L2 DNA repair defects and cancer

DNA repair defects predispose to…

cancer

Germline mutations in DNA repair genes are the most common types of…

inherited cancer-prone syndromes

• up to 10% of breast cancers and 5% of colon cancers

Somatic mutations in DNA repair genes are commonly found in tumours and leukaemias

about 20% of patients with chronic lymphocytic leukaemia have mutations in ATM

DNA damage inducible signalling is important

• activation of cell cycle checkpoints

• activation of txn factors

• activation of programmed cell death (apoptosis)

The most cytotoxic locus of alkylating agents is at the O6 position of guanine

MGMT removes the alkyl group, restoring normal DNA, binding the O6 lesion to itself

• suicide repair mechanism, restoring the DNA to its correct sequence

• the enzyme is no longer useful to the cell

• cancer cells often have high MGMT making them resistant to alkylating agents that rely on insertion of the O6 lesion

Base exclusion repair removes…

bases damaged by alkylation, deamination or oxidation

glycosylases remove a wide range of modified bases from DNA

e.g. OGG1

• generates an apurinic site

endonuclease cuts the DNA and a few bases are removed

APE1

• hydrolyses the AP site

• the gap is filled by polymerase action using the opposite strand as a template

• DNA is religated

subtypes of nuclear exclusion repair

• global genome

• transcription coupled

• recognises and repairs; damage to the DNA which distorts the helical structure e.g. thymine dimers

steps in the nucleotide exclusion repair pathway

• recognition of damaged site

• binding and recruitment of factors

• excision of damaged DNA

• gap filling and religation

Mammalian cells have 2 primary double strand break repair pathways that are

cell cycle dependent

• non-homologous end joining

• homologous recombination using intact sister chromatids

describe non-homologous end joining

• Ku70/80 forms a DNA pk holoenzyme

• DNA pkcs brings ends together

• enzymes artemis

• XRCC4/ligase IV joins the break

• NHEJ proteins are removed from the break in order for ligation to proceed

• DS break repair is complete - results in a loss of nucleotides where errors can be introduced

NHEJ is error prone/mutagenic

High fidelity repair is only possible in S/G2 phase when a…

template strand is available (HR)

The ability to bind DNA, confirmed by the structure of a BRCA2-DNA complex, implicated BRCA2 in binding DNA substrates involved in HRR, which is a…

high-fidelity repair pathway

restore fidelity and integrity of DNA to prevent

mutation/breakage/rearrangement - parental DNA sequence is largely retained

• DNA repair can go wring and genetic errors occur

• DNA repair machinery may be dysfunctional or we can inactivate it

Xeroderma Pigmentosum

photosensitivity, pigment changes, premature skin aging, malignant tumour development - cancer prone

• cellular hypersensitivity to UV radiation, resulting from a defect in DNA repair - no cross sensitivity to X-rays

Ataxia Telangiectasia

• like XP, AT is autosomal recessive but patients are specifically hypersensitive to ionising radiation

• progressive ataxia

cellular characteristics of Ataxia Telangiectasia

• acute radiosensitivity and hypersensitivity to some types of DNA damaging agents

• elevated frequencies of chromosomal breaks and rearrangements

• defect in the homologous recombination repair of DNA double strand breaks

• Defects in the control of multiple cell cycle checkpoints

Defect in AT

• mutation in a gene that codes for a kinase enzyme, named ATM - AT mutated

• A DNA damage-activated kinase; signals damage to cellular machinery

• somatic mutations in ATM are frequent in tumours

We can exploit the key

role of DSB repair

enzymes in maintaining

the integrity of DNA

during cell division

cancer cells are rapidly

dividing, if we prevent

DSB repair, we aim for

cancer cells to attempt to

divide with a DSB in the

genome, a lethal event

for a cell.

We can inhibit either

NHEJ or HRR

DSBs are

lethal to cells

radio and chemotherapy induce DSBs in cells, but

DSB repair can fix breaks and prevent cell killing

NHEJ and HRR could be inhibited, resulting in

failure to repair otherwise lethal DSBs

sensititsing to DSB will result in

enhanced tumour cell killing

tumours could be sensitised to DNA damage by

targeting NHEJ

• Loss of DNA-PK activity

  (NHEJ pathway) could result

  in failure to repair

  otherwise lethal DSBs

  • Sensitising to chemotherapy

  induced DSB results in

  enhanced tumour cell killing

  and patient benefitp

ATM (HRR pathway)

could be inhibited;

failure to repair

otherwise lethal DSBs

Sensitising to DSB will

result in enhanced

tumour cell killing

Base modification and

DNA SSBs repaired by

base excision

repair/single strand break

repair (BER/SSBR)

PARP may be upregulated in

cancer

• inhibitors developed to target this resistance mechanism in cancer and kill tumours

AG014699/ PF-01367338/rucaparib Phase I/II with temozolomide

Profound and sustained PARP inhibition in PBMCs and in tumours

• Some improvement of TMZ efficacy in melanoma + increase in TMZ toxicity

(myelosuppression)

KU-0059436/AZD-2281/ Olaparib

Numerous Ph I/II trials; toxicity issues with topoteca

toxicity increases with efficiacy

• All cells use DNA repair mechanism to overcome

  damage (usually endogenous)

  • Systemic inhibition of repair = systemic sensitisation

  to exogenous damage (chemotherapy)

relationship between a germline mutation and BRCA1 and carcinogenesis breast cancer

• In women with an inherited mutation in BRCA1, during puberty, rise in estrogen leads to

  rapid proliferation of breast cells. During periods of rapid proliferation, cells are more

  susceptible to DNA damage

  • If repair is already compromised by loss of one allele, there is an increased risk of

  acquiring a second mutation in the other allele, especially as the BRCA1 gene has a high

  density of repetitive elements in the gene sequence

  • If both alleles are inactivated, cells develop a "mutator phenotype", and further

  mutations, involving oncogenes and tumour suppressor genes, are likely to develop as

  cells continue to proliferate

  • The mutant cell (purple) will have a growth advantage (eg a p53 mutation will enable

  cells to escape from checkpoint controls)

  • Clonal expansion of these cells, and accumulation of further oncogenic mutations will

  lead to early onset breast cancer

synthetic lethality

BER + HRR

• The pair of genes may encode redundant function in normal cells e.g. different

  components of the DNA repair pathway

  • Though originally defined as the loss of two genes, the hypothesis can be extended to

  loss of gene function e.g. one loss of gene combined with inhibition of function of the

  other gene (product)

  • In oncology, the scenario where one pathway deficiency is tumour-specific and the

  second is induced with a DNA repair inhibitor has significant therapeutic potential