Targeted epigenetic silencing of oncogenic Transcription Factors (PQ18)


Principal Investigator: Pilar Blancafort
Abstract: DESCRIPTION (provided by applicant): Epigenetic modifications play a key role in tumor origin and progression. Oncogenic transcription factors (TFs) are frequently over-expressed in breast cancers, while being silenced in normal epithelial cells. TFs can switch entire transcriptional gene cascades, resulting in tumor initiation and progression. Since most TFs do not have intrinsic enzymatic activities and they lack small-molecule-binding pockets, these targets have been refractory to drug design. The oncogenic TFs Sox2 is over-expressed in breast cancers of advanced stage, while the gene is silenced and hyper-methylated in normal epithelial cells. As a stable repressive mark, DNAme catalyzed by DNA-methyltransferases, is regarded as a key player in epigenetic silencing. DNAme orchestrate other epigenetic modifications, shaping the architecture of the promoter and driving chromatin condensation and gene silencing. A hallmark of DNAme is that it is hereditary and thereby transmitted over cell generations. In many developmentally regulated TFs, such as Sox2, DNAme constitute an epigenetic switch, which changes cells from an active mitogenic state towards a G0/G1 arrest and differentiation. In this application, our objective is to target DNAme into the promoter of Sox2, which is highly expressed in breast cancer cell lines, with levels comparable or superior to embryonic stem cells. To direct specific DNAme, we will fuse engineered DNA-binding proteins made of sequence-specific Zinc Finger (ZF) domains with a catalytically active DNA-methyltransferase domain (Dnmt3a). Our objective is to restore the hereditable epigenetic silencing in the Sox2 promoter of the tumor cell in a pattern that is similar to breast epithelial cells. We hypothesize that ZFs-Dnmt3a fusions are able to target DNAme marks into the Sox2 oncogenic promoter, resulting in transmission of these marks over cell generations. This epigenetic memory will be accompanied by the maintenance of the transcriptional silencing and tumor cell growth inhibition. In Aim1 we propose the construction of 6ZF proteins linked to the Dnmt3a and inactive mutants, to assess whether these engineered proteins deposit specific silencing marks into the Sox2 promoter, resulting in oncogenic silencing. In Aim2 we monitor the longevity of the silencing implemented by the 6ZF- silencers. We will express the 6ZF constructs using inducible vectors to "pulse" and "chase" DNAme in cell culture and breast tumor models. Next, to move the technology towards a pre-clinical phase, we will deliver ATF mRNAs using nanoparticles that will be injected in mouse models of breast cancer (Aim 3). While RNAi technology can be used to knock-down oncogenes, its therapeutic effect is transient because of the short-lived time of the small RNA. The significance of this application is the potential of the ZF agent to induce an endogenous epigenetic reprogramming of the target TF, which is expected to maintain the longevity of the therapeutic effect. Thus, this work will be of vital importance to develop stable, inherited, oncogenic silencing methods, to suppress oncogenic expression in tumor cells.
Funding Period: 2012-08-01 - 2016-05-31
more information: NIH RePORT

Research Grants

Detail Information

Research Grants30

  1. The Shelf Live Evaluation of Investigational Dosage Forms
    Jonathan White; Fiscal Year: 2013
    ..This contract is essential for continued assurance of the quality of drugs undergoing clinical investigation for different types of cancer by Cancer Therapeutics Evaluation Program. ..
  2. UAB / UMN SPORE in Pancreatic Cancer
    Donald J Buchsbaum; Fiscal Year: 2013
    ..The application has strong institutional support from UAB and UMN, excellent pancreatic cancer populations and concurrence with federal guidelines. ..
  3. Wisconsin Center of Excellence in Genomics Science
    Michael Olivier; Fiscal Year: 2013
    ..Data, technology, and software will be widely disseminated by multiple mechanisms including licensing and commercialization activities. ..
  4. NK Cells, Their Receptors and Unrelated Donor Transplant
    Jeffrey S Miller; Fiscal Year: 2013
    ..The program also promises to change practice of allogeneic transplantation, to the greater benefit of patients with advanced leukemia. ..
  5. Structural Cell Biology of DNA Repair Machines
    John A Tainer; Fiscal Year: 2013
    ..abstract_text> ..
  6. Signaling and Progression in Prostate Cancer
    Dan Theodorescu; Fiscal Year: 2013
    ..The long-term objective is to translate our understanding of prostate cancer progression mechanisms into the identification of new drug targets and pre-clinical models that recapitulate key aspects of the human disease. ..
  7. University of Maryland Greenebaum Cancer Center Support Grant
    Kevin J Cullen; Fiscal Year: 2013
    ..Reflecting our remarkable and continued growth, UMGCC seeks to renew its CCSG to enhance and expand its efforts in high-quality and clinically relevant cancer research. ..
    Oliver W Press; Fiscal Year: 2013
    ..We anticipate that the investigations described in this application will allow us to maximize the therapeutic efficacy and minimize the toxicity of myeloablative RIT for hematologic malignancies. ..
  9. Role of 11q23 Chromosome Abnormalities in the Causation of Acute Leukemia
    Carlo M Croce; Fiscal Year: 2013
    ..abstract_text> ..
    Rakesh K Jain; Fiscal Year: 2013
    ..abstract_text> ..
  11. Interrogating Epigenetic Changes in Cancer Genomes
    Tim H M Huang; Fiscal Year: 2013
    ..abstract_text> ..
  12. Mechanistic Pharmacology of Anti-Mitotics and Apoptosis Regulation
    Timothy J Mitchison; Fiscal Year: 2013
    ..In aim 4 we will pursue several approaches towards translating mechanistic understanding from aims 1-3 into improved patient care. ..
  13. SPORE in Soft Tissue Sarcoma
    Samuel Singer; Fiscal Year: 2013
    ..abstract_text> ..
  14. The MIT Center for Single-Cell Dynamics in Cancer (SCDC)
    Scott R Manalis; Fiscal Year: 2013
    ..These facilities and all reagents generated by the cores will be made available to other PS-OCs. ..
  15. M. D. Anderson Cancer Center SPORE in Multiple Myeloma
    ROBERT ZYGMUNT ORLOWSKI; Fiscal Year: 2013
    ..abstract_text> ..