Runx2 Deficiency in Osteoblasts Promotes Myeloma Resistance to Bortezomib By Increasing TSP-1-Dependent TGF-β1 Activation in Bone Marrow

Multiple myeloma (MM) is a plasma cell malignancy that thrives in the bone marrow (BM). The proteasome inhibitor bortezomib (BTZ) is one of the most effective front-line chemotherapeutic drugs for MM; however, 15-20% of high-risk patients do not respond to or become resistant to this drug and the mechanisms of chemoresistance remain unclear. We previously demonstrated that MM cells inhibit Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OBs). In the current study, we investigated the impact of OB-Runx2 deficiency on the outcome of BTZ treatment using our syngeneic mouse model of MM in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2 -/- mice) and OB-Runx2 +/+ mice as control. Five-week-old OB-Runx2 +/+ mice and OB-Runx2 -/- mice were i.v. injected with 5TGM1-Luc murine MM cells (2×10 6). On day 8, after the tumor injection, mice were randomly assigned to treatment for 4 weeks, with either BTZ (i.p. injection, 0.5 mg/kg body weight, twice/week) or PBS. Bioluminescence imaging and serum IgG2bκ (a soluble marker of 5TGM1 MM cells) ELISA showed that BTZ significantly inhibited tumor growth in OB-Runx2 +/+ mice, but not in OB-Runx2 -/- mice. Cytokine array and ELISA showed that in PBS-treated, tumor-bearing OB-Runx2 -/- mice, BM levels of thrombospondin-1 (TSP-1), a matricellular protein that converts latent TGF-β1 to its active form, and active TGF-β1 were significantly higher than levels in tumor-bearing OB-Runx2 +/+ counterparts. Interestingly, BTZ treatment further increased the levels of both TSP-1 and active TGF-β1 in the BM of tumor-bearing OB-Runx2 -/- mice, but it did not affect the level of either TSP-1 or TGF-β1 in the BM of tumor-bearing OB-Runx2 +/+ mice. These results suggest that OB-Runx2 deficiency increases TGF-β1 activation via TSP-1 in BM and BTZ treatment further enhances this effect. SRI31277 is a tripeptide antagonist that blocks TSP-1-mediated activation of TGF-β1 and it has been shown to reduce tumor burden, TGF-β signaling, and osteolytic bone disease in multiple mouse models of MM. To confirm the involvement of TSP-1/TGF-β1 activation in MM BTZ resistance induced by OB-Runx2 deficiency and to test whether blocking TSP1-mediated TGF-β1 activation can alleviate BTZ resistance, 5TGM1-Luc MM tumor-bearing OB-Runx2 +/+ and OB-Runx2 -/- mice were treated with PBS, BTZ, SRI31277 (osmotic pump, 30 mg/kg body weight per day), or BTZ + SRI31277 for 4 weeks. While SRI31277 did not further increase BTZ-induced MM inhibition in OB-Runx2 +/+ mice, SRI31277 treatment significantly reduced the tumor burden in OB-Runx2 -/- mice. Furthermore, treatment with BTZ + SRI31277 augmented the reduction in tumor burden induced by SRI31277 treatment alone. Flow cytometry and Western blot analyses demonstrated that SRI31277 treatment enhanced apoptosis, reduced Ki-67 expression as well as the activity of both canonical (SMAD2/3) and non-canonical (ERK1/2) signaling pathways of TGF-β1 in MM cells in OB-Runx2 -/- mice; and BTZ+SRI31277 treatment augmented these effects. Furthermore, TGF-β has immunosuppressive effects in MM and our data show that SRI31277 overcomes this by reducing myeloid derived suppressor cells, checkpoint, and T cell exhaustion markers and by increasing cytotoxic T cells. In conclusion, OB-Runx2 deficiency, induced by MM cells, promotes MM resistance to BTZ through the upregulation of TGF-β1 activation in the BM and TGF-β1 signaling in MM cells. Importantly, blocking TSP-1-mediated TGF-β1 activation with SRI31277 can reverse this resistance and immune dysfunction. Our findings demonstrate a novel mechanism for BTZ resistance in MM and identify a new target and strategy for overcoming immune dysregulation and chemoresistance in MM. Disclosures Murphy-Ullrich: Millipore Sigma: Patents & Royalties.

Increased circulating LDL cholesterol increases myeloma tumour burden in vivo

Gámez B., Morris EV., Olechnowicz S., Sowman, A., Turner, C. and Edwards CM. Multiple myeloma (MM) is a fatal malignancy characterized by an expansion of malignant plasma cells in the bone marrow (BM) and associated with osteolytic bone disease. MM is preceded by the benign condition, monoclonal gammopathy of undetermined significance (MGUS). Understanding MGUS progression and development of MM bone disease is key for patient management. We and others have previously demonstrated that diet-induced obesity promotes myeloma progression, but the mechanisms underlying this remain unknown. The aim of the current study was to determine the effect of dietary cholesterol on MM development. A 2% cholesterol diet was used to increase circulating LDL in mice. Mice were randomly distributed to either a) cholesterol diet 4 weeks prior to 5TGM1 MM inoculation (pretreatment) or b) cholesterol diet 4 weeks prior to MM inoculation and continued for the entire experiment (continuous). Mice on the continuous cholesterol diet had increased tumour burden, associated with an increase in lipid droplet content of MM cells. No differences in tumour burden were seen in those mice where cholesterol diet was halted at time of MM inoculation. In vitro, myeloma cells cultured with delipidated FBS had a 50% reduction in viability after 72 hours. Rich cholesterol content lipoproteins (LDL) but not VLDL could restore MM cell viability, suggesting that cholesterol is responsible for this lipid-depletion effect. Taken together, our results show that high cholesterol promotes myeloma and results in a higher lipid content in myeloma cells, ultimately increasing BM tumour burden. Pretreatment with a cholesterol diet did not alter disease progression suggesting a direct pro-tumourigenic effect of cholesterol. These results demonstrate both the detrimental effect of cholesterol on myeloma progression and the potential for dietary intervention approaches.

Increased circulating LDL cholesterol increases myeloma tumour burden in vivo

Gámez B., Morris EV., Olechnowicz S., Sowman, A., Turner, C. and Edwards CM. Multiple myeloma (MM) is a fatal malignancy characterized by an expansion of malignantplasma cells in the bone marrow (BM) and associated with osteolytic bone disease. MM ispreceded by the benign condition, monoclonal gammopathy of undetermined significance(MGUS). Understanding MGUS progression and development of MM bone disease is key forpatient management. We and others have previously demonstrated that diet-induced obesitypromotes myeloma progression, but the mechanisms underlying this remain unknown. The aimof the current study was to determine the effect of dietary cholesterol on MM development. A2% cholesterol diet was used to increase circulating LDL in mice. Mice were randomlydistributed to either a) cholesterol diet 4 weeks prior to 5TGM1 MM inoculation (pretreatment)or b) cholesterol diet 4 weeks prior to MM inoculation and continued for the entire experiment(continuous). Mice on the continuous cholesterol diet had increased tumour burden, associatedwith an increase in lipid droplet content of MM cells. No differences in tumour burden wereseen in those mice where cholesterol diet was halted at time of MM inoculation. In vitro,myeloma cells cultured with delipidated FBS had a 50% reduction in viability after 72 hours. Richcholesterol content lipoproteins (LDL) but not VLDL could restore MM cell viability, suggestingthat cholesterol is responsible for this lipid-depletion effect. Taken together, our results showthat high cholesterol promotes myeloma and results in a higher lipid content in myeloma cells,ultimately increasing BM tumour burden. Pretreatment with a cholesterol diet did not alterdisease progression suggesting a direct pro-tumourigenic effect of cholesterol. These resultsdemonstrate both the detrimental effect of cholesterol on myeloma progression and thepotential for dietary intervention approaches.

A Molecular Signature of Circulating MicroRNA Can Predict Osteolytic Bone Disease in Multiple Myeloma

Background: Multiple myeloma bone disease (MMBD) constitutes a common and severe complication of multiple myeloma (MM), impacting the quality of life and survival. We evaluated the clinical value of a panel of 19 miRNAs associated with osteoporosis in MMBD. Methods: miRNAs were isolated from the plasma of 62 newly diagnosed MM patients with or without MMBD. First-strand cDNA was synthesized, and relative quantification was performed using qPCR. Lastly, we carried out extensive biostatistical analysis. Results: Circulating levels of let-7b-5p, miR-143-3p, miR-17-5p, miR-214-3p, and miR-335-5p were significantly higher in the blood plasma of MM patients with MMBD compared to those without. Receiver operating characteristic curve and logistic regression analyses showed that these miRNAs could accurately predict MMBD. Furthermore, a standalone multi-miRNA–based logistic regression model exhibited the best predictive potential regarding MMBD. Two of those miRNAs also have a prognostic role in MM since survival analysis indicated that lower circulating levels of both let-7b-5p and miR-335-5p were associated with significantly worse progression-free survival, independently of the established prognostic factors. Conclusions: Our study proposes a miRNA signature to facilitate MMBD diagnosis, especially in ambiguous cases. Moreover, we provide evidence of the prognostic role of let-7b-5p and miR-335-5p as non-invasive prognostic biomarkers in MM.

Additive Benefits of Radium-223 Dichloride and Bortezomib Combination in a Systemic Multiple Myeloma Mouse Model

Osteolytic bone disease is a hallmark of multiple myeloma (MM) mediated by MM cell proliferation, increased osteoclast activity, and suppressed osteoblast function. The proteasome inhibitor bortezomib targets MM cells and improves bone health in MM patients. Radium-223 dichloride (radium-223), the first targeted alpha therapy approved, specifically targets bone metastases, where it disrupts the activity of both tumor cells and tumor-supporting bone cells in mouse models of breast and prostate cancer bone metastasis. We hypothesized that radium-223 and bortezomib combination treatment would have additive effects on MM. In vitro experiments revealed that the combination treatment inhibited MM cell proliferation and demonstrated additive efficacy. In the systemic, syngeneic 5TGM1 mouse MM model, both bortezomib and radium-223 decreased the osteolytic lesion area, and their combination was more effective than either monotherapy alone. Bortezomib decreased the number of osteoclasts at the tumor–bone interface, and the combination therapy resulted in almost complete eradication of osteoclasts. Furthermore, the combination therapy improved the incorporation of radium-223 into MM-bearing bone. Importantly, the combination therapy decreased tumor burden and restored body weights in MM mice. These results suggest that the combination of radium-223 with bortezomib could constitute a novel, effective therapy for MM and, in particular, myeloma bone disease.

Gamabufotalin Inhibits Osteoclastgenesis and Counteracts Estrogen-Deficient Bone Loss in Mice by Suppressing RANKL-Induced NF-κB and ERK/MAPK Pathways

Osteolytic bone disease is a condition of imbalanced bone homeostasis, characterized mainly by excessive bone-resorptive activity, which could predispose these populations, such as the old and postmenopausal women, to developing high risk of skeletal fragility and fracture. The nature of bone homeostasis is the coordination between the osteoblasts (OBs) and osteoclasts (OCs). Abnormal activation of osteoclasts (OCs) could compromise the bone homeostasis, constantly followed by a clutch of osteolytic diseases, including postmenopausal osteoporosis, osteoarthritis, and rheumatoid arthritis. Thus, it is imperatively urgent to explore effective medical interventions for patients. The traditional Chinese medicine (TCM) gamabufotalin (CS-6) is a newly identified natural product from Chansu and has been utilized for oncologic therapies owing to its good clinical efficacy with less adverse events. Previous study suggested that CS-6 could be a novel anti-osteoporotic agent. Nevertheless, whether CS-6 suppresses RANK-(receptor activator of nuclear factor-κ B ligand)/TRAF6 (TNF receptor-associated factor 6)-mediated downstream signaling activation in OCs, as well as the effects of CS-6 on OC differentiation in vivo, remains elusive. Therefore, in this present study, we aimed to explore the biological effects of CS-6 on osteoclastogenesis and RANKL-induced activation of related signaling pathways, and further to examine the potential therapeutic application in estrogen-deficient bone loss in the mice model. The results of in vitro experiment showed that CS-6 can inhibit RANKL-induced OC formation and the ability of bone resorption in a dose-dependent manner at both the early and late stages of osteoclastogenesis. The gene expression of OC-related key genes such as tartrate-resistant acid phosphatase (TRAP), CTSK, DC-STAMP, MMP9, and β3 integrin was evidently reduced. In addition, CS-6 could mitigate the systemic estrogen-dependent bone loss and pro-inframammary cytokines in mice in vivo. The molecular mechanism analysis suggested that CS-6 can suppress RANKL/TRAF6-induced early activation of NF-κB and ERK/MAPK signaling pathways, which consequently suppressed the transcription activity of c-Fos and NFATc1. Taken together, this present study provided ample evidence that CS-6 has the promise to become a therapeutic candidate in treating osteolytic conditions mediated by elevated OC formation and bone resorption.

Carfilzomib Improves Bone Metabolism in Patients with Advanced Relapsed/Refractory Multiple Myeloma: Results of the CarMMa Study

Carfilzomib with dexamethasone (Kd) is a well-established regimen for the treatment of relapsed/refractory multiple myeloma (RRMM). There is limited information for the effects of Kd on myeloma-related bone disease. This non-interventional study aimed to assess skeletal-related events (SREs) and bone metabolism in patients with RRMM receiving Kd, in the absence of any bone-targeted agent. Twenty-five patients were enrolled with a median of three prior lines of therapy; 72% of them had evidence of osteolytic bone disease at study entry. During Kd treatment, the rate of new SREs was 28%. Kd produced a clinically relevant (≥30%) decrease in C-telopeptide of collagen type-1 (p = 0.048) and of tartrate-resistant acid phosphatase-5b (p = 0.002) at 2 months. This reduction was at least partially due to the reduction in the osteoclast regulator RANKL/osteoprotegerin ratio, at 2 months (p = 0.026). Regarding bone formation, there was a clinically relevant increase in osteocalcin at 6 months (p = 0.03) and in procollagen type I N-propeptide at 8 months post-Kd initiation. Importantly, these bone metabolism changes were independent of myeloma response to treatment. In conclusion, Kd resulted in a low rate of SREs among RRMM patients, along with an early, sustained and clinically relevant decrease in bone resorption, which was accompanied by an increase in bone formation, independently of myeloma response and in the absence of any bone-targeted agent use.

Monoclonal immunoglobulins promote bone loss in multiple myeloma

Most patients with multiple myeloma develop a severe osteolytic bone disease. The myeloma cells secrete immunoglobulins, and the presence of monoclonal immunoglobulins in the patient’s sera is an important diagnostic criterion. Here, we show that immunoglobulins isolated from myeloma patients with bone disease promote osteoclast differentiation when added to human preosteoclasts in vitro, whereas immunoglobulins from patients without bone disease do not. This effect was primarily mediated by immune complexes or aggregates. The function and aggregation behavior of immunoglobulins are partly determined by differential glycosylation of the immunoglobulin-Fc part. Glycosylation analyses revealed that patients with bone disease had significantly less galactose on immunoglobulin G (IgG) compared with patients without bone disease and also less sialic acid on IgG compared with healthy persons. Importantly, we also observed a significant reduction of IgG sialylation in serum of patients upon onset of bone disease. In the 5TGM1 mouse myeloma model, we found decreased numbers of lesions and decreased CTX-1 levels, a marker for osteoclast activity, in mice treated with a sialic acid precursor, N-acetylmannosamine (ManNAc). ManNAc treatment increased IgG-Fc sialylation in the mice. Our data support that deglycosylated immunoglobulins promote bone loss in multiple myeloma and that altering IgG glycosylation may be a therapeutic strategy to reduce bone loss.

An Improved Animal Model of Multiple Myeloma Bone Disease

Multiple myeloma (MM) is a plasma cell malignancy that represents an accumulation of terminally differentiated monoclonal plasma cells (PCs) in the bone marrow (BM), accompanied by increased osteoclasts and decreased osteoblasts in areas adjacent to myeloma cells, leading to MM associated bone disease (MMBD). Osteolytic bone disease is one of the defining features of MM. During the disease, over 90% of patients are developing MMBD. Many of MM animal models have been developed and enable us to interrogate the mechanisms of MM tumorigenesis. Most MMBD models were derived by intratibial injection of myeloma cells. In these models, osteolysis occurs locally at the site where myeloma cells were injected. Mouse myeloma cells, 5TGM1 transplanting C57BL/KaLwRij mouse via the tail vein develops and shows MMBD features close to human MMBD. Even in this model, the MMBD levels on each mouse are widely varied. Lack of appropriate in vivo MMBD model hampers our understanding of the disease and developing therapy. We try to establish a murine model for MMBD to study its pathophysiology and test a novel treatment. 1x106 luciferase-expressing 5TGM1 (5TGM1-Luc) cells were injected into 8-12 week old NOD SCID gamma mouse (NSG) and C57BL/KaLwRij mouse via the tail vein. Myeloma progression was weekly assessed by in vivo bioluminescence (BL) imaging using IVIS-200 (Perkin Elmer). Mice were sacrificed when they showed endpoint signals such as significant weight loss, hindlimb paralysis, etc.. At postmortem, the micro-computer tomography (micro-CT) was performed for bone histo-morphometric analyses using micro CT400, Scano medical, Inc. The median survival was 56 days in NSG, while 42 days in C57BL/KaLwRij. In vivo BL image analysis showed that myeloma slowly develops in NSG mouse in comparison to C57BL/KaLwRij mouse. Histomorphic analyses found that severe osteolytic lesions occur at the lumbar spine in NSG mouse compared to C57BL/KaLwRij mouse, but no significant difference at the femur of both strains. At the lumbar spine, trabecular thickness (p < 0.0004) and trabecular space (p < 0.0014) were significantly increased in NSG mouse compared to C57BL/KaLwRij mouse. On the contrary, trabecular number (p < 0.0002) and bone volume density (p < 0.0005) were significantly decreased in NSG mouse compared to C57BL/KaLwRij mouse. In conclusion, we found that the systemic 5TGM1 injected NSG mouse slowly progresses myeloma and develops more severe MMBD than C57BL/KaLwRij model. This model will serve a better MMBD model to evaluate the therapeutic effects of MMBD targeted drugs. Disclosures No relevant conflicts of interest to declare.

Extracellular Vesicle microRNAs Contribute to the Osteogenic Inhibition of Mesenchymal Stem Cells in Multiple Myeloma

Osteolytic bone disease is the major complication associated with the progression of multiple myeloma (MM). Recently, extracellular vesicles (EVs) have emerged as mediators of MM-associated bone disease by inhibiting the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Here, we investigated a correlation between the EV-mediated osteogenic inhibition and MM vesicle content, focusing on miRNAs. By the use of a MicroRNA Card, we identified a pool of miRNAs, highly expressed in EVs, from MM cell line (MM1.S EVs), expression of which was confirmed in EVs from bone marrow (BM) plasma of patients affected by smoldering myeloma (SMM) and MM. Notably,we found that miR-129-5p, which targets different osteoblast (OBs) differentiation markers, is enriched in MM-EVs compared to SMM-EVs, thus suggesting a selective packaging correlated with pathological grade. We found that miR-129-5p can be transported to hMSCs by MM-EVs and, by the use of miRNA mimics, we investigated its role in recipient cells. Our data demonstrated that the increase of miR-129-5p levels in hMSCs under osteoblastic differentiation stimuli inhibited the expression of the transcription factor Sp1, previously described as a positive modulator of osteoblastic differentiation, and of its target the Alkaline phosphatase (ALPL), thus identifying miR-129-5p among the players of vesicle-mediated bone disease.