Showing papers by "Pablo Tamayo published in 2012"

Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations

Abstract: Medulloblastomas are the most common malignant brain tumours in children. Identifying and understanding the genetic events that drive these tumours is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma on the basis of transcriptional and copy number profiles. Here we use whole-exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas have low mutation rates consistent with other paediatric tumours, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were newly identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR and LDB1. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant, but not wild-type, β-catenin. Together, our study reveals the alteration of WNT, hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic β-catenin signalling in medulloblastoma.

Prognostically relevant gene signatures of high-grade serous ovarian carcinoma

Abstract: Because of the high risk of recurrence in high-grade serous ovarian carcinoma (HGS-OvCa), the development of outcome predictors could be valuable for patient stratification. Using the catalog of The Cancer Genome Atlas (TCGA), we developed subtype and survival gene expression signatures, which, when combined, provide a prognostic model of HGS-OvCa classification, named "Classification of Ovarian Cancer" (CLOVAR). We validated CLOVAR on an independent dataset consisting of 879 HGS-OvCa expression profiles. The worst outcome group, accounting for 23% of all cases, was associated with a median survival of 23 months and a platinum resistance rate of 63%, versus a median survival of 46 months and platinum resistance rate of 23% in other cases. Associating the outcome prediction model with BRCA1/BRCA2 mutation status, residual disease after surgery, and disease stage further optimized outcome classification. Ovarian cancer is a disease in urgent need of more effective therapies. The spectrum of outcomes observed here and their association with CLOVAR signatures suggests variations in underlying tumor biology. Prospective validation of the CLOVAR model in the context of additional prognostic variables may provide a rationale for optimal combination of patient and treatment regimens.

Targeted Tumor-Penetrating siRNA Nanocomplexes for Credentialing the Ovarian Cancer Oncogene ID4

Abstract: .The comprehensive characterization of a large number of cancer genomes will eventually lead to a compendium of genetic alterations in specific cancers. Unfortunately, the number and complexity of identified alterations complicate endeavors to identify biologically relevant mutations critical for tumor maintenance because many of these targets are not amenable to manipulation by small molecules or antibodies. RNA interference provides a direct way to study putative cancer targets; however, specific delivery of therapeutics to the tumor parenchyma remains an intractable problem. We describe a platform for the discovery and initial validation of cancer targets, composed of a systematic effort to identify amplified and essential genes in human cancer cell lines and tumors partnered with a novel modular delivery technology. We developed a tumor-penetrating nanocomplex (TPN) that comprised small interfering RNA (siRNA) complexed with a tandem tumor-penetrating and membrane-translocating peptide, which enabled the specific delivery of siRNA deep into the tumor parenchyma. We used TPN in vivo to evaluateinhibitor of DNA binding 4 (ID4) as a novel oncogene. Treatment of ovarian tumor– bearing mice with ID4-specific TPN suppressed growth of established tumors and significantly improved survival. These observations not only credential ID4 as an oncogene in 32% of high-grade ovarian cancers but also provide a framework for the identification, validation, and understanding of potential therapeutic cancer targets.

Twist1 suppresses senescence programs and thereby accelerates and maintains mutant Kras-induced lung tumorigenesis.

Abstract: KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with KrasG12D to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

An RNA Profile Identifies Two Subsets of Multiple Sclerosis Patients Differing in Disease Activity

Abstract: The multiple sclerosis (MS) patient population is highly heterogeneous in terms of disease course and treatment response. We used a transcriptional profile generated from peripheral blood mononuclear cells to define the structure of an MS patient population. Two subsets of MS subjects (MSA and MSB) were found among 141 untreated subjects. We replicated this structure in two additional groups of MS subjects treated with one of the two first-line disease-modifying treatments in MS: glatiramer acetate (GA) (n = 94) and interferon-β (IFN-β) (n = 128). One of the two subsets of subjects (MSA) was distinguished by higher expression of molecules involved in lymphocyte signaling pathways. Further, subjects in this MSA subset were more likely to have a new inflammatory event while on treatment with either GA or IFN-β (P = 0.0077). We thus report a transcriptional signature that differentiates subjects with MS into two classes with different levels of disease activity.

MicroSCALE Screening Reveals Genetic Modifiers of Therapeutic Response in Melanoma

Abstract: Cell microarrays are a promising tool for performing large-scale functional genomic screening in mammalian cells at reasonable cost, but owing to technical limitations they have been restricted for use with a narrow range of cell lines and short-term assays. Here, we describe MicroSCALE (Microarrays of Spatially Confined Adhesive Lentiviral Features), a cell microarray-based platform that enables application of this technology to a wide range of cell types and longer-term assays. We used MicroSCALE to uncover kinases that when overexpressed partially desensitized B-RAFV600E-mutant melanoma cells to inhibitors of the mitogen-activated protein kinase kinase kinase (MAPKKK) RAF, the MAPKKs MEK1 and 2 (MEK1/2, mitogen-activated protein kinase kinase 1 and 2), mTOR (mammalian target of rapamycin), or PI3K (phosphatidylinositol 3-kinase). These screens indicated that cells treated with inhibitors acting through common mechanisms were affected by a similar profile of overexpressed proteins. In contrast, screens involving inhibitors acting through distinct mechanisms yielded unique profiles, a finding that has potential relevance for small-molecule target identification and combination drugging studies. Further, by integrating large-scale functional screening results with cancer cell line gene expression and pharmacological sensitivity data, we validated the nuclear factor κB pathway as a potential mediator of resistance to MAPK pathway inhibitors. The MicroSCALE platform described here may enable new classes of large-scale, resource-efficient screens that were not previously feasible, including those involving combinations of cell lines, perturbations, and assay outputs or those involving limited numbers of cells and limited or expensive reagents.

Epigenetic inactivation of the tumor suppressor BIN1 drives proliferation of SNF5-deficient tumors

Abstract: Emerging evidence demonstrates that subunits of the SWI/SNF chromatin remodeling complex are specifically mutated at high frequency in a variety of human cancer types. SNF5 (SMARCB1/INI1/BAF47), a core subunit of the SWI/SNF complex, is inactivated in the vast majority of rhabdoid tumors (RT), an aggressive type of pediatric cancer. SNF5-deficient cancers are diploid and genomically stable, suggesting that epigenetically based changes in transcription are key drivers of tumor formation caused by SNF5 loss. However, there is limited understanding of the target genes that drive cancer formation following SNF5 loss. Here we performed comparative expression analyses upon three independent SNF5-deficient cancer data sets from both human and mouse and identify downregulation of the BIN1 tumor suppressor as a conserved event in primary SNF5-deficient cancers. We show that SNF5 recruits the SWI/SNF complex to the BIN1 promoter, and that the marked reduction of BIN1 expression in RT correlates with decreased SWI/SNF occupancy. Functionally, we demonstrate that re-expression of BIN1 specifically compromises the proliferation of SNF5-deficient RT cell lines. Identification of BIN1 as a SNF5 target gene reveals a novel tumor suppressive regulatory mechanism whose disruption can drive cancer formation.

Prediction of response to therapy with ezatiostat in lower risk myelodysplastic syndrome

Abstract: Approximately 70% of all patients with myelodysplastic syndrome (MDS) present with lower-risk disease. Some of these patients will initially respond to treatment with growth factors to improve anemia but will eventually cease to respond, while others will be resistant to growth factor therapy. Eventually, all lower-risk MDS patients require multiple transfusions and long-term therapy. While some patients may respond briefly to hypomethylating agents or lenalidomide, the majority will not, and new therapeutic options are needed for these lower-risk patients. Our previous clinical trials with ezatiostat (ezatiostat hydrochloride, Telentra®, TLK199), a glutathione S-transferase P1-1 inhibitor in clinical development for the treatment of low- to intermediate-risk MDS, have shown significant clinical activity, including multilineage responses as well as durable red-blood-cell transfusion independence. It would be of significant clinical benefit to be able to identify patients most likely to respond to ezatiostat before therapy is initiated. We have previously shown that by using gene expression profiling and grouping by response, it is possible to construct a predictive score that indicates the likelihood that patients without deletion 5q will respond to lenalidomide. The success of that study was based in part on the fact that the profile for response was linked to the biology of the disease. RNA was available on 30 patients enrolled in the trial and analyzed for gene expression on the Illumina HT12v4 whole genome array according to the manufacturer’s protocol. Gene marker analysis was performed. The selection of genes associated with the responders (R) vs. non-responders (NR) phenotype was obtained using a normalized and rescaled mutual information score (NMI). We have shown that an ezatiostat response profile contains two miRNAs that regulate expression of genes known to be implicated in MDS disease pathology. Remarkably, pathway analysis of the response profile revealed that the genes comprising the jun-N-terminal kinase/c-Jun molecular pathway, which is known to be activated by ezatiostat, are under-expressed in patients who respond and over-expressed in patients who were non-responders to the drug, suggesting that both the biology of the disease and the molecular mechanism of action of the drug are positively correlated.

Integrative functional genomics identifies RINT1 as a novel GBM oncogene

Abstract: Large-scale cancer genomics efforts are identifying hundreds of somatic genomic alterations in glioblastoma (GBM). Distinguishing between active driver and neutral passenger alterations requires functional assessment of each gene; therefore, integrating biological weight of evidence with statistical significance for each genomic alteration will enable better prioritization for downstream studies. Here, we demonstrate the feasibility and potential of in vitro functional genomic screens to rapidly and systematically prioritize high-probability candidate genes for in vivo validation. Integration of low-complexity gain- and loss-of-function screens designed on the basis of genomic data identified 6 candidate GBM oncogenes, and RINT1 was validated as a novel GBM oncogene based on its ability to confer tumorigenicity to primary nontransformed murine astrocytes in vivo. Cancer genomics-guided low-complexity genomic screens can quickly provide a functional filter to prioritize high-value targets for further downstream mechanistic and translational studies.

MicroSCALE Screening Reveals Genetic Modifiers of Therapeutic Response in Melanoma

Abstract: Broad Institute of MIT and Harvard (Scientific Planning and Allocation of Resources Committee Grant)

Targeted Tumor-Penetrating siRNA Nanocomplexes for Credentialing the Ovarian Cancer Target ID4

Abstract: The comprehensive characterization of a large number of cancer genomes will eventually lead to a compendium of genetic alterations in specific cancers. Unfortunately, the number and complexity of identified alterations complicate endeavors to identify biologically relevant mutations critical for tumor maintenance, because many of these targets are not amenable to manipulation by small molecules or antibodies. RNAi provides a direct way to study putative cancer targets; however, †To whom correspondence should be addressed. william_hahn@dfci.harvard.edu (W.H.C.); sbhatia@mit.edu (S.N.B.).. *These authors contributed equally. Author contributions: Y.R., H.W.C., G.S.C., B.A.W., J.S.B., D.E.R., W.C.H., and S.N.B. designed the experiments. Y.R., G.V.M., A.A., and S.N.B. designed the peptide screen. Y.R. characterized TPN. G.S.C., J.S.B., B.A.W., D.E.R., and W.C.H. supervised RNAi screens. B.A.W., J.S.B., H.W.C., G.S.C., R.D., P.T., J.P.M., D.E.R., and W.C.H. analyzed the RNAi data. H.W.C., and A.M.K. performed experiments with transformation and shRNAs. B.A.W., H.W.C., and J.S.B. performed expression profiling and analysis. Y.R., H.W.C., W.C.H., and S.N.B. analyzed the integrated RNAi data. J.L. assisted with biodistribution studies. M.S.H. and J.F.L. analyzed the tissue-microarray. V.F. and E.R. provided advice, reagents and analysis on tumor targeting aspects of the work. Y.R., H.W.C., W.C.H., and S.N.B. wrote the manuscript. All authors discussed the results and commented on the manuscript. Competing interests: E.R. is a shareholder of CendR Inc, which licenses the iRGD peptide. A patent has been filed by M.I.T. describing the tumor-penetrating nanocomplexes. Data and materials availability: All data files can be assessed at the Integrative Genomics portal (http://www.broadinstitute.org/IGP/ home). An MTA is required for distribution of the primary FT epithelial cells (A.M.K). NIH Public Access Author Manuscript Sci Transl Med. Author manuscript; available in PMC 2013 April 23. Published in final edited form as: Sci Transl Med. 2012 August 15; 4(147): 147ra112. doi:10.1126/scitranslmed.3003778. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript specific delivery of therapeutics to the tumor parenchyma remains an intractable problem. We describe a platform for the discovery and initial validation of cancer targets, composed of a systematic effort to identify amplified and essential genes in human cancer cell lines and tumors partnered with a novel modular delivery technology. We developed a tumor-penetrating nanocomplex (TPN) comprised of siRNA complexed with a tandem tumor-penetrating and membrane-translocating peptide, which enabled the specific delivery of siRNA deep into the tumor parenchyma. We employed TPN in vivo to evaluate inhibitor of DNA binding 4 (ID4) as a novel oncogene. Treatment of ovarian tumor-bearing mice with ID4-specific TPN suppressed growth of established tumors and significantly improved survival. These observations not only credential ID4 as an oncogene in 32% of high-grade ovarian cancers, but also provide a framework for the identification, validation, and understanding of potential therapeutic cancer targets.

Abstract 4868: Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations within a wide spectrum of genetic heterogeneity

Abstract: Medulloblastoma is a pediatric brain tumor with poor overall survival and adverse long-term effects from current surgical and radiation treatments. Our group has recently used expression and copy number analysis to define six medulloblastoma subtypes, c1-c6. To assess whether these subgroups contain somatic mutations that encode potential therapeutic or diagnostic targets, we performed whole exome hybrid capture and Illumina sequencing of 94 tumor/normal pairs. For each sample, we sequenced 193,094 exons from 18,863 genes to 126X average coverage. Tumors contained a median 5 silent and 11 non-silent candidate mutations, corresponding to 0.34 non-silent mutations per megabase, a low mutation rate consistent with other pediatric tumors. Overall, 16 genes were mutated at statistically significant frequency and several clustered within subtypes. Eleven tumors harbored mutually-exclusive, likely loss-of-function missense mutations within the helicase domains of candidate oncogene DDX3X (n=7) or SMARCA4 (n=4). These mutations were found in five of seven c6 tumors (3 DDX3X, 2 SMARCA4) of which four had known beta-catenin mutations. Two SMARCA4 mutations were identical and two affected adjacent residues. None of the DDX3X mutations were recurrent, however several are proximal when mapped to a tertiary protein model. To confirm these variants, we are performing deep sequencing of these genes using multiplex PCR (Fluidigm) followed by single-molecule real-time sequencing (PacBio). We are also characterizing the functional effect of DDX3X and SMARCA4 mutations alone and in combination with beta-catenin mutations in medulloblastoma cell lines. Other genes with subtype-associated, loss-of-function mutations include DULLARD in 2 of 9 c1 tumors, PTCH1 in 5 of 16 c3 tumors, and MLL2 in 2 of 3 tumors without any copy number alterations as well as a c1 tumor and two c3 tumors. Several genes are mutated across subtypes, notably tumor suppressors TP53 (n=3), GPS2 (n=3) and SOCS4 (n=2). 19 chromatin remodeling genes including cancer genes BRCA2, KDM5/6A, CREBBP, EP300, BRD4, and MLL3/4 are mutated across 17 tumors from all subtypes. While we have uncovered several subtype-associated mutations, 88% of mutated genes are only altered in a single tumor. This analysis demonstrates the diversity of somatic mutation in medulloblastoma, even within copy number subtypes. To better understand infrequently mutated genes, we will attempt to assemble them into commonly altered gene sets and pathways. We also intend to investigate potentially pathogenic germline variations in each case. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4868. doi:1538-7445.AM2012-4868

Prediction of response to therapy with ezatiostat in lower risk myelodysplastic syndrome

Abstract: Approximately 70% of all patients with myelodysplastic syndrome (MDS) present with lower-risk disease. Some of these patients will initially respond to treatment with growth factors to improve anemia but will eventually cease to respond, while others will be resistant to growth factor therapy. Eventually, all lower-risk MDS patients require multiple transfusions and long-term therapy. While some patients may respond briefly to hypomethylating agents or lenalidomide, the majority will not, and new therapeutic options are needed for these lower-risk patients. Our previous clinical trials with ezatiostat (ezatiostat hydrochloride, Telentra®, TLK199), a glutathione S-transferase P1-1 inhibitor in clinical development for the treatment of low- to intermediate-risk MDS, have shown significant clinical activity, including multilineage responses as well as durable red-blood-cell transfusion independence. It would be of significant clinical benefit to be able to identify patients most likely to respond to ezatiostat before therapy is initiated. We have previously shown that by using gene expression profiling and grouping by response, it is possible to construct a predictive score that indicates the likelihood that patients without deletion 5q will respond to lenalidomide. The success of that study was based in part on the fact that the profile for response was linked to the biology of the disease. RNA was available on 30 patients enrolled in the trial and analyzed for gene expression on the Illumina HT12v4 whole genome array according to the manufacturer’s protocol. Gene marker analysis was performed. The selection of genes associated with the responders (R) vs. non-responders (NR) phenotype was obtained using a normalized and rescaled mutual information score (NMI). We have shown that an ezatiostat response profile contains two miRNAs that regulate expression of genes known to be implicated in MDS disease pathology. Remarkably, pathway analysis of the response profile revealed that the genes comprising the jun-N-terminal kinase/c-Jun molecular pathway, which is known to be activated by ezatiostat, are under-expressed in patients who respond and over-expressed in patients who were non-responders to the drug, suggesting that both the biology of the disease and the molecular mechanism of action of the drug are positively correlated.

Abstract 1313: Mutually exclusive somatic mutations in WNT pathway medulloblastomas reveal a critical interaction between DDX3X and SMARCA4

Abstract: Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL DDX3X is an ATP-dependent RNA helicase that belongs to an extended family of D-E-A-D motif (DEAD-box) containing proteins whose functions have been implicated in key biological processes ranging from RNA transcription and splicing to the regulation of translation initiation. Through whole exome sequencing of 94 primary medulloblasotmas, we have identified DDX3X as one of the most common recurrently mutated genes in medulloblastoma. These mutations predominantly occur in WNT pathway medulloblastomas which concurrently harbor stabilized forms of mutant beta-catenin. Interestingly, we have also identified SMARCA4 mutations in WNT pathway tumors that are mutually exclusive of DDX3X mutations. In addition, two WNT pathway tumors that have neither DDX3X nor SMARCA4 gene mutations have mutations in genes known to encode for direct binding partners of DDX3X and SMARCA4 (EIF3E and ACTL6A, respectively). This mutual exclusivity of DDX3X and SMARCA4 mutations suggests either a direct interaction between the DDX3X and SMARCA4 gene products or an overlap of the biological processes regulated by each. Here we reveal the direct interaction between DDX3X and SMARCA4 in normal physiological conditions and further investigate the impact of somatic mutations in DDX3X and SMARCA4 on this interaction in WNT pathway associated medulloblastomas. Our results point to a critical link between RNA helicases and the SWI/SNF chromatin remodeling complex in WNT/beta-catenin signaling and disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1313. doi:1538-7445.AM2012-1313