Drake, Matthew T.

ABSTRACT Objective Data on bone health in patients with mild autonomous cortisol secretion (MACS) are limited and heterogenous. We sought to assess the prevalence of osteoporosis and symptomatic fragility fractures in patients with adrenal adenomas and hypercortisolism, and explore associations with the degree of cortisol excess and frailty. Design This multicenter cross ‐sectional study involved prospective enrollment of adults with nonfunctioning adrenal adenomas (NFA), MACS, Cushing syndrome (CS) and referent subjects without adrenal disorders during the study period (January 2019–September 2022). All participants completed a bone health questionnaire at enrollment and had dual‐energy X ‐ray absorptiometry bone mineral density assessment within 12‐months. Final analyses were adjusted for age, sex, body mass index, smoking status, alcohol use. Results Participants included 117 with NFA (median age 62 years, 80% women), 191 with MACS (median age 63 years, 74% women), 62 with CS (median age 50 years, 92% women), and 101 referent subjects (median age 59 years, 47% women). Both patients with CS (OR: 3.0, 95% CI: 1.1–8.3) and MACS (OR: 2.2, 95% CI: 1.9–4.9) had a higher prevalence of osteoporosis when compared to referents. Among patients with MACS, only those with post 1‐mg dexamethasone suppression test (post‐1mg‐DST) cortisol > 83 nmol/L had a higher prevalence of osteoporosis when compared to referents (OR: 3.2, 95% CI: 1.4–7.5) and NFA (OR: 2.2, 95% CI: 1.1–4.4). For clinical fragility fractures, only patients with CS showed an increased prevalence when compared to referents (OR: 6.1, 95% CI: 1.7–26.4) and NFA (OR: 3.8, 95% CI: 1.3–11.2). Prevalence of clinical fragility fractures was similar to referents in patients with MACS (OR: 1.8, 95% CI: 0.6–6.9) and NFA (OR: 1.5, 95% CI: 0.4–6.1), and was not associated with post‐1mg‐DST cortisol or frailty in the MACS and NFA groups. Conclusion Patients with CS, and those with MACS and post‐1mg‐DST cortisol > 83 nmol/L, exhibited a higher burden of osteoporosis. However, only CS was associated with an elevated risk of symptomatic fragility fractures.

Abstract Gallium-68-DOTA-Tyr3-Octreotate (Ga-68-DOTATATE) positron emission tomography/computed tomography (PET/CT) has recently been shown to have utility for the localization of phosphaturic mesenchymal tumors (PMT) that cause tumor-induced osteomalacia (TIO), a rare renal phosphate-wasting disorder. The aim of this study was to evaluate the accuracy of Ga-68-DOTATATE PET/CT in localizing PMTs causing TIO and to compare its performance with other functional imaging modalities. Prospective recruitment and retrospective chart review of 30 patients with suspected TIO and evaluation with Ga-68-DOTATATE PET/CT between 2017 and 2023 were conducted at a tertiary medical center. True positive (TP) lesions were defined by histological confirmation of PMT. There were 22 TP lesions identified among 18 patients, with a mean SUVmax of 16.8 (±10.9). Sensitivity, specificity, and accuracy of Ga-68-DOTATATE PET/CT were 85.7%, 77.8%, and 83.3% on patient-based analysis, and 84.6%, 56.3%, and 73.8% on lesion-based analysis. Lesions such as subacute fractures, parathyroid adenomas, thymus uptake, vertebral hemangiomas, bone enchondromas, liver hemangiomas, and avascular necrosis were some of the pitfalls in interpretation. Ga-68-DOTATATE PET/CT led to a significant impact on clinical management in 24 (80%) of patients. The presence of DOTATATE-avid fractures was significantly associated with a localizing scan on univariable (OR 15.0, 95% CI 2.80-110, p = .001) and multivariable analysis (OR 9.45, 95% CI 1.33-98.4, p = .003). Ga-68-DOTATATE PET/CT has good accuracy for the localization of TIO, with superior sensitivity compared to F-18-FDG PET/CT. This significantly impacted clinical treatment decisions. Although DOTATATE-avid fractures may be a source of false positives, they may also indicate a higher probability of a localizing study.

OBJECTIVE Hounsfield units (HUs) may better predict biomechanical complications of instrumented fusion than conventional bone quality measures. Typically, noncontrast axial slices are used. This study aims to address the influence of reconstruction plane and contrast administration on measured HUs in patients undergoing lumbar spine imaging. METHODS Patients without prior spine surgery, infection, or tumor were identified for 3 groups: 1) 150 patients with high-resolution noncontrast lumbar CT, 2) 109 patients with noncontrast and contrast-enhanced lumbar CT, and 3) 100 patients with noncontrast lumbar CT and lumbar CT myelograms. Noncontrast HU measures in group 1 were compared between axial, sagittal, and coronal reconstructions. HU measures for groups 2 and 3 were compared on axial slice averages. HU measures between groups were compared using the Wilcoxon signed-rank test to investigate the presence of a statistically significant difference between groups (α = 0.05). Linear regression was also used to determine the degree of correlation between HU measures on noncontrast axial CT slices and HU measures on other sequences. RESULTS The mean patient age was 58.8 ± 15.9 years for group 1 (54.0% male), 65.5 ± 15.9 years for group 2 (61.5% male), and 65.2 ± 14.2 years (53.5% male) for group 3. Comparison of HU measures in group 1 showed significant differences across measurement modalities with the exception of axial average versus coronal average measures (p = 0.257) and sagittal average versus midsagittal measures (p = 0.726). There were significant differences in average axial HUs for each vertebral body between contrast and noncontrast lumbar CT and between CT myelography and noncontrast CT. Linear regression analysis demonstrated an extremely high correlation between measures for all methodologies (r2 = 0.950–0.986, all p < 0.001), and between contrast-enhanced and noncontrast studies (r2 = 0.870, p < 0.001). Measurements on CT myelography were significantly correlated with those in noncontrast studies, although to a lesser degree (r2 = 0.745, p < 0.001). CONCLUSIONS HU measures on noncontrast CT appear similar across multiple different planes, suggesting that a simpler methodology such as single-slice measurement on midsagittal reconstruction may allow for more rapid assessment of underlying bone quality. Consistent with prior work, contrast-enhanced CT sequences appear to reliably mirror underlying bone quality, although CT myelogram measures may vary in a more unpredictable way that precludes their use to interpret underlying bone quality. HU measures on contrast-enhanced studies are on average greater, suggesting the need for higher cutoff values.

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&lt;0.0001, N=3), increased size (median pixel size: HDM 413.2±99.98, Veh 207±8.88, p&lt;0.05, N=3), and increased TAF+ cells (Percent TAF+ cells: HDM 34.77%±10.74%, Veh 11.41%±1.58%, p&lt;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&lt;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&lt;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 (&gt;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.

Abstract Multiple myeloma (MM) is an incurable plasma cell (PC) cancer in which all patients are expected to relapse. Cytotoxic agents such as high-dose melphalan (HDM), which is used as a myeloablative agent prior to autologous stem cell transplant (ASCT) in MM, drive therapy-induced senescence (TIS) in non-cancerous tissues. We hypothesized that HDM similarly activates TIS pathways in surviving MM cells and this response may correlate with patient outcomes post-ASCT. To test this, we developed an in vitro model in which 5TGM1 mouse MM cells are cultured with 10uM melphalan (HDM) or vehicle (Veh) for 6 hours, followed by co-culture in normal media with primary mouse bone marrow stromal cells. Cultures were maintained for 10 days and imaged to quantify cumulative population doublings (CPDs). 5TGM1 were then isolated by FACS to measure telomere-associated DNA damage foci (TAFs), sensitivity to senolytic drugs, and senescence gene expression (RT-qPCR and scRNA-seq). To translate these studies, scRNA-seq was used to analyze CD138+ PCs from MM patient bone marrow post-ASCT. Additionally, longitudinal bone biopsies from MM patients at diagnosis and post-ASCT were stained for CD138+ PCs; PC burden was quantified using artificial intelligence assisted histology (HALO). HDM-5TGM1 exhibited stable growth arrest (decreased CPDs) and sustained DNA damage (increased TAFs) compared to Veh-5TGM1. HDM-5TGM1 were also significantly more sensitive to senolytic ablation (Dasatinib+Quercetin or Fisetin). HDM-5TGM1 showed increased senescence (Cdkn1a, Cdkn1c, Glb1), anti-apoptosis (Bcl2l1), and senescence associated secretory phenotype (Ccl5, Icam1, Mmp13) genes, as well as myeloid markers found to be increased in MM dormancy (Axl, Fcer1g, Mpeg1). Analysis by scRNA-seq revealed that HDM-5TGM1 were G0/G1 arrested, and gene set enrichment analysis (GSEA) confirmed upregulation of senescence pathways with downregulation of DNA replication and cell cycle pathways. Consistent with RT-qPCR results, these cells exhibited myeloid gene expression. Analysis of patient CD138+ PCs post-ASCT by scRNA-seq also revealed a myeloid-like cluster. Of interest, of the 491 genes upregulated in this cluster, 268 were also upregulated in HDM-5TGM1. CD138+ PCs could also be detected in patient bone biopsies post-ASCT, although PC burden was significantly reduced post-ASCT. Surprisingly, the percent reduction in PC burden post-ASCT was greater in relapsed (≤3 years) vs non-relapsed patients. Further, in relapsed patients, PC burden post-ASCT positively correlated with time in remission. Altogether, these findings demonstrate that HDM can induce senescence features and a myeloid-dormancy signature in surviving MM cells. This phenotype is associated with longer durable response, suggesting that activation of TIS may be a strategy to extend PFS. Further, senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent relapse. Citation Format: Angelo Jose Guilatco, Marta Diaz-del-Castillo, Gabriel Alvares Borges, Neal I. Sannuli, Megan L. Ritting, Syed Mohammed Aalam, Nagarajan Kannan, Tamar Tchkonia, James L. Kirkland, Yi Lin, Taxiarchis Kourelis, Matthew T. Drake, Thomas Levin Andersen, Megan Weivoda. Melphalan-treated multiple myeloma cells exhibit a senescent-like dormancy phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2958.

Abstract In their recent population–based study of nearly 100 000 patients from Ontario, Canada, who had suffered a fracture, Vincent et al. characterized postfracture survival rates. Their findings showed that overall survival was decreased particularly following fractures of the hip or vertebrae, and was worse in men and the oldest old. They found that relative survival, particularly after hip fracture, is strikingly poor with less than one-third of men and one-half of women surviving for 5 years following hip fracture, survival which was far less than that seen for patients afflicted by both prostate and breast cancers. Importantly, mortality risk increased immediately after fracture occurrence and was highest within 1 month of fracture. Collectively, their data suggest that a short, critical window may exist immediately following fracture in which application of interventions to improve survival is likely to be of greatest value. Future work is urgently needed to identify the best approaches to employ during this critical time period in order to optimize survival in patients who have suffered a fracture.

Multiple myeloma (MM) is an incurable plasma cell malignancy. Despite therapeutic advances that have vastly improved survival, all patients are expected to relapse. One mechanism that may contribute to MM relapse is tumor dormancy. Dormant MM cells may persist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells may then exit dormancy following treatment to drive disease relapse. Dormancy shares many features with cellular senescence, a state of stable growth arrest induced by cell stress. Chemotherapies that produce genotoxic stress have been associated with therapy-induced senescence (TIS). One such genotoxic agent, melphalan (Mel), is routinely administered to MM patients for myeloablation prior to autologous bone marrow transplant. We hypothesized that High Dose Mel (HDM) induces a senescent-like dormant phenotype in surviving MM cells. To test our hypothesis, we developed an in vitro model for HDM. We identified 10µM Mel as a concentration to induce &gt;90% cell toxicity (HDM) in 5TGM1 mouse MM cells over 10 days. 5TGM1-GFP MM cells were cultured with HDM or vehicle (Veh) for 6 hours; cells were then cultured in normal growth medium in the presence of primary mouse (C57BL/6) bone marrow stromal cells (BMSCs). Co-cultures were imaged by fluorescent microscopy every other day to quantify cumulative population doublings (CPD) and cell morphology over the following 10 days. The effect of HDM on CPDs was also evaluated in the human RPMI-8226 MM cell line. 5TGM1-GFP cells were isolated by FACS on day 10 for evaluation of senescence histological characteristics and gene expression. All studies represent n=3 independent experiments unless otherwise indicated. HDM-5TGM1 cells exhibited a significant reduction in mean CPDs over 10 days compared to Veh (HDM 0.32±0.31, Veh 6.72±0.69, p&lt;0.0001), indicating stable growth arrest in HDM treated cells consistent with senescence. HDM-5TGM1 cells were also significantly larger versus Veh (median size in pixels units: HDM 413.2±99.98, Veh 207±8.88, p&lt;0.05), consistent with classical enlarged morphology in senescent cells. Preliminary assessment (n=1) of HDM-RPMI-8226 cells showed similar growth arrest (mean cumulative doublings: HDM 0.058±0.101, Veh 4.185±0.358) and enlargement (mean size in pixel units: HDM 804.1±6.048, Veh 526.9±2.115), suggesting a translatable mechanism in humans. FACS-sorted 5TGM1-GFP cells were stained for telomere associated foci (TAF), a marker of persistent DNA damage characteristic of senescent cells. HDM-5TGM1 cells showed a higher percentage of TAF+ cells (Percent TAF+ cells: HDM 39.7%±9.1%, Veh 10.6%±0.8%, n=2). Sorted HDM-5TGM1 cells also exhibited significant increases over Veh (p&lt;0.001) in senescence markers ( Cdkn1a, Cdkn1c, Glb1), senescence cell anti-apoptosis pathway genes (SCAPs, Bcl2l1), and senescence associated secretory phenotype (SASP) genes ( Ccl5, Icam1, Mmp13). In addition, HDM-5TGM1 cells showed significant increases in myeloid markers that have previously been shown to be increased in dormant 5TGM1 ( Axl, Fcer1g, Mpeg1). Single cell RNA-sequencing was used to further evaluate senescence gene expression changes in growth arrested HDM-5TGM1 cells. Gene set enrichment analysis (GSEA) of genes differentially expressed in HDM-5TGM1 versus Veh cells was performed for custom senescence phenotyping gene sets (Senescence Up, SenUp; Senescence Growth Arrest, SenGA; Inflammatory SASP, iSASP; SCAPs; and Plasma Cell Senescence; PCSen) as well as mouse ortholog versions of a published senescence and biological aging gene set (SenMayo) and KEGG curated gene sets. GSEA of HDM-5TGM1 versus Veh cells confirmed significant (FDR&lt;0.25) upregulation of iSASP, MayoSen, PCSen, SenUp, SCAPs, and SenGA gene sets. Concurrently, GSEA revealed significant downregulation of KEGG DNA replication, cell cycle, and nucleotide excision repair gene sets, supporting our observations of proliferative arrest and persistent DNA damage TAF. Our findings demonstrate that HDM-MM cells exhibit multiple features of senescence, suggesting a potential mechanistic role for TIS in MM dormancy and highlight a novel therapeutic target that may be present in patients post-HDM. Thus, targeting senescence may be a novel approach to eliminate dormant MM cells and prevent relapse.

Multiple Myeloma (MM) progresses from monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM). Despite similar primary genetic events as MM, MGUS and SMM PCs are non- or low-proliferative. Since oncogene activation and DNA damage drive senescence growth arrest to promote immune clearance of potentially malignant cells, we hypothesized that MGUS and SMM pre-malignant PCs exhibit senescence features. To test this, we performed gene set enrichment analysis (GSEA) of a published human PC gene array dataset (GSE5900) comparing normal, MGUS, and SMM PCs for custom senescence phenotyping gene sets (Senescence Up, SenUp; Senescent Cell Anti-Apoptosis Pathways, SCAPs; Senescence Growth Arrest, SenGA; Inflammatory Senescence Associated Secretory Phenotype, iSASP; Interferon SASP, IFN-SASP) versus biological aging gene sets (SenMayo; CellAge). MGUS and SMM PCs exhibited significant enrichment (NES&gt;1.5, q&lt;0.05) for senescence gene sets SenUp, SenGA, and SCAPs compared to normal PCs, with no enrichment for aging gene sets. MGUS PCs were also enriched for iSASP (NES=1.4, q=0.11), whereas SMM PCs were enriched for IFN-SASP (NES=2.3, q&lt;0.05), characteristic of late-senescence. Thus, both MGUS and SMM PCs show enrichment of senescence gene sets compared to normal PCs with differential SASP profiles. Next, we evaluated MGUS, SMM, and MM patient PCs isolated by magnetic sorting for histological senescence features. PCs were immunostained for LMNB1 and HMGB1, and fluorescent in situ hybridized for α-satellite. Cellular senescence was defined as loss of ≥50% nuclear membrane LMNB1, loss of HMGB1, and ≥2 senescence-associated distensions of satellite (SADS) per nuclei. PCs from MGUS patients (N=12) exhibited increased percentage of senescent PCs (26.9±4.1%, Mean±SEM, p&lt;0.0001) compared to SMM (11.0±1.9%, N=19, p&lt;0.0001) and MM (11.6±1.5%, N=11, p&lt;0.001). A subset analysis of SMM patients for which disease stability was known (≥2 years, N=6-8) showed no differences. These results demonstrate that the percentage of senescent PCs is increased in MGUS but not SMM PCs compared to proliferative disease. To assess PC senescence in the native bone marrow microenvironment (BMME), we evaluated trephine bone biopsies from MGUS and SMM patients that progressed or not to MM ≤10 years, as well as newly diagnosed MM. Biopsies were immunostained for CD138, LMNB1, and HMGB1. Whole biopsy images were analyzed using the artificial intelligence-based software HALO. Cells were classified as senescent if they were double negative and non-senescent if they were double positive for LMNB1 and HMGB1. The percentage of non-senescent PCs increased with progression to MM and positively correlated with total PC burden (R=0.52, p&lt;0.0001), reflective of proliferative disease. Senescent PCs correlated with total senescent BMME (R=0.87, p&lt;0.0001), consistent with a role for senescent cells to drive paracrine senescence. We performed proximity analysis to evaluate the cellular composition within 25um of senescent and non-senescent PCs. As expected, non-senescent PCs exhibited a significantly greater percentage of proximate non-senescent BMME in all groups (p&lt;0.0001). In contrast, senescent PCs in stable MGUS (N=16) and SMM (N=14) were equally surrounded by senescent and non-senescent BMME. In progressing MGUS (N=21) and SMM (N=20), as well as MM (N=18), senescent PCs exhibited significantly reduced senescent BMME in their proximate environment (p&lt;0.05). This suggests that senescent PCs in progressive disease, in contrast to stable disease, fail to drive local paracrine senescence responses. These data demonstrate that PCs from stable MGUS and SMM patients exhibit senescence features, with isolated PCs from stable MGUS showing increased senescence gene expression and histologic features. While stable SMM also exhibits senescence gene expression, they lose morphological features of senescence and show a preferential IFN SASP. Within the bone compartment, senescent PCs in stable MGUS and SMM exhibit the expected proximity to senescent BMME. In contrast, progressing MGUS and SMM, as well as NDMM, show a loss of proximate senescent BMME. Given the role of senescence to drive inflammation to clear potentially tumorigenic cells, these data support that failure to drive paracrine senescence may be key to the immune escape of pre-malignant cells in MGUS and SMM.

Abstract Multiple myeloma (MM) bone disease is a significant cause of morbidity but there is a paucity of data on the impact of malignant plasma cells on adjacent trabecular bone within the BM. Here, we characterize the proteome of trabecular bone tissue from BM biopsies of 56 patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering (SMM), newly diagnosed (NDMM), relapsed MM (RMM), and normal controls. Proteins involved in extracellular matrix (ECM) formation and immunity pathways were decreased in SMM and active MM. Among the proteins most decreased were immunoglobulins, type IV collagen, and TIMP3, suggesting increased immunoparesis and decreased ECM remodelling within trabecular bone. Proteins most increased in SMM/MM were APP (enhances osteoclast activity), ENPP1 (enhances bone mineralization), and MZB1 (required for normal plasmablast differentiation). Pathway analyses showed that proteins involved in gamma -carboxylation, a pathway implicated in osteocalcin function, osteoblast differentiation, and normal hematopoiesis, were also overexpressed in SMM/MM. This study is the first comprehensive proteomic atlas of the BM bone proteome in dysproteinemias. We identify new key proteins and pathways for MM bone disease and potentially impaired hematopoiesis, and show for the first time that gamma -carboxylation pathways are increased in the bone tissue of SMM/MM.

Abstract Multiple myeloma (MM) is a clonal plasma cell (PC) cancer that is preceded by the benign conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). While MGUS/SMM PCs are not proliferative, many of the oncogenes and chromosomal abnormalities in MM PCs are also present in MGUS/SMM. Since oncogenic stress is known to induce cellular senescence, we hypothesized that MGUS/SMM PCs may be in a senescent-like state. Our analysis of a published human dataset (GSE5900) revealed that compared to healthy PCs, MGUS/SMM PCs had significantly increased expression of senescence markers CDKN1A and GADD45A (FDR&lt;0.05). Gene Set Enrichment Analysis (GSEA) showed significant enrichment in MGUS/SMM PCs of our customized senescence phenotyping gene sets (q&lt;0.25). We next evaluated the KaLwRij MGUS mouse model. 10-mo-old female KaLwRij mice were treated with placebo or senolytics (dasatinib+quercetin, D+Q). D+Q treatment significantly reduced PCs compared to placebo, while restoring B cell number and functional gene expression. PCs isolated from D+Q-treated KaLwRij mice exhibited significant reductions in Myc and Il1b expression (p&lt;0.05) versus placebo and a trend towards reduced Trp53 expression (p=0.13), supporting that senescent PCs are targeted by D+Q. Single-cell RNA-seq and single sample (ss)GSEA of 24-mo-old KaLwRij PCs revealed a PC subset enriched for the ‘Plasma Cell Senescence’ gene set, which included genes from our customized gene sets that were differentially expressed in the human PCs dataset GSE5900. Notably, PCs in this subset showed enrichment for ‘Inflammatory SASP’ (median: 15.26%) and ‘Interferon (IFN) SASP’ (39.36%) gene sets. The IFN-SASP is characteristic of late senescence, driven by the accumulation of transposable elements. In the GSE5900 dataset, we found significant enrichment of ‘IFN-SASP’ in SMM (q&lt;0.25) but not MGUS PCs. Further, whole transcriptome RNA-sequencing showed that SMM PCs had increased expression of LINE1 retrotransposon L1HS in relation to normal PCs (FDR&lt;0.1), but not MGUS (FDR=0.83) or MM (FDR=0.5) PCs. Immunostaining confirmed increased cytosolic DNA:RNA hybrids in SMM (median frequency of cells exceeding the intensity threshold = 33%) versus MGUS (16.4%), MM (4.4%), and healthy PCs (1.5%), which is consistent with cytosolic DNA-mediated activation of the IFN SASP in SMM PCs. When these same patients were redistributed according to disease progression, L1HS was higher in patients with progressing SMM and newly diagnosed MM (NDMM) (FDR&lt;0.1) versus patients with stable MGUS (FDR=0.56) or advanced MM (FDR=0.38). Increased DNA:RNA staining was observed in progressing SMM PCs (median frequency = 41%), but the results for NDMM patients (4.4%) and advanced MM (3%) were comparable to healthy or stable patients (3.5%). These data demonstrate that MGUS and SMM PCs exhibit senescence features, suggest mechanisms that may contribute to MM tumorigenesis, and show that pharmacological ablation of senescent cells may prevent progression from MGUS/SMM to MM. Citation Format: Gabriel Alvares Borges, Angelo Jose Guilatco, Christine M. Hachfeld, Ming Ruan, Sonya Royzenblat, Ming Xu, Claire M. Edwards, Marta Diaz-delCastillo, Thomas L. Andersen, Taxiarchis Kourelis, Tamar Tchkonia, James L. Kirkland, Matthew T. Drake, Megan Weivoda. Pre-malignant plasma cells exhibit a senescence-like phenotype and accumulation of transposable elements [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR012.