Effect of MDM2 and vascular endothelial growth factor inhibition on tumor angiogenesis and metastasis in neuroblastoma Danielle M. PattersonDongbing GaoDenae N. TrahanBrett A. Johnson • Andrew LudwigEveline BarbieriZaowen ChenJose Diaz-Miron • Lyubomir VassilevJason M. ShohetEugene S. Kim

Neuroblastoma is the most common pediatric abdominal tumor and principally a p53 wild-type, highly vascular, aggressive tumor, with limited response to anti- VEGF therapies alone. MDM2 is a key inhibitor of p53 and a positive activator of hypoxia-inducible factor-1a (HIF- 1a) and vascular endothelial growth factor (VEGF) activity with an important role in neuroblastoma pathogenesis. We hypothesized that concurrent inhibition of both MDM2 and VEGF signaling would have cooperative anti-tumor effects, potentiating anti-angiogenic strategies for neuro- blastoma and other p53 wild-type tumors. We orthotop- ically implanted SH-SY5Y neuroblastoma cells into nude mice (n = 40) and treated as follows: control, bev- acizumab, Nutlin-3a, combination of bevacizumab plus Nutlin-3a. Expression of HIF-1a and VEGF were measured by qPCR, Western blot, and ELISA. Tumor apoptosis was measured by immunohistochemistry and caspase assay. Angiogenesis was evaluated by immunohistochemistry for vascular markers (CD-31, type-IV collagen, aSMA). Both angiogenesis and metastatic burden were digitally quanti- fied. In vitro, Nutlin-3a suppresses HIF-1a expression with subsequent downregulation of VEGF. Bevacizumab plus Nutlin-3a leads to significant suppression of tumor growth compared to control (P \ 0.01) or either agent alone. Combination treated xenograft tumors display a marked decrease in endothelial cells (P \ 0.0001), perivascular basement membrane (P \ 0.04), and vascular mural cells (P \ 0.004). Nutlin-3a alone and in combination with bevacizumab leads to significant tumor apoptosis (P \ 0.0001 for both) and significant decrease in incidence of metastasis (P \ 0.05) and metastatic burden (P \ 0.03). Bevacizumab plus Nutlin-3a cooperatively inhibits tumor growth and angiogenesis in neuroblastoma in vivo with dramatic effects on tumor vascularity. Concomitantly tar- geting VEGF and p53 pathways potently suppresses tumor growth, and these results support further clinical develop- ment of this approach.