JE 2428 Onco-Pharmacologie

JF Riou

Inhibiteurs de télomérase : les ligands de l'ADN G-Quadruplexe, Approches Actuelles pour le Traitement du Cancer Louvain-la-Neuve, 13 mai 2004

TELOMERE: A CRITICAL COUNTER FOR CELL DIVISION Loss of telomere limits lifespan of diploid cells

Somatic cells

Replicative Senescence

Germinal cells Cancer cells

Immortalization Telomerase

Telomerase

Genetic instability End-to end telomere fusions Division arrest Terminal differentiation Telomerase is Activated in most Cancer cells (~85%)

15 % ALT (Alternative Lengthening of Telomere )

TELOMERE : TTAGGG REPEATS AT THE END OF CHROMOSOMES

G-strand Single-strand DNA overhang (300b)

5’ TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG (TTAGGGTTAGGG) 3’ n

AATCCCAATCCCAATCCCAATCCCAATCCC Double-strand DNA repeats (12-15kb)

C-strand

Protection of chromosomes from: •Recombination •Fusion •Being recognized as damaged DNA

Physiological role of telomerase on telomere structure in normal human cells Masutomi et al (2003) Cell, 114, 241-253 Normal Early PD

hTERT inhibition

Telomerase

5’

3’

5’

hTERT overexpression Telomerase

Telomerase

3’

5’

5’ Telomerase

Mid PD

Late PD

Telomere loss 3’overhang loss Proliferation Senescence

Telomerase

3’

5’

Telomerase

3’

5’

5’

3’

Yes Yes Normal Yes

Yes Premature Slow Premature

3’

Telomerase

Telomerase

Telomerase

5’

Telomerase

Telomerase

3’

Telomerase

Telomerase

No No Normal No

3’

Capping Hypothesis for Telomere Maintenance Critical Telomere length Capped

[no telomerase]

Uncapped telomere

DNA damage signaling

[With telomerase]

Cellular proliferation Senescence or apoptosis induction

Blackburn, Oncogene 2002

T-loop structure

Capped Telomere

Unfolded structure

Telomerase active

Telomere Relication

De Lange Nature Reviews Molecular Cell Biology April 2004

Telomerase is activated and overexpressed in 85% of overall human tumor cells while Telomerase is not expressed (or poorly expressed) in most somatic tissues Initial basis for the telomerase directed therapy of cancers

BLOCK CATALYTIC ACTIVITY OF hTERT Achieve replicative senescence/apoptosis by shortening of telomeres

Telomere length (kb)

Telomerase inhibition Normal cells 20

Tumor cells long telomere 10

5

Telomerase on

Therap. Index

short telomere

Telomerase on

Time

Apoptosis/ senescence

Inhibition of telomerase : 1. Target hTERT (catalytic subunit)

NHEI

Tankyrase

MRE11 RAD50

POT1

hsp90 TERT

GGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAG TRF1 TRF2 CAAUCCCAAUC CCCAATCCCAATCCCAAT TRF1 TRF2

Ku

hTR 5’

3’

2. Target hTR 3. Blocking the substrate

Dyskerin

INTRAMOLECULAR G-QUADRUPLEX STRUCTURE AT TELOMERE:

Central cation

G-QUARTET

G-QUADRUPLEX

INTRAMOLECULAR G-QUADRUPLEX

G-QUADRUPLEX CATION SELECTIVITY : K>Rb>Na>Li=Cs >> Sr>Ba>Ca>Mg

Inhibition of telomerase by G4 ligands Telomerase requires a 3' single stranded protruding end -5’-GGGTTAGGGTTAGGGTTAGGGTTA…(GGGTTA)n-3’ -3’-CCCAATCCCAAT-5’

Reconnaissance des boucles Fixation dans un sillon

-5’-GGGTTAGGGTTAGGGTTA -3’-CCCAATCCCAATCCCAAT

3’

Intercalation "véritable"

Empilement

: Ligand

A STRUCTURE-BASED STRATEGY TO DESIGN INHIBITORS

Thrombine/G-quadruplex XRay Complex

Hit from Trap Telomerase Assay

Thrombine/G-quadruplex Loop-Loop Interactions

Superimposition Model Hit Trap / G-quadruplex Key Features

LAOUI, A, MAILLIET, P., and RIOU, J.F. Rational lead discovery of G-quadruplex DNA ligands. Proc. Am. Assoc. Cancer Res. New Orleans (USA), 24-28 March, 42, 1341 (2001).

Thrombine/G-quadruplex Key Interactions

G-quadruplex Ligands Approaches Potential Anticancer Agents

LIGANDS G4 INHIBITEURS DE TÉLOMÉRASE +

H2N

N

+

N

NH

NH

N H

+

N

N

Acridines

N

Porphyrin

S

Bis-Acridine

S

N N H

N

O

N

N

N

CH3

O

N

N N

N H

N

Triazine

CH3

N N

N H N

O

N N O

N

N H

N

N H

NH

HN

+

N

O

O

N H

N H

H N

O

NH

N

N H

N H

N

N

H2N

N O

+

NH2 +

N

N H N

N

NH

O NH2

Telomestatin

Ethidium

Dibenzophenanthrolines

Enzyme

Blank

Pyrido2,6dicarboxamide derivatives as potent and selective G-quadruplex based telomerase inhibitors

307A 45 30 15 10 1

0.6 0.3 0.1 0.03 µM

N

+

TSG4 TS ITAS

MAILLIET,P. et al Brevet .Aventis-Pharma SA, Premier Dépot: France, 7 Février 2003.

Mailliet, P., et al, (2003).Proc. AACR Meeting. LB28.

O

O N H

N

N

307A

N H

+

Telomestatin is a highly selective G-quadruplex ligand

S

O

N

N

CH3

N O

N O

N N O

N

N O

O

21G (0,5µM) +Telo (5µM)

0,8

1,4

21G (0,5µM)

0,6

1

1,2

d(Fluo)/d(Temp), au

CH3

O

Normalized fluorescence, au

1

1

2

0,8

0,4

3

0,6

4

0,4 0,2 0

0,2

-0,2

Telomestatin

+ Genomic DNA 20µg

30

50

70 Temp, 90 °C

0 30

40

50

60

70

80

90

Temperature, °C

Competition with a 3x103 base molar excess of human genomic DNA

100

G-quadruplex ligands induced telomere shortening and a senescent-like growth arrest after long-term treatment + 115405 (0.4µM)

POPULATION DOUBLING (PD)

100

Control 1

kb

90

115405 (0.4 µM)

9.5

80

Control 2

6.4

70

12459 (0.04 µM)

To 10 14 21 25 28 32 39 42 56 66 70 Days

4.3

60 50

Untreated A549

40 30 20 10 0

0

10

20

30

40

50

60

70

DAYS TREATMENT OF A549 CELLS S N

N

N N H

N

N N

N H

N

Triazine 115405 Riou et al. Proc. Natl. Acad. Sci USA (2002): 99 2672-2677

115405 at 35 PD

TRIAZINES INDUCE APOPTOSIS IN A549 CELLS

80

80

60

60

% Apoptosis

100

2µM 100

40

40

20

20

0

0

C 24 48 72H

12459

20µM

A549 control

C 24 48 72H

115405

Apoptosis in A549 Cells Riou et al. Proc. Natl. Acad. Sci USA (2002): 99 2672-2677

115405 2µM

Are the dual effects of G4 ligands related to telomerase inhibition or telomere capping alteration ? G-quadruplex ligands induced both : 1. Short-term antiproliferative effects : apoptosis 2. Long-term senescence-like growth arrest Induction of resistance for : 1. Short-term treatment : mutagenesis 2. Long-term treatment : adaptation

ESTABLISHMENT OF RESISTANT CELL LINES to 12459

Resistance to short-term treatment A549 lung carcinoma EMS mutagenesis (300µg/ml) on A549

Soft Agar cloning with 5 µM

100 clones

10 µM

100 clones

Selection with 10 µM 12459

% Survival

Selection with 12459 (30 days)

110 100 90 80 70 60 50 40 30 20 10 0 0.1

A549 JFD18 JFD10

1

10

12459 µM 10 clones

5 clones

JFD clones Gomez, D., et al. (2003) J Biol Chem, 278, 50554-62.

30

A549 ng extract 0

JFD10

JFD18

50 100 50 100 50 100 50 100 50 100 50 100

kb 10 9 8 7 6

A549 JFD10 JFD18

Telomerase activity is overexpressed in JFD10 and JFD18 clones and remained to a high level under 12459 treatment.

b

A549

5 4

JFD18

ITAS

12459

– – – + + – – + +

– – + +

3

As a consequence of the stable overexpression of hTERT, telomere length is increased in JFD10 & 18 clones. Gomez, D., et al. (2003) J Biol Chem, 278, 50554-62.

Anaphases bridges in 12459-resistant clones

A549

JFD10

JFD18

JFD18

JFD 10 and JFD18 presented telomere capping alterations Gomez, D., et al. (2003) J Biol Chem, 278, 50554-62.

Antiproliferative effects of 12459 against hTERT or DN-hTERT transfected cells Untreated

Untreated

100

+ 12459 (5µM)

Cloning Efficiency (%)

Cloning Efficiency (%)

+ 12459 (5µM)

90 80 70 60 50 40 30 20

100 90 80 70 60 50 40 30 20 10

10

0

0 A549 HPV

A549 hTERT A549 DN hTERT

Short-term effect of 12459 is independent of hTERT expression

Gomez, D., et al. (2003) J Biol Chem, 278, 50554-62.

JFD18 HPV JFD18 DN hTERT

Over-expression of DN-hTERT Reverse JFD18 resistance

Resistant cells presented an altered telomere capping stabilized by hTERT overexpression JFD18

A549

Telomere capping alteration

Telomere capping normal Telomerase

5’

Telomerase

5’

Resistance to 12459

A549 DN-hTERT

JFD18 DN-hTERT Telomere capping alteration

Telomere capping normal Telomerase

3’

3’

Sensitivity to 12459

5’

Telomerase

Telomerase

3’

Sensitivity to 12459

5’

Telomerase

Sensitivity to 12459

3’

ESTABLISHMENT OF RESISTANT CELL LINES to 12459

Resistance to long-term treatment 100

B 100

90

90

Population Doublings

Population Doublings

A

80 70 60 50 40 30

A549 +12459 0.04 µM

20 10 0

0

10 20 30 40 50 60 70 80 90

Days Treatment

Gomez, D., et al. (2003) Cancer Res, 63, 6149-53.

80 70 60 50 40

0.3

30 0.2

20 10 0

0.15 0.1

A549 JFA 0.05-0.2 µM JFA 0.3 µM

0.05

0 10 20 30 40 50 60 70 80 90 100 110 120

Days Treatment

Telomerase activity is overexpressed in JFA2 and remained to a high level under 12459 treatment. JFA2

A549

Et Br

(CCCTAA)4

kb ITAS

0 -

10 100 200 10 100 200 10 100 200 10 100 200 ng extract - + + + - + + + 12459

A549

A549 0.2µM

JFA2

12 10 8 6 5 4 3

2

ITAS 0

3

10 30 100 0

3

10 30 100

Gomez, D., et al. (2003) Cancer Res, 63, 6149-53.

0

3

10 30 100 ng extract

1 2 3 4

1 2 3 4

40

40 A549

Population Doublings

JFA2

+ 12459 0.2µM

30

+ Telo 1 µM

JFA2

A549 Population Doublings

A549 + Telo 1 µM JFA2

Cross resistance to telomestatin for senescence-like growth arrest and telomere shortening in JFA2

+ Telo 2µM

20

10

+ 12459 0.2µM

30

kb

+ Telo 2µM

12 10 9 8 7

20

6

10

5

4

0

0

10

20

30

Days Treatment

Gomez, D., et al. (2003) Cancer Res, 63, 6149-53.

0

0

10

20

30

Days Treatment

1 2 3 4

Anaphases bridges in JFA2 resistant cell line A549 C A549 JFA2

Hypersensitivity to mitotic poisons in JFA2 resistant cell line Doxorubicin Etoposide Camptothecin Mitomycin C Vinblastin 0

1

Resistance Index (JFA2/A549) Gomez, D., et al. (2003) Cancer Res, 63, 6149-53.

2

Are the dual effects of G4 ligands related to telomerase inhibition or telomere capping alteration ? G-quadruplex ligands induced both : 1. Short-term antiproliferative effects : apoptosis 2. Long-term senescence-like growth arrest Induction of resistance for : 1. Short-term treatment : mutagenesis 2. Long-term treatment : adaptation

Resistant cells presented common features : •Alteration of telomere capping. •Overexpression of hTERT in majority of clones. •Increased telomere length. •No down-regulation of telomerase by G4 treatment (Mechanism?)

G4 ligand 12459 alters the alternative splicing of hTERT A549

Exon: 1

+

2

34 5 6

Alternative Splicing sites PCR primers

*I

†

1

-α -β 2109

Gomez, D.et al. (2004). Nucleic Acids Res, 32, 371-379

7 8 9 10

2531R

11 12

*I2

72 H

-

1

0

0

+Telomestatin

-

-

+BRACO19

CPT 1µM

+

A549

-

12459 10 µM

1

2

48 H

120

2

−β

24 H

β2m

3

+α,+β

control

240

−β

+12459

hTERT−β

+α,+β

4

Relative hTERT transcript

422

Relative hTERT transcript

hTERT+α,+β

bp

12459 (10 µM) 13

14 15 16

*I *I 3 4

hTERT intron 6 contains G-rich sequences able to form G-quadruplexes G4TERT2: GGGGGCCTTGGGGCTCGGCAGGGGTGAAAGGGG G4TERT1: GGGGTGAAAGGGGCCCTGGGCTTGGG VNTR6-1:GGGGTAGGTGGGGATCTGTGGGATTGG 5’

Intron 6

Exon 7

Intron 7

Exon 8

3’

Intron 8

VNTR6-1 VNTR6-2

G4TERT1

0.16

KCl

0.12

0.11

LiCl

LiCl G4TERT1mu NaCl 0

20

40

60

Temperature (°C)

80

Gomez, D.et al. (2004). Nucleic Acids Res, 32, 371-379

0.08

KCl

NaCl

0.09

G4TERT2mu NaCl 0

20

LiCl

0.1

0.1

0.08 0.07

0.13 0.12

0.12

0.09

KCl

0.14

0.11 0.1

0.14

NaCl

Abs

Abs

NaCl

0.15

Abs

0.13

VNTR6-1

G4TERT2

0.18

0.14

40

60

Temperature (°C)

80

0.08 0

20

VNTR6-1mu NaCl

40

60

Temperature (°C)

80

12459 preferentially stabilize hTERT G-quadruplexes 12459 (µM )

GG G

GGG

GG G

GGG

3’

GGG

GGG

GGG

+ G4 ligand 5’GGG

0 .1

G-quadruplex

Test oligomer

.3 1

G4TERT1 G4TERT1mu

+ Complementary oligomer 3’

CCC

G4TERT2

5’

G4TERT2mu

PCR

GGG

3’

GGG CCC

3’ 5’

GG G

GGG

Double-stranded PCR product

GGG

GGG

GGG

5’

GG G

VNTR6-1 VNTR6-1mu

+ 3’ CCC

5’

Annealing inhibition IC50 (µM) on test oligomers

Compound 12459

G4TERT1a

G4TERT1mub

G4TERT2b

G4TERT2mub

VNTR6-1a

VNTR6-1mub

1.75± 0.37

26

2

20

6.25±1.8

20

Telomestatin

5.0 ± 1.7

10

6

10

6.25±0.37

9

BRACO19

5.7±1.1

7.7

>30

>30

>30

>30

1.38±0.28

1.39

1.8

1.8

2.37±0.82

2.3

TMPyP4

Gomez, D.et al. (2004). Nucleic Acids Res, 32, 371-379

3 10 30

12459 resistant cell lines presented hTERT splicing alteration

+α,+β

5

1

JFD10 – +

JFD18 – +

Gomez, D.et al. (2004). Nucleic Acids Res, 32, 371-379

0

A549 JFD9

–

+

–

+

JFD11

–

+

+α,+β −β

2 1 0

+12459

2

3

JFA2

3

+ 12459

Relative hTERT transcript

4

4

A549

A549 12459 : – +

−β Relative hTERT transcript

+α,+β

Relative hTERT transcript

10 9 8 7 6 5 4 3 2 1 0

−β

Conserved G-repeats are found in splicing regulatory regions from human growth hormone and chicken β tropomysin pre-mRNA G4TERT2

G4TERT1

A GGGGGCCTTGGG--GCTCG-GCAGGGGTGA----AAGGGG-----------CCCTGGG--CTT-----------------------GGGTT B GGGGC---TGGGCAGAGCGCGCAGGGTTG------AGGGG-----------AGCAGGGTCCTT-------------------CACAGGGGT C GGGGAA--TGGGT-GCGCA-TCAGGGGTGGCAGGAAGGGGTGACTTTCCCCCGCTGGGAAATAAGAGGAGGAGACTAAGGAGCTCAGGGTT

A : hTERT intron 6 B : β Tropomyosin C : GH-1

G-overhang signal is strongly decreased in the presence of telomestatin in A549-treated cells A549

Telomestatin 5µM 2µM 1µM 0.5µM

A549 Cell Survival (%)

100

A549 16 days Telo

0

2

0 2 (µM)

24 48 24 48 24 48 24 48 H 0 0 5 5 0 0 5 5 Telo (µM)

80 60 40 20 0

0

4

8 12 16 20 Days of culture

24

G-Strand EtBr G-Strand

EtBr

Non-denaturing solution hybridization Gomez, Paterski, Lemarteleur, Shin-ya, Mergny & Riou Submitted

Telomestatin specifically binds to the single-stranded conformation of G-overhang in vitro a

Mg/Na Na Telo (µM)

0 0.1

1 10

Li

K

0.1 1 10 0.1 1 10 0.1 1 10

G-strand

Telo

EtBr

b Relative C-strand hybridization (%)

Mg

Li

Na

K

100

Inhibition 979A

50

0

G-Strand 0 0

0.1 1 Telomestatin (µM)

10

Gomez, Paterski, Lemarteleur, Shin-ya, Mergny & Riou Submitted

EtBr

10

1

Telo 0.1 10

1

0.1 µM

Effect of telomestatin on c-myc and telomeric oligomers forming G-quadruplexes determined by using the PCR-stop assay a

b 21G

Pu22myc

21G 5’-GGGTTAGGGTTAGGGTTAGGG-3’ 3’-AATCCCTTCTGCTCT-5’ Rev21G

100

PCR stop-assay product (%)

5’-GAGGGTGGGGAGGGTGGGGAAG-3’ 3’-ACCCCTTCTGCTCTTCGCTA-5’ RevPu22

pu22myc

50

0

0.01

0.1

1

Telomestatin (µM) Gomez, Paterski, Lemarteleur, Shin-ya, Mergny & Riou Submitted

10

C-myc quadruplex competes with the telomestatin effect at telomeric overhang in vitro and remains attached to the G-overhang in vivo a

A549 + Telo 5µM 24H 48H

A549 untreated Telo 0 pu22myc 0

0 1

0.1 1 10 0.1 1 10 0 0 0 1 1 1

0 0

0 1

0 10

0 0

b

0 0 1 10

µM µM

Telo pu22myc

A549 + Telo 5µM untreated 24H 48H 0 0 5 5 0 0 0 0 0 10 0 10 0 10 0 10

G-strand EtBr

EtBr

c

d

A549 DNA

0 Relative hybridization (%)

Relative hybridization (%)

+ pu22myc 10µM 100

50

0

0.1 1 2 5 Telomestatin (µM)

Gomez, Paterski, Lemarteleur, Shin-ya, Mergny & Riou Submitted

pu22myc (µM) 1

10

100

50

0

A549

24H 48H + Telomestatin (5 µM)

µM µM

Telomestatin induces an effective G-overhang degradation associated with the delayed A549 growth arrest Drug removed at day 16

+Telo 2µM

1

2

1 2

A549 Cell Survival (%)

100

A549 untreated

80

Telo pu22myc

60

G-strand

40

EtBr

20

7 100

0

4

8

12

16

20

Days of culture

days

0 0 2 2 0 0 0 0 0 0 µM 0 10 0 10 0 10 0 10 0 10 µM

100

0

+ Telo 2µM 4 8 12

33

13

10

97 71 62 48 Relative hybridization %

25

G-Strand EtBr

Gomez, Paterski, Lemarteleur, Shin-ya, Mergny & Riou Submitted

C-myc competition recovery markedly decrease at 12 days

A549 Cells Control

G4 ligand

DMS

DMS + G4 ligand

DNA Extraction C-strand hybridization X

X

X

Inhibition

X

Partial inhibition =Protection

EtBr

b

no DMS

Relative hybridization (%)

G-Strand

Telo + DMS

Telo

DMS

a

Control

DMS treatment alters telomestatin effects on G-overhang

100

50

0

Gomez, Paterski, Lemarteleur, Shin-ya, Mergny & Riou Submitted

with DMS

Control

1µM

5µM Telo

TRF2

T-loop

a

G-strand

3’

Capped telomere Telomerase

TRF2

b

G-strand

G-overhang 3’

Replication

O S

O

N

N

N O

telomestatin

N O

N N O

N

N

O O

TRF2

c

ERCC1/XPF O S

O

N

N

G-strand

O

S

O

O

N N

N O

G4 O

N

O

N N

O N

N N

O

N

N

N O

O

N

N

G4 O

O

3’

G-quadruplex Stabilization at G-overhang

G-overhang loss

d

G-strand

TRF2

3’

Uncapped telomere

Telomere fusion Apoptosis Senescence-like

Conclusion • Telomestatin is able to impair telomeric overhang in human cells. • A tight and specific interaction of the ligand with telomeric overhang compatible with the formation of a stable G4 was found. • Prolonged treatment of cells results in a marked decrease of G-overhang which correlated with the onset of senescence. • Telomeric overhangs are important targets for the biological effect of these ligands.