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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.

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

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.

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.

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.

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.

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) 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 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.

Recent significant advances in the treatment of multiple myeloma have resulted in an improvement in median overall survival from 4.6 years, for patients diagnosed between 2001 and 2005, to 6.1 years, for those diagnosed between 2006 and 2010 (Kumar et al., 2014). However, myeloma bone lesions persist in the absence of active disease and continue to be frequent and significant causes of patient morbidity and contribute to mortality. While bisphosphonate therapy in combination with anti-myeloma therapy remains the cornerstone of skeletal disease management in myeloma, open questions regarding the optimal management of patients with myeloma bone disease remain. This article will address when to initiate and stop bone-targeted therapy in patients with monoclonal gammopathies, duration of bisphosphonate treatment in the era of more effective anti-myeloma treatment, the role of bone resorption markers in determining the dosing schedule for anti-resorptive therapy, risks and benefits of long term anti-resorptive therapy, and whether anti-resorptive therapies should be stopped to enhance the potential anabolic effects of proteasome antagonists and other anabolic agents. J. Cell. Physiol. 231: 2374-2379, 2016. © 2016 Wiley Periodicals, Inc.

Introduction: Cancer cells colonized to bone pathologically alter the bone niche to support the cancers' growth and survival. Multiple myeloma (MM) and metastatic solid tumors, particularly breast cancer (BC), activate osteoclasts and suppress osteoblasts leading to many complications and an increased mortality risk. Current strategies primarily target osteoclasts. Known osteoclast activating factor PTHrP is expressed by MM cells and BC cells and may contribute to progression. We hypothesized that alteration of PTH receptor signaling should overcome cancers' inhibition of bone formation and decrease cancer growth in bone. BDDP, a novel PTH analog, is being developed for osteoporosis treatment. We determined the ability of BDDP to reverse osteoblast suppression and slow tumor growth in bone with MM and BC models. Methods: In vitro effects of BDDP on bone genes and bone mineralization were tested in a co-culture assay of osteoblastic MC3T3-E1 cells ± human MM.1s cells. After 3 days in proliferative media, they were cultured in differentiation media. At Day 7, the expression of genes with known roles in osteoblast differentiation were assayed by RT-PCR. At Day 25, nodular calcification was stained and measured by a colorimetric assay. In vivo effects of BDDP were evaluated in an intratibial transplant model. MDA-MB-231 BC cells expressing secreted luciferase were injected into the ipsilateral tibiae of Balb/cnu/nu mice. Mice received either BDDP (40μg/kg/d) or vehicle via osmotic pump for 30 days. After euthanasia, mice were subjected to Faxitron digital X-rays to detect osteolytic lesions and DEXA scans to assess bone mineral content (BMC) and bone mineral density (BMD). Tumor-injected and non-injected tibiae were subjected to micro-QCT for 3D tibiae imaging. Results: MM.1s-treated MC3T3-E1 cells showed approximately a 5-fold reduction in osteoblast differentiation gene RUNX2 and a 3-fold reduction in BGLAP. This effect was attenuated by 100 nM BDDP by approximately 4-fold in both Runx2 and BGLAP. BDDP increased nodular calcification. Nodular calcification was suppressed in MM.1s-treated MC3T3-E1 cells. This effect was reversed by BDDP. BDDP did not directly affect MM.1s and MDA-MB-231 growths in culture but showed dramatic tumor suppression in a MDA-MB-231 intratibial transplant model. Tumor burden assayed by serial serum luciferase assay showed BDDP-treated group to be 95% lower than control, at 3 and 4 weeks post-transplant. Tumor-injected tibiae had lytic lesions in all control mice, while there were no obvious lesions in BDDP-treated mice. There were no significant changes in the trabecular bone of non-injected tibiae, whole body composition, BMC or BMD. Conclusion: BDDP reverses osteoblastic suppression by MM cells in vitro, as well as inhibiting tumor growth and preventing local bone destruction in the BC intratibial transplant model, thereby making BDDP a promising potential treatment strategy for metastatic bone cancers. Citation Format: Teja Vallapuri, Colin Crean, John Chirgwin, G. David Roodman, Attaya Suvannasankha. Effects of a bone-anabolic agent on metastatic bone cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 693.

Multiple Myeloma (MM) is an incurable disease, with a particularly poor prognosis for patients with refractory/relapsed MM (RRMM) or high-risk cytogenetics. Chimeric Antigen Receptor (CAR) T-cell therapy targeting BCMA can induce deep responses in highly pretreated RRMM; however, remissions are not sustained, and the majority of patients eventually relapse. We hypothesized that genomic determinants of MM dictate the expression of surface targets that can be of use for immune-targeting. We analyzed the gene expression of 24 immunotherapeutic targets in 1900 MM patients. In patients with t(14;16), we found that IGF1R, ITGB7, GPRC5D, CD70, TACI and ICAM1 were overexpressed compared to the t(11;14) group. In patients with t(14;20), ITGB7, GPRC5D and TACI were overexpressed, and in patients with t(4;14), IGF1R, GPRC5D, CD70, CD44, BCMA, CD138, FUT3, SLAMF7, CD56 and CD200 were overexpressed compared to the standard risk group. We found that ITGB7, CD86, CD81 and IGF1R were overexpressed in non-hyperdiploid (NHRD) and IGKC, CD138, BCMA, CD74, CD47, BST2, LY9, CD200, CD56, CD1D, ICAM1 and CD19 were overexpressed in hyperdiploid (HRD) patient samples. By stratifying patients using mutated genes, copy number events and chromosomal level changes, we found that GPRC5D, ITGB7, IGF1R and CD70 expression were increased in patients bearing 5-9 and/or 10+ drivers compared to 0-4 drivers. A gene co-expression network analysis enabled the identification of 30 gene modules highly correlating with our targets, suggesting mechanistic insights. Our results provide a means for target selection for precision CAR therapy, by considering both the patient's genomic backgrounds and cancer cell surface profiles. The quest for novel MM targets for immunotherapies remains open, and CAR target discovery driven by specific genetic events remains an active area of investigation.

Bone involvement with cancer frequently occurs and is the third most common site for cancer metastasis. Bone is only exceeded by lung, liver, and lymph nodes as a metastatic site for solid tumor metastasis (Fig. 22.1). For example, up to 90% of patients with advanced prostate cancer have bone metastasis, and 60%–70% of breast cancer patients have bone involvement with advanced disease. Bone involvement also occurs with many other cancers, including lung, kidney, and melanomas. In contrast, bone disease is rare in most hematological malignancies except for multiple myeloma (MM), which is the most frequent cancer to involve bone. Among newly diagnosed MM patients, 65% to 70% present with bone involvement and >90% have bone involvement with advanced disease. Cancer in bone is the leading cause of severe pain for patients with advanced malignancies and is the most frequent cause of cancer-related pain. Further, cancer-related bone destruction can cause systemic muscle weakness that increases the risk of falls and can result in fractures that negatively impact performance status, survival, and quality of life. These severe effects of cancer in bone result from the marked dysregulation of the normal bone remodeling process that is induced by cancer cells and results in severe imbalances in bone formation and resorption. This chapter will provide an overview of the mechanisms by which bone metastases develop and affect normal bone homeostasis, present a brief summary of current imaging techniques to evaluate the presence of cancer in bone, discuss the utility of bone resorption markers to detect and follow patients with bone metastasis, and review current treatments for and prevention of bone metastasis.

Multiple myeloma bone disease (MMBD) is characterized by non-healing lytic bone lesions that persist even after a patient has achieved a hematologic remission. We previously reported that p62 (sequestosome-1) in bone marrow stromal cells (BMSC) is critical for the formation of MM-induced signaling complexes that mediate OB suppression. Importantly, XRK3F2, an inhibitor of the p62-ZZ domain, blunted MM-induced Runx2 suppression in vitro, and induced new bone formation and remodeling in the presence of tumor in vivo. Additionally, we reported that MM cells induce the formation of repressive chromatin on the Runx2 gene in BMSC via direct binding of the transcriptional repressor GFI1, which recruits the histone modifiers, histone deacetylase 1 (HDAC1) and Enhancer of zeste homolog 2 (EZH2). In this study we investigated the mechanism by which blocking p62-ZZ domain-dependent signaling prevents MM-induced suppression of Runx2 in BMSC. XRK3F2 prevented MM-induced upregulation of Gfi1 and repression of the Runx2 gene when present in MM-preOB co-cultures. We also show that p62-ZZ-domain blocking by XRK3F2 also prevented MM conditioned media and TNF plus IL7-mediated Gfi1 mRNA upregulation and the concomitant Runx2 repression, indicating that XRK3F2's prevention of p62-ZZ domain signaling within preOB is involved in the response. Chromatin immunoprecipitation (ChIP) analyses revealed that XRK3F2 decreased MM-induced GFI1 occupancy at the Runx2-P1 promoter and prevented recruitment of HDAC1, thus preserving the transcriptionally permissive chromatin mark H3K9ac on Runx2 and allowing osteogenic differentiation. Furthermore, treatment of MM-exposed preOB with XRK3F2 after MM removal decreased GFI1 enrichment at Runx2-P1 and rescued MM-induced suppression of Runx2 mRNA and its downstream osteogenic gene targets together with increased osteogenic differentiation. Further, primary BMSC (hBMSC) from MM patients (MM-hBMSC) had little ability to increase H3K9ac on the Runx2 promoter in osteogenic conditions when compared to hBMSC from healthy donors (HD). XRK3F2 treatment enriched Runx2 gene H3K9ac levels in MM-hBMSC to the level observed in HD-hBMSC, but did not alter HD-hBMSC H3K9ac. Importantly, XRK3F2 treatment of long-term MM-hBMSC cultures rescued osteogenic differentiation and mineralization. Our data show that blocking p62-ZZ domain-dependent signaling with XRK3F2 can reverse epigenetic-based mechanisms of MM-induced Runx2 suppression and promote osteogenic differentiation.