O1086: Pre-clinical Evidence Supporting Novel Protein Phosphatase 2A (PP2A) Activators as Therapeutics in Hepatoblastoma

Background: Hepatoblastoma is the most common primary liver malignancy in children. The incidence of this devastating disease continues to increase annually, but therapies have not substantially changed in the last twenty years. Investigators have shown that the tumor suppressor, protein phosphatase 2A (PP2A), is downregulated in hepatoblastoma. Previously, our lab demonstrated PP2A activation using the sphingosine analog, FTY720, decreased hepatoblastoma viability in vitro and tumor growth in vivo. FTY720 has significant immunosuppressive properties, so we wished to examine how two novel PP2A activating compounds, ATUX-3364 (3364) and ATUX-8385 (8385), designed to activate PP2A without causing immunosuppression, would affect hepatoblastoma.

Methods: The established human hepatoblastoma cell line, HuH6, and a human hepatoblastoma patient-derived xenograft, PDX67, were treated with increasing doses of 3364 or 8385. PP2A activation was examined using a PP2A activation assay following 24-hour treatment with 3364 or 8385. Viability and proliferation were investigated using alamarBlue® and CellTiter96® assays, respectively. Migration and invasion were examined using wound healing and modified Boyden chamber assays. Real-time PCR (qPCR) examined abundance of mRNA for three stemness markers, Oct4, Nanog, and Sox2, following PP2A activation. The ability of the COA67 cells to form tumorspheres, a marker of cancer cell stemness, was assessed.

Results: Treatment with 3364 or 8385 significantly activated PP2A in HuH6 and PDX67 cells. Viability was significantly decreased following doses of 3364 (Figure 1 A, upper panel) or 8385 (Figure 1 A, lower panel) starting at 5 μM in HuH6 and 0.5 μM in PDX67. Following treatment with either compound, proliferation was similarly significantly decreased in HuH6 cells and PDX67 cells. Treatment with 3364 or 8385 decreased migration and invasion in both HuH6 and PDX67 cells. Treatment with either compound resulted in a significant decrease in mRNA abundance of three stemness markers, Oct4, Nanog, and Sox2 in HuH6 cells (Figure 1 B). Both compounds significantly affected the ability of PDX67 cells to form tumorspheres (Figure 1 C).

Conclusion: The novel PP2A activators, 3364 and 8385, decreased proliferation, viability, and cancer cell stemness in hepatoblastoma. These findings provide evidence for further investigation of these novel PP2A activators as potential therapeutics for hepatoblastoma.