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  • The widespread involvement of HH GLI in

    2021-11-24

    The widespread involvement of HH/GLI in human malignancies has initiated a remarkable effort to identify selective HPIs. As shown in Table 2.1, most of these small molecule inhibitors target the essential effector protein SMO, which should lead to pathway abrogation by eventually decreasing the GLIA/GLIR ratio. This has already been successfully demonstrated in patients with basal cell carcinoma (BCC) and medulloblastoma showing aberrant SMO-dependent HH/GLI signaling (; ; ). However, there is also increasing evidence for SMO-independent stimulation of GLI activity in cancer cells, raising the question of whether SMO antagonists will prove efficient therapeutic drugs in such settings. As SMO function is associated with the primary cilium (Huangfu and Anderson, 2006), this limitation is likely to be relevant for GLI-dependent cancer Fluorescein-12-dUTP lacking this single cellular protrusion, as is the case for most myeloid and lymphoid cells (Pazour and Witman, 2003). In such cases, targeting SMO-independent mechanisms that impinge upon the GLI transcription factors to modulate their activity becomes more relevant.
    SMO-Independent Modulation of GLI Activity
    Hedgehog in Hematopoiesis and Hematopoietic Stem Cells A role of HH in human hematopoiesis was first described in 2001 when observed that primitive CD34+ human blood cells express SHH, PTCH, and SMO. Addition of exogenous SHH to in vitro expansion cultures increased the number of functional progenitor cells and promoted their differentiation. HH inhibition preserved the primitive state of the human progenitors and their capability to repopulate the murine bone marrow when adoptively transferred to NOD/SCID mice. Further, high levels of SHH stimulation caused proliferation of hematopoietic stem and progenitor cells (HSC/Ps) via downstream signaling cascades involving the hematopoietic inducer bone morphogenetic protein 4 (). In a later study, IHH but not SHH was found to be expressed by bone marrow stromal cells (). Consistently, IHH gene transfer into stromal cells enhanced their hematopoietic supporting potential. Expansion of repopulating cells on these IHH-overexpressing stromal cells resulted in increased engraftment in NOD/SCID mice. While in these assays IHH was presented by the stromal cells, it is important to note that it is also intrinsically present on CD34+ cells and may also support the proliferative capacity of the HSC/Ps in an autocrine manner (). Subsequently, several mouse models with modulation of HH signaling pathway members were used to elucidate HH signaling activity in the hematopoietic system. HH signaling activity is increased with impaired activity of the pathway suppressor PTCH1, encoded by the tumor suppressor gene PTCH1. Mice carrying only one allele of Ptch (Ptch+/− mice) exhibited a higher expression of GLI1 and cyclin-D1 in primitive Lin- Sca-1+ c-Kit+(LSK) hematopoietic cells along with an increase of their total number and frequency (). In addition, the heterozygous mice demonstrated an enhanced ability to regenerate the hematopoietic system following its ablation with 5-fluorouracil (5-FU) in both primary and transplant models. However, these phenomena turned out to be transient and the regeneration capacity of the HSCs could not be sustained beyond 8weeks posttransplantation despite continued HH activity (). used a tamoxifen-inducible Ptch1 deletion to demonstrate that Ptch1 is necessary for commitment to the B- and T-cell lineage fate. They did not find any influence of Ptch ablation on the myeloid lineage (). However, transfer assays of Lin- bone marrow cells from tPtch−/− mice into Rag2−/− γ−/− mice lacking mature lymphocytes suggested that the defects in lymphoid lineage commitments were caused by the Ptch defect in the stromal compartment rather than in the progenitors (). To further determine cell-intrinsic versus -extrinsic hematopoietic effects of Hh signaling activation, used inducible, cell-type-specific deletion models of Ptch1. Hematopoietic-specific deletion of Ptch1 using a tamoxifen-inducible Cre-ER recombinase under the control of the stem cell leukemia enhancer did not lead to Hh signaling activation in hematopoietic cells or to any phenotypic effects. In contrast, deletion of Ptch1 in epithelial cells led to the apoptosis of lymphoid progenitors, whereas deletion of Ptch1 in the bone marrow cell niche led to increased numbers in LKS cells and increased mobilization of myeloid progenitors ().