Musculoskeletal diseases are a major health burden. Development of bone-active therapies has been hindered by limited understanding of the cells and genes that regulate the skeleton. We exploited the value of cross-species analysis and developed single-cell methodologies in skeletal tissues to define the critical endosteal compartment that regulates bone turnover. Thirty-four distinct cell types were identified, and disease-relevant cells prioritized using enrichment for rare skeletal disorder genes and bone-mineral-density-associated genes in an extended UK Biobank genome-wide association study. Functional validation was undertaken in over 1,000 genetically modified mouse models. Endothelial cells and vascular smooth muscle cells were identified as new skeletal-disease-relevant cells alongside osteoblast, chondrocyte and osteoclast cell lineages. Hundreds of cell-specific genes with unappreciated roles in skeletal pathophysiology were identified. This comprehensive cellular and molecular framework underpins skeletal physiology and disease and will help prioritize new therapeutic targets to accelerate development of therapies to treat musculoskeletal disease.

Multiple myeloma (MM), the second-most frequent hematologic malignancy, is caused by the neoplastic expansion of clonal plasma cells. Up to 80% of MM patients develop myeloma bone disease (MMBD), a hallmark of which is the development of osteolytic lesions that can lead to skeletal related events such as fractures, spinal cord compression and bone pain. In the Vk*MYC mouse model of MM, MYC is expressed in germinal center B cells, facilitating the formation of malignant plasma cells and development of major characteristics of MM, including MMBD. However, the extent of skeletal changes and underlying cellular mechanisms in the Vk*MYC model has, to date, not been comprehensively described. Here, we provide such comprehensive characterization of MMBD and the associated structural and cellular changes in the femur of mice inoculated with the Vk*MYC cell line Vk14451. Vk*MYC mice developed significant osteolytic lesions in the femur. Serum analysis revealed a notable decrease in the bone formation marker amino terminal propeptide of type I procollagen (P1NP) (-33.3%, p < 0.01) in Vk*MYC mice compared to naïve control mice, while C-telopeptide of type I collagen (CTX-1) levels remained unchanged. Microcomputed tomography (microCT) analysis demonstrated significant deterioration of the trabecular bone structure in Vk*MYC as compared to naïve control mice, evidenced by reductions in bone volume fraction (-49.2%, P < 0.05), bone surface to bone volume ratio (-22.3%, P < 0.001), trabecular number (-46.3%, P < 0.0001) and connectivity density (-79.5%, P < 0.05), as well as increased trabecular separation (+86.3%, P < 0.0001) and thickness (+20.3%, P < 0.001). Cortical bone analysis indicated increased cortical porosity (+164.5%, P < 0.0001) and decreased thickness (-26.9%, P < 0.001) in the Vk*MYC mice. Histological studies revealed a decrease in osteoblast surface relative to bone surface (-49.7%, P < 0.001) and an increase in osteoclast surface relative to bone surface (+53.9%, P < 0.01). Collectively, our findings indicate significant bone loss and development of MMBD in the Vk*MYC mouse model of MM, driven by uncoupled bone remodeling characterized by decreased osteoblast activity and increased osteoclast burden. These data highlight the relevance of the Vk*MYC 14451 syngeneic model of myeloma in studies aiming to explore MM bone disease in immunocompetent C57Blk6 mice.

Rebound bone loss following denosumab discontinuation is an important barrier in the effective long-term treatment of skeletal disorders. This is driven by increased osteoclastic bone resorption following the offset of RANKL inhibition, and sequential osteoclast-directed therapy has been utilized to mitigate this. However, current sequential treatment strategies intervene following the offset of RANKL inhibition and this approach fails to consistently prevent bone loss. Our previous work, using a mouse model of denosumab discontinuation, has shown that the processes that drive the rebound phenomenon occur earlier than when bone loss is detected, namely a rise and overshoot in serum tartrate-resistant acid phosphatase (TRAP). We identified that these changes in serum TRAP may provide an earlier window of opportunity to intervene with sequential therapy following RANKL inhibition withdrawal. Here, we show that early treatment with zoledronate (10 mg/kg, 3 wk following the last dose of OPG:Fc), preceding the rise and overshoot in serum TRAP, effectively mitigates rebound bone density loss through preventing the overshoot in serum TRAP. Further, we show that multiple doses of zoledronate (early treatment and during anticipated BMD loss) is superior in consolidating bone density gains made with RANKL inhibition and preventing rebound BMD loss as measured by DXA. Importantly, we demonstrate the efficacy of early and multi-dose zoledronate strategy in preventing bone loss in both growing and skeletally mature mice. MicroCT analysis showed improved trabecular bone structure in both the femur and lumbar vertebrae with zoledronate treatment compared with control. These increases in bone mass translated to increased fracture resistance in skeletally mature mice. This work provides a novel approach of early and multi-dose sequential treatment strategy following withdrawal of RANKL inhibition, contributing valuable insight into the clinical management of patients who discontinue denosumab therapy. Stopping denosumab leads to loss of bone gained during treatment, due to increased bone resorption when denosumab wears off. Current strategies often fail to prevent this as they cannot stop osteoclasts resorbing bone. Our work using a mouse model has shown that processes that lead to bone loss start earlier than we can detect in the clinic. We show that early and multi-dose zoledronate treatment, another medication used to block osteoclasts, to target these earlier processes can prevent bone loss after stopping denosumab. This approach offers a new strategy for managing bone health in patients stopping denosumab.

Multiple myeloma (MM) remains incurable due to disease relapses, thought to stem from the activation of dormant MM cells. Preventing the awakening or eliminating dormant MM cells is crucial to avoiding disease relapse. In this study, we sought to identify targetable vulnerabilities to eliminate or control dormant cells. First, we used a scRNAseq dataset of dormant MM cells and found that dormant MM cells exhibit upregulation of Notch pathway genes compared to proliferating MM cells. Based on this, we hypothesized that inhibiting Notch signals in the tumor niche should decrease dormant MM cells. To test this notion, we injected immunocompetent mice i.v. with murine 5TGM1 MM cells double-labeled with a fixed genetic reporter and a membrane dye (DiD) retained by non-dividing cells, allowing us to distinguish dormant (GFP+DID+) from proliferating cells (GFP+DID-). After 2wks, we randomized mice to groups receiving vehicle or a bone-targeted Notch inhibitor (BT-GSI). We detected a population of GFP+DiD+ dormant cells that expressed high levels of Axl and were growth arrested throughout the life span of the mice. Short-term treatment with BT-GSI (1wk) reduced tumor burden (75%) and decreased the number of dormant MM cells (70%) via apoptosis. Prolonged BT-GSI treatment (7 wks) eliminated all dormant MM cells and further reduced proliferating cells. These effects were replicated in immunodeficient mice injected with RFP+DID+ human OPM2 MM cells and in human bones infiltrated by OPM2 RFP+DID+ cultured ex vivo. Next, we explored if modulating the tumor niche could influence MM cell dormancy. Since dormant MM cells colocalize with osteoblasts in the endosteal bone surface, we assessed if increasing osteoblasts numbers and their activity prevents the awakening of MM dormant cells or enhances their engagement in dormancy programs. To test this, we injected GFP+DiD+ MM cells and, after two weeks, randomized mice to groups receiving vehicle or Scl-Ab, an anabolic agent that rapidly increases osteoblasts. Scl-Ab (2wks) increased osteoblasts but did not affect the proportion of proliferating or dormant MM cells. Similar results were observed in human bones infiltrated with human MM cells cultured ex vivo treated with Scl-Ab. Next, we pre-treated mice with Scl-Ab (2wks) to generate a microenvironment loaded with osteoblasts before MM cell injection. Despite the increase in osteoblasts, Scl-Ab did not impact the number of dormant or proliferating MM cells. Further, co-administration of Scl-Ab did not interfere with BT-GSI’s ability to eliminate proliferating or dormant MM cells in any of these models. In conclusion, our findings show that 1) Notch signals promote the survival of dormant MM cells, 2) manipulating osteoblasts in the tumor niche does not affect MM cell dormancy, and 3) bone-targeted Notch inhibition is a promising approach to eradicate dormant tumor cells and potentially delay/prevent disease relapse in MM patients. Hayley M. Sabol, Aric Anloague, Japneet Kaur, Sharmin Khan, Bethany Paxton, James Smith, Michelle McDonald, Peter Croucher, C. Lowry Barnes, Elena Ambrogini, Alison Frontier, Frank H. Ebetino, Jesus Delgado-Calle. Notch inhibition as a therapeutic approach to eliminate dormant cancer cells in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5099.

Multiple myeloma (MM) is an incurable hematologic malignancy characterized by the uncontrolled proliferation of bone marrow resident plasma cells (PCs). Two members of the TAM (TYRO3, AXL, and MER) receptor family have previously been implicated in distinct aspects of neoplastic PC biology. AXL expression in MM PCs has been associated with the induction of a dormant, noncycling state within the bone marrow, whereas expression of MER has been implicated in PC proliferation and survival. Here, the generation of single TAM receptor-expressing 5TGM1 murine MM cell lines enabled the individual functional assessment of the effects of Axl and Mer receptor expression on MM development. Axl expression did not affect proliferation, cell cycling, or stromal cell-induced dormancy in vitro. Development of 5TGM1 tumors in C57BL/KaLwRij mice was also unaltered by Axl expression. By contrast, Mer expression conferred an increase in cell proliferation to 5TGM1 cells in vitro and increased 5TGM1 tumor burden in C57BL/KaLwRij mice. The protumorigenic properties of Mer were only observed following intravenous cell delivery into mice with an intact adaptive immune system. Thus, Axl is neither necessary nor sufficient for the induction of MM cancer cell dormancy, whereas MER remains a promising target for therapeutic intervention in patients with MM.

The risk of progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) increases with advancing age, suggesting that progression may be influenced by age-related changes within the bone marrow (BM) microenvironment. We hypothesise that senescent mesenchymal stromal cells (MSCs), which accumulate in the BM with age, may contribute to MGUS progression to MM. Here, we show that, like BM MSCs from aged non-cancer controls, BM MSCs from both MM and MGUS patients exhibit a senescent phenotype characterised by enlarged, flattened morphology, increased β-galactosidase activity and CDKN2A expression, and decreased proliferation rate compared with BM MSCs from healthy young individuals. While coculture with BM MSCs suppresses the proliferative capacity of MM cell lines in vitro, induction of senescence via irradiation or replicative exhaustion in healthy MSCs relieves this suppression, compared with non-senescent MSCs. This may, in part, be attributable to upregulated expression of the BMP antagonist Gremlin1 in senescent MSCs, which facillitates MM cell proliferation. Notably, the risk of progression to MM was significantly elevated in MGUS patients with increased MSC senescence. Collectively, our data provide evidence that age-related accumulation of senescent MSCs may be a driver of MGUS to MM progression.

Rebound bone loss following denosumab discontinuation is an important barrier in the effective long-term treatment of skeletal disorders. This is driven by increased osteoclastic bone resorption following the offset of RANKL inhibition, and sequential osteoclast-directed therapy has been utilised to mitigate this. However, current sequential treatment strategies intervene following the offset of RANKL inhibition and this approach fails to consistently prevent bone loss. Our previous work, using a mouse model of denosumab discontinuation, has shown that the processes that drive the rebound phenomenon occur earlier than when bone loss is detected, namely a rise and overshoot in serum TRAP. We identified that these changes in serum TRAP may provide an earlier window of opportunity to intervene with sequential therapy following RANKL inhibition withdrawal. Here, we show that early treatment with zoledronate (10 mg/kg, 3 weeks following the last dose of OPG:Fc), preceding the rise and overshoot in serum TRAP, effectively mitigates rebound bone density loss through preventing the overshoot in serum TRAP. Further, we show that multiple doses of zoledronate (early treatment and during anticipated BMD loss) is superior in consolidating bone density gains made with RANKL inhibition and preventing rebound BMD loss as measured by DXA. Importantly, we demonstrate the efficacy of early and multi-dose zoledronate strategy in preventing bone loss in both growing and skeletally mature mice. MicroCT analysis showed improved trabecular bone structure in both the femur and lumbar vertebrae with zoledronate treatment compared to control. These increases in bone mass translated to increased fracture resistance in skeletally mature mice. This work provides a novel approach of early and multi-dose sequential treatment strategy following withdrawal of RANKL inhibition, contributing valuable insight into the clinical management of patients who discontinue denosumab therapy.

Summary: Understandably, conventional therapeutic strategies have focused on controlling primary tumors. We ask whether the cost of such strategies is actually an increased likelihood of metastatic relapse.

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF , that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

Supp. Figure 1. Overexpression of HIF-2α and CXCL12 in the HMCLs LP-1 and RPMI-8226

<p>Supp. Figure 4. CXCL12 does not inhibit response to CCL3 in human MM cell lines</p>

PDF file - 50KB, Numbers of MDA-MB-231 cells present in long bones of 12 week old mice 24h after inter-cardiac injection.

Table of genes dysregulated with overexpression of HIF-2alpha in LP-1 cells

Cell fate is commonly studied by profiling the gene expression of single cells to infer developmental trajectories based on expression similarity, RNA velocity, or statistical mechanical properties. However, current approaches do not recover microenvironmental signals from the cellular niche that drive a differentiation trajectory. We resolve this with environment-aware trajectory inference (ENTRAIN), a computational method that integrates trajectory inference methods with ligand-receptor pair gene regulatory networks to identify extracellular signals and evaluate their relative contribution towards a differentiation trajectory. The output from ENTRAIN can be superimposed on spatial data to co-localize cells and molecules in space and time to map cell fate potentials to cell-cell interactions. We validate and benchmark our approach on single-cell bone marrow and spatially resolved embryonic neurogenesis datasets to identify known and novel environmental drivers of cellular differentiation. ENTRAIN is available as a public package at https://github.com/theimagelab/entrain and can be used on both single-cell and spatially resolved datasets.

Abstract Disclosure: A.S. Kim: None. A. Castro-Martinez: None. V. Taylor: None. J. Center: None. C.M. Girgis: None. P.I. Croucher: None. M.M. Mcdonald: None. Denosumab is an effective osteoporosis treatment, preventing bone loss by inhibiting RANKL. However, stopping denosumab leads to rebound bone mineral density(BMD) loss. This is due to accelerated bone resorption by osteoclasts. Serum bone turnover markers such as P1NP and CTX have been utilised in clinical practice to guide sequential therapy following denosumab discontinuation. However, an optimal strategy has not been established. Understanding the temporal changes in osteoclast activity will guide safe, effective sequential therapy following denosumab discontinuation. We hypothesised that serum TRAP5b, a marker of enzymatic activity of osteoclasts, would be a more useful marker in this context to guide sequential treatment following denosumab discontinuation. Seven-week-old female C57BL/6 mice were treated with 2-weeks of thrice-weekly OPG:Fc(10mg/kg) to inhibit RANKL then withdrawn from therapy (OPG-W) or saline(vehicle). Longitudinal BMD and serum TRAP5b were measured throughout the study. Mice were harvested at weeks 2, 8, 11 and 13 to allow a large volume of serum to be collected to concurrently measure serum TRAP5b and the products of collagen formation and breakdown, serum P1NP and CTX respectively . Following OPG:Fc withdrawal, BMD peaked at week 8 in OPG-W mice (92.94 vs75.09mg/cm2, p<0.0001), started to decline at week 10 and normalised to vehicle levels by week 13. Longitudinally, serum TRAP was suppressed by week 2 (0.05 vs 11.90U/L, p<0.0001) and remained suppressed until week 8, following which serum TRAP levels rose progressively to 64% above vehicle levels at week 12 (17.86 vs 10.92U/L, p<0.0001). The rise in TRAP between weeks 8 to 10 preceded the decline in BMD.Serum TRAP was significantly elevated in OPG-W mice at week 11 (15.45 vs 11.46U/L, p=0.01) whereas serum P1NP and CTX remained equivalent to vehicle levels. Serum TRAP, P1NP and CTX were significantly higher in OPG-W mice at week 13 by which time BMD had reached vehicle levels. Our findings show that rebound decline in BMD has already occurred by the time bone turnover markers used in clinical practice (P1NP and CTX) rise above vehicle levels. A significant overshoot in serum TRAP occurs earlier and prior to bone loss and may better inform sequential therapy following denosumab discontinuation. Presentation: Friday, June 16, 2023

Real-time intravital two-photon microscopy showing phagocytosis of a large, granular clump of bisphosphonate by a single tumour-associated macrophage.

Supp. Figure 3. CCR1 expression is upregulated by hypoxia in human MM cell lines

<p>Supp. Figure 5. Cell surface expression of CXCR4 and CCR1 in newly diagnosed MM patients</p>