RESUMO
The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression; however, in-depth single cell characterization of the PDAC TME and its role in response to therapy is lacking. Here, we perform single-cell RNA sequencing on freshly collected human PDAC samples either before or after chemotherapy. Overall, we find a heterogeneous mixture of basal and classical cancer cell subtypes, along with distinct cancer-associated fibroblast and macrophage subpopulations. Strikingly, classical and basal-like cancer cells exhibit similar transcriptional responses to chemotherapy and do not demonstrate a shift towards a basal-like transcriptional program among treated samples. We observe decreased ligand-receptor interactions in treated samples, particularly between TIGIT on CD8 + T cells and its receptor on cancer cells, and identify TIGIT as the major inhibitory checkpoint molecule of CD8 + T cells. Our results suggest that chemotherapy profoundly impacts the PDAC TME and may promote resistance to immunotherapy.
Assuntos
Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Microambiente Tumoral/genética , Ecossistema , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Análise de Sequência de RNA , Neoplasias PancreáticasRESUMO
BACKGROUND: Genetic testing is recommended for all pancreatic ductal adenocarcinoma (PDAC) patients. Prior research demonstrates that multidisciplinary pancreatic cancer clinics (MDPCs) improve treatment- and survival-related outcomes for PDAC patients. However, limited information exists regarding the utility of integrated genetics in the MDPC setting. We hypothesized that incorporating genetics in an MDPC serving both PDAC patients and high-risk individuals (HRI) could: (1) improve compliance with guideline-based genetic testing for PDAC patients, and (2) optimize HRI identification and PDAC surveillance participation to improve early detection and survival. METHODS: Demographics, genetic testing results, and pedigrees were reviewed for PDAC patients and HRI at one institution over 45 months. Genetic testing analyzed 16 PDAC-associated genes at minimum. RESULTS: Overall, 969 MDPC subjects were evaluated during the study period; another 56 PDAC patients were seen outside the MDPC. Among 425 MDPC PDAC patients, 333 (78.4%) completed genetic testing; 29 (8.7%) carried a PDAC-related pathogenic germline variant (PGV). Additionally, 32 (9.6%) met familial pancreatic cancer (FPC) criteria. These PDAC patients had 191 relatives eligible for surveillance or genetic testing. Only 2/56 (3.6%) non-MDPC PDAC patients completed genetic testing (p < 0.01). Among 544 HRI, 253 (46.5%) had a known PGV or a designation of FPC, and were eligible for surveillance at baseline; of the remainder, 15/291 (5.2%) were eligible following genetic testing and PGV identification. CONCLUSION: Integrating genetics into the multidisciplinary setting significantly improved genetic testing compliance by reducing logistical barriers for PDAC patients, and clarified cancer risks for their relatives while conserving clinical resources. Overall, we identified 206 individuals newly eligible for surveillance or genetic testing (191 relatives of MDPC PDAC patients, and 15 HRI from this cohort), enabling continuity of care for PDAC patients and at-risk relatives in one clinic.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Predisposição Genética para Doença , Neoplasias Pancreáticas/patologia , Testes Genéticos , Carcinoma Ductal Pancreático/patologia , Neoplasias PancreáticasRESUMO
The efficacy of the highly selective RET inhibitor selpercatinib is now established in RET-driven cancers, and we sought to characterize the molecular determinants of response and resistance. We find that the pre-treatment genomic landscape does not shape the variability of treatment response except for rare instances of RAS-mediated primary resistance. By contrast, acquired selpercatinib resistance is driven by MAPK pathway reactivation by one of two distinct routes. In some patients, on- and off-target pathway reactivation via secondary RET solvent front mutations or MET amplifications are evident. In other patients, rare RET-wildtype tumor cell populations driven by an alternative mitogenic driver are selected for by treatment. Multiple distinct mechanisms are often observed in the same patient, suggesting polyclonal resistance may be common. Consequently, sequential RET-directed therapy may require combination treatment with inhibitors targeting alternative MAPK effectors, emphasizing the need for prospective characterization of selpercatinib-treated tumors at the time of monotherapy progression.
Assuntos
Neoplasias Pulmonares , Neoplasias da Glândula Tireoide , Humanos , Pulmão , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Estudos Prospectivos , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genéticaRESUMO
BACKGROUND: Cell-free DNA (cfDNA) profiling is increasingly used to guide cancer care, yet mutations are not always identified. The ability to detect somatic mutations in plasma depends on both assay sensitivity and the fraction of circulating DNA in plasma that is tumor-derived (i.e., cfDNA tumor fraction). We hypothesized that cfDNA tumor fraction could inform the interpretation of negative cfDNA results and guide the choice of subsequent assays of greater genomic breadth or depth. METHODS: Plasma samples collected from 118 metastatic cancer patients were analyzed with cf-IMPACT, a modified version of the FDA-authorized MSK-IMPACT tumor test that can detect genomic alterations in 410 cancer-associated genes. Shallow whole genome sequencing (sWGS) was also performed in the same samples to estimate cfDNA tumor fraction based on genome-wide copy number alterations using z-score statistics. Plasma samples with no somatic alterations detected by cf-IMPACT were triaged based on sWGS-estimated tumor fraction for analysis with either a less comprehensive but more sensitive assay (MSK-ACCESS) or broader whole exome sequencing (WES). RESULTS: cfDNA profiling using cf-IMPACT identified somatic mutations in 55/76 (72%) patients for whom MSK-IMPACT tumor profiling data were available. A significantly higher concordance of mutational profiles and tumor mutational burden (TMB) was observed between plasma and tumor profiling for plasma samples with a high tumor fraction (z-score≥5). In the 42 patients from whom tumor data was not available, cf-IMPACT identified mutations in 16/42 (38%). In total, cf-IMPACT analysis of plasma revealed mutations in 71/118 (60%) patients, with clinically actionable alterations identified in 30 (25%), including therapeutic targets of FDA-approved drugs. Of the 47 samples without alterations detected and low tumor fraction (z-score<5), 29 had sufficient material to be re-analyzed using a less comprehensive but more sensitive assay, MSK-ACCESS, which revealed somatic mutations in 14/29 (48%). Conversely, 5 patients without alterations detected by cf-IMPACT and with high tumor fraction (z-score≥5) were analyzed by WES, which identified mutational signatures and alterations in potential oncogenic drivers not covered by the cf-IMPACT panel. Overall, we identified mutations in 90/118 (76%) patients in the entire cohort using the three complementary plasma profiling approaches. CONCLUSIONS: cfDNA tumor fraction can inform the interpretation of negative cfDNA results and guide the selection of subsequent sequencing platforms that are most likely to identify clinically-relevant genomic alterations.
Assuntos
Biomarcadores Tumorais , DNA Tumoral Circulante , Biópsia Líquida/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Variações do Número de Cópias de DNA , Genômica/métodos , Humanos , Mutação , Curva ROC , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.
Assuntos
Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Marcadores Genéticos/genética , Neoplasias/genética , Análise Mutacional de DNA/métodos , Frequência do Gene/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação/genética , Neoplasias/sangue , Neoplasias/patologiaRESUMO
Cancer cells have genetic alterations that often directly affect intracellular protein signaling processes allowing them to bypass control mechanisms for cell death, growth and division. Cancer drugs targeting these alterations often work initially, but resistance is common. Combinations of targeted drugs may overcome or prevent resistance, but their selection requires context-specific knowledge of signaling pathways including complex interactions such as feedback loops and crosstalk. To infer quantitative pathway models, we collected a rich dataset on a melanoma cell line: Following perturbation with 54 drug combinations, we measured 124 (phospho-)protein levels and phenotypic response (cell growth, apoptosis) in a time series from 10 minutes to 67 hours. From these data, we trained time-resolved mathematical models that capture molecular interactions and the coupling of molecular levels to cellular phenotype, which in turn reveal the main direct or indirect molecular responses to each drug. Systematic model simulations identified novel combinations of drugs predicted to reduce the survival of melanoma cells, with partial experimental verification. This particular application of perturbation biology demonstrates the potential impact of combining time-resolved data with modeling for the discovery of new combinations of cancer drugs.
Assuntos
Antineoplásicos/farmacologia , Melanoma , Fosfoproteínas , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimioterapia Combinada , Humanos , Modelos Biológicos , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Biologia de SistemasRESUMO
PURPOSE: The enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell-free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection. METHODS: Tumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra-ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA-cleared tumor sequencing assay, MSK-IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1. RESULTS: Tumor-guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence. CONCLUSION: Plasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow-up schemes, and risk of metastasis.
Assuntos
DNA Tumoral Circulante/genética , Genes do Retinoblastoma/genética , Neoplasias da Retina/genética , Retinoblastoma/genética , Institutos de Câncer , Pré-Escolar , DNA Tumoral Circulante/sangue , Análise Mutacional de DNA/métodos , Éxons/genética , Enucleação Ocular , Estudos de Viabilidade , Técnicas de Genotipagem , Humanos , Lactente , Recém-Nascido , Cidade de Nova Iorque , Neoplasias da Retina/sangue , Neoplasias da Retina/terapia , Retinoblastoma/sangue , Retinoblastoma/terapiaRESUMO
Diffuse gliomas are the most common malignant brain tumours in adults and include glioblastomas and World Health Organization (WHO) grade II and grade III tumours (sometimes referred to as lower-grade gliomas). Genetic tumour profiling is used to classify disease and guide therapy1,2, but involves brain surgery for tissue collection; repeated tumour biopsies may be necessary for accurate genotyping over the course of the disease3-10. While the detection of circulating tumour DNA (ctDNA) in the blood of patients with primary brain tumours remains challenging11,12, sequencing of ctDNA from the cerebrospinal fluid (CSF) may provide an alternative way to genotype gliomas with lower morbidity and cost13,14. We therefore evaluated the representation of the glioma genome in CSF from 85 patients with gliomas who underwent a lumbar puncture because they showed neurological signs or symptoms. Here we show that tumour-derived DNA was detected in CSF from 42 out of 85 patients (49.4%) and was associated with disease burden and adverse outcome. The genomic landscape of glioma in the CSF included a broad spectrum of genetic alterations and closely resembled the genomes of tumour biopsies. Alterations that occur early during tumorigenesis, such as co-deletion of chromosome arms 1p and 19q (1p/19q codeletion) and mutations in the metabolic genes isocitrate dehydrogenase 1 (IDH1) or IDH21,2, were shared in all matched ctDNA-positive CSF-tumour pairs, whereas growth factor receptor signalling pathways showed considerable evolution. The ability to monitor the evolution of the glioma genome through a minimally invasive technique could advance the clinical development and use of genotype-directed therapies for glioma, one of the most aggressive human cancers.
Assuntos
Evolução Molecular , Glioma/líquido cefalorraquidiano , Glioma/genética , Biópsia Líquida , Mutação , Genes Neoplásicos/genética , Genoma Humano/genética , Genômica , Glioblastoma/líquido cefalorraquidiano , Glioblastoma/genética , Glioblastoma/patologia , Glioma/patologia , Humanos , Gradação de TumoresRESUMO
OBJECTIVE: To observe waveform difference among cervical vestibular evoked myogenic potentials (cVEMP) elicited with different types of air conducted sound in normal young Chinese subjects. METHOD: Twenty adult volunteers (40 ears) were recruited as research subjects including 10 males and 10 females aged between 19 and 30.500 Hz Tone Burst, 1000 Hz Tone Burst and Click were employed as stimulus for conventional air conducted sound-cVEMP (ACS-cVEMP) examinations in bilateral ears of each subject. The response rate, threshold, P1 latency, N1 latency, P1-N1 latency interval, amplitude and inter-aural asymmetry were recorded and compared among groups. RESULT: The response rate was 97.5% in 500Hz Tone Burst (39/40), 87.5% in 1 000Hz Tone Burst (35/40)and 67.5% in Click (27/40), There were no statistically significant difference between 500Hz Tone Burst and 1000Hz Tone Burst (P > 0.05) but there were statistically significant difference between click and the other groups (P < 0.05). We collected the waveform parameters (the threshold, P1 latency, N1 latency, P1-N1 latency interval, amplitude) which had statistically significant difference between 500 Hz Tone Burst and the other groups (P < 0.05). The inter-aural asymmetrys had no statistically significant differents among groups. CONCLUSION: The response rate and parameter could be affected by different types of air conducted sound in normal young Chinese subjects. 500 Hz Tone Burst was the best stimulus of type what we have known.
Assuntos
Potenciais Evocados Miogênicos Vestibulares , Vestíbulo do Labirinto/fisiologia , Adulto , Povo Asiático , Feminino , Humanos , Masculino , Pescoço , Adulto JovemRESUMO
Resistance to targeted cancer therapies is an important clinical problem. The discovery of anti-resistance drug combinations is challenging as resistance can arise by diverse escape mechanisms. To address this challenge, we improved and applied the experimental-computational perturbation biology method. Using statistical inference, we build network models from high-throughput measurements of molecular and phenotypic responses to combinatorial targeted perturbations. The models are computationally executed to predict the effects of thousands of untested perturbations. In RAF-inhibitor resistant melanoma cells, we measured 143 proteomic/phenotypic entities under 89 perturbation conditions and predicted c-Myc as an effective therapeutic co-target with BRAF or MEK. Experiments using the BET bromodomain inhibitor JQ1 affecting the level of c-Myc protein and protein kinase inhibitors targeting the ERK pathway confirmed the prediction. In conclusion, we propose an anti-cancer strategy of co-targeting a specific upstream alteration and a general downstream point of vulnerability to prevent or overcome resistance to targeted drugs.
Assuntos
Antineoplásicos/farmacologia , Biologia Computacional/métodos , Técnicas Citológicas/métodos , Resistência a Medicamentos , Melanoma/tratamento farmacológico , Linhagem Celular Tumoral , Combinação de Medicamentos , Redes Reguladoras de Genes , Humanos , Modelos Biológicos , Modelos Teóricos , Quinases raf/antagonistas & inibidoresRESUMO
TGF-ß(1) is a pleotropic growth factor that mediates glomerulosclerosis and podocyte apoptosis, hallmarks of glomerular diseases. The expression of microRNA-21 (miR-21) is regulated by TGF-ß(1), and miR-21 inhibits apoptosis in cancer cells. TGF-ß(1)-transgenic mice exhibit accelerated podocyte loss and glomerulosclerosis. We determined that miR-21 expression increases rapidly in cultured murine podocytes after exposure to TGF-ß(1) and is higher in kidneys of TGF-ß(1)-transgenic mice than wild-type mice. miR-21-deficient TGF-ß(1)-transgenic mice showed increased proteinuria and glomerular extracellular matrix deposition and fewer podocytes per glomerular tuft compared with miR-21 wild-type TGF-ß(1)-transgenic littermates. Similarly, miR-21 expression was increased in streptozotocin-induced diabetic mice, and loss of miR-21 in these mice was associated with increased albuminuria, podocyte depletion, and mesangial expansion. In cultured podocytes, inhibition of miR-21 was accompanied by increases in the rate of cell death, TGF-ß/Smad3-signaling activity, and expression of known proapoptotic miR-21 target genes p53, Pdcd4, Smad7, Tgfbr2, and Timp3. In American-Indian patients with diabetic nephropathy (n=48), albumin-to-creatinine ratio was positively associated with miR-21 expression in glomerular fractions (r=0.6; P<0.001) but not tubulointerstitial fractions (P=0.80). These findings suggest that miR-21 ameliorates TGF-ß(1) and hyperglycemia-induced glomerular injury through repression of proapoptotic signals, thereby inhibiting podocyte loss. This finding is in contrast to observations in murine models of tubulointerstitial kidney injury but consistent with findings in cancer models. The aggravation of glomerular disease in miR-21-deficient mice and the positive association with albumin-to-creatinine ratio in patients with diabetic nephropathy support miR-21 as a feedback inhibitor of TGF-ß signaling and functions.
Assuntos
Albuminúria/metabolismo , Nefropatias Diabéticas/metabolismo , Glomérulos Renais/metabolismo , MicroRNAs/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Animais , Apoptose , Células Cultivadas , Matriz Extracelular/metabolismo , Feminino , Humanos , Glomérulos Renais/patologia , Masculino , Camundongos Endogâmicos DBA , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Smad/metabolismoRESUMO
OBJECTIVE: To study the hydrophilicity and the cell biocompatibility of the poly(3-hydroxybutyrate-co- 3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) coated with a fusion protein polyhydroxyalkanoates granule binding protein (PhaP) fused with Arg-Gly-Asp (RGD) peptide (PhaP-RGD). METHODS: PHBV and PHBHHx films were fabricated by solvent evaporation. Scanning electronic microscope (SEM) was used to study the morphology of the films. PhaP-RGD fusion proteins were expressed and purified by the technology of protein engineering; PHBV and PHBHHx films were immersed in the PhaP-RGD with an amount of 3.5 mg/mL protein/per sample respectively. The hydrophilicity of the surface were detected by the contact angle measurements. Septal cartilage cells obtained from human septal cartilage were cultured in vitro. The 2nd passage chondrocytes were incubated on PHBV unmodified with PhaP-RGD in group A1, PHBV modified with PhaP-RGD in group A2, PHBHHx unmodified with PhaP-RGD in group Bl, PHBHHx modified with PhaP-RGD in group B2, and on the cell culture plates in group C. After cultured for 3 days, the proliferation of cells was detected by the DAPI staining; the proliferation viability of cells was detected by the MTT assay after cultured for 3 and 7 days; after cultured for 7 days, the adhesion and morphology of the cells on the surface of the biomaterial films were observed by SEM and the matrix of the cells was detected through the toluidine blue staining. RESULTS: SEM observation showed that PHBV and PHBHHx films had porous structures. The contact angle of the surface of the PHBV and PHBHHx films modified with PhaP-RGD fusion proteins were significantly reduced when compared with the films unmodified with PhaP-RGD fusion proteins (P < 0.05). Chondrocytes of human nasal septal cartilage incubated on the films could grow in all groups. After 3 days of cultivation in vitro, the cell proliferation and viability of group B2 were the strongest among all groups (P < 0.05); the cell proliferation after cultured for 7 days was significantly stronger than that after cultured for 3 days in groups A1, A2, B1, and B2 (P < 0.05); and the cell proliferation was significantly stronger in groups B1 and B2 than groups A1, A2 and C, in group B2 than group B1, and in group A1 than group A2 (P < 0.05). The results of toluidine blue staining showed that blue metachromasia matrixes were observed in groups A1, A2, B1, and B2; group A1 and group A2 had similar staining degree, and the staining of group B2 was deeper than that of group B1. The adhesion of cells in all groups was good through SEM observation; and the connection of cells formed and stretched into the pores of the materials. CONCLUSION: The biomaterial films of PHBHHx modified with PhaP-RGD fusion protein can promote its biocompatibility with chondrocytes.
Assuntos
Condrócitos/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/farmacologia , Poli-Hidroxialcanoatos/farmacologia , Ácido 3-Hidroxibutírico , Caproatos , Adesão Celular/fisiologia , Técnicas de Cultura de Células , Proliferação de Células , Humanos , Oligopeptídeos , Ácidos Pentanoicos , Propriedades de Superfície/efeitos dos fármacosRESUMO
MOTIVATION: Somatic homozygous deletions of chromosomal regions in cancer, while not necessarily oncogenic, may lead to therapeutic vulnerabilities specific to cancer cells compared with normal cells. A recently reported example is the loss of one of the two isoenzymes in glioblastoma cancer cells such that the use of a specific inhibitor selectively inhibited growth of the cancer cells, which had become fully dependent on the second isoenzyme. We have now made use of the unprecedented conjunction of large-scale cancer genomics profiling of tumor samples in The Cancer Genome Atlas (TCGA) and of tumor-derived cell lines in the Cancer Cell Line Encyclopedia, as well as the availability of integrated pathway information systems, such as Pathway Commons, to systematically search for a comprehensive set of such epistatic vulnerabilities. RESULTS: Based on homozygous deletions affecting metabolic enzymes in 16 TCGA cancer studies and 972 cancer cell lines, we identified 4104 candidate metabolic vulnerabilities present in 1019 tumor samples and 482 cell lines. Up to 44% of these vulnerabilities can be targeted with at least one Food and Drug Administration-approved drug. We suggest focused experiments to test these vulnerabilities and clinical trials based on personalized genomic profiles of those that pass preclinical filters. We conclude that genomic profiling will in the future provide a promising basis for network pharmacology of epistatic vulnerabilities as a promising therapeutic strategy. AVAILABILITY AND IMPLEMENTATION: A web-based tool for exploring all vulnerabilities and their details is available at http://cbio.mskcc.org/cancergenomics/statius/ along with supplemental data files.
Assuntos
Genômica/métodos , Neoplasias/genética , Animais , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Deleção de Genes , Glioblastoma/genética , Humanos , Isoenzimas/genética , Camundongos , Neoplasias/tratamento farmacológico , Medicina de PrecisãoRESUMO
We present a powerful experimental-computational technology for inferring network models that predict the response of cells to perturbations, and that may be useful in the design of combinatorial therapy against cancer. The experiments are systematic series of perturbations of cancer cell lines by targeted drugs, singly or in combination. The response to perturbation is quantified in terms of relative changes in the measured levels of proteins, phospho-proteins and cellular phenotypes such as viability. Computational network models are derived de novo, i.e., without prior knowledge of signaling pathways, and are based on simple non-linear differential equations. The prohibitively large solution space of all possible network models is explored efficiently using a probabilistic algorithm, Belief Propagation (BP), which is three orders of magnitude faster than standard Monte Carlo methods. Explicit executable models are derived for a set of perturbation experiments in SKMEL-133 melanoma cell lines, which are resistant to the therapeutically important inhibitor of RAF kinase. The resulting network models reproduce and extend known pathway biology. They empower potential discoveries of new molecular interactions and predict efficacious novel drug perturbations, such as the inhibition of PLK1, which is verified experimentally. This technology is suitable for application to larger systems in diverse areas of molecular biology.
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Modelos Biológicos , Transdução de Sinais , Biologia de Sistemas , Linhagem Celular Tumoral , Humanos , Método de Monte Carlo , ProbabilidadeRESUMO
Dedifferentiated liposarcoma (DDLS) is a rare but aggressive cancer with high recurrence and low response rates to targeted therapies. Increasing treatment efficacy may require combinations of targeted agents that counteract the effects of multiple abnormalities. To identify a possible multicomponent therapy, we performed a combinatorial drug screen in a DDLS-derived cell line and identified cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor 1 receptor (IGF1R) as synergistic drug targets. We measured the phosphorylation of multiple proteins and cell viability in response to systematic drug combinations and derived computational models of the signaling network. These models predict that the observed synergy in reducing cell viability with CDK4 and IGF1R inhibitors depends on the activity of the AKT pathway. Experiments confirmed that combined inhibition of CDK4 and IGF1R cooperatively suppresses the activation of proteins within the AKT pathway. Consistent with these findings, synergistic reductions in cell viability were also found when combining CDK4 inhibition with inhibition of either AKT or epidermal growth factor receptor (EGFR), another receptor similar to IGF1R that activates AKT. Thus, network models derived from context-specific proteomic measurements of systematically perturbed cancer cells may reveal cancer-specific signaling mechanisms and aid in the design of effective combination therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Lipossarcoma/tratamento farmacológico , Modelos Biológicos , Receptor IGF Tipo 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Simulação por Computador , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Lipossarcoma/enzimologia , Lipossarcoma/genética , Lipossarcoma/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/genéticaRESUMO
MOTIVATION: The interaction between drugs and their targets, often proteins, and between antibodies and their targets, is important for planning and analyzing investigational and therapeutic interventions in many biological systems. Although drug-target and antibody-target datasets are available in separate databases, they are not publicly available in an integrated bioinformatics resource. As medical therapeutics, especially in cancer, increasingly uses targeted drugs and measures their effects on biomolecular profiles, there is an unmet need for a user-friendly toolset that allows researchers to comprehensively and conveniently access and query information about drugs, antibodies and their targets. SUMMARY: The PiHelper framework integrates human drug-target and antibody-target associations from publicly available resources to help meet the needs of researchers in systems pharmacology, perturbation biology and proteomics. PiHelper has utilities to (i) import drug- and antibody-target information; (ii) search the associations either programmatically or through a web user interface (UI); (iii) visualize the data interactively in a network; and (iv) export relationships for use in publications or other analysis tools. AVAILABILITY: PiHelper is a free software under the GNU Lesser General Public License (LGPL) v3.0. Source code and documentation are at http://bit.ly/pihelper. We plan to coordinate contributions from the community by managing future releases.
Assuntos
Anticorpos , Descoberta de Drogas , Software , Bases de Dados Factuais , Internet , ProteômicaRESUMO
Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. We observed that SMAD7, a negative regulator of transforming growth factor-beta (TGF-ß) receptor-I kinase, is markedly reduced in MDS and leads to ineffective hematopoiesis by overactivation of TGF-ß signaling. To determine the cause of SMAD7 reduction in MDS, we analyzed the 3'UTR of the gene and determined that it contains a highly conserved putative binding site for microRNA-21. We observed significantly elevated levels of miR-21 in MDS marrow samples when compared with age-matched controls. miR-21 was shown to directly bind to the 3'UTR of SMAD7 and reduce its expression in hematopoietic cells. Next, we tested the role of miR-21 in regulating TGF-ß signaling in a TGF-ß-overexpressing transgenic mouse model that develops progressive anemia and dysplasia and thus serves as a model of human bone marrow failure. Treatment with a chemically modified miR-21 inhibitor led to significant increases in hematocrit and led to an increase in SMAD7 expression in vivo. Inhibition of miR-21 also led to an increase in erythroid colony formation from primary MDS bone marrow progenitors, demonstrating its ability in stimulating hematopoiesis in vitro. Taken together, these studies demonstrate the role of miR-21 in regulating overactivated TGF-ß signaling in MDS.