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

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

3. 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 bone marrow transplantation may cure the patient;

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

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

6. Nature 1973 243:290-3 “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 Normal individual Leukemic patient Chr. 9 Chr. 22 9; 22 Translocation The Philadelphia chromosome

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

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 ( 35- 40% of adults with ALL can be expected to 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 Chr. 9 Chr. 22 abl bcr Bcr-abl 9; 22 Translocation fuses Bcr and Abl De Klein et al. Nature 300, 765 (1982) Groffen et al. Cell 36, 93 (1984)

13. The Cell, G. Cooper, Fig. 15.25 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

14. 5 ‘ of abl in situ probe3’ of abl in situ probe Fluorescence in situ hybridization (FISH) A tool for diagnosing CML Zymed.com

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

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

17. Src is normally inactive due to intramolecular inhibition

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

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

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

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

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

23. Clinical Course: Phases of CML Chronic phase Median 5–6 years stabilization Accelerated phase Median duration 6–9 months Blast crisis Median survival 3–6 months Advanced phases Provided by: Gleevec.com

24. Blast crisis is thought to involve additional but not well characterized genetic changes 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+ 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) From Gleevec.com

27. Data of the Italian Cooperative Study group on Chronic Myeloid Leukemia. Blood 1998:92 1541–1548 Until recently interferon-alpha treatment Was the gold-standard in CML Even though its mechanism of action IS STILL NOT UNDERSTOOD IFN=interferon-alpha, CHT= conventional chemotherapy

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

29. Gleevec™: in chronic phase CML 0 Months Since Start of Treatment Chronic Phase CML 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1234567 8910 Fraction of patients that responded Major cytogenetic response Complete cytogenetic response 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. 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 Only can be effectively treated if the entire tumor Can be removed surgically Without this median survival 1-2 yrs

32. 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 Research3034 3034–3038, October 2002 With Gleevec treatment ~50% of patients respond Tumors shrink in size and disease symptoms are greatly reduced Long term outcome ?

33. But what does Abl do?

34. 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

35. Abelson has a twin brother SH3 FG SH2kinase Actin- binding FG 89%94%27%34% Abl Arg

36. 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

37. 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, 2001. Hem. Onc.

38. 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 9 179-186

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

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