chronic myeloid leukemia (cml) - the division of biology...
TRANSCRIPT
Chronic Myeloid Leukemia(CML)
Matt WalterMarkey ProgramOctober 21 2014
CML paradigm for cancer treatment
bull Identify primary genetic abnormalitybull Develop an agent that targets that abnormality
bull Monitor for efficacybull Overcoming resistance
Chronic Myeloid Leukemia Session Overview
1 Matt Walter = Hematopoiesis overview2 Dan Link = Signaling animal models3 Peter Westervelt = Clinical overview 4 Steve Oh = Imatinib preclinicalclinical5 Geoff Uy = Imatinib resistance 6 Rizwan Romee = Bone marrow transplant 7 Jerry Radich = Guest speaker on CML8 Last class = round table lunch
Patient interviews
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
Hematopoiesis
Hematopoietic growth factor (HGF) signaling
NEJM (2006) 35419 p2034
ReceptorTyrosine Kinase(RTK)
(ABL non‐Receptor TK)
Frequent targets for mutation in leukemia
Classification of LeukemiasLymphoid Myeloid
CLLChronicLymphocytic Leukemia
CMLChronicMyeloid Leukemia
ALLAcuteLymphocytic Leukemia
AMLAcuteMyeloid Leukemia
10100000 1100000 2100000(~6000 newyear)
5100000
Acute = fast clinical progression Block in differentiation(weeks to months)
TerminallydifferentiatedBlood
ChronicMyeloidLeukemia(CML)
ChronicLymphocyticLeukemia(CLL)
EssentialThrombocytosis(ET)
PolycythemiaVera(PV)
Chronic Myelo‐MonocyticLeukemia(CMML)
Myeloproliferative Neoplasms (MPN)
Chronic Leukemias amp MPNs differentiation occurs
EssentialThrombocytosis(ET) (JAK2)
PolycythemiaVera(PV) (JAK2)
ChronicMyeloidLeukemia(CML) (BCRABL)
Genetics of Myeloproliferative Neoplasms (MPN)
Mastocytosis(KIT)
MyeloidNeoplasm withEosinophilia(PDGFRAB)
Mutationrearrangement of tyrosine kinase genes
CMLbull 141 () average age 45bull Increased blood WBC (gt25kuL) bull Minimalno fibrosis or dysplasia
Westervelt
Granulocyte maturation
Normal peripheral blood
Peter Maslak ASH Image Bank 2011 2011-1083
CML blood
Normal BM
basophil
eosinophil
CML chronic Blastic phase(block in differentiation)
CMLbull Chronic phasendash Driven by BCR‐ABLndash 50 no symptoms found on routine CBC
ndash uarrWBC PB BM spleenndash Fatigue weight loss abdominal fullness bleeding sweats large spleenliver
ndash 75 had accelerated phase 3‐18 months prior to blastic phase
bull Blast phasendash Acquire secondary mutations
ndash 13 = ALL 23 = AMLndash Decreased response to treatment and duration not long lasting
Westervelt
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
CML paradigm for cancer treatment
bull Identify primary genetic abnormalitybull Develop an agent that targets that abnormality
bull Monitor for efficacybull Overcoming resistance
Chronic Myeloid Leukemia Session Overview
1 Matt Walter = Hematopoiesis overview2 Dan Link = Signaling animal models3 Peter Westervelt = Clinical overview 4 Steve Oh = Imatinib preclinicalclinical5 Geoff Uy = Imatinib resistance 6 Rizwan Romee = Bone marrow transplant 7 Jerry Radich = Guest speaker on CML8 Last class = round table lunch
Patient interviews
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
Hematopoiesis
Hematopoietic growth factor (HGF) signaling
NEJM (2006) 35419 p2034
ReceptorTyrosine Kinase(RTK)
(ABL non‐Receptor TK)
Frequent targets for mutation in leukemia
Classification of LeukemiasLymphoid Myeloid
CLLChronicLymphocytic Leukemia
CMLChronicMyeloid Leukemia
ALLAcuteLymphocytic Leukemia
AMLAcuteMyeloid Leukemia
10100000 1100000 2100000(~6000 newyear)
5100000
Acute = fast clinical progression Block in differentiation(weeks to months)
TerminallydifferentiatedBlood
ChronicMyeloidLeukemia(CML)
ChronicLymphocyticLeukemia(CLL)
EssentialThrombocytosis(ET)
PolycythemiaVera(PV)
Chronic Myelo‐MonocyticLeukemia(CMML)
Myeloproliferative Neoplasms (MPN)
Chronic Leukemias amp MPNs differentiation occurs
EssentialThrombocytosis(ET) (JAK2)
PolycythemiaVera(PV) (JAK2)
ChronicMyeloidLeukemia(CML) (BCRABL)
Genetics of Myeloproliferative Neoplasms (MPN)
Mastocytosis(KIT)
MyeloidNeoplasm withEosinophilia(PDGFRAB)
Mutationrearrangement of tyrosine kinase genes
CMLbull 141 () average age 45bull Increased blood WBC (gt25kuL) bull Minimalno fibrosis or dysplasia
Westervelt
Granulocyte maturation
Normal peripheral blood
Peter Maslak ASH Image Bank 2011 2011-1083
CML blood
Normal BM
basophil
eosinophil
CML chronic Blastic phase(block in differentiation)
CMLbull Chronic phasendash Driven by BCR‐ABLndash 50 no symptoms found on routine CBC
ndash uarrWBC PB BM spleenndash Fatigue weight loss abdominal fullness bleeding sweats large spleenliver
ndash 75 had accelerated phase 3‐18 months prior to blastic phase
bull Blast phasendash Acquire secondary mutations
ndash 13 = ALL 23 = AMLndash Decreased response to treatment and duration not long lasting
Westervelt
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Chronic Myeloid Leukemia Session Overview
1 Matt Walter = Hematopoiesis overview2 Dan Link = Signaling animal models3 Peter Westervelt = Clinical overview 4 Steve Oh = Imatinib preclinicalclinical5 Geoff Uy = Imatinib resistance 6 Rizwan Romee = Bone marrow transplant 7 Jerry Radich = Guest speaker on CML8 Last class = round table lunch
Patient interviews
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
Hematopoiesis
Hematopoietic growth factor (HGF) signaling
NEJM (2006) 35419 p2034
ReceptorTyrosine Kinase(RTK)
(ABL non‐Receptor TK)
Frequent targets for mutation in leukemia
Classification of LeukemiasLymphoid Myeloid
CLLChronicLymphocytic Leukemia
CMLChronicMyeloid Leukemia
ALLAcuteLymphocytic Leukemia
AMLAcuteMyeloid Leukemia
10100000 1100000 2100000(~6000 newyear)
5100000
Acute = fast clinical progression Block in differentiation(weeks to months)
TerminallydifferentiatedBlood
ChronicMyeloidLeukemia(CML)
ChronicLymphocyticLeukemia(CLL)
EssentialThrombocytosis(ET)
PolycythemiaVera(PV)
Chronic Myelo‐MonocyticLeukemia(CMML)
Myeloproliferative Neoplasms (MPN)
Chronic Leukemias amp MPNs differentiation occurs
EssentialThrombocytosis(ET) (JAK2)
PolycythemiaVera(PV) (JAK2)
ChronicMyeloidLeukemia(CML) (BCRABL)
Genetics of Myeloproliferative Neoplasms (MPN)
Mastocytosis(KIT)
MyeloidNeoplasm withEosinophilia(PDGFRAB)
Mutationrearrangement of tyrosine kinase genes
CMLbull 141 () average age 45bull Increased blood WBC (gt25kuL) bull Minimalno fibrosis or dysplasia
Westervelt
Granulocyte maturation
Normal peripheral blood
Peter Maslak ASH Image Bank 2011 2011-1083
CML blood
Normal BM
basophil
eosinophil
CML chronic Blastic phase(block in differentiation)
CMLbull Chronic phasendash Driven by BCR‐ABLndash 50 no symptoms found on routine CBC
ndash uarrWBC PB BM spleenndash Fatigue weight loss abdominal fullness bleeding sweats large spleenliver
ndash 75 had accelerated phase 3‐18 months prior to blastic phase
bull Blast phasendash Acquire secondary mutations
ndash 13 = ALL 23 = AMLndash Decreased response to treatment and duration not long lasting
Westervelt
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
Hematopoiesis
Hematopoietic growth factor (HGF) signaling
NEJM (2006) 35419 p2034
ReceptorTyrosine Kinase(RTK)
(ABL non‐Receptor TK)
Frequent targets for mutation in leukemia
Classification of LeukemiasLymphoid Myeloid
CLLChronicLymphocytic Leukemia
CMLChronicMyeloid Leukemia
ALLAcuteLymphocytic Leukemia
AMLAcuteMyeloid Leukemia
10100000 1100000 2100000(~6000 newyear)
5100000
Acute = fast clinical progression Block in differentiation(weeks to months)
TerminallydifferentiatedBlood
ChronicMyeloidLeukemia(CML)
ChronicLymphocyticLeukemia(CLL)
EssentialThrombocytosis(ET)
PolycythemiaVera(PV)
Chronic Myelo‐MonocyticLeukemia(CMML)
Myeloproliferative Neoplasms (MPN)
Chronic Leukemias amp MPNs differentiation occurs
EssentialThrombocytosis(ET) (JAK2)
PolycythemiaVera(PV) (JAK2)
ChronicMyeloidLeukemia(CML) (BCRABL)
Genetics of Myeloproliferative Neoplasms (MPN)
Mastocytosis(KIT)
MyeloidNeoplasm withEosinophilia(PDGFRAB)
Mutationrearrangement of tyrosine kinase genes
CMLbull 141 () average age 45bull Increased blood WBC (gt25kuL) bull Minimalno fibrosis or dysplasia
Westervelt
Granulocyte maturation
Normal peripheral blood
Peter Maslak ASH Image Bank 2011 2011-1083
CML blood
Normal BM
basophil
eosinophil
CML chronic Blastic phase(block in differentiation)
CMLbull Chronic phasendash Driven by BCR‐ABLndash 50 no symptoms found on routine CBC
ndash uarrWBC PB BM spleenndash Fatigue weight loss abdominal fullness bleeding sweats large spleenliver
ndash 75 had accelerated phase 3‐18 months prior to blastic phase
bull Blast phasendash Acquire secondary mutations
ndash 13 = ALL 23 = AMLndash Decreased response to treatment and duration not long lasting
Westervelt
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Hematopoietic growth factor (HGF) signaling
NEJM (2006) 35419 p2034
ReceptorTyrosine Kinase(RTK)
(ABL non‐Receptor TK)
Frequent targets for mutation in leukemia
Classification of LeukemiasLymphoid Myeloid
CLLChronicLymphocytic Leukemia
CMLChronicMyeloid Leukemia
ALLAcuteLymphocytic Leukemia
AMLAcuteMyeloid Leukemia
10100000 1100000 2100000(~6000 newyear)
5100000
Acute = fast clinical progression Block in differentiation(weeks to months)
TerminallydifferentiatedBlood
ChronicMyeloidLeukemia(CML)
ChronicLymphocyticLeukemia(CLL)
EssentialThrombocytosis(ET)
PolycythemiaVera(PV)
Chronic Myelo‐MonocyticLeukemia(CMML)
Myeloproliferative Neoplasms (MPN)
Chronic Leukemias amp MPNs differentiation occurs
EssentialThrombocytosis(ET) (JAK2)
PolycythemiaVera(PV) (JAK2)
ChronicMyeloidLeukemia(CML) (BCRABL)
Genetics of Myeloproliferative Neoplasms (MPN)
Mastocytosis(KIT)
MyeloidNeoplasm withEosinophilia(PDGFRAB)
Mutationrearrangement of tyrosine kinase genes
CMLbull 141 () average age 45bull Increased blood WBC (gt25kuL) bull Minimalno fibrosis or dysplasia
Westervelt
Granulocyte maturation
Normal peripheral blood
Peter Maslak ASH Image Bank 2011 2011-1083
CML blood
Normal BM
basophil
eosinophil
CML chronic Blastic phase(block in differentiation)
CMLbull Chronic phasendash Driven by BCR‐ABLndash 50 no symptoms found on routine CBC
ndash uarrWBC PB BM spleenndash Fatigue weight loss abdominal fullness bleeding sweats large spleenliver
ndash 75 had accelerated phase 3‐18 months prior to blastic phase
bull Blast phasendash Acquire secondary mutations
ndash 13 = ALL 23 = AMLndash Decreased response to treatment and duration not long lasting
Westervelt
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Classification of LeukemiasLymphoid Myeloid
CLLChronicLymphocytic Leukemia
CMLChronicMyeloid Leukemia
ALLAcuteLymphocytic Leukemia
AMLAcuteMyeloid Leukemia
10100000 1100000 2100000(~6000 newyear)
5100000
Acute = fast clinical progression Block in differentiation(weeks to months)
TerminallydifferentiatedBlood
ChronicMyeloidLeukemia(CML)
ChronicLymphocyticLeukemia(CLL)
EssentialThrombocytosis(ET)
PolycythemiaVera(PV)
Chronic Myelo‐MonocyticLeukemia(CMML)
Myeloproliferative Neoplasms (MPN)
Chronic Leukemias amp MPNs differentiation occurs
EssentialThrombocytosis(ET) (JAK2)
PolycythemiaVera(PV) (JAK2)
ChronicMyeloidLeukemia(CML) (BCRABL)
Genetics of Myeloproliferative Neoplasms (MPN)
Mastocytosis(KIT)
MyeloidNeoplasm withEosinophilia(PDGFRAB)
Mutationrearrangement of tyrosine kinase genes
CMLbull 141 () average age 45bull Increased blood WBC (gt25kuL) bull Minimalno fibrosis or dysplasia
Westervelt
Granulocyte maturation
Normal peripheral blood
Peter Maslak ASH Image Bank 2011 2011-1083
CML blood
Normal BM
basophil
eosinophil
CML chronic Blastic phase(block in differentiation)
CMLbull Chronic phasendash Driven by BCR‐ABLndash 50 no symptoms found on routine CBC
ndash uarrWBC PB BM spleenndash Fatigue weight loss abdominal fullness bleeding sweats large spleenliver
ndash 75 had accelerated phase 3‐18 months prior to blastic phase
bull Blast phasendash Acquire secondary mutations
ndash 13 = ALL 23 = AMLndash Decreased response to treatment and duration not long lasting
Westervelt
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
TerminallydifferentiatedBlood
ChronicMyeloidLeukemia(CML)
ChronicLymphocyticLeukemia(CLL)
EssentialThrombocytosis(ET)
PolycythemiaVera(PV)
Chronic Myelo‐MonocyticLeukemia(CMML)
Myeloproliferative Neoplasms (MPN)
Chronic Leukemias amp MPNs differentiation occurs
EssentialThrombocytosis(ET) (JAK2)
PolycythemiaVera(PV) (JAK2)
ChronicMyeloidLeukemia(CML) (BCRABL)
Genetics of Myeloproliferative Neoplasms (MPN)
Mastocytosis(KIT)
MyeloidNeoplasm withEosinophilia(PDGFRAB)
Mutationrearrangement of tyrosine kinase genes
CMLbull 141 () average age 45bull Increased blood WBC (gt25kuL) bull Minimalno fibrosis or dysplasia
Westervelt
Granulocyte maturation
Normal peripheral blood
Peter Maslak ASH Image Bank 2011 2011-1083
CML blood
Normal BM
basophil
eosinophil
CML chronic Blastic phase(block in differentiation)
CMLbull Chronic phasendash Driven by BCR‐ABLndash 50 no symptoms found on routine CBC
ndash uarrWBC PB BM spleenndash Fatigue weight loss abdominal fullness bleeding sweats large spleenliver
ndash 75 had accelerated phase 3‐18 months prior to blastic phase
bull Blast phasendash Acquire secondary mutations
ndash 13 = ALL 23 = AMLndash Decreased response to treatment and duration not long lasting
Westervelt
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
EssentialThrombocytosis(ET) (JAK2)
PolycythemiaVera(PV) (JAK2)
ChronicMyeloidLeukemia(CML) (BCRABL)
Genetics of Myeloproliferative Neoplasms (MPN)
Mastocytosis(KIT)
MyeloidNeoplasm withEosinophilia(PDGFRAB)
Mutationrearrangement of tyrosine kinase genes
CMLbull 141 () average age 45bull Increased blood WBC (gt25kuL) bull Minimalno fibrosis or dysplasia
Westervelt
Granulocyte maturation
Normal peripheral blood
Peter Maslak ASH Image Bank 2011 2011-1083
CML blood
Normal BM
basophil
eosinophil
CML chronic Blastic phase(block in differentiation)
CMLbull Chronic phasendash Driven by BCR‐ABLndash 50 no symptoms found on routine CBC
ndash uarrWBC PB BM spleenndash Fatigue weight loss abdominal fullness bleeding sweats large spleenliver
ndash 75 had accelerated phase 3‐18 months prior to blastic phase
bull Blast phasendash Acquire secondary mutations
ndash 13 = ALL 23 = AMLndash Decreased response to treatment and duration not long lasting
Westervelt
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
CMLbull 141 () average age 45bull Increased blood WBC (gt25kuL) bull Minimalno fibrosis or dysplasia
Westervelt
Granulocyte maturation
Normal peripheral blood
Peter Maslak ASH Image Bank 2011 2011-1083
CML blood
Normal BM
basophil
eosinophil
CML chronic Blastic phase(block in differentiation)
CMLbull Chronic phasendash Driven by BCR‐ABLndash 50 no symptoms found on routine CBC
ndash uarrWBC PB BM spleenndash Fatigue weight loss abdominal fullness bleeding sweats large spleenliver
ndash 75 had accelerated phase 3‐18 months prior to blastic phase
bull Blast phasendash Acquire secondary mutations
ndash 13 = ALL 23 = AMLndash Decreased response to treatment and duration not long lasting
Westervelt
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
CML chronic Blastic phase(block in differentiation)
CMLbull Chronic phasendash Driven by BCR‐ABLndash 50 no symptoms found on routine CBC
ndash uarrWBC PB BM spleenndash Fatigue weight loss abdominal fullness bleeding sweats large spleenliver
ndash 75 had accelerated phase 3‐18 months prior to blastic phase
bull Blast phasendash Acquire secondary mutations
ndash 13 = ALL 23 = AMLndash Decreased response to treatment and duration not long lasting
Westervelt
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
CMLbull Chronic phasendash Driven by BCR‐ABLndash 50 no symptoms found on routine CBC
ndash uarrWBC PB BM spleenndash Fatigue weight loss abdominal fullness bleeding sweats large spleenliver
ndash 75 had accelerated phase 3‐18 months prior to blastic phase
bull Blast phasendash Acquire secondary mutations
ndash 13 = ALL 23 = AMLndash Decreased response to treatment and duration not long lasting
Westervelt
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
BCRABL fusion in CML
bull Cytogenetics = t(922) Philadelphia chromosome + (Ph+) (100 BCR‐ABLtranscript)
bull BCR‐ABL transcript +
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Tjio reports 46 chromosomes = normal
ldquodark agesrdquoTechnical problems
Flemming draws first chromosome
Waldeyer coins ldquochromosomerdquo
Painter reports 48 chromosomes = normal
1 better cultures2 hypotonic solution3 colchicine
Down+21
syndromes
TurnerXOKlinefelterXXY
1890
von Hansemann observesmitotic aberrations in tumor cells
1960
Nowell amp Hungerford = Ph chromosome amp CML
ldquoThe findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemiardquo
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
1973
Rowley reportsPh chr = t(922)
1960
Nowell amp Hungerford = Ph chromosome amp CML
1983‐84 1998‐2001
Druker uses Gleevec to treat CML
Heisterkamp Stephenson Groffen Grosveld clone genes at breakpoints (BCR ABL)
Chromosome Gene Drug
Baltimore v‐Abl1980
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
v‐ABL oncogene
Lymphosarcoma virus‐induced thymic‐independent disease in miceAbelson HT Rabstein LS Cancer Res 1970 Aug30(8)2213‐22
Cell 1979 Dec18(4)955‐62
Cell 1980 Dec22(3)777‐85
v‐Abl
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
ndash p190 = 20‐30 ALL (most B‐cell)ndash p210 (p230) = 99 CML
bull ABL = Loss of N‐terminal Cap region of ABL normally inhibits kinase activity
bull BCR = Gain coil‐coil of BCR allows dimerization of BCR‐ABLndash Results in constitutive activation of the ABL tyrosine kinase
ndash Effects on cellular proliferation survival and motilityDan Link
(coil‐coil)
Heisterkamp Oncogene 2002 21(56)8536‐40
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Bone Marrow Cells
Semi-solid media(methylcellulose)
Culture with specific growth factors for 7-14 days
CFU-GM(GM-CSF)
BFU-E(Epo)
Colony‐forming Cell Assay (progenitors)
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Methylcellulose cultures
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Hematopoietic Stem Cell Assaysbull HSC Definition capable of long‐term (6 months) repopulation of ALL hematopoietic lineages
bull Bone marrow transplantation (gold standard)ndash Xenotransplantation models to assay human HSC
bull Long term culture‐initiating cell (LTC‐IC) assay
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID miceNODSCID gamma (NSG)
(immunodeficient)
Analyze after 3-6 months for blood cells
Mousehematopoietic donor cells (CD452)
congenic recipient mice
(CD451)
Analyze after 3-6 months for blood cells
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Better assays needed to study hematopoiesis
bull Methylcellulose and transplant assays are still the gold standard but is there a faster cheaper way to study these cells
bull How can you prospectively identify the cells and isolate them to study
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Flow cytometry
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
radiation
Murine or Xenotransplantation
Human hematopoietic cells
NODSCID mice(immunodeficient)
Analyze after 3-6 months for blood cells
CD34+ xenotransplant assays = 15000 HSCsCD34+CD38- = 1617 HSCs
Mousehematopoietic cells
congenic mice(CD451CD452)
Kit+Lineage-Sca+ (KLS) cells = 110 HSCsCD150+CD48- (SLAM) cells = 12 HSCs
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
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)
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
929 citations
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
288 single cells of each type
CMP MEP GMPCMP
MEP
GMP No Epo+Tpo
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
200 cells each
Cytospin of 5 pooled colonies
CMP MEP GMP
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
1000 cells on stroma
Single cell sorted
Lineage commitment at CMP single cell level
CMP
MEP
GMP
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Regulation of Hematopoiesis
bull Transcription factorsndash Regulate RNA expression of target genesndash Critical determinants of lineage commitment and terminal differentiation
bull Frequent targets for mutation in leukemia
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
PNAS (2002)9918 p11872
HUMAN PROGENITOR ASSAY
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
NEJM (2006) 35419 p2034
Stem cell
Progenitors
Terminallydifferentiated
Bonemarrow
Blood
What cell is transformed in CML
Link
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Lancet (2007)370 p342
CML therapyOhUyRomeeRadich
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Targeted therapy (chronic phase)bull Imatinib = discovered in high‐throughput screen of kinase inhibitors
NEJM 2003 Oct 9349(15)1399‐401
WesterveltOhUyRadich
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-
Issues
bull Cure vs control of chronic phase CMLbull Resistance in chronic phase CMLbull Treatment of blastic phase CML
- Chronic Myeloid Leukemia(CML)
- CML paradigm for cancer treatment
- Chronic Myeloid Leukemia Session Overview
- Slide Number 4
- Hematopoietic growth factor (HGF) signaling
- Slide Number 6
- Chronic Leukemias amp MPNs differentiation occurs
- Genetics of Myeloproliferative Neoplasms (MPN)
- CML
- Slide Number 10
- CML
- BCRABL fusion in CML
- Slide Number 13
- Slide Number 14
- Slide Number 15
- Slide Number 16
- Slide Number 17
- Slide Number 18
- Methylcellulose cultures
- Hematopoietic Stem Cell Assays
- Slide Number 21
- Better assays needed to study hematopoiesis
- Flow cytometry
- Slide Number 24
- Slide Number 25
- Slide Number 26
- Slide Number 27
- Slide Number 28
- Slide Number 29
- Regulation of Hematopoiesis
- Slide Number 31
- Slide Number 32
- Slide Number 33
- Slide Number 34
- CML therapy
- Targeted therapy (chronic phase)
- Issues
-