Leukaemogenesis inducedbyanactivating b-catenin mutation in osteoblasts

Cells of the osteoblast lineage affect the homing and the number of long-term repopulating haematopoietic stem cells, haematopoietic stem cell mobilization and lineage determination and B cell lymphopoiesis. Osteoblasts were recently implicated in pre-leukaemic conditions in mice. However, a single genetic change in osteoblasts that can induce leukaemogenesis has not been shown. Here we show that an activating mutation of b-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 b-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 b-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. Mice expressing a constitutively activeb-catenin allele in osteoblasts, referred to here as Ctnnb1 (CA, constitutively active; osb, osteoblast specific constitutive activity) are osteopetrotic, and die before 6 weeks of age (Fig. 1a) for unknown reasons. Upon further examination, Ctnnb1 mice were anaemic at 2 weeks of age with peripheral blood monocytosis, neutrophilia, lymphocytopenia and thrombocytopenia (Extended Data Fig. 1a). Erythroid cells were decreased in the marrow and extramedullary haematopoiesis was observed in the liver (Fig. 1c and Extended Data Fig. 1b, l, m). Although the number of myeloid (CD11b/Gr1) cells decreased due to osteopetrosis, their relative percentage increased, indicating a shift in the differentiation of HSCs to the myeloid lineage (Fig. 1d and Extended Data Fig. 1c, d). The haematopoietic stem and progenitor cell (HSPC) population in the bone marrow (LinScac-Kit, LSK) cells decreased twofold in Ctnnb1 mice, but their percentage was twofold greater than in wild-type littermates (Fig. 1e and Extended Data Fig. 1e, f). The long-term repopulating HSC progenitors (LT-HSCs) increased in numbers and percentage, whereas the lymphoid-biased multipotential progenitors, LSK/FLT3, and the granulocyte/monocyte progenitors (GMP) (Extended Data Fig. 1g–j) decreased. The GMP percentage increased (Fig. 1f). Identical abnormalities were observed in the spleen of Ctnnb1 mice (Extended Data Fig. 1n–p). The mutation was introduced in osteoblasts but not in any cells of the haematopoietic compartment (Extended Data Fig. 1q–t) of Ctnnb1 mice. Blasts (12–90%) and dysplastic neutrophils (13–81%) were noted in the blood and there was dense and diffuse infiltration with myeloid and monocytic cells, blasts (30–53% for n 5 12 mice) and dysplastic neutrophils in the marrow and spleen of Ctnnb1 mice (Fig. 1g–k, Extended Data Fig. 2a–c). In the liver, clusters of immature cells with atypical nuclear appearance were seen (Fig. 1l). The increase in immature myeloid cells was confirmed by staining with myeloid markers in bones, spleen and liver (Extended Data Fig. 2d–h). Reduced B-cell lymphopoiesis without changes in T-cell populations was observed in Ctnnb1 mice (Extended Data Fig. 2i–t). Differentiation blockade was demonstrated by the presence of immature myeloid progenitors in Ctnnb1 marrow and differentiation cultures (Fig. 1m, n and Extended Data Fig. 2u–x). These cellular abnormalities fulfil the criteria of AML diagnosis in mice with principle features of human AML. A clonal abnormality involving a Robertsonian translocation Rb(1;19) was identified in myeloid cells of the spleen of a Ctnnb1 mouse (Extended Data Fig. 2y). Recurrent numerical and structural chromosomal alterations were also detected in myeloid cells of the spleen of all mutant mice examined (Fig. 2a and Extended Data Table 1). Frequent abnormalities were detected in chromosome 5, the mouse orthologue of human chromosome 7q associated with common cytogenetic abnormalities in patients with myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML). Whole-exome sequencing identified 4 non-silent somatic mutations in myeloid cells from 3 Ctnnb1 mice (Fig. 2b and Extended Data Fig. 2z), including a recurrent one in Tnfrsf21 and a single somatic mutation in Crb1 previously reported in human AML, but sample size has insufficient statistical power to determine if it is a driver or passenger mutation. Hence, constitutive activation of b-catenin in osteoblasts facilitates clonal progression and is associated with somatic mutations in myeloid progenitors. Transplantation of bone marrow cells from Ctnnb1 leukaemic mice into lethally irradiated wild-type recipients induced all features of haematopoietic dysfunction and AML observed in Ctnnb1 mice including blasts (15–80%) and dysplastic neutrophils (15–75%) in the blood and blasts (30–40%) and abnormal megakaryocytes in the marrow and early lethality (Extended Data Fig. 3a–i). Transplantation of wild-type bone marrow cells to lethally irradiated Ctnnb1 mice also resulted in AML with early lethality (Extended Data Fig. 3j–r). Transplantation of LT-HSCs, but not other haematopoietic populations, from Ctnnb1 mice to sub-lethally irradiated wild-type recipients resulted in AML with early lethality (Fig. 2c, d and Extended