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

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Presentation on theme: " Clonal dominance  Role of alterations in the bone marrow microenvironment Mechanisms of Leukemogenesis in Patients with SCN Daniel C. Link."— Presentation transcript:

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

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

4 Genetics of SCN

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

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  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

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 C C C -Y-Y C C C C -Y-Y G-CSFR d715 -Y-Y -Y-Y -Y-Y

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 d715 G-CSFR allele Stop codon  d715 mice have normal basal granulopoiesis

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

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

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

15 G-CSFR mutations may be an early event during leukemogenesis 0 10 12 16 19 (age-years) SCN G-CSFR SCN G-CSFR Runx1 AML G-CSFR Runx1 -7, 5q- SCN

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

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

18 Competitive Repopulation Assay Wild type d715 Competitive AdvantageNo Competitive Advantage 3-6 Months

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

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

21 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 Neutrophil Monocyte Common Lymphoid Progenitor CFU-GMBFU-E CFU-Meg B cell HSC T cell BM 63.3% 89.1% BM 75.8% 98.6%

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

23 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 Neutrophil Monocyte Common Lymphoid Progenitor CFU-GMBFU-E CFU-Meg B cell HSC T cell BM 63.3% 89.1% BM 75.8% 98.6% 53.3%97.8%

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

25 WTd715 G-CSFSaline Harvest bone marrow at 3 hours RNA expression profiling RNA Expression Profiling Sort Kit+ Sca+ Lineage- (KSL) cells

26

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

28 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

29 Vascular Niche Osteoblast Niche Stem Cell Niches

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

31 Wild-typed715 G-CSFR No G-CSF Single dose G-CSF 7 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

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

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 Untreated G-CSF G-CSF suppresses mature osteoblasts

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

38 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

39 WT or d715 G-CSFR mice G-CSF (1 dose) alone G-CSF (1 dose) + AMD3100 Measure H2AX phosphorylation

40 Normal SCN G-CSF lowG-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 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

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

50 Figure 2A

51 Figure 2b

52 Supplementary Figure 2

53 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

54 2x 10 3-6 Unfractionated or immunophenotypically Flow sorted ALL primary cells NOD/SC ID IL2Rγ null Secondary transplant 2x 10 3-6 equivalent ALL cells 250 cGy Xenotransplantation Assay

55 Patient 3 Figure 3

56

57 Patient 7 Figure 4a-c

58

59 Patient 3 Figure 4d

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…


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