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Supplementary figure 1. MM cells induce apoptosis and activate Notch signaling in osteocytes.

Characterization of 5TGM1 mouse MM cells in vivo and in vitro.

Legend for Supplementary Figures S1-S2.

Supplementary figure 3. Osteocytes regulate cell proliferation and Notch receptor expression in MM cells.

In multiple myeloma, an overabundance of monoclonal plasma cells in the bone marrow induces localized osteolytic lesions that rarely heal due to increased bone resorption and suppressed bone formation. Matrix-embedded osteocytes comprise more than 95% of bone cells and are major regulators of osteoclast and osteoblast activity, but their contribution to multiple myeloma growth and bone disease is unknown. Here, we report that osteocytes in a mouse model of human MM physically interact with multiple myeloma cells in vivo, undergo caspase-3–dependent apoptosis, and express higher RANKL (TNFSF11) and sclerostin levels than osteocytes in control mice. Mechanistic studies revealed that osteocyte apoptosis was initiated by multiple myeloma cell-mediated activation of Notch signaling and was further amplified by multiple myeloma cell-secreted TNF. The induction of apoptosis increased osteocytic Rankl expression, the osteocytic Rankl/Opg (TNFRSF11B) ratio, and the ability of osteocytes to attract osteoclast precursors to induce local bone resorption. Furthermore, osteocytes in contact with multiple myeloma cells expressed high levels of Sost/sclerostin, leading to a reduction in Wnt signaling and subsequent inhibition of osteoblast differentiation. Importantly, direct contact between osteocytes and multiple myeloma cells reciprocally activated Notch signaling and increased Notch receptor expression, particularly Notch3 and 4, stimulating multiple myeloma cell growth. These studies reveal a previously unknown role for bidirectional Notch signaling that enhances MM growth and bone disease, suggesting that targeting osteocyte-multiple myeloma cell interactions through specific Notch receptor blockade may represent a promising treatment strategy in multiple myeloma. Cancer Res; 76(5); 1089–100. ©2016 AACR.

Purpose: This phase I study was conducted to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) for the combination of bortezomib and alvocidib in patients with B-cell malignancies (multiple myeloma, indolent lymphoma, Waldenstrom macroglobulinemia, and mantle cell lymphoma).Experimental Design: Patients received bortezomib (intravenous push), followed by alvocidib (1-hour infusion), on days 1, 4, 8, and 11 of a 21-day treatment cycle. Patients experiencing responses or stable disease continued on treatment at the investigator's discretion. A standard 3+3 dose-escalation design was used to identify the MTD based on DLTs, and pharmacokinetic and pharmacodynamic studies were conducted.Results: A total of 44 patients were enrolled, with 39 patients assessed for response. The MTD was established as 1.3 mg/m2 for bortezomib and 40 mg/m2 for alvocidib. The most common hematologic toxicities included leukopenia, lymphopenia, neutropenia, and thrombocytopenia. The most common nonhematologic toxicities included diarrhea, fatigue, and sensory neuropathy. Three complete remissions (8%) and 10 partial remissions (26%) were observed for a total response rate of 33%. Pharmacokinetic findings with the current dosing regimen were consistent with the comparable literature and the hybrid dosing regimen. Pharmacodynamic study results did not correlate with clinical responses.Conclusions: The combination of bortezomib and alvocidib is tolerable, and an MTD has been established for this schedule. The regimen appears to be efficacious in patients with relapsed/refractory multiple myeloma or indolent non-Hodgkin lymphoma. As the nonhybrid regimen is less cumbersome than the previous hybrid dosing schedule regimen, the current schedule is recommended for successor studies. Clin Cancer Res; 20(22); 5652–62. ©2014 AACR.

Supplementary Table 1

Multiple myeloma patients experience severe bone pain (MMBP) that is undertreated and poorly understood. In this study, we studied MMBP in an intratibial mouse xenograft model that employs JJN3 human multiple myeloma cells. In this model, mice develop MMBP associated in bone with increased sprouting of calcitonin gene-related peptide-positive (CGRP+) sensory nerves and in dorsal root ganglia (DRG) with upregulation of phosphorylated ERK1/2 (pERK1/2) and pCREB, two molecular indicators of neuron excitation. We found that JJN3 cells expressed a vacuolar proton pump (V-ATPase) that induced an acidic bone microenvironment. Inhibition of JJN3-colonized bone acidification by a single injection of the selective V-ATPase inhibitor, bafilomycin A1, decreased MMBP, CGRP+ sensory neuron sprouting, and pERK1/2 and pCREB expression in DRG. CGRP+ sensory nerves also expressed increased levels of the acid-sensing nociceptor ASIC3. Notably, a single injection of the selective ASIC3 antagonist APETx2 dramatically reduced MMBP in the model. Mechanistic investigations in primary DRG neurons cocultured with JJN3 cells showed increased neurite outgrowth and excitation inhibited by bafilomycin A1 or APETx2. Furthermore, combining APETx2 with bafilomycin A1 reduced MMBP to a greater extent than either agent alone. Finally, combining bafilomycin A1 with the osteoclast inhibitor zoledronic acid was sufficient to ameliorate MMBP, which was refractory to zoledronic acid. Overall, our results show that osteoclasts and multiple myeloma cooperate to induce an acidic bone microenvironment that evokes MMBP as a result of the excitation of ASIC3-activated sensory neurons. Furthermore, they present a mechanistic rationale for targeting ASIC3 on neurons along with the multiple myeloma-induced acidic bone microenvironment as a strategy to relieve MMBP in patients. Cancer Res; 77(6); 1283–95. ©2017 AACR.

Supplementary figure 2. MM cells increase Rankl mRNA levels in osteocytes.

Systemic inhibition of Notch with γ-secretase inhibitors (GSI) decreases multiple myeloma tumor growth, but the clinical use of GSI is limited due to its severe gastrointestinal toxicity. In this study, we generated a GSI Notch inhibitor specifically directed to the bone (BT-GSI). BT-GSI administration decreased Notch target gene expression in the bone marrow, but it did not alter Notch signaling in intestinal tissue or induce gut toxicity. In mice with established human or murine multiple myeloma, treatment with BT-GSI decreased tumor burden and prevented the progression of multiple myeloma-induced osteolytic disease by inhibiting bone resorption more effectively than unconjugated GSI at equimolar doses. These findings show that BT-GSI has dual anti-myeloma and anti-resorptive properties, supporting the therapeutic approach of bone-targeted Notch inhibition for the treatment of multiple myeloma and associated bone disease.Significance:Development of a bone-targeted Notch inhibitor reduces multiple myeloma growth and mitigates cancer-induced bone destruction without inducing the gastrointestinal toxicity typically associated with inhibition of Notch.

Supplementary Materials. Supplemental Table 1. C1D1 alvocidib pharmacokinetics by dose Supplemental Figure 1. Sample alvocidib concentration-time profile. Samples were obtained pre-infusion, immediately following infusion, and at 1, 2, 4, 8, 12, and 24 hours post-infusion.

Supplementary figure legends

Supplementary methods

Supplementary Figures S1-S2; Supplementary Tables S1-S2.

Supplementary figures 1, 2, and 3

Systemic inhibition of Notch with γ-secretase inhibitors (GSI) decreases multiple myeloma tumor growth, but the clinical use of GSI is limited due to its severe gastrointestinal toxicity. In this study, we generated a GSI Notch inhibitor specifically directed to the bone (BT-GSI). BT-GSI administration decreased Notch target gene expression in the bone marrow, but it did not alter Notch signaling in intestinal tissue or induce gut toxicity. In mice with established human or murine multiple myeloma, treatment with BT-GSI decreased tumor burden and prevented the progression of multiple myeloma-induced osteolytic disease by inhibiting bone resorption more effectively than unconjugated GSI at equimolar doses. These findings show that BT-GSI has dual anti-myeloma and anti-resorptive properties, supporting the therapeutic approach of bone-targeted Notch inhibition for the treatment of multiple myeloma and associated bone disease. Significance: Development of a bone-targeted Notch inhibitor reduces multiple myeloma growth and mitigates cancer-induced bone destruction without inducing the gastrointestinal toxicity typically associated with inhibition of Notch.

Paget's disease (PD) is characterized by increased numbers of abnormal osteoclasts (OCLs) that drive exuberant bone formation, but the mechanisms responsible for the increased bone formation remain unclear. We previously reported that OCLs from 70% of PD patients express measles virus nucleocapsid protein (MVNP), and that transgenic mice with targeted expression of MVNP in OCLs (MVNP mice) develop bone lesions and abnormal OCLs characteristic of PD. In this report, we examined if OCL‐derived sphingosine‐1‐phosphate (S1P) contributed to the abnormal bone formation in PD, since OCL‐derived S1P can act as a coupling factor to increase normal bone formation via binding S1P‐receptor‐3 (S1PR3) on osteoblasts (OBs). We report that OCLs from MVNP mice and PD patients expressed high levels of sphingosine kinase‐1 (SphK‐1) compared with wild‐type (WT) mouse and normal donor OCLs. SphK‐1 production by MVNP‐OCLs was interleukin‐6 (IL‐6)‐dependent since OCLs from MVNP/IL‐6 −/− mice expressed lower levels of SphK‐1. Immunohistochemistry of bone biopsies from a normal donor, a PD patient, WT and MVNP mice confirmed increased expression levels of SphK‐1 in OCLs and S1PR3 in OBs of the PD patient and MVNP mice compared with normal donor and WT mice. Further, MVNP‐OCLs cocultured with OBs from MVNP or WT mice increased OB‐S1PR3 expression and enhanced expression of OB differentiation markers in MVNP‐OBs precursors compared with WT‐OBs, which was mediated by IL‐6 and insulin‐like growth factor 1 secreted by MVNP‐OCLs. Finally, the addition of an S1PR3 antagonist (VPC23019) to WT or MVNP‐OBs treated with WT and MVNP‐OCL‐conditioned media (CM) blocked enhanced OB differentiation of MVNP‐OBs treated with MVNP‐OCL‐CM. In contrast, the addition of the SIPR3 agonist, VPC24191, to the cultures enhanced osterix and Col‐1A expression in MVNP‐OBs treated with MVNP‐OCL‐CM compared with WT‐OBs treated with WT‐OCL‐CM. These results suggest that IL‐6 produced by PD‐OCLs increases S1P in OCLs and S1PR3 on OBs, to increase bone formation in PD., Model for the effects of sphingosine kinase‐1/sphingosine‐1‐phosphate/S1P‐receptor‐3 (SphK‐1/S1P/S1PR3) in abnormal bone remodeling in Paget's disease (PD). Measles virus nucleocapsid protein (MVNP) in PD‐osteoclasts (OCLs) induces interleukin‐6 (IL‐6), which upregulates insulin‐like growth factor 1 (IGF‐1) and Sphk1. SphK‐1 enhances S1P levels in OCLs, and IL‐6 and IGF‐1 increase S1PR3 on osteoblast (OBs). S1P then increases OB differentiation and bone formation via enhanced S1PR3 expression on OBs.

Multiple myeloma (MM) is the second most common haematological malignancy and is characterized by a clonal proliferation of neoplastic plasma cells within the bone marrow. MM is the most frequent cancer involving the skeleton, causing osteolytic lesions, bone pain and pathological fractures that dramatically decrease MM patients' quality of life and survival. MM bone disease (MBD) results from uncoupling of bone remodelling in which excessive bone resorption is not compensated by new bone formation, due to a persistent suppression of osteoblast activity. Current management of MBD includes antiresorptive agents, bisphosphonates and denosumab, that are only partially effective due to their inability to repair the existing lesions. Thus, research into agents that prevent bone destruction and more importantly repair existing lesions by inducing new bone formation is essential. This review discusses the mechanisms regulating the uncoupled bone remodelling in MM and summarizes current advances in the treatment of MBD. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.

Parathyroid hormone (PTH) signaling downstream of the PTH 1 receptor (Pth1r) results in both bone anabolic and catabolic actions by mechanisms not yet fully understood. In this study, we show that Pth1r signaling upregulates the expression of several components of the Notch pathway and that Notch signals contribute to the catabolic actions of PTH in bone. We found that constitutive genetic activation of PTH receptor signaling in osteocytes (caPth1r(Ot)) or treatment with PTH daily increased the expression of several Notch ligands/receptors in bone. In contrast, sustained elevation of endogenous PTH did not change Notch components expression. Deletion of the PTH receptor or Sclerostin overexpression in osteocytes abolished Notch increases by PTH. Further, deleting the canonical Notch transcription factor Rbpjk in osteocytes decreased bone mass and increased resorption and Rankl expression in caPth1r(Ot) mice. Moreover, pharmacological bone-targeted Notch inhibition potentiated the bone mass gain induced by intermittent PTH by reducing bone resorption and preserving bone formation. Thus, Notch activation lies downstream of anabolic signaling driven by PTH actions in osteocytes, and Notch pharmacological inhibition maximizes the bone anabolic effects of PTH.

Multiple myeloma (MM) remains incurable for most patients due to the emergence of drug resistant clones. Here we report a p53-independent mechanism responsible for Growth Factor Independence-1 (GFI1) support of MM cell survival by its modulation of sphingolipid metabolism to increase the sphingosine-1-phosphate (S1P) level regardless of the p53 status. We found that expression of enzymes that control S1P biosynthesis

Despite recent progress in its treatment, Multiple Myeloma (MM) remains incurable and its associated bone disease persists even after complete remission. Thus, identification of new therapeutic agents that simultaneously suppress MM growth and protect bone is an unmet need. Herein, we examined the effects of Aplidin, a novel anti-cancer marine-derived compound, on MM and bone cells. In vitro, Aplidin potently inhibited MM cell growth and induced apoptosis, effects that were enhanced by dexamethasone (Dex) and bortezomib (Btz). Aplidin modestly reduced osteocyte/osteoblast viability and decreased osteoblast mineralization, effects that were enhanced by Dex and partially prevented by Btz. Further, Aplidin markedly decreased osteoclast precursor numbers and differentiation, and reduced mature osteoclast number and resorption activity. Moreover, Aplidin reduced Dex-induced osteoclast differentiation and further decreased osteoclast number when combined with Btz. Lastly, Aplidin alone, or suboptimal doses of Aplidin combined with Dex or Btz, decreased tumor growth and bone resorption in ex vivo bone organ cultures that reproduce the 3D-organization and the cellular diversity of the MM/bone marrow niche. These results demonstrate that Aplidin has potent anti-myeloma and anti-resorptive properties, and enhances proteasome inhibitors blockade of MM growth and bone destruction.

Graphical abstract, Highlights • The 4T1 mouse breast cancer injected in tibiae induced bone pain. • The 4T1 breast cancer secreted high mobility group box 1 (HMGB1) that promotes axogenesis of sensory neurons. • Bone pain was reduced by HMGB1 antibody and an antagonist for the receptor for advanced glycation end products., Bone pain is a common complication of breast cancer (BC) bone metastasis and is a major cause of increased morbidity and mortality. Although the mechanism of BC-associated bone pain (BCABP) remains poorly understood, involvement of BC products in the pathophysiology of BCABP has been proposed. Aggressive cancers secrete damage-associated molecular patterns (DAMPs) that bind to specific DAMP receptors and modulate cancer microenvironment. A prototypic DAMP, high mobility group box 1 (HMGB1), which acts as a ligand for the receptor for advanced glycation end products (RAGE) and toll-like receptors (TLRs), is increased in its expression in BC patients with poor outcomes. Here we show that 4T1 mouse BC cells colonizing bone up-regulate the expression of molecular pain markers, phosphorylated ERK1/2 (pERK) and pCREB, in the dorsal root ganglia (DRGs) innervating bone and induced BCABP as evaluated by hind-paw mechanical hypersensitivity. Importantly, silencing HMGB1 in 4T1 BC cells by shRNA reduced pERK and pCREB and BCABP with decreased HMGB1 levels in bone. Further, administration of a neutralizing antibody to HMGB1 or an antagonist for RAGE, FPS-ZM1, ameliorated pERK, pCREB and BCABP, while a TLR4 antagonist, TAK242, showed no effects. Consistent with these in vivo results, co-cultures of F11 sensory neuron-like cells with 4T1 BC cells in microfluidic culture platforms increased neurite outgrowth of F11 cells, which was blocked by HMGB1 antibody. Our results show that HMGB1 secreted by BC cells induces BCABP via binding to RAGE of sensory neurons and suggest that the HMGB1/RAGE axis may be a potential novel therapeutic target for BCABP.

In multiple myeloma (MM), Notch signaling, a pathway mediating cell-to-cell communication between cells in the tumor niche, promotes MM proliferation and bone destruction. We previously reported that osteocytes (Ots), the most abundant bone cells, provide a microenvironment conducive for MM progression and bone destruction. Ots activate Notch signaling, increase Notch3 expression, and stimulate proliferation in MM cells. We detected Notch3 in CD138+ cells from MM patients and human and murine MM cell lines. Yet, the role of Notch3 in MM is unknown. Here, we used a shRNA approach to knockdown Notch3 to study its role in MM cells and their communication with Ots. The protein levels of active Notch3 (NICD3) were decreased by 80% in 5TGM1 MM cells transduced with shRNA against Notch3 compared to control shRNA-control cells. In contrast, Notch1, 2, and 4 NICD protein levels remained unchanged compared to controls. Notch3 knockdown decreased Notch target gene and cyclinD1 expression, reduced proliferation by 35%, and modestly increased apoptosis in MM cells. Additionally, Notch3 knockdown decreased Rankl expression and the ability of MM cells to promote osteoclastogenesis in vitro. Consistent with these observations, bioinformatic analysis of the transcriptome of CD138+ cells from newly diagnosed patients revealed upregulated processes related to positive regulation of cell proliferation and osteoclastogenesis in MM patients with high vs. low Notch3 expression. Next, we examined the role of Notch3 in MM-Ots communication. Notch3 knockdown in MM cells partially prevented the upregulation of Notch target genes and cyclinD1 expression and proliferation induced by direct contact with Ots. Inhibition of all Notch receptors with GSI fully prevented osteocyte-induced proliferation and Notch activation, suggesting that in addition to Notch3, other Notch receptors mediate MM-osteocyte communication. Remarkably, shRNA-mediated Notch2 inhibition did not alter MM cell proliferation or communication with osteocytes. Lastly, we analyzed the effects of Notch3 knockdown in MM cells in ex vivo and in vivo models. Using ex vivo bone organ cultures, we found less MM proliferation and lower levels of the resorption marker CTX in conditioned media from bones cultured with shRNA-Notch3 MM cells compared to control bones cultured alone. We injected mice intratibially with shRNA-Notch3 or shRNA-control 5TGM1 MM cells. After 5 weeks, mice bearing shRNA-Notch3 cells had a 50% decrease in tumor burden, 50% reduction in osteolytic lesions, and exhibited 30% more cancellous bone compared to mice bearing control MM cells. Together, these preclinical and clinical findings support that Notch3 signaling is a crucial mediator of homotypic and heterotypic communication in the MM tumor niche. Future studies are needed to evaluate Notch3 in the tumor microenvironment as a therapeutic target for the treatment of MM. Citation Format: Hayley M. Sabol, Tania Amorim, Cody Ashby, David Halladay, Judith Anderson, Meloney Cregor, Megan Sweet, Intawat Nookaew, Noriyoshi Kurihara, G. David Roodman, Teresita Bellido, Jesus Delgado-Calle. Notch3 signaling between myeloma cells and osteocytes in the tumor niche promotes tumor growth and bone destruction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5672.

In multiple myeloma (MM), communication via Notch signaling in the tumor niche stimulates tumor progression and bone destruction. We previously showed that osteocytes activate Notch, increase Notch3 expression, and stimulate proliferation in MM cells. We show here that Notch3 inhibition in MM cells reduced MM proliferation, decreased Rankl expression, and abrogated the ability of MM cells to promote osteoclastogenesis. Further, Notch3 inhibition in MM cells partially prevented the Notch activation and increased proliferation induced by osteocytes, demonstrating that Notch3 mediates MM-osteocyte communication. Consistently, pro-proliferative and pro-osteoclastogenic pathways were upregulated in CD138

Multiple myeloma (MM) is an incurable plasma cell malignancy that evolves from a premalignant condition termed monoclonal gammopathy of undetermined significance (MGUS). MM is the most frequent cancer that involves the skeleton, with 90% of patients eventually developing bone lesions. Bone involvement causes devastating consequences for MM patients, including pathological fractures that occur in 50%–60% of patients and are present in 20% of patients at diagnosis. These fractures cause severe bone pain and increase mortality risk by 20%. MM increases localized bone resorption and suppresses bone formation, causing purely lytic lesions that usually do not repair. MGUS also affects the skeleton, with MGUS patients having a 1.7-fold increased fracture incidence compared to age-matched controls. In this chapter the mechanisms responsible for the impact of MGUS and MM on the skeleton and therapeutic approaches to prevent and treat MGUS- and MM-induced bone disease are discussed.

Background/Objective: Low muscle mass (myopenia), poor muscle quality, myosteatosis, and muscle loss are associated with mortality in solid tumors. However, their impact in hematological malignancies remains unclear. We sought to determine how muscle phenotype relates to survival in patients with multiple myeloma. Methods: We performed a retrospective review of patients with multiple myeloma treated at Indiana University Hospital from 2012-2016. Total skeletal muscle area (SMA) (cm2) and radiodensity were measured on baseline (closest to diagnosis) and last CT scans at the third lumbar vertebrae area. SMA was normalized to height (SMA cm2/m2) to define skeletal muscle index (SKMI). Myopenia was defined as (SKMI)

Multiple Myeloma (MM) induces bone destruction, decreases bone formation, and increases marrow angiogenesis in patients. We reported that osteocytes (Ocys) directly interact with MM cells to increase tumor growth and expression of Ocy-derived factors that promote bone resorption and suppress bone formation. However, the contribution of Ocys to enhanced marrow vascularization in MM is unclear. Since the MM microenvironment is hypoxic, we assessed if hypoxia and/or interactions with MM cells increases pro-angiogenic signaling in Ocys. Hypoxia and/or co-culture with MM cells significantly increased Vegf-a expression in MLOA5-Ocys, and conditioned media (CM) from MLOA5s or MM-MLOA5 co-cultured in hypoxia, significantly increased endothelial tube length compared to normoxic CM. Further, Vegf-a knockdown in MLOA5s or primary Ocys co-cultured with MM cells or neutralizing Vegf-a in MM-Ocy co-culture CM completely blocked the increased endothelial activity. Importantly, Vegf-a-expressing Ocy numbers were significantly increased in MM-injected mouse bones, positively correlating with tumor vessel area. Finally, we demonstrate that direct contact with MM cells increases Ocy Fgf23, which enhanced Vegf-a expression in Ocys. Fgf23 deletion in Ocys blocked these changes. These results suggest hypoxia and MM cells induce a pro-angiogenic phenotype in Ocys via Fgf23 and Vegf-a signaling, which can promote MM-induced marrow vascularization.

Many interventions in global health save lives. One criticism sometimes lobbed at these interventions invokes the spirit of Malthus. The good done, the charge goes, is offset by the harm of spreading the earth's limited resources more thinly: more people, and more misery per person. To the extent this holds, the net benefit of savings lives is lower than it appears at first. On the other hand, if lower mortality, especially in childhood, leads families to have fewer children, life-saving interventions could reduce population. This document critically reviews the evidence. It finds that the impact of life-saving interventions on fertility and population growth varies by context, and is rarely greater than 1:1. In places where lifetime births/woman has been converging to 2 or lower, saving one child's life should lead parents to avert a birth they would otherwise have. The impact of mortality drops on fertility will be nearly 1:1, so population growth will hardly change. In the increasingly exceptional locales where couples appear not to limit fertility much, such as Niger and Mali, the impact of saving a life on total births will be smaller, and may come about mainly through the biological channel of lactational amenorrhea. Here, mortality-drop-fertility-drop ratios of 1:0.5 and 1:0.33 appear more plausible. But in the long-term, it would be surprising if these few countries do not join the rest of the world in the transition to lower and more intentionally controlled fertility.

The Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Myeloma Intergroup has organized an annual workshop focused on minimal residual disease (MRD) testing and immune profiling (IP) in multiple myeloma since 2016. In 2019, the workshop took place as an American Society of Hematology (ASH) Friday Scientific Workshop entitled “Immune Profiling and Minimal Residual Disease Testing in Multiple Myeloma”. This workshop focused on four main topics: the molecular and immunological evolution of plasma cell disorders, the development of new laboratory- and imaging-based MRD assessment approaches, chimeric antigen receptor T-cell therapy research, and the statistical and regulatory issues associated with novel clinical endpoints. In this report, we provide a summary of the workshop and discuss future directions.

Juvenile Paget's disease (JPD) became in 1974 the commonly used name for ultra-rare heritable occurrences of rapid bone remodeling throughout of the skeleton that present in infancy or early childhood as fractures and deformity hallmarked biochemically by marked elevation of serum alkaline phosphatase (ALP) activity (hyperphosphatasemia). Untreated, JPD can kill during childhood or young adult life. In 2002, we reported that homozygous deletion of the gene called tumor necrosis factor receptor superfamily, member 11B (TNFRSF11B) encoding osteoprotegerin (OPG) explained JPD in Navajos. Soon after, other bi-allelic loss-of-function TNFRSF11B defects were identified in JPD worldwide. OPG inhibits osteoclastogenesis and osteoclast activity by decoying receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) away from its receptor RANK. Then, in 2014, we reported JPD in a Bolivian girl caused by a heterozygous activating duplication within TNFRSF11A encoding RANK. Herein, we identify mutation of a third gene underlying JPD. An infant girl began atraumatic fracturing of her lower extremity long-bones. Skull deformity and mild hearing loss followed. Our single investigation of the patient, when she was 15 years-of-age, showed generalized osteosclerosis and hyperostosis. DXA revealed a Z-score of +5.1 at her lumbar spine and T-score of +3.3 at her non-dominant wrist. Biochemical studies were consistent with positive mineral balance and several markers of bone turnover were elevated and included striking hyperphosphatasemia. Iliac crest histopathology was consistent with rapid skeletal remodeling. Measles virus transcripts, common in classic Paget's disease of bone, were not detected in circulating mononuclear cells. Then, reportedly, she responded to several months of alendronate therapy with less skeletal pain and correction of hyperphosphatasemia but had been lost to our follow-up. After we detected no defect in TNFRSF11A or B, trio exome sequencing revealed a de novo heterozygous missense mutation (c.926C>G; p.S309W) within SP7 encoding the osteoblast transcription factor osterix (specificity protein 7, transcription factor SP7). Thus, mutation of SP7 represents a third genetic cause of JPD.

Multiple myeloma (MM) remains incurable for most patients due to the emergence of drug resistant clones. Here we report a p53-independent mechanism responsible for Growth Factor Independence-1 (GFI1) support of MM cell survival by its modulation of sphingolipid metabolism to increase the sphingosine-1-phosphate (S1P) level regardless of the p53 status. We found that expression of enzymes that control S1P biosynthesis, SphK1, dephosphorylation, and SGPP1 were differentially correlated with GFI1 levels in MM cells. We detected GFI1 occupancy on the SGGP1 gene in MM cells in a predicted enhancer region at the 5’ end of intron 1, which correlated with decreased SGGP1 expression and increased S1P levels in GFI1 overexpressing cells, regardless of their p53 status. The high S1P:Ceramide intracellular ratio in MM cells protected c-Myc protein stability in a PP2A-dependent manner. The decreased MM viability by SphK1 inhibition was dependent on the induction of autophagy in both p53WT and p53mut MM. An autophagic blockade prevented GFI1 support for viability only in p53mut MM, demonstrating that GFI1 increases MM cell survival via both p53WT inhibition and upregulation of S1P independently. Therefore, GFI1 may be a key therapeutic target for all types of MM that may significantly benefit patients that are highly resistant to current therapies.

Multiple myeloma (MM) is the most frequent cancer to involve the skeleton with patients developing osteolytic bone lesions due to hyperactivation of osteoclasts and suppression of BMSCs differentiation into functional osteoblasts. Although new therapies for MM have greatly improved survival, MM remains incurable for most patients. Despite the major advances in current anti-MM and anti-resorptive treatments that can significantly improve osteolytic bone lysis, many bone lesions can persist even after therapeutic remission of active disease. Bone marrow mesenchymal stem cells (BMSCs) from MM patients are phenotypically distinct from their healthy counterparts and the mechanisms associated with the long-term osteogenic suppression are largely unknown. In this review we will highlight recent results of transcriptomic profiling studies that provide new insights into the establishment and maintenance of the persistent pathological alterations in MM-BMSCs that occur in MM. We will we discuss the role of genomic instabilities and senescence in propagating the chronically suppressed state and pro-inflammatory phenotype associated with MM-BMSCs. Lastly we describe the role of epigenetic-based mechanisms in regulating osteogenic gene expression to establish and maintain the pro-longed suppression of MM-BMSC differentiation into functional OBs. Keywords: Multiple myeloma bone disease, Bone microenvironment, BMSCs, Osteoblast, Genomic, Senescence, Epigenetic

Bone disease is the most frequent disease-defining clinical feature of multiple myeloma (MM), with 90% of patients developing bone lesions over the course of their disease. For this reason, imaging plays a major role in the management of disease in patients with MM. Although conventional radiography has traditionally been the standard of care, its low sensitivity in detecting osteolytic lesions has called for more advanced imaging modalities. In this review, we discuss the advantages, indications, and applications of whole-body low-dose CT (WBLDCT), 18F-fluorodeoxyglucose (FDG)-PET/CT, MRI, and other novel imaging modalities in the management of disease in patients with plasma cell dyscrasias. We also review the state of the art in treatment of MM bone disease (MMBD) and the role of bisphosphonates and denosumab, a monoclonal antibody that binds and blocks the activity of receptor activator of nuclear factor-kappa B ligand (RANKL), which was recently approved by the U.S. Food and Drug Administration for MMBD.

A large, pivotal, phase 3 trial in patients with newly diagnosed multiple myeloma (MM) demonstrated that denosumab, compared with zoledronic acid, was non-inferior for the prevention of skeletal-related events (SREs), extended the observed median progression-free survival (PFS) by 10.7 months, and showed significantly less renal toxicity. The cost-effectiveness of denosumab vs zoledronic acid in MM in the US was assessed from societal and payer perspectives.The XGEVA Global Economic Model was developed by integrating data from the phase 3 trial comparing the efficacy of denosumab with zoledronic acid for the prevention of SREs in MM. SRE rates were adjusted to reflect the real-world incidence. The model included utility decrements for SREs, administration, serious adverse events (SAEs), and disease progression. Drug, administration, SRE management, SAEs, and anti-MM treatment costs were based on data from published studies. For the societal perspective, the model additionally included SRE-related direct non-medical costs and indirect costs. The net monetary benefit (NMB) was calculated using a willingness-to-pay threshold of US$150,000. One-way deterministic and probabilistic sensitivity analyses were conducted.From a societal perspective, compared with zoledronic acid, the use of denosumab resulted in an incremental cost of US$26,329 and an incremental quality-adjusted life-year (QALY) of 0.2439, translating into a cost per QALY gained of US$107,939 and a NMB of US$10,259 in favor of denosumab. Results were sensitive to SRE rates and PFS parameters.Costs were estimated from multiple sources, which varied by tumor type, patient population, country, and other parameters. PFS and overall survival were extrapolated beyond the follow-up of the primary analysis using fitted parametric curves.Denosumab's efficacy in delaying or preventing SREs, potential to improve PFS, and lack of renal toxicity make it a cost-effective option for the prevention of SREs in MM compared with zoledronic acid.

Multiple myeloma (MM) is an incurable malignancy of mature plasma cells. Despite major advances in the therapeutic armamentarium of MM, only 50% of patients survive more than 5 years after diagnosis, with significantly lower rates (21%) for high-risk patients. Chimeric Antigen Receptor (CAR) T-cell therapy targeting BCMA (B-cell maturation antigen) shows high response rates in relapsed/refractory patients. However, most patients have disease remission that lasts less than 18 months, prompting the search for additional and synergistic therapeutic approaches. We unbiasedly mapped the cell surface proteome of MM by integrating Mass-Spectrometry (MS) and RNA-seq analyses from 7 MM cell lines and 904 primary MM patient samples bearing high-risk cytogenetics. To identify cell surface proteins, we ran a pool of 4,761 proteins and 16,000+ transcripts through five repositories. An integrated scoring database was developed by scoring each ID based on the number of databases (0-5) it was identified in, with 0 if the molecule was not found in any and 5 if the protein was found in all five. We identified 402 proteins with a surface score of 3 or higher in MM cell lines and patient samples by transcriptomics and proteomics. We prioritized the 326 candidates that were more highly expressed in patients. Based on functional enrichment analyses, we found the proteins formed three main networks with immune mechanisms representing the largest cluster (227 out of 326 cell surface proteins) followed by transporters and adhesion proteins.Based on a pipeline we previously established (1), we further selected 97 candidates minimally expressed in normal tissues. This list included current therapeutic targets such as BCMA, SLAMF7, ITGB7 and LY9. Validation in primary patient samples by western blot and flow-cytometric analyses, enabled the identification of 10 top candidates (CCR1, CD320, FCRL3, IL12RB1, ITGA4, LAX1, LILRB4, LRRC8D, SEMA4A, SLAMF6) that resulted most frequently and highly expressed. We found that LAX1, LILRB4 and SEMA4A significantly impact myeloma patient overall survival based on Kaplan-Meier analysis in the MM Research Foundation (MMRF) cohort (2). CCR1, IL12RB1, LILRB4 and SEMA4A were upregulated by the treatment with Bortezomib or Venetoclax that conversely, decreased BCMA expression in MM U266 cells. By stratifying the patient population, we found that the SEMA4A and LAX1 were up-regulated in patients with t(4;14) compared to patients with no cytogenetic abnormality; LILRB4 in patients with t(14;16) and CCR1 patients with t(14;16) and t(14;20). By calculating co-expression levels CCR1-LILRB4 and CCR1-FCRL3 resulted co-expressed in 100% of patients. For safety purposes (3), we excluded candidates with high (>55%) protein abundance in highly-purified normal hematopoietic stem cells and activated T-cells, narrowing down the list to 6 top candidates (CCR1, FCRL3, IL12RB1, LILRB4, LRRC8D, SEMA4A). To define the function of this group of promising cell surface targets, we used a CRISPR/Cas9 inducible system in KMS11 MM cells. We found that knock-out of CCR1, LRRC8D and SEMA4A individually reduces the MM cell growth by ~60%, 50% and 50% respectively, and almost completely abrogates MM cell migration through porous chambers by >80%. By co-culturing irradiated KO and control MM cells with healthy donor T-cells we also found that lack of CCR1 increased T-cell proliferation by 50% compared to controls and enhanced killing of MM cells, suggesting that CCR1 may suppress T-cell mediated immune responses in addition to play a role in MM cell survival and migration. This study suggests the contribution of an altered MM surfaceome to disease development and may lead to potential novel immunotherapeutic approaches for high-risk MM. References 1. Perna F et al., Cancer Cell 2017 3. Dong C et al., in press Oncogene 2021 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Multiple myeloma patients experience severe bone pain (MMBP) that is undertreated and poorly understood. In this study, we studied MMBP in an intratibial mouse xenograft model that employs JJN3 human multiple myeloma cells. In this model, mice develop MMBP associated in bone with increased sprouting of calcitonin gene-related peptide-positive (CGRP+) sensory nerves and in dorsal root ganglia (DRG) with upregulation of phosphorylated ERK1/2 (pERK1/2) and pCREB, two molecular indicators of neuron excitation. We found that JJN3 cells expressed a vacuolar proton pump (V-ATPase) that induced an acidic bone microenvironment. Inhibition of JJN3-colonized bone acidification by a single injection of the selective V-ATPase inhibitor, bafilomycin A1, decreased MMBP, CGRP+ sensory neuron sprouting, and pERK1/2 and pCREB expression in DRG. CGRP+ sensory nerves also expressed increased levels of the acid-sensing nociceptor ASIC3. Notably, a single injection of the selective ASIC3 antagonist APETx2 dramatically reduced MMBP in the model. Mechanistic investigations in primary DRG neurons cocultured with JJN3 cells showed increased neurite outgrowth and excitation inhibited by bafilomycin A1 or APETx2. Furthermore, combining APETx2 with bafilomycin A1 reduced MMBP to a greater extent than either agent alone. Finally, combining bafilomycin A1 with the osteoclast inhibitor zoledronic acid was sufficient to ameliorate MMBP, which was refractory to zoledronic acid. Overall, our results show that osteoclasts and multiple myeloma cooperate to induce an acidic bone microenvironment that evokes MMBP as a result of the excitation of ASIC3-activated sensory neurons. Furthermore, they present a mechanistic rationale for targeting ASIC3 on neurons along with the multiple myeloma-induced acidic bone microenvironment as a strategy to relieve MMBP in patients. Cancer Res; 77(6); 1283–95. ©2017 AACR.

This paper reviews the research on the impacts of incarceration on crime. Where data availability permits, reviewed studies are replicated and reanalyzed. Among three dozen studies I reviewed, I obtained or reconstructed the data and code for eight. Replication and reanalysis revealed significant methodological concerns in seven and led to major reinterpretations of four. I estimate that, at typical policy margins in the United States today, decarceration has zero net impact on crime outside of prison. That estimate is uncertain, but at least as much evidence suggests that decarceration reduces crime as increases it. The crux of the matter is that tougher sentences hardly deter crime, and that while imprisoning people temporarily stops them from committing crime outside prison walls, it also tends to increase their criminality after release. As a result, "tough-on-crime" initiatives can reduce crime in the short run but cause offsetting harm in the long run. A cost-benefit analysis finds that even under a devil's advocate reading of this evidence, in which incarceration does reduce crime in U.S., it is unlikely to increase aggregate welfare.

This paper reviews the research on the impacts of alcohol taxation outcomes such as heavy drinking and mortality. Where data availability permits, reviewed studies are replicated and reanalyzed. Despite weaknesses in the majority of studies, and despite seeming disagreements among the more credible ones — ones based on natural experiments — we can be reasonably confident that taxing alcohol reduces drinking in general and problem drinking in particular. The larger and cleaner the underlying natural experiment, the more apt a study is to detect impacts on drinking. Estimates from the highest-powered study settings, such as in Alaska in 2002 and Finland in 2004, suggest an elasticity of mortality with respect to price of −1 to −3. A 10% price increase in the US would, according to this estimate, save 2,000−6,000 lives and 48,000−130,000 years of life each year.

We report that transgenic mice expressing measles virus nucleocapsid protein (MVNP) in osteoclasts (OCLs) (MVNP mice) are Paget's disease (PD) models and that OCLs from patients with PD and MVNP mice express high levels of OCL-derived IGF1 (OCL-IGF1). To determine OCL-IGF1's role in PD and normal bone remodeling, we generated WT and MVNP mice with targeted deletion of Igf1 in OCLs (Igf1-cKO) and MVNP/Igf1-cKO mice, and we assessed OCL-IGF1's effects on bone mass, bone formation rate, EphB2/EphB4 expression on OCLs and osteoblasts (OBs), and pagetic bone lesions (PDLs). A total of 40% of MVNP mice, but no MVNP/Igf1-cKO mice, had PDLs. Bone volume/tissue volume (BV/TV) was decreased by 60% in lumbar vertebrae and femurs of MVNP/Igf1-cKO versus MVNP mice with PDLs and by 45% versus all MVNP mice tested. Bone formation rates were decreased 50% in Igf1-cKO and MVNP/Igf1-cKO mice versus WT and MVNP mice. MVNP mice had increased EphB2 and EphB4 levels in OCLs/OBs versus WT and MVNP/Igf1-cKO, with none detectable in OCLs/OBs of Igf1-cKO mice. Mechanistically, IL-6 induced the increased OCL-IGF1 in MVNP mice. These results suggest that high OCL-IGF1 levels increase bone formation and PDLs in PD by enhancing EphB2/EphB4 expression in vivo and suggest OCL-IGF1 may contribute to normal bone remodeling.

Multiple myeloma (MM) bone disease is characterized by the development of osteolytic lesions, which cause severe complications affecting the morbidity, mortality, and treatment of myeloma patients. Myeloma tumors seeded within the bone microenvironment promote hyperactivation of osteoclasts and suppression of osteoblast differentiation. Because of this prolonged suppression of bone marrow stromal cells’ (BMSCs) differentiation into functioning osteoblasts, bone lesions in patients persist even in the absence of active disease. Current antiresorptive therapy provides insufficient bone anabolic effects to reliably repair MM lesions. It has become widely accepted that myeloma‐exposed BMSCs have an altered phenotype with pro‐inflammatory, immune‐modulatory, anti‐osteogenic, and pro‐adipogenic properties. In this review, we focus on the role of epigenetic‐based modalities in the establishment and maintenance of myeloma‐induced suppression of osteogenic commitment of BMSCs. We will focus on recent studies demonstrating the involvement of chromatin‐modifying enzymes in transcriptional repression of osteogenic genes in MM‐BMSCs. We will further address the epigenetic plasticity in the differentiation commitment of osteoprogenitor cells and assess the involvement of chromatin modifiers in MSC‐lineage switching from osteogenic to adipogenic in the context of the inflammatory myeloma microenvironment. Lastly, we will discuss the potential of employing small molecule epigenetic inhibitors currently used in the MM research as therapeutics and bone anabolic agents in the prevention or repair of osteolytic lesions in MM. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.