Presentation on theme: "Mechanisms of Leukemogenesis in Patients with SCN Daniel C. Link"— Presentation transcript:
1 Mechanisms of Leukemogenesis in Patients with SCN Daniel C. Link Clonal dominanceRole of alterations in the bone marrow microenvironment
2 Severe Congenital Neutropenia (Kostmann’s Syndrome) Clinical manifestations:Chronic severe neutropenia present at birthAccumulation of granulocytic precursors in the bone marrowRecurrent infectionsTreatment with G-CSFReduces infections and improves survivalMarked propensity to develop acute myeloid leukemia or myelodysplasia
3 Stem CellCFU-GMWhat are the molecular mechanisms for the isolated block in granulopoiesisMyeloblastPromyelocyteBlock in granulocyticdifferentiationMyelocyteWhat is the molecular basis for the marked susceptibility to AMLMetamyelocyteBand NeutrophilSegmented Neutrophil
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/AMLCumulative risk of MDS/AML in SCN: 21% after treatment with G-CSF for 10 yearsCumulative 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 receptorMember of cytokine receptor superfamilyOnly known receptor for G-CSFG-CSF receptor mutations in SCNAcquired heterozygous mutationsStrongly associated with the development of AMLBox 1Box 2C-YG-CSFRd715
10 QuestionsDo 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 codond715 G-CSFR alleled715 mice have normal basal granulopoiesis
12 d715 Tumor Watch100200300400255075WT/WTWT/d715WT/WT + G-CSFWT/d715 + G-CSFTime (days)Percentage survivalThe d715 G-CSFR is not sufficient to induce in mice even with chronic G-CSF stimulation
27 In mutant GR KSL cells, STAT3 activation by G-CSF is attenuated while STAT5 activation is enhanced Stat3 phosphorylationStat5 phosphorylation
28 G-CSFR mutationsClonalDominanceActs at the HSC levelDependent on exogenous G-CSFMediated by exaggerated STAT5 activationWhat are the STAT5 target genes that mediate clonal dominanceWould inhibitors of STAT5 (or their target genes) be effective therapeutic agents in AML.
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 syndromesNormalBMFS (e.g., SCN)G-CSF highG-CSF low
31 G-CSF ROS induction is rapid in vitro (within minutes)Wild-typed715 G-CSFRProlonged G-CSF (≥ 5 days) is associated with marked changes in bone marrow stromal cellsSingle doseG-CSF7 days ofG-CSFNo G-CSFHarvest Bone MarrowFlow CytometryROS in KSL cellsH2AX 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.
35 Hypothesis: Changes in the BM microenvironment induced by G-CSF contribute to DNA damage .G-CSF treatment in miceDecreases osteoblastsDecreases SDF1 expressionThese effects are delayed, first becoming apparent on day of G-CSF
36 G-CSF suppresses mature osteoblasts UntreatedG-CSFAs 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 damageNormalAMD3100G-CSF lowSpecific CXCR4 antagonistDisrupts HSPC/stromal interactionsResults in HSPC mobilization
39 Question: Does disruption of stromal/HSPC interactions sensitize cells to G-CSF induced oxidative DNA damageG-CSF (1 dose) aloneMeasureH2AX phosphorylationG-CSF (1 dose)+AMD3100WT 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 AMLBiomarkers of bone metabolism might predict risk of AMLTreatment 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 perspectiveNature, 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 children5-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 areZelent, Oncogene, 2004Associated with modest number of recurrent genomic CNA (3-6).Del ETV6, del CDKN2A, del PAX5, del 6q, gain Xq
44 Figure 1AHere is an example of an apparent linear architecture with 3 clones. Describe the events….
45 Figure 1BModerately 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.
47 Author commentsCommon 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.
53 Author CommentsClonal 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 transplant2x equivalent ALL cells2xUnfractionated orimmunophenotypicallyFlow sorted ALL primary cellsNOD/SCID IL2RγnullNOD/SCID IL2Rγnull250 cGy250 cGy
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-cPatient 7Here 2 of the clones decreased following serial transsplantation. And one of the smaller clones explanded. And the primary clone not detectable anymore.
59 Figure 4dPatient 3They show some SNP array data for CAN here. There were shred CAN between the samples and some distinct ones.
60 Author SummaryDistinctive 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 CommentsClonal 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…