Hedgehog Signaling in Normal and Malignant Hematopoiesis.

Abstract

The Hedgehog (Hh) signaling pathway is critical for normal development and dictates the self-renewal, proliferation and differentiation of normal stem cells and progenitors. Aberrant reactivation of Hh signaling has been described in a wide variety of human cancers and its role in normal stem cells suggest that pathway dysregulation contributes to oncogenesis and influences the cell fate decisions in cancer stem cells (CSC). Like their normal counterparts, CSC appear to undergo self-renewal as well as give rise to differentiated progeny, and these properties implicate that CSC are responsible for continual tumor cell production that underlies the initiation, maintenance and progression of clinical disease. Myeloid leukemias have long served as the model system for human CSC, but the cellular processes responsible for regulating these rare biologically distinct cell populations have remained unclear. We hypothesized that Hh pathway activation contributes to the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and studied Hh signaling in these settings. Using both RT-PCR for pathway components and a Gli1 reporter assay, we have found that Hh signaling is active in several human AML derived cell lines (Kasumi-1, KG1, KG1a) and primary AML and MDS samples. Approximately 80% (19/24) of primary AML samples tested express the downstream effectors GLI1 or GLI2 indicative of active Hh signaling. Furthermore, inhibition of Hh signaling with the naturally derived SMOOTHENED antagonist cyclopamine reduces the clonogenic growth of KG1 cells implicating the pathway in self-renewal. In contrast, cyclopamine failed to affect colony growth in the HL-60 cell line that lacks expression of Hh pathway signaling components, confirming that the effect of Hh inhibition is specific. In addition, the ectopic expression of Gli1 in KG1 cells partially rescued the effect of cyclopamine on colony formation further demonstrating the specific nature of this compound. We also studied normal CD34+ bone marrow cells and found that they expressed components of Hh pathway by RT-PCR. However, in contrast to KG1 cells, cyclopamine had little effect on the recovery of either normal hematopoietic progenitors or stem cells in an in vitro long-term culture assay. Therefore, it appears that Hh inhibition may preferentially inhibit myeloid leukemias. We further studied the role of Hh pathway activation on normal hematopoiesis and developed a transgenic mouse model in which SMOOTHENED is conditionally over-expressed in the myeloid lineage via Cre recombinase activity regulated by the Lysozyme promoter. Analysis of these mice demonstrated only subtle changes in peripheral blood counts, but further analysis of cells expressing the transgene revealed a significant reduction in the number of mature myeloid cells. This was confirmed by analyzing blood cells for the granulocyte marker Gr1 and pan-myeloid marker Mac1, both of which were significantly reduced in the SMOOTHENED over-expressing cells. These defects are reminiscent of MDS and further suggest that the Hh signaling pathway plays a role in normal hematopoiesis. Therefore, aberrant Hh pathway activation is a feature of myeloid leukemias and inhibitors such as cyclopamine may have a therapeutic role in the treatment of AML and MDS.