Additionally, in CD34+ cord blood engrafted NSG mice treated with Glasdegib, the frequency of human CD45+ cells and the commitment to myeloid and lymphoid cell fates remain similar to that in vehicle-treated controls. This indicates that, unlike leukemic stem cells (LSC), the cell fate decisions of normal human hematopoietic stem cells (HSC) are independent of the hedgehog (Hh) pathway. These findings underscore the specificity of Glasdegib in targeting cancer cells through the selective inhibition of smoothened (SMO), leading to GLI2 downregulation, without affecting the cell fate decisions of normal HSCs, highlighting its niche-dependent effects.

Glasdegib (PF-04449913) acts as a potent inhibitor of the sonic hedgehog (Shh) pathway, evidenced by its ability to suppress Shh-induced luciferase expression in mouse embryonic fibroblasts with an IC50 of 6.8 nM. It also demonstrates a significant reduction in medulloblastoma growth in a Ptch1+/-p53+/- allograft model, attributed to decreased murine Shh target gene expression. Further, in stromal co-culture experiments, Glasdegib effectively reduces breast cancer leukemic stem cells (BC LSC) compared to normal progenitors, as shown by FACS analysis. This reduction in leukemic burden in human BC LSC engrafted RAG2-/-γC-/- mice, treated daily with Glasdegib, is also marked by a significant decrease in spleen weight (p=0.006) and corresponds with a reduction in GLI2 protein expression, identified through nanoproteomic and confocal fluorescence microscopic analysis.