SF1126, a novel pan-PI3K inhibitor, inhibits activation of Mdm2 and increases sensitivity to doxorubicin in wild type p53 neuroblastoma cell lines

LB-294

Neuroblastoma (NB), a heterogeneous and highly malignant cancer of the sympathetic nervous system, is the most common extracranial solid tumor of childhood and is often marked by resistance to chemotherapy. A novel integrin-targeted pan-PI3K inhibitor, SF1126, which consists of LY294002 conjugated to RGD (the arginine-glycine-aspartic acid sequence that binds primarily to the cell surface integrin receptors ανβ3 and ανβ5) was tested in combination with the chemotherapeutic agent doxorubicin (dox) in vitro against a subset of neuroblastoma cell lines containing wt p53 (SH-SY5Y, SH-SKNBE(1) and NB-1691). SF1126 alone (1-20 uM) had marginal effects on cell viability. However, SF1126 significantly enhanced sensitivity to pharmacological levels of dox (0.01-0.5 ug/ml) as evaluated by WST cytotoxicity assays, especially at lower dox concentrations. To investigate the role of p53 and upstream phosphorylation in SF1126-treated cells, we evaluated the phosphorylation status of Akt and Mdm2 and their effects on downstream signaling by immunoblotting. pAkt and pMdm2 were both reduced in a dose and time-course-dependent manner in all 3 lines, suggesting that SF1126 blocked activation of both Akt and its substrate Mdm2. Moreover, p53 levels were increased and survivin levels diminished, suggesting that SF1126 augmented the cytotoxicity of dox by preventing activation of Mdm2, with consequent up-regulation of p53 and down-regulation of survivin. Interestingly, we detected up-regulation of PTEN in one line (NB-1691), a cell line having very low endogenous levels of PTEN; this up-regulation may be due to increased levels of p53. We therefore evaluated the cellular localization of Mdm2 following treatment with SF1126 and found that while Mdm2 was distributed in both the cytoplasmic and nuclear fractions before treatment, it was largely sequestered in the cytoplasm following treatment. These results suggest that SF1126 may enhance the cytotoxicity of doxorubicin in neuroblastoma cells by inhibiting the activation of Mdm2 by Akt, leading to p53-mediated downregulation of survivin and activation of apoptosis. We are investigating the interaction of SF1126 with other chemotherapeutic agents and with other NB p53 phenotypes.