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