RNA Deadenylation Subunit CNOT3 Promotes Myeloid Leukemia By Driving Translation of Oncogenic Targets
Notice bibliographique
Résumé
While somatic alterations in genetic and epigenetic mechanisms in tumorigenesis have been studied extensively, how processes that affect post-transcriptional and translational regulation impact leukemia development is much less well developed. To identify novel regulators of post-transcriptional and translation control in AML, we surveyed several genome-wide CRISPR screens and observed that a number of subunits in the RNA deadenylation CNOT complex were highly ranked among genes essential for survival of human and mouse leukemia cells. The CCR4-NOT (CNOT) complex is one of two major multi-subunit polyA deadenylation complexes which mediate the shortening of the poly(A) tails. The CNOT complex was found to be recruited to m6A transcripts to mediate mRNA degradation. An involvement of the CNOT complex with RNA methylation, mRNA degradation and translation suggest a role of CNOT in bridging these processes to mediate complex gene expression regulation in pathogenesis of AML. Among the subunits of CNOT complex, we found that the highest ranked CNOT3 adaptor subunit demonstrated elevated expression in human AML cell lines (7/9 AML cell lines) and in primary AML patients (8/11 primary samples) assayed by immunoblots. Intracellular staining analysis further demonstrated that CNOT3 is highly expressed in leukemia stem cells-enriched CD34+ compartments in 13 out of 17 tested primary AML samples. Notably, high CNOT3 mRNA was associated with a poor prognosis in 3 independent AML patient cohorts i.e. TCGA-AML, Beat-AML and AMLCG 1999. Depletion of CNOT3 by shRNA-mediate knockdown and CRISPR/Cas9-mediated knockout in a panel of genetically diverse human leukemia cells (MOLM13, MV4-11, HL-60, NB4 and OCI-AML3) resulted in strong inhibition of cell growth, increased myeloid differentiation and apoptosis and significantly delayed leukemia development in vivo (median survival of 42 days, control versus 93.5 days, sh#1 and "not reached", sh#2). CNOT3 ablation also reduced survival and colony forming ability of primary AML patient cells. On the other hand, loss of CNOT3 function in normal cord-blood derived CD34+ (CB-CD34+) robustly induced myeloid differentiation without causing cell death, suggesting a potential therapeutic window to target CNOT3 in leukemia cells. In addition, overexpression of CNOT3 promoted cell growth in both CB-CD34+ and leukemia cells. Taken together, the data strongly indicates that CNOT3 promotes leukemogenesis. To uncover the mechanisms underpinning CNOT3 function in leukemia cells, we employed a CRISPR/Cas9 saturation domain mutagenesis screen by tiling the entire CNOT3 gene with sgRNAs. We identified the NOT box in the C-term domain of CNOT3 to be most critical for AML cell viability. In addition, truncation of the NOT box in CNOT3 abrogated its ability to drive proliferation in leukemia cells. This suggests a functional dependence of leukemia cells on CNOT3 interaction with the deadenylation complex via this domain. Transcriptomic and proteomic profiling of leukemia cells depleted of CNOT3 revealed an increase in p53 pathways, IFN-g immune response and downregulation of genes in G2/M checkpoint and MYC- target genes. We further observed a marked decrease in c-MYC protein upon ablation of CNOT3. We also noted the presence of c-MYC mRNA with longer poly(A) tails in CNOT3 depleted cells. However, there is no significant alternation in total abundancy and half-life of c-MYC mRNA, indicating a post-transcriptional regulation of c-MYC protein expression. In fact, depletion of CNOT3 significantly reduced the abundancy of c-MYC mRNA in the polysome-enriched and actively translated mRNAs, indicating that CNOT3 regulates translation of c-MYC. Immunoprecipitation of CNOT3 and mass spectrometry analysis of co-precipitated proteins captured proteins involved in protein synthesis including translation elongation factors and ribosomal proteins, suggesting a functional interaction of CNOT3 with the translation machinery. Overall, we uncovered a critical role of the CCR4-NOT deadenylation complex in myeloid leukemia and nominated CNOT3 as a potential target for AML therapy.
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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 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,000 |
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 ».