Abstract A03: The EMSY threonine 207 phospho-site is required for EMSY-driven suppression of DNA damage repair
Bibliographic record
Abstract
Abstract Introduction: EMSY, a putative DNA damage repair gene, is amplified in over 10% of high-grade serous ovarian carcinoma (HGSOC) cases. EMSY overexpression has been hypothesized to antagonize BRCA2 via direct interaction and compromise the homology-directed repair (HDR) of DNA double strand breaks. Its role as a transcription factor has been described in a protein kinase AKT1 phosphorylation-dependent manner. The purpose of this study was to decipher EMSY's role in HDR and to assess the importance of its phosphorylation in this context. Experimental procedures: 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. Endogenous immunoprecipitations (IPs) were performed 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 protein kinases were obtained from Active Motif and CellSignaling. Forskolin and H-89 were obtained from Santa Cruz Biotechnology. Summary of the data: EMSY overexpression resulted in decreased HDR activity in all three DR-GFP cell lines, thus supporting the hypothesis that EMSY overexpression impairs HDR. V5-tagged EMSY overexpressing and endogenous immunoprecipitation experiments demonstrated no interaction between EMSY and BRCA2, suggesting EMSY's role in HDR to be BRCA2-independent. We confirmed that EMSY is phosphorylated by AKT1 at serine 209 phospho-site and identified a previously unknown phospho-site at threonine 207. We identified protein kinase A (PKA) as a kinase targeting EMSY T207. 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 PKA and the T207 phospho site for the EMSY-driven HDR suppression. Conclusions: EMSY-overexpressing cells show decreased HDR activity, demonstrating EMSY's relevance to the HDR pathway. Our data support 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. We found a new phospho-site at EMSY T207 and identified PKA as a targeting kinase. Phosphorylation of EMSY at T207, but not S209 phospho-site is necessary for EMSY-driven suppression of HDR. We suggest that an increase in EMSY's T207 phosphorylation in patients bearing EMSY¬-amplified tumors could enhance BRCAness and render these patients more sensitive to drugs effective in HDR-impaired setting, such as PARP inhibitors. Citation Format: Petar Jelinic, Laura Eccles, Jill Tseng, Paulina Cybulska, Simon N. Powell, Douglas Levine. The EMSY threonine 207 phospho-site is required for EMSY-driven suppression of DNA damage repair [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A03.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".