SWI/SNF-mutant cancers depend on catalytic and non-catalytic activity of EZH2

Abstract

Human cancer genome sequencing has recently revealed that genes encoding subunits of SWI/SNF chromatin remodeling complexes are frequently mutated across a wide variety of cancers, and several subunits of the complex have been shown to have bona fide tumor suppressor activity1. However, whether mutations in SWI/SNF subunits result in shared dependencies is unknown. Here we show that EZH2, a catalytic subunit of the Polycomb repressive complex 2 (PRC2), is essential in all tested cancer cell lines and xenografts harboring mutations of the SWI/SNF subunits ARID1A, PBRM1, and SMARCA4, which are several of the most frequently mutated SWI/SNF subunits in human cancer but that co–occurrence of a Ras pathway mutation correlates with abrogation of this dependence. Surprisingly, we demonstrate that SWI/SNF mutant cancer cells are primarily dependent upon a non–catalytic role of EZH2 in stabilization of the PRC2 complex, and only partially dependent on EZH2 histone methyltransferase activity. These results not only reveal a shared dependency of cancers with genetic alterations in SWI/SNF subunits, but also suggest that EZH2 enzymatic inhibitors now in clinical development may not fully suppress the oncogenic activity of EZH2.

Selective Killing of SMARCA2- and SMARCA4-deficient Small Cell Carcinoma of the Ovary, Hypercalcemic Type Cells by Inhibition of EZH2: In Vitro and In Vivo Preclinical Models

“…In vitro data demonstrated that ovarian cell lines with ARID1A mutations did not display antiproliferative effects upon tazemetostat treatment nor show sensitivity to EZH2 knockout in our CRISPR pooled screen. This is in contrast with previous reports that showed in vitro and in vivo sensitivity of ARID1A-mutated lines upon treatment with GSK126, another EZH2 inhibitor, and EZH2 shRNA knockdown (39,40). Given the known off-target effects that accompany RNAi methods, CRISPR knockout is more effective and specific for interpreting gene knockout phenotypes.…”

“…Two-dimensional hierarchical clustering was done in MATLAB R2015a using the "clustergram" function from the Bioinformatics Toolbox (Mathworks). The clustering was done on the top 100, 500, and 1,000 most variable genes across 40 3 (day 14 postinoculation). Mice were assigned into groups using a randomized block design.…”

“…CRISPR pooled screen identifies SCCOHT cell line COV434 as sensitive to EZH2 knockout It has been reported that some tumors with SWI/SNF mutations are dependent on the EZH2 protein itself as a scaffold, but not its catalytic activity (40). Therefore, as we identified ovarian cell lines with loss of expression or mutation in individual SWI/SNF subunits that were insensitive to tazemetostat inhibition of EZH2 catalytic activity, we sought to determine whether they were otherwise sensitive to knockout of EZH2 through CRISPR/Cas9-mediated gene knockout.…”

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2

“…Indeed, in our own study, the MYCN-nonamplified neuroblastoma cell line SK-N-AS was also sensitive to EZH2 knockout, and several of the lines were sensitive to EZH2 chemical inhibition, including the SH-SY-5Y line in vivo. Recently, several studies have revealed specific sensitivity to EZH2 inhibition in cancers with ARID1A or other mutations of the BAF complex (7,68). Resistance to EZH2 inhibition in adult cancers with co-occurrence of BAF complex and RAS pathway mutations was also described (7).…”

“…In fact, genome-scale shRNA screening has already led to the discovery of new dependencies. For example, shRNA screening identified dependencies on the CDK4/cyclin D1 complex in Ewing sarcoma (5) and the PI3K pathway in osteosarcoma (6), and more recently, shRNA screening identified mutations in the SWI/SNF subunits as candidate biomarkers of response to EZH2 inhibition (7). ShRNA screening, however, has the significant limitation of off-target effects due to unintended silencing of transcripts complementary to the seed sequence of the shRNAs (8).…”