ULK1 Presents As a Therapeutic Target to Sensitize Resistant Acute Myeloid Leukemia Cells to Venetoclax
Notice bibliographique
Résumé
Acute myeloid leukemia (AML) is a swiftly progressing hematological malignancy characterized by a rapid accumulation of immature myeloid elements. Despite efforts in identifying novel therapeutic targets and therapies, treatment of AML has not significantly changed over the past 40 years. Recently, venetoclax (VEN), a B-cell lymphoma-2 (BCL-2) inhibitor has been approved for use alongside low-dose chemotherapy. However, some patients exhibit resistance to VEN, limiting its effective use. Our lab has identified Unc-51 like kinase-1 (ULK1) to be abnormally highly expressed in VEN-resistant AML patient cells via gene expression analysis of the BEAT-AML dataset comprising >500 patient expression profiles. ULK1 is an autophagy-related protein required to initiate autophagy, functioning by phosphorylating Beclin1 to induce phagophore formation. To investigate autophagy-mediated VEN-resistance in AML, we have generated VEN-resistant cell lines from MV4-11 and MOLM13 AML cells and found that both have increased activated pULK1 and total ULK1 protein levels via Western blotting (>3-fold, p<0.01). Interestingly, these generated cell lines do not have significantly increased BCL-2 expression compared to their parental line. However, these cells express the BCL-2 family member and anti-apoptotic protein MCL-1 at an increased level compared to their VEN-susceptible counterparts (>2 fold, MV4-11-resistant cells (MVRE) p<0.001, MOLM13-resistant cells (MORE) p=0.02), which may compensate for BCL-2 inhibition via VEN treatment, as MCL-1 is not affected by VEN. Analysis of autophagy-related processes specifically revealed dysregulation in VEN-resistant cells. Confocal analysis using an LC3-autophagy flux reporter construct displayed increased autophagy in resistant cells (~2-fold, p<0.006), which was confirmed further via Western blotting for LC3 and p62 levels, consistent with increased autophagic initiation via overexpression of ULK1. Under ULK1 inhibition via SBP-7455 (SBP) in combination with VEN treatment, resistant cell viability was significantly reduced compared to VEN alone (MV4-11 >95% inhibition, p<0.001, MOLM13 >95% inhibition, p<0.03), with a strong synergistic effect when compared to single treatments of VEN or SBP alone (p<0.001). Apoptosis assays further confirmed this improved cell killing under co-treatment conditions, with a >8-fold increase in apoptotic cells compared to VEN alone in both lines (p<0.0001). Interestingly, after 24-hour co-treatment, both pULK1 (Ser555) and ULK1 protein expression were ablated in resistant cell lines (>90% inhibition). Notably, there was also no MCL-1 expression under co-treatment conditions despite its increased expression in resistant cells, suggesting a shift towards the apoptotic pathway. To limit any off-target effects from the SBP inhibitor, ULK1-knockout cell lines were generated from the MV4-11 and MOLM13-resistant cells using a CRISPR/Cas9 system. The knockout was subsequently confirmed via Western blotting for pULK1, ULK1, and pBeclin (Ser30; the downstream activation target of ULK1). As expected, these KO lines had a decreased IC50 for VEN compared to normal lines (MVRE KO vs. MVRE: 1.8 vs. 6.5 mM; MORE KO vs. MORE: 4.0 vs. 7.5 mM), indicating that ULK1 does play a role in mediating resistance as its absence resulted in increased sensitivity towards VEN. Interestingly, we observed decreased viability in CD34+ AML patient cells under both VEN single and VEN/SBP co-treatment at 72, 96 and 120 hours, with the co-treatment condition appearing to reduce viability at a greater level than VEN alone, similarly to what we have previously demonstrated using VEN-resistant cell line models. CFC assay analysis of these patient samples also displayed decreased proliferative capabilities under both single VEN and combination treatments, but not under SBP treatment alone. Importantly, this combination treatment of VEN and SBP is not toxic to CD34+ normal bone marrow cells, providing a potential therapeutic window. Taken together, these results indicate that ULK1 presents a viable target for further investigation for therapeutic intervention, to re-sensitize resistant AML to venetoclax treatment.
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Prédiction machine sur la base complète
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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) | 0,001 | 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 ».