Smo Antagonist Research Articles

Efcab7 deletion sensitizes mice to the teratogenic effects of gastrulation-stage alcohol exposure

Alcohol exposure during the gastrulation stage of development can disrupt Sonic hedgehog (Shh) signaling and cause eye, craniofacial, and brain defects. One of the genes that regulates Shh signaling is Efcab7, which encodes a protein that facilitates the actions of Smoothened (Smo), a critical component of the Shh pathway. Previous work from our lab has demonstrated that Efcab7 is differentially expressed between two sub-strains of C57BL/6 mice that differ in their sensitivity to gastrulation-stage alcohol exposure. The more alcohol-sensitive C57BL/6 J mice express lower levels of Efcab7 during gastrulation than do the less alcohol-sensitive C57BL/6NHsd mice. The current study examined whether partial or full Efcab7 deletions render mice more sensitive to gastrulation-stage alcohol exposure and affect the sensitivity to other modulators of Shh signaling that cause craniofacial malformations. Efcab7+/- dams were mated with Efcab7+/- sires to produce Efcab7+/+, Efcab7+/-, and Efcab7-/- fetuses. On gestational day 7 (GD 7), they received either alcohol (two doses of 2.9 g/kg, i.p., given 4 hours apart), the Smo antagonist vismodegib (40 mg/kg, or vehicle, p.o.), the Smo agonist SAG (20 mg/kg) or the appropriate vehicles. GD 17 fetuses were collected and examined for ocular and craniofacial dysmorphology. As compared to Efcab7+/+ fetuses, Efcab7-/- fetuses exposed to alcohol or vismodegib treatment had more severe ocular and craniofacial malformations. In contrast, Efcab7-/- fetuses had less severe malformations induced by SAG. These results confirm that Efcab7 can modify responses to Shh agonists and antagonists and further identify Efcab7 as a gene important for the sensitivity to gastrulation-stage alcohol exposure.

Nile Tilapia (Oreochromis niloticus) Patched1 Mutations Disrupt Cardiovascular Development and Vascular Integrity through Smoothened Signaling.

Hedgehog (Hh) signaling is crucial in cardiovascular development and maintenance. However, the biological role of Patched1 (Ptch1), an inhibitory receptor of the Hh signaling pathway, remains elusive. In this study, a Ptch1 ortholog was characterized in Nile tilapia (Oreochromis niloticus), and its function was investigated through CRISPR/Cas9 gene knockout. When one-cell embryos were injected with CRISPR/Cas9 targeting ptch1, the mutation efficiency exceeded 70%. During 0-3 days post fertilization (dpf), no significant differences were observed between the ptch1 mutant group and the control group; at 4 dpf (0 day after hatching), about 10% of the larvae showed an angiogenesis defect and absence of blood flow; from 5 dpf, most larvae exhibited an elongated heart, large pericardial cavity, and blood leakage and coagulation, ultimately dying during the 6-8 dpf period due to the lack of blood circulation. Consistently, multiple differentially expressed genes related to angiogenesis, blood coagulation, and heart development were enriched in the ptch1 mutants. Furthermore, Smoothened (Smo) antagonist (cyclopamine) treatment of the ptch1 mutants greatly rescued the cardiovascular disorders. Collectively, our study suggests that Ptch1 is required for cardiovascular development and vascular integrity via Smo signaling, and excessive Hh signaling is detrimental to cardiovascular development.

Towards modular engineering of cell signalling: Topographically-textured microparticles induce osteogenesis via activation of canonical hedgehog signalling

Polymer microparticles possess great potential as functional building blocks for advanced bottom-up engineering of complex tissues. Tailoring the three-dimensional architectural features of culture substrates has been shown to induce osteogenesis in mesenchymal stem cells in vitro, but the molecular mechanisms underpinning this remain unclear. This study proposes a mechanism linking the activation of Hedgehog signalling to the osteoinductive effect of surface-engineered, topographically-textured polymeric microparticles. In this study, mesenchymal progenitor C3H10T1/2 cells were cultured on smooth and dimpled poly(D,l-lactide) microparticles to assess differences in viability, cellular morphology, proliferation, and expression of a range of Hedgehog signalling components and osteogenesis-related genes. Dimpled microparticles induced osteogenesis and activated the Hedgehog signalling pathway relative to smooth microparticles and 2D-cultured controls without the addition of exogenous biochemical factors. We observed upregulation of the osteogenesis markers Runt-related transcription factor2 (Runx2) and bone gamma-carboxyglutamate protein 2 (Bglap2), as well as the Hedgehog signalling components, glioma associated oncogene homolog 1 (Gli1), Patched1 (Ptch1), and Smoothened (Smo). Treatment with the Smo antagonist KAAD-cyclopamine confirmed the involvement of Smo in Gli1 target gene activation, with a significant reduction in the expression of Gli1, Runx2 and Bglap2 (p ≤ 0.001) following KAAD-cyclopamine treatment. Overall, our study demonstrates the role of the topographical microenvironment in the modulation of Hedgehog signalling, highlighting the potential for tailoring substrate topographical design to offer cell-instructive 3D microenvironments. Topographically-textured microparticles allow the modulation of Hedgehog signalling in vitro without adding exogenous biochemical agonists, thereby eliminating potential confounding artefacts in high-throughput drug screening applications.

Synthesis of Vismodegib Derivatives as Potential Smo Agonist: A Case of Undergraduate Experimental Teaching

The activation of Hh pathway demonstrates therapeutic potential for many diseases. Smo is the main target for the development of Hh pathway modulators. However, compared with Smo antagonist, the development of Smo agonists is lagging behind. Based on our previous work, a series of vismodegib derivatives were designed and synthesized, and their potential to activate the Hh pathway were evaluated via determining the up-regulation of known pathway target genes Gli1, whcih revealed that many target compounds could activate the Hh pathway. XH-16 and XH-17, with the strongest agonistic effect, could upregulate the expression of Gli1 by more than 50% at a concentration of 10 μM. In vitro cytotoxicity against A549 and the MDA-MB-231 cells was evaluated and the results revealed that XH-16 and XH-17 did not exhibit cytotoxicity at a concentration of 50 μM. Molecular docking results confirmed that XH-16 and XH-17 could bind to Smo indicating that their agonistic effect on the Hh pathway may be due to the activation of Smo. XH-16 and XH-17 with novel molecular scaffold could be used as a lead compound for the development of Smo agonists. Moreover, the research process was introduced in a medicinal chemistry experimental course to assist undergraduates in understanding the core of medicinal chemistry and building capabilities for independently carrying out a medicinal chemistry project. And the teaching practice experiences were summed up to provide suggestions for the development of exploratory experimental course.

Exploring the Potential of Anthraquinone-Based Hybrids for Identifying a Novel Generation of Antagonists for the Smoothened Receptor in HH-Dependent Tumour.

Natural products (NPs) are highly profitable pharmacological tools due to their chemical diversity and ability to modulate biological systems. Accessing new chemical entities while retaining the biological relevance of natural chemotypes is a fundamental goal in the design of novel bioactive compounds. Notably, NPs have played a crucial role in understanding Hedgehog (HH) signalling and its pharmacological modulation in anticancer therapy. However, HH antagonists developed so far have shown several limitations, thus growing interest in the design of second-generation HH inhibitors. Through smart manipulation of the NPs core-scaffold, unprecedented and intriguing architectures have been achieved following different design strategies. This study reports the rational design and synthesis of a first and second generation of anthraquinone-based hybrids by combining the rhein scaffold with variously substituted piperazine nuclei that are structurally similar to the active portion of known SMO antagonists, the main transducer of the HH pathway. A thorough functional and biological investigation identified RH2_2 and RH2_6 rhein-based hybrids as valuable candidates for HH inhibition through SMO antagonism, with the consequent suppression of HH-dependent tumour growth. These findings also corroborated the successful application of the NPs-based hybrid design strategy in the development of novel NP-based SMO antagonists.

Resveratrol Inhibits Activation of Microglia after Stroke through Triggering Translocation of Smo to Primary Cilia.

Activated microglia act as a double-edged sword for stroke. In the acute phase of stroke, activated microglia might deteriorate neurological function. Therefore, it is of great clinical transforming potential to explore drugs or methods that can inhibit abnormal activation of microglia in the acute phase of stroke to improve neurological function after stroke. Resveratrol has a potential effect of regulating microglial activation and anti-inflammation. However, the molecular mechanism of resveratrol-inhibiting microglial activation has not been fully clarified. Smoothened (Smo) belongs to the Hedgehog (Hh) signaling pathway. Smo activation is the critical step that transmits the Hh signal across the primary cilia to the cytoplasm. Moreover, activated Smo can improve neurological function via regulating oxidative stress, inflammation, apoptosis, neurogenesis, oligodendrogenesis, axonal remodeling, and so on. More studies have indicated that resveratrol can activate Smo. However, it is currently unknown whether resveratrol inhibits microglial activation via Smo. Therefore, in this study, N9 microglia in vitro and mice in vivo were used to investigate whether resveratrol inhibited microglial activation after oxygen-glucose deprivation/reoxygenation (OGD/R) or middle cerebral artery occlusion/reperfusion (MCAO/R) injury and improved functional outcome via triggering translocation of Smo in primary cilia. We definitively found that microglia had primary cilia; resveratrol partially inhibited activation and inflammation of microglia, improved functional outcome after OGD/R and MCAO/R injury, and triggered translocation of Smo to primary cilia. On the contrary, Smo antagonist cyclopamine canceled the above effects of resveratrol. The study suggested that Smo receptor might be a therapeutic target of resveratrol for contributing to inhibit microglial activation in the acute phase of stroke.

Sonic Hedgehog Promotes Proliferation and Migration of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis via Rho/ROCK Signaling.

Objective To explore the underlying mechanism of the sonic hedgehog (Shh) signaling pathway in promoting cell proliferation and migration in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). Method FLS were collected from 8 patients with RA and 3 patients with osteoarthritis (OA). The expression of smoothened (Smo, the Shh pathway activator) was quantified by real-time PCR and western blot. FLS were incubated with cyclopamine (a Smo antagonist), purmorphamine (a Smo agonist), Y27632 (a Rho/ROCK signaling inhibitor), or a combination of purmorphamine and Y27632, respectively. Cell proliferation was examined using cell counting kit-8 and cell cycle assays while cell migration was measured with Transwell and wound healing assays. Results The expression of Smo was higher in FLS from RA patients than from OA patients (p < 0.05). RA-FLS treated with purmorphamine showed significantly activated proliferation (119.69 vs. 100.0) and migration (252.38 vs. 178.57) compared to untreated cells (both p < 0.001). RA-FLS incubated with cyclopamine or a combination of purmorphamine and Y27632 exhibited significant suppression of proliferation (81.55 vs. 100.0 and 85.84 vs. 100.0) and migration (100 vs. 178.57 and 109.52 vs. 185) ability (all p < 0.001). Conclusion Our results demonstrated that Shh promoted cell growth and migration of FLS in RA patients through the Rho/ROCK signaling pathway.

Desert hedgehog mediates the proliferation of medaka spermatogonia through Smoothened signaling.

Desert hedgehog (DHH) signaling has been reported to be involved in spermatogenesis and the self-renewal of spermatogonial stem cells (SSCs). However, the role of DHH in proliferation of spermatogonia including SSCs remains to be elucidated. Here, we report that Dhh from medaka (Oryizas latipes) (named as OlDhh) could directly mediate the proliferation of spermatogonia via Smoothened (Smo) signaling. Oldhh is 1362 bp in length and encodes 453 amino acid (aa) residues with more than 50% identity with the homologs in other species. It has expression predominantly restricted to testis. The soluble and tag-free 176-aa mature OlDhh (named as mOlDhh) were successfully obtained by fusing with the N-terminal tag of cleavable 6-histidine and small ubiquitin-related modifier and then removing the tag. Notably, mOlDhh significantly promoted the proliferation of SG3 (a spermatogonial stem cell line from medaka testis) in a dose-dependent manner and spermatogonia in testicular organ culture. Furthermore, the proliferation of SG3 in the presence of mOlDhh could be inhibited by Smo antagonist (cyclopamine) resulting in apoptosis. Additionally, mOlDhh significantly upregulated the expression of smo as well as the pluripotent-related genes bcl6b and sall4. These data suggest that Smo is an indispensable downstream component in the Dhh signaling pathway. In conclusion, our findings unambiguously demonstrate that Dhh could directly mediate the proliferation of spermatogonia through Smo signaling. This study provides new knowledge about the proliferation regulation of spermatogonia.

CELL-AUTONOMOUS HEDGEHOG SIGNALING CONTROLS TH17 DIFFERENTIATION TO DRIVE INTESTINAL INFLAMMATION AND IS A DRUGGABLE TARGET FOR THE TREATMENT OF INFLAMMATORY BOWEL DISEASE

Abstract BACKGROUND T helper 17 (Th17) cells play a key role in barrier protection in the gastrointestinal tract but are also key pathological drivers of Inflammatory Bowel Disease (IBD). Although a number of key transcription factors governing Th17 differentiation have been identified, the intracellular signaling pathways regulating Th17 differentiation are poorly understood. Given the highly druggable nature of many intracellular signaling pathways, understanding the signaling pathways involved in Th17 differentiation holds great promise to identify novel drug targets for the treatment of IBD. Hedgehog (Hh) signaling controls cell-fate choices in numerous tissue compartments and is targetable by highly selective, clinically-approved small molecule inhibitors. However the role of Hh signaling in Th17 differentiation and effector function is unstudied. METHODS We generated two conditional knockout mouse models targeting Hh signaling components Smo and Ihh to study Th17 differentiation and effector function in vitro and in vivo in murine T cell adoptive transfer colitis. We supplement this with the use of two small-molecule Smo antagonists for both in vitro and in vivo studies of Th17 function. To underscore the translational relevance of our findings, we have conducted bioinformatic analyses of published gene expression datasets of human rectal biopsies from two large independent cohorts of Ulcerative Colitis patients and healthy controls as well as from sorted human effector T cells from blood and lamina propria of healthy individuals. RESULTS We find that intracellular Hh signaling, independently of extracellular Hh ligands, selectively drives differentiation and effector function of Th17 cells but not of other T helper cell lineages. Using two models of intestinal inflammation, we demonstrate that inhibition of the Hh pathway with either the clinically-approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4+ T cells profoundly diminishes disease severity and Th17-induced pathology in the intestine. Our bioinformatic analyses show that Hh component expression levels are upregulated in human ulcerative colitis patient samples and are closely correlated with expression of Th17 markers/cytokines. Mechanistically we show that the T-cell-intrinsic Indian Hedgehog (Ihh) ligand signals via the signal transducer Smoothened to activate both canonical and non-canonical Hh pathways, through the Gli3 transcription factor and AMPK phosphorylation, respectively. CONCLUSIONS We uncover Hh signaling as a novel pathway controlling Th17 differentiation and pathogenicity in IBD with Gli3 acting as a newly-identified crucial regulatory transcription factor. Our work paves the way for the use of Hh inhibitors for the treatment of IBD.

DOP48 Hedgehog signalling controls Th17 differentiation to drive intestinal inflammation and is a druggable target for the treatment of IBD

Abstract Background T helper 17 (Th17) cells play an important role in barrier protection in the gastrointestinal tract but are also key pathological drivers of Inflammatory Bowel Disease (IBD). Although a number of transcription factors governing Th17 differentiation have been identified, the intracellular signalling pathways regulating Th17 differentiation are poorly understood. Hedgehog (Hh) signalling controls cell-fate choices in numerous tissue compartments and is targetable by highly selective, clinically-approved small molecule inhibitors. However the role of Hh signalling in Th17 differentiation and effector function is unstudied. Methods We generated two conditional knockout mouse models targeting Hh signalling components Smo and Ihh to study Th17 differentiation in vitro by flow cytometry and gene expression analysis. For in vivo studies, T cell adoptive transfer colitis was performed using donor Ihh knockout T cells or heterozygote controls. Histological analysis, mouse weight, colon length/weight measurements, and flow cytometric analysis was performed. We supplement this with the use of two small-molecule Smo antagonists for in vitro and in vivo studies of Th17 function. To underscore the translational relevance of our findings, we conducted bioinformatic analyses of published gene expression datasets of human rectal biopsies from two large independent cohorts of Ulcerative Colitis patients and healthy controls. Results We find that intracellular Hh signalling, independently of extracellular Hh ligands, selectively drives differentiation and effector function of Th17 cells but not of other T helper cell lineages. We demonstrate in vivo that inhibition of the Hh pathway with either the clinically-approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4+ T cells results in a significant decrease in histological and clinical readouts of disease severity as well as a significant reduction in IL-17a+ Th17 cells. Our bioinformatic analyses show that Hh component expression levels are upregulated in human Ulcerative Colitis patient samples and are closely correlated with expression of Th17 markers. Mechanistically we show that the T-cell-intrinsic Indian Hedgehog (Ihh) ligand signals via the signal transducer Smoothened to activate both canonical and non-canonical Hh pathways, through the Gli3 transcription factor and AMPK phosphorylation, respectively. Conclusion We uncover Hh signalling as a novel pathway controlling Th17 differentiation and pathogenicity in IBD with Gli3 acting as a newly-identified crucial regulatory transcription factor. Our work paves the way for the use of Hh inhibitors for the treatment of IBD.

077 Isoform-specific aPKC maintains Hedgehog signaling in the absence of primary cilia

Primary cilia loss is a common feature of advanced cancers. While primary cilia are necessary to initiate Hedgehog (HH)-driven cancers, how HH pathway activity is maintained in advanced cancers devoid of primary cilia is unclear. Here, we find that HH-driven basal cell carcinoma (BCC) and medulloblastoma accumulate mutations in the Alström and Usher syndrome genes. Loss of Alström and Usher syndrome gene expression, which are common underlying causes of deafness and blindness, suppresses ciliogenesis and HH signaling. Loss of primary cilia also enhances atypical protein kinase C iota/lambda (aPKC) expression, a GLI1 kinase necessary for advanced BCC growth. We show that aPKC expression is inversely correlated with primary ciliogenesis and that superficial BCCs display less primary cilia and higher aPKC expression, with the opposite true in nodular BCC subtypes. Surprisingly, a constitutively active isoform of aPKC drives HH pathway activity but not full-length protein. Overexpression of the constitutively active aPKC variant can maintain HH pathway activity in the absence of primary cilia and can drive resistance to the SMO antagonist vismodegib regardless of cilia status. Our results suggest tumors enhance isoform-specific expression of aPKC to prevent mutation-induced cessation of tumor growth and that aPKC may serve as a biomarker for SMO inhibitor sensitivity.

INNV-18. RECURRENT SHH MEDULLOBLASTOMA EXTRANEURAL METASTASES TO THE BONE MARROW WITHOUT CNS INVOLVEMENT

Abstract Medulloblastoma is the most common primary brain tumor in children, but is rare in adults. Because of the scarcity of adult medulloblastoma cases, treatment guidelines in the adult population are not well established, especially regarding surveillance and treatment of recurrent disease. A 47-year-old African American male presented with one month of progressive unsteady gait and dizziness. Imaging revealed a posterior fossa mass and the patient underwent gross total resection. Histology confirmed medulloblastoma and molecular subtyping revealed Sonic Hedgehog (Shh) pathway activation. No metastases were found at the time of diagnosis, and the patient received adjuvant craniospinal radiation therapy. Three years later the patient developed low back pain with severe sciatica affecting his gait and pancytopenia. Imaging revealed diffuse osseous disease without CNS recurrence and bone marrow biopsy confirmed recurrent medulloblastoma. The patient subsequently began reduced chemotherapy consisting of cisplatin and etoposide because of his severe pancytopenia. Due to a lack of response after 4 cycles, the SMO antagonist vismodegib was added. Follow-up imaging revealed improvement/stability of osseous disease, lab work demonstrated resolution of pancytopenia, and repeat bone marrow was without malignancy. Vismodegib was continued as maintenance therapy. Six months after completion of cisplatin/etoposide, follow-up imaging revealed progression of metastatic disease. Shh activated medulloblastomas have demonstrated responses to SMO inhibition. However, these responses are often transient secondary to rapid development of resistance, but may have a role to play in patients who have bone marrow involvement that limits use of cytotoxic therapy or bone marrow transplant. Due to poor survival outcomes associated with bone marrow metastases, aggressive screening for bone marrow metastases at diagnosis and in surveillance should be considered.

Discovery of [1,2,4]triazolo[4,3-a]pyridines as potent Smoothened inhibitors targeting the Hedgehog pathway with improved antitumor activity in vivo

Triple-negative breast cancer (TNBC), a subset of breast cancers, have poorer survival than other breast cancer types. Recent studies have demonstrated that the abnormal Hedgehog (Hh) pathway is activated in TNBC and that these treatment-resistant cancers are sensitive to inhibition of the Hh pathway. Smoothened (Smo) protein is a vital constituent in Hh signaling and an attractive drug target. Vismodegib (VIS) is one of the most widely studied Smo inhibitors. But the clinical application of Smo inhibitors is limited to adult patients with BCC and AML, with many side effects. Therefore, it’s necessary to develop novel Smo inhibitor with better profiles. Twenty [1,2,4]triazolo[4,3-a]pyridines were designed, synthesized and screened as Smo inhibitors. Four of these novel compounds showed directly bound to Smo protein with stronger binding affinity than VIS. The new compounds showed broad anti-proliferative activity against cancer cell lines in vitro, especially triple-negative breast cancer cells. Mechanistic studies demonstrated that TPB15 markedly induced cell cycle arrest and apoptosis in MDA-MB-468 cells. TPB15 blocked Smo translocation into the cilia and reduced Smo protein and mRNA expression. Furthermore, the expression of the downstream regulatory factor glioma-associated oncogene 1 (Gli1) was significantly inhibited. Finally, TPB15 demonstrated greater anti-tumor activity in our animal models than VIS with lower toxicity. Hence, these results support further optimization of this novel scaffold to develop improved Smo antagonists.

Abstract C046: A Smo inhibitor DCBCO1303 with potent Hedgehog signaling pathway antagonist activity is highly active in animal tumor model of medulloblastoma and cholangiocarcinoma

Abstract The hedgehog (Hh) pathway is a critical embryonic signaling cascade regulating cell growth, proliferation and differentiation. Hh signaling is significantly less active in adult tissues, where its primary role appears to be the maintenance of stem cell populations in skin and the central nervous system. Smoothened (Smo), a 7-pass transmembrane receptor with a GPCR-like structure, is a key component of the Hh signaling pathway, the activity of which is suppressed by the 12-pass transmembrane protein Patched (Ptch). Therefore, components of the Hh pathway (such as Shh, SMO, and GLI1/2) are viable therapeutic targets for anti-tumor strategy. We report here the in vivo effects of a potent Smo antagonist DCBCO1303 for the treatment of medulloblastoma and cholagiocarcinoma animal tumor model. DCBCO1303 demonstrated Hh signaling pathway antagonist activity in a 293 cell-based Gli-luciferase inhibition assay upon agonist treatment (IC50 = 3.5 nM), and retains inhibition activity against the Smo wild-type and D473H mutant responsible for resistance to GDC-0449 in medulloblastoma patients with IC50 of 5.1 and 43.7nM, respectively. DCBCO1303 potently suppress Gli1 expression in HEPM cells with IC50 of 2.3 nM. Treatment of a medulloblastoma allograft model with daily oral dosing from 1.25 to 40 mg/kg of DCBCO1303 for 10 days leads to dose responsive antitumor activity with tumor regressions occurring at 40 mg/kg.(Gli 1 mRNA inhibition correlated with tumor growth inhibition). DCBCO1303 was also highly efficacious against the growth of cholangiocarcinoma xenograft tumors. The DCBCO1303 can therefore be evaluated further and serve as a drug development candidate for treatment of Hh pathway-driven cancers. Citation Format: Ying-Shuan E Lee, Mann-Yan Kuo, Pei-Yi Tsai, Chia Wei Liu, Win Yin Wei. A Smo inhibitor DCBCO1303 with potent Hedgehog signaling pathway antagonist activity is highly active in animal tumor model of medulloblastoma and cholangiocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C046. doi:10.1158/1535-7163.TARG-19-C046

Loss of the PTCH1 tumor suppressor defines a new subset of plexiform fibromyxoma

BackgroundPlexiform fibromyxoma (PF) is a rare gastric tumor often confused with gastrointestinal stromal tumor. These so-called “benign” tumors often present with upper GI bleeding and gastric outlet obstruction. It was recently demonstrated that approximately one-third of PF have activation of the GLI1 oncogene, a transcription factor in the hedgehog (Hh) pathway, via a MALAT1-GLI1 fusion protein or GLI1 up-regulation. Despite this discovery, the biology of most PFs remains unknown.MethodsNext generation sequencing (NGS) was performed on formalin-fixed paraffin-embedded (FFPE) samples of PF specimens collected from three institutions (UCSD, NCI and OHSU). Fresh frozen tissue from one tumor was utilized for in vitro assays, including quantitative RT-PCR and cell viability assays following drug treatment.ResultsEight patients with PF were identified and 5 patients’ tumors were analyzed by NGS. An index case had a mono-allelic PTCH1 deletion of exons 15–24 and a second case, identified in a validation cohort, also had a PTCH1 gene loss associated with a suspected long-range chromosome 9 deletion. Building on the role of Hh signaling in PF, PTCH1, a tumor suppressor protein, functions upstream of GLI1. Loss of PTCH1 induces GLI1 activation and downstream gene transcription. Utilizing fresh tissue from the index PF case, RT-qPCR analysis demonstrated expression of Hh pathway components, SMO and GLI1, as well as GLI1 transcriptional targets, CCND1 and HHIP. In turn, short-term in vitro treatment with a Hh pathway inhibitor, sonidegib, resulted in dose-dependent cell killing.ConclusionsFor the first time, we report a novel association between PTCH1 inactivation and the development of plexiform fibromyxoma. Hh pathway inhibition with SMO antagonists may represent a target to study for treating a subset of plexiform fibromyxomas.

Inhibition of Hedgehog Signaling in Fibroblasts, Pancreatic, and Lung Tumor Cells by Oxy186, an Oxysterol Analogue with Drug-Like Properties.

The widespread involvement of the Hedgehog (Hh) signaling pathway in human malignancies has motivated the clinical development of Smoothened (Smo) antagonists, such as vismodegib and sonidegib. However, Smo antagonists have failed to benefit patients suffering from Hh pathway-dependent solid tumors, such as pancreatic, colorectal, or ovarian cancer. Hh-dependent cancers are often driven by activating mutations that occur downstream of Smo and directly activate the transcription factors known as glioma-associated oncogenes (Gli1-3). Hence, the direct targeting of Gli could be a more effective strategy for achieving disease modification compared to Smo antagonism. In this study, we report on the biological and pharmacological evaluation of Oxy186, a semisynthetic oxysterol analogue, as a novel inhibitor of Hh signaling acting downstream of Smo, with encouraging drug-like properties. Oxy186 exhibits strong inhibition of ligand-induced Hh signaling in NIH3T3-E1 fibroblasts, as well as in constitutively activated Hh signaling in Suppressor of Fused (Sufu) null mouse embryonic fibroblast (MEF) cells. Oxy186 also inhibits Gli1 transcriptional activity in NIH3T3-E1 cells expressing exogenous Gli1 and Gli-dependent reporter constructs. Furthermore, Oxy186 suppresses Hh signaling in PANC-1 cells, a human pancreatic ductal adenocarcinoma (PDAC) tumor cell line, as well as PANC-1 cell proliferation in vitro, and in human lung cancer cell lines, A549 and H2039.

Clobetasol Modulates Adult Neural Stem Cell Growth via Canonical Hedgehog Pathway Activation.

Sonic hedgehog (Shh) signaling is a key pathway within the central nervous system (CNS), during both development and adulthood, and its activation via the 7-transmembrane protein Smoothened (Smo) may promote neuroprotection and restoration during neurodegenerative disorders. Shh signaling may also be activated by selected glucocorticoids such as clobetasol, fluocinonide and fluticasone, which therefore act as Smo agonists and hold potential utility for regenerative medicine. However, despite its potential role in neurodegenerative diseases, the impact of Smo-modulation induced by these glucocorticoids on adult neural stem cells (NSCs) and the underlying signaling mechanisms are not yet fully elucidated. The aim of the present study was to evaluate the effects of Smo agonists (i.e., purmorphamine) and antagonists (i.e., cyclopamine) as well as of glucocorticoids (i.e., clobetasol, fluocinonide and fluticasone) on NSCs in terms of proliferation and clonal expansion. Purmorphamine treatment significantly increased NSC proliferation and clonal expansion via GLI-Kruppel family member 1 (Gli1) nuclear translocation and such effects were prevented by cyclopamine co-treatment. Clobetasol treatment exhibited an equivalent pharmacological effect. Moreover, cellular thermal shift assay suggested that clobetasol induces the canonical Smo-dependent activation of Shh signaling, as confirmed by Gli1 nuclear translocation and also by cyclopamine co-treatment, which abolished these effects. Finally, fluocinonide and fluticasone as well as control glucocorticoids (i.e., prednisone, corticosterone and dexamethasone) showed no significant effects on NSCs proliferation and clonal expansion. In conclusion, our data suggest that Shh may represent a druggable target system to drive neuroprotection and promote restorative therapies.

State of the art of Smo antagonists for cancer therapy: advances in the target receptor and new ligand structures.

Since the Hedgehog signaling pathway has been associated with cancer, it has emerged as a therapeutic target for cancer therapy. The main target among the key Hedgehog proteins is the GPCR-like Smo receptor. Therefore, some Smo antagonists that have entered clinical trials, including the US FDA-approved drugs vismodegib and sonidegib, to treat basal cell carcinoma and medulloblastoma. However, early resistance of these drugs has spawned the need to understand the molecular bases of this phenomena. We therefore reviewed details about Smo receptor structures and the best Smo antagonist chemical structures. In addition, we discussed strategies that should be considered to develop new, safer generations of Smo antagonists that avoid current clinical limitations.

Synergistic inhibition of the Hedgehog pathway by newly designed Smo and Gli antagonists bearing the isoflavone scaffold

Aberrant activation of the Hedgehog (Hh) pathway is responsible for the onset and progression of several malignancies. Small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector Gli1 have thus emerged recently as valuable anticancer agents.Here, we have designed, synthesized, and tested new Hh inhibitors taking advantage by the highly versatile and privileged isoflavone scaffold. The introduction of specific substitutions on the isoflavone's ring B allowed the identification of molecules targeting preferentially Smo or Gli1. Biological assays coupled with molecular modeling corroborated the design strategy, and provided new insights into the mechanism of action of these molecules. The combined administration of two different isoflavones behaving as Smo and Gli antagonists, respectively, in primary medulloblastoma (MB) cells highlighted the synergistic effects of these agents, thus paving the way to further and innovative strategies for the pharmacological inhibition of Hh signaling.

Discovery of potent and novel smoothened antagonists via structure-based virtual screening and biological assays

The Hedgehog (Hh) signaling pathway plays a critical role in controlling patterning, growth and cell migration during embryonic development. Aberrant activation of Hh signaling has been linked to tumorigenesis in various cancers, such as basal cell carcinoma (BCC) and medulloblastoma. As a key member of the Hh pathway, the Smoothened (Smo) receptor, a member of the G protein-coupled receptor (GPCR) family, has emerged as an attractive therapeutic target for the treatment and prevention of human cancers. The recent determination of several crystal structures of Smo in complex with different antagonists offers the possibility to perform structure-based virtual screening for discovering potent Smo antagonists with distinct chemical scaffolds. In this study, based on the two Smo crystal complexes with the best capacity to distinguish the known Smo antagonists from decoys, the molecular docking-based virtual screening was conducted to identify promising Smo antagonists from ChemDiv library. A total of 21 structurally novel and diverse compounds were selected for experimental testing, and six of them exhibited significant inhibitory activity against the Hh pathway activation (IC50 < 10 μM) in a GRE (Gli-responsive element) reporter gene assay. Specifically, the most potent compound (compound 20: 47 nM) showed comparable Hh signaling inhibition to vismodegib (46 nM). Compound 20 was further confirmed to be a potent Smo antagonist in a fluorescence based competitive binding assay. Optimization using substructure searching method led to the discovery of 12 analogues of compound 20 with decent Hh pathway inhibition activity, including four compounds with IC50 lower than 1 μM. The important residues uncovered by binding free energy calculation (MM/GBSA) and binding free energy decomposition were highlighted and discussed. These findings suggest that the novel scaffold afforded by compound 20 can be used as a good starting point for further modification/optimization and the clarified interaction patterns may also guide us to find more potent Smo antagonists.