Lecture 5 - MYC Flashcards
Describe how MYC Can Be Induced in Several Ways
MYC (aka c-Myc) is probably the most commonly induced oncogene
MYC gene is frequently amplified in many cancer types, e.g. liver cancer
Burkitt lymphoma is characterised by chromosomal translocations that place MYC gene near an Ig enhancer
MYC gene transcription is stimulated by upstream activators in many cancers e.g. colon cancer, where APC loss allows constitutive actn of b-catenin, which induces MYC gene
MYC protein is stabilised by ERK-mediated phosphorylation e.g. Ras mutn activates MAP kinase pathway, leading to phosphorylation & stabilization of MYC protein by ERK
What is MYC?
MYC is a txn factor
functions as an obligate heterodimer with MAX, using a basic/helix-loop-helix/leucine zipper (bHLHZip) dimerization & DNA binding domain
Can stimulate txn by pols I, II & III
Stimulates pol II pre-initiation complex assembly by recruiting TBP, TFIIH & HATs
Stimulates pol II promoter escape by recruiting P-TEFb
Binds in a sequence-specific manner to target sequences – e boxes
Dimerises with ubiquitous, harmless txn factor MAX
Amalgamation of two types of dimerization domain
Results in two long a helices joined by loop
One helix fits in major groove of DNA
In case of pol II Can stimulate transcription both pre and post initiation
Facilitate pre-initiation complex assembly by recruiting TBP and HATs and basal txn factor TFIIH
Promotes release of polymerase by recruiting P-TEFb
What is TFIIH?
Complex containing two helicases & CDK7 kinase
CDK7 phosphorylates pol II
helicase separates DNA strands and feeds template strand into pol II catalytic site
Helicase unwinds DNA at transcription start site – exposing template strand so it can be copied into RNA
As well as txn, TFIIH is involved in DNA repair
Mutations in helicase subunits can cause rare recessive disease Xeroderma pigmentosum
- extreme photosensitivity
- defective repair of UV-induced DNA damage
- strongly predisposed to skin cancers
- often die young from cancer
Describe how Pol II has a Unique CTD
Largest subunit of pol II (POLR2A) has C-terminal domain (CTD) of 7 residue (heptad) repeats
Not present in pols I or III
Number of repeats varies between species – humans have 52 repeats
CDK7 subunit phosphorylates RNA pol II on long C-terminal domain that extends from its largest subunit
Unique to pol II
Heptad contains multiple phosphoacceptor sites
Phosphorylation status varies during various point of transcription cycle
Describe how Pol II CTD Undergoes a Dynamic Phosphorylation Cycle
pol II with unphosphorylated CTD is recruited to promoters
unphosphorylated CTD binds TBP & helps anchor pol II in PIC
TFIIH phosphorylates Ser5 when txn initiates – lets go of TBP
during elongation, Ser5 undergoes gradual dephosphn & Ser2 gradual phosphn
When pol II recruited to promoters it does so with CTD unphosphorylated – binds to TBP at promoter anchors pol II in position
As transcription initiates the CDK7 subunit of TFIIH phosphorylates the CTD at serine 5
Causing it to release TBP such that pol II can move away from start site and start to transcribe gene
As it moves along the gene the serine 5 phosphorylations gradually get removed by phosphatases
At same time serine 2 of the heptad repeat undergoes progressive phosphorylation by elongation factor PTEF-b which is recruited by MYC
At start of gene pol II has serine 5 phosphorylation and not serine 2 and by end of the gene it is other way round
In middle has phosphorylation of both
These changes in phosphorylation serve as signals to recruit different proteins to docking platform
Describe how CTD Recruits RNA Processing Factors
CTD provides a docking platform that recruits other proteins according to its phosphn state
Unphosphorylated CTD binds TBP, anchoring pol II to the promoter
mRNA capping enzs bind CTD with high Ser5P
CTD with Ser5P & Ser2P recruits elongation & splicing factors
CTD with low Ser5P & high Ser2P recruits proteins involved in mRNA 3’ cleavage & polyadenylation
At start of transcription when heavily phosphorylated on serine 5 the CTD recruits capping enzymes responsibly for placing 5’ cap – important for stabilising mRNAs and enhancing their translation
Very soon after initiation the nascent transcript receives its cap from the capping machinery
When serine 5 phosphates start to be removed and serine 2 ones start to be added the capping machinery dissociates and splicing machinery is recruited
Splicing can commence while transcript is still being synthesised
By the time the end of the gene is reached and serine 2 phosphorylation predominates over serine 5 the CTD recruits enzymes involved in 3’ end formation and polyadenylation of mRNA
Describe how many Genes Display Promoter-Proximal Pausing of Pol II
Estimated to apply to ~30% of
protein-coding genes in human cells e.g. MYC gene
determined by ChIP-seq
Pausing is caused by binding of negative elongation factor (NELF) to pol II
Stops within 30-50 bases of txn start site
Phosphorylation by P-TEFb releases NELF & allows pol II to resume transcribing
By recruiting P-TEFb, MYC protein is able to induce expression of paused genes, including MYC gene
Describe how MYC Induces Multiple Hallmarks of Cancer
MYC induces transcription of genes that promote cell cycle progression, e.g. cyclins, CDKs, E2F, & growth, e.g. rRNA, tRNA, ribosomal proteins & translation factors
MYC induces transcription of the TERT gene encoding telomerase catalytic subunit allowing immortalization of cells
MYC induces transcription of the gene encoding angiogenic factor VEGF stimulating formation of new blood vessels
MYC induces mesenchymal txn factors that suppress E-cadherin expression & promote the epithelial-mesenchymal transition - promotes early stages of invasion and metastasis
MYC promotes aerobic glycolysis & inhibits mitochondrial respiration by induction of genes such as hexokinase 2
MYC induces CD47 & PD-L1, which suppress attack by the innate & adaptive immune systems, respectively
MYC inhibits expression of CDK inhibitors p21, p16 (INK4a) & p15 (INK4b)
Explain why MYC is a challenging drug target
challenges associated with targeting MYC include:-
1. Essential – MYC knockout is embryonic lethal – toxicity concerns 2. Nuclear – inaccessible to therapeutic antibodies 3. Redundancy – other family members may substitute 4. Not an enzyme - no active site to target 5. Good targets have hydrophobic involutions that small cell- permeable drugs can occupy to disrupt protein/protein interactions – interaction surfaces of MYC are large, flat & featureless, making them difficult to target specifically
What is Omomyc?
Omomyc was developed as a dominant negative version of MYC, with 4 substitutions in the leucine zipper region of its dimerization domain
forms inactive complexes with MYC
Omomyc transgene suppresses multiple tumour types in genetically-engineered mouse models, with minimal toxicity – not just MYC-driven tumours
92 amino acid miniprotein version of the bHLH-Zip region of Omomyc can penetrate cells & suppress tumour growth after intravenous delivery to mice with lung cancer xenografts
Synergizes with standard chemotherapy drug paclitaxel, a microtubule inhibitor that blocks cell division
clinical trials predicted to begin in 2020
Explain why Bromodomains are Good Drug Targets
acetylated lysine is recognised by bromodomains
hydrophobic pocket binds acetylated lysine & is an inviting target for small molecule drugs
BET proteins have two bromodomains & an extraterminal domain e.g. BRD4
BRD4 binds acetylated histones & then recruits P-TEFb, allowing promoter escape by paused pol II
Small molecule drug designed to fit into hydrophobic pockets
Bromodomains have pocket where acetylated lysine fits – attractive for drug design
Subgroup of bromodomains found in BET proteins
BRD4 most important example – binds to acetylated lysine through its BET domain and recruits P-TEFb
Can phosphorylate RNA pol II on its serine 2 of its CTD domain, release negative elongation factor, overcome pausing
Drugs designed to fit into pocket
What are BET inhibitors?
JQ1 was developed by modeling candidate ligands in BRD4 bromodomain pocket
high affinity for bromodomains of BRD4 and its relatives in BET subfamily, but not other bromodomains
JQ1 blocks BRD4 binding to acetylated lysine (AcLys)
co-crystal structures showed JQ1 occupying entire AcLys binding pocket
when added to cells, JQ1 displaces BRD4 from chromatin
Several BET inhibitors have been developed besides JQ1
JQ1 example of such a drug
High affinity for bromodomain found in BET proteins but don’t target other bromodomains eg bromodomain in TFIID
Cell-permeable can release BRD4 from chromatin
Entered clinical trials
Key target of BRD4 is MYC
Describe how BET Inhibitors Suppress MYC Expression in Some Tumour Types
MYC gene is a key target of BRD4 in some tumour types, e.g. multiple myeloma
JQ1 suppresses MYC expression in cells from these tumours
can trigger cell cycle arrest, senescence or apoptosis of malignant cells in mouse xenografts & improve survival
several BET inhibitors are in phase I clinical trials for refractory haematological malignancies
initial efficacy is often followed by rapid relapse – combinations likely to be more effective
MYC suffers from promoter-proximal pausing of RNA polymerase II
BRD4 can overcome that pausing by recruiting P-TEFb
BRD4 promotes MYC expression
BET inhibitors that inactivate BRD4 target a number of BRD4 targets but most importantly inhibit MYC expression
In a multiple melanoma cell type treatment with JQ1 is causing clear reduction in expression of MYC protein (see western blot)
Mice injected with multiple melanoma cell type are treated with JQ1 – markedly slowing proliferation of cancer cells
Cancers good at bypassing therapy and prone to relapse so combination therapy may be needed
