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1.
Nature ; 569(7756): E4, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31043737

RESUMO

Further analysis has revealed that the signal reported in Extended Data Fig. 1c of this Letter is attributed to phosphorylethanolamine, not carbamoyl phosphate. A newly developed derivatization method revealed that the level of carbamoyl phosphate in these NSCLC extracts is below the detection threshold of approximately 10 nanomoles. These findings do not alter the overall conclusions of the Letter; see associated Amendment for full details. The Letter has not been corrected online.

2.
N Engl J Med ; 384(22): 2102-2114, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34077643

RESUMO

BACKGROUND: The role of adjuvant treatment in high-risk muscle-invasive urothelial carcinoma after radical surgery is not clear. METHODS: In a phase 3, multicenter, double-blind, randomized, controlled trial, we assigned patients with muscle-invasive urothelial carcinoma who had undergone radical surgery to receive, in a 1:1 ratio, either nivolumab (240 mg intravenously) or placebo every 2 weeks for up to 1 year. Neoadjuvant cisplatin-based chemotherapy before trial entry was allowed. The primary end points were disease-free survival among all the patients (intention-to-treat population) and among patients with a tumor programmed death ligand 1 (PD-L1) expression level of 1% or more. Survival free from recurrence outside the urothelial tract was a secondary end point. RESULTS: A total of 353 patients were assigned to receive nivolumab and 356 to receive placebo. The median disease-free survival in the intention-to-treat population was 20.8 months (95% confidence interval [CI], 16.5 to 27.6) with nivolumab and 10.8 months (95% CI, 8.3 to 13.9) with placebo. The percentage of patients who were alive and disease-free at 6 months was 74.9% with nivolumab and 60.3% with placebo (hazard ratio for disease recurrence or death, 0.70; 98.22% CI, 0.55 to 0.90; P<0.001). Among patients with a PD-L1 expression level of 1% or more, the percentage of patients was 74.5% and 55.7%, respectively (hazard ratio, 0.55; 98.72% CI, 0.35 to 0.85; P<0.001). The median survival free from recurrence outside the urothelial tract in the intention-to-treat population was 22.9 months (95% CI, 19.2 to 33.4) with nivolumab and 13.7 months (95% CI, 8.4 to 20.3) with placebo. The percentage of patients who were alive and free from recurrence outside the urothelial tract at 6 months was 77.0% with nivolumab and 62.7% with placebo (hazard ratio for recurrence outside the urothelial tract or death, 0.72; 95% CI, 0.59 to 0.89). Among patients with a PD-L1 expression level of 1% or more, the percentage of patients was 75.3% and 56.7%, respectively (hazard ratio, 0.55; 95% CI, 0.39 to 0.79). Treatment-related adverse events of grade 3 or higher occurred in 17.9% of the nivolumab group and 7.2% of the placebo group. Two treatment-related deaths due to pneumonitis were noted in the nivolumab group. CONCLUSIONS: In this trial involving patients with high-risk muscle-invasive urothelial carcinoma who had undergone radical surgery, disease-free survival was longer with adjuvant nivolumab than with placebo in the intention-to-treat population and among patients with a PD-L1 expression level of 1% or more. (Funded by Bristol Myers Squibb and Ono Pharmaceutical; CheckMate 274 ClinicalTrials.gov number, NCT02632409.).


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Carcinoma de Células de Transição/tratamento farmacológico , Nivolumabe/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/efeitos adversos , Antígeno B7-H1/metabolismo , Carcinoma de Células de Transição/patologia , Carcinoma de Células de Transição/cirurgia , Quimioterapia Adjuvante , Intervalo Livre de Doença , Método Duplo-Cego , Feminino , Humanos , Análise de Intenção de Tratamento , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Nivolumabe/efeitos adversos , Placebos/uso terapêutico , Qualidade de Vida , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgia
3.
Future Oncol ; 19(6): 413-426, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36919916

RESUMO

WHAT IS THIS SUMMARY ABOUT?: This is a summary of a paper published in a medical journal that describes the results of a study called CheckMate 274. This study looked at a new treatment for muscle-invasive urothelial cancer, a type of cancer found in the urinary tract that has spread from the inner lining of the urinary tract or bladder and into the surrounding muscle wall where it can then spread to other parts of the body. The standard treatment for muscle-invasive urothelial cancer is surgery to remove affected parts of the urinary tract. However, cancer returns in more than half of people after this surgery. Adjuvant therapy is given to people after surgery with muscle-invasive urothelial cancer with a goal to reduce the risk of the cancer coming back; however, at the time this study started, there was no standard adjuvant treatment. WHAT HAPPENED IN THE STUDY?: In the CheckMate 274 study, researchers compared nivolumab with a placebo as an adjuvant treatment for people with muscle-invasive urothelial cancer. The aim of the study was to understand how well nivolumab worked to reduce the chance of the cancer returning after surgery. The study also looked at what side effects (unwanted or unexpected results or conditions that are possibly related to the use of a medication) people had with treatment. WHAT DO THE RESULTS MEAN?: The results showed that people who received nivolumab versus placebo: Survived longer before the cancer was detected again, including people who had programmed death ligand-1 (shortened to PD-L1) on their cancer cells. Survived longer before a secondary cancer outside of the urinary tract was detected. Experienced no differences in health-related quality of life (the impact of the treatment on a person's mental and physical health). Had similar side effects to the people who received nivolumab in other studies. Clinical Trial Registration: NCT02632409 (ClinicalTrials.gov).


Assuntos
Neoplasias Musculares , Neoplasias da Bexiga Urinária , Humanos , Nivolumabe/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Qualidade de Vida , Imunoterapia/métodos , Músculos , Neoplasias Musculares/tratamento farmacológico
4.
Jpn J Clin Oncol ; 53(1): 16-25, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36300304

RESUMO

BACKGROUND: The phase 3 CheckMate 274 trial demonstrated superiority of adjuvant nivolumab over placebo after radical surgery in patients with high-risk muscle-invasive urothelial carcinoma. However, the efficacy and safety of adjuvant nivolumab in Japanese patients with muscle-invasive urothelial carcinoma have not been clarified. METHODS: Patients with muscle-invasive urothelial carcinoma were randomized to adjuvant nivolumab 240 mg or placebo (every 2 weeks via intravenous infusion) up to 120 days after radical surgery in CheckMate 274. RESULTS: Of 49 patients in the Japanese subgroup, 27 and 22 patients were randomized to nivolumab and placebo, respectively. Eleven and 8 patients, respectively, had tumor PD-L1 expression level of 1% or more. The median disease-free survival times in the nivolumab and placebo groups were 29.67 months (95% confidence interval 7.79-not reached) and 9.72 months (95% confidence interval 4.73-not reached), respectively (hazard ratio 0.77, 95% confidence interval 0.35-1.69). The corresponding values in patients with tumor PD-L1 expression level of 1% or more were 29.67 months (95% confidence interval 2.63-not reached) and 25.95 months (95% confidence interval 5.59-not reached) (hazard ratio 1.10, 95% confidence interval 0.31-3.92), respectively. Treatment-related adverse events of Grade 3-4 occurred in 25.9 and 13.6% of patients in the nivolumab and placebo groups, respectively. The most common treatment-related adverse events in the nivolumab group were lipase increased, amylase increased and diarrhea. The changes in quality of life scores from baseline over time were similar in both groups. CONCLUSIONS: The efficacy and safety results in the Japanese subgroup were consistent with the overall population of CheckMate 274.


Assuntos
Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Humanos , Nivolumabe/efeitos adversos , Antígeno B7-H1 , Carcinoma de Células de Transição/tratamento farmacológico , Carcinoma de Células de Transição/cirurgia , Qualidade de Vida , População do Leste Asiático , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/cirurgia , Músculos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico
5.
Nature ; 546(7656): 168-172, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28538732

RESUMO

Metabolic reprogramming by oncogenic signals promotes cancer initiation and progression. The oncogene KRAS and tumour suppressor STK11, which encodes the kinase LKB1, regulate metabolism and are frequently mutated in non-small-cell lung cancer (NSCLC). Concurrent occurrence of oncogenic KRAS and loss of LKB1 (KL) in cells specifies aggressive oncological behaviour. Here we show that human KL cells and tumours share metabolomic signatures of perturbed nitrogen handling. KL cells express the urea cycle enzyme carbamoyl phosphate synthetase-1 (CPS1), which produces carbamoyl phosphate in the mitochondria from ammonia and bicarbonate, initiating nitrogen disposal. Transcription of CPS1 is suppressed by LKB1 through AMPK, and CPS1 expression correlates inversely with LKB1 in human NSCLC. Silencing CPS1 in KL cells induces cell death and reduces tumour growth. Notably, cell death results from pyrimidine depletion rather than ammonia toxicity, as CPS1 enables an unconventional pathway of nitrogen flow from ammonia into pyrimidines. CPS1 loss reduces the pyrimidine to purine ratio, compromises S-phase progression and induces DNA-polymerase stalling and DNA damage. Exogenous pyrimidines reverse DNA damage and rescue growth. The data indicate that the KL oncological genotype imposes a metabolic vulnerability related to a dependence on a cross-compartmental pathway of pyrimidine metabolism in an aggressive subset of NSCLC.


Assuntos
Carbamoil-Fosfato Sintase (Amônia)/metabolismo , DNA/biossíntese , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirimidinas/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/metabolismo , Amônia/metabolismo , Animais , Bicarbonatos/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/deficiência , Carbamoil-Fosfato Sintase (Amônia)/genética , Carbamoil-Fosfato/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Morte Celular , Proliferação de Células , Dano ao DNA/efeitos dos fármacos , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Inativação Gênica , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Masculino , Metabolômica , Camundongos , Mitocôndrias/metabolismo , Nitrogênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Purinas/metabolismo , Pirimidinas/farmacologia , Fase S , Transcrição Gênica , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Eur Urol ; 83(5): 432-440, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36868932

RESUMO

BACKGROUND: The CheckMate 274 trial demonstrated improved disease-free survival (DFS) with adjuvant nivolumab versus placebo in patients with muscle-invasive urothelial carcinoma at high risk of recurrence after radical surgery in both the intent-to-treat population and the subset with tumor programmed death ligand 1 (PD-L1) expression ≥1%. OBJECTIVE: To analyze DFS by combined positive score (CPS), which is based on PD-L1 expression in both tumor and immune cells. DESIGN, SETTING, AND PARTICIPANTS: We randomized a total of 709 patients 1:1 to nivolumab 240 mg or placebo every 2 wk intravenously for ≤1 yr of adjuvant treatment. INTERVENTION: Nivolumab 240 mg. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Primary endpoints were DFS in the intent-to-treat population and patients with tumor PD-L1 expression ≥1% using the tumor cell (TC) score. CPS was determined retrospectively from previously stained slides. Tumor samples with both quantifiable CPS and TC were analyzed. RESULTS AND LIMITATIONS: Of 629 patients evaluable for CPS and TC, 557 (89%) had CPS ≥1, 72 (11%) had CPS <1, 249 (40%) had TC ≥1%, and 380 (60%) had TC <1%. Among patients with TC <1%, 81% (n = 309) had CPS ≥1. DFS was improved with nivolumab versus placebo for patients with TC ≥1% (hazard ratio [HR] 0.50, 95% confidence interval [CI] 0.35-0.71), those with CPS ≥1 (HR 0.62, 95% CI 0.49-0.78), and patients with both TC <1% and CPS ≥1 (HR 0.73, 95% CI 0.54-0.99). CONCLUSION: More patients had CPS ≥1 than TC ≥1%, and most patients who had TC <1% had CPS ≥1. In addition, patients with CPS ≥1 experienced improved DFS with nivolumab. These results may, in part, explain the mechanisms underlying a benefit with adjuvant nivolumab even in patients who had both TC <1% and CPS ≥1. PATIENT SUMMARY: We studied survival time without cancer recurrence (disease-free survival; DFS) for patients treated with nivolumab versus placebo after surgery to remove the bladder or components of the urinary tract for bladder cancer in the CheckMate 274 trial. We assessed the impact of levels of the protein PD-L1 expressed either on tumor cells (tumor cell score; TC) or on both tumor cells and immune cells surrounding the tumor (combined positive score; CPS). DFS was impoved with nivolumab versus placebo for patients with TC ≥1%, CPS ≥1, and for patients with both TC <1% and CPS ≥1. This analysis may help physicians understand which patients would benefit most from treatment with nivolumab.


Assuntos
Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Humanos , Nivolumabe , Antígeno B7-H1/metabolismo , Intervalo Livre de Doença , Neoplasias da Bexiga Urinária/tratamento farmacológico , Carcinoma de Células de Transição/tratamento farmacológico , Estudos Retrospectivos , Recidiva Local de Neoplasia/tratamento farmacológico , Músculos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico
7.
Cancers (Basel) ; 14(17)2022 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-36077617

RESUMO

This phase 1/2a, open-label study (NCT02419417) evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics of BMS-986158, a selective bromodomain and extraterminal domain (BET) inhibitor. Dose escalation was performed with 3 BMS-986158 dosing schedules: A (5 days on, 2 days off; range, 0.75-4.5 mg), B (14 days on, 7 days off; 2.0-3.0 mg), and C (7 days on, 14 days off; 2.0-4.5 mg). Eighty-three patients were enrolled and received ≥1 BMS-986158 dose. Diarrhea (43%) and thrombocytopenia (39%) were the most common treatment-related adverse events (TRAEs). A lower incidence of TRAEs was found with schedules A (72%) and C (72%) vs. B (100%). Stable disease was achieved in 12 (26.1%), 3 (37.5%), and 9 (31.0%) patients on schedules A, B, and C, respectively. Two patients on schedule A with a 4.5-mg starting dose (ovarian cancer, n = 1; nuclear protein in testis [NUT] carcinoma, n = 1) experienced a partial response. BMS-986158 demonstrated rapid-to-moderate absorption (median time to maximum observed plasma concentration, 1-4 h). As expected with an epigenetic modifier, expression changes in select BET-regulated genes occurred with BMS-986158 treatment. Schedule A dosing (5 days on, 2 days off) yielded tolerable safety, preliminary antitumor activity, and a dose-proportional PK profile.

8.
Eur J Cancer ; 160: 61-71, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34802864

RESUMO

BACKGROUND: Docetaxel has immunostimulatory effects that may promote an immunoresponsive prostate tumour microenvironment, providing a rationale for combination with nivolumab (programmed death-1 inhibitor) for metastatic castration-resistant prostate cancer (mCRPC). METHODS: In the non-randomised, multicohort, global phase II CheckMate 9KD trial, 84 patients with chemotherapy-naive mCRPC, ongoing androgen deprivation therapy and ≤2 prior novel hormonal therapies (NHTs) received nivolumab 360 mg and docetaxel 75 mg/m2 every 3 weeks with prednisone 5 mg twice daily (≤10 cycles) and then nivolumab 480 mg every 4 weeks (≤2 years). The co-primary end-points were objective response rate (ORR) and prostate-specific antigen response rate (PSA50-RR; ≥50% decrease from baseline). RESULTS: The confirmed ORR (95% confidence interval [CI]) was 40.0% (25.7-55.7), and the confirmed PSA50-RR (95% CI) was 46.9% (35.7-58.3). The median (95% CI) radiographic progression-free survival (rPFS) and overall survival (OS) were 9.0 (8.0-11.6) and 18.2 (14.6-20.7) months, respectively. In subpopulations with versus without prior NHT, the ORR was 38.7% versus 42.9%, the PSA50-RR was 39.6% versus 60.7%, the median rPFS was 8.5 versus 12.0 months and the median OS was 16.2 months versus not reached. Homologous recombination deficiency status or tumour mutational burden did not appear to impact efficacy. The most common any-grade and grade 3-4 treatment-related adverse events were fatigue (39.3%) and neutropenia (16.7%), respectively. Three treatment-related deaths occurred (1 pneumonitis related to nivolumab; 2 pneumonias related to docetaxel). CONCLUSIONS: Nivolumab plus docetaxel has clinical activity in patients with chemotherapy-naïve mCRPC. Safety was consistent with the individual components. These results support further investigation in the ongoing phase III CheckMate 7DX trial. CLINICALTRIALS. GOV REGISTRATION: NCT03338790.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Docetaxel/uso terapêutico , Nivolumabe/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Estudos de Coortes , Docetaxel/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Nivolumabe/farmacologia
9.
J Immunother Cancer ; 10(8)2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35977756

RESUMO

BACKGROUND: CheckMate 9KD (NCT03338790) is a non-randomized, multicohort, phase 2 trial of nivolumab plus other anticancer treatments for metastatic castration-resistant prostate cancer (mCRPC). We report results from cohorts A1 and A2 of CheckMate 9KD, specifically evaluating nivolumab plus rucaparib. METHODS: CheckMate 9KD enrolled adult patients with histologically confirmed mCRPC, ongoing androgen deprivation therapy, and an Eastern Cooperative Oncology Group performance status of 0-1. Cohort A1 included patients with postchemotherapy mCRPC (1-2 prior taxane-based regimens) and ≤2 prior novel hormonal therapies (eg, abiraterone, enzalutamide, apalutamide); cohort A2 included patients with chemotherapy-naïve mCRPC and prior novel hormonal therapy. Patients received nivolumab 480 mg every 4 weeks plus rucaparib 600 mg two times per day (nivolumab dosing ≤2 years). Coprimary endpoints were objective response rate (ORR) per Prostate Cancer Clinical Trials Working Group 3 and prostate-specific antigen response rate (PSA50-RR; ≥50% PSA reduction) in all-treated patients and patients with homologous recombination deficiency (HRD)-positive tumors, determined before enrollment. Secondary endpoints included radiographic progression-free survival (rPFS), overall survival (OS), and safety. RESULTS: Outcomes (95% CI) among all-treated, HRD-positive, and BRCA1/2-positive populations for cohort A1 were confirmed ORR: 10.3% (3.9-21.2) (n=58), 17.2% (5.8-35.8) (n=29), and 33.3% (7.5-70.1) (n=9); confirmed PSA50-RR: 11.9% (5.9-20.8) (n=84), 18.2% (8.2-32.7) (n=44), and 41.7% (15.2-72.3) (n=12); median rPFS: 4.9 (3.7-5.7) (n=88), 5.8 (3.7-8.4) (n=45), and 5.6 (2.8-15.7) (n=12) months; and median OS: 13.9 (10.4-15.8) (n=88), 15.4 (11.4-18.2) (n=45), and 15.2 (3.0-not estimable) (n=12) months. For cohort A2 they were confirmed ORR: 15.4% (5.9-30.5) (n=39), 25.0% (8.7-49.1) (n=20), and 33.3% (7.5-70.1) (n=9); confirmed PSA50-RR: 27.3% (17.0-39.6) (n=66), 41.9 (24.5-60.9) (n=31), and 84.6% (54.6-98.1) (n=13); median rPFS: 8.1 (5.6-10.9) (n=71), 10.9 (6.7-12.0) (n=34), and 10.9 (5.6-12.0) (n=15) months; and median OS: 20.2 (14.1-22.8) (n=71), 22.7 (14.1-not estimable) (n=34), and 20.2 (11.1-not estimable) (n=15) months. In cohorts A1 and A2, respectively, the most common any-grade and grade 3-4 treatment-related adverse events (TRAEs) were nausea (40.9% and 40.8%) and anemia (20.5% and 14.1%). Discontinuation rates due to TRAEs were 27.3% and 23.9%, respectively. CONCLUSIONS: Nivolumab plus rucaparib is active in patients with HRD-positive postchemotherapy or chemotherapy-naïve mCRPC, particularly those harboring BRCA1/2 mutations. Safety was as expected, with no new signals identified. Whether the addition of nivolumab incrementally improves outcomes versus rucaparib alone cannot be determined from this trial. TRIAL REGISTRATION NUMBER: NCT03338790.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias de Próstata Resistentes à Castração , Adulto , Antagonistas de Androgênios/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Humanos , Indóis , Masculino , Nivolumabe/uso terapêutico , Antígeno Prostático Específico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia
10.
J Biol Chem ; 285(22): 16562-71, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20233725

RESUMO

Mammalian Timeless is a multifunctional protein that performs essential roles in the circadian clock, chromosome cohesion, DNA replication fork protection, and DNA replication/DNA damage checkpoint pathways. The human Timeless exists in a tight complex with a smaller protein called Tipin (Timeless-interacting protein). Here we investigated the mechanism by which the Timeless-Tipin complex functions as a mediator in the ATR-Chk1 DNA damage checkpoint pathway. We find that the Timeless-Tipin complex specifically mediates Chk1 phosphorylation by ATR in response to DNA damage and replication stress through interaction of Tipin with the 34-kDa subunit of replication protein A (RPA). The Tipin-RPA interaction stabilizes Timeless-Tipin and Tipin-Claspin complexes on RPA-coated ssDNA and in doing so promotes Claspin-mediated phosphorylation of Chk1 by ATR. Our results therefore indicate that RPA-covered ssDNA not only supports recruitment and activation of ATR but also, through Tipin and Claspin, it plays an important role in the action of ATR on its critical downstream target Chk1.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína de Replicação A/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte/genética , Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Quinase 1 do Ponto de Checagem , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA , Dimerização , Células HeLa , Humanos , Modelos Biológicos , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Frações Subcelulares/metabolismo
11.
Clin Cancer Res ; 27(15): 4287-4300, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33837006

RESUMO

PURPOSE: To define dominant molecular and cellular features associated with PD-1/PD-L1 blockade resistance in metastatic urothelial cancer. EXPERIMENTAL DESIGN: We pursued an unbiased approach using bulk RNA sequencing data from two clinical trials to discover (IMvigor 210) and validate (CheckMate 275) pretreatment molecular features associated with resistance to PD-1/PD-L1 blockade in metastatic urothelial cancer. We then generated single-cell RNA sequencing (scRNA-seq) data from muscle-invasive bladder cancer specimens to dissect the cellular composition underlying the identified gene signatures. RESULTS: We identified an adaptive immune response gene signature associated with response and a protumorigenic inflammation gene signature associated with resistance to PD-1/PD-L1 blockade. The adaptive immune response:protumorigenic inflammation signature expression ratio, coined the 2IR score, best correlated with clinical outcomes, and was externally validated. Mapping these bulk gene signatures onto scRNA-seq data uncovered their underlying cellular diversity, with prominent expression of the protumorigenic inflammation signature by myeloid phagocytic cells. However, heterogeneity in expression of adaptive immune and protumorigenic inflammation genes was observed among single myeloid phagocytic cells, quantified as the myeloid single cell immune:protumorigenic inflammation ratio (Msc2IR) score. Single myeloid phagocytic cells with low Msc2IR scores demonstrated upregulation of proinflammatory cytokines/chemokines and downregulation of antigen presentation genes, were unrelated to M1 versus M2 polarization, and were enriched in pretreatment blood samples from patients with PD-L1 blockade-resistant metastatic urothelial cancer. CONCLUSIONS: The balance of adaptive immunity and protumorigenic inflammation in individual tumor microenvironments is associated with PD-1/PD-L1 resistance in urothelial cancer with the latter linked to a proinflammatory cellular state of myeloid phagocytic cells detectable in tumor and blood.See related commentary by Drake, p. 4139.


Assuntos
Carcinoma de Células de Transição/tratamento farmacológico , Carcinoma de Células de Transição/genética , Inibidores de Checkpoint Imunológico/uso terapêutico , Células Mieloides/fisiologia , Análise de Sequência de RNA , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Resistencia a Medicamentos Antineoplásicos , Humanos
12.
Mol Cell Biol ; 27(8): 3131-42, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17296725

RESUMO

UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m(2) UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/efeitos da radiação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fase S/efeitos da radiação , Raios Ultravioleta , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Ciclo Celular/genética , Quinase 1 do Ponto de Checagem , DNA/metabolismo , Proteínas de Ligação a DNA , Regulação para Baixo/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Células HeLa , Humanos , Idoxuridina , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Ligação Proteica/efeitos da radiação , Proteínas Quinases/metabolismo , Proteína de Replicação A/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/química , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo
13.
Nat Metab ; 2(12): 1401-1412, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33257855

RESUMO

In non-small-cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and the tumour suppressor STK11 (also known as LKB1) encoding the kinase LKB1 result in aggressive tumours prone to metastasis but with liabilities arising from reprogrammed metabolism. We previously demonstrated perturbed nitrogen metabolism and addiction to an unconventional pathway of pyrimidine synthesis in KRAS/LKB1 co-mutant cancer cells. To gain broader insight into metabolic reprogramming in NSCLC, we analysed tumour metabolomes in a series of genetically engineered mouse models with oncogenic KRAS combined with mutations in LKB1 or p53. Metabolomics and gene expression profiling pointed towards activation of the hexosamine biosynthesis pathway (HBP), another nitrogen-related metabolic pathway, in both mouse and human KRAS/LKB1 co-mutant tumours. KRAS/LKB1 co-mutant cells contain high levels of HBP metabolites, higher flux through the HBP pathway and elevated dependence on the HBP enzyme glutamine-fructose-6-phosphate transaminase [isomerizing] 2 (GFPT2). GFPT2 inhibition selectively reduced KRAS/LKB1 co-mutant tumour cell growth in culture, xenografts and genetically modified mice. Our results define a new metabolic vulnerability in KRAS/LKB1 co-mutant tumours and provide a rationale for targeting GFPT2 in this aggressive NSCLC subtype.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Hexosaminas/biossíntese , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Redes e Vias Metabólicas , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinases Proteína-Quinases Ativadas por AMP , Animais , Azasserina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/antagonistas & inibidores , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Humanos , Neoplasias Pulmonares/mortalidade , Metabolômica , Camundongos , Mutação , Análise de Sobrevida , Ensaio Tumoral de Célula-Tronco
15.
Mol Cell Biol ; 25(8): 3109-16, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15798197

RESUMO

The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. We have found that the human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein.


Assuntos
Ciclo Celular/fisiologia , Ritmo Circadiano/fisiologia , Dano ao DNA , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Quinase 1 do Ponto de Checagem , Montagem e Desmontagem da Cromatina/fisiologia , Criptocromos , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA , Regulação para Baixo/efeitos dos fármacos , Exodesoxirribonucleases/metabolismo , Flavoproteínas/metabolismo , Humanos , Hidroxiureia/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
Mol Cell Biol ; 24(3): 1292-300, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14729973

RESUMO

ATR is an essential protein that functions as a damage sensor and a proximal kinase in the DNA damage checkpoint response in mammalian cells. It is a member of the phosphoinositide 3-kinase-like kinase (PIKK) family, which includes ATM, ATR, and DNA-dependent protein kinase. Recently, it was found that ATM is an oligomeric protein that is converted to an active monomeric form by phosphorylation in trans upon DNA damage, and this raised the possibility that other members of the PIKK family may be regulated in a similar manner. Here we show that ATR is a monomeric protein associated with a smaller protein called ATRIP with moderate affinity. The ATR protein by itself or in the form of the ATR-ATRIP heterodimer binds to naked or replication protein A (RPA)-covered DNAs with comparable affinities. However, the phosphorylation of RPA by ATR is dependent on single-stranded DNA and is stimulated by ATRIP. These findings suggest that the regulation and mechanism of action of ATR are fundamentally different from those of the other PIKK proteins.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Exodesoxirribonucleases , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/efeitos da radiação , Humanos , Peso Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Proteína de Replicação A , Raios Ultravioleta
17.
Cell Rep ; 19(13): 2665-2680, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28658616

RESUMO

Pharmacologic agents that interfere with nucleotide metabolism constitute an important class of anticancer agents. Recent studies have demonstrated that mTOR complex 1 (mTORC1) inhibitors suppress de novo biosynthesis of pyrimidine and purine nucleotides. Here, we demonstrate that mTORC1 itself is suppressed by drugs that reduce intracellular purine nucleotide pools. Cellular treatment with AG2037, an inhibitor of the purine biosynthetic enzyme GARFT, profoundly inhibits mTORC1 activity via a reduction in the level of GTP-bound Rheb, an obligate upstream activator of mTORC1, because of a reduction in intracellular guanine nucleotides. AG2037 treatment provokes both mTORC1 inhibition and robust tumor growth suppression in mice bearing non-small-cell lung cancer (NSCLC) xenografts. These results indicate that alterations in purine nucleotide availability affect mTORC1 activity and suggest that inhibition of mTORC1 contributes to the therapeutic effects of purine biosynthesis inhibitors.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Nucleotídeos de Purina/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Nucleotídeos de Purina/biossíntese
18.
Biosci Rep ; 34(2)2014 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-27919031

RESUMO

The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1-3 follows a sequential ordered Bi-Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in kcat for PKN1 and PKN2, and a decrease in peptide KM for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (Ki<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors.


Assuntos
Trifosfato de Adenosina/química , Ácido Araquidônico/química , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Inibidores de Proteínas Quinases/química , Catálise , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Cinética
19.
Cancer Res ; 74(3): 908-20, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24285728

RESUMO

Myc oncoproteins induce genes driving aerobic glycolysis, including lactate dehydrogenase-A that generates lactate. Here, we report that Myc controls transcription of the lactate transporter SLC16A1/MCT1 and that elevated MCT1 levels are manifest in premalignant and neoplastic Eµ-Myc transgenic B cells and in human malignancies with MYC or MYCN involvement. Notably, disrupting MCT1 function leads to an accumulation of intracellular lactate that rapidly disables tumor cell growth and glycolysis, provoking marked alterations in glycolytic intermediates, reductions in glucose transport, and in levels of ATP, NADPH, and ultimately, glutathione (GSH). Reductions in GSH then lead to increases in hydrogen peroxide, mitochondrial damage, and ultimately, cell death. Finally, forcing glycolysis by metformin treatment augments this response and the efficacy of MCT1 inhibitors, suggesting an attractive combination therapy for MYC/MCT1-expressing malignancies.


Assuntos
Glutationa/biossíntese , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Simportadores/genética , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Glicólise/genética , Homeostase/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Metformina/farmacologia , Camundongos , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Oxirredução , Fosforilação Oxidativa/efeitos dos fármacos , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Transcrição Gênica
20.
Mol Oncol ; 6(3): 284-98, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22217540

RESUMO

PKN3 is an AGC-family protein kinase implicated in growth of metastatic prostate cancer cells with phosphoinositide 3-kinase pathway deregulation. The molecular mechanism, however, by which PKN3 contributes to malignant growth and tumorigenesis is not well understood. Using orthotopic mouse tumor models, we now show that inducible knockdown of PKN3 protein not only blocks metastasis, but also impairs primary prostate and breast tumor growth. Correspondingly, overexpression of exogenous PKN3 in breast cancer cells further increases their malignant behavior and invasiveness in-vitro. Mechanistically, we demonstrate that PKN3 physically interacts with Rho-family GTPases, and preferentially with RhoC, a known mediator of tumor invasion and metastasis in epithelial cancers. Likewise, RhoC predominantly associates with PKN3 compared to its closely related PKN family members. Unlike the majority of Rho GTPases and PKN molecules, which are ubiquitously expressed, both PKN3 and RhoC show limited expression in normal tissues and become upregulated in late-stage malignancies. Since PKN3 catalytic activity is increased in the presence of Rho GTPases, the co-expression and preferential interaction of PKN3 and RhoC in tumor cells are functionally relevant. Our findings provide novel insight into the regulation and function of PKN3 and suggest that the PKN3-RhoC complex represents an attractive therapeutic target in late-stage malignancies.


Assuntos
Neoplasias da Próstata/metabolismo , Proteína Quinase C/metabolismo , Proteínas ras/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Doxiciclina/uso terapêutico , Imunofluorescência , Imunoprecipitação , Masculino , Camundongos , Metástase Neoplásica/genética , Neoplasias da Próstata/patologia , Ligação Proteica , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , RNA Interferente Pequeno , Proteínas ras/genética , Proteína de Ligação a GTP rhoC
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