gdc-0449 and Medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumour in children but also rarely occur in adults. Sonic Hedgehog (SHH) driven MB is associated with aberrant activation of the SHH signalling pathway. SMO inhibitors, sonidegib and vismodegib, have been used as selective antagonist of the hedgehog pathway that acts by binding to SMO, and inhibits activation of the downstream hedgehog target genes. Several clinical trials investigating SMO inhibitors for the treatment of relapsed MB patients have been published.. We conducted a systemic review and meta-analysis among these Phase I and II clinical trials. The pooled effect of SMO inhibitors in relapsed MB were analysed using Reviewer Manager 5.3 software. The clinical efficacy of SMO inhibitors on SHH subtype of MB were measured by the objective response rate. The risk difference was obtained by comparing the ORR between SHH and non-SHH subtypes of MB.. Sonidegib and vismodegib were well tolerated and demonstrated anti-tumour activity in SHH-driven paediatric and adult MB by effectively inhibiting Hh signalling. These results support the ongoing clinical trials using SMO inhibitors in combination with conventional chemotherapies for the treatment of relapsed MB

Topics: Anilides; Antineoplastic Agents; Biphenyl Compounds; Cerebellar Neoplasms; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Hedgehog Proteins; Humans; Medulloblastoma; Pyridines; Signal Transduction; Smoothened Receptor; Treatment Outcome

Medulloblastoma is the most common malignant brain tumor of childhood. It is currently stratified into four molecular variants through the advances in transcriptional profiling. They include: wingless, sonic hedgehog (SHH), Group III, and Group IV. The SHH group is characterized by constitutive activation of the SHH signaling pathway, and genetically characterized by mutations in patched homolog 1 (PTCH1) or other downstream pathway mutations. SHH inhibitors have become of great clinical interest in treating SHH-driven medulloblastoma. Many inhibitors are currently in different stages of development, some already approved for other SHH-driven cancers, such as basal cell carcinoma. In vitro and in vivo medulloblastoma studies have shown efficacy and these findings have been translated into Phase I and II clinical trials. In this review, we present an overview of SHH medulloblastoma, as well as a discussion of currently available SHH inhibitors, and the challenges associated with their use.

Topics: Anilides; Animals; Antineoplastic Agents; Cerebellar Neoplasms; Hedgehog Proteins; Humans; Medulloblastoma; Pyridines

The hedgehog signaling pathway is important in embryogenesis and post natal development. Constitutive activation of the pathway due to mutation of pathway components occurs in ~25% of medulloblastomas and also in basal cell carcinomas. In many other malignancies the therapeutic role for hedgehog inhibition though intriguing, based on preclinical data, is far from assured. Hedgehog inhibition is not an established part of the treatment paradigm of sarcoma but the scientific rationale for a possible benefit is compelling. In chondrosarcoma there is evidence of hedgehog pathway activation and an ontologic comparison between growth plate chondrocyte differentiation and different chondrosarcoma subtypes. Immunostaining epiphyseal growth plate for Indian hedgehog is particularly positive in the zone of pre-hypertrophic chondrocytes which correlates ontologically with conventional chondrosarcoma. In Ewing sarcoma/PNET tumors the Gli1 transcription factor is a direct target of the EWS-FLI1 oncoprotein present in 85% of cases. In many cases of rhabdomyosarcomas there is increased expression of Gli1 (Ragazzini et al., 2004). Additionally, a third of embryonal rhabdomyosarcomas have loss of Chr.9q22 that encompasses the patched locus (Bridge et al., 2000). The potential to treat osteosarcoma by inhibition of Gli2 and the role of the pathway in ovarian fibromas and other connective tissue tumors is also discussed (Nagao et al., 2011; Hirotsu et al., 2010). Emergence of acquired secondary resistance to targeted therapeutics is an important issue that is also relevant to hedgehog inhibition. In this context secondary resistance of medulloblastomas to treatment with a smoothened antagonist in two tumor mouse models is examined.

Topics: Anilides; Animals; Apoptosis; Basal Cell Nevus Syndrome; Carcinoma, Basal Cell; Chondrosarcoma; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Medulloblastoma; Mesenchymal Stem Cells; Osteosarcoma; Pyridines; Sarcoma; Signal Transduction; Zinc Finger Protein Gli2

Acquired resistance to targeted therapies threatens the value of these otherwise very promising agents. The recent description of resistance to the Hedgehog pathway inhibitor vismodegib (GDC-0449) in a medulloblastoma patient who had a dramatic initial response has spurred efforts to understand potential mechanisms of drug resistance. Elucidating these mechanisms will play a significant role in informing strategies to overcome this meaningful limitation.

Topics: Anilides; Animals; Antineoplastic Agents; Basal Cell Nevus Syndrome; Biphenyl Compounds; Carcinoma, Basal Cell; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Medulloblastoma; Mice; Mice, Knockout; Mice, Nude; Molecular Targeted Therapy; Mutation, Missense; Neoplasm Proteins; Patched Receptors; Phosphoinositide-3 Kinase Inhibitors; Pyridines; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Signal Transduction; Skin Neoplasms; Smoothened Receptor; Xenograft Model Antitumor Assays

It has long been known that the hedgehog signaling pathway is crucial for embryonic development, in both Drosophila and vertebrate systems. During the past few years its implication in carcinogenesis has become clear and today it is acknowledged that this pathway plays a role in the malignant transformation of multiple cell types, either owing to the mutation of some of its components or to its erratic activation. New molecular targeted therapies that inhibit the pathway have shown their unquestionable efficiency in several tumours -among which basal cell carcinoma, medulloblastoma, or pancreatic adenocarcinoma. The assessment of these inhibitors in other types of tumours is currently underway with promising results, suggesting that the Sonic hedgehog signalling pathway may become one of the therapeutic targets of the future.

Topics: Anilides; Animals; Carcinoma, Basal Cell; Cerebellar Neoplasms; Embryonic Development; Embryonic Induction; Hedgehog Proteins; Humans; Lung Neoplasms; Medulloblastoma; Molecular Targeted Therapy; Mutation; Neoplasms; Neoplastic Stem Cells; Pancreatic Neoplasms; Patched Receptors; Patient Selection; Pyridines; Receptors, Cell Surface; Signal Transduction; Skin Neoplasms; Small Cell Lung Carcinoma

Vismodegib specifically inhibits Sonic Hedgehog (SHH). We report results of a phase I/II evaluating vismodegib + temozolomide (TMZ) in immunohistochemically defined SHH recurrent/refractory adult medulloblastoma.. TMZ-naïve patients were randomized 2:1 to receive vismodegib + TMZ (arm A) or TMZ (arm B). Patients previously treated with TMZ were enrolled in an exploratory cohort of vismodegib (arm C). If the safety run showed no excessive toxicity, a Simon's 2-stage phase II design was planned to explore the 6-month progression-free survival (PFS-6). Stage II was to proceed if arm A PFS-6 was ≥3/9 at the end of stage I.. A total of 24 patients were included: arm A (10), arm B (5), and arm C (9). Safety analysis showed no excessive toxicity. At the end of stage I, the PFS-6 of arm A was 20% (2/10 patients, 95% unilateral lower confidence limit: 3.7%) and the study was prematurely terminated. The overall response rates (ORR) were 40% (95% CI, 12.2-73.8) and 20% (95% CI, 0.5-71.6) in arm A and B, respectively. In arm C, PFS-6 was 37.5% (95% CI, 8.8-75.5) and ORR was 22.2% (95% CI, 2.8-60.0). Among 11 patients with an expected sensitivity according to new generation sequencing (NGS), 3 had partial response (PR), 4 remained stable disease (SD) while out of 7 potentially resistant patients, 1 had PR and 1 SD.. The addition of vismodegib to TMZ did not add toxicity but failed to improve PFS-6 in SHH recurrent/refractory medulloblastoma. Prediction of sensitivity to vismodegib needs further refinements.

Topics: Anilides; Cerebellar Neoplasms; Hedgehog Proteins; Humans; Medulloblastoma; Pyridines; Temozolomide

Two phase II studies assessed the efficacy of vismodegib, a sonic hedgehog (SHH) pathway inhibitor that binds smoothened (SMO), in pediatric and adult recurrent medulloblastoma (MB).. Adult patients enrolled onto PBTC-025B and pediatric patients enrolled onto PBTC-032 were treated with vismodegib (150 to 300 mg/d). Protocol-defined response, which had to be sustained for 8 weeks, was confirmed by central neuroimaging review. Molecular tests to identify patterns of response and insensitivity were performed when tissue was available.. A total of 31 patients were enrolled onto PBTC-025B, and 12 were enrolled onto PBTC-032. Three patients in PBTC-025B and one in PBTC-032, all with SHH-subgroup MB (SHH-MB), exhibited protocol-defined responses. Progression-free survival (PFS) was longer in those with SHH-MB than in those with non-SHH-MB, and prolonged disease stabilization occurred in 41% of patient cases of SHH-MB. Among those with SHH-MB, loss of heterozygosity of PTCH1 was associated with prolonged PFS, and diffuse staining of P53 was associated with reduced PFS. Whole-exome sequencing identified mutations in SHH genes downstream from SMO in four of four tissue samples from nonresponders and upstream of SMO in two of four patients with favorable responses.. Vismodegib exhibits activity against adult recurrent SHH-MB but not against recurrent non-SHH-MB. Inadequate accrual of pediatric patients precluded conclusions in this population. Molecular analyses support the hypothesis that SMO inhibitor activity depends on the genomic aberrations within the tumor. Such inhibitors should be advanced in SHH-MB studies; however, molecular and genomic work remains imperative to identify target populations that will truly benefit.

Topics: Adult; Anilides; Brain Neoplasms; Female; Hedgehog Proteins; Humans; Male; Medulloblastoma; Middle Aged; Pyridines; Young Adult

To investigate the safety, dose-limiting toxicities, and pharmacokinetics of the smoothened inhibitor vismodegib in children with refractory or relapsed medulloblastoma.. Initially, vismodegib was administered daily at 85 mg/m(2) and escalated to 170 mg/m(2). The study was then revised to investigate a flat-dosing schedule of 150 mg for patients with small body surface area (BSA, 0.67-1.32 m(2)) or 300 mg for those who were larger (BSA, 1.33-2.20 m(2)). Pharmacokinetics were performed during the first course of therapy, and the right knees of all patients were imaged to monitor bone toxicity. Immunohistochemical analysis was done to identify patients with Sonic Hedgehog (SHH)-subtype medulloblastoma.. Thirteen eligible patients were enrolled in the initial study: 6 received 85 mg/m(2) vismodegib, and 7 received 170 mg/m(2). Twenty eligible patients were enrolled in the flat-dosing part of the study: 10 at each dosage level. Three dose-limiting toxicities were observed, but no drug-related bone toxicity was documented. The median (range) vismodegib penetration in the cerebrospinal fluid (CSF) was 0.53 (0.26-0.78), when expressed as a ratio of the concentration of vismodegib in the CSF to that of the unbound drug in plasma. Antitumor activity was seen in 1 of 3 patients with SHH-subtype disease whose tumors were evaluable, and in none of the patients in the other subgroups.. Vismodegib was well tolerated in children with recurrent or refractory medulloblastoma; only two dose-limiting toxicities were observed with flat dosing. The recommended phase II study dose is 150 or 300 mg, depending on the patient's BSA. Clin Cancer Res; 19(22); 6305-12. ©2013 AACR.

Topics: Adolescent; Anilides; Antineoplastic Agents; Cerebellar Neoplasms; Child; Child, Preschool; Dose-Response Relationship, Drug; Female; Humans; Male; Maximum Tolerated Dose; Medulloblastoma; Pyridines; Receptors, G-Protein-Coupled; Smoothened Receptor; Treatment Outcome; Young Adult

Vismodegib (GDC-0449), an orally bioavailable small-molecule inhibitor of Hedgehog signaling, was recently approved by the U.S. Food and Drug Administration for the treatment of basal cell carcinoma that is either metastatic or locally advanced in patients who are not candidates for surgical resection or radiation. Given the absence of previously defined effective drug therapy for this disease, approval was granted primarily on the basis of outcome of a nonrandomized parallel cohort phase II study of 99 patients with advanced basal cell carcinoma, with a primary endpoint of objective response rate. Response rates of 30.3% and 42.9% were observed in metastatic and locally advanced cohorts in this study, respectively, associated with median progression-free survival in both cohorts of 9.5 months. Ongoing clinical investigations include evaluation of the potential efficacy of vismodegib in a variety of diseases and in combination with other agents. The mechanism of action, preclinical and clinical data, and potential utility in other disease contexts are reviewed here.

Topics: Anilides; Carcinoma, Basal Cell; Disease-Free Survival; Drug Approval; Hedgehog Proteins; Humans; Medulloblastoma; Neoplasm Metastasis; Patched Receptors; Pyridines; Receptors, Cell Surface; Signal Transduction; United States; United States Food and Drug Administration

Medulloblastoma is the most common malignant paediatric brain tumour, with ~30% mediated by Sonic hedgehog signalling. Vismodegib-mediated inhibition of the Sonic hedgehog effector Smoothened inhibits tumour growth but causes growth plate fusion at effective doses. Here, we report a nanotherapeutic approach targeting endothelial tumour vasculature to enhance blood-brain barrier crossing. We use fucoidan-based nanocarriers targeting endothelial P-selectin to induce caveolin-1-dependent transcytosis and thus nanocarrier transport into the brain tumour microenvironment in a selective and active manner, the efficiency of which is increased by radiation treatment. In a Sonic hedgehog medulloblastoma animal model, fucoidan-based nanoparticles encapsulating vismodegib exhibit a striking efficacy and marked reduced bone toxicity and drug exposure to healthy brain tissue. Overall, these findings demonstrate a potent strategy for targeted intracranial pharmacodelivery that overcomes the restrictive blood-brain barrier to achieve enhanced tumour-selective penetration and has therapeutic implications for diseases within the central nervous system.

Topics: Animals; Blood-Brain Barrier; Caveolin 1; Cerebellar Neoplasms; Hedgehog Proteins; Medulloblastoma; P-Selectin; Transcytosis; Tumor Microenvironment

Medulloblastoma (MB) is a frequently occurring malignant brain tumor in children, and many of these tumors are identified by the abnormal activation of the Sonic Hedgehog (SHH) pathway. Although the Shh inhibitor GDC0449 initially shows some effectiveness in certain tumors, they eventually recur due to drug resistance mechanisms, highlighting the need for new treatment options. In this study, we explore whether GDC0449 induces autophagy in the human MB cell lines. To investigate the ultrastructural pathology changes of GDC0449-treated Daoy and D283 cells, we employed Transmission Electron Microscopy (TEM) technology to identify the expression of autophagic vacuoles. Our results indicate that GDC0449 only increases autophagy in Daoy cells by increasing the LC3-II/LC3-I ratio and autophagosome formation.We also analyzed Beclin1, LC3, Bax, and Cleaved-caspase3 protein and mRNA expression levels of autophagic and apoptotic markers using fluorescence confocal microscopy, RT-PCR, and Western blot. We found that cell autophagy and apoptosis increased in a dose-dependent manner with GDC0449 treatment. Additionally, we observed increased mammalian target of rapamycin (mTOR) phosphorylation and decreased protein kinase B (AKT/PKB), Ribosomal Protein S6, eIF4E-binding protein (4EBP1) phosphorylation in GDC0449-treated Daoy cells. It was observed that inhibiting autophagy using Beclin1 siRNA significantly blocked the apoptosis-inducing effects of GDC0449, suggesting that GDC0449 mediates its apoptotic effects by inducing autophagy.Our data suggests that GDC0449 inhibits the growth of human MB Daoy cells by autophagy-mediated apoptosis. The mechanism of GDC0449-induced autophagy in Daoy cells may be related to the inhibition of the PI3K/AKT/mTOR signaling pathway.

Topics: Apoptosis; Autophagy; Beclin-1; Cell Line, Tumor; Cerebellar Neoplasms; Child; Hedgehog Proteins; Humans; Medulloblastoma; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases

Topics: Anilides; Biomarkers; Cerebellar Neoplasms; Hedgehog Proteins; Humans; Medulloblastoma; Pyridines

Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and other tumors. Therefore, small molecular inhibitors of Hh pathway are urgently needed. In this study, three new steroidal alkaloids, ⊿

Topics: Alkaloids; Cell Line, Tumor; Cell Proliferation; HEK293 Cells; Humans; Medulloblastoma; Molecular Structure; Smoothened Receptor; Spectrum Analysis; Steroids; Veratrum

We report a nanoparticle formulation of the SHH-pathway inhibitor vismodegib that improves efficacy for medulloblastoma, while reducing toxicity. Limited blood-brain barrier (BBB) penetration and dose-limiting extitle/citraneural toxicities complicate systemic therapies for brain tumors. Vismodegib is FDA-approved for SHH-driven basal cell carcinoma, but implementation for medulloblastoma has been limited by inadequate efficacy and excessive bone toxicity. To address these issues through optimized drug delivery, we formulated vismodegib in polyoxazoline block copolymer micelles (POx-vismo). We then evaluated POx-vismo in transgenic mice that develop SHH-driven medulloblastomas with native vasculature and tumor microenvironment. POx-vismo improved CNS pharmacokinetics and reduced bone toxicity. Mechanistically, the nanoparticle carrier did not enter the CNS, and acted within the vascular compartment to improve drug delivery. Unlike conventional vismodegib, POx-vismo extended survival in medulloblastoma-bearing mice. Our results show the broad potential for non-targeted nanoparticle formulation to improve systemic brain tumor therapy, and specifically to improve vismodegib therapy for SHH-driven cancers.

Topics: Anilides; Animals; Biological Availability; Central Nervous System; Cerebellar Neoplasms; Disease Models, Animal; Drug Carriers; Drug Delivery Systems; Medulloblastoma; Mice; Micelles; Nanoparticles; Oxazoles; Particle Size; Protein Binding; Pyridines; Serum Albumin

The Hedgehog (Hh) pathway plays an indispensable role in bone development and genetic activation of the pathway results in medulloblastoma (MB), the most common malignant brain tumor in children. Inhibitors of Hh pathway (such as vismodegib and sonedigib), which are used to treat MB, cause irreversible defects in bone growth in young children. Cholesterol is required for the activation of the Hh pathway, and statins, inhibitors of cholesterol biosynthesis, suppress MB growth by repressing Hh signaling in tumor cells. Here, we investigate the role of cholesterol biosynthesis in the proliferation and Hh signaling in chondrocytes, and examine the bone development in mice after statin treatment. Statins significantly inhibited MB growth in young mice, but caused no defects in bone development. Conditional deletion of NADP steroid dehydrogenase-like (NSDHL), an enzyme necessary for cholesterol biosynthesis, suppressed cholesterol synthesis in chondrocytes, and disrupted the growth plate in mouse femur and tibia, indicating the important function of intracellular cholesterol in bone development. Hh pathway activation and the proliferation of chondrocytes were inhibited by statin treatment in vitro; however, statins did not impair bone growth in vivo due to insufficient penetration into the bone. Our studies reveal a critical role of cholesterol in bone development, and support the utilization of statins for treatment of MB as well as other Hh pathway-associated malignancies.

Topics: 3-Hydroxysteroid Dehydrogenases; Anilides; Animals; Bone Development; Cell Proliferation; Cholesterol; Chondrocytes; Hedgehog Proteins; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lipogenesis; Medulloblastoma; Mice; Mice, Knockout; Pyridines; Signal Transduction

Aberrant hedgehog (HH) signaling is implicated in the development of various cancer entities such as medulloblastoma. Activation of GLI transcription factors was revealed as the driving force upon pathway activation. Increased phosphorylation of essential effectors such as Smoothened (SMO) and GLI proteins by kinases including Protein Kinase A, Casein Kinase 1, and Glycogen Synthase Kinase 3 β controls effector activity, stability and processing. However, a deeper and more comprehensive understanding of phosphorylation in the signal transduction remains unclear, particularly during early response processes involved in SMO activation and preceding GLI target gene regulation.. We applied temporal quantitative phosphoproteomics to reveal phosphorylation dynamics underlying the short-term chemical activation and inhibition of early hedgehog signaling in HH responsive human medulloblastoma cells. Medulloblastoma cells were treated for 5.0 and 15 min with Smoothened Agonist (SAG) to induce and with vismodegib to inhibit the HH pathway.. Our phosphoproteomic profiling resulted in the quantification of 7700 and 10,000 phosphosites after 5.0 and 15 min treatment, respectively. The data suggest a central role of phosphorylation in the regulation of ciliary assembly, trafficking, and signal transduction already after 5.0 min treatment. ERK/MAPK signaling, besides Protein Kinase A signaling and mTOR signaling, were differentially regulated after short-term treatment. Activation of Polo-like Kinase 1 and inhibition of Casein Kinase 2A1 were characteristic for vismodegib treatment, while SAG treatment induced Aurora Kinase A activity. Distinctive phosphorylation of central players of HH signaling such as SMO, SUFU, GLI2 and GLI3 was observed only after 15 min treatment.. This study provides evidence that phosphorylation triggered in response to SMO modulation dictates the localization of hedgehog pathway components within the primary cilium and affects the regulation of the SMO-SUFU-GLI axis. The data are relevant for the development of targeted therapies of HH-associated cancers including sonic HH-type medulloblastoma. A deeper understanding of the mechanisms of action of SMO inhibitors such as vismodegib may lead to the development of compounds causing fewer adverse effects and lower frequencies of drug resistance. Video Abstract.

Topics: Adaptor Proteins, Signal Transducing; Anilides; BRCA1 Protein; Casein Kinase II; Cell Cycle Proteins; Cerebellar Neoplasms; Cilia; Cytoskeletal Proteins; Enzyme Activation; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Medulloblastoma; Phosphopeptides; Phosphorylation; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proteome; Proteomics; Proto-Oncogene Proteins; Pyridines; Signal Transduction; Substrate Specificity

Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor

Topics: Anilides; Animals; Basal Cell Nevus Syndrome; Cell Line, Tumor; Cell Proliferation; Galectin 1; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Medulloblastoma; Mice; Neoplasms, Experimental; Neural Stem Cells; Patched-1 Receptor; Pyridines

Some patients with metastatic medulloblastoma can be successfully treated with targeted therapy. We report the case of a 42-year-old woman who was diagnosed with sonic hedgehog (SHH)-subgroup medulloblastoma. She was treated with surgery, radiation and chemotherapy. She then developed bone pain. A positron emission tomography (PET) scan confirmed widespread bone metastases from her medulloblastoma. She was started on vismodegib, an oral smoothened inhibitor that targets her tumour type. Her bone pain resolved. A repeat PET scan showed resolution of almost all metastases. Fourteen months after starting vismodegib, her disease recurred and she was transitioned to temozolomide chemotherapy. We document an important case of prolonged response to vismodegib in a patient with systemic SHH-subgroup medulloblastoma metastases.

Topics: Adult; Anilides; Cerebellar Neoplasms; Cerebellum; Female; Follow-Up Studies; Humans; Magnetic Resonance Imaging; Medulloblastoma; Neoplasm Metastasis; Positron Emission Tomography Computed Tomography; Pyridines; Time Factors; Treatment Outcome

Targeting oncogenic pathways holds promise for brain tumor treatment, but inhibition of Sonic Hedgehog (SHH) signaling has failed in SHH-driven medulloblastoma. Cellular diversity within tumors and reduced lineage commitment can undermine targeted therapy by increasing the probability of treatment-resistant populations. Using single-cell RNA-seq and lineage tracing, we analyzed cellular diversity in medulloblastomas in transgenic, medulloblastoma-prone mice, and responses to the SHH-pathway inhibitor vismodegib. In untreated tumors, we find expected stromal cells and tumor-derived cells showing either a spectrum of neural progenitor-differentiation states or glial and stem cell markers. Vismodegib reduces the proliferative population and increases differentiation. However, specific cell types in vismodegib-treated tumors remain proliferative, showing either persistent SHH-pathway activation or stem cell characteristics. Our data show that even in tumors with a single pathway-activating mutation, diverse mechanisms drive tumor growth. This diversity confers early resistance to targeted inhibitor therapy, demonstrating the need to target multiple pathways simultaneously.

Topics: Anilides; Animals; Cell Proliferation; Cerebellar Neoplasms; Cerebellum; Drug Resistance, Neoplasm; Female; Gain of Function Mutation; Hedgehog Proteins; Humans; Male; Medulloblastoma; Mice; Mice, Transgenic; Molecular Targeted Therapy; MyoD Protein; Neoplastic Stem Cells; Pyridines; RNA-Seq; Signal Transduction; Single-Cell Analysis; Smoothened Receptor; Transcription Factor HES-1

The aberrant activation of hedgehog (HH) signaling is a leading cause of the development of medulloblastoma, a pediatric tumor of the cerebellum. The FDA‑approved HH inhibitor, Vismodegib, which targets the transmembrane transducer SMO, has shown limited efficacy in patients with medulloblastoma, due to compensatory mechanisms that maintain an active HH‑GLI signaling status. Thus, the identification of novel actionable mechanisms, directly affecting the activity of the HH‑regulated GLI transcription factors is an important goal for these malignancies. In this study, using gene expression and reporter assays, combined with biochemical and cellular analyses, we demonstrate that mitogen‑activated kinase kinase kinase 1 (MEKK1), the most upstream kinase of the mitogen‑activated protein kinase (MAPK) phosphorylation modules, suppresses HH signaling by associating and phosphorylating GLI1, the most potent HH‑regulated transcription factor. Phosphorylation occurred at multiple residues in the C‑terminal region of GLI1 and was followed by an increased association with the cytoplasmic proteins 14‑3‑3. Of note, the enforced expression of MEKK1 or the exposure of medulloblastoma cells to the MEKK1 activator, Nocodazole, resulted in a marked inhibitory effect on GLI1 activity and tumor cell proliferation and viability. Taken together, the results of this study shed light on a novel regulatory mechanism of HH signaling, with potentially relevant implications in cancer therapy.

Topics: Anilides; Animals; Cell Proliferation; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; MAP Kinase Kinase Kinase 1; Medulloblastoma; Mice; NIH 3T3 Cells; Phosphorylation; Pyridines; RNA, Messenger; Signal Transduction; Transcription Factors; Zinc Finger Protein GLI1

Although most children with medulloblastoma are cured of their disease, Sonic Hedgehog (SHH) subgroup medulloblastoma driven by. The ability of this CK1α activator to block SHH signaling was determined. SSTC3 inhibited SHH activity. Using a newly described small-molecule, SSTC3, we show that CK1a activators could address a significant unmet clinical need for patients with SMO inhibitor-resistant medulloblastoma, including those harboring mutations in TRP53.

Topics: Anilides; Animals; Benzoates; Brain; Casein Kinase Ialpha; Disease Models, Animal; Drug Resistance, Neoplasm; Heterografts; Humans; Medulloblastoma; Mice; N-Myc Proto-Oncogene Protein; Neoplasm Metastasis; Pyridines; Signal Transduction; Smoothened Receptor; Tumor Suppressor Protein p53; Zinc Finger Protein GLI1

Medulloblastoma (MB) is one of the most frequent malignant brain tumors of children, and a large set of these tumors is characterized by aberrant activation of the sonic hedgehog (SHH) pathway. While some tumors initially respond to inhibition of the SHH pathway component Smoothened (SMO), tumors ultimately recur due to downstream resistance mechanisms, indicating a need for novel therapeutic options.. Here we performed a targeted small-molecule screen on a stable, SHH-dependent murine MB cell line (SMB21). Comprehensive isotype profiling of histone deacetylase (HDAC) inhibitors was performed, and effects of HDAC inhibition were evaluated in cell lines both sensitive and resistant to SMO inhibition. Lastly, distinct mouse models of SHH MB were used to demonstrate pharmacologic efficacy in vivo.. A subset of the HDAC inhibitors tested significantly inhibit tumor growth of SMB21 cells by preventing SHH pathway activation. Isotype profiling of HDAC inhibitors, together with genetic approaches suggested that concerted inhibition of multiple class I HDACs is necessary to achieve pathway inhibition. Of note, class I HDAC inhibitors were also efficacious in suppressing growth of diverse SMO inhibitor‒resistant clones of SMB21 cells. Finally, we show that the novel HDAC inhibitor quisinostat targets multiple class I HDACs, is well tolerated in mouse models, and robustly inhibits growth of SHH MB cells in vivo as well as in vitro.. Our data provide strong evidence that quisinostat or other class I HDAC inhibitors might be therapeutically useful for patients with SHH MB, including those resistant to SMO inhibition.

Topics: Anilides; Animals; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Cerebellar Neoplasms; Drug Resistance, Neoplasm; Hedgehog Proteins; High-Throughput Screening Assays; Histone Deacetylase Inhibitors; Hydroxamic Acids; Inhibitory Concentration 50; Medulloblastoma; Mice; Proteins; Pyridines; Repressor Proteins; Signal Transduction; Smoothened Receptor

We report herein the design and synthesis of a series of structural modified dimethylpyridazine compounds as novel hedgehog signaling pathway inhibitors. The bicyclic phthalazine core and 4-methylamino-piperidine moiety of Taladegib were replaced with dimethylpyridazine and different azacycle building blocks, respectively. The in vitro Gli-luciferase assay results demonstrate that the new scaffold still retained potent inhibitory potency. Piperidin-4-amine moiety was found to be the best linker between pharmacophores dimethylpyridazine and fluorine substituted benzoyl group. Furthermore, the optimization of 1-methyl-1H-pyrazol and 4-fluoro-2-(trifluoromethyl)benzamide by different aliphatic or aromatic rings were also investigated and the SAR were described. Several new derivatives were found to show potent Hh signaling inhibitory activity with nanomolar IC

Topics: Animals; Antineoplastic Agents; Binding Sites; Catalytic Domain; Disease Models, Animal; Drug Design; Hedgehog Proteins; Medulloblastoma; Mice; Mice, Knockout; Mice, Nude; Molecular Docking Simulation; Pyridazines; Signal Transduction; Smoothened Receptor; Structure-Activity Relationship; Transplantation, Homologous; Tumor Suppressor Protein p53

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.

Topics: Animals; Antineoplastic Agents; Cells, Cultured; Cerebellar Neoplasms; Drug Design; Hedgehog Proteins; Humans; Isoflavones; Medulloblastoma; Mice; Models, Molecular; Signal Transduction; Smoothened Receptor; Tumor Cells, Cultured; Zinc Finger Protein GLI1

A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology.

Topics: Anilides; Animals; Casein Kinase II; Cell Line, Tumor; Cerebellar Neoplasms; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Kaplan-Meier Estimate; Medulloblastoma; Mice; Mice, Inbred NOD; Mice, Knockout; Mice, Nude; Mice, SCID; Naphthyridines; Neoplasms, Experimental; NIH 3T3 Cells; Phenazines; Phosphoproteins; Proteomics; Pyridines; Signal Transduction; Xenograft Model Antitumor Assays

We report herein the design and synthesis of a series of optimized phthalazine compounds as novel hedgehog signaling pathway inhibitors. The 4-methylamino-piperidine moiety of Taladegib was replaced by different four, five or six-membered azacycle or azaspirocycle building blocks. The in vitro Gli-luciferase assay results demonstrate that the scaffold hopping in this region afforded significant influences on Hh pathway inhibition. Pyrrolidin-3-amine moiety was found to be the best linker between pharmacophores phthalazine and fluorine substituted benzoyl group. Meanwhile the optimization of 1-methyl-1H-pyrazol by different aromatic rings was also investigated and the SAR was described. Many new derivatives were found to show potent Hh signaling inhibitory activity with nanomolar IC

Topics: Animals; Antineoplastic Agents; Cell Proliferation; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Hedgehog Proteins; Medulloblastoma; Mice; Mice, Knockout; Mice, Nude; Molecular Structure; Phthalazines; Rats; Rats, Sprague-Dawley; Signal Transduction; Structure-Activity Relationship

Topics: Animals; DNA; Hedgehog Proteins; Humans; Macrocyclic Compounds; Medulloblastoma; Mice; Mice, Knockout; Models, Molecular; Mutation; Signal Transduction; Structure-Activity Relationship; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1

Medulloblastoma is one of the most prevalent brain tumors in children. Aberrant hedgehog (Hh) pathway signaling is thought to be involved in the initiation and development of medulloblastoma. Vismodegib, the first FDA-approved cancer therapy based on inhibition of aberrant hedgehog signaling, targets smoothened (Smo), a G-protein coupled receptor (GPCR) central to the Hh pathway. Although vismodegib exhibits promising therapeutic efficacy in tumor treatment, concerns have been raised from its nonlinear pharmacokinetic (PK) profiles at high doses partly due to low aqueous solubility. Many patients experience adverse events such as muscle spasms and weight loss. In addition, drug resistance often arises among tumor cells during treatment with vismodegib. There is clearly an urgent need to explore novel Smo antagonists with improved potency and efficacy. Through a scaffold hopping strategy, we have identified a series of novel tetrahydropyrido[4,3-d]pyrimidine derivatives, which exhibited effective inhibition of Hh signaling. Among them, compound 24 is three times more potent than vismodegib in the NIH3T3-GRE-Luc reporter gene assay. Compound 24 has a lower melting point and much greater solubility compared with vismodegib, resulting in linear PK profiles when dosed orally at 10, 30, and 100 mg/kg in rats. Furthermore, compound 24 showed excellent PK profiles with a 72% oral bioavailability in beagle dogs. Compound 24 demonstrated overall favorable in vitro safety profiles with respect to CYP isoform and hERG inhibition. Finally, compound 24 led to significant regression of subcutaneous tumor generated by primary Ptch1-deficient medulloblastoma cells in SCID mouse. In conclusion, tetrahydropyrido[4,3-d]pyrimidine derivatives represent a novel set of Smo inhibitors that could potentially be utilized to treat medulloblastoma and other Hh pathway related malignancies.

Topics: Anilides; Animals; Antineoplastic Agents; Cerebellar Neoplasms; CHO Cells; Cricetulus; Dogs; Drug Design; Female; Humans; Male; Medulloblastoma; Mice; Mice, Inbred ICR; Mice, SCID; Mice, Transgenic; Neoplasm Transplantation; NIH 3T3 Cells; Patched-1 Receptor; Pyridines; Pyrimidines; Random Allocation; Rats, Sprague-Dawley; Smoothened Receptor; Structure-Activity Relationship; Transcriptional Regulator ERG

The SMOOTHENED inhibitor vismodegib is FDA approved for advanced basal cell carcinoma (BCC), and shows promise in clinical trials for SONIC HEDGEHOG (SHH)-subgroup medulloblastoma (MB) patients. Clinical experience with BCC patients shows that continuous exposure to vismodegib is necessary to prevent tumor recurrence, suggesting the existence of a vismodegib-resistant reservoir of tumor-propagating cells. We isolated such tumor-propagating cells from a mouse model of SHH-subgroup MB and grew them as sphere cultures. These cultures were enriched for the MB progenitor marker SOX2 and formed tumors in vivo. Moreover, while their ability to self-renew was resistant to SHH inhibitors, as has been previously suggested, this self-renewal was instead WNT-dependent. We show here that loss of Trp53 activates canonical WNT signaling in these SOX2-enriched cultures. Importantly, a small molecule WNT inhibitor was able to reduce the propagation and growth of SHH-subgroup MB in vivo, in an on-target manner, leading to increased survival. Our results imply that the tumor-propagating cells driving the growth of bulk SHH-dependent MB are themselves WNT dependent. Further, our data suggest combination therapy with WNT and SHH inhibitors as a therapeutic strategy in patients with SHH-subgroup MB, in order to decrease the tumor recurrence commonly observed in patients treated with vismodegib.

Topics: Anilides; Animals; Cell Line, Tumor; Cerebellar Neoplasms; Disease Models, Animal; Hedgehog Proteins; HEK293 Cells; Humans; Male; Medulloblastoma; Mice; Mice, Transgenic; Pyridines; Random Allocation; Small Molecule Libraries; SOXB1 Transcription Factors; Transfection; TRPC Cation Channels; Tumor Suppressor Protein p53; Veratrum Alkaloids; Wnt Proteins; Wnt Signaling Pathway

Topics: Anilides; Antineoplastic Agents; Cerebellar Neoplasms; Child, Preschool; Female; Femur; Growth Plate; Humans; Medulloblastoma; Pyridines; Tibia

The hedgehog (Hh) signaling pathway is activated in many types of cancer and therefore presents an attractive target for new anticancer agents. Here, we show that mebendazole, a benzamidazole with a long history of safe use against nematode infestations and hydatid disease, potently inhibited Hh signaling and slowed the growth of Hh-driven human medulloblastoma cells at clinically attainable concentrations. As an antiparasitic, mebendazole avidly binds nematode tubulin and causes inhibition of intestinal microtubule synthesis. In human cells, mebendazole suppressed the formation of the primary cilium, a microtubule-based organelle that functions as a signaling hub for Hh pathway activation. The inhibition of Hh signaling by mebendazole was unaffected by mutants in the gene that encodes human Smoothened (SMO), which are selectively propagated in cell clones that survive treatment with the Hh inhibitor vismodegib. Combination of vismodegib and mebendazole resulted in additive Hh signaling inhibition. Because mebendazole can be safely administered to adults and children at high doses over extended time periods, we propose that mebendazole could be rapidly repurposed and clinically tested as a prospective therapeutic agent for many tumors that are dependent on Hh signaling.

Topics: Anilides; Animals; Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Repositioning; Drug Synergism; Female; Hedgehog Proteins; HEK293 Cells; Humans; Mebendazole; Medulloblastoma; Mice; Mice, Nude; Mutation; NIH 3T3 Cells; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Xenograft Model Antitumor Assays

Hedgehog (Hh) signaling pathway is a valid therapeutic target in a wide range of malignancies. We focus here on glioblastoma multiforme (GBM), a lethal malignancy of the central nervous system (CNS). By analyzing RNA-sequencing based transcriptomics data on 149 clinical cases of TCGA-GBM database we show here a strong correlation (r = 0.7) between GLI1 and PTCH1 mRNA expression--as a hallmark of the canonical Hh-pathway activity in this malignancy. GLI1 mRNA expression varied in 3 orders of magnitude among the GBM patients of the same cohort showing a single continuous distribution-unlike the discrete high/low-GLI1 mRNA expressing clusters of medulloblastoma (MB). When compared with MB as a reference, the median GLI1 mRNA expression in GBM appeared 14.8 fold lower than that of the "high-Hh" cluster of MB but 5.6 fold higher than that of the "low-Hh" cluster of MB. Next, we demonstrated statistically significant up- and down-regulation of GLI1 mRNA expressions in GBM patient-derived low-passage neurospheres in vitro by sonic hedgehog ligand-enriched conditioned media (shh-CM) and by Hh-inhibitor drug vismodegib respectively. We also showed clinically achievable dose (50 μM) of vismodegib alone to be sufficient to induce apoptosis and cell cycle arrest in these low-passage GBM neurospheres in vitro. Vismodegib showed an effect on the neurospheres, both by down-regulating GLI1 mRNA expression and by inducing apoptosis/cell cycle arrest, irrespective of their relative endogenous levels of GLI1 mRNA expression. We conclude from our study that this single continuous distribution pattern of GLI1 mRNA expression technically puts almost all GBM patients in a single group rather than discrete high- or low-clusters in terms of Hh-pathway activity. That is suggestive of therapies with Hh-pathway inhibitor drugs in this malignancy without a need for further stratification of patients on the basis of relative levels of Hh-pathway activity among them.

Topics: Anilides; Apoptosis; Cell Cycle Checkpoints; Dose-Response Relationship, Drug; Down-Regulation; Gene Expression Regulation, Neoplastic; Glioblastoma; Hedgehog Proteins; Humans; Medulloblastoma; Oncogene Proteins; Pyridines; RNA, Messenger; Signal Transduction; Trans-Activators; Up-Regulation; Zinc Finger Protein GLI1

Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy, and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS-MAPK pathway circumvents Shh pathway dependency, drives tumor growth, and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together, these findings reveal a critical role of the RAS-MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors.

Topics: Anilides; Animals; Carcinoma, Basal Cell; Cell Proliferation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Medulloblastoma; Mice; Mitogen-Activated Protein Kinase Kinases; Pyridines; ras Proteins; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Xenograft Model Antitumor Assays

The altered metabolism of cancer cells has long been viewed as a potential target for therapeutic intervention. In particular, brain tumors often display heightened glycolysis, even in the presence of oxygen. A subset of medulloblastoma, the most prevalent malignant brain tumor in children, arises as a consequence of activating mutations in the Hedgehog (HH) pathway, which has been shown to promote aerobic glycolysis. Therefore, we hypothesized that a low carbohydrate, high fat ketogenic diet would suppress tumor growth in a genetically engineered mouse model of medulloblastoma. However, we found that the ketogenic diet did not slow the growth of spontaneous tumors or allograft flank tumors, and it did not exhibit synergy with a small molecule inhibitor of Smoothened. Serum insulin was significantly reduced in mice fed the ketogenic diet, but no alteration in PI3 kinase activity was observed. These findings indicate that while the ketogenic diet may be effective in inhibiting growth of other tumor types, it does not slow the growth of HH-medulloblastoma in mice.

Topics: Anilides; Animals; Blotting, Western; Cerebellar Neoplasms; Diet, Ketogenic; Glycolysis; Hedgehog Proteins; Hexokinase; Insulin; Magnetic Resonance Imaging; Medulloblastoma; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Patched Receptors; Pyridines; Receptors, Cell Surface; Signal Transduction; Survival Analysis; Tumor Burden; Tumor Suppressor Protein p53

Topics: Anilides; Antineoplastic Agents; Basal Cell Nevus Syndrome; Cerebellar Neoplasms; Clinical Trials as Topic; Cooperative Behavior; Drug Industry; Europe; Humans; Medulloblastoma; Molecular Targeted Therapy; North America; Patched Receptors; Pyridines; Receptors, Cell Surface; Signal Transduction

Medulloblastoma is a cancer of the cerebellum, for which there is currently no approved targeted therapy. Recent transcriptomics approaches have demonstrated that medulloblastoma is composed of molecularly distinct subgroups, one of which is characterized by activation of the Hedgehog pathway, which in mouse models is sufficient to drive medulloblastoma development. There is thus considerable interest in targeting the Hedgehog pathway for therapeutic benefit in medulloblastoma, particularly given the recent approval of the Hedgehog pathway inhibitor vismodegib for metastatic and locally advanced basal cell carcinoma. Like other molecularly targeted therapies, however, there have been reports of acquired resistance to vismodegib, driven by secondary Hedgehog pathway mutations and potentially by activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Given that acquired resistance to vismodegib may occur as a result of inappropriate PI3K pathway activation, we asked if loss of the PI3K pathway regulator, phosphatase and tensin homologue (Pten), which has been reported to occur in patients within the Hedgehog subgroup, would constitute a mechanism of innate resistance to vismodegib in Hedgehog-driven medulloblastoma. We find that Hedgehog pathway inhibition successfully restrains growth of Pten-deficient medulloblastoma in this mouse model, but does not drive tumor regression, as it does in Pten-wild-type medulloblastoma. Combined inhibition of the Hedgehog and PI3K pathways may lead to superior antitumor activity in PTEN-deficient medulloblastoma in the clinic.

Topics: Anilides; Animals; Antineoplastic Agents; Cell Transformation, Neoplastic; Cerebellar Neoplasms; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Female; Gene Deletion; Hedgehog Proteins; Medulloblastoma; Mice; Mice, Nude; Mice, Transgenic; Pregnancy; PTEN Phosphohydrolase; Pyridines; Signal Transduction

Topics: Adult; Anilides; Antineoplastic Agents; Bone Neoplasms; Cerebellar Neoplasms; Humans; Male; Medulloblastoma; Pyridines; Radionuclide Imaging; Remission Induction; Time Factors; Treatment Outcome

Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors. Leading inhibitors in clinical development act by binding to a common site within Smoothened, a critical pathway component. Acquired Smoothened mutations, including SMO(D477G), confer resistance to these inhibitors. Here, we report that itraconazole and arsenic trioxide, two agents in clinical use that inhibit Hedgehog signaling by mechanisms distinct from that of current Smoothened antagonists, retain inhibitory activity in vitro in the context of all reported resistance-conferring Smoothened mutants and GLI2 overexpression. Itraconazole and arsenic trioxide, alone or in combination, inhibit the growth of medulloblastoma and basal cell carcinoma in vivo, and prolong survival of mice with intracranial drug-resistant SMO(D477G) medulloblastoma.

Topics: Anilides; Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Carcinoma, Basal Cell; Drug Resistance, Neoplasm; Hedgehog Proteins; Itraconazole; Medulloblastoma; Mice; Oxides; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor

Dependence of basal cell carcinomas and medulloblastomas on the Hedgehog pathway provides an opportunity for targeted or "personalized" therapy. The recent effectiveness and FDA approval of the first Smoothened inhibitors validates this class of agents, but has revealed drug-resistant tumor variants that bypass Smoothened inhibition. Here, we summarize the effectiveness of Hedgehog pathway inhibitors and highlight promising areas for the development of next generation drug antagonists for Hedgehog-dependent cancers.

Topics: Anilides; Antineoplastic Agents; Carcinoma, Basal Cell; Cerebellar Neoplasms; Drug Resistance, Neoplasm; Hedgehog Proteins; Humans; Medulloblastoma; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Skin Neoplasms; Smoothened Receptor; Transcription Factors; Veratrum Alkaloids; Zinc Finger Protein GLI1

Inappropriate Hedgehog (Hh) signaling has been directly linked to medulloblastoma (MB), a common malignant brain tumor in children. GDC-0449 is an Hh pathway inhibitor (HPI) currently under clinical investigation as an anticancer agent. Treatment of a MB patient with GDC-0449 initially regressed tumors, but this individual ultimately relapsed with a D473H resistance mutation in Smoothened (SMO), the molecular target of GDC-0449. To explore the role of the mutated aspartic acid residue in SMO function, we substituted D473 with every amino acid and found that all functional mutants were resistant to GDC-0449, with positively charged residues conferring potential oncogenic properties. Alanine scan mutagenesis of SMO further identified E518 as a novel prospective mutation site for GDC-0449 resistance. To overcome this form of acquired resistance, we screened a panel of chemically diverse HPIs and identified several antagonists with potent in vitro activity against these GDC-0449-resistant SMO mutants. The bis-amide compound 5 was of particular interest, as it was able to inhibit tumor growth mediated by drug resistant SMO in a murine allograft model of MB. However, focal amplifications of the Hh pathway transcription factor Gli2 and the Hh target gene cyclin D1 (Ccnd1) were observed in two additional resistant models, indicating that resistance may also occur downstream of SMO. Importantly, these HPI resistant MB allografts retained their sensitivity to PI3K inhibition, presenting additional opportunities for the treatment of such tumors.

Topics: Anilides; Animals; Cell Line, Tumor; Cerebellar Neoplasms; Drug Resistance, Neoplasm; HEK293 Cells; Humans; Medulloblastoma; Mice; Mice, Nude; Mice, Transgenic; Mutagenesis, Site-Directed; Mutation; Oncogene Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyridines; Receptors, G-Protein-Coupled; RNA, Messenger; Signal Transduction; Smoothened Receptor; Trans-Activators; Zinc Finger Protein GLI1

Vismodegib (GDC-0449) is a potent and selective inhibitor of the Hedgehog (Hh) pathway that shows antitumor activity in preclinical models driven by mutational or ligand-dependent activation of the Hh pathway. We wished to characterize the pharmacokinetic-pharmacodynamic (PK/PD) relationship of vismodegib in both model systems to guide optimal dose and schedule for vismodegib in the clinic.. Preclinical efficacy and PK/PD studies were carried out with vismodegib in a Ptch(+/-) allograft model of medulloblastoma exhibiting mutational activation of the Hh pathway and patient-derived colorectal cancer (CRC) xenograft models exhibiting ligand-dependent pathway activation. Inhibition of the hedgehog pathway was related to vismodegib levels in plasma and to antitumor efficacy using an integrated population-based PK/PD model.. Oral dosing of vismodegib caused tumor regressions in the Ptch(+/-) allograft model of medulloblastoma at doses ≥25 mg/kg and tumor growth inhibition at doses up to 92 mg/kg dosed twice daily in two ligand-dependent CRC models, D5123, and 1040830. Analysis of Hh pathway activity and PK/PD modeling reveals that vismodegib inhibits Gli1 with a similar IC(50) in both the medulloblastoma and D5123 models (0.165 μmol/L ±11.5% and 0.267 μmol/L ±4.83%, respectively). Pathway modulation was linked to efficacy using an integrated PK/PD model revealing a steep relationship where > 50% of the activity of vismodegib is associated with >80% repression of the Hh pathway.. These results suggest that even small reductions in vismodegib exposure can lead to large changes in antitumor activity and will help guide proper dose selection for vismodegib in the clinic.

Topics: Anilides; Animals; Antineoplastic Agents; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Ligands; Medulloblastoma; Mice; Mice, Nude; Models, Biological; Mutation; Pyridines; Signal Transduction; Transcription Factors; Tumor Burden; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1

The Hedgehog (Hh) signaling pathway is critical for cell growth and differentiation during embryogenesis and early development. While it is mostly quiescent in adults, inappropriate reactivation of the Hh pathway has been shown to be involved in the development of cancer. A number of tumor types rely on overexpression of Hh ligands to activate the pathway in a paracrine manner from the tumor to the surrounding stroma. Alternatively, Hh ligands may act on cancer stem cells in some hematopoietic cancers, such as chronic myelogenous leukemia. However, the role of the Hh pathway is best established in tumors, such as basal cell carcinoma and medulloblastoma, where the pathway is activated via mutations. Understanding the contribution of Hh signaling in these various tumor types will be critical to the development and use of agents targeting this pathway in the clinic. We review here the activity of clinical inhibitors of the Hh pathway, including GDC-0449, a small molecule inhibitor of Smoothened (SMO).

Topics: Adult; Anilides; Antineoplastic Agents; Carcinoma, Basal Cell; Cerebellar Neoplasms; Child; Hedgehog Proteins; Humans; Medulloblastoma; Paracrine Communication; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Skin Neoplasms; Smoothened Receptor

SAR for a wide variety of heterocyclic replacements for a benzimidazole led to the discovery of functionalized 2-pyridyl amides as novel inhibitors of the hedgehog pathway. The 2-pyridyl amides were optimized for potency, PK, and drug-like properties by modifications to the amide portion of the molecule resulting in 31 (GDC-0449). Amide 31 produced complete tumor regression at doses as low as 12.5mg/kg BID in a medulloblastoma allograft mouse model that is wholly dependent on the Hh pathway for growth and is currently in human clinical trials, where it is initially being evaluated for the treatment of BCC.

Topics: Amides; Anilides; Animals; Benzimidazoles; Carcinoma, Basal Cell; Cell Line; Cerebellar Neoplasms; Hedgehog Proteins; Humans; Medulloblastoma; Mice; Mice, Nude; Pyridines; Signal Transduction; Structure-Activity Relationship; Xenograft Model Antitumor Assays

Medulloblastoma is the most common malignant brain tumor in children. Aberrant activation of the hedgehog signaling pathway is strongly implicated in the development of some cases of medulloblastoma. A 26-year-old man with metastatic medulloblastoma that was refractory to multiple therapies was treated with a novel hedgehog pathway inhibitor, GDC-0449; treatment resulted in rapid (although transient) regression of the tumor and reduction of symptoms. Molecular analyses of tumor specimens obtained before treatment suggested that there was activation of the hedgehog pathway, with loss of heterozygosity and somatic mutation of the gene encoding patched homologue 1 (PTCH1), a key negative regulator of hedgehog signaling.

Topics: Adult; Anilides; Antineoplastic Agents; Benzimidazoles; Cerebellar Neoplasms; Gene Expression; Hedgehog Proteins; Humans; Male; Medulloblastoma; Patched Receptors; Patched-1 Receptor; Polymerase Chain Reaction; Pyridines; Receptors, Cell Surface; RNA, Messenger; Signal Transduction; Transcription Factors; Zinc Finger Protein GLI1

The Hedgehog (Hh) signaling pathway is inappropriately activated in certain human cancers, including medulloblastoma, an aggressive brain tumor. GDC-0449, a drug that inhibits Hh signaling by targeting the serpentine receptor Smoothened (SMO), has produced promising anti-tumor responses in early clinical studies of cancers driven by mutations in this pathway. To evaluate the mechanism of resistance in a medulloblastoma patient who had relapsed after an initial response to GDC-0449, we determined the mutational status of Hh signaling genes in the tumor after disease progression. We identified an amino acid substitution at a conserved aspartic acid residue of SMO that had no effect on Hh signaling but disrupted the ability of GDC-0449 to bind SMO and suppress this pathway. A mutation altering the same amino acid also arose in a GDC-0449-resistant mouse model of medulloblastoma. These findings show that acquired mutations in a serpentine receptor with features of a G protein-coupled receptor can serve as a mechanism of drug resistance in human cancer.

Topics: Amino Acid Sequence; Amino Acid Substitution; Anilides; Animals; Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Cinnamates; Drug Resistance, Neoplasm; Hedgehog Proteins; Humans; Medulloblastoma; Mice; Molecular Sequence Data; Mutant Proteins; Mutation, Missense; Neoplasm Metastasis; Patched Receptors; Protein Conformation; Pyridines; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Veratrum Alkaloids

Topics: Adult; Anilides; Animals; Antineoplastic Agents; Benzimidazoles; Brain Neoplasms; Carcinoma, Basal Cell; Drug Resistance, Neoplasm; Hedgehog Proteins; Humans; Male; Medulloblastoma; Mice; Point Mutation; Protein Binding; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Skin Neoplasms; Smoothened Receptor