Pharmacological activation of cAMP pathway protects human Hematopoietic Stem Cells from CAR-T cell induced hemato-toxicity
2Department of Pediatric Hematology-Oncology, Sheba Medical Center
Background: Chimeric antigen receptor T-cell (CAR-T) therapy has transformed cancer treatment by achieving remarkable clinical outcomes in hematological malignancies. However, its therapeutic success is often accompanied by hematotoxicity, primarily driven by cytokine release syndrome (CRS) and its detrimental effects on Hematopoietic Stem and Progenitor Cells (HSPCs). Despite its clinical significance, the mechanisms underlying CAR-T-induced human HSPC apoptosis and dysfunction remain poorly understood, and therapeutic options to mitigate this toxicity are currently limited.
Objective: This study aims to determine whether pharmacological activation of the cAMP pathway can protect human HSPCs function from CAR-T cell-secreted cytotoxic factors.
Methods: A triculture system with human HSPCs, anti-CD19 CAR-T cells, and Nalm6 cells was used to assess direct effects on HSPCs. Transwell experiments and conditioned media (CM) from CAR-T or untransduced T cells co-cultured with Nalm6 cells were used to evaluate indirect effects. Human HSPCs were exposed to these CM, and apoptosis was evaluated. The cytotoxic role of IFN-γ, a key component of CAR-T CM, was examined through direct IFN-γ treatment. Transcriptomic changes will be analyzed by RNA-seq, and functional assays, including colony formation and SCID repopulating assays will assess HSPC functionality. The protective effects of cAMP pathway activation were tested by treating HSPCs with the cAMP pathway activators forskolin and IBMX. Additionally, an in vivo model is being established by humanizing mice and injecting them with CAR-T cells to assess the impact on HSPC frequency and function.
Results: Activated CAR-T cells induced HSPC apoptosis in triculture, transwell, and conditioned media settings. This effect was reversed by treatment with forskolin (FSKN) and IBMX. Notably, FSKN/IBMX did not impact CAR-T cell viability, proliferation, or killing ability. IFN-γ, a key cytotoxic component secreted by CAR-T cells, also induced HSPC apoptosis, which was similarly reversed by FSKN/IBMX. Furthermore, FSKN/IBMX mitigated IFN-γ-induced HSPC dysfunction in the pre-clinical animal model, as evidenced by improved primary and secondary bone marrow engraftment.
Conclusion: cAMP pathway activation with forskolin and IBMX protects human HSPCs from CAR-T-induced apoptosis and dysfunction. This strategy holds potential to improve patient recovery while maintaining the anti-tumor efficacy of CAR-T cells.