Clonal dominance Role of alterations in the bone marrow microenvironment

Mechanisms of Leukemogenesis in Patients with SCN

Daniel C. Link

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

• 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

CFU-GM

Stem Cell

Segmented Neutrophil

Promyelocyte

Myeloblast

Metamyelocyte

Myelocyte

Band Neutrophil

Block in granulocyticdifferentiation

What are the molecular mechanisms for the isolated block in granulopoiesis

What is the molecular basis for the marked susceptibility to AML

Genetics of SCN

All mutations are heterozygous Act in a cell intrinsic fashion to inhibit granulopoiesis

ELANE Mutations

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.

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%

SCN and MDS/AML

Risk of AML/MDS in Bone Marrow Failure Syndromes

G-CSFR Mutations in SCN

• G-CSF receptor– 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 1Box 2

CC

CC

-Y

CC

CC

-Y

G-CSFRG-CSFR d715d715

-Y-Y-Y

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

d715 G-CSFR allele

Stop codon

d715 mice have normal basal granulopoiesis

0 100 200 300 4000

25

50

75

100

WT/WT

WT/d715

WT/WT + G-CSF

WT/d715 + G-CSF

Time (days)

Pe

rce

nta

ge

su

rviv

al

The d715 G-CSFR is not sufficient to induce in mice even with chronic G-CSF stimulation

d715 Tumor Watch

Leukemia?

Growth FactorMutations

Transcription FactorMutations+

d715 G-CSFR PML-RARα+

FLT3 ITD PML-RARα+

Oncogene Cooperativity

Truncations mutations of the G-CSFR contribute to leukemic transformation in SCN.

D715 G-CSFR Tumor Watch

G-CSFR mutations may be an early event during leukemogenesis

0 10 12 16 19 (age-years)

SCNG-CSFR

SCNG-CSFRRunx1

AMLG-CSFRRunx1-7, 5q-

SCN

Clonal Dominance

Clinical Clinical LeukemiaLeukemia

G-CSFR mutations

Likely has to occur in a long-lived self-renewing cell (eg, stem cell)

Competitive Repopulation Assay

Wild typeWild type

1:1 Ratio1:1 Ratio

1,000 cGy

Bone Marrow ChimeraBone Marrow Chimera Syngeneic RecipientSyngeneic Recipientwild type (wild type (Ly5.1Ly5.1))

d715d715

Harvest Bone Marrow

Wild type

d715

Competitive Repopulation AssayCompetitive Repopulation Assay

Wild type

d715

Competitive AdvantageNo Competitive Advantage

3-6 Months

Ly5.2 (d715)Ly5.2 (d715)

B2

20

B2

20

Gr-

1G

r-1

Donor Chimerism Analysis

Ly5.2 (d715)Ly5.2 (d715)

61.8%61.8% 51.0%51.0%

B Lymphocytes Neutrophils

d715 Chimerasd715 Chimeras6 months after transplantation—1:1 ratio

63.5% 46.6% 45.7%50.0%

Red blood cell Platelet

Common Myeloid Progenitor

NeutrophilMonocyte

Common Lymphoid Progenitor

CFU-GM BFU-E CFU-Meg

B cell

HSC

T cell

d715 Chimeras G-CSF (10ug/kg/d x 21 days)

61.1%68.4%

49.7%60.5%

52.6%97.6%

Red blood cell Platelet

Common Myeloid Progenitor

NeutrophilMonocyte

Common Lymphoid Progenitor

CFU-GM BFU-E CFU-Meg

B cell

HSC

T cell

BM63.3%89.1%

BM75.8%98.6%

Long-term d715 G-CSFR chimerism following G-CSF treatment for 21 days

69.2

47.3

76.6

56.9

d715 Chimeras G-CSF (10ug/kg/d x 21 days)

61.1%68.4%

49.7%60.5%

52.6%97.6%

Red blood cell Platelet

Common Myeloid Progenitor

NeutrophilMonocyte

Common Lymphoid Progenitor

CFU-GM BFU-E CFU-Meg

B cell

HSC

T cell

BM63.3%89.1%

BM75.8%98.6%

53.3%53.3%97.8%97.8%

Conclusion

The d715-G-CSFR confers a clonal advantage at the hematopoietic stem cell level in a G-CSF dependent fashion

WT d715

G-CSF Saline G-CSF Saline

Harvest bone marrow at 3 hours

RNA expression profiling

RNA Expression Profiling

Sort Kit+ Sca+ Lineage- (KSL) cells

Differentially regulated genes

Sp

rr2a

En

ah

Bcat1

Tcrg

Pim

2

Cacn

b2

Cd

kn

1a

SO

CS

2

Cis

h

Tn

fsft

1

Zfp

n1a4

Serp

ina3g

Ltb

4r1

0.0

2.5

5.0

7.5

10.0

12.5

15.0

Wt

d715

20

30

40

50

Genes

Rati

o o

f G

-CS

F/S

ali

ne

STAT3 KSL cells

0 25 50 75 100 1250

10

20

30

40

50

*

Time (minutes)

STAT5 KSL cells

0 25 50 75 100 1250

5

10

15

20

WT

d715 G-CSFR

Time (minutes)

In mutant GR KSL cells, STAT3 activation by G-CSF is attenuated while STAT5 activation is enhanced

Stat3 phosphorylation Stat5 phosphorylation

• What are the STAT5 target genes that mediate clonal dominance

• Would inhibitors of STAT5 (or their target genes) be effective therapeutic agents in AML.

G-CSFR mutations

Acts at the HSC level Dependent on exogenous G-CSF Mediated by exaggerated STAT5 activation

Clonal Dominance

Vascular NicheOsteoblast Niche

Stem Cell Niches

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 d715 G-CSFR

No G-CSFSingle dose

G-CSF7 days of

G-CSF

Harvest Bone Marrow

Flow Cytometry•ROS in KSL cells•H2AX phosphorylation in KSL cells

G-CSF ROS induction is rapid in vitro(within 10-60 minutes)

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

WT or d715 G-CSFR mice

G-CSF (7 days) alone

G-CSF (7 days)+

N-acetyl cysteine (NAC)

MeasurementROS

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

Untreated G-CSF

G-CSF suppresses mature osteoblasts

Signaling through the d715 G-CSFR results in marked osteoblast and CXCL12 (SDF1) suppression

Normal

G-CSF low

AMD3100

•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

Question: Does disruption of stromal/HSPC interactions sensitize cells to G-CSF induced oxidative DNA damage

WT or d715 G-CSFR mice

G-CSF (1 dose) alone

G-CSF (1 dose)+

AMD3100

MeasureH2AX

phosphorylation

Normal SCN

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%

Zelent, Oncogene, 2004

Subset of childhood pre-B ALL with ETV6-RUNX1 fusion

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

•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.

Author Comments

2x 10 3-6

Unfractionated or

immunophenotypically

Flow sorted ALL primary cells

NOD/SCID IL2Rγnull

NOD/SCID IL2Rγnull

Secondary transplant2x 10 3-6 equivalent ALL cells

250 cGy 250 cGy

Xenotransplantation Assay

Patient 3

Figure 3

Patient 7

Figure 4a-c

Patient 3

Figure 4d

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…