RESUMEN
Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.
Asunto(s)
Proteínas Hedgehog/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Cromatografía de Afinidad , Proteómica , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
Interaction of cancer cells with their microenvironment generated by stromal cells is essential for tumor cell survival and influences the localization of tumor growth. Here we demonstrate that hedgehog ligands secreted by bone-marrow, nodal and splenic stromal cells function as survival factors for malignant lymphoma and plasmacytoma cells derived from transgenic Emu-Myc mice or isolated from humans with these malignancies. Hedgehog pathway inhibition in lymphomas induced apoptosis through downregulation of Bcl2, but was independent of p53 or Bmi1 expression. Blockage of hedgehog signaling in vivo inhibited expansion of mouse lymphoma cells in a syngeneic mouse model and reduced tumor mass in mice with fully developed disease. Our data indicate that stromally induced hedgehog signaling may provide an important survival signal for B- and plasma-cell malignancies in vitro and in vivo. Disruption of this interaction by hedgehog pathway inhibition could provide a new strategy in lymphoma and multiple myeloma therapy.
Asunto(s)
Proteínas Hedgehog/metabolismo , Linfoma de Células B/metabolismo , Transducción de Señal , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Proteínas Hedgehog/genética , Humanos , Ligandos , Linfoma de Células B/genética , Linfoma de Células B/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Células del Estroma/metabolismo , Tasa de Supervivencia , Transactivadores/genética , Transactivadores/metabolismo , Alcaloides de Veratrum/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína con Dedos de Zinc GLI1RESUMEN
Ortho-biphenyl carboxamides, originally prepared as inhibitors of microsomal triglyceride transfer protein (MTP) have been identified as novel inhibitors of the Hedgehog signaling pathway. Structure-activity relationship studies for this class of compounds reduced MTP inhibitory activity and led to low nanomolar Hedgehog inhibitors. Binding assays revealed that the compounds act as antagonists of Smoothened and show cross-reactivity for both the human and mouse receptor.
Asunto(s)
Amidas/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Amidas/química , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Receptor Smoothened , Relación Estructura-ActividadRESUMEN
Drug resistance poses a great challenge to targeted cancer therapies. In Hedgehog pathway-dependent cancers, the scope of mechanisms enabling resistance to SMO inhibitors is not known. Here, we performed a transposon mutagenesis screen in medulloblastoma and identified multiple modes of resistance. Surprisingly, mutations in ciliogenesis genes represent a frequent cause of resistance, and patient datasets indicate that cilia loss constitutes a clinically relevant category of resistance. Conventionally, primary cilia are thought to enable oncogenic Hedgehog signaling. Paradoxically, we find that cilia loss protects tumor cells from susceptibility to SMO inhibitors and maintains a "persister" state that depends on continuous low output of the Hedgehog program. Persister cells can serve as a reservoir for further tumor evolution, as additional alterations synergize with cilia loss to generate aggressive recurrent tumors. Together, our findings reveal patterns of resistance and provide mechanistic insights for the role of cilia in tumor evolution and drug resistance.Significance: Using a transposon screen and clinical datasets, we identified mutations in ciliogenesis genes as a new class of resistance to SMO inhibitors. Mechanistically, cilia-mutant tumors can either grow slowly in a "persister" state or evolve and progress rapidly in an "aggressive" state. Cancer Discov; 7(12); 1436-49. ©2017 AACR.See related commentary by Goranci-Buzhala et al., p. 1374This article is highlighted in the In This Issue feature, p. 1355.
Asunto(s)
Cilios/genética , Proteínas Hedgehog/genética , Retroelementos/genética , Receptor Smoothened/antagonistas & inhibidores , Animales , Humanos , Ratones , Transducción de SeñalRESUMEN
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.
Asunto(s)
Carcinoma Basocelular/genética , Proteínas Hedgehog/genética , Meduloblastoma/genética , Receptores Acoplados a Proteínas G/biosíntesis , Proteínas ras/genética , Anilidas/administración & dosificación , Animales , Carcinoma Basocelular/tratamiento farmacológico , Carcinoma Basocelular/patología , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Piridinas/administración & dosificación , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Receptor Smoothened , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/biosíntesisRESUMEN
In glioblastoma, phosphatidylinositol 3-kinase (PI3K) signaling is frequently activated by loss of the tumor suppressor phosphatase and tensin homolog (PTEN). However, it is not known whether inhibiting PI3K represents a selective and effective approach for treatment. We interrogated large databases and found that sonic hedgehog (SHH) signaling is activated in PTEN-deficient glioblastoma. We demonstrate that the SHH and PI3K pathways synergize to promote tumor growth and viability in human PTEN-deficient glioblastomas. A combination of PI3K and SHH signaling inhibitors not only suppressed the activation of both pathways but also abrogated S6 kinase (S6K) signaling. Accordingly, targeting both pathways simultaneously resulted in mitotic catastrophe and tumor apoptosis and markedly reduced the growth of PTEN-deficient glioblastomas in vitro and in vivo. The drugs tested here appear to be safe in humans; therefore, this combination may provide a new targeted treatment for glioblastoma.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Proteínas Hedgehog/metabolismo , Fosfohidrolasa PTEN/deficiencia , Fosfatidilinositol 3-Quinasas/metabolismo , Aminopiridinas/administración & dosificación , Animales , Compuestos de Bifenilo/administración & dosificación , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Inhibidores Enzimáticos/administración & dosificación , Glioblastoma/genética , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Ratones , Ratones Desnudos , Morfolinas/administración & dosificación , Fosfohidrolasa PTEN/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/administración & dosificación , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Basal cell carcinoma (BCC) is a distinctive manifestation in nevoid basal cell carcinoma syndrome (NBCCS) patients. Both inherited and acquired mutations of patched 1 (PTCH1), a tumor-suppressor gene controlling the activity of Smoothened (SMO), are the primary cause of the constitutive activation of the Hedgehog (HH) pathway, leading to the emergence of BCCs in NBCCS. LDE225, a distinct, selective antagonist of SMO, showed potent inhibition of basaloid tumor nest formation and mediated regression of preformed basaloid tumors in organ cultures of skin derived from Ptch1 heterozygous knockout mice. In a double-blind, randomized, vehicle-controlled, intraindividual study, a total of 8 NBCCS patients presenting 27 BCCs were treated twice daily with 0.75% LDE225 cream or vehicle for 4 weeks. Application of 0.75% LDE225 cream was well tolerated and showed no skin irritation. Of 13 LDE225-treated BCCs, 3 showed a complete, 9 a partial, and only 1 no clinical response. Except for one partial response, the vehicle produced no clinical response in any of the 14 treated BCCs. Treatment with 0.75% LDE225 cream in NBCCS patients was very well tolerated and caused BCC regression, thus potentially offering an attractive therapeutic alternative to currently available therapies for this indication.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.
Asunto(s)
Antineoplásicos/administración & dosificación , Compuestos de Bifenilo/administración & dosificación , Carcinoma Basocelular/tratamiento farmacológico , Piridinas/administración & dosificación , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/tratamiento farmacológico , Administración Tópica , Animales , Antineoplásicos/efectos adversos , Compuestos de Bifenilo/efectos adversos , Carcinoma Basocelular/patología , Femenino , Cabello/efectos de los fármacos , Cabello/crecimiento & desarrollo , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Receptores Patched , Receptor Patched-1 , Embarazo , Piridinas/efectos adversos , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Neoplasias Cutáneas/patología , Receptor SmoothenedRESUMEN
Aberrant activation of the Hedgehog (Hh) pathway can drive tumorigenesis. To investigate the mechanism by which glioma-associated oncogene family zinc finger-1 (GLI1), a crucial effector of Hh signaling, regulates Hh pathway activation, we searched for GLI1-interacting proteins. We report that the chromatin remodeling protein SNF5 (encoded by SMARCB1, hereafter called SNF5), which is inactivated in human malignant rhabdoid tumors (MRTs), interacts with GLI1. We show that Snf5 localizes to Gli1-regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli-activated gene expression profile in primary MRTs and show that GLI1 drives the growth of SNF5-deficient MRT cells in vitro and in vivo. Therefore, our studies reveal that SNF5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a previously undescribed targetable mechanism contributing to the growth of MRT cells.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Tumor Rabdoide/genética , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/genética , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Humanos , Immunoblotting , Hibridación in Situ , Espectrometría de Masas , Ratones , Análisis por Micromatrices , Proteína SMARCB1 , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1RESUMEN
The malignant brain cancer medulloblastoma is characterized by mutations in Hedgehog (Hh) signaling pathway genes, which lead to constitutive activation of the G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptor Smoothened (Smo). The Smo antagonist NVP-LDE225 inhibits Hh signaling and induces tumor regression in animal models of medulloblastoma. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed several resistance mechanisms. We noted chromosomal amplification of Gli2, a downstream effector of Hh signaling, and, more rarely, point mutations in Smo that led to reactivated Hh signaling and restored tumor growth. Analysis of pathway gene expression signatures also, unexpectedly, identified up-regulation of phosphatidylinositol 3-kinase (PI3K) signaling in resistant tumors as another potential mechanism of resistance. Probing the relevance of increased PI3K signaling, we demonstrated that addition of the PI3K inhibitor NVP-BKM120 or the dual PI3K-mTOR (mammalian target of rapamycin) inhibitor NVP-BEZ235 to the initial treatment with the Smo antagonist markedly delayed the development of resistance. Our findings may be useful in informing treatment strategies for medulloblastoma.
Asunto(s)
Aminopiridinas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Meduloblastoma/enzimología , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Aminopiridinas/uso terapéutico , Animales , Proliferación Celular/efectos de los fármacos , Amplificación de Genes/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Morfolinas/uso terapéutico , Mutación/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteína Gli2 con Dedos de ZincRESUMEN
The blockade of aberrant hedgehog (Hh) signaling has shown promise for therapeutic intervention in cancer. A cell-based phenotypic high-throughput screen was performed, and the lead structure (1) was identified as an inhibitor of the Hh pathway via antagonism of the Smoothened receptor (Smo). Structure-activity relationship studies led to the discovery of a potent and specific Smoothened antagonist N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4'-(trifluoromethoxy)biphenyl-3-carboxamide (5m, NVP-LDE225), which is currently in clinical development.
RESUMEN
Abnormal activation of the Hedgehog (Hh) signaling pathway has been linked to several types of human cancers, and the development of small-molecule inhibitors of this pathway represents a promising route toward novel anticancer therapeutics. A cell-based screen performed in our laboratories identified a new class of Hh pathway inhibitors, 1-amino-4-benzylphthalazines, that act via antagonism of the Smoothened receptor. A variety of analogues were synthesized and their structure-activity relationships determined. This optimization resulted in the discovery of high affinity Smoothened antagonists, one of which was further profiled in vivo. This compound displayed a good pharmacokinetic profile and also afforded tumor regression in a genetic mouse model of medulloblastoma.