Intracellular IL-23 Receptor (IL-23R) Is a Regulator of Mitotic Spindle and Centrosome Formation and Is Essential for AML Viability
Notice bibliographique
Résumé
IL-23 receptor (IL-23R) is a heterodimeric cytokine cell surface receptor that is traditionally expressed on T cells. The cytokine, IL-23, is secreted by dendritic cells and macrophages in response to inflammatory stimuli. Here, we identify a novel function and localization for IL-23R in which intracellular IL-23R is necessary for AML viability and acts as a critical regulator of the mitotic spindle. We analyzed gene ontologies that were upregulated in AML samples compared to normal hematopoietic cells. Amongst upregulated gene ontologies, the mitotic spindle ontology was enriched in AML samples. Further analysis of all protein coding genes and their correlation to the mitotic spindle ontology surprisingly returned IL-23R as a top hit. Given that IL-23R is not known to be expressed in AML, we confirmed the presence of IL-23R protein by immunoblotting in 14 of 20 primary AML patient samples, while it was undetectable in bulk (n=5) and CD34+ sorted (n=3) normal hematopoietic cells. Consistent with previous investigations, we confirmed cell surface localization of IL-23R in double negative T cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were present on the cell surface of AML cells. Instead, IL-23R was detected intracellularly in the cytoplasm and nucleus of AML cell lines, as well as primary AML cells (including the stem cell fraction). We demonstrated intracellular localization using flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. To ensure we were detecting genuine IL-23R protein, we probed for IL-23R using 4 different antibodies targeting 4 different epitopes of the receptor whilst using 4 methods of detection (immunoblotting, flow cytometry, confocal microscopy, and immunoprecipitation). We also demonstrated intracellular localization of the IL-23R heterodimer subunit, IL12Rβ1, which is known to bind the IL-23R subunit to form the fully functional IL-23 receptor. To elucidate the function of intracellular IL-23R, we performed BioID mass spectrometry to identify proteins that interact with IL-23R. Compared to controls, we identified 61 proteins that preferentially interacted with IL-23R. 36 of those 61 proteins are known cytoplasmic or nuclear localized proteins. Pathway analysis of those interacting proteins identified the mitotic spindle as a top pathway corroborating with our bioinformatics analysis. Proximity Ligation Assay (PLA) and confocal microscopy verified that endogenous IL-23R protein interacted with mitotic spindle associated proteins, NUMA, TMEM201, TACC1, and BAG6 in OCI-AML2 cells and primary AML samples. Consistent with our PLA results, we demonstrated IL-23R co-localized with the mitotic spindle and centrosomes in AML cell lines and primary patient samples. Knockdown and knockout of IL-23R in AML cells led to the dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and spindle orientation errors. Knockdown of IL-23R reduced cell proliferation and viability in OCI-AML2, TEX, K562, NB4, and U937 cells compared to non-targeting controls. IL-23R knockdown also reduced the engraftment efficiency of TEX leukemia cells in murine bone marrow. Subsequent knockdowns of IL-23R in primary AML cells demonstrated decreased clonogenic growth and reduced engraftment into the marrow of immune deficient mice. In contrast, knockdown of IL-23R in normal human cord blood cells did not impair their engraftment into murine bone marrow. Finally, we analyzed hematopoietic cells and stem cells in IL-23R -/- mice and found that knockout of IL-23R did not decrease blood counts or the abundance and function of normal hematopoietic cells. In summary, we discovered a novel intracellular localization and function for IL-23R in AML. IL-23R regulates mitotic spindle and centrosome formation and is necessary for AML cell viability. We have thus identified a new biological function for IL-23R and a potential therapeutic target for AML.
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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 | 0,001 | 0,001 |
| 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) | 0,002 | 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 ».