Inhibition of autophagy potentiates the efficacy of Gli inhibitor GANT-61 in MYCN-amplified neuroblastoma cells

Background: Aberrant Hedgehog (Hh) signaling is often associated with neuroblastoma (NB), a childhood malignancy with varying clinical outcomes due to different molecular characteristics. Inhibition of Hh signaling with small molecule inhibitors, particularly with GANT-61, significantly suppresses NB growth. However, NB with MYCN amplification is less sensitive to GANT-61 than those without MYCN amplification. Methods: Autophagic process was examined in two MYCN amplified and two MYCN non-amplified NB cells treated with GANT-61. Subsequently, chemical and genetic approaches were applied with GANT-61 together to evaluate the role of autophagy in GANT-61 induced cell death. Results: Here we show that GANT-61 enhanced autophagy in MYCN amplified NB cells. Both an autophagic inhibitor 3-methyladenine (3-MA) and genetic disruption of ATG5 or ATG7 expression suppressed GANT-61 induced autophagy and significantly increased apoptotic cell death, whereas pre-treatment with an apoptotic inhibitor, Z-VAD-FMK, rescued GANT-61 induced cell death and had no effect on the autophagic process. In the other hand, GANT-61 barely induced autophagy in MYCN non-amplified NB cells, but overexpression of MYCN in MYCN non-amplified NB cells recapitulated GANT-61 induced autophagy seen in MYCN amplified NB cells, suggesting that the level of GANT-61 induced autophagy in NB cells is related to MYCN expression level in cells. Conclusion: Aberrant Hh signaling activation as an oncogenic driver in NB renders inhibition of Hh signaling an effective measure to suppress NB growth. However, our data suggest that enhanced autophagy concomitant with Hh signaling inhibition acts as a pro-survival factor to maintain cell viability, which reduces GANT-61 efficacy. Besides, MYCN amplification is likely involved in the induction of the pro-survival autophagy. Overall, simultaneous inhibition of both Hh signaling and autophagy could be a better way to treat MYCN amplified NB.