Lecture 2 Flashcards
Germline mutation in DNA repair genes are the most common types of which inherited cancer-prone syndromes
up to 10% of breast cancers (BRCA 1 and 2, ATM) and 5% of colon cancers (MMR)
why do other identified DNA repair genes not feature in cancer prone syndromes
Many germline DNA repair defects may be incompatible with life either as homozygotes or heterozygotes (does not apply to somatic mutations)
Some defects may not result in repair deficiency due to redundancy between different repair pathways
Where are somatic mutations in DNA repair genes commonly found
Found in tumours and leukaemia. About 20% of patients with chronic lymphocytic leukaemia have mutations in ATM . About 50% of all cancers carry p53 mutations
Describe the development of chemo-resistance
- Tumour weighing 1 g can contain up to 109 cells
- Exposure of the tumour to a chemotherapeutic agent may kill e.g. 99.9% of the tumour cells
- The residual tumour may only weigh 1 mg, but this contains ~106 surviving tumour cells…
(Maybe -10 cells among the 109 (1/108) of the original cells in the tumour carry a (very rare!) mutation in a gene that confers resistance to the chemotherapeutic agent) - These 10 cells will be among the 106 survivors (1/105 - a 1000-fold enrichment)
Explain how chronic myelogenous leukaemia developed and how its treated
Chronic myelogenous leukaemia (CML) is characterised by the unregulated growth of myeloid leukaemia cells in the bone marrow
- Chr 9/22 translocation (“Philadelphia chromosome”) in CML results in the creation of a new Bcr-Abl fusion gene, encoding a tyrosine kinase unique to the leukaemia cells
- Imatinib (Gleevec) is a drug specifically designed to target the Bcr-Abl kinase, binding to the ATP binding pocket in the catalytic site
What is Gleevec
Revolutionised the treatment of CML, producing long term remissions
However, over time the disease recurs and is now resistant to Gleevec
How does drug resistance in leukaemia identified
Resistance in patients has been identified as due to a set of mutations arising in the catalytic site of the Bcr-Abl gene
This has led to the production of second generation drugs which have been designed to target the mutant enzymes (eg Dasatinib)
How does chemotherapy damage DNA
The majority of chemotherapeutics act by damaging DNA, as does radiotherapy
Chemo and Radio - therapeutics kill cells by damaging DNA
Every individual tumour will have its own unique spectrum of mutation and chromosomal rearrangement
How does the DNA repair pathways do ?
Multiple overlapping pathways exist; these can repair a broad range of different types of DNA damage
DNA damage-inducible signalling also important
○ Activarion of cell cycle checkpoints
○ Activation of transcription factors
○ Activation of programmed cell death (apoptosis)
How do activation of NF-kB & p53 affect DNA damage
Transcription of genes that promote survival
Cell cycle arrest
apoptosis & cell death
How the induction of cell cycle checkpoint affect DNA damage
- activated p53 - causing cell cycle arrest/apoptosis
- may cause RNA repair and therefore cell survival
Defects in which DNA damage-inducible signalling can cause cancer
Aurora B, BUB1,BUB1B,BUB3,MPS1 (Colorectal lung and pancreatic tumours and T cell lymphomas, lymphomas, MVA and PCSS)
ATM (Lymphomas and breast cancer)
ATR (Stomach, endometrial and breast cancer)
CHK1 (Stomach, endometrial, colorectal and lung cancer)
CDK1 & PLK1 (Cancers of the liver, lung, stomach and epidermis)
Aurora A (Several human tumours, for example breast and colorectal cancers)
CDK4 (Melanoma, glioblastoma and osteosarcoma and breast and cervical cancers)
CDK6 (Lymphomas, squamous cell cancer and gloma)
CDK2
CHK2 (Bladder, colon, ovary and other cancer)
What is O6-methylguanine-DNA-methyltransferase (MGMT)
The most cytotoxic locus of alkylating agents is at O6 position of guanine
MGMT removes the alkyl group restoring normal DNA, binding the O6 lesion to itself
This is a suicide repair mechanism, directly restoring the DNA to its correct sequence
The ‘enzyme’ is no longer useful to the cell therefore degraded
Cancer cells often have high MGMT making them resistant to alkylating agents that rely on insertion of the O6-lesion
What occurs during Base excision repair
Removes bases damaged by alkylation, deamination or oxidation
Glycosylases (cleaving N-glycosydic bond) remove a wide range of modified bases from the DNA, e.g OGG1 (8-oxoguanine DNA glycosylase)
[This generates an AP (apurinic/pyrimidinic) site]
Endonuclease cuts the DNA and a few bases are removed. [APE1 (AP endonuclease 1)-hydrolyses the AP site]
The gap is filled by polymerase action using the opposite strand as template - Finally the DNA is religate
how does mismatch repair occur
Binding of mismatch proofreading proteins, DNA scanning detects nick in new DNA strand
Strand is removed and repair DNA synthesis occurs
How does nucleotide excision repair occur
2 subtypes - Global genome, transcription-coupled
- Recognises and repairs damage to the DNA which distorts the helical structure (e.g thymine dimers)
- Steps in repair pathways = Recognition of damaged sites, binding & recruitment of factors, excision of damage DNA, gap filling and religation
what is double strand break repair
Mammalian cells have two primary double strand break repair pathways that are cell cycle-dependent
Non-homologous end joining (NHEJ) - G1
(Ku70/80, DNA-PKcs, Ligase IV, XRCC4, Artemis etc)
Homologous recombination (HR) using intact sister chromatid -S/G2
(RAD51, RAD52, RAD54, RAD51B/C/D, XRCC2/3)
How does non-homologous end joining work
○ Ku80 distal to, Ku70 proximal to the break
○ DNA-PKcs recruited to Ku :becomes active
[Synapsis: 2 DNA ends brought together]
○ Processing to resolve e.g. 3’ and 5’ overhangs, enzymes = Artemis (nuclease activity), Werner syndrome protein (WRN- helicase/exonuclease activity),Human terminal deoxytransferase (TdT- adds nucleotides), Polynucleotide kinase (5’ kinase and 3’ phosphatase)
○ XRCC4/ligase IV joins the break (DNA pol u)
○ NHEJ proteins removed from the break in order for ligation to proceed. Autophosphorylation of DNA-PKcs .
○ DS break repair is complete: however sometimes this results in loss of nucleotides, and therefore errors may be introduced
How did james cleaver discovered DNA repair deficient syndrome lead to cancer
- He grew skin cells from patients with a cancer-prone genetic syndrome called xeroderma pigmentosum, that causes exquisite sensitivity to the damaging effects of sunlight
- He demonstrated that when the cells were exposed to UV, they were unable to carry out DNA repair (Cleaver J.E. Nature, 1968)
- The subsequent isolation and characterisation of mammalian cell lines which were hypersensitive to specific DNA damaging agents led to the identification and cloning of numerous genes involved in DNA repair
- This was the first demonstration of the importance of DNA repair in humans as a defence against carcinogenesis
Herba and Kaposis first described _ in 1874
Xeroderma Pigmentosum
what is Xeroderma pigmentosum
- Rare disorder (one in a million in the UK) transmitted in an autosomal recessive manner (Heterozygotes (XP/xp) have no abnormalities) Median age of onset = 8
- Photosensitivity, pigmentary changes (freckling), premature skin aging, and malignant tumour development
How are people who have xeroderma pigmentosum cancer prone
- 2000-fold increase in the frequencies of melanomas, also basal cell carcinomas, squamous cell carcinomas. 97% of skin cancers develop in exposed areas of skin (head, neck, hands)
- These are due to a cellular hypersensitivity to UV radiation resulting from a defect in DNA repair = no cross sensitivity to X rays
what is the defect which causes people with xeroderma pigmentosum to be cancer prone
- The defect in XP is in nucleotide excision repair (NER). - - Key elements of NER now understood, and the repair process has been reconstituted in vitro.
= 7 XP genes:- XPA through XPG, have been identified.
(Patients have been identified with mutations in all these genes) - Failure to repair UV damage can be seen in the skin tumours of XP patients, which have high levels of mutations in the tumour suppressor gene, p53
what are the clinical features of ataxia telanglectasia
○ Like XP, AT is an autosomal recessive disorder, but in this case, patients cells are specifically hypersensitive to ionising radiation (incidence 1/40,000)
○ Progressive ataxia (Purkinje cells- neurons in brain within cerebellum) Ocular/cutaneous telangiectasia (prominent blood vessels), immune dysfunction, progeric skin
○ > 100-fold increased risk of developing cancers, especially B and T cell lymphoma
what are the cellular characteristics of ataxia telanglectasia
○ 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
Describe the defect in ataxia telangiectasia
○ Mutation in a gene that codes for a kinase enzyme, named ‘ATM’– AT Mutated
○ It is a DNA damage-activated kinase; signals damage to cellular machinery
○ Somatic mutations in ATM are frequent in tumour
how is double strand break repair enzyme explorated
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
How does targeting DNA repair sensitise the cytotoxic therapy
- An additional approach could be to target double strand break repair (DSB)
- DNA-PK (NHEJ) and ATM (HRR) could be inhibited, resulting in failure to repair otherwise lethal DSBs
- Sensitising to DSB will result in enhanced tumour cell killing
- Sensitising to chemotherapy induced DSB results in enhanced tumour cell killing
what is familial breast cancer
○ 1/9 women will develop breast cancer - 50,000 women are diagnosed each year in UK
○ Most commonly diagnosed cancer, and ~1,000 women die of breast cancer every month in UK
what, cellularly, causes familiar breast cancer
○ BRCA 1 and 2 (breast cancer early onset) are defined as tumour suppressor genes. They predispose patients to cancer at specific sites including breast, ovary and pancreas
○ Inheritance of 1 defective allele is sufficient for cancer predisposition. Approximately 70% of women carrying mutant BRCA1 or BRCA2 alleles will develop cancer at some point in their life.
= BRCA1 and BRCA2 play important roles in the repair of DNA double strand breaks
In women with an inherited mutation in BRCA1 during puberty -
rise in oestrogen leads to rapid proliferation of breast cells. During periods of rapid proliferation, cells are more susceptible to DNA damage
Described the relationship between germline mutation in BRCA1 and carcinogenesis
○ 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
After germline mutation in BRCA1 cells develop a mutator ‘phenotype’, what happens to these cells, how can it can lead to cancer
○ A mutant cell 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
What is synthetic lethality
The genes may encode DNA repair function
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
= a situation in which mutations (changes) in two genes together result in cell death, but a mutation in either gene alone does not.
What is an example of synthetic lethality
- PARP & BRCA
PARP inhibition e.g. rucaparib results in elevated DSB. DSB can be repaired by BRCA +ve cells and cells survive (A)
In BRCA -/- cells, broken DNA cannot be repaired – (only) tumor cells are killed by Synthetic Lethality (B)
