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1.
Cell ; 168(5): 817-829.e15, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28215705

RESUMO

Investigating therapeutic "outliers" that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D "knockin" mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.


Assuntos
Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Neoplasias Colorretais/genética , Difenilamina/análogos & derivados , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Linhagem Celular Tumoral , Evolução Clonal , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Difenilamina/farmacologia , Difenilamina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Mutação , Retroviridae
2.
Mol Pharm ; 15(9): 3979-3996, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30040421

RESUMO

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.


Assuntos
Benzodiazepinas/química , Dissulfetos/química , Imunoconjugados/química , Pró-Fármacos/química , Pirróis/química , Linhagem Celular Tumoral , Cisteína/metabolismo , Glutationa/metabolismo , Humanos , Imunoconjugados/metabolismo , Estrutura Molecular
3.
Mol Cell Proteomics ; 15(4): 1220-31, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26711467

RESUMO

The KEAP1/Nrf2 pathway senses and responds to changes in intracellular oxidative stress. Mutations that result in constitutive activation of Nrf2 are present in several human tumors, especially non-small cell lung cancer. Therefore, compounds that inhibit Nrf2 activity might be beneficial in treating patients whose tumors show activation of this pathway. Recent reports suggest that the natural product brusatol can potently and selectively inhibit Nrf2 activity, resulting in cell cytotoxicity, and can be effectively combined with chemotherapeutic agents. Here, we analyzed the effects of brusatol on the cellular proteome in the KEAP1 mutant non-small cell lung cancer cell line A549. Brusatol was found to rapidly and potently decrease the expression of the majority of detected proteins, including Nrf2. The most dramatically decreased proteins are those that display a short half-life, like Nrf2. This effect was confirmed by restricting the analysis to newly synthesized proteins using a labeled methionine analogue. Moreover, brusatol increased the expression of multiple components of the ribosome, suggesting that it regulates the function of this macromolecular complex. Finally, we show that brusatol induces its potent cellular cytotoxicity effects on multiple cancer cell lines in a manner independent of KEAP1/Nrf2 activity and with a profile similar to the protein translation inhibitor silvestrol. In conclusion, our data show that the activity of brusatol is not restricted to Nrf2 but, rather, functions as a global protein synthesis inhibitor.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Espectrometria de Massas/métodos , Fator 2 Relacionado a NF-E2/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Quassinas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Proteômica/métodos
4.
Bioorg Med Chem Lett ; 27(15): 3534-3541, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28606761

RESUMO

Bromodomain-containing protein 9 (BRD9), an epigenetic "reader" of acetylated lysines on post-translationally modified histone proteins, is upregulated in multiple cancer cell lines. To assess the functional role of BRD9 in cancer cell lines, we identified a small-molecule inhibitor of the BRD9 bromodomain. Starting from a pyrrolopyridone lead, we used structure-based drug design to identify a potent and highly selective in vitro tool compound 11, (GNE-375). While this compound showed minimal effects in cell viability or gene expression assays, it showed remarkable potency in preventing the emergence of a drug tolerant population in EGFR mutant PC9 cells treated with EGFR inhibitors. Such tolerance has been linked to an altered epigenetic state, and 11 decreased BRD9 binding to chromatin, and this was associated with decreased expression of ALDH1A1, a gene previously shown to be important in drug tolerance. BRD9 inhibitors may therefore show utility in preventing epigenetically-defined drug resistance.


Assuntos
Resistência a Medicamentos/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Linhagem Celular Tumoral , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Piridonas/química , Piridonas/farmacologia , Retinal Desidrogenase , Fatores de Transcrição/metabolismo
5.
Nature ; 464(7287): 431-5, 2010 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-20130576

RESUMO

Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects. Small molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. Here we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF-MEK-ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.


Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/patologia , Inibidores de Proteínas Quinases/farmacologia , Quinases raf/antagonistas & inibidores , Quinases raf/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Benzamidas/farmacologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Indenos/farmacologia , Indóis/farmacologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/deficiência , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/química , Quinases raf/genética , Proteínas ras/genética , Proteínas ras/metabolismo
6.
Nature ; 466(7308): 869-73, 2010 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-20668451

RESUMO

The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease and for developing targeted therapeutics. Here we report the identification of 2,576 somatic mutations across approximately 1,800 megabases of DNA representing 1,507 coding genes from 441 tumours comprising breast, lung, ovarian and prostate cancer types and subtypes. We found that mutation rates and the sets of mutated genes varied substantially across tumour types and subtypes. Statistical analysis identified 77 significantly mutated genes including protein kinases, G-protein-coupled receptors such as GRM8, BAI3, AGTRL1 (also called APLNR) and LPHN3, and other druggable targets. Integrated analysis of somatic mutations and copy number alterations identified another 35 significantly altered genes including GNAS, indicating an expanded role for galpha subunits in multiple cancer types. Furthermore, our experimental analyses demonstrate the functional roles of mutant GNAO1 (a Galpha subunit) and mutant MAP2K4 (a member of the JNK signalling pathway) in oncogenesis. Our study provides an overview of the mutational spectra across major human cancers and identifies several potential therapeutic targets.


Assuntos
Genes Neoplásicos/genética , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais/genética , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Variações do Número de Cópias de DNA/genética , Análise Mutacional de DNA , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Humanos , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/genética , MAP Quinase Quinase 4/genética , Masculino , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias Ovarianas/classificação , Neoplasias Ovarianas/genética , Neoplasias da Próstata/classificação , Neoplasias da Próstata/genética , Proteínas Quinases/genética , Receptores Acoplados a Proteínas G/genética
7.
Genome Res ; 22(12): 2315-27, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23033341

RESUMO

Lung cancer is a highly heterogeneous disease in terms of both underlying genetic lesions and response to therapeutic treatments. We performed deep whole-genome sequencing and transcriptome sequencing on 19 lung cancer cell lines and three lung tumor/normal pairs. Overall, our data show that cell line models exhibit similar mutation spectra to human tumor samples. Smoker and never-smoker cancer samples exhibit distinguishable patterns of mutations. A number of epigenetic regulators, including KDM6A, ASH1L, SMARCA4, and ATAD2, are frequently altered by mutations or copy number changes. A systematic survey of splice-site mutations identified 106 splice site mutations associated with cancer specific aberrant splicing, including mutations in several known cancer-related genes. RAC1b, an isoform of the RAC1 GTPase that includes one additional exon, was found to be preferentially up-regulated in lung cancer. We further show that its expression is significantly associated with sensitivity to a MAP2K (MEK) inhibitor PD-0325901. Taken together, these data present a comprehensive genomic landscape of a large number of lung cancer samples and further demonstrate that cancer-specific alternative splicing is a widespread phenomenon that has potential utility as therapeutic biomarkers. The detailed characterizations of the lung cancer cell lines also provide genomic context to the vast amount of experimental data gathered for these lines over the decades, and represent highly valuable resources for cancer biology.


Assuntos
Processamento Alternativo , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Neoplasias Pulmonares/genética , Mutação , Transcriptoma , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigenômica , Éxons , Marcadores Genéticos , Heterozigoto , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase , Humanos , Cariotipagem/métodos , Neoplasias Pulmonares/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
8.
Proc Natl Acad Sci U S A ; 109(14): 5299-304, 2012 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-22431598

RESUMO

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.


Assuntos
Nucleotídeos/metabolismo , Proteínas Son Of Sevenless/metabolismo , Proteínas ras/metabolismo , Sítios de Ligação , Linhagem Celular , Humanos , Ligantes , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteínas ras/química
9.
Proc Natl Acad Sci U S A ; 109(47): 19368-73, 2012 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-23134728

RESUMO

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.


Assuntos
Neoplasias/enzimologia , Neoplasias/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutação/genética , Células NIH 3T3 , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
10.
J Biol Chem ; 288(4): 2403-13, 2013 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-23188824

RESUMO

To assess the consequences of endogenous mutant K-Ras, we analyzed the signaling and biological properties of a small panel of isogenic cell lines. These include the cancer cell lines DLD1, HCT116, and Hec1A, in which either the WT or mutant K-ras allele has been disrupted, and SW48 colorectal cancer cells and human mammary epithelial cells in which a single copy of mutant K-ras was introduced at its endogenous genomic locus. We find that single copy mutant K-Ras causes surprisingly modest activation of downstream signaling to ERK and Akt. In contrast, a negative feedback signaling loop to EGFR and N-Ras occurs in some, but not all, of these cell lines. Mutant K-Ras also had relatively minor effects on cell proliferation and cell migration but more dramatic effects on cell transformation as assessed by growth in soft agar. Surprisingly, knock-out of the wild type K-ras allele consistently increased growth in soft agar, suggesting tumor-suppressive properties of this gene under these conditions. Finally, we examined the effects of single copy mutant K-Ras on global gene expression. Although transcriptional programs triggered by mutant K-Ras were generally quite distinct in the different cell lines, there was a small number of genes that were consistently overexpressed, and these could be used to monitor K-Ras inhibition in a panel of human tumor cell lines. We conclude that there are conserved components of mutant K-Ras signaling and phenotypes but that many depend on cell context and environmental cues.


Assuntos
Regulação Neoplásica da Expressão Gênica , Mutação , Proteínas ras/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Perfilação da Expressão Gênica , Genes ras , Heterozigoto , Homozigoto , Humanos , Oncogenes/genética , Fenótipo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transcrição Gênica
11.
Nat Med ; 13(1): 84-8, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17159987

RESUMO

Cancer immunoresistance and immune escape may play important roles in tumor progression and pose obstacles for immunotherapy. Expression of the immunosuppressive protein B7 homolog 1 (B7-H1), also known as programmed death ligand-1 (PD-L1), is increased in many pathological conditions, including cancer. Here we show that expression of the gene encoding B7-H1 increases post transcriptionally in human glioma after loss of phosphatase and tensin homolog (PTEN) and activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway. Tumor specimens from individuals with glioblastoma multiforme (GBM) had levels of B7-H1 protein that correlated with PTEN loss, and tumor-specific T cells lysed human glioma targets expressing wild-type PTEN more effectively than those expressing mutant PTEN. These data identify a previously unrecognized mechanism linking loss of the tumor suppressor PTEN with immunoresistance, mediated in part by B7-H1.


Assuntos
Antígenos CD/genética , Glioma/patologia , PTEN Fosfo-Hidrolase/genética , Análise de Variância , Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígeno B7-H1 , Western Blotting , Caspase 6/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/genética , Glioma/metabolismo , Humanos , Mutação , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Transfecção
12.
Cancer Cell ; 9(5): 341-9, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16697955

RESUMO

The PI3 kinase family of lipid kinases promotes cell growth and survival by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate. To define targets critical for cancers driven by activation of PI3 kinase, we screened a panel of potent and structurally diverse drug-like molecules that target this enzyme family. Surprisingly, a single agent (PI-103) effected proliferative arrest in glioma cells, despite the ability of many compounds to block PI3 kinase signaling through its downstream effector, Akt. The unique cellular activity of PI-103 was traced directly to its ability to inhibit both PI3 kinase alpha and mTOR. PI-103 showed significant activity in xenografted tumors with no observable toxicity. These data demonstrate an emergent efficacy due to combinatorial inhibition of mTOR and PI3 kinase alpha in malignant glioma.


Assuntos
Glioma/tratamento farmacológico , Glioma/enzimologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases , Ativação Enzimática , Receptores ErbB/metabolismo , Glioma/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Compostos Organoplatínicos/farmacologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais , Especificidade por Substrato , Serina-Treonina Quinases TOR , Resultado do Tratamento , Proteína Supressora de Tumor p53/metabolismo
13.
Biochem J ; 452(2): 313-20, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23496764

RESUMO

The mutant forms of KRas, NRas and HRas drive the initiation and progression of a number of human cancers, but less is known about the role of WT (wild-type) Ras alleles and isoforms in cancer. We used zinc-finger nucleases targeting HRas and NRas to modify both alleles of these genes in the mutant KRas-driven Hec1A endometrial cancer cell line, which normally expresses WT copies of these genes. The disruption of either WT isoform of Ras compromised growth-factor-dependent signalling through the ERK (extracellular-signal-regulated kinase) pathway. In addition, the disruption of HRas hindered the activation of Akt and subsequent downstream signalling. This was associated with decreased proliferation, increased apoptosis and decreased anchorage-independent growth in the HRas-disrupted cells. However, xenograft tumour growth was not significantly affected by the disruption of either NRas or HRas. As expected, deleting the mutant allele of KRas abolished tumour growth, whereas deletion of the remaining WT copy of KRas increased the tumorigenic properties of these cells; deleting a single copy of either HRas or NRas did not mimic this effect. The present study demonstrates that the WT copies of HRas, NRas and KRas play unique roles in the context of mutant KRas-driven tumours.


Assuntos
Transformação Celular Neoplásica/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Proteínas ras/química , Proteínas ras/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Transformação Celular Neoplásica/química , Feminino , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética
14.
Angew Chem Int Ed Engl ; 53(15): 3777-9, 2014 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-24590864

RESUMO

One of the first oncogenes to be discovered and one of the most prevalently mutated tumor genes is Ras. Shokat et al. and Gray et al. have succeeded in discovering allosteric inhibitors and inhibitory nucleotide analogues, respectively, of K-Ras proteins that covalently and selectively bind G12C-mutant K-Ras. These strategies of specifically targeting a mutant form of Ras may offer an opportunity for cancer-selective therapies that spare normal tissue.


Assuntos
Proteínas Mutantes/metabolismo , Proteínas ras/metabolismo , Sítios de Ligação , Humanos , Transdução de Sinais
15.
Cancer Res Commun ; 4(2): 540-555, 2024 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-38358346

RESUMO

Type I IFN signaling is a crucial component of antiviral immunity that has been linked to promoting the efficacy of some chemotherapeutic drugs. We developed a reporter system in HCT116 cells that detects activation of the endogenous IFI27 locus, an IFN target gene. We screened a library of annotated compounds in these cells and discovered Aurora kinase inhibitors (AURKi) as strong hits. Type I IFN signaling was found to be the most enriched gene signature after AURKi treatment in HCT116, and this signature was also strongly enriched in other colorectal cancer cell lines. The ability of AURKi to activate IFN in HCT116 was dependent on MAVS and RIG-I, but independent of STING, whose signaling is deficient in these cells. MAVS dependence was recapitulated in other colorectal cancer lines with STING pathway deficiency, whereas in cells with intact STING signaling, the STING pathway was required for IFN induction by AURKi. AURKis were found to induce expression of endogenous retroviruses (ERV). These ERVs were distinct from those induced by the DNA methyltransferase inhibitors (DNMTi), which can induce IFN signaling via ERV induction, suggesting a novel mechanism of action. The antitumor effect of alisertib in mice was accompanied by an induction of IFN expression in HCT116 or CT26 tumors. CT26 tumor growth inhibition by alisertib was absent in NSG mice versus wildtype (WT) mice, and tumors from WT mice with alisertib treatment showed increased in CD8+ T-cell infiltration, suggesting that antitumor efficacy of AURKi depends, at least in part, on an intact immune response. SIGNIFICANCE: Some cancers deactivate STING signaling to avoid consequences of DNA damage from aberrant cell division. The surprising activation of MAVS/RIG-I signaling by AURKi might represent a vulnerability in STING signaling deficient cancers.


Assuntos
Neoplasias Colorretais , Interferon Tipo I , Animais , Camundongos , Retroelementos , Interferon lambda , Aurora Quinases/metabolismo , Interferon Tipo I/metabolismo , Proteína DEAD-box 58/genética , Receptores Imunológicos
16.
Nat Commun ; 15(1): 1842, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38418456

RESUMO

Human papillomavirus (HPV) is a significant contributor to the global cancer burden, and its carcinogenic activity is facilitated in part by the HPV early protein 6 (E6), which interacts with the E3-ligase E6AP, also known as UBE3A, to promote degradation of the tumor suppressor, p53. In this study, we present a single-particle cryoEM structure of the full-length E6AP protein in complex with HPV16 E6 (16E6) and p53, determined at a resolution of ~3.3 Å. Our structure reveals extensive protein-protein interactions between 16E6 and E6AP, explaining their picomolar binding affinity. These findings shed light on the molecular basis of the ternary complex, which has been pursued as a potential therapeutic target for HPV-driven cervical, anal, and oropharyngeal cancers over the last two decades. Understanding the structural and mechanistic underpinnings of this complex is crucial for developing effective therapies to combat HPV-induced cancers. Our findings may help to explain why previous attempts to disrupt this complex have failed to generate therapeutic modalities and suggest that current strategies should be reevaluated.


Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Humanos , Proteína Supressora de Tumor p53/metabolismo , Papillomavirus Humano 16/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Oncogênicas Virais/genética , Genes Supressores de Tumor
17.
Commun Chem ; 7(1): 183, 2024 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-39152201

RESUMO

PTPN2 (protein tyrosine phosphatase non-receptor type 2, or TC-PTP) and PTPN1 are attractive immuno-oncology targets, with the deletion of Ptpn1 and Ptpn2 improving response to immunotherapy in disease models. Targeted protein degradation has emerged as a promising approach to drug challenging targets including phosphatases. We developed potent PTPN2/N1 dual heterobifunctional degraders (Cmpd-1 and Cmpd-2) which facilitate efficient complex assembly with E3 ubiquitin ligase CRL4CRBN, and mediate potent PTPN2/N1 degradation in cells and mice. To provide mechanistic insights into the cooperative complex formation introduced by degraders, we employed a combination of structural approaches. Our crystal structure reveals how PTPN2 is recognized by the tri-substituted thiophene moiety of the degrader. We further determined a high-resolution structure of DDB1-CRBN/Cmpd-1/PTPN2 using single-particle cryo-electron microscopy (cryo-EM). This structure reveals that the degrader induces proximity between CRBN and PTPN2, albeit the large conformational heterogeneity of this ternary complex. The molecular dynamic (MD)-simulations constructed based on the cryo-EM structure exhibited a large rigid body movement of PTPN2 and illustrated the dynamic interactions between PTPN2 and CRBN. Together, our study demonstrates the development of PTPN2/N1 heterobifunctional degraders with potential applications in cancer immunotherapy. Furthermore, the developed structural workflow could help to understand the dynamic nature of degrader-induced cooperative ternary complexes.

18.
Cancer Cell ; 2(6): 507-14, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12498719

RESUMO

The Nf1 tumor suppressor encodes a GTPase-activating protein for Ras. Previous work has implicated hyperactive Ras in the aberrant growth of Nf1-deficient cells; however, there are limited data on which effectors modulate specific phenotypes. To address this, we generated myeloid cell lines by infecting fetal liver cells with a retrovirus encoding a truncated allele of c-Myb. Granulocyte-macrophage colony stimulating factor (GM-CSF) promoted the survival of wild-type Myb cells in a dose-dependent manner. By contrast, Nf1-deficient myeloid cells deprived of growth factors, were resistant to apoptosis due to hyperactivation of the phosphoinositide-3-OH kinase/protein kinase B cascade. Nf1(-/-) cells also demonstrated growth factor-independent proliferation and upregulation of GM-CSF mRNA production that were dependent upon Raf/MEK/ERK signaling. These data link specific Ras effectors with discrete cellular phenotypes in Nf1-deficient cells.


Assuntos
Genes ras/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurofibromina 1/deficiência , Proteínas Proto-Oncogênicas/metabolismo , Animais , Apoptose , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feto , Genes myb , Genes ras/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Hepatócitos/fisiologia , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Células Mieloides/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/análise , Transdução de Sinais , Transfecção
19.
Mol Cancer Res ; 7(4): 511-22, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19372580

RESUMO

Breast cancers can be divided into subtypes with important implications for prognosis and treatment. We set out to characterize the genetic alterations observed in different breast cancer subtypes and to identify specific candidate genes and pathways associated with subtype biology. mRNA expression levels of estrogen receptor, progesterone receptor, and HER2 were shown to predict marker status determined by immunohistochemistry and to be effective at assigning samples to subtypes. HER2(+) cancers were shown to have the greatest frequency of high-level amplification (independent of the ERBB2 amplicon itself), but triple-negative cancers had the highest overall frequencies of copy gain. Triple-negative cancers also were shown to have more frequent loss of phosphatase and tensin homologue and mutation of RB1, which may contribute to genomic instability. We identified and validated seven regions of copy number alteration associated with different subtypes, and used integrative bioinformatics analysis to identify candidate oncogenes and tumor suppressors, including ERBB2, GRB7, MYST2, PPM1D, CCND1, HDAC2, FOXA1, and RASA1. We tested the candidate oncogene MYST2 and showed that it enhances the anchorage-independent growth of breast cancer cells. The genome-wide and region-specific differences between subtypes suggest the differential activation of oncogenic pathways.


Assuntos
Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Amplificação de Genes , Instabilidade Genômica , Oncogenes/fisiologia , Transdução de Sinais , Adulto , Idoso , Western Blotting , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/secundário , Carcinoma Lobular/genética , Carcinoma Lobular/metabolismo , Carcinoma Lobular/secundário , Ensaio de Unidades Formadoras de Colônias , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/farmacologia , Células Tumorais Cultivadas
20.
BMC Med ; 8: 42, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20594338

RESUMO

Recent work has shown that it is possible to target regulatory elements to DNA sequences of an investigator's choosing, increasing the armamentarium for probing gene function. In this review, we discuss the development and use of designer zinc finger proteins (ZFPs) as sequence specific tools. While the main focus of this review is to discuss the attachment of the FokI nuclease to ZFPs and the ability of the resulting fusion protein (termed zinc finger nucleases (ZFNs)) to genomically manipulate a gene of interest, we will also cover the utility of other functional domains, such as transcriptional activators and repressors, and highlight how these are being used as discovery and therapeutic tools.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Dedos de Zinco , Produtos Biológicos/química , Produtos Biológicos/genética , Produtos Biológicos/metabolismo , Produtos Biológicos/uso terapêutico , Pesquisa Biomédica/tendências , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/uso terapêutico , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/uso terapêutico , Regulação da Expressão Gênica , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico
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