Synergistic Activation of p53 Family of Proteins by the Combination of Nutlin 3a and SAHA in Acute Myelogenous Leukemia.

Nutlin 3a binds to murine double minute 2 (MDM2) a negative regulator of p53 and potently increases p53 protein levels by inhibiting MDM2 mediated ubiquitilation. Our group has shown activity of Nutlin 3a in acute myelogenous leukemia (AML) and an intact p53 pathway appeared to be necessary for this activity (Blood. 2005;106:3150-9). The lysine residues on p53 that are ubiquitilated by MDM2 are also sites for acetylation, a process that stabilizes p53 for efficient transcription of p53 target proteins. SAHA is a wide spectrum Histone de-acetylase inhibitor (HDACi) and is known to induce acetylation of non-histone proteins. We postulated that the combination of Nutlin 3a and SAHA will lead to increased acetylation of p53, enhanced transcription of pro-apoptotic targets of p53 and synergistic activity in AML. As Nutlin 3a has been reported to disrupt interaction of MDM2 with other p53 family members notably p73 (Oncogene. 2008;27:997-1003) and HDACi can induce pro-apoptotic p73 (Oncogene. 2004;23:4807-17), we further postulated that the combination may be effective in AML cells with defective p53 pathway.

In this study we investigated the effects of Nutlin 3a/SAHA combination on AML cell lines. OCI-AML-3 (p53 wild-type) cells were pre-treated with Nutlin 3a for 6 hrs and treated with Nutlin 3a+SAHA for a further 48 hrs. SAHA and Nutlin 3a induced apoptosis synergistically even in low drug concentrations (2.5 μM Nutlin 3a and 0.5 μM SAHA). In OCI-AML-3 cells, combination treatment resulted in increased protein levels of p53, increased p53 acetylation at Lys373/Lys382and induction of pro-apoptotic p53 transcriptional target protein, Noxa..

Interestingly, synergistic apoptosis induction with Nutlin 3a+SAHA was seen in p53 null (HL-60) and mutant (NB4 and OCI-AML-2) cells after 72 hrs. In HL-60 cells, treatment with Nutlin 3a+SAHA, resulted in increase in protein levels of p73. Noxa is also a transcriptional target of p73 and the treatment with Nutlin 3a+SAHA combination upregulated protein levels of Noxa in HL60 cells. Experiments with “knock-down” of p73 and Noxa and combination of Nutlin 3a with other HDACi are in progress. Though SAHA is known to be active against cancer cell lines with P-glycoprotein (Pgp) expression, we plan to exclude the possibility of modulation of P-gp by Nutlin 3a contributing to this synergy. Co-culture with normal bone marrow mesenchymal stromal cells (MSCs) confers chemo-resistance to leukemia cells. Treatment with the combination of Nutlin 3a and SAHA for 72 hrs effectively induced apoptosis in OCI-AML-3 cells co-cultured with MSCs.

The combination of MDM2 inhibitor, Nutlin 3a and HDACi, SAHA show promising synergistic activity against AML cells irrespective of their p53 status and can potentially overcome the resistance mediated by the “microenvironment”.

Off Label Use: This is an in vitro study of SAHA (FDA approved) and the investigative agent Nutlin. No clinical data included..