Leveraging Increased Nucleoside Kinase Activity to Selectively Deplete Mitochondrial DNA (mtDNA), Impair Oxidative Phosphorylation, and Target AML Cells
Notice bibliographique
Résumé
Abstract Mitochondrial DNA (mtDNA) replication requires adequate nucleotide pools from the mitochondria and cytoplasm to support DNA biosynthesis. Gene expression profiling of 542 AML patient samples (GSE13159) demonstrated that 55% of AML patients had upregulated mtDNA biosynthesis pathway expression compared to 73 normal hematopoietic cells (mononuclear cells isolated from peripheral blood and bone marrow). We also identified upregulation of pathways which support mitochondrial nucleotide pools, which include mitochondrial nucleotide transporters and a subset of cytoplasmic nucleotide salvage enzymes, which phosphorylate nucleosides to nucleotides. Upregulation of nucleoside kinases in a subset of primary AML samples compared to normal hematopoietic progenitor cells (normal G-CSF (granulocyte-colony stimulating factor) mobilized peripheral blood stem cells (PBSC's)) was confirmed by immunoblotting. These results suggest that AML cells import cytoplasmic nucleotides to support mitochondrial DNA biogenesis. To determine if cytoplasmic nucleoside kinases regulate mtDNA content, we knocked down nucleoside kinases in AML cells. Partial target knockdown of DCK (deoxycytidine kinase) and CMPK1 (cytidine/uridine monophosphate kinase 1) reduced mtDNA content (60+8%, and 62+13%, respectively compared to controls, 5 and 7 days post-shRNA transduction), indicating a role in mtDNA biogenesis. As expected, knockdown of mtDNA replication factors POLG and TFAM reduced mtDNA content in AML cells. The cytidine nucleoside analog, 2'3'-dideoxycytidine (ddC) is activated by DCK and CMPK1 to produce its triphosphate form, ddC-triphosphate (ddC-TP). To assess nucleoside kinase activity, primary AML and normal hematopoietic cells were treated with ddC and total levels of ddC and ddC-TP were measured by mass spectrometry. Levels of ddC did not differ between AML and normal, but ddC-TP levels was increased in AML samples > 7-fold compared to normal (p< 0.05, one-way ANOVA). Previously we and others demonstrated that AML cells and stem cells have increased mitochondrial biogenesis and reliance on oxidative phosphorylation due to decreased spare reserve capacity and an inability to upregulate glycolysis. ddC-TP inhibits the sole mtDNA polymerase POLG, but not nuclear DNA polymerases. Given the increased activity of nucleoside kinases in AML cells over normal, we examined the effects of ddC treatment on mtDNA content and cellular bioenergetics. AML and normal cells were treated with increasing concentrations of ddC. At increasing times after treatment, ddC depleted mtDNA levels > 85% at 0.5 uM, 3 day treatment in OCI-AML2 and TEX cells as assessed by qPCR. ddC decreased protein expression of mtDNA encoded electron transport chain (ETC) subunits COXI and COX II, but not nuclear encoded subunit COXIV) and reduced basal oxygen consumption. ddC also decreased proliferation of AML cell lines (OCI-AML2, TEX, HL-60, K562) (> 95% reduction at 0.5uM, 10 days). Knockdown of DCK abrogated the effects of ddC on AML cell proliferation. We next examined the effects of ddC in primary human leukemia cells (AML = 7, CML blast crisis = 1, CMML-2 = 1) and normal hematopoietic progenitor cells (n=8). ddC preferentially inhibited mtDNA biosynthesis and reduced viability in a subset of primary cells (6 of 9 AML) compared to normal PBSC's (n=8). Sensitivity to ddC positively correlated with mtDNA depletion. Finally, we evaluated the efficacy and toxicity of ddC in mouse models of human AML. ddC (35 mg/kg daily i.p. x 11 days) caused tumor regression in an OCI-AML2 xenograft model without toxicity (changes body weight, behavior, serum chemistries). In OCI-AML2 cells isolated from treated mice, ddC reduced mtDNA by 95% and mtDNA-encoded ETC proteins by 90%. In addition, ddC (75 mg/kg i.p x 6 weeks) significantly reduced human AML bone marrow engraftment in primary AML (n=2, P<0.0001, n=1,P<0.05, t-test) and secondary AML (n=1, 14 vs 3%, P<0.01, t-test) without toxicity. Thus, AML cells have increased nucleoside kinase activity that is functionally important for mtDNA biogenesis. We leveraged this unique biological vulnerability to preferentially activate ddC, deplete mtDNA and selectively target oxidative phosphorylation in AML. Disclosures Schimmer: Novartis: Honoraria.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
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 ».