Inhibition of PAK6-Mediated Survival and Cell Cycle Controls Selectively Targets Therapy-Resistant CML Stem/Progenitor Cells
Notice bibliographique
Résumé
The resistance of chronic myeloid leukemia (CML) leukemic stem cells (LSC) to ABL tyrosine kinase inhibitor (TKI) monotherapy remains a challenge in curing CML. We have recently identified miR-185 as a useful biomarker to predict therapy response in treatment-naïve CML stem/progenitor cells. We also demonstrated that restored miR-185 expression in LSCs impaired survival, sensitizing them to TKIs in vitro and in preclinical patient-derived xenotransplantation models, indicating that miR-185 is a critical regulator mediating TKI response/resistance of CML stem/progenitor cells. PAK6, a serine/threonine-protein kinase, was uncovered as a target gene of miR-185 by RNA-seq and was found to be upregulated in CD34+ TKI-nonresponder cells vs. TKI-responders, but its biological functions in CML are largely unknown. To investigate the biological effects of inhibiting PAK6 activity in TKI-resistant cells, we tested a pre-clinically validated pan-PAK inhibitor (PF-3758309) in vitro. PF-3758309 significantly reduced the growth of IM-resistant cell lines, including K562-resistant cells, BV173 blast cells (IC50 25-70 nM) and CD34+ TKI-nonresponder cells, as assessed by viability and CFC assays, and increased their apoptosis; these effects were significantly enhanced by TKIs (~2-fold, P<0.05). PF-3758309 alone, or in combination with a TKI, did not have obvious inhibitory effects on CD34+ normal bone marrow. These results were further confirmed in IM-resistant cells using a lentiviral knockdown system that specifically inhibits PAK6. Interestingly, PF-3758309 alone, or in combination with a TKI, greatly reduced mitochondrial activity in CD34+ TKI-nonresponder cells, as shown in functional assessments with MitoTracker, a dye that accumulates in active mitochondria, the site of OXPHOS; this was not seen with TKI alone (P<0.002). Similarly, CellROX analysis confirmed a reduction in ROS levels upon PF-3758309 treatment, or a combination of PF-3758309 with TKI, in these cells. In addtion, MDM2, a critical negative regulator of the p53 tumor suppressor, was identified as one of substrates of PAK6, by PhosphoSitePlus analysis. Its expression was found to be correlated with PF-3758309 treatment in CML cells based on CellMinerDB univariate analyses using gene-small-molecule association data from the CTRPv2 database. Indeed, Western blot analysis showed that PAK6 knockdown in K562 and IM-resistant cells led to a reduction in MDM2 protein levels. Furthermore, MDM2 downregulation by PAK6 inhibition corresponded to an increase in p21 levels, suggesting a mechanism of MDM2-mediated p21 regulation independent of p53, as these cells are p53-null. Most interestingly, PAK6 knockdown in IM-resistant cells leads to G2/M phase accumulation and increased senescence levels (2-fold, P<0.05), detected by senescence-associated β-galactosidase staining. PAK6 knockdown induced senescence was further supported by observations of enlarged cell size (p<0.05) and increased granulation, as well as changes in senescence-associated protein markers, including p21, p27, MMP-3 and the DNA damage marker pH2Ax, by Western blot analysis. Hence, our findings indicate that dual targeting of miR-185-PAK6-mediated survival, cell cycle and metabolic pathways, along with BCR-ABL, selectively eradicates drug-resistant CML stem/progenitors. Specifically, PAK6 plays roles in MDM2/p21-mediated apoptosis, senescence and cell cycle controls, offering a valuable therapeutic strategy for improved treatment and care. Disclosures No relevant conflicts of interest to declare.
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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 | 0,001 |
| 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 ».