1. hyperproliferation of white blood cells
Leukemia is characterized by
hyperproliferation
of immature white blood cells
normal person
Leukemic patient
red blood cells
white blood cell
hyperproliferation of white blood cells

2. Different types of leukemia affect different stem cell types
and distinct stages in
their development
Molecular Cell Biology
Lodish et al. Fig. 24.1

3. 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 bone marrow transplantation
may cure the patient

4. Chronic myelogenous leukemia (CML)
Arises in a particular bone marrow stem cell =
The granulocyte precursor
Gives rise to neutrophils , basophils & megakaryocytes.
Neutrophils-- fight infection by phagocytosis
Basophils-- release immune modulators, e.g., histamines, Prostaglandins
Platelets- cell fragments of megakaryocytes.

5. CML arises in a stem cell that is a granulocyte precursor
Molecular Cell Biology
Lodish et al. Fig. 24.1

6. Upon receiving the Lasker Award
Nature :290-3
“A new consistent
chromosomal
abnormality in
CML identified by
quinacrine fluorescence
and Giemsa staining.”
Rowley JD.
Janet Rowley in 1998
Upon receiving the Lasker Award

7. A chromosomal translocation
triggers CML
Leukemic patient
Normal individual
Chr. 22
Chr. 9
9; 22 Translocation
The Philadelphia
chromosome

8. A characteristic karyotype indicates CML
Karyotype courtesy of
L. J. Beauregard,
Eastern Maine Medical Center

9. 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 very poor
(Only % of adults with ALL survive 2 years)
Bone marrow transplant
the only long term treatment

10. Abelson was first identified as the oncogene
carried by Abelson leukemia virus,
which causes pre-B cell Lymphoma in mice
Abelson and Rabstein, Cancer Res 30, 2213 (1970)

11. The v-abl containing
retrovirus was recovered
from a tumor found
in mice infected by
Moloney Leukemia virus

12. The Philadelphia chromosome
translocation fuses
the bcr and abl genes
normal individual
Leukemic patient
bcr
Bcr-abl
Chr. 22
abl
Chr. 9
9; 22 Translocation
fuses Bcr and Abl
De Klein et al. Nature 300, 765 (1982)
Groffen et al. Cell 36, 93 (1984)

13. 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
The Cell, G. Cooper, Fig

14. Fluorescence in situ hybridization (FISH) A tool for diagnosing CML
Zymed.com
5 ‘ of abl in situ probe
3’ of abl in situ probe

15. Abelson kinase A fatty-acid modified and actin-binding
non-receptor tyrosine kinase
Actin-binding
Myristate
SH2 F G
SH3
kinase

16. Oncogenic versions of Abelson
Abl
Actin-binding
SH2 F G
SH3
kinase
v-abl
Gag F G
Bcr-Abl
Bcr F G

17. Src is normally
inactive due to
intramolecular
inhibition

18. The structure of Abl reveals a novel mode of intramolecular inhibition
Nagar et al.
Cell 112:859 (2003)

19. Distinct yet analogous modes of regulation
Src and Abl
Distinct yet analogous modes of regulation
Harrison Cell 112, 737 (2003)

20. A multistep mechanism for activating Src
Harrison Cell 112, 737 (2003)

21. A proposed mechanism for activating Abl
Harrison Cell 112, 737 (2003)

22. Abl’s Kinase Domain In complex With the inhibitor Gleevac
Kuriyan lab website

23. Clinical Course: Phases of CML
Advanced phases
Chronic phase
Median 5–6 years stabilization
Accelerated phase
Median duration 6–9 months
Blast crisis
Median survival 3–6 months
Clinical Course: Phases of CML
CML progresses through 3 phases characterized by increasing refractoriness to therapy and
worsening clinical features and laboratory findings. These stages include chronic phase,
accelerated phase, and blast crisis. Although the majority of patients present in chronic phase
and then progress to accelerated phase, 25% to 40% of patients progress directly from chronic
phase to the terminal blast crisis phase without evidence of a transitional accelerated phase.1,2
Chronic phase. In the chronic phase, there are less than 10% blasts in peripheral blood and bone marrow, and the white blood cell (WBC) count at presentation is typically elevated to 20 x 109/L. Signs and symptoms may be mild initially and develop as the disease progresses. The chronic phase of CML may last 5 to 6 years before the disease accelerates.1,3
Accelerated phase. There are more than 10% to 15% (but less than 30%) blasts in either peripheral blood or bone marrow. Symptoms may increase and include unexplained fever, bone pain, splenomegaly, and hepatomegaly. Basophilia, decreased platelet counts, and cytogenetic progression may also be observed. The accelerated phase may last 6 to 9 months.1,3
Blast crisis. There are more than 30% blasts in peripheral blood or bone marrow and symptomatology is increased, especially relating to anemia and infection, central nervous system (CNS) disease, lymphadenopathy, and bleeding. Approximately 50% of patients have myeloid blast crisis, 25% have lymphoid blast crisis, and 25% are mixed.3 Patients with CML in blast crisis have a poor prognosis owing to the lack of effective therapy. This phase is rapidly fatal, with a median survival of 3 to 6 months.2,3
References
1. Hill JM, Meehan KR. Chronic myelogenous leukemia. Curable with early diagnosis and treatment. Postgrad Med ;106: ,
2. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology ;13:
3. Cortes JE, Talpaz M, Kantarjian H. Chronic myelogenous leukemia: a review. Am J Med. 1996;100:
Provided by: Gleevec.com

24. Blast crisis is thought to involve additional
genetic changes that are only beginning
to be characterized
Suggested events:
•Mutations in p53
•MSI2/HOXA9 fusion protein
•AML1/EVI-1 fusion protein
•Ras mutations

25. Therapy for CML: how do you evaluate whether the drug is working?
Hematologic Response Cytogenetic Response
– Complete: – Major:
Normal peripheral blood count Complete: 0% Ph+
No immature cells Partial 1-35% Ph+
– Minor:
36%–95% Ph+
Goals of Therapy for CML: Response Criteria
Response criteria in CML have been defined in terms of hematologic and cytogenetic responses.1,2
The first goal of therapy in patients with CML is to reduce the WBC count to near-normal levels.
Hematologic responses are defined by whether (1) peripheral cell counts have normalized and (2) immature myeloid cells are no longer present. Hematologic improvement must be maintained for 1 month to qualify as response. Disappearance of signs (eg, splenomegaly or hepatomegaly) and symptoms of disease is also required.
The second goal is to achieve a cytogenetic remission by reducing or eliminating cells that carry the Ph chromosome.
Cytogenetic responses are based on bone marrow analyses of dividing myeloid cells in metaphase to quantify the number of detectable Ph+ cells. Molecular techniques such as fluorescence in situ hybridization (FISH) and quantitative reverse transcription polymerase chain reaction (RT-PCR) are also used for detection of bcr-abl transcripts.2
Achieving a cytogenetic response has been associated with improved survival in interferon-alpha (IFN-) patients.3
A therapy that is well tolerated and has no or few side effects is also an important parameter.
References
1. Faderl S, Talpaz M, Estrov Z, et al. Chronic myelogenous leukemia: biology and therapy. Ann Intern Med ;131:
2. Sawyers CL. Chronic myeloid leukemia. N Engl J Med ;340:
3. Kantarjian HM, Smith TL, O’Brien S, et al. Prolonged survival in chronic myelogenous leukemia after cytogenetic response to interferon- therapy. Ann Intern Med ;122:
Modified from Gleevec.com

26. Therapeutic Options for CML
Allogeneic stem cell transplantation (SCT)
Interferon-alpha (IFN-)–based treatments
Chemotherapy with hydroxyurea, busulfan
Gleevec™ (imatinib mesylate, = STI571)
Therapeutic Options for CML
The current treatment options for patients with CML include allogeneic stem cell transplantation (SCT), IFN-–based regimens, chemotherapy with hydroxyurea or busulfan, and Gleevec™ (imatinib mesylate, formerly STI571).
Allogeneic SCT is currently the only therapy with the potential to “cure” CML. Access to the procedure is limited by availability of a suitable donor and by patient age (many centers do not accept candidates >55 years of age). Transplantation within 1 year of diagnosis is preferred.1 Approximately 15% to 20% of patients with CML meet these criteria for SCT.2 Five-year survival for patients transplanted during the chronic phase of the disease ranges from 54% to 70%; survival rates decrease and morbidity and mortality increase dramatically with advanced stages of disease.
IFN- induces hematologic responses in most patients and cytogenetic responses in some patients. The effects are dose-related; higher doses of IFN- correlate with higher rates of hematologic and cytogenetic response as well as more severe adverse events.3,4 Approximately 25% of patients discontinued therapy because of major side effects.5
Chemotherapy is of limited value in inducing cytogenetic responses, but it is effective in lowering WBC counts. These agents are generally considered to be palliative.6
Gleevec is a tyrosine kinase inhibitor that has shown very promising results in phase I and phase II clinical trials.
References
1. Faderl S, Talpaz M, Estrov Z, et al. Chronic myelogenous leukemia: biology and therapy. Ann Intern Med ;131:
2. Sawyers CL. Chronic myeloid leukemia. N Engl J Med ;340:
3. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology ;13:
4. Silver RT, Woolf SH, Hehlmann R, et al. An evidence-based analysis of the effect of busulfan, hydroxyurea, interferon, and allogeneic bone marrow transplantation in treating the chronic phase of chronic myeloid leukemia: developed for the American Society of Hematology. Blood ;94:
5. Guilhot F, Chastang C, Michallet M, et al. Interferon alfa-2b combined with cytarabine versus interferon alone in chronic myelogenous leukemia. N Engl J Med ;337:
6. Hill JM, Meehan KR. Chronic myelogenous leukemia. Curable with early diagnosis and treatment. Postgrad Med ;106: ,
From Gleevec.com

27. Until recently interferon-alpha treatment Was the gold-standard in CML
Even though its mechanism of action
IS STILL NOT UNDERSTOOD
Data of the Italian Cooperative Study group on
Chronic Myeloid Leukemia. Blood 1998: –1548
IFN=interferon-alpha, CHT= conventional chemotherapy

28. Abl’s Kinase Domain In complex With the inhibitor Gleevac
Kuriyan lab website

29. Months Since Start of Treatment
Gleevec™: in chronic phase CML
Chronic Phase CML
1.0
Major cytogenetic response
0.9
0.8
Complete cytogenetic
response
0.7
Fraction of patients
that responded
0.6
0.5
0.4
Gleevec™: Rapid Hematologic and Cytogenetic Response1
More than 50% of patients with MCR achieved their response within 3 months (median = 2.9 months) in chronic phase CML. In accelerated phase and blast crisis phase CML, similar results were obtained.
The median time to a hematologic response was 1 month, with a range of 0.9 to 9.3 months in the accelerated phase CML. The median time to a hematologic response was also 1 month for the other phases of CML.
Reference
1. Data on file. Novartis Pharmaceuticals Corporation, East Hanover, NJ.
0.3
0.2
0.1
0.0 1 2 3 4 5 6 7 8 9 10
Months Since Start of Treatment
Data: Novartis Pharmaceuticals Corporation

30. Drug was discontinued for adverse events
in 1% of patients in chronic phase,
2% in accelerated phase,
and 5% in blast crisis

31. Unfortunately, natural selection is
a powerful process
“We now know of over 30 different mutations
that can cause BCR-ABL to become
resistant to imatinib,” says Dr. Charles Sawyers
of UCLA’s Jonsson Cancer Center. In patients
with newly diagnosed disease, we are seeing
resistance to imatinib in about 4 percent of patients per year.
The further the disease has progressed before
initiating imatinib treatment,
the greater the chances are that resistance will arise.”

32. A possible solution: a new generation of kinase inhibitors that
Still inhibit Gleevec-resistant tumors
aka Dasatinib
Inject Luciferase-expressing tumor cells
Science :

33. FDA approved for patients with relapses
NCI Cancer Bulletin October 5, 2006
aka Dasatinib
Inject Luciferase-expressing tumor cells

34. Gleevec also has promise in other tumors
e.g., Gastrointestinal Stromal Tumors
90% of malignant GISTs harbor a mutation in c-kit
leading to KIT receptor autophosphorylation and
ligand-independent activation
Does not respond to chemotherapy (<10% response)
Only can be effectively treated if the entire tumor
Can be removed surgically
Without this median survival 1-2 yrs

35. With Gleevec treatment ~50% of patients respond Tumors shrink in size
and disease symptoms are greatly reduced
Report from the FDA
Approval Summary: Imatinib Mesylate in the Treatment
of Metastatic and/or Unresectable Malignant
Gastrointestinal Stromal Tumors
Dagher et al.
Clinical Cancer Research –3038,
October 2002

36. With Gleevec treatment ~50% of patients respond Tumors shrink in size
and disease symptoms are greatly reduced
Long term outcome ?
Many patients who initially respond develop
secondary resistance to Gleevec and relapse
Cause: second site mutations in c-kit

37. Many patients who initially respond develop
secondary resistance to Gleevec and relapse
Cause: second site mutations in c-kit
Approach: Develop new drugs
targeted against c-kit
e.g., AMG706, SU11248
Current Oncology Reports (2005) 7:

38. An alternate approach:
broader spectrum inhibitors that hit multiple targets
Sunitinib: targets Abl/PDGF Receptor, Src,
and VEGF Receptor
FDA approved after
Phase III clinical trial reveal
efficacy in GIST patients whose
Tumors are resistant to Gleevec
NCI Cancer Bulletin Oct

39. But what does Abl normally do?

40. Insights from the mouse model
• abl mutant mice are viable
but runted and 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

41. Abelson has a twin brother
Abl
Actin-binding
SH3
SH2
kinase F G
34%
89%
94%
27% F G
Arg

42. 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
Wild-type
abl; arg

43. BCR-Abl affects multiple cell functions
Proliferation & differentiation
Stem cell turnover S G 2 M 1 G0
BCR-Abl
Cytoskeleton/
adhesion defects
Apoptosis
Adapted from Jörgensen, Hem. Onc.

44. Abl may play roles in the nucleus in response to DNA damage
•ATM can phosphorylate Abl
in response to DNA damage
•Abl may stabilize p53
Van Etten, TICB

45. Abl may transmit signals in response to cell matrix adhesion
* Proteins that bind or
are phosphorylated by
Bcr-Abl
Salesse and Verfaille
Oncogene 21, 8605 (2002)

46. In the fruit fly
Abl transmits
signals from
axon guidance
receptors to the
cytoskeleton