Abstract 3360: Targeted catalytic inhibition of EZH2 synergizes with low-dose HDACi in malignant rhabdoid tumors

Abstract

Malignant Rhabdoid Tumor (MRT) is a rare pediatric cancer of the kidney and CNS that is resistant to current treatment protocols. MRT is genetically characterized by homozygous inactivation of SMARCB1, a critical subunit of the SWI/SNF chromatin-remodeling complex. Next-generation sequencing data suggests that inactivation of SMARCB1 is the primary driver mutation, implicating epigenetic deregulation in the pathogenesis of MRT. Recently, we showed that sustained treatment of MRT cell lines with low-dose Panobinostat (LBH589), inhibited tumor growth by driving multi-lineage differentiation in vitro and in vivo. Furthermore, re-expression of physiological levels of SMARCB1 in G401 MRT cells phenocopied the low-dose LBH589 treatment and led to growth inhibition, senescence and terminal differentiation in vitro and in vivo. Enhancer of Zeste homolog 2 (EZH2), a core subunit of the Polycomb Repressive Complex 2 (PRC2), 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 LBH589 treatment and re-expression of SMARCB1 in G401 MRT cells. Sustained siRNA knockdown of EZH2 in G401 cells resulted in reduced cell growth and changes in mRNA expression similar to those observed following low-dose LBH589 treatment and SMARCB1 re-expression. Treatment of MRT cells with the EZH2-catalytic domain inhibitor, GSK-126, had no effect on EZH2 expression and only partially reduced H3K27me3 and cell growth at doses 1nM-10μM suggesting important non-catalytic EZH2 function. However, MRT cells treated in combination with low-dose LBH589 and GSK-126, lost EZH2 and H3K27me3 expression and exhibited significantly reduced cell growth in vitro compared to single agent controls, revealing a synergistic relationship. Similar effects were observed in an in vivo xenograft model, with low-dose LBH589 and GSK-126 treatment leading to a marked reduction in tumor growth, not observed with single agent treatment. This data suggests EZH2 is an important mediator of MRT proliferation and differentiation and provides evidence for dual therapeutic targeting of EZH2 with low-dose HDACi in MRT. Citation Format: Dean Popovski, Elizabeth M. Algar, Catherine R. Cochrane, Anette Szczepny, W. Samantha Jayasekara, David M. Ashley, Peter Downie, D. Neil Watkins, Jason E. Cain. Targeted catalytic inhibition of EZH2 synergizes with low-dose HDACi in malignant rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2017-3360

Targeting EZH2 for the treatment of soft tissue sarcomas

“…Additionally, the PRC2-SWI/SNF interactions are essential in the regulation of gene expression in several signalling pathways, such as RB, Cyclin D1, MYC and hedgehog, which are impaired in many cancer types [6] . In the presented results, EZH2 depletion led to decreased activity of the oncogenic molecules such as E2F and c-Myc involved in signal transduction pathways [39] , proposing indirect implications of EZH2 to oncogenesis. When interpreting the outcomes, there is also a need for contextualizing these with relation to the tumour microenvironment (TME), which consists not only of cancer cells but also of extracellular matrix, fibroblasts, adipocytes, endothelia, and immunomodulators such as T and NK cells, tumour-associated macrophages and dendritic cells.…”

“…Silencing of EZH2 by shRNAs in SMARCB1-deficient paediatric ATRT cell lines (BT12, BT16 and patientderived UPN 737) has been shown to reduce cell proliferation and concomitantly induce cellular senescence and apoptosis, with the biggest effects seen with combination EZH2-silencing by shRNA and administration of DZNep. It was also demonstrated that inhibition of EZH2 affected crucial pathways and molecules involved in cell cycle regulation with downregulation of E2F and c-Myc [39] . Furthermore, in an MRT cell line model, sustained knockdown of EZH2 through siRNA manifested as reduction of cellular growth and SMARCB1 restoration [40] .…”

“…In ATRTs, use of DZNep resulted in anti-proliferative and pro-apoptotic effects, increased sensitivity of cancerous cells to radiation and decreased ability to self-renewal and to form tumour spheres [39] . Another study showed that cell viability was significantly impaired in vitro, but administration of DZNep in vivo did not change the survival time relative to control or systemic-treated/combination-treated xenografts [58] .…”