Hallmarks of CML

Question 1

What 3 things are required for a translocation event?

Answer 1

• DNA double strand break (DSB)
• Appropriate DNA DSB repair mechanism e.g. homologous recombination
• A homologous template DNA to repair the damage which requires temporal and spacial juxtaposition

Question 2

What causes recurrent formation of BCR-ABL1 fusion oncogene? (4)

Answer 2

• Neves et al, 1999 showed that BCR and ABL1 are closely localised in late S/G2 phase
• DNA DSBs are often formed during DNA replication due to stalling and collapse of replication forks when they encounter single strand breaks
• Homologous recombination is the favoured repair pathway in S phase
• All contributes to a translocation rearrangement

Question 3

How many BCR-ABL1 fusion proteins are possible in CML? (2)

Answer 3

• 3 based on 3 breakpoints in BCR
• Contribution of ABL1 is always the same

Question 4

How are CML patients monitored for relapse?

Answer 4

MRD monitoring by qPCR across the fusion site of the BCR-ABL1 transcript

Question 5

Why is it important to identify the type of fusion gene at diagnosis? (2)

Answer 5

• Type of fusion influences the clinical phenotype and prognosis
• Patients are monitored by qPCR following treatment and the PCR primers are designed to anneal either side of the BCR-ABL1 junction which differs for the 3 types of fusion (MRD monitoring)

Question 6

Where is the double strand breakpoint in the ABL1 gene? (2)

Answer 6

• Can occur in 3 locations but usually between exons 1b and 1a
• Alternative splicing means contribution of the ABL1 gene to the fusion oncogene is the same for all 3 fusions

Question 7

How many exons does ABL1 have?

Answer 7

11

Question 8

How many exons does BCR have?

Answer 8

23

Question 9

What are the 3 fusion proteins seen in CML?

Answer 9

• p210
• p190
• p230

Question 10

What determines the type of fusion protein generated by BCR-ABL1 fusion?

Answer 10

The breakpoint in BCR

Question 11

How is the p210 fusion protein formed? (3)

Answer 11

• Break occurs in the BCR major region between exons 12 and 16
• Splicing results in exon 13 or 14 only ever being fused to ABL1 gene
• Most common fusion gene accounting for 99% of all CML cases

Question 12

How is the p190 fusion protein formed? (3)

Answer 12

• Break occurs in the BCR minor region between exons 1 and 2
• Detected at low levels in 90% of CML cases due to alternative splicing of the p210 transcript
• p190 fusion proteins from genomic fusion are observed in acute lymphoid leukaemia

Question 13

How is the p230 fusion protein formed? (4)

Answer 13

• Break in BCR occurs between exons 19 and 21
• Extremely rare
• Slightly worse prognosis than p210 fusion
• Phenotype is similar to CNL (another MPN) so genetic investigation is needed to correctly classify

Question 14

What other MPN is the morphology of CML similar to?

Answer 14

• Essential Thrombocythaemia (ET)
• Can be confused prior to genetic investigation

Question 15

How does BCR-ABL1 fusion protein cause leukaemia? (3)

Answer 15

• ABL1 is a tyrosine kinase
• The clamp domain changes conformation to turn the kinase domain on and off by blocking the kinase domain to prevent it docking ATP for phosphorylation of other proteins
• Fusion to BCR means the clamp can’t inactivate the kinase domain of ABL1 rendering it constitutively active

Question 16

What are the features of the BCR-ABL1 fusion protein? (5)

Answer 16

• Proline rich region which can interact with adaptor proteins
• DNA and actin binding domains
• Tyrosine kinase domain
• Clamp domain
• Domain which regulates cellular localisation (nucleus or cytoplasm)

Question 17

How does the clamp region regulate kinase activity? (6)

Answer 17

• Positioning of the activation loop in the active site in the open form allows ABL1 to receive a phosphate group from ATP which it can transfer to other proteins (phosphorylation)
• This stabilises the proline rich regions which aids interaction with adaptor proteins
• The activation loop is usually in the closed form which prevents ABL1 accepting phosphate groups from ATP
• This destabilises the proline rich region so ABL1 can’t dock with other proteins (turned off)
• Phosphorylation of the clamp region is regulated by PDGF which regulates the conformation of the activation loop
• BCR fusion to ABL1 holds the activation loop permanently in the open form

Question 18

How is CML treated? (6)

Answer 18

• Used to use tyrosine kinase inhibitors
• Imatinib is now prescribed to all CML patients
• Competes with ATP for the binding pocket on the fusion protein and binds permanently which tucks away the activation loop and shuts off the kinase activity of the fusion protein
• 90% remission rates
• Minimal off target interactions
• 1 pill a day

Question 19

What is the downside of Imatinib? (2)

Answer 19

• Expensive (£20 000 per patient per year)
• Mutations in the BCR-ABL1 kinase domain may cause resistance to Imatinib (there are backup drugs in this case)

Question 20

How does BCR-ABL1 activate mitogenic signalling? (2)

Answer 20

• Results in activation of pathways including JAK-STAT, PI3K, C-Myc and Ras-MAPK
• Therefore BCR-ABL1 causes sustained proliferative signalling and evasion of growth suppression resulting in bone marrow hypercellularity and leukocytosis

Question 21

How is BCR-ABL1 involved in Ras-MAPK signalling? (5)

Answer 21

• BCR-ABL1 can autophosphorylate tyrosine residue Y177 which recruits GRB2
• Then recruits SOS
• Complex forms at the plasma membrane and activates Ras
• Activation of Ras-MAPK cascade
• Results in phosphorylation of RB/E2F complex which inactivates it and allows cell cycle progression

Question 22

How does BCR-ABL1 cause suppression of apoptosis? (3)

Answer 22

• Activates Ras which activates Bcl-2 (anti-apoptotic, prevents release of cytochrome C from the mitochondria)
• Activates JAK-STAT which activates Bcl-XL (anti-apoptotic)
• Therefore can resist cell death, resulting in bone marrow hypercellularity and leukocytosis

Question 23

How does BCR-ABL1 cause altered cell adhesion? (5)

Answer 23

• Adhesion to bone marrow stroma suppresses haematopoietic proliferation (evasion of growth suppression)
• Mediated by beta-1 integrins
• CML cells have beta-1 integrin variant which has anti-adhesion properties
• BCR-ABL1 phosphorylates Crk1 which promotes cellular motility
• Therefore activates invasion and metastasis as reduced adhesion causes enhanced motility, blasts are able to leave the bone marrow

Question 24

How does BCR-ABL1 cause loss of genome stability? (4)

Answer 24

• Telomeres are shorter than expected in CML and shorten with disease progression
• Progressive shortening leads to chromosome instability which elevates mutation rates
• BCR-ABL1 linked to decreased BRCA1 expression which causes dysregulation of the mitotic spindle (= aneuploidy) and loss of homologous recombination for double strand breaks (= high levels of DNA damage)
• CML cells therefore don’t have replicative immortality but do have genomic instability

Question 25

How are platelets involved in CML? (3)

Answer 25

• Thrombocytosis is common in CML
• Platelets can ‘cloak’ tumour cells to facilitate metastasis and evade the immune system
• Platelets release pro-angiogenic factors to drive angiogenesis of the bone marrow