Bortezomib Impairs Myeloma Cells (MM) Homologous Recombination Through Inhibition of the E2-Ubiquitin-Conjugating Enzyme UBC13
Notice bibliographique
Résumé
Abstract Abstract 1834 Background: Chromosomal instability is a defining feature of clonal MM plasma cells resulting in the perpetual accumulation of genomic aberrations. In addition to its role in protein homeostasis the ubiquitin-proteasome system is also involved in the regulation of DNA damage repair proteins. We have recently reported that proteasome inhibition induces a BRCAness state in MM cells (MM) resulting in a contextual synthetic lethality when combined with PARP inhibitors. We now report on the mechanisms by which bortezomib impairs homologous recombination (HR) mediated repair of DNA double stranded breaks (DNA-DSBs). Methods and results: Using the DR-GFP/SceI reporter assay in which cells are transfected with a plasmid tandemly expressing mutated GFP genes (5'-Sce/GFP – truncated IGFP-3') and followed by infection with an adenovirus (AdNGUS24i) expressing the yeast Sce endonuclease, we have confirmed that treatment with bortezomib does severely impair HR in MM cells. At the transcriptional level, proteasome inhibition transiently (6–12 hours post treatment) reduced the mRNA expression of several HR and DNA damage repair genes (BRCA1, BRCA2, RAD51 and FANCD2) followed by recovery to baseline levels by 24 hours. This transient downregulation of these DNA damage repair genes was also confirmed in a promoter luciferase reporter screen. At the post-trancriptional level, examining the dynamics and kinetics of DNA-DSBs repair induced by radiation therapy or PARP inhibition, we have demonstrated that bortezomib does not alter the initial phase of DNA damage sensing (MRN complex recruitment, ATM and H2AX phosphorylation and MDC1 activation) but does impair the Lys63-poly-ubiquitylation of histones γH2AX and H2A. This histone ubiquitylation is a required modification for the second wave of DNA repair proteins recruitment and retention of BRCA1 and RAD51 at the sites of DNA-DSBs. Furthermore, we have shown that bortezomib treatment depleted the nuclear pools of ubiquitin and abrogated the poly-ubiquitylation (lack of co-localization poly-Ub FK2 foci with γH2AX), but not the phosphorylation and foci formation of histone H2AX. Therefore, bortezomib appears to impair histones (H2AX and H2A) poly-ubiquitylation, a process that is dependent on the activation and recruitment of the E3 ubiquitin-protein ligases RNF8 and RNF-168 as well as the ubiquitin-E2 conjugating enzyme UBC13 to the sites of DNA-DSBs. Western blot analysis of the nuclear and cytoplasmic fractions of cellular extracts, revealed that bortezomib dramatically reduced the nuclear accumulation of UBC13 in response to DNA-DSBs as well as the formation of UBC13-Ubiquitin thiolester bonds formation and hence impaired the transfer of ubiquitin to nucleosomal histones. Conclusion: Our studies demonstrate that proteasome inhibition with bortezomib severely impairs homology-mediated repair of DNA breaks in MM cells at the post-transcriptional level by altering the nuclear accumulation and function of the E2-ubiquitin conjugating enzyme UBC13. These results explain the observed clinical synergy between bortezomib and several DNA damaging agents and support further clinical investigation of the combination of these class of drugs in MM patients. Disclosures: Neri: Celgene: Honoraria, Research Funding. Bahlis:Celgene: Honoraria, Speakers Bureau.
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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) | 0,000 | 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) | 0,002 | 0,001 |
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 ».