An Ongoing Story of Discovery: Pathophysiology of Chronic Myeloproliferative Disorders

1. An Ongoing Story of Discovery:Pathophysiology of Chronic Myeloproliferative Disorders Katy Moran MD August 30, 2005

2. “Imagination is more important than knowledge, for knowledge is limited while imagination embraces the entire world.” Albert Einstein

3. First, some cases. . .

4. Case #1: 63 yo woman presents to clinic with increasing abdominal girth, physical – hepatosplenomegaly. CBC reveals a Hct 52% and Platelet count 900,000 cells/mm3. Diagnosis? Polycythemia vera Case #2: 46 yo man presents to clinic with painful unilateral swelling of the right lower extremity for 48 hours. No known risk factors for DVT, ultrasound reveals femoral vein DVT. CBC reveals platelet count 1,200,000 cells/mm3. Diagnosis? Essential thrombocytosis Case #3: 65 yo man of Jewish ancestry presents with fatigue, low grade fever. Mild pancytopenia and teardrop-shaped rbcs are noted on blood smear. Bone marrow biopsy shows atypical megakaryocytes and stromal stranding. Diagnosis? Agnogenic Myeloid Metaplasia ≈ Idiopathic Myelofibrosis Case #4: 55 yo man presents with complaints of generalized fatigue, weight loss and abdominal discomfort with early satiety. Physical exam – afebrile, thin, massive splenomegaly. No adenopathy is identified, liver is normal in size. CBC reveals neutrophilic leukocytosis. Diagnosis? Chronic myelogenous leukemia

5. Tefferi, A. N Engl J Med 2000;342:1255-1265

7. Chronic Myeloproliferative Disorders • Common features: • Overproduction of one or more formed elements in the blood in the absence of an obvious stimulus • Clonal disorders arising in a single, multipotent progenitor or stem cell  proliferates  dominates the marrow and blood • Extramedullary hematopoiesis • Hypercellular marrow • Hyperplastic megakaryocytes  myelofibrosis • Clinical tendency toward thrombotic and hemorrhagic complications

8. 1892 Louis Vasquez of Paris described a pt with cyanotic polycythemia, autopsy massive enlargement liver and spleen • 1903 William Osler at Johns Hopkins reported four patients with polycythemia, two with splenomegaly • Osler-Vasquez disease ( polycythemia vera) • 1951 William Dameshek writes an article in Blood grouping PV, idiopathic myelofibrosis, ET, CML, and ‘erythroleukemia’ into a general category termed myeloproliferative disorders • “Perhaps it is possible…not that the various conditions listed are different, but that they are closely interrelated. It is possible that these various conditions – “myeloproliferative disorders”-are all somewhat variable manifestations of proliferative activity of the bone marrow cells, perhaps due to a hitherto undiscovered stimulus.”

9. Dameshek W. Some Speculations on the Myeloproliferative Syndromes. Blood 1951. Adaptation from Table 1:  Myelostimulatory Factor (s) 

10. “Myelostimulatory Factor” • Highly potent since it causes not only normal bone marrow to become highly proliferative but also causes activation of sites embryonic or potential hematopoeisis such as spleen and liver • Theorized of a hormonal or steroid type of factor

11. “In the middle of difficulty lies opportunity.” Albert Einstein

12. 1974 NEJM Prchal and Axelrad demonstrate that in patients with PV erythroid progenitor cells from marrow or peripheral blood proliferate in serum-containing culture in the absence of exogenous erythropoietin termed “Endogenous Erythroid Colony” formation • 1977 J Clin Invest Zanjani shows this phenomenon really is hypersensitivity to erythropoietin in the culture serum rather than a erythropoietin independent respose • 1989 Cell D’Andrea – Cloning of EPO receptor • No recognizable intracellular signals/pathway compared with other known receptors such as insulin

13. 1989 Research continues on a new class of receptors, called type I cytokine receptors • GM-CSF, multiple interleukin receptors, and others are identified • Mechanism via novel kinase/signal transduction pathway • 1992 Cell Valezquez describe this novel pathway as JAK receptor/signal transducer and activator of transcription (STAT) • JAK – “Just another kinase” • Janus kinase – named for Roman god of gates and passages • Studies in 1992-1994 demonstrate hypersensitivity of PV erythroid progenitor cells with a variety of growth factors such as IL-3, GM-CSF, IGF-1 • ? Downstream effect

14. Tyrosine Kinases • Enzymes that catalyze transfer of phosphate from ATP to tyrosine residues in polypeptides • 2 Classes • Receptor TK – Transmembrane Protein with extracellular domain • Nonreceptor TK – Intracellular - found in cytosol, nucleus

16. Janus Kinase Protein • Kinase domain (JH1)+ catalytically inactive pseudokinase domain (JH2) which acts as a regulator • Intermediate between membrane receptors and signaling molecules • Cytoplasmic region of a membrane receptor – when receptor is activated (for example a cytokine binds) JAK is phosphorylated and activated initiating signalling cascade via the STAT molecules • STAT molecules enter the nucleus  transcription

17. Four members of JAK family • JAK 1 • JAK 2 • Activated particularly when receptor binds to hematopoietic growth factors, including erythropoietin, GM-CSF, G-CSF, and thrombopoietin • JAK 3 • TYK 2 (tyrosine kinase 2)

19. Region of JH2 interacts with the activation loop of the kinase domain. A specific site mutation in the JH2 domain results in constitutive kinase activity of JH1 • Mutation has been mapped to position 617 on the pseudokinase domain • Guanine to thiamine substitution –>Amino acid Δvaline to phenylalanine • Termed V617F

20. Addition of pseudokinase JH2 domain greatly reduces the level of autoactivation  Expression of an isolated JAK-2 JH1 kinase domain leads to its constitutive activity  Goldman, J. M. N Engl J Med 2005;352:1744-1746

21. Schwartz, R. N Engl J Med 2002;347:462-463

22. Mutation found only in hematopoietic cells • Acquired somatic mutation • Present in DNA from granulocytes but absent in T cells • Mechanism for loss of heterozygosity at chromosome 9p • Deletion of telomeric part of wild-type chromosome 9p • Events during mitotic recombination Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005; 352: 1779-1790.

23. Mechanism of Loss of Heterozygosity at Chromosome 9p

24. What are the implications of this mutation among the chronic myeloproliferative disorders?

25. * Carriers of the mutation had more complications such as fibrosis, hemorrhage, and thrombosis and were more likely to receive cytoreductive therapy.

26. Adaptation from Table 1: Jones A, et al. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 2005 (in press).

27. Further evidence of V617 mutation contribution to CMPDs • Introduction of mutant clone into irradiated mice led to substantial erythrocytosis • Erythroid progenitor cells carrying the mutation were able grow in the absence of exogenous erythropoietin • Homozygosity • Arise from recombination of chromatids during mitosis rather than a second mutation the mutant heterozygous line • Loss of heterozygosity results in a proliferative advantage • Individuals with one mutant and one wild type gene have reduced cellular autonomous JAK2 activity and growth factor independent behavior compared with homozygous individuals James C, Ugo V, Le Couedic J-P, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythemia vera. Nature (in press). Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative diseases. Lancet. 2005; 365: 1054-1061

28. Duration of disease was significantly longer among homozygotes compared to heterozygotes • Patients testing negative for the mutation had the shortest duration of disease • Homozygous – mean 48 months • Heterozygous – mean 23 months • Wild type – mean 15 months • Phenotype may be expressed without the mutation • Suggests acquiring the mutation and then homozygosity are likely stepwise processes Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005; 352: 1779-1790.

30. In patients that are found to be positive for this mutation by genetic testing, diagnostic and possibly prognostic information may be obtained • Specific therapeutic target at the level of the mutant kinase • More questions. . .

31. “If the facts don't fit the theory, change the facts.” Albert Einstein

32. How does one mutation give rise to these various disorders? • Additional genetic alterations? Pre-existing or acquired after the JAK2? • Dependent on the subtype of progenitor cell in which the mutation first arises? • What is the mechanism for disease in patients who do not carry the V617 mutation? • Some answers may lie in further exploration of genes that are activated by STAT (signal transducer and activator of transcription) cascade • Recently, members of the JAK and STAT families have been implicated in cellular decisions on whether to proliferate or enter apoptosis • One family of genes called suppressor of cytokine signaling (SOCS) encode proteins that bind to JAKs and receptor sites and then BLOCK further signaling Receptor  JAK  STAT SOCS  Programmed blockade of further JAK signals

33. Why do some patients progress from indolent CMPDs such as PV to acute leukemia? • Rational approach to therapy? • Tyrosine kinases as potential targets • Broad spectrum of malignancy mediated via this family of proteins • Examples: Fms-like tyrosine kinase 3 (FLT3) in acute myeloid leukemia, epidermal growth factor receptor in subset NSCLC, c-KIT mutation in GIST

34. JAKs mediate intracellular signaling in other pathways and diseases • Leptin receptor • Growth hormone receptor • Interleukin receptors • Cardiovascular signaling systems • Inherited JAK3 deficiency has been implicated in cases of severe combined immunodeficiency • Developing inhibitors that act specifically on V617F without causing side effects in other signaling systems may be challenging

35. Summary • Advances in the field of molecular/cell biology and specifically describing JAK2 have provided a valuable window into the mechanism of chronic myeloproliferative diseases including PV, ET, and IMF among others • This information has diagnostic and prognostic clinical relevance • Tyrosine kinases are vital proteins which have broad implications • Ongoing research in this field will impact how medicine is practiced for years to come

36. “If we knew what we were doing, it wouldn't be called research, would it?” Albert Einstein

37. References • Tefferi, A. N Engl J Med 2000;342:1255-1265 • Dameshek W. Some Speculations on the Myeloproliferative Syndromes. Blood 1951. • Goldman, J. M. N Engl J Med 2005;352:1744-1746 • Schwartz, R. N Engl J Med 2002;347:462-463 • Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative diseases. Lancet. 2005; 365: 1054-1061. • Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocytosis, and myeloid metaplasia with myelofibrosis. Cancer Cell (in press). • James C, Ugo V, Le Couedic J-P, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythemia vera. Nature (in press). • Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005; 352: 1779-1790. • Jones A, et al. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 2005 • MKSAP Review Hematology and Oncology • Up to Date • Krause, DS, Etten RA. Tyrosine Kinases as Targets for Cancer Therapy. N. Engl J Med. 2005; 353: 172-187. • Tefferi A, Gilliland DG. The JAK2 Tyrosine Kinase Mutation in MPD: Status report. Mayo Clin. Proc. July 2005: 80 (7): 947-958. • Kaushansky K. On the molecular origins of the chronic myeloproliferative disorders: it all makes sense. Blood. June 2005. 105: 4187-4190.