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cancer > 22. theraputic stratergies > Flashcards

22. theraputic stratergies Flashcards

1
Q

what is oncogene addiction

A

when the cell relies on a dominant oncogene and they will die without this

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2
Q

give an example of an oncogene which as cancer is addicted to

A

BCR-abl

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3
Q

what is mutated in 80% of lung cancer?

A

k-ras

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4
Q

what do lots of haematopoietic cancers upregulate?

A

myc - this is a TF

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5
Q

you can carry out experiments were a cancer is expressing myc and then switch this expression off, what can be seen?

A

haematopoietic cancers went into senescence

>p16 levels rapidly rose (myc is supressing this in some way)

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6
Q

how does Myc function

A

heterodimer with Max

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7
Q

describe myc and max, are they good therapeutic targets?

A

> long alpha helices to interact with each other
alpha helix to interact specifically with DNA.
- no, not much to target, although blocking protein-DNA and protein-protein interactions are being developed

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8
Q

in lung cancer, what mutation occurs in 10% of K-ras and how can this be targeted?

A

> mutants introduce a cysteine into an important part of the protein
drug can irreversible covalently bind this cysteine and block these forms
- this adds selection pressure for resistance

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9
Q

in terms of oncogene addition what do we need to do for the future?

A

identify better oncogenes which tumour are addicted to target and combine these with existing therapies

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10
Q

what is synthetic lethality?

A

two genes:

  • both WT - no affect
  • KO either separately - no affect
  • KO both - death
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11
Q

Rb null cells are deregulated in proliferation. name a target of E2F that can be targeted and potentially with what?

A

topoisomerase II - this is upregulated in Rb null cells

>etoposide

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12
Q

what does topoisomerase II do?

A

unwinds DNA and prevents it becoming tangled - especially in replicating cells

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13
Q

what does etoposide do?

A

It binds topoisomerase II and holds it in its DNA cleaving complex this prevents break from reforming and results in double stranded DNA breaks

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14
Q

what happens when you treat Rb null cells with etoposide?

A

they have more double strand DNA breaks than they cannot repair - this results in apoptosis

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15
Q

what are more single stranded breaks seen in Rb null cells treated with etoposide?

A

these cells upregulate topoisomerase II

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16
Q

what is PARP?

A

poly(ADP ribose) polymerase

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17
Q

what does PARP do?

A

poly-ADP ribosylates proteins, it builds up huge chains of ADP ribose onto proteins
>has a role in transcription where it poly-ADP ribosylates histones and this allows histones to open up and allow TF to bind
>it predominantly binds to single strand breaks

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18
Q

in what cells is PARP synethically lethal?

A

cells defective in homologous recombination repair e.g. BRCA mutant

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19
Q

when there is a single strand break in DNA one of the first things that is recruited to this break is PARP. what then happens?

A

PARP recruits repair complex around itself and repairs the damage

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20
Q

if there is a ssDNA break in DNA and replication is occurring, when the machinery gets to this point what occurs? what type of cells cannot do this? and what do they do?

A

this looks like a double strand break and so homologous recombination pathway is triggered, replication continues
>BRCA mutants, they use PARP

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21
Q

what happens when you inhibit PARP in normal cells?

A

they use homologous recombination

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22
Q

what is the second proposed mechanism of how PARP inhibition in BRCA mutants causes synthetic lethality?

A

> PARP binds single strand break trapped to this strand in inhibited form
normal cells use HR to move around blockage and continue replication
these are ss breaks that BRCA mutants cant repair

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23
Q

why is using targeting by synthetic lethality good?

A

this target is only lethal in cells where the tumour suppresser is mutated
>normal tissue is resistant and so this gives you a bigger therapeutic window to achieve efficacy

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24
Q

what can synthetic lethality also be used in?

A

activated oncogenes - look for genes that when inhibited function in cells with oncogenes you get a death response

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25
Q

RNAi library was transfected into cells with activated k-ras, what gene was identified as synthetically lethal? when this genes is inhibited in WT Ras cells what happens? what also causes decreased in K-Ras cells?

A

GATA2
>these cells do not die
>K-ras KO

26
Q

give two examples of two things that are downstream of GATA2? how is this used therapeutically?

A
  • proteasome complex
  • Rho signalling
    >GATA2 is a transcription factor and is not drugable. some of the things downstream of it are.
27
Q

what two types of inhibitors can be used with K-ras cells?

A
  • proteasomes inhibitor (bortezomid)
  • Rho kinase inhibitor (Fasudil)
    >either alone works well
    >when used together and they target two braches of GATA2 pathway this works better
28
Q

if we cannot target something in synthetic lethality what can we target?

A

> something downstream

29
Q

name 3 ways to perform gene correction, and what may these be used for?

A
  • zine finger nucleases
  • TALENs
  • CRISPR/Cas9
    >edit the genome to remove key oncogenes or install key tumour suppresser
30
Q

what did a nature paper do in terms of cancer and gene correction

A

used Cas9 system to remove mutations in human cancer, put these cells back into patients to try and improve immune response for that tumour

31
Q

why is gene editing in tumours going to be hard?

A

delivering Cas9 bits to the appropriate site

>viral delivery is currently the only method we have

32
Q

name a DNA viral vector

A

adenovirus

33
Q

name 2 RNA viral vectors

A

MLV and HIV

34
Q

discuss DNA viral vectors

A
  • don’t integrate into DNA
  • transient
  • might be lost in replication
  • due to this they are give lost of times and so immune response will be developed against them
35
Q

what may happen if someone is given adenovirus too many times ?

A

they might have an anaphylactic shock and die

36
Q

discuss RNA viral vectors

A
  • integrate into genome
  • implications on where virus integrates
    MLV integrates randomly
37
Q

what have RNA viruses been useful in

A

delivering p53 to p53 null cells

38
Q

name the three proteins encoded for by viral vectors

A

GAG
POL
ENV

39
Q

what specificity does ENV give?

A

specific tissue type - binds to sodium phosphorus symporter which is present on many cells in our body and it is internalised into cells

40
Q

how large is MLV viral genome? and what implication does this have?

A

8kb

>this is relevant in dictating the load in which this virus can carry

41
Q

describe retroviral life cycle

A
  • binds receptor and is internalised
  • RT makes DNA and integrates into genome
  • RNA used to make viral particles and packaged into virus
42
Q

if you want to convert MLV into a delivery system for things like Cas9 or a tumour suppresser, what is the simplest thing to do?

A

remove everything in the virus apart from LTR and the packaging sequence (comes straight after 1st LTR)
>add a strong promoter in front of gene in virus

43
Q

what is the packaging sequence?

A

specific 3D stem loop structure which GAG proteins bind and locks the viral genome into GAG encased capsid
>ensures genome is incorporated into the viral particle

44
Q

when you have the recombinant viral genome, then what do you do?

A

convert it to DNA

insert into plasmid - this needs origin and antibiotic resistance

45
Q

what are packaging cells?

A
  • typically mammalian cells
  • have promoter that drives ENV
  • and promoter that drives GAG and POL
  • proteins come together and release viral particle from cell
46
Q

once you have plasmid encoding viral genome what do you do?

A

transfect this into packaging cells
>recombinant plasmid produces RNA genome
>this has packaging sequence and so is incorporated into viral particles and released from cells

47
Q

why are packaging cells used?

A
  • this is one of the safest ways to use HIV which is a good virus for viral therapy
  • don’t want WT HIV particles contaminating recombinant particles
48
Q

why are GAG and POL on difference chromosomes from ENV?

A

so that there is no way that they can come together and make a viral genome
>in addition neither of these have packaging sequence
>only particles produces are out recombinant ones

49
Q

how are the recombinant viral particles generated?

A

harvest the supernatant of tissue culture cells

50
Q

how can we optimise tropism of viral delivery? give an example

A

using different packaging cells that have different ENV proteins which target different cells
>if we want to target lung cells then using the Evn from Ebola virus is very efficient

51
Q

what is changing the tropism of a virus called?

A

Pseudotyping

52
Q

what is another parameter you can optimise in viral delivery? give an example, and a way this can enhance safety

A

the promoter that you use to drive gene expression
e.g. EMV is a strong promoter that does not have tissue specificity
>tissue specific promoter could be used to enhance safety

53
Q

what give you very good tissue specific expression?

A

combing tropism with tissue specific promoter

54
Q

what is a big issue with viral gene therapy?

A

integration - if you want long term expression then you want integration.

55
Q

what is the adeno-associated viruses?

A

this is a DNA virus that integrates into a single site on chromosome 17
>as sign that is known to be benign

56
Q

what has a biotech company in china managed to do?

A

delivered a WT copy of p53 to patients with head and neck cancer
>success associated with this was limited

57
Q

describe another way that viruses can be used as therpautics?

A

viruses themselves can be manipulates to specifically target the tumour tissue
>dampen down immune response to allow these to have therapeutic affect

58
Q

what is E6 function in HPV?

A

targets p53 and recruits Ub machinery to cause p53 degradation

59
Q

what does the equivalent dominant oncoproteins of adenovirus do?

A

E1a binds Rb and E1b binds p53

60
Q

how can adenovirus be used therapeutically?

A

> they lacks E1b, still have E1a to binds Rb and cause proliferation.
lots of viral particles will be made in S phase.
in WT cells p53 will top replication in cells.
50% of tumours have mutant p53
these cells will die due to errors in fast viral driven replication as virus takes over cells
this has shown reasonable results in the clinic but was shelved due to financial reasons

cancer (24 decks)

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