CML pt 2 Flashcards
In CML, what is possible? what does this impact?
3 fusions are possible and these have impact on expected prognosis based on 3 breakpoints in BCR
ABL1 breaks in the same region for all
What does Alternate splicing id BCR-ABL1 transcript result in?
Splicing of the BCR-ABL1 transcript results in fusion of BCR to exon a2, regardless of where the break in ABL1 occurred.
Due to alternate splicing, contribustion of ABL1 to BCR/ABL1 is always the same
WHat does the BCR breakpoint determine?
determines the type of fusion protein generated.
What is essential for CML diagnosis?
BCR-ABL1 fusion oncogene.
How many types of BCR/ABL1 fusion are there? How are they classified?
3 different types
Classified according to the molecular weight of the encoded protein
Each fusion oncoprotein is encoded by a different fusion oncogene.
Why is is essential to identify type of fusion gene at diagnosis?
Type of fusion influences the clinical phenotyoe and prognosis
Following treatment, patients are monitored by qPCR. Primers are designed to anneal either side of the BCR/ABL1 junction.
what is MRD?
Minimal Residual Disease
MRD monitoring by pPCR across fusion site
What are ABL1 genomic rearrangements that cause CML?
Usually result in breakage of the ABL1 gene in a single locus located between 1b and 1a. Meaning contribution from the ABL1 gene to the fusion oncogene is the same for all three fusions.
What are the diffrence between the three fusions in BCR gene?
Due to location of the other break in the BCR gene.
ABL1 gene - draw it!
9q34
11 exons
Transcript: ~5.5Kb
1130aa protein
BCR gene - draw it!
22q11.2
23 exons
Transcript: ~6.8Kb
1271aa protein
What are the three different break point clusters in BCR gene? what do they give rise to?
Between exons 1 and 2, between exons 12 and 16 and between 19 and 21.
Threee different fusion transcripts.
What are the three proteins encoded by the fusion transcripts
P210 (210kDa)
P190 (190kDa)
230 (230kDa)
How is P210 formed?
when the ABL1 gene is fused to the in the BCR Major break point cluster, which is located between exons 12 and 16. Due to splicing exons 12, 15 and 16 are removed from the fusion transcript by splicing, which means exons 13 or 14 of BCR are only ever fused to the ABL1 gene.
Whats the most common fusion gene seen in CML?
210
99% of all cases.
The two transcripts that are generated are both referred to as P210 as the proteins generated are of vertually indistinguishable size.
How is p190 formed? WHy is it complicated?
when breaks occur in BCR at the minor region, which is located between exons 1 and 2.
To make matters more complictaed the transcript that encodes the p190 protein is actually detected 90% of CML cases – but at low levels, and is thought to be due to alternate splicing of p210 transcript.
Where are P190 more commonly observed? What does this mean?
P190 fusions proteins that are expressed from the corresponding genomic fusion, are actually more commonly observed in cases of acute lymphoid leukaemia.
This is why the classification of leukaemia requires a multidisciplinary approach – we need the haematological and histological evidence in order to provide context for genetic data.
How is P230 fusion protein generated?
when the break in BCR occurs between exons 19 and 21.
This is extremely rare
Whats prognosis like for patients with P230
Slightly worse for patient in comprison to those with P210.
Phenotyoe similar to those with CNL.
Genetic investigation of blood malignancies are essential to correctly classify disease.
What other disease is the morphology of CML similar to?
very similar to another MPN – Essential Thrombocythemia (ET) and prior to a genetic investigation – the two can be confused.
What is BCR fusion thought to do?
BCR fusion is thought to inhibit the interaction of SH2/3 with the kinase, resulting in a constitutive activity. Mediated through dimerisation via the BCR protein unit
What determines cellular localisation of the fusion protein?
Nuclear import and export signals dictate cellular localisation of the fusion protein, which is largely cytoplasmic, but capable of entering the nucleus.
What does the BCR/ABL1 fusion protein consist of?
NH3
BCR
SH3
SH2
Kinase domain
Tyrosine kinase
Actin binding
DNA binding
Region which mediates interaction with adaptor proteins (proline rich)
SH2/3 clamp
Interaction with C-terminal end of the kinase domain. In this conformation (SH2/3 clamp blocking kinase domain) ATP and substrate proteins are blocked from the active site due to conformational changes in the kinase domain.
Charactersitics of BCR/ABL1 fusion protein
DNA binding and actin binding capability
Specific motif able to facilitate the fusion proteins binding to toher proteins in the cell.
What is BCR/ABL1
A tyrosine kinase
Phorphorylation of Try residue 117 has been
What has been shown to be crucial to the function of oncoprotein?
. Phosphorylation of Tyr reside 117 has been shown to be crucial to the function of the oncoprotein and is therefore essential for leukaemogenesis
Function of the clamp in normal protein
this domain is mobile and change conformation to turn the kinase domain on and off. Normally i.e. in ABL1 (not the fusion), this domain is arranged in such a way to block the kinase domain of the protein, and thus prevent it from docking with ATP – which you should all know is essential for the phosphorylation of other proteins by kinases. The function of the ABL1 protein is therefore tightly regulated…
WHat happens to clamp when BCR has fused to ABL1
the clamp is not longer able to hold the kinase domain in an inactive state… The result is a constitutively active kinase domain..
How is this fusion sufficient to cause CML (Leukaemia)
Think of ABL1 like a light switch – which is normally off and only turned on once certainly tightly controlled pathways have been activated. Once BCR has been fused to it – the switch is turned on PERMENANTLY. Control is lost! And this is sufficient to cause the leukaemia
ABL1 open conformation
Precise conformational positioning is needed to position ATP appropriately for Kinase activity – OPEN CONFORMATION
In this conformation, Activation loop is able to receive a phosphate group from ATP, that is donated to a substrate protein
Phosphorylation of the activation loop stabilizes protein conformation facilitate substrate interaction
in other words - the Activation loop in the active site of the kinase domain is positioned is such a way that it is able to receive a phosphate group from ATP, and is thus able to doc with other protein and transfer this group to them in a reaction known as phosphorylation.
What is the positioning of the activation loop in open form also thought to do?
is also thought to stabilize the proline rich region of the protein, which helps to interact with adaptor proteins
How is the activation loop usually held?
What is this though to help?
in a closed form, which prevents it from accepting phosphate groups from ATP.
This conformation also helps to destabilise the proline rich domain which helps ABL1 to doc with other proteins. The result is that ABL1 is basically turned off.
What is though to regulate conformation of activation loop?
Under normal circumstances, conformational changes in Abl to present the activation loop are induced by Platelet derived growth factor (PDGF) through phosphorylation of residues in the clamp region
This is why when BCR is fused to the protein – the activation loop is held permanently in the open form.
WHat drug is prescrbed to all patients with CML
Imatinib
What does Imatinib do? How specfic is it?
Binds to the ATP binding pocket of Abl1 kinase, forcing the activation loop into the inactive form that cannot accept a phosphate group from ATP. Kinase inactivation.
This drug competes with ATP for it’s binding pocket in the fusion protein, and binds to it irreversibly. Once bound, the activation loop is tucked away within the protein – which prevents it from accepting a phosphate group from ATP – and thus shuts off the kinase activity of the protein.
Imatinib has a highly specific interaction with few off target integrations
How often do patient take Imatinib and how much does it cost
≈90% remission rates in chronic phase CML, achieve within 6 to 18 months of the start of treatment
1 pill per day, cost approx. £20,000 per patient per year and accounts for 1% of all cancers in the UK
What might cause resistance to Imatinib
Mutations in the kinase domain of BCR/ABL1 may cause resistance to imatinib.
3 ways BCR/ABL1 protein interactions dictate malignant transformation? (i.e cause Leukaemia)
3 ways fusion protein promotes uncontrolled cell growth and proliferation
Altered adhesion to BM stroma
Constitutive activation of mitogenic signalling
Inhibition of apoptosis.
Alitered adhesion to bone marrow stroma
it is able to reduce the affinity that stem cells have for the bone marrow stroma. The interaction between the stoma and stem cells normally inhibits their proliferation, so by releasing cells from this interaction, leads to enhanced proliferation. It’s also thought to facilitate the abnormal stem cells to leave the bone marrow and end up in the peripheral blood.
Proliferation of haematopoietic cells is inhibited by adhesion to the BM stroma
Abl1 normally acts in the intracellular transduction of stromal inhibitory signals
Constituative activation of mitogenic signalling
Kinase activity of Abl1 is constitutively activated when fused to BCR
There are several substrates of BCR/ABl1 involved in the signal transduction of external growth factors
Name the 4 proto-oncogenes which are phosphorylated consistency due to the constitutively active ABL1 kinase domain on the fusion protein.
KIT, JAK, MYC and RAS protein.
They have important roles in cell proliferation signalling.
What is invovled in the BCR-ABL1 fusion portein ability to protect cells from apoptosis.
RAS.
RAS activates BCL2 (a protein) which has anti-apoptotic activity.
SO what is the BCR-APL1 fusion protein able to do?
Enhance proliferation and at same time protect abnormal cells from dying.
What Imatinibs invovlement in acivity of BCR-ABL1 fusion gene?
Imatinib turns off the activity of BCR-ABL1. Over time the masses of abnormal cells formed by abnormal proliferation - eventually apoptose, and the cellularity of the peripheral blood and bone marrow gradually return to normal.
What is used to monitor these patients?
FISH is used to monitor the bone marrow down to about 1% disease load.
Patients are then switch to qPCR – and fusion transcript levels are then monitored down to less than 1 10th or even 1 100th of a percent.
Patients are monitored regularly for evidence of up-regulation of transcript levels – which is a hallmark of relapse – most likely caused by a mutation in the BCR-ABL1 fusion protein, which means that imatinib is no longer able to bind.
Describe what could happen in a replased CML patient
Through karyotype analysis
Original markers of disease
Evidence of clonal evolution.
- translocation between long arm of chromosome 5 and short arm chromosome 12
- 3 copies of 19 (commonly observed in CML blast phase)
Name common additonal abnormalities seen in transforming CML
- monosomy 7 or deletion of the q arm.
- trisomy 8, which is thought to be a mechanism by which the cMYC oncogene is unregulated
- isochromosome 17q, which effectively deletes one copy of the TP53 TS, on 17p
- trisomy 19
- trisomy 21 – which is also thought to up regulate key oncogenes.
- gain of additional copies of the Philadelphia chromosome – which is thought to result in higher levels of BCR-ABL1 expression – which drives the cancer ever forward
Further reading
t(9;22) is “recurrent”
Other translocations are possible
WHY IS t(9;22) OBSERVED MORE FREQUENTLY?
