Title: An Ongoing Story of Discovery: Pathophysiology of Chronic Myeloproliferative Disorders
1An 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 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
5Tefferi, A. N Engl J Med 20003421255-1265
6Disease Characteristics Transformation
CML Genetic translocation Philadelphia chromosome t(922) resulting in fusion of bcr-abl oncogene gt90 will transform to acute leukemia if untreated
Polycythemia vera Elevated red cell mass, hypercellular marrow, independent of erythropoietin 10 myelofibrosis _at_10 yrs 25 myelofibrosis _at_ 25 yrs
Essential thrombocytosis Clonal or autonomous thrombocytosis lt5 will transform to acute leukemia
Agnogenic myeloid metaplasia (Chronic idiopathic myelofibrosis) Bone marrow fibrosis not associated with CML or MDS Mean survival lt5 yrs
Atypical Atypical CML, chronic neutrophilic leukemia, systemic mast cell disease, chronic eosinophilic leukemia Variable
7Chronic 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 possiblenot 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.
9Dameshek W. Some Speculations on the
Myeloproliferative Syndromes. Blood 1951.
Adaptation from Table 1
Syndrome Erythroblasts Granulocyte Mega-karyocytes Fibroblasts Spleen and liver
Chronic Granulocytic Leukemia (CML) /- to
PV to to to
Agnogenic Myeloid Metaplasia /- /- to
Mega-karyocytic Leukemia /- /- to
? 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 DAndrea 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
14Tyrosine 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
15(No Transcript)
16Janus 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)
18(No Transcript)
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 gtAmino acid ?
valine to phenylalanine - Termed V617F
20Addition 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 20053521744-1746
21Schwartz, R. N Engl J Med 2002347462-463
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.
23Mechanism of Loss of Heterozygosity at Chromosome
9p
24- What are the implications of this mutation among
the chronic myeloproliferative disorders?
25Study Purpose PV ET MF
Cambridge, UK Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative diseases. Lancet. 2005 365 1054-1061. Focused on the key role of JAK2 in signal transduction from multiple hematopoietic growth factor receptors 97 N73 57 N51 50 N16
Boston 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). DNA sequence analysis of activation loops and autoinhibitory domains of 85 tyrosine kinases 74 N164 32 N115 35 N46
Paris 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). Endogenous erythroid colonies inhibitors 88 N45 small small
Switzerland-Italy 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. Observed patients with PV had loss of heterozygosity in chromosome 9p that included the site of the JAK2 gene 65 N128 23 N93 57 N23
Carriers of the mutation had more complications
such as fibrosis, hemorrhage, and thrombosis and
were more likely to receive cytoreductive therapy.
26Adaptation from Table 1 Jones A, et al.
Widespread occurrence of the JAK2 V617F mutation
in chronic myeloproliferative disorders. Blood
2005 (in press).
Disease Subtype N V617F Positive number () V617F Negative number () V617F homozygotes number ( of mutants)
PV 72 58 (81) 14 (19) 24 (41)
ET 59 24 (41) 35 (59) 4 (17)
IMF 35 15 (43) 20 (67) 10 (67)
Idiopathic Hyper-eosinophilic syndrome 134 2 (1.5) 132 (99) 2 (100)
Mastocytosis 28 0 - -
CML-like MPDs 99 17 (17) 82 (93) 8 (47)
Unclassified MPD 53 12 (25) 40 (75) 7 (54)
Total 480 129 (27) 351 (73) 55 (43)
27Further 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.
29(No Transcript)
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
35Summary
- 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
37References
- Tefferi, A. N Engl J Med 20003421255-1265
- Dameshek W. Some Speculations on the
Myeloproliferative Syndromes. Blood 1951. - Goldman, J. M. N Engl J Med 20053521744-1746
- Schwartz, R. N Engl J Med 2002347462-463
- 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.