The Arginine Methyltransferase PRMT5 Regulates Maintenance and TKI Resistance of Self-Renewing FLT3-ITD AML LSC
Notice bibliographique
Résumé
FLT3-ITD mutations are commonly observed in acute myeloid leukemia (AML) and associated with adverse prognosis. FLT3 tyrosine kinase inhibitors (TKIs) demonstrate clinical efficacy but fail to eliminate leukemia stem cells (LSC) leading to treatment failure and relapse. There is a pressing need to understand mechanisms of FLT3-ITD AML LSC persistence and develop strategies to enhance their elimination. Here we show that the type II protein arginine methyltransferase PRMT5 is a critical regulator of FLT3-ITD AML cell maintenance and TKI resistance. To assess the role of epigenetic regulators in FTL3-ITD AML cell maintenance we screened FLT3-ITD AML cell lines using a well-curated collection of epigenetic probes (SGC Toronto). We identified inhibitors of the type II protein arginine methyltransferase PRMT5 to be amongst the top hits. We confirmed dose and time-dependent inhibition of FLT3-ITD AML cells by the PRMT5 inhibitors (PRMT5i), GSK-591 and LLY-283, and showed that combination of PRMT5i with FLT3 TKI (Quizartinib, Giltertinib) synergistically inhibited FLT3-ITD AML cells. Similar results were seen with CRISPR-Cas targeting of PRMT5 in FLT3-ITD AML cells. We further show that PRMT5i reduced the growth of patient-derived primary human FLT3-ITD AML HSPC and enhanced AML HSPC targeting in combination with TKI in vitro, and that the combination of PRMT5i and TKI also significantly enhanced inhibition of AML HSPC growth in PDX models in vivo. PRMT5i also showed activity against primary CD34+ cells from patients FLT3-ITD AML who had relapsed following TKI treatment. Finally, we show that PRMT5i significantly reduced LSC numbers and secondary transplant capacity in a genetic mouse model with FLT3-ITD+ and Tet2 knockout, and enhanced survival of mice after the completion of treatment, with enhanced effect seen in combination with TKI. PRMT5i did not significantly inhibit the growth of normal CD34+ cells in vitro or of normal murine hematopoiesis in vivo. PRMT5 catalyzes the formation of symmetric dimethyl arginine and regulates several histone and nonhistone proteins functioning in RNA processing, transcription, signal transduction and cell cycle regulation. There is growing evidence for an important role for PRMT5 in leukemic and normal hematopoiesis. Ectopic FLT3-ITD expression led to enhanced PRMT5 expression in FLT3-WT cells, whereas FLT3 knockout led to reduced PRMT5 expression in FLT3-ITD cells, supporting a role for FLT3-ITD in modulating PRMT5 expression. FLT3-ITD-mediated PRMT5 regulation appears to be kinase-independent since TKI treatment did not affect PRMT5 expression. RNASeq analysis showed increased p53, inflammatory and apoptosis gene signatures and reduced cell cycling signatures in AML LSK cells following combination treatment. PRMT5i treatment led to enhanced suppression of residual P-Akt, P-MAPK and P-STAT5 levels in TKI-treated AML cells. PRMT5 inhibition and knockdown led to reduced FLT3-ITD protein expression, related to enhanced proteasomal degradation. Finally, we observed that PRMT5i induced an increase in alternative splicing events in FLT3-ITD AML LSK cells, and that TKI treatment while not altering RNA splicing by itself led to a synergistic increase in alternative splicing in combination with PRMT5i treatment. Combination treatment leads to reduced levels of the SR protein kinase and phospho-SR protein levels in FTL3-ITD cells, and depleted phospho-SR proteins from nuclear speckles. We observed increased alternative splicing of genes involved in p53 regulation, Ras/MAPK signaling, growth factor signaling, RNA processing, chromatin modification and transcriptional regulation with combination treatment. We confirmed that combination treatment led to increase in an alternatively spliced MDM4 isoform which was associated with increased p53 and p53 target gene expression. We conclude that PRMT5 plays a critical role in maintenance of self-renewing FLT3-ITD AML LSC and in their persistence following TKI treatment, and that combined PRMT5i and TKI treatment effectively depletes FLT3-ITD AML LSC with disease regenerating capacity. We identify mechanisms underlying the synergistic effects of the combination of PRMT5i with TKI, including depletion of FLT3-ITD protein, activation of the p53 pathway, enhanced inhibition of STAT5, MAPK and AKT signaling, and increased alternative splicing events affecting key LSC maintenance pathways.
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| 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) | 0,000 | 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 tête enseignante, 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 ».