Mdm2 Inhibitor Nutlin-3 Enhances the Cytotoxic Synergism Induced by the Combination of MEK1 Inhibitor and Arsenic Trioxide (ATO) in AML Cells.
Authors: Lunghi Paolo , Mazzera Laura , Rizzoli Vittorio , Bonati Antonio 
Published: April 2020

Abstract
We recently reported that PD184352 (PD) (Pfizer), a highly selective inhibitor of MEK1 phosphorylation and activation, sensitizes primary acute myelogenous leukemia (AML) to ATO-induced apoptosis via p73, a p53 paralogue, and Bad pro-apoptotic pathways activation (Blood107: 4549–4554, 2006). We also demonstrated that high doses of ATO (2μM) induced p53 accumulation (more than two-fold increase compared with control) in 11 out of 21 patients (52%), indicating a possible contribution of p53 pathway in apoptosis induction of dual treated blasts. TP53 mutations are quite rare in AML (5–10%) and MDM2 (murine double minute 2), its principal negative regulator, has been found to be frequently overexpressed in AML, a process that can actively enhance tumorigenic potential and resistance to apoptosis. The aim of this study was to investigate whether Nutlin-3, a potent and selective small-molecule MDM2 antagonist, can potentiate the apoptotic effect of the PD plus ATO combination in AML cells that retain a functional p53. Apoptosis was evaluated, after 24 and 48 hours of treatment, by measurement of sub-G1 DNA content, annexin V binding and mitochondrial transmembrane potential assays. We first analyzed the pharmacologic interactions between Nutlin-3, PD and ATO (0, 0.25, 0.5, 1, 2, 5, or 10 μM) using a fixed-ratio (1:1:1) experimental design in OCI-AML-3 cell line that has wild-type p53. We found that the three-drugs combination showed cytotoxic synergism stronger than PD plus ATO combination indicating that the inhibition of the p53-MDM2 interaction can positively influence the pro-apoptotic efficacy of dual-treated (PD plus ATO) cells: the averaged Combination Index (CI) values calculated from the ED50 (50% effective dose), ED75 and ED90, in Nutlin-3 plus PD plus ATO versus PD plus ATO treated cells were 0.36± 0.03 and 0.66± 0.02 respectively (CI values equal to 0.85 or less indicate synergism). In order to investigate the molecular effectors involved in Nutlin-3-PD-ATO or PD-ATO-induced apoptosis we first studied the kinetics (2h, 12h, 24h and 48h) of p53, p73 and phospho-Bad at Ser112 in OCI-AML-3. In the absence of Nutlin-3 ATO, even at high doses, did not promote a p53 accumulation whereas modulated the expression of the p73 gene by inducing the pro-apoptotic and anti-proliferative TAp73 and the anti-apoptotic and pro-proliferative ΔNp73 isoforms, thereby failing to elevate the TA/ΔNp73 ratio. Conversely, treatment with PD reduced the level of ΔNp73 and blunted the ATO-mediated up-regulation of ΔNp73 thus causing an increase in the TA/ΔNp73 ratio of dual-treated cells. In the presence of Nutlin-3, p53 accumulated and the triple combination of Nutlin-3, PD and ATO enhanced the loss of mitochondrial membrane potential occurred in PD plus ATO treatment. Furthermore, pretreatment with MEK1 inhibitor strongly increased the expression of dephosphorylated Bad and blunted the ATO-mediated phosphorylation of Bad at Ser112, thereby activating its pro-apoptotic functions. These findings suggest that the pro-apoptotic p73 and Bad pathways, both involved in PD plus ATO efficacy, can be potentiated by the rescue of p53 pathway in AML cells that possess a functional p53 pathway.