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2.
Signal Transduct Target Ther ; 7(1): 51, 2022 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-35185150

RESUMO

Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2-selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Leucemia Mieloide Aguda , Sistema de Sinalização das MAP Quinases , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2 , Sulfonamidas , Proteínas ras , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia
3.
Int J Toxicol ; 40(1): 26-39, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33176523

RESUMO

Predictive indicators of testicular toxicity could improve drug development by allowing early in-life screening for this adverse effect before it becomes severe. We hypothesized that circulating microRNAs (miRNAs) could serve as testicular toxicity biomarkers in dogs. Herein, we describe the results of an exploratory study conducted to discover biomarkers of drug-induced testicular injury. Following a dose-selection study using the testicular toxicant ethylene glycol monomethyl ether (EGME), we chose a dose of 50 mg/kg/d EGME to avoid systemic toxicity and treated 2 groups of dogs (castrated, non-castrated) for 14 to 28 days. Castrated animals were used as negative controls to identify biomarkers specific for testicular toxicity because EGME can cause toxicity to organ systems in addition to the testis. Blood was collected daily during the dosing period, followed by recovery for 29 to 43 days with less frequent sampling. Dosing was well tolerated, resulting in mild-to-moderate degeneration in testes and epididymides. Global profiling of serum miRNAs at selected dosing and recovery time points was completed by small RNA sequencing. Bioinformatics data analysis using linear modeling demonstrated several circulating miRNAs that were differentially abundant during the dosing period compared with baseline and/or castrated control samples. Confirmatory reverse transcription quantitative polymerase chain reaction data in these animals was unable to detect sustained alterations of miRNAs in serum, except for 1 potential candidate cfa-miR-146b. Taken together, we report the results of a comprehensive exploratory study and suggest future directions for follow-up research to address the challenge of developing diagnostic biomarkers of testicular toxicity.


Assuntos
MicroRNA Circulante/efeitos dos fármacos , Desenvolvimento de Medicamentos/métodos , Etilenoglicóis/toxicidade , Marcadores Genéticos/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Cães , Masculino
4.
Clin Cancer Res ; 26(23): 6335-6349, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32943458

RESUMO

PURPOSE: Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action. EXPERIMENTAL DESIGN: Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen. RESULTS: Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti-PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti-PD-L1. CONCLUSIONS: STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Neoplasias do Colo/terapia , Neoplasias/terapia , Oligonucleotídeos/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Microambiente Tumoral/imunologia , Ensaios Clínicos Fase I como Assunto , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Quimioterapia Combinada , Humanos , Imunomodulação , Macrófagos/imunologia , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Fator de Transcrição STAT3/genética , Linfócitos T/imunologia , Células Tumorais Cultivadas
5.
Clin Epigenetics ; 12(1): 84, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32539856

RESUMO

BACKGROUND: Alzheimer's disease (AD) is a chronic progressive neurodegenerative disease impacting an estimated 44 million adults worldwide. The causal pathology of AD (accumulation of amyloid-beta and tau), precedes hallmark symptoms of dementia by more than a decade, necessitating development of early diagnostic markers of disease onset, particularly for new drugs that aim to modify disease processes. To evaluate differentially methylated positions (DMPs) as novel blood-based biomarkers of AD, we used a subset of 653 individuals with peripheral blood (PB) samples in the Alzheimer's disease Neuroimaging Initiative (ADNI) consortium. The selected cohort of AD, mild cognitive impairment (MCI), and age-matched healthy controls (CN) all had imaging, genetics, transcriptomics, cerebrospinal protein markers, and comprehensive clinical records, providing a rich resource of concurrent multi-omics and phenotypic information on a well-phenotyped subset of ADNI participants. RESULTS: In this manuscript, we report cross-diagnosis differential peripheral DNA methylation in a cohort of AD, MCI, and age-matched CN individuals with longitudinal DNA methylation measurements. Epigenome-wide association studies (EWAS) were performed using a mixed model with repeated measures over time with a P value cutoff of 1 × 10-5 to test contrasts of pairwise differential peripheral methylation in AD vs CN, AD vs MCI, and MCI vs CN. The most highly significant differentially methylated loci also tracked with Mini Mental State Examination (MMSE) scores. Differentially methylated loci were enriched near brain and neurodegeneration-related genes (e.g., BDNF, BIN1, APOC1) validated using the genotype tissue expression project portal (GTex). CONCLUSIONS: Our work shows that peripheral differential methylation between age-matched subjects with AD relative to healthy controls will provide opportunities to further investigate and validate differential methylation as a surrogate of disease. Given the inaccessibility of brain tissue, the PB-associated methylation marks may help identify the stage of disease and progression phenotype, information that would be central to bringing forward successful drugs for AD.


Assuntos
Doença de Alzheimer/diagnóstico por imagem , Disfunção Cognitiva/diagnóstico por imagem , Metilação de DNA/genética , Neuroimagem/métodos , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Disfunção Cognitiva/sangue , Disfunção Cognitiva/líquido cefalorraquidiano , Diagnóstico Diferencial , Progressão da Doença , Diagnóstico Precoce , Epigenômica/métodos , Feminino , Genótipo , Humanos , Estudos Longitudinais , Masculino , Testes de Estado Mental e Demência/normas , Fenótipo , Transcriptoma/genética
7.
Cancer Res ; 78(23): 6691-6702, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30297535

RESUMO

: PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity. SIGNIFICANCE: These findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells.


Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Neoplasias da Mama/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/química , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , Ensaios Antitumorais Modelo de Xenoenxerto
8.
J Immunother Cancer ; 5(1): 63, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28807001

RESUMO

BACKGROUND: T-cell checkpoint blockade and MEK inhibitor combinations are under clinical investigation. Despite progress elucidating the immuno-modulatory effects of MEK inhibitors as standalone therapies, the impact of MEK inhibition on the activity of T-cell checkpoint inhibitors remains incompletely understood. Here we sought to characterize the combined effects of MEK inhibition and anti-CTLA-4 mAb (anti-CTLA-4) therapy, examining effects on both T-cells and tumor microenvironment (TME). METHODS: In mice, the effects of MEK inhibition, via selumetinib, and anti-CTLA-4 on immune responses to keyhole limpet haemocyanin (KLH) immunization were monitored using ex vivo functional assays with splenocytes. In a KRAS-mutant CT26 mouse colorectal cancer model, the impact on the tumor microenvironment (TME) and the spleen were evaluated by flow cytometry. The TME was further examined by gene expression and immunohistochemical analyses. The combination and sequencing of selumetinib and anti-CTLA-4 were also evaluated in efficacy studies using the CT26 mouse syngeneic model. RESULTS: Anti-CTLA-4 enhanced the generation of KLH specific immunity following KLH immunization in vivo; selumetinib was found to reduce, but did not prevent, this enhancement of immune response by anti-CTLA-4 in vivo. In the CT26 mouse model, anti-CTLA-4 treatment led to higher expression levels of the immunosuppressive mediators, Cox-2 and Arg1 in the TME. Combination of anti-CTLA-4 with selumetinib negated this up-regulation of Cox-2 and Arg1, reduced the frequency of CD11+ Ly6G+ myeloid cells, and led to the accumulation of differentiating monocytes at the Ly6C+ MHC+ intermediate state in the tumor. We also report that MEK inhibition had limited impact on anti-CTLA-4-mediated increases in T-cell infiltration and T-cell activation in CT26 tumors. Finally, we show that pre-treatment, but not concurrent treatment, with selumetinib enhanced the anti-tumor activity of anti-CTLA-4 in the CT26 model. CONCLUSION: These data provide evidence that MEK inhibition can lead to changes in myeloid cells and immunosuppressive factors in the tumor, thus potentially conditioning the TME to facilitate improved response to anti-CTLA-4 treatment. In summary, the use of MEK inhibitors to alter the TME as an approach to enhance the activities of immune checkpoint inhibitors warrants further investigation in clinical trials.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Benzimidazóis/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Benzimidazóis/farmacologia , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Reprogramação Celular/efeitos dos fármacos , Neoplasias Colorretais/genética , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Cancer Res ; 77(4): 827-838, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-27965317

RESUMO

Personalized therapy is a major goal of modern oncology, as patient responses vary greatly even within a histologically defined cancer subtype. This is especially true in acute myeloid leukemia (AML), which exhibits striking heterogeneity in molecular segmentation. When calibrated to cell-specific data, executable network models can reveal subtle differences in signaling that help explain differences in drug response. Furthermore, they can suggest drug combinations to increase efficacy and combat acquired resistance. Here, we experimentally tested dynamic proteomic changes and phenotypic responses in diverse AML cell lines treated with pan-PIM kinase inhibitor and fms-related tyrosine kinase 3 (FLT3) inhibitor as single agents and in combination. We constructed cell-specific executable models of the signaling axis, connecting genetic aberrations in FLT3, tyrosine kinase 2 (TYK2), platelet-derived growth factor receptor alpha (PDGFRA), and fibroblast growth factor receptor 1 (FGFR1) to cell proliferation and apoptosis via the PIM and PI3K kinases. The models capture key differences in signaling that later enabled them to accurately predict the unique proteomic changes and phenotypic responses of each cell line. Furthermore, using cell-specific models, we tailored combination therapies to individual cell lines and successfully validated their efficacy experimentally. Specifically, we showed that cells mildly responsive to PIM inhibition exhibited increased sensitivity in combination with PIK3CA inhibition. We also used the model to infer the origin of PIM resistance engineered through prolonged drug treatment of MOLM16 cell lines and successfully validated experimentally our prediction that this resistance can be overcome with AKT1/2 inhibition. Cancer Res; 77(4); 827-38. ©2016 AACR.


Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Transdução de Sinais/fisiologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Compostos de Bifenilo/uso terapêutico , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Simulação por Computador , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Humanos , Leucemia Mieloide Aguda/fisiopatologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-pim-1/fisiologia , Transdução de Sinais/efeitos dos fármacos , Tiazolidinas/uso terapêutico
10.
Clin Cancer Res ; 23(6): 1531-1541, 2017 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-27663586

RESUMO

Purpose: The emergence of EGFR inhibitors such as gefitinib, erlotinib, and osimertinib has provided novel treatment opportunities in EGFR-driven non-small cell lung cancer (NSCLC). However, most patients with EGFR-driven cancers treated with these inhibitors eventually relapse. Recent efforts have identified the canonical Wnt pathway as a mechanism of protection from EGFR inhibition and that inhibiting tankyrase, a key player in this pathway, is a potential therapeutic strategy for the treatment of EGFR-driven tumors.Experimental Design: We performed a preclinical evaluation of tankyrase inhibitor AZ1366 in combination with multiple EGFR-inhibitors across NSCLC lines, characterizing its antitumor activity, impingement on canonical Wnt signaling, and effects on gene expression. We performed pharmacokinetic and pharmacodynamic profiling of AZ1366 in mice and evaluated its therapeutic activity in an orthotopic NSCLC model.Results: In combination with EGFR inhibitors, AZ1366 synergistically suppressed proliferation of multiple NSCLC lines and amplified global transcriptional changes brought about by EGFR inhibition. Its ability to work synergistically with EGFR inhibition coincided with its ability to modulate the canonical Wnt pathway. Pharmacokinetic and pharmacodynamic profiling of AZ1366-treated orthotopic tumors demonstrated clinically relevant serum drug levels and intratumoral target inhibition. Finally, coadministration of an EGFR inhibitor and AZ1366 provided better tumor control and improved survival for Wnt-responsive lung cancers in an orthotopic mouse model.Conclusions: Tankyrase inhibition is a potent route of tumor control in EGFR-dependent NSCLC with confirmed dependence on canonical Wnt signaling. These data strongly support further evaluation of tankyrase inhibition as a cotreatment strategy with EGFR inhibition in an identifiable subset of EGFR-driven NSCLC. Clin Cancer Res; 23(6); 1531-41. ©2016 AACR.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Inibidores Enzimáticos/administração & dosagem , Receptores ErbB/antagonistas & inibidores , Tanquirases/antagonistas & inibidores , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Gefitinibe , Humanos , Camundongos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Quinazolinas/administração & dosagem , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Oncotarget ; 7(8): 9163-74, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26824321

RESUMO

Agents that target components of the PI3K/AKT/mTOR pathway are under investigation for the treatment of diffuse large B cell lymphoma (DLBCL). Given the highly heterogeneous nature of DLBCL, it is not clear whether all subtypes of DLBCL will be susceptible to PI3K pathway inhibition, or which kinase within this pathway is the most favorable target. Pharmacological profiling of a panel of DLBCL cell lines revealed a subset of DLBCL that was resistant to AKT inhibition. Strikingly, sensitivity to AKT inhibitors correlated with the ability of these inhibitors to block phosphorylation of S6K1 and ribosomal protein S6. Cell lines resistant to AKT inhibition activated S6K1 independent of AKT either through upregulation of PIM2 or through activation by B cell receptor (BCR) signaling components. Finally, combined inhibition of AKT and BTK, PIM2, or S6K1 proved to be an effective strategy to overcome resistance to AKT inhibition in DLBCL.


Assuntos
Antineoplásicos/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular Tumoral , Humanos , Linfoma Difuso de Grandes Células B/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína S6 Ribossômica/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
Bioorg Med Chem Lett ; 25(24): 5743-7, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26546219

RESUMO

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZeneca's collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.


Assuntos
Centrossomo/metabolismo , Ftalazinas/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Administração Oral , Animais , Sítios de Ligação , Células CACO-2 , Centrossomo/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Células HeLa , Humanos , Microssomos/metabolismo , Conformação Molecular , Simulação de Dinâmica Molecular , Ftalazinas/administração & dosagem , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Estrutura Terciária de Proteína , Ratos , Tanquirases/antagonistas & inibidores , Tanquirases/metabolismo
13.
Cancer Cell ; 27(1): 57-71, 2015 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-25584894

RESUMO

A functional genomics study revealed that the activity of acetyl-CoA synthetase 2 (ACSS2) contributes to cancer cell growth under low-oxygen and lipid-depleted conditions. Comparative metabolomics and lipidomics demonstrated that acetate is used as a nutritional source by cancer cells in an ACSS2-dependent manner, and supplied a significant fraction of the carbon within the fatty acid and phospholipid pools. ACSS2 expression is upregulated under metabolically stressed conditions and ACSS2 silencing reduced the growth of tumor xenografts. ACSS2 exhibits copy-number gain in human breast tumors, and ACSS2 expression correlates with disease progression. These results signify a critical role for acetate consumption in the production of lipid biomass within the harsh tumor microenvironment.


Assuntos
Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Ácidos Graxos/metabolismo , Neoplasias/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Hipóxia , Células MCF-7 , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias/genética , Neoplasias/metabolismo , Estresse Fisiológico
14.
J Natl Cancer Inst ; 107(2)2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25505253

RESUMO

BACKGROUND: PIM1 kinase is coexpressed with c-MYC in human prostate cancers (PCs) and dramatically enhances c-MYC-induced tumorigenicity. Here we examine the effects of a novel oral PIM inhibitor, AZD1208, on prostate tumorigenesis and recurrence. METHODS: A mouse c-MYC/Pim1-transduced tissue recombination PC model, Myc-CaP allografts, and human PC xenografts were treated with AZD1208 (n = 5-11 per group). Androgen-sensitive and castrate-resistant prostate cancer (CRPC) models were studied as well as the effects of hypoxia and radiation. RNA sequencing was used to analyze drug-induced gene expression changes. Results were analyzed with χ(2) test. Student's t test and nonparametric Mann-Whitney rank sum U Test. All statistical tests were two-sided. RESULTS: AZD1208 inhibited tumorigenesis in tissue recombinants, Myc-CaP, and human PC xenograft models. PIM inhibition decreased c-MYC/Pim1 graft growth by 54.3 ± 39% (P < .001), decreased cellular proliferation by 46 ± 14% (P = .016), and increased apoptosis by 326 ± 170% (P = .039). AZD1208 suppressed multiple protumorigenic pathways, including the MYC gene program. However, it also downregulated the p53 pathway. Hypoxia and radiation induced PIM1 in prostate cancer cells, and AZD1208 functioned as a radiation sensitizer. Recurrent tumors postcastration responded transiently to either AZD1208 or radiation treatment, and combination treatment resulted in more sustained inhibition of tumor growth. Cell lines established from recurrent, AZD1208-resistant tumors again revealed downregulation of the p53 pathway. Irradiated AZD1208-treated tumors robustly upregulated p53, providing a possible mechanistic explanation for the effectiveness of combination therapy. Finally, an AZD1208-resistant gene signature was found to be associated with biochemical recurrence in PC patients. CONCLUSIONS: PIM inhibition is a potential treatment for MYC-driven prostate cancers including CRPC, and its effectiveness may be enhanced by activators of the p53 pathway, such as radiation.


Assuntos
Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Tiazolidinas/farmacologia , Administração Oral , Aloenxertos , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/administração & dosagem , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Masculino , Camundongos , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Tiazolidinas/administração & dosagem , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Oncotarget ; 6(4): 2407-20, 2015 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-25537515

RESUMO

Acquired resistance to PI3K/mTOR/Akt pathway inhibitors is often associated with compensatory feedback loops involving the activation of oncogenes. Here, we have generated everolimus resistance in ER+ breast cancer cells and in long-term estrogen deprived (LTED) models that mimic progression on anti-estrogens. This allowed us to uncover MYC as a driver of mTOR inhibitor resistance. We demonstrate that both everolimus resistance and acute treatment of everolimus can lead to the upregulation of MYC mRNA, protein expression and, consequently, the enrichment of MYC signatures as revealed by RNA sequencing data. Depletion of MYC resulted in resensitization to everolimus, confirming its functional importance in this setting. Furthermore, ChIP assays demonstrate that MYC upregulation in the everolimus resistant lines is mediated by increased association of the BRD4 transcription factor with the MYC gene. Finally, JQ1, a BRD4 inhibitor combined with everolimus exhibited increased tumor growth inhibition in 3D Matrigel models and an in vivo xenograft model. These data suggest that MYC plays an important role in mediating resistance to everolimus in ER+ and ER+/LTED models. Furthermore, given the regulation ofMYCby BRD4 in this setting, these data have implications for increased therapeutic potential of combining epigenetic agents with mTOR inhibitors to effectively downregulate otherwise difficult to target transcription factors such as MYC.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Epigênese Genética/efeitos dos fármacos , Everolimo/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Antineoplásicos/farmacologia , Azepinas/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Células MCF-7 , Camundongos Nus , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Oncotarget ; 5(13): 4990-5001, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24970801

RESUMO

Diffuse large B cell lymphoma is generally treated by chemotherapy and there is an unmet medical need for novel targeted therapies or combination therapies. Using in vitro screening, we have identified the combination of ibrutinib, an inhibitor of the tyrosine kinase BTK, and AZD2014, an mTOR catalytic inhibitor, as being highly synergistic in killing ABC-subtype DLBCL cell lines. Simultaneous inhibition of BTK and mTOR causes apoptosis both in vitro and in vivo and results in tumor regression in a xenograft model. We identify two parallel mechanisms that underlie apoptosis in this setting: cooperative inhibition of cap-dependent translation, and the inhibition of an NF-κB/IL10/STAT3 autocrine loop. Combined disruption of these pathways is required for apoptosis. These data represent a rational basis for the dual inhibition of BTK and mTOR as a potential treatment for ABC-subtype DLBCL.


Assuntos
Apoptose/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Complexos Multiproteicos/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Animais , Benzamidas , Western Blotting , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Células HEK293 , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos SCID , Morfolinas/farmacologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Piperidinas , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Lipid Res ; 53(8): 1459-71, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22628619

RESUMO

Dysregulation of ceramide synthesis has been associated with metabolic disorders such as atherosclerosis and diabetes. We examined the changes in lipid homeostasis and gene expression in Huh7 hepatocytes when the synthesis of ceramide is perturbed by knocking down serine pal mitoyltransferase subunits 1, 2, and 3 (SPTLC123) or dihydroceramide desaturase 1 (DEGS1). Although knocking down all SPTLC subunits is necessary to reduce total ceramides significantly, depleting DEGS1 is sufficient to produce a similar outcome. Lipidomic analysis of distribution and speciation of multiple lipid classes indicates an increase in phospholipids in SPTLC123-silenced cells, whereas DEGS1 depletion leads to the accumulation of sphingolipid intermediates, free fatty acids, and diacylglycerol. When cer amide synthesis is disrupted, the transcriptional profiles indicate inhibition in biosynthetic processes, downregulation of genes involved in general endomembrane trafficking, and upregulation of endocytosis and endosomal recycling. SPTLC123 silencing strongly affects the expression of genes involved with lipid metabolism. Changes in amino acid, sugar, and nucleotide metabolism, as well as vesicle trafficking between organelles, are more prominent in DEGS1-silenced cells. These studies are the first to provide a direct and comprehensive understanding at the lipidomic and transcriptomic levels of how Huh7 hepatocytes respond to changes in the inhibition of ceramide synthesis.


Assuntos
Ceramidas/biossíntese , Ceramidas/metabolismo , Inativação Gênica , Homeostase/genética , Oxirredutases/genética , Serina C-Palmitoiltransferase/genética , Transcriptoma/genética , Linhagem Celular , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Oxirredutases/deficiência , Serina C-Palmitoiltransferase/deficiência , Transcrição Gênica/genética
18.
PLoS One ; 3(8): e2857, 2008 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-18682726

RESUMO

The mouse Xin repeat-containing proteins (mXinalpha and mXinbeta) localize to the intercalated disc in the heart. mXinalpha is able to bundle actin filaments and to interact with beta-catenin, suggesting a role in linking the actin cytoskeleton to N-cadherin/beta-catenin adhesion. mXinalpha-null mouse hearts display progressively ultrastructural alterations at the intercalated discs, and develop cardiac hypertrophy and cardiomyopathy with conduction defects. The up-regulation of mXinbeta in mXinalpha-deficient mice suggests a partial compensation for the loss of mXinalpha. To elucidate the evolutionary relationship between these proteins and to identify the origin of Xin, a phylogenetic analysis was done with 40 vertebrate Xins. Our results show that the ancestral Xin originated prior to the emergence of lamprey and subsequently underwent gene duplication early in the vertebrate lineage. A subsequent teleost-specific genome duplication resulted in most teleosts encoding at least three genes. All Xins contain a highly conserved beta-catenin-binding domain within the Xin repeat region. Similar to mouse Xins, chicken, frog and zebrafish Xins also co-localized with beta-catenin to structures that appear to be the intercalated disc. A putative DNA-binding domain in the N-terminus of all Xins is strongly conserved, whereas the previously characterized Mena/VASP-binding domain is a derived trait found only in Xinalphas from placental mammals. In the C-terminus, Xinalphas and Xinbetas are more divergent relative to each other but each isoform from mammals shows a high degree of within-isoform sequence identity. This suggests different but conserved functions for mammalian Xinalpha and Xinbeta. Interestingly, the origin of Xin ca. 550 million years ago coincides with the genesis of heart chambers with complete endothelial and myocardial layers. We postulate that the emergence of the Xin paralogs and their functional differentiation may have played a key role in the evolutionary development of the heart.


Assuntos
Proteínas de Ligação a DNA/genética , Coração/fisiologia , Proteínas Nucleares/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Evolução Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Filogenia , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Tempo , beta Catenina/química , beta Catenina/fisiologia
19.
Methods Mol Biol ; 366: 183-201, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17568125

RESUMO

To understand how cardiac gene expression is regulated, the identification and characterization of cis-regulatory elements and their trans-acting factors by gel mobility shift assay (GMSA) or gel retardation assay are essential and common steps. In addition to providing a general protocol for GMSA, this chapter describes some applications of this assay to characterize cardiac-specific and ubiquitous trans-acting factors bound to regulatory elements [novel TCTG(G/C) direct repeat and A/T-rich region] of the rat cardiac troponin T promoter. In GMSA, the specificity of the binding of trans-acting factor to labeled DNA probe should be verified by the addition of unlabeled probe in the reaction mixture. The migratory property of DNA-protein complexes formed by protein extracts prepared from different tissues can be compared to determine the tissue specificity of trans-acting factors. GMSA, coupled with specific antibody to trans-acting factor (antibody supershift assay), is used to identify proteins present in the DNA-protein complex. The gel-shift competition assay with an unlabeled probe containing a slightly different sequence is a powerful technique used to assess the sequence specificity and relative binding affinity of a DNA-protein interaction. GMSA with SDS-PAGE fractionated proteins allows for the determination of the apparent molecular mass of bound trans-acting factor.


Assuntos
Ensaio de Desvio de Mobilidade Eletroforética/métodos , Miocárdio/metabolismo , Regiões Promotoras Genéticas , Elementos Reguladores de Transcrição/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Mucosa Gástrica/metabolismo , Fígado/metabolismo , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Ligação Proteica , Ratos , Homologia de Sequência do Ácido Nucleico , Troponina T/genética
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