The mTORC1-complex is negatively regulated by TSC1 and TSC2. Activation of Hedgehog signaling is strictly dependent on communication between Smoothened and the Hedgehog-signaling effector and transcription factor, GLI2, in the primary cilium. Details about this communication are not known, and we wanted to explore this further. Here we report that in Tsc2 -/- MEFs constitutively activated mTORC1 led to mis-localization of Smoothened to the plasma membrane, combined with increased concentration of GLI2 in the cilia and reduced Hedgehog signaling, measured by reduced expression of the Hedgehog target gene, Gli1 Inhibition of mTORC1 rescued the cellular localization of Smoothened to the cilia, reduced the cilia concentration of GLI2, and restored Hedgehog signaling. Our results reveal evidence for a two-step activation process of GLI2. The first step includes GLI2 stabilization and cilium localization, whereas the second step includes communication with cilia-localized Smoothened. We found that mTORC1 inhibits the second step. This is the first demonstration that mTORC1 is involved in the regulation of Hedgehog signaling.

OBJECTIVE: Smoothened (SMO), a key component of the hedgehog signaling pathway, represents a therapeutic target for triple negative breast cancer (TNBC), yet the chemotherapy response rate in TNBC patients is only 40-50%, underscoring the urgent need for the development of novel drugs to effectively treat this condition. The novel compound TPB15, an SMO inhibitor derived from [1,2,4] triazolo [4,3-α] pyridines, demonstrated superior anti-TNBC activity and lower toxicity compared to the first SMO inhibitor vismodegib in both in vitro and in vivo. However, the compound\'s pharmacokinetic properties remain unclear. The present work aims to develop a simple HPLC-MS/MS method to profile the pharmacokinetics and bioavailability of TPB15 in rats as a ground work for further clinical research.
METHODS: Separation was performed on an Agilent ZORBAX StableBond C18 column by gradient elution using acetonitrile and 0.1% formic acid as mobile phase at a flow rate of 0.3 mL/min. Multiple reaction monitoring(MRM) in positive mode with the transitions of m/z 454.2 → 100.0, 248.1 → 121.1 was employed to determine TPB15 and internal standard tinidazole, respectively. The specificity, intra- and inter- day precision and accuracy, extraction recovery, stability, matrix effect, dilution integrity and carryover of the method was validated. The pharmacokinetics and bioavailability  study of TPB15 were carried out on rats through intravenous injection at the dose of 5 mg/kg and oral gavage at the dose of 25 mg/kg, and the pharmacokinetics parameters were calculated by the non-compartment analysis using the pharmacokinetics software DAS 2.1.1.
RESULTS: The values of specificity, intra- and inter- day precision and accuracy, extraction recovery, stability, matrix effect, dilution integrity and carryover satisfied the acceptable limits. The lower limit of quantification of this method was 10 ng/mL with a linear range of 10-2000 ng/mL. The validated method was then applied to pharmacokinetics and bioavailability studies in rat by dosing with gavage (25 mg/kg) and intravenous injection(5 mg/kg), and the oral bioavailability of TBP15 in rat was calculated as 16.4 ± 3.5%. The pharmacokinetic parameters were calculated as following: maximum of plasma concentration (Cmax) (PO: 2787.17 ± 279.45 µg/L), Time to maximum plasma concentration (Tmax) (PO: 4.20 ± 0.90 h), the area under the concentration-time curve 0 to time (AUC0-t) (PO: 17,373.03 ± 2585.18 ng/mL·h, IV: 21,129.79 ± 3360.84 ng/mL·h), the area under the concentration-time curve 0 to infinity (AUC0-∞) (PO: 17,443.85 ± 2597.63 ng/mL·h, IV: 17,443.85 ± 2597.63 ng/mL·h), terminal elimination half-life (t1/2) (PO: 7.26 ± 2.16 h, IV: 4.78 ± 1.09 h).
CONCLUSIONS: TPB15, a promising candidate for treating TNBC, has demonstrated outstanding efficacy and safety in vitro and in vivo. This study established a simple, sensitive, and rapid HPLC-MS/MS bioanalytical method, developed and validated in accordance with FDA and EMA guidelines, for conducting pharmacokinetic and bioavailability studies of TPB15. The results revealed a favorable pharmacokinetic profile owing to its long t1/2. Nevertheless, the next phase of research should include formulation screening to enhance bioavailability, as well as clinical trials, metabolism pathway analysis, and assessment of potential drug-drug interactions.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

Sonic hedgehog subgroup of medulloblastoma (SHH-MB) is characterized by aberrant activation of the SHH signaling pathway. An inhibition of the positive SHH regulator Smoothened (SMO) has demonstrated promising clinical efficacy. Yet, primary and acquired resistance to SMO inhibitors limit their efficacy. An understanding of underlying molecular mechanisms of resistance to therapy is warranted to bridge this unmet need. Here, we make use of genome-wide CRISPR-Cas9 knockout screens in murine SMB21 and human DAOY cells, in order to unravel genetic dependencies and drug-related genetic interactors that could serve as alternative therapeutic targets for SHH-MB. Our screens reinforce SMB21 cells as a faithful model system for SHH-MB, as opposed to DAOY cells, and identify members of the epigenetic machinery including DNA methyltransferase 1 (DNMT1) as druggable targets in SHH-dependent tumors. We show that Dnmt1 plays a crucial role in normal murine cerebellar development and is required for SHH-MB growth in vivo. Additionally, DNMT1 pharmacological inhibition alone and in combination with SMO inhibition effectively inhibits tumor growth in murine and human SHH-MB cell models and prolongs survival of SHH-MB mouse models by inhibiting SHH signaling output downstream of SMO. In conclusion, our data highlight the potential of inhibiting epigenetic regulators as a novel therapeutic avenue in SMO-inhibitor sensitive as well as resistant SHH-MBs.

Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 μM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 μM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1-/- mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.

The purpose of this study was to investigate the effects of BOC on glioblastoma cells and its underlying mechanisms. In vitro, BOC-knockdown was performed in glioma cell lines. CCK-8 and Transwell were used to assess the impact of BOC on the viability, invasion, and migration of gliobma cells. RNA-seq technology was employed to analyze the differential gene expression between BOC-knockdown glioma cells and the control group, and qRT-PCR was used to validate the expression of downstream differential genes. SMO-overexpression was performed to investigate the effects of SMO on glioma cells. A BOC-knockdown mouse subcutaneous tumor model was to verify the effects of BOC on mouse tumors. Tissue microarray technology was used to detect the expression of BOC and SMO in samples of normal human brain tissue and glioma tissue. In vitro, BOC-knockdown inhibited the viability, invasion, and migration of glioma cells, as well as downregulated the expression of downstream differential genes SMO, EGFR, HRAS, and MRAS. Conversely, SMO-overexpression upregulated the viability, invasion, and migration abilities of BOC-knockdown cells. In vivo, BOC-knockdown suppressed tumor growth in mice and downregulated the expression of downstream differential genes SMO, EGFR, HRAS, and MRAS. Tissue microarray results showed that both BOC and SMO were highly expressed in glioma tissues. BOC is aberrantly overexpressed in glioma patients and promotes glioma development. Mechanistically, BOC activates the Hedgehog (Hh) and RAS signaling pathways by upregulating the expression of SMO, EGFR, HRAS, and MRAS, thereby facilitating the Proliferation, invasion and migration of glioma cells.

Asthma is the most prevalent chronic inflammatory disease of the airways in children. The most prevalent phenotype of asthma is eosinophilic asthma, which is driven by a Th2 immune response and can be effectively managed by inhaled corticosteroid therapy. However, there are phenotypes of asthma with Th17 immune response that are insensitive to corticosteroid therapy and manifest a more severe phenotype. The treatment of this corticosteroid-insensitive asthma is currently immature and requires further attention. The objective of this study is to elucidate the regulation of the Hedgehog signaling pathway in Th17 cell differentiation in asthma. The study demonstrated that both Smo and Gli3, key components of the Hedgehog signaling pathway, were upregulated in Th17 polarization in vitro and in a Th17-dominant asthma model in vivo. Inhibiting Smo with a small molecule inhibitor or genetically knocking down Gli3 was found to suppress Th17 polarization. Smo was found to increase in Th1, Th2, Th17 and Treg polarization, while Gli3 specifically increased in Th17 polarization. ChIP-qPCR analyses indicated that Gli3 can directly interact with IL-6 in T cells, inducing STAT3 phosphorylation and promoting Th17 cell differentiation. Furthermore, the study demonstrated a correlation between elevated Gli3 expression and IL-17A and IL-6 expression in children with asthma. In conclusion, the study demonstrated that the Hedgehog signaling pathway plays an important role in the pathogenesis of asthma, as it regulates the differentiation of Th17 cells through the IL-6/STAT3 signaling. This may provide a potential therapeutic target for corticosteroid-insensitive asthma driven by Th17 cells.

OBJECTIVE: Apart from the role of the retinoblastoma gene, the genomic events associated with poor outcomes in patients with ophthalmic tumors are poorly understood.
METHODS: We retrospectively analyzed 48 patients with six types of ophthalmic tumors. We searched for high-frequency mutated genes and susceptibility genes in these patients using combined exome and transcriptome analysis.
RESULTS: We identified four clearly causative genes (TP53, PTCH1, SMO, BAP1). Susceptibility gene analysis identified hotspot genes, including RUNX1, APC, IDH2, and BRCA2, and high-frequency gene analysis identified several genes, including TP53, TTN, and MUC16. Transcriptome analysis identified 5868 differentially expressed genes, of which TOP2A and ZWINT were upregulated in all samples, while CFD, ELANE, HBA1, and HBB were downregulated. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the phosphoinositide 3-kinase (PI3K)-Akt and Transcriptional misregulation in cancer signaling pathways may be involved in ophthalmic tumorigenesis.
CONCLUSIONS: TP53 is clearly involved in ophthalmic tumorigenesis, especially in basal cell carcinoma, and the PI3K-Akt signaling pathway may be an essential pathway involved in ophthalmic tumorigenesis. RUNX1, SMO, TOP2A, and ZWINT are also highly likely to be involved in ophthalmic tumorigenesis, but further functional experiments are needed to verify the mechanisms of these genes in regulating tumorigenesis.

In this issue of Cell Chemical Biology, Liu et al.1 report the identification of Q29, a synthetic diterpenoid that blocks covalent cholesterol modification of smoothened (SMO) and inhibits hedgehog signaling. Q29 is capable of suppressing tumor cell growth, both in vitro and in vivo, and overcoming resistance to SMO inhibitors.

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.