Background
Stem cells
Adult somatic stem cells are defined as undifferentiated cells with three key properties: long life, multipotency, and the capacity to self-renew. 1 However, recent evidence has shown that although their phenotype is tightly defined, these cells are functionally heterogeneous. 2 The properties of stem cell self-renewal and survival are controlled by highly conserved embryonic signaling pathways, including the hedgehog (Hh), epithelial-mesenchymal transition, WNT, and Notch signaling pathways, the HOX transcription factors, and the BMI1/polycomb transcriptional regulators. 3
Cancer stem cells
There is experimental evidence from a number of malignancies to suggest the presence of a small population of very primitive cells that share many of the properties of somatic stem cells. These cells have been termed cancer stem cells (CSCs). 1
Whilst there is still much to learn in respect of the CSC, various experimental models have shown these cells to be quiescent, resistant to therapy, capable of self-renewal, and the initiation of tumors in secondary transplanted hosts. 4 Malignancies with evidence of CSC origin include hematological cancers 4 , 5 and solid tumors including: prostate, 6 pancreas, 7 lung, 8 and certain neurological malignancies. 9
Although the concept of the CSC is largely accepted, the model by which it is capable of generating a tumor, or leukemia, remains contentious. 10 The stochastic model argues tumors are biologically homogeneous, with CSC behavior being determined by intrinsic or extrinsic factors; tumor heterogeneity arising because these factors are unpredictable. 11 In contrast, the hierarchy model argues CSCs are highly organized in unidirectional cellular hierarchies (the pattern of normal tissue growth). In the hierarchical model, the CSCs are biologically unique. 12 The fundamental difference between the models lies in which cell, or cells, are capable of behaving as a CSC with discrimination relying on accurate, well-defined experimental design. 13
The CSC hypothesis is of considerable clinical importance, potentially explaining minimal residual disease, relapse and disease progression, and highlighting the need to target these cells in order to effect a cure. 14 CSCs have been shown to be innately less sensitive to treatment, to continually develop genomic and epigenomic changes, and to uniquely interact with the stem cell niche. 14 , 15 The influence each of these factors has on the CSCs treatment-resistant phenotype is however, unclear.
The pathways involved in self-renewal are of intense interest, with many, if not all being implicated in neoplastic proliferation when deregulated. 15 It is the behavior of one of these pathways, the Hh signaling pathway, which is the focus of this review.
Hh signaling pathway
The Hh signaling pathway was initially discovered in 1980 by Nüsslein-Volhard and Weischaus whilst studying embryonic patterning in the Drosophila fruit fly, with absence of the Hh protein giving the Drosophila a characteristic “hairy” or “prickly” appearance. 16 , 17 Subsequent work has shown the Hh pathway to be highly conserved across species and vitally important in embryogenesis, performing the function of patterning during the early stages of development through the expansion and contraction of stem cell numbers. In adult organisms, through its ability to affect stem cell behavior in responsive tissues, it is involved in aspects of tissue maintenance and regeneration – proliferation, apoptosis, chromatin modeling, and self-renewal, acting in concert with other stimuli and the stem cell niche. 18
Canonical signaling
Classically, the Hh signaling pathway is believed to be ligand-dependent. Three Hh ligands (Sonic [SHH], Indian [IHH], and Desert [DHH]) have been identified in vertebrates, affecting stem cell behavior in a time- and concentration-dependent manner. 19 SHH is widely expressed, particularly during embryogenesis, with SHH deficiency being embryonically lethal. 17 IHH is produced in hematopoietic cells, bone, and cartilage, 20 whilst DHH is found in the peripheral nervous system and testes. 21 Hh ligands are initially synthesized as an inactive 45 kDa precursor, undergoing post-translational modifications to form a 19 kDa amino-terminal active signaling molecule. 22 This cholesterol and palmitoyl modification, catalyzed by Hh acyltransferase, 23 not only enhances ligand activity but also modifies its diffusion capacity. 24 The Hh ligands bind to the 12 trans-membrane receptor protein Patched 1 (PTCH1), causing its internalization and removing its repression of the 7-span trans-membrane protein Smoothened (SMO), allowing pathway activity. 25 In vertebrates, activity of the Hh pathway appears intrinsically related to primary immotile cilia; in the absence of ligand, PTCH1 is located within the primary cilia. Following ligand binding, and the internalization of PTCH1, SMO is able to concentrate in the primary cilia where it interacts with the GLI transcription factors shifting the balance toward pathway activation. 25 Whilst the intricacies of this interaction remain poorly understood, studies suggest these receptors do not physically interact, rather PTCH1 is thought to regulate SMO through an intermediary, with studies suggesting that oxysterols, including vitamin D3, are involved. 26 SMO subsequently causes accumulation of the full length active form of the zinc transcription factors GLI-2 and GLI-3 in the nucleus, and potentiates the activity of other positive regulators of the pathway including serine threonine kinase 36 (STK36) and kinesin family member 7 (KIF7), resulting in transcription of key downstream targets such as GLI-1 and PTCH1 , regulators of chromatin formation, cell cycle activity, cell mobility, and apoptosis, eg, bone morphogenetic protein 4, forkhead box protein M1, and WNT2a , 27 Figure 1A . Of the transcription factors, GLI-1 functions as a positive regulator, GLI-3 a transcriptional repressor, and GLI-2 both a positive and negative transcriptional regulator determined by post-transcriptional and post-translational modifications. 28 It is the balance between these transcription factors which determines pathway activity. 29
Alternatively, the Hh ligands can bind to a number of membrane-associated glycoproteins: Patched 2 (PTCH2), Hh-interacting protein (HHIP), and Dispatched (DISP). PTCH2, although structurally similar to PTCH1, has distinct functional properties and notably different tissue distribution, PTCH1 being broadly expressed whilst PTCH2 is primarily restricted to the skin and testes. 30 , 31 HHIP is an endogenous Hh ligand inhibitor, with a binding affinity comparable to PTCH1, preventing pathway activation. 32 DISP, a 12 trans-membrane receptor protein, is not involved in Hh ligand synthesis or processing but rather facilitates ligand movement, thereby modulating canonical pathway activity. 33
In the inactive state the transcription factors GLI-2 and GLI-3 are retained in the cytoplasm in a protein complex associated with the inhibitory molecule, suppressor of fused (SUFU) 34 and non-specifically phosphorylated by casein kinase (CKI), glycogen synthase 3β (GSK3β), and protein kinase A (PKA). This complex subsequently undergoes E3 ubiquitin-mediated proteolysis by the Skp1-Cullin1-F-box protein (SCF β-TRCP ) to the truncated repressor form which, on translocating to the nucleus, strongly inhibits the Hh pathway, 35 Figure 1B . It is the complex interplay between the active and inactive state of the pathway and the positive and negative feedback loops that maintain the careful balance of Hh signaling in normal tissue.
Non-canonical signaling
The notion of non-canonical signaling has arisen following observations that the pathway response does not always appear to follow the classical canonical signaling paradigm, although the evidence is not conclusive. Three scenarios of non-canonical Hh signaling have been described, Figure 2 : direct interaction with components of other pathways, atypical interaction of components, and activity independent of GLI-mediated transcription, defined as Type I (PTCH-dependent) and Type II (SMO-dependent). 36 In reality, it is likely the canonical and non-canonical pathways act in parallel.
Downstream targets
There are numerous downstream targets of the Hh pathway: BCL-2, the ATP-binding cassette transporter family members; the multi-drug resistance protein-1 and components of the epithelial-mesenchymal transition, WNT, and Notch signaling pathways, Figure 3 . There is already evidence to show up-regulation and involvement of many of these pathways in chemo-resistance in human malignancies. 37 , 38 Additionally, studies have highlighted several of these downstream targets to be over-expressed in both acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), with expression linked to chemo-resistance and poorer survival. 39
Hh signaling in hematopoiesis
In vertebrates, hematopoiesis is broadly divided into two major phases, primitive (embryonic) and definitive. 40 The Hh signaling pathway has a complex role in both embryonic and adult hematopoiesis. This role appears to be dependent on developmental stage, cell lineage, and whether the hematopoietic system is under regenerative pressure. 27 , 41 Whilst evidence has shown it to be vital for early hematopoietic development, 20 there remains controversy over its role in normal hematopoiesis in adult organisms, 41 – 43 although some of this may be explained by experimental method. Interestingly, recent early phase clinical trials looking at SMO inhibition have shown little or no hematopoietic toxicity potentially indicating Hh signaling may be dispensable in certain situations. 44 Importantly, abrogation of canonical Hh signaling by knockout of SMO does not adversely affect steady-state normal hematopoiesis. 42 , 45
Hh signaling in malignancy
Crucially, abnormal Hh signaling has been associated with diverse human malignancies including basal cell carcinoma, 46 medulloblastoma, 47 pancreatic, 48 and lung cancer. 49 Interestingly, data suggest different mechanisms of action in the various tumor environments. Constitutive pathway activation through loss-of-function mutations, 46 epigenetic modifications, 50 or reduced expression of the negative regulators PTCH, HHIP, and SUFU 51 or gain-of-function mutations and epigenetic changes 52 in the positive regulator SMO have been observed in a number of solid malignancies. 53 To date, no mutations have been identified in hematological malignancies; however, epigenetic modifications have been observed in a cohort of pediatric AML patients, correlating with disease status. 54 Ligand-dependent canonical pathway activation involves autocrine or paracrine Hh signaling. 55 Autocrine Hh signaling has been identified in multiple myeloma (MM), 56 prostate, 57 and lung cancer. 58 Paracrine Hh signaling has been observed in lymphoma, 59 colon, and pancreatic cancer. 60
Hh signaling in myeloid malignancies
In myeloid malignancies, Hh signaling has been found to be vital in the maintenance and expansion of the CSC or “leukemic stem cell” (LSC), either as a survival and proliferation signal or through direction of the LSC fate. 27 , 61 – 64
CML is a clonal myeloproliferative disease driven by the Philadelphia chromosome which encodes the constitutively active BCR-ABL tyrosine kinase. For CML to develop, this mutation must originate in a multi-potent hematopoietic stem cell (HSC), producing the leukemia-initiating cell or LSC. 65 Acquisition of BCR-ABL by an HSC results in a number of functional changes, including increased proliferation, differentiation block, inhibition of apoptosis, and altered cell adhesion and stromal interactions, producing the clinical phenotype of CML. 66
The development of tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of CML, with the majority of patients now achieving long-term survival with a good quality of life. 67 TKI therapy, however, is not without side effects, nor is it effective in all patients; TKI resistance and disease persistence despite TKI therapy remain ongoing issues. Persistent disease is believed to be due to lack of dependence on BCR-ABL signaling for survival and TKI resistance in CML stem cells. 68
Hh signaling appears to be intimately involved in the persistence and expansion of these CML stem cells. 35 , 43 , 61 Two different SMO -deficient murine models have shown supporting results – reduced incidence of leukemia in primary and secondary transplant recipients and a prolonged latency in primary transplantation. 43 , 61 Additionally, these groups demonstrated improved survival, with an accompanying reduction in the LSC population following treatment with the SMO antagonist cyclopamine; combination therapy with a TKI and cyclopamine resulting in the largest reduction in LSCs in vitro and in vivo. These findings are supported by studies using clinical grade SMO inhibitors alone, and combined with TKIs, showing an improved survival, with a reduced incidence of leukemia in secondary transplant recipients and a marked reduction in measures of self-renewal. 69 Supporting work in NOD scid gamma mice, looking at molecular targets, has shown targeting the Hh pathway with dasatinib and GDC-0449 resulted in reduced expression of GLI-1, GLI-2, BCL-2, and Cyclin D2, and increased expression of p21, pATM, pChk2, and γH2AX. 70 Interestingly, low PTCH1 expression has been found to be an independent predictor of imatinib failure and reduced overall survival. 71
AML is an extremely heterogeneous clonal disorder. Whilst there is clear evidence to support the CSC theory in AML, 4 , 72 recent work has suggested the LSC population is phenotypically variable, and may not be confined to a single clonal subpopulation. Further, whether this LSC arises following progenitor cell acquisition of abnormal self-renewal potential or from an HSC remains unclear. 4 , 5 , 72
There is increasing evidence showing the Hh pathway is deregulated in AML. Leukemic cell lines and primary AML cells express components of the Hh pathway, SHH and GLI-1 . 73 GLI-1 expression correlating with cytogenetic risk, inferior event-free survival and a reduced overall survival, with GLI-1 conferring drug resistance through UGT1A-dependent glucuronidation. 74 Further, high GLI-1 expression predicts poor remission status and reduced overall survival in secondary AML. 75 GLI-2 has been shown to be a negative prognostic indicator in a number of microarrays. Additionally, aberrant Hh signaling has been linked to drug resistance with inhibition of the pathway restoring chemosensitivity in AML. 36 , 62 Further, pathway inhibition with PF-04449913 sensitized AML cell lines and primary cells to the standard chemotherapy agent cytarabine; additional inhibition of SMO modulated cell cycle and self-renewal signaling. 76 In pediatric AML the hypo- and hypermethylation of pathway promoters were highly associated with AML diagnosis and relapse. 54 The complex interplay between the intrinsic and extrinsic signals governing LSC behavior may, however, mean targeting a single pathway, such as the Hh pathway, is not sufficient to eradicate these CSCs. For example, in an MLL-AF9 -mediated murine model of myeloid leukemia Hh signaling was completely dispensable for the development of acute leukemia. 42 , 45
The role of the Hh pathway in myelodysplastic syndrome (MDS) is not so well understood. However, there is increasing evidence to show it is deregulated. Analysis of primary MDS samples found overexpression of SHH, DHH, PTCH1 , and SMO . 77 Further, there was a correlation between ligand expression and the progenitor/stem cell marker c-KIT. 77
Interestingly, work has shown the Hh pathway to act on the microenvironmental stromal cells, thereby regulating HSC behavior, 64 with the Hh ligands acting in an autocrine and/or paracrine manner. 59 , 62 , 78 Moreover, HHIP was highly expressed in primary stromal cells, but not in primary peripheral blood, bone marrow, or cord blood CD34+ selected cells, with stromal HHIP expression suppressing leukemic cell proliferation. 79 Further, studies have demonstrated HHIP expression in AML and MDS-derived bone marrow stromal cells to be markedly reduced, with these cells supporting the proliferation of leukemic cells; pretreatment with azacitidine increased stromal cell HHIP expression, with a parallel reduction in leukemic cell proliferation in co-culture. 79
Myeloproliferative neoplasms is a term encompassing several BCR-ABL-negative neoplasms – myelofibrosis (MF), polycythemia vera, and essential thrombocythemia – characterized by stem cell-derived clonal myeloproliferation and the Janus kinase 2 (JAK2V617F) 80 or Calreticulin mutation. 81 There are limited data available on the Hh pathway in these malignancies. GLI-1 and PTCH1 are increased 20–100-fold in primary myeloproliferative neoplasms samples, with pathway activity demonstrated in a murine bone marrow transplant model of MF using a GLI-luciferase reporter system. Combining the SMO antagonist LDE225 with INC424, a JAK1/2 dual kinase inhibitor, in this murine model saw a reduction in mutant allele burden and an improved clinical phenotype. 82 Another murine model of MF (Gata1 low ) has shown alterations in the Hh pathway at the gene level in the bone marrow and spleen, components of the Hh pathway coordinating with TGFβ, p53, and mTOR-related genes to produce the biological phenotype of MF. 83 However, a recent Phase II clinical trial using IPI-926 in MF saw the majority of patients taken off therapy due to side effects or no response. 84 How the Hh inhibitors work in combination with other novel drugs remains to be fully ascertained.
Hh signaling in lymphoid disorders
Hh signaling is vital for normal B- and T-cell development; with Hh ligands, produced by either the bone marrow or lymphoid organ microenvironment, determining lymphoid cell behavior. 63
B-cell disorders
B-cell acute lymphoblastic leukemia (B-ALL) is thought to develop from the malignant transformation of immature hematopoietic progenitor cells. Unlike AML, in which the CSC hypothesis was first demonstrated and is widely accepted, the B-ALL LSC has yet to be isolated or characterized, 85 blasts from all stages of maturation being able to reconstitute and establish a leukemic phenotype in vivo. 86 Interestingly however, up-regulation of components of the Hh pathway has been observed in precursor B-ALL, 87 SMO inhibition reducing in vitro and in vivo measures of self-renewal. 88
B-cell chronic lymphocytic leukemia (B-CLL) is a clonal disorder of mature, differentiated lymphocytes. Although not a CSC disease, both components of the Hh pathway ( GLI-1, GLI-2 , and SUFU ) and key downstream targets ( BCL-2, BCL-XL ) are increased, and importantly correlate with clinical outcome. 89 Expression of BCL-2 is increased in the presence of active Hh signaling and down-regulated upon inhibition of the pathway. 59 Further, cyclopamine increased apoptosis of B-CLL cells in vitro, in stromal co-culture conditions and in combination with fludarabine. 59 , 89 Gene expression of components of the Hh pathway is extremely variable in CLL patients, with expression correlating with response to SMO inhibition. Sixty percent of treated patient samples responded to at least one SMO antagonist; likelihood of response correlating with elevated GLI-1 and PTCH1 expression and the presence of trisomy 12 (a poor prognostic indicator). This work also showed evidence of autocrine DHH signaling, potentially enabling non-canonical signaling in B-CLL cells. 90 In contrast, another study found PTCH1, SMO , and GLI-1 to be reduced in primary CLL samples, although there was considerable heterogeneity – about 25% showing high transcript levels compared to normal B-lymphocytes. GANT61, a direct GLI inhibitor, induced apoptosis in a time- and concentration-dependent manner in association with STAT3 phosphorylation. 91 A further study found SMO and GLI-1 to be significantly down-regulated in B-CLL cells compared to normal B cells. 92 Moreover, whilst cyclopamine and knockdown of SMO had only minor specific effects on B-CLL cell survival in vitro, GANT61 caused significant apoptosis in B-CLL cells but not normal B cells. 92 Combined treatment with GANT61 and fludarabine causing increased apoptosis compared to either agent alone. Interestingly however, whilst CLL viability improved in the presence of the stromal cell lines HS-5 and M210-B4, known to produce Hh ligands, 59 , 92 the same effect was not achieved with soluble SHH indicating other factors are involved. So, whilst these results support previous reports of Hh pathway activity in CLL, they would support redundancy of canonical Hh signaling in vitro.
The term lymphoma encompasses a broad spectrum of diseases from the relatively indolent to the aggressive and rapidly fatal; each has a complex and heterogeneous pathogenesis. Broadly there are two categories, Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). Components of the Hh pathway and key downstream targets (BCL-2 and BCL-XL) are expressed in a variety of NHL cell lines and primary tissue, 59 , 93 with expression of the downstream targets being influenced by the Hh pathway.
Whilst an E μ- myc murine model of Burkitt’s lymphoma, an extremely aggressive form of NHL, demonstrated the importance of stromal co-culture for lymphoma cell survival and expansion, stroma could be replaced with soluble SHH or IHH. 59 Moreover, Burkitt’s cells underwent apoptosis in the absence of Hh signaling both in vitro and in vivo.
In diffuse large B-cell lymphoma (DLBCL), an aggressive form of NHL, response to Hh signaling is dependent on subtype, predominantly inducing cell cycle arrest in germinal center B-cell type, and apoptosis in the activated B-cell type. 93 In addition to responding to the Hh ligands, these DLBCL cells were also shown to synthesize and secrete Hh ligands, supporting a role for autocrine signaling. 94 Pharmacological and silencing techniques have shown expression of the AKT genes to be modulated by the Hh pathway in DLBCL at the transcriptional level, 95 with another study showing that the Hh pathway activates the NF-kβ pathway. 96
In another form of aggressive NHL, mantle cell lymphoma, a therapy-resistant murine model showed up- regulation of the GLI transcription factors at the gene level, 97 confirming previous work showing the GLI transcription factors to be over-expressed in mantle cell lymphoma, both in cell lines and primary lymphoma cells, compared to normal B cells. 98 Further, targeting the GLI transcription factors with antisense oligonucleotides down-regulated BCL-2 and Cyclin D1 resulting in decreased proliferation and increased susceptibility to chemotherapy. 98
Whilst these represent high grade forms of NHL, the Hh pathway has also been found to be deregulated and amenable to therapeutic intervention in low-grade lymphomas. In Waldenstrom’s macroglobulinemia, a characteristically indolent form of lymphoma secreting immunoglobulin M (IgM), GLI-2 has been found to influence IgM levels. Further, GANT61 resulted in a significant reduction in IgM secretion across a number of Waldenstrom’s macroglobulinemia cell lines potentially through the interleukin-6 receptor; this effect was not seen with cyclopamine. 99
In classical HL, whilst the Hh ligands and GLI transcription factors GLI-1 and -2 were expressed at a relatively low level, GLI-3 was highly expressed in all cell lines. Furthermore, immunohistochemistry for GLI-3 showed strong, uniform nuclear expression in virtually all Hodgkin/Reed-Stenberg cells whilst expression was variable in nodular lymphocyte predominant HL and NHL. 100 Interestingly GLI-3 in thymic stromal cells has been shown to regulate T-cell selection and thymocyte differentiation; 101 whether it is responsible for a similar role in HL remains to be determined.
MM is a CSC disorder, arising from the proliferation of a clonal population of plasma cells, and associated with the production of monoclonal immunoglobulin. Despite a variety of therapeutic approaches MM is incurable with a relapsing natural history; it is the MM CSC which is believed to be resistant to standard therapies, including lenalidomide, bortezomib, dexamethasone, and cyclophosphamide.
In MM, Hh pathway activity has been implicated in the maintenance and differentiation of the CSC. 56 , 78 Pathway inhibition, with cyclopamine, or neutralization, using the monoclonal antibody 5E1, resulted in contraction of the CSC compartment, whereas exogenous Hh ligand caused expansion. 78 In addition, using LDE225 to target canonical signaling, and Forskolin a GLI-1 modulating compound, thereby bypassing PTCH1 and SMO and targeting non-canonical signaling, investigators have shown both mechanisms are amenable to therapeutic intervention in vitro in cell lines and patient samples. 56 Supporting evidence, using LDE225, confirmed differentiation was significantly induced and de-differentiation blocked in both cell lines and primary cells in vitro. 102 Moreover, there was a marked discrepancy in Hh pathway component expression, activity, and cyclopamine sensitivity between the stem cell and differentiated cell compartments. 78 Interestingly, Hh pathway gene expression was significantly altered in vivo suggesting the cell microenvironment markedly affects expression of Hh pathway components. 78
T-cell disorders
T-cell disorders, characterized by the abnormal proliferation of T-cell precursors, are generally aggressive, and far rarer than B-cell disorders. Interestingly, in comparison to B-ALL and B-cell lymphomas there are significant similarities between T-ALL and T-cell lymphomas; many arguing these entities are variations of the same disease, the two conditions often being treated in the same way. 103
The Hh pathway has been shown to be important in T-cell development; 104 it has also been implicated in the etiology of T-cell malignancies. In ALK + anaplastic large cell lymphoma cell lines and primary tissue, SHH was amplified. In addition, GLI-1 expression at both the gene and protein level was increased; both SHH and GLI1 expression being influenced by the PI3K/AKT pathway although the mechanism remains unclear. 105 Supporting the importance of these components in cell survival, inhibition of SHH signaling with cyclopamine or silencing GLI-1 with ribonucleic acid interference resulted in cell cycle arrest and apoptosis. 105
Epigenetic modifications of the Hh pathway
Epigenetic modifications are stable, heritable alterations in gene expression caused without a change in coding DNA but rather DNA methylation and histone modification. 106 The bromodomain and extra-terminal domain (BET) protein family, responsible for reading differentially acetylated histones and thereby communicating changes in transcription potential, comprises four distinct genes, BRD2, -3, and -4 which are ubiquitously expressed and BRDT which is restricted to the testes. Importantly, there is increasing evidence to support the epigenetic modification of components of the Hh pathway. Work has predominantly focused on neural tissue and medulloblastoma, finding GLI-1 and GLI-2, but not GLI-3, to be acetylated, with a careful regulatory balance controlling GLI acetylation, and thereby transcriptional activity. 107 Transcriptional activity occurs through histone deacetylation, whilst the binding of REN KCTD11 , an endogenous histone deacetylase inhibitor, to SCF (Β-TrCP) 3-like E3 ubiquitin ligase complex, promotes GLI acetylation inhibiting transcription. 108 Recent work has shown BET proteins to further modulate GLI transcription and thereby pathway activity. 109 Targeting BRD4 with JQ1, a small-molecule bromodomain inhibitor, preventing BRD4 acetyl-lysine binding resulted in a global down-regulation of GLI associated genes in both patient and genetically engineered mouse model-derived Hh-driven tumors. 109 It remains to be seen whether this will translate into clinical practice. Further, how epigenetic modification of other components of the pathway will affect Hh antagonism in clinical practice and whether these too will be amenable to manipulation remains to be investigated.
Targeting the Hh pathway in hematological malignancies
There is increasing evidence to support the role of the Hh signaling pathway in hematological malignancies, with components representing viable therapeutic targets, making it a very attractive potential treatment strategy. Further, from a drug development perspective, inhibition of SMO and GLI-1 can be readily achieved, Table 1 .
The rationale for Hh inhibition in hematological malignancies is to target the LSC population, eradicating these therapy resistant cells, potentially affecting a cure. However, whilst there is evidence that the Hh pathway is important for LSC maintenance and expansion in CML and MM, its role in other diseases such as AML, MF, CLL, and lymphoma appears more complex, representing a delicate balance between the diseased cells and the stromal microenvironment. Whether Hh antagonism will be therapeutically useful in hematologic malignancies not only depends on its ability to target the diseased cells but also on the anticipated level of toxicity, both hematologic and systemic. To date, there appears to be little hematologic toxicity reported, particularly in solid tumor studies. 130
Whilst experimental data suggest Hh antagonism may address the persistence of CSC and the protective effect of the tumor microenvironment in both myeloid and lymphoid malignancies, clinical trials are only in the early stages, Table 2 . Interestingly, it is only the SMO antagonists which are currently being trialed clinically. Excitingly however, several of these clinical grade SMO inhibitors are now in Phase II trial; with a number of Phase III trials in solid tumors and the licensing of vismodegib for use in basal cell carcinoma in 2012 hinting these early results could translate into clinical practice.
In hematological malignancies, results of a Phase Ia study using PF-04449913 in refractory, resistant, or intolerant myeloid malignancies were presented in 2011. 131 Thirty-two patients were enrolled, with early indications of efficacy seen across all diseases. 131 Notably, one patient with AML achieved a complete remission albeit with incomplete blood recovery and five had a >50% reduction in bone marrow blast counts. Additionally, one patient with MF achieved a >50% reduction in extramedullary disease and five attained stable disease. Results of the current Phase II trials are eagerly awaited.
Conclusion
Deregulated expression of components of the Hh signaling pathway is found throughout the huge range of hematological malignancies; the pathway appearing to either determine CSC behavior directly or indirectly via the stromal microenvironment.
As our knowledge and understanding of the Hh and other conserved embryonic pathways in hematologic malignancies increases, our ability to manipulate and potentially use these as therapeutic targets can only increase. Further, the sheer number of compounds coming onto the market, targeting both canonical and non-canonical mechanisms and the array of clinical trials, makes this an exciting time looking to tailor therapy and minimize treatment-related side effects.
Disclosure
M Copland has received research funding from Bristol-Myers Squibb and Novartis, honoraria from/attended Advisory Boards for Bristol-Myers Squibb, Pfizer, Ariad and Novartis, Bristol-Myers Squibb and Pfizer and travel funding from Bristol-Myers Squibb and Novartis. V Campbell is funded by the Wellcome Trust via the STMTI programme. M Copland is funded by Leukaemia and Lymphoma Research (Grant No: 11017). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in this manuscript apart from those discussed.
References
1 Nguyen LV Vanner R Dirks P Eaves CJ Cancer stem cells: an evolving concept Nat Rev Cancer 2012 12 2 133 143 22237392 10.1038/nrc3184
2 Copley MR Beer PA Eaves CJ Hematopoietic stem cell heterogeneity takes center stage Cell Stem Cell 2012 10 6 690 697 22704509 10.1016/j.stem.2012.05.006
3 Zon LI Intrinsic and extrinsic control of haematopoietic stem-cell self-renewal Nature 2008 453 7193 306 313 18480811 10.1038/nature07038
4 Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell Nat Med 1997 3 7 730 737 9212098 10.1038/nm0797-730
5 Goardon N Marchi E Atzberger A Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia Cancer Cell 2011 19 1 138 152 21251617 10.1016/j.ccr.2010.12.012
6 Collins AT Berry PA Hyde C Stower MJ Maitland NJ Prospective identification of tumorigenic prostate cancer stem cells Cancer Res 2005 65 23 10946 10951 16322242 10.1158/0008-5472.CAN-05-2018
7 Buglino JA Resh MD Palmitoylation of hedgehog proteins Vitam Horm 2012 88 229 252 22391306 10.1016/B978-0-12-394622-5.00010-9 PMC4214369
8 O’Flaherty JD Barr M Fennell D The cancer stem-cell hypothesis: its emerging role in lung cancer biology and its relevance for future therapy J Thorac Oncol 2012 7 12 1880 1890 23154562 10.1097/JTO.0b013e31826bfbc6
9 Singh SK Clarke ID Terasaki M Identification of a cancer stem cell in human brain tumors Cancer Res 2003 63 18 5821 5828 14522905
10 Valent P Bonnet D De Maria R Cancer stem cell definitions and terminology: the devil is in the details Nat Rev Cancer 2012 12 11 767 775 23051844 10.1038/nrc3368
11 Wang JC Dick JE Cancer stem cells: lessons from leukemia Trends Cell Biol 2005 15 9 494 501 16084092 10.1016/j.tcb.2005.07.004
12 Dick JE Stem cell concepts renew cancer research Blood 2008 112 13 4793 4807 19064739 10.1182/blood-2008-08-077941
13 Clevers H The cancer stem cell: premises, promises and challenges Nat Med 2011 17 3 313 319 21386835 10.1038/nm.2304
14 Reya T Morrison SJ Clarke MF Weissman IL Stem cells, cancer, and cancer stem cells Nature 2001 414 6859 105 111 11689955 10.1038/35102167
15 Alison MR Lim SM Nicholson LJ Cancer stem cells: problems for therapy? J Pathol 2011 223 2 147 161 21125672 10.1002/path.2793
16 Nusslein-Volhard C Wieschaus E Mutations affecting segment number and polarity in Drosophila Nature 1980 287 5785 795 801 6776413 10.1038/287795a0
17 Ingham PW McMahon AP Hedgehog signaling in animal development: paradigms and principles Genes Dev 2001 15 23 3059 3087 11731473 10.1101/gad.938601
18 Katoh Y Katoh M Hedgehog target genes: mechanisms of carcinogenesis induced by aberrant hedgehog signaling activation Curr Mol Med 2009 9 7 873 886 19860666 10.2174/156652409789105570
19 Ingham PW Placzek M Orchestrating ontogenesis: variations on a theme by sonic hedgehog Nat Rev Genet 2006 7 11 841 850 17047684 10.1038/nrg1969
20 Cridland SO Keys JR Papathanasiou P Perkins AC Indian hedgehog supports definitive erythropoiesis Blood Cells Mol Dis 2009 43 2 149 155 19443245 10.1016/j.bcmd.2009.04.004
21 Bitgood MJ Shen L McMahon AP Sertoli cell signaling by Desert hedgehog regulates the male germline Curr Biol 1996 6 3 298 304 8805249 10.1016/s0960-9822(02)00480-3
22 Lee JJ Ekker SC von Kessler DP Autoproteolysis in hedgehog protein biogenesis Science 1994 266 5190 1528 1537 7985023 10.1126/science.7985023
23 Buglino JA Resh MD Hhat is a palmitoylacyltransferase with specificity for N-palmitoylation of Sonic Hedgehog J Biol Chem 2008 283 32 22076 22088 18534984 10.1074/jbc.M803901200 PMC2494920
24 Porter JA von Kessler DP Ekker SC The product of hedgehog autoproteolytic cleavage active in local and long-range signalling Nature 1995 374 6520 363 366 7885476 10.1038/374363a0
25 Rohatgi R Milenkovic L Scott MP Patched1 regulates hedgehog signaling at the primary cilium Science 2007 317 5836 372 376 17641202 10.1126/science.1139740
26 Taipale J Cooper MK Maiti T Beachy PA Patched acts catalytically to suppress the activity of Smoothened Nature 2002 418 6900 892 897 12192414 10.1038/nature00989
27 Bhardwaj G Murdoch B Wu D Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation Nat Immunol 2001 2 2 172 180 11175816 10.1038/84282
28 Sasaki H Nishizaki Y Hui C Nakafuku M Kondoh H Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and Gli3 as primary mediators of Shh signaling Development 1999 126 17 3915 3924 10433919 10.1242/dev.126.17.3915
29 Ruiz i Altaba A Mas C Stecca B The Gli code: an information nexus regulating cell fate, stemness and cancer Trends Cell Biol 2007 17 9 438 447 17845852 10.1016/j.tcb.2007.06.007 PMC2601665
30 Carpenter D Stone DM Brush J Characterization of two patched receptors for the vertebrate hedgehog protein family Proc Natl Acad Sci U S A 1998 95 23 13630 13634 9811851 10.1073/pnas.95.23.13630 PMC24870
31 Rahnama F Toftgard R Zaphiropoulos PG Distinct roles of PTCH2 splice variants in Hedgehog signalling Biochem J 2004 378 Pt 2 325 334 14613484 10.1042/BJ20031200 PMC1223965
32 Chuang PT McMahon AP Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein Nature 1999 397 6720 617 621 10050855 10.1038/17611
33 Kawakami T Kawcak T Li YJ Mouse dispatched mutants fail to distribute hedgehog proteins and are defective in hedgehog signaling Development 2002 129 24 5753 5765 12421714 10.1242/dev.00178
34 Chen MH Wilson CW Li YJ Cilium-independent regulation of Gli protein function by Sufu in Hedgehog signaling is evolutionarily conserved Genes Dev 2009 23 16 1910 1928 19684112 10.1101/gad.1794109 PMC2725943
35 Irvine DA Copland M Targeting hedgehog in hematologic malignancy Blood 2012 119 10 2196 2204 22223823 10.1182/blood-2011-10-383752
36 Jenkins D Hedgehog signalling: emerging evidence for non-canonical pathways Cellular Signal 2009 21 7 1023 1034 10.1016/j.cellsig.2009.01.033 19399989
37 Reed JC Bcl-2 family proteins: strategies for overcoming chemoresistance in cancer Adv Pharmacol 1997 41 501 532 9204157 10.1016/s1054-3589(08)61070-4
38 Ling V Charles F Kettering Prize. P-glycoprotein and resistance to anticancer drugs Cancer 1992 69 10 2603 2609 1348966 10.1002/1097-0142(19920515)69:10<2603::aid-cncr2820691034>3.0.co;2-e
39 Kurinna S Konopleva M Palla SL Bcl2 phosphorylation and active PKC alpha are associated with poor survival in AML Leukemia 2006 20 7 1316 1319 16642043 10.1038/sj.leu.2404248
40 Keller G Lacaud G Robertson S Development of the hematopoietic system in the mouse Exp Hematol 1999 27 5 777 787 10340392 10.1016/s0301-472x(99)00024-7
41 Trowbridge JJ Scott MP Bhatia M Hedgehog modulates cell cycle regulators in stem cells to control hematopoietic regeneration Proc Natl Acad Sci U S A 2006 103 38 14134 14139 16968775 10.1073/pnas.0604568103 PMC1599924
42 Gao J Graves S Koch U Hedgehog signaling is dispensable for adult hematopoietic stem cell function Cell Stem Cell 2009 4 6 548 558 19497283 10.1016/j.stem.2009.03.015 PMC2914688
43 Dierks C Beigi R Guo GR Expansion of Bcr-Abl-positive leukemic stem cells is dependent on Hedgehog pathway activation Cancer Cell 2008 14 3 238 249 18772113 10.1016/j.ccr.2008.08.003
44 Cohen DJ Targeting the hedgehog pathway: role in cancer and clinical implications of its inhibition Hematol Oncol Clin North Am 2012 26 3 565 588 22520980 10.1016/j.hoc.2012.01.005
45 Hofmann I Stover EH Cullen DE Hedgehog signaling is dispensable for adult murine hematopoietic stem cell function and hematopoiesis Cell Stem Cell 2009 4 6 559 567 19497284 10.1016/j.stem.2009.03.016 PMC3065323
46 Xie J Murone M Luoh SM Activating Smoothened mutations in sporadic basal-cell carcinoma Nature 1998 391 6662 90 92 9422511 10.1038/34201
47 Berman DM Karhadkar SS Hallahan AR Medulloblastoma growth inhibition by hedgehog pathway blockade Science 2002 297 5586 1559 1561 12202832 10.1126/science.1073733
48 Thayer SP di Magliano MP Heiser PW Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis Nature 2003 425 6960 851 856 14520413 10.1038/nature02009 PMC3688051
49 Park KS Martelotto LG Peifer M A crucial requirement for Hedgehog signaling in small cell lung cancer Nat Med 2011 17 11 1504 1508 21983857 10.1038/nm.2473 PMC3380617
50 Martin ST Sato N Dhara S Aberrant methylation of the Human Hedgehog interacting protein (HHIP) gene in pancreatic neoplasms Cancer Biol Ther 2005 4 7 728 733 15970691 10.4161/cbt.4.7.1802
51 Slade I Murray A Hanks S Heterogeneity of familial medulloblastoma and contribution of germline PTCH1 and SUFU mutations to sporadic medulloblastoma Fam Cancer 2011 10 2 337 342 21188540 10.1007/s10689-010-9411-0
52 Shahi MH Lorente A Castresana JS Hedgehog signalling in medulloblastoma, glioblastoma and neuroblastoma Oncol Rep 2008 19 3 681 688 18288402
53 Car D Sabol M Musani V Ozretic P Levanat S Epigenetic regulation of the Hedgehog-Gli signaling pathway in cancer Period Biol 2010 112 4 419 423
54 Ochs M Farrar J Considine M Genome wide promoter methylation patterns predict AML subtype outcomes and identify novel pathways characterizing diagnostic and relapsed disease in children Presented at: 54th ASH Annual Meeting and Exposition December 8–11, 2012 Atlanta. GA, USA
55 Scales SJ de Sauvage FJ Mechanisms of Hedgehog pathway activation in cancer and implications for therapy Trends Pharmacol Sci 2009 30 6 303 312 19443052 10.1016/j.tips.2009.03.007
56 Blotta S Jakubikova J Calimeri T Canonical and noncanonical Hedgehog pathway in the pathogenesis of multiple myeloma Blood 2012 120 25 5002 5013 22821765 10.1182/blood-2011-07-368142 PMC3525024
57 Karhadkar SS Bova GS Abdallah N Hedgehog signalling in prostate regeneration, neoplasia and metastasis Nature 2004 431 7009 707 712 15361885 10.1038/nature02962
58 Watkins DN Berman DM Burkholder SG Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer Nature 2003 422 6929 313 317 12629553 10.1038/nature01493
59 Dierks C Grbic J Zirlik K Essential role of stromally induced hedgehog signaling in B-cell malignancies Nat Med 2007 13 8 944 951 17632527 10.1038/nm1614
60 Theunissen JW de Sauvage FJ Paracrine Hedgehog signaling in cancer Cancer Res 2009 69 15 6007 6010 19638582 10.1158/0008-5472.CAN-09-0756
61 Zhao C Chen A Jamieson CH Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia Nature 2009 458 7239 776 779 19169242 10.1038/nature07737 PMC2946231
62 Kobune M Takimoto R Murase K Drug resistance is dramatically restored by hedgehog inhibitors in CD34+ leukemic cells Cancer Sci 2009 100 5 948 955 19245435 10.1111/j.1349-7006.2009.01111.x PMC11158794
63 Kobune M Kato J Kawano Y Adenoviral vector-mediated transfer of the Indian hedgehog gene modulates lymphomyelopoiesis in vivo Stem Cells 2008 26 2 534 542 17962696 10.1634/stemcells.2007-0741
64 Kobune M Ito Y Kawano Y Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID-beta2m−/− repopulating cells Blood 2004 104 4 1002 1009 15105288 10.1182/blood-2003-09-3347
65 Deininger MW Goldman JM Melo JV The molecular biology of chronic myeloid leukemia Blood 2000 96 10 3343 3356 11071626
66 Hochhaus A Hughes T Clinical resistance to imatinib: mechanisms and implications Hematol Oncol Clin North Am 2004 18 3 641 656 15271397 10.1016/j.hoc.2004.03.001
67 Hochhaus A O’Brien SG Guilhot F Six-year follow-up of patients receiving imatinib for the first-line treatment of chronic myeloid leukemia Leukemia 2009 23 6 1054 1061 19282833 10.1038/leu.2009.38
68 Hamilton A Helgason GV Schemionek M Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival Blood 2012 119 6 1501 1510 22184410 10.1182/blood-2010-12-326843 PMC3286213
69 Irvine DA Zhang B Allan EK Combination of the hedgehog pathway inhibitor LDE225 and nilotinib eliminates chronic myeloid leukemia stem and progenitor cells Presented at: 51st ASH Annual Meeting and Exposition December 5–8, 2009 New Orleans, LA
70 Okabe S Tauchi T Tanaka Y Katagiri S Ohyashiki K Effects of the hedgehog inhibitor GDC-0449, alone or in combination with dasatinib, on BCR-ABL-positive leukemia Cells Stem Cells Dev 2012 21 16 2939 2948 22642671 10.1089/scd.2012.0016
71 Alonso-Dominguez JM Grinfeld J Alikian M PTCH1 expression at diagnosis reliably predicts treatment failure in imatinib-treated chronic myeloid leukaemia patients Presented at: 54th ASH Annual Meeting and Exposition December 8–11, 2012 Atlanta, GA
72 Lapidot T Sirard C Vormoor J A cell initiating human acute myeloid leukaemia after transplantation into SCID mice Nature 1994 367 6464 645 648 7509044 10.1038/367645a0
73 Bai LY Chiu CF Lin CW Differential expression of Sonic hedgehog and Gli1 in hematological malignancies Leukemia 2008 22 1 226 228 17928882 10.1038/sj.leu.2404978
74 Zahreddine HA Culjkovic-Kraljacic B Assouline S The sonic hedgehog factor GLI1 imparts drug resistance through inducible glucuronidation Nature 2014 511 7507 90 93 24870236 10.1038/nature13283 PMC4138053
75 Campbell V Tholouli E Quigley MT Evidence that activated hedgehog signaling predicts for poor clinical outcome in acute myeloid leukemia Presented at: 54th ASH Annual Meeting and Exposition December 8–11, 2012 Atlanta, GA
76 Fukushima N Minami Y Hayakawa F Treatment with hedgehog inhibitor, PF-04449913, attenuates leukemia-initiation potential in acute myeloid leukemia cells Presented at: 55th ASH Annual Meeting and Exposition December 7–10, 2013 New Orleans, LA
77 Xavier JM Duarte ASS Pericole FV Hedgehog pathway is deregulated in myelodysplastic syndrome progenitor bone marrow cells Presented at: 55th ASH Annual Meeting and Exposition December 7–10, 2013 New Orleans, LA
78 Peacock CD Wang Q Gesell GS Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma Proc Natl Acad Sci U S A 2007 104 10 4048 4053 17360475 10.1073/pnas.0611682104 PMC1805487
79 Kobune M Iyama S Kikuchi S Stromal cells expressing hedgehog-interacting protein regulate the proliferation of myeloid neoplasms Blood Cancer J 2012 2 e87 22961059 10.1038/bcj.2012.36 PMC3461706
80 Levine RL Wadleigh M Cools J Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis Cancer Cell 2005 7 4 387 397 15837627 10.1016/j.ccr.2005.03.023
81 Klampfl T Gisslinger H Harutyunyan AS Somatic mutations of calreticulin in myeloproliferative neoplasms N Engl J Med 2013 369 25 2379 2390 24325356 10.1056/NEJMoa1311347
82 Bhagwat N Keller MD Rampal RK Improved efficacy of combination of JAK2 and hedgehog inhibitors in myelofibrosis Presented at: 55th ASH Annual Meeting and Exposition December 7–10, 2013 New Orleans, LA
83 Zingariello M Martelli F Ciaffoni F Characterization of the TGF-beta1 signaling abnormalities in the Gata1low mouse model of myelofibrosis Blood 2013 121 17 3345 3363 23462118 10.1182/blood-2012-06-439661 PMC3637011
84 Sasaki KG Gotlib JR Mesa RA A phase 2 study of IPI-926, an oral hedgehog inhibitor, in patients with myelofibrosis J Clin Oncol 2014 32 5s (suppl; abstr 7111) 10.3109/10428194.2014.984703 PMC4919663 25641433
85 Bernt KM Armstrong SA Leukemia stem cells and human acute lymphoblastic leukemia Semin Hematol 2009 46 1 33 38 19100366 10.1053/j.seminhematol.2008.09.010 PMC4031465
86 le Viseur C Hotfilder M Bomken S In childhood acute lymphoblastic leukemia, blasts at different stages of immunophenotypic maturation have stem cell properties Cancer Cell 2008 14 1 47 58 18598943 10.1016/j.ccr.2008.05.015 PMC2572185
87 Lang F Badura S Ruthardt M Rieger MA Ottmann OG Modulation of leukemic stem cell self-renewal and cell fate decisions by inhibition of hedgehog signalling in human acute lymphoblastic leukemia (ALL) Presented at: 54th ASH Annual Meeting and Exposition December 8–11, 2012 Atlanta, GA
88 Lin TL Wang QH Brown P Self-renewal of acute lymphocytic leukemia cells is limited by the Hedgehog pathway inhibitors cyclopamine and IPI-926 PloS One 2010 5 12 e15262 21203400 10.1371/journal.pone.0015262 PMC3011010
89 Hegde GV Peterson KJ Emanuel K Hedgehog-induced survival of B-cell chronic lymphocytic leukemia cells in a stromal cell microenvironment: a potential new therapeutic target Mol Cancer Res 2008 6 12 1928 1936 19074837 10.1158/1541-7786.MCR-08-0142
90 Decker S Zirlik K Djebatchie L Trisomy 12 and elevated GLI1 and PTCH1 transcript levels are biomarkers for Hedgehog-inhibitor responsiveness in CLL Blood 2012 119 4 997 1007 22130798 10.1182/blood-2011-06-359075
91 Wang C Lu K Wang X GLI1 Inhibitor GANT61 Deregulates stat3 Phosphorylation In Chronic Lymphocytic Leukemia Cells Blood 2013 122 21
92 Desch P Asslaber D Kern D Inhibition of GLI, but not Smoothened, induces apoptosis in chronic lymphocytic leukemia cells Oncogene 2010 29 35 4885 4895 20603613 10.1038/onc.2010.243
93 Patole SP Patole AS Rhen DS Patterned carbon nanotube growth using an electron beam sensitive direct writable catalyst Nanotechnology 2009 20 31 315302 19597250 10.1088/0957-4484/20/31/315302
94 Singh RR Kim JE Davuluri Y Hedgehog signaling pathway is activated in diffuse large B-cell lymphoma and contributes to tumor cell survival and proliferation Leukemia 2010 24 5 1025 1036 20200556 10.1038/leu.2010.35
95 Agarwal NK Qu CJ Kunkalla K Liu YD Vega F GLI1 directly regulates the transcription of AKT genes in diffuse large B-cell lymphoma Blood 2012 120 21 Presented at: 54th ASH Annual Meeting and Exposition December 8–11, 2012 Atlanta, GA, USA
96 Qu CJ Liu YD Kunkalla K Agarwal NK Vega F Smoothened (SMO) activates NF-Kb pathway through activation of PKC beta/CARMA1 and TRAF6 stabilization in diffuse large B-cell lymphoma Blood 2012 120 21 Presented at: 54th ASH Annual Meeting and Exposition December 8–11, 2012 Atlanta, GA, USA
97 Hegde GV Nordgren TM Munger CM Novel therapy for therapy-resistant mantle cell lymphoma: multipronged approach with targeting of hedgehog signaling Int J Cancer 2012 131 12 2951 2960 22511234 10.1002/ijc.27602
98 Hegde GV Munger CM Emanuel K Targeting of sonic hedgehog-GLI signaling: a potential strategy to improve therapy for mantle cell lymphoma Mol Cancer Ther 2008 7 6 1450 1460 18524848 10.1158/1535-7163.MCT-07-2118
99 Amarsaikhan N Dennison JR Boi SK Neil MS Elsawa SF GLI transcription factors modulate IgM secretion in waldenstrom macroglobulinemia Blood 2013 122 21
100 Greaves WO Kim JE Singh RR Glioma-associated oncogene homologue 3, a hedgehog transcription factor, is highly expressed in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma Hum Pathol 2011 42 11 1643 1652 21531006 10.1016/j.humpath.2010.12.023 PMC3165071
101 Hager-Theodorides AL Dessens JT Outram SV Crompton T The transcription factor Gli3 regulates differentiation of fetal CD4- CD8-double-negative thymocytes Blood 2005 106 4 1296 1304 15855276 10.1182/blood-2005-03-0998 PMC1274277
102 Yang SB D R Loh YS Blockade of the hedgehog signaling pathway by the novel agent NVP-LDE225 induces differentiation, prevents de-differentiation and inhibits proliferation of multiple myeloma stem cells in vitro Blood 2012 120 120 Presented at: 54th ASH Annual Meeting and Exposition December 8–11, 2012 Atlanta, GA, USA
103 Hoelzer D Gokbuget N T-cell lymphoblastic lymphoma and T-cell acute lymphoblastic leukemia: a separate entity? Clin Lymphoma Myeloma 2009 9 Suppl 3 S214 S221 19778844 10.3816/CLM.2009.s.015
104 El Andaloussi A Graves S Meng F Hedgehog signaling controls thymocyte progenitor homeostasis and differentiation in the thymus Nat Immunol 2006 7 4 418 426 16518394 10.1038/ni1313
105 Singh RR Cho-Vega JH Davuluri Y Sonic hedgehog signaling pathway is activated in ALK-positive anaplastic large cell lymphoma Cancer Res 2009 69 6 2550 2558 19244133 10.1158/0008-5472.CAN-08-1808
106 Shih AH Abdel-Wahab O Patel JP Levine RL The role of mutations in epigenetic regulators in myeloid malignancies Nat Rev Cancer 2012 12 9 599 612 22898539 10.1038/nrc3343
107 Canettieri G Di Marcotullio L Coni S Greco A Gulino A Turning off the switch in medulloblastoma: the inhibitory acetylation of an oncogene Cell Cycle 2010 9 11 2047 2048 20505339 10.4161/cc.9.11.11860
108 Canettieri G Di Marcotullio L Greco A Histone deacetylase and Cullin3-REN(KCTD11) ubiquitin ligase interplay regulates Hedgehog signalling through Gli acetylation Nat Cell Biol 2010 12 2 132 142 20081843 10.1038/ncb2013
109 Tang Y Gholamin S Schubert S Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition Nat Med 2014 20 7 732 740 24973920 10.1038/nm.3613 PMC4108909
110 Kim J Lee JJ Gardner D Beachy PA Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector Proc Natl Acad Sci U S A 2010 107 30 13432 13437 20624968 10.1073/pnas.1006822107 PMC2922148
111 Cooper MK Porter JA Young KE Beachy PA Teratogen-mediated inhibition of target tissue response to Shh signaling Science 1998 280 5369 1603 1607 9616123 10.1126/science.280.5369.1603
112 Firestone AJ Weinger JS Maldonado M Small-molecule inhibitors of the AAA plus ATPase motor cytoplasmic dynein Nature 2012 484 7392 125 129 22425997 10.1038/nature10936 PMC3321072
113 Chen JK Taipale J Cooper MK Beachy PA Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened Genes Dev 2002 16 21 2743 2748 12414725 10.1101/gad.1025302 PMC187469
114 Lauth M Bergstrom A Shimokawa T Toftgard R Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists Proc Natl Acad Sci U S A 2007 104 20 8455 8460 17494766 10.1073/pnas.0609699104 PMC1866313
115 Hyman JM Firestone AJ Heine VM Small-molecule inhibitors reveal multiple strategies for Hedgehog pathway blockade Proc Natl Acad Sci U S A 2009 106 33 14132 14137 19666565 10.1073/pnas.0907134106 PMC2721821
116 Kim J Tang JY Gong R Itraconazole, a commonly used antifungal that inhibits Hedgehog pathway activity and cancer growth Cancer Cell 2010 17 4 388 399 20385363 10.1016/j.ccr.2010.02.027 PMC4039177
117 Lee J Wu X Pasca di Magliano M A small-molecule antagonist of the hedgehog signaling pathway Chembiochem 2007 8 16 1916 1919 17886323 10.1002/cbic.200700403
118 Manetti F Faure H Roudaut H Virtual screening-based discovery and mechanistic characterization of the acylthiourea MRT-10 family as smoothened antagonists Mol Pharmacol 2010 78 4 658 665 20664000 10.1124/mol.110.065102
119 Rohner A Spilker ME Lam JL Effective targeting of hedgehog signaling in a medulloblastoma model with PF-5274857, a potent and selective smoothened antagonist that penetrates the blood-brain barrier Mol Cancer Ther 2012 11 1 57 65 22084163 10.1158/1535-7163.MCT-11-0691
120 Stanton BZ Peng LF Small-molecule modulators of the Sonic Hedgehog signaling pathway Mol Biosyst 2010 6 1 44 54 20024066 10.1039/b910196a
121 Petrova E Rios-Esteves J Ouerfelli O Glickman JF Resh MD Inhibitors of Hedgehog acyltransferase block Sonic Hedgehog signaling Nat Chem Biol 2013 9 4 247 249 23416332 10.1038/nchembio.1184 PMC3604071
122 Chen JK Taipale J Young KE Maiti T Beachy PA Small molecule modulation of Smoothened activity Proc Natl Acad Sci U S A 2002 99 22 14071 14076 12391318 10.1073/pnas.182542899 PMC137838
123 Wang Y Arvanites AC Davidow L Selective identification of hedgehog pathway antagonists by direct analysis of smoothened ciliary translocation ACS Chem Biol 2012 7 6 1040 1048 22554036 10.1021/cb300028a PMC3905677
124 Incardona JP Gaffield W Lange Y Cyclopamine inhibition of sonic hedgehog signal transduction is not mediated through effects on cholesterol transport Developmental Biology 2000 224 2 440 452 10926779 10.1006/dbio.2000.9775
125 Gendreau SB Hawkins D Ho C Preclinical characterization of BMS-833923 (XL139), a hedgehog (HH) pathway inhibitor in early clinical development Mol Cancer Ther 2009 8 B192
126 Wong H Alicke B West KA Pharmacokinetic-pharmacodynamic analysis of vismodegib in preclinical models of mutational and ligand-dependent Hedgehog pathway activation Clin Cancer Res 2011 17 14 4682 4692 21610148 10.1158/1078-0432.CCR-11-0975
127 Peluso MO Campbell VT Harari JA Impact of the smoothened inhibitor, IPI-926, on smoothened ciliary localization and hedgehog pathway activity PloS One 2014 9 3 e90534 24608250 10.1371/journal.pone.0090534 PMC3946503
128 Pan SF Wu X Jiang JQ Discovery of NVP-LDE225, a potent and selective smoothened antagonist ACS Med Chem Lett 2010 1 3 130 134 24900187 10.1021/ml1000307 PMC4007689
129 Munchhof MJ Li Q Shavnya A Discovery of PF-04449913, a potent and orally bioavailable inhibitor of smoothened ACS Med Chem Lett 2012 3 2 106 111 24900436 10.1021/ml2002423 PMC4025788
130 Ohashi T Oguro Y Tanaka T Discovery of the investigational drug TAK-441, a pyrrolo[3,2-c]pyridine derivative, as a highly potent and orally active hedgehog signaling inhibitor: Modification of the core skeleton for improved solubility Bioorg Med Chem 2012 20 18 5507 5517 22898254 10.1016/j.bmc.2012.07.034
131 Jamieson C Cortes JE Oehler V Phase 1 dose-escalation study of PF-04449913, an oral hedgehog (Hh) inhibitor, in patients with select hematologic malignancies Blood 2011 118 21 195 196 Presented at: 53rd ASH Annual Meeting and Exposition December 10–13, 2011 San Diego, CA, USA