Targeting GLI1 and GLI2 with small molecule inhibitors to suppress GLI-dependent transcription and tumor growth.

Aberrant activation of Hedgehog (HH) signaling in cancer is the result of genetic alterations of upstream pathway components (canonical) or other oncogenic mechanisms (noncanonical), that ultimately concur to activate the zinc-finger transcription factors GLI1 and GLI2. Therefore, inhibition of GLI activity is a good therapeutic option to suppress both canonical and noncanonical activation of the HH pathway. However, only a few GLI inhibitors are available, and none of them have the profile required for clinical development due to poor metabolic stability and aqueous solubility, and high hydrophobicity. Two promising quinoline inhibitors of GLI were selected by virtual screening and subjected to hit-to-lead optimization, thus leading to the identification of the 4-methoxy-8-hydroxyquinoline derivative JC19. This molecule impaired GLI1 and GLI2 activities in several cellular models interfering with the binding of GLI1 and GLI2 to DNA. JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in several cancer cell lines in vitro. Specificity towards GLI1 and GLI2 was demonstrated by lower activity of JC19 in GLI1- or GLI2-depleted cancer cells. JC19 showed excellent metabolic stability and high passive permeability. Notably, JC19 inhibited GLI1-dependent melanoma xenograft growth in vivo, with no evidence of toxic effects in mice. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2.

ERK2 MAP kinase regulates SUFU binding by multisite phosphorylation of GLI1.

Crosstalk between the Hedgehog and MAPK signaling pathways occurs in several types of cancer and contributes to clinical resistance to Hedgehog pathway inhibitors. Here we show that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. ERK2 phosphorylates GLI1 on three evolutionarily conserved target sites (S102, S116, and S130) located near the high-affinity binding site for SUFU; these phosphorylations cooperate to weaken the affinity of GLI1-SUFU binding by over 25-fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily conserved SUFU affinity-modulating region. In cultured mammalian cells, GLI1 variants containing phosphomimetic substitutions of S102, S116, and S130 displayed an increased ability to drive transcription. We conclude that multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.

GLI1-targeting drugs induce replication stress and homologous recombination deficiency and synergize with PARP-targeted therapies in triple negative breast cancer cells.

Triple negative breast cancer (TNBC), an aggressive and highly metastatic subtype of breast cancer. Glioma-associated oncogene 1 (GLI1) is a transcription factor and effector of the Hedgehog (Hh) signaling pathway, and is predictive of poor survival for TNBC patients. A nanostring DNA Damage Response (DDR) mRNA panel was used to identify GLI1-induced regulation of DDR genes. Western blots, immunohistochemistry and immunofluorescence were used to evaluate protein expression. Colony assays and mammosphere formation assays were utilized to assess survival of cancer cells. Flow cytometry analyses were employed to evaluate changes in the cell cycle profile, and DNA fiber assays were used to analyze alterations in replication dynamics in TNBC cells. The UALCAN portal and Ensemble programs were used for computational analysis of TCGA data. CompuSyn software was used to calculate combination index (CI) values to assess synergism in drug combination experiments. Inhibition of GLI1 in TNBC cells transcriptionally downregulate expression of FANCD2 and its foci formation, and causes a homologous recombination repair (HR) deficiency. As HR-deficient cancer cells are sensitive to PARP-targeted therapies, we evaluated a combination of the GLI1 inhibitor, GANT61, and a PARP inhibitor (olaparib) in TNBC cells. Combination of GANT61 and olaparib elevated DNA damage levels and these drug combinations caused synergistic lethality to TNBC cells. Aberrantly activated GLI1 regulates HR-mediated DNA repair by transcriptionally regulating FANCD2 to overcome chemotherapy-induced replication stress and DNA damage, and it contributes to resistance of TNBC cells to therapeutics.

Targeting CD99 Compromises the Oncogenic Effects of the Chimera EWS-FLI1 by Inducing Reexpression of Zyxin and Inhibition of GLI1 Activity.

Ewing sarcoma, a highly aggressive pediatric tumor, is driven by EWS-FLI1, an oncogenic transcription factor that remodels the tumor genetic landscape. Epigenetic mechanisms play a pivotal role in Ewing sarcoma pathogenesis, and the therapeutic value of compounds targeting epigenetic pathways is being identified in preclinical models. Here, we showed that modulation of CD99, a cell surface molecule highly expressed in Ewing sarcoma cells, may alter transcriptional dysregulation in Ewing sarcoma through control of the zyxin-GLI1 axis. Zyxin is transcriptionally repressed, but GLI1 expression is maintained by EWS-FLI1. We demonstrated that targeting CD99 with antibodies, including the human diabody C7, or genetically inhibiting CD99 is sufficient to increase zyxin expression and induce its dynamic nuclear accumulation. Nuclear zyxin functionally affects GLI1, inhibiting targets such as NKX2-2, cyclin D1, and PTCH1 and upregulating GAS1, a tumor suppressor protein negatively regulated by SHH/GLI1 signaling. We used a battery of functional assays to demonstrate (i) the relationship between CD99/zyxin and tumor cell growth/migration and (ii) how CD99 deprivation from the Ewing sarcoma cell surface is sufficient to specifically affect the expression of some crucial EWS-FLI1 targets, both in vitro and in vivo, even in the presence of EWS-FLI1. This article reveals that the CD99/zyxin/GLI1 axis is promising therapeutic target for reducing Ewing sarcoma malignancy.

Mebendazole Mediates Proteasomal Degradation of GLI Transcription Factors in Acute Myeloid Leukemia.

The prognosis of elderly AML patients is still poor due to chemotherapy resistance. The Hedgehog (HH) pathway is important for leukemic transformation because of aberrant activation of GLI transcription factors. MBZ is a well-tolerated anthelmintic that exhibits strong antitumor effects. Herein, we show that MBZ induced strong, dose-dependent anti-leukemic effects on AML cells, including the sensitization of AML cells to chemotherapy with cytarabine. MBZ strongly reduced intracellular protein levels of GLI1/GLI2 transcription factors. Consequently, MBZ reduced the GLI promoter activity as observed in luciferase-based reporter assays in AML cell lines. Further analysis revealed that MBZ mediates its anti-leukemic effects by promoting the proteasomal degradation of GLI transcription factors via inhibition of HSP70/90 chaperone activity. Extensive molecular dynamics simulations were performed on the MBZ-HSP90 complex, showing a stable binding interaction at the ATP binding site. Importantly, two patients with refractory AML were treated with MBZ in an off-label setting and MBZ effectively reduced the GLI signaling activity in a modified plasma inhibitory assay, resulting in a decrease in peripheral blood blast counts in one patient. Our data prove that MBZ is an effective GLI inhibitor that should be evaluated in combination to conventional chemotherapy in the clinical setting.

Hedgehog/GLI1 Transcriptionally Regulates FANCD2 in Ovarian Tumor Cells: Its Inhibition Induces HR-Deficiency and Synergistic Lethality with PARP Inhibition.

Ovarian cancer (OC) is one of the most lethal type of cancer in women due to a lack of effective targeted therapies and high rates of treatment resistance and disease recurrence. Recently Poly (ADP-ribose) polymerase inhibitors (PARPi) have shown promise as chemotherapeutic agents; however, their efficacy is limited to a small fraction of patients with BRCA mutations. Here we show a novel function for the Hedgehog (Hh) transcription factor Glioma associated protein 1 (GLI1) in regulation of key Fanconi anemia (FA) gene, FANCD2 in OC cells. GLI1 inhibition in HR-proficient OC cells induces HR deficiency (BRCAness), replication stress and synergistic lethality when combined with PARP inhibition. Treatment of OC cells with combination of GLI1 and PARP inhibitors shows enhanced DNA damage, synergy in cytotoxicity, and strong in vivo anticancer responses.

GLI1 reduces drug sensitivity by regulating cell cycle through PI3K/AKT/GSK3/CDK pathway in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a hematological malignancy with high incidence and recurrence rates. Gene expression profiling has revealed that transcriptional overexpression of glioma-associated oncogene 1 (GLI1), a vital gene in the Hedgehog (Hh) signaling pathway, occurs in poor-prognosis AML, and high levels of phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1) and AKT3 predict shorter overall survival in AML patients. In this study, we discovered that GLI1 overexpression promotes cell proliferation and reduces chemotherapy sensitivity in AML cells while knocking down GLI1 has the opposite effect. Moreover, GLI1 promoted cell cycle progression and led to elevated protein levels of cyclins and cyclin-dependent kinases (CDKs) in AML cells. By luciferase assays and co-immunoprecipitation, we demonstrated that the PI3K/AKT pathway is directly activated by GLI1. GLI1 overexpression significantly accelerates tumor growth and upregulated p-AKT, CDK4, and cyclinD3 in vivo. Notably, the GLI1 inhibitor GANT61 and the CDK4/6 inhibitor PD 0332991 had synergistic effects in promoting Ara-c sensitivity in AML cell lines and patient samples. Collectively, our data demonstrate that GLI1 reduces drug sensitivity by regulating cell cycle through the PI3K/AKT/GSK3/CDK pathway, providing a new perspective for involving GLI1 and CDK4/6 inhibitors in relapsed/refractory (RR) patient treatment.

CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.

GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1-edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative-real time PCR (q-rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer-related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT-61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage-specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.

HEDGEHOG/GLI Modulates the PRR11-SKA2 Bidirectional Transcription Unit in Lung Squamous Cell Carcinomas.

We previously demonstrated that proline-rich protein 11 (PRR11) and spindle and kinetochore associated 2 (SKA2) constituted a head-to-head gene pair driven by a prototypical bidirectional promoter. This gene pair synergistically promoted the development of non-small cell lung cancer. However, the signaling pathways leading to the ectopic expression of this gene pair remains obscure. In the present study, we first analyzed the lung squamous cell carcinoma (LSCC) relevant RNA sequencing data from The Cancer Genome Atlas (TCGA) database using the correlation analysis of gene expression and gene set enrichment analysis (GSEA), which revealed that the PRR11-SKA2 correlated gene list highly resembled the Hedgehog (Hh) pathway activation-related gene set. Subsequently, GLI1/2 inhibitor GANT-61 or GLI1/2-siRNA inhibited the Hh pathway of LSCC cells, concomitantly decreasing the expression levels of PRR11 and SKA2. Furthermore, the mRNA expression profile of LSCC cells treated with GANT-61 was detected using RNA sequencing, displaying 397 differentially expressed genes (203 upregulated genes and 194 downregulated genes). Out of them, one gene set, including BIRC5, NCAPG, CCNB2, and BUB1, was involved in cell division and interacted with both PRR11 and SKA2. These genes were verified as the downregulated genes via RT-PCR and their high expression significantly correlated with the shorter overall survival of LSCC patients. Taken together, our results indicate that GLI1/2 mediates the expression of the PRR11-SKA2-centric gene set that serves as an unfavorable prognostic indicator for LSCC patients, potentializing new combinatorial diagnostic and therapeutic strategies in LSCC.

Targeting the GLI family of transcription factors for the development of anti-cancer drugs.

INTRODUCTION: GLI1 is a transcription factor that has been identified as a downstream effector for multiple tumorigenic signaling pathways. These include the Hedgehog, RAS-RAF-MEK-ERK, and PI3K-AKT-mTOR pathways, which have all been separately validated as individual anti-cancer drug targets. The identification of GLI1 as a key transcriptional regulator for each of these pathways highlights its promise as a therapeutic target. Small molecule GLI1 inhibitors are potentially efficacious against human malignancies arising from multiple oncogenic mechanisms. AREAS COVERED: This review provides an overview of the key oncogenic cellular pathways that regulate GLI1 transcriptional activity. It also provides a detailed account of small molecule GLI1 inhibitors that are currently under development as potential anti-cancer chemotherapeutics. EXPERT OPINION: Interest in developing inhibitors of GLI1-mediated transcription has significantly increased as its role in multiple oncogenic signaling pathways has been elucidated. To date, it has proven difficult to directly target GLI1 with small molecules, and the majority of compounds that inhibit GLI1 activity function through indirect mechanisms. To date, no direct-acting GLI1 inhibitor has entered clinical trials. The identification and development of new scaffolds that can bind and directly inhibit GLI1 are essential to further advance this class of chemotherapeutics.

Gli affects the stemness and prognosis of epithelial ovarian cancer via homeobox protein NANOG.

Gli proteins are key transcription factors of the Hedgehog (HH) signaling pathway, which is associated with tumorigenesis and drug resistance. However, the role of the HH signaling pathway in epithelial ovarian cancer (EOC) remains unclear. Studies have demonstrated that in some tumors, homeobox protein NANOG (NANOG), a known stem cell marker, is a downstream effector of Gli. However, limited research has been conducted on the association between Gli and NANOG in EOC, particularly regarding their roles in the tumor stemness, such as tumor development, drug resistance and patient prognosis. Thus, the aim of the present study was to explore the aforementioned issues. In this study, Gli1, Gli2 and NANOG expression in EOC tissues was assessed using immunohistochemistry. Gene expression was also assessed using western blotting and reverse transcription­quantitative PCR in SKOV3 cells treated with a Gli inhibitor and an HH agonist. Furthermore, cell proliferation, colony­forming ability and cisplatin sensitivity were assessed using Cell Counting Kit­8 and colony formation assays. The results showed that both Gli1 and NANOG were associated with cisplatin resistance and EOC disease stage, while the nuclear expression of Gli2 was significantly associated with cisplatin resistance. Together, the expression of Gli and NANOG predicted poor patient prognosis. Targeting Gli with GANT61 impeded tumor proliferation, reversed cisplatin resistance and colony formation, and reduced NANOG expression. To conclude, Gli and NANOG may be effective indicators of platinum resistance and prognosis in EOC. Targeting Gli may reduce the stemness of ovarian cancer cell, which may be achieved via indirect targeting of NANOG.

The Hedgehog signaling pathway promotes chemotherapy resistance via multidrug resistance protein 1 in ovarian cancer.

Various studies have revealed that the Hedgehog (Hh) signaling pathway promotes ovarian cancer invasion, migration and drug resistance. Previous studies by our group have identified a set of genes, including multidrug resistance gene 1 (MDR1), that are regulated by Hh signaling in ovarian cancer. However, the association between Hh signaling activation and MDR1 expression requires further validation. In the present study, reverse transcription­quantitative PCR or western blot assays were used to evaluate the mRNA and protein expression levels of MDR1, Sonic Hh (Shh), glioma­associated oncogene 2 (Gli2), Gli1 and γ­phosphorylated H2A.X variant histone (γ­H2AX). MTT and colony­formation assays were performed to determine the effect of cisplatin (DDP) after inhibiting the Hh pathway in ovarian cancer cells. The results indicated that MDR1, Gli2 and Shh levels were much higher in SK­OV­3 cells with acquired DDP resistance than in native SK­OV­3 cells. ES­2 cells with overexpression of Gli2 were capable of efficiently forming colonies in the presence of low DDP concentrations. By contrast, Gli2 knockdown in SK­OV­3 cells decreased the colony­forming ability under the same concentration of DDP. As determined by MTT assays, knockdown of Gli2 or targeting of the Hh signaling pathway with either Gli­antagonist 61 (GANT61) or cyclopamine, in combination with DDP treatment, diminished the viability of ES­2 and SK­OV­3 cells, whereas Gli2 overexpression increased the viability of ES­2 cells in the presence of DDP. Knockdown of Gli2 or targeting the Hh signaling pathway with GANT61 also increased γ­H2AX levels but decreased the expression of MDR1 in the presence of DDP. MDR1 expression is regulated by the Hh signaling pathway and is likely a downstream transcription factor of Gli2. In conclusion, targeting the Hh signaling pathway increases the sensitivity of ovarian cancer to DDP. MDR1 is a target gene of the Hh signaling pathway and this pathway may affect chemoresistance of ovarian cancer to DDP via MDR1.

Microcystin-leucine-arginine induces liver fibrosis by activating the Hedgehog pathway in hepatic stellate cells.

Microcystin-leucine-arginine (MC-LR), produced by cyanobacteria, accumulates in the liver through blood circulation. We investigated the impact of MC-LR on liver fibrosis. Mice received a daily injection of MC-LR at various concentrations for 14 consecutive days aa and then mouse liver was obtained for histopathological and immunoblot analysis. Next, a human hepatic stellate cell line (LX-2) was treated with MC-LR at various concentrations followed by measurement of cell viability, cell cycle and relevant protein expression levels. Our data confirmed the induction of mouse liver fibrosis after exposure to MC-LR at 15 µg/kg and 30 µg/kg. Furthermore, we demonstrated that LX-2 cells could uptake MC-LR, resulting in cell proliferation and differentiation through impacting the Hedgehog signaling after the treatment of MC-LR at 50 nM. Our data supported that MC-LR could induce liver fibrosis by modulating the expression of the transcription factor Gli2 in the Hedgehog signaling in hepatic stellate cells.

The SHH/GLI signaling pathway: a therapeutic target for medulloblastoma.

INTRODUCTION: Medulloblastoma (MB) is a heterogeneous tumor of the cerebellum that is divided into four main subgroups with distinct molecular and clinical features. Sonic Hedgehog MB (SHH-MB) is the most genetically understood and occurs predominantly in childhood. Current therapies consist of aggressive and non-targeted multimodal approaches that are often ineffective and cause long-term complications. These problems intensify the need to develop molecularly targeted therapies to improve outcome and reduce treatment-related morbidities. In this scenario, Hedgehog (HH) signaling, a developmental pathway whose deregulation is involved in the pathogenesis of several malignancies, has emerged as an attractive druggable pathway for SHH-MB therapy. AREAS COVERED: This review provides an overview of the advancements in the HH antagonist research field. We place an emphasis on Smoothened (SMO) and glioma-associated oncogene homolog (GLI) inhibitors and immunotherapy approaches that are validated in preclinical SHH-MB models and that have therapeutic potential for MB patients. Literature from Pubmed and data reported on ClinicalTrial.gov up to August 2020 were considered. EXPERT OPINION: Extensive-omics analysis has enhanced our knowledge and has transformed the way that MB is studied and managed. The clinical use of SMO antagonists has yet to be determined, however, future GLI inhibitors and multitargeting approaches are promising.

Crosstalk between Hedgehog pathway and the glucocorticoid receptor pathway as a basis for combination therapy in T-cell acute lymphoblastic leukemia.

Notwithstanding intensified therapy, a considerable fraction of T-cell acute lymphoblastic leukemia (T-ALL) patients face a dismal prognosis due to primary resistance to treatment and relapse, raising the need for more efficient and targeted therapies. Hedgehog (HH) signaling is a major developmental pathway frequently deregulated in cancer, for which a role in T-ALL is emerging. Mounting evidence suggests that ligand-independent activation of HH pathway occurs in cancer including T-ALL, emphasizing the necessity of dissecting the complex interplay between HH and other signaling pathways regulating activation. In this work, we present a therapeutically relevant crosstalk between HH signaling and the glucocorticoid receptor (NR3C1) pathway acting at the level of GLI1 transcription factor. GLI inhibitor GANT61 and dexamethasone were shown to exert a synergistic anti-leukemic effect in vitro in T-ALL cell lines and patient-derived xenografts. Mechanistically, dexamethasone-activated NR3C1 impaired GLI1 function by dynamically modulating the recruitment of PCAF acetyltransferase and HDAC1 deacetylase. Increased GLI1 acetylation was associated with compromised transcriptional activity and reduced protein stability. In summary, our study identifies a novel crosstalk between GLI1 and NR3C1 signaling pathway which could be exploited in HH-dependent malignancies to increase therapeutic efficacy.

A Gli inhibitor GANT61 suppresses cell proliferation, promotes cell apoptosis and induces G1/G0 cycle retardation with a dose- and time-dependent manner through inhibiting Notch pathway in multiple myeloma.

PURPOSE: This study aimed to explore the effect of GANT61 on regulating cell proliferation, cell apoptosis and cell cycle, and to investigate whether GANT61 would function in multiple myeloma (MM) via inhibiting Notch pathway. Methods: RPMI-8226 and U266 cells were treated by GANT61 (0, 2.5, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0 µmol/L) for 18, 24 and 36 hours (h), and cell proliferation was detected by Cell Counting Kit 8. Then these cells were treated by GANT61 at 0, 2.5, 5.0, 10.0 µmol/L for 24 h or treated by 10.0 µmol/L GANT61 for 0, 18, 24 and 36 h, and cell apoptosis rate, apoptosis markers and cell cycle were detected by AV/PI, Western blot, and PI staining. Notch1, Jagged1, Jagged2 and Hes1 expressions were detected by qPCR and Western blot. Further rescue experiments were conducted by upregulating Notch1. Results: In RPMI-8226 and U266 cells, GANT61 inhibited cell proliferation, increased cell apoptosis rate and cell percentage of G1/G0 phase while decreased cell percentage of S phase in a dose- and time-dependent manner. Besides, GANT61 inhibited Notch1, Jagged1, Jagged2 and Hes1 expressions in a dose- and time-dependent manner as well. In rescue experiments, Notch1 upregulation attenuated the inhibition of cell proliferation, promotion of cell apoptosis, induction of G1/G0 cycle retardation and repression of Notch signaling pathway induced by GANT61 treatment in RPMI-8226 and U266 cells. Conclusions: GANT61 suppresses cell proliferation, promotes cell apoptosis and induces G1/G0 cycle retardation with a dose- and time-dependent manner through inhibiting Notch pathway in MM. ABBREVIATIONS: MM: Multiple myeloma; Hh: Hedgehog; EMT: epithelial mesenchymal transition; AML: acute myeloid leukemia; GANT61: GLI antagonist; DMSO: dimethyl sulfoxide; CCK-8: Cell Counting Kit 8; C-Caspase 3: Cleaved Caspase 3; Bcl-2: B-cell lymphoma-2; RT-qPCR: real-time quantitative polymerase chain reaction; OD: optical density; PTCH1: Patched1.

Sonic Hedgehog-Gli1 Signaling and Cellular Retinoic Acid Binding Protein 1 Gene Regulation in Motor Neuron Differentiation and Diseases.

Cellular retinoic acid-binding protein 1 (CRABP1) is highly expressed in motor neurons. Degenerated motor neuron-like MN1 cells are engineered by introducing SODG93A or AR-65Q to model degenerated amyotrophic lateral sclerosis (ALS) or spinal bulbar muscular atrophy neurons. Retinoic acid (RA)/sonic hedgehog (Shh)-induced embryonic stem cells differentiation into motor neurons are employed to study up-regulation of Crabp1 by Shh. In SODG93A or AR-65Q MN1 neurons, CRABP1 level is reduced, revealing a correlation of motor neuron degeneration with Crabp1 down-regulation. Up-regulation of Crabp1 by Shh is mediated by glioma-associated oncogene homolog 1 (Gli1) that binds the Gli target sequence in Crabp1's neuron-specific regulatory region upstream of minimal promoter. Gli1 binding triggers chromatin juxtaposition with minimal promoter, activating transcription. Motor neuron differentiation and Crabp1 up-regulation are both inhibited by blunting Shh with Gli inhibitor GANT61. Expression data mining of ALS and spinal muscular atrophy (SMA) motor neurons shows reduced CRABP1, coincided with reduction in Shh-Gli1 signaling components. This study reports motor neuron degeneration correlated with down-regulation in Crabp1 and Shh-Gli signaling. Shh-Gli up-regulation of Crabp1 involves specific chromatin remodeling. The physiological and pathological implication of this regulatory pathway in motor neuron degeneration is supported by gene expression data of ALS and SMA patients.

Up-regulation of GLI1 in vincristine-resistant rhabdomyosarcoma and Ewing sarcoma.

BACKGROUND: The clinical significance of GLI1 expression either through canonical Hedgehog signal transduction or through non-canonical mechanisms in rhabdomyosarcoma (RMS) or Ewing sarcoma (EWS) is incompletely understood. We tested a role for Hedgehog (HH) signal transduction and GL11 expression in development of vincristine (VCR) resistance in RMS and EWS. METHODS: We characterized baseline expression and activity of HH pathway components in 5 RMS (RD, Rh18, Ruch-2, Rh30, and Rh41) and 5 EWS (CHLA9, CHLA10, TC32, CHLA258, and TC71) cell lines. We then established VCR-resistant RMS and EWS cell lines by exposing cells to serially increasing concentrations of VCR and determining the IC50. We defined resistance as a ≥ 30-fold increase in IC50 compared with parental cells. We determined changes in gene expression in the VCR-resistant cells compared with parental cells using an 86-gene cancer drug resistance array that included GLI1 and tested the effect of GLI1 inhibition with GANT61 or GLI1 siRNA on VCR resistance. RESULTS: We found evidence for HH pathway activity and GLI1 expression in RMS and EWS cell lines at baseline, and evidence that GLI1 contributes to survival and proliferation of these sarcoma cells. We were able to establish 4 VCR-resistant cell lines (Ruch-2VR, Rh30VR, Rh41VR, and TC71VR). GLI1 was significantly up-regulated in the Rh30VR, Rh41VR, and TC71VR cells. The only other gene in the drug resistance panel that was significantly up-regulated in each of these VCR-resistant cell lines compared with their corresponding parental cells was the GLI1 direct target and multidrug resistance gene, ATP-binding cassette sub-family B member 1 (MDR1). We established major vault protein (MVP), which was up-regulated in both vincristine-resistant alveolar RMS cell lines (Rh30VR and Rh41VR), as another direct target of GLI1 during development of drug resistance. Treatment of the VCR-resistant cell lines with the small molecule inhibitor GANT61 or GLI1 siRNA together with VCR significantly decreased cell viability at doses that did not reduce viability individually. CONCLUSIONS: These experiments demonstrate that GLI1 up-regulation contributes to VCR resistance in RMS and EWS cell lines and suggest that targeting GLI1 may benefit patients with RMS or EWS by reducing multidrug resistance.

Bicyclic Imidazolium Inhibitors of Gli Transcription Factor Activity.

Gli transcription factors within the Hedgehog (Hh) signaling pathway direct key events in mammalian development and promote a number of human cancers. Current therapies for Gli-driven tumors target Smoothened (SMO), a G protein-coupled receptor-like protein that functions upstream in the Hh pathway. Although these drugs can have remarkable clinical efficacy, mutations in SMO and downstream Hh pathway components frequently lead to chemoresistance. In principle, therapies that act at the level of Gli proteins, through direct or indirect mechanisms, would be more efficacious. We therefore screened 325 120 compounds for their ability to block the constitutive Gli activity induced by loss of Suppressor of Fused (SUFU), a scaffolding protein that directly inhibits Gli function. Our studies reveal a family of bicyclic imidazolium derivatives that can inhibit Gli-dependent transcription without affecting the ciliary trafficking or proteolytic processing of these transcription factors. We anticipate that these chemical antagonists will be valuable tools for investigating the mechanisms of Gli regulation and developing new strategies for targeting Gli-driven cancers.

Hedgehog Inhibitors Suppress Osteoclastogenesis in In Vitro Cultures, and Deletion of Smo in Macrophage/Osteoclast Lineage Prevents Age-Related Bone Loss.

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on Hh functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0-48 h (early stage of osteoclast differentiation) or 48-96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.