Unit 5 - From Oncogenes and TSGs to Drugs Flashcards
genetic disease
irreversible
SNP
gross chr rearrangement
epigenetic
reversible - affects ways in which genes can be transcribed, how many copies of mRNA you can make
DNA methylation - affecting gene exp
histone modifications - methylation, acetylation - affecting gene exp
Point mutations
single nucleotide base changes
present in DNA, transcribed into RNA - can result in the encoded protein
nonsense mutation
altered codon encodes a termination codon
inappropriate termination of translation
shortened (truncated) protein
missense mutation
altered codon encodes a different AA
protein will contain an incorrect AA - missense mutation
could result in a non-functional (most) or hyperactive protein
silent mutation
altered codon encodes for the same AA
gross chr rearrangement
increased/decreased copy numbeer and gene expression
how is DNA organised
into chromatin by DNA binding proteins (histones)
Nucleosomes and histones
Protein in middle - DNA around
tails are piece of protein of histones - stick out - highly modified - charged
changes in chromatin conformation
what is RB
a transcriptional repressor
RB pathway
RB binds to the transcriptional activator E2F
E2F promote the expression of genes involved in cell proliferation
mutations in both alleles of RB1 lead to the retinal cancer -retinoblastoma
RB1 is a tumour suppressor
RB pathway is de-regulated in virtually every human cancer
role of INK4 family
2 types of genes altered in cancer cells
oncogenes e.g. myc, ras, abl
protein products act as ACCELERATORS of cell division or promote the cancer phenotype
tumour suppressor genes (TSG) e.g. RB, p53, BRCA1, BRCA2
protein products normally act as BRAKES on cell division or counteract the cancer phenotype
inheritance pattern - oncogenes vs TSGs
oncogenes = dominant
TSGs = recessive
what is RAS
a proto-oncogene and a central node of multiple pathways relevant to cancer
Mediates signalling through tyrosine kinase receptors
In order to activate another pathway
Survival - cell cycle progression - when active, promotes phenotypes related to cancer
normal vs mutant RAS gene
this mutant protein lacks GTPase activity, so it is active (on) all the time
mutations in RAS gene - what does the gene encode
what does mutation lead to
encodes RAS GTPase protein
leads to production of an altered RAS protein that binds GTP but cannot break it down to GDP
so RAS protein is active (on) all the time
RAS signalling pathway is continuously activated
cell proliferation is stimulated - promotes tumour formation
prevalence of mutations in RAS
c-MYC and Burkitt’s lymphoma
cancer of what type of cell
type of mutation
results in
cancer of lymphocytes - common in parts of Africa
caused by translocation of gene for c-MYC transcription factor
c-MYC gene translocated from chr 8 → chr 14
enhanced production of c-myc protein
stimulates cell proliferation - tumour formation
how does Myc regulate proliferation
through CDKs
Myc is a TRANSCRIPTION FACTOR
Protein that binds to DNA in order to promote transcription
Works with MAX to activate transcription of genes
Transcribe - cyclin D and CDK4 (promote cell cycle progression)
Excess of kinase it binds and sequesters the KIP protein - causes its degradation - cyclin kinase inhibitor - inhibits cyclin E
MYC + MAX = transcriptional activator
However when myc binds MIZ1 it is a transcriptional repressor
MYC + MAX =
transcriptional activator
but when myc binds MIZ1 it is a transcriptional repressor
MYC promotes function of
CDK4
promotes inhibitor
Li-Fraumeni Syndrome
pattern of inheritance
rare cancer-prone syndrome
inherit 1 mutated copy (allele) of p53
somatic mutations in other copy (allele) of p53 gene
early onset of variety of cancers - blood, breast, bone, lung, skin
both copies (alleles) of a TSG must be inactivated for a phenotype to result
p53 gene codes for p53 protein - named bacuase protein is 53 kDa - transcription factor
how are cellular stress signals mediated
by the p53 transcription factor
Downstream of a lot of signalling - tells cells we’re under stress - lack of O2, loss of signalling factors etc
how does a mutated p53 react in response to DNA damage
loss of ability to arrest cell cycle progression after DNA damage
cell continues to divide in the presence of DNA damage
increase in mutations in genome - genome instability
cells lacking p53 also fail to undergo apoptosis (cell death) after DNA damage
- because transcription of certain gene products required for apoptosis does not occur
- also become resistant to some chemotherapeutic agents
what is required to cause most cancers
multiple lesions
e.g. model of progression of colorectal carcinoma
sequence of genetic events in progression of normal epithelial cell to carcinoma
tumours are __________
heterogenous
can spread
cycling and resting cells
genetic info can vary in cells of the same tumour
therapeutic potential
MOA of nitrogen mustards
in use
DNA alkylation
cyclophosphamide
antimetabolites MOA
in use
folic acid analogue active on leukaemia
MOA = DNA and RNA synthesis
IN USE = metotrexate, 5-FU, gemcitabine
cellular screening for cytotoxic compounds - mechanisms
in use
mechanisms target essential cellular components and processes - DNA, microtubules, enzymes
in use = cisplatinum, doxorubicine, taxanes
targeted therapy
in use
molecular target defined upfront and then drugs that act through that target are identified
potential for better selectivity versus cancer cells
in use - Gleevec, Avastin
history of chemotherapeutics
a chemotherapeutic agent (drug) =
a substance that has been demonstrated to give benefit to cancer patients in controlled clinical studies
clinical benefit is defined according to the specific disease - cure, prolonged lifespan (survival time), improved side effects/increased quality of life
Pro-drug
drug is not the active substance
drug needs to be activated modified by either tumour cell or by the host organism
4 classifications of therapeutics
chemical type/nature
mechanism
molecular target
cellular/tumour response
chemical type/nature
small molecule
de novo chemical synthesis
natural products
antibody
molecular target
kinase inhibitors
topoisomerase inhibitors
mechanism
alkylating agents - bind to DNA
antimetabolites
cell cycle and/or mitotic inhibitors
anti-angiogenesis
endocrine agents
cellular/tumour response
cytotoxic or cytostatic
curative - goal of chemo
early stage sensitive tumours e.g. testicular cancers, lymphomas
adjuvant goals of chemo
after surgery or radiotherapy to minimise recurrences - mostly solid tumours
neoadjuvant goals of chemo
before surgery to reduce tumour size
activty of a drug is defined by
its therapeutic window
TW is the ratio between toxic conc and active conc
lethal dose50/effective dose50
generally a single digit number
toxicological liabilities - mechanism related and drug specific
mechanism related - affecting normal proliferating cells - GI and bone marrow
drug specific i.e. neurotoxicity of taxanes, cardiotoxicity of doxorubicine
taxanes
neurotoxicity
doxorubicine
cardiotoxicity
alkylating agents
what do they do
e.g.
covalently modify DNA
cyclophosphamides, cisplatin
intercalating agents
what do they do
e.g.
bind with bases and to minor groove of the DNA, NOT to backbone of DNA
doxorubicin
antimetabolites
what do they do
e.g.
inhibit supply of dNTPs
block DNA (and RNA) synthesis
5-flourouracil, gemcitabine
drug approved for pancreatic cancer
gemcitabine
active metabolites inhibit RNR (ribonucleotide reductase) and are incorporated into the DNA during replication
active in a broad range of tumours
microtubule dynamics as a target of cancer therapy
example of drug resistance mechanisms developed by tumours
Decrease accumulation
Decrease activation
Change in met causing increase in activation
** target of drug is transformed - kinase inhibitors
how cells respond to anticancer drugs
benefit of combination of drugs as treatment
2+ drugs delivered to a patient
increase cell killing by using different mechanisms
minimise risks of resistance
reduce peaks of toxicities of single drugs
molecular targeted therapeutics - MTT
- What are the signalling pathways involved in getting to cancerous phenotypes*
- Learn which are key proteins important for mediating process*
Bcr-Abl
type of mutation
results in
reciprocal translocation between chr 9-22 causes the expression of chimeric Bcr-Abl protein with tyrosine kinase activity
loss of -ve regulation
gain of protein-protein interaction domains
POTENT ONCOGENE - sufficient for cellular transformation by activating multiple molecular pathways
present in 95% of patients with CML, 15-30% with ALL, 2% with AML
Gleevec/Imatinib
first successful MTT
complete remission of Bcr-Abl+ leukaemia
95% 5 year survival
before Gleevec was available, 50% of patients progressed to the more advanced stages of Ph+ CML after only 3-5 years and survival was generally shorter for these patients
how does resistance to Gleevec therapy arise
from mutations in Bcr-Abl kinase domain
mutations in the ATP pocket strongly reduce the affinity for Gleevec
2nd generation of compounds that inhibit mutant forms has been developed
targeting tumour suppressors - what is it necessary to do
synthetic/combined lethality
PARP
poly (ADP-ribose) polymerase
Allow chromatin to be more relaxed
Enzyme - chain of adiporibose on chromatin histones - allow chromatin to be more relaxed to allow DNA repair protein to be relocated to help in DNA repair
mode of action of PARP inhibitors
SS breaks are not repaired
BRCA mutations and PARP inhibitors
BRCA1 and 2 - TSGs often mutated in breast and ovarian cancers
they have deficient HR DNA repair - cells are dependent of Base Excision repair
PARP inhibitors block the repair of DNA SS breaks and BE repair
normal cells are insensitive to PARP inhibitors
BRCA cells are 1000x more sensitive to PARP inhibitors
mechanisms of resistance to PARPi
oncology drug discovery process
screening funnel
considerations for choosing a good target in oncology - biological
BIOLOGICAL
NB disease progression
essential for tumour growth - target in tumour cells, external target
specificity fro tumour - only expressed in tumour, hyperactive in tumour (activating mutations in target, normal -ve regulation is lost in tumour)
different cellular response in tumour vs normal cells - genetic background strongly influence outcome of treatment
defined patient population
considerations for choosing a good target in oncology - technical
druggable - a small molecule can block target function
enzymes - normally have a catalytic pocket
protein-protein interactions - only if small defined surface is involved
availability of cellular and animal models
availability of specific technologies and reagents - targeted chemical libraries, screening methods
business considerations for oncogotherapeutics
compound collections for screening can be
biochemical kinetic assays
HTS
high throughput screening
virtual screening for new ligands
- target structure known
- computational docking of molecules into target’s active site
- ID of potential binders
- experimental test predictions
functions of cell proliferation assays
to verify the antiproliferative activity of selected cpds
to compare potency among different cpds
to compare potency among different cell lines
IC50 and IC90
RB pathway
no absolute specificity
off target events may be prevalent
E.g. CDK4 inhibitors
But microtubule inhibitors can also block cell cycle progression - what you’re looking for is a marker that is as close as possible to action of target e.g. P of RB
xenografted mouse models
immunosuppressed animal (nu/nu)
human cancer cells implanted subcuteneously
orthotropic implanted mouse models
human tumour cells surgically implanted in their normal contest
patient derived xenografts (PDX)
immunosuppressed animals (nu/nu)
small tumours derived from patients
spontaneous (induced) models
DMBA rats
female are dosed intragastrically with DMBA
after approx 2 months animals develop mammary carcinoma
transgenic mouse models - MMTV/v-Ha-Ras
expressing v-Ha-Ras oncogene in the mammary and salivary epithelium
mice develop malignant adenocarcinomas of the mammary and salivary gland with 16-24 weeks of age
transgenic mouse models - TRAMP
transgenic adenocarcinoma mouse prostate, Probasin-SV40 T antigen
expressing the SV40 T antigen in the prostatic epithelium
TRAMP mice develop prostatic adenocarcinomas by 18 weeks of age
by 24-30 weeks of age metastasis are commonly detected in the lymph nodes and lungs
transgenic mouse models - p53 knockout
mice carry a null mutation in the p53 tumour suppressor gene
mice are prone to spontaneous development of different tumours before they reach 20 weeks, particularly lymphomas and sarcomas
xenograft of human solid tumours
xenografts - advantages and disadvantages
transgenic, spontaneous models, PDX
advantages and disadvantages
phase 1, 2 and 3 of clinical studies
Phase I - 2 years
phase I testing
phase II testing - 2+ years
phase III testing - 2+ years
probability of success - oncology vs non-oncology
