Week 2

Question 1

Intracellular signaling

Answer 1

Set of linked biochemical events connecting a stimulus with a response

Question 2

Communication between cells controls cell behaviour and survival

Answer 2

Differnation signals ->Quiescence differentiation
Growth factors-> Proliferation
Death signals->Cell death (apoptosis)

Question 3

Signal molecules can bind intracellular or cell surface receptors

Answer 3

Hydrophilic signals: these bind to the receptors on the cell surface membrane
Small hydrophobic signals: that can bypass the cell membrane and go directly into the cell and bind to a receptor within the cells eg hormones

Question 4

Receptor signalling cascades and intracellular proteins

Answer 4

A signal binds to a receptor
This activates lots of downstream proteins which then:
-activate multiple downstream factors to allow for multiple different effects
-acts as signal amplifiers to allow for greater effects
One of the ultimate effects can be gene expression, metabolism or an effect on cell cytoskeleton

Question 5

Phosphorylation and cell signalling

Answer 5

Phosphorylation is mediated by kinases which act to catalyse phosphorylation
Phosphatases removes phosphorylation to return the protein back to normal
Can occur on 3 residues: threonine, tyrosine, serine residues
Different kinases have different specificity for what residue they phosphorylate
Phosphorylation is an example of post transcriptional modification of the protein
Phosphorylation can act as a binding site on the protein for phospho reader proteins, it might increase/supres the activity of the protein

Question 6

Growth factors and receptor tyrosine kinase

Answer 6

Platelet derived growth factor PDGF
Epidermal growth factor EGF
Insulin
Insulin like growth factor IGF
Transforming growth factor TGFa and B
Nerve growth factor NGF
Vascular endothelial growth factor VEGF
Macrophage-colony-stimulating factor MCSF
Ephrins

Question 7

Receptor tyrosine kinases: an important class of cell surface receptor

Answer 7

Membrane bound receptors
Phosphorylate things on tyrosine residues
All have a kinase domain and extracellular domain
When in an inactive state they exist as monomers
When in active state they exist as dimers
Most are homodimers (identical monomer) but some can heterodimerise

Question 8

Growth factor binding to tyrosine kinases

Answer 8

Causes:
-conformational change
-dimerisation
-activation of tyrosine kinase activity of the cytoplasmic domain
-transphosphorylation- tyrosine kinase on left phosphorylated one on right vice versa

Question 9

Growth factors and receptor tyrosine kinases II

Answer 9

After transphosphorylation you additionally get autophosphorylation of the RTK to further activate tyrosine kinases activity
Various cytoplasmic proteins bind to the phosphorylated receptor
This allows for 2 things to happen:
-some recruited proteins become activated by tyrosine phosphorylation
-facilitates interactions between recruited proteins or with other factors localised at the plasma membrane
Theres considerable overlap in cytoplasmic signalling pathways activated by different growth factor receptors (eg PDGF and FGF receptors)

Question 10

Summary of MAP (mitogen activated protein) kinase pathway

Answer 10

One of molecules that can activate MAPK pathway is EGF
It binds to the EGF receptor which is then activated and can recruit downstream proteins such as GRB2
GRB2 acts to activate other downstream proteins such as RAS
RAS becomes activated and binds to other factors which results in a downstream cascade allowing for the activation of other kinases -> impact on cell growth
I.e MAPK pathways drive cell growth and one of the signals that drive it is EGF

Question 11

Grb2 and Sos

Answer 11

Grb2: growth factor receptor bound protein 2, contains SH2 domain that binds P-Tyr residues on RTKs
Grb2 binds to Sos
Assembly of receptor-Grb2-Sos complex enables recruitment of RAS
If you dont have Sos no recruitment of RAS
If you dont have phosphorylation of RTK then don’t recruit RAS either or Sos

Question 12

The RAS family

Answer 12

3 Ras genes: h-Ras, k-Ras, n-Ras
Encode “G-proteins” (GTP- coupled signal transducing protein)
Contains small lipid group that attaches Ras to membrane
RAS is most commonly mutated oncogene, implicated in 20-30% human cancers
Function:
-in inactive form binds GDP
-if you get a signal it exchanges the GDP to GTP and becomes active
-it can then pass the signal downstream

Question 13

The RAS-GDP/RAS-GTP cycle

Answer 13

Inactive RAS binds to GDP
When we have a growth signal eg EGF it results in RAS exchanging GDP to GTP, it does this with help of other proteins called guanine nucleotide exchange factors GEFS which help speed up the exchange
This results in activation of RAS and the signal is then transduced allowing for an effect eg gene expression
Then RAS turns itself off
-able to do this because it has a GTPase which hydrolyses GTP for GDP, this is stimulated by other proteins called GTPase-activating proteins
A lot of mutations in RAS stop its ability to inactivate itself- cancer

Question 14

RAS and Sos

Answer 14

Important thing about the Grb/SOS receptor is that they act as a GEF (guanine nucleotide exchange factor) for RAS so they ie turn RAS on and so we get signal transduction downstream

Question 15

RAS in signal transduction

Answer 15

Activated RAS binds another protein called RAF
RAF allows the cell to activate downstream kinases such as MEK kinase via phosphorylation
MEK then goes on to phosphorylate other signals eg ERK
Activation of these 2 sets of kinases MEK and ERK ultimately lead to activation of lots of transcription factors eg C-MYC, C-jun, C-fos which then act to drive cell proliferation
MEK and ERK are both serine or threonine (not tyrosine) kinases

Question 16

Turning off the MAPK pathway

Answer 16

Many ways to turn off MAPK pathway to stop cell growth:
-remove signal eg stop expressing EGF
-switch of receptor by Tyr phosphatase remove phosphorylation signal so receptors become inactive
-GTPase activating proteins drive inactivation of RAS and therefore stop signal
-dephosphorylation of targets by ser/thr phosphatases
Ultimate effect cell growth switched off
In tumours this is often deregulated eg RAS mutation

Question 17

MAPK mutations and cancer

Answer 17

RAS is mutated 90% of prostate cancer
Commonly mutated in ovarian cancer too
RAS mutations in cancer always involve point mutations at specific sites (codons 12,13,21)
-eg G/T transversion (gly->val), typical carcinogen induced mutations in smokers or workers with occupational carcinogen exposure
RAF mutations common in melanoma (80%), lung and colorectal cancers:
-basal level kinase activity elevated 2-12 fold
-V600D and E mutations (most common)- mutants stimulate proliferation independent of upstream signals because mutation elevates activity RAF to turn on downstream signal

Question 18

Summary of the PI3 (phospatidylinositol) kinase pathway

Answer 18

This pathway can be turned on by lots of signals eg insulin
One of the key molecules that is involved in this pathway is a lipid called PI : PI(3,4)P3, PI(4,5)P2
PI binds to membrane and when you have activation of the receptor you recruit and then activate a kinase called PI3 kinase
PI3 kinase phosphorylates PI to give it PIP3
Once we have 3 phosphorylations we can recruit downstream proteins called PDK1 and AKT
AKT is activated via phosphorylation
AKT is then able to activate a number of targets with the ultimate effect of driving cell growth, proteins synthesis and suppression of apoptosis

Question 19

PI (phosphatidylinositol) and PI3-kinase

Answer 19

PI (phosphatidylinositol) is a phospholipid found in eukaryotic cell membranes
PI is phosphorylated to form PI(4)P which is in turn phosphorylated to form PI(4,5)P2
PI3K is activated by binding to phosphorylated Tyr residues on RTKs
PI3-K catalyses phosphorylation from PI(4,5)P2 to PI(3,4,5)P3

Question 20

PDK1, AKT and mTOR

Answer 20

PI(3,4,5)P3 acts as a docking site for 2 proteins, PDK1 and AKT
Upon binding PDK1 phosphorylates and activates AKT
mTOR (as part of mTORC2) also plays an important role in activating AKT
AKT activates mTOR (as part of mTORC1) to stimulate cell growth via protein production/preventing protein degradation

Question 21

Turning off the PI3-kinase pathway

Answer 21

Normal cells would want to turn off PI3K pathway once they’ve synthesised the appropriate proteins or undergone cell growth
The way pathway is tuned off:
-remove signal
-switch off receptor by Tyr phosphatase
-dephosphorylate PI(3,4,5)P3 to PIP2, PTEN (lipid phosphatases)
-dephosphorylation of targets by ser/thr phosphatases

Question 22

Targeted treatments of these pathways

Answer 22

“Stratified” or “personalised” medicine;
-a more effective way of treating cancer by grouping patients according to their genetic mutation(s) and then targeting the signalling pathway to which it contributes

Question 23

Receptor tyrosine kinases and cancer

Answer 23

Amino acid changes or partial deletions:
-constitutive tyrosine kinase activity
-loss of “receptor domain”
-growth factor independent signalling
Gene amplification and overexpression:
-increased expression of normal receptors- allows more signal binding which results in response amplification
-overexpression of aberrant receptors
Increased signaling

Question 24

Targeting RTKs

Answer 24

Target antibodies to receptor:
-inhibit ligand binding
-inhibit dimerisation
-result in degradation of receptor
-induce cell killing of tumour cells
Small molecule inhibitors of RTK:
-molecules need to get inside the cells todo this

Question 25

HER2 and breast cancer

Answer 25

HER2 belongs to the epidermal growth factor receptor EGFR family of RTKs Orphan receptor (no known ligand). Heterodimerises with other EGFR family members Amplification found in 30% breast cancers Overexpression/increased copy number: -poorer prognosis -constitutive signalling

Question 26

Targeting RTKs with antibodies

Answer 26

Trastuzumab (herceptin): does not block dimerisation, blocks internalisation, partial block of downstream signalling, promotes antibody dependent cytotoxicity Pertuzumab: blocks dimerisation and activation, effective in cancers where HER2 is activated but not overexpressed Cetuxumab, panitumumab: blocks ligand (EGF) binding, steric hindrance, promotes antibody-dependent cytotoxicity

Question 27

HER2 targeted treatments

Answer 27

Trastuzumab: inhibits internalisation/cleavage of HER, Ab-dependent cell toxicity, indication( metastatic breast cancer, adjuvant early breast cancer, advanced gastric cancer) Pertuzumab: inhibits ligand binding and dimerisation, indication (metastatic breast cancers, neoadjuvant breast cancer) Lapatinib: reversible ATP competitor (small molecule), targets EGFR as well, indication (advanced breast cancer) Trastuzumab-emtansine: same as Trastuzumab plus MT inhibition and cell lysis. Indication (advanced breast cancer)

Question 28

EGFR targeted treatments

Answer 28

Cetuximab: inhibits ligand dependent activation. Indication (colorectal cancer, colorectal cancer with WT KRAS, head and neck with radiotherapy) Panitumumab: inhibits ligand dependent activations, colorectal cancer, colorectal cancer with WT KRAS Patients with KRAS mutations do not respond to EGFR antibodies, KRAS mutations usually mutually exclusive with EGFR mutations Erlotinib (gefitinib): reversible ATP competitor (small molecule), response defined by certain mutations . Indications (lung cancer, pancreatic cancer)

Question 29

Resistance to EGFR inhibitors

Answer 29

Eg amplification of EGFR in glioblastoma, breast and lung cancer Resistance to erlotinib and gefitinib: Thr790Met mutation prevents binding of inhibitor

Question 30

BCR-ABL: a fusion kinase

Answer 30

BCR is a protein that regulates the activation of GTP in the cell, its a Tyr kinase It works with ABL (cytoplasmic Tyr kinase promotes cell survival and cell growth upon activation ) which is usually inactive in the cell because it has a self inactivation domain which stops activation of the kinase In certain tumours especially hematopoietic tumours you get a fusion between the gene that encodes BCR and gene that encodes ABL resulting in BCR-ABL fusion kinase During the fusion parts of the 2 genes are broken so get missing parts of each: usually miss some GTPase activation function of BCR and lose inhibitor function ABL So ABL and BCR Much more active Then get another kinase domain from ABL and BCR cross talking which further activate one another becomes even more active

Question 31

BCR-ABL fusion

Answer 31

Coiled-coil increases tyrosine kinase activity Fusion kinase activates ABL signalling pathways Fusion inhibits the SH3 regulatory domain leading to constitutive kinase activity BCR recruits ABL to cytoplasmic signalling pathways including RAS pathway

Question 32

The (9,22) translocation leads to expression of BCR-ABL in CML

Answer 32

Resultant chromosome is known as the Philadelphia chromosome t(9;22)-(q34;q11) The main tumour we see as a result of BCR-ABL fusion in chronic myeloid leukaemia The reasons why BCR-ABL inhibitors good target for CML is that its completely abnormal and exclusively expressed in tumour cells

Question 33

BCR-ABL inhibitors

Answer 33

Gleevec (imatinib mesylate; Novartis) -specific inhibitor of the BCR-ABL tyrosine kinase Found by random screening for inhibitors of other tyrosine kinases Competitive inhibitor of ATP binding to active site inhibits tyrosine kinases activity Has dramatic effects in patients 80% patients with CML: responses in 53/54 patients at upper doses, 29 cytological responses including 7 complete cytogenetic remissions, remissions durable (18 months) chronic phase Some patients experience resistance to imatinib and relapse: -development of point mutations within try kinase domain of BCR-ABL -tendency to relapse with resistant BCR-ABL mutants if treated in acute phase (blast crisis) -second generation Tyr kinase inhibitors have been developed

Question 34

What is an oncogene

Answer 34

A gene that has the potential to cause cancer when mutated or overexpressed Several hundred oncogenes have been identified Derived from a normal cellular gene termed proto oncogene “Activated oncogene” Variant with abnormal expression or activity that positively contributes to cancer formation

Question 35

Proto-oncogenes and oncogenes

Answer 35

Protooncogenes are normal cellular genes that control critical processes such as proliferation, survival and differentiation Typically function as growth factors, receptors, signal transduction molecules and transcription factors Activated oncogenes over express these normal proteins or produce altered proteins with altered (usually constitutive) activity that contribute to the hallmarks of cancer

Question 36

Key features of oncogenes

Answer 36

Usually constitutively active i.e gain of function mutation A single altered copy (allele) is sufficient ie dominant effect Gain of function drives cell transformation

Question 37

Mechanisms of oncogene activation

Answer 37

Point mutations-> protein with altered characteristics eg EGFR, RAS, BRAF Amplification of a genomic DNA region that includes the proto oncogene-> overexpression of the gene and increased amounts of protein eg MYCN., EGFR Chromosome translocation that brings a proto oncogene under the control of a different promoter-> inappropriate gene and protein expression eg c-myc, BCL-2 Chromosome translocation that joins the 2 genes together-> creates a chimeric fusion gene and protein with novel characteristics eg BCR, ABL

Question 38

Oncogenic single nucleotide variants (SNVs) seen in cancer

Answer 38

EGFR- L858R (leucine 858 to arginine) KRAS- G12C (glycine 12 to cysteine) BRAF- V600E(valine 600 to glutamic acid) PIK3CA- H1047R (histidine 1047 to arginine) All lead to altered protein activity

Question 39

Receptor tyrosine kinases

Answer 39

58 human receptor tyrosine kinases RTKs Examples include EGFR (ErbB1, Her1), ErbB2 (her2), VEGFR, ROS, RET Control cell growth, survival, motility, differentiation, metabolism Share a similar protein structure of an extracellular ligand binding domain a transmembrane helix and an intracellular tyrosine kinase TK domain Dysregulation leads to many human diseases, especially cancer

Question 40

Common EGFR mutations

Answer 40

In Frame deletion eg del 747-752, increases dimerisation Missense mutation eg L858R (leu>arg), increases kinase activity 50fold When the receptor has one of these 2 mutations it becomes constitutively active and signals through downstream pathway constantly

Question 41

RAS is frequently mutated in cancers

Answer 41

RAS a molecular switch involved in signal transduction Different RAS genes are associated with different cancers Different cancers show variable patterns of KRAS, NRAS and HRAS mutations

Question 42

Oncogenic RAS mutations invariably involve Missense mutations affecting one of 3 codons (G12, G13, or Q61)

Answer 42

Mutations occur at just 3 amino acids in the entire protein sequence glycine 12, 13 and glutamine 61 This is because if we look at the 3D structure of RAS G12, G13, Q61 are all part of the same pocket in ED structure the GTP binding site

Question 43

How do these mutations affect RAS

Answer 43

RAS is involved in a signalling cascade from the RTK which ultimately results in changes in gene expression RAS is switched between an on/off conformation depending on whether it is bound to GDP or GTP -when it is bound to GTP, RAS active -when its bound to GDP inactive Mutations at G12, G13, Q61 at GTP binding site decrease GTP hydrolysis and so lock RAS in active GTP bound state This leads to constitutive activation of RAS/MAPK pathway

Question 44

Mutations in RAS and EGFR are typical of those seen in many oncogenes

Answer 44

Mutations are clustered in specific regions of the gene (hotspots) These regions encode important functional domains in the proteins Most mutations are Missense mutations (amino acid substitution) resulting in a gain of function

Question 45

Oncogene amplification in cancer

Answer 45

MYCN- neuroblastoma -20% C-MYC- small cell lung cancer- 15-20%, breast cancer 20%, ovarian cancer 20-30%, oesophageal cancer- 38% Cyclin D1- breast cancer -20%, oesophageal cancer 25% EGFR- glioblastoma -40% All lead to protein overexpression from the amplified gene

Question 46

MYCN amplification in neuroblastoma

Answer 46

MYCN amplification detected by FISH

Question 47

N-Myc protein expression is increased in neuroblastomas with MYCN gene amplification

Answer 47

If you get a tumour sample and take to path lab they look for expression of the MYCN protein using immunohistochemistry Lots n-myc in MYCN amplification

Question 48

MYCN amplification as a prognostic factor

Answer 48

MYCN amplification frequently associated with aggressive diseases, metastatic potential, therapeutic resistance and poor patient outcomes

Question 49

ERBB2/HER2 amplification in breast cancer

Answer 49

Associated with poor prognosis and reduced survival.

Question 50

Translocations leading to changes in oncogene expression

Answer 50

T(8;14) C-myc. Burkitt lymphoma T(14;18) BCL2 follicular lymphoma T(3;14) BCL6. Diffuse large B cell lymphoma T(1;14) TAL1 T cell acute lymphoblastic leukaemia

Question 51

Burkitt lymphoma BL

Answer 51

Highly aggressive b cell lymphoma derived from mature B cells Frequently presents as a fascial tumour One of fastest growing tumours

Question 52

Translocations involving c-MYC are a hallmark of Burkitt lymphoma

Answer 52

All BL tumour cells carry a chromosomal translocation involving c-MYC gene on chromosome 8 and one of the immunoglobulin gene loci T(8;14) MYC-IgH 85% T(2;8) MYC-IgK T(8;22) MYC-IgL

Question 53

Identification of t(8;14)(q24;q32)

Answer 53

How can we detect translocation Using karyotyping-chromosomes stained with a dye and matched up based on size and banding pattern Chromosome 8- where MYCN gene normally located Chromosome 14- where normal immunoglobulin heavy chain is located Theres a bit missing at end of C8 and extra on C14. C8-C14 translocation and as result place MYC gene next to immunoglobulin gene on this chomosome

Question 54

T(8;14) translocation places IgH enhancer adjacent to c-MYC

Answer 54

When you translocate onto C14 next to IgH enhancer you get overexpression of MYC Because IgH enhancer very powerful in B cells as they need to produce lots of antibodies So when you put MYC gene next to enhancer and promoter you end up with super high level of expression of MYC Ki67 marker of cell proliferation

Question 55

C-MYC is a transcription factor that regulates >600 cellular genes

Answer 55

Genes required for: -cell growth -proliferation -ribosomal synthesis -protein synthesis -metabolism -energy generation These are all hallmark of cancer So if you deregulate MYC because it controls many of the proteins involved in different features of cancer the cell becomes cancerous

Question 56

C-MYC functions in cancer cells

Answer 56

Cell growth Cell cycel Apoptosis Energy production Anabolic metabolism DNA replication RNA biology and accumulation

Question 57

Translocations leading to fusion genes

Answer 57

T(9;22) BCR, cABL chronic myeloid leukaemia T(8;21) RUNX, ETO acute myeloid leukaemia Inv(2) EML4, ALK non small cell lung cancer T(5;1) SQSTM1, NTRK1 non small cell lung cancer and many others

Question 58

BCR-ABL is a hallmark of CML

Answer 58

All CML cells have this translocation in it Translocation between C9 and C22 -Philadelphia chromosome t(9;22)-(q34;q11) Novel BCR-ABL fusion protein

Question 59

Proto-oncogene to oncogene

Answer 59

4 main mechanisms Mutation in coding sequence- hyperactive protein made in normal amounts Gene amplification- normal protein greatly overproduced Chromosome rearrangement- nearby regulatory DAN sequence causes normal protein to be overproduced, fusion to actively transcribed gene greatly overproduces fusion protein; or fusion protein is hyperactive

Question 60

Why is this important

Answer 60

Genomic analysis of cancer cells: Better understanding of the function of oncogenes Detailed information about mutational landscape of cancers Enables improved cancer diagnostic tests Better prognostic information Important for designing new targeted cancer treatments

Question 61

Era of precision cancer therapy

Answer 61

Move away from ‘one size fits all’ approach Develop inhibitors of key oncogenes that drive tumour cell growth Improve patient outcomes and reduce side effects Examples include: BCR-ABL- imatanib, EGFR- gefetinib, afatanib, osimertinib, HER2- Trastuzumab, RAS- sotorasib Ib-small molecule inhibitor. Ab-monoclonal antibody

Question 62

BCR-ABL: a paradigm for targeted therapy

Answer 62

Imatinib (gleevec) is a specific inhibitor of the BCR- ABL tyrosine kinase Competitive inhibitor of ATP binding to active site, inhibiting tyrosine kinase activity Treated CML patients show 83% 10 year survival

Question 63

EGFR targeted therapies

Answer 63

First generation TKI eg gefitinib, erlotinib Competitive ATP mimics, reversible binding, moderatively increase life expectancy Second generation TKI eg afatinib, dacomitinib Irreversible binding in the ATP pocket, binds to mutated version which causes resistance Third generation TKI: osimertinib, binds more avidly to EGFR T790M mutants than wild type EGFR