Smoothened Research Articles

Synthesis and molecular docking simulations of novel azepines based on quinazolinone moiety as prospective antimicrobial and antitumor hedgehog signaling inhibitors

A series of novel azepine derivatives based on quinazolinone moiety was synthesized through the reaction of quinazolinone chalcones (2a–d) either with 2-amino aniline in acidic medium to give diazepines (3a–d) or with 2-aminophenol to offer oxazepine (4a–d). The structure of the synthesized compounds was confirmed via melting points, elemental analyses, and different spectroscopic techniques. Moreover, these newly compounds mode of action was investigated in-silico using molecular docking against the outer membrane protein A (OMPA), exo-1,3-beta-glucanase for their antimicrobial activity, and against Smoothened (SMO), transcription factor glioma-associated homology (SUFU/GLI-1), the main proteins of Hedgehog signaling pathway to inspect their anticancer potential. Our results showed that, diazepine (3a) and oxazepine (4a) offered the highest binding energy against the target OMPA/ exo-1,3-beta-glucanase proteins and exhibited the potent antimicrobial activities against E. coli, P. aeruginosa, S. aureus, B. subtilis, C. Albicans and A. flavus. As well, diazepine (3a) and oxazepine (4a) achieved the best results among the other compounds, in their binding energy against the target SMO, SUFU/GLI-1 proteins. The in-vitro cytotoxic study was done for them on panel of cancer cell lines HCT-116, HepG2, and MCF-7 and normal cell line WI-38. Conclusively, it was revealed that molecular docking in-silico simulations and the in-vitro experiments were agreed. As a result, our findings elucidated that diazepine (3a) and oxazepine (4a), have the potential to be used as antimicrobial agents and as possible cancer treatment medications.

Effectiveness of cannabidiol (CBD) on histopathological changes and gene expression in hepatocellular carcinoma (HCC) model in male rats: the role of Hedgehog (Hh) signaling pathway.

The third most prevalent malignancy to cause mortality is hepatocellular carcinoma (HCC). The Hedgehog (Hh) signaling pathway is activated by binding to the transmembrane receptor Patched-1 (PTCH-1), which depresses the transmembrane G protein-coupled receptor Smoothened (SMO). This study was performed to examine the preventative and therapeutic effects of cannabidiol in adult rats exposed to diethyl nitrosamine (DENA)-induced HCC.A total of 50 male rats were divided into five groups of 10 rats each. Group I was the control group. Group II received intraperitoneal (IP) injections of DENA for 14 weeks. Group III included rats that received cannabidiol (CBD) orally (3-30mg/kg) for 2weeks and DENA injections for 14 weeks. Group IV rats received oral CBD for 2weeks before 14weeks of DENA injections. Group V included rats that received CBD orally for 2weeks after their last injection of DENA. Measurements were made for alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and alpha fetoprotein (AFP). Following total RNA extraction, Smo, Hhip, Ptch-1, and Gli-1 expressions were measured using quantitative real-time polymerase chain reaction (qRT-PCR). A histopathological analysis of liver tissues was performed.The liver enzymes, oxidant-antioxidant state, morphological, and molecular parameters of the adult male rat model of DENA-induced HCC showed a beneficial improvement after CBDadministration. In conclusion, by focusing on the Hh signaling system, administration of CBD showed a beneficial improvement in the liver enzymes, oxidant-antioxidant status, morphological, and molecular parameters in the DENA-induced HCC in adult male rats.

Dual-Hit Strategy for Therapeutic Targeting of Pancreatic Cancer in Patient-Derived Xenograft Tumors.

Paracrine activation of pro-fibrotic hedgehog (HH) signaling in pancreatic ductal adenocarcinoma (PDAC) results in stromal amplification that compromises tumor drug delivery, efficacy, and patient survival. Interdiction of HH-mediated tumor-stroma crosstalk with smoothened (SMO) inhibitors (SHHi) "primes" PDAC patient-derived xenograft (PDX) tumors for increased drug delivery by transiently increasing vascular patency/permeability, and thereby macromolecule delivery. However, patient tumor isolates vary in their responsiveness, and responders show co-induction of epithelial-mesenchymal transition (EMT). We aimed to identify the signal derangements responsible for EMT induction and reverse them and devise approaches to stratify SHHi-responsive tumors noninvasively based on clinically-quantifiable parameters. Animals underwent diffusion-weighted magnetic resonance (DW-MR) imaging for measurement of intratumor diffusivity. In parallel, tissue-level deposition of nanoparticle probes was quantified as a marker of vascular permeability/perfusion. Transcriptomic and bioinformatic analysis was employed to investigate SHHi-induced gene reprogramming and identify key "nodes" responsible for EMT induction. Multiple patient tumor isolates responded to short-term SHH inhibitor exposure with increased vascular patency and permeability, with proportionate increases in tumor diffusivity. Nonresponding PDXs did not. SHHi-treated tumors showed elevated FGF drive and distinctly higher nuclear localization of fibroblast growth factor receptor (FGFR1) in EMT-polarized tumor cells. Pan-FGFR inhibitor NVP-BGJ398 (Infigratinib) reversed the SHHi-induced EMT marker expression and nuclear FGFR1 accumulation without compromising the enhanced permeability effect. This dual-hit strategy of SMO and FGFR inhibition provides a clinically-translatable approach to compromise the profound impermeability of PDAC tumors. Furthermore, clinical deployment of DW-MR imaging could fulfill the essential clinical-translational requirement for patient stratification.

Itraconazole halts hepatocellular carcinoma progression by modulating sonic hedgehog signaling in rats: A novel therapeutic approach

Liver cancer stands as the fourth leading global cause of death, and its prognosis remains grim due to the limited effectiveness of current medical interventions. Among the various pathways implicated in the development of hepatocellular carcinoma (HCC), the hedgehog signaling pathway has emerged as a crucial player. Itraconazole, a relatively safe and cost-effective antifungal medication, has gained attention for its potential as an anticancer agent. Its primary mode of action involves inhibiting the hedgehog pathway, yet its impact on HCC has not been elucidated. The main objective of this study was to investigate the effect of itraconazole on diethylnitrosamine-induced early-stage HCC in rats. Our findings revealed that itraconazole exhibited a multifaceted arsenal against HCC by downregulating the expression of key components of the hedgehog pathway, shh, smoothened (SMO), and GLI family zinc finger 1 (GLI1), and GLI2. Additionally, itraconazole extended survival and improved liver tissue structure, attributed mainly to its inhibitory effects on hedgehog signaling. Besides, itraconazole demonstrated a regulatory effect on Notch1, and Wnt/β-catenin signaling molecules. Consequently, itraconazole displayed diverse anticancer properties, including anti-inflammatory, antiangiogenic, antiproliferative, and apoptotic effects, as well as the potential to induce autophagy. Moreover, itraconazole exhibited a promise to impede the transformation of epithelial cells into a more mesenchymal-like phenotype. Overall, this study emphasizes the significance of targeting the hedgehog pathway with itraconazole as a promising avenue for further exploration in clinical studies related to HCC treatment.

Combined transcriptomics and proteomics studies on the effect of electrical stimulation on spinal cord injury in rats

Electrical stimulation (ES) of the spinal cord is a promising therapy for functional rehabilitation after spinal cord injury (SCI). However, the specific mechanism of action is poorly understood. We designed and applied an implanted ES device in the SCI area in rats and determined the effect of ES on the treatment of motor dysfunction after SCI using behavioral scores. Additionally, we examined the molecular characteristics of the samples using proteomic and transcriptomic sequencing. The differential molecules between groups were identified using statistical analyses. Molecular, network, and pathway-based analyses were used to identify group-specific biological features. ES (0.5 mA, 0.1 ms, 50 Hz) had a positive effect on motor dysfunction and neuronal regeneration in rats after SCI. Six samples (three independent replicates in each group) were used for transcriptome sequencing; we obtained 1026 differential genes, comprising 274 upregulated genes and 752 downregulated genes. A total of 10 samples were obtained: four samples in the ES group and six samples in the SCI group; for the proteome sequencing, 48 differential proteins were identified, including 45 up-regulated and three down-regulated proteins. Combined transcriptomic and proteomic studies have shown that the main enrichment pathway is the hedgehog signaling pathway. Western blot results showed that the expression levels of Sonic hedgehog (SHH) (P < 0.001), Smoothened (SMO) (P = 0.0338), and GLI-1 (P < 0.01) proteins in the ES treatment group were significantly higher than those in the SCI group. The immunofluorescence results showed significantly increased expression of SHH (P = 0.0181), SMO (P = 0.021), and GLI-1 (P = 0.0126) in the ES group compared with that in the SCI group. In conclusion, ES after SCI had a positive effect on motor dysfunction and anti-inflammatory effects in rats. Moreover, transcriptomic and proteomic sequencing also provided unique perspectives on the complex relationships between ES on SCI, where the SHH signaling pathway plays a critical role. Our study provides a significant theoretical foundation for the clinical implementation of ES therapy in patients with SCI.

A novel BRAF::PTPRN2 fusion in meningioma: a case report

Gene fusion events have been linked to oncogenesis in many cancers. However, gene fusions in meningioma are understudied compared to somatic mutations, chromosomal gains/losses, and epigenetic changes. Fusions involving B-raf proto-oncogene, serine/threonine kinase (BRAF) are subtypes of oncogenic BRAF genetic abnormalities that have been reported in certain cases of brain tumors, such as pilocytic astrocytomas. However, BRAF fusions have not been recognized in meningioma. We present the case of an adult female presenting with episodic partial seizures characterized by déjà vu, confusion, and cognitive changes. Brain imaging revealed a cavernous sinus and sphenoid wing mass and she underwent resection. Histopathology revealed a World Health Organization (WHO) grade 1 meningioma. Genetic profiling with next generation sequencing and microarray analysis revealed an in-frame BRAF::PTPRN2 fusion affecting the BRAF kinase domain as well as chromothripsis of chromosome 7q resulting in multiple segmental gains and losses including amplifications of cyclin dependent kinase 6 (CDK6), tyrosine protein-kinase Met (MET), and smoothened (SMO). Elevated pERK staining in tumor cells provided evidence of activated mitogen-activated protein kinase (MAPK) signaling. This report raises the possibility that gene fusion events may be involved in meningioma pathogenesis and warrant further investigation.

ACTB::GLI1 Gene Fusion Small Bowel Mesenteric Tumor; a Case Report and Review of the Literature

Abstract Introduction/Objective GLI1 encodes a transcription factor that targets cell cycle regulators affecting stem cell proliferation. Activation of the GLI (glioma-associated oncogene homologue 1). GLI activity in adult tissues is restricted but has been identified in various neoplasms, as a result of mutations in the PTCH (patched) or SMOH (smoothened) genes, encoding components of the sonic hedgehog pathway, or by amplification of GLI. The B-actin (ACTB) gene, encoding an essential cytoskeletal component, is under the control of a conserved, strong, and complex promoter that assures a high level of expression in non-muscle cells. ACTB::GLI1 gene fusions have been reported in the tongue as the pathognomonic genetic abnormality defining an unusual subset of actin-positive, perivascular tumors with t (7;12) translocation. Methods/Case Report We report a case of 53-year-old male presented to the hospital with dizziness and bloody stools. CT scan of the abdomen and pelvis revealed a 10.5 x 7.7 cm large complex cystic and solid mass of the right lower quadrant of the abdomen adjacent to the cecum and terminal ileum. The patient has a past medical history of testicular germ cell tumor and underwent orchiectomy with adjuvant chemotherapy 30 years ago. The mass was excised and histological examination of the mass showed an unencapsulated, relatively circumscribed neoplasm composed of sheets of monomorphic bland spindle to ovoid cells. Immunohistochemical (IHC) stains, including pan- CK, SALL4, c-Kit, CD34, SMA, MDM2, HMB45, and MART1, were all negative. NGS sarcoma fusion panel was done and revealed ACTB::GLI1 gene fusion. The tumor was finally diagnosed as GLI1 -rearranged enteric tumor. Results (if a Case Study enter NA) NA Conclusion our case, in addition to, previously reported cases in the literature support the existence of a newly identified, seemingly discrete group of soft tissue tumors at different body locations. The morphology of these tumors is somewhat variable, making identifying and diagnosing these tumors more challenging. In addition to the morphologic variance, the diagnosis of these tumors in unusual locations is not uncommon, with some variations in IHC staining patterns. Identifying the molecular pattern of these tumors and the biological mechanism will facilitate more effective treatment options for patients, including targeted therapy.

Fully activated structure of the sterol-bound Smoothened GPCR-Gi protein complex.

Smoothened (SMO) is an oncoprotein and signal transducer in the Hedgehog signaling pathway that regulates cellular differentiation and embryogenesis. As a member of the Frizzled (Class F) family of G protein-coupled receptors (GPCRs), SMO biochemically and functionally interacts with Gi family proteins. However, key molecular features of fully activated, G protein-coupled SMO remain elusive. We present the atomistic structure of activated human SMO complexed with the heterotrimeric Gi protein and two sterol ligands, equilibrated at 310 K in a full lipid bilayer at physiological salt concentration and pH. In contrast to previous experimental structures, our equilibrated SMO complex exhibits complete breaking of the pi-cation interaction between R4516.32 and W5357.55, a hallmark of Class F receptor activation. The Gi protein couples to SMO at seven strong anchor points similar to those in Class A GPCRs: intracellular loop 1, intracellular loop 2, transmembrane helix 6, and helix 8. On the path to full activation, we find that the extracellular cysteine-rich domain (CRD) undergoes a dramatic tilt, following a trajectory suggested by positions of the CRD in active and inactive experimental SMO structures. Strikingly, a sterol ligand bound to a shallow transmembrane domain (TMD) site in the initial structure migrates to a deep TMD pocket found exclusively in activator-bound SMO complexes. Thus, our results indicate that SMO interacts with Gi prior to full activation to break the molecular lock, form anchors with Gi subunits, tilt the CRD, and facilitate migration of a sterol ligand in the TMD to an activated position.

Elucidating the Role of miRNA-326 Modulating Hedgehog Signaling in Pancreatic Carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the lethal malignancies worldwide characterized by poor prognosis. MicroRNAs (miRNAs) function as the key regulators in carcinogenesis and may act as noninvasive biomarkers in various malignancies including PDAC. The present study aimed to elucidate the role of miR-326, a known modulator of hedgehog (Hh) pathway in PDAC. miR-326 circulating levels were assessed in 105 PDAC patients, 31 with chronic pancreatitis (CP) and 36 healthy controls by quantitative Polymerase chain reaction. The expression of miR-326 and smoothened (SMO) was checked in surgical PDAC tissue. SMO protein expression was analyzed by immunohistochemistry in different groups. Finally, the role of miR-326 as a modulator of Hh pathway was assessed in vitro. Our results demonstrate that miR-326 is downregulated in both blood and tissue of PDAC patients as compared with controls. In contrast, the target gene/protein expression of SMO is upregulated in PDAC. Moreover, the tumor stromal expression of SMO was found to be clinically associated with lymph-node metastasis and vascular encasement in PDAC. Overexpression of miR-326 in Panc1 cell line was found to induce downregulation of SMO suggesting the tumor suppressor role of miR-326 in PDAC. Taken together, miR-326 acts as a tumor suppressor in PDAC by modulating Hh pathway. It may be a promising target for the development of efficient drug therapies for the treatment of PDAC.

Asymmetric Synthesis of Cyclopamine, a Hedgehog (Hh) Signaling Pathway Inhibitor.

Cyclopamine is a teratogenic steroidal alkaloid, which inhibits the Hedgehog (Hh) signaling pathway by targeting the Smoothened (Smo) receptor. Suppression of Hh signaling with synthetic small molecules has been pursued as a therapeutic approach for the treatment of cancer. We report herein the asymmetric synthesis of cyclopamine based on a two-stage relay strategy. Stage-I: total synthesis of veratramine through a convergent approach, wherein a crucial photoinduced excited-state Nazarov reaction was applied to construct the basic [6-6-5-6] skeleton of C-nor-D-homo-steroid. Stage-II: conversion of veratramine to cyclopamine was achieved through a sequence of chemo-selective redox manipulations.

Deciphering the Role of RAS Pathway Mutations in the Biology of Human Acute Myeloid Leukemia Using In Vivo Models

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy for which the mutational profile of the leukemic cells is the most important prognostic factor for disease-free survival and overall survival. The failure of chemotherapy to completely eradicate the leukemic clones is the most common cause of death in AML. The genetic events that can contribute to chemoresistance are therefore important factors for the clinical outcome, but the relative importance of these events on chemoresistance and persistence of leukemia stem cells (LSC) remains poorly defined. MLL rearranged AMLs account for 5-10% of all AMLs and the RAS pathway is the most frequently mutated pathway in this sub-group of AMLs. The goal of this project is to study the role and biology of RAS pathway mutations in clonal evolution in human MLL-rearranged AML. Methods We have previously shown that human cord blood CD34+ cells transduced with a retrovirus encoding the MLL-AF9 fusion gene with an EGFP maker generate human AML when injected into immunodeficient NSG-SGM3 mice. Using a similar experimental design, we added a second hit to the CD34 transduced cells before their injection into NSG-SGM3 mice. MLL-AF9 cells were electroporated with Cas9 enzyme for the control and with Cas9 enzyme plus a single guide RNA (sgRNA) designed to induce the KRAS G13D or the NRAS G12D mutation. Mice were sacrificed when they showed sign of disease and the bone marrow and spleen were collected and analyzed. Kaplan-Meier curves, flow cytometry analyses, RNA and exome sequencing and secondary transplantation were performed on the samples. Results Mice injected with MLL-AF9 + KRAS G13D got sick around day 80 after injection and around day 60 for MLL-AF9 NRAS G12D, whereas it took ~150 days for control mice (MLL-AF9 KRAS WT) to get sick (fig 1). In multiple experiment, the latency was roughly cut by half by the KRAS G13D and NRAS G12D mutations. The cell surface phenotype and morphology were not significantly different between MLL-AF9 WT and MLL-AF9 KRAS/NRAS mutated. The variant allele frequency (VAF) for the KRAS mutation invariably increased from the day of injection to the day of sacrifice, where the percentages varied between 20-40%, which is typically observed in AML patients. In contrast, the VAF for NRAS mutated cells was lower (3-9%) but seems to be very aggressive into mice. We performed limiting dilution analyses in secondary mice to quantify the LSC frequencies in MLL-AF9 WT and KRAS G13D / NRAS G12D leukemia. The average frequencies of control MLL-AF9 AMLs were between 1/510 000 to 1/1 450 000, while MLL-AF9+KRAS G13D AMLs frequencies were &amp;gt;1/45 000 and 1/7 800 for MLL-AF9+NRAS G12D AMLs, demonstrating that these 2 mutations increase the LSC frequency by &amp;gt;20-100 fold in our modelled MLL-AF9 AML. Using transcriptome analyses, we looked at the gene expression changes induced by the KRAS G13D mutation in comparison to MLL-AF9 leukemias. These experiments are in progress for the NRAS G13D mutation. Many more genes were overexpressed in MLL-AF9 KRAS G13D vs MLL-AF9 KRAS WT than down regulated. Genes from the hedgehog (Hh) pathway such as Patched1 (PTCH1) and Smoothened (SMO) were amongst the genes the most differentially overexpressed by the KRAS mutation. It is particularly interesting because they have been shown important in stem cell biology and could explain the increase in LSC. Some SMO inhibitors have been tested in clinical trial and Glasdegib is FDA approved in combination with low dose cytarabine in elderly patients. Its effect on MLL leukemias or on KRAS mutated AMLs has not been tested/reported in a clinical study. Experiments are currently in progress to test the effect of Glasgebib on engraftment of MLL-AF9 KRAS mutated cells in our experimental setting. Single cell RNA sequencing data on the three conditions will be presented at the meeting. Given the success of generating these two point mutations, we are currently generating more RAS pathway mutations, including other KRAS mutations, PNTP11 and NF1 mutations. Conclusion These experiments showed: 1) It is possible to induce 2 oncogenic hits in human primary cells and get leukemia in vivo; 2) the KRAS G13D and NRAS G12D mutations shorten the latency of the disease and 3) increase the LSC frequency in secondary mice; 4) a possible involvement of the Hh pathway on stemness/LSC in RAS mutated cells; 5) our experimental approach is robust and very promising to decipher the RAS pathway in human MLL leukemias.

Putative Role of Cholesterol in Shaping the Structural and Functional Dynamics of Smoothened (SMO).

The smoothened (SMO) receptor belongs to the superfamily of class F G protein-coupled receptors (GPCRs) and is a potential drug target in several types of cancer. It has two ligand binding sites, respectively, in the cysteine-rich domain (CRD) and the transmembrane domain (TMD). It has been shown that cholesterol is important for its activation and function. However, the molecular-level understanding of SMO dynamics in the presence of cholesterol has not been explored in sufficient detail. In this work, we have carried out atomistic molecular dynamics simulations totaling 3.6 μs to analyze the effect of cholesterol binding to TMD and/or CRD on the structure and dynamics of the SMO receptor. Our results show that the presence of cholesterol in the CRD and TMD, respectively, alters the conformational dynamics of SMO differently. We reported that the reorganization of the D-R-E network at the extracellular end of the TMD is important for the high activity of SMO. In general, the transmembrane helices 5, 6, and 7 and helix 8 are most affected, which, in turn, leads to changes in the CRD and intracellular cytoplasmic domain (ICD) dynamics patterns depending on the presence or absence of cholesterol in the CRD and/or the TMD. We have also reported that the interaction of membrane lipids with SMO is different in different SMO states. These results agree with the experimental structural observations and data of cholesterol-bound and unbound structures of SMO and add to our molecular understanding of the SMO-cholesterol interaction.

Clinicopathological relevance of BRAF and SMO mutations in Chinese patients with ameloblastoma

Ameloblastomas are benign tumors associated with recurrence and morbidity. Mutations in the V-Raf murine sarcoma viral oncogene homolog B1 (BRAF) and smoothened (SMO) genes have been identified in ameloblastomas. The mutation rate of BRAF in different regions and mutation sites of SMO remains controversial. This study assesses the relationship between these mutations and clinicopathological features of ameloblastoma in Chinese patients. Ameloblastomas were surgically removed from 30 patients and BRAF gene polymorphisms were detected using DNA sequencing analysis. We also examined the relationship between BRAF or SMO mutations and clinicopathological parameters. BRAF V600E mutation was detected in 18 of 30 ameloblastomas. No significant correlation was found between BRAF V600E mutation and age, sex, location, histologic type, capsular invasion, or tumor recurrence. Five cases carried an SMO T364N mutation in exon 5, whereas six carried an SMO S590 T mutation in exon 10. The SMO mutation rate in patients aged ≤ 20 years was significantly higher than that in patients aged > 20 years. SMO T364N and S590 T mutations were closely related to age and capsular invasion. These findings indicate that BRAF V600E, SMO T364N, and SMO S590 T mutations play important roles in the pathogenesis of ameloblastoma. Key highlights The BRAF V600E mutation was highly prevalent in Chinese patients with ameloblastoma. SMO T364N and S590 T mutations are closely related to age and capsular invasion. The BRAF V600E, SMO T364N, and SMO S590 T mutations play important roles in the pathogenesis of ameloblastoma.

Ligand-dependent hedgehog signaling maintains an undifferentiated, malignant osteosarcoma phenotype

TP53 and RB1 loss-of-function mutations are common in osteosarcoma. During development, combined loss of TP53 and RB1 function leads to downregulation of autophagy and the aberrant formation of primary cilia, cellular organelles essential for the transmission of canonical Hedgehog (Hh) signaling. Excess cilia formation then leads to hypersensitivity to Hedgehog (Hh) ligand signaling. In mouse and human models, we now show that osteosarcomas with mutations in TP53 and RB1 exhibit enhanced ligand-dependent Hh pathway activation through Smoothened (SMO), a transmembrane signaling molecule required for activation of the canonical Hh pathway. This dependence is mediated by hypersensitivity to Hh ligand and is accompanied by impaired autophagy and increased primary cilia formation and expression of Hh ligand in vivo. Using a conditional genetic mouse model of Trp53 and Rb1 inactivation in osteoblast progenitors, we further show that deletion of Smo converts the highly malignant osteosarcoma phenotype to benign, well differentiated bone tumors. Conversely, conditional overexpression of SHH ligand, or a gain-of-function SMO mutant in committed osteoblast progenitors during development blocks terminal bone differentiation. Finally, we demonstrate that the SMO antagonist sonidegib (LDE225) induces growth arrest and terminal differentiation in vivo in osteosarcomas that express primary cilia and Hh ligand combined with mutations in TP53. These results provide a mechanistic framework for aberrant Hh signaling in osteosarcoma based on defining mutations in the tumor suppressor, TP53.

Increasing Ciliary ARL13B Expression Drives Active and Inhibitor-Resistant Smoothened and GLI into Glioma Primary Cilia.

ADP-ribosylation factor-like protein 13B (ARL13B), a regulatory GTPase and guanine exchange factor (GEF), enriches in primary cilia and promotes tumorigenesis in part by regulating Smoothened (SMO), GLI, and Sonic Hedgehog (SHH) signaling. Gliomas with increased ARL13B, SMO, and GLI2 expression are more aggressive, but the relationship to cilia is unclear. Previous studies have showed that increasing ARL13B in glioblastoma cells promoted ciliary SMO accumulation, independent of exogenous SHH addition. Here, we show that SMO accumulation is due to increased ciliary, but not extraciliary, ARL13B. Increasing ARL13B expression promotes the accumulation of both activated SMO and GLI2 in glioma cilia. ARL13B-driven increases in ciliary SMO and GLI2 are resistant to SMO inhibitors, GDC-0449, and cyclopamine. Surprisingly, ARL13B-induced changes in ciliary SMO/GLI2 did not correlate with canonical changes in downstream SHH pathway genes. However, glioma cell lines whose cilia overexpress WT but not guanine exchange factor-deficient ARL13B, display reduced INPP5e, a ciliary membrane component whose depletion may favor SMO/GLI2 enrichment. Glioma cells overexpressing ARL13B also display reduced ciliary intraflagellar transport 88 (IFT88), suggesting that altered retrograde transport could further promote SMO/GLI accumulation. Collectively, our data suggest that factors increasing ARL13B expression in glioma cells may promote both changes in ciliary membrane characteristics and IFT proteins, leading to the accumulation of drug-resistant SMO and GLI. The downstream targets and consequences of these ciliary changes require further investigation.

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.

P20.16.B FIRST-LINE TREATMENT OF MEDULLOBLASTOMA IN ADOLESCENTS AND ADULTS - UPDATE ON THE EORTC 1634-BTG/NOA-23 PHASE II TRIAL

Abstract BACKGROUND Medulloblastoma is a common brain malignancy in children. Patients after puberty are rare and bear an intermediate prognosis. Standard treatment consists of maximal resection plus radio-chemotherapy. Treatment toxicity is high and produces disabling long-term side effects. The sonic hedgehog (SHH) subgroup is highly overrepresented in the post-pubertal and adult population and can be targeted by smoothened (SMO) inhibitors. No practice-changing prospective randomized data have been generated in adults so far. MATERIAL AND METHODS EORTC 1634-BTG/NOA-23 trial randomizes patients between standard-dose vs. reduced-dosed craniospinal radiotherapy and SHH-subgroup patients between the SMO inhibitor sonidegib (OdomzoTM, Sun Pharmaceuticals Industries, Inc., New York, USA) and standard chemotherapy. Based on data that suggest equal efficacy, a reduction of toxicity with dose-reduced CSI while maintaining efficacy is investigated by using 35.2 Gy in the standard vs. 24.6 Gy in the experimental arm. Chemotherapy consists of vincristine during radiotherapy, followed by a maximum of six cycles of lomustine, cisplatin and vincristine of six-weekly cycles. Sonidegib is added for patients with SHH-activated medulloblastoma. An increase in efficacy in the SHH subgroup is the primary efficacy endpoint. A number of translational research projects are performed alongside the trial. RESULTS The trial aims at developing a personalized, genotype-based, intensity-modulated therapy for post-pubertal and adult patients with newly diagnosed medulloblastoma. We are investigating tumor tissue, blood, and cerebrospinal fluid as well as magnetic resonance imaging and radiotherapy plans to generate information that helps to further improve treatment outcomes. Long-term follow-up is performed to monitor classic side effects of therapy, but also health-related quality of life, cognition, social and professional outcome, and reproduction and fertility. The 1634-BTG/NOA-23 trial started recruitment in November 2022. Updated information on recruitment as well as on demographic data of recruited patients will be presented at the EANO annual meeting. CONCLUSION EORTC 1634-BTG/NOA-23 is the first trial worldwide that prospectively enrolls post-pubertal and adult patients with medulloblastoma according to their molecular subgroup in a randomized design. Translational subprojects will enable us to evaluate events on several layers to gain deeper insights into subgrouping, risk stratification, therapeutic targets, resistance mechanisms, and the toxicity of treatments. The trial will therefore generate unprecedented data that will be translated into treatment changes in post-pubertal patients with medulloblastoma and will help to design future clinical trials.

P04.04.A GENOME-WIDE CRISPR/CAS9 KNOCKOUT SCREENS REVEAL EPIGENETIC REGULATORS AS DRUGGABLE TARGETS FOR SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract BACKGROUND Sonic hedgehog medulloblastoma (SHH-MB) is a malignant pediatric brain tumor that accounts for 30% of all MBs and is characterized by constitutive activation of the SHH signaling pathway. Besides the standard-of-care therapies, current therapies are targeting Smoothened (SMO) receptor, in order to inhibit the pathway. However, acquired or de novo resistance against SMO inhibition limits its efficacy. Therefore, our aim is to unravel novel therapeutic targets for SHH-MB, potentially beyond the SHH pathway. MATERIAL AND METHODS We performed genome-wide CRISPR/Cas9 knockout screens, in order to decipher cancer dependencies and synthetic lethal interactors for SHH-MB. The hits identified were first validated genetically and pharmacologically in in vitro assays, while further molecular analyses including RNAseq and differential methylation were also conducted. Moreover, the identified hits were evaluated in in vivo mouse models representing normal cerebellar, as well as SHH-MB development. RESULTS Functional genomics identified members of the epigenetic machinery, such as Dnmt1 and Smarca5 as context-specific dependencies for SHH-MB. We prove that DNMT1 inhibition is efficacious against SMO-resistant and sensitive SHH-MB cell lines, by inhibiting SHH pathway output, as shown via RNAseq. A further knockout drug screen unraveled novel synthetic lethal interactors for DNMT1 inhibitors. Genetic ablation and pharmacological inhibition of epigenetic regulators prolongs survival of established mouse models of SHH-MB. CONCLUSION Summarizing, our data indicate inhibitors of epigenetic regulators as novel therapeutic targets for SHH-MB irrespective of mutational drivers within the SHH pathway. We further propose combinatorial treatment approaches for this tumor entity that involve DNMT1 inhibitors.

The energetics and ion coupling of cholesterol transport through Patched1.

Patched1 (PTCH1) is a tumor suppressor protein of the mammalian Hedgehog (HH) signaling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating ciliary cholesterol accessibility. Using extensive molecular dynamics simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15 to 20 kilojoule per mole for cholesterol export. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between cation binding sites, transmembrane motions, and PTCH1 activity. Using complementary simulations of Dispatched1, we find that transition between "inward-open" and solvent "occluded" states is accompanied by Na+-induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion coupling stoichiometries of PTCH1 transport mechanisms, whereby one to three Na+ or two to three K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.

Polymeric micelles for cutaneous delivery of the hedgehog pathway inhibitor TAK-441: Formulation development and cutaneous biodistribution in porcine and human skin

TAK-441 is a potent inhibitor of the hedgehog pathway (IC50 4.4 nM) developed for the treatment of basal cell carcinoma that is active against the vismodegib-resistant Smoothened receptor D473H mutant. The objective of this study was to develop a micelle-based formulation of TAK-441 using D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) and to investigate its cutaneous delivery and biodistribution. The micelles were prepared using solvent evaporation and incorporation of TAK-441 in the TPGS micelles increased aqueous solubility ∼40-fold. The optimal formulation, a 3% HPMC hydrogel of TAK-441 loaded TPGS micelles, retained ∼92% of the initial TAK-441 content (2.5 mgTAK-441/g) after storage at 4 °C for 6 months. Finite dose experiments using human skin demonstrated that this formulation resulted in significantly greater cutaneous deposition of TAK-441 after 12 h than a non-micelle control formulation, (0.40 ± 0.11 µg/cm2 and 0.05 ± 0.02 µg/cm2, respectively) – no transdermal permeation was observed. The cutaneous biodistribution profile demonstrated that TAK-441 was predominantly delivered to the viable epidermis and upper dermis. Delivery from the HPMC hydrogel formulation resulted in TAK-441 epidermal concentrations that were several thousand-fold higher than the IC50, with almost negligible transdermal permeation, thereby decreasing the risk of systemic side effects in vivo.