Abstract 258: Is P53 the up-and-coming predictive biomarker for volasertib treatment in NSCLC
Authors: Van den Bossche Jolien , Lardon Filip , Deben Christophe , De Pauw Ines , Deschoolmeester Vanessa , Smits Evelien , Specenier Pol , Pauwels Patrick , Peeters Marc , Wouters An 
Published: July 2016

Abstract
Introduction: Polo-like kinase (Plk1), a key regulator of cell division, is considered as an attractive target for mitotic intervention. The observed Plk1 overexpression in several tumor types, including non-small cell lung cancer (NSCLC), has led to the development of small molecule inhibitors. Moreover, previous studies suggested an interplay between Plk1 and the tumor suppressor P53. This led to the hypothesis that the P53 status might be predictive for the response to Plk1 inhibition. Therefore, we investigated the cytotoxic effect of volasertib in NSCLC cell lines with a different P53 background, under normoxia and hypoxia.
Material and methods: A549 (TP53 wild type) was transduced with a P53 shRNA lentiviral vector to obtain the P53 deficient sub-cell line A549-920 or with an empty vector as control (A549-NTC). In addition, NCI-H1975 cells were used as a TP53 mutant (R273H) cell line. Cells were treated with 0-85nM volasertib (24-72h). Cell survival was assessed using the sulphorhodamine B assay and IC50-values were calculated using WinNonlin software. The effect of Plk1 inhibition (0-20nM) on cell cycle distribution (24h) and apoptosis induction (48h) was determined flow cytometrically. In addition, we investigated the potential of volasertib to inhibit cell migration using a Transwell system. All experiments were performed under both normoxia and hypoxia (&lt;0.1% O2).
Results: Plk1 inhibition established a dose-dependent growth inhibition under both normoxia and hypoxia. Interestingly, a reduced sensitivity to volasertib treatment (24h) was observed in P53 deficient cells (A549-920, IC50: 27.59±5.77 nM) compared to P53 wild type cells (A549-NTC, IC50: 17.87±0.40 nM) (p&lt;0.001). Except for the NCI-H1975 cells, a decreased effect of volasertib was observed under hypoxia (p&lt;0.001). After treatment, a G2/M phase block was induced in all cell lines (p&lt;0.004), albeit more pronounced in A549-920 cells compared to A549-NTC cells (p = 0.06). Under hypoxia, this mitotic arrest was only detected using high volasertib concentrations (12.5–20nM) (p&lt;0.04). Furthermore, a sub-G1 peak could be observed after treatment with high volasertib concentrations, suggesting apoptosis induction. Indeed, an increase in PI-stained cells was detected 48h after Plk1 inhibition in all cell lines (p&lt;0.042). Remarkably, induction of cell death was higher in P53 wild type cells compared to P53 deficient cells (p&lt;0.02). Transwell experiments, investigating the potential of volasertib to inhibit cell migration, are currently being analyzed.
Conclusion: Our results show that there is a difference in response to Plk1 inhibition in cancer cells with and without functional P53. While P53 deficient/mutant cells were mostly arrested in the G2/M phase, cell death was induced in P53 wild type cells. These data lead to the hypothesis that P53 might be a predictive marker for response to Plk1 inhibition.
Citation Format: Jolien Van den Bossche, Filip Lardon, Christophe Deben, Ines De Pauw, Vanessa Deschoolmeester, Evelien Smits, Pol Specenier, Patrick Pauwels, Marc Peeters, An Wouters. Is P53 the up-and-coming predictive biomarker for volasertib treatment in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 258.