Oncogenes and Chromosomal Aberrations

The Story of Abl and the Philadelphia Chromosome

Wikipedia

The Hematopoietic System

myeloid lymphoid

self renewal

self renewal self renewal

granulocytes

Leukemia – malignancy of the white blood cells and their precursors.

myelogenous leukemias lymphocytic leukemias

self renewal

self renewal self renewal

Molecular Cell BiologyLodish et al. Fig. 24.1

Leukemia is characterized by hyper-proliferation of immature white blood

cells

Acute (fast, children and adults)Chronic (slow, adults)

Figure 8.32 The Biology of Cancer (© Garland Science 2007)

Chronic Myelogenous Leukemia (CML)

elevated numbers of differentiated neutrophils

less differentiated blast cells are “taking over”

the blood is full of blast cells

basophil

blast

neutrophils and precursors

promyelocyte

Molecular Cell BiologyLodish et al. Fig. 24.1

CML arises in a stem cell that isa granulocyte precursor.

Chronic Myelogenous Leukemia (CML)

Annual incidence: 1/100,000 people

(~15% of all leukemias)Median age: 30-60 yrs

Median survival: 4 yrs with conventional chemotherapy

6 yrs with aIFN therapy; Allogeneic (human) bone marrow transplantation

may cure the patient.

CML - Pathology

• Chronic Phase– Accumulation of myeloid cells

• bone marrow• peripheral blood• spleen and liver

• elsewhere• Accelerated Phase

– Further genetic changes in the stem cell leading eventually to acute transformation (i.e.

acute leukemia) and death

“The findings suggest a causal relationship between the chromosome abnormality observed and chronic granulocytic leukemia.”

Peter Nowell

1960 Nowell and Hungerford find that one copy of chromosome 22 is extremely short in CML

patients

The tiny Philadelphia chromosome is a clear and consistent marker of CML.

Nowell and Hungerford, University of Pennsylvania, Philadelphia

Janet Rowley in 1998Upon receiving the Lasker Award

What caused this chromosome aberration?

A chromosomal translocation triggers CML

healthy individual leukemic patient

Chr. 9

Chr. 22

9; 22 TranslocationThe Philadelphia

chromosome

Karyotype courtesy of L. J. Beauregard,Eastern Maine Medical Center

A characteristic karyotype indicates CML

Acute Lymphoblastic Leukemia (ALL)

Affects precursor of leukocytes(B and T cells)

Ph+ chromosomes in 20% of adult ALL2-5% of childhood ALL

In adults prognosis is very poor(Only 35- 40% of adults with ALL survive 2 years)

Bone marrow transplant the only long term treatment

Figure 2.8a The Biology of Cancer (© Garland Science 2007)

tumors exhibiting a pre-B lymphocyte marker

The v-abl containing retrovirus was recovered from a tumor

found in mice infected by Moloney Leukemia virus

Abelson and Rabstein, Cancer Res 30, 2213 (1970)

The Abelson viral oncogene

v-abl is cloned

Rosenberg and Witte, 1978-1980

The v-abl sequence is used as a probe to find homologous cellular sequences.

Next step: using v-abl and c-abl as probes to map c-abl to a chrmosome.

How?

Mouse-human somatic hybrids.

mouse cell linehuman cells

abl is mapped to chromosome 9

healthy individual leukemic patient

Chr. 9

Chr. 22abl

9; 22 Translocation

De Klein et al. Nature 300, 765 (1982)

abl

and is translocated to the Philadelphia chromosome in CML patients

near the translocation site of CML patients.Does abl translocate in CML patients?

mouse cellsHuman CML cells

The Philadelphia chromosome translocation fuses the bcr and abl genes

Chr. 9

Chr. 22abl

bcr bcr-abl

9; 22 Translocationfuses bcr and abl

Groffen et al. Cell 36, 93 (1984)

healthy individual leukemic patient

bcr= (breakpoint cluster region)

Figure 4.15a The Biology of Cancer (© Garland Science 2007)

The translocation results in production of a fusion protein that joins the amino-terminal end of the Bcr protein to most

of the Abl protein

Figure 4.15b The Biology of Cancer (© Garland Science 2007)

The translocation results in production of a fusion protein that joins the amino-terminal end of the Bcr protein to most

of the Abl protein

Daley et al. Science (1990)

Bcr-Abl is sufficient to

cause leukemia !!

Fluorescence In Situ Hybridization (FISH)a tool for diagnosing CML

abl bcr

fusion 9abl/bcr

fusion 22bcr/abl

ablbcr

Fluorescence In Situ Hybridization (FISH)a tool for diagnosing CML

BCR ABL

The current methd: PCR.

Nagar JN(2007)

Abelson kinase

A fatty-acid modified and actin-binding non-receptor tyrosine kinase

So, what makes Bcr-Abl sufficient to cause leukemia?

Src is normally inactive due to intramolecular inhibition.

c-Abl

SH3F G

SH2

kinase

Actin-binding

F GGagv-Abl

F GBcr

Bcr-Abl

myristate

How does Bcr-Abl cause cancer?

Nagar et al. Cell 112:859 (2003)

The structure of Abl reveals a novel mode of intramolecular inhibition

the N-terminal myristate binds to a pocket on the kinase domain

Harrison Cell 112, 737 (2003)

Src and AblDistinct yet analogous modes of regulation

A multistep mechanism for activating Src

Harrison Cell 112, 737 (2003)

A proposed mechanism for activating Abl

Harrison Cell 112, 737 (2003)

c-Abl

SH3F G

SH2

kinase

Actin-binding

F GGagv-abl

F GBcr

Bcr-Abl

myristate

Abl w/o a myr residue is oncogenic

But what does Abl normally do?

Insights From the Mouse Model

• abl mutant mice are viable but have a shortened lifespan.They also have problems with:

male fertility B cell maturation

osteoblasts and bone formation

• Truncation of C-terminus leaving an intact kinase has same phenotype as the null mutant

Abelson Has a Twin Brother

SH3

F G

SH2 kinaseActin-

binding

F G

89% 94% 27%34%

Abl

Arg

Are Abl and Arg redundant?

• arg mutant mice have behavioral defects (Arg is expressed in the brain at high levels)

• abl; arg double mutants have defects in neural tube and are embryonic lethal

Wild-type abl; arg

modified from Weisberg et al. Nature Reviews Cancer 7 (2007)

Constitutively active Abl promotes proliferation and survival even in the

absence of growth factors.

Abl at the Cellular Level

Focal adhesion proteins are phosphorylated by Abl.

Rac is required for Bcr-Abl–dependent proliferation and invasiveness.

Skorski et al. PNAS, 95 (1998)

24h

black: cells expressing Bcr-Ablwhite: cells expressing Bcr-Abl and a DN Rac mutant

adhesion

motility

Calbiochem

Bcr-Abl

Cytoskeleton/adhesion defects

SG2

M

1GG0

Apoptosis

Stem cell turnoverProliferation & differentiation

Bcr-Abl affects multiple cell functions.

Adapted from Jörgensen, 2001. Hem. Onc.

Why are increased motility and survival and reduced adhesion relevant for

leukemias?

Gabrilovich Nature Reviews Immunology 4 (2004)

Imatinib (Gleevec)or, why should we do basic studies?

In chronic phase 30% of patients have no detectable sign of disease, 50% in remission

Also has some effect on patients in blast crisis for whom other treatments are not effective

Plus—relatively few serious side-effects

STI571

Gleevec blocks the ATP binding site of the kinase domain

Table 4.5 The Biology of Cancer (© Garland Science 2007)