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1.
Cell ; 155(5): 1049-1060, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-24267890

RESUMO

The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD.


Assuntos
RNA Polimerase II/química , RNA Polimerase II/metabolismo , Ativação Transcricional , Células HeLa , Humanos , Hidrogéis/química , Hidrogéis/metabolismo , Repetições de Microssatélites , Fosforilação , Polimerização , Estrutura Terciária de Proteína , Proteína EWS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Fatores Associados à Proteína de Ligação a TATA/química , Fatores Associados à Proteína de Ligação a TATA/metabolismo
2.
Cell ; 149(4): 768-79, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22579282

RESUMO

Cellular granules lacking boundary membranes harbor RNAs and their associated proteins and play diverse roles controlling the timing and location of protein synthesis. Formation of such granules was emulated by treatment of mouse brain extracts and human cell lysates with a biotinylated isoxazole (b-isox) chemical. Deep sequencing of the associated RNAs revealed an enrichment for mRNAs known to be recruited to neuronal granules used for dendritic transport and localized translation at synapses. Precipitated mRNAs contain extended 3' UTR sequences and an enrichment in binding sites for known granule-associated proteins. Hydrogels composed of the low complexity (LC) sequence domain of FUS recruited and retained the same mRNAs as were selectively precipitated by the b-isox chemical. Phosphorylation of the LC domain of FUS prevented hydrogel retention, offering a conceptual means of dynamic, signal-dependent control of RNA granule assembly.


Assuntos
Encéfalo/citologia , RNA/análise , RNA/metabolismo , Ribonucleoproteínas/química , Animais , Biotinilação , Encéfalo/metabolismo , Linhagem Celular , Sistema Livre de Células , Humanos , Isoxazóis/metabolismo , Camundongos , Transporte de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo
3.
Cell ; 149(4): 753-67, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22579281

RESUMO

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Proteínas de Ligação a RNA/análise , RNA/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Sistema Livre de Células , Grânulos Citoplasmáticos/química , Células-Tronco Embrionárias/metabolismo , Masculino , Camundongos , Modelos Moleculares , Células NIH 3T3 , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Testículo/citologia , Testículo/metabolismo , Difração de Raios X
4.
Cell ; 142(1): 39-51, 2010 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-20603013

RESUMO

An in vivo screen was performed in search of chemicals capable of enhancing neuron formation in the hippocampus of adult mice. Eight of 1000 small molecules tested enhanced neuron formation in the subgranular zone of the dentate gyrus. Among these was an aminopropyl carbazole, designated P7C3, endowed with favorable pharmacological properties. In vivo studies gave evidence that P7C3 exerts its proneurogenic activity by protecting newborn neurons from apoptosis. Mice missing the gene encoding neuronal PAS domain protein 3 (NPAS3) are devoid of hippocampal neurogenesis and display malformation and electrophysiological dysfunction of the dentate gyrus. Prolonged administration of P7C3 to npas3(-/-) mice corrected these deficits by normalizing levels of apoptosis of newborn hippocampal neurons. Prolonged administration of P7C3 to aged rats also enhanced neurogenesis in the dentate gyrus, impeded neuron death, and preserved cognitive capacity as a function of terminal aging. PAPERCLIP:


Assuntos
Carbazóis/farmacologia , Avaliação Pré-Clínica de Medicamentos , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Fármacos Neuroprotetores/farmacologia , Envelhecimento/efeitos dos fármacos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carbazóis/química , Cognição/efeitos dos fármacos , Giro Denteado/citologia , Giro Denteado/fisiologia , Feminino , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Fármacos Neuroprotetores/química , Ratos
5.
Eur Respir J ; 57(3)2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32972983

RESUMO

Pulmonary arterial hypertension (PAH) is a destructive disease of the pulmonary vasculature often leading to right heart failure and death. Current therapeutic intervention strategies only slow disease progression. The role of aberrant hypoxia-inducible factor (HIF)2α stability and function in the initiation and development of pulmonary hypertension (PH) has been an area of intense interest for nearly two decades.Here we determine the effect of a novel HIF2α inhibitor (PT2567) on PH disease initiation and progression, using two pre-clinical models of PH. Haemodynamic measurements were performed, followed by collection of heart, lung and blood for pathological, gene expression and biochemical analysis. Blood outgrowth endothelial cells from idiopathic PAH patients were used to determine the impact of HIF2α-inhibition on endothelial function.Global inhibition of HIF2a reduced pulmonary vascular haemodynamics and pulmonary vascular remodelling in both su5416/hypoxia prevention and intervention models. PT2567 intervention reduced the expression of PH-associated target genes in both lung and cardiac tissues and restored plasma nitrite concentration. Treatment of monocrotaline-exposed rodents with PT2567 reduced the impact on cardiovascular haemodynamics and promoted a survival advantage. In vitro, loss of HIF2α signalling in human pulmonary arterial endothelial cells suppresses target genes associated with inflammation, and PT2567 reduced the hyperproliferative phenotype and overactive arginase activity in blood outgrowth endothelial cells from idiopathic PAH patients. These data suggest that targeting HIF2α hetero-dimerisation with an orally bioavailable compound could offer a new therapeutic approach for PAH. Future studies are required to determine the role of HIF in the heterogeneous PAH population.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Células Cultivadas , Células Endoteliais , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Artéria Pulmonar
6.
Cell Chem Biol ; 31(1): 139-149.e14, 2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-37967558

RESUMO

A novel class of benzoxaboroles was reported to induce cancer cell death but the mechanism was unknown. Using a forward genetics platform, we discovered mutations in cleavage and polyadenylation specific factor 3 (CPSF3) that reduce benzoxaborole binding and confer resistance. CPSF3 is the endonuclease responsible for pre-mRNA 3'-end processing, which is also important for RNA polymerase II transcription termination. Benzoxaboroles inhibit this endonuclease activity of CPSF3 in vitro and also curb transcriptional termination in cells, which results in the downregulation of numerous constitutively expressed genes. Furthermore, we used X-ray crystallography to demonstrate that benzoxaboroles bind to the active site of CPSF3 in a manner distinct from the other known inhibitors of CPSF3. The benzoxaborole compound impeded the growth of cancer cell lines derived from different lineages. Our results suggest benzoxaboroles may represent a promising lead as CPSF3 inhibitors for clinical development.


Assuntos
Antineoplásicos , Compostos de Boro , Fator de Especificidade de Clivagem e Poliadenilação , Endonucleases , Precursores de RNA , Processamento Pós-Transcricional do RNA , Fator de Especificidade de Clivagem e Poliadenilação/antagonistas & inibidores , Fator de Especificidade de Clivagem e Poliadenilação/química , Endonucleases/antagonistas & inibidores , Precursores de RNA/genética , Precursores de RNA/metabolismo , Compostos de Boro/química , Compostos de Boro/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Humanos , Linhagem Celular Tumoral
7.
bioRxiv ; 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39211113

RESUMO

Cancer cell proliferation requires precise control of E2F1 activity; excess activity promotes apoptosis. Here, we developed cell-permeable and bioavailable macrocycles that selectively kill small cell lung cancer (SCLC) cells with inherent high E2F1 activity by blocking RxL-mediated interactions of cyclin A and cyclin B with select substrates. Genome-wide CRISPR/Cas9 knockout and random mutagenesis screens found that cyclin A/B RxL macrocyclic inhibitors (cyclin A/Bi) induced apoptosis paradoxically by cyclin B- and Cdk2-dependent spindle assembly checkpoint activation (SAC). Mechanistically, cyclin A/Bi hyperactivate E2F1 and cyclin B by blocking their RxL-interactions with cyclin A and Myt1, respectively, ultimately leading to SAC activation and mitotic cell death. Base editor screens identified cyclin B variants that confer cyclin A/Bi resistance including several variants that disrupted cyclin B:Cdk interactions. Unexpectedly but consistent with our base editor and knockout screens, cyclin A/Bi induced the formation of neo-morphic Cdk2-cyclin B complexes that promote SAC activation and apoptosis. Finally, orally-bioavailable cyclin A/Bi robustly inhibited tumor growth in chemotherapy-resistant patient-derived xenograft models of SCLC. This work uncovers gain-of-function mechanisms by which cyclin A/Bi induce apoptosis in cancers with high E2F activity, and suggests cyclin A/Bi as a therapeutic strategy for SCLC and other cancers driven by high E2F activity.

8.
bioRxiv ; 2023 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-36865268

RESUMO

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and in some cases, new therapeutic leads. In select cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

9.
Cell Chem Biol ; 30(11): 1453-1467.e8, 2023 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-37607550

RESUMO

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and new therapeutic leads. In selected cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.


Assuntos
Antineoplásicos , Neoplasias do Colo , Humanos , Reparo de Erro de Pareamento de DNA , Antineoplásicos/farmacologia , Mutagênese , Citotoxinas
10.
J Med Chem ; 62(15): 6876-6893, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31282155

RESUMO

The hypoxia-inducible factor 2α (HIF-2α) is a key oncogenic driver in clear cell renal cell carcinoma (ccRCC). Our first HIF-2α inhibitor PT2385 demonstrated promising proof of concept clinical activity in heavily pretreated advanced ccRCC patients. However, PT2385 was restricted by variable and dose-limited pharmacokinetics resulting from extensive metabolism of PT2385 to its glucuronide metabolite. Herein we describe the discovery of second-generation HIF-2α inhibitor PT2977 with increased potency and improved pharmacokinetic profile achieved by reduction of phase 2 metabolism. Structural modification by changing the geminal difluoro group in PT2385 to a vicinal difluoro group resulted in enhanced potency, decreased lipophilicity, and significantly improved pharmacokinetic properties. In a phase 1 dose-escalation study, the clinical pharmacokinetics for PT2977 supports the hypothesis that attenuating the rate of glucuronidation would improve exposure and reduce variability in patients. Early evidence of clinical activity shows promise for PT2977 in the treatment of ccRCC.


Assuntos
Antineoplásicos/uso terapêutico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Carcinoma de Células Renais/tratamento farmacológico , Indanos/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Sulfonas/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/metabolismo , Cães , Relação Dose-Resposta a Droga , Feminino , Haplorrinos , Humanos , Indanos/síntese química , Indanos/farmacologia , Neoplasias Renais/metabolismo , Camundongos , Camundongos SCID , Ratos , Sulfonas/síntese química , Sulfonas/farmacologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
11.
J Med Chem ; 61(21): 9691-9721, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30289716

RESUMO

HIF-2α, a member of the HIF family of transcription factors, is a key oncogenic driver in cancers such as clear cell renal cell carcinoma (ccRCC). A signature feature of these cancers is the overaccumulation of HIF-2α protein, often by inactivation of the E3 ligase VHL (von Hippel-Lindau). Herein we disclose our structure based drug design (SBDD) approach that culminated in the identification of PT2385, the first HIF-2α antagonist to enter clinical trials. Highlights include the use of a putative n → π*Ar interaction to guide early analog design, the conformational restriction of an essential hydroxyl moiety, and the remarkable impact of fluorination near the hydroxyl group. Evaluation of select compounds from two structural classes in a sequence of PK/PD, efficacy, PK, and metabolite profiling identified 10i (PT2385, luciferase EC50 = 27 nM) as the clinical candidate. Finally, a retrospective crystallographic analysis describes the structural perturbations necessary for efficient antagonism.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Carcinoma de Células Renais/patologia , Desenho de Fármacos , Indanos/química , Indanos/farmacologia , Neoplasias Renais/patologia , Sulfonas/química , Sulfonas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Linhagem Celular Tumoral , Cães , Indanos/farmacocinética , Camundongos , Modelos Moleculares , Conformação Proteica , Ratos , Relação Estrutura-Atividade , Sulfonas/farmacocinética , Distribuição Tecidual
12.
Oncogene ; 24(20): 3246-56, 2005 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-15735695

RESUMO

A variety of experimental evidence including findings in various mouse models indicates that the BCR-ABL oncogene is the cause of chronic myeloid leukemia (CML). Since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we determined whether this is an active process mediated by the leukemia cells. The lipocalin 24p3 was reported to be secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells. Here, we show that BCR-ABL+ mouse hematopoietic cells induced persistent expression and secretion of 24p3. Importantly, BCR-ABL+ hematopoietic cells were resistant to the apoptotic effects of 24p3. The expression of the Bcr-Abl oncoprotein and its tyrosine kinase were required for induction of 24p3 expression. Co-culture studies showed that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Antisense 24p3/siRNA expression reduced the level of 24p3 protein in both BCR-ABL+ cells and in conditioned medium (CM) obtained from these cells. CM from BCR-ABL+ cells expressing antisense 24p3/siRNA had reduced apoptotic activity for target cells; 24p3 antibody also reduced the apoptotic activity of the CM. Leukemic mice induced by BCR-ABL+ cells expressing either antisense 24p3 or 24p3 siRNA had increased levels of normal hematopoiesis and reduced invasion of leukemia cells in marrow and spleen tissues. These findings indicate that suppression of normal hematopoiesis in BCR-ABL-induced leukemia is an active process involving secretion of the cell death-inducing factor 24p3 by mouse leukemia cells, raising the possibility that similar factors are involved in BCR-ABL+ CML.


Assuntos
Proteínas de Fase Aguda/fisiologia , Proteínas de Fusão bcr-abl/fisiologia , Hematopoese/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Oncogênicas/fisiologia , Animais , Apoptose , Western Blotting , Células da Medula Óssea/metabolismo , Morte Celular , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interleucina-3/metabolismo , Lentivirus/genética , Leucemia/metabolismo , Lipocalina-2 , Lipocalinas , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos NOD , Camundongos SCID , Oligonucleotídeos Antissenso/farmacologia , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/metabolismo , Tetraciclina/farmacologia , Fatores de Tempo
13.
Cancer Res ; 62(2): 386-90, 2002 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-11809685

RESUMO

The BCR protein is involved in the inhibition of oncogenic activity of the Bcr-Abl oncoprotein. This inhibition is believed to be the result of binding to the SH2 domain of Bcr-Abl in a non-phosphotyrosine-dependent manner. We showed that the Arg to Leu mutation in the Phe-Leu-Val-Arg-Glu-Ser (FLVRES) sequence of the SH2 domain, known to interfere with phosphotyrosine sequence binding, did not block the binding of Bcr first exon sequences to the Abl SH2 domain. We examined the structural-functional properties of a first exon mutant of BCR lacking the oligomerization domain, termed Bcr(64-413), that encodes the Ser-Thr protein kinase activity of Bcr. The autokinase product contained a M(r) 45,000-47,000 and 55,000 protein. Both species were detected by a Bcr phosphoserine 354 sequence-specific antibody. In contrast, the S354A mutant of Bcr(64-413), although maintaining autokinase activity, produced only the M(r) 45,000-47,000 kinase product. Abl SH2 binding experiments indicated that the M(r) 55,000 species of Bcr(64-413) but not the M(r) 45,000-47,000 species bound strongly to glutathime transferase-Abl SH2. The S354A mutant of Bcr(64-413) did not bind to glutathime transferase-Abl SH2. An adenovirus encoding Bcr(64-413) S354A did not induce cell death in CML cell lines in contrast to wild-type Bcr(64-413). Our findings indicate that Ser-354 of Bcr is part of a gating mechanism, which, after its phosphorylation, allows structural changes to occur in the Bcr protein. This altered phosphoserine form of the Bcr protein selectively binds to the Abl SH2 domain of the oncoprotein, which we propose down-regulates the activity of the Bcr-Abl tyrosine kinase.


Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas Oncogênicas/fisiologia , Fosfosserina/metabolismo , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Células COS , Éxons , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Células K562 , Peso Molecular , Mutação , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosfosserina/imunologia , Proteínas Proto-Oncogênicas c-bcr , Relação Estrutura-Atividade , Transfecção , Domínios de Homologia de src/genética , Domínios de Homologia de src/fisiologia
14.
Cancer Res ; 76(18): 5491-500, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27635045

RESUMO

More than 90% of clear cell renal cell carcinomas (ccRCC) exhibit inactivation of the von Hippel-Lindau (pVHL) tumor suppressor, establishing it as the major underlying cause of this malignancy. pVHL inactivation results in stabilization of the hypoxia-inducible transcription factors, HIF1α and HIF2α, leading to expression of a genetic program essential for the initiation and progression of ccRCC. Herein, we describe the potent, selective, and orally active small-molecule inhibitor PT2385 as a specific antagonist of HIF2α that allosterically blocks its dimerization with the HIF1α/2α transcriptional dimerization partner ARNT/HIF1ß. PT2385 inhibited the expression of HIF2α-dependent genes, including VEGF-A, PAI-1, and cyclin D1 in ccRCC cell lines and tumor xenografts. Treatment of tumor-bearing mice with PT2385 caused dramatic tumor regressions, validating HIF2α as a pivotal oncogenic driver in ccRCC. Notably, unlike other anticancer agents that inhibit VEGF receptor signaling, PT2385 exhibited no adverse effect on cardiovascular performance. Thus, PT2385 represents a novel class of therapeutics for the treatment of RCC with potent preclincal efficacy as well as improved tolerability relative to current agents that target the VEGF pathway. Cancer Res; 76(18); 5491-500. ©2016 AACR.


Assuntos
Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Animais , Antineoplásicos/química , Calorimetria , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Oncogene ; 21(47): 7137-46, 2002 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-12370803

RESUMO

We have previously shown that the Jak2 tyrosine kinase is activated in Bcr-Abl positive cell lines and blood cells from CML blast crisis patients by tyrosine phosphorylation. We are searching for downstream targets of Jak2 in Bcr-Abl positive cells. It is known that c-Myc expression is required for the oncogenic effects of Bcr-Abl, and that over-expression of c-Myc complements the transformation defect of the Bcr-Abl SH2 deletion mutant. Moreover, the Bcr-Abl SH2 deletion mutant and an Abl C-terminal deletion mutant are deficient in activating c-Myc expression. Since the Jak2 binds to the C-terminal domain of Bcr-Abl and optimal Jak2 activation requires the SH2 domain, we tested whether Jak2 was involved in c-Myc protein induction by Bcr-Abl. We treated the 32Dp210 Bcr-Abl cells with the Jak2 specific tyrosine kinase inhibitor, AG490, and found that this drug, like the Abl tyrosine kinase inhibitor STI-571, inhibited c-Myc protein induction by Bcr-Abl. Treatment of 32Dp210 Bcr-Abl cells with AG490 also inhibited c-MYC RNA expression. It is also known that c-Myc protein is a labile protein that is increased in amounts in response to various growth factors by a mechanism not involving new Myc protein formation. Treatment of 32Dp210 Bcr-Abl cells with both the proteasome inhibitor MG132 and AG490 blocked the reduction of the c-Myc protein observed by AG490 alone. An adaptor protein SH2-Bbeta is involved in the enhancement of the tyrosine kinase activity of Jak2 following ligand/receptor interaction. In this regard we showed that the Jak2/Bcr-Abl complex contains SH2-Bbeta. Expression of the SH2-Bbeta R555E mutant in 32Dp210 Bcr-Abl cells reduced c-Myc expression about 40% compared to a vector control. Interestingly, we found the reduction of the c-Myc protein in several clones of dominant-negative (DN) Jak2 expressing K562 cells correlated very well with the reduction of tumor growth of these cells in nude mice as compared to vector transfected K562 cells. Both STI-571 and AG490 also induced apoptosis in 32Dp210 cells. Of interest, IL-3 containing medium reversed the STI-571 induced apoptosis of 32Dp210 cells but did not reverse the induction of apoptosis by AG490, which strongly supports the specificity of the inhibitory effects of AG490 on the Jak2 tyrosine kinase. In summary, our findings indicate that Jak2 mediates the increase in c-Myc expression that is induced by Bcr-Abl. Our results indicate that activated Jak2 not only mediates an increase of c-MYC RNA expression but also interferes with proteasome-dependent degradation of c-Myc protein.


Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Regulação da Expressão Gênica , Genes myc , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Linhagem Celular , Humanos , Janus Quinase 2 , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leupeptinas/farmacologia , Mutagênese , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/antagonistas & inibidores , RNA Mensageiro/metabolismo , Fator de Transcrição STAT5 , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Tirfostinas/farmacologia , Proteínas de Xenopus/metabolismo
16.
Mol Cell Proteomics ; 6(4): 669-76, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17208939

RESUMO

IMAC in combination with mass spectrometry is a promising approach for global analysis of protein phosphorylation. Nevertheless this approach suffers from two shortcomings: inadequate efficiency of IMAC and poor fragmentation of phosphopeptides in the mass spectrometer. Here we report optimization of the IMAC procedure using (32)P-labeled tryptic peptides and development of MS/MS/MS (MS3) for identifying phosphopeptide sequences and phosphorylation sites. The improved IMAC method allowed recovery of phosphorylated tryptic peptides up to approximately 77% with only minor retention of unphosphorylated peptides. MS3 led to efficient fragmentation of the peptide backbone in phosphopeptides for sequence assignment. Proteomics of mitochondrial phosphoproteins using the resulting IMAC protocol and MS3 revealed 84 phosphorylation sites in 62 proteins, most of which have not been reported before. These results revealed diverse phosphorylation pathways involved in the regulation of mitochondrial functions. Integration of the optimized batchwise IMAC protocol with MS3 offers a relatively simple and more efficient approach for proteomics of protein phosphorylation.


Assuntos
Proteínas Mitocondriais/isolamento & purificação , Fosfoproteínas/isolamento & purificação , Proteoma/isolamento & purificação , Proteômica/métodos , Animais , Cromatografia de Afinidade/métodos , Camundongos , Mitocôndrias Hepáticas/química , Proteínas Mitocondriais/química , Mapeamento de Peptídeos , Fosfoproteínas/química , Fosforilação , Espectrometria de Massas em Tandem
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