Molecular Pathogenesis of Severe Congenital Neutropenia

1
Molecular Pathogenesis of Severe Congenital
Neutropenia

• Daniel C. Link
• Washington University

2
Severe Congenital Neutropenia (Kostmanns
Syndrome)

• First described by Kostmann in 1950.
• 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 predisposition to develop AML/MDS
• Cumulative incidence of 21 after 10 years G-CSF

3
Genetic Basis of SCN
4
ELA2 Mutations

• Marked diversity of mutations Over 50 mutations
  identified (80 missense, 10 truncations, 10
  splicing)
• Heterozygous-suggesting dominant mechanism of
  action
• Act in a cell-intrinsic fashion

5
Models of Disease Pathogenesis(SCN with ELA2
mutations)

• Dysregulated NE protease model
• Lack of a consistent affect of the different ELA2
  mutations on NE protease activity
• NE cellular mistrafficking model
• Dysregulation of LEF1
• LEF1 expression is reduced in myeloid cells of
  patients with SCN but not CN
• Restoration of LEF1 expression rescues the defect
  in granulocytic differentiation
• Relationship to ELA2 mutations unclear

6
Hypothesis

• Mutations in the ELA2 gene lead to the production
  of misfolded NE, an induction of the unfolded
  protein response (UPR), and the subsequent
  apoptosis of granulocytic precursors resulting in
  neutropenia.

7
The Unfolded Protein Response
8
Unfolded Protein Response

• Adaptive cellular program designed to handle
  misfolded proteins in the ER
• The UPR has two outcomes
• Successful handling of misfolded proteins.
• Induction of protein chaperones to facilitate
  protein folding
• Redox maintenance
• General inhibition of protein translation
• Degradation of misfolded protein (ERAD)
• Induction of apoptosis

9
UPR in Human Disease
10
Unfolded Protein Responseand ELA2 Mutations

• Level of protein expression
• Degree of protein misfolding

NE is expressed at extremely high levels at a
discrete stage of granulocytic differentiation
(promyelocytes)

• NE is predicted to have high surface
  hydrophobicity, suggesting an inherent propensity
  of protein misfolding
• Many of the NE mutations are predicted to cause
  structural changes and loss of protein stability

11
Induction of XBP-1 splicing

• Unspliced (US) XBP-1 mRNA retains 26 base-pair
  intron with a PstI site
• Spliced (S) XBP-1 mRNA is resistant to PstI
  digestion
• Ratio of processed to unprocessed XBP-1 is a
  measure of ER stress

12
Induction BiP (GRP78) mRNA
CN
CN/ SCN
SCN

• Real time RT-PCR for BiP, ?-actin, and NE
  performed
• Magnitude of BiP mRNA induction correlates with
  disease severity

13
Induction of Apoptosis

• Expression of mutant NE induces apoptosis in
  protease independent fashion

14
UPR Model of Disease Pathogenesis
15
Induction of Apoptosis

• Expression of CHOP, a gene associated with
  UPR-induced apoptosis, is increased

16
UPR Activation in Primary SCN Myeloid Precursors
Normal
SCN
CD16
CD15

• Strategy to sort promyelocytes/myelocytes from
  human bone marrow samples
• 87-96 of cells were promyelocytes or myelocytes

17
UPR Activation in Primary SCN Myeloid Precursors

• BM samples from 8 patients with SCN, 7 healthy
  controls, and 2 healthy volunteers treated with
  G-CSF
• Two-fold increase in percentage of spliced XBP-1
• 5.6-fold increase in BiP mRNA (relative to NE)
  expression

18
NE localization in SCN Myeloid Precursors
SCN
S97L
A32V
A28T
Normal

• Normal

NE
MPO
NE MPO To-Pro-3
19
NE localization in SCN Myeloid Precursors
SCN
Severe Idiopathic
A32V
ELA2 normal
Normal

• Normal

NE
MPO
NE MPO To-Pro-3
20
Defining the UPR transcriptome in human
granulocytic precursors
Tunicamycin
Sort Promyelo-cytes
RNA Profiling
16 hrs
UPR Transcriptome Human Promyelocytes (26 genes)

Normalhuman BM
Saline
UPR target genes (literature)
21
Primary Promyelocyte Samples

• 5 healthy volunteers
• 2 healthy volunteers treated with G-CSF for 5
  days (5 µg/kg/day)
• 8 patients with SCN (all with ELA2 mutations)
• 4 patients with SDS (neutropenia control)

22
SCN Promyelocytes Display a UPR Transcriptional
Signature
23
CLGN
BiP/Grp78 2.3-fold induction
CLGN 10.7-fold induction
CLGN Encodes a protein with ER chaperone
activity previously reported to be expressed in
testes.
24
Summary of Evidence Supporting the UPR model of
Disease Pathogenesis

• Expression of mutant but not wild type ELA2 in
  myeloid cells induces the UPR
• Primary granulocytic cells from patients with SCN
  display evidence of UPR activation
• G6PC3 deficient cells display UPR activation

25
Mouse Models of SCN(Ela2 mutations)
219
H41
D88
S174
WT mNE
M
V72M mNE
V72
X
G193X mNE
G 193
26
Generation of NEG193X transgenic mice
27
Expression of G193X mRNA
28
Expression of G193X NE protein
29
Induction of the UPR
Isolate Kit lineage- cells (WT or G193X ELA2
mice) Culture 3-4 days (Kit ligand and
G-CSF) Assess UPR Activation ATF6 BiP/GRP78 XBP1
splicing
30
Basal Granulopoiesis
31
Ongoing Experiments

• Characterize stress granulopoiesis in G193X Ela2
  mice
• Assess UPR activation in myeloid cells from G193X
  Ela2 mice
• Cross G193X Ela2 mice with Perk-/- mice
• Perk haploinsufficiency worsens UPR-induced cell
  death
• Cross G193X Ela2 mice with ATF6-/- mice

32
Acknowledgments
Link Lab David Grenda Jun Xia Mark Murakami
Suparna Nuana Ghada Kunter Kyle Eash Jill
Woloszynek Matt Christopher Fulu Liu Alyssa
Gregory Priya Gopalan Adam Greenbaum Nancy
Link Kevin ODell

• Collaborators
• David Dale
• Larry Boxer
• Mary Dinauer
• Adriana Vlachos
• Akiko Shimamura