Patricia Kannouche

Altération des ADN polymérases: Impact sur la stabilité

génétique et effets pléiotropiques

Gustave Roussy Cancer CampusLaboratory of Genetic Stability and Oncogenesis

CNRS-UMR 8200Villejuif, France

Patricia Kannouche

Carrefour de PathologieSymposium SFP/INSERM

Anomalies de la réparation et de la réplication de l’ADN: des bases fondamentales aux applications cliniques

Palais des Congrès, Jeudi 7 novembre 2019

Presentation

I- Overview on DNA replication

II- Identification and characterization of somatic and germinal mutations

in POLE or POLD1 genes in tumors

III- Non-cancer diseases with germinal mutations in DNA polymerase genes

Epidemiological data showed that DNA replication errors may account for two-

thirds of the mutations found in human cancers (Tomasetti et al., Science 2015,

2017)

Classical DNA polymerases and associated subunits

➢ The human genome encodes at least 14 DNA polymerases — a surprisingly large number !

DNA polymerases in human genome

Adapted from Loeb and Monnat, 2008

Pola Pold Pole Polg

DNA polymerase catalysis and structure

Switch between Polymerizing mode to Editing mode

Fork progression

Ongoing DNA Replication

Adapted from Garcia-Muse and Aguileras, NRMCB 2018

Déterminants de la fidélité de la réplication

Adapté de McCulloch and Kunkel, Cell Res 2008



G

T

parentalnéosynthétisé

GMSH2/MSH6

MLH1/PMS2

G

T

EXO1

G

PCNA polδ

reconnaissance

excision

resynthèse

Fidélité de la réplication de l’ADN

Taille du génome humain: env 3 . 109 paires de base


G

T

parentalnéosynthétisé

GMSH2/MSH6

MLH1/PMS2

G

T

EXO1

G

PCNA polδ

reconnaissance

excision

resynthèse


12

➢ Tumors with somatic mutations in exonuclease domain of DNA polymerase epsilon exhibit overall mutation frequencies often exceeding 100 mutations/Mb.

➢ Tumors with somatic mutations in the exonuclease domain of DNA polymerase delta, or deficient in MMR : 10-100 mutations/Mb.

➢ Tumors with somatic mutations in the exonuclease domain of DNA polymerase epsilon and deficient in MMR (bMMRD) : ~ 600 new mutations/generation.

DNA replication-associated errors are repaired by two components: polymerase proofreading and mismatch repair.


Campbell et al, Cell 2017

Replication repair deficiency drives a mutator phenotype

in many adults cancers

New hereditary colorectal cancer syndrome

“Polymerase Proofreading Associated Polyposis” (PPAP)

➢ Germline pathogenic variants in human POLE or POLD1 exonuclease domains

➢ Multiple polyposis and CRC following an autosomal dominant pattern of

inheritance

➢ Contributions of POLD1 and POLE genetic defects to early onset

of familial colorectal cancer: 0.2 and 0.6%, respectively

➢ Larger phenotypic spectrum :

- endometrial cancer

- ovarian and brain tumors

- pancreatic and small intestine cancer

- melanoma.

Rayner et et al, Nature Review Cancer 2016

POLEPOLD1

POLE and POLD1 mutations reported in CRC and EC

➢ POLE is altered much more frequently than POLD1 in hypermutated MSS tumors

➢ Most somatic POLD1 mutations are found in MSI tumors.

➢ Some mutations are observed at a vastly greater frequency than others.

POLE

POLD1

Yellow: ssDNA

Location of cancer-associated Polε mutations

within the exonuclease domain

Barbari, et al., 2018

Functional assays of POLE variants in yeast model

POLE P286R/P301R has a robust DNA polymerase activity

superior to that of the wild-type or proofreading-deficient yPolε

Xing et al, Nature Comms 2019

Biochemical studies on POLE P286R

POLE P286R/P301R retains weak 3ʹ→5ʹ exonuclease activity

Model explaining the ultramutator phenotype in tumors harboring the

cancer-associated P286R mutation in POLE

Xing et al, Nature Comms 2019

Relationship between the incidence of individual POLE variants in sporadic

tumors and their mutator effects deduced from in vivo functional assays

P236R

F367S

P236H

S459F

D275VL424V

P436R


Adapted from Poulos et al., PLoS Genet. 2018

POLE p.P286R

Accumulation of mutations related to Signature 10

100-fold increase in C>A transversions in T[C>A]T context

30-fold increase in C>T transitions in T[C>T]G context

Serine to Tyrosine/ Leucine

Arginine to Isoleucine/Glutamine

Glutamic Acid to Stop Codon

Strong bias for particular amino acid changes

0%

10%

20%

30%

Distinctive pattern of missense/truncation mutations in

oncoproteins and tumour suppressors

PTEN pR130Q

MSH6 pE946*

ARID1A pR1989*PIK3CA pR88Q

APC pQ1338* P53 pR213*

Mutational signatures in POLE tumors

It is extremely important to distinguish among causative, non-polymorphic germline variants, and

somatic pathogenic, non-passenger variants with prognosis meaning.

Landscape of drivers and passengers in POLE and POLD1 genes

Mutations dans les gènes POLE ou POLD1 non-

liées à une prédisposition au cancer

IMAGe syndrome

Clinical features closely resembling IMAGe syndrome (associated with GOF mutations

in CDKN1C)

Intrauterine growth restriction [IUGR]

Metaphyseal dysplasia

Adrenal hypoplasia congenita

Genitourinary anomalies in males

Distinctive facial features

Variable immune dysfunction

Intronic variant (c.1686+32C>G, altering splicing) as part of a common haplotype, in

combination with different loss-of-function variants in trans.

Biallelic mutations in POLE

Logan et al, AJHG 2018

This mutation causes a distinct multisystem disorder that includes:

-subcutaneous lipodystrophy,

-deafness,

-mandibular hypoplasia

-hypogonadism in males

-Progeroid features

-Insulin resistance

MDPL syndromeAn in-frame deletion at the polymerase active site of POLD1 causes a

multisystem disorder with lipodystrophy

Clinical characteristics of individuals with MDPL syndrome

no detectable polymerase activity, but

robust exonuclease activity

Biochemical characteristics

Fibrosis

Weedon et al., Nature Genetics 2013

To date, about 25 cases with POLD1-related MDPL syndrome have been reported worldwide, albeit almost entirely of European originMale-to-female ratio 7:15. Most of patients with p.Ser605del, 3 patients with p.Arg507Cys, 1 patient with novel mutation p.Ile1070Ans, 2 related female patients with Glu1067Lys

Heterozygous mutation in POLD1

Polg, l’ADN polymérase mitochondriale

Small circular DNA

A schematic diagram of a mitochondrial DNA

replication intermediate

Copeland’s Lab

More than 180 disease mutations for the POLG gene http://tools.niehs.nih.gov/polg/

Symptoms associated with different POLG syndromes

Farnum et al., BBA 2014

MERCI POUR VOTRE ATTENTION

Minimizing and balancing leading and lagging strand mismatches

Patricia Kannouche

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