Mesenchymal cell populations contribute to microenvironments regulating stem cells and the growth of malignant cells. Osteolineage cells participate in the hematopoietic stem cell niche. Here, we report that deletion of the miRNA processing endonuclease Dicer1 selectively in mesenchymal osteoprogenitors induces markedly disordered hematopoiesis. Hematopoietic changes affected multiple lineages recapitulating key features of human myelodysplastic syndrome (MDS) including the development of acute myelogenous leukemia. These changes were microenvironment dependent and induced by specific cells in the osteolineage. Dicer1−/− osteoprogenitors expressed reduced levels of Sbds, the gene mutated in the human bone marrow failure and leukemia predisposition Shwachman-Bodian-Diamond Syndrome. Deletion of Sbds in osteoprogenitors largely phenocopied Dicer1 deletion. These data demonstrate that differentiation stage-specific perturbations in osteolineage cells can induce complex hematological disorders and indicate the central role individual cellular elements of ‘estroma’ can play in tissue homeostasis. They reveal that primary changes in the hematopoietic microenvironment can initiate secondary neoplastic disease.

Drug targeting of adult stem cells has been proposed as a strategy for regenerative medicine, but very few drugs are known to target stem cell populations in vivo. Mesenchymal stem/progenitor cells (MSCs) are a multipotent population of cells that can differentiate into muscle, bone, fat, and other cell types in context-specific manners. Bortezomib (Bzb) is a clinically available proteasome inhibitor used in the treatment of multiple myeloma. Here, we show that Bzb induces MSCs to preferentially undergo osteoblastic differentiation, in part by modulation of the bone-specifying transcription factor runt-related transcription factor 2 (Runx-2) in mice. Mice implanted with MSCs showed increased ectopic ossicle and bone formation when recipients received low doses of Bzb. Furthermore, this treatment increased bone formation and rescued bone loss in a mouse model of osteoporosis. Thus, we show that a tissue-resident adult stem cell population in vivo can be pharmacologically modified to promote a regenerative function in adult animals.

Understanding the pathogenesis of cancer-related bone disease is crucial to the discovery of new therapies. Here we identify activin A, a TGF-β family member, as a therapeutically amenable target exploited by multiple myeloma (MM) to alter its microenvironmental niche favoring osteolysis. Increased bone marrow plasma activin A levels were found in MM patients with osteolytic disease. MM cell engagement of marrow stromal cells enhanced activin A secretion via adhesion-mediated JNK activation. Activin A, in turn, inhibited osteoblast differentiation via SMAD2-dependent distalless homeobox-5 down-regulation. Targeting activin A by a soluble decoy receptor reversed osteoblast inhibition, ameliorated MM bone disease, and inhibited tumor growth in an in vivo humanized MM model, setting the stage for testing in human clinical trials.osteoblasts | osteoclasts | tumor niche

Purpose: The increasing incidence of osteonecrosis of the jaw and its possible association with high cumulative doses of bisphosphonate led us to study the effects of high doses of zoledronic acid (ZA) on bone remodeling. Experimental Design: Five-week-old C57BL6 mice were treated with saline or ZA weekly for 3 weeks at increasing doses (0.05-1 mg/Kg). Effects of ZA on bone remodeling were studied using standard assays. Results: We observed an increase in bone mineral density and content in treated animals at doses of 0.05 mg/Kg, which was not further enhanced at higher doses of ZA. Trabecular bone volume at the proximal tibia and the distal femur assessed by histomorphometry and microCT, respectively, increased significantly in ZA-treated groups. There was however no difference between 0.5 and 1 mg/kg, suggesting a ceiling effect for ZA. ZA led to decreased numbers of osteoclasts and osteoblasts per bone perimeter that paralleled a significant reduction of serum levels of TRAC5b and osteocalcin in vivo. Effects on osteoblasts were confirmed in in vitro assays. Mechanical testing of the femur showed increased brittleness in ZA-treated mice. Conclusions: High doses of ZA inhibit both osteoclast and osteoblasts function and bone remodeling in vivo interfering with bone mechanical properties. No dose response was noted beyond 0.5 mg/kg suggesting that lower doses of ZA may be adequate in inhibiting bone resorption. Our data may help inform future studies of ZA use with respect to alternate and lower doses in the treatment of patients with cancer bone disease. (Clin Cancer Res 2009;15(18):5829-39)

Purpose: The incidence of bone metastasis in advanced breast cancer (BrCa) exceeds 70%. Bortezomib, a proteasome inhibitor used for the treatment of multiple myeloma, also promotes bone formation. We tested the hypothesis that proteasome inhibitors can ameliorate BrCa osteolytic disease.Experimental Design: To address the potentially beneficial effect of bortezomib in reducing tumor growth in the skeleton and counteracting bone osteolysis, human MDA-MB-231 BrCa cells were injected into the tibia of mice to model bone tumor growth for in vivo assessment of treatment regimens before and after tumor growth.Results: Controls exhibited tumor growth, destroying trabecular and cortical bone and invading muscle. Bortezomib treatment initiated following inoculation of tumor cells strikingly reduced tumor growth, restricted tumor cells mainly to the marrow cavity, and almost completely inhibited osteolysis in the bone microenvironment over a 3-to 4-week period as shown by [ 18 F]fluorodeoxyglucose positron emission tomography, micro-computed tomography scanning, radiography, and histology. Thus, proteasome inhibition is effective in killing tumor cells within the bone. Pretreatment with bortezomib for 3 weeks before inoculation of tumor cells was also effective in reducing osteolysis. Our in vitro and in vivo studies indicate that mechanisms by which bortezomib inhibits tumor growth and reduces osteolysis result from inhibited cell proliferation, necrosis, and decreased expression of factors that promote BrCa tumor progression in bone.Conclusion: These findings provide a basis for a novel strategy to treat patients with BrCa osteolytic lesions, and represent an approach for protecting the entire skeleton from metastatic bone disease. Clin Cancer Res; 16(20); 4978-89. ©2010 AACR.Metastatic osteolytic disease is prevalent in cancer patients. In advanced breast cancer (BrCa), 70% of women develop osteolytic lesions, resulting in pain, pathologic fracture, and increased morbidity. Dysfunction of the ubiquitin-proteasome system is associated with tumor growth and metastatic disease, providing the rationale for development of proteasome inhibitors as antineoplastic therapies (1, 2). The proteasome is a ubiquitous enzyme complex that plays a critical role in the degradation of proteins involved in cell cycle regulation, apoptosis, and angiogenesis (2, 3). Bortezomib, a selective proteasome inhibitor used to treat multiple myeloma, has a potent anabolic effect on bone (4-9). Bortezomib alters the bone marrow microenvironment by increasing the number and differentiation of resident mesenchymal stem cells into osteoblasts, thereby increasing bone formation rates within 4 weeks in normal mice and resulting in trabecular bone formation in bone loss model (7). A similar enhancement of osteoblast differentiation is found in myeloma patients treated with bortezomib who show sustained increases in circulating osteocalcin, a marker of bone formation (6, 10). Thus, bortezomib treatment represents a novel and clinically feasible app...

SUMMARY WTX is an X-linked tumor suppressor targeted by somatic mutations in Wilms tumor, a pediatric kidney cancer, and by germline inactivation in osteopathia striata with cranial sclerosis, a bone overgrowth syndrome. Here, we show that Wtx deletion in mice causes neonatal lethality, somatic overgrowth, and malformation of multiple mesenchyme-derived tissues, including bone, fat, kidney, heart, and spleen. Inactivation of Wtx at different developmental stages and in primary mesenchymal progenitor cells (MPCs) reveals that bone mass increase and adipose tissue deficiency are due to altered lineage fate decisions coupled with delayed terminal differentiation. Specification defects in MPCs result from aberrant β-catenin activation, whereas alternative pathways contribute to the subsequently delayed differentiation of lineage-restricted cells. Thus, Wtx is a regulator of MPC commitment and differentiation with stage-specific functions in inhibiting canonical Wnt signaling. Furthermore, the constellation of anomalies in Wtx null mice suggests that this tumor suppressor broadly regulates MPCs in multiple tissues.

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A distinct feature of multiple myeloma (MM) is the tight interaction between malignant plasma cells and their bone microenvironment, creating a niche suitable for MM growth. In particular, MM cells inhibit osteoblast (OB) differentiation and stimulate osteoclast (OC) function, resulting in imbalanced bone remodeling and osteolytic bone disease. Here we studied a novel cytokine, activin A, identified from a broad range of cytokines, in the development of MM bone disease. We next asked whether activin A inhibition could restore bone balance and suppress tumor growth. Activin, a member of the TNF-α superfamily, is a pleiotropic cytokine involved in bone remodeling. Here, we observed, that MM patients with multiple osteolytic lesions had a 4-fold increase in activin A expression levels in bone marrow plasma compared to MM patients with one or less osteolytic lesions and non-MM patients (average 123.6 ± 136 vs 26.4 ± 21.4 vs 30.6 ± 25.1 pg/ml respectively, p<0.05). Interestingly, our data demonstrate that the main source of activin in the MM niche are bone marrow stromal cells (BMSCs), followed by OCs, and OBs (average levels in 72h culture supernatant are 1884, 1300, 299 pg/ml, respectively). In contrast, MM cells did not secrete activin, but stimulated its secretion in coculture by BMSC (by 1.3 to 2 fold increase). Activin A stimulated OC differentiation in synergy with RANKL and M-CSF via induction of a three-fold increase in precursor cell proliferation. Moreover, activin A had a potent inhibitory effect on OB differentiation as verified by ALP activity (reduced by 30% compared to control, p<0.05) and OB function, assessed with alizarin red (80% inhibition, p< 0.01). To test the role of targeting activin A with therapeutic intent, we used both a neutralizing antibody and a soluble receptor fusion, RAP-011 (Acceleron Pharma Inc., Cambridge). In effect, both strategies enhanced OB differentiation and activity (5 fold increase in calcium deposition at day 21, p<0.05). This was confirmed by quantitative-PCR analysis of ALP and osteocalcin gene expression. Importantly, RAP-011 promoted OB differentiation even in the presence of INA6 MM cells and reversed the inhibitory effects of the stroma-dependent MOLP5 MM cells as well as patient derived MM cells on OB. Enhanced OB differentiation by RAP-011 resulted in inhibition of MM cell proliferation compared to BMSCs and mature OB. These data thus suggest that manipulating the bone niche may result in reduced tumor growth. To further verify if these results translated in reduced tumor growth in vivo, we used the SCID-hu mouse model consisting of INA6 MM cells injected in a subcutaneously implanted fetal human bone. RAP-011 treatment resulted in a decrease in the number of osteolytic lesions assessed by CT imaging accompanied by improved bone density. These effects translated in reduced MM cell growth, analyzed by soluble human IL6 receptor levels, in the treated group compared to the control (p<0.02). These data therefore suggest that activin A is involved in development of MM bone disease and can be effectively targeted by a novel clinical grade compound. RAP-011 demonstrated bone-anabolic effects via inhibition of OC formation and stimulation of OB differentiation resulting in restoration of bone balance within the MM niche, which translated in in-vivo inhibition of MM cell growth. These effects of RAP-011 support the use of the human ACE-011 as an attractive approach for the treatment of MM.