Abstract 3198: HDAC inhibitor panobinostat as a selective agent, synergizes with chemotherapeutics in medulloblastoma and neuroblastoma
Authors: Witte Amanda B. , Durston Mary , Zhao Ping , Nagulapally Abhinav B. , Bond Jeffrey , Saulnier Sholler Giselle L. 
Published: July 2016

Background: Embryonal tumors of the central nervous system continue to present therapeutic challenges with high rates of relapse and poor prognosis at advanced stages. Medulloblastoma (MB) accounts for over 15% of pediatric brain tumors, with a five-year survival rate of 62%. Neuroblastoma (NB) is the most common extracranial solid tumor in children, accounting for 15% of pediatric cancer deaths.

Among many pathways that contribute to MB and NB potency, several histone deacetylases (HDACs) have been shown to prevent cell differentiation. In cells expressing wt-p53, HDAC inhibitors induce nuclear relocalization of p53 to the nucleus to induce expression of the cell cycle inhibitor p21/Waf1/Cip1. As most NB tumors express wt-p53, HDAC inhibitors are promising candidates for therapy. Panobinostat has been shown to induce differentiation, cell cycle arrest, and apoptosis in NB cells. This effect is partially due to down-regulation of CHK1, a pathway by which cancer cells can develop resistance to conventional chemotherapeutic drugs.

This study proposes panobinostat coupled with conventional chemotherapeutics such as doxorubicin, etoposide, and velcade, as an effective target against the HDAC pathway in MB and NB.

Methods: Established MB and NB cell lines and patient derived cell lines (MB: DAOY, D283, D341, 384MED, 458MED, 487MED, 556MED, 581MED, 721MED; NB: BE(2)-C, CHLA-90, SMS-KCNR, SH-SY5Y, MGT8-117-08, BIO-036-08) were used to quantify panobinostat's effects. Cell viability was measured using Calcein AM fluorescent assay at doses of 0.39-50 nM. Isobologram analysis of panobinostat in combination with doxorubicin, etoposide, and velcade were generated using Calcein AM. Western blot was used to measure HIF1 alpha, CHK1, and acetyl H4, Caspase 3 and PARP levels. ATP level per cell was measured using CyQuant fluorescent DNA assay with CellTiter-Glo luminescent cell viability assay.

Results: Cell viability assays show cytotoxicity of panobinostat in MB and NB cell lines, with IC50 values from 2-10 nM in MB cells, 5-20 nM in established NB cell lines and 23-91 nM in patient derived NB lines. ATP/cell activity was inhibited in MB and NB cells following treatment with panobinostat alone. Isobologram experiments suggest synergistic cytotoxicity of panobinostat in combination with doxorubicin, etoposide, and velcade in NB cell lines BE(2)-C and SMS-KCNR. Western blot analysis indicates caspase-mediated apoptosis occurs, with inhibition of overexpressed HDAC proteins among multiple classes in MB and NB cells.

Conclusions: This study indicates that panobinostat targets the HDAC inhibition pathway of MB and NB cells. Additionally, panobinostat synergizes with doxorubicin, etoposide, and velcade in inducing apoptosis in MB and NB cells. This study provides rationale for initiation of a clinical trial in treating MB and NB patients with panobinostat in combination with conventional chemotherapeutics.

Citation Format: Amanda B. Witte, Mary Durston, Ping Zhao, Abhinav B. Nagulapally, Jeffrey Bond, Giselle L. Saulnier Sholler. HDAC inhibitor panobinostat as a selective agent, synergizes with chemotherapeutics in medulloblastoma and neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3198.