molecular mechanisms of therapy-induced cancer allan.pdf · 2016. 9. 18. · radiation induced...
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Molecular Mechanisms of Therapy-Induced Cancer
James M. Allan
1
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Mechanisms of therapy-induced transformation
2
EXPOSURE(chemotherapy/radiotherapy)
CONSTITUTIONAL GENETICS
THERAPY-INDUCED CANCER
MODIFIERS(smoking, diet, age etc)
MECHANISMS?Somatic
alterations
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Chemotherapy-induced AML
3
ALKYLATING AGENTS(procarbazine, dacarbazine, temozolomide,
cyclophosphamide, carmustine etc)
•Risk increases 1-2 yr, peaks 5-10 yr, then decreases
•Preceding myelodysplastic syndrome (4-7 hits)
•Chromosomal deletions or monosomies (Chr. 5/7)
•DNA mismatch repair defective with high mutation rate (after methylating agents)
TOPOISOMERASE INHIBITORS(etoposide, teniposide,
doxorubicin, daunorubicin etc)
•Shorter induction period (median 2-3 yr)(1-2 hits)
•Balanced chromosomal translocations (MLL at 11q23)(MLL breaks map to topoisomerase binding sites)
Relative risk of developing a therapy-associated cancer after Hodgkin lymphoma
Mechanisms of AML pathogenesis after methylating chemotherapy are well defined
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Methylating agents select for DNA
mismatch repair loss
azathioprine, 6-thioguanine, 6-mercaptopurine
mutationrate
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Molecular mechanisms of transformation
DNA damage Gene-specific effects Altered phenotype
O6-methylguanine/6-thiopurine
Loss of DNA MMR(MLH1, MSH2, MSH6,
PMS1, PMS2 etc)
Mutation acquisition/anti-apoptosis
DNA strand breaksat topoisomerase
binding sites
MLL fusion gene Dysregulated gene expression
IONISING RADIATION?
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Radiation induced cellular damage
MODELS !
Radiation exposure induces DNA double-strand breaks and DNA base– cytotoxic AND mutagenic –
Clustered DNA damage difficult to repair and can be converted to DNA DSBs.
DNA DSBs difficult to repair with high fidelity – translocations, loss (deletion) and gain (amplification) of genetic material.
How do we identify genetic lesions induced by radiation?
- Strand break induction- Hydrolysis of water
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Radiogenic cancer in Hodgkin lymphoma
survivors
7
Risk of second cancer associated with radiation exposure
Positively correlated with dose
Inversely correlated with age
Most common second cancers
Breast
Thyroid
Contribution to risk of chemotherapy is unclear (site-specific?)
Responsible for 20% of the mortality in HL survivors
In vitro model system based on the MCF10A breast epithelial cell line
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MCF10A
47, XX, i(1)(q10),+del(1)(q12q32), der(3)t(3;9)(p14;p21), der(8)t(8;8)(q22;p23), der(9)t(3;9;5)(p14;p21;q23).
- reciprocal translocation between chromosomes 3 and 9 (deletion of CDKN2A/2B)- non-reciprocal duplication of the end of the q arm of chromosome 5 to the derivative
chromosome 9- Isochromosome 1 and 8q duplication
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SNP array karyotyping
9
Lo
g2
Ratio
Copy
nu
mber
Log2 ratio of 0 = 2 copies (diploid)
Chromosome 8
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Immortality conferred by CDKN2A deletion
10
Chromosome 9 breakpoint
Chromosome 3 breakpoint
CDKN2A
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Irradiation protocol
MCF-10 A
X-irradiation
Cell quiescence
Cell recovery
Cells cryopreserved Cells forRe-irradiation
5Gy and 10Gy fractions to a cumulative dose of 80Gy +/- 10nM 17-b oestradiol
Cell Passage
Recovered cells
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Radiogenic copy number alterations
0
5
10
15
20
25
5 Gy 10 Gy Ctrl 5 Gy 10 Gy Ctrl
Copy n
um
ber
altera
tions
+ 10nM 17-b oestradiol
c-MYC
121086420
121086420
121086420
121086420
q24.21
Parental
MCF-10A
40 Gy
60 Gy
80 Gy
Chromosome 8
c-MYC
Copy
Number
- has a pivotal function in growth control, differentiation and apoptosis.- over-expression has potent oncogenic activity.- dysregulation is a hallmark of many cancer types.
c-MYC……..
>50kb discernible
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~59 Mb
3 of 15
clones
12 of 15
clones
c-MYC
q24.21
Chromosome 8
dup(8)(q12q24)
Evolution of the c-MYC locus in irradiated cells
q24.21
c-MYC
PVT1
0.5
1.0
1.5
-0.5
-1.0
-1.5
0
Log2 r
atio
predicted region
of breakpoint
original duplication
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Evolution of the c-MYC locus in irradiated cells
c-MYC
CEP8
der(8)t(8;8)(q22;p23)
8
c-MYC
CEP8
dup(8)(q12q24)
der(8)t(8;8)(q22;p23)
der(8)t(8;8)(q22;p23)8
dup(8)(q12q24)der(8)t(8;8)(q22;p23)
Parental MCF-10A
8(q21-q24)
8(p)
8(p)
8(qter-q22)
8(p)
8(q)
8(p)
8(q)
8(q)
8(q)
8(qter-q22)
80Gy Population
~59 Mb
c-MYC
q24.21
dup(8)(q12q24)
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Evolution of the c-MYC locus in irradiated cells
c-MYCq24.21Chromosome 8
1 23
4 5
6
Radiation-induced
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c-MYC is prone to alteration in irradiated cells
Triplication
der(8)t(8;8)(q22;p23)
c-MYC
CEP8
Duplication
der(8)t(8;8)(q22;p23)
c-MYC
CEP8
Translocation
der(8)t(8;8)(q22;p23)
c-MYC
CEP8
8
Rare events – not part of the major clone
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Radiation selects for c-MYC amplified cells
4 c-MYC copies (MCF-10A)
3 c-MYC copies (MCF-10A)
>4 c-MYC copies (MCF-10A)
c-MYC (67 kDa)
GAPDH (37 kDa)
MCF-10A Cell population
c-MYC x3 CEP8 x2
c-MYC x4 CEP8 x2
c-MYC >4 CEP8 x2
p=0.0027, two-way ANOVA
Multiple copies (<10) of c-MYC
Amplification correlated with expression
Amplified cells are radioresistant
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Breast cancer (BC) in HL survivors
o Breast cancer after radiotherapy for Hodgkin lymphomao N=18o Mean age at BC diagnosis 37 years, range 28-47 yearso Mean age at HL diagnosis 21 years, range 16-29o Mean latency between HL and BC 15 years, range 7-24 yearso Mean cumulative RT dose 19.9 Gy, range 1.2-42.7
o Breast cancer without a radiation aetiologyo N=33o Mean age at BC diagnosis 40 years, range 32-49 years.
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c-MYC amplification in BC
19
Mean c-MYC copy number
Mean c-MYC/centromere 8 ratio
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Heterogeneous c-MYC amplification
20
c-MYCCEP8
c-MYCCEP8
3:2 5:1 6:1
7:2 13:2 ?:2
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Radiogenic cancer has high c-MYC expression
C
BA
D
RAD13
Histoscore 0
RAD9
Histoscore 70.4
RAD6
Histoscore 158.8
+ve for c-MYC expressionAll cases
Does radiation induce c-MYC amplification in non-immortalised primary cells?
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22
PRIMARY Human Mammary Epithelial Cells
(HuMEC)
X-irradiation 2Gy
Cell recovery
X-irradiation
1GyX-irradiation
2Gy
Total dose
3GyTotal dose
4Gy
Cell
senescenceCell
senescence
Primary human mammary epithelial cells
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Primary human mammary epithelial cells
c-MYC x4 CEP8 x4
IGH x4
Tetraploid
c-MYC Amp CEP8 x3
IGH x4
Polyploid Amplified
c-MYC x7 CEP8 x2
IGH x2
Diploid Amplified
HuMEC FISH
Non-amplified Diploid
c-MYC x2 CEP8 x2
IGH x2
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Model for radiogenic breast cancer
BREAST
BLOOD VESSEL LOBULE OR DUCT
endothelial cell
lumen
epithelial cell
lumen
vascular smooth muscle
extracellularmatrix
fibroblast
myoepithelial cell
RADIATION
c-MYC amplification
radiogenic adenocarcinoma of the breast
Elevated c-MYC expression drives cell proliferation and replicative stress
radiogenic angiosarcoma of the breast
macrophage
radiation-induced
atypical vascular
lesion
(non-malignant)
No c-MYC amplification
no c-MYC amplification
attenuated PRDM1(BLIMP1)
expressionpost-radiation
rs4946728
6q21
OR Het 3.20 [0.89–11.52]
OR Hom 11.4 [3.23–40.25]
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Molecular mechanisms of transformation
DNA damage Gene-specific effects Altered phenotype
O6-methylguanine/6-thiopurine
Loss of DNA MMR(MLH1, MSH2, MSH6,
PMS1, PMS2 etc)
Mutation acquisition
DNA strand breaks MLL fusion geneDysregulated gene
expression
base damage/strand breaks (clustered?)
c-MYC amplification Transcriptionalreprogramming
IONISING RADIATION?
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Risk modifiers?
26
Critical to minimise healthy tissue exposure
MU
TATI
ON
T
CEL
L D
EATH
DOSE
TOXICITY
MUTN
Identify modifiers that change the relationship between mutation and cell death
MUTATION CELL DEATH
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Chemotherapy-induced mutation
27
0
40
80
120
160
0 20 40 60 80 100
Mf
(x10
-6)
Cytotoxicity (%)
TK locus
0
40
80
120
160
0 20 40 60 80 100
Cytotoxicity (%)
HPRT locus
MNU
Ara-C
MNU
Ara-C
0
40
80
120
160
0 20 40 60 80 100
Mf
(x10-6
)
Cytotoxicity (%)
TK locus
0
40
80
120
160
0 20 40 60 80 100
Cytotoxicity (%)
HPRT locus
WT
MSH2deficient
0,01
0,1
1
10
100
0 10 20 30 40 50
Surv
ival F
ractio
n (
%)
MNU (mM)
TK6
0,01
0,1
1
10
100
0 10 20 30 40 50
Surv
ival F
ractio
n (
%)
MNU (mM)
TK6
TK6 MSH2i
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Loss of MSH2 increases the mutagenic window
28
MU
TATI
ON
CEL
L D
EATH
DOSE
TOXICITY
MUTN
Identifying radiation risk modifiers?- Secondary particles, bystander effects (likely mutagenic, are they also cytotoxic?)- Proliferative index of the exposed healthy tissue (dividing cells fix more DNA to mutation)- Volume of healthy tissue exposed (probability of SMN = mutations per cell X no. cells)- Constitutional genetics (eg. PRDM1 MAF 0.22; RB1 MAF 10-5, TP53 rare)- Acquired somatic alterations (DNA repair loss or apoptotic signalling dysfunction)- Dose fractionation
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29
Chicago
Ken Onel
Indiana
Lois Travis
Newcastle
Sarah Fordham
Victoria Forster
Nicola Sunter
Helen Marr
Mohammed Nahari
Mark Wade
Felicity May
Sarah Fordham Mark Wade