Abstract 4399: Exploring the postmitotic roles of STAG2 mutations in solid tumors
Authors: Guan Mary , Tan Mengyao , Parolia Abhijit , Kregel Steve , Cieslik Marcin , Chinnaiyan Arul 
Published: August 2019

Mutations in the cohesin complex are associated with cancer, in particular Ewing’s Sarcoma (EWS) and bladder cancer. The STAG2 subunit of cohesin is essential for its loading onto chromatin and proper function of the complex. In EWS, STAG2 is mutated at a rate of 22% in the form of inactivating loss of function mutations. How STAG2 functions as a tumor suppressor is not fully understood, and few whole-genome experiments have been performed examining the role of STAG2 in EWS. We hypothesize that in EWS, where the EWSR1-FLI1 fusion is pathognomonic, the genetic epistasis of EWSR1-FLI1 and STAG2 is due to the suppressive role of STAG2 on EWSR1-FLI1.

In this study, we used co-immunoprecipitation (co-IP) to examine a ternary interaction between STAG2 and EWSR1-FLI1, as well as STAG2 and EZH2. We then induced KD of STAG2 in EWS cells after 72h of siRNA treatment or 5 days of dox induction with shRNA to examine the phenotypic and transcriptional effects of STAG2 loss. As the H3K27me3 signature was enriched upon STAG2 KD, we performed IC50 drug screens with EZH2 treatment on STAG2 WT vs KD EWS cells. Finally, a STAG2 ChIP-seq was completed in EWS cells.

No physical interaction was detected between STAG2 and EWSR1-FLI1 in A673 EWS cells via endogenous co-IP. In A673 EWS cells, no significant difference in proliferation was observed after 5 days of shNT vs shSTAG2 induction. Turning to the transcriptome, differentially expressed genes after 72h of siSTAG2 KD in three EWS cell lines were identified using RNA-seq and verified using qPCR. No change in expression of EWSR1, FLI1, or EWSR1-FLI1 was seen after siSTAG2 treatment compared to siNT treatment. STAG2 is also found at the borders of topologically associated domains (TADs), and groups have shown that loss of STAG2 increases intra-TAD interactions. One particular GSEA signature common to all three cell lines was enrichment in genes with H3K27me3. This led us to hypothesize that in EWS, loss of cohesin-STAG2 could lead to new intra-TAD interactions via H3K27 methylation by EZH2, destabilizing enhancer-promoter interactions and maintaining the stemness of EWS cells to drive oncogenesis. Western blot of H3K27me3 and EZH2 levels after 5 and 7 days of sh-STAG2 KD did not show differences in total levels of H3K27me3 or EZH2. Treatment with Tazemetostat, an EZH2-inhibitor, resulted in no significant change in IC50 in shNT compared to shSTAG2 A673 cells. ChIP-seq and ChIP-qPCR in EWS cells demonstrated enrichment in known STAG2 targets in mouse, Myc and Sox2.

In summary, our results suggest STAG2 does not physically interact with EWSR1-FLI1 to mediate its expression or expression of its target genes. Instead, STAG2 may act upon chromatin to significantly rewire the cancer transcriptome in EWS in an EZH2 independent manner, thus displaying differential dependency on certain transcriptional regulators. This could shed new light on medical therapy for EWS – which currently does not exist – through targeting of STAG2-activated pathways.

Citation Format: Mary Guan, Mengyao Tan, Abhijit Parolia, Steve Kregel, Marcin Cieslik, Arul Chinnaiyan. Exploring the postmitotic roles of STAG2 mutations in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4399.