clonal dominance role of alterations in the bone marrow microenvironment mechanisms of...
TRANSCRIPT
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Clonal dominance Role of alterations in the bone marrow microenvironment
Mechanisms of Leukemogenesis in Patients with SCN
Daniel C. Link
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Severe Congenital Neutropenia
(Kostmann’s Syndrome)
• Clinical manifestations:– Chronic severe neutropenia present at birth– Accumulation of granulocytic precursors in the bone
marrow– Recurrent infections
• Treatment with G-CSF– Reduces infections and improves survival
• Marked propensity to develop acute myeloid leukemia or myelodysplasia
• Clinical manifestations:– Chronic severe neutropenia present at birth– Accumulation of granulocytic precursors in the bone
marrow– Recurrent infections
• Treatment with G-CSF– Reduces infections and improves survival
• Marked propensity to develop acute myeloid leukemia or myelodysplasia
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CFU-GM
Stem Cell
Segmented Neutrophil
Promyelocyte
Myeloblast
Metamyelocyte
Myelocyte
Band Neutrophil
Block in granulocyticdifferentiation
What are the molecular mechanisms for the isolated block in granulopoiesis
What is the molecular basis for the marked susceptibility to AML
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Genetics of SCN
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All mutations are heterozygous Act in a cell intrinsic fashion to inhibit granulopoiesis
ELANE Mutations
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Molecular Pathogenesis of SCN associated with ELANE Mutations
Working hypothesis: ELANE mutations lead to the production of misfolded neutrophil elastase, induction of the unfolded protein response, and the subsequent apoptosis of granulocytic precursors resulting in neutropenia.
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Cumulative risk of MDS/AML in SCN: 21% after treatment with G-CSF for 10 years Cumulative risk of leukemia (all types) up to age 40: 0.15%
SCN and MDS/AML
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Risk of AML/MDS in Bone Marrow Failure Syndromes
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G-CSFR Mutations in SCN
• G-CSF receptor– Member of cytokine
receptor superfamily– Only known receptor for
G-CSF
• G-CSF receptor mutations in SCN– Acquired heterozygous
mutations– Strongly associated with
the development of AML
Box 1Box 2
CC
CC
-Y
CC
CC
-Y
G-CSFRG-CSFR d715d715
-Y-Y-Y
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Questions
• Do the G-CSFR mutations contribute to leukemic transformation?
• And if so, – How do the G-CSFR mutations gain clonal
dominance? – What are the molecular mechanisms
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d715 “Knock-in” Mice
WT G-CSFR gene
Targeting vector
d715 G-CSFR allele
Stop codon
d715 mice have normal basal granulopoiesis
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0 100 200 300 4000
25
50
75
100
WT/WT
WT/d715
WT/WT + G-CSF
WT/d715 + G-CSF
Time (days)
Pe
rce
nta
ge
su
rviv
al
The d715 G-CSFR is not sufficient to induce in mice even with chronic G-CSF stimulation
d715 Tumor Watch
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Leukemia?
Growth FactorMutations
Transcription FactorMutations+
d715 G-CSFR PML-RARα+
FLT3 ITD PML-RARα+
Oncogene Cooperativity
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Truncations mutations of the G-CSFR contribute to leukemic transformation in SCN.
D715 G-CSFR Tumor Watch
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G-CSFR mutations may be an early event during leukemogenesis
0 10 12 16 19 (age-years)
SCNG-CSFR
SCNG-CSFRRunx1
AMLG-CSFRRunx1-7, 5q-
SCN
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Clonal Dominance
Clinical Clinical LeukemiaLeukemia
G-CSFR mutations
Likely has to occur in a long-lived self-renewing cell (eg, stem cell)
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Competitive Repopulation Assay
Wild typeWild type
1:1 Ratio1:1 Ratio
1,000 cGy
Bone Marrow ChimeraBone Marrow Chimera Syngeneic RecipientSyngeneic Recipientwild type (wild type (Ly5.1Ly5.1))
d715d715
Harvest Bone Marrow
Wild type
d715
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Competitive Repopulation AssayCompetitive Repopulation Assay
Wild type
d715
Competitive AdvantageNo Competitive Advantage
3-6 Months
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Ly5.2 (d715)Ly5.2 (d715)
B2
20
B2
20
Gr-
1G
r-1
Donor Chimerism Analysis
Ly5.2 (d715)Ly5.2 (d715)
61.8%61.8% 51.0%51.0%
B Lymphocytes Neutrophils
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d715 Chimerasd715 Chimeras6 months after transplantation—1:1 ratio
63.5% 46.6% 45.7%50.0%
Red blood cell Platelet
Common Myeloid Progenitor
NeutrophilMonocyte
Common Lymphoid Progenitor
CFU-GM BFU-E CFU-Meg
B cell
HSC
T cell
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d715 Chimeras G-CSF (10ug/kg/d x 21 days)
61.1%68.4%
49.7%60.5%
52.6%97.6%
Red blood cell Platelet
Common Myeloid Progenitor
NeutrophilMonocyte
Common Lymphoid Progenitor
CFU-GM BFU-E CFU-Meg
B cell
HSC
T cell
BM63.3%89.1%
BM75.8%98.6%
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Long-term d715 G-CSFR chimerism following G-CSF treatment for 21 days
69.2
47.3
76.6
56.9
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d715 Chimeras G-CSF (10ug/kg/d x 21 days)
61.1%68.4%
49.7%60.5%
52.6%97.6%
Red blood cell Platelet
Common Myeloid Progenitor
NeutrophilMonocyte
Common Lymphoid Progenitor
CFU-GM BFU-E CFU-Meg
B cell
HSC
T cell
BM63.3%89.1%
BM75.8%98.6%
53.3%53.3%97.8%97.8%
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Conclusion
The d715-G-CSFR confers a clonal advantage at the hematopoietic stem cell level in a G-CSF dependent fashion
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WT d715
G-CSF Saline G-CSF Saline
Harvest bone marrow at 3 hours
RNA expression profiling
RNA Expression Profiling
Sort Kit+ Sca+ Lineage- (KSL) cells
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Differentially regulated genes
Sp
rr2a
En
ah
Bcat1
Tcrg
Pim
2
Cacn
b2
Cd
kn
1a
SO
CS
2
Cis
h
Tn
fsft
1
Zfp
n1a4
Serp
ina3g
Ltb
4r1
0.0
2.5
5.0
7.5
10.0
12.5
15.0
Wt
d715
20
30
40
50
Genes
Rati
o o
f G
-CS
F/S
ali
ne
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STAT3 KSL cells
0 25 50 75 100 1250
10
20
30
40
50
*
Time (minutes)
STAT5 KSL cells
0 25 50 75 100 1250
5
10
15
20
WT
d715 G-CSFR
Time (minutes)
In mutant GR KSL cells, STAT3 activation by G-CSF is attenuated while STAT5 activation is enhanced
Stat3 phosphorylation Stat5 phosphorylation
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• What are the STAT5 target genes that mediate clonal dominance
• Would inhibitors of STAT5 (or their target genes) be effective therapeutic agents in AML.
G-CSFR mutations
Acts at the HSC level Dependent on exogenous G-CSF Mediated by exaggerated STAT5 activation
Clonal Dominance
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Vascular NicheOsteoblast Niche
Stem Cell Niches
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Chronic disruption of the stem cell niche in the bone marrow may contribute to the high rate of leukemic transformation in bone marrow failure syndromes
Normal
G-CSF low
BMFS (e.g., SCN)
G-CSF high
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Wild-type d715 G-CSFR
No G-CSFSingle dose
G-CSF7 days of
G-CSF
Harvest Bone Marrow
Flow Cytometry•ROS in KSL cells•H2AX phosphorylation in KSL cells
G-CSF ROS induction is rapid in vitro(within 10-60 minutes)
Prolonged G-CSF (≥ 5 days) is associated with marked changes in bone marrow stromal cells
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ROS Induction is increased in d715 KSL cells after 7 days of GCSF Rx
ROS
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H2Ax Phosphorylation Enhanced in d715 KSL cells after 7 days of GCSF Rx
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NAC attenuates G-CSF induced H2AX phosphorylation
WT or d715 G-CSFR mice
G-CSF (7 days) alone
G-CSF (7 days)+
N-acetyl cysteine (NAC)
MeasurementROS
H2AX-P
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Hypothesis: Changes in the BM microenvironment induced by G-CSF contribute to DNA damage
G-CSF treatment in mice• Decreases osteoblasts• Decreases SDF1 expression• These effects are delayed, first becoming apparent on day of G-CSF
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Untreated G-CSF
G-CSF suppresses mature osteoblasts
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Signaling through the d715 G-CSFR results in marked osteoblast and CXCL12 (SDF1) suppression
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Normal
G-CSF low
AMD3100
•Specific CXCR4 antagonist
•Disrupts HSPC/stromal interactions
•Results in HSPC mobilization
Question: Does disruption of stromal/HSPC interactions sensitize cells to G-CSF induced oxidative DNA damage
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Question: Does disruption of stromal/HSPC interactions sensitize cells to G-CSF induced oxidative DNA damage
WT or d715 G-CSFR mice
G-CSF (1 dose) alone
G-CSF (1 dose)+
AMD3100
MeasureH2AX
phosphorylation
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Normal SCN
G-CSF low G-CSF high
Lowering G-CSF levels (by treating the underlying neutropenia) may reduce the risk of AML Biomarkers of bone metabolism might predict risk of AML Treatment with G-CSF, by disrupting the stem cell niche, may sensitize leukemic cells to chemotherapy
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Nature, Jan 20, 2011
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Pre-B ALL is the most common pediatric cancer – 30% of all cancers in children
5-year survival rate of 80%
Structural abnormalities:
t(12;21) ETV6/RUNX1 : 20-25%t(1;19) E2A/PBX1 translocation: 5 %t(4;11) MLL/AF4 rearrangement : 5%t(9;22) BCR/ABL translocation (Philadelphia chromosome): 3-4%t(8;14) MYC/IGH translocation : 1%
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Zelent, Oncogene, 2004
Subset of childhood pre-B ALL with ETV6-RUNX1 fusion
Associated with modest number of recurrent genomic CNA (3-6).Del ETV6, del CDKN2A, del PAX5, del 6q, gain Xq
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Figure 1A
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Figure 1B
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Figure 1C
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Author comments• Common or highly recurrent CNA are not
acquired in any particular order.
• Sub-clones with highest number of CNA were not necessarily numerically dominant.
• CNA involving the same gene could be simultaneously present in distinct sub-clones and must therefore arise more than once, independently.
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Supplementary Figure 3
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Supplementary Figure 3
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Figure 2A
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Figure 2b
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Supplementary Figure 2
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•Clonal architecture at relapse is different from that of diagnosis in most patients.
•Relapse seem to derive from either major or minor clones at diagnosis but with a suggestion that more than one sub-clone might contribute to relapse.
•The dominant sub-clone in relapse itself continues to genetically diversify.
Author Comments
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2x 10 3-6
Unfractionated or
immunophenotypically
Flow sorted ALL primary cells
NOD/SCID IL2Rγnull
NOD/SCID IL2Rγnull
Secondary transplant2x 10 3-6 equivalent ALL cells
250 cGy 250 cGy
Xenotransplantation Assay
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Patient 3
Figure 3
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Patient 7
Figure 4a-c
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Patient 3
Figure 4d
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Author Summary
• Distinctive genotypes are associated with variable capacity for leukemia propagation.
• The relevance of the xenotransplantation to study clonal expansion is questionable. Studies of clonal evolution in patients with ALL (e.g., at diagnosis and at relapse) are more relevant.
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Comments
• Clonal diversity is underestimated in this study (only few CNAs were measured. Not particularly sensitive assay with 1% detectable threshold.
• To study the full complexity of subclonal architecture will require whole genome genome sequencing at single cell level ( or colonies from leukemic cells).
• Implications for targeted therapy in cancer…