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Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts

Nature volume 506, pages 240–244 (2014)Cite this article

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Abstract

Cells of the osteoblast lineage affect the homing1,2 and the number of long-term repopulating haematopoietic stem cells3,4, haematopoietic stem cell mobilization and lineage determination and B cell lymphopoiesis5,6,7. Osteoblasts were recently implicated in pre-leukaemic conditions in mice8,9. However, a single genetic change in osteoblasts that can induce leukaemogenesis has not been shown. Here we show that an activating mutation of β-catenin in mouse osteoblasts alters the differentiation potential of myeloid and lymphoid progenitors leading to development of acute myeloid leukaemia with common chromosomal aberrations and cell autonomous progression. Activated β-catenin stimulates expression of the Notch ligand jagged 1 in osteoblasts. Subsequent activation of Notch signalling in haematopoietic stem cell progenitors induces the malignant changes. Genetic or pharmacological inhibition of Notch signalling ameliorates acute myeloid leukaemia and demonstrates the pathogenic role of the Notch pathway. In 38% of patients with myelodysplastic syndromes or acute myeloid leukaemia, increased β-catenin signalling and nuclear accumulation was identified in osteoblasts and these patients showed increased Notch signalling in haematopoietic cells. These findings demonstrate that genetic alterations in osteoblasts can induce acute myeloid leukaemia, identify molecular signals leading to this transformation and suggest a potential novel pharmacotherapeutic approach to acute myeloid leukaemia.

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Figure 1: Anaemia and myeloid lineage expansion in Ctnnb1CAosb mice.
Figure 2: AML in Ctnnb1CAosb mice.
Figure 3: Inactivation of jagged 1 in osteoblasts prevents AML in Ctnnb1CAosb mice.
Figure 4: Nuclear accumulation of β-catenin in osteoblasts and increased Notch signalling in human MDS/AML.

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Accession codes

Accessions

Gene Expression Omnibus

Sequence Read Archive

Data deposits

Microarray and aCGH data were deposited in Gene Expression Omnibus (accession numbers GSE43242, GSE51690) and exome sequencing data were deposited in Sequence Read Archive (accession number SRP031981).

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Acknowledgements

The authors are grateful to M. Taketo for providing the Catnb+/lox(ex3) mice; R. Dalla-Favera and G. Karsenty for helpful discussions and critical reading of the manuscript; M. Van Den Brink for providing funding support; D. McMahon and C. A. Zhang for help with statistical analysis; and J. Sha, F. N. Emmons, I. Linkov and J. Pichard for technical assistance. The histology and metabolic unit facility of the Diabetes and Endocrinology Research Center (DERC, NIDDK DK063608-07), the Molecular Pathology facility of the Herbert Irving Cancer Center of Columbia University Medical Center provided help with histological analysis and W. Fantl and G. Nolan for provided flow cytometry conditions for the nuclear β-catenin antibody. This work was supported by the National Institutes of Health (R01 AR054447, and P01 AG032959 and R01 AR055931 to S.K.) and by the Division of Hematologic Oncology, Memorial Sloan-Kettering Cancer Center, New York (to E.B.) and by a European Union Marie Curie Fellowship INTERBONE−FP7−PEOPLE−2011−IRSES−295181 (to A.B.).

Author information

Authors and Affiliations

  1. Department of Medicine, Division of Endocrinology, College of Physicians & Surgeons, Columbia University, New York, 10032, New York, USA

    Aruna Kode, John S. Manavalan, Ioanna Mosialou, Na Luo, Andrea Brum & Stavroula Kousteni

  2. Department of Pathology and Cell Biology, College of Physicians & Surgeons, Columbia University, New York, 10032, New York, USA

    Govind Bhagat

  3. Department of Genetics and Development College of Physicians & Surgeons, Columbia University, New York, 10032, New York, USA

    Chozha V. Rathinam

  4. Department of Biomedical Informatics and Center for Computational Biology and Bioinformatics, Columbia University, New York, 10032, New York, USA

    Hossein Khiabanian, Albert Lee & Raul Rabadan

  5. Department of Pathology & Institute for Cancer Genetics Irving Cancer Research Center, Columbia University, New York, 10032, New York, USA

    Vundavalli V. Murty

  6. Herbert Irving Comprehensive Cancer Center and Department of Biomedical Informatics, Biomedical Informatics Shared Resource, College of Physicians & Surgeons, Columbia University, New York, 10032, New York, USA

    Richard Friedman

  7. Department of Internal Medicine, Erasmus MC, Dr. Molewaterplein 50, NL-3015 GE Rotterdam, The Netherlands,

    Andrea Brum

  8. Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, 10021, New York, USA

    David Park & Julie Teruya-Feldstein

  9. Myelodysplastic Syndromes Center, Columbia University, New York, 10032, New York, USA

    Naomi Galili & Azra Raza

  10. Departments of Medicine Hematology & Oncology Columbia University, New York, 10032, New York, USA

    Siddhartha Mukherjee

  11. Department of Medicine, Leukemia Service, Memorial Sloan-Kettering Cancer Center, New York, 10021, New York, USA

    Ellin Berman

  12. Department of Physiology & Cellular Biophysics, College of Physicians & Surgeons, Columbia University, New York, 10032, New York, USA

    Stavroula Kousteni

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Contributions

A.K. and S.K. initiated the study and designed the experiments. A.K., J.S.M. and S.K. analysed the data. A.K. carried out most of the experimental work with the help of J.S.M., I.M. and N.L. J.S.M. performed the flow cytometry analysis. H.K., A.L. and R.R. performed whole-exome sequencing analysis. I.M. confirmed exome mutations. N.L. performed immunofluorescence. C.V.R. reviewed and discussed haematopoiesis data and bone marrow transplantations. G.B., D.P. and J.T.-F. performed histology in mouse samples. J.T.-F. and D.P. performed histology in human samples. A.R., S.M., N.G., J.T.-F. and E.B. provided human AML and MDS samples and reviewed and discussed human bone marrow and bone biopsy data. M.V. performed G-banding karyotype analysis. R.F. analysed microarray data. A.B. assisted with mouse flow cytometry experiments. A.K. and S.K. wrote the manuscript. S.K. directed the research. All authors discussed and commented on the manuscript.

Corresponding author

Correspondence to Stavroula Kousteni.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Anaemia, peripheral blood leukocytosis and monocytosis and deregulated haematopoiesis specific activation of β-catenin in osteoblasts of Ctnnb1CAosb mice.

a, Haematopoietic parameters. White blood cells (WBC), red blood cells (RBC), haemoglobin (HB), haematocrit (HCT), platelets, lymphocytes (LY), neutrophils (NE) and monocytes (MO) in 2-week-old mice. b–k, Bone marrow samples. b, Erythroid cell numbers. c, Representative flow cytometry image showing monocytic/granulocytic (CD11b/Gr1+) subset. d, Numbers of the CD11b+/Gr1+ subset. e, Distribution of LSK (Lineage−Sca+C-kit+) population. f, LSK numbers. g, h, Frequency (g) and percentage (h) of LT-HSCs and ST-HSCs. i, Numbers of LSK+/ FLT3+ cells. j, Myeloid progenitor profile by CD34 versus FcgRII/III analysis of electronically gated Lin−Sca-1−c-Kit+ bone marrow cells. k, Numbers of myeloid progenitor populations. l, Spleen weight. m, Extramedullar haematopoiesis in the liver of 3-weeks-old Ctnnb1CAosb mice indicated by megakaryocytes (black arrow), myeloid (white arrow) and rare erythroid precursors (dotted arrow). n–p Percentage of Ter119+ (n), CD11b+/Gr1+ (o) and myeloid progenitor (p) populations in the spleen. q, PCR analysis of genomic DNA from osteoblasts and indicated haematopoieic populations from wild-type and Ctnnb1CAosb mice. r–t, Real-time PCR analysis of β-catenin targets in bone marrow CD45+CD34+CD31+ cells (r), spleen (s) and bones (t). In a, n = 6; b–k, n–p, n = 8; l, m, n = 5 and r–t, n = 4 mice per group. Results are mean ± s.d. and show a representative from five (a–p) or 2 (q–t) independent experiments. *P < 0.05 versus wild type. MNC, mononuclear cells.

Extended Data Figure 2 Multi-organ infiltration with blasts and dysplastic cells and myeloid differentiation block in Ctnnb1CAosb mice.

a, Blast infiltration (solid arrows) and neutrophil hypersegmentation (open arrow and magnified panels) in the blood of Ctnnb1CAosb mice. Images at ×40 or ×100 magnification. b, Blast infiltration (solid arrows) and micro-megakaryocytes with hyperchromatic nuclei (white arrows) in the bone marrow of Ctnnb1CAosb mice. Images at ×60 magnification. c, Blast infiltration (solid arrows and magnified panel) and presence of dysplastic megakaryocytes (yellow arrows and magnified panel) in the spleen of Ctnnb1CAosb mice. Image at ×400 magnification. d–f, Myeloperoxidase (MPO) staining of long bone (d), spleen (e), and liver (f) showing massive invasion of myeloid cells. g, CD117 (c-kit) staining of bone sections showing CD117+ blasts in Ctnnb1CAosb mice. h, CD13 staining of bone sections showing myeloid/monocytic infiltration in Ctnnb1CAosb mice. In d–h images at ×60 magnification. i–l, B-cell progenitors numbers in the bone marrow (i, j), spleen (k) and lymph nodes (l). m–t, Proportion of T cells. u, Lack of myeloid cell differentiation in Ctnnb1CAosb bone marrow cells following treatment with cytokines. v–x, Percentage of immature myeloid cells in ex vivo bone marrow cultures treated with cytokines. y, Robertsonian translocation between chromosomes 1 and 19 in 2 of 30 metaphases of the spleen of an 18-day-old Ctnnb1CAosb mouse. Inset shows the same abnormality in another cell. z, Whole-exome sequencing of myeloid malignancies (CD11b+/Gr1+) from 3 Ctnnb1CAosb mice and 3 germline normal (tail) samples. In i–x, n = 6 mice per group. *P < 0.05 versus wild type. Results are mean ± s.d. and show a representative of five (i–t) or three (u–x) independent experiments.

Extended Data Figure 3 Cell autonomous AML development by bone marrow and LT-HSCs cells of Ctnnb1CAosb mice.

a, Engraftment efficiency of CD45.2 Ctnnb1CAosb bone marrow cells in lethally irradiated CD45.1 wild-type mice 7 weeks following transplantation. b–f, Percentage of indicated populations in bone marrow of transplanted mice. g, Blasts in blood (15–80%, solid arrows) of lethally irradiated CD45.1 wild-type mice transplanted with CD45.2 Ctnnb1CAosb marrow cells 7 weeks following transplantation. h, Blasts (solid arrows) and dysplastic megakaryocytes (open arrow) in bone marrow of transplanted mice. Images at ×60 magnification. i, Lethality curves. j, Engraftment efficiency of CD45.1 wild-type bone marrow cells in lethally irradiated CD45.2 Ctnnb1CAosb mice. k–o, Increased percentage of LSK cells (k), myeloid progenitors (l), and CD11b+/Gr1+ (m) cells and decreases in erythroid cells (n) and B-lymphopoiesis (o) in the bone marrow of transplanted mice. p, q, Blasts in the blood (p) and bone marrow (q) (black arrows) of transplanted mice. Images were taken at ×100 (o) and ×60 (p) magnification. r, Lethality curves. s, Engraftment efficiency of indicated bone marrow haematopoietic populations from 4-week-old CD45.2 Ctnnb1CAosb or wild-type mice in sublethally irradiated CD45.1 wild-type mice after 4 weeks (for LT-HSCs, due to lethality) and 8 weeks (for other populations) of transplantation. t, Blood counts in wild type mice transplanted mice. u–w, Lack of blasts in the blood of wild-type mice transplanted with indicated haematopoietic cells from Ctnnb1CAosb mice. x, Disease development in wild-type mice transplanted with indicated haematopoietic cells from Ctnnb1CAosb mice. y, Splenomegaly in wild-type mice transplanted with LT-HSCs from Ctnnb1CAosb. z, Spleen size and weight in wild-type mice transplanted with indicated haematopoietic populations from WT/ Ctnnb1CAosb mice. n = 6 mice per group. Results are mean ± s.d. and show a representative of two independent experiments. *P < 0.05 wild type–wild type versus wild type– Ctnnb1CAosb or versus Ctnnb1CAosb–wild type transplanted group.

Extended Data Figure 4 Newborn Ctnnb1CAosb mice show MDS but fetal HSCs from Ctnnb1CAosb mice do not transfer AML.

a–e, Increased percentage of LSK cells (a), GMPs (b), and CD11b+/Gr1+ (c) cells and decreases in erythroid (d) cells and B-lymphopoiesis (e) in the liver of newborn (P1) Ctnnb1CAosb mice. f–j, Increased percentage of LSK cells (f), GMPs (g), and CD11b+/Gr1+ (h) cells and decreases in erythroid cells (i) and B-lymphopoiesis (j) in the bone marrow of newborn (P1) Ctnnb1CAosb mice. k–m, Liver (k), bone marrow (l) and spleen (m) of of newborn (P1) Ctnnb1CAosb mice showing microhypolobated megakaryocytes (open arrows), Pelger Huet neutrophils (yellow arrows) or blasts (solid arrows). Images at ×100 magnification. n, Percentage of immature myeloid cells in the bone marrow of newborn mice. o–t, Flow cytometry and Giemsa-stained cytospins showing lack of changes in the percentage of immature myeloid cells in ex vivo cultures of bone marrow cells from P1 stage Ctnnb1CAosb mice and treated with indicated cytokines. u, Engraftment efficiency of CD45.2 Ctnnb1CAosb LSK cells obtained from the liver of embryonic day 18.5 embryos in sublethally irradiated CD45.1 wild-type mice. v, Normal peripheral blood measurements in transplanted mice. w, Lack of blasts in the blood of transplanted wild-type mice. Images at ×100 magnification. n = 6 mice per group. Results are mean ± s.d. and represent at least two independent experiments. *P < 0.05 versus wild type.

Extended Data Figure 5 Inhibition of increased Notch signalling normalizes blood counts and rescues haematopoietic defects in Ctnnb1CAosb mice.

a, Microarray analysis of calvaria-derived osteoblasts from Ctnnb1CAosb mice. AML and Notch-related genes in Ctnnb1CAosb osteoblasts and with P < 0.05 and fold change of ± 20% in one comparison. Genes that are up- or downregulated relative to wild type are shown. b, Flow cytometry analysis of jagged 1expression in osteoblasts (MFI: mean fluorescent intensity). c, Luciferase activity in HEK293T cells co-transfected with β-catenin, Lef1 and Jag1-Luc reporter constructs (−4112/+130) and (−2100/+130). Results show fold induction over respective Jag1-Luc reporter constructs. *P < 0.05 versus respective Jag1-Luc. Results are mean ± s.d. d, ChIP in primary osteoblasts using anti-β-catenin antibody. Primers spanned the putative TCF/LEF binding sites (indicated) on the Jag1 promoter. e, Expression of Notch1 and Notch2 in LSK+ cells. f–g, Expression of Notch targets in LSK+ subpopulations. h, i, Normal intestinal architecture (h) and PAS staining (i) showing lack of goblet cell (arrows) metaplasia in DBZ-treated mice. Images at ×60 magnification. j, Peripheral blood counts and bone marrow cellularity in wild-type and Ctnnb1CAosb mice treated daily with vehicle or DBZ (2 µmol per kg body weight) for 10 days. k–p, Percentage of LSK cells (k), LSK+ subpopulations (l), myeloid progenitors (m), CD11b+/Gr1+ population (n), erythroid cells (o) and LSK+/FLT3+ population (p) in the bone marrow. m, Percentage of erythroid cells and p, LSK+/FLT3+ population in the bone marrow. q–s, Percentage of myeloid progenitor populations (q), CD11b+/Gr1+ (r), cells and erythroid (s), cells in the spleen. In a n = 3 mice per group and in b, n = 4 mice per group. In c, d, results represent two independent experiments. In e–g N = 4 mice per group, and *P < 0.05 versus wild type. In h–s n = 8 mice per group and *P < 0.05 versus wild type and #P < 0.05 Ctnnb1CAosb vehicle versus DBZ-treated Ctnnb1CAosb group. Results are mean ± s.d. and show a representative of two independent experiments.

Extended Data Figure 6 Jag1 inactivation in osteoblasts prevents AML of Ctnnb1CAosb mice.

a–d, Expression of Notch transcriptional targets in bone marrow LSK subpopulations. Rescue of changes in the proportions of LSK (e) and erythroid (f) cells in the bone marrow of Ctnnb1CAosb /Jag1osb+/− mice. g, Improvement of B-lymphopoiesis in Ctnnb1CAosb;Jag1osb+/− mice. Normal (h) bone marrow, spleen (i) and liver (j) histology in Ctnnb1CAosb;Jag1osb+/− mice. k, Long bone sections. Images at ×4 magnification. In a–d, n = 4 and in e–k n = 8 mice per group. *P < 0.05 versus wild type and #P < 0.05 versus Ctnnb1CAosb;Jag1osb+/− mice. Results are mean ± s.d. and show a representative of three (a–d) and two (e–k) independent experiments.

Extended Data Figure 7 Inhibition of Notch signalling reverses AML in Ctnnb1CAosb mice.

a, Lack of blasts (solid arrows) and, normal neutrophils (right panel) in blood of DBZ-treated Ctnnb1CAosb mice. b–c, Normal megakaryocytes in the bone marrow (b) and spleen (c). d, Normal spleen histology in DBZ-treated Ctnnb1CAosb mice. Yellow arrows indicate abnormal cells with large nucleoli and dotted arrow indicates abnormal megakaryocytes in Ctnnb1CAosb mice; white arrow indicates normal megakaryocytes in DBZ-treated Ctnnb1CAosb mice. e, Lack of monocyte infiltration in the liver of DBZ-treated Ctnnb1CAosb mice. Arrow indicates cluster of mononuclear cells. f–h, MPO staining of bone marrow (f), spleen (g) and liver (h). i, Per cent of cells staining with MPO in the indicated tissues. j, Increased survival in DBZ-treated Ctnnb1CAosb mice. In a, b, images taken at ×100 magnification. In c–g images taken at ×60 magnification. k–l, Proportion of B-cell populations in the bone marrow (k) and spleen (l). m, Long bone sections. Images at ×4 magnification. n = 6 mice per group. *P < 0.05 versus wild type and #P < 0.05 Ctnnb1CAosb vehicle versus DBZ-treated Ctnnb1CAosb group. Results are mean ± s.d. and show a representative from two independent experiments.

Extended Data Figure 8 Nuclear accumulation of β-catenin in osteoblasts and increased Notch signalling in 38.3% of patients with MDS/AML and identification of underlying pre-AML conditions by nuclear localization of β-catenin in osteoblasts.

a–f, Double immunofluorescence staining with β-catenin and RUNX2 in osteoblasts from bone marrow biopsies from 6 MDS/AML patients harbouring nuclear accumulation of β-catenin in osteoblasts and showing nuclear accumulation of HEY1 in the corresponding patients (×60 magnification). g, h, During screening assumed healthy controls, 2 individuals were identified with nuclear β-catenin in their osteoblasts. Re-evaluation showed underlying hematologic disorder, Case 1: MDS RAEB-1, Case 2: Jak2 positive myelofibrosis. g, Double immunofluorescence staining with β-catenin and RUNX2 in osteoblasts from bone marrow biopsies of the 2 cases (×60 magnification). h, β-catenin cellular localization in cases 1 and 2 with associated cytogenetic abnormalities. NL: normal cytogenetics. In the fourth column percentages indicate osteoblasts with nuclear localization of β-catenin.

Extended Data Figure 9 Membrane accumulation of β-catenin in osteoblasts in 61.7% of patients with MDS/AML and in healthy subjects and nuclear accumulation of β-catenin in osteoblasts in 38.3% of patients with MDS/AML identified by flow cytometry.

Double immunofluorecsence staining with β-catenin and Runx2 in osteoblasts from bone marrow biopsies from a–c, 3 MDS/AML patients and d–g, 4 healthy subjects harbouring membrane localization of β-catenin in osteoblasts. h–j, Flow cytometry using a non-phospho β-catenin antibody detecting nuclear/activated β-catenin. Representative plots showing nuclear (h), versus non-nuclear (i), localization of β -catenin in osteoblasts from individual MDS/AML patients and, non-nuclear localization of β-catenin in osteoblasts from 5 healthy subjects as CD34−/Lin−OCN+ cells, (OCN, osteocalcin an osteoblast-specific protein used for isolation of live osteoblastic cells).

Extended Data Table 1 Structural chromosomal alterations detected by aCGH in Ctnnb1CAosb mice
Full size table

Supplementary information

Supplementary Table 1

This file shows the nuclear accumulation of β-catenin in osteoblasts of 38.29% MDS/AML patients. β-catenin cellular localization in patients with MDS, AML, and AML arising from prior MDS with associated cytogenetic abnormalities. NL: normal cytogenetics; NE: cytogenetics non-evaluable; ND: cytogenetics not determined. Percentages in parentheses in the 4th column indicate percentages of osteoblasts with nuclear localization of β-catenin; Y denotes nuclear localization of β-catenin in osteoblasts and N denotes membrane localization of β-catenin in osteoblasts. In the latter samples nuclear localization of β-catenin was not detected in any of the osteoblasts. (XLSX 14 kb)

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Kode, A., Manavalan, J., Mosialou, I. et al. Leukaemogenesis induced by an activating β-catenin mutation in osteoblasts. Nature 506, 240–244 (2014). https://doi.org/10.1038/nature12883

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