Tyrosine kinase in cancer

Question 1

How many TKs are there in the human genome?

Answer 1

~90

Question 2

What are the two types of TKs?

Answer 2

Cytoplasmic and receptor TKs

Question 3

what induces activation of RTKs?

Answer 3

oligomerisation (dimerisation etc)

Question 4

What changes occur to an RTK following its activation

Answer 4

Following ligand binding, a conformational change induces the activation of the TK domain. This leads to transphosphorylation of the partner receptors cytoplasmic domain.

Question 5

How do most cytoplasmic TKs associate with receptors that lack intrinsic TK activity

Answer 5

Non-covalently.

Question 6

How do cytoplasmic TKs become activated.

Answer 6

Similar RTKs, oligomerisation induces transphosphorylation of TKs which then phosphorylate the receptor itself.

Question 7

Give 5 ways in which tyrosine kinases get inappropriately activated in malignant disease

Answer 7

1. Mutation resulting in constitutive activation or hypersensitivity
2. By fusion to other proteins
3. By receptor overexpression leading to reduced threshold for signalling
4. By activation of upstream signalling pathways
5. By autocrine production of growth factors

Question 8

Give an example of a mutation resulting in constitutive activation or hypersensitivity

Answer 8

EGFR in lung cancer, FLT3 in AML, JAK2 in myeloproliferative neoplasms

Question 9

Give an example of a fusion event to another protein that causes malignant disease

Answer 9

BCR-ABL in CML, EML-ALK in lung cancer

Question 10

Give an example of receptor overexpression leading to a reduced threshold

Answer 10

EGFR, HER2 in various cancers

Question 11

Give an example of upstream signalling activation pathways that lead to malignancy

Answer 11

B-cell receptor and subsequent BTK activation

Question 12

Give an example of autocrine production of growth factors that leads to malignancy

Answer 12

IGF1 production by numerous tumours

Question 13

How are chromosomal translocations detected?

Answer 13

Cytogenetics, FISH or PCR which spans the break point of the fusion.

Question 14

How do chromosomal translocations induce malignancy?

Answer 14

Through inappropriate activation of target gene(s) or in the wrong cellular location (changes in protein localisation due to fusion may induce the activity of inappropriate pathways

Question 15

Which hallmarks of cancer does TK activation mainly contribute to

Answer 15

Sustained proliferation and reduced sensitivity to cell death/antiproliferative signals.

Question 16

What are the ideal characteristics of a TK inhibitor

Answer 16

1. Target is activated by genomic alteration (tumour is more likely to be dependent on this type of lesion)
2. Target is present in all the cells of the tumour, not just sub-clones
3. The candidate has good drug-like properties (pharmacokinetics etc)

Question 17

What is a small molecule

Answer 17

A a low weight chemical which can pass through the plasma membrane and get into the cell to give its effect

Question 18

How do most small molecule inhibitors have their effect on TKs?

Answer 18

Compete for ATP binding, and less commonly or substrate binding.

Question 19

How do monoclonal antibodies target RTKs

Answer 19

Prevent ligand binding and/or induce receptor downregulation/internalisation or trigger ADCC

Question 20

How does chronic myeloid leukaemia present clinically?

Answer 20

Excessive production of mature, differentiated white blood cells (neutrophils). Enlargement of the spleen and liver

Question 21

What is the pathological hallmark of CML?

Answer 21

The philadelphia chromosome

Question 22

What is the Philadelphia chromosome?

Answer 22

An abnormal chromosome 22. This is a translocation between genes on chromosome 9 and 22. Sequences of the BCR gene runs into almost the entire coding region of ABL (a tyrosine kinase).

Question 23

In what cell of haematopoiesis does the BCR-ABL fusion event occur, and what is the effect of this.

Answer 23

In the initial haematopoietic stem cell. Main effect is to cause excessive proliferation - biased to neutrophils

Question 24

How does WT ABL kinase act? How does this change after fusion to BCR

Answer 24

Cytosolic kinase. Involved in DNA damage response. Highly regulated, quiescent kinase.
Complete disregulation, not confined to the nucleus so found throughout the cytosol and the ABL part phosphorylates multiple signalling pathway constantly. It also decreases apoptosis and increases susceptibility to DNA damage which increases likelyhood of developing mutations without the cell dying.

Question 25

Which phase represents the ML stage in which only the BCR-ABL fusion mutant is present

Answer 25

Chronic ML

Question 26

How does chronic (CML) progress into acute myeloin leukaemia (AML)

Answer 26

Amplify multiple chromosomes (Double Ph, Chr17, 19 abn)

Question 27

How can ATP binding site targeted drugs be specific for a single kinase?

Answer 27

Whilst the ATP binding site is almost identical between each kinase. The entrance to the pocket is very different across kinase families.

Question 28

What conformation suggests a kinase is active? how does this compare to inactive?

Answer 28

Inactive - DFG three amino acid sequence is inside the pocket when inactive. When active the DFG sequence flips outside the ATP binding pocket.

Question 29

What 4 types of interaction between drugs and TKs are there?

Answer 29

1 - bind the inactive conformation
Type 2 - Binds the active confirmation
Type 3 - Binds the adjacent ATP pocket
Type 4 - Binds the substrate binding site or the allosteric site causing a conformational change to the ATP binding pocket

Question 30

True or false- TKIs can be stopped after going into long term remission?

Answer 30

False - Only around 10-15% of CML patients can stop their therapy. In most cases once the treatment is stopped the amount of fusion protein present increases.

Question 31

How does clinical resistance to CML drugs occur and what does this tell you about the BCR-ABL fusion gene

Answer 31

New mutations occur in BCR-ABL, which confirms that this oncogene is critical for the development of the tumour to be maintained.
Also, to a lesser extent some patients gain BCR-ABL amplification.

Question 32

How do you tackle resistance to Imatinib

Answer 32

Increase the does of imatinib - especially in amplifcation
OR
Switch to a different tyrosine kinase inhibitor - eg dasatinib, nilotinib

Question 33

What treatment can be offered if the initial methods of tackling resistance fail to work?

Answer 33

Use alternative modality eg allogenic stem cell transplant
OR
Reduce emergence of resistance by treatment with combination

Question 34

What are the pros and cons for selectivity for a TK inhibitor

Answer 34

High specificity potentially validates the target and reduces off-target toxicity
Lack of selectivity may broaden the therapeutic indications, inhibit tumour growth by several mechanisms - may reveal novel pathophysiological mechanisms.