RESUMO
Diffuse midline gliomas (DMGs) are universally lethal malignancies occurring chiefly during childhood and involving midline structures of the central nervous system, including thalamus, pons, and spinal cord. These molecularly related cancers are characterized by high prevalence of the histone H3K27M mutation. In search of effective therapeutic options, we examined multiple DMG cultures in sequential quantitative high-throughput screens (HTS) of 2706 approved and investigational drugs. This effort generated 19,936 single-agent dose responses that inspired a series of HTS-enabled drug combination assessments encompassing 9195 drug-drug examinations. Top combinations were validated across patient-derived cell cultures representing the major DMG genotypes. In vivo testing in patient-derived xenograft models validated the combination of the multi-histone deacetylase (HDAC) inhibitor panobinostat and the proteasome inhibitor marizomib as a promising therapeutic approach. Transcriptional and metabolomic surveys revealed substantial alterations to key metabolic processes and the cellular unfolded protein response after treatment with panobinostat and marizomib. Mitigation of drug-induced cytotoxicity and basal mitochondrial respiration with exogenous application of nicotinamide mononucleotide (NMN) or exacerbation of these phenotypes when blocking nicotinamide adenine dinucleotide (NAD+) production via nicotinamide phosphoribosyltransferase (NAMPT) inhibition demonstrated that metabolic catastrophe drives the combination-induced cytotoxicity. This study provides a comprehensive single-agent and combinatorial drug screen for DMG and identifies concomitant HDAC and proteasome inhibition as a promising therapeutic strategy that underscores underrecognized metabolic vulnerabilities in DMG.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Glioma/tratamento farmacológico , Ensaios de Triagem em Larga Escala/métodos , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias do Tronco Encefálico/tratamento farmacológico , Morte Celular , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Glioma/genética , Glioma/metabolismo , Humanos , Lactonas/farmacologia , Lactonas/uso terapêutico , Masculino , Metabolômica , Camundongos , Panobinostat/farmacologia , Panobinostat/uso terapêutico , Pirróis/farmacologia , Pirróis/uso terapêutico , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recurrent respiratory papillomatosis (RRP) is a benign neoplasm of the larynx caused mainly by human papillomavirus type 6 or 11 and its standard treatment involves repeated surgical debulking of the laryngeal tumors. However, significant morbidity and occasional mortality due to multiple recurrences occur. Conditional reprogramming (CR) was used to establish a HPV-6 positive culture from an RRP patient, named GUMC-403. High-throughput screening was performed at the National Center for Advanced Technology (NCATS) to identify potential drugs to treat this rare but morbid disease. GUMC-403â¯cells were screened against the NPC library of >2800 approved drugs and the MIPE library of >1900 investigational drugs to identify new uses for FDA-approved drugs or drugs that have undergone significant research and development. From the two libraries, we identified a total of 13 drugs that induced significant cytotoxicity in RRP cells at IC50 values that were clinically achievable. We validated the efficacy of the drugs in vitro using CR 2D and 3D models and further refined our list of drugs to panobinostat, dinaciclib and forskolin as potential therapies for RRP patients.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Infecções por Papillomavirus/tratamento farmacológico , Infecções Respiratórias/tratamento farmacológico , Animais , Biópsia , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Suscetibilidade a Doenças , Relação Dose-Resposta a Droga , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Papillomavirus Humano 6/fisiologia , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/etiologia , Infecções por Papillomavirus/virologia , Infecções Respiratórias/etiologiaRESUMO
The identification of drug combinations as alternatives to single-agent therapeutics has traditionally been a slow, largely manual process. In the last 10 years, high-throughput screening platforms have been developed that enable routine screening of thousands of drug pairs in an in vitro setting. In this chapter, we describe the workflow involved in screening a single agent versus a library of mechanistically annotated, investigation, and approved drugs using a full dose-response matrix scheme using viability as the readout. We provide details of the automation required to run the screen and the informatics required to process data from screening robot and subsequent analysis and visualization of the datasets.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Acústica/instrumentação , Animais , Sobrevivência Celular/efeitos dos fármacos , Combinação de Medicamentos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Desenho de Equipamento , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Software , Fluxo de TrabalhoRESUMO
Glioblastoma (GBM) is a lethal brain cancer with a median survival time of approximately 15 months following treatment. Common in vitro GBM models for drug screening are adherent and do not recapitulate the features of human GBM in vivo. Here we report the genomic characterization of nine patient-derived, spheroid GBM cell lines that recapitulate human GBM characteristics in orthotopic xenograft models. Genomic sequencing revealed that the spheroid lines contain alterations in GBM driver genes such as PTEN, CDKN2A, and NF1. Two spheroid cell lines, JHH-136 and JHH-520, were utilized in a high-throughput drug screen for cell viability using a 1912-member compound library. Drug mechanisms that were cytotoxic in both cell lines were Hsp90 and proteasome inhibitors. JHH-136 was uniquely sensitive to topoisomerase 1 inhibitors, while JHH-520 was uniquely sensitive to Mek inhibitors. Drug combination screening revealed that PI3 kinase inhibitors combined with Mek or proteasome inhibitors were synergistic. However, animal studies to test these drug combinations in vivo revealed that Mek inhibition alone was superior to the combination treatments. These data show that these GBM spheroid lines are amenable to high-throughput drug screening and that this dataset may deliver promising therapeutic leads for future GBM preclinical studies.
Assuntos
Antineoplásicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Glioblastoma/patologia , Mutação , Esferoides Celulares/efeitos dos fármacos , Antineoplásicos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Células Tumorais CultivadasRESUMO
Despite relative success of therapy for Hodgkin's lymphoma (HL), novel therapeutic agents are needed for patients with refractory or relapsed disease. Recently, anti-PD1 immunotherapy or treatment with the anti-CD30 toxin conjugate brentuximab vedotin (BV) have been associated with remissions; however, the median responses of complete responses (CRs) with the latter were only 6.7 mo. To obtain curative therapy, other effective agents, based on HL biology, would have to be given in combination with BV. Hodgkin's Reed-Sternberg (HRS) cells secrete cytokines including IL-6 and -13, leading to constitutive activation of JAK/STAT signaling. In the present study the JAK1/2 inhibitor ruxolitinib reduced phosphorylation of STAT3 and STAT6 and expression of c-Myc in the HL cell line HDLM-2. These changes were enhanced when, on the basis of a matrix screen of drug combinations, ruxolitinib was combined with the Bcl-2/Bcl-xL inhibitor Navitoclax. The combination augmented expression of Bik, Puma, and Bax, and attenuated Bcl-xL expression and the phosphorylation of Bad. The use of the two-agent combination of either ruxolitinib or Navitoclax with BV or the three-agent combination strongly activated Bax and increased activities of cytochrome c and caspase-9 and -3 that, in turn, led to cleavage of poly(ADP ribose) polymerase and Mcl-1. Either ruxolitinib combined with Navitoclax or BV alone prolonged survival but did not cure HDLM-2 tumor-bearing mice, whereas BV combined with ruxolitinib and/or with Navitoclax resulted in a sustained, complete elimination of the HDLM-2 HL. These studies provide scientific support for a clinical trial to evaluate BV combined with ruxolitinib in select patients with HL.
Assuntos
Compostos de Anilina/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Doença de Hodgkin/tratamento farmacológico , Imunoconjugados/uso terapêutico , Pirazóis/uso terapêutico , Sulfonamidas/uso terapêutico , Compostos de Anilina/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Brentuximab Vedotin , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Dosagem de Genes , Doença de Hodgkin/enzimologia , Doença de Hodgkin/patologia , Humanos , Imunoconjugados/farmacologia , Janus Quinase 2/genética , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nitrilas , Fosforilação/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Resultado do Tratamento , Proteína bcl-X/metabolismoRESUMO
Phenotypic assays have a proven track record for generating leads that become first-in-class therapies. Whole cell assays that inform on a phenotype or mechanism also possess great potential in drug repositioning studies by illuminating new activities for the existing pharmacopeia. The National Center for Advancing Translational Sciences (NCATS) pharmaceutical collection (NPC) is the largest reported collection of approved small molecule therapeutics that is available for screening in a high-throughput setting. Via a wide-ranging collaborative effort, this library was analyzed in the Open Innovation Drug Discovery (OIDD) phenotypic assay modules publicly offered by Lilly. The results of these tests are publically available online at www.ncats.nih.gov/expertise/preclinical/pd2 and via the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/) (AID 1117321). Phenotypic outcomes for numerous drugs were confirmed, including sulfonylureas as insulin secretagogues and the anti-angiogenesis actions of multikinase inhibitors sorafenib, axitinib and pazopanib. Several novel outcomes were also noted including the Wnt potentiating activities of rotenone and the antifolate class of drugs, and the anti-angiogenic activity of cetaben.
Assuntos
Reposicionamento de Medicamentos , Linhagem Celular Tumoral , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Fenótipo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Tumor cell subpopulations called cancer stem cells (CSCs) or tumor-initiating cells (TICs) have self-renewal potential and are thought to drive metastasis and tumor formation. Data suggest that these cells are resistant to current chemotherapy and radiation therapy treatments, leading to cancer recurrence. Therefore, finding new drugs and/or drug combinations that cause death of both the differentiated tumor cells as well as CSC populations is a critical unmet medical need. Here, we describe how cancer-derived CSCs are generated from cancer cell lines using stem cell growth media and nonadherent conditions in quantities that enable high-throughput screening (HTS). A cell growth assay in a 1536-well microplate format was developed with these CSCs and used to screen a focused collection of oncology drugs and clinical candidates to find compounds that are cytotoxic against these highly aggressive cells. A hit selection process that included potency and efficacy measurements during the primary screen allowed us to efficiently identify compounds with potent cytotoxic effects against spheroid-derived CSCs. Overall, this research demonstrates one of the first miniaturized HTS assays using CSCs. The procedures described here should enable further testing of the effect of compounds on CSCs and help determine which pathways need to be targeted to kill them.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Células-Tronco Neoplásicas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Proteína Homeobox Nanog , Neoplasias/tratamento farmacológicoRESUMO
The NCI Developmental Therapeutics Program Human Tumor cell line data set is a publicly available database that contains cellular assay screening data for over 40 000 compounds tested in 60 human tumor cell lines. The database also contains microarray assay gene expression data for the cell lines, and so it provides an excellent information resource particularly for testing data mining methods that bridge chemical, biological, and genomic information. In this paper we describe a formal knowledge discovery approach to characterizing and data mining this set and report the results of some of our initial experiments in mining the set from a chemoinformatics perspective.