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1.
J Biol Chem ; 298(11): 102539, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36179791

RESUMO

Recent studies have reported that the peroxisome proliferator-activated receptor gamma (PPARγ) pathway is activated in approximately 40% of patients with muscle-invasive bladder cancer. This led us to investigate pharmacological repression of PPARγ as a possible intervention strategy. Here, we characterize PPARγ antagonists and inverse agonists and find that the former behave as silent ligands, whereas inverse agonists (T0070907 and SR10221) repress downstream PPARγ target genes leading to growth inhibition in bladder cancer cell lines. To understand the mechanism, we determined the ternary crystal structure of PPARγ bound to T0070907 and the corepressor (co-R) peptide NCOR1. The structure shows that the AF-2 helix 12 (H12) rearranges to bind inside the ligand-binding domain, where it forms stabilizing interactions with the compound. This dramatic movement in H12 unveils a large interface for co-R binding. In contrast, the crystal structure of PPARγ bound to a SR10221 analog shows more subtle structural differences, where the compound binds and pushes H12 away from the ligand-binding domain to allow co-R binding. Interestingly, we found that both classes of compound promote recruitment of co-R proteins in biochemical assays but with distinct conformational changes in H12. We validate our structural models using both site-directed mutagenesis and chemical probes. Our findings offer new mechanistic insights into pharmacological modulation of PPARγ signaling.


Assuntos
PPAR gama , Neoplasias da Bexiga Urinária , Humanos , PPAR gama/metabolismo , Ligantes , Benzamidas/farmacologia
2.
Nature ; 526(7572): 273-276, 2015 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-26416749

RESUMO

Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.


Assuntos
Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Neoplásicos/genética , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Animais , Proteínas de Ciclo Celular , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos SCID , Proteínas Nucleares/antagonistas & inibidores , Compostos Policíclicos/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
3.
Blood ; 121(13): 2533-41, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23361907

RESUMO

The t(6;11)(q27;q23) is a recurrent chromosomal rearrangement that encodes the MLLAF6 fusion oncoprotein and is observed in patients with diverse hematologic malignancies. The presence of the t(6;11)(q27;q23) has been linked to poor overall survival in patients with AML. In this study, we demonstrate that MLL-AF6 requires continued activity of the histone-methyltransferase DOT1L to maintain expression of the MLL-AF6-driven oncogenic gene-expression program. Using gene-expression analysis and genome-wide chromatin immunoprecipitation studies followed by next generation sequencing, we found that MLL-fusion target genes display markedly high levels of histone 3 at lysine 79 (H3K79) dimethylation in murine MLL-AF6 leukemias as well as in ML2, a human myelomonocytic leukemia cell line bearing the t(6;11)(q27;q23) translocation. Targeted disruption of Dot1l using a conditional knockout mouse model inhibited leukemogenesis mediated by the MLL-AF6 fusion oncogene. Moreover, both murine MLL-AF6-transformed cells as well as the human MLL-AF6-positive ML2 leukemia cell line displayed specific sensitivity to EPZ0004777, a recently described, selective, small-molecule inhibitor of Dot1l. Dot1l inhibition resulted in significantly decreased proliferation, decreased expression of MLL-AF6 target genes, and cell cycle arrest of MLL-AF6-transformed cells. These results indicate that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation.


Assuntos
Transformação Celular Neoplásica/genética , Histona-Lisina N-Metiltransferase/genética , Cinesinas/genética , Metiltransferases/fisiologia , Proteína de Leucina Linfoide-Mieloide/genética , Miosinas/genética , Proteínas de Fusão Oncogênica/genética , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/fisiologia , Lisina/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Compostos de Fenilureia/farmacologia
4.
Mol Cancer Ther ; 21(6): 890-902, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35642432

RESUMO

Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both  ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy-resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089). SUMMARY: H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors.


Assuntos
Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ensaios Clínicos como Assunto , Receptor alfa de Estrogênio/genética , Feminino , Fulvestranto/uso terapêutico , Humanos , Indazóis , Recidiva Local de Neoplasia , Piridinas
5.
Nat Commun ; 10(1): 137, 2019 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-30635584

RESUMO

Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Antígenos de Histocompatibilidade Menor/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Splicing de RNA/efeitos dos fármacos , Proteína bcl-X/genética , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Sinergismo Farmacológico , Compostos de Epóxi/farmacologia , Feminino , Humanos , Neoplasias Pulmonares/genética , Macrolídeos/farmacologia , Melanoma/genética , Camundongos , Camundongos Nus , Interferência de RNA , Splicing de RNA/genética , RNA Interferente Pequeno/genética , Spliceossomos/efeitos dos fármacos , Spliceossomos/genética , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cancer Discov ; 8(9): 1176-1193, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29991605

RESUMO

Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs.Significance: Nearly 30% of endocrine therapy-resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176-93. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Antagonistas do Receptor de Estrogênio/administração & dosagem , Receptor alfa de Estrogênio/antagonistas & inibidores , Indazóis/administração & dosagem , Mutação , Administração Oral , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Antagonistas do Receptor de Estrogênio/química , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Indazóis/química , Indazóis/farmacologia , Células MCF-7 , Camundongos , Conformação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Int J Dev Biol ; 47(5): 355-61, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12895030

RESUMO

We have assessed the type and amount of neural tissue induced in the chick gastrula ectoblast with increasing duration of contact with the Hensen node inducer. At least 4 h contact is necessary to induce the ectoblast in the area pellucida, 9 h in the area opaca and even longer at the margin of overgrowth. In the area pellucida, the inductive response shifts from archencephalic type at 4-6 h contact to deuterencephalic type after 9 h contact. The induced tissue volume and cell number increased as graft-host contact increased from 4-9 h, and then decreased with longer contact. We suggest that in addition to the period of contact and the grafting position on competent ectoblast, the rate of cell proliferation may control the axial specificity and morphological organization of the induced neural tissue.


Assuntos
Neurônios/citologia , Organizadores Embrionários/patologia , Animais , Divisão Celular , Embrião de Galinha , Ectoderma/patologia , Embrião não Mamífero/patologia , Indução Embrionária , Mesoderma/metabolismo , Modelos Anatômicos , Modelos Biológicos , Fatores de Tempo , Transplantes
8.
Cancer Cell ; 26(6): 896-908, 2014 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-25464900

RESUMO

Homeotic (HOX) genes are dysregulated in multiple malignancies, including several AML subtypes. We demonstrate that H3K79 dimethylation (H3K79me2) is converted to monomethylation (H3K79me1) at HOX loci as hematopoietic cells mature, thus coinciding with a decrease in HOX gene expression. We show that H3K79 methyltransferase activity as well as H3K79me1-to-H3K79me2 conversion is regulated by the DOT1L cofactor AF10. AF10 inactivation reverses leukemia-associated epigenetic profiles, precludes abnormal HOXA gene expression, and impairs the transforming ability of MLL-AF9, MLL-AF6, and NUP98-NSD1 fusions-mechanistically distinct HOX-activating oncogenes. Furthermore, NUP98-NSD1-transformed cells are sensitive to small-molecule inhibition of DOT1L. Our findings demonstrate that pharmacological inhibition of the DOT1L/AF10 complex may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression.


Assuntos
Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Metiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Células da Medula Óssea/metabolismo , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Metilação , Metiltransferases/antagonistas & inibidores , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Neoplasias Experimentais , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Compostos de Fenilureia/farmacologia
9.
Dev Growth Differ ; 44(2): 169-80, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11940103

RESUMO

One to eight post-nodal fragments (PN) or Hensen's nodes (HN) from full primitive streak stage chick embryos were transplanted onto the area pellucida or area opaca of stage 4 embryos and cultured for 20 h. Thirteen morphological and numerical parameters were affected in the host embryos and analyzed by multiple logistic regression for parametric hierarchy. In the area pellucida, both PN and HN transplants inhibited cell population growth while only PN caudalized the host axis and induced supernumerary primitive streaks expressing the mesoderm-specific gene Brachyury. In the area opaca, neither grafts influenced host axis morphogenesis, but PN inhibited the cell population growth significantly. Tracking [(3)H]TdR labeled grafts showed that PN cells migrated towards the host axis and participated in the formation of supernumerary somites and hearts. When placed near the host axis, PN caudalized it and inhibited cell population growth.


Assuntos
Indução Embrionária/fisiologia , Proteínas Fetais , Transplante de Tecido Fetal/fisiologia , Gástrula/fisiologia , Organizadores Embrionários/transplante , Proteínas com Domínio T/metabolismo , Animais , Blastoderma/citologia , Blastoderma/ultraestrutura , Divisão Celular/fisiologia , Embrião de Galinha , Marcação por Isótopo , Modelos Logísticos , Organizadores Embrionários/embriologia , Coloração e Rotulagem , Proteínas com Domínio T/genética , Transplantes
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