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Background and aims: Erythropoietin (EPO) is a pleiotropic cytokine, which besides its classical role in driving erythropoiesis, displays tissue protective and immunomodulatory activities. EPO also induces bone loss. While hematopoiesis is mediated via the homodimeric EPO receptor (EPOR), tissue protection is conferred via a heteromer composed of EPOR and CD131. Cibinetide (CIB), a non-erythropoietic analogue of EPO, specifically binds to the heteromeric receptor and confers tissue protection. Our published findings that EPO stimulates osteoclast precursors and entrains a decrease in bone density, raise questions regarding the underlying molecular mechanisms. Here, we evaluated the role of the heteromeric complex in bone metabolism using CIB alone and in combination with EPO in vivo and in vitro. Results: CIB injections to 12-week-old female mice (120 µg/kg thrice weekly for 4 weeks) resulted in a significant increase in tissue mineral density in cortical bone by 5.8% (1416.4±39.27 vs 1338.74±16.56 mgHA/cm 3) and in trabecular bone by 5.2% (1056.52±30.94 vs 1004.13±16.91 mg HA/cm 3) (n=10 in each group, p< 0.05 versus saline-injected controls), as measured by microCT (Figure 1A). To evaluate the capacity of CIB to attenuate EPO mediated bone loss, we administered CIB (300 µg/kg) for 5 consecutive days, to 13-week-old female mice that also received 2 injections of 120U EPO on days 1 and 4. Flow cytometry analysis revealed a 1.8-fold reduction in the number of osteoclast progenitors, defined as Lin -CD11b −CD115 +Ly6C hi, in the EPO + CIB injected mice, compared to the mice injected with EPO alone (n=7 in each group, p< 0.05). Hemoglobin levels and TER119 + bone marrow (BM) erythroid progenitors were similar in both groups. In vitro, EPO administration to BM-derived macrophages (BMDM) enhanced osteoclastogenesis, whereas CIB had an opposite, dose-dependent effect. Combining CIB with EPO inhibited osteoclastogenesis in BMDM, suggesting that CIB overrides the pro-osteoclastogenic effect of EPO (Figure 1B). Conclusions: Our findings highlight the increasing complexity of EPOR signaling in bone and pave the way for clinical translation through potential combination therapy of EPO and CIB in anemic and in cancer patients. Adjunctive administration of CIB may prevent or attenuate bone loss while preserving the erythropoietic actions of EPO. This study was supported by a grant from the Dotan Hemato-oncology Fund, the Cancer Biology Research Center, Tel Aviv University to DN and YG. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background and aims: Immunotherapy with anti-CD20-specific antibodies (e.g. rituximab), has become the standard of care for B cell lymphoproliferative disorders and many autoimmune diseases. Despite previously demonstrated role for B cells in bone metabolism, the effect of anti-CD20-mediated B cell depletion on bone mass in human patients has not been thoroughly studied. For example, in rheumatological patients the effect of rituximab on bone mass yielded conflicting results, while in lymphoma patients it has not yet been described. Here, we describe the effect of treatment with anti-CD20-specific antibodies on bone mass in a cohort of patients with follicular lymphoma and propose a plausible mechanism using murine model. Methods: To assess the effect of rituximab on bone mass in lymphoma patients, we retrospectively studied the bone mass in patients with follicular lymphoma (FL) during the maintenance phase of chemoimunotherapy, i.e. when rituximab is administered as monotherapy. FL patients on no maintenance (historical controls) or patients with marginal zone lymphoma were include as a control group. Cross-sectional X-ray imaging (CT/PET-CT), performed at the completion of the induction phase and 6-12 months thereafter, were used to serially assess bone density. Wild-type female C57BL/6J mice and mouse anti-mouse CD20 antibody (Genentech) were used for the animal experimental system. Murine bone structure was assessed by the microCT method. Immunophenotyping of the bone marrow (BM), spleen and peripheral blood cells was performed. ELISA and "real-time" quantitative PCR were used to measure the levels of key mediators of bone remodeling, e.g. RANKL, OPG and TNFα. Standard osteoclastogenic assay was used to assesses the osteoclastogenic potential of BM cells. Results: Rituximab treatment prevented the decline in bone mass observed in patients who did not receive active maintenance therapy, both in the lumbar spine (-2.6% vs -8%) and femoral head (-0.5% vs -5.1%) (n=12 patients in each group, p Conclusions: While many lymphoma patients may suffer from bone loss due to advanced age and glucocorticoid administration, our data suggest additional favorable effect of anti-CD20 treatment in bone preservation. Importantly, our murine studies indicate that B cell depletion has a direct effect on bone remodeling, primarily by reducing the osteoclastogenic signals, thus potentially diminishing bone resorption. This novel unrecognized effect should be taken into consideration when maintenance treatment is considered. MM and DN contributed equally to this work. Figure Disclosures No relevant conflicts of interest to declare.

Background and aims: Erythropoietin (EPO) is the key regulator of red blood cell production, commonly used in clinical practice to treat certain forms of anemia. Our studies and those of others have demonstrated that EPO administration induces substantial trabecular bone loss. We proposed that EPO-induced bone loss is partially mediated by subsets of bone marrow (BM) B cells that express EPO-R. Mechanistically, EPO upregulates the surface expression of RANKL by BM B cells and augments B cell-derived osteoclastogenesis in vitro. We showed that the latter is likely mediated by pro-B cells expressing the MCS-F receptor (CD115) and capable of transdifferentiation to osteoclasts (Abstract # 1007, EHA 2017). Here we address the role of B cell-specific EPO-R in EPO-induced bone loss (i.e. at supra-physiological EPO levels). Moreover, we demonstrate, for the first time, the occurrence of B cell-derived osteoclastogenesis in vivo, a finding of critical importance in the field of osteohematology. Methods: In order to trace the B cell lineage from its earliest precursors, we used the MB1-Cre mouse line combined with either the R26R-EYFP or the EPO-Rfl/fl mice for lineage tracing and B cell-specific EPO-R knockdown, respectively. Sequential fluorescence and light microscopy were used for the demonstration of B cell-derived osteoclastogenesis in vivo. Human recombinant EPO was administered in vivo at a dose of 180IU thrice weekly for two weeks. Immunophenotyping of BM B cell populations was assessed by multi-color flow cytometry. Results: Using female MB1-Cre; EPO-Rfl/fl (cKD) mice, we found that B cell-specific EPO-R knockdown attenuated the profound EPO-induced trabecular bone loss in the proximal part of the femoral distal metaphysis (proximal BV/TV 0.034±0.012% vs 0.007±0.003% in the cKD vs control mice, p Using the MB1-Cre;R26R-EYFP murine model for B cell lineage tracing, we could demonstrate that some of the TRAP+/ β3 integrin+ bone lining cells were also positive for EYFP (Figure 2). This demonstrates the B cell origin of some of the osteoclasts in vivo. Conclusions: Our work highlights B cells as an important extra-erythropoietic target of EPO-EPO-R signaling that regulates bone homeostasis and might also indirectly affect EPO-stimulated erythropoietic response. The relevance and the mechanisms of the latter phenomenon merits further investigation. Importantly, we present here, for the first time, histological evidence for B cell-derived osteoclastogenesis in vivo, thus opening novel research avenues. DN and YG Equal contribution Funded by the German Israel Foundation, Grant # 01021017 to YG, DN, MR and BW and by the Israel Science Foundation (ISF) Grant No. 343/17 to DN. Disclosures Mittelman: Novartis: Honoraria, Research Funding, Speakers Bureau.

Bone turnover is regulated by the coupled actions of osteoblasts, the bone-forming cells, and monocyte-derived osteoclasts, which mediate bone resorption. B cells were also shown to regulate bone metabolism, chiefly via paracrine signals. Depending on their state and/or mode of activation, B cells may inhibit or enhance osteoclastogenesis. In mammals, B cell development and maturation occurs in the bone marrow (BM), spleen, and other peripheral lymphoid tissues. In the BM, Pro-B cells sequentially differentiate into Pre-B and immature B cells. Whether BM B cells can transdifferentiate into osteoclasts remains controversial, since osteoclast differentiation from residual monocytic precursors in the cultures was not excluded in the earlier studies. Osteoclasts and B cells arise from distinct myeloid and lymphoid progenitors, respectively, which are downstream of a common multipotent progenitor cell. Monocyte differentiation into osteoclasts relies on monocyte-macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). Here, we investigated the possibility that BM B-cells contribute to bone loss by transdifferentiating into bone-resorbing osteoclasts. We found that B220+CD19+ cells can transdifferentiate into multinucleated tartrate-resistant acid phosphatase (TRAP) positive osteoclasts in the presence of RANKL and M-CSF. Our results show that Pro-B cells (B220+CD19+CD43HighIGM-), but not Pre-B cells (B220+CD19+CD43LowIGM-), nor immature B cells (B220+CD19+CD43-IGM+), could transdifferentiate into osteoclasts (16%±3.7 vs. 0.79%±0.28 and 0.48%±0.13 osteoclasts area, respectively). Moreover, among the Pro-B cells, only those expressing M-CSF receptor (CD115) could transdifferentiate into functional osteoclasts (18%±6.55 vs. 0.11%±0.05 osteoclasts area, respectively, Figure 1A and B). To unequivocally establish the generation of osteoclasts from B-cells, we next utilized a mouse model in which all B cell lineage-derived progenies express EYFP. We found that B cells isolated from BM of CD19-Cre:EYFP mice differentiated into TRAP+ multinucleated osteoclasts that were also positive for EYFP (Figure1C). Erythropoietin (EPO) is a crucial kidney-derived hormone responsible for erythropoiesis. Once thought to act solely on the erythroid compartment to potentiate red blood cell production, it became evident that EPO receptors are also found on the monocytic lineage (monocytes, macrophages and dendritic cells). In that respect, we have reported that EPO directly stimulates bone loss via activation of EPO-R signaling in the monocytic lineage (Hiram-Bab et al., 2015). Here we report that B cells express EPO-R, and that EPO enhances Pro-B cell differentiation into osteoclasts by 70% (p Conceivably, in other scenarios, e.g., sex hormone deficiency, certain hematological cancers, and treatment with anti-CD20, increased B cell lymphocytes contribute to the bone loss phenotype due to transdifferentiation of B-cell precursors into osteoclasts. Taken together, our data suggest a new physio-pathological role for BM B cell precursors in bone metabolism, via their capacity to differentiate into functional osteoclasts, and a possible role for EPO in this process. Figure 1 Osteoclastogenesis in vitro from sorted B cells. (A) TRAP staining of osteoclasts derived from the indicated sorted cells originating from BM (10,000 cells per well) and cultured with M-CSF and RANKL. Left - Pro-B cells expressing CD115 (B220+CD19+CD43HighIgM-CD115+). Right - Pro-B cells not expressing CD115 (B220+CD19+CD43HighIgM-CD115-). Data are mean±SEM of osteoclast area, n=5 mice in each group; *p Disclosures No relevant conflicts of interest to declare.

Background and rationale: Identifying risk factors for bone loss is of utmost importance in the prevention of osteoporotic bone fractures. We and others have recently described the dramatic erythropoietin (EPO)-induced bone loss. Recombinant EPO and its long-acting derivative, darbepoetin alpha (DA), are frequently and interchangeably used in clinical practice for similar indications including diseases (e.g. multiple myeloma) in which bone mass may be compromised already, as a part of the disease process. Here, we examined in a murine model, the dose response to the erythroid stimulating agents (ESA) EPO and DA, in the erythroid and bone compartments. Methods:C57B6/JRcchsd 9-week old female mice were treated for two weeks with either EPO or DA using two dose intensities. EPO and DA were administered subcutaneously thrice or once weekly, respectively, for a total weekly dose of 27IU/mouse or 540IU/mouse, hereafter defined correspondingly as intermediate (ID) and high dose (HD) subgroups. Bone morphometry of the distal femoral metaphysis was assessed using microCT. Flow cytometry was used for immunophenotyping of bone marrow (BM) cells. One-way ANOVA was used to compare variables among the treatment groups. Results: First, we found that ID-DA induced bone loss to a similar extent as ID-EPO as reflected by a significant (p0.05 for EPO versus DA groups). In view of the established role of B cells in bone metabolism, we determined the effects of EPO and DA on BM B cell compartment. HD-EPO but not ID-EPO induced a "maturation arrest"-like state in the bone marrow B cell population, increasing the proportion of B220+/CD19-/CD43high/IgM- pre-pro-B two-fold and B220+/CD19+/CD43high/IgM- pro-B cells 2.8-fold (p Conclusion:Applying clinically comparable dosing regimen, both recombinant EPO and DA exert a similar, dose-dependent bone loss, not directly proportional to the erythroid response. However, DA, in contrast to EPO, spares the preosteoblast and bone marrow B cell populations. These finding suggest a different mechanism of action of these closely related ESA preparations on the skeletal and immune systems thus warranting further exploration. Finally, our findings advocate for initiating and maintaining patients with the lowest possible doses of EPO/DA to minimize untoward skeletal and other extra-erythropoietic effects, while achieving the desired correction of anemia. This study was supported by a grant from the Israel Cancer Association to DN, YG and MM. Disclosures No relevant conflicts of interest to declare.

The negative effect of hypoxia on bone metabolism is well established but its mechanism of action is not fully understood. Hypoxia triggers the production of erythropoietin (EPO), a hormone most recognized for its hematopoietic function. An increasing number of roles unrelated to red blood cell production have been attributed to EPO, many of which are mediated by non-erythroid cells. In light of the controversy on the effect of increased serum levels of EPO on the skeleton, we investigated here the effect of the hormone on bone metabolism using EPO overexpressing (Tg6), and EPO-administered adult mice. In these models, the increase in EPO levels is similar to its physiologic increase at high altitude without the confounding direct effect of hypoxia on bone cells. Using microcomputed tomography, histology and serum markers we found that high EPO levels result in a severe trabecular bone loss (-32 to -61% decrease in bone density in Tg6 and EPO-injected animals, respectively; p EPO has direct stimulatory effects in vitro on both macrophages and preosteoclasts thus coupling immune and skeletal systems, Activation of purified BM-derived macrophages with EPO, led to a 33% increase (p=0.01) in phagocytosis of 5T33 multiple myeloma cells. EPO strongly stimulated osteoclastogenesis (TRAP staining, Figure 1B) and pit resorption (measured in calcium-phosphate-coated wells) in a cell-autonomous manner. Furthermore, our data indicate that EPO receptor (EPO-R) signaling in osteoclast precursors involves the Jak2 and PI3K pathways, but is independent of the MAPK/MEK pathway. In addition to the direct effect of EPO on monocyte derived cells, high EPO also led to a 1.6 fold (p=0.03) increase in the transcript levels of the osteoclastogenic cytokine RANKL in total bone marrow. Notably, the major sources of RANKL are B cells and osteoblasts, and our data indicate that both cell types express EPO-R, suggesting that they are direct targets of EPO. Accordingly we found an EPO-related increase in membrane bound RANKL on B cells (1.8-fold, p=0.0003) and osteoblasts (1.16-fold, p=0.001) isolated from bone marrow (Figure 1C). To test whether EPO-R on osteoblasts also mediates the attenuation of bone formation, we treated isolated osteoblasts with EPO. However, EPO treatment in vitro did not inhibit osteoblast differentiation and activity, as demonstrated by RT-qPCR of marker genes and mineralization assays. Taken together, these findings demonstrate that EPO strongly regulates bone mass by stimulating osteoclastogenesis and attenuating bone formation. The osteoclastogenic action of EPO is mediated both directly by EPO-R on the monocytic lineage and indirectly via EPO-R signaling on B cells and osteoblasts. Regarding bone formation, the negative effect of EPO on bone formation rate in vivo was not reproduced in isolated cells, thus implying the involvement of cells beyond the osteoblastic lineage. We also propose a new mechanism for hypoxia-induced bone loss that involves EPO action on monocytic, B cell and osteoblastic lineages. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells and monoclonal gammopathy. It is associated with various clinical manifestations including bone disease and anemia. Clinical introduction of recombinant human erythropoietin (rHuEPO) and its derivatives has been a breakthrough in treating patients with anemia, mainly those with chronic kidney disease and cancer patients on chemotherapy. EPO was found to have additional biological effects, including potentiation of both humoral and cell-mediated immune responses. Our studies and those of others, have shown that EPO treatment for anemia in MM patients was associated with general improved immunological functions and immune-mediated modulation (suppression) of MM. A role for EPO in bone metabolism was previously suggested, but its effects were often controversial and the underlying mechanisms remain to be resolved. Objectives To determine the immunological and skeletal effects as well as the underlying mechanisms of EPO signaling in the 5T33 MM mouse model. Results EPO administration led to a 50% decrease in the levels of the MM pathological κ light chain while attenuating the decrease in normal IgA. These immunoglobulin changes were accompanied by a 70% elevation (p=0.015) of B220+IGM+IgD- immature B cells in the BM of EPO-treated MM mice, compared to diluent-injected MM mice (9.8%±0.91 versus 5.68%±1.19 of B220-gated cells, respectively). We also found a 45% increase (p=0.01) in the level of BM macrophages (F4/80+CD11b+) following EPO administration in both healthy and MM mice. Moreover, stimulation in vitro of purified BM-derived macrophages with EPO, led to a 33% increase (p=0.01) in phagocytosis of the 5T33 MM cells. Th17 cells play an important role in MM pathobiology. Our data demonstrate that in MM mice, rHuEPO administration induces a 70% decrease (p=0.021) in the expression level of RORγt, a Th17 hallmark transcription factor. These findings were associated with a 4-fold increase (p Bone disease is a major component of MM and is associated with significant morbidity. High-resolution CT analysis of the distal femurs confirmed bone loss in 5T33 MM mice. The femur bone density of 5T33 MM mice was 30% lower (p=0.04) than that of their healthy counterparts (3.1%±0.003 versus 4.4%±0.008, respectively). Unexpectedly, EPO treatment of MM mice, in comparison to diluent-injected MM mice, induced a 35% reduction (p=0.004) in trabecular bone density (3.1%±0.003 and 2%±0.007, respectively) and 41% (p Conclusions We show that in MM, EPO acts as a double-edged sword, by stimulating the immune response against MM on one hand, but also accelerating bone resorption on the other, which further alters the already compromised bone tissue. A better understanding of the osteoimmunological roles of EPO may advocate for the use of rHuEPO along with targeted bone protective treatment in MM patients, to attenuate the anemia and MM progression, while also preventing bone damage. ND-U and SH-B have contributed equally. Supported by the Multiple Myeloma Research Foundation (DN). Disclosures: No relevant conflicts of interest to declare.

Abstract 5013 Erythropoietin (Epo), mainly produced by the adult kidney, is the major hormone that promotes erythropoiesis. As such, clinical introduction of recombinant human Epo (rHuEpo) and its derivatives (Epoetins), has been a breakthrough in treating patients with anemia, mainly those with chronic kidney failure and cancer patients on chemotherapy. In contrast to 'red flags' suggesting possible detrimental effects of Epo in certain cases of cancer, the hormone was found by numerous studies to have beneficial effects such as neuroprotective and cardio-protective effects. Immune-mediated anti-cancer effects were also documented, as exemplified in multiple myeloma (MM) patients and mouse models. These effects, at least in part, are mediated by Epo receptors (EpoRs) present on cells other than the erythroid lineage. In this respect, we have recently identified functional EpoRs in murine bone marrow-derived macrophages. MM is characterized by clonal proliferation of malignant plasma cells that produce a pathological paraprotein, accompanied by a decrease in the levels of the normal immunoglobulins. Typically this disease is associated with lytic bone lesions. In 5T33 MM, an experimental murine model of MM (producing an IgG2bk paraprotein), mice that were treated with rHuEpo, displayed a 50% decrease in the levels of the pathological K light chain. Notably, the decline in the levels of normal IgA, typically observed in the MM mice, was attenuated in the Epo-treated MM mice. Administration of rHuEpo led to increased levels of liver (three fold increase) and splenic (ten fold increase) macrophages as determined by the number of F4/80/CD11b expressing cells. These findings were associated with an increase in the transcript levels of the Th1 cytokine IFN-g in the bone marrow of Epo-treated 5T33 MM mice as compared to diluent-treated 5T33 MM mice. As MM is typically associated with a Th2 response, Epo may have a beneficial effect in MM, by shifting the balance towards a Th1 response. Myeloma-associated bone disease is considered a major cause of morbidity in MM patients. Using high-resolution computed tomography analysis of recovered femurs we confirmed that the 5T33 MM mice indeed exhibit bone disease. Femur trabecular bone density (bone volume/total volume) of 5T33 MM mice was 43% lower (p=0. 04) than that of their healthy control counterparts (3. 1%±0. 003 versus 4. 4%±0. 008, respectively). This decrease in bone density reflects a 38% increase (p=0. 001) in the total femur volume (4. 38mm3±0. 13 and 2. 72mm3±0. 95 in the 5T33 MM mice, compared to that of healthy mice) and no significant change in trabecular bone volume. Unexpectedly, Epo treatment of the MM mice induced a 34% reduction (p=0. 004) in trabecular bone density compared to the diluent-treated MM mice (2%±0. 007 and 3. 1%±0. 003, respectively) and 41% (p=1E-6) reduction in trabecular number (1. 54mm−1±0. 266 and 2. 62mm−1±0. 23, respectively). This was accompanied by a 19% increase (p=0. 006) in trabecular bone thickness in the Epo-treated MM mice (0. 043mm±0. 005 and 0. 036mm±0. 003, respectively), suggesting a mechanical compensatory mechanism in response to the extensive bone loss. Taken together, our data demonstrate that Epo's impact extends well beyond erythropoiesis. The administration of rHuEpo may be considered as an adjunct treatment to specific anti-MM therapy in cases which require elevation of the erythrocyte counts and an enhancement of the immune system. Yet, a bone protection regimen (bisphosphonates?) should be considered. This study is supported by the Multiple Myeloma Research Foundation, to DN. Disclosures: No relevant conflicts of interest to declare.