Targeted catalytic inhibition of EZH2 synergizes with low-dose panobinostat in malignant rhabdoid tumor.

Abstract

Malignant Rhabdoid Tumor (MRT) is a rare pediatric cancer predominantly occurring in the kidney and CNS that is highly resistant to current treatment protocols. MRT is almost exclusively characterized by homozygous inactivation of SMARCB1, a critical subunit of the SWI/SNF chromatin-remodeling complex, implicating epigenetic deregulation in the pathogenesis of the disease. Recently, we showed that sustained treatment of human MRT cell lines with the Histone deacetylase inhibitor, Panobinostat, at low-dose, inhibited tumor growth by driving multi-lineage differentiation in vitro and in vivo. Furthermore, re-expression of SMARCB1 in MRT cells phenocopied low-dose Panobinostat treatment and led to growth inhibition, senescence and terminal differentiation in vitro and in vivo, suggesting similar mechanistic functionality. Enhancer of Zeste homolog 2 (EZH2), a core subunit of the Polycomb Repressive Complex 2, confers transcriptional silencing via the addition of methyl groups to Lysine 27 of Histone 3 (H3K27me3), and is a transcriptional target of SMARCB1. EZH2 expression and H3K27me3 were drastically reduced following sustained low-dose Panobinostat treatment and re-expression of SMARCB1 in MRT cells. Sustained siRNA knockdown of EZH2 in G401 cells resulted in reduced cell growth and changes in mRNA expression, mimicking low-dose Panobinostat treatment and SMARCB1 re-expression. Treatment of MRT cells with the EZH2-catalytic domain inhibitors, GSK126, GSK343 and UNC1999, had no effect on EZH2 expression and only partially reduced cell growth despite dose-dependent reductions in H3K27me3 implying important non-catalytic EZH2 function. Remarkably, co-treatment of MRT with low-dose Panobinostat and GSK126 resulted in reduced EZH2 and H3K27me3 expression, significantly reduced cell growth and increased differentiation in vitro and in vivo compared to single agent controls, demonstrating a synergistic relationship. This data suggests EZH2 is an important mediator of MRT proliferation and differentiation and provides evidence for improved efficacy of dual therapeutic targeting of EZH2 with low-dose Panobinostat.