Cancer genome data has been growing in both size and complexity, primarily driven by advances in ... more Cancer genome data has been growing in both size and complexity, primarily driven by advances in next-generation sequencing technologies, such as Pan-cancer data from TCGA, ICGC, and single-cell sequencing. Yet, discerning the functional role of individual genomic lesions remains a substantial challenge due to the complexity and scale of the data. Previously, we introduced REVEALER, which identifies groups of genomic alterations that significantly associate with target functional profiles or phenotypes, such as pathway activation, gene dependency, or drug response. In this paper, we present a new mathematical formulation of the algorithm. This version (REVEALER 2.0) is considerably more powerful than the original, allowing for rapid processing and analysis of much larger datasets and facilitating higher-resolution discoveries at the level of individual alleles. REVEALER 2.0 employs the Conditional Information Coefficient (CIC) to pinpoint features that are either complementary or mu...
International Journal of Radiation Oncology Biology Physics, Sep 1, 2014
embryo fibroblasts (MEFs). The stress response to ionizing radiation AE YTR107 was assessed in wi... more embryo fibroblasts (MEFs). The stress response to ionizing radiation AE YTR107 was assessed in wild-type and NPM-null MEFs and 6 NSCLC cell lines by quantifying pNPM1, gamma-H2AX, and Rad51 foci, neutral comet tail moment, and colony formation. In vivo efficacy of YTR107 radiosensitization was tested in a syngeneic xenograft model of tumor growth delay. NPM1 levels in a human-derived NSCLC tissue microarray (TMA) were determined by immunohistochemistry. Results: We observed that proliferating NPM1-null MEFs, but not wildtype MEFs, have high levels of gamma-H2AX foci, a characteristic of replication stress. In addition, NPM1-null MEFs exhibit increased radiation sensitivity via a defect in Rad51-mediated repair of DNA DSBs. We further determined that YTR107-mediated radiosensitization requires NPM1. The DNA damage response pathway is continuously activated in NSCLC due to constant replication stress. Because NPM1 expression minimizes replication stress, we further hypothesized that NSCLC tumors may have significant expression of NPM1. This was validated by analyzing a NSCLC TMA that demonstrated very low NPM1 expression in matched normal lung parenchyma but significantly increased NPM1 expression in Stage III NSCLC cores. We then observed that YTR107-mediated targeting of NPM1 resulted in radiosensitization in 6 NSCLC cell lines in vitro and in a syngeneic tumor model in vivo. Conclusions: These data demonstrate genetic evidence of NPM1 contribution to repair of radiation-induced DNA damage and suggest that NPM1 represents a potential therapeutic target for radiosensitization of NSCLC.
International Journal of Radiation Oncology*Biology*Physics, 2015
Purpose/Objective(s): 11 C-acetate positron emission tomography (PET) imaging allows for the dete... more Purpose/Objective(s): 11 C-acetate positron emission tomography (PET) imaging allows for the detection of occult metastatic disease that may otherwise go undetected with standard imaging for prostate cancer. The aim of this study was to evaluate the ability of the standard Radiation Therapy Oncology Group (RTOG) pelvic radiation field to cover all sites of lymph node disease in patients with node-positive prostate cancer as determined by 11 C-acetate PET imaging. Materials/Methods: A retrospective analysis was conducted on 125 male patients with prostate cancer who underwent 11 C-acetate PET scans at our institution between 2007 and 2014. Patients with non-lymph node metastatic disease and/or those who were negative for nodal disease on PET imaging, as determined by the interpreting radiologist, were excluded from this study. On the node-positive scans, individual lymph nodes were characterized by location, size on corresponding computed tomography-based attenuation correction (CTAC) imaging, and standardized uptake value (SUV) on 11 C-acetate PET. Anatomic location of each positive node was then transposed to the standard RTOG pelvic radiation field. Results: A total of 176 11 C-acetate PET scans (range: 1e5 scans) on 125 men were reviewed. The majority of patients were white (123, 98%) with a mean age of 66 years. Of the 176 scans, 55 scans (from 53 men) met criteria for inclusion in the study. Thirty-six patients had a prior prostatectomy (68%), 9 had brachytherapy (16%), and 8 had external beam radiotherapy (15%). Median prostate-specific antigen at diagnosis was 8.23 (1.41e85). Gleason score (GS) was available for 45 patients: 11 were GS 6 or less (24%), 19 were GS 7 (42%), 7 were GS 8 (16%), and 8 were GS 9e10 (18%). The 55 included scans contained a total of 161 positive lymph nodes. Of the positive individual nodes, 76.5% were less than 1 cm. The most frequent sites of lymph node involvement were in the distribution of the external iliacs (33.5%), common iliacs (16.8%), and para-aortics (18.0%). Additionally, 11.2% of positive nodes were identified as nodes of Cloquet. In relation to the RTOG pelvic radiation field, 48.4% of positive nodes were within the treatment field while 51.6% fell outside the field. Of the nodes that were outside of the radiation field, 34.9% were para-aortic, 22.9% were proximal common iliac, 15.7% were distal external iliac, and 21.7% were nodes of Cloquet. Conclusion: Based on 11 C-acetate PET imaging, the RTOG pelvic radiation field will miss nearly half of all positive lymph nodes. Clinicians should use caution when using the standard RTOG contouring atlas in patients with node-positive prostate cancer. 11 C-acetate PET imaging may be useful in defining target volumes for patients with node-positive prostate cancer.
A resampling-based method for class discovery and visualization of gene expression microarray data
In this paper we present a new methodology of class discovery and clustering validation tailored ... more In this paper we present a new methodology of class discovery and clustering validation tailored to the task of analyzing gene expression data. The method can best be thought of as an analysis approach, to guide and assist in the use of any of a wide range of available clustering algorithms. We call the new methodology consensus clustering, and in conjunction with resampling techniques, it provides for a method to represent the consensus across multiple runs of a clustering algorithm and to assess the stability of the discovered clusters. The method can also be used to represent the consensus over multiple runs of a clustering algorithm with random restart (such as K-means, model-based Bayesian clustering, SOM, etc.), so as to account for its sensitivity to the initial conditions. Finally, it provides for a vi- sualization tool to inspect cluster number, membership, and boundaries. We present the results of our experiments on both simulated data and real gene expression data aimed a...
MDSCs are immune cells of myeloid lineage that plays a key role in promoting tumor growth. The ex... more MDSCs are immune cells of myeloid lineage that plays a key role in promoting tumor growth. The expansion of MDSCs in tumor-bearing hosts reduces the efficacy of checkpoint inhibitors and CAR-T therapies, and hence strategies that deplete or block the recruitment of MDSCs have shown benefit in improving responses to immunotherapy in various cancers, including NB. Ibrutinib, an irreversible molecular inhibitor of BTK, has been widely studied in B cell malignancies, and recently, this drug is repurposed for the treatment of solid tumors. Herein we report that BTK is highly expressed in both granulocytic and monocytic murine MDSCs isolated from mice bearing NB tumors, and its increased expression correlates with a poor relapse-free survival probability of NB patients. Moreover, in vitro treatment of murine MDSCs with ibrutinib altered NO production, decreased mRNA expression of Ido, Arg, Tgfβ, and displayed defects in T-cell suppression. Consistent with these findings, in vivo inhibitio...
Hundreds of genetically characterized cell lines are available for the discovery of genotype-spec... more Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo.
Proceedings of the National Academy of Sciences, 2005
Snf5 (Ini1/Baf47/Smarcb1), a core member of the Swi/Snf chromatin remodeling complex, is a potent... more Snf5 (Ini1/Baf47/Smarcb1), a core member of the Swi/Snf chromatin remodeling complex, is a potent tumor suppressor whose mechanism of action is largely unknown. Biallelic loss of Snf5 leads to the onset of aggressive cancers in both humans and mice. We have developed an innovative and widely applicable analytical technique for cross-species validation of cancer models and show that the gene expression profiles of our Snf5 murine models closely resemble those of human Snf5-deficient rhabdoid tumors. We exploit this system to produce what we believe to be the first report documenting the effects on gene expression of inactivating a Swi/Snf subunit in normal mammalian cells and to identify the transcriptional pathways regulated by Snf5. We demonstrate that the tumor suppressor activity of Snf5 depends on its regulation of cell cycle progression; Snf5 inactivation leads to aberrant up-regulation of E2F targets and increased levels of p53 that are accompanied by apoptosis, polyploidy, an...
Systematic efforts to sequence the cancer genome have identified large numbers of mutations and c... more Systematic efforts to sequence the cancer genome have identified large numbers of mutations and copy number alterations in human cancers. However, elucidating the functional consequences of these variants, and their interactions to drive or maintain oncogenic states, remains a challenge in cancer research. We developed REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene dependency of oncogenic pathways or sensitivity to a drug treatment. We used REVEALER to uncover complementary genomic alterations associated with the transcriptional activation of β-catenin and NRF2, MEK-inhibitor sensitivity, and KRAS dependency. REVEALER successfully identified both known and new associations, demonstrating the power of combining functional profiles with extensive characterization of genomic alterations in cancer genomes.
Systematic efforts to sequence the cancer genome have identified large numbers of mutations and c... more Systematic efforts to sequence the cancer genome have identified large numbers of mutations and copy number alterations in human cancers. However, elucidating the functional consequences of these variants, and their interactions to drive or maintain oncogenic states, remains a challenge in cancer research. We developed REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene dependency of oncogenic pathways or sensitivity to a drug treatment. We used REVEALER to uncover complementary genomic alterations associated with the transcriptional activation of β-catenin and NRF2, MEK-inhibitor sensitivity, and KRAS dependency. REVEALER successfully identified both known and new associations, demonstrating the power of combining functional profiles with extensive characterization of genomic alterations in cancer genomes.
In a substantial fraction of cancers TERT promoter (TERTp) mutations drive expression of the cata... more In a substantial fraction of cancers TERT promoter (TERTp) mutations drive expression of the catalytic subunit of telomerase, contributing to their proliferative immortality. We conducted a pan-cancer analysis of cell lines and find a TERTp mutation expression signature dominated by epithelial-to-mesenchymal transition (EMT) and MAPK signaling. These data indicate that TERTp mutants are likely to generate distinctive tumor microenvironments and intercellular interactions. Analysis of high throughput screening tests of 546 small molecules on cell line growth indicated that TERTp mutants displayed heightened sensitivity to specific drugs, including RAS pathway inhibitors, and we found that inhibition of MEK1 and 2, key RAS/MAPKpathway effectors, inhibited TERT mRNA expression. Consistent with an enrichment of mesenchymal states in TERTp mutants, cell lines and some patient tumors displayed low expression of the central adherens junction protein E-cadherin, and we provide evidence that its expression in these cells is regulated by MEK1/2. Several mesenchymal transcription factors displayed elevated expression in TERTp mutants including ZEB1 and 2, TWIST1 and 2 and SNAI1. Of note, the developmental transcription factor SNAI2/SLUG was conspicuously elevated in a significant majority of TERTp mutant cell lines, and knockdown experiments suggest that it promotes TERT expression. Implications: Cancers harboring TERT promoter mutations are often more lethal, but the basis for this higher mortality remains unknown. Our study identifies that TERTp mutants, as a class, associate with a distinct gene and protein expression signature likely to impact their biological and clinical behavior and provide new directions for investigating treatment approaches for these cancers.
The systematic sequencing of the cancer genome has led to the identification of numerous genetic ... more The systematic sequencing of the cancer genome has led to the identification of numerous genetic alterations in cancer. However, a deeper understanding of the functional consequences of these alterations is necessary to guide appropriate therapeutic strategies. Here, we describe Onco-GPS (OncoGenic Positioning System), a data-driven analysis framework to organize individual tumor samples with shared oncogenic alterations onto a reference map defined by their underlying cellular states. We applied the methodology to the RAS pathway and identified nine distinct components that reflect transcriptional activities downstream of RAS and defined several functional states associated with patterns of transcriptional component activation that associates with genomic hallmarks and response to genetic and pharmacological perturbations. These results show that the Onco-GPS is an effective approach to explore the complex landscape of oncogenic cellular states across cancers, and an analytic frame...
Transcriptional profiling of 60 human cancer lines has been used to assess chemosensitivity to 23... more Transcriptional profiling of 60 human cancer lines has been used to assess chemosensitivity to 232 chemical compounds. Significance and context Pharmacogenomics aims to predict an individual patient's response to particular drugs on the basis of their genotype, in order to design tailor-made treatments. These are particularly needed in cancer, where the ease with which drug resistance develops is most variable and unpredictable. A known effective drug is expected to kill the tumor, but most of these drugs can also have serious side effects on the patient's metabolism. The development of oligonucleotide microarrays that enable analysis of the expression of many genes simultaneously will make it easier to predict which cellular functions are likely to be affected by a drug. Key results Staunton et al. have developed a new algorithm for analyzing gene expression with respect to the drug sensitivity of the cells. The authors combined information from the analysis of gene expression in untreated cells and from drug-sensitivity testing (232 drugs out of 5,084 initially assayed) in 60 tumor cell lines to find sets of genes (classifiers) that describe a cell's sensitivity. Generation of the classifiers was based on training sets, and experimental results from the remaining cell lines were used to test the accuracy of the predictions. Statistical analysis showed that the classification based on gene expression is significantly nonrandom. The validity of the classifiers was evaluated on a test set with 38% of the expression-based classifiers performing with an accuracy of 64-92%. This indicates that there is a significant group of drugs for which expression data have adequate predictive power.
Microarray DNA chip technology has been used for the first time to demonstrate the feasibility of... more Microarray DNA chip technology has been used for the first time to demonstrate the feasibility of cancer classification on the basis of gene-expression profile analysis. Significance and context New modalities for cancer therapy will be derived from the development of treatment strategies that are specifically adapted to the tumor type, thereby maximizing efficacy and minimizing toxicity. This requires improved methods for cancer classification. At present, classification is carried out by histopathological analysis of tumor samples, using techniques that are time-consuming and imperfect. Improved classification and subclass definition will depend on the identification of sets of molecular markers. Golub and colleagues from the Lander lab at the Massachusetts Institute of Technology have applied microarray technology to cancer-class discovery and class prediction. They chose human acute leukemias as a test case for such an approach. Acute leukemias can be divided into those of lymphoid (ALL) or myeloid (AML) origin. ALL and AML are currently diagnosed by immunochemistry and cytogenetic analysis and require distinct clinical treatment protocols. This study clearly shows that future cancer classification could be based on gene-expression profiles.
High-Throughput RNA Interference Screening Identifies Synthetic Lethality Between Oncogenic KRAS Dependency and Suppression of STK33
Blood
Activating RAS mutations are among the most common pathogenetic events in a broad spectrum of hem... more Activating RAS mutations are among the most common pathogenetic events in a broad spectrum of hematologic malignancies and epithelial tumors. However, oncogenic RAS has thus far not proven to be a tractable target for therapeutic intervention. An alternative to direct targeting of known oncogenes is to perform “synthetic lethality” screens to identify genes that are selectively required for cell viability in the context of specific cancer-causing mutations. Using this approach, we have discovered a synthetic lethal interaction between mutant KRAS, the most frequently mutated oncogene in human cancer, and inactivation of the gene encoding the STK33 serine/threonine protein kinase. To identify genes that are essential for cell viability in the context of mutant KRAS, we performed high-throughput loss-of-function RNA interference (RNAi) screens in eight human cancer cell lines (mutant KRAS, n=4; wildtype KRAS, n=4), representing seven different tumor types (acute myeloid leukemia, mult...
Gene Expression Studies May Identify Lower Risk Myelodysplastic Syndrome Patients Likely to Respond to Therapy with Ezatiostat Hydrochloride (TLK199)
Blood
2779 Interpretation of gene expression studies in MDS have been especially challenging due to the... more 2779 Interpretation of gene expression studies in MDS have been especially challenging due to the heterogeneity of the cell lineages that comprise the malignant clone. In attempting to overcome these difficulties we have used a bedside-to-bench approach to define an expression signature that may identify patients likely to respond. Ezatiostat hydrochloride (TLK199) is an inhibitor of glutathione S-transferase, an enzyme that is over expressed in many cancers, and has been shown in vitro to stimulate growth and differentiation of hematopoietic progenitor cells and to induce apoptosis in leukemia cells. Based on multilineage responses in low-Int1 MDS patients in our phase 2 study of oral TLK199, a multi institutional phase 2 study was conducted in low-Int1 patients. Response was evaluated by International Working Group (IWG 2006) criteria. Pre-therapy bone marrow mononuclear cells of patients treated with TLK199 were analyzed for gene expression on the Illumina HT12v4 whole genome arr...
An Erythroid Differentiation Gene Expression Signature Predicts Response to Lenalidomide in Myelodysplasia
Blood
Lenalidomide is an effective new agent for the treatment of Myelodysplastic Syndrome (MDS). In ph... more Lenalidomide is an effective new agent for the treatment of Myelodysplastic Syndrome (MDS). In phase II clinical trials, patients treated with Lenalidomide achieved unprecedented rates of hematologic and cytogenetic responses, particularly in patients with interstitial deletions of chromosome 5q. More than 80% of patients with MDS do not have 5q deletions, though approximately 25% of these patients respond to Lenalidomide.3–5 Neutropenia and thrombocytopenia are the primary toxicities of Lenalidomide, and are severe in some cases. MDS is a highly heterogeneous disorder, and no diagnostic assays predict which patients without 5q deletions will respond to Lenalidomide. Using gene expression profiling, we identified a molecular signature that predicts Lenalidomide response. Biologic characterization of the predictive genes demonstrated that the response signature is comprised of a cohesive set of erythroid-specific genes: patients who respond have a lower expression of the erythroid si...
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Papers by PABLO TAMAYO