genetic polymorphism of thiopurine methyltransferase and

20
Genetic Polymorphism of Thiopurine Methyltransferase and Thiopurine Therapy Dr. William Evans 1 The screen versions of these slides have full details of copyright and acknowledgements Genetic Polymorphism of Thiopurine Methyltransferase and Thiopurine Therapy From Molecular Genetics to Clinical Medicine 1 Dr. William Evans St. Jude Children's Research Hospital and the University of Tennessee, College of Pharmacy and Medicine USA Genetic polymorphism of thiopurine methyltransferase and thiopurine therapy • Azathioprine, mercaptopurine, thioguanine From molecular genetics to clinical medicine 2 Inactivated by a polymorphic enzyme Toxicity determined by TPMT genotype Mercaptopurine metabolism in hematopoietic tissues TGN (active) TPMT XO HPRT MP 3 Evans et al., SJCRH, 2000 MeMP 6Tu (inactive)

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Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

1The screen versions of these slides have full details of copyright and acknowledgements

Genetic Polymorphism of Thiopurine Methyltransferase

and Thiopurine Therapy

From Molecular Genetics to Clinical Medicine

1

Dr. William Evans

St. Jude Children's Research Hospital and the University of Tennessee, College of Pharmacy and Medicine

USA

Genetic polymorphism of thiopurine methyltransferase and thiopurine therapy

• Azathioprine, mercaptopurine, thioguanine

From molecular genetics to clinical medicine

2

p , p p , g

• Inactivated by a polymorphic enzyme

• Toxicity determined by TPMT genotype

Mercaptopurine metabolism in hematopoietic tissues

TGN (active)

TPMT XO

HPRTMP

3Evans et al., SJCRH, 2000

MeMP 6Tu(inactive)

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

2The screen versions of these slides have full details of copyright and acknowledgements

AZA

Azathioprine metabolism

TGN (active)

TPMT XO

HPRTMP

4

MeMP 6Tu(inactive)

TPMT methylates (inactivates) 6MP

5

Contribution of genetic polymorphisms to drug metabolism

Phase I Phase II

6Evans WE and Relling MV, Science 286:487-91, 1999

1Proportions based on estimated number of drug substrates

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

3The screen versions of these slides have full details of copyright and acknowledgements

TPMT

Me-MP

TPMT TPMT TPMT

HPRT

MP

A

7

TPMTMe-TG

Krynetski et al., Mol Pharmacol 47(6):1141-7, 1995

TPMT

TG

HPRT

B

How does MP exert its antileukemic effects?

Incorporation into DNA:

• Inhibits DNA replication (e.g., RNaseH, TopoII)

• Triggers apoptosis via;

8

– MSH2 dependent mechanism (p53 dependent)

– HMGB1/GAPDH/Hsp70/Erp60

Inhibition of DNPS:

• Via MeMPR

6MP + PRPP → TGN↑

DNA6MP

THF

TSGARtf AICARtfTHF

5,10-CH2

MTHFR5-CH3THF

Homocysteine

MTX

De novopurine synthesis

PRPP

9How 6MP kills leukemia cells: TG incorporation into DNA

DHF

DHFR

MTXglu

glu

GGH

MTXglu5

gluglu

n=7

MTXgluRFC

diffusionMTXglu

MRP

MethionineMTX

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

4The screen versions of these slides have full details of copyright and acknowledgements

Local structural modifications of duplex DNA by thioguanine incorporation (NMR Structure, JBC 2003)

10

11Somerville et al., JBC, 2003

Thioguanine in DNA alters the DNA repair enzyme topoisomerase II function

ThioG substitution effects on topo II cleavage

e D

NA

clea

vageTopo II

Topo II + Etoposide

12FASEB J 14:2339-44, 2000

Rea

lativ

e

Top strand Bottom strand

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

5The screen versions of these slides have full details of copyright and acknowledgements

Duplex* StructureNuclearextract

Relative RNase H cleavage (%,15 min)

V (X=dG)

X=dG X=dGS

E.Coli RNase HMolt4CEMN l 6

10076 ± 272 ± 458 ± 2

VI (X=dGS)

15 255’..UGGCGGCCGUAGCGCGGUGGUCCCACCUGA..3’

C3’-TCG XC CACCA- 5’

13 ± 1 8 ± 16 ± 16 ± 1

Thioguanine incorporation in DNA alters RNaseH function

13

Nalm6HeLa

E.Coli RNase HCEMNalm6P12HeLa

58 ± 289 ± 1

10081 ± 281 ± 283 ± 174 ± 4

VII (X=dG)

VIII (X=dGS)

* In all experiments duplex concentration C0 = 0.2x10-5M

5’.. UAGCGCGGUGGUC..3’C3’-TCG XC CACCA- 5’

6 ± 119 ± 2

54 ± 6 69 ± 269 ± 366 ± 274 ± 3

Krynetskaia et al., Mol Pharm, 1999

14Science august 1996, 273

MSH2 facilitates activation of Chk2

CHEK2P

P P

MDM2P

MSH3 CHK2MSH2

Damaged base

HMGB1

HSP70

ERp60

GAPDHDamaged

base

Hmgb1-/-

%

Alternative mechanism to trigger apoptosis in MSH2- ALL

15

P Pp53

p53 target Gene expression

Apoptosis

p53P

G1 and G2 arrestKrynetski et al., Ca Res 2003

MP µM

Viab

ility

The absence of Hmgb1 alters sensitivity to MP; Viability of Hmgb1-/-

(C1) and Hmgb1+/+ (VA1) cells (MEFs) after 5 days of MP treatment, determined by MTT assay; ◊ , C1 cells; ●, VA1 cells. Each point represents the results of three parallel experiments (mean ± SE);In contrast, Hmgb1-/- cells were not more resistant to vincristine

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

6The screen versions of these slides have full details of copyright and acknowledgements

MeTIMP

6MP 6MP + PRPP → TIMP → TGNTPMT

THF

TS

MTX

GARtf AICARtfTHF5,10-CH2

MTHFR5-CH3THF

Homocysteine De novopurine synthesis

PRPP

16MeTIMP inhibits DNPS

DHF

DHFR

MTXglu

MTX glu

GGH

MTXglu5

gluglu

n=7

MTXgluRFC

diffusionMTXglu

MRP

Methionine

Antileukemic effects correlated with inhibition of DNPS

60%70%80%90%

100%

of p

atie

nts

No inhibitionPartial inhibitionFull inhibition

-40%-30%-20%-10%

0%

ukoc

yte

coun

ts

y 0

to d

ay 3

Inhibition of DNPS in ALL cells after in vivo MP treatment

No inhibitionPartial inhibitionFull inhibition

17

0%10%20%30%40%50%

MP LDMTX+ MP

HDMTX+ MP

Perc

enta

ge

-70%-60%-50%

Cha

nge

in le

from

day

Note: 6MP alone had much lower effect on DNPS than MTX or MTX+MP

Multiple mechanisms by which MP exerts antileukemic effects

Incorporation into DNA: Inhibits DNA replication (e.g., RNaseH, TopoII)

Triggers apoptosis via MSH2 dependent mechanism (p53 dependent) D d

18

CHK2MSH3 MSH2

HMGB1

HSP70

ERp60

GAPDH

HMGB1/GAPDH/Hsp70/Erp60

Inhibition of DNPS: via MeMPR

mechanism (p53 dependent)

Damaged base

Damaged base

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

7The screen versions of these slides have full details of copyright and acknowledgements

Why is TPMT genetic polymorphism important for MP?

19

Mercaptopurine metabolism in hematopoietic tissues

TGN (active)

TPMT XO

HPRTMP

20

MeMP 6Tu(inactive)

Evans et al., SJCRH, 2000

21Lenard et al., Lancet, 336:225-9, 1990

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

8The screen versions of these slides have full details of copyright and acknowledgements

10

5

TPMTH

TPMTH

TPMT L

TPMT H

Human RBC TMPT

f sub

ject

s ni

ts o

f act

ivity

298 unrelated adults

22

5

0

TPMT H

TPMT L

TPMT L

Weinshilboum and Sladek Am J Hum Gen 32(5):651-62, 1980

TPMT activity, unit S/ML RBC

% o

fpe

r 0.5

u

sm (%

)

Enzyme activity in humansHigh

TPMT inactivates 6MP

TPMT genetic polymorphism determines mercaptopurine (6MP) metabolism

Our cancer prototype

23

Poly

mor

phi

TPMT activity ml-1 red blood cells

Medium

Low

6MP meMPTPMT (inactive)

TGN(active)

Post-docs: TPMT*2 Gene Krynetski (GK) ‘95TPMT*3A Ezra Tai & GK ‘96TPMT*3C Trina Leonechen & GK ‘97 Sladek and Weinchilboum, 1981

Inheritance of TPMT activity

Gene (PCR)

mRNA (Northern)

24Evans et al., SJCRH, 2000

Enzyme: (Western)

# Patients

Phenotypes: Low deficient

intermediate High

Enzyme activity

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

9The screen versions of these slides have full details of copyright and acknowledgements

Pulse-chase analysis of rh TPMT

in COS-1 cellsstable

T1/2α = 0.35 h (T1/2 β =41 h)

*1

*2

25Tai et al., Pharmacogenetics 9(5):641-50 1999

T1/2 = 0.67 h

T1/2 = 5.5 h

T1/2 = 11.2 h

*3A

*3B

*3C

TPMT

Deficient Heterozygous Homozygous

TPMT

AB

vity

C

26Tai et al., Proc Natl Acad Sci 94(12):6444-9, 1997

TPMT protein (density of western blot/2x106 RBC)

Eryt

hroc

yte

TPM

T ac

ti(U

/mL

pRB

C)

PCR based genotyping method

27Yates et al., Ann. Int. Med 126(8):608-14, 1997

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

10The screen versions of these slides have full details of copyright and acknowledgements

28Yates et al., Ann. Int. Med 126(8):608-14, 1997

45 112 184 93 133 53 75 86 45 2075

ATG

ATG G238C

PNAS, 95

TPMT*1

TPMT*2

Human TPMT gene and mutant alleles

(Wild type)

( Activity)

29Evans et al., SJCRH, 2000

(Ala Pro)

ATG A719G(Tyr Cys)

ATG G460A(Ala Thr)

A719G(Tyr Cys)

AJHG, 96

CPT, 98AIM, 97

TPMT*3A

TPMT*3C

( Activity)

( Activity)

>95% concordance between TPMT genotype and phenotype

Disclosure: US Patent (1995) for molecular diagnostics (genotyping) based on these 3 TPMT SNPs and alleles.

Sensitivity = 90%; Specificity = 99% Schaefeler et al., PGEN, 2004 (n=1214)

30

25

20

15

10MT

activ

ity, U

/mL

30

5

0

Yates et al., Ann. Int. Med 126(8):608-14, 1997

TPM

Thiopurine S-methyltransferase (TPMT) activity in patients with different TPMT genotypes determined by mutation – specific polymerase chain reaction methods

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

11The screen versions of these slides have full details of copyright and acknowledgements

Estimated TPMT allelic frequencies for the African-American

and Caucasian populations

31Hon et al., Human Molecular Genetics 8(2):371-6, 1999

TPMT variant alleles in various world populations

Norway3A,3C

Korea3C,6

UK3A,3C,2

Mayo/Europe3A,3C,2, 3B,3D,4,5

France3A,3C,2,7

China3CCaucasians:

3A,3C,2African American:

St. Jude

32

Ghana3C

Thailand3C

S.W. Asia3A

African American: 3A,3C,2,8

Kenya3C

9% ND ND 5-9%

17% 0% 0% 81 89%

TPMT non-functional mutant alleles

ATG G238C(Ala→Pro)

TPMT*2

TPMT*3A

%of mutant alleles

African-Am 1 Afr. 2 Asian 3 Caucasian 4

33

17% 0% 0% 81-89%

52% ~100% ~100% 5-11%

ATG

ATG

G640A(Ala→Thr)

A719G(Tyr→Cys)

A719G(Tyr→Cys)

TPMT*3C

(1) S.E USA, (2) Ghanaias, Kenyan, (3) Japanese, Chinese (4) US, UK, France

Ann. Int. Med, Yates et al., 1997; CPT, Otterness et al., 1997; Br. J. Pharmacol, de la moureyreet al., 1998; Hum. Mol. Gen., Hon et al., Ameyaw et al., 1999; Pharmacogenetics, Collied et al., 1999

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

12The screen versions of these slides have full details of copyright and acknowledgements

TPMT*1

TPMT*3ATPMT*3BTPMT*3C

TPMT*2

34

Very rare TPMT alleles

The importance of TMPT haplotype for common SNPs

TPMT*1(wild type)

TPMT*3A

Genotypes Haplotype structures Phenotypes

35

VERY RAREAllele freq: ~ 1 / 120,000TPMT*3B

TPMT*3C

36

Pharmacogenetics 2002, 12:1-7

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

13The screen versions of these slides have full details of copyright and acknowledgements

Drug exposure determined by TPMT genotype

5000

4000

3000

37/genotype

deficient heterozygote wild-type

TPMT phenotype

2000

1000

0

Krynetski and evans, Pharm Res 16(3):342-9, 1999

Hematological toxicity determined by TPMT genotype

ce

Mercaptopurine therapy intolerance and heterozygosity at the thiopurine S-methyltrasferase gene locus

38Relling et al., JNCI, 1999 years

Cum

ulat

ive

inci

den

1.0

0.9

0.8

0.7

0.6

azat

hiop

rine

ther

apy

Wild-type TPMT

TPMT genotype and tolerance of azathioprine therapy

Azathioprine 2-3 mg/kg po qd 67 patients

39

0 20 40 60 80 100 120 140 160 180

Time on azathioprine therapy (weeks)

0.5

0.4

0.3

0.2

0.1

0.0

Patie

nts

rem

aini

ng o

n a Wild-type TPMTmedian 39 weeks

Mutant TPMTmedian 2 weeks

Black, McLeod, Capell et al., Ann Intern Med 1998

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

14The screen versions of these slides have full details of copyright and acknowledgements

TMPT genotype in thiopurine intolerant patients

ulat

ion

100

40J. Clin. Oncol. 2001

General population

Patients intolerant to thiopurines

MUT/MUT

% o

f pop

u

0

50

>20%

~70%

10

8

6

4

2

0

Freq

uenc

y (%

)

Thiopurine methyltransferase (TMPT) genetic polymorphism and 6MP dose requirement

wt/wt

wt/mm/m

41Evans et al., SJCRH, 2000

00 5 10 15 20 25 30

TPMT activity (units/ml pRBC)500

250

0

6MP

dosa

ge

(mg/

m2 /W

K)

TPMT genotypewt/wtwt/mm/m

6MP dosage reduction in TPMT deficient all patient

6MP dosage RBC 6TGN

WK

(PO

) Pmol/8

42TPMT deficient

(<1) n=1NL TPMT

(7.5-37) n=30

MG

/M2 /W

8 x 108R

BC

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

15The screen versions of these slides have full details of copyright and acknowledgements

Fatal toxicity from azathioprine in a TPMT-deficient patient

WBC

A thi i

43

Peripheral blood leucocyte count (WBC) and azathioprine (AZA) dose in TPMT deficient heart transplant recipient who developed fatal toxicity

Schuetz et al., Lancet 341:436, 1993

Azathioprine

Dosing 6MP without pharmacogenetics

Conventional

TreatmentMercptopurine dose Systemic exposure Toxicity

MP

(mg/

m2/

wee

k-1 )

TGN

pm

ol/8

x 1

08 R

BC

Cum

ulat

ive

inci

denc

e

v/v wt/v wt/wtv/v wt/v wt/wt

44Cheok and Evans, Nat Rev Cancer 6:117, 2006

Individualized

yearsv/v wt/v wt/wtv/v wt/v wt/wt

MP

(mg/

m2/

wee

k-1 )

TGN

pm

ol/8

x 1

08 R

BC

Cum

ulat

ive

inci

denc

e

yearsv/v wt/v wt/wtv/v wt/v wt/wt

NB: 6MP dose reduction based on TPMT genotype did NOT influence ALL relapse

45Relling et al., Blood, 2006

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

16The screen versions of these slides have full details of copyright and acknowledgements

TPMT-KO mouse recapitulates human TPMT phenotypes

TPMT-KO

Capecchi,M. Evans & Smithies

46Hartford, Ca Res 2007

0

5

10

15

20

-/- -/+ +/+

units

/ml p

RB

Cs

Interactions of genetics polymorphisms and treatment may result in adverse effects

47Evans et al., SJCRH, 2000

TPMT deficiency associated with higher risk of radiation-Induced brain tumors in patients

receiving 6MP + CNS radiation

48Relling et al., Lancet, 354:34-39, 1999

Estimated cumulative incidence of radiation- associated secondary malignant brain tumor for seven children in total XII who received preventive cranial radiotherapy and had genetic defects in thiopurine methyltransferase compared with 45 with wild-type status

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

17The screen versions of these slides have full details of copyright and acknowledgements

Thioguanine in DNA alters the DNA repair enzyme topoisomerase II function

NA

clea

vage

ThioG substitution effects on topo II cleavageTopo II

Topo II + Etoposide

49FASEB J 14:2339-44, 2000

Rel

ativ

e D

N

Top strand Bottom strand

TPMT and ITPA(inosine triphosphatase) exhibit genetic Polymorphism (SNPs)

TPMT

THIO-GDP

THIO-GTP

Methyl-thio-ITP

Thio-ITP6-thio-uric acidITPAThio-IDP

K

50

HPRT

TPMT TPMT

Mercaptopurine

Methyl-mercaptopurine Methyl-thio-IMP Methyl-thio-GMP

THIO-GMPThio-IMP

XO

TPMT

IMPDH GMPH

Thio IDP

K

↑ MMPN whenITPA is variant

(non-functional) and TPMT is wild type

ITPA genotype and median concentrations of 6MP metabolites in RBC during continuation treatment with MP

[wt TPMT] (Total XIIIB)

51(G Stocco et al., 2008)

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

18The screen versions of these slides have full details of copyright and acknowledgements

ITPA ?

Effects of ITPA when

TPMT is non-functional

but MP dose adjusted

for TPMT genotype?

TPMT

THIO-GDP

THIO-GTP

Methyl-thio-ITP

Thio-ITP6-thio-uric acid K

52

ITPA

TPMT

?

HPRT

TPMT

Mercaptopurine

Methyl-mercaptopurine Methyl-thio-IMP Methyl-thio-GMP

THIO-GMPThio-IMP

XO

TPMT

IMPDH GMPH

Thio-IDP

K

ITPA & TPMT genotypes and concentrations of mercaptopurine metabolites in RBC during continuation

phase treatment with MP (Total XIIIB)

TGN MeMPN

8x10

8 er

ythr

ocyt

es)

A B

0080

010

00

030

000

8x10

8 er

ythr

ocyt

es)

53TPMT Wild type / ITPA Wild type

(G Stocco et al., 2008)

TGN

met

abol

ites

(pm

ol/ 8

TPMT Wild type /ITPA Variant

TPMT Variant / ITPA Wild type

TPMT Wild type / ITPA Wild type

TPMT Variant / ITPA Variant

200

400

60

010

000

2000

MM

PN m

etab

olite

s (p

mol

/

TPMT Wild type /ITPA Variant

TPMT Variant / ITPA Wild type

TPMT Variant / ITPA Variant

MP dose NOTadjusted for TPMT

MP dose adjusted for TPMT

2

3

4

5

atio

of t

oxic

ity

54

TPMT: W/W W/V W/W W/V

ITPA: W/W W/V

6MP inducted toxicity (fever and neutropenia)

0

1Odd

s r

W/W W/V

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

19The screen versions of these slides have full details of copyright and acknowledgements

Potential of pharmacogenomics

1 Genetic profile for non-response

or toxicity

55

2Treat with alternative

drug or dose

Treat with conventional drug or dose

Genetic profile for favorable response

56Evans WE and Relling MV, Science 286:487-91, 1999

57Evans et al., SJCRH, 2000

Genetic Polymorphism of ThiopurineMethyltransferase and Thiopurine Therapy

Dr. William Evans

20The screen versions of these slides have full details of copyright and acknowledgements

58