Mechanisms of Leukemogenesis in Patients with SCN Daniel C. Link

1. Mechanisms of Leukemogenesis in Patients with SCNDaniel 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-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 C C C C C C C C Box 1 -Y -Y Box 2 -Y -Y -Y G-CSFR d715 G-CSFR Mutations in SCN

10. 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 Questions

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

12. 100 WT/WT WT/d715 75 WT/WT + G-CSF WT/d715 + G-CSF 50 Percentage survival 25 0 0 100 200 300 400 Time (days) d715 Tumor Watch The d715 G-CSFR is not sufficient to induce in mice even with chronic G-CSF stimulation

13. Transcription Factor Mutations Growth Factor Mutations + + FLT3 ITD PML-RARα + d715 G-CSFR PML-RARα Oncogene Cooperativity 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 0 10 12 16 19 (age-years) AML G-CSFR Runx1 -7, 5q- SCN G-CSFR Runx1 SCN G-CSFR SCN

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

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

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

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

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

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

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

23. HSC Common Myeloid Progenitor Common Lymphoid Progenitor BFU-E CFU-GM BM 75.8% 98.6% BM 63.3% 89.1% CFU-Meg Red blood cell Platelet Monocyte T cell B cell Neutrophil d715 ChimerasG-CSF (10ug/kg/d x 21 days) 53.3% 97.8% 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 Profiling WT 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 • What are the STAT5 target genes that mediate clonal dominance • Would inhibitors of STAT5 (or their target genes) be effective therapeutic agents in AML. • Acts at the HSC level • Dependent on exogenous G-CSF • Mediated by exaggerated STAT5 activation

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 low G-CSF high

31. G-CSF ROS induction is rapid in vitro (within 10-60 minutes) Wild-type d715 G-CSFR Single dose G-CSF 7 days of G-CSF No G-CSF Prolonged G-CSF (≥ 5 days) is associated with marked changes in bone marrow stromal cells 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 ROS

33. H2Ax Phosphorylation Enhanced in d715 KSL cells after 7 days of GCSF Rx

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

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. 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

41. Nature, Jan 20, 2011

42. 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%

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

45. Figure 1B

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