Lecture 2. Deregulating the Drivers of Cancer

Question 1

Describe how oncogenes can be activated

Answer 1

1. Point mutations in coding sequence can alter activity e.g. point mutns lock Ras in GTP-bound active form, resulting in constitutive signals that promote development of colon cancer
2. Chromosomal rearrangements can introduce aberrant control elements causing abnormal expression e.g. in Burkitt’s lymphoma, an Ig enhancer becomes positioned near MYC gene, raising its expression in B cells
3. Chromosomal amplifications can increase copy number & hence expression levels e.g. MYC gene is frequently amplified in breast cancers
4. Elevated expression can result from changes to upstream regulators e.g. MYC gene is induced in colon cancers by b-catenin, following loss of APC

Question 2

Describe how tumour suppressors are inactivated

Answer 2

1. Tumour suppressor genes are often silenced by epigenetic regulation e.g. MMR genes are frequently silenced by DNA methylation in colon cancer
2. Chromosomal rearrangements can delete part or all of a tumour suppressor gene e.g. APC gene is commonly deleted in colon cancers
3. Point mutations in coding sequence can alter activity
   e. g. point mutns in p53 are common in many cancer types

Question 3

Describe how p53 mutations arise

Answer 3

P53 gene is deleted or mutated in >50% of human cancers – highest frequency of any known gene
E.g. p53 is mutated in >90% of oesophageal cancers
In normal oesophagus from organ donors, 5-35% of epithelial cells have p53 mutations
Most p53 mutns arise somatically, but germ-line mutns occur in some cancer-prone families - Li-Fraumeni syndrome
Most p53 mutns are missense substitutions – deletions are less common than for other tumour suppressor genes
Substitutions generally occur within DNA-binding domain of p53 & certain residues are hot-spots

Missense mutns may confer oncogenic activity
Mouse models & Li-Fraumeni syndrome patients with missense mutations in p53 have earlier cancer onset than p53 nulls
These point mutations are selected for – not only do they inactivate p53 but they confer oncogenic activity
No p53 preferable no missense p53 in some cases

Question 4

How is mutant p53 destabilised?

Answer 4

Missense mutns can alter p53 conformation & expose hydrophobic residues – such mutants are stabilised by the chaperone protein hsp90
Hsp90 inhibitors have been developed that destabilise mutant p53 proteins E.g. ganetespib is an hsp90 inhibitor in advanced clinical trials for lung cancer
Exposing hydrophobic residues compromises stability of protein
Activity of ganetespib sensitive to status of p53
Ganetespib improves survival of mice with mutant p53 but not of p53 null mice

Question 5

Describe the oncogenic activity of mutant p53

Answer 5

mutant p53 may have oncogenic effects through more than one mechanism
p53 forms tetramers (pairs of dimers) – dominant negative effects occur when mutant monomers bind & inactivate WT monomers in heterozygous cells
Gain of function by missense mutants can also result in oncogenic effects
E.g. some oncogenic p53 mutants can bind txn factor ETS2 & induce its target genes e.g. MLL1 – WT p53 does not interact with ETS2 or its targets
induction of MLL1 seems important for oncogenic activity of p53 gain-of-function mutants – results in aberrant transcription patterns
P53 mutant has exposed residues that allow it to make interactions – eg with txn factor ETS2
Can recruit co-activators and activate genes that are not activated by wt p53
MLL1 important in promoter recognition
cPG islands CG dinucleotides recognised by MLL1

Question 6

Describe why MLL1 is an Important Epigenetic Regulator

Answer 6

Most vertebrate promoters lie within ~1-2kb regions
enriched for CG dinucleotides - “CpG Islands”
Unmethylated CpG is recognised by MLL1 & MLL2 histone methyltransferases in a complex called COMPASS
MLL1 & MLL2 trimethylate lysine 4 of histone H3 – marks promoters as active
As well as binding unmethylated CpG DNA, MLL1 & 2 bind H3K4me3 on histone tails – allows mark to spread to adjacent nucleosomes

Question 7

Describe how H3K4me3 Marks Active Promoters

Answer 7

H3K4me3 is recognized by TAF3 subunit of TFIID
TFIID is a complex of TATA-binding protein (TBP) and TBP-associated factors (TAFs)
TFIID recruitment to promoters begins assembly of transcription complex & is often the rate-limiting step in gene activation

Question 8

Describe how TFIID Recognizes Acetylated Lysines

Answer 8

Acetylated lysines are associated with active promoters & are recognised by bromodomains
TAF1 Subunit of TFIID has a double bromodomain allowing simultaneous recognition of two close acetylated lysines - increases specificity

Question 9

Describe how several subunits of TFIID Contribute to Promoter Recognition

Answer 9

TBP binds TATA box DNA
TAF3 binds H3K4me3
TAF1 binds diacetylated histone H4 and initiator (Inr) DNA
TAF6 binds downstream promoter element (DPE) DNA
Provides versatility for different promoter arrangements e.g. TATA-less promoters
No single feature defines all promoters

Question 10

Describe how HDAC inhibitors are used as epigenetic therapy

Answer 10

Histone acetylation promotes gene txn - many txn activators recruit histone acetyltransferases (HATs) to promoters
As well as attracting proteins with bromodomains, acetylation removes positive charge of lysine side chains & so weakens histone binding to negatively-charged DNA - this increases accessibility
histone deacetylases (HDACs) reduce promoter accessibility & inhibit txn
HDAC inhibitors raise histone acetylation & can induce silent tumour suppressor genes E.g. vorinostat, which occupies HDAC active site & blocks substrate access 
Vorinostat is used to treat cutaneous T-cell lymphoma & is in clinical trials for other cancer types

Many txn activators function by recruiting HATs
This is a mark to recruit proteins with bromodomains eg TFIID
Has effect of opening up chromatin
HDACs often used by proteins wanting to repress gene transcription – this is one route of silencing tumour suppressors

Question 11

Describe how DNMT inhibitors are used as epigenetic therapy

Answer 11

First approved epigenetic drug was 5-azacytidine, licensed in 2004 for patients with myelodysplastic syndromes (MDS), cancers where blood cells fail to mature
5-azacytidine is a nucleoside analogue that is incorporated into DNA
Inactivates DNA methyltransferase (DNMT) enzymes responsible for methylating DNA
DNA methylation can silence genes by blocking binding of txn factors e.g. MLL1
5-azacytidine can restore expression of methylated tumour suppressor genes
changes expression of many genes – toxicity problems
Improves survival of MDS patients, but has poor penetration of solid tumours
Problems of toxicity and penetration of solid tumours – works better on blood cancers

Question 12

Describe how EZH2 is used as an epigenetic regulator

Answer 12

Inhibitors of HDACs & DNMTs are the only epigenetic drugs that are currently licensed for routine use
Many clinical trials are ongoing for inhibitors of other epigenetic regulators e.g. EZH2
EZH2 trimethylates histone H3 at lysine 27 (H3K27), a mark of inactive heterochromatin
Methylation of lysine side-chains blocks their acetylation
H3K27me3 stimulates EZH2 activity, allowing spread of mark across adjacent nucleosomes
EZH2 can also recruit HDACs & DNMTs, bolstering repression
Involved in silencing genes on inactive X chromosome in females
Overexpressed in several cancer types e.g. prostate metastases
Prostate cancer patients with low EZH2 levels have less chance of fatal relapse after surgery – suggests an oncogenic role

In lymphomas, EZH2 is frequently activated by catalytic domain mutns
EZH2 inhibitor drugs have been developed that eradicate human lymphomas grown in mice
EZH2 inhibitors are in clinical trials for refractory lymphomas & advanced solid tumours

Question 13

Describe how EZH2 is Antagonized by SWI/SNF Tumour Suppressor

Answer 13

EZH2 activity is antagonized by H3K27 demethylase & also by SWI/SNF, a chromatin remodelling complex
SWI/SNF can increase accessibility of DNA by sliding or ejecting nucleosomes
SWI/SNF subunit genes are mutated or deleted in ~20% of cancers
Loss of SWI/SNF subunits or H3K27 demethylase is common in bladder cancers & can result in silencing of EZH2 targets
Illustrates how different molecular changes can give the same result

Question 14

Describe how MLL1 Gene is Translocated in Mixed Lineage Leukaemias

Answer 14

in mixed lineage leukaemias, chromosomal translocations often fuse MLL1 gene to genes encoding other transcription factors

fusion products aberrantly activate txn of HOX genes & this inhibits differentiation

leukaemogenesis by MLL1 fusion proteins requires binding to COMPASS subunit menin

structural studies allowed design of small molecules that block menin binding to MLL1

inhibit aberrant HOX expression & induce differentiation of leukaemia cells in mice