Marty O’Neill II Carmen Banea Genetic Mutations that Lead to Chronic Myelogenous Leukemia: Causes and Treatments Marty O’Neill II Carmen Banea

What is CML? Chronic Myelogenous Leukemia (CML) is defined as, “a malignant cancer of the bone marrow. It causes rapid growth of the blood-forming cells (known as myeloid precursors) in the bone marrow, peripheral blood, and body tissues.” [2] CML represents about 14% of all occurrences of leukemias. Patients who have CML are said to be in one of the following three phases (in order of occurrence): the chronic phase (between 1 and 15% blasts) the accelerated phase (between 15% - 30% blasts) the blast phase (more than 30% blasts). [5]

Symptoms and Diagnosis CML can be discovered in a routine physical examination 70% of those diagnosed with CML had symptoms including: fatigue, abdominal discomfort, weight loss, and sweating Philadelphia Chromosome is indicator that patient has CML

Epidemiology of CML* Median age range at presentation is 45-55 years Incidence increases with age Up to 30% of patients are aged >60 years Slightly higher incidence in males Male-to-female ratio—1.3:1 At presentation 50% diagnosed by routine laboratory tests 85% diagnosed during chronic phase Globally, CML has an incidence of 1 to 2 cases per 100,000 population and is responsible for 15% to 20% of all adult leukemia. The median age at presentation is 53 years, with a median range of 45 to 55 years. The incidence of CML increases with age; up to 30% of patients are 60 years of age or older at presentation, which may influence the selection of treatment options in this population. CML is less common in children, with approximately 10% of patients less than 20 years of age. CML occurs somewhat more frequently in males, with a male-to-female ratio of 1.3:1. Approximately 50% of patients are asymptomatic at diagnosis, with CML discovered through routine laboratory blood tests. Eighty-five percent of patients are diagnosed during the chronic phase of disease. Sawyers. N Engl J Med. 1999;340:1330. Faderl et al. Ann Intern Med. 1999;131:207. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology (Huntingt). 1999;13:169-180. Faderl S, Talpaz M, Estrov Z, Kantarjian HM. Chronic myelogenous leukemia: biology and therapy. Ann Intern Med. 1999;131:207-219. Hill JM, Meehan KR. Chronic myelogenous leukemia. Curable with early diagnosis and treatment. Postgrad Med. 1999;106:149-152, 157-159. Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999;340:1330-1340.

The Ph Chromosome and the bcr-abl Gene: The t(9;22) Translocation* Chromosome 9 q+ Chromosome 9 Philadelphia Chromosome (or 22q-) Chromosome 22 The Ph chromosome is the result of a reciprocal translocation, t(9;22)(q34;q11), between the long arms of chromosomes 9 and 22. A segment of the abl gene (Abelson mouse leukemia proto-oncogene) on chromosome 9q34 coding for a nonreceptor tyrosine kinase is translocated to the bcr gene (breakpoint cluster region) on chromosome 22q11 to form an abnormal hybrid bcr-abl gene. The bcr-abl gene is transcribed into a hybrid messenger RNA; the translation product of this RNA is an abnormal fusion protein tyrosine kinase. The Ph chromosome was first described in 1960 as a shortened chromosome 22 present in myeloid cells from patients with CML. This was the first report of a human cancer associated with a specific genetic abnormality. Ninety-five percent of patients with CML have the Ph chromosome—hence, this chromosome is the hallmark of CML. The Ph chromosome can be detected in BM cells in metaphase by standard cytogenetic techniques. The Ph chromosome is present in all myeloid cell lineages, including erythrocytes, granulocytes, monocytes, and megakaryocytes, as well as some cells of lymphocytic lineage, indicating that malignant transformation to CML originates at the stem cell level. Irradiated mice that received BM infected with a retrovirus carrying the p210 Bcr-Abl kinase encoded by the Ph chromosome developed hematologic malignancies, including a myoproliferative disease similar to chronic phase CML. bcr-abl bcr FUSION PROTEIN WITH CONSTITUTIVE TYROSINE KINASE ACTIVITY abl Melo. Blood. 1996;88:2375. Pasternak et al. J Cancer Res Clin Oncol. 1998;124:643. Daley GQ, Van Etten RA, Baltimore D. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science. 1990;247:824-830. Osarogiagbon UR, McGlave PB. Chronic myelogenous leukemia. Curr Opin Hematol. 1999;6:241-246. Faderl S, Talpaz M, Estrov Z, O’Brien S, Kurzrock R, Kantarjian HM. The biology of chronic myeloid leukemia. N Engl J Med. 1999;341:164-172. Melo JV. The diversity of BCR-ABL fusion proteins and their relationship to leukemia phenotype. Blood. 1996;88:2375-2384. Pasternak G, Hochhaus A, Schultheis B, Hehlmann R. Chronic myelogenous leukemia: molecular and cellular aspects. J Cancer Res Clin Oncol. 1998;124:643-660. Sawyers CL. Chronic myeloid leukemia. N Engl J Med. 1999;340:1330-1340.

Genetic Mutations Can occur as exposures to radiation, chemicals, etc Known from 1981 or before that Benzene (a byproduct of the use of laser printers and copy machines) caused the mutation leading to CML in humans Not enough has been done to protect employees who work in the presence of Benzene

Treatments Transplantation is the only known cure. Chemo Therapy Imatinib (STI-571) BMS-354825 and AMN107 (still under study)

Normal Bcr-Abl Signaling* The kinase domain activates a substrate protein, eg, PI3 kinase, by phosphorylation This activated substrate initiates a signaling cascade culminating in cell proliferation and survival Substrate Bcr-Abl Effector P P ADP Bcr-Abl is a disregulated tyrosine kinase protein capable of both auto- and substrate phosphorylation. Highly plastic structure with both open (active) and closed (autoinhibited) stages. State is regulated both intramolecularly and by other proteins. Bcr-Abl signaling begins when adenosine triphosphate (ATP) binds to the kinase domain. This allows the binding of the substrate to be phosphorylated. Following phosphate transfer, the phosphosubstrate is competent to bind to and activate downstream effector molecules. P ATP P P P SIGNALING ADP = adenosine diphosphate; ATP = adenosine triphosphate; P = phosphate. Savage and Antman. N Engl J Med. 2002;346:683 Scheijen and Griffin. Oncogene. 2002;21:3314. Hantschel O, Superti-Furga G. Regulation of the c-Abl and Bcr-Abl tyrosine kinases. Nat Rev Mol Cell Biol. 2004;5:33-44. Savage DG, Antman KH. Imatinib mesylate–a new oral targeted therapy. N Engl J Med. 2002;346:683-693. Scheijen B, Griffin JD. Tyrosine kinase oncogenes in normal hematopoiesis and hematological disease. Oncogene. 2002;21:3314-3333.

Imatinib Mesylate: Mechanism of Action* Imatinib mesylate occupies the ATP binding pocket of the Abl kinase domain This prevents substrate phosphorylation and signaling A lack of signaling inhibits proliferation and survival Bcr-Abl P ATP At the molecular level, imatinib mesylate targets a specific part of the tyrosine kinase region of Bcr-Abl. Imatinib mesylate is an ATP-mimetic agent that binds Bcr-Abl with greater affinity than ATP at its ATP-binding site. The tyrosine kinase activity of Bcr-Abl is dependent on its ATPase activity. Bcr-Abl–bound imatinib mesylate prevents ATP binding and hydrolysis and hence the tyrosine kinase action of Bcr-Abl. This blocks the downstream substrate of Bcr-Abl, which is dependent on phosphorylation for activation. This in turn blocks downstream signal transduction pathways activated by Bcr-Abl. Imatinib mesylate SIGNALING Savage and Antman. N Engl J Med. 2002;346:683. Savage DG, Antman KH. Imatinib mesylate–a new oral targeted therapy. N Engl J Med. 2002;346:683-693. Scheijen B, Griffin JD. Tyrosine kinase oncogenes in normal hematopoiesis and hematological disease. Oncogene. 2002;21:3314-3333.

Imatinib Mesylate in Chronic Phase CML Following IFN- Failure: Overall Survival* 1.0 0.9 0.8 0.7 0.6 Survival probability 0.5 0.4 Seventy-five percent of patients remain alive on imatinib mesylate therapy after a median follow-up of 45 months. Patients who received imatinib mesylate had significantly improved survival rates, with an estimated 4-year survival rate of 86% vs 43% (P<0.0001). The estimated 4-year transformation-free survival was 80%. Total Dead 0.3 261 31 Imatinib mesylate 0.2 251 193 Others 0.1 (P<0.0001) 24 48 72 96 Months Kantarjian et al. Blood. 2004;104;1979. Copyright American Society of Hematology, used with permission. Kantarjian HM, Cortes JE, O’Brien S, et al. Long-term survival benefit and improved complete cytogenetic and molecular response rates with imatinib mesylate in Philadelphia chromosome-positive, chronic-phase chronic myeloid leukemia after failure of interferon-alpha. Blood. 2004;104:1979-1988.

Probably not really linear Case Study: WBC Probably not really linear Begins Imatinib Treatment White Blood Count progression in subject beginning one year before being diagnosed, and continuing throughout the first year of treatment with STI-571.

Case Study: Probable Cause of CML Worked in a Copy Center for five years directly before diagnosis Probable cause of genetic mutations that led to CML was exposure to benzene Nothing has been done to further examine or address this problem in that copy center From: http://www.seton.com/

Conclusions CML research has been ongoing for over 150 years Better methods of treatment have been found that targets the CML cells on a molecular level The causes of CML are still not completely known, but benzene has been known for at least 25 years to lead to CML New regulations and education programs are needed to protect employees who work in the presence of benzene

References [1] D’Antonio, J. Chronic Myelogenous Leukemia. Clinical Journal of Oncology Nursing. 9(5): 535-8. [2] Faderl et al. Oncology (Huntingt). 1999; 13:169. [3] Genetic Science Learning Center at the University of Utah. http://gslc.genetics.utah.edu/. [4] National Marrow Donor Program overview slide presentation. http://www.marrow.org/NMDP/SLIDESET/sld031.htm. [5] Medline Plus Medical Encyclopedia. http://nlm.nih.gov. [6] Pasternak et al. Chronic Meylogenous Leukemia: Molecular and Cellular Aspects. J Cancer Res Clin Oncol. 1998; 643-60. [7] Rinsky et al. Leukemia in Benzene Workers. Am J Ind Med. 1981; 2(3): 217-45. [8] Smith, MT & Zhang, L. Biomarkers of Leukemia Risk: Benzene as a Model. Environ Health Perspect. 1998 Aug; 106 Suppl 4:937-46. [9] STI-571 in Chronic Myelogenous Leukaemia. British Journal of Haematology. 2002; 15-24. * Some slides in this presentation were borrowed or adapted from IMPACT “The Era of Molecular Therapy: Focus on Chronic Myelogenous Leukemia”