The role of the hedgehog signalling pathway in acute myeloid leukaemia

Acute myeloid leukaemia (AML) encompasses a group of aggressive haematological neoplasms. It is a cancer stem cell (CSC) disorder. The Hedgehog (Hh) signalling pathway is one of the self-renewal pathways, highly conserved across species and important in determining stem cell fate, affecting a number of clinically important downstream targets including the Bcl-2 family. Abnormal Hh signalling has been associated with a diverse range of human malignancies. In myeloid malignancies, Hh signalling has been found to be vital in the maintenance and expansion of the CSC. Primary immotile cilia regulate canonical Hh signal transduction. These highly specialised organelles are present in single-celled eukaryotes through to humans, with defective primary cilia expression being linked to disease. It is unclear whether haematopoietic cells, normal or malignant, express primary cilia and the role the Hh pathway plays in the pathophysiology of AML. We sought to answer these questions, and further, to determine whether the Hh signalling pathway represented a therapeutic target in AML. Analysis of primary human AML mononuclear cells (MNCs) (n=76) showed the Hh pathway to be deregulated. SMO was significantly deregulated (p10% of cells by immunohistochemistry (IHC) on formalin fixed paraffin embedded (FFPE) primary human bone marrow trephines (BMTs) compared to none of our normal controls (n=10) (p<0.0001). GLI-1 expression was independent of SMO and PTCH-1. SHH was significantly down-regulated (p<0.001) within the blast population whilst secreted SHH, measured by enzyme-linked immunosorbant assay, was up-regulated suggesting paracrine activity. Impaired post-translational modification of SHH was demonstrated with protein located within the nuclei by IHC and immunocytochemistry (ICC). Nuclear expression of SHH was limited to primitive (CD34+) haematopoietic cells and absent from mature haematopoietic (CD14+, CD15+) cells. This correlated with a 20-fold reduction in HHAT, the acetyltransferase involved in Hh processing, in normal primitive haematopoietic cells compared to normal MNCs (p<0.01). There was no correlation between subtype, mutational profile or clinical outcome and any of the components of the Hh pathway. Primary immotile cilia were identified in all AML (n=23), and 20% of normal (n=10) primary human FFPE BMTs by ICC. Primary cilia were not identified in AML cell lines (n=7) or primitive (n=4) or mature haematopoietic cells (n=6) isolated from peripheral blood, suggesting they are lost once cells migrate from the bone marrow (BM) microenvironment. In vitro, SMO inhibition with 20μM cyclopamine reduced cell proliferation by trypan blue exclusion in select, genetically diverse AML cell lines (HL-60, Kasumi-1, KG1a, MOLM-13, MV4-11, OCI-AML3 and THP-1). No change in early or late apoptosis was seen in HL-60, KG1a, MV4-11, OCI-AML3 and THP-1 by flow cytometry (FACS). In contrast an increase in the number of dead cells by trypan blue exclusion was seen in Kasumi-1 (p<0.01) and MOLM-13 (p<0.05) with apoptosis confirmed by FACS (both p<0.05). Culture of the OCI-AML3 cell line with cyclopamine led to cell cycle arrest with an increase in G0-G1 cells (p<0.05), and a 3-fold reduction in cell division by CFSE (p=ns); striking morphological changes were seen with an increase in cytoplasm, granules and vacuoles and loss of nucleoli, with FACS analysis demonstrating increased expression of CD11b (p<0.001) and CD11c (p