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

Upregulation of cytokines and chemokines is a frequent finding in multiple myeloma (MM). CCL3 (also known as MIP-1α) is a pro-inflammatory chemokine whose levels in the MM microenvironment correlate with osteolytic lesions and tumor burden. CCL3 and its receptors, CCR1 and CCR5, contribute to the development of bone disease in MM by supporting tumor growth and regulating osteoclast (OC) differentiation. Here, we identify inhibition of osteoblast (OB) function as an additional pathogenic mechanism in CCL3-induced bone disease. MM-derived and exogenous CCL3 represses mineralization and osteocalcin production by primary human bone marrow stromal cells and HS27A cells. Our results suggest that CCL3 effects on OBs are mediated by ERK activation and subsequent downregulation of the osteogenic transcription factor osterix. CCR1 inhibition reduced ERK phosphorylation and restored both osterix and osteocalcin expression in the presence of CCL3. Finally, treating SCID-hu mice with a small molecule CCR1 inhibitor suggests an upregulation of osteocalcin expression along with OC downregulation. Our results show that CCL3, in addition to its known catabolic activity, reduces bone formation by inhibiting OB function and therefore contributes to OB/OC uncoupling in MM.

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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)

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Cyclin dependent kinases (CDKs) and their cyclin complexes play a crucial role in cell cycle control and transcriptional regulation. In multiple myeloma (MM), the abnormal activation of different CDKs and their cyclin partners, especially CDK4/cyclin D1 and CDK6/Cyclin D2, mediate uncontrolled cell cycle progression. Therefore CDKs represent promising novel therapeutic targets for MM. Additionally the cytokine dependent PI3K/Akt signaling pathway mediates growth, survival, drug resistance, migration and cell cycle regulation in MM. Activated Akt in turn phosphorylates downstream target molecules like glycogen synthase kinase (GSK)-3 β impacting growth and survival. Here we investigated the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519 in MM. In vitro kinase assays demonstrated more potent inhibition of CDK 1, 2, 4, 5 and 9 compared to CDK 3, 6, and 7. AT7519 also demonstrated potent inhibitory activity against GSK-3 β. No significant inhibitory effects against other kinases were observed. We next investigated the growth inhibitory effect of AT7519 on MM cell lines. Maximal cytotoxicity was observed in 48 hour culture with IC50 values ranging from 0.5μM (MM.1S, U266) to 4 μM (MM1R). AT7519 was also effective against primary tumor cells from MM patients with no significant cytotoxicity noted in peripheral blood mononuclear cells from healthy volunteers. To delineate the underlying mechanism of cytotoxicity induced by AT7519, cell cycle analysis using PI staining in MM.1S cell line was performed. No significant accumulation of cells in a particular phase of cell cycle was noted; however, AT7519 showed an increased sub-G1 population, indicative of apoptosis, which was confirmed by Annexin V+PI+ staining and associated with caspase-8-9 and -3 cleavage. Importantly, we found that AT7519 markedly inhibited phosphorylation (serine 2 and serine 5 sites) of the carboxyl terminal domain of RNA polymerase II (RNA pol II) within 6 hours of treatment. Non-cell cycle CDKs including CDK9 are responsible for phosphorylation and activation of RNA pol II. Similarly, AT7519 also inhibited phosphorylation of GSK-3β while no significant effects on CDK expression levels were evident at early time points. To investigate whether there was a correlation between inhibition of phosphorylation of GSK-3β and RNA pol II, MM.1S cells were cultured with α-amanitin, a specific inhibitor of RNA pol II. Although phosphorylation of RNA pol II was significantly inhibited, phosphorylation of GSK-3β was not altered by amanitin (10 μM for up to 24 hours). These results suggest that GSK-3β and RNA pol II dephosphorylation at serine 2 and serine 5 may be two independent mechanisms by which AT7519 induces apoptosis in MM cells. Ongoing studies are confirming the role of GSK-3 β in AT7519 induced cytotoxicity of MM cells. Finally, the in vivo efficacy of AT7519 was examined using a xenograft mouse model of human MM. Mice treated with AT7519 demonstrated slower tumor growth compared to the control group without adverse effects. Moreover, AT7519 resulted in a significant prolongation in median overall survival in treated mice (40 days in the treatment group versus 27.5 days in the control cohort, p = 0.0324). In conclusion, these results show significant anti-MM activity of AT7519, and provide the rationale for its clinical evaluation in MM.

739 A common feature of bone disease in cancer is hyperactivity of osteoclasts (OC). However, osteoblast (OB) inhibition is critical to the development of osteolytic lesions. Tumor-OC interactions have been extensively studied, the mechanism of OB inhibition however still remains elusive. Several chemokines are upregulated in the multiple myeloma (MM)/bone microenvironment. CCL3 (MIP-1αa), in particular, mediates MM cell migration and stimulates OC differentiation, directly, by stimulating precursor cell fusion, and indirectly, by inducing OB secretion of RANKL. Here, we investigate whether CCL3 interferes with OB differentiation and activity. In vitro osteoblastogenesis consists of differentiation of alkaline phosphatase (ALP) positive cells, followed by secretion and mineralization of the extracellular matrix. We observed that ALP-positive cells express both CCL3 receptors, CCR1 and CCR5, late in differentiation (40% and 32%, respectively, isotype control 9%). No significant CCL3 secretion was detected in OB culture supernatant, suggesting that paracrine CCL3 may primarily affect matrix mineralization. Indeed, exogenous CCL3 (25 to 100 ng/ml) did not modify OB number, instead it decreased calcium deposition (10% decrease at 25ng/ml, 33% decrease at 50 and 100 ng/ml, p<0.05). We next assessed the expression levels of proteins critical to matrix formation and mineralization including osteopontin, bone-sialoprotein and osteocalcin. Osteopontin expression was not affected by CCL3, while both bone-sialoprotein and osteocalcin were downregulated. Importantly, either continuous exposure to CCL3 or 24h stimulation of mature OB impaired RNA expression of osteocalcin (30% to 80%) and bone-sialoprotein (26% to 60%) but not ALP, confirming that CCL3 interferes with OB function rather than formation. We also observed a correlation between osteocalcin expression by IHC on BM biopsies and CCL3 levels in BM serum of MM patients. These data suggest that MM-derived CCL3 interferes with bone mineralization by inhibiting osteocalcin expression. Using neutralizing antibodies against CCL3 we restored both osteocalcin and bone sialoprotein RNA expression levels in the presence of CCL3. Importantly, pretreatment with a small molecule CCR1 inhibitor, MLN3897 (Millennium Pharmaceuticals) completely abrogated the inhibition on osteocalcin and partially reversed bone-sialoprotein expression, suggesting CCR1 as the main mediator of CCL3 effects. We further verified these results in an in-vivo setting of MM bone disease, using the SCID-hu model. CB17 SCID mice bearing a human fetal bone implant were engrafted with CCL3-expressing INA6 MM cells and treated orally with MLN3897 for a total of 49 doses. After 4 weeks of treatment the bones were harvested and stained for TRAP activity, hematoxylin-eosin and osteocalcin. The number of OC/400x field was significantly reduced in the treated group (2.7 vs 1.9, p<0.05), thus confirming in vivo the anti-osteoclastogenic effect of CCR1 inhibition. Moreover, in the presence of INA6 MM cells osteocalcin levels were downregulated compared to non-injected bones and treatment with the CCR1 inhibitor partially restored osteocalcin expression. These results suggest a new role for the CCL3/CCR1 pathway in the development of osteolytic lesions in MM, as inhibitor of OB function other than OC growth factor. Targeting this pathway represents a promising strategy for the treatment of bone disease. Disclosures: Veiby: Millennium Pharmaceuticals: Employment. Anderson:Millennium: Research Funding. Raje:Astrazeneca, Novartis, Celgene: Research Funding.

Uncontrolled proliferation and development of drug resistance in multiple myeloma (MM) cells are consequences of the numerous genetic aberrations which are further stimulated in the context of bone marrow microenvironment. Thus, inhibition of complementary pro-survival signaling and transcriptional networks rather than individual pathway is required for induction of optimal cytotoxicity in MM cells. Recent studies have shown that cyclin-dependent kinases inhibitors (CDKIs), designed to block cell cycle progression through inhibition of CDK/cyclin complexes, block transcription through suppression of RNA polymerase II phosphorylation at its C-terminal domain (CTD), resulting in downregulation of cell proliferation (cyclins: D, A, B1, pRb) and anti-apoptotic proteins (i.e. Mcl-1, survivin, XIAP). Here we examined the anti-MM activity of RGB 286638, a novel multi-targeted small molecule inhibitor, originally designed to induce broad cell cycle suppression via multiple CDK inhibition. Treatment with RGB 286638 triggered a dose-dependent cytotoxicity in conventional drug-sensitive (MM.1S, RPMI 8226, U266, OPM2), and resistant (MM.1R, Dox-40, LR5) MM cell lines, as well as primary tumor cells from MM patients. Induction of apoptosis was evidenced by Annexin V/PI staining, and confirmed by PARP and caspase cleavage. Additionally, RGB 286638 overcame the proliferative advantage conferred by MM patient-derived bone marrow stromal cells (BMSCs) and cytokines (IL-6, IGF-1) on MM cells. To determine molecular mechanisms responsible for RGB 286638-induced cytotoxicity, we assessed the cell cycle profile, which revealed G2/M arrest followed by increased sub-G1 phase. Importantly, RGB 286638 inhibited phosphorylation of RNA polymerase II in a dose- and a time-dependent fashion, followed by suppression of CDK1/cyclin B, CDK4, 6/Cyclin D1, D3, and CDK2/Cyclin E complexes associated with rapid down-regulation of Rb phosphorylation. Interestingly, RGB 286638 effectively reduced IL-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation (Tyr705) and Janus kinase 2 (JAK2) phosphorylation (Tyr 1007/1008), suggesting that RGB 286638 is a possible JAK2 inhibitor. Based on sufficient in vitro cytotoxicity, we examined anti-tumor activity of RGB 286638 in vivo using a human MM cell xenograft model in SCID mice and demonstrated that RGB 286638 inhibited tumor growth and prolonged survival. In conclusion, our data demonstrate preclinical activity and provide the rational to test RGB 286638 in the treatment of MM.