Presentation Abstract

Abstract Number:

4431

Presentation Title:

A novel derivative of the Cdk inhibitor roscovitine that induces apoptosis in CLL and overcomes stromal cell-mediated protection

Presentation Time:

Tuesday, Apr 20, 2010, 2:00 PM - 5:00 PM

Location: Exhibit Hall A-C, Poster Section 22

PosterSection:

22

Poster Board Number:

20

Author Block:

Rong Chen1, Yuling Chen1, Simon R. Green2, William G. Wierda1, William Plunkett1. 1UT M. D.Anderson Cancer Center, Houston, TX; 2Cyclacel Ltd., Dundee, United Kingdom

Abstract Body:

Cmpd 5 is a derivative of R-roscovitine (CYC-202, seliciclib) with increased potency that selectively inhibits Cdk2, Cdk5 and Cdk9. In chronic lymphocytic leukemia (CLL) a disease that is addicted to the over-expression of anti-apoptotic proteins for survival, inhibition of Cdk9 by Cmpd 5 reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription. These actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1, and induced apoptosis in CLL cells in vitro. Cmpd 5 was about 30 times more potent than its parent compound R-roscovitine with an IC50 of 0.86 µM after a 24-hr incubation. Although cell death was initiated after a 4-hr incubation in a time course study of Cmpd 5-induced apoptosis, the maximum cell death was not reached until 10-12 hr. In addition, it was known that co-culture of CLL cells with the marrow and lymphoid stromal cells may be responsible for resistance to fludarabine therapy. We evaluated Cmpd 5 in overcoming such stromal cell-mediated protection. Cmpd 5 killed the CLL cells similarly in the presence or absence of the human stromal cell line StromaNKtert without toxicity to the stromal cells, whereas the stromalcells clearly protected the CLL cells from the toxicity of fludarabine. Since Mcl-1 is the major target of Cmpd 5 in CLL, and the action of Cmpd 5 relies on the intrinsically rapid turn-over rate of Mcl-1, we compared the protein levels and the half-life of Mcl-1 in CLL cells in the presence orabsence of the stromal cell layer. After an overnight incubation of CLL cells with the StromaNKtert cells, there was a 3-4 fold induction of Mcl-1 transcript and protein. However, there was no difference in the Cmpd 5-induced decay rate of either the Mcl-1 mRNA or protein, indicating that stromal cells did not alter the stability of Mcl-1. Therefore, the elevated Mcl-1 level likely indicated increased biosynthesis, rather than enhanced stability. Thus, the activation of the survival pathways and induction of the biosynthesis of Mcl-1 by the stromal cells may protect CLL cells from fludarabine-induced apoptosis but loss of the short-lived anti-apoptotic proteins was not affected, explaining the lack of stromal cell protection for CLL cells from Cmpd 5. Similar time-dependence of cell death induction as well as stromal cell response was also observed with other inhibitors of transcription including flavopiridol, R-roscovitine and Actinomycin D, implying a class effect. Taken together, our data suggested that Cmpd 5 is a promising candidate for clinical development for CLL.

American Association for Cancer Research615 Chestnut St. 17th Floor

Philadelphia, PA 19106

Page 1 of 1Abstract Print View

A novel derivative of the Cdk inhibitor roscovitine that induces

apoptosis in CLL and overcomes stromal cell-mediated protection

Rong Chen1, Yuling Chen1, Simon R. Green2, William G. Wierda1

and William Plunkett11The University of Texas M.D. Anderson Cancer Center, Houston, TX

and 2Cyclacel Ltd, Dundee, UK

•

Inhibition of Cdk9 by Compound 5 reduced phosphorylation of RNA pol II and blocked transcription.

•

These actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1 and XIAP, and induced apoptosis in CLL cells in vitro.

•

Cmpd5 was about 30 times more potent than its parent compound R-roscovitine.•

Compound 5 followed a similar time course of cell death induction as the other transcription inhibitors: cell death was initiated after a 4-hr incubation and reached the maximum at 10-12 hr.

•

Compound 5 and other transcription inhibitors overcame stromal cell-mediated protection.

•

Stromal cells did not increase the stability of Mcl-1. Thus, the stromal cells may protect CLL cells from fludarabine-induced apoptosis but loss of the short-lived anti-apoptotic proteins was not affected, explaining the lack of stromal cell

protection for CLL cells from transcription inhibitors.

•

Taken together, our data suggested that Compound 5 is a promising candidate for clinical development for CLL.

Conclusions

Introduction

•

Chronic lymphocytic leukemia (CLL) is characterized as being dependent on the over-expression of anti-apoptotic proteins for survival.

•

In such a biologic context, agents that aim at antagonizing or diminishing the anti-apoptotic proteins would release the pro-death signals to commit cells to apoptosis. This has been a focus of new therapeutics in CLL.

•

We have proposed a strategy that directly target this pathogenesis of CLL by inhibiting the synthesis of anti-apoptotic proteins using cyclin dependent

kinases (Cdk) inhibitors that block transcription.

•

Cdk7 and Cdk9 phosphorylate the Ser5 and Ser2 sites, respectively, on the C-

terminal domain (CTD) of RNA polymerase II (pol II) to promote transcription initiation and elongation.

•

Cdk9 inhibitors such as flavopiridol (Alvocidib), R-roscovitine (CYC-202

, seliciclib)

and SNS-032, which block transcription, have shown activities in CLL in vitro and in clinical trials. These compounds transcriptionally reduced short-

lived anti-apoptotic proteins, which was associated with induction of apoptosis.•

This investigation focused on Compound 5, a novel derivative of R-roscovitine with increased potency that selectively inhibits Cdk2, Cdk5 and Cdk9.

Compound 5 reduced mRNA levels of Mcl-1, XIAP and Bcl-2

Hypothesis

Transcription inhibitors overcame stromal-mediated protection

# 4431

Inhibition of transcription by Compound 5 would preferentially reduce the short-

lived anti-apoptotic proteins, which would lead to induction of apoptosis in CLL.

Compound 5 inhibited RNA synthesis: [3H]uridine incorporation

siRNA western repeat

Inhibition of transcription in CLL cells by Compound 5 Compound 5 reduced protein levels of Mcl-1 and XIAP, but not Bcl-2

Compound 5 reduced expression of short-lived anti-apoptotic proteins

Time-

and concentration-

dependent induction of apoptosis by Compound 5Comparison to other transcription inhibitors

Mechanism of action of Cdk9 inhibitors in CLL

Transcription inhibitors kill the CLL cells at the presence of stromal cell protection

Stromal cells did not increase Mcl-1 mRNA and protein half -life

Incubating with stromal cells increased Mcl-1 mRNA and protein level

25

25

25

PP

P

RNA Pol II2

52

52

5

RNA Pol II

RNA

25

25

25

PP

PP

PP

RNA Pol II

RNA

SNS-032, flavopiridol, roscovitine, Compound 5

Cdk7 Cdk9 CycT

transcription initiation transcription elongation

CycH

Kinase Flavopiridol IC50

(nM)R-roscovitine

IC50

(nM)SNS-032 IC50

(nM)Cmpd

5 IC50

(nM)Cdk1/cyclin B 41 2700 480 578

Cdk2/cyclin A 100 710 385

Cdk2/cyclin E 170 100 48

Cdk4/cyclin D 65 >10 μM 925 232

Cdk5/p35 ~100 160 340 21

Cdk6/cyclin D ~100 >100 μM >1000 N.A.

Cdk7/cyclin H ~300 490 62 193

Cdk9/cyclin T 6 600 4 26

Additional human kinases

PKCδ

890PKCε

480DYRK1A 3100

ERK2 1170190 kinases

>1000LKB1 63

Cdk7/9 inhibitors block transcription

inhibit Pol II CTD phosphorylation

Inhibit RNA synthesis

Intrinsically short-lived mRNAs, such as Mcl-1, XIAP

Intrinsically short-lived anti-apoptotic proteins

Caspase activation

Apoptosis

Time, h

6

24 Cmpd

5, μM 0 0.1 0.25 0.5 1 1.5 0 0.1 0.25 0.5

1 1.5

pSer5-Pol II

pSer2-Pol II

Total RNA Pol II

Cmpd5 inhibited RNA Pol II phosphorylation

0.00 0.25 0.50 0.75 1.00 1.25 1.500

20

40

60

80

100

1206 hr24 hr

Cmpd5, μM

Urid

ine

Inco

rpor

atio

n(%

of C

ontro

l)

ActinBcl-2

XIAP

Mcl-1

Time, h 6 24 Cmpd

5, μM 0 0.1 0.25 0.5 1 1.5 0 0.1 0.25 0.5 1 1.5

PARP

% Death by Annexin/PI 25 27 36 53 80 80 30 35 54 79 93 93

0 2 4 6 8 10 12 14 16 18 20 22 240

20

40

60

80

100

Cmpd5 1.5μMRoscovitine 50μM

Dactinomycin 200 nMFlavopiridol 1μM

SNS-032 0.3μM

ctrl

Hours

% S

urvi

val

CtrlCmpd

5Rosc

ovitin

eDac

tinomyc

inFlav

opiridol

SNS-032

Fludara

bine

0

20

40

60

80

100

n=3

% S

trom

al V

iabi

lity

CtrlCmpd5

Rosco

vitine

Dactin

omycin

Flavopir

idolSNS-03

2

Fludara

bine, 48

h

0

20

40

60

80

100CLL onlywith stromal

n=5

% C

ell D

eath

0 1 2 3 4 50.00

0.25

0.50

0.75

1.00

control 2.8 h+stromal 1.8 h

n=4

Hours

Mcl

-1/C

ontro

l

0 1 2 3 4 50.00

0.25

0.50

0.75

1.00

control 3.2 h+stromal 1.5 h

n=5

Hours

Mcl

-1/C

ontro

l

Ctrl stromal Ctrl stromal0

1

2

3

4

mRNA protein

Mcl

-1

Transcription inhibitors were not toxic to the stromal cells

Since Mcl-1 is the major target of Cmpd

5 in CLL, and the action of Cmpd

5 relies on the intrinsically rapid turn-over rate of Mcl-1, we compared the protein levels and the half-life of Mcl-1 in CLL cells in the presence or absence of the stromal cell layer.

Comparison of Compound 5 with other Cdk inhibitors

0

25

50

75

100

Cmpd5Roscovitine

Flavopiridol

0.05

IC5062.0 nM0.20 μM0.52 μM0.86 μM30.0 μM

SNS-032Dactinomycin

0.1

0.2

0.4

0.8

1.6

3.2 10 20 40 80 160

320

0.02

50.

0125

n=5-10

Drug, μM

% S

urvi

val

mRNA protein

6 hr

0.00 0.25 0.50 0.75 1.00 1.25 1.500

25

50

75

100

125 Mcl-1XIAPBcl-2

Cmpd5, μM

mR

NA

(% o

f Con

trol)

24 hr

0.00 0.25 0.50 0.75 1.00 1.25 1.500

25

50

75

100

125 Mcl-1XIAPBcl-2

Cmpd5, μM

mR

NA

(% o

f Con

trol)

6 hr

0.00 0.25 0.50 0.75 1.00 1.25 1.500

25

50

75

100

125

pSer2

pSer5

Cmpd5, μM

P-Po

l II/T

otal

(%

of C

ontro

l)24 hr

0.00 0.25 0.50 0.75 1.00 1.25 1.500

25

50

75

100

125

pSer2

pSer5

Cmpd5, μM

mR

NA

(% o

f Con

trol) 6 hr

0.00 0.25 0.50 0.75 1.00 1.25 1.500

25

50

75

100

125

XIAP

Mcl-1

Bcl-2

Cmpd5, μM

Prot

ein

(% o

f Con

trol)

24 hr

0.00 0.25 0.50 0.75 1.00 1.25 1.500

25

50

75

100

XIAP

Mcl-1

Bcl-2

Cmpd5, μM

Prot

ein

(% o

f Con

trol)

n=8

n=5

n=8

n=8

n=4