Arsenic Trioxide (ATO) and MEK1 Inhibitor Activate Apoptotic p73 Pathway in Primary Acute Myelogenous Leukemia Blasts.

We recently reported that PD184352 (PD) (Pfizer), a highly selective inhibitor of MEK1 phosphorylation and activation, strikingly enhances ATO-mediated apoptosis in NB4, K562 cell lines (Blood104: 519–525, 2004), and in primary acute myelogenous leukemia (AML) cases. The aim of this study was to investigate whether the p53-related gene p73 is a molecular target of the combined treatment in AML blasts. P73 exists as multiple TA (transactivation competent, pro-apoptotic and anti-proliferative) p73 COOH-terminal splicing isoforms (α,β,γ,δ,ε,ζ) of which the two major forms are p73α and p73β. In addition, dominant negative (ΔN) variants are expressed from a second promoter, that possess anti-apoptotic and pro-proliferative potential. In order to evaluate whether the combined treatment modulates p73 isoforms in AML blasts, the protein expression of TA-p73α, TA-p73β and ΔN-p73α were evaluated before and after the treatment with PD and/or ATO (1–2 μM) in 13 cases of AML and the TA/ΔN-p73 ratio was calculated. The basal expression of TA-p73α and TA-p73β were clearly evident in 11/13 and 12/13 cases respectively. In addition ΔN-p73α expression was detectable in 11/13 cases. Interestingly, in the PD+ATO responsive cases (11/13) we found that MEK1 inhibitor reduced the levels of ΔNp73 proteins and promoted the accumulation of endogenous TA-p73α and/or TA-p73β elevating the TA/ΔN ratio. ATO alone promoted the increase of both TA and ΔN-p73 proteins expression failing, or resulting less efficient than PD or PD+ATO in elevating TA/ΔN ratio. The lack of p73 proteins expression or the inability of PD in elevating TA/ΔN-p73 ratio resulted in loss of PD+ATO efficacy treatment. We then evaluated whether the changes in TA/ΔN-p73 ratio observed in PD+ATO vs ATO treated cells resulted in an increased expression of pro-apoptotic p73/p53 target genes, Bax, PUMA (p53 Up-regulated Modulator of Apoptosis), and P53AIP1 (p53-regulated Apoptosis-Inducing Protein 1) a primary effector gene of wild type p53 and TAp73-induced apoptosis whose overexpression induces massive mithocondrial apoptotic cell death. Interestingly, after a 48 hours treatment we found that Bax and PUMA proteins accumulated to a greater extent when cells were challenged with ATO treatment than with PD+ATO in 6/9 AML whereas p53AIP1 expression was greatly enhanced after PD+ATO treatment compared to ATO alone (2 or more fold increase). Conversely no differences in p53AIP1, Bax and PUMA expression between ATO and PD+ATO treatment were observed in no responsive cases. Interestingly, both p73 and p53 proteins, accumulated in AML blasts treated with 2μM of ATO, whereas lower concentration of ATO (1μM) induced only p73. In the responsive cases the combined treatment also led to an increased poly (ADP-ribose) polymerase (PARP) fragmentation that reflected the increased apoptosis. Finally, the selective downregulation of ΔNp73 by using specific siRNA, sensitized both K562 and NB4 leukemic cell lines to ATO-induced apoptosis suggesting that it may contribute to the ATO-resistance in leukemia cells. Our results support ATO plus PD combination as potential anti-leukemic treatment.