Showing papers by "Pablo Tamayo published in 2015"

The Molecular Signatures Database Hallmark Gene Set Collection

Abstract: The Molecular Signatures Database (MSigDB) is one of the most widely used and comprehensive databases of gene sets for performing gene set enrichment analysis. Since its creation, MSigDB has grown beyond its roots in metabolic disease and cancer to include >10,000 gene sets. These better represent a wider range of biological processes and diseases, but the utility of the database is reduced by increased redundancy across, and heterogeneity within, gene sets. To address this challenge, here we use a combination of automated approaches and expert curation to develop a collection of “hallmark” gene sets as part of MSigDB. Each hallmark in this collection consists of a “refined” gene set, derived from multiple “founder” sets, that conveys a specific biological state or process and displays coherent expression. The hallmarks effectively summarize most of the relevant information of the original founder sets and, by reducing both variation and redundancy, provide more refined and concise inputs for gene set enrichment analysis.

KRAS Genomic Status Predicts the Sensitivity of Ovarian Cancer Cells to Decitabine

Abstract: Decitabine, a cancer therapeutic that inhibits DNA methylation, produces variable antitumor response rates in patients with solid tumors that might be leveraged clinically with identification of a predictive biomarker. In this study, we profiled the response of human ovarian, melanoma, and breast cancer cells treated with decitabine, finding that RAS/MEK/ERK pathway activation and DNMT1 expression correlated with cytotoxic activity. Further, we showed that KRAS genomic status predicted decitabine sensitivity in low-grade and high-grade serous ovarian cancer cells. Pretreatment with decitabine decreased the cytotoxic activity of MEK inhibitors in KRAS-mutant ovarian cancer cells, with reciprocal downregulation of DNMT1 and MEK/ERK phosphorylation. In parallel with these responses, decitabine also upregulated the proapoptotic BCL-2 family member BNIP3, which is known to be regulated by MEK and ERK, and heightened the activity of proapoptotic small-molecule navitoclax, a BCL-2 family inhibitor. In a xenograft model of KRAS-mutant ovarian cancer, combining decitabine and navitoclax heightened antitumor activity beyond administration of either compound alone. Our results define the RAS/MEK/DNMT1 pathway as a determinant of sensitivity to DNA methyltransferase inhibition, specifically implicating KRAS status as a biomarker of drug response in ovarian cancer.

A Genome-Wide siRNA Screen in Mammalian Cells for Regulators of S6 Phosphorylation

Abstract: mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms.

Constellation Map: Downstream visualization and interpretation of gene set enrichment results [version 1; referees: 2 approved]

Abstract: Summary: Gene set enrichment analysis (GSEA) approaches are widely used to identify coordinately regulated genes associated with phenotypes of interest. Here, we present Constellation Map, a tool to visualize and interpret the results when enrichment analyses yield a long list of significantly enriched gene sets. Constellation Map identifies commonalities that explain the enrichment of multiple top-scoring gene sets and maps the relationships between them. Constellation Map can help investigators take full advantage of GSEA and facilitates the biological interpretation of enrichment results. Availability: Constellation Map is freely available as a GenePattern module at http://www.genepattern.org.

Constellation Map: Downstream visualization and interpretation of gene set enrichment results.

Abstract: Summary: Gene set enrichment analysis (GSEA) approaches are widely used to identify coordinately regulated genes associated with phenotypes of interest. Here, we present Constellation Map, a tool to visualize and interpret the results when enrichment analyses yield a long list of significantly enriched gene sets. Constellation Map identifies commonalities that explain the enrichment of multiple top-scoring gene sets and maps the relationships between them. Constellation Map can help investigators take full advantage of GSEA and facilitates the biological interpretation of enrichment results. Availability: Constellation Map is freely available as a GenePattern module at http://www.genepattern.org .

Mb-27pathway analysis of a human neural stem cell model of aggressive medulloblastoma reveals ckd inbhibition as a potential therapeutic modality

Abstract: Medulloblastoma, the most common malignant brain tumor in children, is divided into multiple subgroups with different associated mutations and clinical prognoses. To facilitate studying the biology and developing new therapeutic options, we created a model of aggressive medulloblastoma using human neural stem cells. Neural stem cells were obtained from the cerebellar anlage of first trimester fetal autopsy specimens and transfected using lentiviral vectors with different combinations of oncogenes associated with poor prognosis medulloblastoma: dominant-negative p53, hTERT, c-MYC, and constitutively active AKT. Cerebellar derived stem cells transformed with all four oncogenes formed fast growing, aggressive tumors that were histologically similar to primary anaplastic medulloblastoma tumors. We compared the expression profile of primary medulloblastoma tumors to our model, and confirmed that our model most closely matches the most aggressive and clinically devastating subgroup of medulloblastoma. The expression profile of our model was compared with a drug sensitivity database to identify therapeutic agents that could target our model. One class of drugs identified was cyclin-dependent kinase inhibitors. Because MYC also regulates the expression of CDKs 4/6, we hypothesized that flavopiridol (alvocidib), a cyclin dependent kinase inhibitor currently in clinical trials for a variety of tumor types would be effective against MYC-driven medulloblastoma. Our model and the medulloblastoma cell lines D425Med and D283Med were sensitive to treatment with flavopiridol. The IC50 of flavopiridol was less than 50nM for all cell lines tested. Flavopiridol decreased the growth and proliferation of both cell lines and our neurosphere model as measured by MTS assay and BrdU incorporation (p < 0.001)). Flavopiridol also included apoptosis as determined by staining for cleaved caspase-3 and flow cytometry (p < 0.01). The data shown here demonstrate that our human neurosphere model of medulloblastoma can facilitate the identification of agents with therapeutic efficacy against aggressive medulloblastoma.

Abstract PR04: A melanoma transcriptional state distinction influences sensitivity to MAPK pathway inhibitors

Abstract: The deployment of cancer therapeutics that exploit oncogenic dependencies has yielded remarkable advances in patient treatment. However, the therapeutic benefit of these approaches is transient and the majority of patients develop resistance within several months. BRAF V600E -mutant malignant melanoma provides an illustrative example of this phenomenon: treatment with RAF and MEK inhibitors yields clinical responses in 50-80% of patients. However, 10-20% fail to respond to treatment (intrinsic resistance) and patients that do respond become drug resistant within ∼9 months (acquired resistance), presenting a formidable and unsolved clinical challenge. It remains incompletely understood why a subset of BRAF V600 -mutant melanoma patients (10-20%) fail to respond to MAPK-pathway inhibition. Here, we show that RAF inhibitor sensitive and resistant BRAF V600 -mutant melanomas display distinct transcriptional profiles. RAF-inhibitor sensitive cell lines are distinguishable by expression and activity of the melanocytic lineage transcription factor MITF, whereas intrinsically drug-resistant cell lines are defined by expression of the receptor tyrosine kinase AXL and elevated levels of NF-κB signaling. In vitro, these signatures were sufficient to predict MAPK-pathway inhibitor responsiveness in independent panels of melanoma cell lines. MITF-low, AXL/NF-κB high melanomas were resistant to single-agent RAF, MEK and ERK and combined RAF/MEK inhibition. In treatment-naive patient biopsies, markers of the drug sensitive transcriptional states were associated with improved therapeutic responses to combined RAF/MEK inhibitors in BRAF V600 -mutant melanoma. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF and AXL/NF-κB activity can influence intrinsic resistance to MAPK pathway inhibitors in BRAF V600 -mutant melanoma. More broadly, these data suggest that the transcriptional context in which an oncogenic event arises can have a profound impact on the establishment of oncogene-dependencies and associated drug susceptibilities. Citation Format: Cory Johannessen, David Konieczkowski, Omar Abudayyeh, Jong Wook Kim, Zachary Cooper, Adriano Piris, Dennie Frederick, Michal Barzily-Rokni, Ravid Straussman, Rizwan Haq, David Fisher, Jill Mesirov, William Hahn, Keith Flaherty, Jennifer Wargo, Pablo Tamayo, Levi Garraway. A melanoma transcriptional state distinction influences sensitivity to MAPK pathway inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR04.