title: The role of the hedgehog signalling pathway in acute myeloid leukaemia
Creators: name: Campbell, Victoria L.
Publisher: name: University of Glasgow
Year: 2018
Subjects: subject: Acute myeloid leukaemia, hedgehog signalling, cilia.

description: 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 (p&lt;0.0001) with two divergent cohorts identified. GLI-1, indicative of pathway activity, was detected in 52.6% of samples. Sanger sequencing (n=36) did not identify a mutation in SMO to account for the pathway deregulation though a number of silent and missense mutations were identified (mean 1; range 1-3). 25% of our AML samples (9/37) showed expression of GLI-1 in &gt;10% 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&lt;0.0001). GLI-1 expression was independent of SMO and PTCH-1. SHH was significantly down-regulated (p&lt;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&lt;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&lt;0.01) and MOLM-13 (p&lt;0.05) with apoptosis confirmed by FACS (both p&lt;0.05). Culture of the OCI-AML3 cell line with cyclopamine led to cell cycle arrest with an increase in G0-G1 cells (p&lt;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&lt;0.001) and CD11c (p&lt;0.0001) consistent with a more mature phenotype. Early haematopoietic markers NAB2, GATA1, EGR2, SCL, IRF8 and EGR1 were down-regulated whilst PU1, GCSF and MPO involved in differentiation and maturation were up-regulated (p=ns) in cyclopamine treated cells. 
Colony forming cell (CFC) assays showed a statistically significant reduction in the more pluripotent CFU-GM (p=0.006) colonies and an increase in omnipotent CFU-G (p=0.013) colonies following culture with cyclopamine 20μM. 
There is extensive evidence supporting Bcl-2 is altered in malignancy; its role in AML cell maintenance and survival, is well recognised. Further, Bcl-2 is a key downstream target of the Hh signalling pathway. Complete linkage analysis found key members of the Bcl-2 family and cell cycle regulators to cluster with components of the Hh signalling pathway. Sensitivity to the BH3 mimetic ABT-199 was not solely dependent on the expression of Bcl-2, rather a complex interplay between the pro-apoptotic and anti-apoptotic family members. Targeting Bcl-2 had a variable effect on KG1a, MOLM-13, MV4-11, OCI-AML3 and THP-1 cells; combination treatment with ABT-199 and Ara-C showed a highly synergistic effect on cell death in MOLM-13 cells (Chou-Talalay CI&lt;0.47 (range 0.13-0.47) and DRI&gt;2 (range 2-70) for all dose combinations). 
We show the Hh signalling pathway to be deregulated in AML, expression of components of the pathway changing with myeloid maturation. We demonstrate primary cilia on haematopoietic cells within the BM, with an increased frequency observed in AML. Their absence when cells migrate from the BM fits with their function and suggests a ‘switching off’ of canonical signalling on maturation. In the absence of primary cilia, we show the Hh pathway remains active, suggesting a role for non-canonical signalling in AML. Pharmacological inhibition led to both apoptosis and differentiation; preliminary results suggest the effect is dependent upon the degree of SMO inhibition. We believe further work is required to determine the role of the Hh signalling pathway in normal and malignant haematopoiesis but that our data, considered especially in context with other recently published studies provides a rationale to continue to explore SMO or downstream Bcl-2 pathway inhibition as potential therapies in AML.