Genetics of Chronic Myelogenous Leukemia (CML)

Question 1

What is the oncogenic mechanism and treatment for CML?

Answer 1

• Genomic translocation t(9;22) creates new gene encoding constitutively active tyrosine kinase
• Targeted molecular therapy based on mechanism - tyrosine kinase inhibitors - Imantinib (Gleevac)

Question 2

What are important things to remember about how CML develops?

Answer 2

• CML arises from defects in normal neutrophil differentiation pathway
• BCR-ABL genomic translocation = genetic basis for defects
• Fundamental defect in CML, BCR-ABL fusion protein = exploited to create therapy

Question 3

What are the most abundant white cells in circulation?

Answer 3

Neutrophils!

Question 4

What do we need to regulate proliferation of neutrophils?

Answer 4

• It’s critical for fighting infection
• Short lived
• Generated continuously in the bone marrow
• Job of neutrophil lineage to generate neutrophils in a regulated fashion

Question 5

What should you remember about stem cells?

Answer 5

• Undergoes self-renewal
• Pluripotent - capable of generating all lineages
• Capable of proliferation

Question 6

What should you remember about progenitor cells?

Answer 6

• Capable of proliferation but not self renewal
• Multipotent - can generate more than one lineage
• Capacity becomes narrower as progresses down pathway

Question 7

What should you remember about committed cells?

Answer 7

• Do not proliferate

- Only one fate, to generate the next step in the path to the neutrophil

Question 8

Self renewal and proliferation are tightly regulated by. . .

Answer 8

. . .extracellular signaling from bone marrow!

Question 9

How does differentiation of the neutrophil lineage occur?

Answer 9

-Bone marrow secretes factors specific for different lineages
-Factors bind receptors on progenitor cells
-SIgnal activates cell-type specific transcription factors
Ex: Progenitor cell - GMP
Cytokine - G-CSF
Transcription factor - CEBPalpha
Result - Neutrophil differentiation

Question 10

What spurs the neutrophil lineage to go into CML chronic phase?

Answer 10

-BCR-ABL1 genomic translocation in hematopoietic stem cell
-Creates genetic chimera of parts of BCR (breakpoint cluster region) gene and ABL1 (ableson tyrosine kinase) gene
-BCR-ABL1 fusion protein is a constitutively active tyrosine kinase that activates proliferation and blocks apoptosis in absence of extracellular signals
-Mutation arises in hematopoietic stem cell but is passed down to all progeny
Expression fo BCR-ABL fusion protein from genomic translocation disrupts normal neutrophil differentiation.

Question 11

What is special about BCR-ABL1 progenitor cells?

Answer 11

• Proliferate more than other cells, survive longer!
• Have more opportunity to acquire more mutations which can make cells more oncogenic
• DO NOT self renew
• Continue to differentiate

Question 12

What is the CML chronic phase like?

Answer 12

• Expansion of progenitor and committed cells
• Mature cells still produced
• Disease relatively mild
• BCR-ABL1 translocation in hematopoietic stem cell –> Increased proliferation and survival of progenitor cells, opportunity to acquire more mutations

Question 13

What is the blast phase like?

Answer 13

-GMP acquires ability tot self renew, acquires block to differentiation –> huge expansion of blasts, 30% extramedullary

Question 14

What should you remember about CML accelerated and blast phases?

Answer 14

• BCR-ABL1 alone does not cause progression from chronic to blast phase
• Additional changes needed:
• –GMP acquire self renewal capacity - Wnt beta cattiness signaling is activated
• –Differentiation is blocked - translation of CEBPalpha is inhibited

Question 15

What is the outcome of CML accelerated and blast phases?

Answer 15

• Extreme expansion of blasts
• Production of functional mature cells blocked
• Severe disease

Question 16

What is the mechanism of the BCR-ABL1 translocation?

Answer 16

• Double stranded breaks occur in two chromosomes at specific breakpoints
• -Two most common:
• –p210 in CML, most common
• –p190 in some ALL, less common
• -Why these sites is not understood
• Mechanism used is non-homologous end joining (NHEJ)
• -Common DNA repair mechanisms
• -Doesn’t require extensive homology
• Ends of different chromosomes are joined

Question 17

What is BCR?

Answer 17

• Key functional domain for normal protein - Ser/Thr kinase domain
• Key functional domains for oncogenic fusion protein coiled-coil domain tyrosine 177
• Normal function - role in inhibition of some inflammatory responses

Question 18

What is ABL1?

Answer 18

-Key functional domain for both normal and oncogenic fusion protein is tyrosine kinase
-In normal protein, kinase is held inactive unless activated by external signals
-Long chain fatty acid myristate maintains inhibition
-Normal functions: role in DNA repair cytoskeletal organization
NEITHER BCR or ABL ALONE ARE ONCOGENIC!!

Question 19

What is the difference between the ABL and BCR-ABL1 kinases?

Answer 19

ABL - require extracellular signaling - then protein opens up conformation and this makes it possible for the kinase to dimerize
BCR-ABL1 - dimerization can occur WITHOUT AN EXTRACELLULAR SIGNAL –> this causes phosphorylation of tyrosine 177 (occurs in CML cells)

Question 20

ABL tyrosine kinase phosphorylates Y177 in CML. Phosphorylation causes:

Answer 20

• Dysregulated proliferation

- Protection against apoptosis

Question 21

Is BCR a tyrosine kinase?

Answer 21

NO

Question 22

How is CML treated?

Answer 22

• Targeting it BCR-ABL fusion protein
• CML is treated with BCR-ABL specific tyrosine kinase inhibitors
• Most BCR-ABL mutated cells can be eliminated
• Transition to blast phase can be blocked

Question 23

What is Imatinib?

Answer 23

• Prototype of BCR-ABL specific tyrosine kinase inhibitors
• It specifically inactivates ABL1 kinase by blocking ATP binding
• Prevents selective advantage that comes from activated oncogenic signaling pathways
• Inactivation of BCR-ABL in mutant cells also causes apoptosis (cells became dependent on BCR-ABL for proliferation and anti-apoptosis signaling)
• Loss of BCR-ABL mutant cells allows normal cells to repopulate

Question 24

How effective is Imatinib?

Answer 24

-5 yr survival for individuals with newly diagnosed chronic phase CML, treated with tyrosine kinase inhibitors - 85-90%
Natural history of disease w/out treatment:
—Chronic phase lasts

Question 25

If BCR-ABL translocation arises in HSC, why does leukemia arise in the neutrophil lineage?

Answer 25

Specific signaling opportunities created by BCR-ABL protein benefit neutrophil progenitor more than others

1. Some lymphocytic leukemias do arise from BCR-ABL translocation but he fusion is at a different site, so many initiate different signaling
2. In CML, a subset of B cells do carry the BCR-ABL translocation but are not leukemic

Question 26

What are the limitations of Imatinib?

Answer 26

1. Many develop secondary resistance to Imatinib primarily due to mutations in BCR-ABL (trying to create 2nd and 3rd line tyrosine kinase inhibitors)
2. BCR-ABL1 tyrosine kinase inhibitors not effective against blast phase disease
3. CML stem cells are resistant to tyrosine kinase inhibitors (affects even successful treatment, even a few stem cells are sufficient to restart disease if treatment stops
   - -> so Imatinib must be taken for life!!