A Novel TWIST2-ID2 Axis That Modulates The Growth and Imatinib Sensitivity Of CML CD34+ Cells
Notice bibliographique
Résumé
Abstract TWIST is a basic helix-loop-helix transcription factor that specifies Drosophila mesoderm development. In mammals there are 2 members, TWIST1 and TWIST2. TWIST2 is a regulator of osteoblasts and muscle development and plays a critical role in the epithelial-mesenchymal transition process, as well as in cancer initiation and metastasis. Twist2-deficient mice develop a myeloproliferative disease. These findings led us to query a potential role of TWIST2 in normal and leukemic (CML) human hematopoietic cells. RT-PCR and immuno-fluorescence analysis of CD34+ bone marrow (BM) cells obtained from healthy donors demonstrated their expression of TWIST2 transcripts and protein. Lentiviral vector-mediated knockdown of TWIST2 with 2 independent shRNA sequences enhanced the erythroid and granulopoietic colony-forming activity of transduced normal BM cells ∼2-fold compared with control transduced cells (n=3, p<0.05). Interestingly, ChIP studies showed that TWIST2 can bind directly to the DNA promoter for ID2 in CD34-enriched BM cells and knockdown of TWIST2 reduced ID2 expression by 50%. In lin-CD34+ cells from 14 chronic myeloid leukemia (CML) patients, we found both TWIST2 and ID2 transcripts to be 5 and 6 fold lower than those from 6 healthy BM donors (p<0.05), with similar findings for TWIST2 and ID2 protein in the same cells. BCR-ABL1-transduced Baf/3 cells also showed a reduction in Twist2 expression. Conversely, TWIST2 expression became elevated when K562 cells were treated with Imatinib mesylate (IM). We then generated a lentiviral vector encoding TWIST2 which proved capable of inhibiting the growth of K562 and MEG-01 cells as well as CFC production from CML CD34+ cells (n=11, p<0.05). Overexpression of TWIST2 in MEG-01 cells also reduced their tumorigenic ability in subcutaneously injected nude mice (0/8 for TWIST2 group, 7/8 for control group). In addition, increased TWIST2 sensitized the IM response of K562 cells and IM-resistant CD34+ cells from CML patients (2 in chronic phase and 2 in blast crisis). Correspondingly, knockdown of TWIST2 in K562 cells enhanced their cloning efficiency by 15% and made them IM-resistant. To obtain further insight into these biological effects of TWIST2, we generated several TWIST2 mutant cDNAs, including ones with a N-terminal truncation (ΔN), a C-terminal truncation (ΔC), a F86P dimerization mutant and a b- DNA binding mutant. Analysis of the effects of these mutants when overexpressed in CML cells and cell lines showed TWIST2 dimer formation was critical for the effects obtained with wild-type TWIST2, whereas the DNA binding domain could modulate these effects but was not essential, and the N-terminal and C-terminal domains were dispensable. We also found that overexpression of TWIST2 enhanced ID2 expression in CML CD34+ cells (n=3), as well as K562 and MEG-01 cells, and ChIP analyses confirmed the binding of TWIST2 to ID2 promoter DNA from K562 and MEG-01 cells. Using ID2 promoter-driven luciferase reporter and a mutant derivative (with only the E-box sequence altered), we found that TWIST2 could activate the wild-type promoter but not the mutated one in both K562 and MEG-01 cells. Finally, we co-transduced CML cells from 3 patients with TWIST2 and shRNA against ID2 and found that this reversed the suppressed production of CFC obtained with TWIST2 alone. Similarly in K562 cells this treatment partially restored their growth rate and IM resistance. Taken together, we report a novel TWIST2-ID2 regulatory axis in normal hematopoietic progenitor cells, which can also modulate the growth and IM response of CML progenitor cells. These findings provide a baseline for the future development of more effective therapy of CML. 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 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 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 ».