RESUMO
Control of the G1/S phase transition by the Retinoblastoma (RB) tumor suppressor is critical for the proliferation of normal cells in tissues, and its inactivation is one of the most fundamental events leading to cancer. Cyclin-dependent kinase (CDK) phosphorylation inactivates RB to promote cell cycle-regulated gene expression. Here we show that, upon stress, the p38 stress-activated protein kinase (SAPK) maximizes cell survival by downregulating E2F gene expression through the targeting of RB. RB undergoes selective phosphorylation by p38 in its N terminus; these phosphorylations render RB insensitive to the inactivation by CDKs. p38 phosphorylation of RB increases its affinity toward the E2F transcription factor, represses gene expression, and delays cell-cycle progression. Remarkably, introduction of a RB phosphomimetic mutant in cancer cells reduces colony formation and decreases their proliferative and tumorigenic potential in mice.
Assuntos
Neoplasias da Mama/genética , Quinases Ciclina-Dependentes/genética , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica , Proteína do Retinoblastoma/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Quinases Ciclina-Dependentes/metabolismo , Fatores de Transcrição E2F/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Camundongos , Mimetismo Molecular , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína do Retinoblastoma/química , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date. Using a genome-wide CRISPR/Cas9 screen, we identified LRRC8A, which encodes a chloride channel subunit, as the gene most associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl- efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na+/K+/2Cl- cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions.
Assuntos
Tamanho Celular , Canais de Cloreto/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transporte Biológico , Sistemas CRISPR-Cas , Morte Celular , Sobrevivência Celular , Células HeLa , Humanos , Pressão Osmótica , Fosforilação , Potássio/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sódio/metabolismoRESUMO
Nuclear architecture is decisive for the assembly of transcriptional responses. However, how chromosome organization is dynamically modulated to permit rapid and transient transcriptional changes in response to environmental challenges remains unclear. Here we show that hyperosmotic stress disrupts different levels of chromosome organization, ranging from A/B compartment changes to reduction in the number and insulation of topologically associating domains (TADs). Concomitantly, transcription is greatly affected, TAD borders weaken, and RNA Polymerase II runs off from hundreds of transcription end sites. Stress alters the binding profiles of architectural proteins, which explains the disappearance of local chromatin organization. These processes are dynamic, and cells rapidly reconstitute their default chromatin conformation after stress removal, uncovering an intrinsic organization. Transcription is not required for local chromatin reorganization, while compartment recovery is partially transcription-dependent. Thus, nuclear organization in mammalian cells can be rapidly modulated by environmental changes in a reversible manner.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Pressão Osmótica , RNA Polimerase II/metabolismo , Transcrição Gênica , Linhagem Celular , HumanosRESUMO
AIMS: Familial partial lipodystrophic syndrome 3 (FPLD3) is associated with mutations in the transcription factor PPARγ. One of these mutations, the P467L, confers a dominant negative effect. We and others have previously investigated the pathophysiology associated with this mutation using a humanized mouse model that recapitulates most of the clinical symptoms observed in patients who have been phenotyped under different experimental conditions. One of the key clinical manifestations observed, both in humans and mouse models, is the ectopic accumulation of fat in the liver. With this study we aim to dissect the molecular mechanisms that contribute to the excessive accumulation of lipids in the liver and characterize the negative effect of this PPARγ mutation on the activity of PPARα in vivo when activated by fibrates. MATERIAL AND METHODS: P465L-PPAR mutant and wild-type mice were divided into 8 experimental groups, 4 different conditions per genotype. Briefly, mice were fed a chow diet or a high-fat diet (HFD 45% Kcal from fat) for a period of 28 days and treated with WY14643 or vehicle for five days before culling. At the end of the experiment, tissues and plasma were collected. We performed extensive gene expression, fatty acid composition and histological analysis in the livers. The serum collected was used to measure several metabolites and to perform basic lipoprotein profile. RESULTS: P465L mice showed increased levels of insulin and free fatty acids (FFA) as well as increased liver steatosis. They also exhibit decreased levels of very low density lipoproteins (VLDL) when fed an HFD. We also provide evidence of impaired expression of a number of well-established PPARα target genes in the P465L mutant livers. CONCLUSION: Our data demonstrate that P465L confers partial resistance to the hypolipidemic action of fibrates. These results show that the fatty liver phenotype observed in P465L mutant mice is not only the consequence of dysfunctional adipose tissue, but also involves defective liver metabolism. All in all, the deleterious effects of P465L-PPARγ mutation may be magnified by their collateral negative effect on PPARα function.
Assuntos
Resistência a Medicamentos/genética , Fígado Gorduroso/tratamento farmacológico , Ácidos Fíbricos/uso terapêutico , Hipolipemiantes/uso terapêutico , Mutação de Sentido Incorreto , PPAR gama/genética , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Fígado Gorduroso/sangue , Fígado Gorduroso/genética , Hiperlipidemias/sangue , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/genética , Leucina/genética , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto/fisiologia , Prolina/genéticaRESUMO
The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2(as) phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1-a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling-dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR-sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Dano ao DNA/efeitos da radiação , Proteínas Nucleares/metabolismo , Radiação Ionizante , Hidrolases Anidrido Ácido , Ciclo Celular , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11 , FosforilaçãoRESUMO
Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis are the main therapeutic option for patients with advanced non-small cell lung cancer (NSCLC) without a druggable oncogenic alteration. Nevertheless, only a portion of patients benefit from this type of treatment. Here, we assessed the value of shallow whole-genome sequencing (sWGS) on plasma samples to monitor ICI benefit. We applied sWGS on cell-free DNA (cfDNA) extracted from plasma samples of 45 patients with metastatic NSCLC treated with ICIs. Over 150 samples were obtained before ICI treatment initiation and at several time points throughout treatment. From sWGS data, we computed the tumor fraction (TFx) and somatic copy number alteration (SCNA) burden and associated them with ICI benefit and clinical features. TFx at baseline correlated with metastatic lesions at the bone and the liver, and high TFx (≥ 10%) associated with ICI benefit. Moreover, its assessment in on-treatment samples was able to better predict clinical efficacy, regardless of the TFx levels at baseline. Finally, for a subset of patients for whom SCNA burden could be computed, increased burden correlated with diminished benefit following ICI treatment. Thus, our data indicate that the analysis of cfDNA by sWGS enables the monitoring of two potential biomarkers-TFx and SCNA burden-of ICI benefit in a cost-effective manner, facilitating multiple serial-sample analyses. Larger cohorts will be needed to establish its clinical potential.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , DNA Tumoral Circulante/genética , Biomarcadores Tumorais/genética , Resultado do Tratamento , Antígeno B7-H1RESUMO
MPM is an aggressive disease with an immunosuppressive tumor microenvironment, and interest in exploring immunotherapy in this disease has been increasing. In the first line of treatment, the combination of nivolumab and ipilimumab demonstrated an improvement in survival over chemotherapy. The presence of TILs has been recognized as a marker of antitumor immune response to chemotherapy in solid tumors. The aim of our study is to identify the effect of treatment on immune cells and the immune gene profile in MPM. We investigated the changes in expression of TILs in 10 human MPM paired tumor tissues using immunohistochemistry and gene expression analysis from paired untreated and treated samples. In this small series, we demonstrated that during the evolution of disease without any treatment there was an increase in the inflammatory component in tumor samples. After systemic treatment there was a decrease in the number of TILs. We observed that after systemic treatment or disease progression immune gene signatures were suppressed. Our integrated analysis of paired samples with immune profile and genomic changes on MPM suggested that during the evolution of the disease the immune system tends to switch, turning off with treatment.
RESUMO
Immunotherapy resistance in non-small cell lung cancer (NSCLC) may be mediated by an immunosuppressive microenvironment, which can be shaped by the mutational landscape of the tumor. Here, we observed genetic alterations in the PTEN/PI3K/AKT/mTOR pathway and/or loss of PTEN expression in >25% of patients with NSCLC, with higher frequency in lung squamous carcinomas (LUSC). Patients with PTEN-low tumors had higher levels of PD-L1 and PD-L2 and showed worse progression-free survival when treated with immunotherapy. Development of a Pten-null LUSC mouse model revealed that tumors with PTEN loss were refractory to antiprogrammed cell death protein 1 (anti-PD-1), highly metastatic and fibrotic, and secreted TGFß/CXCL10 to promote conversion of CD4+ lymphocytes into regulatory T cells (Treg). Human and mouse PTEN-low tumors were enriched in Tregs and expressed higher levels of immunosuppressive genes. Importantly, treatment of mice bearing Pten-null tumors with TLR agonists and anti-TGFß antibody aimed to alter this immunosuppressive microenvironment and led to tumor rejection and immunologic memory in 100% of mice. These results demonstrate that lack of PTEN causes immunotherapy resistance in LUSCs by establishing an immunosuppressive tumor microenvironment that can be reversed therapeutically. SIGNIFICANCE: PTEN loss leads to the development of an immunosuppressive microenvironment in lung cancer that confers resistance to anti-PD-1 therapy, which can be overcome by targeting PTEN loss-mediated immunosuppression.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares , PTEN Fosfo-Hidrolase , Linfócitos T Reguladores , Animais , Humanos , Camundongos , Antígeno B7-H1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Imunoterapia/métodos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Microambiente Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêuticoRESUMO
Although immunotherapy (IO) has changed the paradigm for the treatment of patients with advanced non-small cell lung cancers (aNSCLC), only around 30% to 50% of treated patients experience a long-term benefit from IO. Furthermore, the identification of the 30 to 50% of patients who respond remains a major challenge, as programmed Death-Ligand 1 (PD-L1) is currently the only biomarker used to predict the outcome of IO in NSCLC patients despite its limited efficacy. Considering the dynamic complexity of the immune system-tumor microenvironment (TME) and its interaction with the host's and patient's behavior, it is unlikely that a single biomarker will accurately predict a patient's outcomes. In this scenario, Artificial Intelligence (AI) and Machine Learning (ML) are becoming essential to the development of powerful decision-making tools that are able to deal with this high-complexity and provide individualized predictions to better match treatments to individual patients and thus improve patient outcomes and reduce the economic burden of aNSCLC on healthcare systems. I3LUNG is an international, multicenter, retrospective and prospective, observational study of patients with aNSCLC treated with IO, entirely funded by European Union (EU) under the Horizon 2020 (H2020) program. Using AI-based tools, the aim of this study is to promote individualized treatment in aNSCLC, with the goals of improving survival and quality of life, minimizing or preventing undue toxicity and promoting efficient resource allocation. The final objective of the project is the construction of a novel, integrated, AI-assisted data storage and elaboration platform to guide IO administration in aNSCLC, ensuring easy access and cost-effective use by healthcare providers and patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/patologia , União Europeia , Inteligência Artificial , Estudos Retrospectivos , Estudos Prospectivos , Qualidade de Vida , Carcinoma Pulmonar de Células não Pequenas/patologia , Biomarcadores , Imunoterapia , Pulmão/patologia , Antígeno B7-H1 , Microambiente TumoralRESUMO
In recent years, immunotherapy-based regimens have been included into the treatment's algorithm of several cancer types. Programmed death-1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) interact with their ligands found on the surface of antigen presenting cells (APC) or tumor cells (PD-L1/2 and CD80/86). Through these interactions, stimulatory or inhibitory signals are established. Immune checkpoint inhibitors (ICIs), block these interactions, and when administered not only as monotherapy but also as part of combination regimens, have shown to improve survival results in multiple advanced cancers leading to an increasing number of patients treated with ICI and, as a consequence, a rise in the number of patients developing immune-related adverse events (irAEs). Presence of irAEs has been associated with greater benefit from treatment, especially when blocking PD-L1. Recent data suggests that treatment benefit persists after discontinuation of ICIs due to a treatment related adverse event, regardless of the grade. Patients experiencing grade 3-4 irAEs are at risk of toxicity recurrence after reintroducing immunotherapy and therefore, the decision to resume the treatment is challenging. In these cases, a multidisciplinary approach is always needed and several factors should be considered. Management of severe toxicities may require systemic corticosteroids which can impact on T-cell function. Due to their immunosuppressive properties, it is necessary to deeper determine how corticosteroids influence responses. In terms of overall survival (OS), the use of steroids as therapy for irAEs seems not to reduce OS and several studies have reported durable responses in patients experiencing autoimmune toxicities treated with corticosteroids.
RESUMO
BACKGROUND: Immune checkpoint inhibitors (ICIs) targeting the programmed cell death 1/programmed death-ligand 1 axis have transformed the management of advanced non-small cell lung cancer (NSCLC). However, many patients do not benefit from this type of treatment, and thus several molecular biomarkers of benefit have been explored. The value of somatic copy number alterations (SCNAs) burden remains elusive. PATIENTS AND METHODS: We assembled a cohort of 109 patients with NSCLC treated with ICIs and available tumor samples. We performed shallow whole-genome sequencing on 89 patients to determine genome-wide SCNAs and targeted gene expression analysis on 63 patients to study immune infiltration. We analyzed SCNAs burden in different ways (ie, the fraction of the genome altered or number of events) and studied their association with ICIs benefit based on survival analysis. We correlated SCNAs burden and immune infiltration on 35 patients of our cohort and on patients with lung adenocarcinoma from The Cancer Genome Atlas (TCGA). RESULTS: High SCNAs burden, computed in diverse ways, is negatively associated with ICIs progression-free survival (PFS), with the fraction of the genome altered (FGA) by arm and chromosome events showing the strongest association with PFS (p=0.002) (n=77). Nevertheless, we found differences in SCNAs across some clinicopathological features (sample site origin). A multivariate analysis adjusted for relevant characteristics showed that the FGA of arm and chromosome alterations was strongly associated with PFS (HR=2.21, p=3.3 x 10-5). Finally, we confirmed that SCNAs burden negatively correlates with tumor immune infiltration (n=35), although this correlation was not found for the males studied. Similar results were observed in the TCGA cohort. CONCLUSIONS: SCNAs burden is a potential biomarker of benefit to ICIs in patients with NSCLC, although there appear to be some nuances worth consideration. Further studies will be needed to establish its role as a biomarker of benefit to ICIs.
Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Aberrações Cromossômicas , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Masculino , Análise de SobrevidaRESUMO
Lung cancer remains the leading cause of cancer-related death worldwide. We identify DSTYK, a dual serine/threonine and tyrosine non-receptor protein kinase, as a novel actionable target altered in non-small cell lung cancer (NSCLC). We also show DSTYK's association with a lower overall survival (OS) and poorer progression-free survival (PFS) in multiple patient cohorts. Abrogation of DSTYK in lung cancer experimental systems prevents mTOR-dependent cytoprotective autophagy, impairs lysosomal biogenesis and maturation, and induces accumulation of autophagosomes. Moreover, DSTYK inhibition severely affects mitochondrial fitness. We demonstrate in vivo that inhibition of DSTYK sensitizes lung cancer cells to TNF-α-mediated CD8+-killing and immune-resistant lung tumors to anti-PD-1 treatment. Finally, in a series of lung cancer patients, DSTYK copy number gain predicts lack of response to the immunotherapy. In summary, we have uncovered DSTYK as new therapeutic target in lung cancer. Prioritization of this novel target for drug development and clinical testing may expand the percentage of NSCLC patients benefiting from immune-based treatments.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Serina , Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Treonina , Fator de Necrose Tumoral alfa/metabolismo , TirosinaRESUMO
Retinoic acid (RA) induces embryonic stem cell differentiation. The effects of RA are mediated by retinoic acid receptors (RARs) that promote epigenetic changes controlling gene transcription. We show here that RARγ, in the absence of the ligand RA, is required for deposition of the histone variant H2A.Z and the polycomb group protein Suz12 at RA target genes, and that in embryonic stem cells both RARγ and Suz12 exist in a multi-protein complex in the absence of ligand. Addition of RA causes removal of H2A.Z and Suz12 from RARγ target genes when the genes are transcriptionally activated.
Assuntos
Células-Tronco Embrionárias/fisiologia , Histonas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Proteínas Repressoras/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Camundongos , Camundongos Knockout , Complexo Repressor Polycomb 2 , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/genética , Proteínas Repressoras/genética , Ácido Retinoico 4 Hidroxilase , Tretinoína/metabolismo , Tretinoína/farmacologia , Receptor gama de Ácido RetinoicoRESUMO
CheckMate 743 trial demonstrated survival benefit of immunotherapy in first line in MPM with some differences in the efficacy of chemotherapy according to histology. The objective of this study is to characterize the impact of chemotherapy according to histology in patients diagnosed with MPM at our institution. Clinical records of all MPM patients diagnosed at Vall d'Hebron University Hospital between November 2002 and April 2020 were reviewed. Associations between clinical variables and outcomes were assessed with Cox regression models. Survival data were calculated by the Kaplan-Meier method. 189 patients were included with 76% of tumors classified as epithelioid subtype. First line chemotherapy was offered to 85% of patients. Median survival in overall population was 21.3 months (95% CI 17.2-24.3). We found that patients with epithelioid tumors had better overall survival (OS) and progression free survival (PFS). Median OS of epithelioid patients treated with first line chemotherapy was 26.7 months versus 15.0 months in non-epithelioid patients (HR 2.25 CI 95% 1.4-3.4; p < 0.001). Median PFS for patients with epithelioid tumors treated with chemotherapy was 4.8 months versus 3.6 months in non-epithelioid (HR 1.5 CI 95% 1.0-2.3; p = 0.03). The improvement of outcomes in patients with epithelioid histology was detected in patients treated with cisplatin or carboplatin. Histology was not a predictive factor for the platinum agent sensitivity (p of interaction PFS = 0.09, p of interaction OS = 0.65). In our series, patients with non-epithelioid tumors presented worse prognosis. Although epithelioid tumors exposed to cisplatin had higher PFS, histology was not a clear predictor of chemotherapy efficacy.
Assuntos
Antineoplásicos/uso terapêutico , Mesotelioma Maligno/tratamento farmacológico , Neoplasias Pleurais/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Mesotelioma Maligno/epidemiologia , Mesotelioma Maligno/patologia , Pessoa de Meia-Idade , Pleura/efeitos dos fármacos , Pleura/patologia , Neoplasias Pleurais/epidemiologia , Neoplasias Pleurais/patologia , Estudos Retrospectivos , Análise de Sobrevida , Resultado do TratamentoRESUMO
Immune checkpoint inhibitors (ICIs) have transformed non-small cell lung cancer (NSCLC) treatment. Unfortunately, only some patients benefit from these therapies. Thus, certain clinicopathological characteristics of the patients have been proposed as biomarkers of ICIs response. We assembled a retrospective cohort of 262 NSCLC patients treated with ICIs, compiled relevant clinicopathological characteristics, and studied their associations with treatment outcome using Cox proportional-hazards survival models. Additionally, we investigated the interrelations between clinicopathological features and devised a method to create a compendium associated with ICIs response by selecting those that provide non-redundant information. In multivariate analyses, ECOG performance status (hazard ratio (HR) 1.37 (95% CI 1.11 to 1.68), p < 0.005), LDH (HR 1.24 (95% CI 1.03 to 1.48), p = 0.02)) and PD-L1 negativity were associated with decreased PFS (HR 1.92 (95% CI 1.03 to 3.58), p < 0.04), whereas presentation of immune-related adverse events (irAEs) (HR 0.35 (95% CI 0.22 to 0.55, p < 0.005) or females (HR 0.52 (95% CI 0.33 to 0.80, p < 0.005) had longer progression-free survival. Additionally, numerous clinicopathological indicators were found to be interrelated. Thus, we searched for features that provide non-redundant information, and found the combination of LDH levels, irAEs, and gender to have a better association with ICIs treatment response (cross-validated c-index = 0.66). We concluded that several clinicopathological features showed prognostic value in our real-world cohort. However, some are interrelated, and compendiums of features should therefore consider these interactions. Joint assessment of LDH, irAEs, and gender may be a good prognostic compendium.
RESUMO
Immunotherapy has transformed advanced non-small cell lung cancer (NSCLC) treatment strategies and has led to unprecedented long-lasting responses in some patients. However, the molecular determinants driving these long-term responses remain elusive. To address this issue, we performed an integrative analysis of genomic and transcriptomic features of long-term immune checkpoint inhibitors (ICIs)-associated responders. We assembled a cohort of 47 patients with NSCLC receiving ICIs that was enriched in long-term responders [>18 months of progression-free survival (PFS)]. We performed whole-exome sequencing from tumor samples, estimated the tumor mutational burden (TMB), and inferred the somatic copy number alterations (SCNAs). We also obtained gene transcription data for a subset of patients using Nanostring, which we used to assess the tumor immune infiltration status and PD-L1 expression. Our results indicate that there is an association between TMB and benefit to ICIs, which is driven by those patients with long-term response. Additionally, high SCNAs burden is associated with poor response and negatively correlates with the presence of several immune cell types (B cells, natural killers, regulatory T cells or effector CD8 T cells). Also, CD274 (PD-L1) expression is increased in patients with benefit, mainly in those with long-term response. In our cohort, combined assessment of TMB and SCNAs burden enabled identification of long-term responders (considering PFS and overall survival). Notably, the association between TMB, SCNAs burden, and PD-L1 expression with the outcomes of ICIs treatment was validated in two public datasets of ICI-treated patients with NSCLC. Thus, our data indicate that TMB is associated with long-term benefit following ICIs treatment in NSCLC and that TMB, SCNAs burden, and PD-L1 are complementary determinants of response to ICIs.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Variações do Número de Cópias de DNA , Feminino , Humanos , Imunoterapia , Neoplasias Pulmonares/genética , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Transcriptoma , Sequenciamento do ExomaRESUMO
T(3) regulates energy metabolism by stimulating metabolic rate and decreasing metabolic efficiency. The discovery of mitochondrial uncoupling protein 3 (UCP3), its homology to UCP1, and regulation by T(3) rendered it a possible molecular determinant of the action of T(3) on energy metabolism, but data are controversial. This controversy may in part be attributable to discrepancies observed between the regulation by T(3) of UCP3 expression in rats, humans, and mice. To clarify this issue, we studied 1) the induction kinetics of the UCP3 gene by T(3) in rat skeletal muscle, 2) the influence of fatty acids, and 3) the structure and regulation of the various UCP3 promoters by T(3). Within 8 h of single-dose T(3) administration, hypothyroid rats showed a rise in serum fatty acid levels concomitant with a rapid increase in UCP3 expression in gastrocnemius muscle, followed by inductions of peroxisome proliferator activated receptor delta (PPARdelta) (within 24 h) and PPAR target gene expression (after 24 h). This T(3)-induced early UCP3 expression depended on fatty acid-PPAR signaling because depleting serum fatty acid levels abolished its expression, restorable by administration of the PPARdelta agonist L165,041 (4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxy]acetic acid). In transfected rat L6 myoblasts, only the rat UCP3 promoter positively responded to T(3) and L165,041 together in the presence of MyoD, thyroid hormone receptor beta1 (TRbeta1), PPARdelta, or PPARdelta plus the TR dimerization partner retinoid X receptor alpha. All promoters share a response element common to TR and PPAR (TRE 1), but the observed species differences may be attributable to different localizations of the MyoD response element, which in the rat maps to exon 1.
Assuntos
Ácidos Graxos/metabolismo , Canais Iônicos/genética , Proteínas Mitocondriais/genética , Transcrição Gênica/fisiologia , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologia , Acetatos/farmacologia , Animais , Carnitina O-Palmitoiltransferase/genética , Éxons/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Hipotireoidismo/tratamento farmacológico , Hipotireoidismo/metabolismo , Canais Iônicos/metabolismo , Masculino , Camundongos , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/fisiologia , Proteína MyoD/genética , PPAR delta/agonistas , PPAR delta/genética , PPAR delta/metabolismo , Palmitoil-CoA Hidrolase/genética , Fenóis/farmacologia , Fenoxiacetatos , Regiões Promotoras Genéticas/fisiologia , Ratos , Ratos Wistar , Receptor X Retinoide alfa/genética , Especificidade da Espécie , Receptores beta dos Hormônios Tireóideos/genética , Transcrição Gênica/efeitos dos fármacos , Transfecção , Proteína Desacopladora 3 , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Cdk7, the CDK-activating kinase and transcription factor IIH component, is a target of inhibitors that kill cancer cells by exploiting tumor-specific transcriptional dependencies. However, whereas selective inhibition of analog-sensitive (AS) Cdk7 in colon cancer-derived cells arrests division and disrupts transcription, it does not by itself trigger apoptosis efficiently. Here, we show that p53 activation by 5-fluorouracil or nutlin-3 synergizes with a reversible Cdk7as inhibitor to induce cell death. Synthetic lethality was recapitulated with covalent inhibitors of wild-type Cdk7, THZ1, or the more selective YKL-1-116. The effects were allele specific; a CDK7as mutation conferred both sensitivity to bulky adenine analogs and resistance to covalent inhibitors. Non-transformed colon epithelial cells were resistant to these combinations, as were cancer-derived cells with p53-inactivating mutations. Apoptosis was dependent on death receptor DR5, a p53 transcriptional target whose expression was refractory to Cdk7 inhibition. Therefore, p53 activation induces transcriptional dependency to sensitize cancer cells to Cdk7 inhibition.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Fenilenodiaminas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Imidazóis/farmacologia , Piperazinas/farmacologia , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Promoter-proximal pausing by RNA polymerase II (Pol II) ensures gene-specific regulation and RNA quality control. Structural considerations suggested a requirement for initiation-factor eviction in elongation-factor engagement and pausing of transcription complexes. Here we show that selective inhibition of Cdk7--part of TFIIH--increases TFIIE retention, prevents DRB sensitivity-inducing factor (DSIF) recruitment and attenuates pausing in human cells. Pause release depends on Cdk9-cyclin T1 (P-TEFb); Cdk7 is also required for Cdk9-activating phosphorylation and Cdk9-dependent downstream events--Pol II C-terminal domain Ser2 phosphorylation and histone H2B ubiquitylation--in vivo. Cdk7 inhibition, moreover, impairs Pol II transcript 3'-end formation. Cdk7 thus acts through TFIIE and DSIF to establish, and through P-TEFb to relieve, barriers to elongation: incoherent feedforward that might create a window to recruit RNA-processing machinery. Therefore, cyclin-dependent kinases govern Pol II handoff from initiation to elongation factors and cotranscriptional RNA maturation.
Assuntos
Quinases Ciclina-Dependentes/fisiologia , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética/fisiologia , Iniciação da Transcrição Genética/fisiologia , Imunoprecipitação da Cromatina , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Células HCT116 , Histonas/metabolismo , Humanos , Immunoblotting , Proteínas Nucleares/metabolismo , Fosforilação , Fatores de Transcrição/metabolismo , Fatores de Transcrição TFII/metabolismo , Fatores de Elongação da Transcrição , Ubiquitinação , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Peroxisome proliferator activated receptor-gamma co-activator-1alpha (PGC-1alpha) is a transcriptional co-activator that coordinately regulates the expression of distinct sets of metabolism-related genes in different tissues. Here we show that PGC-1alpha expression is reduced in skeletal muscles from mice lacking the sirtuin family deacetylase SIRT1. Conversely, SIRT1 activation or overexpression in differentiated C2C12 myotubes increased PGC-1alpha mRNA expression. The transcription-promoting effects of SIRT1 occurred through stimulation of PGC-1alpha promoter activity and were enhanced by co-transfection of myogenic factors, such as myocyte enhancer factor 2 (MEF2) and, especially, myogenic determining factor (MyoD). SIRT1 bound to the proximal promoter region of the PGC-1alpha gene, an interaction potentiated by MEF2C or MyoD, which also interact with this region. In the presence of MyoD, SIRT1 promoted a positive autoregulatory PGC-1alpha expression loop, such that overexpression of PGC-1alpha increased PGC-1alpha promoter activity in the presence of co-expressed MyoD and SIRT1. Chromatin immunoprecipitation showed that SIRT1 interacts with PGC-1alpha promoter and increases PGC-1alpha recruitment to its own promoter region. Immunoprecipitation assays further showed that SIRT1-PGC-1alpha interactions are enhanced by MyoD. Collectively, these data indicate that SIRT1 controls PGC-1alpha gene expression in skeletal muscle and that MyoD is a key mediator of this action. The involvement of MyoD in SIRT1-dependent PGC-1alpha expression may help to explain the ability of SIRT1 to drive muscle-specific gene expression and metabolism. Autoregulatory control of PGC-1alpha gene transcription seems to be a pivotal mechanism for conferring a transcription-activating response to SIRT1 in skeletal muscle.