Perifosine plus nutlin-3 combination shows a synergistic anti-leukaemic activity.

Perifosine, which belongs to the novel class of antitumoral agents alkylphospholipids, is currently undergoing phase II clinical evaluation (van Blitterswijk & Verheij, 2008). Although the mechanisms by which perifosine exerts its antineoplastic activity need to be fully elucidated, it has been shown that perifosine inactivates the phosphatidylinositol 3-kinase/Akt pathway (van Blitterswijk & Verheij, 2008). While previous studies have demonstrated that perifosine shows cytotoxic activity against different types of haematological malignancies, when used either alone or in combination with chemotherapeutic drugs (van Blitterswijk & Verheij, 2008), its anti-leukaemic potential has not been investigated in combination with innovative compounds. Therefore, the effect of perifosine (Cayman Chemical, Ann Arbor, MI, USA) was analyzed in a panel of p53wild-type (SKW6.4, OCI, MOLM), p53mutated (BJAB, MAVER) and p53null (HL-60) cell lines, of both B lymphoid (SKW6.4, BJAB and MAVER) and myeloid (OCI, MOLM, HL-60) origin, all purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) or obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Perifosine was used alone or in combination with nutlin-3 (Cayman Chemical), a potent and selective small molecule inhibitor of the p53-MDM2 interaction, which induces a strong non genotoxic activation of the p53 pathway (Vassilev, 2007). In preliminary experiments, perifosine alone dose-dependently induced cytotoxicity, evaluated as previously described (Borgatti et al, 1997), in all leukaemic cell lines. The simultaneous addition of high concentrations of perifosine plus nutlin-3 (10 μmol/l each) induced a significant (P < 0·05) increase of cytotoxicity with respect to each agonist used alone in p53wild-type SKW6.4, OCI and MOLM cell lines, but not in p53mutated BJAB and MAVER and p53null cell lines, with the exception of MAVER cell line at 24 h. In order to evaluate whether the combined effect of perifosine plus nutlin-3 was synergistic, cultures were treated with serial concentrations of perifosine and nutlin-3 at a constant perifosine:nutlin-3 ratio, for 24 and 48 h and data were analyzed by the method of Chou and Talalay (1984). Synergy of perifosine plus nutlin-3 combination treatment (average combination index values <1) was clearly observed in all p53wild-type cell lines. Similarly, perifosine plus nutlin-3 (10 μmol/l each) induced enhanced cytotoxicity with respect to the treatments with the single agents also in some cases of p53wild-type primary acute myeloid leukaemia (AML, M1 and M5 type) and B chronic lymphocytic leukaemia (B-CLL) samples, but not in p53mutated or p53null cell lines. In order to investigate the molecular basis for the synergistic activity exerted by the combination of perifosine plus nutlin-3 in p53wild-type leukaemic cell lines, we have performed Western blot analysis to examine changes in the protein levels of p53 and of its major transcriptional targets (p21, Bax, MDM2), as well as MDM4/MDMX, an homolog of MDM2 which plays a non-redundant role with respect to MDM2 in down-regulating the activity of p53 and is not a transcriptional target of p53 (Meulmeester & Jochemsen, 2008). Nutlin-3 alone induced a marked accumulation of p53 protein, which was not affected by the concomitant presence of perifosine. In parallel, nutlin-3 induced an early and robust induction of both p21 and Bax, which started at 6 h and lasted for the duration of the experiment (48 h). Of note, the accumulation of both p21 and Bax was significantly potentiated by the concomitant treatment with perifosine. On the contrary, the combination of perifosine plus nutlin-3 induced completely different effects on MDM2 and MDM4/MDMX. In fact, while nutlin-3 alone significantly up-regulated the levels of expression of MDM2, which represents a major transcriptional target of p53 (Vassilev, 2007), the simultaneous addition of perifosine significantly counteracted the nutlin-3-mediated induction of MDM2. At variance to MDM2, nutlin-3 not only did not induce MDM4/MDMX, but rather promoted its degradation, an effect that is considered an important part of the mechanism of action of nutlin-3-mediated cytotoxicity (Secchiero et al, 2008). Of interest, the combination of perifosine plus nutlin-3 resulted in a more rapid degradation of MDM4/MDMX with respect to cultures treated with nutlin-3 alone. The sustained increase in Bax protein levels observed in response to the nutlin-3 plus perifosine combination with respect to nutlin-3 alone strongly suggests that Bax induction probably represents a major molecular mechanism mediating the combined cytotoxicity of nutlin-3 plus perifosine. However, other apparently more subtle effects of the nutlin-3 plus perifosine combination were also noticed. The induction of MDM2, which represents a major transcriptional target and a key negative regulator of p53, was significantly weaker in cultures treated with nutlin-3 plus perifosine with respect to cultures treated with nutlin-3 alone. As p53 protein showed a comparable accumulation in samples treated with nutlin-3 or with nutlin-3 plus perifosine, and the best characterized mechanism of action of MDM2 is to down-regulate the transcriptional activity of p53 (Vassilev, 2007), the weaker induction of MDM2 in cultures treated with perifosine plus nutlin-3 probably accounts for the increased levels of other p53 transcriptional targets, p21 and Bax in SKW6.4 cells treated with the drug combination. A likely explanation for the differential ability of perifosine plus nutlin-3 to super-induce p21 and Bax while counteracting the accumulation of MDM2 is that perifosine blocks the Akt pathway (van Blitterswijk & Verheij, 2008), which has an important role in stabilizing both MDM2 (Zhou et al, 2001) and MDM4/MDMX (Secchiero et al, 2008). In this respect, also the ability of perifosine to potentiate the nutlin-3-mediated down-regulation of MDM4/MDMX is particularly noteworthy since it demonstrated that the levels of MDM4/MDMX represent a major molecular determinant in the cytotoxic response to nutlin-3, at least in solid tumours (Secchiero et al, 2008). In conclusion, our present study suggests that the combined treatment of perifosine plus nutlin-3 might offer a novel therapeutic strategy for p53wild-type haematological malignancies, which represent >80% of all haematological malignancies at diagnosis (Mitani et al, 2007).