DISCLOSURE. I have the following financial relationships:

Transcription

1 DISCLOSURE I have the following financial relationships: Consultant for: Fate Therapeutics, GlaxoSmithKline, Bone Therapeutics, G1 Therapeutics Contracted Research for: GlaxoSmithKline Royalties from: Fate Therapeutics Stock Holder: Fate Therapeutics

2 Marrow Microenvironmental Abnormalities and MDS David Scadden Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital Harvard Stem Cell Institute Department of Stem Cell and Regenerative Biology, Harvard University Disclosure: Fate Therapeutics, GSK, Bone Therapeutics

3 Topics: 1. Bone marrow niche concept and composition 2. Niche features relevant to MDS 3. MDS microenvironment studies in human

4 Niche hypothesis: stem cells live in a specialized microenvironment or niche and depend upon it for selfrenewal and regulated differentiation Proposed features: 1.Restriction on stem cell entry into cell cycle and differentiation programs 2.Integration of signals reflecting tissue and organismal state 3.Imposition of stem cell features on daughter cells 4.Mechanism for limiting mutational errors. R. Schofield. Blood Cells 4, 7-25; 1978.

5 HSC niche is perivascular

6 Osteolineage and its effects on hematopoiesis Osteo stem/proge nitor Osteoprogenitor Pre-osteoblast Osteoblast Osteocyte Mx1 Nestin Prx1 Osterix Col1a1(2.3kb) Osteocalcin DMP1 With perturbation HSPC number and function B progenitor number and function HSPC localization T progenitor number and function

7 Niche in Ecology

8 Adhesion molecules Niche in Ecology Growth factors

9 Levine, J. M. & HilleRisLambers, J. (2012). Nature Education Knowledge 3(10):59 Niche effects on inhabitant diversity Uniform niche Variegated niche

10 Sub-population number Time (mutations) will increase this variable Niche breadth If this variable also increases, possible support for abnormal hematopoietic clones go up

11 Contribution of niche to neoplasia depends on niche population characteristics Niche Hematopoietic Cancer cell

12 Are niche mesenchymal cell dynamics like hematopoiesis? or pancreatic islets? Rare cell turnover Replenished by mature cell division ~10 4 total self-renewing HSC / cells day Cell dynamics can be defined by cell labeling (pulse) tracked over time (chase)

13 Cell dynamics can be defined by cell labeling (pulse) tracked over time (chase) Chase Measure: Pulse 1.Rate of cell loss 2.Replenishment Time in this case, by like cells Cell type specific genetic tag or 3. Replenishment by other cells

14 3D image L1 cavity Genetic pulse-chase to define osteoblast lifetime Day 0 iocn calvaria Day 14 Day 30 Day m Y x Z Dongsu Park, Henry Kronenberg, Charles Lin

15 Osteoblasts have a 60 day lifespan

16 MSC Osteoprogenitor Pre-osteoblast Osteoblast Osteocyte Mx1 Osterix Col1a1 Osteocalcin DMP Selfrenewing 90d labeling 60d labeling Responsive to injury Minimally proliferative Nonproliferative Dongsu Park Migratory Migratory Migratory Park D., et al. Cell Stem Cell, 2012

17 Conclusions (1): 1.Mesenchymal populations in the bone marrow have turnover rates measured in weeks to months 2.They follow a stem/progenitor model for replenishment 3.Genetic alteration in stem/progenitor cells could result in field changes affecting parenchymal cells

18 Effect of niche cell dysfunction MSC Osteoprogenitor Pre-osteoblast Osteoblast Osteocyte DGCR8 X Drosha SBDS X Dicer1 X Dicer1 Mx1 Nestin Osterix Col1a1 Osteocalcin DMP Myelodysplasia No phenotype Hematopoietic cells Hematopoietic cells Raaijmakers et a. Nature 2010; 464,

19 Hematopoietic defects were microenvironment dependent Myelodysplasia reverted to normal B Myelodysplasia induced in the Dicer-/- host

20 Acute myeloid leukemia with soft tisssue infiltration in Dicer1 deleted mice Tumor X400 Bone marrow X400 Spleen X400

21 Molecular characterization of the leukemic infiltrate Intact Dicer1 in infiltrating cells Dicer1 was not deleted Tumor Evidence for multiple mutations and clonality by CGH (Charles Lee: DFHCC Cytogenetics Core) T1 T2 T3 qc7 Chr 4 qc1 Chr 14 qc1 Chr 14 qa3.3 Chr X qa5 Chr 2 qb3 Chr 18 WT qd2.1 qc3 qc3 qa5 qf2 qd1 DNA PCR Common amplified region in Ch14C1 by CGH in 2/3 tumors

22 Niche oncogenesis model Progeny Genotype normal Phenotype normal Progeny Genotype normal Phenotype dysplastic Progeny Genotype abnormal Phenotype dysplastic Stem cell Genotype mutated Phenotype malignant Stem cell Genotype normal Phenotype normal Stem cell Genotype normal Phenotype abnormal Stem cell Genotype mutated Phenotype abnormal Stem cell Genotype mutated Phenotype malignant Secondary event Tertiary event Niche normal Niche abnormal Niche abnormal Niche abnormal Partial niche Initiating event dependence remains?

23 Niche oncogenesis model Progeny Genotype normal Phenotype normal Progeny Genotype normal Phenotype dysplastic Progeny Genotype abnormal Phenotype dysplastic Stem cell Genotype mutated Phenotype malignant Stem cell Genotype normal Phenotype normal Stem cell Genotype normal Phenotype abnormal Stem cell Genotype mutated Phenotype abnormal Stem cell Genotype mutated Phenotype malignant Niche normal Niche abnormal Niche abnormal Niche abnormal Cooperating alterations?

24 Conclusions (2): 1. Specific mesenchymal cell types play critical roles in maintaining the integrity of hematopoiesis 2. Genetic perturbations of specific subtypes of mesenchymal cells can enable outgrowth of dysplastic and neoplastic hematopoietic cells

25 Niche cells CAN cause disease and niche cell dynamics are consistent with acquired lesions causing field defects DO THEY cause dysplastic and neoplastic disease? Caveats in investigation: 1.Cultured stromal cells are notoriously unstable 2.Distinct stromal cells play distinct functional roles: studying cells in bulk may be limited by noise 3.Validation of causality is experimentally challenging

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27 Clinical samples demonstrating genetic abnormalities in cultured stromal cells Blau O et al. Blood 2011, 118; MDS and 51 AML patients evaluated by cytogenetics, FISH and PCR of NPM1 and FLT3 15 of 94 (16%) MDS/AML patients (0/36 controls) had chromosomal abnormalities in MSC Abnormalities distinct from those in hematopoietic cells No MSC abnormal karyotype seen in those with favorable cytogenetic hematopoietic cells

28 Blau O et al. Blood 2011, 118;5583.

29 Caveats in investigation: 1.Cultured stromal cells are notoriously unstable 2.Distinct stromal cells play distinct functional roles: studying fresh cells in bulk may be limited by noise 3.Validation of causality is experimentally challenging

30 Defining human stromal subsets by flow cytometry Single Double Youmna Kfoury

31 Distinct gene expression signatures for cell subsets Single Double

32 Engraftment of human MDS cells in mice enhanced by stromal cell lines Li X, et al. Blood Cancer J 2013, 4;e113. NSG mice transplanted with CD34+ cells from 23 patients with MDS None engrafted unless co-transplanted with the HS27a cell line Efficiency 95% with HS27a and karyotype consistent with original sample Cell line HS5 did not enable engraftment of CD34+ patient cells

33 Human bone marrow in mice to model disease Blood 2012,119:

34 Sub-population number Pairing of parenchymal and mesenchymal features enables malignant clone emergence Time is likely to increase both variables Niche breadth

35 Acknowledgements Dongsu Park Bong Ihn Koh Joel Spencer Charles Lin Henry Kronenberg Marc Raaijmakers Sid Mukherjee Shangqin Guo Youmna Kfoury Peter Kharchenko Guillermo Garcia-Manero Michael Andreef Ben Ebert Ross Levine Donna Neuberg Eyal Attar Funding: NHLBI, NIDDK, Harvard Stem Cell Institute, Ellison Medical Foundation

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