Download Chronic Myeloid Leukemia (CML)

Chronic Myeloid Leukemia
(CML)
Matt Walter
Markey Program
October 21, 2014
CML: paradigm for cancer treatment
• Identify primary genetic abnormality
• Develop an agent that targets that abnormality
• Monitor for efficacy
• Overcoming resistance
Chronic Myeloid Leukemia Session Overview
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Matt Walter = Hematopoiesis overview
Dan Link = Signaling, animal models
Peter Westervelt = Clinical overview *
Steve Oh = Imatinib preclinical/clinical
Geoff Uy = Imatinib resistance *
Rizwan Romee = Bone marrow transplant *
Jerry Radich = Guest speaker on CML
Last class = round table lunch
* Patient interviews
Hematopoiesis
Stem cell
Bone
marrow
Blood
Progenitors
Terminally
differentiated
NEJM (2006) 354;19, p.2034
Hematopoietic growth factor (HGF) signaling
Receptor
Tyrosine Kinase
(RTK)
(ABL, non‐
Receptor TK)
*Frequent targets for mutation in leukemia
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NEJM (2006) 354;19, p.2034
Classification of Leukemias
Lymphoid
Myeloid
CLL
ALL
CML
AML
Chronic
Lymphocytic Leukemia
Acute
Lymphocytic Leukemia
Chronic
Myeloid Leukemia
Acute
Myeloid Leukemia
10/100,000
1/100,000
2/100,000
(~6,000 new/year)
5/100,000
Acute = fast clinical progression. Block in differentiation
(weeks to months)
Chronic Leukemias & MPNs: differentiation occurs
Terminally
differentiated
Blood
Essential
Thrombocytosis
(ET)
Chronic Myelo‐
Monocytic
Leukemia
(CMML)
Polycythemia
Vera
(PV)
Chronic
Myeloid
Leukemia
(CML)
Myeloproliferative Neoplasms (MPN)
Chronic
Lymphocytic
Leukemia
(CLL)
Genetics of Myeloproliferative Neoplasms (MPN)
Myeloid
Neoplasm with
Eosinophilia
(PDGFRA/B)
Essential
Thrombocytosis
(ET) (JAK2)
Polycythemia
Vera
(PV) (JAK2)
Chronic
Myeloid
Leukemia
(CML) (BCR:ABL)
Mutation/rearrangement of tyrosine kinase genes
Mastocytosis
(KIT)
Westervelt
CML
• 1.4:1 (♂:♀), average age 45
• Increased blood WBC (>25k/uL) • Minimal/no fibrosis or dysplasia
Normal peripheral blood
Granulocyte maturation
basophil
eosinophil
Normal BM
Peter Maslak, ASH Image Bank 2011; 2011-1083
CML blood
CML chronic
Blastic phase
(block in differentiation)
Westervelt
CML
• Chronic phase
– Driven by BCR‐ABL
– 50% no symptoms, found on routine CBC
– ↑WBC PB, BM, spleen
– Fatigue, weight loss, abdominal fullness, bleeding, sweats, large spleen/liver
– 75% had accelerated phase 3‐18 months prior to blastic phase
• Blast phase
– Acquire secondary mutations
– 1/3 = ALL, 2/3 = AML
– Decreased response to treatment and duration not long lasting
BCR:ABL fusion in CML
• Cytogenetics = t(9;22), Philadelphia chromosome + (Ph+), (100% BCR‐ABL
transcript)
• BCR‐ABL transcript +
Flemming draws first chromosome
Tjio reports 46 chromosomes = normal
syndromes
Waldeyer coins “chromosome”
Painter reports 48 chromosomes = normal
1890
von Hansemann observes
mitotic aberrations in tumor cells
“dark ages”
Technical problems
1. better cultures
2. hypotonic solution
3. colchicine
Down
+21 Turner
XO
Klinefelter
XXY
1960
Nowell & Hungerford = Ph chromosome & CML
“The findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemia”.
Chromosome Gene Drug
Baltimore, v‐Abl
1980
1960
1973
Nowell & Hungerford = Ph chromosome & CML
Rowley reports
Ph chr. = t(9;22) 1983‐84
Heisterkamp, Stephenson, Groffen, Grosveld clone genes at breakpoints (BCR, ABL)
1998‐2001
Druker uses Gleevec to treat CML
v‐ABL oncogene
Lymphosarcoma: virus‐induced thymic‐independent disease in mice.
Abelson HT, Rabstein LS. Cancer Res. 1970 Aug;30(8):2213‐22 v‐Abl
Cell. 1979 Dec;18(4):955‐62
Cell. 1980 Dec;22(3):777‐85
(coil‐coil)
– p190 = 20‐30% ALL (most B‐cell)
– p210 (p230) = 99% CML
• ABL = Loss of N‐terminal Cap region of ABL, normally inhibits kinase activity
• BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABL
– Results in constitutive activation of the ABL tyrosine kinase.
– Effects on cellular proliferation, survival and motility
Dan Link
Heisterkamp, Oncogene, 2002 ;21(56):8536‐40.
What cell is transformed in CML?
Stem cell
Bone
marrow
Blood
Progenitors
Terminally
differentiated
Link
NEJM (2006) 354;19, p.2034
Colony‐forming Cell Assay (progenitors)
Bone Marrow Cells
Culture with specific
growth factors for
7-14 days
CFU-GM
(GM-CSF)
Semi-solid media
(methylcellulose)
BFU-E
(Epo)
Methylcellulose cultures
Hematopoietic Stem Cell Assays
• HSC Definition: capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
• Bone marrow transplantation (gold standard)
9– Xenotransplantation models to assay human HSC
• Long term culture‐initiating cell (LTC‐IC) assay
Murine or Xenotransplantation
radiation
Human
hematopoietic
cells
Mouse
hematopoietic
donor cells
(CD45.2)
NOD/SCID mice
NOD/SCID gamma (NSG)
(immunodeficient)
congenic
recipient mice
(CD45.1)
Analyze
after 3-6
months for
blood cells
Analyze
after 3-6
months for
blood cells
Better assays needed to study hematopoiesis
• Methylcellulose and transplant assays are still the gold standard, but is there a faster, cheaper way to study these cells?
• How can you prospectively identify the cells and isolate them to study?
Flow cytometry
Murine or Xenotransplantation
radiation
Human
hematopoietic
cells
NOD/SCID mice
(immunodeficient)
Analyze
after 3-6
months for
blood cells
CD34+ xenotransplant assays = 1:5000 HSCs
CD34+/CD38- = 1:617 HSCs
Mouse
hematopoietic
cells
congenic mice
(CD45.1/CD45.2)
Kit+/Lineage-/Sca+ (KLS) cells = 1:10 HSCs
CD150+/CD48- (SLAM) cells = 1:2 HSCs
GOALS: 1) prospectively identify cells using flow cytometry (Weissman group = 1st mouse, 2nd human hematopoiesis). 2) Define what cell is transformed in CML (chronic phase and blast crisis). Stem cell
Bone
marrow
Blood
Progenitors
Terminally
differentiated
929 citations
CMP
MEP
GMP
MEP
288 single cells of each type
No Epo+Tpo
CMP GMP
200 cells each
CMP
Cytospin of 5 pooled colonies
MEP
GMP
1000 cells on stroma
GMP
CMP
MEP
Single cell sorted
Lineage commitment at CMP single cell level: Regulation of Hematopoiesis
• Transcription factors
– Regulate RNA expression of target genes
– Critical determinants of lineage commitment and terminal differentiation
• Frequent targets for mutation in leukemia
HUMAN PROGENITOR ASSAY
PNAS (2002)99;18, p.11872
What cell is transformed in CML?
Stem cell
Bone
marrow
Blood
Progenitors
Terminally
differentiated
Link
NEJM (2006) 354;19, p.2034
Oh
Uy
Romee
Radich
CML therapy
Lancet (2007)370, p.342
Westervelt
Oh
Uy
Radich
Targeted therapy (chronic phase)
• Imatinib = discovered in high‐throughput screen of kinase inhibitors.
NEJM. 2003 Oct 9;349(15):1399‐401 Issues
• Cure vs. control of chronic phase CML
• Resistance in chronic phase CML
• Treatment of blastic phase CML