Transcript Hematopoiesis - Siteman Cancer Center

Mechanisms of Leukemogenesis
in Patients with SCN
Daniel C. Link
 Clonal dominance
 Role of alterations in the bone
marrow microenvironment
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
Stem Cell
CFU-GM
Myeloblast
What are the molecular
mechanisms for the isolated
block in granulopoiesis
Promyelocyte
Myelocyte
Metamyelocyte
Band Neutrophil
Segmented Neutrophil
Block in granulocytic
differentiation
What is the molecular basis
for the marked
susceptibility to AML
Genetics of SCN
ELANE Mutations
 All mutations are heterozygous
 Act in a cell intrinsic fashion to inhibit granulopoiesis
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.
SCN and MDS/AML
 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%
Risk of AML/MDS in Bone
Marrow Failure Syndromes
G-CSFR Mutations in SCN
• G-CSF receptor
C
C
C
C
– 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 1
Box 2
-Y
-Y
-Y
-Y
G-CSFR
C
C
C
C
-Y
d715
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
d715 “Knock-in” Mice
WT G-CSFR gene
Targeting vector
Stop codon
d715 G-CSFR allele
 d715 mice have normal basal granulopoiesis
d715 Tumor Watch
Percentage survival
100
75
WT/WT
WT/d715
50
WT/WT + G-CSF
WT/d715 + G-CSF
25
0
0
100
200
300
400
Time (days)
The d715 G-CSFR is not sufficient to induce in mice
even with chronic G-CSF stimulation
Oncogene Cooperativity
Growth Factor
Mutations
+
Transcription Factor
Mutations
FLT3 ITD
+
PML-RAR
+
PML-RAR
d715 G-CSFR
Leukemia?
D715 G-CSFR Tumor Watch
 Truncations mutations of the G-CSFR
contribute to leukemic transformation in SCN.
G-CSFR mutations may be an
early event during leukemogenesis
0
10
SCN
12
SCN
G-CSFR
16
SCN
G-CSFR
Runx1
19 (age-years)
AML
G-CSFR
Runx1
-7, 5q-
Clonal Dominance
G-CSFR mutations
Clinical
Leukemia
Likely has to occur in a long-lived self-renewing cell (eg, stem cell)
Competitive Repopulation Assay
Wild type
Wild type
Harvest
Bone Marrow
d715
1:1 Ratio
d715
1,000 cGy
Bone Marrow Chimera
Syngeneic Recipient
wild type (Ly5.1)
Competitive Repopulation Assay
Wild type
d715
3-6 Months
No Competitive Advantage
Competitive Advantage
Donor Chimerism Analysis
B Lymphocytes
Neutrophils
51.0%
Gr-1
B220
61.8%
Ly5.2 (d715)
Ly5.2 (d715)
d715 Chimeras
6 months after transplantation—1:1 ratio
HSC
Common Lymphoid Progenitor
Common Myeloid Progenitor
BFU-E
B cell
T cell
63.5% 46.6%
Red blood cell
CFU-Meg
CFU-GM
Platelet
Monocyte
Neutrophil
50.0%
45.7%
d715 Chimeras
G-CSF (10ug/kg/d x 21 days)
HSC
Common Lymphoid Progenitor
Common Myeloid Progenitor
BFU-E
BM
63.3%
89.1%
B cell
T cell
61.1% 49.7%
68.4% 60.5%
Red blood cell
CFU-Meg
CFU-GM
Platelet
Monocyte
BM
75.8%
98.6%
Neutrophil
52.6%
97.6%
Long-term d715 G-CSFR chimerism following
G-CSF treatment for 21 days
69.2
76.6
47.3
56.9
d715 Chimeras
G-CSF (10ug/kg/d x 21 days)
HSC
Common Lymphoid Progenitor
Common Myeloid Progenitor
BFU-E
BM
63.3%
89.1%
B cell
T cell
61.1% 49.7%
68.4% 60.5%
53.3%
97.8%
Red blood cell
CFU-Meg
CFU-GM
Platelet
Monocyte
BM
75.8%
98.6%
Neutrophil
52.6%
97.6%
Conclusion
The d715-G-CSFR confers a clonal
advantage at the hematopoietic stem cell
level in a G-CSF dependent fashion
RNA Expression Profiling
WT
G-CSF
d715
Saline
G-CSF
Harvest bone marrow at 3 hours
Sort Kit+ Sca+ Lineage- (KSL) cells
RNA expression profiling
Saline
Genes
Ltb4r1
Serpina3g
Zfpn1a4
Tnfsft1
Cish
SOCS2
Cdkn1a
Cacnb2
Pim2
Tcrg
Bcat1
Enah
Sprr2a
Ratio of G-CSF/Saline
Differentially regulated genes
50
40
30
20
15.0
12.5
Wt
10.0
d715
7.5
5.0
2.5
0.0
In mutant GR KSL cells, STAT3 activation by G-CSF is
attenuated while STAT5 activation is enhanced
Stat3 phosphorylation
Stat5 phosphorylation
G-CSFR mutations
Clonal
Dominance
Acts at
leveltarget genes that mediate clonal
• What
arethe
theHSC
STAT5
dominance
Dependent on exogenous G-CSF
 Mediated by exaggerated STAT5 activation
• Would inhibitors of STAT5 (or their target genes) be
effective therapeutic agents in AML.
Stem Cell Niches
Osteoblast Niche
Vascular Niche
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
Wild-type
No G-CSF
d715 G-CSFR
Single dose
G-CSF
G-CSF ROS
induction is rapid
in vitro
(within 10-60
minutes)
7 days of
G-CSF
Harvest Bone Marrow
Flow Cytometry
•ROS in KSL cells
•H2AX phosphorylation
in KSL cells
Prolonged G-CSF
(≥ 5 days) is
associated with
marked changes in
bone marrow
stromal cells
ROS Induction is increased in d715 KSL cells after
7 days of GCSF Rx
ROS
H2Ax Phosphorylation Enhanced in d715 KSL cells
after 7 days of GCSF Rx
NAC attenuates G-CSF induced H2AX phosphorylation
G-CSF (7 days) alone
WT or d715 G-CSFR mice
G-CSF (7 days)
+
N-acetyl cysteine (NAC)
Measurement
ROS
H2AX-P
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
G-CSF suppresses mature osteoblasts
Untreated
G-CSF
Signaling through the d715 G-CSFR results in marked
osteoblast and CXCL12 (SDF1) suppression
Question: Does disruption of stromal/HSPC interactions
sensitize cells to G-CSF induced oxidative DNA damage
Normal
AMD3100
G-CSF low
•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
G-CSF (1 dose) alone
WT or d715 G-CSFR mice
G-CSF (1 dose)
+
AMD3100
Measure
H2AX
phosphorylation
SCN
Normal
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
Nature, Jan 20,
2011
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%
Subset of childhood pre-B ALL with ETV6-RUNX1 fusion
Zelent, Oncogene, 2004
Associated with modest number of recurrent genomic CNA (3-6).
Del ETV6, del CDKN2A, del PAX5, del 6q, gain Xq
Figure 1A
Figure 1B
Figure 1C
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.
Supplementary Figure 3
Supplementary Figure 3
Figure 2A
Figure 2b
Supplementary Figure 2
Author Comments
•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.
Xenotransplantation Assay
Secondary transplant
2x 10 3-6 equivalent ALL cells
2x 10 3-6
Unfractionated or
immunophenotypically
Flow sorted ALL primary cells
NOD/SC
ID
null
IL2Rγ
250 cGy
NOD/SC
ID
null
IL2Rγ
250 cGy
Figure 3
Patient 3
Figure 4a-c
Patient 7
Figure 4d
Patient 3
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.
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…