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.

Complement factor H (CFH) is an immune regulator that inhibits the complement system. Here, we identify CFH as a secreted and regulated factor in human bone marrow skeletal stem cells (hBMSCs) culture during osteoblast (OB) differentiation. To explore its role in bone formation, we investigated the effects of CFH on OB differentiation and bone homeostasis. CFH-deficient hBMSCs exhibited impaired OB differentiation, whereas CFH overexpression or supplementation enhanced OB differentiation in hBMSCs. In vivo, CFH-deficient (CFH-/-) mice displayed reduced trabecular and cortical bone mass, decreased bone formation, and diminished bone strength. Primary CFH-/- mouse bone marrow MSCs (mBMSCs) showed reduced osteogenesis but enhanced osteoclast differentiation, consistent with lower serum levels of the bone formation marker P1NP and elevated bone resorption markers TRAPc and CTX-1. Furthermore, CFH-/- mice exhibited delayed fracture healing and accelerated bone loss following ovariectomy or high-fat diet feeding. Clinically, CFH levels in bone marrow plasma were negatively correlated with fracture risk in patients. Notably, plasma CFH levels were positively associated with bone mineral density (BMD) and were significantly reduced in patients with osteoporosis. These findings establish CFH as a key regulator of osteogenesis and bone homeostasis, with potential implications for bone-related disorders.

Currently, no consensus exists on either terminology, definition, or the biological significance of the phenomena intratrabecular tunneling. Despite this, observations of intratrabecular tunneling are frequently reported in literature covering diseases or treatments involving parathyroid hormone or kidneys. A few attempts to quantify this phenomena have been made mainly based on bone resorption, despite the concurrent presence of bone formation. This study demonstrates that intratrabecular tunneling is a previously unrecognized physiological mode of intratrabecular bone remodeling occurring across conditions and ages. Similar to intracortical remodeling, it is induced by PTH-treatment and creates an extensive interconnected tunnel-network that hollows out the trabeculae. This study primarily utilizes iliac crest bone biopsies collected from a clinical trial where patients with hypoparathyroidism were randomized to receive daily injections with either 100μg rhPTH(1-84) or placebo as add-on to conventional therapy for 6 months. In addition, further bone biopsies were collected from a 24-month open-label extension study, including patients receiving either only conventional treatment, continued rhPTH-treatment or discontinued rhPTH-treatment. Histomorphometry demonstrated that PTH-treatment induced an 18-fold and 36-fold increase in intratrabecular porosity after 6 and 30 months of treatment, respectively. After 6-months of PTH- versus conventional treatment, a median 7.7% versus 0.0% were eroded pores, 69.1% versus 0.0% were eroded-formative pores, 12.9% versus 0.0% were formative pores and 0.9% versus 81.2% were quiescent pores. PTH-treatment withdrawal normalized the intratrabecular remodeling to levels similar to conventional therapy. The intratrabecular remodeling mainly occurred in plates and junctions of trabeculae, and not in trabecular rods. Intratrabecular remodeling parameters showed a positive correlation with PTH-induced trabecular mineralization and a negative correlation with active vitamin D-supplementary doses. Dynamics of the PTH-induced intratrabecular remodeling could be tracked using time-lapsed synchrotron radiation μCT in a rabbit model and the complexity of the trabecular strain environment was confirmed with micro-finite-element analysis.

Stromal progenitor cells of bone marrow origin are non-hematopoietic cells that give rise to osteoblasts and adipocytes in the postnatal organism. Marrow stromal cells (also known as mesenchymal stem cells - MSCs) are currently being employed in a large number of clinical trials for regenerative purposes post in vitro expansion. However, the clinical outcome has been variable, which might in part be due to the heterogeneity of the cells and the lack of a defined cell product with a molecular signature that favors tissue regeneration. In this study, we determined the cellular heterogeneity of primary stromal cultures and examined how inter-donor variation in subpopulation composition contributes to the differentiation potential of primary cultures. We profiled 136 014 stromal progenitors from 26 donors and identified 5 subpopulations that were linked to distinct bone-related pathways and genetic traits of bone mineral density and morphology. Abundance of one cluster characterized by high expression of ITGA11 (integrin alpha-11) and genes related to matrix function, collagen organization, and elevated expression upon lineage commitment was positively correlated with osteoblastic differentiation capacity in vitro. In addition, ITGA11 protein expression in progenitor cells was a predictive marker for matrix mineralization in vitro and ectopic bone formation in vivo. Sorting stromal progenitors into ITGA11high and ITGA11low cells established cultures with high and low osteoblastic differentiation potential and revealed transcriptional differences reflective of the subpopulation-specific signature, which was not affected by siRNA-mediated knockdown of ITGA11 expression. Our findings corroborate the presence of an extensive donor-dependent cellular heterogeneity that persists in cultured stromal cells, and that ITGA11 can be employed as a marker for isolating cells with high bone-forming potential, a feature likely to benefit clinical trials of bone regeneration.

Intermittent PTH treatment has been used as both an osteoanabolic treatment in osteoporosis and a hormone replacement in hypoparathyroidism for many years. This scoping review compiles and reinterprets studies using histomorphometry supported by bone turnover markers to investigate the elusive cellular effect of intermittent PTH treatment locally within the bone, while illuminating knowledge gaps. Intermittent PTH increases both osteoclast and osteoblast activity within the first 6 months of treatment. Based on the combination of systemic bone turnover markers and histomorphometry we suggest that in osteoporosis, the activity of the individual osteoclast increases within the first 18 months of treatment. During the initial 6 months, osteoblast activation increases bone formation, whereafter bone formation returns to baseline after 7–18 months of treatment. Based on the studies available after 24 months of treatment, more osteoclasts populate the bone surfaces, but the individual osteoclast may potentially be less active. At the same time, osteoblastic bone formation appears to be reactivated. In hypoparathyroidism, treatment up to 72–120 months increases bone formation and normalizes it to a level of matched healthy controls, while the osteoclast remains largely uninvestigated. The increase in bone forming surfaces in both osteoporosis and hypoparathyroidism may partly be achieved by rejuvenating arrested eroded surfaces accumulated during disease. Contrary to early beliefs, modeling-based bone formation (MBF) is not a major contributor to PTH-induced bone formation. Rather osteoclast-initiated bone formations such as remodeling-based bone formation (RBF) and overflow remodeling-based bone formation (oRBF) are the predominate modes of bone formation, underlining a need for further investigations into possible effects of previous osteoclast-inhibiting anti-resorptive treatment.

Larsen syndrome is a rare genetic condition characterized by facial dysmorphism and skeletal deformities. It is caused by heterozygous pathogenic variants in the Filamin B encoding gene (FLNB). FLNB is a cytoskeletal protein that plays a key role in bone morphogenesis; however, the skeletal phenotype of Larsen syndrome has not been described in detail. Here, we studied the skeletal presentation in two subjects with Larsen syndrome. A case-study including a 63-year-old women and her 33-year-old daughter with Larsen syndrome, both carrying a novel FLNB c.688G > T, p.(Val230Phe) variant. The bone morphologic evaluation included, radiographs, bone mineral density assessment, and high-resolution peripheral quantitative tomography (HR-pQCT). In addition, a transiliac crest bone biopsy from the mother was evaluated by µCT, histomorphometry, and in situ examination of FLNB expression within physiological human bone remodeling sites of controls. Both women were diagnosed with severe osteoporosis (T-score < -5). The HR-pQCT analysis showed a low trabecular bone volume, as well as a low cortical thickness compared to a healthy cohort. Histomorphometry and µCT analysis of the iliac bone biopsy confirmed low cortical thickness, and revealed a high density of small eroded and quiescent intracortical pores. The trabecular bone remodeling was not affected, while cortical remodeling events accumulated as small eroded pores and quiescent pores with an improved infilling. The FLNB variant is associated with low bone mineral density reflecting severe osteoporosis and an altered trabecular and cortical bone structure, while bone turnover was less affected at the time of analysis.

Neovascular age-related macular degeneration and diabetic macular edema are leading causes of vision loss evoked by retinal neovascularization and vascular leakage. The glycoprotein microfibrillar-associated protein 4 (MFAP4) is an integrin aVb3/5/6 ligand present in the extracellular matrix. Single-cell transcriptomics reveal MFAP4 expression in cell types in close proximity to vascular endothelial cells, including choroidal vascular mural cells, retinal astrocytes, and M & uuml;ller cells. Binding of the anti-MFAP4 antibody, hAS0326, makes MFAP4 inaccessible for integrin receptor interaction, and thereby hAS0326 blocked endothelial cell motility in vitro. Intravitreal hAS0326 inhibited retinal vascular lesion area and neovessel volume in a laser-induced choroidal neovascularization mouse model, vascular permeability in streptozotocin-induced retinopathy, and vascular leakage area in a chronic non-human primate model of DL-2-aminoadipic acid-induced retinopathy. 12 weeks in the latter model. Moreover, hAS0326 treatment tion of integrin binding. Our data suggest that hAS0326 constitutes a promising treatment of neovascularization and vascular leakage in retinal diseases.

An increasing number of studies have characterized the bone as an endocrine organ, and that bone secreted factors may not only regulate local bone remodeling, but also other tissues and whole-body metabolic functions. The precise nature of these regulatory factors and their roles at bridging the bone, bone marrow adipose tissue, extramedullary body fat and whole-body energy homeostasis are being explored. In this study, we report that KIAA1199, a secreted factor produced from bone and bone marrow, previously described as an inhibitor of bone formation, also plays a role at promoting adipogenesis. KIAA1199-deficient mice exhibit reduced bone marrow adipose tissue, subcutaneous and visceral fat tissue mass, blood cholesterol, triglycerides, free fatty acids and glycerol, as well as improved insulin sensitivity in skeletal muscle, liver and fat. Moreover, these mice are protected from the detrimental effects of high-fat diet feeding, with decreased obesity, lower blood glucose and glucose tolerance, as well as decreased adipose tissue inflammation, insulin resistance and hepatic steatosis. In human studies, plasma levels of KIAA1199 or its expression levels in adipose tissue are positively correlated with insulin resistance and blood levels of cholesterol, triglycerides, free fatty acids, glycerol, fasting glucose and HOMA-IR. Mechanistically, KIAA1199 mediates its effects on adipogenesis through modulating osteopontin-integrin and AKT / ERK signaling. These findings provide evidence for the role of bone secreted factors on coupling bone, fat and whole-body energy homeostasis.

Myeloma bone disease (MBD), a common complication in multiple myeloma (MM), causes increased risk of fractures leading to morbidity and impaired quality of life for patients. Proteasome inhibitors, a cancer-targeting therapy for MM, have been shown to have a beneficial off-target bone anabolic effect. However, side effects and toxicities of proteasome inhibitors limits their long-term use, especially in patients in disease remission. Ixazomib is an oral proteasome inhibitor with anti-myeloma effect, but a less severe toxicity profile compared to other approved proteasome inhibitors. To investigate the effect of ixazomib on MBD, we conducted a single-center clinical study where 30 patients with MM in remission received ixazomib, and evaluated bone-specific changes through serum markers, imaging, cell cultures, and bone histomorphometry. We have previously shown that short-term ixazomib treatment (3 months) induces increased trabecular bone volume and formation of enlarged bone structural units (BSU) without changing osteoblast number or activity. Here, we present evidence that long-term (24 months) ixazomib treatment inhibits the activation of new bone remodeling events through attenuation of both bone resorption and formation. The initial gains in percentage of superficial trabecular BSU bone volume remained stable and the proportion of large BSUs containing woven bone decreased, suggesting improved bone mineralization over time. Overall, our results indicate that long-term ixazomib treatment led to prolonged bone formation events during the initial treatment phase, followed by inhibition of new bone resorption and its coupled bone formation, preserving the gained bone and possibly preventing advancement of MBD in patients with MM in remission. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov ID NCT04028115.

Objective: Fracture risk is increased in longstanding type 2 diabetes (T2D). High-resolution peripheral quantitative CT scans have demonstrated higher cortical porosity in T2D complicated by microvascular disease (MVD). We investigated if cortical bone resorption is followed by inadequate bone formation in individuals with T2D complicated by MVD. Methods: Thirty-five adult men and women with T2D were recruited from outpatient clinics and through public advertisement. All participants had at least one previous measure of c-peptide >700, a negative GAD antibody test, and 13 had known microvascular disease status. Trans iliac crest bone biopsies were collected for histomorphometric analysis. Glucose control was assessed using HbA1c. Additionally, trans iliac bone specimens from 10 individuals without T2D were included as controls. Results: Following quality assessment, samples from 30 T2D and 10 controls were used for histomorphometric analyses of cortical bone remodelling. The final study population included 23 men and 7 postmenopausal women with a mean age of 65.8 years for the T2D-MVD group (CI95% 61.2-70.3) and 65.2 years in the T2D + MVD group (CI95% 59.6-70.9), and a mean T2D disease duration of 16.9 years. Seventeen had MVD (57 %). The controls included 5 men and 5 women with a mean age of 64.7 years (CI95% 58.5-70.9). The area, diameter, and density of cortical pores were the same in cases with and without MVD, but the pore diameter was lower than controls. While T2D had significantly more eroded-formative pores compared to controls, there were no significant differences in the proportion of eroded and formative pores between the groups. In quiescent pores/ osteons, the osteon diameter and wall thickness were larger in T2D groups than controls. Conclusion: Cortical bone porosity was not increased in individuals with T2D complicated by MVD. However, an enhanced prevalence of eroded-formative pores and increased osteon diameter concur with a slightly prolonged reversal-resorption phase in T2D irrespective of the presence of MVD.

IntroductionIntestinal failure-associated liver disease is a potentially life-threatening complication in patients with intestinal failure (IF) receiving home parenteral support (HPS). The aim of this study was to evaluate liver stiffness and steatosis non-invasively in adult patients with IF receiving long-term HPS.MethodsPatients with IF on a stable HPS prescription (≥4 days/week for ≥10 weeks) were included in this cross-sectional study. Liver stiffness measurements (LSMs) were assessed by transient elastography, and steatosis by MRI-proton density fat fraction (MRI-PDFF). Blood samples were analyzed for liver function. Data were compared to sex- and age-matched controls.ResultsA total of 33 patients (median age, 57 years; BMI, 23 kg/m2) and 14 controls (median age, 58 years; BMI, 24 kg/m2) were examined. Patients had higher LSMs compared to controls (5.6 kPa vs. 4.3 kPa; p = 0.0079). Elevated LSMs (>7 kPa) were seen in 37% of patients vs. 0% in controls (p = 0.0085). LSMs were associated with HPS energy (p = 0.026) and lipid content (p = 0.029). Median MRI-PDFF was 1.8% in patients and 1.5% in controls (p = 0.43). Nineteen percent of patients exhibited elevated MRI-PDFF (≥8%) vs. 7% in controls (p = 0.41). Alkaline phosphatase and gamma-glutamyltransferase levels >1.5x the ULN were observed in 21 and 45% of patients, respectively. Overall, these results indicate that in this adult IF population, elevated LSMs and a cholestatic pattern in liver enzymes were more prevalent than steatosis as assessed by MRI-PDFF. Patients at risk of increased liver stiffness appeared to have severe IF as indicated by high HPS energy and lipid requirements.

Multiple myeloma (MM) is a plasma cell (PC) malignancy that is preceded by monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering multiple myeloma (SMM). MGUS and SMM PCs exhibit the same primary oncogenic abnormalities as MM but lack the end-organ damage that defines proliferative disease, suggesting that clonal PCs in these precursor conditions could exhibit senescence or senescence-like growth arrest. Herein we identified monoclonal gammopathy patient-derived PCs that exhibit senescence features and found that senescent PCs were significantly increased in MGUS patients compared to SMM or MM. Spatial analysis of senescent PCs in stable MGUS and SMM patient biopsies demonstrated the activation of local paracrine senescence in the bone marrow microenvironment. Stable MGUS and SMM patients also exhibited disease-specific senescence-associated secretory phenotype (SASP) signatures that significantly correlated with PC burden and clonal antibody. In contrast, progressing MGUS, SMM, and new MM patients lacked local paracrine senescence responses and robust activation of disease specific SASP signatures. Overall, these data suggest that failure to activate tumor-specific paracrine senescence responses is key to disease progression in monoclonal gammopathies.

Solar UVB light causes damage to the outermost layer of skin. This insult induces rapid local responses, such as dermal inflammation, keratinocyte cell death, and epidermal thickening, all of which have traditionally been associated with DNA damage response signaling. Another stress response that is activated by UVB light is the ribotoxic stress response (RSR), which depends on the ribosome-associated mitogen-activated protein 3 kinases (MAP3K) ZAKα and culminates in p38 and JNK activation. Using ZAK knockout mice, we here show that it is the RSR that is responsible for the early manifestation of UVB-induced skin inflammation and keratinocyte death and subsequent proliferation in vivo. We also show that the RSR controls both p38-mediated pyroptotic and JNK-mediated apoptotic programmed cell death of human keratinocytes in vitro. In sum, our work highlights that skin cells rely on a cytoplasmic and ribosomal stress signal rather than a nuclear and DNA-templated signal for rapid inflammatory responses to UV exposure.

Denosumab is a monoclonal anti-RANKL antibody that inhibits bone resorption, increases bone mass, and reduces fracture risk. Denosumab discontinuation causes an extensive wave of rebound resorption, but the cellular mechanisms remain poorly characterized. We utilized in situ hybridization (ISH) as a direct approach to identify the cells that activate osteoclastogenesis through the RANKL/OPG pathway. ISH was performed across species, skeletal sites, and following recombinant OPG (OPG:Fc) and parathyroid hormone 1-34 (PTH) treatment of mice. OPG:Fc treatment in mice induced an increased expression of RANKL mRNA mainly in trabecular, but not endocortical bone surface cells. Additionally, a decreased expression of OPG mRNA was detected in bone surface cells and osteocytes of both compartments. A similar but more pronounced effect on RANKL and OPG expression was seen one hour after PTH treatment. These findings suggest that bone surface cells and osteocytes conjointly regulate the activation of osteoclastogenesis, and that OPG:Fc treatment induces a local accumulation of osteoclastogenic activation sites, ready to recruit and activate osteoclasts upon treatment discontinuation. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data from murine bone marrow stromal cells revealed that Tnfsf11+ cells expressed high levels of Mmp13, Limch1, and Wif1, confirming their osteoprogenitor status. ISH confirmed co-expression of Mmp13 and Tnfsf11 in bone surface cells of both vehicle- and OPG:Fc-treated mice. Under physiological conditions of human/mouse bone, RANKL is expressed mainly by osteoprogenitors proximate to the osteoclasts, while OPG is expressed mainly by osteocytes and bone-forming osteoblasts.

Bone formation is critical to maintain bone integrity. Here, we studied the importance of intact energy metabolism for bone formation in humans. The skeletal impact of impaired oxidative phosphorylation (OXPHOS) was investigated in adult individuals with genetically defective mitochondrial DNA translation (m.3243A>G). Although impaired mitochondrial ATP production in m.3243A>G human bone marrow stromal cells (hBMSC) was compensated by increased glycolytic ATP production (unchanged net ATP production), both in vitro osteoblast differentiation and in vivo ectopic bone formation were decreased. The impaired OXPHOS was associated with mitochondrial stress and disruption of the pro-osteogenic transcriptional program characteristic of hBMSC. Supporting OXPHOS pharmacologically in hBMSC restored mitochondrial ATP production, their transcriptional program and metabolism, leading to upregulation of osteogenic genes and restoration of bone formation capacity. These findings demonstrate a mitochondrial regulation mechanism of the osteogenic capacity of hBMSCs and identify OXPHOS as a potential target for increasing bone formation. ### Competing Interest Statement J.S (Founder and CEO) and H.R. (CSO) are employees of Khondrion, a mitochondrial medicine company. During the final stages of this manuscript preparation, M.F. became an employee of Novo Nordisk A/S (1st June 2024) and P.F.G. an employee of Agilent Technologies Inc (1st August 2024). All experiments, data collection and analysis of this study, as well as the conclusions of this study were done prior to both M.F. and P.F.G. current employments (October 2023). Therefore, ensuring no influence of their current employments on the content of this article. Furthermore, the authors P.F.G., M.F., A.L.F., H.R., and J.S. have a pending patent application related to this work. The patent application was filed after the experiments, data collection and analysis presented in this manuscript were completed, and did not influence the research process, content or conclusions of this article. The other authors declare no competing interests.

Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy. Despite therapeutic advances that have vastly improved survival, all patients are expected to relapse. Current standard of care includes autologous stem cell transplant (ASCT) with high-dose melphalan (HDM) administered as a conditioning agent. Cytotoxic chemotherapies such as melphalan have been shown to drive therapy-induced senescence (TIS) in several tissues. We hypothesize that HDM activates TIS pathways in surviving MM cells and that this may correlate with patient outcomes post-ASCT. We developed an in vitro HDM model using 5TGM1 mouse MM cells treated with vehicle (Veh) or HDM (10µM, IC90) for 6h, followed by co-culture with primary mouse bone marrow stromal cells. Image analysis was performed to quantify proliferation by cumulative population doublings (CPDs) over 10 days, after which 5TGM1 cells were evaluated for cell size, telomere-associated DNA damage foci (TAFs), sensitivity to senolysis, and senescence gene expression (scRNA-seq and RT-qPCR). The effect of HDM on CPDs and cell size was also evaluated in human MM cell lines RPMI-8226 and H929. scRNA-seq was used to analyze CD138+ PCs from MM patients at various timepoints post-ASCT. Longitudinal MM patient iliac crest biopsies (diagnosis, post-ASCT; N=20) were stained for CD138+ PCs; PC burden was quantified by AI-assisted histology. Lastly, we established an in vivo model of induction therapy (bortezomib) followed by HDM/SCT in Vk*MYC mouse MM engrafted C57BL/6 mice. Tumor burden was tracked by serum protein electrophoresis for M-spike using submandibular bleeds. Bone marrow was then flushed from long bones and analyzed by flow cytometry and immunofluorescence. HDM-5TGM1 showed decreased CPDs (HDM 0.32±0.31, Veh 6.72±0.69, p<0.0001, N=3), increased size (median pixel size: HDM 413.2±99.98, Veh 207±8.88, p<0.05, N=3), and increased TAF+ cells (Percent TAF+ cells: HDM 34.77%±10.74%, Veh 11.41%±1.58%, p<0.05, N=3) consistent with known TIS features. Preliminary assessment (N=1) of human MM cell lines showed similar growth arrest (RPMI-8226 CPDs: HDM 0.058±0.101, Veh 4.185±0.358; H929 CPDs: HDM 0±0, Veh 4.61±0.243) and size increase (RPMI-8226 mean pixel size: HDM 804.1±6.048, Veh 526.9±2.115; H929 mean pixel size: HDM 371.2±26.95, Veh 130.3±41.43), suggesting a translatable mechanism. HDM-5TGM1 cells were more sensitive to senolysis by dasatinib+quercetin (viability: HDM 54.9%±0.09%, Veh 76.0%±0.05%, N=2). scRNA-seq analysis showed that HDM-5TGM1 were enriched for gene sets related to senescence pathways, including the published set SenMayo (FDR<0.25). HDM-5TGM1 cells also exhibited myeloid gene expression consistent with previous publications on MM dormancy. RT-qPCR confirmed significant increases in HDM-5TGM1 (p<0.001, N=3) in senescence markers (Cdkn1a, Cdkn1c, Glb1), senescent cell anti-apoptosis pathway genes (SCAPs, Bcl2l1), senescence associated secretory phenotype (SASP) genes (Ccl5, Icam1, Mmp13), and myeloid markers (Axl, Fcer1g, Mpeg1). scRNA-seq of patient CD138+ PCs confirmed a myeloid-like cluster that gained prominence in relapsed patients with resistant disease. CD138+ PCs were detected in patient bone biopsies post-ASCT. Interestingly, patients who relapsed (≤3 years, N=9) had a greater percent reduction in PC burden from diagnosis to post-ASCT when compared to patients that did not relapse (N=11) in that time (p=0.02). Further, CD138+ PC burden post-ASCT positively correlated with time in remission in relapsed patients (R=0.697, p=0.03). Bortezomib reduced serum M-spike in Vk*MYC-engrafted mice (p=0.0003); M-spike was further reduced by HDM/SCT compared to Veh-treated mice (p=0.0007). Flow cytometry confirmed a decrease in Vk*MYC cells after HDM/SCT (p=0.0043), and the percentage of Vk*MYC cells with greater size and complexity was increased compared to Veh-treated mice (p=0.0043), consistent with our in vitro data. Vk*MYC cells from HDM-treated mice also trended for more TAF+ cells (>3 TAFs) compared to Veh-treated mice (p=0.0714). Our findings suggest that HDM can induce a TIS and myeloid signature in surviving MM cells. Greater MM burden post-ASCT appears to be associated with longer durable response, again suggesting that these cells may be in growth arrest consistent with TIS. Finally, senolytics may be a novel approach to eliminate surviving MM cells and prevent relapse.

Multiple Myeloma (MM) is the second most common hematological malignancy and is characterized by clonal expansion of malignant plasma cells in the bone marrow. In spite of recent advances in the field of MM, the disease has remained incurable. MM is preceded by a premalignant state known as monoclonal gammopathy of undetermined significance (MGUS), with a risk of progression to MM of 1% per year. Establishing a scalable approach that refines the identification of MGUS patients at high risk of progression to MM can transform the clinical management of the disease, improve the patient's quality of life, and will have significant socioeconomic implications. Here, we provide evidence that changes in the bone marrow adipose tissue (BMAT) provide an early sign for progression from MGUS to MM. We employed AI-assisted histological analysis of unstained bone marrow biopsies from MGUS subjects with or without progression to MM within 10 years (n = 24, n = 17 respectively). Although the BMAT fraction was not different between the two groups, bone marrow adipocyte (BMAd) density was decreased in MGUS patients who developed MM, compared to non-progressing MGUS patients. Importantly, the distribution profile for BMAd size and roundness was significantly different between the two groups, indicating a shift toward increased BMAd size and roundness in MGUS patients who developed MM. These early changes in the BMAT could serve as valuable early indicators for the transition from MGUS to MM, potentially enabling timely interventions and personalized treatment strategies. Finally, the AI-based approach for histological characterization of unstained bone marrow biopsies is cost-effective and fast, rendering its clinical implementation feasible.