Abstract 1872: Pharmacological inhibition of WIP1 sensitizes AML cells to MDM2 inhibitors
Authors: Fontana Maria Chiara , Nanni Jacopo , Simonetti Giorgia , Rorà Andrea Ghelli Luserna di , Padella Antonella , Bruno Samantha , Papayannidis Cristina , Marconi Giovanni , Abbenante Mariachiara , Cavo Michele , Martinelli Giovanni 
Published: August 2018

Abstract
Introduction: The wild-type (wt) p53-inducible protein phosphatase WIP1 (gene PPM1D), is frequently mutated in solid tumors and in blood of healthy-individuals, but its role is still unknown in Acute Myeloid Leukemia (AML). WIP1 suppresses p53 after cell-stress. The aim of this study is to investigate whether the inhibition of WIP1 (GSK2830371) could increase the sensitivity to MDM2 inhibitors (Nutlin-3a) in order to obtain a novel therapeutic strategy for AML patients restoring p53 activity.
Methods: Whole Exome Sequencing (WES) and targeted Next Generation Sequencing (NGS) have been performed to investigate the mutational state of PPM1D in newly diagnosed AML patients. We performed in vitro WST-1 cell-viability and Annexin V-PI apoptosis assays in AML cell lines (MOLM-13, MV-4-11) and AML primary cells.
Results: WIP1i and Nut-3a reduced the cell viability as single agent in all the treated cells in a time and dosage-dependent manner. The IC50 values in TP53-wt cells showed that MOLM-13 (10 υM) are more sensitive than MV-4-11 (20 υM) to WIP1i. Combination index analyses confirmed that WIP1i and Nut-3a had a synergistic effect (C.I.&lt;1) in both cell lines. Annexin V/PI staining performed after treatment with different concentration of Nut-3a (0.5 υM for MOLM-13, 15 υM for MV4-11) and WIP1 inhibitor (10 υM for MOLM-13, 20 υM for MV4-11) demonstrated that the combination significantly increased the number of apoptotic cells in comparison to single agents. In particular, after 48 hours of incubation with Nut-3a and WIP1i as single agents, the percentage of apoptotic cells was 34.4% and 21.4% for MOLM-13 and 17% and 18% for MV-4-11, respectively; while the combination induced 71.6% and 54.5% of apoptotic cells. The decrease of cells in G2/M phase induced by single agents, was enhanced by the drugs combination in both cell lines and, in particular, MV-4-11 showed an increasing percentage of S phase cells allowing us to hypothesize that the apoptotic death induced by the combination of drugs occurs when cells are blocked in S phase. AML primary cells were also tested, showing a similar pattern when the two drugs are combined. Finally, by WES and targeted-NGS, 7 mutations have been detected in 2/37 and 4/96 patients. One patient harbored a truncating mutation on exon 6, known to be gain of function, also confirmed by Western Blot (WB), increasing the inhibition of p53; while the other mutations affected the exon 1 and have not been described yet.
Conclusions: This pre-clinical study allows us to affirm for the first time that WIP1i potentiates the sensitivity to Nut-3a in AML. Further studies on TP53-mutated and wt cells will help to confirm these preliminary data, Gene Expression Profile and WB will identify the proteins playing a relevant role in the biological alterations induced by WIP1i. Supported by: ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project.
Citation Format: Maria Chiara Fontana, Jacopo Nanni, Giorgia Simonetti, Andrea Ghelli Luserna di Rorà, Antonella Padella, Samantha Bruno, Cristina Papayannidis, Giovanni Marconi, Mariachiara Abbenante, Michele Cavo, Giovanni Martinelli. Pharmacological inhibition of WIP1 sensitizes AML cells to MDM2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1872.