Targeting the DNA Repair Targeting the DNA Repair

Enzyme TDG to Enhance Enzyme TDG to Enhance

Sensitivity of Cancer Cells to Sensitivity of Cancer Cells to

AntiAnti--Leukemic AgentsLeukemic Agents

Laura Cosentino, PhD

Fox Chase Cancer Center

Disclosure: I have no conflict of interest concerning this presentation.

DNA Repair and Cancer

� Genomic instability is a hallmark of tumorigenesis and it is defined as an

increased propensity for genomic alterations

� DNA repair pathways have a central role in maintaining genomic stability by

repairing DNA damages or replication errors

� DNA repair may be an important determinant of drug sensitivity

� We focus on DNA repair as a molecular target to improve cancer therapy

Hanahan and Weinberg, Cell 2011

1. Mismatch Repair (MMR)

2. Nucleotide Excision Repair (NER)

3. Base Excision Repair (BER)

Base Excision Repair Machinery

� BER is responsible for repairing damage induced by endogenous metabolic processes (methylation, deamination, ROS and hydrolysis)

(Wood et al, Science 2001)

� BER-specific enzymes are

DNA N-glycosylases and

AP endonuclease

� Our laboratory studies

Thymine DNA Glycosylase (TDG)

DNA glycosylase

AP

endonuclease

dRpase

DNA polymerase and

ligase

DNA glycosylase

Thymine DNA Glycosylase (TDG)

� TDG recognizes G�T mispairs at CpG sites

� CpG sites are hotspots for mutations due to the “spontaneous”deamination of 5-MeC to T, causing a G/T mismatch

� TDG is involved in restoration of C instead of 5-MeC, leading to overall demethylation in key regions of the epigenome

Adapted from Fitzgerald and Drohat, JBC 2008

Mechanisms of active DNA demethylation

Dalton and Bellacosa, Epigenomics, 2012

Disruption of the balanced epigenetic network may have significant impact on

chromatin structure and transcriptional activity.

TDG in Epigenetic Gene Regulation

Cortellino et al, Cell, 2011

Leukemia as an epigenetic disease

Normal

Leukemia as an epigenetic disease

Normal

TDG Mut

Leukemia

� KNOWN: DNMT inhibitor (Decitabine) yields promising results in

hematopoietic malignancies (Galm et al, Blood 2006)

� QUESTION: Can we target TDG for therapeutic purposes?

Cytotoxic enhancement to BDM by TDG inactivation

Untreated 10 µM 50 µM 200 µM

BDM

Untreated 10 µM 50 µM 200 µM

BDM

TDG wt VS. TDG -/- MEFs

Cortellino et al, Cell, 2011

Beeharry et al, PLoSONE, 2012

Bendamustine (BDM)

� Nitrogen mustard

� Used for:

Chronic lymphocytic

leukemia

Non-Hodgkin’s lymphoma

Multiple myeloma

TDG is involved in cytotoxicity induced by BDM

TDG is involved in cytotoxicity induced by BDM

HOW?

TDG is involved in cytotoxicity induced by BDM

HOW?

Repair of DNA damage?

Epigenetic effect?

TDG is involved in cytotoxicity induced by BDM

HOW?

Repair of DNA damage?

Epigenetic effect?

TDG is involved in cytotoxicity induced by BDM

HOW?

Repair of DNA damage?

Epigenetic effect?

Both?

The concept of TDG inhibition

DRUG

DRUG

DRUG

Development of a high-throughput TDG repair assay

Assay modified from Svilar D et al, J Vis Exp. 2012

0

500

1000

1500

2000

2500

0 1000 2000 3000 4000 5000 6000 7000 8000

Time

AU

U251 hTDG nucl

U251 parent 1 nucl

negative control

A549 U251

wt +TDG wt +TDG

- 60 KDa

- 50 KDa

- 40 KDa

0

500

1000

1500

2000

2500

0 1000 2000 3000 4000 5000 6000 7000 8000

Time

AU

U251 hTDG nucl

U251 parent 1 nucl

negative control

A549 U251

wt +TDG wt +TDG

- 60 KDa

- 50 KDa

- 40 KDa

A549 U251

wt +TDG wt +TDG

- 60 KDa

- 50 KDa

- 40 KDa

A549 U251

wt +TDG wt +TDG

- 60 KDa

- 50 KDa

- 40 KDa

A549 U251

wt +TDG

A549 U251

wt +TDG wt +TDG

- 60 KDa

- 50 KDa

- 40 KDa

TDG+APE137ºC

T

G

G

T

FAM

Dabcyl

� TDG is a single turnover enzyme

(Koff , dissociation E from P, is

rate limiting)

� APE increases TDG turnover by

42- and 26-fold for G:T and G:U

substrates

� TDG and APE are in vast excess

over substrate, therefore

conditions approximate single

turnover kinetics

� Equilibration of TDG with potential

inhibitors will allow screening in

pre-steady state conditions

Optimization of a high-throughput TDG repair assay

-500

0

500

1000

1500

2000

2500

3000

500 1000 1500 2000 2500 3000 3500

Time

AU

+ ENZYMES

- ENZYMES

-

+

Optimization of a high-throughput TDG repair assay:

Determination of the Z’ factor

0

500

1000

1500

2000

2500

3000

3500

0 1000 2000 3000

Time

AU

TDG+APE1 0.05 mM EDTA 0.1 mM EDTA

0.2 mM EDTA 1 mM EDTA 5 mM EDTA

25 mM EDTA

Optimization of a high-throughput TDG repair assay:

Determination of the Z’ factor

0

500

1000

1500

2000

2500

3000

3500

0 1000 2000 3000

Time

AU

TDG+APE1 0.05 mM EDTA 0.1 mM EDTA

0.2 mM EDTA 1 mM EDTA 5 mM EDTA

25 mM EDTA

Sample NumberA

U

Dynamic Range

Z’ factor is a characteristic parameter for the quality of the screening itself, without

intervention of test compounds (Zhang et al, J Biomol Screen 1999)

Z' factor = 0.79

1>Z≥ 0.5 Separation band is large and the assay is excellent

Optimization of a high-throughput TDG repair assay:

determination of the Z’ factor

Activity/B

ackg

round

Control

Inhibitor

Control

Inhibitor

Control

Inhibitor

Conclusions

� TDG has a dual role in both DNA repair (genomic stability) and active DNA

demethylation & transcriptional regulation (epigenomic stability)

� TDG loss / activity inhibition enhances tumor killing

� We have developed and optimized a homogeneous fluorescence-based

high-throughput assay to evaluate inhibition of TDG

� Inhibitor concentrations in the same range of substrate concentration

should give a drastic reduction of fluorescence generation, facilitating

inhibitor discovery

� Pharmacological inhibition of TDG could lead to significant sensitization to

known anti-leukemic agents, including bendamustine (BDM)

� This study could lead to the development of investigator-initiated

preclinical and clinical trials

Alfonso Bellacosa

Valentina Doneddu

Pietro Mancuso

Rossella Tricarico

Hélène Leger

Andy Andrews

Timothy Yen

Neil Beeharry

Robert W Sobol

(University of Pittsburgh

Cancer Institute)

FCCC Facilities

Emmanuelle Nicolas

Margret B. Einarson

Yan Zhou

Betsy Bove