RESUMEN
Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Familia 4 del Citocromo P450/deficiencia , Familia 4 del Citocromo P450/genética , Descubrimiento de Drogas , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Glucocorticoides/farmacología , Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, including autophagy, and aberrant activation of such receptors is a common feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors and restores autophagy in non-small-cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Beclina-1 , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Receptores ErbB/genética , Xenoinjertos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , FosforilaciónRESUMEN
Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Indoles/farmacología , Neoplasias Pulmonares/metabolismo , Triazinas/farmacología , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras , Línea Celular Tumoral , Proteína Coatómero/metabolismo , Femenino , Genes ras , Xenoinjertos , Humanos , Neoplasias Pulmonares/patología , Lisosomas/metabolismo , Ratones , Terapia Molecular Dirigida , Proteína con Dominio Pirina 3 de la Familia NLR , Trasplante de Neoplasias , Fosforilación OxidativaRESUMEN
Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes, including signal transduction and gene expression patterns, arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here, we quantified metabolic features, mostly from the 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression, and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral/metabolismo , Metaboloma/fisiología , Biomarcadores de Tumor/metabolismo , Cromatografía de Gases y Espectrometría de Masas/métodos , Regulación Neoplásica de la Expresión Génica/fisiología , Glucosa/metabolismo , Glutamina/metabolismo , Humanos , Redes y Vías Metabólicas/genética , Metabolómica/métodos , Neoplasias/metabolismoRESUMEN
The scientific community has entered an era of big data. However, with big data comes big responsibilities, and best practices for how data are contributed to databases have not kept pace with the collection, aggregation, and analysis of big data. Here, we rigorously assess the quantity of data for specific leaf area (SLA) available within the largest and most frequently used global plant trait database, the TRY Plant Trait Database, exploring how much of the data were applicable (i.e., original, representative, logical, and comparable) and traceable (i.e., published, cited, and consistent). Over three-quarters of the SLA data in TRY either lacked applicability or traceability, leaving only 22.9% of the original data usable compared with the 64.9% typically deemed usable by standard data cleaning protocols. The remaining usable data differed markedly from the original for many species, which led to altered interpretation of ecological analyses. Though the data we consider here make up only 4.5% of SLA data within TRY, similar issues of applicability and traceability likely apply to SLA data for other species as well as other commonly measured, uploaded, and downloaded plant traits. We end with suggested steps forward for global ecological databases, including suggestions for both uploaders to and curators of databases with the hope that, through addressing the issues raised here, we can increase data quality and integrity within the ecological community.
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Hojas de la Planta , Plantas , Macrodatos , Bases de Datos Factuales , FenotipoRESUMEN
MOTIVATION: Novel approaches are needed for discovery of targeted therapies for non-small-cell lung cancer (NSCLC) that are specific to certain patients. Whole genome RNAi screening of lung cancer cell lines provides an ideal source for determining candidate drug targets. RESULTS: Unsupervised learning algorithms uncovered patterns of differential vulnerability across lung cancer cell lines to loss of functionally related genes. Such genetic vulnerabilities represent candidate targets for therapy and are found to be involved in splicing, translation and protein folding. In particular, many NSCLC cell lines were especially sensitive to the loss of components of the LSm2-8 protein complex or the CCT/TRiC chaperonin. Different vulnerabilities were also found for different cell line subgroups. Furthermore, the predicted vulnerability of a single adenocarcinoma cell line to loss of the Wnt pathway was experimentally validated with screening of small-molecule Wnt inhibitors against an extensive cell line panel. AVAILABILITY AND IMPLEMENTATION: The clustering algorithm is implemented in Python and is freely available at https://bitbucket.org/youngjh/nsclc_paper CONTACT: marcotte@icmb.utexas.edu or jon.young@utexas.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Carcinoma de Pulmón de Células no Pequeñas/genética , ADN de Neoplasias , Neoplasias Pulmonares/genética , Algoritmos , Análisis por Conglomerados , Regulación Neoplásica de la Expresión Génica , HumanosRESUMEN
Systematic efforts are underway to decipher the genetic changes associated with tumor initiation and progression. However, widespread clinical application of this information is hampered by an inability to identify critical genetic events across the spectrum of human tumors with adequate sensitivity and scalability. Here, we have adapted high-throughput genotyping to query 238 known oncogene mutations across 1,000 human tumor samples. This approach established robust mutation distributions spanning 17 cancer types. Of 17 oncogenes analyzed, we found 14 to be mutated at least once, and 298 (30%) samples carried at least one mutation. Moreover, we identified previously unrecognized oncogene mutations in several tumor types and observed an unexpectedly high number of co-occurring mutations. These results offer a new dimension in tumor genetics, where mutations involving multiple cancer genes may be interrogated simultaneously and in 'real time' to guide cancer classification and rational therapeutic intervention.
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Análisis Mutacional de ADN/métodos , Mutación , Neoplasias/genética , Oncogenes , Perfilación de la Expresión Génica , Genoma Humano , Genotipo , HumanosRESUMEN
A small-molecule mimetic of Smac/Diablo that specifically counters the apoptosis-inhibiting activity of IAP proteins has been shown to enhance apoptosis induced by cell surface death receptors as well as chemotherapeutic drugs. Survey of a panel of 50 human non-small-cell lung cancer cell lines has revealed, surprisingly, that roughly one-quarter of these lines are sensitive to the treatment of Smac mimetic alone, suggesting that an apoptotic signal has been turned on in these cells and is held in check by IAP proteins. This signal has now been identified as the autocrine-secreted cytokine tumor necrosis factor alpha (TNFalpha). In response to autocrine TNFalpha signaling, the Smac mimetic promotes formation of a RIPK1-dependent caspase-8-activating complex, leading to apoptosis.
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Apoptosis , Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Mitocondriales/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Caspasa 8/metabolismo , Línea Celular Tumoral , Humanos , Ratones , Ratones Desnudos , Modelos Biológicos , Trasplante de Neoplasias , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
We describe here the existence of a heregulin-HER3 autocrine loop, and the contribution of heregulin-dependent, HER2-mediated HER3 activation to gefitinib insensitivity in non-small cell lung cancer (NSCLC). ADAM17 protein, a major ErbB ligand sheddase, is upregulated in NSCLC and is required not only for heregulin-dependent HER3 signaling, but also for EGFR ligand-dependent signaling in NSCLC cell lines. A selective ADAM inhibitor, INCB3619, prevents the processing and activation of multiple ErbB ligands, including heregulin. In addition, INCB3619 inhibits gefitinib-resistant HER3 signaling and enhances gefitinib inhibition of EGFR signaling in NSCLC. These results show that ADAM inhibition affects multiple ErbB pathways in NSCLC and thus offers an excellent opportunity for pharmacological intervention, either alone or in combination with other drugs.
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Proteínas ADAM/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/metabolismo , Piperidinas/farmacología , Receptor ErbB-3/metabolismo , Transducción de Señal/efectos de los fármacos , Compuestos de Espiro/farmacología , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Receptores ErbB/genética , Femenino , Gefitinib , Expresión Génica/genética , Humanos , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Biológicos , Paclitaxel/farmacología , Piperidinas/uso terapéutico , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Compuestos de Espiro/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Small cell lung cancer (SCLC) is a recalcitrant cancer of neuroendocrine (NE) origin. Changes in therapeutic approaches against SCLC have been lacking over the decades. Here, we use preclinical models to identify a new therapeutic vulnerability in SCLC consisting of the targetable Jumonji lysine demethylase (KDM) family. We show that Jumonji demethylase inhibitors block malignant growth and that etoposide-resistant SCLC cell lines are particularly sensitive to Jumonji inhibition. Mechanistically, small molecule-mediated inhibition of Jumonji KDMs activates endoplasmic reticulum (ER) stress genes, upregulates ER stress signaling, and triggers apoptotic cell death. Furthermore, Jumonji inhibitors decrease protein levels of SCLC NE markers INSM1 and Secretogranin-3 and of driver transcription factors ASCL1 and NEUROD1. Genetic knockdown of KDM4A, a Jumonji demethylase highly expressed in SCLC and a known regulator of ER stress genes, induces ER stress response genes, decreases INSM1, Secretogranin-3, and NEUROD1 and inhibits proliferation of SCLC in vitro and in vivo. Lastly, we demonstrate that two different small molecule Jumonji KDM inhibitors (pan-inhibitor JIB-04 and KDM4 inhibitor SD70) block the growth of SCLC tumor xenografts in vivo. Our study highlights the translational potential of Jumonji KDM inhibitors against SCLC, a clinically feasible approach in light of recently opened clinical trials evaluating this drug class, and establishes KDM4A as a relevant target across SCLC subtypes.
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BACKGROUND: Ionizing radiation composed of accelerated ions of high atomic number (Z) and energy (HZE) deposits energy and creates damage in cells in a discrete manner as compared to the random deposition of energy and damage seen with low energy radiations such as γ- or x-rays. Such radiations can be highly effective at cell killing, transformation, and oncogenesis, all of which are concerns for the manned space program and for the burgeoning field of HZE particle radiotherapy for cancer. Furthermore, there are differences in the extent to which cells or tissues respond to such exposures that may be unrelated to absorbed dose. Therefore, we asked whether the energy deposition patterns produced by different radiation types would cause different molecular responses. We performed transcriptome profiling using human bronchial epithelial cells (HBECs) after exposure to γ-rays and to two different HZE particles (28Si and 56Fe) with different energy transfer properties to characterize the molecular response to HZE particles and γ-rays as a function of dose, energy deposition pattern, and time post-irradiation. RESULTS: Clonogenic assay indicated that the relative biological effectiveness (RBE) for 56Fe was 3.91 and for 28Si was 1.38 at 34% cell survival. Unsupervised clustering analysis of gene expression segregated samples according to the radiation species followed by the time after irradiation, whereas dose was not a significant parameter for segregation of radiation response. While a subset of genes associated with p53-signaling, such as CDKN1A, TRIM22 and BTG2 showed very similar responses to all radiation qualities, distinct expression changes were associated with the different radiation species. Gene enrichment analysis categorized the differentially expressed genes into functional groups related to cell death and cell cycle regulation for all radiation types, while gene pathway analysis revealed that the pro-inflammatory Acute Phase Response Signaling was specifically induced after HZE particle irradiation. A 73 gene signature capable of predicting with 96% accuracy the radiation species to which cells were exposed, was developed. CONCLUSIONS: These data suggest that the molecular response to the radiation species used here is a function of the energy deposition characteristics of the radiation species. This novel molecular response to HZE particles may have implications for radiotherapy including particle selection for therapy and risk for second cancers, risk for cancers from diagnostic radiation exposures, as well as NASA's efforts to develop more accurate lung cancer risk estimates for astronaut safety. Lastly, irrespective of the source of radiation, the gene expression changes observed set the stage for functional studies of initiation or progression of radiation-induced lung carcinogenesis.
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Bronquios/citología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Rayos gamma/efectos adversos , Perfilación de la Expresión Génica , Línea Celular , Supervivencia Celular/efectos de la radiación , Células Epiteliales/citología , Humanos , Transferencia Lineal de Energía , Efectividad Biológica RelativaRESUMEN
Caveolae are a major membrane domain common to most cells. One of the defining features of this domain is the protein caveolin. The exact function of caveolin, however, is not clear. One possible function is to attract adapter molecules to caveolae in a manner similar to how clathrin attracts molecules to coated pits. Here, we characterize a candidate adapter molecule called SRBC. SRBC binds PKCdelta and is a member of the STICK (substrates that interact with C-kinase) superfamily of PKC-binding proteins. We also show it co-immunoprecipitates with caveolin-1. A leucine zipper in SRBC is essential for both co-precipitation with caveolin and localization to caveolae. SRBC remains associated with caveolin when caveolae bud to form vesicles (cavicles) that travel on microtubules to different regions of the cell. In the absence of SRBC, intracellular cavicle traffic is markedly impaired. We conclude that SRBC (sdr-related gene product that binds to c-kinase) and two other family members [PTRF (Pol I and transcription release factor) and SDPR] function as caveolin adapter molecules that regulate caveolae function.
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Caveolas/metabolismo , Caveolinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caveolinas/genética , Línea Celular , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestructura , Fibroblastos/citología , Fibroblastos/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Proteínas de Unión a Fosfato , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinasa C/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Distribución TisularRESUMEN
Abundant evidence suggests that a unifying principle governing the molecular pathology of cancer is the co-dependent aberrant regulation of core machinery driving proliferation and suppressing apoptosis. Anomalous proteins engaged in support of this tumorigenic regulatory environment most probably represent optimal intervention targets in a heterogeneous population of cancer cells. The advent of RNA-mediated interference (RNAi)-based functional genomics provides the opportunity to derive unbiased comprehensive collections of validated gene targets supporting critical biological systems outside the framework of preconceived notions of mechanistic relationships. We have combined a high-throughput cell-based one-well/one-gene screening platform with a genome-wide synthetic library of chemically synthesized small interfering RNAs for systematic interrogation of the molecular underpinnings of cancer cell chemoresponsiveness. NCI-H1155, a human non-small-cell lung cancer line, was employed in a paclitaxel-dependent synthetic lethal screen designed to identify gene targets that specifically reduce cell viability in the presence of otherwise sublethal concentrations of paclitaxel. Using a stringent objective statistical algorithm to reduce false discovery rates below 5%, we isolated a panel of 87 genes that represent major focal points of the autonomous response of cancer cells to the abrogation of microtubule dynamics. Here we show that several of these targets sensitize lung cancer cells to paclitaxel concentrations 1,000-fold lower than otherwise required for a significant response, and we identify mechanistic relationships between cancer-associated aberrant gene expression programmes and the basic cellular machinery required for robust mitotic progression.
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Genes Letales/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Paclitaxel/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Análisis Mutacional de ADN , Relación Dosis-Respuesta a Droga , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/tratamiento farmacológico , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Mitosis/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Huso Acromático/patologíaRESUMEN
Smac mimetics target cancer cells in a TNFalpha-dependent manner, partly via proteasome degradation of cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2. Degradation of cIAPs triggers the release of receptor interacting protein kinase (RIPK1) from TNF receptor I (TNFR1) to form a caspase-8 activating complex together with the adaptor protein Fas-associated death domain (FADD). We report here a means through which cancer cells mediate resistance to Smac mimetic/TNFalpha-induced apoptosis and corresponding strategies to overcome such resistance. These human cancer cell lines evades Smac mimetic-induced apoptosis by up-regulation of cIAP2, which although initially degraded, rebounds and is refractory to subsequent degradation. cIAP2 is induced by TNFalpha via NF-kappaB and modulation of the NF-kappaB signal renders otherwise resistant cells sensitive to Smac mimetics. In addition, other signaling pathways, including phosphatidyl inositol-3 kinase (PI3K), have the potential to concurrently regulate cIAP2. Using the PI3K inhibitor, LY294002, cIAP2 up-regulation was suppressed and resistance to Smac mimetics-induced apoptosis was also overcome.
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Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Materiales Biomiméticos/farmacología , Resistencia a Antineoplásicos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Mitocondriales/fisiología , Proteínas Reguladoras de la Apoptosis , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Secuencia de Bases , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Cromonas/farmacología , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/fisiología , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Imidazoles/farmacología , Proteínas Inhibidoras de la Apoptosis/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Morfolinas/farmacología , FN-kappa B/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Quinoxalinas/farmacología , ARN Interferente Pequeño/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitina-Proteína Ligasas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The transcription factor achaete-scute complex homolog 1 (ASCL1) is a lineage oncogene that is central for the growth and survival of small cell lung cancers (SCLC) and neuroendocrine non-small cell lung cancers (NSCLC-NE) that express it. Targeting ASCL1, or its downstream pathways, remains a challenge. However, a potential clue to overcoming this challenage has been information that SCLC and NSCLC-NE that express ASCL1 exhibit extremely low ERK1/2 activity, and efforts to increase ERK1/2 activity lead to inhibition of SCLC growth and surival. Of course, this is in dramatic contrast to the majority of NSCLCs where high activity of the ERK pathway plays a major role in cancer pathogenesis. A major knowledge gap is defining the mechanism(s) underlying the low ERK1/2 activity in SCLC, determining if ERK1/2 activity and ASCL1 function are inter-related, and if manipulating ERK1/2 activity provides a new therapeutic strategy for SCLC. We first found that expression of ERK signaling and ASCL1 have an inverse relationship in NE lung cancers: knocking down ASCL1 in SCLCs and NE-NSCLCs increased active ERK1/2, while inhibition of residual SCLC/NSCLC-NE ERK1/2 activity with a MEK inhibitor increased ASCL1 expression. To determine the effects of ERK activity on expression of other genes, we obtained RNA-seq from ASCL1-expressing lung tumor cells treated with an ERK pathway MEK inhibitor and identified down-regulated genes (such as SPRY4, ETV5, DUSP6, SPRED1) that potentially could influence SCLC/NSCLC-NE tumor cell survival. This led us to discover that genes regulated by MEK inhibition suppress ERK activation and CHIP-seq demonstrated these are bound by ASCL1. In addition, SPRY4, DUSP6, SPRED1 are known suppressors of the ERK1/2 pathway, while ETV5 regulates DUSP6. Survival of NE lung tumors was inhibited by activation of ERK1/2 and a subset of ASCL1-high NE lung tumors expressed DUSP6. Because the dual specificity phosphatase 6 (DUSP6) is an ERK1/2-selective phosphatase that inactivates these kinases and has a pharmacologic inhibitor, we focused mechanistic studies on DUSP6. These studies showed: Inhibition of DUSP6 increased active ERK1/2, which accumulated in the nucleus; pharmacologic and genetic inhibition of DUSP6 affected proliferation and survival of ASCL1-high NE lung cancers; and that knockout of DUSP6 "cured" some SCLCs while in others resistance rapidly developed indicating a bypass mechanism was activated. Thus, our findings fill this knowledge gap and indicate that combined expression of ASCL1, DUSP6 and low phospho-ERK1/2 identify some neuroendocrine lung cancers for which DUSP6 may be a therapeutic target.
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AIM: Time outdoors and contact with nature are positively associated with a broad range of children's health outcomes. Pediatricians are uniquely positioned to promote active play in nature (APN) but may face challenges to do so during well child visits. The objective of this study was to understand barriers to children's APN, before and during the COVID-19 pandemic, and how health care providers could promote APN. METHODS: Focus groups were conducted with 14 pediatric providers and interviews with 14 parents (7 in English, 7 in Spanish) of children ages 3 to 10 on public insurance. Dedoose was used for coding and content analysis. We contextualized this work within the WHO's Commission on Social Determinants of Health conceptual framework. RESULTS: Parents mentioned a range of material circumstances (time, finances, family circumstances, access to safe outdoor play spaces and age-appropriate activities) and behavioral/psychosocial factors (previous experiences in nature, safety, and weather concerns), many of which were exacerbated by the pandemic, that serve as barriers to children's APN. Providers said they were motivated to talk to families about children's APN but mentioned barriers to this conversation such as time, other pressing priorities for the visit, and lack of resources to give families. CONCLUSIONS: Many pre-pandemic barriers to APN were exacerbated by the COVID-19 pandemic. Well-child visits may be an effective setting to discuss the benefits of APN during and beyond the pandemic, and there is a need for contextually appropriate resources for pediatric providers and families.
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COVID-19 , Niño , Preescolar , Comunicación , Personal de Salud , Humanos , Pandemias , Padres/psicologíaRESUMEN
Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB), and introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype recapitulated in human STK11/LKB1 mutant NSCLCs. Systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expanded tumor-associated TCF1+PD-1+CD8 T cells, restoring therapeutic response to PD-1 ICB in KPL tumors. This was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. NSCLC-affected individuals with identified STK11/LKB1 mutations receiving bemcentinib and pembrolizumab demonstrated objective clinical response to combination therapy. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Linfocitos T CD8-positivos/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Receptor de Muerte Celular Programada 1/genética , Proteínas Serina-Treonina Quinasas/genética , Tirosina Quinasa del Receptor AxlRESUMEN
Most non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors.
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Neoplasias Encefálicas/secundario , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Resistencia a Antineoplásicos , Receptores ErbB/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Activación Enzimática , Receptores ErbB/genética , Femenino , Humanos , Hibridación Fluorescente in Situ , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-met/genética , Tasa de SupervivenciaRESUMEN
BACKGROUND: New therapeutics targeting epidermal growth factor receptor (EGFR) have significantly improved tumor responses to therapy in non-small cell lung cancer (NSCLC) patients. Molecular testing for EGFR mutations informs important therapeutic decisions in clinical practice. In this study, we sought to validate the clinical relevance of sequencing-based EGFR mutation testing combined with cytological analysis using body fluid specimens. METHODS: Two NSCLC cell lines were used in sensitivity analyses. In addition, we performed cytological analyses and directly sequencing of exons 18-21, for 32 specimens. The absence of EGFR mutations determined by direct sequencing in 14 specimens was confirmed by real-time PCR. Changes made to patients' therapeutic strategies after reports of EGFR mutation status were investigated by querying electronic medical records. RESULTS: Sensitivity studies showed that detection of in-frame deletions in exon 19 and point mutations in exon 21 was possible in specimens containing 10% and 5% mutant DNA, respectively. In clinical practice, EGFR mutations were detected in 18 of 32 specimens (56.3%). Twelve patients with EGFR mutations detected by direct sequencing were started on treatment with EGFR tyrosine kinase inhibitor (TKI) after reports of EGFR mutation. EGFR-TKI therapy was discontinued for two patients with TKI-resistant T790M mutation. The results of real-time PCR were consistent with those of direct sequencing in 13 of 14 specimens (92.9%) in which no mutation was detected by direct sequencing. CONCLUSIONS: Combined direct sequencing and cytological analysis of body fluid specimen might be clinically useful and sensitive test for the detection of EGFR mutations in NSCLC patients.
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Líquidos Corporales/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Análisis Mutacional de ADN/métodos , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Línea Celular Tumoral , Técnicas Citológicas , Femenino , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los ResultadosRESUMEN
Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). Here we optimized and leveraged this property to identify drug resistance mutations, developing a technique we term LentiMutate. This technique was validated by identifying clinically relevant EGFR resistance mutations, then applied to two additional clinical anticancer drugs: imatinib, a BCR-ABL inhibitor, and AMG 510, a KRAS G12C inhibitor. Novel deletions in BCR-ABL1 conferred resistance to imatinib. In KRAS-G12C or wild-type KRAS, point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations conferred resistance to AMG 510. LentiMutate should prove highly valuable for clinical and preclinical cancer-drug development. SIGNIFICANCE: LentiMutate can evaluate a drug's on-target activity and can nominate resistance mutations before they occur in patients, which could accelerate and refine drug development to increase the survival of patients with cancer.