Abstract A03: The EMSY threonine 207 phospho-site is required for EMSY-driven suppression of DNA damage repair
Notice bibliographique
Résumé
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.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,001 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,013 | 0,005 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».