Retinoids are an important component of therapy at the stage of minimal residual disease for advanced neuroblastoma. However, 40-50% of patients treated with 13-cis-retinoic acid still relapse, indicating the necessity for more effective retinoid therapy. Fenretinide (4-HPR) and vorinostat (SAHA), were used in early phase single agent paediatric oncology trials, but only stabilised disease. We used human neuroblastoma cell lines, siRNA knockdown and xenograft tumour models to evaluate 4-HPR + SAHA combination treatment for therapeutic synergy and biomarkers of response.
At clinically relevant concentrations of 4-HPR (3µM) + SAHA (0.5µM) the combination therapy exerted potent cytopathic effects in multiple neuroblastoma cell lines (combination index < 1). The proportion of apoptotic cells was markedly increased in neuroblastoma cells, compared with non-malignant MRC-5 cells. The cytopathic effect of 4-HPR + SAHA was much greater than 13-cis-retinoic acid + SAHA. In vivo xenograft experiments of BE(2)-C cells injected into the flank of athymic nude mice treated with 4-HPR (1.45mg/kg, i.v.) + SAHA (35mg/kg, i.p.) also demonstrated therapeutic synergy. To identify biomarkers of sensitivity to 4-HPR + SAHA, we evaluated the transcriptional changes in neuroblastoma cells treated with the combination. The four candidate biomarker genes for which changes in expression level best correlated with the cytopathic responses were RARα, RARβ, Thymosin-beta-4-X (Tβ4), and Synaptogyrin-3 (SYNGR3). High expression of both Tβ4 and SYNGR3 was associated with a significantly better patient outcome by logrank analysis among 650 neuroblastoma patients. We performed siRNA knockdown of Tβ4 in neuroblastoma cells treated with 4-HPR + SAHA and found treatment synergy was lost without Tβ4 expression, indicating Tβ4 was necessary for the 4-HPR + SAHA cytopathic effect. Our data suggest that 4-HPR + SAHA is an effective anticancer combination therapy in vitro and in vivo in neuroblastoma.