Impact of the overexpression of the tyrosine kinase receptor RET in the hematopoietic potential of induced pluripotent stem cells (iPSCs).

INTRODUCTION: Previous studies have suggested that the tyrosine kinase receptor RET plays a significant role in the hematopoietic potential in mice and could also be used to expand cord-blood derived hematopoietic stem cells (HSCs). The role of RET in human iPSC-derived hematopoiesis has not been tested so far. METHODS: To test the implication of RET on the hematopoietic potential of iPSCs, we activated its pathway with the lentiviral overexpression of RETWT or RETC634Y mutation in normal iPSCs. An iPSC derived from a patient harboring the RETC634Y mutation (iRETC634Y) and its CRISPR-corrected isogenic control iPSC (iRETCTRL) were also used. The hematopoietic potential was tested using 2D cultures and evaluated regarding the phenotype and the clonogenic potential of generated cells. RESULTS: Hematopoietic differentiation from iPSCs with RET overexpression (WT or C634Y) led to a significant reduction in the number and in the clonogenic potential of primitive hematopoietic cells (CD34+/CD38-/CD49f+) as compared to control iPSCs. Similarly, the hematopoietic potential of iRETC634Y was reduced as compared to iRETCTRL. Transcriptomic analyses revealed a specific activated expression profile for iRETC634Y compared to its control with evidence of overexpression of genes which are part of the MAPK network with negative hematopoietic regulator activities. CONCLUSION: RET activation in iPSCs is associated with an inhibitory activity in iPSC-derived hematopoiesis, potentially related to MAPK activation.

Chimeric antigen-receptor (CAR)-engineered natural killer (NK) cells targeting chronic myeloid Leukemia (CML) blast crisis

Background The use of tyrosine kinase inhibitors (TKIs) has dramatically modified the therapy of chronic myeloid leukemia (CML), generating durable remissions and prolonging survival in TKI-responders. However, progression to blast crisis (BC) still occurs especially in TKI-resistant patients and represents a clinical challenge. We and others have identified IL2RA/CD25 as a typical cell surface marker of BC-CML and reported that its overexpression is correlated with the progression of CML from CP-CML to BC-CML (Imeri et al, Cells 2023). Here we show the experimental development of a third-generation CAR-NK therapy strategy against the CD25 based on the scFV of the clinically approved monoclonal humanized antibody, Basiliximab. Methods: As NK cell model, we have used the NK92 cell line which has a well-established and clinically demonstrated NK cell activity. We have lentivirally transduced NK92 cells with the CAR construct containing a selectable gene (GFP). After FACS-sorting of GFP-positive cells, phenotypical characterization was performed by FACS. The expression of the CAR-CD25 at the surface of the cells was demonstrated an anti-Fab antibody and double-positive (Fab/GFP) cells were further purified. The functionality of the cells was evaluated using CD107a degranulation assay after 3h co-culture and ELISA for IFN-gamma release. We have in parallel engineered K562 cells expressing CD25 by lentiviral transduction (K562-CD25) as well as also a second target cell line (RAJI) using the same strategy. Annexin V staining of target K562 cells was used for in vitro cytotoxicity assessments. For in vivo assays, NSG mice were intraperitoneally (IP) injected with K562-CD25 cells expressing Luciferase at Day-3 (3.10 6 cells/mouse, n = 13). At Days 0, 3, and 7, mice were treated by IP injection of either irradiated CD25 CAR-NK92 cells (10 .10 6/mouse n=6) or irradiated Wild-type (WT)-NK92 cells (n=5). The clinical evolution of mice transplanted mice was followed weekly by luminescence (IVIS 200). Results: After cell sorting, we obtained more than 90% of double-positive NK92 CAR+/GFP+ cells. Lentiviral transduction did not affect the activatory or inhibitory signals of NK92 cells. No statistical differences were observed between CD25 CAR-NK92 and WT- NK92 cells for the expression of NKp30, NKp46, KirDl2-3, TIGIT, and DNAM. However, we observed a strong increase in the Granzyme B and Perforin in CD25 CAR-NK92 cells after co-culture with K562-CD25 as compared to WT NK92 cells (p<0.001). Importantly, we have found increased levels of degranulation after co-culture of target K562-CD25 with CD25-CAR-NK92 cells (40%) as compared to cells co-cultured with WT NK92 alone (20% ) suggesting strongly the occurrence of an additional specific effect due to CAR-CD25. IFN-gamma levels after co-culture of CAR CD25 NK92 cells were also found to be significantly increased ( 400 pg/ml) in as compared to co-cultures of target cells with WT-NK92 (200 pg/ml)(p<0.0001). Similarly,in vitro cytotoxicity assays showed induction of higher levels of apoptosis in target cells (K562-CD25 and Raji-CD25) when co-cultured with CD25 CAR-NK92 as compared to NK92 WT (p<0.0001). In in vivo experiments, we have analyzed K562-CD25 leukemia-bearing mice treated with CAR-NK92 cells (n=6) or WT-NK92 cells (n=5). These experiments analyzed at D+30 post-transplant showed stronger anti-leukemia effect of CAR-NK therapy by IVIS imaging with a survival rate of 84% for mice treated with CD25 CAR-NK92 versus 40% for those treated with WT-NK92. All control mice transplanted with K562-CD25 cells and left untreated died by D+20. Conclusion: We show here for the first time the potential use of an NK cell-mediated CAR therapy strategy targeting CD25 which has been shown to be upregulated in CML blast crisis. The experimental data show a significantly increased and selective in vitro and in vivo cytotoxicity of CD25 CAR-NK92 cells against CD25-expressing leukemia cells as compared to WT-NK92 cells. These results suggest that targeting CD25 by a CD25 CAR based on Basilixiamb's scFV might be an interesting tool in BC-CML and in all acute leukemias overexpressing CD25. In order to translate these findings to NK cells derived from induced pluripotent stem cells (iPSCs), we have produced iPSCs expressing CAR-CD25 constructs and experiments are underway to evaluate the therapeutic potential of iPSC-derived CD25 CAR-NK cells in CML blast crisis or AML models.

Chimeric antigen-receptor (CAR) engineered natural killer cells in a chronic myeloid leukemia (CML) blast crisis model.

During the last two decades, the introduction of tyrosine kinase inhibitors (TKIs) to the therapy has changed the natural history of CML but progression into accelerated and blast phase (AP/BP) occurs in 3-5% of cases, especially in patients resistant to several lines of TKIs. In TKI-refractory patients in advanced phases, the only curative option is hematopoietic stem cell transplantation. We and others have shown the relevance of the expression of the Interleukin-2-Receptor α subunit (IL2RA/CD25) as a biomarker of CML progression, suggesting its potential use as a therapeutic target for CAR-based therapies. Here we show the development of a CAR-NK therapy model able to target efficiently a blast crisis cell line (K562). The design of the CAR was based on the scFv of the clinically approved anti-CD25 monoclonal antibody (Basiliximab). The CAR construct was integrated into NK92 cells resulting in the generation of CD25 CAR-NK92 cells. Target K562 cells were engineered by lentiviral gene transfer of CD25. In vitro functionality experiments and in vivo leukemogenicity experiments in NSG mice transplanted by K562-CD25 cells showed the efficacy and specificity of this strategy. These proof-of-concept studies could represent a first step for further development of this technology in refractory/relapsed (R/R) CML patients in BP as well as in R/R acute myeloblastic leukemias (AML).