Human iPSC-Derived NK Cells with Knock-in of the BCL2 G101V Mutation Are Resistant to Venetoclax and Demonstrate Improved Anti-AML Activity In Vivo
Notice bibliographique
Résumé
Treatment options for acute myeloid leukemia (AML) patients who are unfit for intensive chemotherapy are limited. Both targeted therapy and cellular therapy have been investigated, typically as separate treatments. For patients with AML unfit for intensive induction chemotherapy, venetoclax, a BCL-2 inhibitor, containing regimens have significantly improved treatment outcomes. However, patients treated long-term with venetoclax can develop resistance. Based on the characterization of the BCL2 G101V mutation that mediates resistance to venetoclax, we investigated the ability of homozygous BCL2 G101V knock-in in induced pluripotent stem cell (iPSC)-derived natural killer (NK) cells to promote resistance to venetoclax in these effector cells to enable concurrent treatment with venetoclax and the engineered iPSC-derived NK cells that we hypothesize will provide improved NK cell-mediated killing of AML. Our group has pioneered the production of genetically modified NK cells from human iPSCs. iPSCs provide an advantage of a stable platform for genome engineering and previous studies demonstrate we can engineer iPSCs to express or delete genes of interest to derive genetically modified iPSC-NK cells with improved anti-tumor activity. To generate iPSC-derived NK cells with resistance to venetoclax, we employed CRISPR-Cas9 technology to knock-in the BCL2 G101V mutation (BCL2 G101V) in iPSCs. iPSCs homozygous for BCL2 G101V were selected and differentiated to NK cells. Homozygous BCL2 G101V engineered iPSC-NK cells demonstrated resistance to venetoclax compared to wildtype (WT) iPSC-NK cells in vitro. BCL2 G101V iPSC-NK cells were 94-fold more resistant to venetoclax compared to wildtype iPSC NK cells with an EC50 of 6018 nM, above the serum levels of patients receiving venetoclax. Analysis of cell surface proteins demonstrated that both sets of iPSC-NK cells had a typical NK cell phenotype with no differences in expression of receptors analyzed. There was no difference seen in cytotoxicity against K562 tumor cells between BCL2 G101V iPSC-NK cells and WT-iPSC NK cells. Additional functional analyses demonstrated that activity of the BCL2 G101V iPSC NK cells was preserved upon exposure to venetoclax. Cytotoxic activity, as measured by CD107a expression on WT iPSC-NK cells stimulated by MOLM13 AML tumors cells, was reduced nearly 3-fold upon addition of venetoclax. In contrast, activation of BCL2 G101V iPSC NK cells was not significantly affected by addition of venetoclax. This resistance to venetoclax was confirmed in longer term 36-hour Incucyte cytotoxicity assays where BCL2 G101V iPSC-NK cells demonstrated more than 10-fold increase in anti-AML activity compared to the WT-iPSC-NK cells. We then conducted in vivo studies to test the WT and engineered NK cells with and without addition of venetoclax in an AML xenograft model using NSG mice (Figure Panels A-D). These studies used MOLM13 AML cells made resistant to venetoclax and demonstrated that without venetoclax treatment, both the WT and the BCL2 G101V iPSC NK cells mediate effective anti-AML killing. With addition of venetoclax, the WT NK cells were unable to effectively kill the AML cells while the BCL2 G101V iPSC cells mediated potent anti-AML activity and demonstrated significantly improved survival when the NK cells are given in combination with venetoclax. Specifically, in this system, the mice treated with WT iPSC-NK cells had a median survival of 22.8 days while the median survival of mice treated with the BCL2 G101V iPSC NK cells was not reached (p<0.01) Intriguingly, BCL2 G101V iPSC NK cells also demonstrated improved activity against MOLM13 cells resistant to venetoclax. Together our results demonstrate that iPSC-NK cells can be engineered to generate venetoclax-resistance for use in combination with concurrent venetoclax therapy to markedly improve treatment of AML. Furthermore, this work demonstrates that novel drug resistance mechanisms can be introduced via genome engineering into iPSC-derived NK cells as a new strategy to produce improved cell products for “off-the-shelf” therapy.
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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) | 0,003 | 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 ».