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. Wikipedia To understand leukemia we need to examine development of the Hematopoietic System myeloidlymphoid self renewal granulocytes

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

4. 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

5. 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.

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

7. “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

8. 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

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

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

11. 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 35- 40% of adults with ALL survive 2 years) Bone marrow transplant the only long term treatment

12. Table 4.5 The Biology of Cancer (© Garland Science 2007) Chromosomal rearrangements are a hallmark of leukemia, being present in 70-90% of cases

13. Table 4.5 The Biology of Cancer (© Garland Science 2007) Why is this the case?

14. Molecular Biology of the Cell Alberts et al We cannot dedicate all 25,000 genes in the genome just to make antibodies. What’s the solution? Put antibodies together by a mix-and match approach!

15. Molecular Biology of the Cell Alberts et al requires rearranging the DNA

16. Molecular Biology of the Cell Alberts et al requires rearranging the DNA

17. Molecular Biology of the Cell Alberts et al The result: an antibody light chain

18. Since there are multiple types of each gene segment, there are thousands of possible V-D-J combinations Each B cell gets a unique combination

20. As we have seen, sometimes this goes wrong, and other genes are juxtaposed to the Ig or TCR genes

21. Rearrangement mistakes can also juxtapose Other genes with oncogenic consequences

22. 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)

23. 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)

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

25. The Cell, G. Cooper, Fig. 15.25 In CML 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

26. In fact, different breakpoints in bcr Lead to slightly different Bcr-Abl fusion proteins That are found in different cancers

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

28. 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

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

30. Abelson kinase The front end looks a lot like Src!! SH3 FG SH2 kinase Actin- binding Myristate

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

32. What’s changed?? FG Gag Abl v-abl SH3 FG SH2 kinase Actin- binding FG Bcr Bcr-Abl

33. Remember this? Src is normally inactive due to intramolecular inhibition

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

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

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

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

38. But what does Abl normally do?

39. 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

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 Why so mild??

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

42. Are Abl and Arg redundant? arg mutant mice have behavioral defects (Arg is expressed in the brain at high levels)

43. 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

44. Focal adhesion proteins are phosphorylated by Abl (mediator of integrin signaling)

45. Abl phosphorylates regulators of the actin cytoskeleton

46. Of course it’s even more complicated Than that! Bradley and Koleske jcs.biologists.org/cgi/content/ full/122/19/3441/FIG3

47. Abl can also directly regulate Cytoskeletal events Using its C-terminal region to bundle actin filaments and Link them to microtubules

48. But does this all help us understand and treat leukemia? white blood cell Leukemic patientnormal person red blood cells hyperproliferation of white blood cells

49. 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.

50. 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

51. To understand this, we must start by learning more about the clinical progression 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

52. 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 Deletion of the Ikaros transcription factor

53. Therapy for CML: how do you evaluate whether a 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

54. 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

55. 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

56. STI571 Gleevec blocks the ATP binding site of the kinase domain

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

58. 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 Gleevec™: in chronic phase CML

59. 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 This is an awesome drug! Most patients survive 10+ years

60. Drug was discontinued for adverse events in 1% of patients in chronic phase, 2% in accelerated phase, and 5% in blast crisis Of course there are side effects...

61. $40,000-50,000 per year!! And the cost.....

62. “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%of patients per year. The further the disease has progressed before initiating imatinib treatment, the greater the chances are that resistance will arise.” Unfortunately, natural selection is a powerful process

63. About 17% of all patients develop resistance in 5 years Unfortunately, natural selection is a powerful process Science 331: 1542-44 March 25 2011

64. A possible solution: a new generation of kinase inhibitors that Still inhibit Gleevec-resistant tumors Inject Luciferase-expressing tumor cells Science 2004 305:399-401 aka Dasatinib

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

66. Dasatinib FDA approved for patients with relapses NCI Cancer Bulletin October 5, 2006 Leads to 73% progression free survival for 3 years

67. Dasatinib FDA approved for patients with relapses NCI Cancer Bulletin October 5, 2006 Phase II trials suggest Dasatinib effective in Blast-Crisis Patients with Gleevec-resistant tumors NCI Cancer Bulletin May 2 2007 Phase II trials suggest Dasatinib effective in ALL patients with Gleevec-resistant tumors NCI Cancer Bulletin May 29 2007

68. This is becoming a general approach Science 331: 1542-44 March 25 2011

69. How much do you think that costs?

70. For CML Add another $70,000/year!

71. 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 http://www.answers.com/topic/gist-2-jpg-1

72. 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

73. 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

74. April 17, 2008 Gleevec treatment also reduces risk or recurrence After surgical removal of GISTs 97% of patients treated with Gleevec had no recurrence after 1 year Versus 83% of those receiving placebo

75. GIST Gleevecxtends median survival from 15 months to 5 years

76. But..... Long term outcome ? Many patients who initially respond develop secondary resistance to Gleevec and relapse Cause: second site mutations in c-kit!

77. GIST Long term outcome ? 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: 293-299

78. An alternate approach: broader spectrum inhibitors that hit multiple targets Sunitinib: targets Abl/PDGF Receptor, Src, and VEGF Receptor NCI Cancer Bulletin Oct. 31 2006 FDA approved after Phase III clinical trial reveal efficacy in GIST patients whose Tumors are resistant to Gleevec George Demetri, MD