December 2012

Curriculum Vitae

Pilar BlancafortAssociate ProfessorBreast Cancer Biology & EpigeneticsSchool of Anatomy, Physiology, and Human Biology M309The University of Western Australia35 Stirling HighwayCrawley, WAAustraliaPhone: +61 8 64883293Fax: +61 8 6488 1051

Education:

Ph.D. in Biochemistry, Universite de Montreal (Canada) supervised by Robert Cedergren. August 5th, 1999.B.Sc., Molecular Biology and Biochemistry, Universitat de Barcelona (Spain), September 1993.

Languages:

CatalanSpanishFrenchEnglishPortuguese (reading only)

Professional Experience:

Associate Professor, Cancer Biology, School of Anatomy, Physiology and Human Biology, The University of Western Australia, September 2012-presentAssociate Professor of Pharmacology, University of North Carolina at Chapel Hill, January 2010-September 2012Assistant Professor of Pharmacology, University of North Carolina at Chapel Hill, January 2005-January 2010Senior Research Associate in Dr. Carlos Barbas III laboratory. Department of Molecular Biology, The Scripps Research Institute, January 2004Research Associate in Dr. Carlos Barbas III laboratory. Department of Molecular Biology, The Scripps Research Institute, August 1999

Editor in Chief, Natural Products against cancer (Versita & Springer), January 2012-presentMember, Center for stem cell therapy and regenerative medicine, UWA, September 2012-presentMember, American Association for Cancer Research, AACR, January 2007Member, Lineberger Comprehensive Cancer Center, January 2005

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Honors and Awards:Provocative question NCI PQ18 R01 award, August 2012, Epigenetic silencing of oncogenic transcription factors in breast cancer.Susan Swerling invited Lecture, “Targeted Therapy for Cancer: Moving Beyond Kinase Inhibitors.” Dana Farber Cancer Institute, Harvard Medical School, May 16, 2013.Winner Research Competition at UWA “Novel anti-cancer peptides for the treatment of basal-like breast cancers”, November 2012Department of Defense Breast Cancer Idea Award, 2010 UCRF Cancer Research Pilot Core Award, 2009UCRF Innovation Research Award, 2008Golfers Against Cancer Research Award, 2008Carolina Center of Cancer Nanotechnology Excellence (C-CCNE) Pilot Grant Award, 2007Department of Defense Breast Cancer Idea Award, 2006University Research Council Award, UNC-CH, 2005American Lung Discovery Award, 2005GI SPORE Research Award, 2005V-Foundation Award for Cancer Research (breast cancer fellowship in honor of Julie Stewart), 2005Winner, Simon-Pierre Noel Price for the best graduate student presentation in the department of Biochemistry, University of Montreal, 1998 FES (Faculté des Études Supérieures) fellowship to support foreign students in the University of Montreal, 1994Fellowship from Foundation Jean Branchet (ULB, Bruxelles) to support foreign students at the ULB (Universite libre de Bruxelles), Belgium, 1993.EU ERASMUS fellowship to finish last year of B.Sc. in ULB (Universite libre de Bruxelles), Belgium, 1992. Personal project with Dr. Alain Ghysen

Bibliography:

Books and Chapters:

Blancafort P, Juárez-Moreno K, Stolzenburg S, Beltran AS (2011) Engineering Transcription Factors in Breast Cancer Stem Cells. Breast Cancer - Carcinogenesis, Cell Growth and Signalling Pathways ISBN 978-953-307-714-7, InTech, Available from: http://wwwintechopencom/articles/show/title/engineering-transcription-factors-in-breast-cancer-stem-cells

Hassiotou F., Geddes D., Blancafort P., Filgueira L., Hartmann P.E. (2013). Breastmilk stem cells: Recent advances and future prospects. IN: Regenerative medicine using redundant abdominal fat and menstrual blood. Niranjan Bhattacharya, Phillip Stubblefield (eds.), Springer Verlag International (In press - expected publication date: September 2013).

Submitted/in review:

Beltran A.S., Graves, L.M., Blancafort, P. Interference peptides (iPeps): novel synthetic interfaces to target “undruggable” oncogenic transcription factors. Current Biology (2013).Significance: This paper describes a novel technology that uses small synthetic peptides (interference peptides), ipeps, to block epitopes necessary for protein-protein interactions, which

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maintain cooperativity between oncogenic transcription factors in breast cancer. We show that delivery of ipeps with cell penetrating peptide against oncogenes overexpressed in triple negative breast cancer cells results in potent and selective apoptosis of breast cancer cells.

Stolzenburg, S., Beltran, AS, Rivenbark, AG, Rots, MG, Scanlan-Swift, T, Strahl, BS, Ford, E. Lister R, Blancafort P. Hereditable oncogenic silencing in breast tumors by programmable sequence specific methyltransferases. Submitted to Letters to Nature.

Significance: This paper uses inducible Zinc-finger methyltransferases in animal models of breast cancer to show inherited, stable propagation of DNA methylation in targeted oncogenes. The transmission of DNA methylation originally nucleated by artificial methyltransferases is propagated in vivo in the breast cancers implants even in absence of expression of the zinc finger methyltrasferase. This paper proofs the concept that targeted methylation in oncogenic promoters results in long-lasting tumor suppression and provides an opportunity to target cancers for which no cure is available.

In Press:

Juárez-Moreno K, Erices R, Beltran AS, Stolzenburg S, Cuello-Fredes M, Owen, GI, Qian H, Blancafort P (2013). Breaking through an epigenetic wall: Re-activation of OCT4 by KRAB-containing designer zinc finger transcription factors. Epigenetics. In Press.

Van der Gun BTF, Huisman C., Stolzenburg S., Kazemier HG, Ruiters, MHJ., Blancafort, P. Rots MG (2013), Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer.BJC. In Press.

Refereed papers and articles

Original research:

Wang Y, Su H-h, Yang Y, Zhang L, Blancafort P, Huang L. Systemic Delivery of Modified mRNA Encoding Herpes Simplex Virus 1 Thymidine Kinase for Targeted Cancer Gene Therapy. Molecular Therapy. (2012); Dec 11. doi: 10.1038/mt.2012.250. [Epub ahead of print]

Blancafort P, Jin J, Frye S (2012). Writing and re-writing the epigenetic code of cancer cells: from engineered proteins to small molecules. Mol Pharmacology Minireview. Available on line http://molpharm.aspetjournals.org

Hassiotou F, Beltran A, Chetwynd E, Stuebe AM, Twigger AJ, Metzger P, Trengove N, Tat Lai C, Filgueira L, Blancafort P, Hartmann PE. Breastmilk is a Novel Source of Stem Cells with Multi-Lineage Differentiation Potential. Stem Cells. (2012) Aug 3. doi: 10.1002/stem.1188. [Epub ahead of print]

Stolzenburg S, Rots MG, Beltran AS, Rivenbark AS, Yuan X, Strahl BS, Blancafort P (2012) Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer. Nucleic Acids Research. (2012); 40:6725-40.

Significance: This paper was selected for the Highlights of the Journal (and the cover),

which comprises 5% of the papers based on innovation and scientific excellence.

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December 2012

Lara H, Wang Y, Beltran AS, Juarez-Moreno K, Yuan X, Kato S, Leisewitz AV, Cuello-Fredes M, Licea AF, Connolly DC, Huang L, Blancafort P. Targeting serous epithelial ovarian cancer with designer zinc finger transcription factors. J Biol Chem. (2012); 287:29873-86.

Rivenbark AG, Beltran AS, Yuan X, Rots MG, Strahl BD, Blancafort P. Epigenetic Reprogramming of Cancer Cells via Targeted DNA Methylation. Epigenetics (2012); 7:350-60.

Huang X, Narayanaswamy R, Fenn K, Szpakowski S, Sasaki C, Costa J, Blancafort P, Lizardi PM. Sequence-Specific Biosensors Report Drug-Induced Changes in Epigenetic Silencing in Living Cells. DNA Cell Biol. (2012) Feb 7.

Beltran, A and Blancafort, P. Reactivation of Maspin in Non-Small Cell Lung Carcinoma cells (NSCLC) by Artificial Transcription Factors (ATFs). Epigenetics. 2011 Feb 20;6(2). [EPub ahead of Print]

Beltran, A. S., Russo, A, Lara, H, Fan, C, Lizardi, P. M, Blancafort, P. Suppression of breast tumor growth and metastasis by an engineered transcription factor. PLoS One. 2011. 6: 9 e24595

Beltran AS, Rivenbark AG, Richardson BT, Yuan X, Quian H, Hunt JP, Zimmerman E, Graves LM, Blancafort P. Generation of tumor-initiating cells by exogenous delivery of OCT4 transcription factor. Breast Cancer Res. 2011 Sep 27;13(5):R94

Beltran, A., Blancafort, P . Remodeling genomes with Artificial Transcription Factors and chromatin remodeling drugs. Zinc Finger Protocols. Humana Press. Methods Mol Biol. 2010;649: 163-82.

Beltran, A., Sun, X., Lizardi,PM, Blancafort, P. Reprogramming epigenetic silencing: Artificial Transcription Factors synergize with chromatin remodeling drugs to re-activate the tumor suppressor mammary serine protease inhibitor maspin. Mol Cancer Ther. 2008;7:1080-90.

Blancafort P, Beltran AS. Rational Design, Selection and Specificity of Artificial Transcription Factors (ATFs): The Influence of Chromatin in Target Gene Regulation. Comb Chem High Throughput Screen. (2008);11:146-58.

Blancafort, P., Tschan, M.P., Edrmann, D., Barbas III, C.F. Modulation of drug resistance by artificial transcription factors. Mol Cancer Ther. (2008); 7:688-97.

Beltran, A., Parikh, S., Liu, Y., Cuevas, BD, Johnson GL, Fustcher, BW and Blancafort, P. Reactivation of a dormant tumor suppressor by designed transcription factors. Oncogene. (2007);26:2791-8.

Beltran, A., Liu, Y., Parikh, S., Brenda, T., and Blancafort, P . Interrogating genomes with combinatorial transcription factor libraries: asking zinc finger questions. Assay Drug Dev Technol. (2006); 4:317-331

Dreier, B., Fuller, R.P, Segal, D.J., Lund, C., Blancafort, P., Huber, A., Koksch, B., and Barbas, C.F.III. Development of zinc finger domains for recognition of the 5’-CNN-3’ family DNA sequences and their use in the construction of artificial transcription factors. J. Biol. Chem. (2005); 280: 35588-97.

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Blancafort, P . , Chen, E., Gonzalez, B., Bergquist, S., Zijlstra, A., Guthy, D., Brachat, A., Brakenhoff, R., Quigley, J., Edrmann, D., and Barbas C.F.III. Genetic reprogramming of tumor cells by zinc finger transcription factors. Proc. Natl. Acad. Sci. (2005); 102:11716-21.

Magnenat, L., Blancafort, P. and Barbas III, C.F. In vivo library selection and designed affinity manuration of polydactyl zinc finger transcription factors for ICAM–1 gene regulation. J. Mol. Biol. (2004); 341:635-49.

Blancafort, P, Segal, D.J., and Barbas III, C.F. Designing transcription factor architectures for drug design. Mol Pharmacol. (2004); 66:1361-71.

Lund, C.V, Blancafort, P., Popkov, M. and Barbas III, C.F. Promoter-targeted Phage Display Selections with Preassembled Synthetic Zinc Finger Libraries for Endogenous Gene Regulation. J.Mol. Biol. (2004); 340:599-613.

Blancafort,P., Magnenat, L. and Barbas III, C.F. Scanning the human genome with combinatorial transcription factor libraries. Nat Biotechnol. (2003);21:269-74.

Segal, D.J., Beerli R.R, Blancafort, P., Dreider, B., Effertz, K., Huber, A., Koksch, B., Magnenat, L., Valente, D., and Barbas III., C.F. (2003). Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins. Biochemistry. 2003; 42:2137-48.

Blancafort, P., Klinck, R., Steinberg, S., Scott, J.K. and Cedergren, R. The recognition of a non-canonical base pair by a zinc finger protein. Chem Biol. (1999); 6:585-97.

Blancafort, P . , Ferbeyre, G., Sariol, C., and Cedergren, R. Pol I-driven integrative expression vectors for yeast. Journal of Biotechnology (1997);56:41-7

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Published abstracts:Ferbeyre, G., Bratty, J., Blancafort, P ., and Cedergren, R. (1995). Yeast as a model for hammerhead ribozyme action. Yeast, 11:15-48A.

Patents:Blancafort, P. and Barbas III, C.F. "Zinc finger libraries". International Patent Application Serial No. PCT/US03/03705 (2001).

Blancafort P, Beltran AS. “Interference Peptides, ipeps. Pending patent application.

Conference presentations:

Blancafort P. Susan Swerling invited Lecture, “Targeted Therapy for Cancer: Moving Beyond Kinase Inhibitors.” Dana Farber Cancer Institute, Harvard Medical School, May 16, 2013

Hassiotou F., Trengove N., Tat Lai C., Filgueira L., Blancafort P., Hartmann P.E., (2012). Breastmilk stem cells: An overview of the current knowledge. Breastfeeding and Lactation Symposium, Vienna, Austria, April 2012 (Plenary Speaker).

Blancafort, P. Transcriptional regulation of breast cancer stem cell fate. American Society for Investigative Pathology. Breast Cancer Workshop. Washington, DC. April 13th, 2011.

Blancafort, P. Selection of Artificial Transcription Factors modulating breast cancer metastasis. Phage and Yeast Display Conference. Boston, MA. May 9-10 2011.

Stolzenburg, S, Blancafort P. Reprogramming the epigenetic code of breast cancer cells. Era of Hope meeting, Department of defense Breast cancer meeting, Orlando Florida, August 2-5, 2011.

Blancafort P. Reprogramming Epigenetic Silencing with Artificial Transcription Factors. The Chilean Society for Cell Biology meeting. Pucon, Chile. October 29nd –November 2nd 2010.

Blancafort P. Reprogramming Epigenetic Silencing with Artificial Transcription Factors. Department of Pharmacology, UNC. October 5th, 2010.

Blancafort, P. Reprogramming self-renewal in breast cancer. Centre de Regulacio Genetica de Barcelona, CRG, Barcelona, Spain. July 31st, 2009.

Blancafort, P. Epigenetic reprogramming of tumor and stem cells by Artificial Transcription Factors. Molecular therapeutics of Cancer Research Conference. Stanford University July 23-25, 2009.

Blancafort, P. Remodeling genomes of tumor and stem cells by Artificial Transcription Factors. November 5th, 2008. Loyola University. Host: Bruce Cuevas.

Blancafort, P. Reprogramming epigenetic silencing with Artificial Transcription factors. April 15th, 2008. AACR, experimental and molecular cancer therapeutics minisymposium. San Diego, CA. Abstract selected for a 10-min presentation.

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Blancafort, P. Targeting breast and lung cancer metastases with zinc finger engines. NIEHS, February 12, 2008. Host: Dr. John Roberts.

Blancafort, P. Reprogramming epigenetic silencing in tumor/stem cells with Artificial Transcription factors. April first, 2008. Department of Biochemistry, The University of North Carolina at Chapel Hill. Host: Dr. L. Parise.

Blancafort, P. Reprogramming epigenetic silencing with Artificial Transcription factors. June 29 th, 2007. University of Pennsylvania and Penn Genomic Institute, Summer Mini-Symposium. Host: Dr. Jim Eberwine.

Blancafort, P. Exploring the Dark Side of the human genome with Artificial Transcription Factors (ATFs). June 19th, 2007. University of Montreal, Department of Biochemistry. Host: Dr. Sylvie Mader.

Blancafort, P. Reprogramming breast tumor cells with artificial transcription factors. June 16 th, 2006. University of California, Davis Genome Center. Host: Dr. David Segal.

Blancafort, P. Genetic reprogramming of tumor cells by artificial transcription factors. May 24th, 2005. Yale University, dept. of pathology. Host: Dr. Paul Lizardi.

Blancafort, P, Magnenat, L. and Barbas, CF III. Endogenous gene regulation and gene discovery with polydactyl zinc finger transcription factors, Protein Engineering section, the 227 th ACS National Meeting, Anaheim, CA March 28th 2004.

Blancafort, P. Zinc finger libraries for gene discovery. Invited speaker. Banting and Best Department of Medical Research. Department of Chemistry, University of Toronto, Toronto, Canada. Host: Dr. C. Boone. May 20, (2003).

Blancafort, P. Scanning the human genome with Zinc finger libraries. Invited speaker. Department of Chemistry, University of California, Irvine. Host: Dr. G. A. Weiss. May 5, (2003).

Blancafort, P. Zinc finger libraries and targeted regulation of endogenous genes. Understanding Phage Display. Morris j. Centre for Dialogue, Simon Fraser University, Vancouver, Canada, January 17, (2003).

Blancafort, P. Making conformation-specific RNA binding zinc fingers. 4th Annual Montreal Joint Center for Structural Biology Workshop. Biotechnology Research Institute, Montreal, Canada. May 22 (1998).

Blancafort, P., Selection in vitro de doigts de zinc liant l’ARN. Concours Simon Pierre Noel, Departement de Biochimie, Universite de montreal, Montreal, Canada (1998).

Blancafort, P., Modeles d'interaction proteine-ARN issus de la selection in vitro de banques aleatoires de proteines. Concours Simon-Pierre Noel, Departement de Biochimie, Universite de montreal, Montreal, Canada (1996).

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Posters (most relevant to this work):

Epigenetic reprogramming of breast tumors by engineered zinc finger DNA methyltransferases (2012). Stolzenburg, S, Beltran AS, Rivenbark AG, Swift-Scanlan T, Strahl BS, Blancafort, P. Max-Planck Freiburg Epigenetics meeting. MPI of Immunology and Epigenetics, Freiburg, Germany, December 2012.

Hassiotou F., Beltran A., Trengove N., Tat Lai C., Hartmann P.E., Filgueira L., Blancafort P. (2012). Embryonic transcription factor upregulation during normal lactation and breast oncogenesis. International Society for Stem Cell Research, Yokohama, Japan, June 2012.

Hassiotou F., Trengove N., Tat Lai C., Blancafort P., Filgueira L., Hartmann P.E. (2012). Breastmilk is a novel source of stem cells from the lactating breast with multi-lineage differentiation potential. International Society for Stem Cell Research, Yokohama, Japan, June 2012.

Hassiotou F., Beltran, A., Trengove N., Tat Lai C., Filgueira L., Hartmann P.E., Blancafort, P. (2012). Embryonic transcription factor upregulation during normal lactation and breast oncogenesis. Experimental Biology, San Diego, US, April 2012.

Beltran AS, Rivenbark, AG, Richardson, BT, Blancafort P (2011). Generation of tumor initiating cells by exogenous delivery of the Oct4 transcription factor. American Society for Investigative Pathology. Annual meeting. Washington, DC. April 13th, 2011

Stolzenburg, S, Beltran AS, Blancafort P (2011). Reprogramming the epigenetic code of breast cancer cells. Era of Hope meeting, Department of defense Breast cancer meeting, Orlando Florida, August 2-5, 2011.

Beltran AS, Blancafort P. Discovery of Novel Artificial Transcription Factors Regulating Breast Cancer Cell Invasion and Progression (2008). Baltimore Convention Center. Baltimore June 25–28, 2008.

Blancafort, P, Magnenat, L. and Barbas III, C.F. Scanning the human genome with transcription factor libraries. American Association for Cancer Research. 94th Annual meeting, Washington D.C July 11–14 (2003).

Blancafort, P, Magnenat, L. and Barbas III, C.F. Scanning the human genome with transcription factor libraries. Drug target validation: gene suppression. Keystone symposia, Granlibakken Resort, Tahoe City, California, USA. January 17-22, (2003). Blancafort, P., Magnenat, L., Lund, C. and Barbas III, C.F. (2002). Zinc fingers as a tools to study mammalian genomes. Angiogenesis in cancer and other diseases. Keystone symposia, Banff, Alberta, CA. February 7-8-13, (2002).

Blancafort, P., Klinck, R., Scott, J.K and Cedergren, R. The “zinc-fingering” of RNA. Proteins that bind RNA conference. Seven Mile Beach Island on the Jersey Cape, Avalon, New Jersey. May 3-7 (1998).

Ferbeyre, G., Bratty, J., Blancafort, P., and Cedergren, R. Yeast as a model for Hammerhead ribozyme action. 17th int.Conf. on yeast genetics and molecular biology. Lisboa, Portugal (1995).

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Blancafort, P., Ferbeyre, G., and Cedergren, R. Expression of small RNAs in yeast using ribosomal minigenes. Rencontre Scientifique Louis-Philippe Bouthillier, Departement de Biochimie, Hotel Far Hills Inn, Val Morin, Quebec, Canada (1995).

Blancafort, P., Sirinivassan, J., and Cedergren, R. Selection of decapeptides binding to the GAGA tetraloop. The exploration of the sequence space: selecting functional molecules from random pools. Indiana University, Bloomington, IN (1995).

Blancafort, P., Sirinivassan, J., and Cedergren, R. Selection of decapeptides binding to the GAGA tetraloop. 9th CIAR Evolutionary Biology Meeting. Val Morin, Quebec, Canada (1995).

Teaching Activities:

Present Graduate students: Sabine Stolzenburg.. Thesis Title: Transcriptional regulation of Sox2 by Artificial Transcription

Factors. Expected completion: June 2011.

Previous graduate students: Angela Russo: Thesis Title: Reprogramming metastatic behavior with Artificial Transcription

Factors. Completion: June 2009.

2005-2012 Teaching Commitments PHO 739: Course Director. Reprogramming of stem and somatic cells, 1hr/week- 5-6 students per class.

PHO 730: Course Co-Director. Seminars in Pharmacology, 4 hr/week, 12 students/class

PHO 701: Techniques in Gene Manipulation (1 hr lecture), Nuclear Receptors (1 hr lecture). 25 students/class

2004 Mentored Ph.D student Sharon Bergquist, The Scripps Research Institute2003 Mentored summer High school teacher Paul Messier, The Scripps Research Institute2002 Mentored College student Leanna Lagpacan, The Scripps Research Institute1994-1997 Teaching assistant, biochemistry undergraduate laboratory, Faculté des Arts et des

Sciences, Université de Montréal

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Grants:

CURRENT SUPPORT

1R01CA170370-01 Role: Principal Investigator, Dr. Pilar Blancafort, Ph.D.Title: Targeted epigenetic silencing of oncogenic Transcription Factors (PQ 18)Time commitment: 4.0 calendar months or 33% effortSupporting Agency: National Institutes of HealthAgency Contracting/Grants Officer: Paul Okano; Phone: 301-496-9326; Email: okanop@mail.nih.govPerformance Period: 07/01/2012 – 06/30/2016Annual funding: $207,500Project description: The goal of this project is to construct six-zinc finger DNA binding domains liked to a DNA- methyltransferase enzyme to promote stable inherited silencing of SOX2.

Specific Aims:

Aim 1.To target DNA methylation into the proximal promoter region of Sox2 to silence the promoter using ZF-based Epigenetic Silencers. The 6ZFs targeting the Sox2 promoter will be linked to the Dnmt3a catalytic domain and inactive mutants, and delivered in tumor cell lines expressing high Sox2 using retroviral vectors. 1a.To investigate the capability of the ZFs-Dnmt3a constructs to bind and down-regulate the Sox2 promoter. 1b.To quantify DNAme frequencies in the Sox2 promoter by sodium bisulfate sequencing and Illumina DNA methylation Chips. 1c.To analyze changes in H3K9me levels and recruitment of PolII in the Sox2 promoter by Chromatin Immunoprecipitation (ChIP). 1d.To analyze the effect of the ZF silencers in the growth suppression phenotype of the tumor cells by cell proliferation, cell cycle, and colony formation assays.

Aim 2.To determine if the epigenetic and gene expression changes are transmitted over cell divisions. 2a.To investigate the transmission of DNAme and the durability of the transcriptional silencing in cell culture models in vitro using inducible retroviral vectors. 2b.To investigate the longevity of the epigenetic silencing in vivo in orthotopic breast cancer implants upon induction/suppression of ZFs-Dnmt3a expression.

Aim3.To develop tumor-specific nanoparticles encapsulating the 6ZF-Dnmt3a mRNAs. 3a. To investigate the efficiency of delivery and efficiency of silencing of Sox2 in breast cancer cell lines in vitro. 3b. To deliver tumor-specific nanoparticles by systemic injection in nude mice bearing subcutaneous or orthotopic breast cancer implants (xenografts). Tumor growth will be monitored by caliper measurements and Sox2 down-regulation verified by methylation assays, IF, and q-RTPCR.

Department of Defense Breast cancer Idea AwardRole: Principal Investigator, Dr. Pilar Blancafort, Ph.D. Investigator/ Dr. Brian Strahl, Ph.D (partnering PI)Title: Re-writing the histone code of breast cancerTime commitment: 15% Supporting Agency: Department of DefenseAgency Contracting/Grants Officer: Melissa Green, Science Officer; Phone 301-619-0523Performance Period: 01/01/2010 – 12/31/2012Annual funding: Blancafort Allocation: $407,000

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Project description: The goal of this project is to generate novel epigenetic remodeling enzymes able to silence the promoter of the oncogenic transcription factor SOX2 in breast cancer cells.Specific Aims:(Aim 1) To design and construct Designed Epigenetic Remodeling Factors (DERFs) able to trigger epigenetic silencing of the endogenous SOX2 promoter in basal breast cancer cell lines enriched in cancer stem cells (SUM102 and SUM159 cell lines). a) To generate highly specific six-Zinc Finger (6ZFs) DNA-Binding Domains (DBDs) binding different 18-bp sequences in the SOX2 human proximal promoter (Blancafort). b) To generate fusions of the engineered 6ZFs with specific panel of Histone methyl- and demethyltransferases (Strahl). c) To assess the capability of these fusions to regulate endogenous SOX2 expression by real-time/western analysis (Blancafort) and to trigger specific epigenetic silencing by ChIP assay (Strahl). Year 1.

(Aim 2) To analyze the phenotype of ATF-transduced cells a) To analyze the proliferative capabilities of ATF-transduced cells by self-renewal assays anchorage-independent growth assays (Blancafort). b) To study the capability of ATF-transduced cells to sensitize the cancer stem cell lines to chemotherapy agents (taxol, doxorubicin, cis-platin) by cell viability assays (Blancafort) Year 1.

(Aim 3) To investigate if DERFs characterized in Aim 1 and 2 are able to modulate tumor formation in a xenograft/nude mouse model (Blancafort, Strahl). Year 2.

5R01CA125273-05Role: Principal Investigator, Dr. Pilar Blancafort, Ph.D.Title: Re-activation of maspin tumor suppressor gene by designed transcription factorsTime commitment: 3.0 calendar months or 25% effortSupporting Agency: NIH/National Cancer InstituteAgency Contracting/Grants Officer: Snyderwine, Elizabeth G; Phone: 301-435-1878Performance Period: 05/01/2007 – 02/29/2013Annual funding: $190,000 Project description: The major goals of this project is to design, construct and characterize artificial transcription factors made of 6 zinc finger domains that are able to re-activate the mammary serine protease inhibitor (maspin) gene in metastatic breast cancer cells.

Specific Aims: (Aim 1) To isolate and characterize ATFs able to up-regulate the maspin promoter in a model cell line MCF7. 1a. To design 6-ZF-containing ATFs binding specific sequences on the maspin gene promoter. 1b. To study the in vitro specificity of the designed proteins. 1c. To study the capability of the designed ATFs to transactivate the maspin promoter.

(Aim 2) To study the ATF activity and function in several breast cancer cell lines: MCF-7, MDA-MB-231and MDA-MB-468 1a. To investigate the capability of the ATFs to up-regulate the endogenous maspin in these cell lines. 2b. To study if ATF activation synergizes with methyltransferase and HDAC inhibitors in cell lines where the maspin promoter is silenced and methylated: MCF-7 and MDA-MB-231. 2c. To study if ATF overexpression induces apoptosis and down-regulates cell invasion 2d. To study the genome-wide specificity of the ATFs.

(Aim 3) To study the capability of the ATFs, by themselves or in combination with 5-aza-2'-deoxycytidine (5-Aza-2'dC) and/or suberoylanide hydroxamic acid (SAHA), to reduce tumor growth and metastatic potential of MDA-MB-231 cell line in an orthotopic model in nude mice.

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Role: Principal Investigator, Dr. Pilar Blancafort, Ph.D.Title: Re-activation of maspin tumor suppressor gene by designed transcription factors (ARRA Funding)Time commitment: 0% effort.Supporting Agency: NIH/National Cancer InstituteAgency Contracting/Grants Officer: Snyderwine, Elizabeth G., Phone: 301-435-1878Performance Period: 09/30/2009 – 09/29/2012Annual funding: $100,000Project description: The major goal of this project is to develop novel nanoparticle delivery systems to encapsulate Artificial Transcription Factors designed to up-regulate the tumor suppressor gene maspin in a mouse model of breast cancer.

Specific Aims:(Aim 1) To generate tumor-specific nanoparticles encapsulating the ATF-encoding DNA and to assess the anti-proliferative capabilities in breast cancer cells.

(Aim 2) To deliver the tumor-specific nanoparticles in breast and metastasis models and assess anti-cancer activity.

PREVIOUS RESEARCH SUPPORT

2P50CA058223-14 (Earp, H.S.) 09/21/2006 - 07/31/2010 10% effortNIH/NCI $12,497SPORE in Breast CancerDr. Blancafort receives salary support from the Career Development Core of theSpecialized Program of Research Excellence at the Lineberger Comprehensive Cancer Ctr. This SPORE is the integration of public health, clinical and molecular sciences to better investigate etiology, prevention and early detection in breast cancer and to devise ways to reduce breast cancer mortality.

1-R01-GM080242-01 (PI: Lizardi; CoPI: Blancafort)04/01/2007-03/31/2009 10% effort

Yale University/NIH $77,141Probes for Detection of DNA Accessibility in ChromatinThe Blancafort Laboratory will be responsible for the construction of 6 zinc finger DNA-binding

domains, the sub-cloning of those domains in several bacterial and mammalian-expression vectors and for the characterization of those domains both in vitro and in vivo.

Idea Award (Blancafort) 04/18/06 – 04/17/09 25% effort Department of Defense $94,622

Discovery of Novel Artificial Transcription Factors Regulating Breast Cancer Cell Invasion and Progression

The major goals of this project are the selection of ATFs that induce cell invasion in breast epithelial cells.

We want to study if ATF, delivered in highly invasive breast cells, can modulate metastasis in vivo using a mouse model.

LCCC (UNC University Cancer Research Fund) Internal UNC award

02/01/2008-06/30/2009 0% effort

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$60,000Targeting lung tumors with zinc finger-linked enzymatic engines

GAC (Golfers Against Cancer award) Internal UNC award 02/01/2008-06/30/2009 0% effort

$40,000Targeting lung tumors with zinc finger-linked enzymatic engines

Discovery Award (Blancafort) 07/01/05-06/30/07 10% effort American Lung Association $100,000 Discovery of novel artificial transcription factors regulating lung cancer cell invasion and progression.The major goals of this project are the isolation transcription factors controlling motility and invasion in vitro in lung cancer cells.

Professional Services:

To Discipline

Peer reviewed in the following Journals:

PLoS ONE (2012) Nucleic Acids Research (2012) The Journal of American Society of Pathology (2012) Epigenetics (2012) Nature Protocols (2009) PNAS (2009) Cancer Research (2009) Epigenetics (2009) Gene Therapy (2008) Oncogene (2007-2008) Molecular Cancer therapeutics (2008) Pharmacogenomics (2008)

Services in the Pharmacology Department, UNC-CH:

Thesis Committees for Oral/Written exams: Nicole Vincent, (Advisor: Dr. G. Johnson) 2009-2012 Katy Cappell (Advisor: Dr. A. Whitehurst) 2009-2011 John Bauman, (Advisor: Dr. R. Juliano) 2009 Chairman, Eric Zimmerman (Advisor: Dr. L. Graves) 2007 Member, Asya Borikova (Advisor: Dr. G. Johnson) 2007 Member, Angela Russo, 2007 Admission Committees in Pharmacology: 2005-2006 Faculty Recruitment Committee, 2008

Services outside Pharmacology, UNC-CH: BBSP Focus group member, 2009-2012

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BBSP Focus group member, 2008 BBSP Faculty panel member, 2008 IBMS Admission Committee, 2007

Thesis Committees: Star Li’s External thesis member, 2008 (School of Pharmacy, UNC-CH; Advisor: Dr. Leaf

Huang) Yang Liu External thesis member, 2012 (School of Pharmacy; UNC-CH Advisor: Dr. Leaf

Huang) Colin Lickwar External thesis member, 2012 (School of Biology; UNC-CH Advisor: Dr. Jason

Lieb)

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Teaching Statement

I am committed to teach both undergraduate and graduate programs. Santiago-Ramon y Cajal, a Spanish Neurobiologist once said: "No se enseña bien sino lo que no se hace y quien no investiga no enseña a investigar”, “We can only teach Research if only we indeed proceed to perform Research”. I always loved to do Research and still this days to be in contact with the cells in real action is what inspires me to teach. I tell my students to pay attention (observe) carefully the cells in real action, they tell us everything. My ideal of teaching, especially to graduate students, is to teach “how to think” and thus to develop scientific rationale based on empirical, observational and experimental facts.

I have been teaching several lectures at UNC-CH, such as Pharmacology graduate courses in “Techniques of gene manipulation”, which includes Gene Therapy & molecular cancer therapeutics, and “Nuclear Receptors” as part of the PHO701 curriculum. In these lectures, I teach students state-of-the art techniques to deliver biological macromolecules into cell and tissue systems, such as DNA, RNA and polypeptides, with special emphasis on cancer. In the “Nuclear Receptors” lectures, I focus on the biology of Zinc Finger (ZF) transcription factors in important diseases like breast and ovarian cancers. I give students structural insights of DNA-binding proteins, which ultimately are at the basis of genomic specificity and biological function. Related to that, is the emphasis in chromatin structure and epigenetic modifications, which influence the binding site of a transcription factor in the context of a complex genome.

Together with Dr. Adrienne Cox, I am a course co-director of Seminars in Recent Advances of Pharmacology (PHO730). This is an interactive class that teaches students:

1) To critically evaluate and learn from the published literature regarding the development of a scientific hypothesis, appropriate experimental design and methods, and what conclusions that can be drawn from a given set of data.

2) To gain experience in evaluating the main conclusions of a paper, to be able to formulate critiques and to look at what could be done differently.

3) To understand in depth a variety of methods and techniques (strengths and limitations, when to use them, what alternatives are available).

4) To gain experience in oral presentation of scientific data, and to practice answering questions during the course of the presentation.

5) To practice effective time management.

In 2010, I began a new elective in Pharmacology named: “Reprogramming of somatic and stem cells”, which I believe is a unique initiative at UNC focused on the applications of stem cells in Pharmacology and human disease, such as cancer, aging, and tissue regeneration. The field of reprogramming is extremely young and began in 2006 with a seminal paper of the Yamanaka group, which showed that ectopic delivery of four Transcription Factors in somatic cells lead to the generation of induced embryonic pluripotent stem cells. Since 2006, this field has grown and irradiated to several areas of Biomedicine and Pharmacology. In this class, our spirit is to introduce students to the main types of stem cells: 1) How to reprogram or direct the epigenetic landscape of these cells towards specific lineages; 2) Teach students how dysregulation of stem cell functions might be at the basis of diseases, such as cancer.

Mechanistically my idea of teaching is interactive. I’d like to make students answer questions, outline the essential components, and create opportunities for students to actively participate in the class. I believe that

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ultimately our mission as teachers is to drive enthusiasm, inspire students as well as route students towards their scientific paths. I will always be accessible to my students as much as I am capable. My classes are a dynamic experience. In my presentations I try to give students an essential and clear infrastructure to understand fundamental aspects in the field, from which they can further read. My presentations are clear, precise, simple, and timed. I also like to give a few slides that are integrative and associate the class with other fundamental fields. I think it is our function as teachers to give an integrative view in order to provide students with “the bigger picture”. Ultimately, my goal is to wake up the student’s own scientific curiosity and allow them to think beyond the class.

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