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 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).