genetics and epigenetics of blood stem cell function grant challen, ph.d. / challen lab division of...
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Genetics and Epigenetics of Blood Genetics and Epigenetics of Blood Stem Cell FunctionStem Cell Function
Genetics and Epigenetics of Blood Genetics and Epigenetics of Blood Stem Cell FunctionStem Cell Function
Grant Challen, Ph.D. / Challen LabGrant Challen, Ph.D. / Challen LabDivision of OncologyDivision of OncologyDepartment of Internal MedicineDepartment of Internal MedicineWashington University in St. LouisWashington University in St. Louis
Grant Challen, Ph.D. / Challen LabGrant Challen, Ph.D. / Challen LabDivision of OncologyDivision of OncologyDepartment of Internal MedicineDepartment of Internal MedicineWashington University in St. LouisWashington University in St. Louis
B-cells
T-cellsNK-cells
Monocytes
Granulocytes
Platelets
Erythrocytes
LT-HSC
Hematopoietic Stem Cells
• Regenerate the blood
• Long-term self-renewal
• Multi-lineage differentiation
Megakaryocytes
The Importance of HSCs in Basic Research and Clinical Practice
• Bone Marrow Transplantationo Most clinically successful stem cell therapyo In USA more than 18,000 patients require BMT each year
• HSC development / cancer mechanismso Many of the genes pathways critical for HSC function are also involved in hematopoietic
malignancies (e.g. leukemia, lymphoma)o Understanding the normal functions of these genes in HSC biology will help deduce the
effect of mutations in disease
• Paradigm for other stem cell systemso Well-defined system – markers, assays etc…o Many of the discoveries in HSC biology translate to other somatic stem cell systems
Rossi et al., Cell Stem Cell, 2012
HSCs are Tightly Regulated by Intrinsic and Extrinsic Factors
Rossi et al., Cell Stem Cell, 2012
B-cells
T-cellsNK-cells
Monocytes
Granulocytes
Platelets
Erythrocytes
LT-HSC
MYELOID
LYMPHOID
Clonal Diversity
Model
Heterogeneity in the HSC Compartment
Dykstra et al., Cell Stem Cell 2007
Sieburg et al.,Blood 2006
Contrasting functional outputs from phenotypically similar HSCs
HSC Activity
Gradient of Activity Within the SP
Goodell et al., Nature Medicine 1997
Can Hoechst dye efflux discriminate
functionally distinct HSC subtypes?
0 750 1,500 2,250 3,000 3,750
1
2
3
4
52 weeks after transplant
WBM
WBM
Tri-lineage?
Tri-lineage?
Single Cell Transplantation
Long-Term Self
Renewal?
L-SP U-SP
18 / 65 15 / 76
27.7% 19.7%
En
gra
ftm
ent
Myeloid B-cells T-cells
Lin
eag
esPeripheral Blood 12-weeks Post-Transplant
Lower-SPKLS Upper-SPKLS
* ** * * ** *Myeloid-biased Lymphoid-biased
Myeloid B-cells T-cells
L-SP
U-SP
Secondary Transplants - 12-weeks
Lineage bias is a stable phenotype
SP powerfully discriminates these activities
Lineage Bias
Myeloid
B-Cells
T-Cells
Myeloid bias with age results from predominance of My-HSC type rather than intrinsic change
Physiological Relevance
•Aging -
• Old HSCs show myeloid bias
• Could this result from proportional changes in the HSC subtypes?
Molecular Regulation of HSC Subtypes
•Microarray Analysis -
• TGF signaling pathway enriched in My-HSCs
• Both inhibitory and stimulatory for HSCs
• Due to different effects on HSC subtypes?
My-HSCs Ly-HSCs
PBS TGF1 PBS TGF1
in vivo injection – 12-hoursin vitro culture – 5-hours
Effect of TGF1 on HSCs in vitro
Myeloid-biased HSC Lymphoid-biased HSC
Difference mainly due to effect on CFU-GM colonies
Myeloid
Lymphoid
MYELOID
LYMPHOID
TGF1 response
Proliferation
Self-renewalDye stain
• Stable
• Unionized
Aging
Artur Pappenheim 1905
“Stamzelle”
Paul Erlich“Dualists”
Neumann, Maximow“Unitarians”
Ramalho-Santos & WillenbringCell Stem Cell 2007
Differences – MolecularFunctional Phenotypic Epigenetic
Lower-SPKLS
434 Genes
Upper-SPKLS
351 Genes
DNA methyltransferase 3a (Dnmt3a)
Epigenetic Factors are Differentially Expressed in HSC Subtypes
Jarid1a
Jarid1b
Jarid1c
Suz12
Jmjd1c
Ehmt1
Ehmt2
Epc1
Phc3
Nsd1
• de novo AML ~22%
• MDS ~10%
• T-cell lymphoma ~11%
• T-ALL ~18%
DNMT3A Mutations in Hematopoietic Malignancies
Mutated Gene Function Disease
IDH1, IDH2 isocitrate dehydrogenase MPN, MDS, AML
TET2 methylcytosine dioxygenase MPN, MDS, AML
EZH2 H3K27me3 methyltransferase MPN, MDS, AML, ALL
ASXL1 chromatin-binding protein MPN, MDS, AML
MLL H3K4me3 methyltransferase AML, ALL
DNMT3A DNA methyltransferase AML, MDS, T-ALL
Epigenetic Mutations in Hematopoietic Diseases
DNA Methylation
Histone Modifications
DNA MethylationDNA Methylation
• Addition of methyl group to CpG dinucleotides
• Functions – >
Silencing “foreign” DNA
> X-chromosome inactivation
> Genomic imprinting
> Gene silencing / activation
• Epigenetic regulation of gene transcription – CpG Islands
• Leukemia – > Global and gene-specific aberrant methylation
> Hypermethylation and silencing of tumor suppressor genes
> Amenable to pharmacalogical reversion (5-aza-D / decitabine)
DNA Methyltransferase EnzymesDNA Methyltransferase Enzymes
• Dnmt1 = “maintenance” methyltransferase
• Dnmt3a / Dnmt3b = “de novo” methyltransferases
Jones & Liang, Nature Genetics, 2009
LT-HSCs
Full-Length Dnmt3a is Highly Expressed in HSCs
Dnmt3a may have unique functions
in HSCs
Conditional Deletion of Dnmt3a Does Not Affect Steady-State Hematopoiesis
Mx1-cre:Dnmt3afl/flpIpC Injections
6 injections every other day
Mx1-cre:Dnmt3a /
Dnmt3a Male Mice = pIpC injections = 5-FU injection
cre-cre+
cre-cre+ cre-
cre+
WBCs RBCs Platelets
Kaneda et al., 2004, Nature
Testing HSC Potential in vivo
CD45.2 conditional knockout donors
CD45.1 wild-type competitors
250 purified HSCs
200,000 whole bone marrow cells
CD45.1 Recipients
4 weeksCheck
5-6 weeksDeletion
pIpC Injections
4 week intervals
Monitor
1o 2o
CD45.1 Recipients
CD45.1 wild-type competitors
200,000 whole bone marrow cells
250 purified HSCs
4 weeks
10 Marrow
FACS
8 weeks
12 weeks
16 weeks
Repeat for serial transplantation
%D
onor
-Der
ived
B
lood
Cel
ls
1o Transplant 2o Transplant
# of
Don
or-D
eriv
ed
HS
Cs
/ mou
se (
x103 )
Dnmt3a-KO HSCs show greater contribution to peripheral blood
Mice transplanted with Dnmt3a-KO HSCs have an expanded HSC pool in the bone marrow
1o 2o
Enhanced Activity in Serial Transplants Reflects Expanded HSC Pool
Expanded Dnmt3a-KO HSCs phenotypically
resemble normal HSCs
Mechanism for Accumulation of Dnmt3a-KO Mechanism for Accumulation of Dnmt3a-KO HSCs in the Bone Marrow?HSCs in the Bone Marrow?
Ste
m C
ells
Pro
gen
ito
rs
• Apoptosis?
• Proliferation?
X
X
Dnmt3a-KO HSCs Do Not Show Proportional Differentiation With HSC Content in Serial Transplantation
Loss of de novo DNA Methylation Skews the Balance Between Normal HSC Self-Renewal and Differentiation
Normal HSC Dnmt3a-KO HSC
16-weeks post-transplant: Blood donor cell chimerism by flow cytometry
Total animal WBC count by CBC
Number of donor-derived HSCs in the bone marrow
Amplification per HSC (~self-renewal) = number of CD45.2+ HSCs in the bone marrow / number of original input donor HSCs
Differentiation per HSC = total WBC count of recipient mouse X Donor cell engraftment in peripheral blood / number of donor HSCs in the bone marrow
DIFFERENTIATION AMPLIFICATION
Single CD45.2+ SPKLS/CD150+ from transplanted mice sorted into individual wells of 96-well Methocult plates
Enhanced HSC Activity is Cell Autonomous
Transgene Deletion PCRs in HSC Clones
Gene Expression Changes in Dnmt3a-KO HSCs
Methylation Profiling Dnmt3a-KO HSCs
MS-HPLCMS-HPLCRRBSRRBS
Control HSC MethylationD
nm
t3a
-KO
HS
C M
eth
yla
tio
n
Hypomethylation of HSC multipotency genes
Dnmt3a-KO B-cells Show Incomplete Repression of “HSC Genes”
B-cells
HSCsMS-HPLCMS-HPLC
DREAMDREAM
Mycn, Ptpn14, Src, Vwf, Vldlr, Prdm16
Vasn, Runx1
Hypomethylation and expression of
HSC genes in differentiated cells
Dnmt3a represses the “stem cell program” in HSCs to permit lineage
differentiaton
Dnmt3a-KO HSCs Cannot Silence “HSC Genes” For Efficient Long-Term HSC Differentiation
Venezia et al., 2004, PLoS
Vasn, Runx1, Nr4a2
LT-HSCs
• Upregulated “HSC multipotency” genes
• Both hyper- and hypo-methylation in HSCs
• Hypo-methylation and incomplete
repression of “HSC genes” in KO B-cells
Challen, Nature Genetics, in press
Signal for differentiation
Vasn - HSCs Vasn – B-cells
Co
ntr
ol
Dn
mt3
a-K
O
Pathogenesis
Dnmt3a
Dnmt3aX
SummarySummary
Clinical SignificanceClinical Significance• DNMT3A mutations prevalent in MDS, AML, T-cell lymphoma, T-ALL• Targets of Dnmt3a methylation represent potential for personalized medicine or prognostic indicators
• The HSC pool is composed of distinct subtypes which can be discriminated based on Hoechst efflux
• Dnmt3a is required to epigenetically silence the stem cell genetic network in HSCs to allow efficient differentiation
Future DirectionsFuture Directions• Interaction between Dnmt3a / DNA methylation and other epigenetic modifications
• Identify co-operating mutations in mouse models of Dnmt3a pathology
Goodell lab - BCM• Peggy Goodell• Jonathan Berg • Allison Rosen• Mira Jeong• Min Liu• Chris Benton• Wei Li• Deqiang Sun
Funding $$$ Funding $$$ NIH – NIDDK R00DK084259
American Society of Hematology
Alex’s Lemonade Stand
Children’s Discovery Institute
Challen Lab – Wash U• Andy Martens• Cates Mallaney
ASXL1•Additional sex combs like 1 (Drosophila)
• Chromatin binding protein, polycomb-like properties
• H2AK119 deubiquitase activity
• Loss of function mutations – o 10-15% of myeloproliferative neoplasms (MPN)
o 15-25% of myeldysplastic syndrome (MDS)
o 10-15% of acute myeloid leukemia (AML)
The goals of this study were to determine the effects of ASXL1 mutations on ASXL1 expression as well as the transcriptional and biological effects of perturbations in ASXL1 which might contribute
towards myeloid transformation
Leukemia ASXL1 mutations are loss-of-function
ASXL1 and BAP1 physically interact in human hematopoietic cells but BAP1 loss does not result in increased HoxA gene
expression
ASXL1 loss is associated with loss of H3K27me3 and increased expression of
genes poised for transcription
Rescue of leukemic cell lines with ectopic expression of ASXL1
Rescue of leukemic cell lines with ectopic expression of ASXL1
ASXL1 interacts with the PRC2 complex in hematopoietic cells
ASXL1 silencing co-operates with NRasG12D in vivo in a
mouse model of AML
Summary / Conclusions
• ASXL1 mutations in myeloid leukemia patients and myeloid cell lines are loss-of-function.
• Loss of ASXL1 leads to reduced H3K27me3 repressive chromatin and increased HOXA gene expression.
• ASXL1 physically interacts with PRC2 and recruits to target genes
Subsequent epigenomic studies of human malignancies will likely uncover novel routes to malignant transformation in different
malignancies, and therapeutic strategies that reverse epigenetic alterations may be of specific benefit in patients with mutations in
epigenetic modifiers