Polymerase ε Mutations Accelerate Mutation Rates in Colorectal and Endometrial Cancer
David A. Wheeler & TCGA Network
TCGA 2nd Annual Symposium November 28, 2012
HumanGenomeSequencingCenter
Mutation rates classify CRC patients
silent non-silent
Patient Hypermutated
“Ultramutated”
2
Mutation frequencies in DNA repair genes in colorectal cancers
1-10/Mb 10-100/Mb >100/Mb
3Categories according to RD Wood and colleagues: http://sciencepark.mdanderson.org/labs/wood/DNA_Repair_Genes.html
POLE Mutations in Colorectal Cancer
4
1 2286
Exonuclease Polymerase
R150X
R231X
V411L
S459FL1255V
V1368MR762W
P286HF367S
P436R
A189T
POLE
ZNBFinger
S459F
P1421S
T4 exonuclease mutagenesis
• rII- reversion rates – Sensitive to 1/109-10
• Mutator phenotype also studied in bacteria, yeast, mice
LJ Reha-Krantz, BBA 1804: 1049 (2010)
Polymerase
Exonuclease
5
6
Pole, Pold1 exonuclease KO in mice
• Pole and Pold1 exonuclease KO exhibit mutator phenotype
• Homozygous mutants rapidly die of cancer
– Polee/e intestinal and lymphoma
– Pold1e/e lymphoma and lung
TA Albertson et al. PNAS 106: 17101 (2009)
7
Endometrial
V411L
Colorectal P286R,H,L
S459F F367S
POLE
Distribution of mutations in POLE
Eve Shinbrot, HGSC; cBIO Portal
8
Mutation properties of colorectal tumors
Log
Mut
atio
nFr
eque
ncy
Rel
ativ
eM
utat
ion
Freq
uenc
y MSS low MSI hyper
MSS ultra
POLE mutations in CRC and Endometrial Cancer
9
“Polymerase B” domain family sequence similarity over “a billion” years of evolution
T4 p
hage
Exo Pol
Human POLE
10
POLE mutation clusters
11
12
Asymmetric roles of major replicative DNA polymerases
Leading strand
Lagging strand
Mutation profile is skewed at sites enriched for origins of replication*
*C.-L. Chen et al. Mol. Biol Evolution 28: 2327 (2011)
• 60:40 bias of CA pattern on leading strand • Caveat:
– Whole exome sequence data is limited in resolution – Need to replicate in whole genome
Nils Weinhold, Niki Schultz, MSKCC 13
14
Pole, Pold1 exonuclease KO in mice
• Pole and Pold1 exonuclease KO exhibit mutator phenotype
• Homozygous mutants rapidly die of cancer
– Polee/e intestinal and lymphoma
– Pold1e/e lymphoma and lung
TA Albertson et al. PNAS 106: 17101 (2009)
POLD1: no exonuclease domain mutations in ultramutated patients
Colorectal Endometriod (14 patients) (17 patients)
Eve Shinbrot, HGSC; cBIO Portal 15
16
Mutation frequencies are anticorrelated with expression level in ultramutated patients
Mut
atio
ns p
er M
b
Colorectal
mRNA Expression [log] mRNA Expression [log]
Endometroid
Nils Weinhold, Niki Schultz, MSKCC
• ~2X reduction in mutation rate on highly transcribed genes
UCEC Progression free survival favors ultramutated patients
17
Conclusions and future directions
• Rare exonuclease-mutation in POLE leads to an ultramutator phenotype in colorectal and endometrioid cancers.
• The ultramutator phenotype defines a new subtype of these tumors that may have unique prognostic features and interesting biological properties. – Need further mutation profiling in colorectal and endometriod cohorts
with clinical outcomes. • Ultramutator patients exhibit a signature of transcription coupled repair. • Absence POLD1 ultramutators suggests it may perform an essential
function in this new subtype of colorectal and endometrioid cancers (role in TCR?).
• Strand-specific mutation pattern associated with putative origins of replication in humans is first suggestive evidence for confirmation of yeast model of replication in a higher eukaryote.
• Whole genome sequencing should help to separate the effects of transcriptional repair and strand-specific mutation effects.
18
19
Acknowledgements
• Human Genome Sequencing Center, Baylor College of Med
– Richard Gibbs – Donna Muzny – Jeffrey Read – Jennifer Drummond – Nipun Kakkar – Kyle Chang – Lisa Trevino
• Dan Duncan Cancer Center, Baylor College of Med
– Chad Creighton – Larry Donehower
• Memorial Sloan Kettering Cancer Center
– Nils Weinhold – Niki Schultz – Chris Sanders – Doug Levine
• The Genome Institute, Washington University
• MD Anderson Cancer Center • Gordon Mils • Stan Hamilton
• Broad Institute of MIT and Harvard Mike Lawrence Gaddy Getz
• National Human Genome Research Institute
• National Cancer Institute • The TCGA Network
20
21
CRC-total samples MMR
Endometrial-total samples MMR
22
Exonuclease motifs of B family DNA polymerases
* *
23
Nucleotide context of POLE mutations
a
Colorectal cancer MSS standard
MSI hyper
MSS ultra
C ->
A
C ->
G
C ->
T
T ->
A
T ->
C
T ->
G
b TCT
24
Nucleotide context of POLE mutations
c
Endometrial cancer MSS standard
MSI hyper
MSS ultra d
C ->
A
C ->
G
C ->
T
T ->
A
T ->
C
T ->
G
25
Colorectal
Colorectal
Endometrial
26
P286R/H
F367S
V411L
S459F
human T4 Human POLE
vs T4 POL human
T4
human T4
human T4
human T4
T4
T4
human T4
human
human
IN P
RO
GR
ES
S
27
P286!
S459!
V411!
### ###
28
http://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi
P286!
P326!
POLD1!POLE!
V411! S459!
470! C312!
POLE!
POLD1!
29
Detailed mutations in POLE
30
Detailed mutations in POLE. CRC
31
Significantly Mutated Genes Mike Lawrence, Gaddy Getz, Broad
32
1-10/Mb 10-100/Mb >100/Mb
33
1-10/Mb 10-100/Mb >100/Mb
34
Mutation spectrum changes with increased MMR
• Transversions that reverse paired bases appear less frequently in hypermutated patients
Hypermutated
Standard Mutation
Mike Lawrence, Broad Institute
35
Flip transversions in ultramutated
Mutations (Log10)
Frac
tion
of C
A ->
GT
(Log
10)
Mike Lawrence, Broad Institute
36
http://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi
38
P286R/H
F367S
V411L S459F
Eukaryotic POLE alignments • All mutator positions are
invariant across eukaryotes
39
MSS Ultra
Mutation properties of endometroid tumors
Log
Mut
atio
n Fr
eque
ncy
Rel
ativ
e M
utat
ion
Freq
uenc
y
MSS Low MSI Hyper