Oncogenes and Chromosomal Aberrations The Story of Abl and the Philadelphia Chromosome

Wikipedia The Hematopoietic System myeloidlymphoid self renewal granulocytes

Leukemia – malignancy of the white blood cells and their precursors. myelogenous leukemias lymphocytic leukemias self renewal

Molecular Cell Biology Lodish et al. Fig 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 Biology Lodish et al. Fig CML arises in a stem cell that is a granulocyte precursor.

Chronic Myelogenous Leukemia (CML) Annual incidence: 1/100,000 people (~15% of all leukemias) Median age: 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 1998 Upon receiving the Lasker Award What caused this chromosome aberration?

A chromosomal translocation triggers CML healthy individualleukemic patient Chr. 9 Chr. 22 9; 22 Translocation The 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 ALL 2-5% of childhood ALL In adults prognosis is very poor (Only % 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,

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. 22 abl 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 cells Human CML cells

The Philadelphia chromosome translocation fuses the bcr and abl genes Chr. 9 Chr. 22 abl bcr bcr-abl 9; 22 Translocation fuses 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 ablbcr

fusion 9 abl/bcr fusion 22 bcr/abl abl bcr Fluorescence In Situ Hybridization (FISH) a tool for diagnosing CML BCRABL 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 SH3 FG SH2 kinase Actin- binding FG Gag v-Abl FG Bcr 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 Abl Distinct 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 SH3 FG SH2 kinase Actin- binding FG Gag v-abl FG Bcr 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 FG SH2kinase Actin- binding FG 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-Abl white: cells expressing Bcr-Abl and a DN Rac mutant adhesion motility Calbiochem

Bcr-Abl Cytoskeleton/ adhesion defects S G 2 M 1 G G0G0 Apoptosis Stem cell turnover Proliferation & differentiation Bcr-Abl affects multiple cell functions. Adapted from Jörgensen, 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)