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p21-activated kinases (PAKs) are serine/threonine protein kinases that serve as important mediators of Rac and Cdc42 GTPase function as well as pathways required for Ras-driven tumorigenesis. PAK1 has been implicated in signaling by growth factor receptors and morphogenetic processes that control cell polarity, invasion, and actin cytoskeleton organization. To better understand the role of PAK1 in tumorigenesis, PAK1 genomic copy number and expression were determined for a large panel of breast, lung, and head and neck tumors. PAK1 genomic amplification at 11q13 was prevalent in luminal breast cancer, and PAK1 protein expression was associated with lymph node metastasis. Breast cancer cells with PAK1 genomic amplification rapidly underwent apoptosis after inhibition of this kinase. Strong nuclear and cytoplasmic PAK1 expression was also prevalent in squamous nonsmall cell lung carcinomas (NSCLCs), and selective PAK1 inhibition was associated with delayed cell-cycle progression in vitro and in vivo. NSCLC cells were profiled using a library of pathway-targeted small-molecule inhibitors, and several synergistic combination therapies, including combination with antagonists of inhibitor of apoptosis proteins, were revealed for PAK1. Dual inhibition of PAK1 and X chromosome-linked inhibitor of apoptosis efficiently increased effector caspase activation and apoptosis of NSCLC cells. Together, our results provide evidence for dysregulation of PAK1 in breast and squamous NSCLCs and a role for PAK1 in cellular survival and proliferation in these indications.
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Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacocinética , Quinases Ativadas por p21/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfonodos/enzimologia , Linfonodos/patologia , Metástase Linfática , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Quinases Ativadas por p21/metabolismoRESUMO
Rare DRAM2 coding variants cause retinal dystrophy with early macular involvement via unknown mechanisms. We found that DRAM2 is ubiquitously expressed in the human eye and expression changes were observed in eyes with more common maculopathy such as Age-related Macular Degeneration (AMD). To gain insights into pathogenicity of DRAM2-related retinopathy, we used a combination of in vitro and in vivo models. We found that DRAM2 loss in human pluripotent stem cell (hPSC)-derived retinal organoids caused the presence of additional mesenchymal cells. Interestingly, Dram2 loss in mice also caused increased proliferation of cells from the choroid in vitro and exacerbated choroidal neovascular lesions in vivo. Furthermore, we observed that DRAM2 loss in human retinal pigment epithelial (RPE) cells resulted in increased susceptibility to stress-induced cell death in vitro and that Dram2 loss in mice caused age-related photoreceptor degeneration. This highlights the complexity of DRAM2 function, as its loss in choroidal cells provided a proliferative advantage, whereas its loss in post-mitotic cells, such as photoreceptor and RPE cells, increased degeneration susceptibility. Different models such as human pluripotent stem cell-derived systems and mice can be leveraged to study and model human retinal dystrophies; however, cell type and species-specific expression must be taken into account when selecting relevant systems.
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Mutations in the GBA1 gene encoding glucocerebrosidase (GCase) are linked to Gaucher (GD) and Parkinson's Disease (PD). Since some GD and PD patients develop ocular phenotypes, we determined whether ocular phenotypes might result from impaired GCase activity and the corresponding accumulation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph) in the Gba1D409V/D409V knock-in (Gba KI/KI; "KI") mouse. Gba KI mice developed age-dependent pupil dilation deficits to an anti-muscarinic agent; histologically, the iris covered the anterior part of the lens with adhesions between the iris and the anterior surface of the lens (posterior synechia). This may prevent pupil dilation in general, beyond an un-responsiveness of the iris to anti-muscarinics. Gba KI mice displayed atrophy and pigment dispersion of the iris, and occlusion of the iridocorneal angle by pigment-laden cells, reminiscent of secondary open angle glaucoma. Gba KI mice showed progressive thinning of the retina consistent with retinal degeneration. GluSph levels were increased in the anterior and posterior segments of the eye, suggesting that accumulation of lipids in the eye may contribute to degeneration in this compartment. We conclude that the Gba KI model provides robust and reproducible eye phenotypes which may be used to test for efficacy and establish biomarkers for GBA1-related therapies.
Assuntos
Doença de Gaucher , Glaucoma de Ângulo Aberto , Glucosilceramidase , Mutação de Sentido Incorreto , Doença de Parkinson , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Doença de Gaucher/enzimologia , Doença de Gaucher/genética , Doença de Gaucher/patologia , Técnicas de Introdução de Genes , Glaucoma de Ângulo Aberto/enzimologia , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/patologia , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/patologiaRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Geographic atrophy (GA), the advanced form of dry age-related macular degeneration (AMD), is characterized by progressive loss of retinal pigment epithelium cells and photoreceptors in the setting of characteristic extracellular deposits and remains a serious unmet medical need. While genetic predisposition to AMD is dominated by polymorphisms in complement genes, it remains unclear how complement activation contributes to retinal atrophy. Here we demonstrate that complement is activated on photoreceptor outer segments (POS) in the retina peripheral to atrophic lesions associated with GA. When exposed to human serum following outer blood-retinal barrier breakdown, POS act as potent activators of the classical and alternative complement pathway. In mouse models of retinal degeneration, classical and alternative pathway complement activation on photoreceptors contributed to the loss of photoreceptor function. This was dependent on C5a-mediated recruitment of peripheral blood monocytes but independent of resident microglia. Genetic or pharmacologic inhibition of both classical and alternative complement C3 and C5 convertases was required to reduce progressive degeneration of photoreceptor rods and cones. Our study implicates systemic classical and alternative complement proteins and peripheral blood monocytes as critical effectors of localized retinal degeneration with potential relevance for the contribution of complement activation to GA.
Assuntos
Ativação do Complemento/genética , Atrofia Geográfica/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Atrofia/patologia , Ativação do Complemento/fisiologia , Complemento C3/genética , Complemento C3/fisiologia , Complemento C4/genética , Complemento C4/fisiologia , Atrofia Geográfica/genética , Humanos , Degeneração Macular/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/metabolismoRESUMO
Age-related macular degeneration (AMD), a leading cause of vision impairment in the ageing population, is characterized by irreversible loss of retinal pigment epithelial (RPE) cells and photoreceptors and can be associated with choroidal neovascularization. Mononuclear phagocytes are often present in AMD lesions, but the processes that direct myeloid cell recruitment remain unclear. Here, we identify IL-33 as a key regulator of inflammation and photoreceptor degeneration after retina stress or injury. IL-33(+) Müller cells were more abundant and IL-33 cytokine was elevated in advanced AMD cases compared with age-matched controls with no AMD. In rodents, retina stress resulted in release of bioactive IL-33 that in turn increased inflammatory chemokine and cytokine expression in activated Müller cells. Deletion of ST2, the IL-33 receptor α chain, or treatment with a soluble IL-33 decoy receptor significantly reduced release of inflammatory mediators from Müller cells, inhibited accumulation of mononuclear phagocytes in the outer retina, and protected photoreceptor rods and cones after a retina insult. This study demonstrates a central role for IL-33 in regulating mononuclear phagocyte recruitment to the photoreceptor layer and positions IL-33 signaling as a potential therapeutic target in macular degenerative diseases.
Assuntos
Imunidade Inata , Interleucina-33/metabolismo , Degeneração Macular/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Estudos de Casos e Controles , Núcleo Celular/imunologia , Citocinas/metabolismo , Células Ependimogliais/imunologia , Células Ependimogliais/patologia , Feminino , Humanos , Técnicas In Vitro , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33/química , Interleucina-33/deficiência , Interleucina-33/genética , Macula Lutea/imunologia , Macula Lutea/patologia , Degeneração Macular/genética , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Processamento de Proteína Pós-Traducional , Ratos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Interleucina/deficiência , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/patologiaRESUMO
We have recently reported a series of tetrahydroquinazoline (THQ) mTOR inhibitors that produced a clinical candidate 1 (GDC-0349). Through insightful design, we hoped to discover and synthesize a new series of small molecule inhibitors that could attenuate CYP3A4 time-dependent inhibition commonly observed with the THQ scaffold, maintain or improve aqueous solubility and oral absorption, reduce free drug clearance, and selectively increase mTOR potency. Through key in vitro and in vivo studies, we demonstrate that a pyrimidoaminotropane based core was able to address each of these goals. This effort culminated in the discovery of 20 (GNE-555), a highly potent, selective, metabolically stable, and efficacious mTOR inhibitor.
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Inibidores Enzimáticos/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Tropanos/farmacologia , Cromatografia Líquida , Inibidores Enzimáticos/química , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Tropanos/químicaRESUMO
Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.
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CDK8 is a cyclin-dependent kinase that mediates transcriptional control of pathways linked to both cancer and stem cells. In this study, we show that CDK8 is required for both tumor growth and maintenance of tumor dedifferentiation in vivo and uncover a common role for CDK8 in controlling cancer and stem cell function. Acute CDK8 loss in vivo strongly inhibited tumor growth and promoted differentiation. Transcriptional profiling identified a set of embryonic stem cell-related genes that are activated by CDK8 in cancer. Consistent with this, we found that CDK8 expression correlated to the embryonic stem cell pluripotency state and loss of CDK8 caused embryonic stem cells to differentiate. This effect was, at least partially, mediated by the ability of CDK8 to regulate MYC protein and downstream MYC target gene expression. Similar regulation of MYC target genes by CDK8 was observed in colon tumor cells, and increased expression of a CDK8-regulated, embryonic stem cell MYC target gene signature was associated with loss of differentiation and poor outcome in primary human colon cancers. Together, these observations reveal that CDK8 acts, at least in part, through MYC to maintain both tumors and embryonic stem cells in an undifferentiated state. This raises the intriguing possibility that targeting CDK8 therapeutically may specifically inhibit the stem-like properties of cancer cells.
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Desdiferenciação Celular/fisiologia , Quinase 8 Dependente de Ciclina/metabolismo , Células-Tronco Embrionárias/enzimologia , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/patologia , Células-Tronco Pluripotentes/enzimologia , Animais , Western Blotting , Linhagem Celular Tumoral , Separação Celular , Células-Tronco Embrionárias/citologia , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes myc , Humanos , Imuno-Histoquímica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/citologia , TransfecçãoRESUMO
The mammalian target of rapamycin (mTOR) is a central node in a complex signaling network that is regulated by several pathways deregulated in human cancers, including the PI3K/Akt and MAPK pathways. Targeting mTOR therefore presents an opportunity for therapeutic intervention. However, mTOR inhibition with rapamycin analogs or kinase inhibitors reduces cell growth but does not induce apoptosis, and the clinical benefit of rapamycin analogs has been modest. In this study we show that mTOR kinase inhibitors can potentiate apoptosis when used in combination with upstream targeted agents such as PI3K and MEK inhibitors. This increased apoptosis is dependent on genetic background, and correlates with active growth factor survival pathways. In PI3K mutant tumors, mTOR inhibition leads to partial reactivation of Akt which allows cells to survive, whereas in KRAS mutant tumors, this same reactivation of Akt occurs but is not required for cell survival. These data suggest the use of selected rational combinations of mTOR kinase inhibitors with other targeted inhibitors in specific tumor genotypes to achieve the maximal cytotoxic response by inhibiting two nodes in the activated signaling network.
Assuntos
Apoptose/efeitos dos fármacos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Western Blotting , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , HumanosRESUMO
Breast cancer has been redefined into three clinically relevant subclasses: (i) estrogen/progesterone receptor positive (ER+/PR+), (ii) HER2/ERRB2 positive, and (iii) those lacking expression of all three markers (triple negative or basal-like). While targeted therapies for ER+/PR+ and HER2+ tumors have revolutionized patient treatment and increased lifespan, an urgent need exists for identifying novel targets for triple-negative breast cancers. Here, we used integrative genomic analysis to identify candidate oncogenes in triple-negative breast tumors and assess their function through loss of function screening. Using this approach, we identify lactate dehydrogenase B (LDHB), a component of glycolytic metabolism, as an essential gene in triple-negative breast cancer. Loss of LDHB abrogated cell proliferation in vitro and arrested tumor growth in fully formed tumors in vivo. We find that LDHB and other related glycolysis genes are specifically upregulated in basal-like/triple-negative breast cancers as compared with other subtypes, suggesting that these tumors are distinctly glycolytic. Consistent with this, triple-negative breast cancer cell lines were more dependent on glycolysis for growth than luminal cell lines. Finally, we find that patients with breast cancer and high LDHB expression in their tumors had a poor clinical outcome. While previous studies have focused on the ubiquitous role of LDHA in tumor metabolism and growth, our data reveal that LDHB is upregulated and required only in certain cancer genotypes. These findings suggest that targeting LDHB or other components of lactate metabolism would be of clinical benefit in triple-negative breast cancer.
Assuntos
Neoplasias da Mama/genética , Lactato Desidrogenases/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Lactato Desidrogenases/biossíntese , Células MCF-7 , Camundongos , Camundongos Nus , Prognóstico , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Transplante HeterólogoRESUMO
Selective inhibitors of mammalian target of rapamycin (mTOR) kinase based upon saturated heterocycles fused to a pyrimidine core were designed and synthesized. Each series produced compounds with K(i) < 10 nM for the mTOR kinase and >500-fold selectivity over closely related PI3 kinases. This potency translated into strong pathway inhibition, as measured by phosphorylation of mTOR substrate proteins and antiproliferative activity in cell lines with a constitutively active PI3K pathway. Two compounds exhibiting suitable mouse PK were profiled in in vivo tumor models and were shown to suppress mTORC1 and mTORC2 signaling for over 12 h when dosed orally. Both compounds were additionally shown to suppress tumor growth in vivo in a PC3 prostate cancer model over a 14 day study.
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Antineoplásicos/síntese química , Complexos Multiproteicos/antagonistas & inibidores , Pirimidinas/síntese química , Pirróis/síntese química , Quinazolinas/síntese química , Serina-Treonina Quinases TOR/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Nus , Simulação de Acoplamento Molecular , Transplante de Neoplasias , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Neoplasias da Próstata , Pirimidinas/química , Pirimidinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
A series of inhibitors of mTOR kinase based on a quaternary-substituted dihydrofuropyrimidine was designed and synthesized. The most potent compounds in this series inhibited mTOR kinase with K(i) < 1.0 nM and were highly (>100×) selective for mTOR over the closely related PI3 kinases. Compounds in this series showed inhibition of the pathway and antiproliferative activity in cell-based assays. Furthermore, these compounds had excellent mouse PK, and showed a robust PK-PD relationship in a mouse model of cancer.
Assuntos
Antineoplásicos/síntese química , Furanos/síntese química , Pirimidinas/síntese química , Serina-Treonina Quinases TOR/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Disponibilidade Biológica , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Furanos/farmacocinética , Furanos/farmacologia , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Transplante de Neoplasias , Inibidores de Fosfoinositídeo-3 Quinase , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Especificidade da Espécie , Estereoisomerismo , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
PURPOSE: Oncogenic activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is prevalent in breast cancer and has been associated with resistance to HER2 inhibitors in the clinic. We therefore investigated the combinatorial activity of GDC-0941, a novel class I PI3K inhibitor, with standard-of-care therapies for HER2-amplified breast cancer. EXPERIMENTAL DESIGN: Three-dimensional laminin-rich extracellular matrix cultures of human breast cancer cells were utilized to provide a physiologically relevant approach to analyze the efficacy and molecular mechanism of combination therapies ex vivo. Combination studies were done using GDC-0941 with trastuzumab (Herceptin), pertuzumab, lapatinib (Tykerb), and docetaxel, the principal therapeutic agents that are either approved or being evaluated for treatment of early HER2-positive breast cancer. RESULTS: Significant GDC-0941 activity (EC(50) <1 micromol/L) was observed for >70% of breast cancer cell lines that were examined in three-dimensional laminin-rich extracellular matrix culture. Differential responsiveness to GDC-0941 as a single agent was observed for luminal breast cancer cells upon stimulation with the HER3 ligand, heregulin. Combined treatment of GDC-0941, trastuzumab, and pertuzumab resulted in growth inhibition, altered acinar morphology, and suppression of AKT mitogen-activated protein kinase (MAPK) / extracellular signed-regulated kinase (ERK) kinase and MEK effector signaling pathways for HER2-amplified cells in both normal and heregulin-supplemented media. The GDC-0941 and lapatinib combination further showed that inhibition of HER2 activity was essential for maximum combinatorial efficacy. PI3K inhibition also rendered HER2-amplified BT-474M1 cells and tumor xenografts more sensitive to docetaxel. CONCLUSIONS: GDC-0941 is efficacious in preclinical models of breast cancer. The addition of GDC-0941 to HER2-directed treatment could augment clinical benefit in breast cancer patients.
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Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Indazóis/uso terapêutico , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Sulfonamidas/uso terapêutico , Animais , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Docetaxel , Humanos , Lapatinib , Camundongos , Camundongos Nus , Inibidores de Fosfoinositídeo-3 Quinase , Quinazolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Taxoides/farmacologia , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: The pathways underlying basal-like breast cancer are poorly understood, and as yet, there is no approved targeted therapy for this disease. We investigated the role of mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors as targeted therapies for basal-like breast cancer. EXPERIMENTAL DESIGN: We used pharmacogenomic analysis of a large panel of breast cancer cell lines with detailed accompanying molecular information to identify molecular predictors of response to a potent and selective inhibitor of MEK and also to define molecular mechanisms underlying combined MEK and PI3K targeting in basal-like breast cancer. Hypotheses were confirmed by testing in multiple tumor xenograft models. RESULTS: We found that basal-like breast cancer models have an activated RAS-like transcriptional program and show greater sensitivity to a selective inhibitor of MEK compared with models representative of other breast cancer subtypes. We also showed that loss of PTEN is a negative predictor of response to MEK inhibition, that treatment with a selective MEK inhibitor caused up-regulation of PI3K pathway signaling, and that dual blockade of both PI3K and MEK/extracellular signal-regulated kinase signaling synergized to potently impair the growth of basal-like breast cancer models in vitro and in vivo. CONCLUSIONS: Our studies suggest that single-agent MEK inhibition is a promising therapeutic modality for basal-like breast cancers with intact PTEN, and also provide a basis for rational combination of MEK and PI3K inhibitors in basal-like cancers with both intact and deleted PTEN.