Cancer Genomes Flashcards
1
Q
How can we acquire mutations?
A
- Exogenous sources - UV, cigarette smoke, radiation (that form adducts)
- Endogenous sources - chemical/enzymatic processes - depurination, deamination, oxidation, methtylation
- DNA replication - misincorporation of bases, replication slippage, DSBs at replication forks
2
Q
What mechanisms does our body use to protect against cancer?
A
- Protection mechanisms - e.g., physical shielding, stem cell properties; enzymatic detoxification
- Mechanisms to ensure accuracy in DNA replication - e.g., DNA proofreading
- DNA Damage repair mechanisms - e.g., HDR, NHEJ, MMR, NER, BER
3
Q
How do germline and somatic mutations differ in their cause of cancer?
A
- Germline mutations - inherited predisposition to cancer - if a genetic defect in a protective/repair mechanism - this will influence the likelihood of developing cancer
- Somatic mutations - sporadic cancer
4
Q
What are the types of genes involved with cancer?
A
- Oncogenes
- Tumour suppressor genes - ‘gatekeeper genes’
- Genes involved with maintaining genome integrity - ‘caretaker genes’
5
Q
What are the features of an oncogene & how are protooncogenes activated - examples?
A
Oncogenes are dominantly acting - require mutation of one allele only
- Mainly occur sporadically in somatic cells
Proto-oncogenes are activated by gain-of-function mutations - by:
- over-expression (gene amplification/chromosome translocation) or alteration in protein structure (chromosome translocation/point mutation)
- Roles of proto-oncogene: - promote proliferative signalling (Ras), inhibit apoptosis (BCL2)
6
Q
Are mutated oncogenes often inherited through the germine?
A
NO - very rarely
- Because mutations would be lethal in development
- More common in sporadic cancers in somatic cells
7
Q
What are the features of tumour suppressor genes (TSGs), and how are they activated?
A
TSGs are recessive at molecular level
- So require mutation of both alleles
- Loss-of-function mutations activate TSGs - contribute to cancer - generally ‘inactivated’ by deletions/point mutations or epigenetic silencing (methylation)
- Normal role of TSG - e.g., suppress proliferative signalling - retinoblasta
- To oppose oncogene action
8
Q
Are TSG mutations found in the germline or somatic cells?
A
BOTH - Inheritance of mutant TSG results in hereditary predisposition to cancer
9
Q
What two types of genes can contribute to cancer?
A
Indirectly active genes - don’t interact with DNA - but may be involved with carcinogen activation/detoxification
Directly active genes - genes that directly impact our DNA structure - e.g., involved in DNA proofreading/repair pathways
- Mutations in these genes accelerate the rate at which mutations accumulate - as tissue divides
10
Q
What three levels can mutations occur at - and what are some examples of each?
A
- DNA level - indirect/directly active genes
- Chromatin level - e.g., histone variants, chromatin remodelling, 3-D chromatin organisation, DNA methylation machinery
- Chromosome level - genes involved in chromosome stability, telomere maintenance (BFB cycles), disorders in prematrure ageing, POT1 mutations, genes in mitotic spindle assemly - anueploidy (st james’ lectures)
11
Q
What is a mutational signature?
A
- Imprints of specific mutagenic processes, repair mechanisms/repair mechanism defects
12
Q
What types of analysis can be done to investigate cancer genomes?
A
- DNA sequence and expression analysis
- DNA methylation status
- Histone modification status
- 3-D organisation of chromosomes
13
Q
By investigating cancer genomes what information can be found out?
A
- ‘Driver’ / passenger genes
- Common pathways
- Characterisation of mutational signatures
- Understanding of mechanisms of aberrant regulation
- Understanding of tumour evolution
- Clincal application
14
Q
What is meant by ‘driver’/’passenger’ mutations?
A
‘Driver’ mutations:
- Directly involved in cancer progression
- Under selection
- Mutations found in hotspots (for oncogenes)
- Bias in mutation type
‘Passenger’ mutations:
- Mutations that do not influence cancer progression
- Not affected by selection
- Mutation type reflects the mutagenic process and repair mechanism
- Often look at intronic regions
- Provide information on cancers mutational signatures
15
Q
What types of damage can endogenous chemical processes do to DNA?
A
- Strand breakage - (one/both)
- Hydrolytic processes - deamination (loss of amino groups) or loss of bases (leaving abasic site)
OR - alter base structure:
- Base modificaton - oxidation
- Base cross-linking - same/diff strand
16
Q
How common is each endogenous hydrolytic processes and how are they delt with?
A
- Depurination - most common base loss/alterations
- Depyrimidation - less common
- DDR fixes these
- Deamination - remove amino group from C, A and G - delt with efficiently by BER
17
Q
Why is deamination of 5-methyl-C problematic?
A
- 5 me-3 has role in gene expression (associated with transcriptional repression)
- Is becasue 5 me-3 is deaminated to thymine - which is a natural DNA base - so is much less efficiently fixed by BER
- So is a major source of point mutations - at CpG sequences - (30% of p53 mutations arise at CpGs)
- 5 me-3 is also a favoured target for benzo[a]pyrene - hydrocarbon in tobacco smoke
18
Q
How are ROSs generated?
A
- From byproducts of mitochondrial reactions - when oxygen is not completely reduced
- Inflammed tissues
- Spontaneous oxidation of lipids
- Oxygen-utilising enzymes in peroxisomes
19
Q
How can ROSs cause damage?
A
- They can covalently bond with DNA bases
- Induce SSBs/DSBs
- Induce abasic sites - apurinic/apyrimidinic
- Induce protein-DNA cross-links
20
Q
What are two examples of mutagenic oxidation reactions and how are they detected?
A
8-oxo-G
- Deoxyguanosine (dG) is oxidated to 8-oxo-G
- 8-oxo-G can pair with A or C - which replaces a G:C base pair with T:A - after DNA replication
- G to T transversion mutations
5-methyl-C
- Oxidation of 5-methyl-C forms deoxythymidine glycol - which blocks DNA polymerase
Detected through urine (8-oxo-G/thymine glycol)
- Smaller animals = higher metabolic rate = increased level of oxidation = higher ROS in urine
21
Q
What causes increased levels of 8-oxo-G/thymine glycol?
A
- Inflamed and neoplastic tissues: Inflammation - phagocytes kill cells - releasing oxidants - oxidation, nitration, deamination, halogenation of bases
- Smokers - chronic inflammation of lungs = 50% increase in oxidised bases in urine
- Tumours - increased metabolism = increased oxidation - more 8-oxo-G than normal tissues
22
Q
How can aberrant DNA methylation cause mutations?
A
S-adenosylmethionine (SAM)
- Cytosine bases are methylated - using SAM donor
- SAM is donor in non-enzymatic reactions - SAM inappropriately methylates DNA - distorting double-helix & DNA-protein interactions
23
Q
How do exogenous/endogenous agents relatively affect damage?
A
- Endogenous - 100,000 base damage per cell genome per day - but less severe
- Exogenous - much more rare - but generate ROSs that create SSBs/DSBs - impact is bulkier
24
Q
How do X-rays cause mutations?
A
X-rays (ionising radiation): generate ROS that create SS/DSBs
25
How does UV cause mutations?
**UV**: leads to formation of covalent bonds between two adjacent pyrimidines on the same strand (6-4PPs/CPDs - CC-TT changes - melanomas/p53 mutations)
26
How do alkylating agents cause mutations?
**Alkylating agents**: very mutagenic (used to induce tumours in labs/chemotherapy) - lead to destabilisation of bond to deoxyRibose - loss of base = misread in DNA replication
27
How does tobacco smoke cause mutations?
**Tobacco smoke**: BP is oxidised to ultimate carcinogen (BPDE) by CYPs - BPDE attacks other molecules in same cells e.g., lung epithelial cells forming bulky adducts with G residues
28
How can alcohol cause mutations - direct/indirectly?
**Alcohol**:
1. Directly mutagenic - ethanol is oxidised to acetaldehyde (ADH) (mutagenic) forming smaller adducts with G. Increased ADH/reduced ALDH = increased risk of oral/oesophagael cancer (East Asians)
2. Indirectly mutagenic - ethanol is cytotoxic so kills cells - causing inflammation - oxidation (ROS)
29
How can cooked meat cause mutations?
**Cooked Meat**: forms heterocyclic amines (HCAs) e.g., PhIP - oxidised by CYPs - forming large adduct by cutting out ring or oxidising exocyclic amine group - ROS - colon/breast carcinomas & lymphomas in mice
30
How can Aflatoxin B cause mutations?
**Aflatoxin B (AFB1)**: - aspergillus mould on rice/grains - AFB1 oxidised to 8,9-oxide - adduct - liver cancer - China (humid - regional differences) - p53
31
What are CYPs?
- Superfamily of enzymes - **biosynthesise metabolites**
- Aid in **oxidation** of compounds and detoxification of xenobiotics
- Turn **exogenous agents into mutagens** in the body - through oxidation - e.g., BP to BPDE
32
What are the three mechanisms for protecting the genome form attack by mutagens?
- Physical shielding
- Stem cell compartment
- Enzymatic detoxification
33
How do melanosomes protect us?
- **Melanosomes** carry melanin - have transferred from melanocytes to keratinocytes in basal layers of epidermis
- Melanosomes are assemlbed over keratinocytes - to protect them from UV - without this they receive 4X DNA damage
- People with red hair compared to dark brown/black hair - 4-fold increase in melanoma
34
How are stem cells protected from DNA damage?
- Their **physical location** (anatomical barrier) in tissue is far from contact with mutagen
- **Infrequent division**
- Via **transit-amplifying cells** - that divide rapidly before differentiating - so that damage occured at this stage is flushed out before differentiation
- Stem cells more readily undergo **apoptosis** - upon DNA damage - some cancer stem cells resistant to apoptosis
- Express high levels of **Mrd1 pump** - that actively pumps out mutagenic compounds
- **Asymmetric Replication mechanism** - newly replicated strand is alloctaed to daughter cell - unreplicated parent strand is allocated to stem cell
35
How are stem cells protected in gastrointestinal crypts?
- Layer of mucous in crypts - protects from mutagens
- Stem cells spawn large no. of transit amplifying cells which divide every 12 hours - then produce enterocytes
- Enterocytes apoptose after 5-7 days
- Lgr5 = marker for stem cells - Lgr5-GTP transgene in mice
36
How do stem cells re-populate?
- Conversion of transit-amplifying cell back to a stem cell
- Convert from asymetric division to symmetric division
37
How was it discovered that stem cells can be targets for mutagenesis in cancer?
- Evidence - cell exposed to initiator (mutagen) - and cell 'remembered' the event - suggesting it must be a long-lived cell - e.g., stem cell
- Treatment with (5-FU - that kills cycling cells) did not prevent cancer progression - it must be cell that divides infrequently
- CML - translocation in common progenitor cell
- Transit-amplifying cell can be targeted - they will carry mutation back to stem cell - cytotoxic substances could be carcinogenic via this mechanism
38
How does stem cells asymmetric replication mechanism protect them?
- Conserved strand is always passed onto the new stem cell at cell division
- The same conserved strand is always used as template for DNA replication
- Prevents transit-amplifying cells with mutations being passed down to differentiating cells during cell replication
39
What may happen if stem cell population is depleted?
- Remaining stem cells may undergo symmetric divison - where a recently synthesised strand is used as conserved strand
- Here - a mutation in newly synthesised strand could be introduced into the stem cell compartment
40
How can enzymatic processes protect DNA from mutagens?
- Superoxide dismutase, catalase - detoxify ROS
- Vit C ,Vit E, Bilirubin, urate - free radical scavengers that detoxify ROS
- **Glutathionine-S-transferase (GST)** - links electrophilic compounds with glutathionine - detoxifies them
41
How can loss of Glutathionine S-transferase (GST) affect cancer susceptibility?
- Loss of GST - **increases susceptibility** to mutagenesis - early in tumourigenesis
- Common in **prostate cancer** - 90% have shutdown GST
- **GSTT1/GSTM1** in myelodyplasia (MDS) - bone marrow - have null genes - T1 enzyme detoxifies compounds - provokes MDS
42
How can an individual cancer risk differ due to enzymatic differences?
Difference in:
- **Xenobiotic detoxification**- enzymes that inadvertantly convert otherwise non-reactive compounds into chemically reactive mutagens (CYPs;NAT1 - breast adenomas) during detoxification
- Enzymes that detoxify **mutagenic compounds** (e.g., GSTs - lung cancer)
43
How can GSTT1/GSTM1 affect effectiveness of chemotherapy?
- Enzymes that detoxify xenobiotics - can also act as to detoxify chemotherapy drugs
- If GSTT1/GSTM1 alleles were active - not effectively treated; if null - effectively treated
- Thus - genetic defects can lead to increased cancer risk - but can be beneficial for chemotherapeutic treatment as a result
44
How are DNA replication errors repaired?
- Base changes and replication slippage - proofreading activity and MMR
- DSBs at replication forks - HDR (HR) and NHEJ
45
How is endogenous chemical damage to DNA corrected?
Endogenous chemical damage
e.g., Hydrolytic damage - depurination, depyrimidation, deamination
- Oxidative damage - base modification
- Aberrant DNA methylation
**- BER and direct repair**
46
How is endogenous enzymatic damage to DNA repaired?
Endogenous enzymatic DNA damage - e.g., cytosine deaminases
**- BER**
47
How is exogenous damage repaired?
- Ionising radiation (x-rays) - DSBs -**HDR and NHEJ**
- Non-ionising radiation (UV) - **NER** - if there is adduct on DNA
- Chemicals (alkylation - large adducts) - **NER; direct repair**
48
What is direct repair, and how is it achieved with MGMT?
- Simplest form of repair - restores normal base structure
- **MGMT flips damaged base** out of double-helix - then removes alkyl group
- **Stoichiometric** reaction - C145 becomes irreversibly alkylated
- If not repaired - can lead to G to A mutation
49
Why is MGMT particularly relevant in cancer?
- Mammalian cells only express a single MGMT enzyme - but highly expressed in embyros
- MGMT is relevant in determining tissue-specific susceptibility to cancer in mouse models
- Promoter methylation silences MGMT
- Alkylating agents used as chemotherapies are much more effective in patients where MGMT is silenced - no direct repair
50
Where is direct removal of large adducts important, and how does it work?
- In inflammed tissue - formed by inadvertant oxidation if unsaturated lipids - produce highly reactive lipid - expoayldehydes and peroxides
- Found in ulceratice colitis
- Adducts are removed by hABH2 - homolog of AlkB enzyme
- hABH2 also removes methyl and ethyl groups
51
How do BER and MMR differ?
- BER generally detects smaller regions, detecting altered bases - from **endogenous sources** - e.g., ROS and depurination
- MMR corrects errors arising during **DNA replication**
52
How do the DNA glycosylases used in BER compare?
DNA glycosylases recognise specific abnormal base in BER - cleaves base
- **Uracil DNA N-glycosylase**- (U arising from C deamination) - efficient
- **T:G glycosylase** - arising from deamination of 5me-C - less efficient glycosylase
- **8-oxo-G DNA glycosylase** (OGG1) recognises oxoG - IMPORTANT - if not corrected = copied by translesion polymerase - mutation
53
Difference between short and long patch BER?
**Short patch:** gap is filled by DNA polymerase-β and ligated (polymerase lacks proofreading)
**Long patch:** single base is inserted by DNA polymerase-β and extended by DNA polymerase δ or ε - displaced bit of DNA is cleaved and the join is ligated
54
What is NER used to repair?
- Repairs much bulkier lesions from exogenous agents - e.g., UV/chemical carcinogens
55
What are the two pathways in NER?
Multisubunit complex - 2 pathways:
1. **Transcription-coupled repair** - when RNA polymerase is stalled at site of DNA damage
- Preferntial/more efficient when damaged bases are found in transcribed genes - template strand is corrected
2. **Global genomic repair (GGR)** - Repairs damage regardless of its location in transcribed or non-transcribed regions
56
What is a mutational signature to show that transcription-coupled repair has been operative in a region?
- If a region has lots more mutations in non-template strand - it indicated that transcription coupled repair has been operative - mutational signature
57
What do error-prone polymerases do?
- DNA replication through a 'still-unrepaired' stretch of template strand DNA
- Distributive enzymes - they dissociate from the DNA template after incorporation of a few nucleotides - have different degrees of accuracy over diff regions
They can:
- Add nucleotides
- Extend nascent DNA- using primer
- Incorporate a base oppposite a bulky adduct that has not been removed
58
Where are Error-prone polymerases particularly relevant - why?
- In UV-induced DNA damage
- T-T dimers are generally repaired accurately
XPV gene product
- Recognises TT dimers and inserts AA on opposite strand - but misincorporates CC - leading to C to T transitions
- Mutational signature - UV exposure - characterised by CC to TT mutations
59
What does polymerase κ do?
- Replicates past bulky adducts through templates containing 8-oxo-dG
- Incorporates A more often than C opposite 8-oxo-dG
- Therefore - is critical to remove 8-oxo-dG by BER before translesion DNA synthesis (TLS) machinery can get to it
60
Why is DNA polymerase β relevant?
- Is overexpressed in some cancers - Ovarian carcinoma
- Replaces nucleotides removed during BER
- Lacks proofreading
61
How are errors in DNA replication mitigated and corrected?
**Proofreading** - mechanism to ensure accuracy
**Mismatch Repair (MMR)** - mechanism to correct any errors that are missed by proofreading
62
Where do cancers with mutations in proofreading/MMR tend to be found?
In highly proliferative tissue - lots of DNA replication
63
Which DNA polymerases have/don't have proofreading activity? And what is their misincorporation rate?
Polymerase α (primase) - no proofreading activity
Polymerases ε and δ (leading/lagging strands) have efficient proofreading
- **Proofreading** overlooks ~ 1 in 100 misincorporated bases
- **DNA copying error** rate - 1 in 10^5
= overall error rate of DNA synthesis - 1 in 10^7
64
What is microsatellite instability?
Microsatellites are very short repeats
- Microsatelltile instability is **replication slippage** at microsatellites
65
How can DSBs occur during DNA replication? And how many?
10 DSBs per cell genome - each passage through S phase
Occurs near replication fork - **replication stress** = replication fork collapse
- Chemically-modified bases - may cause polymerase stalling
- Over-expression of oncogenes - may cause replication stress
66
How was the importance of proofreading initially studied and what are the associated genes in humans?
- **In mice:** Homozygote mutant polymerase δ mice = higher incidence
- In humans: identifed mutations in genes POLE/POLD1 (encoding polymerase ε or δ) as germline variants = higher penetrance
- POLD1 = endometrial cancer
67
What does high penetrance mean?
Means that individuals with the mutant allele have a high chance of developing the associated disease
68
What is a mutator phenotype and when would you expect to develop one?
Presence of lots of mutations scattered throughout the genome - e.g., what you'd expect if mutations in proofreading - lots of base substitution mutations
- Highly proliferative tissues most susceptible - lots of DNA replication
- Another **mutational signature**
69
How do POLE/POLD1 mutations differ?
**POLE:** catalytic subunit of DNA polymerase ε
- Leading strand
- Proofreading activity - exonuclease domain
- Mutations - exonuclease domain
**POLD1:** catalytic subunit of DNA polymerase δ
- Lagging strand
- Participates in MMR/BER
- Mutations to exonuclease domain
70
What is the function of MMR?
**MMR - Mismatch Repair**
Proofreading isn't foolproof - so: MMR:
- Fixes errors missed by proofreading
- Corrects substitution errors and small replication slippages
- Carried out by three types of protein dimer
- ONLY fixes DNA replication errors - not errors independent of DNA replication - e.g., deamination of 5me-C (BER)
71
How does MMR distinguish each strand?
- Recognises more nicks - that are common on freshly-synthesied strand
- Undermethylation of new strand - compared to template strand
72
How does MMR work?
- MSH6/MSH2 scans for mismatch
- MLH1/PSM2 scans for nicks
- Triggers degradation of this strand back through nick
- Allows for repair DNA synthesis to follow
73
Where is MMR particularly important and what is its relevance in human cancer?
- At **microsatellites**
- Defective MMR causes microsatellite instability - MIN/SI (small insertions/deletions at these repeats)
- Base substitutions, expansions and contractions - mutational signature
- Mutation rate is 1000x in cells with defective MMR machinery
- **Lynch syndrome** (colon cancer) - defective MMR - accelerated cancer progression - mutations accumulate more rapidly
- Mainly mutations in MSH2 and MLH1 but some in MSH6 and PMS2
74
How can defective MMR be detected?
- By analysing a selection of standard microsatellite DNA markers
- Look for distribution of peaks that have moved compare to normal situation - whether repeat has changed in length or not
75
How are MMR defects typically caused in sporadic cancers?
- Caused by point mutation or epigenetic silencing (methylation) of **MLH1** gene
- MLH1 silencing often occurs early in colon/endometrial cancers
- E.g., TGF-β receptor - inhibits colorectal cell proliferation - cancer cells become resistant to TGF-β so grow too rapidly (MMR defects)
76
How can genes affected by microsatellite instability be analysed?
**Analysed**:
- Patterns of single-nucleotide variations in MMR and proofreading pathways
- Genomic distribution and sequence properties of affected microsatellites
- Catalog of genomic loci with frequent MSI
- Microsatellites that were frequently affected by MSI events in many individual tumours - these are likely to represent driver genes
77
What did MSI analysis show about the types of mutations associated with MMR genes?
That diff MMR genes were inactivated by diff mechanisms:
- **MLH1** - promoter methylation - epigenetic - Plays **Primary role** in MSI phenotype
- MSH3/MSH6 - frameshifting DNA slippage - MSI events - most likely a victim of MLH1 inactivation/ point mutation - complementary mechanism
78
What did proofreading analysis show about POLE mutations?
- Large no. of point mutations (SNV) - POLE (polymerase ε)
- But POLE-mutated genomes with functional MMR **did not have microstatellite instability (MSI)**
- Some genomes had mutations in POLE & MLH1 - but did **not have high single-nucleotide variation (SNV)**- suggesting POLE mutations are acquired **later** in tumourigenesis
- Most of inactivated MMR genes - had POLE mutations - suggesting **defective proofreading led to accumulation of mutations in MMR genes**
- **Cascade** - loss of function of one gene can exacerbate situation - cause loss of function in another gene
79
What did analysis of MSI loci suggest about frameshift events?
- Frameshifts in coding genes - disrupt gene function
- Highly recurrent MSI events - predicted to contribute to cancer - rather than 'bystander' events - hence - most likely to be suppressing a tumour suppressor gene - higher **frameshift-to-inframe ratio - selective advantage - positive selection**
- Non-recurrent MSI events in coding regions - less likely to represent frameshift MSI events than ones found in non-coding regions
- I.e. negative selection of frameshifting MSI events on coding sequences
80
What was used to map a tumour genome?
Massively parallel pyrosequencing - (high throughput sequencing)
- Compare reference genome against tumour genome
- Short sequence reads - shorter than Sanger
81
What mutations were found in the melanoma tumour genome?
- BRAFL597R
- Heterozygous mutant
82
Features of malignant melanoma?
- Highly aggressive
- Resistant to chemotherapy
- Caused by UV - melanin
83
What was discovered about the genes/pathway affecting melanoma?
- BRAF - primary gene in tumours (RAF family)
- RAS-RAF-MEK-MAP kinase pathway
- Since - shown that 80% of melanomas have **mutually exclusive** activating mutations in BRAF and NRAS
- Mutant BRAF had increased kinase activity - ERK1/ERK2
- Mutant BRAF induced transformation of MIH3T3 cells
- BRAF mutations happen **early** in melanoma development
- Most mutations are in activation segment (AS) - **V600E mutations**
84
What selective inhibitor was used to treat melanoma?
- Selective inhibitor of oncogenic B-Raf kinase (BRAF inhibitor)
- PLX4720 (vemurafenib) - used to treat patients with BRAFV600E mutation
85
What is the RAF inhibitor Paradox?
BRAF inhibitors **accelerated** cancer progression in patients that did not have the BRAFV600E mutation
- V600E RAF inhibitors are only effective in melanomas that have the V600E mutation
86
What RAF mechanisms were uncovered to investigate the RAF inhibitor paradox?
- Wild-type RAF functions as a **dimer**
- B-RAF monomer will be in an **inactive closed conformation -** where the N-terminus inhibits its catalytic C-terminus
- Recruitment of BRAF to RAS-CTP - leads to **altered conformation** in the BRAF N-terminus
- RAS binding mediates the **dimerisation** of BRAF (with itself of CRAF)
- NtA domain of BRAF **'transactivates' CRAF**
- Transactivation results in phosphorylation of activation loop of CRAF - producing an actove CRAF kinase that can phosphorylate MEK
- **CRITICALLY** - activation of wild-type RAF requires **dimerisation** - which is dependent on RAS interaction
87
How does the V600E mutation affect the function of RAF?
- V600E mutation **mimics phosphorylation** of the **activation loop** of the kinase domain
- Therefore - this mutant form of BRAF is **constiuitively active** and **independent of dimerisation** - and **independent of Ras signalling**
- Thus, **critically** - BRAF mutant can act as a **monomer**
- Thus, high levels of signalling from active mutant BRAFV600E - leads to high levels of feedback inhibition & very low levels of active Ras
- As levels of Ras are low - mutant BRAFV600E is not recruited to RAS - and therefore doesn't dimerise - hence functioning as monomer
88
How does the selective inhibitor for BRAF work?
BRAFV600E inhibitor binds to monomeric mutant BRAF - and blocks its activity
89
How can RAF inhibitors lead to activation of dimeric RAF?
- Inhibitor can bind to a dimeric subunit of RAF (wt or mutant) - it inhibits the subunit to which it binds - but activates the other subunit - **allosterically activates**
- BRAF inhibitors - therefore lead to **increased downstream signalling** and accelerated cell proliferation in any setting that is associated with an increase in **RAF dimers**
- Thus regions with RAS mutations (and consequently high BRAF dimers) - will be stimulated by BRAF inhibitors
- However, signalling from mutant RAS is **independent** of upstream growth factor signalling - and is therefore **not affected by feedback inhibiton**
90
When given inhibitor, how are the effects different for BRAF/RAS mutations?
- BRAF - Inhibitor **reduced** phosphorylation of substrates - inhibition
- RAS mutant - **increase** in phosphorylation - independent of growth factor signalling
**Monomeric V600E RAF is inhibited but dimeric RAF forms are activated by the inhibitor**
91
How can the resistance to RAF inhibitors increase?
By increase dimeric forms of RAF: 3 ways:
1. **RAS activation** (activating NRAS mutation) increases RAF dimerisation - constituitively active RAS is independent of upstream signalling and feedback inhibition
2. **Mutant BRAFV600E** may mutate to acquire **splice variants** enhanced ability to dimerise - BRAF dimerization is independent of upstream signalling from RAS
3. **CRAF Overexpression** - enhancing dimerisation and downstream signalling in presence of BRAFV600E inhibitor - BRAF dimerization is independent of upstream RAS signalling
92
What are paradox breaker drugs and how do they work?
Aim to **uncouple effects** of inhibitor on monomeric and dimeric forms of BRAFV600E
- Paradox breaker inhibitors **weaken/disrupt the RAF dimerisation interface**
- Interaction of **PLX7904 and L505** moves the αC helix of RAF - causing disruption to RAF dimer interface
- So paradox-breaker inhibitors do not activate RAF in RAS mutant cells because inhibitors do not allow for RAF dimerisation
93
How do first generation and second generation (paradox breaker) inhibitors compare?
First generation - **enhance** RAF dimerisation
Second generation (paradox breaker) inhibitors (PLX7904) - **do not enhance** RAF dimerisation
- These also overcome first generation RAF inhibitor resistance
94
How has it shown to be possible to engineer first generation RAF inhibitors into paradox breakers?
- By modifying tail - to engineer PLX7904 onto first generation inhibitor
- Elegant study - Zhang et al., 2015
95
How are wild-type/mutant RAF different?
Wild-type RAF - functions as dimer
Mutant RAF - functions as monomer - is why first generation RAF inhibitors work - but activate dimeric form
96
What type of genes are good/bad for analysing mutational signatures?
- GOOD: **Passenger genes**- because they reflect an **unbiased representation**of that mutagen
- BAD: **driver genes**- are under positive selection - **bias** representation - found in regions that will encode the critical AAs
97
How was p53 used to find a mutational signature for tobacco smoke?
- p53 mutations associated with smoking carried more **G to T transversions** than p53 mutations found in other cancers
- Showed that G to T transversions were a '**signature**' of exposure to carcinogens derived from tobacco smoke
98
What is the mutational signature for UV exposure?
**CC to TT mutations & fewer mutations on transcribed strand (TC-NER)**
- UV-induced pyrimidine dimers are repaired via NER - but if not 100% effective - C to T transitions arise due to activity of error-prone polymerases copying across CC dimers - (**TC-NER**)
- So C to T and **CC to TT mutations** are frequent due to UV is high at **CpG dinucleotides**
- Becasue of TC-NER - stalling of DNA polymerase when encountering bulky adduct - the transcribed strand is repaired more efficiently than damage on the non-transcribed strand
- Therefore, another signature is: **fewer mutations on the transcribed strand**
99
What do different numbers of mutations on the transcribed vs non-transcribed strands tell us?
That **transition-coupled nucleotide excision repair** has been used - which is an indication of an **exogenous mutagen**
100
What enzymes play a role in deaminating cytosine to Uracil?
**AID** - off-target chromosomal deamination leads to B-cell tumourigenesis?
**APOBEC3B** - leads to doubling of C to T base changes
101
Features of APOBEC3
- Expressed in many human cancer cell lines
- Only deaminase family member with **constituitive nuclear localisation**
- **General mutagenic factor** in many human cancers
- Overexpression of APOBEC3 - had many **C to T transitions** - deamination of C to U can lead to the insertion of A opposite U during DNA replication - sometimes U is clipped out by DNA glycosylase and error-prone polymerase inserts wrong base opposite
- Mutations were mainly at **TCA motifs** - and were clustered - lots together
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Name 4 different mutational signatures - examples
**Exogenous/endogenous mutagen** - e.g., tocacco smoke; UV light; base deamination
'Normal' intrinsic infidelity of **replication machinery** - e.g., polymerase errors overlooked by proofreading
**Enzymatic modifications** of DNA e.g., APOBEC3 mutagenesis
**Defective proofreading and DNA repair** - e.g., MMR; NER
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What does each mutational signature relate to? And what is signature 1
A distinct biological process - e.g., process 1 leads to signature 1
Signature 1: derives from spontaneous deamination of 5me-C - associated with C to T transitions in context of CpGs
- Total no. of signature 1 increases with age
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How can mutational signatures change through life?
- Some can be constant and 'weak' throughout life - e.g., deamination of 5meC
- Others can be high at specific stages - e.g., exogenous agents - tobacco smoke, UV
- Others may be operational at various times - but at more moderate levels - e.g., APOBEC3
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What two mutational signatures are associated with APOBEC3?
Signature 2: TCA > TTA (C to T transitions)
Signature 13: TCA > TGA (reflects activity of error-prone translesion polymerase)
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What is signature 7 associated with?
Results from UV-induced DNA damage and repair
- Lots of C to T transitions in dinucleotide contexts (CC to TT)
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What signatures are associated with defective MMR?
Signatures 6 and 15
- Lots of small deletions and insertions
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What signatures are associated with tobacco smoke?
Signatures 2, 5, 13 and 16
- Signature 4 - only found in cancers with direct exposure to smoke (C>A/G>T base substitutions)
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What suggests a high load of passenger mutations?
High number of synonymous point mutations in coding regions
- i.e. mutations that do not change the encoded AA
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Why can you expect different no./rates of mutations in different regions?
- Is a reflection of chromatin organisation and access to DNA repair machinery
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How did they analyse whether genes were under selection (drivers)
- Use normal distribution to determine a 'normal' amount of nonsilent mutations in that region
- Compare this with how many nonsilent mutations were actually found
- If this was significantly higher (using p-value) then it suggests this is highly unlikely to be by chance - and that this region is under selection - drivers
**- Observed > expected**
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What novel driver gene in melanoma was discovered via genome-wide analysis, and where did its mutations lie?
**RAC1** - GTPase - involved in regulation of cellular adhesion and migration
- Mutations were located in a **hotspot** - **gain-of-function** - **oncogene**
- P29S mutation stabilises inactive GDP-bound state - and favours active GTP-bound state
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What does it suggest if mutations are located in a hotspot?
- **Hotspot** - i.e. many tumours had mutations in the same place
- Strong indication that these mutations are **gain-of-function** (very few ways in which any given gene can become activated)
- And that it is an **oncogene**
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How can tumour suppressor genes (TSGs) be inactivated?
- Mutations in TSGs result in **protein truncation** - have higher likelihood of conferring a fitness advantage than missense mutations in same gene
- E.g., loss-of-function mutations (for TSGs) can be **nonsense**, **splice-site** and **frameshifts** - are associated with a higher loss-of-function burden than expected by chance
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What TSGs were identified in melanoma?
- p16^INK4a and ARID2
- **ARID2** - componenet of chromatin-remodelling complex - (first mutated chromatin remodelling in melanoma)
- Then carried our 'targeted search' to look for loss-of-function mutations in other chromatin remodelling components in melanoma
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What is the most frequently mutated chromatin remodeller in human cancer and what was discovered about them?
**SWI/SNF** remodeller - >20% human cancer
- SWI/SNF mutations had very few other genetic mutations - suggests SWI/SNF mutations gave a strong selective advantage for cancer initiation/progression
- **Both loss/gain-of function** mutations found
- Loss-of-function - implementation; gain-of-function - regulation (sliding activity - accessibility)
- **Pleiotropic effects** - mutations in chromatin remodelling here - has knock-on effects on many other genes - as chromatin remodelling is critical for gene regulation
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What was found about the top 15 known driver mutations in cancer genomes?
- Of the 15 driver mutations - only mutations found in promoter regions was **TERT mutations**
- The rest were in protein-coding regions
- Also showed that p53 (TSG) had many sites of mutations, whereas KRAS/BRAF (oncogenes) mutations were all in same hotspot
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How do TSG mutations and Oncogenic mutations differ?
- TSGs have many sites of mutations
- Oncogenes tend to all be located in a hotspot
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What did they conclude about how pathways are affected by mutations - from analysis on coding/non-coding driver genes?
- Chromatin remodelling and proliferation - coding mutations
- Developmental pathways - coding and noncoding mutations
- Splicing - mainly non-coding - (new found non-coding drivers)
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How were tumours first classified?
**By no. of alterations - deletions/duplications in specific regions of** chromosome
- 4 clusters - cluster 1: few copy no. alterations; cluster 4 - lots of copy no. alterations - cluster 2/3 on between
- Cluster 1, 2, 3 - were endometroid tumours
- Cluster 4 - contains most serous tumours - agressive (12% of endometroid)
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What are endometroid cancers? Features
Cancers resting in the endometrium
- Much rarer (10% of tumours) - more agressive = type 2
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What about endometroid tumours did not correlate with traditional tumour histology/grade?
- Subset of endometroid tumours contain **distinct patterns of somatic copy number alterations** (SCNAs) that **do not correlate** with traditional tumour histology/grade
- But resemble those of serous tumours - so need to be treated accordingly
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What are the characteristics of tumours with POLE (proofreading) mutations?
- **Very many mutations** - littered throughout genome (ultramutated)
- Tumours did **NOT** have large copy-number alterations - and were in endometroid classification
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What are the characteristics of tumours with MRR defects (microsatellite instability (MSI) mutations?
- Large no. of **point mutations** (not as many as POLE)
- Characterised by microsatellite instability (MSI) and epigenetic silencing (DNA methylation of MLH1)
- Most tumours did **not** have large no. of copy-number alterations
- Were in endometroid classification
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What are the characteristics of tumours with POLE and MSI mutations?
- Had mutations in tumour suppressor PTEN
- But most had wild-type p53
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What did tumours with high copy-number alterations show?
- Had **fewer point mutations** and **normal proofreading/MMR**
- But were characterised by **p53 mutations**
**Critically** - although most of these tumours had been classified as being 'serous' histology - some were 'endometroid' - posssible that these represent patients who would originally be classified as having 'type 1' cancer (with good prognosis) but whose cancer would in fact return - (i.e. not all serous histology)
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What suggested the possibility of using overlapping treatments for different tumour types?
- Tumours showing similar patterns of **copy number alterations**
- And similar patterns of mutation of key driver genes
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What was the aim of the experiment invesitgating kidney cancer and what did they find?
Mutation A: Find genes involved in conversion of normal tissue to cancer tissue (early)
Mutation B, C and D: Genes associated with progression of primary tumour but not metastasis (in different regions of tumour)
Mutation F: genes associated with metastasis
- Found region 4 of primary tumour - appeared to represent two clonal populations - (with mutant 'green' & 'yellow' genes) - metastasis appeared out of 'green' genes - then mapped these genes onto pathway
- Noted that some TSGs were inactivated by diff mechanisms in diff regions of primary tumour and in diff metastases - example of **convergent evolution** - leading to loss of function
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What did deep sequencing with CLL show?
Deep sequencing - comparison of mutations in individual reads
- Mutations placed along timecourse of tumour dev - and diff mutations processes are 'fitted' to data
- Detects major events in tumour evolution - e.g., transitions into a new subclone
- E.g., CLL - initially signature 9 - somatic hypermutation mutations responsible; later on signature 5 (unknown process) was more predominant
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What did analysis of the mutational landscape of normal tissues show?
In colon, endometrial, skin and oesophagus:
- Many glands represented **clonal populations** that contained mutations in **driver** cancer genes
- So - many of our normal tissues are riddled with clonal expansions of driver mutations
- But normal endometrial glands were **not** characterised by defects in proofreading/MMR, MSI or copy-no. alteratioins (which are frequently found in endometrial cancer)
- So these processes are what lead to full blown cancer
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What are copy-number alterations?
- Large deletions of specific regions on chromosomes
