O2186: Pharmacologic PP2A activation as an adjunct to doxorubicin treatment in neuroblastoma

Background: Neuroblastoma is the most common childhood extracranial solid tumor. While overall survival has improved, patients affected by metastatic or recurrent disease have an estimated survival rate below 40% despite multifaceted therapies. Anthracyclines, including doxorubicin, continue to be a mainstay of treatment for neuroblastoma, but drug resistance remains a challenge. Membrane bound transport proteins such as MDR1, an ATP-binding cassette (ABC) transporter, function to pump chemotherapeutics out of the cell thereby contributing to therapeutic resistance. MDR1 is associated with chemoresistance to doxorubicin in hepatocellular carcinoma and prostate cancer. Further, high levels of MDR1 RNA in neuroblastoma patient samples are correlated with chemotherapeutic resistance. Protein phosphatase 2A (PP2A) is a tumor suppressor that functions to dephosphorylate MDR1, promoting its degradation. PP2A-induced degradation of drug efflux pumps may enhance chemotherapeutic response, and in breast cancer, PP2A activation combined with doxorubicin treatment reduced cell viability and tumor growth. We have previously shown that PP2A is downregulated in neuroblastoma. Because of the effects of PP2A activation on drug efflux pumps in other cancer cell types, we hypothesized that pharmacologic reactivation of PP2A in neuroblastoma would enhance doxorubicin cytotoxicity through modulation of drug efflux pumps.

Methods: SK-N-BE(2) human neuroblastoma cells were treated with doxorubicin in combination with a novel PP2A activating compound, ATUX-1215, supplied by our collaborator. We used flow cytometry to evaluate intracellular doxorubicin accumulation and immunoblotting to examine protein expression of drug efflux pumps. Cell viability was assessed using colorimetric assay, after which the lethal dose 50% (LD50) and combination indices (CI) were calculated, and isobolograms constructed to evaluate synergy.

Results: Flow cytometry showed that treatment with ATUX-1215 significantly increased intracellular doxorubicin accumulation in the cells (5.9% vs 2.0% ATUX-1215 and doxorubicin vs doxorubicin alone, p< 0.001, Figure 1a). Immunoblotting revealed decreased protein expression of multiple drug efflux pumps including MDR1, ABCG2, and MRP1 in neuroblastoma cells treated with ATUX-1215 and doxorubicin compared to cells treated with doxorubicin alone (Figure 1b). Cell viability was significantly decreased with the combination treatment, and isobolograms indicated synergy (CI < 1) when cells were treated with doxorubicin in combination with ATUX-1215 (Figure 1c).

Conclusion: Pharmacologic PP2A activation in human neuroblastoma cells increases intracellular accumulation of doxorubicin resulting in a synergistic effect on cell viability. These findings are likely secondary to decreased expression of drug efflux pumps seen with PP2A activation. Further exploration of PP2A activators as a chemotherapeutic adjunct in neuroblastoma is warranted.