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