Sialic acid blockade inhibits the metastatic spread of prostate cancer to bone.

BACKGROUND: Bone metastasis is a common consequence of advanced prostate cancer. Bisphosphonates can be used to manage symptoms, but there are currently no curative treatments available. Altered tumour cell glycosylation is a hallmark of cancer and is an important driver of a malignant phenotype. In prostate cancer, the sialyltransferase ST6GAL1 is upregulated, and studies show ST6GAL1-mediated aberrant sialylation of N-glycans promotes prostate tumour growth and disease progression. METHODS: Here, we monitor ST6GAL1 in tumour and serum samples from men with aggressive prostate cancer and using in vitro and in vivo models we investigate the role of ST6GAL1 in prostate cancer bone metastasis. FINDINGS: ST6GAL1 is upregulated in patients with prostate cancer with tumours that have spread to the bone and can promote prostate cancer bone metastasis in vivo. The mechanisms involved are multi-faceted and involve modification of the pre-metastatic niche towards bone resorption to promote the vicious cycle, promoting the development of M2 like macrophages, and the regulation of immunosuppressive sialoglycans. Furthermore, using syngeneic mouse models, we show that inhibiting sialylation can block the spread of prostate tumours to bone. INTERPRETATION: Our study identifies an important role for ST6GAL1 and α2-6 sialylated N-glycans in prostate cancer bone metastasis, provides proof-of-concept data to show that inhibiting sialylation can suppress the spread of prostate tumours to bone, and highlights sialic acid blockade as an exciting new strategy to develop new therapies for patients with advanced prostate cancer. FUNDING: Prostate Cancer Research and the Mark Foundation For Cancer Research, the Medical Research Council and Prostate Cancer UK.

Metastatic Infiltration of Nervous Tissue and Periosteal Nerve Sprouting in Multiple Myeloma-Induced Bone Pain in Mice and Human.

Multiple myeloma (MM) is a neoplasia of B plasma cells that often induces bone pain. However, the mechanisms underlying myeloma-induced bone pain (MIBP) are mostly unknown. Using a syngeneic MM mouse model, we show that periosteal nerve sprouting of calcitonin gene-related peptide (CGRP+) and growth associated protein 43 (GAP43+) fibers occurs concurrent to the onset of nociception and its blockade provides transient pain relief. MM patient samples also showed increased periosteal innervation. Mechanistically, we investigated MM induced gene expression changes in the dorsal root ganglia (DRG) innervating the MM-bearing bone of male mice and found alterations in pathways associated with cell cycle, immune response and neuronal signaling. The MM transcriptional signature was consistent with metastatic MM infiltration to the DRG, a never-before described feature of the disease that we further demonstrated histologically. In the DRG, MM cells caused loss of vascularization and neuronal injury, which may contribute to late-stage MIBP. Interestingly, the transcriptional signature of a MM patient was consistent with MM cell infiltration to the DRG. Overall, our results suggest that MM induces a plethora of peripheral nervous system alterations that may contribute to the failure of current analgesics and suggest neuroprotective drugs as appropriate strategies to treat early onset MIBP.SIGNIFICANCE STATEMENT Multiple myeloma (MM) is a painful bone marrow cancer that significantly impairs the quality of life of the patients. Analgesic therapies for myeloma-induced bone pain (MIBP) are limited and often ineffective, and the mechanisms of MIBP remain unknown. In this manuscript, we describe cancer-induced periosteal nerve sprouting in a mouse model of MIBP, where we also encounter metastasis to the dorsal root ganglia (DRG), a never-before described feature of the disease. Concomitant to myeloma infiltration, the lumbar DRGs presented blood vessel damage and transcriptional alterations, which may mediate MIBP. Explorative studies on human tissue support our preclinical findings. Understanding the mechanisms of MIBP is crucial to develop targeted analgesic with better efficacy and fewer side effects for this patient population.

HSV1716 Prevents Myeloma Cell Regrowth When Combined with Bortezomib In Vitro and Significantly Reduces Systemic Tumor Growth in Mouse Models.

Multiple myeloma remains largely incurable due to refractory disease; therefore, novel treatment strategies that are safe and well-tolerated are required. Here, we studied the modified herpes simplex virus HSV1716 (SEPREHVIR®), which only replicates in transformed cells. Myeloma cell lines and primary patient cells were infected with HSV1716 and assessed for cell death using propidium iodide (PI) and Annexin-V staining and markers of apoptosis and autophagy by qPCR. Myeloma cell death was associated with dual PI and Annexin-V positivity and increased expression of apoptotic genes, including CASP1, CASP8, CASP9, BAX, BID, and FASL. The combination of HSV1716 and bortezomib treatments prevented myeloma cell regrowth for up to 25 days compared to only transient cell growth suppression with bortezomib treatment. The viral efficacy was tested in a xenograft (JJN-3 cells in NSG mice) and syngeneic (murine 5TGM1 cells in C57BL/KaLwRijHsd mice) systemic models of myeloma. After 6 or 7 days, the post-tumor implantation mice were treated intravenously with the vehicle or HSV1716 (1 × 107 plaque forming units/1 or 2 times per week). Both murine models treated with HSV1716 had significantly lower tumor burden rates compared to the controls. In conclusion, HSV1716 has potent anti-myeloma effects and may represent a novel therapy for multiple myeloma.

The strategy and clinical relevance of in vitro models of MAP resistance in osteosarcoma: a systematic review.

Over the last 40 years osteosarcoma (OS) survival has stagnated with patients commonly resistant to neoadjuvant MAP chemotherapy involving high dose methotrexate, adriamycin (doxorubicin) and platinum (cisplatin). Due to the rarity of OS, the generation of relevant cell models as tools for drug discovery is paramount to tackling this issue. Four literature databases were systematically searched using pre-determined search terms to identify MAP resistant OS cell lines and patients. Drug exposure strategies used to develop cell models of resistance and the impact of these on the differential expression of resistance associated genes, proteins and non-coding RNAs are reported. A comparison to clinical studies in relation to chemotherapy response, relapse and metastasis was then made. The search retrieved 1891 papers of which 52 were relevant. Commonly, cell lines were derived from Caucasian patients with epithelial or fibroblastic subtypes. The strategy for model development varied with most opting for continuous over pulsed chemotherapy exposure. A diverse resistance level was observed between models (2.2-338 fold) with 63% of models exceeding clinically reported resistance levels which may affect the expression of chemoresistance factors. In vitro p-glycoprotein overexpression is a key resistance mechanism; however, from the available literature to date this does not translate to innate resistance in patients. The selection of models with a lower fold resistance may better reflect the clinical situation. A comparison of standardised strategies in models and variants should be performed to determine their impact on resistance markers. Clinical studies are required to determine the impact of resistance markers identified in vitro in poor responders to MAP treatment, specifically with respect to innate and acquired resistance. A shift from seeking disputed and undruggable mechanisms to clinically relevant resistance mechanisms may identify key resistance markers that can be targeted for patient benefit after a 40-year wait.

Myeloma Bone Disease: The Osteoblast in the Spotlight.

Lytic bone disease remains a life-altering complication of multiple myeloma, with up to 90% of sufferers experiencing skeletal events at some point in their cancer journey. This tumour-induced bone disease is driven by an upregulation of bone resorption (via increased osteoclast (OC) activity) and a downregulation of bone formation (via reduced osteoblast (OB) activity), leading to phenotypic osteolysis. Treatments are limited, and currently exclusively target OCs. Despite existing bone targeting therapies, patients successfully achieving remission from their cancer can still be left with chronic pain, poor mobility, and reduced quality of life as a result of bone disease. As such, the field is desperately in need of new and improved bone-modulating therapeutic agents. One such option is the use of bone anabolics, drugs that are gaining traction in the osteoporosis field following successful clinical trials. The prospect of using these therapies in relation to myeloma is an attractive option, as they aim to stimulate OBs, as opposed to existing therapeutics that do little to orchestrate new bone formation. The preclinical application of bone anabolics in myeloma mouse models has demonstrated positive outcomes for bone repair and fracture resistance. Here, we review the role of the OB in the pathophysiology of myeloma-induced bone disease and explore whether novel OB targeted therapies could improve outcomes for patients.

The P2RX7B splice variant modulates osteosarcoma cell behaviour and metastatic properties.

Background: Osteosarcoma (OS) is the most common type of primary bone cancer affecting children and adolescents. OS has a high propensity to spread meaning the disease is often incurable and fatal. There have been no improvements in survival rates for decades. This highlights an urgent need for the development of novel therapeutic strategies. Here, we report in vitro and in vivo data that demonstrates the role of purinergic signalling, specifically, the B isoform of the purinergic receptor P2RX7 (P2RX7B), in OS progression and metastasis. Methods: TE85 and MNNG-HOS OS cells were transfected with P2RX7B. These cell lines were then characterised and assessed for proliferation, cell adhesion, migration and invasion in vitro. We used these cells to perform both paratibial and tail vein injected mouse studies where the primary tumour, bone and lungs were analysed. We used RNA-seq to identify responsive pathways relating to P2RX7B. Results: Our data shows that P2RX7B expression confers a survival advantage in TE85 + P2RX7B and MNNG-HOS + P2RX7B human OS cell lines in vitro that is minimised following treatment with A740003, a specific P2RX7 antagonist. P2RX7B expression reduced cell adhesion and P2RX7B activation promoted invasion and migration in vitro, demonstrating a metastatic phenotype. Using an in vivo OS xenograft model, MNNG-HOS + P2RX7B tumours exhibited cancer-associated ectopic bone formation that was abrogated with A740003 treatment. A pro-metastatic phenotype was further demonstrated in vivo as expression of P2RX7B in primary tumour cells increased the propensity of tumour cells to metastasise to the lungs. RNA-seq identified a novel gene axis, FN1/LOX/PDGFB/IGFBP3/BMP4, downregulated in response to A740003 treatment. Conclusion: Our data illustrates a role for P2RX7B in OS tumour growth, progression and metastasis. We show that P2RX7B is a future therapeutic target in human OS.

Multiple myeloma-A painful disease of the bone marrow.

Multiple myeloma is a bone marrow neoplasia with an incidence of 6/100,000/year in Europe. While the disease remains incurable, the development of novel treatments such as autologous stem cell transplantation, proteasome inhibitors and monoclonal antibodies has led to an increasing subset of patients living with long-term myeloma. However, more than two thirds of patients suffer from bone pain, often described as severe, and knowledge on the pain mechanisms and its effect on their health-related quality of life (HRQoL) is limited. In this review, we discuss the mechanisms of myeloma bone disease, the currently available anti-myeloma treatments and the lessons learnt from clinical studies regarding HRQoL in myeloma patients. Moreover, we discuss the mechanisms of cancer-induced bone pain and the knowledge that animal models of myeloma-induced bone pain can provide to identify novel analgesic targets. To date, information regarding bone pain and HRQoL in myeloma patients is still scarce and an effort should be made to use standardised questionnaires to assess patient-reported outcomes that allow inter-study comparisons of the available clinical data.

The E3 ligase HUWE1 inhibition as a therapeutic strategy to target MYC in multiple myeloma.

Proteasome inhibitors have provided a significant advance in the treatment of multiple myeloma (MM). Consequently, there is increasing interest in developing strategies to target E3 ligases, de-ubiquitinases, and/or ubiquitin receptors within the ubiquitin proteasome pathway, with an aim to achieve more specificity and reduced side-effects. Previous studies have shown a role for the E3 ligase HUWE1 in modulating c-MYC, an oncogene frequently dysregulated in MM. Here we investigated HUWE1 in MM. We identified elevated expression of HUWE1 in MM compared with normal cells. Small molecule-mediated inhibition of HUWE1 resulted in growth arrest of MM cell lines without significantly effecting the growth of normal bone marrow cells, suggesting a favorable therapeutic index. Studies using a HUWE1 knockdown model showed similar growth inhibition. HUWE1 expression positively correlated with MYC expression in MM bone marrow cells and correspondingly, genetic knockdown and biochemical inhibition of HUWE1 reduced MYC expression in MM cell lines. Proteomic identification of HUWE1 substrates revealed a strong association of HUWE1 with metabolic processes in MM cells. Intracellular glutamine levels are decreased in the absence of HUWE1 and may contribute to MYC degradation. Finally, HUWE1 depletion in combination with lenalidomide resulted in synergistic anti-MM activity in both in vitro and in vivo models. Taken together, our data demonstrate an important role of HUWE1 in MM cell growth and provides preclinical rationale for therapeutic strategies targeting HUWE1 in MM.

JZL184, A Monoacylglycerol Lipase Inhibitor, Induces Bone Loss in a Multiple Myeloma Model of Immunocompetent Mice.

Multiple myeloma (MM) patients develop osteolysis characterised by excessive osteoclastic bone destruction and lack of osteoblast bone formation. Pharmacological manipulation of monoacylglycerol lipase (MAGL), an enzyme responsible for the degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced skeletal tumour burden and osteolysis associated with osteosarcoma and advanced breast and prostate cancers. MM and hematopoietic, immune and bone marrow cells express high levels of type 2 cannabinoid receptor and osteoblasts secrete 2-AG. However, the effects of MAGL manipulation on MM have not been investigated. Here, we report that treatment of pre-osteoclasts with non-cytotoxic concentrations of JZL184, a verified MAGL inhibitor, enhanced MM- and RANKL-induced osteoclast formation and size in vitro. Exposure of osteoblasts to JZL184 in the presence of MM cell-derived factors reduced osteoblast growth but had no effect on the ability of these cells to mature or form bone nodules. In vivo, administration of JZL184 induced a modest, yet significant, bone loss at both trabecular and cortical compartments of long bones of immunocompetent mice inoculated with the syngeneic 5TGM1-GFP MM cells. Notably, JZL184 failed to inhibit the in vitro growth of a panel of mouse and human MM cell lines, or reduce tumour burden in mice. Thus, MAGL inhibitors such as JZL184 can exacerbate MM-induced bone loss.

Differential Pain-Related Behaviors and Bone Disease in Immunocompetent Mouse Models of Myeloma.

Bone pain is a serious and debilitating symptom of multiple myeloma (MM) that impairs the quality of life of patients. The underlying mechanisms of the pain are unknown and understudied, and there is a need for immunocompetent preclinical models of myeloma-induced bone pain. The aim of this study was to provide the first in-depth behavioral characterization of an immunocompetent mouse model of MM presenting the clinical disease features: osteolytic bone disease and bone pain. We hypothesized that a widely used syngeneic model of MM, established by systemic inoculation of green fluorescent protein-tagged myeloma cells (5TGM1-GFP) in immunocompetent C57Bl/KaLwRijHsd (BKAL) mice, would present pain-related behaviors. Disease phenotype was confirmed by splenomegaly, high serum paraprotein, and tumor infiltration in the bone marrow of the hind limbs; however, myeloma-bearing mice did not present pain-related behaviors or substantial bone disease. Thus, we investigated an alternative model in which 5TGM1-GFP cells were directly inoculated into the intrafemoral medullary cavity. This localized myeloma model presented the hallmarks of the disease, including high serum paraprotein, tumor growth, and osteolytic bone lesions. Compared with control mice, myeloma-bearing mice presented myeloma-induced pain-related behaviors, a phenotype that was reversed by systemic morphine treatment. Micro-computed tomography analyses of the myeloma-inoculated femurs showed bone disease in cortical and trabecular bone. Repeated systemic bisphosphonate treatment induced an amelioration of the nociceptive phenotype, but did not completely reverse it. Furthermore, intrafemorally injected mice presented a profound denervation of the myeloma-bearing bones, a previously unknown feature of the disease. This study reports the intrafemoral inoculation of 5TGM1-GFP cells as a robust immunocompetent model of myeloma-induced bone pain, with consistent bone loss. Moreover, the data suggest that myeloma-induced bone pain is caused by a combinatorial mechanism including osteolysis and bone marrow denervation. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

TGFß Inhibition Stimulates Collagen Maturation to Enhance Bone Repair and Fracture Resistance in a Murine Myeloma Model.

Multiple myeloma is a plasma cell malignancy that causes debilitating bone disease and fractures, in which TGFß plays a central role. Current treatments do not repair existing damage and fractures remain a common occurrence. We developed a novel low tumor phase murine model mimicking the plateau phase in patients as we hypothesized this would be an ideal time to treat with a bone anabolic. Using in vivo µCT we show substantial and rapid bone lesion repair (and prevention) driven by SD-208 (TGFß receptor I kinase inhibitor) and chemotherapy (bortezomib and lenalidomide) in mice with human U266-GFP-luc myeloma. We discovered that lesion repair occurred via an intramembranous fracture repair-like mechanism and that SD-208 enhanced collagen matrix maturation to significantly improve fracture resistance. Lesion healing was associated with VEGFA expression in woven bone, reduced osteocyte-derived PTHrP, increased osteoblasts, decreased osteoclasts, and lower serum tartrate-resistant acid phosphatase 5b (TRACP-5b). SD-208 also completely prevented bone lesion development in mice with aggressive JJN3 tumors, and was more effective than an anti-TGFß neutralizing antibody (1D11). We also discovered that SD-208 promoted osteoblastic differentiation (and overcame the TGFß-induced block in osteoblastogenesis) in myeloma patient bone marrow stromal cells in vitro, comparable to normal donors. The improved bone quality and fracture-resistance with SD-208 provides incentive for clinical translation to improve myeloma patient quality of life by reducing fracture risk and fatality. © 2019 American Society for Bone and Mineral Research.

Targeted magnetic nanoparticle hyperthermia for the treatment of oral cancer.

INTRODUCTION: Patients with oral squamous cell carcinoma currently experience a five-year survival rate of approximately 60% with conventional surgical, chemotherapy and radiotherapy treatments. Magnetic hyperthermia offers an alternative treatment method by utilising the heating properties of magnetic nanoparticles to produce thermal ablation of the tumour site when exposed to an alternating magnetic field. In this study, we investigate in vitro if targeted magnetic hyperthermia offers a potential treatment for oral squamous cell carcinoma. MATERIALS AND METHODS: Magnetic iron oxide nanoparticles, with a biocompatible silica coating, were produced and conjugated with antibodies to target integrin αvß6, a well-characterised oral squamous cell carcinoma biomarker. Utilising the heating properties of the magnetic nanoparticles, we exposed them to an alternating magnetic field to produce thermo ablation of tumour cells either negative for or overexpressing integrin αvß6. RESULTS: The cell surface biomarker, αvß6 integrin, was upregulated in tissue biopsies from oral squamous cell carcinoma patients compared to normal tissue. Functionalisation of the silica coating with anti-αvß6 antibodies enabled direct targeting of the nanoparticles to αvß6 overexpressing cells and applying thermal therapy significantly increased killing of the targeted tumour cells compared to control cells. CONCLUSION: Combining antibody-targeting magnetic nanoparticles with thermal ablation offers a promising therapy for the targeted treatment of oral squamous cell carcinoma.

Preventing and Repairing Myeloma Bone Disease by Combining Conventional Antiresorptive Treatment With a Bone Anabolic Agent in Murine Models.

Multiple myeloma is a plasma cell malignancy, which develops in the bone marrow and frequently leads to severe bone destruction. Current antiresorptive therapies to treat the bone disease do little to repair damaged bone; therefore, new treatment strategies incorporating bone anabolic therapies are urgently required. We hypothesized that combination therapy using the standard of care antiresorptive zoledronic acid (Zol) with a bone anabolic (anti-TGFß/1D11) would be more effective at treating myeloma-induced bone disease than Zol therapy alone. JJN3 myeloma-bearing mice (n = 8/group) treated with combined Zol and 1D11 resulted in a 48% increase (p ≤ 0.001) in trabecular bone volume (BV/TV) compared with Zol alone and a 65% increase (p ≤ 0.0001) compared with 1D11 alone. Our most significant finding was the substantial repair of U266-induced osteolytic bone lesions with combination therapy (n = 8/group), which resulted in a significant reduction in lesion area compared with vehicle (p ≤ 0.01) or Zol alone (p ≤ 0.01). These results demonstrate that combined antiresorptive and bone anabolic therapy is significantly more effective at preventing myeloma-induced bone disease than Zol alone. Furthermore, we demonstrate that combined therapy is able to repair established myelomatous bone lesions. This is a highly translational strategy that could significantly improve bone outcomes and quality of life for patients with myeloma. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

ARQ-197, a small-molecule inhibitor of c-Met, reduces tumour burden and prevents myeloma-induced bone disease in vivo.

The receptor tyrosine kinase c-Met, its ligand HGF, and components of the downstream signalling pathway, have all been implicated in the pathogenesis of myeloma, both as modulators of plasma cell proliferation and as agents driving osteoclast differentiation and osteoblast inhibition thus, all these contribute substantially to the bone destruction typically caused by myeloma. Patients with elevated levels of HGF have a poor prognosis, therefore, targeting these entities in such patients may be of substantial benefit. We hypothesized that ARQ-197 (Tivantinib), a small molecule c-Met inhibitor, would reduce myeloma cell growth and prevent myeloma-associated bone disease in a murine model. In vitro we assessed the effects of ARQ-197 on myeloma cell proliferation, cytotoxicity and c-Met protein expression in human myeloma cell lines. In vivo we injected NOD/SCID-γ mice with PBS (non-tumour bearing) or JJN3 cells and treated them with either ARQ-197 or vehicle. In vitro exposure of JJN3, U266 or NCI-H929 cells to ARQ-197 resulted in a significant inhibition of cell proliferation and an induction of cell death by necrosis, probably caused by significantly reduced levels of phosphorylated c-Met. In vivo ARQ-197 treatment of JJN3 tumour-bearing mice resulted in a significant reduction in tumour burden, tumour cell proliferation, bone lesion number, trabecular bone loss and prevented significant decreases in the bone formation rate on the cortico-endosteal bone surface compared to the vehicle group. However, no significant differences on bone parameters were observed in non-tumour mice treated with ARQ-197 compared to vehicle, implying that in tumour-bearing mice the effects of ARQ-197 on bone cells was indirect. In summary, these res ults suggest that ARQ-197 could be a promising therapeutic in myeloma patients, leading to both a reduction in tumour burden and an inhibition of myeloma-induced bone disease.

New agents in the Treatment of Myeloma Bone Disease.

Patients with multiple myeloma develop a devastating bone disease driven by the uncoupling of bone remodelling, excess osteoclastic bone resorption and diminished osteoblastic bone formation. The bone phenotype is typified by focal osteolytic lesions leading to pathological fractures, hypercalcaemia and other catastrophic bone events such as spinal cord compression. This causes bone pain, impaired functional status, decreased quality of life and increased mortality. Early in the disease, malignant plasma cells occupy a niche environment that encompasses their interaction with other key cellular components of the bone marrow microenvironment. Through these interactions, osteoclast-activating factors and osteoblast inhibitory factors are produced, which together uncouple the dynamic process of bone remodelling, leading to net bone loss and focal osteolytic lesions. Current management includes antiresorptive therapies, i.e. bisphosphonates, palliative support and orthopaedic interventions. Bisphosphonates are the mainstay of treatment for myeloma bone disease (MBD), but are only partially effective and do have some significant disadvantages; for example, they do not lead to the repair of existing bone destruction. Thus, newer agents to prevent bone destruction and also promote bone formation and repair existing lesions are warranted. This review summarises novel ways that MBD is being therapeutically targeted.

The Pharmacological Profile of a Novel Highly Potent Bisphosphonate, OX14 (1-Fluoro-2-(Imidazo-[1,2-α]Pyridin-3-yl)-Ethyl-Bisphosphonate).

Bisphosphonates are widely used in the treatment of clinical disorders characterized by increased bone resorption, including osteoporosis, Paget's disease, and the skeletal complications of malignancy. The antiresorptive potency of the nitrogen-containing bisphosphonates on bone in vivo is now recognized to depend upon two key properties, namely mineral binding affinity and inhibitory activity on farnesyl pyrophosphate synthase (FPPS), and these properties vary independently of each other in individual bisphosphonates. The better understanding of structure activity relationships among the bisphosphonates has enabled us to design a series of novel bisphosphonates with a range of mineral binding properties and antiresorptive potencies. Among these is a highly potent bisphosphonate, 1-fluoro-2-(imidazo-[1,2 alpha]pyridin-3-yl)-ethyl-bisphosphonate, also known as OX14, which is a strong inhibitor of FPPS, but has lower binding affinity for bone mineral than most of the commonly studied bisphosphonates. The aim of this work was to characterize OX14 pharmacologically in relation to several of the bisphosphonates currently used clinically. When OX14 was compared to zoledronate (ZOL), risedronate (RIS), and minodronate (MIN), it was as potent at inhibiting FPPS in vitro but had significantly lower binding affinity to hydroxyapatite (HAP) columns than ALN, ZOL, RIS, and MIN. When injected i.v. into growing Sprague Dawley rats, OX14 was excreted into the urine to a greater extent than the other bisphosphonates, indicating reduced short-term skeletal uptake and retention. In studies in both Sprague Dawley rats and C57BL/6J mice, OX14 inhibited bone resorption, with an antiresorptive potency equivalent to or greater than the comparator bisphosphonates. In the JJN3-NSG murine model of myeloma-induced bone disease, OX14 significantly prevented the formation of osteolytic lesions (p < 0.05). In summary, OX14 is a new, highly potent bisphosphonate with lower bone binding affinity than other clinically relevant bisphosphonates. This renders OX14 an interesting potential candidate for further development for its potential skeletal and nonskeletal benefits. © 2017 American Society for Bone and Mineral Research.

Osteoclasts control reactivation of dormant myeloma cells by remodelling the endosteal niche.

Multiple myeloma is largely incurable, despite development of therapies that target myeloma cell-intrinsic pathways. Disease relapse is thought to originate from dormant myeloma cells, localized in specialized niches, which resist therapy and repopulate the tumour. However, little is known about the niche, and how it exerts cell-extrinsic control over myeloma cell dormancy and reactivation. In this study, we track individual myeloma cells by intravital imaging as they colonize the endosteal niche, enter a dormant state and subsequently become activated to form colonies. We demonstrate that dormancy is a reversible state that is switched 'on' by engagement with bone-lining cells or osteoblasts, and switched 'off' by osteoclasts remodelling the endosteal niche. Dormant myeloma cells are resistant to chemotherapy that targets dividing cells. The demonstration that the endosteal niche is pivotal in controlling myeloma cell dormancy highlights the potential for targeting cell-extrinsic mechanisms to overcome cell-intrinsic drug resistance and prevent disease relapse.

NOD/SCID-GAMMA mice are an ideal strain to assess the efficacy of therapeutic agents used in the treatment of myeloma bone disease.

Animal models of multiple myeloma vary in terms of consistency of onset, degree of tumour burden and degree of myeloma bone disease. Here we describe five pre-clinical models of myeloma in NOD/SCID-GAMMA mice to specifically study the effects of therapeutic agents on myeloma bone disease. Groups of 7-8 week old female irradiated NOD/SCID-GAMMA mice were injected intravenously via the tail vein with either 1x106 JJN3, U266, XG-1 or OPM-2 human myeloma cell lines or patient-derived myeloma cells. At the first signs of morbidity in each tumour group all animals were sacrificed. Tumour load was measured by histological analysis, and bone disease was assessed by micro-CT and standard histomorphometric methods. Mice injected with JJN3, U266 or OPM-2 cells showed high tumour bone marrow infiltration of the long bones with low variability, resulting in osteolytic lesions. In contrast, mice injected with XG-1 or patient-derived myeloma cells showed lower tumour bone marrow infiltration and less bone disease with high variability. Injection of JJN3 cells into NOD/SCID-GAMMA mice resulted in an aggressive, short-term model of myeloma with mice exhibiting signs of morbidity 3 weeks later. Treating these mice with zoledronic acid at the time of tumour cell injection or once tumour was established prevented JJN3-induced bone disease but did not reduce tumour burden, whereas, carfilzomib treatment given once tumour was established significantly reduced tumour burden. Injection of U266, XG-1, OPM-2 and patient-derived myeloma cells resulted in less aggressive longer-term models of myeloma with mice exhibiting signs of morbidity 8 weeks later. Treating U266-induced disease with zoledronic acid prevented the formation of osteolytic lesions and trabecular bone loss as well as reducing tumour burden whereas, carfilzomib treatment only reduced tumour burden. In summary, JJN3, U266 or OPM-2 cells injected into NOD/SCID-GAMMA mice provide robust models to study anti-myeloma therapies, particularly those targeting myeloma bone disease.

Myeloma bone disease: pathogenesis, current treatments and future targets.

INTRODUCTION: Patients with myeloma develop localized and generalized bone loss leading to hypercalcaemia, accelerated osteoporosis, vertebral wedge fractures, other pathological fractures, spinal cord compression and bone pain. Bone loss is mediated by a variety of biological modifiers including osteoclast-activating factors (OAF) and osteoblast (OB) inhibitory factors produced either directly by malignant plasma cells (MPCs) or as a consequence of their interaction with the bone marrow microenvironment (BMM). Raised levels of OAFs such as receptor activator of nuclear factor-kappa B ligand (RANKL), macrophage inflammatory protein 1 alpha, tumour necrosis factor-alpha and interleukin 6 stimulate bone resorption by recruiting additional osteoclasts. Via opposing mechanisms, increases in OB inhibitory factors, such as dickkopf-1 (Dkk-1), soluble frizzled-related protein-3 and hepatocyte growth factor (HGF), suppress bone formation by inhibiting the differentiation and recruitment of OBs. These changes result in an uncoupling of physiological bone remodelling, leading to myeloma bone disease (MBD). Moreover, the altered BMM provides a fertile ground for the growth and survival of MPCs. Current clinical management of MBD is both reactive (to pain and fractures) and preventive, with bisphosphonates (BPs) being the mainstay of pharmacological treatment. However, side effects and uncertainties associated with BPs warrant the search for more targeted treatments for MBD. This review will summarize recent developments in understanding the intimate relationship between MBD and the BMM and the novel ways in which they are being therapeutically targeted. SOURCES OF DATA: All data included were sourced and referenced from PubMed. AREAS OF AGREEMENT: The clinical utility of BP therapy is well established. However, there is general acknowledgement that BPs are only partially successful in the treatment of MBD. The number of skeletal events attributable to myeloma are reduced by BPs but not totally eliminated. Furthermore, existing damage is not repaired. It is widely recognized that more effective treatments are needed. AREAS OF CONTROVERSY: There remains controversy concerning the duration of BP therapy. Whether denosumab is a viable alternative to BP therapy is also contested. Many of the new therapeutic strategies discussed are yet to translate to clinical practice and demonstrate equal efficacy or superiority to BP therapy. It also remains controversial whether reported anti-tumour effects of bone-modulating therapies are clinically significant. GROWING POINTS: The potential clinical utility of bone anabolic therapies including agents such as anti-Dkk-1, anti-sclerostin and anti-HGF is becoming increasingly recognized. AREAS TIMELY FOR DEVELOPING RESEARCH: Further research effectively targeting the mediators of MBD, targeting both bone resorption and bone formation, is urgently needed. This should translate promptly to clinical trials of combination therapy comprising anti-resorptives and bone anabolic therapies to demonstrate efficacy and improved outcomes over BPs.