S. R. Govindu1, A. P. Shah1, A. F. Espinoza1, R. Patel1, S. A. Vasudevan1, S. E. Woodfield1 1Divisions of Pediatric Surgery and Surgical Research, Michael E. DeBakey Department of Surgery, Pediatric Surgical Oncology Laboratory, Dan L. Duncan Cancer Center, Baylor College Of Medicine, Houston, TX, USA
Introduction:
Hepatoblastoma (HB) is the most common pediatric primary tumor of the liver. Relapsed and treatment refractory cases have a survival rate of less than 50% due to limited treatment options. Previous work showed NFE2L2/NRF2 hot-spot mutations in 11% of HB patients, all with high-risk tumors. NRF2 is a transcription factor that directly activates multiple target genes including NQO1, and carcinogenic effects of mutant NFE2L2 are dependent on mTOR. Previous literature suggests upregulation of phosphorylated mTOR in 96% of HB cases. Thus, we tested HB cell lines with the mTOR inhibitor sapanisertib to establish efficacy and mechanism of action.
Methods:
Expression of NQO1 was first assayed in HB cell lines (HepG2, HepT1, B6-2, HB17) with qRT-PCR experiments. Sapanisertib sensitivity of these cell lines were then tested with cytotoxicity (MTT) assays. Immunoblotting assays were used to assess changes in expression of total and phosphorylated mTOR and AKT with sapanisertib treatment.
Results:
HepT1 (NFE2L2 mutant), B6-2, and HB17 cell lines with high expression of NQO1 showed strong sensitivity with IC50 values of 0.08, 0.44, and 0.04 μM, respectively. HepG2 (NFE2L2 wild-type) with low expression of NQO1 showed resistance to sapanisertib up to 10 μM. Thus, sensitivity to sapanisertib correlated with NQO1 expression. Further studies with HepT1 cells treated with a combination of sapanisertib and cisplatin indicated that combinatorial therapeutic regimens can lead to a significant decrease in viability of NFE2L2 mutant cells. Immunoblotting showed decreased expression of phosphorylated AKT for HepT1 (NFE2L2 mutant) and no expression of phosphorylated AKT for HepG2 (NFE2L2 wild-type) treated with sapanisertib.
Conclusion:
Sapanisertib demonstrates strong in vitro efficacy in HB. This work suggests mTOR inhibition may act through a NRF2/NQO1 mediated mechanism. Further work with this drug may lead to clinical trials with HB patients.