1. Mechanisms of Leukemogenesis in Patients with SCN Daniel C. Link
Clonal dominance
Role of alterations in the bone marrow microenvironment

2. 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

3. Stem Cell
CFU-GM
What are the molecular mechanisms for the isolated block in granulopoiesis
Myeloblast
Promyelocyte
Block in granulocytic
differentiation
Myelocyte
What is the molecular basis for the marked susceptibility to AML
Metamyelocyte
Band Neutrophil
Segmented Neutrophil

4. Genetics of SCN

5. ELANE Mutations All mutations are heterozygous
Act in a cell intrinsic fashion to inhibit granulopoiesis

6. 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.

7. SCN and MDS/AML
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%

8. Risk of AML/MDS in Bone Marrow Failure Syndromes

9. 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 1
Box 2 C -Y
G-CSFR
d715

10. 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

11. d715 “Knock-in” Mice WT G-CSFR gene Targeting vector
Stop codon
d715 G-CSFR allele
d715 mice have normal basal granulopoiesis

12. d715 Tumor Watch
100
200
300
400 25 50 75
WT/WT
WT/d715
WT/WT + G-CSF
WT/d715 + G-CSF
Time (days)
Percentage survival
The d715 G-CSFR is not sufficient to induce in mice even with chronic G-CSF stimulation

13. Oncogene Cooperativity
Growth Factor
Mutations
Transcription Factor +
FLT3 ITD
PML-RARα +
d715 G-CSFR
PML-RARα +
Leukemia?

14. D715 G-CSFR Tumor Watch
Truncations mutations of the G-CSFR contribute to leukemic transformation in SCN.

15. G-CSFR mutations may be an early event during leukemogenesis
(age-years)
SCN
SCN
G-CSFR
SCN
G-CSFR
Runx1
AML
G-CSFR
Runx1
-7, 5q-

16. Clonal Dominance G-CSFR mutations Clinical Leukemia
Likely has to occur in a long-lived self-renewing cell (eg, stem cell)

17. Competitive Repopulation Assay
Wild type
1:1 Ratio
Harvest
Bone Marrow
Wild type
d715
d715
1,000 cGy
Bone Marrow Chimera
Syngeneic Recipient
wild type (Ly5.1)

18. Competitive Repopulation Assay
Wild type
d715
3-6 Months
No Competitive Advantage
Competitive Advantage

19. Donor Chimerism Analysis
B Lymphocytes
Neutrophils
B220
Gr-1
61.8%
51.0%
Ly5.2 (d715)
Ly5.2 (d715)

20. 6 months after transplantation—1:1 ratio
d715 Chimeras
6 months after transplantation—1:1 ratio
Red blood cell
Platelet
Common Myeloid Progenitor
Neutrophil
Monocyte
Common Lymphoid Progenitor
CFU-GM
BFU-E
CFU-Meg
B cell
HSC
T cell
63.5%
46.6%
50.0%
45.7%

21. d715 Chimeras G-CSF (10ug/kg/d x 21 days)
Red blood cell
Platelet
Common Myeloid Progenitor
Neutrophil
Monocyte
Common Lymphoid Progenitor
CFU-GM
BFU-E
CFU-Meg
B cell
HSC
T cell BM
63.3%
89.1%
75.8%
98.6%
61.1%
68.4%
49.7%
60.5%
52.6%
97.6%

22. Long-term d715 G-CSFR chimerism following G-CSF treatment for 21 days
69.2
76.6
47.3
56.9

23. d715 Chimeras G-CSF (10ug/kg/d x 21 days)
53.3%
97.8%
Red blood cell
Platelet
Common Myeloid Progenitor
Neutrophil
Monocyte
Common Lymphoid Progenitor
CFU-GM
BFU-E
CFU-Meg
B cell
HSC
T cell BM
63.3%
89.1%
75.8%
98.6%
61.1%
68.4%
49.7%
60.5%
52.6%
97.6%

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

25. RNA Expression ProfilingWT
d715
G-CSF Saline
G-CSF Saline
Harvest bone marrow at 3 hours
Sort Kit+ Sca+ Lineage- (KSL) cells
RNA expression profiling

27. In mutant GR KSL cells, STAT3 activation by G-CSF is attenuated while STAT5 activation is enhanced
Stat3 phosphorylation
Stat5 phosphorylation

28. G-CSFR mutations
Clonal
Dominance
Acts at the HSC level
Dependent on exogenous G-CSF
Mediated by exaggerated STAT5 activation
What are the STAT5 target genes that mediate clonal dominance
Would inhibitors of STAT5 (or their target genes) be effective therapeutic agents in AML.

29. Stem Cell Niches
Osteoblast Niche
Vascular Niche

30. 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
BMFS (e.g., SCN)
G-CSF high
G-CSF low

31. G-CSF ROS induction is rapid in vitro
(within minutes)
Wild-type
d715 G-CSFR
Prolonged G-CSF (≥ 5 days) is associated with marked changes in bone marrow stromal cells
Single dose
G-CSF
7 days of
G-CSF
No G-CSF
Harvest Bone Marrow
Flow Cytometry
ROS in KSL cells
H2AX phosphorylation in KSL cells

32. ROS Induction is increased in d715 KSL cells after 7 days of GCSF Rx

33. H2Ax Phosphorylation Enhanced in d715 KSL cells after 7 days of GCSF Rx
We next measured H2AX phosphorylation. H2AX phosphorylation is an early and sensitive marker of DNA damage. <click> In wild type mice, no increase in H2AX-P was observed after short of prolonged G-CSF administration. <cllick> In d715 G-CSFR mice, the basal level of H2AX-P was low. Again, short term G-CSF treatment did not significantly induced H2AX-P, while 7 days of G-CSF resulted in a significant increase.

34. NAC attenuates G-CSF induced H2AX phosphorylation
G-CSF (7 days) alone
Measurement
ROS
H2AX-P
G-CSF (7 days) +
N-acetyl cysteine (NAC)
WT or d715 G-CSFR mice

35. 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

36. G-CSF suppresses mature osteoblasts
Untreated
G-CSF
As I mentioned, osteoblast lineage cells are a key cellular component of the stem cell niche. We and others showed that G-CSF treatment is associated with marked suppression of osteoblasts in the bone marrow. Shown here is representative photomicrograph of the femur of a transgenic mouse that expresses EGFP in osteoblasts. The green cells line the endosteal and trabecular bone surfaces.
<click> Treatment of mice with G-CSF for 7 days results in a marked loss of osteoblasts in the bone marrow.

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

38. Question: Does disruption of stromal/HSPC interactions sensitize cells to G-CSF induced oxidative DNA damage
Normal
AMD3100
G-CSF low
Specific CXCR4 antagonist
Disrupts HSPC/stromal interactions
Results in HSPC mobilization

39. Question: Does disruption of stromal/HSPC interactions sensitize cells to G-CSF induced oxidative DNA damage
G-CSF (1 dose) alone
Measure
H2AX phosphorylation
G-CSF (1 dose) +
AMD3100
WT or d715 G-CSFR mice

40. SCN Normal 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

41. The article I am going to present is titled, Genetic variegation of clonal architecture and prropogating cells in leukemia, published this month in nature. This is a relative short article and so I am going to intially talk about the background to put this article in perspective
Nature, Jan 20, 2011

42. 5-year survival rate of 80% Structural abnormalities:
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%
In addition to hyperdiploidy and hypodiploidy there are several specific chromosoomal translocation descrbed in childhood ALL.

43. Subset of childhood pre-B ALL with ETV6-RUNX1 fusion
ETV6-RUNX1 fusion as we have discussed is predominantly a prenatal event and a presumed initiating event. This si asssociated with modest number of recurrent genomic CAN. Del of ETV6 is the most common present in present in 97% of the cases. Other common events are
Zelent, Oncogene, 2004
Associated with modest number of recurrent genomic CNA (3-6).
Del ETV6, del CDKN2A, del PAX5, del 6q, gain Xq

44. Figure 1A
Here is an example of an apparent linear architecture with 3 clones. Describe the events….

45. Figure 1B
Moderately complex architecture with 5 subclones… Loss of ETV6… Loss of both CDK and then a loss of ETV6. Here there is a gain of RUNX and then a loss of ETV6.

46. Figure 1C

47. 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.

48. Supplementary Figure 3

49. Supplementary Figure 3

50. Figure 2A

51. Figure 2b

52. Supplementary Figure 2

53. Author Comments
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.

54. Xenotransplantation Assay
Secondary transplant
2x equivalent ALL cells 2x
Unfractionated or
immunophenotypically
Flow sorted ALL primary cells
NOD/SCID IL2Rγnull
NOD/SCID IL2Rγnull
250 cGy
250 cGy

55. Figure 3
Patient 3

56. Here is the summary table of different serial transsplants done with patient no,3 sample. The diagnostic samples clonesa re in the top row. One of the clones is mostly not detecctable, some clones are lost and the others are preserved.

57. Figure 4a-c
Patient 7
Here 2 of the clones decreased following serial transsplantation. And one of the smaller clones explanded. And the primary clone not detectable anymore.

59. Figure 4d
Patient 3
They show some SNP array data for CAN here. There were shred CAN between the samples and some distinct ones.

60. 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.

61. 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…