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

Expression of GLUT1 and GLUT4 receptors in xenograft tumors with either WT or mutant KRAS.

<p>Proteins were extracted from untreated tumors and 10μg protein was loaded in each lane. Levels of GLUT1 and GLUT4 proteins were determined by western blot and tubulin was used as a loading control. n = 3 for each tumor.</p

Induction of β-oxidation by ixazomib treatment in SW48 WT tumors.

<p>metabolites involved in fatty acid beta oxidation pathway. (A) Table summarizing free fatty acids in tumor samples. The numbers indicate the fold change in drug treated tumors over vehicle treated tumors at 1 and 8hrs time points. Each data point represents an average of tumors from five different animals. Table summarizing the levels of acetyl Co-A and 3 Hydroxy butyrate at 1hr after ixazomib treatment. The numbers indicate fold change in ixazomib treated tumors over vehicle treated tumors, n = 5 per data point. Changes in CPT-1 protein level following ixazomib treatment in SW48 and SW48-G13D tumors. The vehicle samples were collected at 4hrs after dosing and the ixazomib treated samples were collected at different time points (as indicated in the figure) after the drug treatment. The color coding is as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144825#pone.0144825.g004" target="_blank">Fig 4</a>.</p

KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical <i>In Vivo</i> Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism

<div><p>In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines <i>in vitro</i>. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and <i>KRAS</i> genotype; tumors with wild-type (WT) <i>KRAS</i> were more sensitive to ixazomib than tumors harboring <i>KRAS</i> activating mutations. To confirm the association between <i>KRAS</i> genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib <i>in vivo</i>. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between <i>KRAS</i> genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.</p></div

Baseline metabolic differences between SW48 KRAS WT and G13D mutant tumors.

<p>A-C: Glutathione metabolism pathways (A), glycogen metabolism (B) and relative expression of pathway metabolites in KRAS WT vs KRAS mutant tumors treated with vehicle for 1 and 8 hrs. The numbers indicate the fold change of each metabolite in KRAS mutant tumors compared to KRAS WT tumors and are the average of metabolite levels from 5 different tumors. C: Relative expression of free long chain fatty acids, acetyl coA, acetylcarnitine and citrate in KRAS mutant vs KRAS WT tumors. Green boxes indicates a ratio <1, with dark green boxes as highly significant (p ≤0.05) and light green boxes as less significant (0.051, with dark red boxes as highly significant (p ≤0.05) and light red boxes as less significant (0.05</p