Cystatin M/E ameliorates bone resorption through increasing osteoclastic cell estrogen influx.

In multiple myeloma (MM), increased osteoclast differentiation leads to the formation of osteolytic lesions in most MM patients. Bisphosphonates, such as zoledronic acid (ZA), are used to ameliorate bone resorption, but due to risk of serious side effects as well as the lack of repair of existing lesions, novel anti-bone resorption agents are required. Previously, the absence of osteolytic lesions in MM was strongly associated with elevated levels of cystatin M/E (CST6), a cysteine protease inhibitor, secreted by MM cells. In this study, both MM- and ovariectomy (OVX)-induced osteoporotic mouse models were used to compare the effects of recombinant mouse CST6 (rmCst6) and ZA on preventing bone loss. µCT showed that rmCst6 and ZA had similar effects on improving percent bone volume, and inhibited differentiation of non-adherent bone marrow cells into mature osteoclasts. Single-cell RNA sequencing showed that rmCst6 and not ZA treatment reduced bone marrow macrophage percentage in the MM mouse model compared to controls. Protein and mRNA arrays showed that both rmCst6 and ZA significantly inhibit OVX-induced expression of inflammatory cytokines. For OVX mice, ERα protein expression in bone was brought to sham surgery level by only rmCst6 treatments. rmCst6 significantly increased mRNA and protein levels of ERα and significantly increased total intracellular estrogen concentrations for ex vivo osteoclast precursor cell cultures. Based on these results, we conclude that CST6 improves MM or OVX bone loss models by increasing the expression of estrogen receptors as well as the intracellular estrogen concentration in osteoclast precursors, inhibiting their maturation.

A Risk Stratification System in Myeloma Patients with Autologous Stem Cell Transplantation.

Autologous stem cell transplantation (ASCT) has been a mainstay in myeloma treatment for over three decades, but patient prognosis post-ASCT varies significantly. In a retrospective study of 5259 patients with multiple myeloma (MM) at the University of Arkansas for Medical Sciences undergoing ASCT with a median 57-month follow-up, we divided the dataset into training (70%) and validation (30%) subsets. Employing univariable and multivariable Cox analyses, we systematically assessed 29 clinical variables, identifying crucial adverse prognostic factors, such as extended duration between MM diagnosis and ASCT, elevated serum ferritin, and reduced transferrin levels. These factors could enhance existing prognostic models. Additionally, we pinpointed significant poor prognosis markers like high serum calcium and low platelet counts, though they are applicable to a smaller patient population. Utilizing seven easily accessible high-risk variables, we devised a four-stage system (ATM4S) with primary stage borders determined through K-adaptive partitioning. This staging system underwent validation in both the training dataset and an independent cohort of 514 ASCT-treated MM patients from the University of Iowa. We also explored cytogenetic risk factors within this staging system, emphasizing its potential clinical utility for refining prognostic assessments and guiding personalized treatment approaches.

Bispecific BCMA/CD24 CAR-T cells control multiple myeloma growth.

Anti-multiple myeloma B cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T-cell therapies represent a promising treatment strategy with high response rates in myeloma. However, durable cures following anti-BCMA CAR-T cell treatment of myeloma are rare. One potential reason is that a small subset of minimal residual myeloma cells seeds relapse. Residual myeloma cells following BCMA-CAR-T-mediated treatment show less-differentiated features and express stem-like genes, including CD24. CD24-positive myeloma cells represent a large fraction of residual myeloma cells after BCMA-CAR-T therapy. In this work, we develop CD24-CAR-T cells and test their ability to eliminate myeloma cells. We find that CD24-CAR-T cells block the CD24-Siglec-10 pathway, thereby enhancing macrophage phagocytic clearance of myeloma cells. Additionally, CD24-CAR-T cells polarize macrophages to a M1-like phenotype. A dual-targeted BCMA-CD24-CAR-T exhibits improved efficacy compared to monospecific BCMA-CAR-T-cell therapy. This work presents an immunotherapeutic approach that targets myeloma cells and promotes tumor cell clearance by macrophages.

Three years of maintenance with VRD in multiple myeloma: results of total therapy IIIB with a 15-year follow-up.

ABSTRACT: The total therapy (TT) IIIB phase 2 study incorporated bortezomib into tandem melphalan-based hematopoietic stem cell transplantation with dexamethasone, thalidomide, cisplatin, doxorubicin, cyclophosphamide, and etoposide for induction/consolidation and bortezomib, lenalidomide, and dexamethasone (VRD) for maintenance in patients with newly diagnosed multiple myeloma (MM). This updated analysis presents a 15.4-year median follow-up. Of 177 patients, 21% patients had gene expression profile (GEP)-defined high-risk MM. 15-year progression free survival (PFS) was 27.9%. Median PFS was better in GEP-defined low-risk patients at 7.8 years and in International Staging System stage 1 patients at 8.7 years. Overall, median OS was 9.1 years, and 15-year overall survival (OS) was 35.9%. GEP-defined low-risk patients' median OS was 11.2 years, and that of GEP-defined high-risk patients was 2.8 years. There was no difference in OS between TT IIIB and TT IIIA. This study includes the longest follow-up of patients treated with maintenance VRD reported to date. In patients with GEP-defined low-risk, nearly half and one-third of patients without ongoing treatment showed no signs of progression at 10 and 15 years, respectively. One-third of patients survived more than 15 years, but 3 years of VRD maintenance did not improve outcomes for patients with GEP-defined high-risk MM. The study was registered on www.clinicaltrials.gov as #NCT00572169.

BCMA- and CST6-specific CAR T cells lyse multiple myeloma cells and suppress murine osteolytic lesions.

We have previously demonstrated that cystatin E/M (CST6), which is elevated in a subset of patients with multiple myeloma (MM) lacking osteolytic lesions (OLs), suppresses MM bone disease by blocking osteoclast differentiation and function. CST6 is a secreted type 2 cystatin, a cysteine protease inhibitor that regulates lysosomal cysteine proteases and the asparaginyl endopeptidase legumain. Here, we developed B cell maturation antigen (BCMA) CST6 chimeric antigen receptor T cells (CAR-T cells), which lysed MM cells and released CST6 proteins. Our in vitro studies show that these CAR-T cells suppressed the differentiation and formation of tartrate-resistant acid phosphatase-positive (TRAP+) osteoclasts. Using xenografted MM mice, bioluminescence images showed that both BCMA-CAR-T and BCMA-CST6-CAR-T cells inhibited MM growth to a similar extent. Reconstructed micro-computed tomography images revealed that BCMA-CST6-CAR-T cells, but not BCMA-CAR-T cells, prevented MM-induced bone damage and decreased osteoclast numbers. Our results provide a CAR-T strategy that targets tumor cells directly and delivers an inhibitor of bone resorption.

High NEK2 expression in myeloid progenitors suppresses T cell immunity in multiple myeloma.

Multiple myeloma (MM) growth is supported by an immune-tolerant bone marrow microenvironment. Here, we find that loss of Never in mitosis gene A (NIMA)-related kinase 2 (NEK2) in tumor microenvironmental cells is associated with MM growth suppression. The absence of NEK2 leads to both fewer tumor-associated macrophages (TAMs) and inhibitory T cells. NEK2 expression in myeloid progenitor cells promotes the generation of functional TAMs when stimulated with MM conditional medium. Clinically, high NEK2 expression in MM cells is associated with increased CD8+ T effector memory cells, while low NEK2 is associated with an IFN-γ gene signature and activated T cell response. Inhibition of NEK2 upregulates PD-L1 expression in MM cells and myeloid cells. In a mouse model, the combination of NEK2 inhibitor INH154 with PD-L1 blockade effectively eliminates MM cells and prolongs survival. Our results provide strong evidence that NEK2 inhibition may overcome tumor immune escape and support its further clinical development.

Application of R2-ISS risk stratification to patients with multiple myeloma treated with autologous stem cell transplants at UAMS.

The Second Revision of the International Staging System (R2-ISS) was published in 2022 and has been validated in several cohorts of patients with multiple myeloma (MM). In this study, we investigated a total of 860 patients with MM who received an upfront autologous stem cell transplantation between 2001 and 2021. The median age of the patients was 60 years, with a median overall survival (OS) of 123 months and median progression-free survival (PFS) of 70 months. We collected the variables included in the ISS, R-ISS, and R2-ISS systems as well as additional standard variables. Our analyses demonstrated that all 3 ISS series systems (ISS, R-ISS, and R2-ISS) exhibited robust discrimination in terms of both OS and PFS among our study cohort. The ISS system effectively stratified patients into 3 risk groups, whereas the R-ISS system accurately identified patients at extremely high or low risk. The R2-ISS system further refined risk stratification by dividing patients into 4 more balanced risk groups. Furthermore, we specifically focused on identifying variables that distinguished patients with OS < 3 years and OS > 10 years within the low-risk R2-ISS stages (I and II) and high-risk R2-ISS stages (III and IV). Our findings revealed that age, hemoglobin, and 1p deletion significantly influenced the classification of patients in the low-risk R2-ISS stage. Additionally, serum light chain, platelet count, age, and the presence of the t(14;16) translocation were found to affect high-risk classification.

A gene signature can predict risk of MGUS progressing to multiple myeloma.

Multiple myeloma is preceded by monoclonal gammopathy of undetermined significance (MGUS). Serum markers are currently used to stratify MGUS patients into clinical risk groups. A molecular signature predicting MGUS progression has not been produced. We have explored the use of gene expression profiling to risk-stratify MGUS and developed an optimized signature based on large samples with long-term follow-up. Microarrays of plasma cell mRNA from 334 MGUS with stable disease and 40 MGUS that progressed to MM within 10 years, was used to define a molecular signature of MGUS risk. After a three-fold cross-validation analysis, the top thirty-six genes that appeared in each validation and maximized the concordance between risk score and MGUS progression were included in the gene signature (GS36). The GS36 accurately predicted MGUS progression (C-statistic is 0.928). An optimal cut-point for risk of progression by the GS36 score was found to be 0.7, which identified a subset of 61 patients with a 10-year progression probability of 54.1%. The remainder of the 313 patients had a probability of progression of only 2.2%. The sensitivity and specificity were 82.5% and 91.6%. Furthermore, combination of GS36, free light chain ratio and immunoparesis identified a subset of MGUS patients with 82.4% risk of progression to MM within 10 years. A gene expression signature combined with serum markers created a highly robust model for predicting risk of MGUS progression. These findings strongly support the inclusion of genomic analysis in the management of MGUS to identify patients who may benefit from more frequent monitoring.

Geographic and Racial Disparities in Access to Chimeric Antigen Receptor-T Cells and Bispecific Antibodies Trials for Multiple Myeloma.

Importance: The use of chimeric antigen receptor-T cell (CAR-T) therapy and bispecific antibodies in multiple myeloma is expanding, with encouraging early results. It is unknown if the current geographic distribution of CAR-T therapy and bispecific antibodies in multiple myeloma allows access for patients in need, especially for Black populations, which have a higher incidence of multiple myeloma. Objective: To investigate if the current geographic distribution of CAR-T cell therapy and bispecific antibodies for multiple myeloma allows equitable access for Black patients with multiple myeloma. Design, Setting, and Participants: This cross-sectional study of data from CAR-T therapy and bispecific antibodies multiple myeloma clinical trials for all available studies listed in ClinicalTrials.gov until January 31, 2022. Only studies with 1 or more open sites in the US were analyzed. Data were analyzed February 2022. Results: A total of 162 clinical trials were found, and 69 analyzed-7896 participants were either enrolled or expected to enroll, with 4386 participants (55.5%) enrolled or to be enrolled in CAR-T therapies clinical trials. The vast majority of clinical trials (66 [96%]) were sponsored by industry, and there were 140 clinical trials sites. The mean number of sites per trial was 8.1 (7.8 for CAR-T trials [range, 1-30 trials] vs 8.7 for bispecific antibodies [range, 1-26 trials]). Only 35.9% of Black patients lived in a county with an open trial. For the 10 states with the highest proportion of Black residents (ranging from 18.6% to 41.4%), 6 of those states (60%) had no (3 states) or less than 3 clinical trial openings (3 states) for either a CAR-T or bispecific antibody study. Conclusions and Relevance: In this cross-sectional study, we found that the geographic distribution of clinical trials for CAR-T and bispecific antibodies may contribute to disparities in access to the most advanced clinical trials for new multiple myeloma therapies. Since most of the ongoing trials were sponsored by industry, regulating the distribution of clinical trial sites may reduce these inequities.

CST6 suppresses osteolytic bone disease in multiple myeloma by blocking osteoclast differentiation.

Osteolytic bone disease is a hallmark of multiple myeloma (MM). A significant fraction (~20%) of MM patients do not develop osteolytic lesions (OLs). The molecular basis for the absence of bone disease in MM is not understood. We combined PET-CT and gene expression profiling (GEP) of purified BM CD138+ MM cells from 512 newly diagnosed MM patients to reveal that elevated expression of cystatin M/E (CST6) was significantly associated with the absence of OL in MM. An enzyme-linked immunosorbent assay revealed a strong correlation between CST6 levels in BM serum/plasma and CST6 mRNA expression. Both recombinant CST6 protein and BM serum from patients with high CST6 significantly inhibited the activity of the osteoclast-specific protease cathepsin K and blocked osteoclast differentiation and function. Recombinant CST6 inhibited bone destruction in ex vivo and in vivo myeloma models. Single-cell RNA-Seq showed that CST6 attenuates polarization of monocytes to osteoclast precursors. Furthermore, CST6 protein blocks osteoclast differentiation by suppressing cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 following RANKL stimulation. Secretion by MM cells of CST6, an inhibitor of osteoclast differentiation and function, suppresses osteolytic bone disease in MM and probably other diseases associated with osteoclast-mediated bone loss.

Feasibility of Outpatient Stem Cell Transplantation in Multiple Myeloma and Risk Factors Predictive of Hospital Admission.

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) remains the standard of care for multiple myeloma (MM) patients. Although outpatient ASCT has been shown to be safe and feasible, the procedure is overall rare with most patients in the US undergoing inpatient ASCT. Furthermore, hospitalization rates for patients that undergo outpatient ASCT remain high. Adequate markers that predict hospitalization during outpatient ASCT are lacking, yet would be of great clinical value to select patients that are suited to outpatient ASCT. In this study we aimed to elucidate differences between planned outpatient and inpatient ASCT and further evaluated clinical characteristics that are significantly associated with hospitalization during planned outpatient hospitalization. Factors that were significantly associated with a planned inpatient ASCT included an advanced MM disease stage, worse performance status as well as non-Caucasian race, while low albumin levels and female gender were significantly associated with hospitalization during outpatient ASCT. The results of this analysis provide crucial knowledge of factors that are associated with planned inpatient ASCT and hospitalization during outpatient ASCT and could guide the treating physician in decision-making and further facilitate outpatient transplantation.

Clinical implications of loss of bone marrow minimal residual disease negativity in multiple myeloma.

Multiple myeloma (MM) patients frequently attain a bone marrow (BM) minimal residual disease (MRD) negativity status in response to treatment. We identified 568 patients who achieved BM MRD negativity following autologous stem cell transplantation (ASCT) and maintenance combination therapy with an immunomodulatory agent and a proteasome inhibitor. BM MRD was evaluated by next-generation flow cytometry (sensitivity of 10-5 cells) at 3- to 6-month intervals. With a median follow-up of 9.9 years from diagnosis (range, 0.4-30.9), 61% of patients maintained MRD negativity, whereas 39% experienced MRD conversion at a median of 6.3 years (range, 1.4-25). The highest risk of MRD conversion occurred within the first 5 years after treatment and was observed more often in patients with abnormal metaphase cytogenetic abnormalities (95% vs 84%; P = .001). MRD conversion was associated with a high risk of relapse and preceded it by a median of 1.0 years (range, 0-4.9). However, 27% of MRD conversion-positive patients had not yet experienced a clinical relapse, with a median follow-up of 9.3 years (range, 2.2-21.2). Landmark analyses using time from ASCT revealed patients with MRD conversion during the first 3 years had an inferior overall and progression-free survival compared with patients with sustained MRD negativity. MRD conversion correctly predicted relapse in 70%, demonstrating the utility of serial BM MRD assessment to complement standard laboratory and imaging to make informed salvage therapy decisions.

Predicting risk of progression in relapsed multiple myeloma using traditional risk models, focal lesion assessment with PET-CT and minimal residual disease status.

Not available.

Salvage Autologous Stem Cell Transplantation in Daratumumab-Refractory Multiple Myeloma.

Daratumumab, a CD38-targeting monoclonal antibody, has significantly improved survival rates in multiple myeloma (MM), yet patients who progress on Daratumumab have dismal clinical outcomes with an overall median of less than 10 months. While emerging novel modalities have shown promising results, the current study explores the use of high-dose chemotherapy followed by autologous stem cell transplantation (ASCT) in heavily pretreated Daratumumab-refractory MM patients. We retrospectively investigated the outcome of 69 consecutive patients who received upfront ASCT. The median progression-free survival (PFS) for the entire patient cohort was 7.2 months with a median overall survival (OS) of 19.3 months. For patients with ≥very good partial response (VGPR), median PFS and OS improved to 9 months and 34 months, respectively. Achievement of MRD negativity in ≥VGPR did not further improve the outcome. A better performance status, younger age, longer time interval from initial MM diagnosis/initial ASCT to salvage ASCT and low-risk GEP70 were all associated with improved PFS and OS after salvage ASCT. Our results suggest a role for salvage ASCT in selected heavily pretreated and Daratumumab-refractory patients.

TRIP13 modulates protein deubiquitination and accelerates tumor development and progression of B cell malignancies.

Multiple myeloma (MM), a terminally differentiated B cell malignancy, remains difficult to cure. Understanding the molecular mechanisms underlying the progression of MM may identify therapeutic targets and lead to a fundamental shift in treatment of the disease. Deubiquitination, like ubiquitination, is a highly regulated process, implicated in almost every cellular process. Multiple deubiquitinating enzymes (DUBs) have been identified, but their regulation is poorly defined. Here, we determined that TRIP13 increases cellular deubiquitination. Overexpression of TRIP13 in mice and cultured cells resulted in excess cellular deubiquitination by enhancing the association of the DUB USP7 with its substrates. We show that TRIP13 is an oncogenic protein because it accelerates B cell tumor development in transgenic mice. TRIP13-induced resistance to proteasome inhibition can be overcome by a USP7 inhibitor in vitro and in vivo. These findings suggest that TRIP13 expression plays a critical role in B cell lymphoma and MM by regulating deubiquitination of critical oncogenic (NEK2) and tumor suppressor (PTEN, p53) proteins. High TRIP13 identifies a high-risk patient group amenable to adjuvant anti-USP7 therapy.

Genetic Analysis of Multiple Myeloma Identifies Cytogenetic Alterations Implicated in Disease Complexity and Progression.

Multiple myeloma (MM) is a genetically heterogeneous disease characterized by genomic chaos making it difficult to distinguish driver from passenger mutations. In this study, we integrated data from whole genome gene expression profiling (GEP) microarrays and CytoScan HD high-resolution genomic arrays to integrate GEP with copy number variations (CNV) to more precisely define molecular alterations in MM important for disease initiation, progression and poor clinical outcome. We utilized gene expression arrays from 351 MM samples and CytoScan HD arrays from 97 MM samples to identify eight CNV events that represent possible MM drivers. By integrating GEP and CNV data we divided the MM into eight unique subgroups and demonstrated that patients within one of the eight distinct subgroups exhibited common and unique protein network signatures that can be utilized to identify new therapeutic interventions based on pathway dysregulation. Data also point to the central role of 1q gains and the upregulated expression of ANP32E, DTL, IFI16, UBE2Q1, and UBE2T as potential drivers of MM aggressiveness. The data presented here utilized a novel approach to identify potential driver CNV events in MM, the creation of an improved definition of the molecular basis of MM and the identification of potential new points of therapeutic intervention.