midwest dna repair symposium16thmwdr.clas.wayne.edu/2014midwestdna-repair... · symposium may 17...

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Midwest DNA Repair Symposium May 17-18 Wayne State University

16th Annual

Midwest DNA Repair

Symposium

May 17 and 18, 2014

Wayne State University

Spencer M. Partrich Auditorium

McGregor Memorial Conference Center

Detroit, MI

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16th Annual

Midwest DNA Repair

Symposium

Program Contents

Sponsors p.3

Keynote Speakers p.4

Scientific Program p.6

List of Abstracts p.12

Abstracts p.16

Author Index p. 73

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16th Annual

Midwest DNA Repair Symposium

We are grateful to our sponsors

SPONSORS

$2,000 Level

$1,000 Level

Molecular Therapeutics unit

$500 Level

Travel Award

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KEYNOTE SPEAKERS:

Joann Sweasy, Department of Therapeutic Radiology and

Department of Human Genetics, Yale University School of Medicine

Base excision repair (BER) plays a fundamental role in the

preservation of genome integrity. This pathway repairs

approximately 20,000 DNA base lesions per cell per day. DNA

polymerase beta (Pol β) is a key enzyme in the BER pathway,

that prevents accumulation of intermediate DNA repair

substrates, such as single nucleotide gaps, that lead to genomic

instability. Because mice deficient in POLB are not viable, the

function of this polymerase is not known within the context of

the whole organism. The Y265C variant of Pol β catalyzes DNA

synthesis at a significantly slower rate than wild-type Pol β

(WT). Here we show that knock-in mice expressing the Y265C

Pol β variant develop autoimmune pathology strongly resembling Systemic Lupus

Erythematosus (SLE). SLE is a multi-organ disease, characterized by the presence of

autoantibodies that form immune complexes and lead to tissue damage. As in SLE, the

POL BY265C/C mice exhibit increased levels of dermatitis, antinuclear antibody (ANA),

and renal disease compared to their WT siblings. Of note, the immunoglobulin heavy

chain junctions from the POL BY265C/C mice have shorter lengths, and somatic

hypermutation is dramatically increased. These results indicate that compromised Pol

β function during the generation of immune diversity leads to lupus-like disease in

mice, and raise the possibility that compromised DNA repair could be an underlying

cause of autoimmune disease in humans.

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Thomas Glover, Department of Human Genetics and Department

of Pathology, University of Michigan

Copy number variants (CNVs), defined as deletions or

duplications of 50 bp to over a Mb, arise at a high

mutation rate and are a major factor in genomic

structural variation, numerous genetic disorders and

cancer. However, there is limited understanding of the

mechanisms by which many CNVs arise, both in terms of

initial DNA insult and aberrant repair, and the risk factors

involved. Over the past seven years, we have

demonstrated that CNVs are readily induced by agents

that lead to replication stress in cultured human cells.

Both simple and complex CNVs are induced that are

characterized by microhomologous breakpoint junctions

indicative of erroneous nonhomologous repair. They

closely resemble the “nonrecurrent” class of CNVs and

provide a model system for mechanisms involved in their formation. Genomic

hotspots for these CNVs were found, some of which coincided with common fragile

sites (CFSs). Working with large sets of de novo CNVs and metaphase breaks from

cultured cell lines, we have recently demonstrated that CNVs and CFSs are different

manifestations of replication stress at the same human loci. A combination of gene

size, active transcription and late replication acting in a cell-type specific manner

impacts sites of instability and allows predictions of instability hotspots in any cell

type. A unified model emerges in which large active transcription units drive extreme

locus instability due to a high susceptibility to fork failure, especially double fork-

failures followed by template switching or MMBIR. These findings define CNV

hotspots and CFSs as loci where a dangerous combination of large active transcription

units and late replication drives extreme local genomic instability under replication

stress.

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Scientific Program

16th Annual Midwest DNA Repair Symposium

May 17-18, 2014

Scientific Sessions, Lunch, Breakfast: Spencer M Partrich Auditorium

Banquet, Posters, Reception: McGregor Memorial Conference Center

Saturday, May 17, 2014

11:00-1:00 Registration Spencer M Partrich Atrium

11:00-12:00 Lunch (boxed) Spencer M Partrich Atrium

Poster set-up McGregor Memorial Conference Center

12:00-12:15 Welcome

Dr. Wayne Raskind, Dean, College of Liberal Arts and Sciences (CLAS)

Introduction: Diane Cabelof

Keynote Speaker

12:15-1:15 Joann Sweasy (Introduction: Dr. G. Andrés Cisneros)

“DNA Repair and Autoimmunity”

Oral Symposium 1 DNA Repair and Human Disease Associations

Session Chair: Ahmad Heydari

1:15-1:30 A recurrent cancer-associated DNA polymerase epsilon mutation causes

an exceptionally strong mutator phenotype

Daniel P. Kane and Polina V. Shcherbakova

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska

Medical Center

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1:30-1:45 Targeting translesion DNA synthesis to sensitize ovarian cancer stem cells

to cisplatin

Qi-En Wang, Amit Kumar Srivastava, Chunhua Han, Ran Zhao, Tiantian Cui, Altaf

A. Wani

Department of Radiology, The Ohio State University

1:45-2:00 Folate restriction provides protection against colon carcinogenesis in DNA

polymerase β haploinsufficient mice: modulation of mTOR pathway

impacting cancer and aging

Safa Beydoun, Archana Unnikrishnan, Michael FitzGerald, Lisa Ventrella-

Lucente, Sukayna Ismail, Ali Fardous, Ahmad Heydari.

Department of Nutrition and Food Science, Wayne State University

2:00-2:15 Role of DNA polymerase β in premature senescence of Down syndrome

Aqila A. Ahmed, MieJung Park-York, Diane C. Cabelof

Department of Nutrition and Food Science, Wayne State University

2:15-2:30 Stem cell mutagenesis and carcinogenesis in the colon of wild type and

immune compromised mice after treatment with azoxymethane and/or

dextran sodium sulfate

Ryan Whetstone, Shih-Fan Kuan, Barry Gold

University of Pittsburgh

BREAK

2:30-3:00

Keynote Speaker 3:00-4:00 Dr. Thomas Glover (Introduction: Dr. Henry Heng)

“Unifying the Mechanisms of Replication Stress-Induced Fragile sites,

CNVs and Complex Chromosomal Rearrangements”

Coffee/Snack Break

4:00-4:15

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Oral Symposium 2 DSB repair/Replication Forks/Chromosomal Instability

Session Chair: Ashok Bhagwat

4:15-4:30 Chromosome fragmentation and non-homologous end joining lead to

genome chaos and cancer evolution

Joshua B Stevens, Guo Liu, Batoul Y. Abdallah, Steven D. Horne, Karen J. Ye,

Christine J. Ye, Henry H. Heng

Center for Molecular Medicine and Genetics, Department of Internal Medicine, and

Department of Pathology, Wayne State University School of Medicine

4:30-4:45 RAD54 family translocases counter genotoxic effects of RAD51

overexpression in human tumor cells

Jennifer M Mason, Kritika Dusad, Hillary Logan, Brian Budke, Megan Wu,

Jennifer Grubb, Ralph R. Weichselbaum, Philip P. Connell, Douglas K. Bishop

Department of Radiation and Cellular Oncology, Ludwig Center for Metastasis

Research, Department of Molecular Genetics and Cell Biology, University of

Chicago

4:45-5:00 Dissecting the telomeric and non-telomeric roles of human CST using a

STN1 separation of function mutant

Anukana Bhattacharjee, Jason Stewart, Mary F. Chaiken, Shih-Jui Hsu, Carolyn

Price

College of Medicine, University of Cincinnati

5:00-5:15 Exploring the role of Rad6 in repair of platinum-induced DNA lesions

Brittany Haynes, Matthew Sanders, Malathy Shekhar

Department of Oncology, Wayne State University and Karmanos Cancer Institute

5:15-5:30 Two replication fork maintenance pathways fuse inverted repeats to

rearrange chromosomes

Tae Moon Kim, Lingchuan Hu, Mi Young Son, Sung-A Kim, Cory L. Holland,

Satoshi Tateishi, Dong Hyun Kim, P. Renee Yew, Cristina Montagna, Lavinia C.

Dumitrache, Paul Hasty

Department of Molecular Medicine/Institute of Biotechnology, The Barshop

Institute of Longevity and Aging Studies, the University of Texas Health Science

Center at San Antonio

CHECK-IN

5:30-5:45 Judges for poster session pick up assignments from Diane

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Poster Session McGregor Memorial Conference Center

5:30-6:50

Banquet McGregor Memorial Conference Center

7:00

Reception McGregor Memorial Conference Center

9:00

Sunday May 18 2014

8:00-9:00 Continental Breakfast Spencer M Partrich Atrium

Oral Symposium 3 Ubiquitination/Pathway Coordination/Repair Kinetics

Session Chair: Malathy Shekhar

9:00-9:15 A mutant poisoning approach to determine if λ exonuclease trimers use a

sequential or non-sequential mechanism for processive digestion of

dsDNA substrates

Xinlei Pan, Charles Bell

The Ohio State University

9:15-9:30 Kinetic mechanism for the excision of εA by AlkA

Erin L. Miller, Patrick J. O’Brien

Biological Chemistry Department, University of Michigan

9:30-9:45 Transient kinetics and simulation revealed conformational changes

associated with human translesion DNA polymerase kappa

Linlin Zhao, Matthew G. Pence, Robert L. Eoff, Shuai Yuan, Catinca A. Fercu, F.

Peter Guengerich

Department of Chemistry, Central Michigan University; Department of

Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of

Medicine; Department of Biochemistry and Molecular Biology, University of

Arkansas for Medical Sciences

9:45-10:00 UbcH7 regulates 53BP1 stability and DSB repair

Xiangzi Han, Lei Zhang, Jinsil Chung, Amanda Tran, James W. Jacobberger, Ruth

Keri, Hannah Gilmore, Yowei Zhang

Department of Pharmacology, Department of Genetics and Genome Sciences,

School of Medicine, Case Western Reserve University

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10:00-10:15 Novel insights into homologous recombination and cancer genetics: the

RAD51 paralog, RAD51C, functionally interacts with PALB2 and BRCA2

Jung-Young Park, Thiyam R. Singh, Nicolas Nassar, Fan Zhang, Marcel Freund,

Helmut Hanenberg, Amom Ruhikanta Meetei, Paul R. Andreassen

Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s

Research Foundation

Coffee/SnackBreak

10:15-10:30

Oral Symposium 4 Novel Approaches in DNA Repair/Intermediate repair products

Session Chair: G. Andrés Cisneros

10:30-10:45 Glioma-associated isocitrate dehydrogenase 1 mutation R132H inhibits

repair of DNA double strand breaks in human astrocytes

Robert Koncar, Lindsey Romick-Rosendale, Susanne Wells, Timothy Chan, El

Mustapha Rahassi

Division of Hematology Oncology, Department of Molecular and Cellular

Physiology, University of Cincinnati; Department of Experimental Hematology and

Cancer Biology, Cincinnati Children’s Hospital Medical Center; Department of

Radiation Oncology, Memorial Sloan-Kettering Cancer Center

10:45-11:00 Kinetic study of chromosomal double-strand breaks with diverse break

structures using high-resolution tecnhiques

Zhuobin Liang, Sivakumar Nallasivam, Thomas E. Wilson

Department of pathology, Department of Human Genetics, Department of

Molecular, Cellular and Developmental Biology, University of Michigan

11:00-11:15 Cellular processing of the small, excised, damage-containing DNA

oligonucleotide (sedDNA) products of nucleotide excision repair

Michael G. Kemp, Shobhan Gaddameedhi, Jun-Hyuk Choi, Jinchuan Hu, Aziz

Sancar

Department of Biochemistry & Biophysics, University of North Carolina School of

Medicine

11:15-11:30 Genome-wide splicing kinetics suggests differential rates of splicing and

turnover of introns

Jayendra Prasad, Karan Bedi, Brian Magnuson, Jerry Oomen, Artur Veloso,

Michelle Paulsen, Thomas E. Wilson, Mats Ljungman

Department of Radiation Oncology, Department of Human Genetics

Bioinformatics Program, Translational Oncology Program, University of Michigan

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11:30-11:40 Break

11:40-11:45 17th Annual Midwest DNA Repair

11:45-12:15 Funding and Future Directions for DNA Repair

Introductions: Dr. Christopher States

Dr. Leroy Worth, NIH, Environmental Health Sciences

12:30 Awards Presentation

Graduate Student Poster, 1st and 2nd

Postdoctoral Poster, 1st and 2nd

Graduate Student Platform, 1st and 2nd

Postdoctoral Platform, 1st and 2nd

Young Investigator EMGS travel award (Peter Stambrook, presenting)

Thank you for your participation! Safe travels home.

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ABSTRACTS

Platform # Title

1. A recurrent cancer-associated DNA polymerase epsilon mutation causes an exceptionally

strong mutator phenotype

2. Targeting translesion DNA synthesis to sensitize ovarian cancer stem cells to cisplatin

3. Folate restriction provides protection against colon carcinogenesis in DNA Polymerase β

Haploinsufficient mice; And modulates the mTOR pathway impacting cancer and aging.

4. Role of DNA polymerase β in premature senescence of Down syndrome

5. Stem Cell Mutagenesis and Carcinogenesis in the Colon of Wild Type and Immune

Compromised Mice after Treatment with Azoxymethane and/or Dextran Sodium Sulfate

6. Chromosome fragmentation and non-homologous end joining lead to genome chaos and

cancer evolution

7. RAD54 family translocases counter genotoxic effects of RAD51 overexpression in human

tumor cells

8. Dissecting the Telomeric and Non-telomeric Roles of Human CST Using a STN1

Separation of Function Mutant

9. Exploring the Role of Rad6 in Repair of Platinum-induced DNA Lesions

10. Two replication fork maintenance pathways fuse inverted repeats to rearrange chromosomes

11. A mutant poisoning approach to determine if λ Exonuclease trimers use a sequential or non-

sequential mechanism for processive digestion of dsDNA substrates

12. Kinetic mechanism for the excision of εA by AlkA

13. Transient kinetics and simulation revealed conformational changes associated with human

translesion DNA polymerase kappa

14. UbcH7 regulates 53BP1 stability and DSB repair

15. Novel insights into homologous recombination and cancer genetics: The RAD51 paralog,

RAD51C, functionally interacts with PALB2 and BRCA2

16. Glioma-associated Isocitrate Dehydrogenase 1 mutation R132H inhibits repair of DNA

double strand breaks in human astrocytes

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17. Kinetic study of chromosomal double-strand breaks with diverse break structures using

high-resolution techniques

18. Cellular processing of the small, excised, damage-containing DNA oligonucleotide

(sedDNA) products of nucleotide excision repair

19. Genome-wide splicing kinetics suggests differential rates of splicing and turnover of introns

Poster # Title

1. Targeted inhibition of Replication Protein A increases Replication Stress in Cancer Cells

and Suppresses Tumor Growth

2. Translesion syntheses across O6-guanine-butylene-O6-guanine DNA interstrand cross-links

3. Functional Analysis of Conserved Amino Acids in DNA Ligase I

4. A Novel Mechanism of Hydroxyurea-Induced Cell Death in the Fission Yeast

Schizosaccharomycespombe

5. The melanocortin 1 receptor (MC1R) pathway protects against ROS-induced oxidative

stress in human melanocytes.

6. Stimulating and Sustaining ATR activity in G2/M checkpoint through ATM

Phosphorylation of RPA

7. Mre11-Cyclin Dependent Kinase 2 Interaction in the DNA Double-Strand Break Response

8. Contributions of Positively Charged DNA Binding Residues to Searching and Catalysis by

Human Alkyladenine DNA Glycosylase

9. Role of downstream mismatch repair proteins in the processing of cisplatin interstrand cross

links.

10. Developing Small Molecule Inhibitors Targeting Nucleotide Excision Repair Protein XPA

for Platinum based Combination Chemotherapy

11. Arsenic Inhibits DNA Mismatch Repair by Altering PCNA Function

12. Exploring global transcription elongation using BruDRB-Seq

13. Synthesis and evaluation of small molecule inhibitors of replication protein A

14. Catalytic Insights into Human DNA Ligase III

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15. USP7 deubiquitinates XPC in response to ultraviolet light irradiation

16. Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision

repair

17. Multiple Types of DNA Damage in Germinal Center-derived Human B cell Lymphomas

and Leukemias Expressing AID

18. Functional characterization of the N-terminal DNA-binding domain of Redβ: a unique

single-strand annealing protein

19. Improved promoter and enhancer signal detection in nascent RNA using BruUV-seq

20. Metabolomic profiling of triple negative breast cancer cells treated with Rad6 inhibitor

21. Sub-complexes of DNA Repair Proteins Assessed by Proximity Ligation

22. G-quadruplex sequences stall Klenow polymerase at the TCF3/PBX1 major break points in

t(1;19) translocations potentially leading to genomic instability.

23. Drug treatment fuels genome-mediated cancer evolution

24. Integrin alpha6beta4 promotes DNA repair-mediate transcriptional activation

25. Chronic maternal exposure to cigarette smoke induces a fetal DNA repair response without

a phase I xenobiotic metabolism response

26. Ab Initio QM/MM Calculations Show an Intersystem Crossing in the Hydrogen Abstraction

Step in Dealkylation Catalyzed by AlkB

27. DNA REPAIR ACTIVITIES OF MYCOBACTERIAL RMPs ARE DEFINED BY TWO

ALTERNATIVE DNA BINDING MODES.

28. A Homology Model of ABH1 Protein

29. Functional Consequences of SNPs in the RPA1 Gene

30. Nucleotide Excision Repair and Lung Cancer in Appalachian Kentucky

31. DESENSITIZATION OF BASAL CELL CARCINOMA TO THE ANTI-TUMORAL

EFFECT OF VITAMIN D: ROLE OF REDD1.

32. SAD-6: an SNF2-family protein involved in meiotic silencing by unpaired DNA

33. Investigating a DNA homology search process in the Model Organism Neurospora crassa

34. Low-dose ionizing radiation induces CNVs in cultured cells.

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35. Genome instability, the ultimate driver for cancer evolution

36. Small Molecule Inhibitors of ERCC1-XPF: A Novel Approach for Combination

Chemotherapy

37. Targeting the ERCC1/XPF Nuclease for Cancer Therapy

38. DNMT inhibitors sensitize breast cancer to radiation

39. Targeting translesion DNA synthesis to sensitize ovarian cancer stem cells to cisplatin

40. Role of DNA polymerase β in premature senescence of Down syndrome

41. Stem Cell Mutagenesis and Carcinogenesis in the Colon of Wild Type and Immune

Compromised Mice after Treatment with Azoxymethane and/or Dextran Sodium Sulfate

42. RAD54 family translocases counter genotoxic effects of RAD51 overexpression in human

tumor cells

43. Dissecting the Telomeric and Non-telomeric Roles of Human CST Using a STN1


44. A mutant poisoning approach to determine if λ Exonuclease trimers use a sequential or non-

sequential mechanism for processive digestion of dsDNA substrates

45. Kinetic mechanism for the excision of εA by AlkA

46. Glioma-associated Isocitrate Dehydrogenase 1 mutation R132H inhibits repair of DNA

double strand breaks in human astrocytes

47. Cellular processing of the small, excised, damage-containing DNA oligonucleotide

(sedDNA) products of nucleotide excision repair

48. Genome-wide splicing kinetics suggests differential rates of splicing and turnover of introns

49. Kinetic study of chromosomal double-strand breaks with diverse break structures using

high-resolution techniques

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Platform #1

Title: A recurrent cancer-associated DNA polymerase epsilon mutation causes an

exceptionally strong mutator phenotype

Author: Daniel P. Kane, Polina V. Shcherbakova

Affiliations: Eppley Institute for Research in Cancer and Allied Diseases, University of

Nebraska Medical Center, Omaha, Nebraska

Abstract: Exonucleolytic proofreading and DNA mismatch repair (MMR) act in series to

maintain high-fidelity DNA replication and avoid mutagenesis. MMR defects

elevate overall mutation rate and are associated with increased cancer incidence.

Hypermutable colorectal and endometrial tumors with functional MMR were

recently reported to carry amino acid substitutions in the exonuclease domain of

DNA polymerase (pol) epsilon. This created a notion that loss of the proofreading

activity of pol epsilon is an initiating cause of some sporadic human cancers;

however, the functional consequences have not yet been assessed experimentally

for any of the cancer-associated pol epsilon mutations. We report here the in vivo

functional analysis of the P286R variant that is the most frequently observed

mutation in both colorectal and endometrial. Remarkably, despite its location in

the ExoI motif essential for exonucleolytic proofreading, the P286R variant

caused an extremely strong mutator phenotype, exceeding that of proofreading-

deficient pol epsilon mutants by two orders of magnitude. This explains the

unique recurrent appearance of the P286R mutation in tumors and argues that it

acts at some level other than loss of exonuclease activity to elevate cancer risk.

We present biochemical and genetic data that provide insight into the enigmatic

mutagenesis pathway activated by the P286R change. We further show that

heterozygosity for the variant allele causes a strong mutator effect comparable to

that of complete MMR deficiency, providing an explanation for why loss of

heterozygosity is not required for the development of pol epsilon-mutant human

tumors.

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Platform #2/ Poster #39

Title: Targeting translesion DNA synthesis to sensitize ovarian cancer stem cells to

cisplatin

Authors: Qi-En Wang, Amit Kumar Srivastava, Chunhua Han, Ran Zhao, Tiantian Cui,

Altaf A. Wani

Affiliations: Department of Radiology, The Ohio State University.

Abstract: Ovarian cancer is the leading cause of gynecologic cancer deaths in the United

States. Cancer stem cells (CSCs) with enhanced tumorigenicity and

chemoresistance are believed to be responsible for treatment failure and tumor

relapse in ovarian cancer patients. However, it is still unclear how CSCs survive

DNA-damaging agent treatment. Here we analyzed the formation and removal of

cisplatin-induced 1,2-intrastrand crosslinks (Pt-GG) in CSCs, which were isolated

from ovarian cancer cell lines based on the dual expression of surface marker

CD44 and CD117. Surprisingly, our results did not support the previous

hypothesis that inefficient formation of DNA damage and enhanced DNA repair

capacity after cisplatin treatment are contributors to the cisplatin resistance of

ovarian CSCs. We further analyzed the status of the translesion DNA synthesis

(TLS), a DNA damage tolerance machinery than can help cells survive cisplatin

treatment, in both ovarian CSCs and their corresponding bulk cancer cells. Our

results revealed an enhanced expression of TLS polymerase η (Polη) at both

mRNA and protein levels in ovarian CSCs. In addition, we also observed an

increased level of monoubiquitylated PCNA (ub-PCNA), which is a key

regulatory element in TLS, in ovarian CSCs. These data indicate that CSCs may

have intrinsically enhanced TLS. To investigate the contribution of enhanced

TLS to the survival of CSCs upon cisplatin treatment, we analyzed the abundance

of CD44+CD117+ cells in various ovarian cancer cell lines after downregulation

of Polη and cisplatin treatment, as well as the sensitivity of ovarian CSCs to

cisplatin upon Polη knockdown. Our data demonstrated that downregulation of

Polη blocked cisplatin-induced CSC enrichment through the enhancement of

cisplatin-induced apoptosis in CSCs. Taken together, our data indicate that

ovarian CSCs may have intrinsically enhanced TLS, which facilitates CSCs to

survive cisplatin treatment, and leading to tumor relapse.

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Platform #3

Title: Folate restriction provides protection against colon carcinogenesis in DNA

Polymerase β Haploinsufficient mice; And modulates the mTOR pathway

impacting cancer and aging.

Authors: Safa Beydoun, Archana Unnikrishnan, Michael FitzGerald, Lisa Ventrella-

Lucente, Sukayna Ismail, Ali Fardous, Ahmad R. Heydari

Affiliations: Department of Nutrition and Food Science Wayne State University Detroit, Mi

48202

Abstract: Folate, an essential water soluble vitamin and a cofactor in one-carbon

metabolism has been associated with the etiology of many chronic diseases such

as cardiovascular disease, neurological degeneration, neural tube defect and

gastrointestinal cancers. Studies from our lab show that folate restriction (FR)

compromises the BER pathway by initiating repair without complete resolution

of the damage, resulting in accumulation of repair intermediates such as single

strand breaks. Interestingly, FR confers a protective phenotype in our BER-

deficient mice, with these animals showing significantly reduced development of

aberrant crypt foci in the colon of mice exposed to folate restriction. A diet

resulting in 90% reduction in serum folate levels modulated nutrient sensing

pathways, thereby impacting the etiology of cancer in mice. Mechanistically, our

data indicate that FR modulates nutrient sensing networks, specifically mTOR

Complex 1 (mTORC1). FR attenuates ATP levels; elevates NAD⁺ and the

NAD⁺/NADH ratio; and decreases the plasma branched-chain amino acids

leucine and isoleucine. As a result of altered purine biosynthesis, FR also

promotes accumulation of AICAR, a potent activator of AMPK. Consequently,

FR appears to increase AMPK phosphorylation and REDD1 expression, but

decreases IPMK expression; all these lead to attenuated mTORC1 kinase activity,

and thus reduced translation and proliferation and enhanced autophagy-a potential

mechanistic rationale for the anti-aging/cancer phenotype of FR. Furthermore, FR

appears to modulate survival in mice independent of food-level restriction. Our

aging colony shows an 86% survival rate for FR mice as compared to 60%

survival for a folate-supplemented group at 800 days of life.

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Platform #4/ Poster # 40

Title: Role of DNA polymerase β in premature senescence of Down syndrome

Authors: Aqila A Ahmed, MieJung Park-York, Diane C Cabelof

Affiliations: Department of Nutrition and Food Science, Wayne State University

Abstract: Down syndrome (DS) is a condition of intellectual disability characterized by

accelerated aging. Many premature aging disorders are causatively linked to

DNA repair pathways. However, accelerated aging in DS has not been directly

connected to a DNA repair defect in spite of evidence accumulated over decades

that a specific repair pathway, DNA base excision repair (BER) is reduced in DS.

We hypothesize that loss of BER in DS is causative for the observed accelerated

aging. To evaluate whether cells from DS donors senesce prematurely in

response to stress, DS and nonDS primary fibroblasts were exposed to 100μM

H2O2 for 5 days; there was a two-fold increase in stress-induced senescence in DS

fibroblasts. This accelerated senescence was accompanied by an increase in p16

expression (1.8 fold increase, p<0.01), demonstrating a predisposition for cells

from DS donors to senesce early. We also evaluated and verified the gene dosage

effect of Trisomy 21 on key oxidative stress genes localized to chromosome 21.

In the absence of exogenous oxidative stress (H2O2) there was a significant

upregulation of both CBS and SOD1 in DS donors when compared to nonDS

controls. When exogenous oxidative stress was added there was a significant

down regulation of CBS in DS cells. Bach1, a repressor of the oxidative stress

response and a Chr21 localized gene, suppresses senescence under normal

conditions. Inhibition of Bach1 by Chr21-localized miR 155 could block

senescence suppression. Our data in DS cells shows a reduction in Bach1 in fetal

samples. We hypothesized that a gene-dosage effect of a chromosome 21-linked

miRNA (miR-155) inhibited BER inducibility and promoted a damage-induced

senescence phenotype. To test this hypothesis, we ectopically overexpressed

miR-155 in MEFs; both transient and stable miR-155 overexpression

downregulated DNA polymerase beta (~50%, p<0.001). To confirm that this

occurred at the level of polB transactivation, the core polB promoter was

transfected into MEFs, resulting in a significant (50%) reduction in polB

promoter activity both at baseline and in response to MMS and H2O2 (p<0.05) in

the miR-155 overexpressing cells. Bach1 expression was also downregulated in

miR 155 cells thus allowing us to suggest that miR155 may play a key role in

inhibition of BER and polB and promotion of senescence.

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Title: Stem Cell Mutagenesis and Carcinogenesis in the Colon of Wild Type and

Immune Compromised Mice after Treatment with Azoxymethane and/or Dextran

Sodium Sulfate

Authors: Ryan Whetstone, Shih-Fan Kuan, Barry Gold

Affiliations: University of Pittsburgh

Abstract: The induction of colon tumors and colonic stem cell mutations were evaluated

using an acute inflammation model of azoxymethane (AOM) and dextran sodium

sulfate (DSS) in wild type, TCRβ-/-, TCRδ-/- and TCRβ-/-TCRδ-/- C57Bl/6

mice. The incidence of colon tumors after AOM (15 mg/kg) followed by DSS

(1.5% in drinking water for 7 days) was: WT > TCRδ-/- > TCRβ-/-~TCRβ-/-

TCRδ-/- mice. Neither AOM or DSS alone resulted in tumors by 90 days. The

mutation frequency in colonic stem cells of wild type and TCRβ-/- mice was

evaluated after treatment with AOM 10 mg/kg) and/or DSS (2% in drinking

water for 7 days) using an enzymatic assay that monitors crypts fully populated

by cells with mutated (defective) glucose-6-phosphate dehydrogenase (G6PD)

activity. The mutation frequency in the untreated and DSS treated mice was < 10-

5. AOM alone yielded fully mutant crypts in both wild type and TCRβ-/- mice

with mutation frequencies of ~4x10-4 and 2x10-4, respectively. There was a

statistically significance decrease in the mutation frequency in the TCRβ-/-mice.

The combined treatment of AOM+DSS afforded fully mutated crypts in both

strains but with a lower M.F. than AOM alone that is attributed to the destruction

of AOM mutated crypts by the subsequent treatment with DSS. The

carcinogenicity results using the higher exposure to DSS (2% vs. 1.5%)

eliminated the tumor susceptibility difference between the WT and TCRβ-/-mice.

The results show that tumor incidence is related to DSS exposure, although DSS,

plays no role in stem mutagenesis.

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Platform #6

Title: Chromosome fragmentation and non-homologous end joining lead to genome

chaos and cancer evolution

Author: Joshua B. Stevens, Guo Liu 1, Batoul Y. Abdallah

1, Steven D. Horne

1, Karen J.

Ye 1, Christine J. Ye

2, Henry H. Heng

1,3

Affiliations: 1 Center for Molecular Medicine and Genetics, Wayne State University School of

Medicine 2 Department of Internal Medcine, Wayne State University School of

Medicine 3 Department of Pathology

Abstract: Chromosomal alterations are primary contributors to cancer evolution, which is

underscored by the identification of unique, massive genomic alterations in the

majority of tumors by whole genome sequencing. These alterations often involve

recombination of multi-megabase portions of multiple chromosomes into a single

derivative chromosome. Although many hypotheses about the formation and

function of these genomes exist, the mechanism(s) behind them remain unclear.

For chaotic genomes to occur, megabase sized chromosomal pieces must be

produced and recombined to form. We show that in incomplete chromosome

fragmentation (a form of mitotic cell death) that occurs as a response to stress

produces fragments that are then rejoined. Double knockout of Ku70 and Ku80

inhibits non-homologous end joining (NHEJ) and reduces the frequency of

genome chaos as does inhibition of ligase IV activity by the inhibitor SCR7,

suggesting NHEJ plays a significant role in genome chaos. These chaotic

genomes (and their subtypes including chromothripsis, chromoplexy, and others)

rapidly and drastically alter the genome context. Alteration of genome context

results in increased diversity of replicate transcriptome profiles in cell

populations with elevated frequencies of genome chaos suggesting that genome

chaos contributes to cancer evolution by increasing transcriptome diversity of

cellular populations. Though often associated with suppression of cancer, cell

death and DNA repair mechanisms are paradoxically associated with genome

chaos resulting in the formation of new genome defined systems that play a

significant role in cancer evolution.

22



Title: RAD54 family translocases counter genotoxic effects of RAD51 overexpression

in human tumor cells

Authors: Jennifer M Mason 1, Kritika Dusad

1, Hillary Logan

1, Brian Budke

1, Megan Wu

1,

Jennifer Grubb1, Ralph R. Weichselbaum

1,2, Philip P. Connell

1, and Douglas K.

Bishop 1,3

Affiliations: 1Department of Radiation and Cellular Oncology,

2Ludwig Center for Metastasis

Research, 3Department of Molecular Genetics and Cell Biology, University of

Chicago, Chicago, IL

Abstract: The DNA repair protein RAD51 forms filaments on tracts of ssDNA leading to

repair via homologous recombination (HR). RAD51 can also bind directly to

undamaged dsDNA, although this activity is not associated with repair. The

RAD54 family DNA translocases specifically dissociate RAD51 from dsDNA. In

budding yeast, translocase mutants accumulate non-damage-associated RAD51

foci, resulting in genome instability and reduced viability. Using human tumor

cells, we show that simultaneous depletion of translocases RAD54L and

RAD54B and/or induction of RAD51 overexpression results in enhanced

accumulation of non-damage-associated RAD51 complexes. Furthermore, we

demonstrate depletion of RAD54 translocases results in reduced proliferation,

increased replication fork stalling, and mitotic defects. These results imply that

RAD54L and RAD54B counteract genome-destabilizing activities of RAD51 in

human tumor cells. In addition, treatment of human tumor cells with the RAD51-

stimulatory compound, RS-1, results in accumulation of non-damage associated

RAD51 complexes resulting in cell death. Finally, we demonstrate treatment with

RS-1 significantly reduces tumor size in a xenograft mouse model. Thus,

inhibition of RAD54 family translocases and/or direct stabilization of toxic

RAD51 complexes may be effective in the treatment of cancer.

23



Title: Dissecting the Telomeric and Non-telomeric Roles of Human CST Using a STN1


Authors: Anukana Bhattacharjee, Jason Stewart, Mary F. Chaiken, Shih-Jui Hsu, Carolyn

Price

Affiliations: College of Medicine, University of Cincinnati

Abstract Human CST (CTC1-STN1-TEN1) is an RPA-like, ssDNA-binding complex that

plays a novel role in telomere replication. Human STN1 and TEN1 crystal

structures are strikingly similar to RPA2 and RPA3 respectively and bear OB fold

domains which are important for ssDNA binding and protein-protein interactions.

Although no crystal structures are available, structure prediction with CTC1

shows similarity with RPA1 in having multiple OB fold domains. CTC1 and

STN1 were originally identified as a DNA polymerase alpha stimulatory factor

(AAF) that enhances pol α processivity and template affinity. Our group has

shown that, similar to the budding yeast CST (Cdc13-Stn1-Ten1) complex,

human CST functions later in telomere replication after telomerase mediated

elongation of the G-rich strand, to promote fill in synthesis of the complementary

C-Strand. Human CST also facilitates the earlier replication of the telomere

duplex DNA. Unlike yeast CST, the human complex seems to have no direct role

in telomere protection. Depletion of STN1 leads to an increase in multi-telomere

signals (MTS), increased length of the telomeric G-overhang and a lag in

telomere duplex replication. CST is not only important for telomere replication,

but also plays roles in genome wide replication rescue. Depletion of STN1 leads

to an increase in anaphase bridges in the absence of telomeric fusions.

Furthermore, we found that depletion of STN1 leads to a decrease in nucleotide

(EdU) uptake following hydroxyurea (HU)-induced fork stalling. We also

observed a concomitant decrease in new origin firing. These findings indicate that

CST plays a more general role in genome-wide replication restart. Our results

suggest that human CST functions as a specialized DNA replication factor that

promotes the rescue of stalled replication within the telomere duplex region and

across the genome. To further dissect the telomeric verses genome wide roles of

CST, we created a cell line stably expressing a mutated form of STN1 with three

amino acid changes in the conserved OB domain. We and others have shown that

expression of this mutant STN1 in STN1 knockdown cells greatly reduces the

DNA binding capacity of CST. The STN1 mutant rescues the telomeric C-strand

fill in function and genome-wide replication restart defects associated with the

STN1 knockdown but increases the anaphase bridges and MTS. Thus the OB-

fold mutant demonstrates a separation of function. We postulate that the final

mechanism whereby CST rescues telomeric or non-telomeric replication

following different forms of replication stress may ultimately be the same, e.g. by

interaction with pol α. However, our data clearly indicates that the transactions

involved in the replication rescue must involve different set of protein-DNA or

protein-protein interactions.

24


Platform #9

Title: Exploring the Role of Rad6 in Repair of Platinum-induced DNA Lesions

Authors: Brittany Haynes, Matthew Sanders Malathy Shekhar

Affiliations: Department of Oncology, Wayne State University and Karmanos Cancer Institute

Abstract: TNBCs share several histologic features with BRCA1-related breast cancer,

which have aberrant DNA repair. Thus DNA repair pathways are thought to play

a significant role in TNBC development and therapy response. Platinum (Pt)-

based compounds induce toxic interstrand DNA crosslinks (ICLs), which require

the BRCA/Fanconi anemia (FA) and Rad6 postreplication DNA repair (PRR)

pathways for repair. Rad6 regulates PCNA ubiquitination, a critical event for

PRR, and also promotes FancD2 ubiquitination, a critical event for FA. We have

recently identified a small molecule inhibitor (SMI) of Rad6 that targets its

ubiquitin conjugating activity. We hypothesize that targeting Rad6 will be

beneficial to TNBCs treated with Pt-based compounds by preventing acquisition

of resistance and overcoming Pt resistance. MTT assays showed that MDA-MB-

231 TNBC cells have intrinsic resistance to cisplatin (IC50 12.5 μM), and

treatment with the Rad6 SMI enhances cisplatin sensitivity (IC50 reduced to 2.8

μM). Western blot analysis showed that cisplatin-induced DNA damage response

leads to PCNA and FancD2 monoubiqutitination. Treatment of MDA-MB-231

cells with the Rad6 SMI attenuated PCNA and FancD2 monoubiquitination.

Cisplatin-induced PCNA and FancD2 foci formation were diminished in cells

treated with the Rad6 SMI.We also evaluated the restart of cisplatin-induced

stalled replication forks in MDA-MB-231 cells treated with Rad6 SMI by

IdU/CldU double labeling. Our data showed that restarting of forks is observed in

vehicle and nontarget siRNA treated MDA-MB-231 cells, but rarely in cells

treated with Rad6 SMI or Rad6B siRNA. These data reveal an important role for

Rad6 in ICL repair and platinum response/resistance in TNBC.

25


Platform #10

Title: Two replication fork maintenance pathways fuse inverted repeats to rearrange

chromosomes

Authors: Tae Moon Kim, Lingchuan Hu, Mi Young Son, Sung-A Kim, Cory L. Holland,

Satoshi Tateishi, Dong Hyun Kim, P. Renee Yew, Cristina Montagna, Lavinia C.

Dumitrache, & Paul Hasty

Affiliations: Department of Molecular Medicine/Institute of Biotechnology, The Barshop

Institute of Longevity and Aging Studies, The University of Texas Health

Science Center at San Antonio

Abstract: Replication fork maintenance pathways maintain chromosome integrity, but their

faulty application at nonallelic repeats could generate rearrangements causing

cancer. Potential causal mechanisms are homologous recombination (HR) and

error-free postreplication repair (EF-PRR). HR repairs DNA double-strand breaks

(DSBs) and single-ended DSBs within replication. To facilitate HR, the

recombinase RAD51 and mediator BRCA2 form a filament on the DNA strand at

a break to enable annealing to the complementary sister chromatid while the

RecQ helicase, BLM (Bloom syndrome mutated) suppresses crossing over to

prevent recombination. HR also stabilizes and restarts replication forks without a

DSB. EF-PRR bypasses DNA incongruities that impede replication by

ubiquitinating PCNA (proliferating cell nuclear antigen) using the RAD6–

RAD18 and UBC13–MMS2–RAD5 ubiquitin ligase complexes. Some

components are common to both HR and EF-PRR. Here we describe two

pathways that spontaneously fuse inverted repeats to generate unstable

chromosomal rearrangements in wild-type mouse embryonic stem (ES) cells.

Gamma-radiation induced a BLM-regulated pathway that selectively fused

identical repeats while ultraviolet light induced a RAD18-dependent pathway that

efficiently fused mismatched repeats. Moreover, TREX2 (a 3’-> 5’ exonuclease)

suppressed identical repeat fusion but enhanced mismatched repeat fusion, clearly

separating these pathways. TREX2 associated with UBC13 and enhanced PCNA

ubiquitination in response to ultraviolet light, consistent with it being a novel

member of EF-PRR. RAD18 and TREX2 also suppressed replication fork stalling

in response to nucleotide depletion. Interestingly, replication fork stalling induced

fusion for identical and mismatched repeats, implicating faulty replication as a

causal mechanism for both pathways.

26



Title: A mutant poisoning approach to determine if λ Exonuclease trimers use a

sequential or non-sequential mechanism for processive digestion of dsDNA

substrates

Authors: Xinlei Pan, Charles Bell

Affiliations: The Ohio State University

Abstract: λ Exonuclease is a highly processive 5’-3’ exonuclease that binds double-

stranded DNA ends and digests the 5’ end into mononucleotides. The digestion

product, a 3’ single-stranded overhang DNA, can serve as the substrate for

pairing proteins in homologous recombination. λ Exonuclease forms a

homotrimeric ring with a tapered central channel for tracking along the DNA.

Two mechanisms could be envisioned for enzymes that form oligomeric rings to

use their multiple active sites: the sequential mechanism, where all active sites in

the oligomer are engaged in catalysis sequentially; and the non-sequential

mechanism, where the substrate DNA locks onto one active site for multiple

rounds of catalysis. To understand how the λ Exonuclease trimer uses its three

active sites, we used a “mutant poisoning” approach, where inactive subunits are

mixed with active subunits to form hybrid trimers. In the sequential mechanism,

one inactive subunit will cause the trimer to lose all activity; whereas in the non-

sequential mechanism, the hybrid trimers remain active. A K131A mutant of λ

Exonuclease, which is completely inactive for nucleotide hydrolysis, but

maintains its DNA binding ability, was introduced. Nickel spin pull down assays,

where K131A mutant with a Histidine tag was mixed with untagged wild-type λ

Exonuclease and run on a nickel column, confirmed the formation of hybrid

trimers. The λ Exonuclease activity was measured by determining DNA digestion

rate. The results showed that the λ Exonuclease hybrid trimers still remain highly

active, suggesting that a non-sequential mechanism of active sites is used during

λ Exonuclease catalysis.

27



Title: Kinetic mechanism for the excision of εA by AlkA

Authors: Erin L. Miller, Patrick J. O'Brien

Affiliations: Biological Chemistry Department, University of Michigan, Ann Arbor, MI

Abstract: A wide variety of DNA nucleobase lesions are generated when cells are exposed

to alkylating agents. Cellular repair pathways, including the base excision repair

(BER) pathway, have evolved to combat such damage. The bacterial 3-

methyladenine DNA glycosylase II (AlkA) and the human alkyladenine DNA

glycosylase (AAG) are independently evolved enzymes that initiate BER by

flipping a lesion out of the DNA duplex and hydrolyzing the N-glycosidic bond.

Both glycosylases have broad substrate ranges that include alkylated purines and

1-N6-ethenoadenine (εA). This study looks to characterize the kinetic mechanism

of AlkA catalyzed εA excision. As εA is excised by both glycosylases, direct

comparisons can thus be made between the kinetic mechanisms of AlkA and

AAG. Using in vitro kinetics experiments, we show that AlkA rapidly forms a

specific recognition complex with εA and that the hydrolysis of the N-glycosidic

bond is rate limiting. Stopped flow was used to directly monitor the DNA binding

and nucleotide flipping steps for AlkA. Binding and flipping occur very rapidly

in both the forward and reverse directions, indicating that the AlkA flipped out

recognition complex is relatively unstable. In contrast, AAG has been shown to

stabilize the flipped out εA complex. Additionally, while the rate of εA excision

is identical between the two proteins, AAG binds εA 1000-fold more tightly than

AlkA. Thus, AAG is the more efficient enzyme for εA excision. These data

expand our knowledge of base excision by two glycosylases that have identical

maximal repair rates, but use different mechanisms of substrate recognition.

28


Platform #13

Title: Transient kinetics and simulation revealed conformational changes associated

with human translesion DNA polymerase kappa

Authors: Linlin Zhao, Matthew G. Pence, Robert L. Eoff, Shuai Yuan, Catinca A. Fercu,

and F. Peter Guengerich

Affiliations: Department of Chemistry, Central Michigan University, Mount Pleasant,

Michigan 48859 (USA) Department of Biochemistry and Center in Molecular

Toxicology, Vanderbilt University School of Medicine, Nashville, TN, 37232

Department of Biochemistry and Molecular Biology, University of Arkansas for

Medical Sciences, Little Rock, AR, 72205 USA

Abstract: Human DNA polymerase kappa (hpol kappa) is one of the tranlesion DNA

polymerases critical for maintaining human genome integrity. To investigate the

role of conformational dynamics during hpol kappa catalysis, we created mutants

containing a single Trp residue as a fluorescence probe. A series of pre-steady

state and steady state kinetic analyses were performed using WT pol kappa and

mutants. Stopped-flow kinetic assays revealed a decrease in Trp fluorescence for

the G:dCTP pair but not for any mispairs. The decrease in fluorescence was not

rate-limiting and is considered to be related to a conformational change necessary

for correct nucleotidyl transfer. When a free 3´-hydroxyl was present on the

primer, the Trp fluorescence change returned to the baseline level at a rate similar

to the observed kcat, suggesting that this change occurs during or after

nucleotidyl transfer. However, kpol was fast, indicating that the slow

fluorescence step follows phosphodiester bond formation and is rate-limiting.

Pyrophosphate formation and release were fast and are likely to precede the

slower relaxation step. These results and simulation from global fitting of four

sets of data revealed a conformational change after the phosphodiester formation

as the rate limiting step for hpol kappa catalysis.

29


Platform #14

Title: UbcH7 regulates 53BP1 stability and DSB repair

Authors: Xiangzi Han1, Lei Zhang

1, Jinsil Chung

1, Amanda Tran

1, James W. Jacobberger

2,

Ruth Keri1, Hannah Gilmore

3 and Youwei Zhang

1,4

Affiliations: 1 Department of Pharmacology,

4 Department of Genetics and Genome Sciences,

School of Medicine, Case Western Reserve University, Cleveland, OH 44106,

USA

Abstract: DNA double strand break (DSB) repair is not only key to the genome stability but

is also an important anticancer target. Here we report the identification of UbcH7

(also known as Ube2L3), an ubiquitin E2 enzyme, as a novel player in DSB

repair. UbcH7 regulates ubiquitination and proteasome-dependent degradation of

53BP1. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end

resection. Therefore, UbcH7 depleted cells display increased non-homologous

end-joining (NHEJ) and reduced homologous recombination (HR) for DSB

repair. Accordingly, UbcH7 depleted cells are much more sensitive to DNA

damage than control cells likely because they mainly used the error-prone NHEJ

pathway to repair DSBs. Our studies reveal a novel layer of regulation of the

DSB repair choice and propose an innovative approach to enhance the effect of

radiotherapy or chemotherapy through stabilizing 53BP1.

30


Platform #15

Title: NOVEL INSIGHTS INTO HOMOLOGOUS RECOMBINATION AND

CANCER GENETICS: THE RAD51 PARALOG, RAD51C, FUNCTIONALLY

INTERACTS WITH PALB2 AND BRCA2

Authors: Jung-Young Park, Thiyam R. Singh, Nicolas Nassar,Fan Zhang, Marcel Freund,

Helmut Hanenberg, Amom Ruhikanta Meetei, Paul R. Andreassen

Affiliations: Division of Experimental Hematology and Cancer Biolgy, Cincinnati Children's

Research Foundation

Abstract: While the RAD51 paralogs, including RAD51B/C/D and XRCC2/3, are required

for DNA repair by homologous recombination (HR), little is known about their

function in this process. Adding to the uncertainty about their role, the prevailing

model is that they only form protein complexes among themselves, including

RAD51B/C/D-XRCC2 and RAD51C-XRCC3 complexes. RAD51C is of

particular interest, since RAD51C has been reported as a breast/ovarian cancer

susceptibility gene and a Fanconi anemia (FA) gene. To better understand the

function of RAD51C in HR, we have characterized RAD51C-containing protein

complexes either from untreated cells or following exposure to mitomycin C

(MMC). Surprisingly, in addition to associations with RAD51 and other RAD51

paralogs, the only other interactions found were with two components of the core

machinery for HR, PALB2 and BRCA2. Of further interest, like RAD51C,

PALB2 and BRCA2 are also associated with breast/ovarian cancer and FA.

Importantly, the C-terminal WD40 domain of PALB2 directly binds RAD51C,

XRCC3, RAD51, and BRCA2. Thus, the RAD51 paralogs, RAD51C and

XRCC3, appear to function in association with the core machinery for HR. Also,

the PALB2 WD40 domain may coordinate the function of all of these proteins in

HR. In support of this possibility, mutations of PALB2 or RAD51C found in

cancer or FA patients disrupt interactions of BRCA2, RAD51, and XRCC3 with

PALB2 or RAD51C, and compromise DNA repair. Our work yields novel insight

into mechanisms involved in HR and establishes the first system for functional

characterization of PALB2 missense mutations identified in cancer patients.

31



Title: Glioma-associated Isocitrate Dehydrogenase 1 mutation R132H inhibits repair of

DNA double strand breaks in human astrocytes

Authors: Robert Koncar1,2,

Lindsey Romick-Rosendale3, Susanne Wells

3, Timothy Chan

4,

El Mustapha Bahassi1

Affiliations: 1Division of Hematology Oncology;

2Department of Molecular and Cellular

Physiology, University of Cincinnati, Cincinnati, OH; 3Department of

Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital

Medical Center, Cincinnati, OH; 4Department of Radiation Oncology, Memorial

Sloan-Kettering Cancer Center, New York, New York

Abstract: BACKGROUND AND PURPOSE

Malignant gliomas are associated with a dismal prognosis and nearly universal

recurrence following treatment. Approximately 70-90% of grades II-III gliomas

contain a single base substitution in one Isocitrate dehydrogenase 1 (IDH1)

allele, usually at codon 132, affecting the protein’s catalytic domain. IDH1

converts isocitrate to α-ketoglutarate (αKG). However, mutant IDH1, instead,

converts αKG to 2-hydroxyglutarate. IDH1 mutation occurs early in

gliomagenesis and gliomas with the mutation respond better to treatment.

However, the role of the mutant enzyme in tumor development is unclear. We

hypothesized that mutant IDH1 impairs DNA damage repair to promote

tumorigenesis.

METHODS

Immortalized human astrocytes were transduced with an R132H mutant IDH1

gene and treated with 3Gy ionizing radiation or 20uM etoposide for 12 hours.

DNA damage was assessed by Western blot for γH2AX. Reactive oxygen species

(ROS) was measured with 2′,7′-Dichlorofluorescein diacetateusing flow

cytometry.

RESULTS

We detected higher levels of γH2AX in IDH1 mutant astrocytes compared to

controls at 3 hours (p<0.001) and 6.5 hours (p<0.05) post-irradiation and 3 hours

post-etoposide treatment, compared to IDH1 wild-type astrocytes. There was no

significant difference between IDH1 mutant and wild-type astrocyte ROS levels

at rest or following H2O2 treatment indicating a defect in DNA repair mechanisms

rather than increased ROS levels may be responsible for the genomic instability

that initiates tumor development.

CONCLUSIONS

Mutant IDH1 impairs repair of DNA double strand breaks in astrocytes.

Identifying the mechanism by which these cells accumulate damaged DNA will

help design new therapeutic strategies for treatment of malignantgliomas.

32



Title: Kinetic study of chromosomal double-strand breaks with diverse break structures

using high-resolution techniques

Authors: Zhuobin Liang, Sivakumar Nallasivam, Thomas E. Wilson

Affiliations: Department of Pathology; Department of Human Genetics; Department of

Molecular, Cellular and Developmental Biology, University of Michigan, Ann

Arbor, MI

Abstract: Non-homologous end joining (NHEJ) is the dominant double-strand break (DSB)

repair pathway in cells with limited or no 5’ resection. DSBs often harbor diverse

break structures that can complicate rejoining and lead to mutations. To better

understand how overhang polarity affects repair, we engineered an efficient

system to induce site-specific 5’-overhanging DSBs (5’ DSBs) in the S.

cerevisiae genome using zinc finger nucleases (ZFNs). Improved activity of our

ZFN system allows us to study for the first time the repair kinetics of 5’ DSBs by

chromatin immunoprecipitation and next-generation sequencing. Surprisingly,

NHEJ factors, including Yku80, Pol4 and Dnl4, had significantly higher

recruitment to ZFN-induced 5’ DSBs as compared to HO-induced 3’ DSBs in the

same locus. Consistently, NHEJ efficiency was also higher at ZFN-induced 5’

DSBs. Exonucleases involved in 5’ resection, such as Exo1, have been

demonstrated in vitro to have varying activity on substrates with different

overhang polarities. We thus hypothesize that 3’ and 5’ DSBs have different

kinetics of end-processing affecting the stability and/or activity of NHEJ. We are

in the process of analyzing the kinetics of end-processing using our newly

developed ligation-mediated qPCR at single-nucleotide resolution. In addition,

we demonstrate that yeast Tyrosyl-DNA phosphodiesterase 1 (Tdp1) was

recruited at a low level exclusively to 5’ DSBs and that its recruitment was

antagonized by Ku. Conversely, overexpression of Tdp1 weakly compromised

NHEJ. These findings suggest that Tdp1 competes with NHEJ at 5’ DSBs.

Moreover, sequencing of chromosomal 5’-DSB joints has not to date revealed

evidence for Tdp1-mediated suppression of insertional mutagenesis as observed

in plasmid studies. In summary, our study provides new insights of how overhang

polarity at genomic DSBs influences end-processing and repair outcomes.

33



Title: Cellular processing of the small, excised, damage-containing DNA

oligonucleotide (sedDNA) products of nucleotide excision repair

Authors: Michael G. Kemp, Shobhan Gaddameedhi, Jun-Hyuk Choi, Jinchuan Hu, and

Aziz Sancar

Affiliations: Department of Biochemistry & Biophysics University of North Carolina School

of Medicine Chapel Hill, North Carolina 27599

Abstract: This year marks the 50th anniversary of the discovery of nucleotide excision

repair (NER), which is a versatile system capable of removing a wide variety of

helix-distorting, bulky adducts from genomic DNA. The primary product of NER

is a ~30 nt-long oligonucleotide that contains the DNA lesion, the fate of which is

unknown. Our laboratory has recently developed methods to isolate, detect, and

characterize these small, excised, damage-containing DNA oligonucleotide

(sedDNA) products of NER in vivo in UV-irradiated human cells and mouse

tissues (Hu et al, JBC 2013; Choi et al, NAR 2014). We have found that this

methodology is applicable to a wide variety of DNA damaging agents, including

UV, benzo[a]pyrene, and cisplatin, and is capable of detecting and quantifying

excision repair events within minutes of damage induction. This sedDNA assay

should therefore be considered a new and powerful approach for studying

nucleotide excision repair in vivo. Current studies show that the primary, full-

length sedDNA products of NER remain largely associated with chromatin and in

complex with the repair/transcription factor TFIIH. Following release from

TFIIH, the sedDNAs become bound by the single-stranded DNA binding protein

RPA and undergo limited nucleolytic degradation. The inhibition of excision gap

filling activities (DNA synthesis and ligation) prevents the release of sedDNAs

from RPA and slows the rate of DNA damage removal. These findings suggest

that gap filling is tightly coordinated with sedDNA release from RPA, which

frees RPA to function in new rounds of NER and maintain genome integrity.

34



Title: Genome-wide splicing kinetics suggests differential rates of splicing and turnover

of introns

Authors: Jayendra Prasad, Karan Bedi, Brian Magnuson, Jerry Oomen, ArturVeloso,

Michelle Paulsen, Thomas E. Wilson and Mats Ljungman

Affiliations: Department of Radation Oncology, Department of Human Genetics

Bioinformatics Program, Translational Oncology Program

Abstract: Splicing is a process wherein the spliceosome recognizes splice junctions in

nascent transcripts, excises the introns and ligates neighboring exons. Higher

eukaryotic systems have multiple introns in each gene and neither the fate of the

introns nor the kinetics with which they are spliced has been investigated on a

genome-wide scale. Here, using a new method, BruChase-seq, we demonstrate

that splicing kinetics differ between introns even when located on the same

transcript. The differences in rates was considerable and independent of gene and

intron size. Strikingly, we also found a number of spliced introns with

significantly prolonged half-lives suggesting that these introns may serve a

cellular function. The introns having these features partially overlapped across

cell lines suggesting that the functional elements (sequences) are conserved.

Collectively, we show using BruChase-seq that splicing kinetics and half-lives of

individual introns differ dramatically suggesting that these events play

physiological roles in gene regulation.

35


POSTER #1

Title: Targeted inhibition of Replication Protein A increases Replication Stress in

Cancer Cells and Suppresses Tumor Growth

Presenting

Author:

Jason Glanzer

Other

Author(s):

Shengqin Liu, Ling Wang, Adam Mosel, Aimin Peng, Greg G. Oakley

Affiliations: University of Nebraska Medical Center Eppley Institute for Research in Cancer

Abstract: The ATR/Chk1 pathway is a critical surveillance network that maintains

genomic integrity during DNA replication by stabilizing the replication forks

during normal replication to avoid replication stress. One of the many

differences between normal cells and cancer cells is the amount of replication

stress that occurs during replication. Cancer cells with activated oncogenes

generate increased levels of replication stress. This creates an increased

dependency on the ATR/Chk1 pathway in cancer cells and opens up an

opportunity to preferentially kill cancer cells by inhibiting this pathway. In

support of this idea, we have identified a small molecule, HAMNO ((1Z)-1-[(2-

hydroxyanilino)methylidene]naphthalen-2-one), a novel protein interaction

inhibitor of a protein involved in the ATR/Chk1 pathway, Replication Protein A

(RPA). HAMNO selectively binds the N-terminal domain of RPA70, effectively

inhibiting critical RPA protein interactions dependent on this domain. HAMNO

prevents etoposide-induced ATR phosphorylation of RPA32 Ser33

phosphorylation. HAMNO treatment alone induces DNA replication stress in

cancer cells that are already experiencing replication stress but not in normal

cells as detected by S-phase specific H2AX phosphorylation. HAMNO acts

synergistically with etoposide in killing cancer cells in vitro and inhibits tumor

growth in vivo. Thus, HAMNO and other protein-protein inhibitors of RPA have

potential use in cancer therapy, providing selectivity towards cancer cells by

targeting the cellular response to replication stress.

36


POSTER #2

Title: Translesion syntheses across O6-guanine-butylene-O6-guanine DNA interstrand

cross-links

Presenting

Author:

Daniel Kool #

Other

Author(s):

Derek K. O'Flaherty § Anne M. Noronha § Christopher J. Wilds § Storm J.

Shriver # Martin Egli ¶ Linlin Zhao #

Affiliations: # Department of Chemistry, Central Michigan University, Mount Pleasant,

Michigan 48859 (USA) § Department of Chemistry and Biochemistry,

Concordia University, Montreal, Quebec H4B1R6 (Canada) ¶ Department of

Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee

37232-0146 (USA)

Abstract: DNA damage includes a variety of DNA modifications, such as DNA base

adducts, abasic sites, and intra- and interstrand cross-links. DNA interstrand

cross-links (ICLs) are two strands of DNA that are covalently linked. ICLs are

especially cytotoxic because they interfere with replication, transcription, and

recombination by preventing strand separation. One of the pathways to repair

the ICLs involves an unhooked intermediate produced by endonucleolytic

incisions adjacent to the ICL followed by translesion DNA polymerases

bypassing the lesion. Acrolein derived minor groove ICLs formed between the

exocyclic nitrogens of deoxyguanosines were previously used to test the lesion

bypass capability of translesion DNA polymerases. Due to the reactivity of the

O6-guanine atom towards various alkylating agents, O6-guanine DNA ICLs can

be potentially formed by reactions of DNA with anti-cancer drug 1,4-butanediol

dimethanesulfonate. However the miscoding potential of these O6-guanine

major groove ICLs remain elusive. We are investigating the replication

dependent ICL repair pathway for major groove DNA ICLs with O6-2′-

deoxyguanosine-butyl-O6-2′-deoxyguanosine using several lesion bypass DNA

polymerases, including Sulfolobus solfataricus DNA polymerase IV (Dpo4) and

human DNA polymerase κ. Experimental methods used include primer

extension and single base extension assays using annealed primer-template

duplexes containing a site-specifically modified O6-2′-deoxyguanosine-butyl-

O6-2′-deoxyguanosine ICL. These studies will further our understanding of

repair efficiency of major groove ICLs, which could potentially be useful for

understanding the efficacy of certain anti-cancer drugs.

37


POSTER #3

Title: Functional Analysis of Conserved Amino Acids in DNA Ligase I

Presenting

Author:

Tom J. Jurkiw

Other

Author(s):

Mark R. Taylor and Patrick J. O'Brien

Affiliations: University of Michigan Department of Biological Chemistry

Abstract: The DNA ligase family in mammals consists of DNA ligase I, DNA ligase III

and DNA ligase IV, with DNA ligase I (LIG1) being the main ligase in the

nucleus functioning in replication and single-strand break repair. LIG1 binds to

single-stranded nicks in DNA where, in the presence of ATP and MgCl2, the

enzyme is able to catalyze the ligation of the DNA nick in a multi-step reaction.

Previous studies have helped to elucidate the kinetic mechanism of the overall

ligation reaction, but the roles of specific amino acid residues in the reaction

have not been studied in human LIG1 in detail. Studies using the Chlorella virus

DNA ligase have identified conserved residues that, when mutated, affect

catalysis, either having a universal impact or an impact on specific steps during

ligation. In order to see if these same residues have relevance in human LIG1,

we made mutations in the conserved residue E621, located in the active site, and

in conserved residues R879 and K882, located in the OB fold domain. The

active site E621 mutation, while hypothesized to affect metal coordination,

abrogated ligation efficiency, resulting in a single-turnover rate several

magnitudes of order lower than wildtype. The OB fold double mutant was

hypothesized to result in added flexibility to the domain, allowing LIG1 to

catalyze ligation at a pre-adenylylated nick, but interestingly did not appear to

differ significantly from wildtype LIG1.

38


POSTER #4

Title: A Novel Mechanism of Hydroxyurea-Induced Cell Death in the Fission Yeast

Schizosaccharomycespombe.

Presenting

Author:

Amanpreet Singh

Other

Author(s):

Yong-jie Xu

Affiliations: Department of Biochemistry and Molecular Biology, Boonshoft School of

Medicine, Wright State University, Dayton, OH.

Abstract: Hydroxyurea (HU) is an inhibitor of ribonucleotide reductase (RNR) that has

been used to treat various diseases such as chronic myelogenous leukemia,

sickle cell anemia, and psoriasis. It is generally believed that its cytotoxicity is

directly caused by the DNA replication stress. Recent studies in E. coli and S.

cerevisae suggest that HU may also kill the cells by generating reactive oxygen

species (ROS). However, direct evidence remains lacking in eukaryotes,

partially because the cellular effects of ROS production and replication stress

overlap. Mutants in which the two effects can be separated will be valuable in

dissecting the mechanisms of HU-induced cell death. Here, we provide evidence

for HU-induced ROS production in a fission yeast hem13 mutant. First, the HU

sensitivity can be suppressed completely in mutant cells under anaerobic

conditions or by co-incubation with antioxidants. Second, while overexpression

of the RNR small subunit can suppress the HU sensitivity of checkpoint

mutants, it fails to rescue the hem13 mutant. Third, combination of the

checkpoint mutants with the hem13 mutation has an additive effect. Fourth, the

mutant is not sensitive to other DNA damaging agents. Finally, the levels of

ROS in HU-treated cells are increased as determined by using fluorescent dye

2',7'-dichlorodihydrofluorescein diacetate. Since hemin can rescue the mutant’s

HU sensitivity, we believe that defect in heme synthesis generates a minimal

oxidative stress, which can be exacerbated by the HU-induced ROS production.

Our study may have great implications to the treatment of cancer and fungal

diseases.

39


POSTER #5

Title: The melanocortin 1 receptor (MC1R) pathway protects against ROS-induced

oxidative stress in human melanocytes.

Presenting

Author:

Alexandra Amaro-Ortiz

Other

Author(s):

Claci Ayers John A. D’Orazio,

Affiliations: Markey Cancer Center, Graduate Center for Toxicology, Department of

Pediatrics, University of Kentucky College of Medicine Lexington, KY, USA,

Department of Agriculture, University of Kentucky College of Art and Science

Lexington KY.

Abstract: The generation of free radicals and reactive oxidative species (ROS) are thought

to be major consequences of UV exposure, leading to cellular damage and

mutagenesis. Antioxidant enzymes are critically important in the removal of

ROS in the cell. The melanocortin 1 receptor (MC1R) signaling pathway is well

known for its role against UV resistance. Moreover, MC1R activates epidermal

melanocytes cell differentiation and cell survival. Therefore, MC1R signaling

may be an exploitable target against skin carcinogenesis. We hypothesize that

MC1R signaling regulates the expression of antioxidant enzymes, particularly

manganese superoxide dismutase (MnSOD). We used the human melanoma cell

line A375, known to harbor a loss-of-function signaling mutation in MC1R, to

determine effects of cAMP stimulation on MnSOD expression. We found

accumulation of MnSOD in the mitochondria after pharmacologic induction of

cAMP with forskolin. Furthermore, addition of an oxidative agent such as

H2O2 enhanced expression of MnSOD at the protein level as early as one hour

after MC1R stimulation. Because of this timing, we hypothesize that MC1R-

mediated MnSOD increases may not involve transcriptional up-regulation but

rather MnSOD transport and/or stability. To address the mechanism, we

transfected cells with a FLAG-tagged MnSOD and performed

immunoprecipitation to determine cAMP’s effect on possible binding partners.

Our results suggest that the MC1R signaling has a protective role in the

regulation of the oxidative injury by enhancing MnSOD in human melanocytes.

Furthermore, the protective effect of MC1R signaling may be dependent on the

levels of exogenous ROS in the cells. The MC1R/cAMP signaling pathway

holds promise as a novel preventive mechanism against UV-mediated oxidative

skin injury and melanoma.

40


POSTER #6

Title: Stimulating and Sustaining ATR activity in G2/M checkpoint through ATM

Phosphorylation of RPA

Presenting

Author:

Shengqin Liu

Other

Author(s):

Adam Mosel, Nima Mirmoghtadaei, Jason G. Glanzer and Greg G. Oakley

Affiliations: University of Nebraska Medical Center Department of Oral Biology and Eppley

Cancer Center, Omaha, NE

Abstract: Ataxia telangiectasia and Rad3 related (ATR) protein is the key regulator of

G2/M checkpoint. Both loading and activation of ATR requires Replication

Protein A (RPA). Simultaneously, RPA is phosphorylated at the N-terminus of

the RPA2 subunit by ATR/ATM in response to DNA damage. To understand

the role RPA phosphorylation plays in the DNA damage response, we generated

wild type and S4A/S8A-RPA2 phosphomutant cell lines with endogenous

RPA2 expression stably knocked down. S4A/S8A-RPA2 phosphomutant cells

showed a defective G2/M checkpoint and failed to halt mitotic entry after

etoposide treatment in G2 of the cell cycle. Stimulation and maintenance of the

ATR-Chk1 signaling pathway, but not the initial activation of the pathway is

compromised in the RPA phosphomutant expressing cells. The stimulation of

ATR activity requires the chromatin binding and interaction of TopBP1 with

ATR. The loss of RPA2 phosphorylation results in decreased TopBP1

chromatin binding. Using ATM, DNA-PK and ATR inhibitors, ATM was

identified as the kinase responsible for phosphorylation of Ser4/Ser8 of RPA2.

Inhibiting ATM in wild type cells lead to the identical phenotype exhibited by

cells expressing the RPA2 phosphomutant. Thus, ATM and ATR work together

to maintain the G2/M checkpoint through interaction and modulation of RPA.

41


POSTER #7

Title: Mre11-Cyclin Dependent Kinase 2 Interaction in the DNA Double-Strand

Break Response

Presenting

Author:

Mary J. Morgan(1)

Other

Author(s):

Todd A. Festerling(1,2), Jeffrey Buis(1), and David O. Ferguson(1)

Affiliations: 1) Department of Pathology, University of Michigan Medical School, Ann

Arbor, MI 48109 2) Graduate Program in Toxicology, University of Michigan

School of Public Health, Ann Arbor, MI 48109

Abstract: In eukaryotes, MRN (Mre11/Rad50/NBS1) is required for resection to initiate

homologous recombination (HR). Although required, the nuclease activities of

Mre11 alone are not sufficient for resection; BRCA1 and CtIP are needed as

well. CtIP is the factor primarily responsible for cell-cycle regulation of

resection, while the major cyclin-dependent kinase (CDK) in S-phase is CDK2

bound to Cyclin A. The phosphorylation of CtIP by CDK2 allows for assembly

of the MRN-CtIP-BRCA1 resection complex. This complex provides maximum

resection capacity for HR during the S and G2 cell cycle phases. Mre11 controls

these events through a direct interaction with CDK2 which is required for CtIP

phosphorylation and BRCA1 interaction in normally dividing cells. This

observation demonstrates that MRN has important functions both in the DDR

and in regulating the normal cell cycle. The DDR has been extensively

elucidated, but CDK2’s role within the DDR remains unclear. Thus, the present

research project investigates 1) the impact of DNA damage on the Mre11-

CDK2/Cyclin A interaction, 2) whether changes in the interaction are ATM-

dependent, and 3) whether alterations in the activity of CDK2 indicate DDR

participation. To examine the effect of DNA damage on the Mre11-

CDK2/Cyclin A complex, we exposed mammalian cells to ionizing radiation

and evaluated the interaction status of endogenous Mre11 and CDK2/Cyclin A.

Our data indicate that upon induction of DNA damage, the Mre11-

CDK2/Cyclin A complex rapidly dissociates in a manner that requires ATM

kinase activity and is correlative to a reduction in CDK2 activity.

42


POSTER #8

Title: Contributions of Positively Charged DNA Binding Residues to Searching and

Catalysis by Human Alkyladenine DNA Glycosylase

Presenting

Author:

Yaru Zhang

Other

Author(s):

Patrick J. O'Brien

Affiliations: Chemical Biology Program and Biological Chemistry Department, University

of Michigan, MI 48109

Abstract: Human alkyladenine DNA glycosylase (AAG) initiates the base excision repair

pathway by excising alkylated or deaminated purine lesions. AAG is thought to

use facilitated diffusion to efficiently search nonspecific DNA to find rare sites

of damage. Crystal structures of AAG in complex with damaged DNA reveals a

positively charged DNA binding surface that nicely accommodates the bend of

the extrahelical recognition complex, but it is not known whether nonspecific

DNA interactions would take advantage of the same set of electrostatic

interactions. We have individually mutated the 5 arginine and 3 lysine residues

that are near to the DNA binding interface and evaluated their contributions to

both in vitro and in vivo DNA repair. The results establish that catalytic

specificity, kcat/KM, is positively correlated with processivity, suggesting that

most residues contribute to both specific binding and nonspecific binding. A

single mutant, K210M, behaves like a separation-of-function mutant with

decreased processivity and wild-type catalytic specificity, suggesting that

nonspecific DNA binding interactions may extend beyond the specific DNA

binding site. The mutants were then tested for their ability to complement the

MMS sensitivity of a mag1 yeast deletion strain, revealing a positive correlation

between catalytic specificity and cell survival. This is consistent with the model

that cell survival requires efficient capture of cytotoxic lesions. Survival of cells

with searching deficient mutant proteins could be rescued by overexpressing the

mutant proteins. Thus, it appears that chromosomal access is not restricted and

sites of damage are readily accessible to a searching protein.

43


POSTER #9

Title: Role of downstream mismatch repair proteins in the processing of cisplatin

interstrand cross links.

Presenting

Author:

Akshada Sawant

Other

Author(s):

Anbarasi Kothandapani, Robert Sobol, Anatoly Zhitkovich, and Steve Patrick

Affiliations: University of Toledo medical center. Univerisity of Pittsburgh. Brown

University Karmanos cancer institute, Wayne state university.

Abstract: Cisplatin is a widely used chemotherapeutic agent which is used in the

treatment of a variety of human malignancies. Cisplatin causes its cytotoxic

effects via formation of DNA adducts which disrupt various cellular processes,

ultimately leading to apoptosis. The adducts formed by cisplatin are of two

types; Intrastrand adducts (~90%) and interstrand crosslinks (ICLs) (~10%).

Despite the fewer interstrand cross links formed, these cross links are

considered to be highly cytotoxic. In our previous studies, we have shown that

cisplatin ICLs are processed by base excision repair (BER) proteins and this

processing is mutagenic and leads to activation and recruitment of mismatch

repair (MMR) proteins. Mismatch repair recognition proteins are required to

maintain a cisplatin sensitive phenotype. In addition, we show that, MLH1, a

downstream MMR protein, is essential for maintaining cisplatin sensitivity.

Loss of MLH1 resulted in the development of resistance to the drug.

Furthermore, cells displayed increased ICL repair and double strand break

repair capacity in the absence of MLH1. Similar results were observed in the

case of clinically relevant MLH1 mutants lacking ATPase activity. These

results indicate that mismatch repair processing adjacent to the cisplatin ICLs,

which is dependent on the ATPase activity, is essential for maintaining cisplatin

sensitivity. Our next aim was to study the pathways that can be targeted to

increase cisplatin sensitivity in the resistant cells. Our preliminary data reveal

that ATR and ATM kinases can be potential targets important for sensitizing

BER deficient cells to cisplatin.

44


POSTER #10

Title: Developing Small Molecule Inhibitors Targeting Nucleotide Excision Repair

Protein XPA for Platinum based Combination Chemotherapy

Presenting

Author:

Akaash Mishra

Other

Author(s):

Derek S. Woods, SilvanaDormi and John J. Turchi

Affiliations: Department of Biochemistry and Molecular Biology, Indiana University School

of Medicine, Indianapolis, IN 46202. Department of Medicine, Division of

Hematology Oncology, Indiana University School of Medicine, Indianapolis, IN

46202.

Abstract: Resistance to platinum (Pt)-based chemotherapeutic agents has been a major

limitation for successful treatment of epithelial ovarian cancers (EOC).

Enhanced DNA repair is a major contributor for Pt-resistance. Repair of Pt-

DNA adducts occurs primarily via nucleotide excision repair (NER) and

homologous recombination repair (HRR). Germ-line mutations in BRCA1/2

predispose women to hereditary ovarian cancers that are HRR deficient. In order

to exploit the concept of synthetic lethality in Pt-based combination therapies;

we have targeted the NER pathway in HRR deficient cancers, such as BRCA1

or BRCA2 null ovarian cancer. Towards this end we have recently identified

NER inhibitors targeting the DNA binding activity of the

XerodermaPigmentosum Group A (XPA) protein, a critical component of the

NER pathway. XPA binding to damaged DNA duplex is essential for DNA

damage recognition and verification in NER and has been described as the rate-

limiting step in NER-catalyzed repair. We have employed Electrophoretic

Mobility Shift Assays to identify and characterize third-generation XPA small

molecule inhibitors. The data demonstrate a 100-fold increase in potency with

IC50 values of 1µM. Analysis of the third generation inhibitors has revealed

structure-activity relationships that define the chemical and structural features

necessary for interaction with XPA and cellular permeability. Based on these in

vitro findings, we will pursue cellular cytotoxicity and sensitization to Pt

treatment in BRCA1 null and wild type ovarian cancer cell lines, which will

form the basis for in vivo xenograft studies in mouse models for EOC.

45


POSTER #11

Title: Arsenic Inhibits DNA Mismatch Repair by Altering PCNA Function

Presenting

Author:

Janice Ortega1

Other

Author(s):

Dan Tong2, Christine Kim1, Liya Gu1, and Guo-Min Li1

Affiliations: 1Graduate Center for Toxicology and Markey Cancer Center, University of

Kentucky College of Medicine, Lexington, Kentucky 2 Wuhan University,

Wuhan, China

Abstract: Chronic exposure to arsenic is associated with the development of certain types

of cancer, including lung and skin cancers. The exact mechanism by which

arsenic induces tumorigenesis is unknown. Previous studies have shown that

arsenic enhances the expression of epidermal growth factor receptor (EGFR),

which phosphorylates the proliferating cell nuclear antigen (PCNA), an

important mismatch repair (MMR) component. We therefore hypothesize that

arsenic induces tumorigenesis through its ability to inactivate the MMR system.

We demonstrate here, that cells exposed to arsenic express high levels of EGFR

and an increased level of phosphorylated PCNA. Interestingly, nuclear extracts

treated with arsenic or derived from arsenic-treated cells are defective in MMR;

and the arsenic-induced MMR deficiency could be reversed when non-

phosphorylated recombinant PCNA was added to the reaction. These results

suggest that arsenic inhibits MMR by promoting PCNA phosphorylation

through the activation of EGFR. Our work therefore reveals a novel mechanism

by which arsenic induces carcinogenesis.

46


POSTER #12

Title: Exploring global transcription elongation using BruDRB-Seq

Presenting

Author:

Brian Magnuson

Other

Author(s):

ArturVeloso, Killeen S. Kirkconnell, Michelle Paulsen, Thomas E. Wilson, and

Mats Ljungman

Affiliations: Department of Radiation Oncology, University of Michigan Department of

Environmental Health Science, School of Public Health, University of Michigan

Department of Human Genetics, University of Michigan Department of

Computational Medicine and Bioinformatics, University of Michigan

Department of Pathology, University of Michigan Translational Oncology

Program, University of Michigan

Abstract: Transcription of DNA to RNA proceeds in three general steps: initiation,

elongation, and termination. The elongation process can take from minutes to

hours depending on the length of a gene and associated elongation factors.

Therefore, this step plays a critical, though sometimes overlooked, role in

timing – and therefore regulation – of gene expression. The reversible

transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole

(DRB) does not inhibit transcription initiation, but rather blocks the transition

from initiation to elongation by preventing RNA polymerase II phosphorylation.

Initiated RNAPII is released upon DRB removal. By measuring nascent RNA

synthesis with Bru-Seq following a given period of recovery after DRB

treatment, we were able to calculate transcription elongation rates across the

genome. BruDRB-Seq revealed that elongation rates vary among genes

expressed in a given cell line. Elongation was measured in multiple cell lines

and about half of genes exhibited similar rates across these cell lines, falling into

the distinct categories of fast or slow, and the rest tended to exhibit variable

and/or intermediate rates. We found that a number of sequence-specific, gene

neighborhood, and epigenetic factors correlate with elongation rate, including:

exon density, distance from nearby transcription units, and histone methylation.

BruDRB-Seq is a novel approach to measure transcription elongation and we

anticipate it to be valuable in future studies of the mechanisms of transcription

regulation.

47


POSTER #13

Title: SYNTHESIS AND EVALUATION OF SMALL MOLECULE INHIBITORS

OF REPLICATION PROTEIN A

Presenting

Author:

SilvanaDormi

Other

Author(s):

Akaash Mishra Derek Woods John J. Turchi

Affiliations: Department of Biochemistry & Molecular Biology, Indiana University School

of Medicine, Indianapolis, IN 46202 Department of Medicine, Indiana

University School of Medicine, Indianapolis, IN 46202 NERxBioSciences Inc.,

351 W. 10th St., Suite 510, Indianapolis, IN 46202

Abstract: Cisplatin and carboplatin impart their chemotherapeutic effect by forming Pt-

DNA adducts that block DNA replication and transcription, culminating in

apoptosis. Repair of those Pt-DNA adducts via nucleotide excision repair

(NER) or homologous recombination repair (HRR) can substantially reduce the

effectiveness of the Pt therapy, contributing to cellular resistance. Hence,

inhibition of these repair pathways holds the potential to sensitize resistant

cancer cells to Pt treatment. Unlike most therapies, which are focused on

enzyme-substrate interactions, our approach addresses protein-DNA disruption,

and it is based on the hypothesis that targeting the NER pathway in HRR

deficient cancers in combination with cisplatin therapy will provide increased

efficacy with minimal toxicity. Replication protein A (RPA), a single-stranded

DNA binding protein that plays a fundamental role in the NER pathway, is the

subject of the research herein presented. After having identified a small

molecule inhibitor (SMI) of RPA with promising in vitro and cellular activity,

we synthesized analogs of the lead compound and evaluated their ability to be

used in combination therapy. Structure-activity relationship (SAR) studies led

us to the selection of an optimized lead, which showed single agent activity in

A2780 epithelial ovarian cancer cells and in a xenograft lung cancer mouse

model. These data demonstrate the utility of RPA inhibition in vivo and the

potential in the development of a novel class of anticancer therapeutics that

target protein-DNA interactions.

48


POSTER #14

Title: Catalytic Insights into Human DNA Ligase III

Presenting

Author:

Justin R. McNally

Other

Author(s):

Patrick J. O'Brien

Affiliations: Department of Biological Chemistry, University of Michigan

Abstract: The three ATP-dependent human DNA ligases, ligase I, III, and IV, are

essential for the completion of DNA replication, repair, and recombination

pathways. Human DNA ligase III (Lig3) is unique among the human DNA

ligases due to the existence of four alternative-splice isoforms. Individual

isoforms are expressed in specific tissues and organelles, making Lig3 the only

human DNA ligase localized to both the mitochondria and the nucleus. In the

mitochondria, Lig3 plays a crucial role in DNA replication and maintenance of

the genome. However, the role of Lig3 in the nucleus is less understood. It has

been postulated that Lig3 may participate in erroneous nuclear ligation events,

possibly leading to reciprocal chromosomal translocations and/or chromosomal

fusions capable of inducing tumorigenesis. Although there are obvious

structural differences between human DNA ligase I (Lig1) and the human DNA

Lig3 isoforms, the extent of their physiological differences has yet to be

established. A direct biochemical comparison of these enzymes may provide

insight as to how the Lig3 isoforms are catalytically different from Lig1 and

from one another. To conduct a thorough characterization of the Lig3 variants,

suitable reaction conditions were identified, ensuring prolonged enzymatic

stability and activity at physiological temperature and ionic strength. Under

steady-state conditions, we investigated the Mg2+, ATP and nicked duplex

DNA substrate dependencies specific to the Lig3b isoform. Subsequent work

will focus on catalytic properties of the Lig3 isozymes, focusing on the

enzymatic contributions of the Lig3 N-terminal zinc finger, as well as the Lig3a

specific binding partner XRCC1.

49


POSTER #15

Title: USP7 deubiquitinates XPC in response to ultraviolet light irradiation

Presenting

Author:

Jinshan He1

Other

Author(s):

Qianzheng Zhu1, Nidhi Sharma1, Gulzar Wani1, Chunhua Han1, Jiang Qian1,

Kyle Pentz1, Qi-en Wang1 and Altaf A. Wani1,2, 3

Affiliations: 1Department of Radiology, 2Department of Molecular and Cellular

Biochemistry, 3James Cancer Hospital and Solove Research Institute, The Ohio

State University, Columbus, OH 43210

Abstract: Ultraviolet light (UV)-induced Xeroderma pigmentosum complementation

group C (XPC) protein ubiquitination is mediated by an E3 ubiquitin ligase

complex containing UV damaged-DNA binding protein. Here, we report that

ubiquitin specific protease 7 (USP7) deubiquitinates XPC during NER. We

have demonstrated that transiently compromising cellular USP7, by siRNA

leads to accumulation of ubiquitinated forms of XPC. However, complete USP7

disruption causes an ubiquitin-mediated XPC degradation upon cellular

irradiation. We show that USP7 interacts with XPC in vitro and in vivo.

Overexpression of wild-type USP7, but not its catalytically inactive or

interaction-defective mutants, reduces ubiquitinated forms of XPC. Importantly,

USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination

assays in vitro. We further showed that valosin-containing protein (VCP)/p97 is

required for UV-induced XPC degradation in USP7-deficient cells. VCP/p97 is

readily recruited to DNA damage sites and co-localizes with XPC. Inhibition of

VCP/p97 causes an accumulation of ubiquitinated XPC on DNA damaged

chromatin. Moreover, USP7 disruption severely impairs the repair of

cyclobutane pyrimidine dimers (CPD) and, to a lesser extent, affects the repair

of 6-4 photoproducts (6-4PP). Taken together, our findings have uncovered an

important role of USP7 in regulating NER via deubiquitinating XPC and by

preventing its VCP/p97-regulated proteolysis (This work was supported by

grants from NIH).

50


POSTER #16

Title: Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in

nucleotide excision repair

Presenting

Author:

Chunhua Han

Other

Author(s):

Gulzar Wani, Ran Zhao, Jiang Qian, Nidhi Sharma, Jinshan He, Qianzheng

Zhu, Qi-En Wang, Altaf A. Wani

Affiliations: Department of Radiology, The Ohio State University Wexner Medical Center.

Abstract: Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair

endonuclease, which cleaves DNA strands on the 3’ side of the DNA damage

during nucleotide excision repair (NER). In addition, XPG plays a crucial role

in initiating DNA repair synthesis through recruitment of PCNA to the repair

sites. However, the fate of XPG protein subsequent to the excision of DNA

damage has remained unresolved. Here, we show that XPG is degraded through

proteasome-mediated proteolysis upon induction of bulky lesions from

exposures to UV irradiation and cisplatin. NER process is required for XPG

degradation because both UV and cisplatin treatment-induced XPG degradation

is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In

addition, the NER-related XPG degradation requires Cdt2, a component of an

E3 ubiquitin ligase, CRL4Cdt2. Micropore local UV irradiation and in situ

Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-

damage sites and interacts with XPG in the presence of PCNA. Importantly,

Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of

DNA polymerase δ and DNA repair synthesis. Collectively, our data supports

the idea of PCNA recruitment to damage sites in conjunction with XPG,

recognition of the PCNA-bound XPG by CRL4Cdt2 for specific ubiquitylation

and protein degradation. Thus, XPG removal clears the space needed at the

damage site for the subsequent recruitment of DNA pol δ and initiation of DNA

synthesis. (This work was supported by grants from NIH).

51


POSTER #17

Title: Multiple Types of DNA Damage in Germinal Center-derived Human B cell

Lymphomas and Leukemias Expressing AID

Presenting

Author:

Sophia Shalhout

Other

Author(s):

Angela Sosin2, Alberto Martin3, Thomas Holland4, Ayad Al-Katib2, Ashok S.

Bhagwat1

Affiliations: 1Department of Chemistry, Wayne State University, Detroit, MI 48202, USA;

2Department of Internal Medicine, Wayne State University School of Medicine,

Detroit, MI 48201; 3Department of Immunology, University of Toronto,

Toronto, ON, Canada; 4Department of Immunology and Microbiology, Wayne

State University School of Medicine, Detroit, MI 48201, USA.

Abstract: An overwhelming majority of human B lymphocyte malignancies derive from

cells that have undergone the germinal center reaction and are associated with

the expression of AID, an enzyme that converts cytosines in DNA to uracil and

is required for antibody maturation through somatic hypermutation and class-

switch recombination. Several studies have shown that these lymphoma cells

contain a higher frequency of point mutations in many genes and chromosome

translocations, but no study has yet studied genomic uracils, the likely direct

consequence of AID expression in these cancers. We show here that human B

cell lymphoma and leukemia cell lines and patient samples that overexpress

AID accumulate unprecedented levels of uracils. These uracils are present at

many loci beyond the immunoglobulin genes and unexpectedly a majority of

the uracil are not in U•G mismatches. The high genomic uracil levels in these

tumors may result from an imbalance between expression of AID and UNG

genes. These cells also contain elevated levels of other types of DNA damage

including abasic sites, and single- and double-strand breaks, and have reduced

viability. B-cell lymphomas continuously accumulate DNA lesions therefore

compromising genomic integrity, due to the action of an endogenous agent, the

enzyme AID.

52


POSTER #18

Title: Functional characterization of the N-terminal DNA-binding domain of Redβ: a

unique single-strand annealing protein

Presenting

Author:

Christopher E. Smith

Other

Author(s):

Charles E. Bell

Affiliations: The Ohio State University

Abstract: Bacteriophage λ encodes a two-component synaptase-exonuclease (Syn-Exo)

system used for generating end-to-end concatamers of λ genome before

packaging. Redα (λ exo) is a processive 5’-3’ exonuclease that digests linear

dsDNA, yielding a 3’ ssDNA overhang. Redβ is a single-strand annealing

protein (SSAP) that binds to the resulting 3’ overhang and anneals it to a

complementary ssDNA. The current model for Redβ DNA binding and

annealing describes β binding weakly to ssDNA as an oligomeric ring of 10-15

subunits, and forming a very tight complex with newly annealed duplex in the

form of a helical filament. Redβ serves as a model to study the unique DNA

repair mechanism of single-strand annealing, which is conserved in higher

eukaryotes. We have identified a protease-resistant fragment of Redβ (1-177),

which is a target for structure determination via x-ray crystallography. We

predict the N-terminal fragment forms the DNA binding domain, while a more

flexible C-terminal tail modulates interaction with the partner exonuclease. Here

we show Redβ(FL) and Redβ(177) are both able to assemble into oligomeric

structures, but their functional properties differ significantly. Using a

fluorescence-based assay, we found Redβ(FL) preferentially binds to

sequentially-added complementary oligonucleotides, while Redβ(177) binds

more tightly to ss oligonucleotides. Utilizing a Ni-affinity pulldown assay,

Redβ(177) fails to interact with λ exonuclease. Further, we found Redβ(177) is

unable to recombine a PCR product or ss oligonucleotide with a target plasmid

containing regions of homology in vivo. Our results provide insight into how

SSAP perform their DNA binding and pairing function.

53


POSTER #19

Title: Improved promoter and enhancer signal detection in nascent RNA using

BruUV-seq

Presenting

Author:

Killeen S Kirkconnell

Other

Author(s):

Artur Veloso, Brian Magnuson, Leonardo Carmo de Andrade Lima, Michelle

T. Paulsen, Emily A. Ljungman, Karan Bedi, Jayendra Prasad, Thomas E.

Wilson and Mats Ljungman

Affiliations: University of Michigan

Abstract (250

words or less):

We recently developed Bru-seq, a method based on metabolic labeling of

nascent RNA with bromouridine to assess transcription rates genome-wide.

Here we present BruUV-seq, which utilizes UV light to introduce

transcription-blocking DNA lesions prior to labeling and deep sequencing. By

inhibiting transcription elongation but not initiation, UV light enhances

sequencing reads near promoters and enhancers. BruUV-seq, in combination

with Bru-seq, revealed unannotated transcription start site (TSS) usage,

multiple genes being transcribed from a single TSS, and the BCR-ABL1 gene

fusion in K562 leukemia cells. While the majority of expressed genes utilized

a single TSS, we were able to identify genes with 2 to 5 active TSS. A strong

correlation was observed between the BruUV-seq signals in the first 5 kb of

genes with the Bru-seq data over the length of genes, suggesting that BruUV-

seq could be used as a surrogate for estimating nascent transcription rates.

Indeed, relative changes in transcription rates measured after TNF treatment

of HF-1 fibroblasts using the BruUV-seq signal from the TSS peaks were

similar to those measured using the Bru-seq signal from the whole gene.

Furthermore, TNF induced changes in gene expression were accompanied by

changes in eRNA production. Taken together, BrUV-seq is a powerful new

approach that can obtain very detailed information on TSS utilization, relative

nascent transcription rates, and eRNA production genome-wide.

54


POSTER #20

Title: Metabolomic profiling of triple negative breast cancer cells treated with Rad6

inhibitor

Presenting

Author:

Nadia Saadat

Other

Author(s):

Yanhua Zhang, Brittany Haynes, Smiti Gupta, Guangzhao Mao,

MalathyShekhar

Affiliations: Wayne State University

Abstract: Rad6 is a fundamental component of the postreplication DNA repair pathway,

which allows cells to complete DNA replication despite the presence of

replication-blocking lesions induced by DNA damaging drugs. Rad6 is an

ubiquitin conjugating enzyme, and this activity is essential for Rad6 function.

Normal breast tissues have low levels of Rad6 expression. However, increases

in Rad6 expression are observed in breast hyperplasias, with overexpression in

invasive and metastatic breast cancers. Rad6 silencing in metastatic breast

cancer cell lines inhibits tumor growth, reverses EMT phenotype, and tumor

progression. These data implicate the potential benefits of targeting Rad6.

Through structure-guided pharmacophore modeling and in silico analysis, we

have developed a small molecule inhibitor (SMI 9) that selectively inhibits

Rad6 ubiquitin conjugating activity. Treatment of triple negative breast cancer

cells with SMI 9 inhibits cell proliferation and migration. To overcome the

solubility issues of SMI 9, we have developed a gold nanoparticle-based

platform for delivering SMI 9. MTT assays showed that SMI 9-GNPs and free

SMI 9 have comparable, if not superior, anti-proliferative effects on MDA-MB-

231 and SUM1315 breast cancer cells. To identify potential markers of drug

response, we performed metabolomic analysis of cells treated with blank or

SMI 9-GNPs. Principle component analysis score plots showed cell-specific

differences in metabolomic profiles, and loading plots identified potential

spectral regions that were affected by drug treatment. We are in the process of

identifying the targeted metabolites.

55


POSTER #21

Title: Sub-complexes of DNA Repair Proteins Assessed by Proximity Ligation

Presenting

Author:

Pamela VanderVere-Carozza

Other

Author(s):

John J. Turchi

Affiliations: Department of Medicine Indiana University School of Medicine, Indianapolis

IN, 46202 Department of Biochemistry and Molecular Biology, Indiana

University School of Medicine, Indianapolis IN, 46202

Abstract: Repair of damage to chromosomal DNA is orchestrated by a complex array of

proteins and their interactions. The nucleotide excision repair (NER) pathway

catalyzes the removal of bulky DNA damage including UV-photoproducts and

intrastrand DNA lesions induced by platinum-based cancer chemotherapeutics.

The non-homologous end joining (NHEJ) pathway can repair a simple DNA

double strand break by directly ligating the ends without the need for a

homologous template. Many of the interactions in these pathways have been

identified and characterized using immunoprecipitation, yeast two-hybrid

assays, co-purification and co-localization using immunofluorescence. To

interrogate a number of these interactions, we employed a proximity ligation

assay (PLA) which provides excellent resolution and quantification of specific

protein-protein associations in individual cells. The XPA-RPA interaction has

been extensively characterized and occurs independent of exogenous DNA

damage. PLA analysis confirmed this interaction using multiple antibodies to

detect the interaction in numerous human cancer cell lines. Interestingly, the

analysis of a number of other NER protein interactions was detected

independent of exogenous DNA damage. These data suggest that sub-

complexes of NER proteins exist in cells independent of exogenous DNA

damage and that assembly of an active NER complex involves pre-formed sub-

complexes. We selected the Ku70-DNA-PKcs interactions to monitor the NHEJ

pathway interactions. Again, a robust signal was detected independent of

exogenously induced DNA DSBs. Overall these results suggest that sub-

complexes of DNA repair proteins exist within cells independent of DNA

damage and may serve as a reservoir of complexes to be deployed to the sites of

damage.

56


POSTER #22

Title: G-quadruplex sequences stall Klenow polymerase at the TCF3/PBX1 major

break points in t(1;19) translocations potentially leading to genomic instability.

Presenting

Author:

Jonathan Williams

Other

Author(s):

Erik Larson

Affiliations: Illinois State University

Abstract: Translocations are initiated by DNA breaks, which can occur upon replication

stalling, DNA damage, or other forms of genetic instability. The t(1;19)

TCF3/PBX1 translocation is responsible for 25% of all pre-B cell leukemia, and

the resulting fusion protein has also been detected in prostate and small cell

lung cancers. It is not clear why some regions of the genome are prone to DNA

breaks and translocations. However, the notion that non-B form DNA structures

contribute to genetic instability is enjoying increasing experimental support. To

further test this model we have analyzed the TCF3 and PBX1 break point

clusters for sequence motifs that can adopt G-quadruplex DNA (G4 DNA)

structures. G4 DNA is four stranded and folds from guanine repeats under

physiological conditions. This occurs when the guanine repeats are freed from

complement during transcription or replication. Using Circular Dichroism we

identified intramolecular G4 DNA structures, which map to positions 3’ and 5’

of break point clusters in TCF3 and PBX1. Using mobility shift assays,

structure formation was potassium dependent, consistent with the ionic

requirements for G4 folding. When these same sequences were positioned to

serve as the template for DNA synthesis, we observed Klenow polymerase

stalling at the guanine-rich repeats. We suggest that the break points responsible

for the t(1;19) TCF3/PBX1 translocation occur because of replication blockage

at G4 structures, implying a mechanism for genetic instability at the TCF3

locus.

57


POSTER #23

Title: Drug treatment fuels genome-mediated cancer evolution

Presenting

Author:

Steven D. Horne

Other

Author(s):

Guo Liu, Joshua B. Stevens, Ph.D., Batoul Y. Abdallah, and Henry H.Q. Heng,

Ph.D.

Affiliations: Center for Molecular Medicine and Genetics, Wayne State University School of

Medicine, Detroit, MI

Abstract: The central paradox associated with current cancer therapeutic strategies is

initially effective treatment, which eliminates a high tumor cell count,

consistently results in successful drug resistance. Mathematical and

evolutionary modeling have previously suggested that therapeutic intervention

can provide selective pressure for the expansion of resistant variants. Drug-

related stress has been associated with genome chaos, a common phenomenon

in cancer characterized as rapid, stochastic genomic shattering and

reorganization. Since cancer represents an evolutionary process, analysis within

the context of genome-mediated cancer evolution can shed light on this key

problem of therapeutics. We propose that genomic change is a general response

to therapeutics. Drug-induced karyotypic alteration has been linked with

transcriptomic elevation, implying that drug-induced genomic change would

paradoxically provide an advantage for cancer cells through an increase of

genome heterogeneity or evolutionary potential for selection. In vivo and in

vitro models were tested using different therapeutic approaches, and surviving

cells displayed altered karyotypes for each case. To determine whether drug-

induced genome change can provide a long-term advantage to cancer cell

survival, a karyotypically stable colon cancer cell line was treated with

chemotherapy, and growth patterns were followed in a series of in vitro single-

cell and population-based experiments. Outlier treated cells displayed faster

growth rates than untreated cells, and population-based data support that these

outliers may drive cancer progression post-therapy. This macro-evolutionary

based, general mechanism of cancer drug resistance challenges the current

therapeutic aim of maximizing cancer cell death and has great implications in

the development and administration of future therapeutic strategies.

58


POSTER #24

Title: Integrin alpha6beta4 promotes DNA repair-mediate transcriptional activation

Presenting

Author:

Brittany L. Carpenter

Other

Author(s):

Min Chen Kathleen L. O'Connor

Affiliations: University of Kentucky Markey Cancer Center Department of Molecular and

Cellular Biochemistry

Abstract: Pancreatic carcinoma has the highest death to incidence ratio of all cancers.

Integrin alpha6beta4 is overexpressed in pancreatic carcinoma, and promotes

cancer progression in part by stimulating transcriptional changes. These changes

include upregulation of ligands for the epidermal growth factor receptor,

amphiregulin and epiregulin, which are major regulators of malignancy. Based

on our previous observations that integrin alpha6beta4 alters the overall

transcriptome by targeting genes for DNA demethylation, we hypothesized that

integrin alpha6beta4 controls expression of Areg and Ereg by targeting their

respective promoters for DNA demethylation. To test this hypothesis, pancreatic

cancer cells were treated with 5-aza-2’deoxycytidine (DAC), which resulted in

increased expression of Areg and Ereg. Subsequent treatment and removal of

DAC caused stable overexpression of Areg and Ereg in low integrin

alpha6beta4-expressing cells. Similarly, treatment with the hypermethylating

agent S-adenosylmethionine decreased expression of both ligands, supporting

our hypothesis that reversible epigenetic changes are responsible for Areg and

Ereg overexpression. DNA repair has been implicated in active DNA

demethylation. To determine if DNA repair is required for Areg and Ereg

expression, pancreatic cancer cells were treated with Gemcitabine, a

chemotherapeutic that inhibits GADD45a-mediated base-excision repair (BER),

which downregulated Areg and Ereg. We also found that siRNA-mediated

knockdown or cDNA-mediated overexpression of GADD45a repressed or

elevated Areg and Ereg, respectively. Parp inhibition also suppressed

transcription of Areg and Ereg in cells with high integrin alpha6beta4. These

data indicate that integrin alpha6beta4 promotes a malignant phenotype by

regulating transcriptional expression of Ereg and Areg through DNA repair-

mediated DNA demethylation.

59


POSTER #25

Title: Chronic maternal exposure to cigarette smoke induces a fetal DNA repair

response without a phase I xenobiotic metabolism response

Presenting

Author:

Smith, SC

Other

Author(s):

Webb C, Greene RM, Pisano MM

Affiliations: Birth Defects Center University of Louisville School of Dentistry

Abstract: Fetuses from dams exposed to cigarette smoke during gestation often exhibit

low birth weight and birth defects. Additionally, an increased likelihood of

oncogenesis looms over children exposed to cigarette smoke in utero. The

underlying mechanisms responsible for these adverse developmental outcomes

remain unclear. The constituents of cigarette smoke include polycyclic aromatic

hydrocarbons (PAHs). Phase I xenboiotic metabolism is inducible in the

developing fetus in response to PAH exposure. To determine whether chronic

maternal cigarette smoke exposure alters the xenobiotic metabolism response in

the exposed embryo, C57Bl/6 mouse dams were exposed to main- and

sidestream smoke from gestation day (gd) 1 through 17. Cigarette smoke

exposure (CSE) resulted in a significant decrease in fetal weight and length on

gd 18. Cyp1a1, canonically induced by aryl hydrocarbon receptor (AhR)

activation as part of phase I xenobiotic metabolism, was not expressed in

hepatic tissue from either the CSE or sham exposed fetuses, nor was the

expression of AhR different in CSE fetuses. PolΚ and Ercc1, however, were

induced in response to CSE, suggestive of a repair response to DNA damage.

Primary and immortalized mouse fibroblasts exposed to cigarette smoke

condensate (CSC) also induced PolΚ, and exhibited an increased micronuclei

frequency and polyploidy. Collectively, these findings suggest that the altered

fetal metabolic response to PAHs may promote an increased susceptibility to

damage-induced chromosomal aberrations.

60


POSTER #26

Title: Ab Initio QM/MM Calculations Show an Intersystem Crossing in the Hydrogen

Abstraction Step in Dealkylation Catalyzed by AlkB

Presenting

Author:

Dong Fang

Other

Author(s):

Richard L. Lord , G. Andrés Cisneros

Affiliations: Department of Chemistry, Wayne State University

Abstract: AlkB is a bacterial enzyme that catalyzes the dealkylation of alkylated DNA

bases. The rate-limiting step is known to be the abstraction of an H atom from

the alkyl group on the damaged base by a FeIV-oxo species in the active site.

We have used hybrid ab initio quantum mechanical/molecular mechanical

methods to study this step in AlkB. Instead of forming an FeIII-oxyl radical

from FeIV-oxo near the C–H activation transition state, the reactant is found to

be an FeIII-oxyl with an intermediate-spin Fe (S = 3/2) ferromagnetically

coupled to the oxyl radical, which we explore in detail using molecular orbital

and quantum topological analyses. The minimum energy pathway remains on

the quintet surface, but there is a transition between ISFeIII-oxyl and the state

with a high-spin Fe (S = 5/2) antiferromagnetically coupled to the oxyl radical.

These findings provide clarity for the evolution of the well-known π and σ

channels on the quintet surface in the enzyme environment. Additionally, an

energy decomposition analysis reveals nine catalytically important residues for

the C–H activation step, some of which are conserved in two human

homologues. These conserved residues are proposed as targets for experimental

mutagenesis studies.

61


POSTER #27

Title: DNA REPAIR ACTIVITIES OF MYCOBACTERIAL RMPs ARE DEFINED

BY TWO ALTERNATIVE DNA BINDING MODES.

Presenting

Author:

Mikhail Ryzhikov

Other

Author(s):

Sergey Korolev

Affiliations: Department of Biochemistry and Molecular Biology, Saint Louis University

School of Medicine St. Louis, MO

Abstract: Recombination mediator proteins (RMPs) are important for maintaining

genomic stability in all organisms after formation of DNA double-stranded

breaks (DSBs). RMPs support two distinct reactions: homologous

recombination by loading RecA-like recombinases onto ssDNA and annealing

of complementary ssDNA strands. Both reactions are inhibited by SSBs (single-

stranded DNA binding proteins) and RMPs overcome such inhibition during

DNA repair. The E. coli RecO requires interaction with SSB’s C-terminal (SSB-

Ct) to bind DNA to initiate both reactions. We previously demonstrated that

Mycobacterium smegmatis RecO does not bind SSB-Ct and its DNA binding

and annealing activities are stimulated by zinc. Here we show that

mycobacterial RecO initiates recombination independently of zinc and this

reaction is also independent of interaction with SSB-Ct. In spite of a lack of

SSB-Ct interaction, mycobacterial RecO in the presence and absence of RecR

does not displace SSB from ssDNA, suggesting that all three proteins form a

complex ssDNA. We hypothesize that the mycobacterial RecO and RecOR

complexes with SSB represent critical intermediates in annealing and

recombination. Such complexes can explain the ability of RMPs to sense stalled

replication and to support two alternative reactions in the same pathway of DSB

repair.

62


POSTER #28

Title: A Homology Model of ABH1 Protein

Presenting

Author:

Pavel Silvestrov

Other

Author(s):

G. Andrés Cisneros*

Affiliations: Department of Chemistry, Wayne State University, Detroit, MI 48202

Abstract: ABH1 is a member of the AlkB family of proteins. These proteins have an

important role in repairing DNA from damage induced by alkylating agents. In

particular, they catalyze direct dealkylation of DNA and RNA bases.

Homologues of AlkB differ in their affinity to single or double stranded DNA

and RNA, however, the residues comprising the dealkylase active site are

conserved. The jelly-roll fold is characteristic to the structures of these

enzymes. Experimental studies have shown that ABH1 can act as a lyase at AP

sites of DNA in addition to its dealkylase function. As there is currently no

experimentally determined structure of ABH1, we carried out homology

modeling. Using homologous proteins as templates, the structure of ABH1 was

predicted in the Rosetta suite of programs. ABH1 is close to AlkB in

phylogenetic tree, and, thus, AlkB was used as a template. Duffy-binding-like

domain was also used as a template since it has high alignment scores for parts

of the sequence of ABH1. Here we present a homology model of the full ABH1

protein including the proposed AP lyase domain. MD simulations of the

predicted structures were performed. K25 is predicted to form a covalent bond

with DNA upon lyase catalysis.

63


POSTER #29

Title: Functional Consequences of SNPs in the RPA1 Gene

Presenting

Author:

Layne Weatherford

Other

Author(s):

George Brush

Affiliations: Cancer Biology Graduate Program Department of Oncology

Abstract: Replication protein A (RPA) is a single-stranded DNA-binding protein that is

required for DNA replication, repair, and recombination. A mutation in the

large subunit of RPA (RPA1) has been shown to increase cancer risk in a mouse

model system. We will examine functional consequences of natural RPA1 SNPs

in two different systems, with attention paid to cancer-related phenotypes. In the

first system, we have created the corresponding mutations in yeast and tested

the strains for mutation frequency and sensitivity to DNA damaging agents and

a replication inhibitor. Some strains containing a variant of RFA1 (yeast

orthologue of RPA1) show slight sensitivity to ionizing radiation. Several

strains containing an RFA1 variant display a slightly higher mutation frequency

than wild type RFA1. In a preliminary study, we found some strains containing

RFA1 variants to show an increased rate of UV-induced mutagenesis. In these

cases, the mutant residue may compromise RFA1 function, at least in regard to

its role in DNA repair. For the second system, we will follow SV40 DNA

replication in vitro, a cell-free assay that relies on human DNA replication

proteins. We will use this system to determine if human SNPs affect DNA

replication efficiency. In addition, we will use this assay combined with

sequencing analysis as a novel method for determining mutation frequencies.

64


POSTER #30

Title: Nucleotide Excision Repair and Lung Cancer in Appalachian Kentucky

Presenting

Author:

Nathaniel Holcomb

Other

Author(s):

Nathaniel Holcomb1, Mamta Goswami1, Gary Gairola1, David K. Orren1, Eric

Durbin2, Tamas S. Gal2, Isaac Hands3, Bront Davis3, Brent Hallahan4, Brent

Shelton5 Jeffrey Boyles5, Susanne Arnold6, and Isabel Mellon1

Affiliations: 1. The Graduate Center for Toxicology, College of Medicine 2. Division of

Biomedical Informatics, College of Public Health 3. Cancer Research

Informatics Shared Resource Facility, Markey Cancer Center 4. Biospecimen

Shared Resource Facility, Markey Cancer Center 5. Department of Biostatistics,

College of Public Health 6. Department of Medicine, College of Medicine

Abstract: Persistent DNA damage can result in mutations that drive carcinogenesis.

Cellular repair pathways act to remove DNA damage, minimizing genetic

changes and suppressing cancer development. Exposure to tobacco smoke

generates DNA damage causative for development of lung and other cancers;

many of the lesions are targeted specifically by the nucleotide excision repair

(NER) pathway. Thus, the efficiency of an individual’s NER system is likely to

impact cancer susceptibility, particularly in regards to tobacco smoke exposure

and lung cancer development. Although most lung cancers are related to

tobacco smoke exposure, many smokers never develop lung cancer, suggesting

additional factors involved in smoking-induced lung cancer. Chronic arsenic

exposure has been linked to lung cancer in humans. Our lab has discovered that

exposure to arsenic or cigarette smoke condensate inhibits NER efficiency in

vitro and significantly reduced the abundance of a key NER protein, XPC.

Appalachian Kentucky has an extremely high incidence of lung cancer, not

fully explained by smoking rates. Individuals in the region have elevated

exposure to trace metals (arsenic, chromium, and nickel). An epidemiological

lung cancer case-control study was initiated to collect biological and

environmental samples and personal data. We hypothesize that NER is

suppressed in individuals who develop lung cancer and we are testing this

hypothesis by measuring and comparing NER efficiency in lymphocytes

isolated from subjects in the study. We also hypothesize that exposure to trace

metals and/or tobacco smoke can inhibit the NER pathway in individuals which

in turn can lead to the development of lung cancer.

65


POSTER #31

Title: DESENSITIZATION OF BASAL CELL CARCINOMA TO THE ANTI-

TUMORAL EFFECT OF VITAMIN D: ROLE OF REDD1.

Presenting

Author:

RawiaKhasawneh

Other

Author(s):

Dr. Ahmad Heydari Dr. ArchanaUnnikrishnan Dr. Bassel Mahmoud Dr.

IltefatHamzavi

Affiliations: Wayne State University, Henry Ford Hospital

Abstract: The relationship between Vitamin D and Non-melanoma skin Cancer is not

clear. Most of epidemiological studies are inconsistent and inconclusive, and

Mechanistic studies are lacking. Vitamin D is a seco-steroid hormone, whose

nuclear receptor (VRD) is a nuclear transcription factor. It was suggested that

vitamin D possesses anti-proliferation and pro-differentiation effect by

negatively regulating important signaling pathways. In particular, REDD1

(regulated in development and DNA damage response 1) which is a VDR target

gene inhibits mTOR pathway impacting cellular growth. In response to DNA

damage, REDD1 transcription is up-regulated by elevated levels of P53,

resulting in inhibition of mTOR pathway. Subsequently, reduction of P53

protein occurs as a feedback inhibition at the translation level. This case-control

study aimed to explore the impact of vitamin D status on the onset and

progression and possibly treatment of basal cell carcinoma. Three tissue

samples were collected from 20 BCC patients (Cancer, Proximal, and Distal),

and 6 cancer-free individuals from southeast Michigan. BCC and its feeding

cells seemed to up-regulate vitamin D activation enzymes, VDR, and its co-

activators proposing higher local activity of vitamin D. Despite the significantly

increased protein levels of REDD1 in the cancer tissue, our data showed that

VRD’s ability to down-regulate mTOR pathway through REDD1 was

diminished. Interestingly, Vitamin D negative regulation of Hedgehog-Gli

pathways was also lost in the cancer tissue. Kras mutation was detected in all

specimens obtained from cases participants. In conclusion, we propose that the

anticipated role of vitamin D is not conserved in BCC tissue.

66


POSTER #32

Title: SAD-6: an SNF2-family protein involved in meiotic silencing by unpaired

DNA

Presenting

Author:

Dilini A. Samarajeewa

Other

Author(s):

Nick A. Rhoades, Hua Xiao, Patrick K. T. Shiu, Kevin A. Edwards, and

Thomas M. Hammond

Affiliations: School of Biological Sciences, Illinois State University, Normal, Illinois,

61790. Division of Biological Sciences, University of Missouri, Columbia,

Missouri, 65211.

Abstract: Meiotic silencing by unpaired DNA (MSUD) is a process that detects and

silences unpaired DNA between homologous chromosomes for the duration of

meiosis. It is believed that gene silencing works through an RNA-interference-

related pathway via the production of a theoretical aberrant RNA molecule.

However, the nature of the unpaired DNA scanning mechanism and the proteins

involved in the nuclear aspects of MSUD are completely unknown. Rad54 is a

protein involved in repairing double stranded DNA breaks by homologous

recombination in yeast. SAD-6 is a putative SNF2-family protein closely related

to Rad54 in Neurospora crassa. We found that MSUD is significantly

suppressed by the deletion of sad-6 locus, suggesting that it is required for the

process. Moreover, confocal microscopy studies have confirmed that GFP

tagged SAD-6 protein is localized in the nucleus during meiosis suggesting that

SAD-6 could be a protein involved in the unpaired DNA detection process of

MSUD. Further studies will seek to identify the theoretical aberrant RNA and

determine if SAD-6 is required for its production.

67


POSTER #33

Title: Investigating a DNA homology search process in the Model Organism

Neurospora crassa

Presenting

Author:

Pegan Sauls

Other

Author(s):

Zach Smith, Kevin J. Sharp, and Thomas M. Hammond

Affiliations: Illinois State University

Abstract: When Neurospora crassa’s cells undergo meiosis, its genome undergoes a

quality control process called Meiotic Silencing by Unpaired DNA (MUSD).

MSUD scans homologous chromosomes for segments of DNA that are

unpaired. This can occur when an ectopic copy of a gene is added to a

chromosome. Since an ectopic gene copy is unpaired, it will be detected by

MSUD and silenced for the duration of meiosis. While many of the protein

players of the silencing process have been identified, those involved in the

scanning process are unknown. In our research, we have taken a genetic marker

and integrated it into different locations of the N.crassa genome. We have

discovered that unpaired genes are not always recognized by MSUD.

Essentially, recognition appears to be dependent on the distance between the

unpaired genes and their homologs, with small distances leading to unpaired

genes escaping detection. Our current work seeks to identify and characterize

the proteins involved in the detection of unpaired genes, which should help

explain the imprecision in the unpaired DNA identification process.

68


POSTER #34

Title: Low-dose ionizing radiation induces CNVs in cultured cells.

Presenting

Author:

Martin F. Arlt

Other

Author(s):

Sountharia Rajendran, Shanda R. Birkeland, Thomas E. Wilson, Thomas W.

Glover

Affiliations: Department of Human Genetics, University of Michigan; Department of

Pathology, University of Michigan

Abstract: Copy number variants (CNVs) are deletions and duplications that can exceed 1

Mb in size. Despite their importance to human genetic variationand disease,

little is known about the molecularmechanisms and environmental risk

factorsthat impact CNV formation.While it is clear that replication stress

canlead to de novo CNVs, for example, followingtreatment of cultured

mammalian cells with aphidicolin(APH) and hydroxyurea (HU), the effectof

different types of mutagens on CNV inductionis unknown. We have

investigated the effects of ionizing radiation (IR) on CNV formation in normal

human fibroblasts. Wefound that IR in the range of 1.5–3.0 Gy

effectivelyinduces de novo CNV mutations in these cells. The IR-induced

CNVsare found throughout the genome, with the samehotspot regions seen after

APH- and HU-inducedreplication stress. IR produces duplications at ahigher

frequency relative to deletions than doAPH and HU. At most hotspots, these

duplicationsare physically shifted from the regions typicallydeleted after APH

or HU, suggestingdifferent pathways involved in their formation.CNV

breakpoint junctions from irradiated samplesare characterized by

microhomology, bluntends, and insertions like those seen in spontaneousand

APH/HU-induced CNVs and most nonrecurrentCNVs in vivo. The similarity to

APH/HU-induced CNVs suggests that low-dose IR inducesCNVs through a

replication-dependent mechanism,as opposed to replication-independentrepair

of DSBs. Consistent with this mechanism, fewer CNVs were induced by IR

when cellswere held for 48 hr before replating after irradiation.These results

predict that any environmentalDNA damaging agent that impairs replicationis

capable of creating CNVs.

69


POSTER #35

Title: Genome instability, the ultimate driver for cancer evolution

Presenting

Author:

Henry H Heng

Other

Author(s):

Joshua Stevens, Batoul Abdallah, Steven Horne, Karen Ye, Guo Liu

Affiliations: Center for Molecular Medicine and Genetics, and Department of Pathology,

Wayne State University School of Medicine, Detroit, MI.

Abstract: The highly complex diverse genomic profile of cancer challenges current gene

mutation/pathways centered cancer research. Each specific gene mutation only

has limited predictive ability due to such a high level of complexity in each

tumor. Failure to find highly penetrant mutational drivers of cancer necessitates

searching for common drivers of cancer evolution, specifically by focusing on

the genomic organization instead of cataloging gene mutations. The genome,

rather than genes, is the platform of macro-cellular evolution meaning that

genome diversity is a pre-condition for cancer evolution. Genomic instability,

the engine of genome diversity, is the key to understanding and control cancer.

Here we discuss multiple aspects of genome instability including its

definition(s), measures, its relationship(s) with DNA repair pathways, and its

ultimate importance for cancer evolution. We then apply the genome theory

(where the karyotype rather than individual genes defines the system

inheritance) and evolutionary theory (with interaction between both punctuated

and stepwise phases) to propose a general mechanism that uses genome

instability to unify the diverse molecular causes of cancer. References: 1).

Genome chaos: survival strategy during crisis. Cell Cycle. 13:528-37 (2014). 2).

Unstable genomes elevate transcriptome dynamics. Int J Cancer. 134:2074-87

(2014). 3). Single cell heterogeneity: why unstable genomes are incompatible

with average profiles. Cell Cycle. 12:3640-9 (2013). 4). Chromosomal

instability (CIN): what it is and why it is crucial to cancer evolution. Cancer

Metastasis Rev. 32:325-40 (2013). 5). Evolutionary mechanisms and diversity

in cancer. Adv Cancer Res. 112:217-53 (2011).

70


POSTER #36

Title: Small Molecule Inhibitors of ERCC1-XPF: A Novel Approach for Combination

Chemotherapy

Presenting

Author:

Steve M. Patrick

Other

Author(s):

Sanjeevani Arora, Vivian Kalman-Maltese, Jeffrey Sarver, Paul Erhardt and Q.

Ping Dou

Affiliations: Wayne State University/Karmanos Cancer Institute University of Toledo Health

Science Campus

Abstract: We have established a high throughput screen to identify inhibitors of the

ERCC1-XPF endonuclease. ERCC1-XPF is a critical component of the DNA

repair pathways that remove bulky DNA lesions and interstrand crosslinks from

the genome. In this role, ERCC1-XPF has a significant impact on the efficacy of

DNA damaging chemotherapeutic drugs and has been shown to affect drug

resistance. Recent literature has suggested that ERCC1 is a prognostic indicator

of chemotherapy response in lung cancer, ERCC1 deficient cells synergize with

PARP inhibitors and ERCC1 deficient cells synergize with proteins in the ATR

signaling pathway to yield a synthetic lethal interaction. These data highlight

the significance of targeting ERCC1-XPF endonuclease with small molecules to

provide novel combination therapies to achieve better patient outcomes in a

variety of cancers. In our initial screen of the NCI diversity set, we identified 2

Hits that had IC50 values in the low nanomolar range against ERCC1-XPF

while having minimal effect against two non-related endonucleases (XPG and

HhaI). In the 10-15 micromolar range, these Hits were shown to inhibit DNA

repair in cell culture and potentiate cisplatin efficacy in colony survival assays.

A structural similarity search to Hit 1 identified a natural compound from green

tea, (-)-epigallocatechin-3-gallate (EGCG), that had nanomolar potency (IC50:

50-100 nM) in the cell free assay and low micromolar (5-10 uM) activity in cell

culture. Our future studies will focus on screening structural analogs of EGCG

that are more potent ERCC1-XPF inhibitors that potentiate cisplatin effects in

ovarian and lung cancer xenografts.

71


POSTER #37

Title: Targeting the ERCC1/XPF Nuclease for Cancer Therapy

Presenting

Author:

Derek Woods

Other

Author(s):

Shehnaz Khan, Vivian Kalman-Maltese, Kate Marchal, Steve M. Patrick, John

J. Turchi

Affiliations: NERx Biosciences, Inc.

Abstract: Cisplatin is one of the most widely prescribed chemotherapeutic agents used to

treat cancer. Cisplatin functions by damaging DNA and the effectiveness of

cisplatin in killing cancer cells is often limited by the repair of cisplatin-DNA

damage by the nucleotide excision repair (NER) and the homologous

recombination repair (HRR) pathways. The ERCC1/XPF structure-specific

nuclease functions in both of these pathways and is responsible for incision of

damaged DNA 5’ of the lesion. Expression of both ERCC1 and XPF has been

correlated with cisplatin response in ovarian and lung cancers. For this reason,

the inhibition of ERCC1/XPF nuclease activity holds the potential to enhance

cisplatin efficacy in non-responding patients that have high ERCC1 expression.

In pursuit of novel anti-cancer agents, we completed an in vitro high throughput

screen of drug like molecules for inhibitors of ERCC1/XPF. Hits were validated

in a second, gel-based assay and inhibition was confirmed to have a titratable

effect with IC50 values in the low nanomolar range. Hits were tested for

specificity of ERCC1/XPF inhibition against XPG and the HhaI restriction

enzyme. As expected, specificity varied among the validated hits. Hits were also

show to sensitize the A2780 cell line to cisplatin consistent with predicted

ERCC1/XPF inhibition. Together these results support the pursuit of

ERCC1/XPF inhibitors. Future studies will focus on determining the method of

action for these inhibitors and in vivo xenograft studies to test efficacy of leads

as adjuvant agents.

72


POSTER #38

Title: DNMT inhibitors sensitize breast cancer to radiation

Presenting

Author:

Steven Zielske, Ph.D.

Other

Author(s):

Deborah A. Antwih, Kristina M. Gabbara

Affiliations: Department of Radiation Oncology, Wayne State University and Karmanos

Cancer Institute, Detroit, Michigan

Abstract: Epigenetic regulation has been shown to play a key role in the development of

many malignancies. One of the major obstacles in treating many tumor

malignancies is radiation resistance. DNA methyltransferase (DNMT) inhibitors

can restore gene expression silenced by DNA methylation. We investigated the

effects of DNMT inhibitors on radiation sensitivity in breast cancer cells. Two

breast cancer cell lines were incubated with or without 5-azacytidine for two

days before treatment with ionizing radiation and subsequently plated for

colony formation. The survival fraction of cells treated with radiation and 5-

azacytidine was lower, showing a radiation enhancement factor of 1.8 compared

to those treated with ionizing radiation alone. We further evaluated additional

DNMT inhibitors. To determine the effects of DNMT inhibitors on DNMT

levels, cells were treated with 5-azacytidine, decitabine, and zebularine. At the

end of treatment, cells were collected and lysed for Western blotting analysis to

evaluate the level of DNMT1, 3a and 3b. Pretreatment of cells with DNMT

inhibitors resulted in decreased DNMT1, and 3a, while there was no change in

DNMT3b. Each inhibitor had differential ability to reduce DNMT levels

depending on cell line. 5-Methylcytosine (5-mC) levels were quantified by

ELISA and found to be progressively reduced over time in DNMT inhibitor-

treated cells. Our results show reduced levels of 5-mC in cells treated with

DNMT inhibitors and strong radiosensitization. These data suggest that there is

a synergistic effect caused by DNMT inhibitors and ionizing radiation in the

treatment of breast cancer cells, thereby enhancing radiation toxicity.

73


AUTHOR INDEX

Abdallah, Batoul Y (8) Ahmed, Aqila A (7)

Al-Katib, Ayad (51) Amaro-Ortiz, Alexandra (39)

Andreassen, Paul R (10, 30) Antwih, Deborah A (72)

Arlt, Martin F (68) Arnold, Susanne (64)

Arora, Sanjeevani (70) Ayers, Claci (39)

Bedi, Karan (10, 34, 53) Bell, Charles E (9, 26, 52)

Beydoun, Safa (7, 18) Bhagwat, Ashok S (8, 51)

Bhattacharjee, Anukana (8, 23) Birkeland, Shanda R (68)

Bishop, Douglas K (8, 22) Boyles, Jeffrey (64)

Brush, George (63) Budke, Brian (8, 22)

Buis, Jeffrey (41) Cabelof, Diane C (6, 7, 19)

Carmo de Andrade Lima, Leonardo (53) Carpenter, Brittany L (58)

Chaiken, Mary F (8, 23) Chan, Timothy (10, 31)

Chen, Min (58) Choi, Jun-Hyuk (10, 33)

Chung, Jinsil (9, 29) Cisneros, G. Andrés (6, 10, 60, 62)

Connell, Philip P (8, 22) Cui, Tiantian (7, 17)

D’Orazio, John A (39) Davis, Bront (64)

Dormi, Silvana (44, 47) Dou, Q. Ping (70)

Dumitrache, Lavinia C (8, 25) Durbin, Eric (64)

Dusad, Kritika (8, 22) Edwards, Kevin A (66)

Egli, Martin (36) Eoff, Robert L (9, 28)

Erhard, Paul (70) Fang, Dong (60)

Fardous, Ali (7, 18) Fercu, Catinca A (9, 28)

Ferguson, David O (41) Festerling, Todd A (41)

FitzGerald, Michael (7, 18) Freund, Marcel (10, 30)

Gabbara, Kristina M (72) Gaddameedhi, Shobhan (10, 33)

Gairola, Gary (64) Gal, Tamas S (64)

Gilmore, Hannah (9, 29) Glanzer, Jason G (35, 40)

Glover, Thomas W (5, 7, 68) Gold, Barry (7, 20)

Goswami, Mamta (64) Greene, RM (59)

Grubb, Jennifer (8, 22) Gu, Liya (45)

Guengerich, F. Peter (9, 28) Gupta, Smiti (54)

Hallahan, Brent (64) Hammond, Thomas M (66, 67)

Hamzavi, Iltefat (65) Han, Chunhua (7, 17, 49, 50)

Han, Xiangzi (9, 29) Hands, Isaac (64)

Hanenberg, Helmut (10, 30) Hasty, Paul (8, 25)

Haynes, Brittany (8, 24, 54) He, Jinshan (49, 50)

Heng, Henry H (7, 8, 21, 57, 69) Heydari, Ahmad (6, 7, 18, 65)

Holcomb, Nathaniel (64) Holland, Cory L (8, 25)

Holland, Thomas (51) Horne, Steven D (8, 21, 57, 69)

Hsu, Shih-Jui (8, 23) Hu, Jinchuan (10, 33)

Hu, Lingchuan (8, 25) Ismail, Sukayna (7, 18)

Jacobberger, James W (9, 29) Jurkiw, Tom J (37)

Kalman-Maltese, Vivian (70, 71) Kane, Daniel P (6, 16)

74


Kemp, Michael G (10, 33) Keri, Ruth (9, 29)

Khan, Shehnaz (71) Khasawneh, Rawia (65)

Kim, Christine (45) Kim, Dong Hyun (8, 25)

Kim, Sung-A (8, 25) Kim, Tae Moon (8, 25)

Kirkconnell, Killeen S (46, 53) Koncar, Robert (10, 31)

Kool, Daniel (36) Korolev, Sergey (61)

Kothandapani, Anbarasi (43) Kuan, Shih-Fan (7, 20)

Larson, Erik (56) Li, Guo-Min (45)

Liang, Zhuobin (10, 32) Liu, Guo (8, 21, 57, 69)

Liu, Shengqin (35, 40) Ljungman, Emily A (53)

Ljungman, Mats (10, 34, 46, 53) Logan, Hillary (8, 22)

Lord, Richard L (60) Magnuson, Brian (10, 34, 46, 53)

Mahmoud, Bassel (65) Mao, Guangzhao (54)

Marchal, Kate (71) Martin, Alberto (51)

Mason, Jennifer M (8, 22) McNally, Justin R (48)

Meetei, Amom Ruhikanta (10, 30) Mellon, Isabel (64)

Miller, Erin L (9, 27) Mirmoghtadaei, Nima (40)

Mishra, Akaash (44, 47) Montagna, Cristina (8, 25)

Morgan, Mary J (41) Mosel, Adam (35, 40)

Nallasivam, Sivakumar (10, 32) Nassar, Nicolas (10, 30)

Noronha, Anne M (36) Oakley, Greg G (35, 40)

O'Brien, Patrick J (9, 27, 37, 42, 48) O'Connor, Kathleen L (58)

O'Flaherty, Derek K (36) Oomen, Jerry (10, 34)

Orren, David K (64) Ortega, Janice (45)

Pan, Xinlei (9, 26) Park, Jung-Young (10, 30)

Park-York, MieJung (7, 19) Patrick, Steve M (43, 70, 71)

Paulsen, Michelle T (10, 34, 46, 53) Pence, Matthew G (9, 28)

Peng, Aimin (35) Pentz, Kyle (49)

Pisano, MM (59) Prasad, Jayendra (10, 34, 53)

Price, Carolyn (8, 23) Qian, Jiang (49, 50)

Rahassi, El Mustapha (10) Rajendran, Sountharia (68)

Rhoades, Nick A (66) Romick-Rosendale, Lindsey (10, 31)

Ryzhikov, Mikhail (61) Saadat, Nadia (54)

Samarajeewa, Dilini A (66) Sancar, Aziz (10, 33)

Sanders, Matthew (8, 24) Sarver, Jeffrey (70)

Sauls, Pegan (67) Sawant, Akshada (43)

Shalhout, Sophia (51) Sharma, Nidhi (49, 50)

Sharp, Kevin J (67) Shcherbakova, Polina V (6, 16)

Shekhar, Malathy (8, 9, 24, 54) Shelton, Brent (64)

Shiu, Patrick K T (66) Shriver. Storm J (36)

Silvestrov, Pavel (62) Singh, Amanpreet (38)

Singh, Thiyam R (10, 30) Smith, Christopher E (52)

Smith, SC (59) Smith, Zach (67)

Sobol, Robert (43) Son, Mi Young (8, 25)

Sosin, Angela (51) Srivastava, Amit Kumar (7, 17)

Stevens, Joshua B (8, 21, 57, 69) Stewart, Jason (8, 23)

75


Sweasy, Joann (4, 6) Tateishi, Satoshi (8, 25)

Taylor, Mark R (37) Tong, Dan (45)

Tran, Amanda (9, 29) Turchi, John J (44, 47, 55, 71)

Unnikrishnan, Archana (7, 18, 65) VanderVere-Carozza, Pamela (55)

Veloso, Artur (10, 34, 46, 53) Ventrella-Lucente, Lisa (7, 18)

Wang, Ling (35) Wang, Qi-En (7, 17, 49, 50)

Wani, Altaf A (7, 17, 49, 50) Wani, Gulzar (49, 50)

Weatherford, Layne (63) Webb, C (59)

Weichselbaum, Ralph R (8, 22) Wells, Susanne (10, 31)

Whetstone, Ryan (7, 20) Wilds, Christopher J (36)

Williams, Jonathan (56) Wilson, Thomas E (10, 32, 34, 46, 53, 68)

Woods, Derek S (44, 47, 71) Worth, Leroy (11)

Wu, Megan (8, 22) Xiao, Hua (66)

Xu, Yong-jie (38) Ye, Christine J (8, 21)

Ye, Karen J (8, 21, 69) Yew, P. Renee (8, 25)

Yuan, Shuai (9, 28) Zhang, Fan (10, 30)

Zhang, Lei (9, 29) Zhang, Yanhua (54)

Zhang, Yaru (42) Zhang, Yowei (9)

Zhao, Linlin (9, 28, 36) Zhao, Ran (7, 17, 50)

Zhitkovich, Anatoly (43) Zhu, Qianzheng (49, 50)

Zielske, Steven (72)

midwest dna repair symposium16thmwdr.clas.wayne.edu/2014midwestdna-repair... · symposium may 17...

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