Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to D-2-hydroxyglutarate (D-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or D-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.isocitrate dehydrogenase | cartilage tumor | hedgehog E nchondromas, one of the most common benign tumors occurring in bone, are present in more than 3% of the population (1, 2). They are composed of cells derived from chondrocytes and occur as solitary lesions or multiple lesions in enchondromatosis syndromes (Ollier disease or Maffucci syndrome-in the latter, enchondromas are associated with vascular malformations). Clinical problems caused by enchondromas include pain, fractures, and skeletal deformity. There is a potential for malignant progression to chondrosarcoma that may be greater than 50% in some cases of multiple enchondromatosis (i.e., Maffucci syndrome) (3-7). Many chondrosarcomas are thought to derive from enchondromas, and such sarcomas are termed central chondrosarcomas (3).The hedgehog (Hh) signaling pathway is constitutively active in enchondromas and chondrosarcomas (8,9). Hh is important in growth-plate chondrocyte differentiation, where it cooperates with parathyroid hormone-like hormone in a negative feedback loop to inhibit the differentiation of proliferative growth-plate chondrocytes (6,(10)(11)(12)(13)(14). Disruption of this feedback loop can result in either skeletal dysplasias with abnormal bone growth or enchondromas; 5% of enchondromas harbor mutation in parathyroid hormone-like hormone receptor (PTHR1), resulting in activation of Hh signaling (6,(10)(11)(12)(13)(14), and expression of a mutant PTHR1 or overexpression of the Hh-regulated transcription factor Gli2 under the Col2a1 promoter causes enchondroma-like cartilage lesions to develop adjacent to the growth-plate ...

The osteoclast is a multinucleated monocyte/macrophage lineage cell that degrades bone. Here we used lineage tracing studies, labeling cells expressing Cx3cr1, Csf1r, or Flt3 to identify the precursors of osteoclast in mice. We identified an erythromyeloid progenitor (EMP)-derived osteoclast precursor population. Yolk-sac macrophages of EMP origin produced neonatal osteoclasts that can create a space for postnatal bone marrow hematopoiesis. Furthermore, EMPs gave rise to long-lasting osteoclast precursors that contributed to postnatal bone remodeling in both physiological and pathological settings. Our single cell RNA-sequencing data showed that EMP-derived osteoclast precursors arose independently from hematopoietic stem cell (HSC) lineage and the data from fate tracking of EMP-and HSC-lineage provided a possibility of cell-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The cell of origin for most mesenchymal tumors is unclear. One cell type that contributes to this lineages is the pericyte, a cell expressing Ng2/Cspg4. Using lineage tracing, we demonstrated that bone and soft tissue sarcomas driven by the deletion of the Trp53 tumor suppressor, or desmoid tumors driven by a mutation in Apc can derive from cells expressing Ng2/Cspg4. Deletion of the Trp53 tumor suppressor gene in these cells resulted in the bone and soft tissue sarcomas that closely resemble human sarcomas, while stabilizing β-catenin in this same cell type caused desmoid tumors. Comparing expression between Ng2/Cspg4 expressing pericytes lacking Trp53 and sarcomas that arose from deletion of Trp53, showed inhibition of β-catenin signaling in the sarcomas. Activation of β-catenin inhibited the formation and growth of sarcomas. Thus, pericytes can be a cell of origin for mesenchymal tumors, and β-catenin dysregulation plays an important role in the neoplastic phenotype.

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SUMMARYCellular heterogeneity is frequently observed in cancer, but the biological significance of heterogeneous tumor clones is not well defined. Using multicolor reporters and CRISPR-Cas9 barcoding, we trace clonal dynamics in a mouse model of sarcoma. We show that primary tumor growth is associated with a reduction in clonal heterogeneity. Local recurrence of tumors following surgery or radiation therapy is driven by multiple clones. In contrast, advanced metastasis to the lungs is driven by clonal selection of a single metastatic clone (MC). Using RNA sequencing (RNA-seq) and in vivo assays, we identify candidate suppressors of metastasis, namely, Rasd1, Reck, and Aldh1a2. These genes are downregulated in MCs of the primary tumors prior to the formation of metastases. Overexpression of these suppressors of metastasis impair the ability of sarcoma cells to colonize the lungs. Overall, this study reveals clonal dynamics during each step of tumor progression, from initiation to growth, recurrence, and distant metastasis.

During enchondral ossification, mesenchymal cells express genes regulating the intracellular biosynthesis of cholesterol and lipids. Here, we have investigated conditional deletion of Scap or of Insig1 and Insig2 (Scap inhibits intracellular biosynthesis and Insig proteins activate intracellular biosynthesis). Mesenchymal condensation and chondrogenesis was disrupted in mice lacking Scap in mesenchymal progenitors, whereas mice lacking the Insig genes in mesenchymal progenitors had short limbs, but normal chondrogenesis. Mice lacking Scap in chondrocytes showed severe dwarfism, with ectopic hypertrophic cells, whereas deletion of Insig genes in chondrocytes caused a mild dwarfism and shortening of the hypertrophic zone. In vitro studies showed that intracellular cholesterol in chondrocytes can derive from exogenous and endogenous sources, but that exogenous sources cannot completely overcome the phenotypic effect of Scap deficiency. Genes encoding cholesterol biosynthetic proteins are regulated by Hedgehog (Hh) signaling, and Hh signaling is also regulated by intracellular cholesterol in chondrocytes, suggesting a feedback loop in chondrocyte differentiation. Precise regulation of intracellular biosynthesis is required for chondrocyte homeostasis and long bone growth, and these data support pharmacological modulation of cholesterol biosynthesis as a therapy for select cartilage pathologies.

Tumor-propagating cells (TPCs) are believed to drive cancer initiation, progression and recurrence. These cells are characterized by enhanced tumorigenicity and self-renewal. The ability to identify such cells in primary human sarcomas relies on the dye exclusion ability of tumor side population (SP) cells. Here, we performed a high-throughput cell surface antigen screen and found that CD146 is enriched in the SP population. In vivo serial transplantation assays showed that CD146+ cells are highly tumorigenic, capable of self-renewal and thus enriches for the TPC population. In addition, depletion of SP cells from the CD146+ population show that CD146+ cells and SP cells are a distinct and overlapping TPC populations. Gene expression profiling of CD146+ and SP cells revealed multiple pathways commonly upregulated in both of these populations. Inhibition of one of these upregulated pathways, Notch signaling, significantly reduced tumor growth and self-renewal. Our data demonstrate that CD146 is an effective cell surface marker for enriching TPCs in primary human sarcomas. Targeting differentially activated pathways in TPCs may provide new therapeutic strategies for treating sarcoma.

Skeletal stem/progenitor cells (SSPC) are critical regulators of bone homeostasis by providing a continuous supply of osteoblasts throughout life. In response to inductive signals, SSPC proliferate before osteoblast differentiation. Proliferation requires the duplication of all cellular components before cell division. This imposes a unique biosynthetic requirement for amino acids that can be used for biomass production. Thus, the ability to sense and respond to amino acid availability is likely a major determinant for proliferation. Using a cellular and genetic approach, we demonstrate the amino acid sensor GCN2 is required to support the robust proliferative capacity of SSPC during bone homeostasis. GCN2 ablation results in decreased postnatal bone mass due primarily to reduced osteoblast numbers. Decreased osteoblast numbers is likely attributed to reduced SSPC proliferation as loss of GCN2 specifically affected proliferation in cultured bone marrow stromal cells (BMSCs) without impacting osteoblast differentiation in vitro. Mechanistically, GCN2 regulates proliferation by increasing amino acid uptake downstream of the transcriptional effector ATF4. Collectively, these data suggest amino acid sensing through the GCN2/ATF4 pathway is indispensable for robust SSPC proliferation necessary for bone homeostasis.