens.</p></div

Plk1 inhibition leads to prolonged mitotic arrest followed by detection of DNA damage in HT-29 cells.

<p>Cells were treated with increasing concentrations of MLN0905 and mitotic arrest (via pHisH3) and DNA damage (via γH2AX) were analyzed at indicated times. A) Immunoblotting was used to demonstrate prolonged mitotic arrest precedes DNA damage (representative blot shown from two independent experiments). B) The same samples used in A) were quantified for DNA damage (γH2AX) using FACS analysis. C) In a separate experiment immunofluorescent chemistry was also used to demonstrate mitotic arrest (pHisH3) precedes DNA damage (γH2AX) (representative images shown from two independent experiments; 200 nM MLN0905 used).</p

<i>In vivo</i>, Plk1 inhibition leads to mitotic arrest, DNA damage and senescence.

<p>A) HCT116 tumor bearing mice were dosed orally once a day with 23.3 mg/kg of MLN0905. Tumor tissues were harvested and stained for pHisH3 (green staining) and γH2AX (brown staining). Representative tumor sections are shown with either vehicle or MLN0905 treatment 24 hours after the first dose. pHisH3 and γH2AX signals were quantified (see graph, shown is mean ±SD) and arrows indicate when doses were delivered. B) Tumor tissues were harvested at the indicated times and stained for β-galactosidase activity. Images shown indicate β-galactosidase staining in the MLN0905 treated tumors but not in the time-matched vehicle controls. Staining was quantified and is represented as % tumor area positive (see graph, shown is mean ±SD; *p≤0.04, two-tailed paired t-test).</p

Plk1 inhibition leads to mitotic arrest and DNA damage in a wide range of human cancer cell lines.

<p>Cells were treated with MLN0905 (Calu-6, 10 nM; DLD-1, 90 nM; A549, 30 nM; SW480, 20 nM) for the indicated times and immunoblotting was used to monitor mitotic arrest (pHisH3) and DNA damage (γH2AX).</p

Plk1 inhibition leads to transient mitotic arrest followed by DNA damage in HCT116 cells.

<p>Cells were treated with increasing concentrations of MLN0905 and at indicated times analyzed for mitotic arrest (via pHisH3) and DNA damage (via γH2AX). A) Immunoblotting indicates transient mitotic arrest leads to DNA damage (representative blot shown from two independent experiments). B) The same samples used in A) were quantified for DNA damage (γH2AX staining) using FACS analysis. C) In a separate experiment immunofluorescent chemistry was also used to demonstrate mitotic arrest precedes DNA damage (representative images shown from two independent experiments; 50 nM MLN0905 used).</p

Plk1 inhibition leads to senescence in multiple cell lines.

<p>A) Cells were treated for two weeks with MLN0905 (50 nM for HCT116, 30 nM for A549, 20 nM for SW480 and HT-29) and then stained for β-galactosidase activity. B) β-galactosidase staining was then quantified in senescent cells and represented as % area (shown is mean ±SD; *p = 0.0159, **0.0033 and ***0.032 by a two-tailed Mann-Whitney test).</p

Discovery of a Potent and Orally Bioavailable Benzolactam-Derived Inhibitor of Polo-Like Kinase 1 (MLN0905)

This article describes the discovery of a series of potent inhibitors of Polo-like kinase 1 (PLK1). Optimization of this benzolactam-derived chemical series produced an orally bioavailable inhibitor of PLK1 (<b>12c</b>, MLN0905). In vivo pharmacokinetic–pharmacodynamic experiments demonstrated prolonged mitotic arrest after oral administration of <b>12c</b> to tumor bearing nude mice. A subsequent efficacy study in nude mice achieved tumor growth inhibition or regression in a human colon tumor (HT29) xenograft model