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1.
Immunity ; 40(4): 477-89, 2014 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-24745332

RESUMO

We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.


Assuntos
Benzenoacetamidas/farmacologia , Compostos Benzidrílicos/farmacologia , Digoxina/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Redes Reguladoras de Genes/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Esclerose Múltipla/tratamento farmacológico , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Subpopulações de Linfócitos T/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Androstenóis/química , Animais , Benzenoacetamidas/química , Compostos Benzidrílicos/química , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Citocinas/metabolismo , Digoxina/química , Encefalomielite Autoimune Experimental/imunologia , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla/imunologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Fragmentos de Peptídeos/imunologia , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Biologia de Sistemas , Subpopulações de Linfócitos T/imunologia , Células Th17/imunologia , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos
2.
Nature ; 538(7623): 114-117, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27680702

RESUMO

The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5-Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1-TEAD inhibitor. These findings indicate that clinically available XPO1 inhibitors are a promising therapeutic strategy for a considerable cohort of patients with lung cancer when coupled to genomics-guided patient selection and observation.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Carioferinas/antagonistas & inibidores , Carioferinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Feminino , Proteínas Relacionadas à Folistatina/genética , Genes Letais/genética , Via de Sinalização Hippo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Porfirinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Verteporfina , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAP , Proteína Exportina 1
3.
Blood ; 129(16): 2233-2245, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28096095

RESUMO

Dysregulated oncogenic serine/threonine kinases play a pathological role in diverse forms of malignancies, including multiple myeloma (MM), and thus represent potential therapeutic targets. Here, we evaluated the biological and functional role of p21-activated kinase 4 (PAK4) and its potential as a new target in MM for clinical applications. PAK4 promoted MM cell growth and survival via activation of MM survival signaling pathways, including the MEK-extracellular signal-regulated kinase pathway. Furthermore, treatment with orally bioavailable PAK4 allosteric modulator (KPT-9274) significantly impacted MM cell growth and survival in a large panel of MM cell lines and primary MM cells alone and in the presence of bone marrow microenvironment. Intriguingly, we have identified FGFR3 as a novel binding partner of PAK4 and observed significant activity of KPT-9274 against t(4;14)-positive MM cells. This set of data supports PAK4 as an oncogene in myeloma and provide the rationale for the clinical evaluation of PAK4 modulator in myeloma.


Assuntos
Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Quinases Ativadas por p21/genética , Regulação Alostérica , Animais , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/enzimologia , Células da Medula Óssea/patologia , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 4 , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/patologia , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/patologia , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Translocação Genética , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/metabolismo
4.
Int J Mol Sci ; 21(1)2019 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-31905765

RESUMO

Lenvatinib is a multitargeted tyrosine kinase inhibitor (TKI) that shows improved median progression-free survival (PFS) in patients with thyroid carcinomas. However, virtually all patients ultimately progress, indicating the need for a better understanding of the mechanisms of resistance. Here, we examined the molecular profile of anaplastic thyroid cancer cells (8505C) exposed to lenvatinib and found that long-term exposure to lenvatinib caused phenotypic changes. Consistent with change toward mesenchymal morphology, activation of pro-survival signaling, nuclear exporter protein exportin 1 (XPO1) and Rho GTPase effector p21 activated kinases (PAK) was also observed. RNA-seq analysis showed that prolonged lenvatinib treatment caused alterations in numerous cellular pathways and several oncogenes such as CEACAM (carcinoembryonic antigen-related cell adhesion molecule) and NUPR1 (Nuclear protein 1) were also upregulated. Further, we evaluated the impact of XPO1 and PAK4 inhibition in the presence or absence of lenvatinib. Targeted inhibition of XPO1 and PAK4 could sensitize the 8505C cells to lenvatinib. Both XPO1 and PAK4 inhibitors, when combined with lenvatinib, showed superior anti-tumor activity in 8505C sub-cutaneous xenograft. These studies bring forward novel drug combinations to complement lenvatinib for treating anaplastic thyroid cancer. Such combinations may possibly reduce the chances of lenvatinib resistance in thyroid cancer patients.


Assuntos
Antineoplásicos/farmacologia , Carioferinas/antagonistas & inibidores , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Transcriptoma/efeitos dos fármacos , Quinases Ativadas por p21/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Quimioterapia Combinada , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Carioferinas/metabolismo , Camundongos Endogâmicos ICR , Camundongos SCID , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas/uso terapêutico , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Transcriptoma/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/metabolismo , Proteína Exportina 1
5.
Int J Mol Sci ; 20(19)2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31569391

RESUMO

Gastric cancer remains an unmet clinical problem in urgent need of newer and effective treatments. Here we show that the nuclear export protein, Exportin 1 (XPO1, chromosome region maintenance 1 or CRM1), is a promising molecular target in gastric cancer. We demonstrate significant overexpression of XPO1 in a cohort of histologically diverse gastric cancer patients with primary and metastatic disease. XPO1 RNA interference suppressed gastric cancer cell growth. Anti-tumor activity was observed with specific inhibitor of nuclear export (SINE) compounds (selinexor/XPOVIO), second-generation compound KPT-8602/eltanexor, KPT-185 and +ve control Leptomycin B in three distinct gastric cancer cell lines. SINE compounds inhibited gastric cancer cell proliferation, disrupted spheroid formation, induced apoptosis and halted cell cycle progression at the G1/S phase. Anti-tumor activity was concurrent with nuclear retention of tumor suppressor proteins and inhibition of colony formation. In combination studies, SINE compounds enhanced the efficacy of nab-paclitaxel in vitro and in vivo. More significantly, using non-coding RNA sequencing studies, we demonstrate for the first time that SINE compounds can alter the expression of non-coding RNAs (microRNAs and piwiRNAs). SINE treatment caused statistically significant downregulation of oncogenic miR-33b-3p in two distinct cell lines. These studies demonstrate the therapeutic significance of XPO1 in gastric cancer that warrants further clinical investigation.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Neoplasias Gástricas/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Sobrevivência Celular , Humanos , Paclitaxel/química , Paclitaxel/farmacologia , Proteína Exportina 1
6.
Genomics ; 2017 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-29055713

RESUMO

The p-21 Activated Kinase 4 (PAK4) protein kinase is implicated in many cancers, including breast cancer. Overexpression of PAK4 is sufficient to cause mouse mammary epithelial cells (iMMECs) to become tumorigenic. To gain insight into the long-term gene expression changes that occur downstream to PAK4, we performed Next Generation Sequencing of RNA collected from PAK4 overexpressing iMMECs and wild-type iMMECs. We identified a list of genes whose expression levels were altered in response to PAK4 overexpression in iMMECs. Some of these genes, including FoxC2 and ParvB, are consistent with a role for PAK4 in cancer. In addition, PAK4 regulates many genes that are frequently associated with the inflammatory response, raising the possibility that there is a connection between PAK4, inflammation, and the tumor microenvironment. This study delineates the PAK4 transcriptome profile in transformed mammary cells and can provide translational utility in other types of cancers as well.

7.
Gut ; 66(8): 1358-1368, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-27196599

RESUMO

OBJECTIVES: Oesophageal squamous cell carcinoma (OSCC) is an aggressive malignancy and the major histological subtype of oesophageal cancer. Although recent large-scale genomic analysis has improved the description of the genetic abnormalities of OSCC, few targetable genomic lesions have been identified, and no molecular therapy is available. This study aims to identify druggable candidates in this tumour. DESIGN: High-throughput small-molecule inhibitor screening was performed to identify potent anti-OSCC compounds. Whole-transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) were conducted to decipher the mechanisms of action of CDK7 inhibition in OSCC. A variety of in vitro and in vivo cellular assays were performed to determine the effects of candidate genes on OSCC malignant phenotypes. RESULTS: The unbiased high-throughput small-molecule inhibitor screening led us to discover a highly potent anti-OSCC compound, THZ1, a specific CDK7 inhibitor. RNA-Seq revealed that low-dose THZ1 treatment caused selective inhibition of a number of oncogenic transcripts. Notably, further characterisation of the genomic features of these THZ1-sensitive transcripts demonstrated that they were frequently associated with super-enhancer (SE). Moreover, SE analysis alone uncovered many OSCC lineage-specific master regulators. Finally, integrative analysis of both THZ1-sensitive and SE-associated transcripts identified a number of novel OSCC oncogenes, including PAK4, RUNX1, DNAJB1, SREBF2 and YAP1, with PAK4 being a potential druggable kinase. CONCLUSIONS: Our integrative approaches led to a catalogue of SE-associated master regulators and oncogenic transcripts, which may significantly promote both the understanding of OSCC biology and the development of more innovative therapies.


Assuntos
Acrilamidas/farmacologia , Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Expressão Gênica/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Pirimidinas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Esofágicas/tratamento farmacológico , Feminino , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP40/genética , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Transplante de Neoplasias , Oncogenes/genética , Fosfoproteínas/genética , Análise de Sequência de RNA , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Fatores de Transcrição , Transcriptoma , Proteínas de Sinalização YAP , Quinases Ativadas por p21/genética , Quinase Ativadora de Quinase Dependente de Ciclina
8.
Kidney Int ; 92(4): 922-933, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28545714

RESUMO

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common hereditary renal disease with no currently available targeted therapies. Based on the established connection between ß-catenin signaling and renal ciliopathies, and on data from our and other laboratories showing striking similarities of this disease and cancer, we evaluated the use of an orally bioavailable small molecule, KPT-9274 (a dual inhibitor of the protein kinase PAK4 and nicotinamide phosphoribosyl transferase), for treatment of ADPKD. Treatment of PKD-derived cells with this compound not only reduces PAK4 steady-state protein levels and regulates ß-catenin signaling, but also inhibits nicotinamide phosphoribosyl transferase, the rate-limiting enzyme in a key NAD salvage pathway. KPT-9274 can attenuate cellular proliferation and induce apoptosis associated with a decrease in active (phosphorylated) PAK4 and ß-catenin in several Pkd1-null murine cell lines, with a less pronounced effect on the corresponding phenotypically normal cells. Additionally, KPT-9274 shows inhibition of cystogenesis in an ex vivo model of cyclic AMP-induced cystogenesis as well as in the early stage Pkd1flox/flox:Pkhd1-Cre mouse model, the latter showing confirmation of specific anti-proliferative, apoptotic, and on-target effects. NAD biosynthetic attenuation by KPT-9274, while critical for highly proliferative cancer cells, does not appear to be important in the slower growing cystic epithelial cells during cystogenesis. KPT-9274 was not toxic in our ADPKD animal model or in other cancer models. Thus, this small molecule inhibitor could be evaluated in a clinical trial as a viable therapy of ADPKD.


Assuntos
Acrilamidas/farmacologia , Aminopiridinas/farmacologia , Citocinas/metabolismo , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Rim Policístico Autossômico Dominante/tratamento farmacológico , Quinases Ativadas por p21/metabolismo , Acrilamidas/uso terapêutico , Aminopiridinas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais , Feminino , Humanos , Rim/citologia , Masculino , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Fosforilação , Rim Policístico Autossômico Dominante/patologia , Receptores de Superfície Celular/genética , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPP/genética , beta Catenina/metabolismo
10.
J Cell Physiol ; 231(12): 2711-9, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-26991404

RESUMO

Previous work has shown that the three-dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT-185, KPT-330/selinexor, and KPT-8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment-naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non-lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711-2719, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Núcleo Celular/metabolismo , Imageamento Tridimensional , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Telômero/metabolismo , Idoso , Linhagem Celular Tumoral , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Proteína Exportina 1
11.
J Immunol ; 192(6): 2564-75, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24516202

RESUMO

IL-17-producing CD4(+)Th17 cells, CD8(+)Tc17 cells, and γδ T cells play critical roles in the pathogenesis of autoimmune psoriasis. RORγt is required for the differentiation of Th17 cells and expression of IL-17. In this article, we describe a novel, potent, and selective RORγt inverse agonist (TMP778), and its inactive diastereomer (TMP776). This chemistry, for the first time to our knowledge, provides a unique and powerful set of tools to probe RORγt-dependent functions. TMP778, but not TMP776, blocked human Th17 and Tc17 cell differentiation and also acutely modulated IL-17A production and inflammatory Th17-signature gene expression (Il17a, Il17f, Il22, Il26, Ccr6, and Il23) in mature human Th17 effector/memory T cells. In addition, TMP778, but not TMP776, inhibited IL-17A production in both human and mouse γδ T cells. IL-23-induced IL-17A production was also blocked by TMP778 treatment. In vivo targeting of RORγt in mice via TMP778 administration reduced imiquimod-induced psoriasis-like cutaneous inflammation. Further, TMP778 selectively regulated Th17-signature gene expression in mononuclear cells isolated from both the blood and affected skin of psoriasis patients. In summary, to our knowledge, we are the first to demonstrate that RORγt inverse agonists: 1) inhibit Tc17 cell differentiation, as well as IL-17 production by γδ T cells and CD8(+) Tc17 cells; 2) block imiquimod-induced cutaneous inflammation; 3) inhibit Th17 signature gene expression by cells isolated from psoriatic patient samples; and 4) block IL-23-induced IL-17A expression. Thus, RORγt is a tractable drug target for the treatment of cutaneous inflammatory disorders, which may afford additional therapeutic benefit over existing modalities that target only IL-17A.


Assuntos
Dermatite/prevenção & controle , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Células Th17/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Adulto , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Dermatite/imunologia , Dermatite/metabolismo , Relação Dose-Resposta a Droga , Feminino , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Células Jurkat , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Psoríase/sangue , Psoríase/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/imunologia , Pele/metabolismo , Pele/patologia , Bibliotecas de Moléculas Pequenas/química , Células Th17/imunologia , Células Th17/metabolismo , Transcriptoma/imunologia
12.
Semin Cancer Biol ; 27: 74-86, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24755012

RESUMO

Clinical targeting of multi-dimensional proteins such as the proteasome has been efficacious in recent years. Inhibitors such as bortezomib and carfilzomib have been used successfully to treat multiple myeloma despite early skepticism surrounding unsubstantiated toxic side effects. Another target of this magnitude is ready to emerge as a clinically viable option for targeting various neoplasias. This target, XPO1 (exportin-1 also known as Chromosome Region Maintenance 1 (CRM1)), is the transport protein responsible for nuclear export of many of the major tumor suppressor proteins and cell growth regulators. Up-regulation of XPO1 protein, a common occurrence in a variety of cancers, can lead to aberrant cytoplasmic localization and degradation of tumor suppressors such as p53 and FOXO. Therefore, inhibition of XPO1 using specific small molecules collectively called Selective Inhibitors of Nuclear Export (SINE) could potentially restore normal tumor suppressor function and have universal application for the treatment of cancer. This review will discuss the current pre-clinical data on SINE compounds in both hematological and solid malignancies. Cancer treatment through direct inhibition of the proteasome and the nuclear export machinery should instill optimism for further targeting of critical cellular pathways.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Neoplasias/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Modelos Animais de Doenças , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Pesquisa Translacional Biomédica
13.
Immunology ; 145(3): 347-56, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25604624

RESUMO

The orphan nuclear receptor, retinoic acid receptor-related orphan nuclear receptor γt (RORγt), is required for the development and pathogenic function of interleukin-17A-secreting CD4(+) T helper type 17 (Th17) cells. Whereas small molecule RORγt antagonists impair Th17 cell development and attenuate autoimmune inflammation in vivo, the broader effects of these inhibitors on RORγt-dependent gene expression in vivo has yet to be characterized. We show that the RORγt inverse agonist TMP778 acts potently and selectively to block mouse Th17 cell differentiation in vitro and to impair Th17 cell development in vivo upon immunization with the myelin antigen MOG35-55 plus complete Freund's adjuvant. Importantly, we show that TMP778 acts in vivo to repress the expression of more than 150 genes, most of which fall outside the canonical Th17 transcriptional signature and are linked to a variety of inflammatory pathologies in humans. Interestingly, more than 30 genes are related with SMAD3, a transcription factor involved in the Th17 cell differentiation. These results reveal novel disease-associated genes regulated by RORγt during inflammation in vivo, and provide an early read on potential disease indications and safety concerns associated with pharmacological targeting of RORγt.


Assuntos
Diferenciação Celular/imunologia , Expressão Gênica/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Feminino , Adjuvante de Freund/imunologia , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Imunização/métodos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fragmentos de Peptídeos/imunologia , Células Th17/efeitos dos fármacos , Células Th17/metabolismo
14.
J Virol ; 88(17): 10228-43, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24965445

RESUMO

UNLABELLED: Influenza is a global health concern, causing death, morbidity, and economic losses. Chemotherapeutics that target influenza virus are available; however, rapid emergence of drug-resistant strains is common. Therapeutic targeting of host proteins hijacked by influenza virus to facilitate replication is an antiviral strategy to reduce the development of drug resistance. Nuclear export of influenza virus ribonucleoprotein (vRNP) from infected cells has been shown to be mediated by exportin 1 (XPO1) interaction with viral nuclear export protein tethered to vRNP. RNA interference screening has identified XPO1 as a host proinfluenza factor where XPO1 silencing results in reduced influenza virus replication. The Streptomyces metabolite XPO1 inhibitor leptomycin B (LMB) has been shown to limit influenza virus replication in vitro; however, LMB is toxic in vivo, which makes it unsuitable for therapeutic use. In this study, we tested the anti-influenza virus activity of a new class of orally available small-molecule selective inhibitors of nuclear export, specifically, the XPO1 antagonist KPT-335 (verdinexor). Verdinexor was shown to potently and selectively inhibit vRNP export and effectively inhibited the replication of various influenza virus A and B strains in vitro, including pandemic H1N1 virus, highly pathogenic H5N1 avian influenza virus, and the recently emerged H7N9 strain. In vivo, prophylactic and therapeutic administration of verdinexor protected mice against disease pathology following a challenge with influenza virus A/California/04/09 or A/Philippines/2/82-X79, as well as reduced lung viral loads and proinflammatory cytokine expression, while having minimal toxicity. These studies show that verdinexor acts as a novel anti-influenza virus therapeutic agent. IMPORTANCE: Antiviral drugs represent important means of influenza virus control. However, substantial resistance to currently approved influenza therapeutic drugs has developed. New antiviral approaches are required to address drug resistance and reduce the burden of influenza virus-related disease. This study addressed critical preclinical studies for the development of verdinexor (KPT-335) as a novel antiviral drug. Verdinexor blocks progeny influenza virus genome nuclear export, thus effectively inhibiting virus replication. Verdinexor was found to limit the replication of various strains of influenza A and B viruses, including a pandemic H1N1 influenza virus strain, a highly pathogenic H5N1 avian influenza virus strain, and a recently emerging H7N9 influenza virus strain. Importantly, oral verdinexor treatments, given prophylactically or therapeutically, were efficacious in limiting lung virus burdens in influenza virus-infected mice, in addition to limiting lung proinflammatory cytokine expression, pathology, and death. Thus, this study demonstrated that verdinexor is efficacious against influenza virus infection in vitro and in vivo.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antivirais/metabolismo , Inibidores Enzimáticos/metabolismo , Vírus da Influenza A/fisiologia , Vírus da Influenza B/fisiologia , Carioferinas/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Linhagem Celular , Quimioprevenção/métodos , Modelos Animais de Doenças , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Proteína Exportina 1
15.
Nat Chem Biol ; 9(5): 319-25, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23524983

RESUMO

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.


Assuntos
Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Zinco/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/síntese química , Histona Desacetilases/genética , Humanos , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Oxidiazóis/química , Relação Estrutura-Atividade , Zinco/metabolismo
16.
bioRxiv ; 2023 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-37034616

RESUMO

KRASG12C inhibitors have revolutionized the treatment landscape for cancer patients harboring the G12C mutant isoform of KRAS. With the recent FDA approval of sotorasib and adagrasib, patients now have access to more promising treatment options. However, patients who receive these agents as a monotherapy usually develop drug resistance. Thus, there is a need to develop logical combination strategies that can delay or prevent the onset of resistance and simultaneously enhance the antitumor effectiveness of the treatment regimen. In this study, we aimed at pharmacologically targeting PAK4 by KPT9274 in combination with KRASG12C inhibitors in KRASG12C mutant pancreatic ductal adenocarcinoma (PDAC) and nonâ€"small cell lung cancer (NSCLC) preclinical models. PAK4 is a hub molecule that links several major signaling pathways and is known for its tumorigenic role in mutant Ras-driven cancers. We assessed the cytotoxicity of PAK4 and KRASG12C inhibitors combination in KRASG12C mutant 2D and 3D cellular models. KPT9274 synergized with both sotorasib and adagrasib in inhibiting the growth of KRASG12C mutant cancer cells. The combination was able to reduce the clonogenic potential of KRASG12C mutant PDAC cells. We also evaluated the antitumor activity of the combination in a KRASG12C mutant PDAC cell line-derived xenograft (CDX) model. Oral administration of a sub-optimal dose of KPT9274 in combination with sotorasib (at one-fourth of MTD) demonstrated significant inhibition of the tumor burden ( p = 0.002). Similarly, potent antitumor efficacy was observed in an NSCLC CDX model where KPT9274, acting as an adjuvant, prevented tumor relapse following the discontinuation of sotorasib treatment ( p = 0.0001). KPT9274 and sotorasib combination also resulted in enhanced survival. This is the first study showing that KRASG12C inhibitors can synergize with PAK4 inhibitor KPT9274 both in vitro and in vivo resulting in remarkably enhanced antitumor activity and survival outcomes. Significance: KRASG12C inhibitors demonstrate limited durable response in patients with KRASG12C mutations. In this study, combining PAK4 inhibitor KPT9274 with KRASG12C inhibitors has resulted in potent antitumor effects in preclinical cancer models of PDAC and NSCLC. Our results bring forward a novel combination therapy for cancer patients that do not respond or develop resistance to KRASG12C inhibitor treatment.

17.
Mol Cancer Ther ; 22(12): 1422-1433, 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-37703579

RESUMO

KRASG12C inhibitors, such as sotorasib and adagrasib, have revolutionized cancer treatment for patients with KRASG12C-mutant tumors. However, patients receiving these agents as monotherapy often develop drug resistance. To address this issue, we evaluated the combination of the PAK4 inhibitor KPT9274 and KRASG12C inhibitors in preclinical models of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). PAK4 is a hub molecule that links several major signaling pathways and is known for its tumorigenic role in mutant Ras-driven cancers. We found that cancer cells resistant to KRASG12C inhibitor were sensitive to KPT9274-induced growth inhibition. Furthermore, KPT9274 synergized with sotorasib and adagrasib to inhibit the growth of KRASG12C-mutant cancer cells and reduce their clonogenic potential. Mechanistically, this combination suppressed cell growth signaling and downregulated cell-cycle markers. In a PDAC cell line-derived xenograft (CDX) model, the combination of a suboptimal dose of KPT9274 with sotorasib significantly reduced the tumor burden (P= 0.002). Similarly, potent antitumor efficacy was observed in an NSCLC CDX model, in which KPT9274, given as maintenance therapy, prevented tumor relapse following the discontinuation of sotorasib treatment (P= 0.0001). Moreover, the combination of KPT9274 and sotorasib enhances survival. In conclusion, this is the first study to demonstrate that KRASG12C inhibitors can synergize with the PAK4 inhibitor KPT9274 and combining KRASG12C inhibitors with KPT9274 can lead to remarkably enhanced antitumor activity and survival benefits, providing a novel combination therapy for patients with cancer who do not respond or develop resistance to KRASG12C inhibitor treatment.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma Ductal Pancreático , Neoplasias Pulmonares , Neoplasias Pancreáticas , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases Ativadas por p21/genética , Neoplasias Pancreáticas
18.
J Hematol Oncol ; 15(1): 103, 2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35922861

RESUMO

Patients with higher-risk myelodysplastic syndromes (MDS) refractory to hypomethylating agents (HMAs) have limited therapeutic options and an expected overall survival (OS) of 3-5 months. Eltanexor is an investigational oral selective inhibitor of nuclear export with low central nervous system penetrance and an acceptable tolerability profile. Preclinical studies suggest that myeloid malignancies are sensitive to nuclear export inhibition. Eltanexor exhibited efficacy in hematologic models, supporting exploration in a clinical trial. This phase 1/2 study (NCT02649790) assessed single-agent activity of eltanexor in patients with higher-risk MDS and 5-19% myeloblasts. Two starting doses of eltanexor were evaluated: 20 mg (n = 15), 10 mg (n = 5), both administered on days 1-5 each week of a 28-day cycle. Twenty patients with primary HMA-refractory MDS, with a median age of 77 years (range 62-89), and a median of two prior treatment regimens (range 1-4) were enrolled. Of these, 15 were evaluated for efficacy and 20 for safety. The overall response rate (ORR) was 53.3%, with seven patients (46.7%) achieving marrow complete remission (mCR) and one additional patient achieving hematologic improvement (HI). In the 10 mg group, three patients (60%) reached mCR and two (40%) stable disease (SD), while for 20 mg, four patients (40%) had mCR and two (20%) SD. A total of three patients (20%) had HI and became transfusion independent ≥ 8 weeks. Median OS for the efficacy-evaluable patients (n = 15) was 9.86 months (7.98, NE). Overall, the most frequently reported treatment-related adverse events were nausea (45%), diarrhea (35%), decreased appetite (35%), fatigue and neutropenia (both 30%). Single-agent oral eltanexor was active, safe, and well tolerated in patients with higher-risk, primary HMA-refractory MDS.


Assuntos
Antineoplásicos , Síndromes Mielodisplásicas , Administração Oral , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Humanos , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/patologia , Neutropenia/induzido quimicamente , Resultado do Tratamento
19.
Cancers (Basel) ; 14(1)2021 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-35008323

RESUMO

Diffuse large B-cell lymphoma (DLBCL), grade 3b follicular lymphoma (FL), and mantle cell lymphoma (MCL) are aggressive non-Hodgkin's lymphomas (NHL). Cure rates are suboptimal and novel treatment strategies are needed to improve outcomes. Here, we show that p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyl transferase (NAMPT) is critical for lymphoma subsistence. Dual targeting of PAK4-NAMPT by the Phase I small molecule KPT-9274 suppressed cell proliferation in DLBCL, FL, and MCL. Growth inhibition was concurrent with apoptosis induction alongside activation of pro-apoptotic proteins and reduced pro-survival markers. We observed NAD suppression, ATP reduction, and consequent cellular metabolic collapse in lymphoma cells due to KPT-9274 treatment. KPT-9274 in combination with standard-of-care chemotherapeutics led to superior inhibition of cell proliferation. In vivo, KPT-9274 could markedly suppress the growth of WSU-DLCL2 (DLBCL), Z-138, and JeKo-1 (MCL) sub-cutaneous xenografts, and a remarkable increase in host life span was shown, with a 50% cure of a systemic WSU-FSCCL (FL) model. Residual tumor analysis confirmed a reduction in total and phosphorylated PAK4 and activation of the pro-apoptotic cascade. This study, using various preclinical experimental models, demonstrates the therapeutic potential of targeting PAK4-NAMPT in DLBCL, FL, and MCL. The orally bioavailable, safe, and efficacious PAK4-NAMPT dual inhibitor KPT-9274 warrants further clinical investigation.

20.
Mol Cancer Ther ; 20(10): 1836-1845, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34253597

RESUMO

Metastatic pancreatic neuroendocrine tumors (PNET) remain an unmet clinical problem. Chronologic treatment in PNETs includes observation (watchful protocol), surgery, targeted therapy, and chemotherapy. However, increasing evidence illustrates that the outcomes of targeted therapeutic options for the treatment of advanced PNETs show minimal response. The FDA-approved mTOR inhibitor everolimus does not shrink these tumors. It only delays disease progression in a subset of patients, while a significant fraction acquires resistance and shows disease progression. Thus, there is a need for more effective targeted approaches to sensitize PNETs to everolimus for better treatment outcomes. Previously, we showed that mTOR regulator p21 activated kinase 4 (PAK4) and nicotinamide adenine dinucleotide biosynthesis enzyme nicotinamide phosphoribosyl transferase (NAMPT) were aberrantly expressed in PNET tissue and promoted everolimus resistance. In this report, we demonstrate that PAK4-NAMPT dual inhibitor KPT-9274 can synergize with everolimus (growth inhibition, colony suppression, and glucose uptake assays). KPT-9274-everolimus disrupted spheroid formation in multiple PNET models. Molecular analysis showed alteration of mTORC2 through downregulation of RICTOR as a mechanism supporting synergy with everolimus in vitro KPT-9274 suppressed ß-catenin activity via inhibition of PAK4, highlighting the cross-talk between Rho GTPases and Wnt signaling in PNETs. KPT-9274, given at 150 mg/kg in combination with sub-MTD everolimus (2.5 mg/kg), significantly suppressed two PNET-derived xenografts. These studies bring forward a well-grounded strategy for advanced PNETs that fail to respond to single-agent everolimus.


Assuntos
Acrilamidas/farmacologia , Aminopiridinas/farmacologia , Citocinas/antagonistas & inibidores , Everolimo/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Tumores Neuroendócrinos/tratamento farmacológico , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Quinases Ativadas por p21/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Quimioterapia Combinada , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
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