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Grant Details

Grant Number: 5R21CA177519-03 Interpret this number
Primary Investigator: Harismendy, Olivier
Organization: University Of California, San Diego
Project Title: In Vivo Detection and Genome-Wide Location Analysis of DNA-Adducts
Fiscal Year: 2015


Abstract

DESCRIPTION (provided by applicant): DNA adducts are the hallmark and most common form of DNA damage in the cell. They result from environmental carcinogen exposure (such as UV) or during chemotherapy using DNA modifying agents like cisplatin (cDDP) or alkylators such as chlorambucil (CLB). While mechanisms underlying sensitivity, agent homeostasis, detoxification, DNA repair and apoptosis, have been well investigated, the central molecular event, the formation of adducts, is not well understood in vivo. Evidence suggests that the epigenetic landscape and the structure of the chromatin influences the formation of adducts and mediates drug sensitivity. Therefore, there is a need to better identify DNA adducts and understand the association between the epigenetic marks in the cell. Currently there is no method to determine the exact location of DNA adducts in vivo nor at a high-resolution across the genome. In order to address this, we propose to develop a method, TdT-Seq, that will identify these adducts genome-wide at the single base pair resolution. The expertise of the investigators include knowledge in cancer biology and platinum drug pharmacology (Drs. Howell and Abada) as well as experience in high-throughput genomic assays and computational analysis (Dr. Harismendy); expertise that will be needed to successfully develop the assay. The TdT-Seq assay relies on adduct-mediated inhibition of the DNA polymerase in vitro. The resulting single strand DNA will be captured by a specific TdT mediated ligation, enriched, then sequenced in high throughput. We propose to establish the technical validity of the assay by determining 1) sensitivity at various cDDP concentrations and read depth, 2) specificity by the development of a locus specific method (Strand Specific Adduct Detection) and independent analysis of 50 adduct loci, and 3) quantativity using increasing cDDP concentrations and known spike-in controls. We will also perform specific experiments to establish TdT-Seq's use for clinical cancer research. In particular, we will optimize the protocol for the identification of UVor chlorambucil (CLB) induced adducts to broaden its applicability. We will also develop the protocol for low amounts of DNA originating from mouse tissues or heterogeneous tissue specimens. Finally, we will analyze the ability of TdT-Seq to measure the kinetics of DNA repair using genetically modified cell lines. TdT-Seq's development will therefore lead to a robust and innovative assay, with demonstrated performance and utility for cancer research. TdT-Seq will generate an entirely new type of data, which can be used in combination of other whole genome datasets from the ENCODE or TCGA consortium to provide a more precise and comprehensive description of the mechanism of DNA damage and repair in vivo in various cell types and cancers. The long-term benefits of such research include the prediction of drug sensitivity or the study of epigenetic modifying compounds to rationalize combinations for optimal drug efficacy.



Publications

Autophagy gene haploinsufficiency drives chromosome instability, increases migration, and promotes early ovarian tumors.
Authors: Delaney J.R. , Patel C.B. , Bapat J. , Jones C.M. , Ramos-Zapatero M. , Ortell K.K. , Tanios R. , Haghighiabyaneh M. , Axelrod J. , DeStefano J.W. , et al. .
Source: PLoS genetics, 2020 01; 16(1), p. e1008558.
EPub date: 2020-01-10.
PMID: 31923184
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Activation of hedgehog signaling associates with early disease progression in chronic lymphocytic leukemia.
Authors: Ghia E.M. , Rassenti L.Z. , Neuberg D.S. , Blanco A. , Yousif F. , Smith E.N. , McPherson J.D. , Hudson T.J. , HALT Pan-Leukemia Gene Panel Consortium , Harismendy O. , et al. .
Source: Blood, 2019-06-20; 133(25), p. 2651-2663.
EPub date: 2019-03-28.
PMID: 30923040
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PinAPL-Py: A comprehensive web-application for the analysis of CRISPR/Cas9 screens.
Authors: Spahn P.N. , Bath T. , Weiss R.J. , Kim J. , Esko J.D. , Lewis N.E. , Harismendy O. .
Source: Scientific reports, 2017-11-20; 7(1), p. 15854.
EPub date: 2017-11-20.
PMID: 29158538
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Biochemical, Molecular, and Clinical Characterization of Succinate Dehydrogenase Subunit A Variants of Unknown Significance.
Authors: Bannon A.E. , Kent J. , Forquer I. , Town A. , Klug L.R. , McCann K. , Beadling C. , Harismendy O. , Sicklick J.K. , Corless C. , et al. .
Source: Clinical cancer research : an official journal of the American Association for Cancer Research, 2017-11-01; 23(21), p. 6733-6743.
EPub date: 2017-07-19.
PMID: 28724664
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Haploinsufficiency networks identify targetable patterns of allelic deficiency in low mutation ovarian cancer.
Authors: Delaney J.R. , Patel C.B. , Willis K.M. , Haghighiabyaneh M. , Axelrod J. , Tancioni I. , Lu D. , Bapat J. , Young S. , Cadassou O. , et al. .
Source: Nature communications, 2017-02-15; 8, p. 14423.
EPub date: 2017-02-15.
PMID: 28198375
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Duodenal-Jejunal Flexure GI Stromal Tumor Frequently Heralds Somatic NF1 and Notch Pathway Mutations.
Authors: Burgoyne A.M. , De Siena M. , Alkhuziem M. , Tang C.M. , Medina B. , Fanta P.T. , Belinsky M.G. , von Mehren M. , Thorson J.A. , Madlensky L. , et al. .
Source: JCO precision oncology, 2017; 2017, .
EPub date: 2017-08-15.
PMID: 29938249
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FGFR1 and NTRK3 actionable alterations in "Wild-Type" gastrointestinal stromal tumors.
Authors: Shi E. , Chmielecki J. , Tang C.M. , Wang K. , Heinrich M.C. , Kang G. , Corless C.L. , Hong D. , Fero K.E. , Murphy J.D. , et al. .
Source: Journal of translational medicine, 2016-12-14; 14(1), p. 339.
EPub date: 2016-12-14.
PMID: 27974047
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Nrf2 Induces IL-17D to Mediate Tumor and Virus Surveillance.
Authors: Saddawi-Konefka R. , Seelige R. , Gross E.T. , Levy E. , Searles S.C. , Washington A. , Santosa E.K. , Liu B. , O'Sullivan T.E. , Harismendy O. , et al. .
Source: Cell reports, 2016-08-30; 16(9), p. 2348-58.
EPub date: 2016-08-18.
PMID: 27545889
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Immune DNA signature of T-cell infiltration in breast tumor exomes.
Authors: Levy E. , Marty R. , Gárate Calderón V. , Woo B. , Dow M. , Armisen R. , Carter H. , Harismendy O. .
Source: Scientific reports, 2016-07-25; 6, p. 30064.
EPub date: 2016-07-25.
PMID: 27452728
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A strategy to combine pathway-targeted low toxicity drugs in ovarian cancer.
Authors: Delaney J.R. , Patel C. , McCabe K.E. , Lu D. , Davis M.A. , Tancioni I. , von Schalscha T. , Bartakova A. , Haft C. , Schlaepfer D.D. , et al. .
Source: Oncotarget, 2015-10-13; 6(31), p. 31104-18.
PMID: 26418751
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BAP1 mutation is a frequent somatic event in peritoneal malignant mesothelioma.
Authors: Alakus H. , Yost S.E. , Woo B. , French R. , Lin G.Y. , Jepsen K. , Frazer K.A. , Lowy A.M. , Harismendy O. .
Source: Journal of translational medicine, 2015-04-16; 13, p. 122.
EPub date: 2015-04-16.
PMID: 25889843
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