genetic polymorphism of thiopurine methyltransferase and
TRANSCRIPT
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