RESUMEN

This Letter is being retracted owing to issues with Fig. 1d and Supplementary Fig. 31b, and the unavailability of original data for these figures that raise concerns regarding the integrity of the figures. Nature published two previous corrections related to this Letter1,2. These issues in aggregate undermine the confidence in the integrity of this study. Authors Michael Foley, Monica Schenone, Nicola J. Tolliday, Todd R. Golub, Steven A. Carr, Alykhan F. Shamji, Andrew M. Stern and Stuart L. Schreiber agree with the Retraction. Authors Lakshmi Raj, Takao Ide, Aditi U. Gurkar, Anna Mandinova and Sam W. Lee disagree with the Retraction. Author Xiaoyu Li did not respond.

RESUMEN

While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.

Asunto(s)

Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Terapia Molecular Dirigida , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Diferenciación Celular , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , Proteínas de Homeodominio/genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Células Mieloides/patología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Pirimidinas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

High cancer death rates indicate the need for new anticancer therapeutic agents. Approaches to discovering new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identified phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds through phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the gene PDE3A, encoding phosphodiesterase 3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells, whereas others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrated that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggestive of a neomorphic activity. Coexpression of SLFN12 with PDE3A correlates with DNMDP sensitivity, whereas depletion of SLFN12 results in decreased DNMDP sensitivity. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery.

Asunto(s)

Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Citotoxinas/farmacología , Neoplasias/terapia , Piridazinas/química , Piridazinas/farmacología , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citotoxinas/química , Citotoxinas/aislamiento & purificación , Sistemas de Liberación de Medicamentos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genómica , Humanos , Immunoblotting

RESUMEN

Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876's mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets.

RESUMEN

Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells in which the G1-S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2-M checkpoints mediated by the ATR (ataxia telangiectasia and Rad3 related) and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR-Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs.

Asunto(s)

Neoplasias/genética , Proteínas Quinasas/biosíntesis , Proteína p53 Supresora de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/biosíntesis , Catálisis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular , Células HeLa , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteínas Quinasas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/administración & dosificación

RESUMEN

Insulin constitutes a principal evolutionarily conserved hormonal axis for maintaining glucose homeostasis; dysregulation of this axis causes diabetes. PGC-1α (peroxisome-proliferator-activated receptor-γ coactivator-1α) links insulin signalling to the expression of glucose and lipid metabolic genes. The histone acetyltransferase GCN5 (general control non-repressed protein 5) acetylates PGC-1α and suppresses its transcriptional activity, whereas sirtuin 1 deacetylates and activates PGC-1α. Although insulin is a mitogenic signal in proliferative cells, whether components of the cell cycle machinery contribute to its metabolic action is poorly understood. Here we report that in mice insulin activates cyclin D1-cyclin-dependent kinase 4 (Cdk4), which, in turn, increases GCN5 acetyltransferase activity and suppresses hepatic glucose production independently of cell cycle progression. Through a cell-based high-throughput chemical screen, we identify a Cdk4 inhibitor that potently decreases PGC-1α acetylation. Insulin/GSK-3ß (glycogen synthase kinase 3-beta) signalling induces cyclin D1 protein stability by sequestering cyclin D1 in the nucleus. In parallel, dietary amino acids increase hepatic cyclin D1 messenger RNA transcripts. Activated cyclin D1-Cdk4 kinase phosphorylates and activates GCN5, which then acetylates and inhibits PGC-1α activity on gluconeogenic genes. Loss of hepatic cyclin D1 results in increased gluconeogenesis and hyperglycaemia. In diabetic models, cyclin D1-Cdk4 is chronically elevated and refractory to fasting/feeding transitions; nevertheless further activation of this kinase normalizes glycaemia. Our findings show that insulin uses components of the cell cycle machinery in post-mitotic cells to control glucose homeostasis independently of cell division.

Asunto(s)

Ciclo Celular , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Transducción de Señal , Acetilación , Aminoácidos/farmacología , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Ciclina D1/deficiencia , Ciclina D1/genética , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Diabetes Mellitus/metabolismo , Activación Enzimática , Ayuno , Eliminación de Gen , Gluconeogénesis/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Histona Acetiltransferasas/metabolismo , Homeostasis , Humanos , Hiperglucemia/metabolismo , Hiperinsulinismo/metabolismo , Masculino , Ratones , Fosforilación , ARN Mensajero/análisis , ARN Mensajero/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos

RESUMEN

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drug's antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential.

Asunto(s)

Apoptosis , Fenotiazinas/química , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteína Fosfatasa 2/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Supervivencia Celular , Cromatografía de Afinidad , Modelos Animales de Enfermedad , Antagonistas de Dopamina/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Espectrometría de Masas , Ratones , Perfenazina/química , Fosforilación , Pigmentación , Proteómica , Receptores Notch/metabolismo , Factores de Tiempo , Pez Cebra

RESUMEN

Pancreatic and duodenal homeobox 1 (PDX1), a member of the homeodomain-containing transcription factor family, is a key transcription factor important for both pancreas development and mature ß cell function. The ectopic overexpression of Pdx1, Neurog3, and MafA in mice reprograms acinar cells to insulin-producing cells. We developed a quantitative PCR-based gene expression assay to screen more than 60,000 compounds for expression of each of these genes in the human PANC-1 ductal carcinoma cell line. We identified BRD7552, which upregulated PDX1 expression in both primary human islets and ductal cells, and induced epigenetic changes in the PDX1 promoter consistent with transcriptional activation. Prolonged compound treatment induced both insulin mRNA and protein and also enhanced insulin expression induced by the three-gene combination. These results provide a proof of principle for identifying small molecules that induce expression of transcription factors to control cellular reprogramming.

Asunto(s)

Carcinoma Ductal/genética , Carcinoma Ductal Pancreático/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Transactivadores/genética , Animales , Línea Celular Tumoral , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Ratones , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos

RESUMEN

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.

Asunto(s)

Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Leucemia , Células Madre Neoplásicas/efectos de los fármacos , Línea Celular Tumoral , Células Madre Hematopoyéticas , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lovastatina/farmacología , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/fisiología

RESUMEN

RAD54, an important homologous recombination protein, is a member of the SWI2/SNF2 family of ATPase-dependent DNA translocases. In vitro, RAD54 stimulates RAD51-mediated DNA strand exchange and promotes branch migration of Holliday junctions. It is thought that an ATPase-dependent DNA translocation is required for both of these RAD54 activities. Here we identified, by high-throughput screening, a specific RAD54 inhibitor, streptonigrin (SN), and used it to investigate the mechanisms of RAD54 activities. We found that SN specifically targets the RAD54 ATPase, but not DNA binding, through direct interaction with RAD54 and generation of reactive oxygen species. Consistent with the dependence of branch migration (BM) on the ATPase-dependent DNA translocation of RAD54, SN inhibited RAD54 BM. Surprisingly, the ability of RAD54 to stimulate RAD51 DNA strand exchange was not significantly affected by SN, indicating a relatively smaller role of RAD54 DNA translocation in this process. Thus, the use of SN enabled us to identify important differences in the effect of the RAD54 ATPase and DNA translocation on two major activities of RAD54, BM of Holliday junctions and stimulation of DNA pairing.

Asunto(s)

Antibióticos Antineoplásicos/farmacología , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/metabolismo , ADN de Hongos/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Estreptonigrina/farmacología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , ADN Cruciforme/genética , ADN Cruciforme/metabolismo , ADN de Hongos/genética , Recombinación Homóloga/efectos de los fármacos , Recombinación Homóloga/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.

Asunto(s)

Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Antígenos CD40/antagonistas & inhibidores , Antígenos CD40/genética , Evaluación Preclínica de Medicamentos , Alelos , Animales , Antígenos CD19/genética , Artritis Reumatoide/patología , Linfocitos B/citología , Linfocitos B/metabolismo , Antígenos CD40/metabolismo , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Sitios de Carácter Cuantitativo/genética , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología

RESUMEN

While cytotoxic chemotherapy remains the hallmark of cancer treatment, intensive regimens fall short in many malignancies, including high-risk neuroblastoma. One alternative strategy is to therapeutically promote tumor differentiation. We created a gene expression signature to measure neuroblast maturation, adapted it to a high-throughput platform, and screened a diversity oriented synthesis-generated small-molecule library for differentiation inducers. We identified BRD8430, containing a nine-membered lactam, an ortho-amino anilide functionality, and three chiral centers, as a selective class I histone deacetylase (HDAC) inhibitor (HDAC1 > 2 > 3). Further investigation demonstrated that selective HDAC1/HDAC2 inhibition using compounds or RNA interference induced differentiation and decreased viability in neuroblastoma cell lines. Combined treatment with 13-cis retinoic acid augmented these effects and enhanced activation of retinoic acid signaling. Therefore, by applying a chemical genomic screening approach, we identified selective HDAC1/HDAC2 inhibition as a strategy to induce neuroblastoma differentiation.

Asunto(s)

Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Lactamas/farmacología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/enzimología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Inhibidores de Histona Desacetilasas/química , Humanos , Lactamas/química , Neuroblastoma/genética , Neuroblastoma/patología , Tretinoina/metabolismo

RESUMEN

Integration of flexible data-analysis tools with cheminformatics methods is a prerequisite for successful identification and validation of "hits" in high-throughput screening (HTS) campaigns. We have designed, developed, and implemented a suite of robust yet flexible cheminformatics tools to support HTS activities at the Broad Institute, three of which are described herein. The "hit-calling" tool allows a researcher to set a hit threshold that can be varied during downstream analysis. The results from the hit-calling exercise are reported to a database for record keeping and further data analysis. The "cherry-picking" tool enables creation of an optimized list of hits for confirmatory and follow-up assays from an HTS hit list. This tool allows filtering by computed chemical property and by substructure. In addition, similarity searches can be performed on hits of interest and sets of related compounds can be selected. The third tool, an "S/SAR viewer," has been designed specifically for the Broad Institute's diversity-oriented synthesis (DOS) collection. The compounds in this collection are rich in chiral centers and the full complement of all possible stereoisomers of a given compound are present in the collection. The S/SAR viewer allows rapid identification of both structure/activity relationships and stereo-structure/activity relationships present in HTS data from the DOS collection. Together, these tools enable the prioritization and analysis of hits from diverse compound collections, and enable informed decisions for follow-up biology and chemistry efforts.

Asunto(s)

Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento , Relación Estructura-Actividad , Algoritmos , Técnicas Químicas Combinatorias , Bases de Datos Factuales , Humanos

RESUMEN

The SWI/SNF-like adenosine triphosphate (ATP)-dependent chromatin remodeling complex, esBAF, is both necessary and, in some contexts, sufficient to induce the pluripotent state. Furthermore, mutations in various BAF subunits are associated with cancer. Little is known regarding the precise mechanism(s) by which this complex exerts its activities. Thus, it is unclear which protein interactions would be important to disrupt to isolate a relevant readout of mechanism. To address this, we developed a gene expression-based assay to identify inhibitors of the native esBAF complex. Specifically, a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay was developed in mouse embryonic stem (ES) cells to monitor expression of Bmi1, a developmentally important gene repressed by the esBAF complex. The assay was miniaturized to a 384-well format and used to screen a diverse collection of compounds, including novel products of diversity-oriented synthesis (DOS). Confirmed hits were validated using a knock-in ES cell reporter line in which luciferase is inserted into the Bmi1 locus. Several of the validated hits regulate a panel of target genes in a manner similar to the BAF chromatin-remodeling complex. Together these data indicate that expression-based screening using qRT-PCR is a successful approach to identify compounds targeting the regulation of key developmental genes in ES cells.

Asunto(s)

Ensamble y Desensamble de Cromatina/efectos de los fármacos , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Animales , Línea Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Complejo Represivo Polycomb 1/genética , Proteínas Proto-Oncogénicas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo

RESUMEN

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.

Asunto(s)

Azepinas/farmacología , Descubrimiento de Drogas , Leucemia Megacarioblástica Aguda/tratamiento farmacológico , Megacariocitos/metabolismo , Poliploidía , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Aurora Quinasa A , Aurora Quinasas , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Leucemia Megacarioblástica Aguda/genética , Megacariocitos/citología , Megacariocitos/patología , Ratones , Ratones Endogámicos C57BL , Mapas de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Quinasas Asociadas a rho/metabolismo

RESUMEN

The cellular content of mitochondria changes dynamically during development and in response to external stimuli, but the underlying mechanisms remain obscure. To systematically identify molecular probes and pathways that control mitochondrial abundance, we developed a high-throughput imaging assay that tracks both the per cell mitochondrial content and the cell size in confluent human umbilical vein endothelial cells. We screened 28,786 small molecules and observed that hundreds of small molecules are capable of increasing or decreasing the cellular content of mitochondria in a manner proportionate to cell size, revealing stereotyped control of these parameters. However, only a handful of compounds dissociate this relationship. We focus on one such compound, BRD6897, and demonstrate through secondary assays that it increases the cellular content of mitochondria as evidenced by fluorescence microscopy, mitochondrial protein content, and respiration, even after rigorous correction for cell size, cell volume, or total protein content. BRD6897 increases uncoupled respiration 1.6-fold in two different, non-dividing cell types. Based on electron microscopy, BRD6897 does not alter the percent of cytoplasmic area occupied by mitochondria, but instead, induces a striking increase in the electron density of existing mitochondria. The mechanism is independent of known transcriptional programs and is likely to be related to a blockade in the turnover of mitochondrial proteins. At present the molecular target of BRD6897 remains to be elucidated, but if identified, could reveal an important additional mechanism that governs mitochondrial biogenesis and turnover.

Asunto(s)

Tamaño de la Célula , Células Endoteliales de la Vena Umbilical Humana/química , Células Endoteliales de la Vena Umbilical Humana/citología , Mitocondrias/química , Animales , Línea Celular , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Ensayos Analíticos de Alto Rendimiento , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología

RESUMEN

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α-specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3ß has been well studied in cancer development, these studies support a role for GSK-3α in AML.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Glucógeno Sintasa Quinasa 3/metabolismo , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/metabolismo , Neoplasias/metabolismo , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Perfilación de la Expresión Génica , Genómica , Células HL-60 , Humanos , Interferencia de ARN , Tecnología Farmacéutica , Células U937

RESUMEN

Malignant transformation, driven by gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes, results in cell deregulation that is frequently associated with enhanced cellular stress (for example, oxidative, replicative, metabolic and proteotoxic stress, and DNA damage). Adaptation to this stress phenotype is required for cancer cells to survive, and consequently cancer cells may become dependent upon non-oncogenes that do not ordinarily perform such a vital function in normal cells. Thus, targeting these non-oncogene dependencies in the context of a transformed genotype may result in a synthetic lethal interaction and the selective death of cancer cells. Here we used a cell-based small-molecule screening and quantitative proteomics approach that resulted in the unbiased identification of a small molecule that selectively kills cancer cells but not normal cells. Piperlongumine increases the level of reactive oxygen species (ROS) and apoptotic cell death in both cancer cells and normal cells engineered to have a cancer genotype, irrespective of p53 status, but it has little effect on either rapidly or slowly dividing primary normal cells. Significant antitumour effects are observed in piperlongumine-treated mouse xenograft tumour models, with no apparent toxicity in normal mice. Moreover, piperlongumine potently inhibits the growth of spontaneously formed malignant breast tumours and their associated metastases in mice. Our results demonstrate the ability of a small molecule to induce apoptosis selectively in cells that have a cancer genotype, by targeting a non-oncogene co-dependency acquired through the expression of the cancer genotype in response to transformation-induced oxidative stress.

Asunto(s)

Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Dioxolanos/farmacología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Dioxolanos/efectos adversos , Dioxolanos/química , Genotipo , Ratones , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Bibliotecas de Moléculas Pequeñas/química , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Activating K-RAS mutations are the most frequent oncogenic mutations in human cancer. Numerous downstream signaling pathways have been shown to be deregulated by oncogenic K-ras. However, to date there are still no effective targeted therapies for this genetically defined subset of patients. Here we report the results of a small molecule, synthetic lethal screen using mouse embryonic fibroblasts derived from a mouse model harboring a conditional oncogenic K-ras(G12D) allele. Among the >50,000 compounds screened, we identified a class of drugs with selective activity against oncogenic K-ras-expressing cells. The most potent member of this class, lanperisone, acts by inducing nonapoptotic cell death in a cell cycle- and translation-independent manner. The mechanism of cell killing involves the induction of reactive oxygen species that are inefficiently scavenged in K-ras mutant cells, leading to oxidative stress and cell death. In mice, treatment with lanperisone suppresses the growth of K-ras-driven tumors without overt toxicity. Our findings establish the specific antitumor activity of lanperisone and reveal oxidative stress pathways as potential targets in Ras-mediated malignancies.

Asunto(s)

Neoplasias/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Proteínas ras , Animales , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Fibroblastos/citología , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Especies Reactivas de Oxígeno/agonistas