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Cancer Genomes Flashcards

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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
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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
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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
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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’
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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)

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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

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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
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8
Q

Are TSG mutations found in the germline or somatic cells?

A

BOTH - Inheritance of mutant TSG results in hereditary predisposition to cancer

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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

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10
Q

What three levels can mutations occur at - and what are some examples of each?

A
  1. DNA level - indirect/directly active genes
  2. Chromatin level - e.g., histone variants, chromatin remodelling, 3-D chromatin organisation, DNA methylation machinery
  3. 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)
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11
Q

What is a mutational signature?

A
  • Imprints of specific mutagenic processes, repair mechanisms/repair mechanism defects
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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
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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
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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

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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

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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
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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
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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
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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
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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

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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
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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

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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
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24
Q

How do X-rays cause mutations?

A

X-rays (ionising radiation): generate ROS that create SS/DSBs

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25
Q

How does UV cause mutations?

A

UV: leads to formation of covalent bonds between two adjacent pyrimidines on the same strand (6-4PPs/CPDs - CC-TT changes - melanomas/p53 mutations)

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26
Q

How do alkylating agents cause mutations?

A

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

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27
Q

How does tobacco smoke cause mutations?

A

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

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28
Q

How can alcohol cause mutations - direct/indirectly?

A

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)

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29
Q

How can cooked meat cause mutations?

A

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

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30
Q

How can Aflatoxin B cause mutations?

A

Aflatoxin B (AFB1): - aspergillus mould on rice/grains - AFB1 oxidised to 8,9-oxide - adduct - liver cancer - China (humid - regional differences) - p53

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31
Q

What are CYPs?

A
  • 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
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32
Q

What are the three mechanisms for protecting the genome form attack by mutagens?

A
  • Physical shielding
  • Stem cell compartment
  • Enzymatic detoxification
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33
Q

How do melanosomes protect us?

A
  • 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
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34
Q

How are stem cells protected from DNA damage?

A
  • 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
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35
Q

How are stem cells protected in gastrointestinal crypts?

A
  • 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
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36
Q

How do stem cells re-populate?

A
  • Conversion of transit-amplifying cell back to a stem cell
  • Convert from asymetric division to symmetric division
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37
Q

How was it discovered that stem cells can be targets for mutagenesis in cancer?

A
  • 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
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38
Q

How does stem cells asymmetric replication mechanism protect them?

A
  • 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
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39
Q

What may happen if stem cell population is depleted?

A
  • 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
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40
Q

How can enzymatic processes protect DNA from mutagens?

A
  • 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
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41
Q

How can loss of Glutathionine S-transferase (GST) affect cancer susceptibility?

A
  • 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
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42
Q

How can an individual cancer risk differ due to enzymatic differences?

A

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)
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43
Q

How can GSTT1/GSTM1 affect effectiveness of chemotherapy?

A
  • 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
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44
Q

How are DNA replication errors repaired?

A
  • Base changes and replication slippage - proofreading activity and MMR
  • DSBs at replication forks - HDR (HR) and NHEJ
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45
Q

How is endogenous chemical damage to DNA corrected?

A

Endogenous chemical damage
e.g., Hydrolytic damage - depurination, depyrimidation, deamination
- Oxidative damage - base modification
- Aberrant DNA methylation

- BER and direct repair

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46
Q

How is endogenous enzymatic damage to DNA repaired?

A

Endogenous enzymatic DNA damage - e.g., cytosine deaminases
- BER

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47
Q

How is exogenous damage repaired?

A
  • 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
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48
Q

What is direct repair, and how is it achieved with MGMT?

A
  • 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
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49
Q

Why is MGMT particularly relevant in cancer?

A
  • 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
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50
Q

Where is direct removal of large adducts important, and how does it work?

A
  • 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
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51
Q

How do BER and MMR differ?

A
  • BER generally detects smaller regions, detecting altered bases - from endogenous sources - e.g., ROS and depurination
  • MMR corrects errors arising during DNA replication
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52
Q

How do the DNA glycosylases used in BER compare?

A

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

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53
Q

Difference between short and long patch BER?

A

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
Q

What is NER used to repair?

A
  • Repairs much bulkier lesions from exogenous agents - e.g., UV/chemical carcinogens
55
Q

What are the two pathways in NER?

A

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

  1. Global genomic repair (GGR) - Repairs damage regardless of its location in transcribed or non-transcribed regions
56
Q

What is a mutational signature to show that transcription-coupled repair has been operative in a region?

A
  • If a region has lots more mutations in non-template strand - it indicated that transcription coupled repair has been operative - mutational signature
57
Q

What do error-prone polymerases do?

A
  • 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
Q

Where are Error-prone polymerases particularly relevant - why?

A
  • 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
Q

What does polymerase κ do?

A
  • 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
Q

Why is DNA polymerase β relevant?

A
  • Is overexpressed in some cancers - Ovarian carcinoma
  • Replaces nucleotides removed during BER
  • Lacks proofreading
61
Q

How are errors in DNA replication mitigated and corrected?

A

Proofreading - mechanism to ensure accuracy
Mismatch Repair (MMR) - mechanism to correct any errors that are missed by proofreading

62
Q

Where do cancers with mutations in proofreading/MMR tend to be found?

A

In highly proliferative tissue - lots of DNA replication

63
Q

Which DNA polymerases have/don’t have proofreading activity? And what is their misincorporation rate?

A

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
Q

What is microsatellite instability?

A

Microsatellites are very short repeats
- Microsatelltile instability is replication slippage at microsatellites

65
Q

How can DSBs occur during DNA replication? And how many?

A

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
Q

How was the importance of proofreading initially studied and what are the associated genes in humans?

A
  • 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
Q

What does high penetrance mean?

A

Means that individuals with the mutant allele have a high chance of developing the associated disease

68
Q

What is a mutator phenotype and when would you expect to develop one?

A

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
Q

How do POLE/POLD1 mutations differ?

A

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
Q

What is the function of MMR?

A

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
Q

How does MMR distinguish each strand?

A
  • Recognises more nicks - that are common on freshly-synthesied strand
  • Undermethylation of new strand - compared to template strand
72
Q

How does MMR work?

A
  • 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
Q

Where is MMR particularly important and what is its relevance in human cancer?

A
  • 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
Q

How can defective MMR be detected?

A
  • 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
Q

How are MMR defects typically caused in sporadic cancers?

A
  • 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
Q

How can genes affected by microsatellite instability be analysed?

A

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
Q

What did MSI analysis show about the types of mutations associated with MMR genes?

A

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
Q

What did proofreading analysis show about POLE mutations?

A
  • 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
Q

What did analysis of MSI loci suggest about frameshift events?

A
  • 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
Q

What was used to map a tumour genome?

A

Massively parallel pyrosequencing - (high throughput sequencing)
- Compare reference genome against tumour genome
- Short sequence reads - shorter than Sanger

81
Q

What mutations were found in the melanoma tumour genome?

A
  • BRAFL597R
  • Heterozygous mutant
82
Q

Features of malignant melanoma?

A
  • Highly aggressive
  • Resistant to chemotherapy
  • Caused by UV - melanin
83
Q

What was discovered about the genes/pathway affecting melanoma?

A
  • 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
Q

What selective inhibitor was used to treat melanoma?

A
  • Selective inhibitor of oncogenic B-Raf kinase (BRAF inhibitor)
  • PLX4720 (vemurafenib) - used to treat patients with BRAFV600E mutation
85
Q

What is the RAF inhibitor Paradox?

A

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
Q

What RAF mechanisms were uncovered to investigate the RAF inhibitor paradox?

A
  • 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
Q

How does the V600E mutation affect the function of RAF?

A
  • 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
Q

How does the selective inhibitor for BRAF work?

A

BRAFV600E inhibitor binds to monomeric mutant BRAF - and blocks its activity

89
Q

How can RAF inhibitors lead to activation of dimeric RAF?

A
  • 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
Q

When given inhibitor, how are the effects different for BRAF/RAS mutations?

A
  • 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
Q

How can the resistance to RAF inhibitors increase?

A

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
Q

What are paradox breaker drugs and how do they work?

A

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
Q

How do first generation and second generation (paradox breaker) inhibitors compare?

A

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
Q

How has it shown to be possible to engineer first generation RAF inhibitors into paradox breakers?

A
  • By modifying tail - to engineer PLX7904 onto first generation inhibitor
  • Elegant study - Zhang et al., 2015
95
Q

How are wild-type/mutant RAF different?

A

Wild-type RAF - functions as dimer
Mutant RAF - functions as monomer - is why first generation RAF inhibitors work - but activate dimeric form

96
Q

What type of genes are good/bad for analysing mutational signatures?

A
  • GOOD: Passenger genes- because they reflect an unbiased representationof that mutagen
  • BAD: driver genes- are under positive selection - bias representation - found in regions that will encode the critical AAs
97
Q

How was p53 used to find a mutational signature for tobacco smoke?

A
  • 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
Q

What is the mutational signature for UV exposure?

A

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
Q

What do different numbers of mutations on the transcribed vs non-transcribed strands tell us?

A

That transition-coupled nucleotide excision repair has been used - which is an indication of an exogenous mutagen

100
Q

What enzymes play a role in deaminating cytosine to Uracil?

A

AID - off-target chromosomal deamination leads to B-cell tumourigenesis?
APOBEC3B - leads to doubling of C to T base changes

101
Q

Features of APOBEC3

A
  • 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
102
Q

Name 4 different mutational signatures - examples

A

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

103
Q

What does each mutational signature relate to? And what is signature 1

A

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

104
Q

How can mutational signatures change through life?

A
  • 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
105
Q

What two mutational signatures are associated with APOBEC3?

A

Signature 2: TCA > TTA (C to T transitions)

Signature 13: TCA > TGA (reflects activity of error-prone translesion polymerase)

106
Q

What is signature 7 associated with?

A

Results from UV-induced DNA damage and repair
- Lots of C to T transitions in dinucleotide contexts (CC to TT)

107
Q

What signatures are associated with defective MMR?

A

Signatures 6 and 15
- Lots of small deletions and insertions

108
Q

What signatures are associated with tobacco smoke?

A

Signatures 2, 5, 13 and 16
- Signature 4 - only found in cancers with direct exposure to smoke (C>A/G>T base substitutions)

109
Q

What suggests a high load of passenger mutations?

A

High number of synonymous point mutations in coding regions
- i.e. mutations that do not change the encoded AA

110
Q

Why can you expect different no./rates of mutations in different regions?

A
  • Is a reflection of chromatin organisation and access to DNA repair machinery
111
Q

How did they analyse whether genes were under selection (drivers)

A
  • 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
112
Q

What novel driver gene in melanoma was discovered via genome-wide analysis, and where did its mutations lie?

A

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

113
Q

What does it suggest if mutations are located in a hotspot?

A
  • 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
114
Q

How can tumour suppressor genes (TSGs) be inactivated?

A
  • 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
115
Q

What TSGs were identified in melanoma?

A
  • 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
116
Q

What is the most frequently mutated chromatin remodeller in human cancer and what was discovered about them?

A

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

117
Q

What was found about the top 15 known driver mutations in cancer genomes?

A
  • 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
118
Q

How do TSG mutations and Oncogenic mutations differ?

A
  • TSGs have many sites of mutations
  • Oncogenes tend to all be located in a hotspot
119
Q

What did they conclude about how pathways are affected by mutations - from analysis on coding/non-coding driver genes?

A
  • Chromatin remodelling and proliferation - coding mutations
  • Developmental pathways - coding and noncoding mutations
  • Splicing - mainly non-coding - (new found non-coding drivers)
120
Q

How were tumours first classified?

A

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)

121
Q

What are endometroid cancers? Features

A

Cancers resting in the endometrium
- Much rarer (10% of tumours) - more agressive = type 2

122
Q

What about endometroid tumours did not correlate with traditional tumour histology/grade?

A
  • 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
123
Q

What are the characteristics of tumours with POLE (proofreading) mutations?

A
  • Very many mutations - littered throughout genome (ultramutated)
  • Tumours did NOT have large copy-number alterations - and were in endometroid classification
124
Q

What are the characteristics of tumours with MRR defects (microsatellite instability (MSI) mutations?

A
  • 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
125
Q

What are the characteristics of tumours with POLE and MSI mutations?

A
  • Had mutations in tumour suppressor PTEN
  • But most had wild-type p53
126
Q

What did tumours with high copy-number alterations show?

A
  • 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)

127
Q

What suggested the possibility of using overlapping treatments for different tumour types?

A
  • Tumours showing similar patterns of copy number alterations
  • And similar patterns of mutation of key driver genes
128
Q

What was the aim of the experiment invesitgating kidney cancer and what did they find?

A

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
129
Q

What did deep sequencing with CLL show?

A

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

130
Q

What did analysis of the mutational landscape of normal tissues show?

A

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
131
Q

What are copy-number alterations?

A
  • Large deletions of specific regions on chromosomes

Advanced Genetics (4 decks)

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