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.

OBJECTIVE: Foramen magnum (FM) meningiomas pose significant surgical challenges and have high morbidity and mortality rates. This study aimed to investigate the distribution of clinically actionable mutations in FM meningiomas and identify clinical characteristics associated with specific mutational profiles.
METHODS: The authors conducted targeted next-generation sequencing of 62 FM meningiomas from three international institutions, covering all relevant meningioma genes (AKT1, KLF4, NF2, POLR2A, PIK3CA, SMO, TERT promoter, and TRAF7). Patients with a radiation-induced meningioma or neurofibromatosis type 2 (NF2) were excluded from the study. Additionally, patient and tumor characteristics, including age, sex, radiological features, and tumor location, were retrospectively collected and evaluated.
RESULTS: The study cohort consisted of 46 female and 16 male patients. Clinically significant driver mutations were detected in 58 patients (93.5%). The most commonly observed alteration was TRAF7 mutations (26, 41.9%), followed by AKT1E17K mutations (19, 30.6%). Both mutations were significantly associated with an anterolateral tumor location relative to the brainstem (p = 0.0078). NF2 mutations were present in 11 cases (17.7%) and were associated with posterior tumor location, in contrast to tumors with TRAF7 and AKT1E17K mutations. Other common mutations in FM meningiomas included POLR2A mutations (8, 12.9%; 6 POLR2AQ403K and 2 POLR2AH439_L440del), KLF4K409Q mutations (7, 11.3%), and PIK3CA mutations (4, 6.5%; 2 PIK3CAH1047R and 2 PIK3CAE545K). POLR2A and KLF4 mutations exclusively occurred in female patients and showed no significant association with specific tumor locations. All tumors harboring AKT1E17K and POLR2A mutations displayed meningothelial histology. Ten tumors exhibited intratumoral calcification, which was significantly more frequent in NF2-mutant compared with AKT1-mutant FM meningiomas (p = 0.047).
CONCLUSIONS: These findings provide important insights into the molecular genetics and clinicopathological characteristics of FM meningiomas. The identification of specific genetic alterations associated with tumor location, volume, calcification, histology, and sex at diagnosis may have implications for personalized treatment strategies in the future.

Ameloblastoma (AB) is an odontogenic tumor that arises from ameloblast-lineage cells. Although relatively uncommon and rarely metastatic, AB tumors are locally invasive and destructive to the jawbone and surrounding structures. Standard-of-care surgical resection often leads to disfigurement, and many tumors will locally recur, necessitating increasingly challenging surgeries. Recent genomic studies of AB have uncovered oncogenic driver mutations, including in the mitogen-activated protein kinase (MAPK) and Hedgehog signaling pathways. Medical therapies targeting those drivers would be a highly desirable alternative or addition to surgery; however, a paucity of existing AB cell lines has stymied clinical translation. To bridge this gap, here we report the establishment of 6 new AB cell lines-generated by \"conditional reprogramming\"-and their genomic characterization that reveals driver mutations in FGFR2, KRAS, NRAS, BRAF, PIK3CA, and SMO. Furthermore, in proof-of-principle studies, we use the new cell lines to investigate AB oncogene dependency and drug sensitivity. Among our findings, AB cells with KRAS or NRAS mutation (MAPK pathway) are exquisitely sensitive to MEK inhibition, which propels ameloblast differentiation. AB cells with activating SMO-L412F mutation (Hedgehog pathway) are insensitive to vismodegib; however, a distinct small-molecule SMO inhibitor, BMS-833923, significantly reduces both downstream Hedgehog signaling and tumor cell viability. The novel cell line resource enables preclinical studies and promises to speed the translation of new molecularly targeted therapies for the management of ameloblastoma and related odontogenic neoplasms.

Leukemia stem cells (LSCs) are responsible for leukemia initiation and targeting LSCs is one strategy to treat this disease. This study aims to target LSCs using multi-siRNA loaded antibodies modified with mesoporous silica nanoparticles (MSNs). Here, both siRNAGLI1 and siRNASMO were loaded in an anti-CD34 antibody modified with MSNs, and then, the MSN@siRNAGLI1@Antibody + MSNs@siRNASMO@Antibody cocktail was used to target LSCs. Expression levels of BCL-2 in LSCs were significantly reduced whereas Bax expression was significantly increased after treatment with nano-drug carriers. In addition, these nano-drug carriers also effectively induced the apoptosis of LSCs. The MSNs@siRNAGLI1@Antibody + MSNs@siRNASMO@Antibody cocktail significantly inhibited LSCs. In short, we constructed two target MSN nano-drug carriers where loaded siRNAs can be used in a chemotherapeutic drug cocktail to improve the treatment of leukemia.

Background and aim. Ameloblastoma is a benign, intraosseous, progressively growing, epithelial, odontogenic neoplasm. BRAF and SMO mutations have been reported in ameloblastoma. In this study, we evaluated BRAF V600E and SMO L412F mutations; and assessed the relationship between BRAF V600E mutant expression and the clinicopathological features in Japanese patients with ameloblastoma. Methods. We examined 24 formalin-fixed paraffin-embedded samples. All specimens were from patients with mandibular ameloblastoma: 20 were conventional ameloblastoma and 4 were unicystic ameloblastoma. The BRAF V600E mutation was assessed by Sanger sequencing and immunohistochemistry, and the SMO L412F mutation was assessed only by Sanger sequencing. Results. Twenty of the 24 (83%) ameloblastoma samples carried the BRAF V600E mutation; 22 of the 24 (92%) samples were immunohistochemically positive for BRAF V600E. However, the SMO L412F mutation was not detected in any of them. The BRAF V600E mutation status did not correlate with the clinicopathological features, such as age, sex, location, method, recurrence, and subtype. Conclusion. BRAF inhibitors could be a potential treatment option for Japanese patients with ameloblastoma, harboring the BRAF V600E mutation.

Cholesterol is an abundant lipid in mammalian plasma membranes that regulates the reception of the Hedgehog (Hh) signal in target cells. In vertebrates, cell-surface organelles called primary cilia function as compartments for the propagation of Hh signals. Recent structural, biochemical, and cell-biological studies have led to the model that Patched-1 (PTCH1), the receptor for Hh ligands, uses its transporter-like activity to lower cholesterol accessibility in the membrane surrounding primary cilia. Cholesterol restriction at cilia may represent the long-sought-after mechanism by which PTCH1 inhibits Smoothened (SMO), a cholesterol-responsive transmembrane protein of the G protein-coupled receptor superfamily that transmits the Hh signal across the membrane.Protein probes based on microbial cholesterol-binding proteins revealed that PTCH1 controls only a subset of the total cholesterol molecules, a biochemically defined fraction called accessible cholesterol. The accessible cholesterol pool coexists (and exchanges) with a pool of sequestered cholesterol, which is bound to phospholipids like sphingomyelin. In this chapter, we describe how to measure the accessible and sequestered cholesterol pools in live cells with protein-based probes. We discuss how to purify and fluorescently label these probes for use in flow cytometry and microscopy-based measurements of the cholesterol pools. Additionally, we describe how to modulate accessible cholesterol levels to determine if this pool regulates Hh signaling (or any other cellular process of interest).

Smoothened (SMO) antagonist Vismodegib effectively inhibits the Hedgehog pathway in proliferating cancer cells. In early stage of treatment, Vismodegib exhibited promising outcomes to regress the tumors cells, but ultimately relapsed due to the drug resistive mutations in SMO mostly occurring before (primary mutations G497W) or after (acquired mutations D473H/Y) anti-SMO therapy. This study investigates the unprecedented insights of structural and functional mechanism hindering the binding of Vismodegib with sensitive and resistant mutant variants of SMO (SMOMut ). Along with the basic dynamic understanding of Vismodegib-SMO complexes, network propagation theory based on heat diffusion principles is first time applied here to identify the modules of residues influenced by the individual mutations. The allosteric modulation by GLY497 residue in Vismodegib bound SMO wild-type (SMOWT ) conformation depicts the interconnections of intermediate residues of SMO with the atom of Vismodegib and identify two important motifs (E-X-P-L) and (Q-A-N-V-T-I-G) mediating this allosteric regulation. In this study a novel computational framework based on the heat diffusion principle is also developed, which identify significant residues of allosteric site causing drug resistivity in SMOMut . This framework could also be useful for assessing the potential allosteric sites of different other proteins. Moreover, previously reported novel inhibitor \"ZINC12368305,\" which is proven to make an energetically favorable complex with SMOWT is chosen as a control sample to assess the impact of receptor mutation on its binding and subsequently identify the important factors that govern binding disparity between Vismodegib and ZINC12368305 bound SMOWT/Mut conformations.

OBJECTIVE: To investigate the clinical features, pathologic manifestations, and biologic behaviors of a variant of ameloblastoma with basal cell features (AM-BC).
METHODS: Following retrospective review of the clinical and pathological data of six cases of AM-BC, we described their histological and immunohistochemical (IHC) features and discussed the biologic behaviors, prognoses, pathogenesis, and clinical relevance of AM-BC. Direct sequencing of polymerase chain reaction products was also performed in all cases.
RESULTS: The six cases of AM-BC involved four women and two men, aged 22-82 years. Four lesions occurred in the maxilla and two in the mandible. Histologically, the basal cells tended to be arranged as unequally sized follicles, strands, or cords of odontogenic epithelium in the connective tissue stroma. Little or no stellate reticulum was present in the central portion of the nest. Expression of CKs was consistent with other histological variants of ameloblastoma (AM), but AM-BC had significantly higher p53 and Ki-67 (p < 0.05) labeling indices than other histological variants of AM. Two patients had BRAF gene mutations.
CONCLUSIONS: Ameloblastoma with basal cell features is a very rare variant of AM. Our study showed the differences and relationships that exist between AM-BC and other variants of AM, which could enhance understanding of AM-BC.

Up-regulation of the Hedgehog (Hh) pathway is implicated in the genesis of a wide range of tumors including triple negative breast cancer (TNBC). Sonidegib is a potent and selective oral inhibitor of Smo, a key component of the Hh signaling pathway. We designed a phase I clinical study to explore the combination of sonidegib plus docetaxel (fixed dose at 75 mg/m2) in advanced TNBC patients. The primary objective was to ascertain the combination\'s maximum tolerated dose and the recommended phase II dose (RP2D), based on dose limiting toxicities (DLTs) in the first 2 cycles. A standard \"3 + 3\" design was followed including three dose levels (DL) of sonidegib: 400 mg (DL1), 600 mg (DL2), and 800 mg (DL3). Twelve patients were included. Sonidegib 800 mg orally q.d. plus docetaxel 75 mg/m2 given intravenously on day 1 of 21-day cycles was established as the RP2D. No DLTs were observed at any DL. The median number of administered cycles at DL3 was 8 (range: 6 to 9). Grade 3 adverse events (AEs) at DL3 were neutropenia (66.7%), CPK increase (33.3%), leukopenia (33.3%), and paresthesia (33.3%), grade 4 AEs were not reported at this DL. At the RP2D, the combination showed antitumor activity in three out of 10 patients with measurable disease. Median time to progression for the overall study was 42.5 days (95% Confidence Interval: 29-155), and 188 days at DL3. No drug-to-drug interactions between sonidegib and docetaxel were found in the PK assessment. Trial Registration: EudraCT study number: 2013-001750-96. Study GEICAM/2012-12. TRIAL REGISTRATION: EudraCT study number: 2013-001750-96. Study GEICAM/2012-12. ClinicalTrials.gov: NCT02027376.

BACKGROUND: The smoothened (SMO) receptor is an essential component of the Sonic hedgehog (SHH) signalling, which is associated with the development of skin basal cell carcinoma (BCC). SMO inhibitors are indicated for BCC patients when surgical treatment or radiation therapy are not possible. Unfortunately, SMO inhibitors are not always well tolerated due to severe side effects, and their therapeutical success is limited by resistance mutations.
METHODS: We investigated how common are resistance-causing mutations in two genomic databases which are not linked to BCC or other cancers, namely 1000 Genomes and ExAC. To examine the potential for combination therapy or other treatments, we further performed knowledge-based simulations of SHH signalling, in the presence or absence of SMO and PI3K/Akt inhibitors.
RESULTS: The database analysis revealed that of 18 known mutations associated with Vismodegib-resistance, three were identified in the databases. Treatment of individuals carrying such mutations is thus liable to fail a priori. Analysis of the simulations suggested that a combined inhibition of SMO and the PI3K/Akt signalling pathway may provide an effective reduction in tumour proliferation. However, the inhibition dosage of SMO and PI3K/Akt depended on the activity of phosphodiesterases (PDEs). Under high PDEs activities, SMO became the most important control node of the network. By applying PDEs inhibition, the control potential of SMO decreased and PI3K appeared as a significant factor in controlling tumour proliferation.
CONCLUSIONS: Our systems biology approach employs knowledge-based computer simulations to help interpret the large amount of data available in public databases, and provides application-oriented solutions for improved cancer resistance treatments.