A Novel FBXO45-Gef-H1 Axis Controls Oncogenic Signaling in B-Cell Lymphoma
Notice bibliographique
Résumé
Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.
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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 ».