Bone Marrow Failure is a PreLeukemic Condition

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Bone Marrow Failure is a Pre-Leukemic Condition

• High frequency of chromosome 7 abnormalities in
  AML arising in the setting of bone marrow
  failure (35-68) versus de novo AML (5)

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Severe Congenital Neutropenia (Kostmanns
Syndrome)

• First described by Kostmann in 1956.
• Clinical manifestations
• Chronic severe neutropenia present at birth
• Accumulation of granulocytic precursors in the
  bone marrow
• Rare (1 in 100,000)
• Inherited in a sporadic, autosomal dominant, and
  autosomal recessive fashion
• Treatment with G-CSF is effective

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Bone Marrow Examination
Normal
SCN
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Stem Cell
CFU-GM
Myeloblast
Granulocytic Differentiation
Promyelocyte
Myelocyte
Metamyelocyte
Band Neutrophil
Segmented Neutrophil
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Cardinal Features of SCN

• Isolated block in granulocytic differentiation
• Severe chronic neutropenia
• Markedly increased risk of developing AML or MDS
• Cumulative risk of MDS/AML is 11.7 in SCN
• 9 for patients on G-CSF for lt6 years
• 23 for patients on G-CSF between 6 10 years
• 33 for patients on G-CSF gt10 years

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Cell Culture Studies in SCN

• Cell intrinsic defect in the granulocytic
  differentiation of progenitor cells
• Increased apoptosis of granulocytic cells upon
  growth factor deprivation

• Decreased responsiveness of progenitors to
  granulocyte colony-stimulating factor (G-CSF)
• Treatment with G-CSF is effective in increasing
  neutrophil counts in most patients

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G-CSF

• Hematopoietic cytokine that regulates neutrophil
  production, differentiation, and function.
• Widely used in the clinical setting to treat
  neutropenia
• G-CSF and G-CSF receptor deficient mice display
  severe isolated neutropenia

• G-CSF is the principal cytokine regulating
  granulopoiesis.

• Is altered G-CSF signaling responsible for SCN?

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G-CSF Receptor
Ig-like
C C C C
CRH
Extracellular Domain
WSXWS
Fn3
Fn3
Transmembrane Domain
Fn3
Box 1
Box 2
Y 704
Cytoplasmic Domain
Y 729
Y 744
Y 764
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G-CSF Receptor Signaling
G-CSF
Jak
c-rel
p38
STATs
Y
Ras
JNK
Lyn
Y
Y
Y
Erk1/2/5
PI
k
3
VAV
SHC
GRB2/SOS
SHIP
Akt
Bmx
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• Proliferative signals are enhanced by the mutant
  G-CSFR

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• Granulocytic differentiation by the mutant
  G-CSFR is impaired

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G-CSFR Mutations in SCN

• G-CSFR mutations are acquired!

• 30 of SCN patients have G-CSF receptor
  mutations
• These mutations are strongly associated with
  development of MDS and AML
• Collectively, 17/21 patients with MDS/AML have
  G-CSFR mutations.
• Mutations are rare in de novo AML
• 1 of 58 cases tested

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Questions

• Do G-CSFR mutations contribute to impaired
  granulopoiesis in patients with SCN?
• How do cells expressing the mutant G-CSFR gain
  clonal dominance?
• Why are premature truncation mutations of the
  G-CSFR only seen in the setting of SCN?
• Do G-CSFR mutations contribute to leukemogenesis?
• Should the detection of mutant G-CSFR lead
  immediately to transplant for these patients?

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18 kb
Wt G-CSFR allele
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15
17
14
13
11
E

Neo
Neo
Targeting vector
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15
17
E

Neo
Mt G-CSFR allele
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16
15
17
14
13
Probe
6 kb
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Blood Analysis in d715 G-CSFR Mice
d715 G-CSFR mice are not neutropenic
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Questions

• Do G-CSFR mutations contribute to impaired
  granulopoiesis in patients with SCN?
• How do cells expressing the mutant G-CSFR gain
  clonal dominance?
• Why are premature truncation mutations of the
  G-CSFR only seen in the setting of SCN?
• Do G-CSFR mutations contribute to leukemogenesis?
• Should the detection of mutant G-CSFR lead
  immediately to transplant for these patients?

No!
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Hematopoiesis
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Competitive Repopulation Assay
1,000 cGy
Wild type host (Ly 5.1)
Donor
(d715 Ly 5.2)
Hematologic Recovery
and
(6 months)
(Wild-type Ly 5.1)
11 Ratio of Bone Marrow Cells
Bone Marrow Chimera
(5 x 106 cells)
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Flow Cytometry Assay
61.8
51.0
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d715 Chimeras 6 months after transplantation11
tranplantation ratio
63.5
46.6
50.0
45.7
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d715 Chimeras G-CSF (10ug/kg/d x 21 days)
BM 63.3 89.1
BM 75.8 98.6
T cell
Neutrophil
61.1 68.4
49.7 60.5
52.6 97.6
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Analysis of Stem Cell Compartment
c-Kit
Sca-1
Untreated
G-CSF treated
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d715 Chimeras G-CSF (10ug/kg/d x 21 days)
53.3 97.8
61.1 68.4
49.7 60.5
52.6 97.6
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Questions

• Do G-CSFR mutations contribute to impaired
  granulopoiesis in patients with SCN?
• How do cells expressing the mutant G-CSFR gain
  clonal dominance?
• Why are premature truncation mutations of the
  G-CSFR only seen in the setting of SCN?
• Do G-CSFR mutations contribute to leukemogenesis?
• Should the detection of mutant G-CSFR lead
  immediately to transplant for these patients?

No!
The G-CSFR mutations confer a growth and/or
survival advantage at the hematopoietic stem cell
level.
The G-CSFR mutations are dependent upon G-CSF.
In SCN systemic levels of G-CSF are high.
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Two-hit Model of Acute Leukemia
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• Hypothesis Activating mutations in one or more
  cytokine signaling genes occur in every patient
  with AML

PTPN11 (SHP-2)
Rasgrp1 (RAS-GAP)
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Chimeric Transcription Factor
RTK Signal
Leukemia?
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d715 Tumor Watch
The d715 G-CSFR is not sufficient to induce AML
or MDS in mice even with chronic G-CSF stimulation
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Cooperativity between d715 and PML-RARa
Too early to tell!
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Questions

• Do G-CSFR mutations contribute to impaired
  granulopoiesis in patients with SCN?
• How do cells expressing the mutant G-CSFR gain
  clonal dominance?
• Why are premature truncation mutations of the
  G-CSFR only seen in the setting of SCN?
• Do G-CSFR mutations contribute to leukemogenesis?
  
• Should the detection of mutant G-CSFR lead
  immediately to transplant for these patients?

No!
The G-CSFR mutations confer a growth and/or
survival advantage at the hematopoietic stem cell
level.
The G-CSFR mutations are dependent upon G-CSF.
In SCN systemic levels of G-CSF are high.
?
?
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• Inherited mutations of the ELA2 gene are found
  in
• 100 of cases of cyclic neutropenia
• 38-87 of cases of SCN

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Somatic Mosaicism of an ELA2 Gene Mutation
WT
MT
WT
WT
MT
MT
T-lymphocytes
Neutrophils
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Somatic Mosaicism of an ELA2 Gene Mutation
WT
MT
WT
WT
MT
MT
T-lymphocytes
Neutrophils
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ELA2 mutations in SCNSummary of Genetic Data

• Inherited heterozygous mutations in the majority
  of cases of SCN
• Selective loss of ELA2 mutant neutrophils in a
  case of paternal mosaicism

• Hypothesis ELA2 mutations act in a dominant
  (gain-of-function) and cell-intrinsic fashion to
  induce a block in granulocytic differentiation

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What is Neutrophil Elastase?

• Serine protease expressed at the promyelocyte
  stage of granulocytic differentiation.
• Many recognized and potential substrates,
  including coagulation proteins, growth factors,
  and extracellular matrix components
• NE deficient mice have normal granulopoiesis

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Biochemical Characterization of Mutant NE

• No consistent effect on NE proteolytic activity
  against a number of peptide substrates
• No consistent effect on subcellular localization
• No consistent effect on ability to interact with
  serpins
• Enforced expression in cell lines does not induce
  apoptosis

Li et al., JBC, 27614230, 2001
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7.6 kb
B
B
N
N
N
H
WT NE
1
2
4
3
5
Probe
B
B

Targeting
PGK-NEO
Vector
5.2 kb
B
B
B

NEO-targeted
PGK-NEO
Allele
3.6 kb
B
B
B

Targeted
Allele
1
2
4
3
5
LoxP
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Peripheral Blood Counts
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Weekly blood counts
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Morphologic Analysis of Bone Marrow
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Apoptosis
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Stress Granulopoiesis Response
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V72M NE Transgenic Mice

• Competitive repopulation assay
• Normal contribution of heterozygous V72M NE cells
  to granulopoiesis
• Cross with G-CSF receptor mutant mice
• No effect on granulopoiesis
• Tumor Watch
• No AML with a median follow-up of 15 months

• Expression of the V72M murine NE mutant is not
  sufficient to induce an SCN phenotype or AML in
  mice.

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V72M NE Transgenic Mice

• The V72M mutation may not have the same effect on
  murine NE structure and/or function as is does on
  human NE.
• The murine hematopoietic environment may not
  present the necessary NE target proteins for the
  development of an SCN phenotype.
• Expression of mutant NE may not be sufficient to
  induce an SCN phenotype.

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