Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT) Activity Selectively Targets Human Acute Myeloid Leukemia Stem Cells
Notice bibliographique
Résumé
Abstract Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow associated with poor clinical outcomes. Conventional chemotherapies are effective in debulking the leukemic burden in most AML patients. However, a small population of disease-sustaining leukemic stem cells (LSCs) frequently persists and contributes to relapsed disease. Novel therapies that eradicate LSCs have the potential to improve clinical outcomes in AML. To discover novel anti-LSC agents, we performed a high-throughput flow cytometry-based drug screen of 1,220 compounds against a primary AML sample (8227). This sample harbors distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation is restricted to the CD34+CD38- fraction. Through this screen, we identified compounds that selectively depleted the CD34+CD38- fraction including four structurally-unrelated NAMPT inhibitors (FK866, STF-118804, GMX1778, and KPT-9274). These inhibitors also depleted the LSC-enriched CD34+CD38- fraction in two other primary AML samples, indicating that the effect was not unique to 8227 cells. To further evaluate their impact on LSCs in 8227, we measured the expression of 104 genes that were previously found to be differentially expressed between LSC+ and LSC- cell fractions isolated from patient samples. Treatment with NAMPT inhibitors reduced the correlation between the measured LSC gene signature and the LSC+ reference profile, providing additional evidence for their anti-LSC activity. To determine whether the selective loss of CD34+CD38- cells was due to cell death or differentiation, we sorted subsets of 8227 cells based on CD34 and CD38 expression and treated each fraction with FK866. NAMPT inhibition preferentially triggered apoptosis as measured by Annexin V staining in the CD34+CD38- and CD34+CD38+ fractions over the CD34- fraction. We did not observe significant changes in the expression of CD34, CD38, or other myeloid differentiation markers (CD14 and CD15) in the remaining viable cells. Our subsequent mechanistic studies focused on KPT-9274 because it is the furthest along in clinical development. NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway that converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), a direct NAD+ precursor. To confirm a decrease in intracellular NAD+ with KPT-9274 treatment, we introduced expression of genetically-encoded biosensors for measuring NAD+ levels in different cellular compartments in an AML cell line. KPT-9274 treatment for 15 hours lowered the free NAD+ pool in the cytosol and mitochondria but not in nucleus. To determine whether the drop in NAD+ was necessary for the effects of KPT-9274 on LSCs, we supplemented the primary AML samples with nicotinamide riboside (NR) which can be directly converted to NMN, thereby bypassing the requirement for NAMPT activity to generate NAD+. The addition of NR completely rescued the effects of KPT-9274 on the CD34+CD38- fraction. Niacin can also generate NAD+ through an alternative pathway that depends on nicotinic acid phosphoribosyltransferase (NAPRT). However, niacin supplementation failed to rescue the effects of NAMPT inhibition which correlated with the lack of NAPRT expression in LSC-enriched CD34+CD38- cells. Next, we studied the effects of KPT-9274 on normal CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from human cord blood. Although HSPCs were sensitive to the pro-apoptotic effects of KPT-9274, their survival was fully rescued by both NR and niacin. The rescue by niacin correlated with a higher expression of NAPRT in HSPCs. As the blood concentration of niacin is ~1,000 fold higher than that of NR, KPT-9274 is predicted to have a favorable therapeutic window in vivo. To demonstrate its in vivo activity, we treated immunodeficient NOD/SCID/IL2Rγ-null (NSG) mice engrafted with a luciferase-tagged AML cell line (OCI-AML3) with KPT-9274 at a dose of 150 or 250 mg/kg/day or vehicle control for 50 consecutive days by oral administration. KPT-9274 treatment significantly lowered leukemia burden and prolonged survival in both dosing cohorts. In summary, our results indicate that NAMPT inhibition represents an effective approach to target human LSCs through reduction in intracellular NAD+ levels and induction of apoptosis. Our data provide the preclinical rationale for investigating the use of KPT-9274 in AML clinical trials. Disclosures Chan: Genentech: Research Funding; Celgene: Research Funding; AbbVie: Research Funding.
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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 ».