Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia

Abstract

Mesenchymal cells contribute to the 'stroma' of most normal and malignant tissues, with specific mesenchymal cells participating in the regulatory niches of stem cells. By examining how mesenchymal osteolineage cells modulate haematopoiesis, here we show that deletion of Dicer1 specifically in mouse osteoprogenitors, but not in mature osteoblasts, disrupts the integrity of haematopoiesis. Myelodysplasia resulted and acute myelogenous leukaemia emerged that had acquired several genetic abnormalities while having intact Dicer1. Examining gene expression altered in osteoprogenitors as a result of Dicer1 deletion showed reduced expression of Sbds, the gene mutated in Schwachman-Bodian-Diamond syndrome-a human bone marrow failure and leukaemia pre-disposition condition. Deletion of Sbds in mouse osteoprogenitors induced bone marrow dysfunction with myelodysplasia. Therefore, perturbation of specific mesenchymal subsets of stromal cells can disorder differentiation, proliferation and apoptosis of heterologous cells, and disrupt tissue homeostasis. Furthermore, primary stromal dysfunction can result in secondary neoplastic disease, supporting the concept of niche-induced oncogenesis.

Figure 1. Impaired osteoblastic differentiation in OCD ^fl/fl mice

a, endogenous osterix expression in osteolineage cells. Dicer1 deletion was demonstrated by b, genomic PCR and c,d, gene expression of primary osteolineage cells (n=5) e, dicer1 gene expression in hematopoietic subsets excluding dicer1 deletion from hematopoietic cells in OCD ^fl/fl mice (n=4) f–h impaired osteogenic differentiation capacity of OCD ^fl/fl bone marrow stromal cells as shown by reduced CFU-Alk (f,g) (n=2, performed in quadruplicate) and decreased osteocalcin gene expression by in situ hybridization (h). Data are mean ± s.e.m. * p≤0.05, **p≤0.01. OSX=osterix, LKS=lineage⁻ckit⁺Sca⁻ cells, CFU-ALK=colony forming unit alkaline phosphatase. For further details see supplementary information.

Figure 2. Myelodysplasia in OCD ^fl/fl mice

a, Leukopenia with variable anemia (p=0.16) and thrombocytopenia (p=0.08) in OCD ^fl/fl mice (n=10). b, blood smears showing dysplastic hyperlobulated nuclei in granulocytes c, bone marrow sections showing micro-megakaryocytes with hyperchromatic nuclei d, increased apoptosis of hematopoietic progenitor cells in OCD ^fl/fl mice. (n=4) e, increased proliferation of hematopoietic progenitor cells as shown by in vivo BRDU labeling (n=4). Data are mean ± s.e.m. * p≤0.05, **p≤0.01. RBC=red blood cells, LKS= lineage ⁻C-kit⁺ Sca1⁺ cells LKS-SLAM= lineage ⁻C-kit⁺ Sca1⁺ CD150⁺ CD48⁻ cells L-K+= lineage⁻c-kit⁺ cells L-K-int=lineage⁻ Ckit ^intermediate, BRDU= bromodeoxyuridine. For further details see supplementary information.

Figure 3. Myelodysplasia in OCD ^fl/fl mice is induced by the bone marrow microenvironment

a, bone marrow cells of OCD ^fl/fl or littermate OCD ^fl/+ mice (n=2) were transplanted into lethally irradiated WT (B6.SJL) mice (n=4 per OCD mouse) demonstrating complete normalization of leukopenia (b), granulocyte (c) and megakaryocyte (d, open arrows ) morphology (open arrows) and bone marrow vascularity (d, closed arrow) 16 weeks post-transplant. Conversely, when e, wildtype B6.SJL cells were transplanted into OCD ^fl/+ (n=8) or OCD ^fl/fl (n=8) mice, hematopoiesis in mutant mice at 8 weeks dysplayed f, leucopenia g, dysgranulopoiesis with giant platelets (indicated by arrows) and h, increased bone marrow vascularity with dysplastic megakaryopoiesis (arrows). Data are mean ± s.e.m. * p≤0.05, **p≤0.01. For further details see supplementary information.

Figure 4. Myeloid sarcoma and acute myelogenous leukemia in OCD ^fl/fl mice

a, tumors infiltrating soft tissue in 2–4 weeks old mice. Data from one representative animal. b, tumor sections showing predominantly myeloblasts admixed with maturing granulocytes, typical of myeloid sarcoma and confirmed by immunostaining with CD13. c, exclusion of Dicer1 deletion in myeloid sarcoma cells by genomic PCR. Bone from the same animal as positive control. d, genomic aberrancies detected in myeloid sarcomas (n=3) as detected by array-based CGH. A common amplified region was identified on chromosome 14qC1 in two tumors. Green lines represent amplifications, red line represents deletion e, myelocytosis, f, anemia, g, splenomegaly with h, infiltration of CD13⁺ blasts in the spleen and i, bone marrow, and j–k, monocytic blasts in a background of dysplastic granulocytes in the peripheral blood. HB=hemoglobin, PB=peripheral blood

Figure 5. Targeted deletion of the Sbds gene from osteoprogenitor cells recapitulates many features of the OCD ^fl/fl phenotype

a, genetic model of Sbds deletion from osteoprogenitor cells b, leukopenia (n=8) c, dysplasia of neutrophils in peripheral blood d, dysplasia of megakaryocytes (micro-megakaryocytes) e, hypervascularity of the bone marrow f, increased intramedullary apoptosis of hematopoietic progenitor cells(n=5). Data are mean ± s.e.m. * p≤0.05, **p≤0.01.