Abstract PR01: EMSY impairs DNA damage repair in a phosphorylation-dependent manner.
Authors: Jelinic Petar , Eccles Laura , Powell Simon N. , Levine Douglas A. 
Published: May 2016

Background: EMSY, a putative DNA damage repair gene, is amplified in over 10% of high-grade serous ovarian carcinoma (HGSOC) cases. EMSY was initially identified as a BRCA2-interacting protein. Its overexpression has been hypothesized to antagonize BRCA2 and therefore compromise the homology-directed repair (HDR) of DNA double strand breaks; however, this has not been proven. Although EMSY's role in HDR has yet to be elucidated, it is known to be phosphorylated by the protein kinase AKT1. The purpose of this study was to decipher EMSY's role in HDR and to assess the importance of its phosphorylation in this context. In addition, we sought to test whether EMSY overexpressing cells are sensitive to HDR-targeted therapies such as PARP inhibitors.

Methods: We measured HDR activity in several cell lines (U2OS osteosarcoma, H1299 non-small cell lung carcinoma and OVCAR8 HGSOC) using the DR-GFP reporter assay and RAD51 foci assessment according to standard protocols. Endogenous immunoprecipitations (IPs) were performed in 293T, HeLa and OVCAR8 cells with low stringency lysis buffer and protein A/G-plus agarose. V5-tagged EMSY constructs were made using the Invitrogen's Gateway TOPO cloning system. These constructs were further used to create EMSY phospho-mutants. Cells were transfected by either electroporation or FuGene reagent. For the in vitro kinase assays, EMSY constructs were sub-cloned and expressed in BL21 STAR bacteria and purified using Invitrogen's Champion pET102 Expression kit. Recombinant active protein kinase A (PKA) and AKT1 were obtained from Active Motif and CellSignaling, respectively. Colony formation assay was used to assess the sensitivity of EMSY-overexpressing cells to PARP inhibitors (SelleckChem).

Results: V5-IP experiments in V5-tagged EMSY overexpressing 293T cells demonstrated no interaction between EMSY and BRCA2. This was further confirmed by endogenous immunoprecipitation in three cell lines: 293T, HeLa and OVCAR8. EMSY overexpression resulted in decreased HDR activity in all three DR-GFP cell lines (U2OS, H1299 and OVCAR8), thus supporting the hypothesis that EMSY-overexpression impairs HDR. We confirmed that EMSY is phosphorylated by AKT1 at serine 209 phospho-site and identified a previously unknown phospho-site at threonine 207. By using kinase prediction bioinformatic tools, we identified protein kinase A (PKA) as a potential kinase targeting EMSY T207. This was confirmed by in vitro kinase assay. Furthermore, by performing both DR-GFP assay and RAD51 foci assessment in OVCAR8 cells that overexpress WT EMSY or either phospho-mutant, we demonstrated that mutant EMSY-S209A affects HDR activity similar to the WT EMSY while EMSY-T207A does not. This suggests the importance of the T207 phospho site for the EMSY-driven HDR suppression. Finally, EMSY-overexpressing cells were more sensitive to the PARP inhibitors olaparib and veliparib when compared to mock cells.

Conclusions: Our data demonstrates that BRCA2 and EMSY do not interact. This supports the notion that EMSY-driven HDR impairment is BRCA2-interaction-independent and challenges the currently held impression that EMSY overexpression mimics the BRCA2-depleted phenotype via direct interaction. EMSY-overexpressing cells show decreased HDR activity, demonstrating EMSY's relevance to the HDR pathway. We found a new phospho-site at EMSY's T207 and identified PKA as a targeting kinase. Moreover, phosphorylation of EMSY at T207, but not S209 phospho-site is necessary for EMSY-driven suppression of HDR. Finally, EMSY-overexpressing cells are sensitive to PARP inhibition suggesting that the patients bearing EMSY-amplified ovarian cancer may benefit from HDR-targeted therapies.

Citation Format: Petar Jelinic, Laura Eccles, Simon N. Powell, Douglas A. Levine. EMSY impairs DNA damage repair in a phosphorylation-dependent manner. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR01.