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1.
Blood ; 135(1): 41-55, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31697823

RESUMO

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (<9 months from diagnosis), 65% in early relapse (9-36 months), and 32% in late relapse (>36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

2.
Leukemia ; 33(10): 2365-2378, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30940905

RESUMO

Bone marrow (BM) niche responds to chemotherapy-induced cytokines secreted from acute lymphoblastic leukemia (ALL) cells and protects the residual cells from chemotherapeutics in vivo. However, the underlying molecular mechanisms for the induction of cytokines by chemotherapy remain unknown. Here, we found that chemotherapeutic drugs (e.g., Ara-C, DNR, 6-MP) induced the expression of niche-protecting cytokines (GDF15, CCL3 and CCL4) in both ALL cell lines and primary cells in vitro. The ATM and NF-κB pathways were activated after chemotherapy treatment, and the pharmacological or genetic inhibition of these pathways significantly reversed the cytokine upregulation. Besides, chemotherapy-induced NF-κB activation was dependent on ATM-TRAF6 signaling, and NF-κB transcription factor p65 directly regulated the cytokines expression. Furthermore, we found that both pharmacological and genetic perturbation of ATM and p65 significantly decreased the residual ALL cells after Ara-C treatment in ALL xenograft mouse models. Together, these results demonstrated that ATM-dependent NF-κB activation mediated the cytokines induction by chemotherapy and ALL resistance to chemotherapeutics. Inhibition of ATM-dependent NF-κB pathway can sensitize ALL to chemotherapeutics, providing a new strategy to eradicate residual chemo-resistant ALL cells.

3.
FASEB J ; 33(3): 4525-4537, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30702927

RESUMO

It has been shown that 5-amino-4-imidazolecarboxamide riboside (AICAr) can inhibit cell proliferation and induce apoptosis in childhood acute lymphoblastic leukemia (ALL) cells. Although AICAr could regulate cellular energy metabolism by activating AMPK, the cytotoxic mechanisms of AICAr are still unclear. Here, we knocked out TP53 or PRKAA1 gene (encoding AMPKα1) in NALM-6 and Reh cells by using the clustered regularly interspaced short palindromic repeats/Cas9 system and found that AICAr-induced proliferation inhibition was independent of AMPK activation but dependent on p53. Liquid chromatography-mass spectrometry analysis of nucleotide metabolites indicated that AICAr caused an increase in adenosine triphosphate, deoxyadenosine triphosphate, and deoxyguanosine triphosphate levels by up-regulating purine biosynthesis, while AICAr led to a decrease in cytidine triphosphate, uridine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate levels because of reduced phosphoribosyl pyrophosphate production, which consequently impaired the pyrimidine biosynthesis. Ribonucleoside triphosphate (NTP) pool imbalances suppressed the rRNA transcription efficiency. Furthermore, deoxy-ribonucleoside triphosphate (dNTP) pool imbalances induced DNA replication stress and DNA double-strand breaks, followed by cell cycle arrest and apoptosis in ALL cells. Exogenous uridine could rebalance the NTP and dNTP pools by supplementing pyrimidine and then attenuate AICAr-induced cytotoxicity. Our data indicate that RNA transcription inhibition and DNA replication stress induced by NTP and dNTP pool imbalances might play a key role in AICAr-mediated cytotoxic effects on ALL cells, suggesting a potential clinical application of AICAr in future ALL therapy.-Du, L., Yang, F., Fang, H., Sun, H., Chen, Y., Xu, Y., Li, H., Zheng, L., Zhou, B.-B. S. AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.

4.
J Cell Mol Med ; 22(12): 6202-6212, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30255549

RESUMO

Relapse-specific mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1), a rate-limiting purine biosynthesis enzyme, confer significant drug resistances to combination chemotherapy in acute lymphoblastic leukemia (ALL). It is of particular interest to identify drugs to overcome these resistances. In this study, we found that PRPS1 mutant ALL cells specifically showed more chemosensitivity to 5-Fluorouracil (5-FU) than control cells, attributed to increased apoptosis of PRPS1 mutant cells by 5-FU. Mechanistically, PRPS1 mutants increase the level of intracellular phosphoribosyl pyrophosphate (PRPP), which causes the apt conversion of 5-FU to FUMP and FUTP in Reh cells, to promote 5-FU-induced DNA damage and apoptosis. Our study not only provides mechanistic rationale for re-targeting drug resistant cells in ALL, but also implicates that ALL patients who harbor relapse-specific mutations of PRPS1 might benefit from 5-FU-based chemotherapy in clinical settings.


Assuntos
Fluoruracila/farmacologia , Fosforribosil Pirofosfato/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Ribose-Fosfato Pirofosfoquinase/genética , Animais , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Células Jurkat , Lentivirus/genética , Camundongos , Fosforribosil Pirofosfato/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia
5.
J Exp Clin Cancer Res ; 37(1): 204, 2018 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-30157922

RESUMO

BACKGROUND: Considerable efforts have been devoted toward the uncovering of the molecular mechanisms underlying the maintenance of hematopoietic stem cells (HSCs) by the normal bone marrow (BM) niche. Previously, we demonstrated that a chemotherapy-induced niche, which is mainly composed of mesenchymal stem cells (MSCs), protects the residual B-cell acute lymphoblastic leukemia (B-ALL) cells from the insult of chemotherapeutic drugs. However, the roles of chemotherapy-induced niche on HSCs functions in B-ALL remain unclear. METHODS: We established an oncogenic N-MYC-driven B-ALL mouse model, which were subsequently treated with common chemotherapy drug cytarabine (Ara-C) and daunorubicin (DNR). After treatment, the structures of the BM niche were imaged by immunofluorescence staining. Then, the self-renewal and differentiation capability of the MSCs in the BM after Ara-C and DNR treatment were studied by ex vivo culture and gene expression analysis with RNA-seq and qRT-PCR. The effects of chemotherapy-induced niche on the hematopoietic reconstitution of HSCs were determined with series transplantation assay. Furthermore, the cell cycle, ROS level, mitochondrial membrane potential and cell apoptosis of HSCs were detected by flow cytometry. RESULTS: The MSCs, which is the main component of chemotherapy-induced BM niche, have decreased self-renewal capability and are prone to differentiate into adipocytes and chondrocytes. The results of gene expression analysis with RNA-seq showed that the MSCs have reduced levels of cytokines, including SCF, CXCL12, ANGPT1, VCAM1, and IL7. Furthermore, the chemotherapy-induced niche perturbed the hematopoietic reconstitution of HSCs in our N-MYC-driven B-ALL mouse model by promoting HSCs to enter cell cycle and increasing intracellular ROS levels and mitochondrial membrane potential of HSCs, which lead to the cell apoptosis of HSCs. CONCLUSIONS: Chemotherapy-induced BM niche perturbs the hematopoietic reconstitution of HSCs by increasing intracellular ROS level and inducing cell apoptosis.


Assuntos
Citarabina/administração & dosagem , Células-Tronco Hematopoéticas/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Modelos Animais de Doenças , Citometria de Fluxo , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/patologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Espécies Reativas de Oxigênio/metabolismo , Nicho de Células-Tronco/genética
6.
Oncotarget ; 9(2): 2268-2278, 2018 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-29416770

RESUMO

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Thiopurine is a widely used drug in the maintaining treatment of ALL. After a period of chemotherapy, 20% of pediatric patients and over 50% of adult patients will relapse. To investigate the mechanisms of drug resistance in vitro, we established the thiopurine resistant cell lines Reh-6MPR (6-MP Resistant cell) and Reh-6TGR (6-TG Resistant cell) by stepwise selection of the ALL cell line Reh. Cell viability assay revealed that 6MPR and 6TGR cells were almost 1000-fold more resistant to thiopurine comparing with the control Reh cells, and thiopurine conversion was significantly impaired in the resistant cells. Mechanistically, a same novel hypoxanthine phosphoribosyl transferase 1 (HPRT1) mutation c.495_496insA (p.V165fs) was found by whole exome sequencing in both resistant cells. The HPRT1 mutation dramaticly decreased the production of [13C5,15N4]-IMP from [13C5,15N4]-hypoxanthine (HX), showed a loss-of-funciton mechanism. Notably, re-expression the wildtype HPRT1 in Reh-6MPR cell can reverse the drug resistance and thiopurine conversion in Reh-6MPR cells. These results highlight the importance of HPRT1's activity in thiopurine resistance.

7.
Nat Med ; 21(6): 563-71, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25962120

RESUMO

Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Among chemotherapeutics, thiopurines are key drugs in ALL combination therapy. Using whole-exome sequencing, we identified relapse-specific mutations in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapsed childhood B cell ALL (B-ALL) cases. All individuals who harbored PRPS1 mutations relapsed early during treatment, and mutated ALL clones expanded exponentially before clinical relapse. Our functional analyses of PRPS1 mutants uncovered a new chemotherapy-resistance mechanism involving reduced feedback inhibition of de novo purine biosynthesis and competitive inhibition of thiopurine activation. Notably, the de novo purine synthesis inhibitor lometrexol effectively abrogated PRPS1 mutant-driven drug resistance. These results highlight the importance of constitutive activation of the de novo purine synthesis pathway in thiopurine resistance, and they offer therapeutic strategies for the treatment of relapsed and thiopurine-resistant ALL.


Assuntos
Retroalimentação Fisiológica/efeitos dos fármacos , Leucemia de Células B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Ribose-Fosfato Pirofosfoquinase/genética , Adolescente , Criança , Pré-Escolar , Exoma/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/patologia , Masculino , Mercaptopurina/administração & dosagem , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Purinas/biossíntese , Recidiva , Ribose-Fosfato Pirofosfoquinase/química , Tetra-Hidrofolatos/administração & dosagem
8.
Cancer Res ; 71(12): 4236-46, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21540235

RESUMO

Poorly differentiated tumors in non-small cell lung cancer (NSCLC) have been associated with shorter patient survival and shorter time to recurrence following treatment. Here, we integrate multiple experimental models with clinicopathologic analysis of patient tumors to delineate a cellular hierarchy in NSCLC. We show that the oncofetal protein 5T4 is expressed on tumor-initiating cells and associated with worse clinical outcome in NSCLC. Coexpression of 5T4 and factors involved in the epithelial-to-mesenchymal transition were observed in undifferentiated but not in differentiated tumor cells. Despite heterogeneous expression of 5T4 in NSCLC patient-derived xenografts, treatment with an anti-5T4 antibody-drug conjugate resulted in complete and sustained tumor regression. Thus, the aggressive growth of heterogeneous solid tumors can be blocked by therapeutic agents that target a subpopulation of cells near the top of the cellular hierarchy.


Assuntos
Antígenos de Neoplasias/análise , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Imunotoxinas/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Glicoproteínas de Membrana/análise , Células-Tronco Neoplásicas/imunologia , Animais , Antígeno CD24/análise , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Humanos , Receptores de Hialuronatos/análise , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Glicoproteínas de Membrana/fisiologia , Camundongos
9.
Nat Rev Drug Discov ; 8(10): 806-23, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19794444

RESUMO

The hypothesis that cancer is driven by tumour-initiating cells (popularly known as cancer stem cells) has recently attracted a great deal of attention, owing to the promise of a novel cellular target for the treatment of haematopoietic and solid malignancies. Furthermore, it seems that tumour-initiating cells might be resistant to many conventional cancer therapies, which might explain the limitations of these agents in curing human malignancies. Although much work is still needed to identify and characterize tumour-initiating cells, efforts are now being directed towards identifying therapeutic strategies that could target these cells. This Review considers recent advances in the cancer stem cell field, focusing on the challenges and opportunities for anticancer drug discovery.


Assuntos
Antineoplásicos/uso terapêutico , Descoberta de Drogas/tendências , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia
10.
Clin Cancer Res ; 15(20): 6314-20, 2009 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-19808869

RESUMO

Damage to genetic material represents a persistent and ubiquitous threat to genomic stability. Once DNA damage is detected, a multifaceted signaling network is activated that halts the cell cycle, initiates repair, and in some instances induces apoptotic cell death. In this article, we will review DNA damage surveillance networks, which maintain the stability of our genome, and discuss the efforts underway to identify chemotherapeutic compounds targeting the core components of DNA double-strand breaks (DSB) response pathway. The majority of tumor cells have defects in maintaining genomic stability owing to the loss of an appropriate response to DNA damage. New anticancer agents are exploiting this vulnerability of cancer cells to enhance therapeutic indexes, with limited normal tissue toxicity. Recently inhibitors of the checkpoint kinases Chk1 and Chk2 have been shown to sensitize tumor cells to DNA damaging agents. In addition, the treatment of BRCA1- or BRCA2-deficient tumor cells with poly(ADP-ribose) polymerase (PARP) inhibitors also leads to specific tumor killing. Due to the numerous roles of p53 in genomic stability and its defects in many human cancers, therapeutic agents that restore p53 activity in tumors are the subject of multiple clinical trials. In this article we highlight the proteins mentioned above and catalog several additional players in the DNA damage response pathway, including ATM, DNA-PK, and the MRN complex, which might be amenable to pharmacological interventions and lead to new approaches to sensitize cancer cells to radio- and chemotherapy. The challenge is how to identify those patients most receptive to these treatments.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Neoplasias/genética , Proteínas de Ciclo Celular/metabolismo , Sistemas de Liberação de Medicamentos , Genes cdc , Instabilidade Genômica , Humanos , Modelos Genéticos , Transdução de Sinais
11.
J Biol Chem ; 283(12): 8046-54, 2008 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-18182388

RESUMO

The Notch pathway regulates the development of many tissues and cell types and is involved in a variety of human diseases, making it an attractive potential therapeutic target. This promise has been limited by the absence of potent inhibitors or agonists that are specific for individual human Notch receptors (NOTCH1-4). Using an unbiased functional screening, we identified monoclonal antibodies that specifically inhibit or induce activating proteolytic cleavages in NOTCH3. Remarkably, the most potent inhibitory and activating antibodies bind to overlapping epitopes within a juxtamembrane negative regulatory region that protects NOTCH3 from proteolysis and activation in its resting autoinhibited state. The inhibitory antibodies revert phenotypes conveyed on 293T cells by NOTCH3 signaling, such as increased cellular proliferation, survival, and motility, whereas the activating antibody mimics some of the effects of ligand-induced Notch activation. These findings provide insights into the mechanisms of Notch autoinhibition and activation and pave the way for the further development of specific antibody-based modulators of the Notch receptors, which are likely to be of utility in a wide range of experimental and therapeutic settings.


Assuntos
Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptores Notch/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Movimento Celular/genética , Movimento Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Receptor Notch3 , Receptores Notch/genética , Receptores Notch/imunologia , Receptores Notch/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia
12.
Cancer Cell ; 10(1): 39-50, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16843264

RESUMO

We describe here the existence of a heregulin-HER3 autocrine loop, and the contribution of heregulin-dependent, HER2-mediated HER3 activation to gefitinib insensitivity in non-small cell lung cancer (NSCLC). ADAM17 protein, a major ErbB ligand sheddase, is upregulated in NSCLC and is required not only for heregulin-dependent HER3 signaling, but also for EGFR ligand-dependent signaling in NSCLC cell lines. A selective ADAM inhibitor, INCB3619, prevents the processing and activation of multiple ErbB ligands, including heregulin. In addition, INCB3619 inhibits gefitinib-resistant HER3 signaling and enhances gefitinib inhibition of EGFR signaling in NSCLC. These results show that ADAM inhibition affects multiple ErbB pathways in NSCLC and thus offers an excellent opportunity for pharmacological intervention, either alone or in combination with other drugs.


Assuntos
Proteínas ADAM/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/metabolismo , Piperidinas/farmacologia , Receptor ErbB-3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Compostos de Espiro/farmacologia , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/genética , Feminino , Gefitinibe , Expressão Gênica/genética , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Paclitaxel/farmacologia , Piperidinas/uso terapêutico , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Compostos de Espiro/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Ann N Y Acad Sci ; 1059: 56-60, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16382043

RESUMO

ADAMs (a disintegrin and metalloproteases) are zinc-dependent transmembrane metalloproteases that shed the extracellular domains of membrane-bound growth factors, cytokines, and receptors. Recently, ADAMs have emerged in ErbB signaling pathways as sheddases or multiple ErbB ligands. As the ErbB pathway is a validated target for anticancer drugs, upstream activators of ErbB ligands, their sheddases, become new drug targets in the ErbB pathway. We have identified selective small molecule inhibitors of ADAM proteases that block shedding and activation of multiple ErbB ligands, and we are planning to test the compounds in the clinic.


Assuntos
Proteínas ADAM/antagonistas & inibidores , Receptores ErbB/metabolismo , Neoplasias/metabolismo , Proteínas ADAM/metabolismo , Animais , Humanos , Ligantes , Camundongos , Neoplasias/tratamento farmacológico , Ligação Proteica , Transporte Proteico , Transdução de Sinais
14.
Expert Opin Investig Drugs ; 14(6): 591-606, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16004590

RESUMO

A disintegrin and metalloproteases (ADAMs) are zinc-dependent trans-membrane metalloproteases that shed the extracellular domains of membrane-bound growth factors, cytokines and receptors. Key functions of ADAMs have emerged in ErbB signalling pathways as being sheddases for multiple ErbB ligands. As the ErbB pathway is a validated target for anti-cancer drugs, the upstream activators of ErbB ligands, their sheddases, now enter the spotlight as new drug targets in the ErbB pathway. ADAMs are involved not only in tumour cell proliferation but also in angiogenesis and metastasis. Therefore, strategies targeting ADAMs might be an important complement to existing anti-ErbB approaches.


Assuntos
Proteínas ADAM/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Neoplasias/metabolismo , Transdução de Sinais/fisiologia , Proteínas ADAM/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Neoplasias/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos
15.
Mol Cancer Ther ; 3(10): 1221-7, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15486189

RESUMO

Inhibitors of the G(2) DNA damage checkpoint can selectively sensitize cancer cells with mutated p53 to killing by DNA-damaging agents. Isogranulatimide is a G(2) checkpoint inhibitor containing a unique indole/maleimide/imidazole skeleton identified in a phenotypic cell-based screen; however, the mechanism of action of isogranulatimide is unknown. Using natural and synthetic isogranulatimide analogues, we show that the imide nitrogen and a basic nitrogen at position 14 or 15 in the imidazole ring are important for checkpoint inhibition. Isogranulatimide shows structural resemblance to the aglycon of UCN-01, a potent bisindolemaleimide inhibitor of protein kinase C beta (IC(50), 0.001 micromol/L) and of the checkpoint kinase Chk1 (IC(50), 0.007 micromol/L). In vitro kinase assays show that isogranulatimide inhibits Chk1 (IC(50), 0.1 micromol/L) but not protein kinase C beta. Of 13 additional protein kinases tested, isogranulatimide significantly inhibits only glycogen synthase kinase-3beta (IC(50), 0.5 micromol/L). We determined the crystal structure of the Chk1 catalytic domain complexed with isogranulatimide. Like UCN-01, isogranulatimide binds in the ATP-binding pocket of Chk1 and hydrogen bonds with the backbone carbonyl oxygen of Glu(85) and the amide nitrogen of Cys(87). Unlike UCN-01, the basic N15 of isogranulatimide interacts with Glu(17), causing a conformation change in the kinase glycine-rich loop that may contribute importantly to inhibition. The mechanism by which isogranulatimide inhibits Chk1 and its favorable kinase selectivity profile make it a promising candidate for modulating checkpoint responses in tumors for therapeutic benefit.


Assuntos
Antineoplásicos/farmacologia , Dano ao DNA , Fase G2 , Imidazóis/farmacologia , Indóis/farmacologia , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Domínio Catalítico , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Cisteína/química , Relação Dose-Resposta a Droga , Glutamina/química , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Indóis/antagonistas & inibidores , Concentração Inibidora 50 , Maleimidas/antagonistas & inibidores , Modelos Químicos , Modelos Moleculares , Nitrogênio/química , Fenótipo , Ligação Proteica , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Estrutura Terciária de Proteína , Fatores de Tempo
17.
Prog Cell Cycle Res ; 5: 413-21, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14593735

RESUMO

The DNA damage response includes not only checkpoint and apoptosis, but also direct activation of DNA repair networks. Downstream in the DNA damage response pathway are Chk1, an essential checkpoint kinase, and Chk2, which plays a critical role in p53-dependent apoptosis. Chk1 inhibition is expected to lead to chemosensitization of tumors, while Chk2 inhibition could protect normal sensitive tissues from some chemotherapeutic agents. Drugs targeting Chk1 and Chk2 have the potential to significantly improve the therapeutic window of DNA damaging agents available in the clinic.


Assuntos
Dano ao DNA/efeitos dos fármacos , Desenho de Drogas , Genes cdc/efeitos dos fármacos , Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases , Animais , Antineoplásicos/farmacologia , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Dano ao DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Genes cdc/fisiologia , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Quinases/metabolismo
18.
Cancer Biol Ther ; 2(4 Suppl 1): S16-22, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14508077

RESUMO

The DNA damage response includes not only cell cycle arrest and apoptosis, but also direct activation of DNA repair networks. Four DNA checkpoint kinases ATM, ATR, Chk1 and Chk2 have been identified in the mammalian DNA damage response signal transduction pathway. In this article, we review and discuss current knowledge and thinking about checkpoint kinases, and their potential as cancer drug targets. Particular emphasis is given to various therapeutic hypotheses and their promise for improving current cancer therapies.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA , Humanos , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor
20.
J Biol Chem ; 277(48): 46609-15, 2002 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-12244092

RESUMO

Chk1 is a serine-threonine kinase that plays an important role in the DNA damage response, including G(2)/M cell cycle control. UCN-01 (7-hydroxystaurosporine), currently in clinical trials, has recently been shown to be a potent Chk1 inhibitor that abrogates the G(2)/M checkpoint induced by DNA-damaging agents. To understand the structural basis of Chk1 inhibition by UCN-01, we determined the crystal structure of the Chk1 kinase domain in complex with UCN-01. Chk1 structures with staurosporine and its analog SB-218078 were also determined. All three compounds bind in the ATP-binding pocket of Chk1, producing only slight changes in the protein conformation. Selectivity of UCN-01 toward Chk1 over cyclin-dependent kinases can be explained by the presence of a hydroxyl group in the lactam moiety interacting with the ATP-binding pocket. Hydrophobic interactions and hydrogen-bonding interactions were observed in the structures between UCN-01 and the Chk1 kinase domain. The high structural complementarity of these interactions is consistent with the potency and selectivity of UCN-01.


Assuntos
Alcaloides/farmacologia , Inibidores de Proteínas Quinases , Alcaloides/química , Sequência de Aminoácidos , Quinase 1 do Ponto de Checagem , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Quinases/química , Estaurosporina/análogos & derivados , Relação Estrutura-Atividade
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