Role In Multiple Myeloma Research Articles

Type I-conventional dendritic cells support the progression of multiple myeloma in the bone marrow.

Type I conventional dendritic cells (cDC1s) play a key role in priming anti-tumor cytotoxic T cells and inducing immune tolerance for self-antigens and tumor antigens. However, it remains unclear whether cDC1 has a protective or pathogenic role in multiple myeloma. We investigated a role of cDC1 in myeloma progression. A myeloma mouse model was performed by intravenous transplantation of Vk*MYC myeloma cells into XCR1-Diphtheria toxin receptor (DTR) knock-in or wild-type mice. Following injection with Diphtheria toxin (DT), monoclonal (M)-proteins and myeloma cells were analyzed by ELISA and flow cytometry. Here we show that inducible depletion of cDC1 after myeloma transplantation markedly suppressed the progression of myeloma in the bone marrow and extramedullary sites, such as the spleen. cDC1 appeared in the bone marrow and spleen of myeloma-transplanted mice, which highly expressed CD103 and lowly produced interleukin (IL)-12. Consequently, the frequencies of exhausted CD8 T cells and regulatory T cells significantly decreased in the bone marrow of cDC1-depleted mice. cDC1 supports the progression of myeloma inducing exhausted CD8 T cells and regulatory T cells.

Clinical implications of residual normal plasma cells within bone marrow across various disease stages in multiple myeloma.

Residual normal plasma cells (NPCs), which compete with tumor plasma cells, play an important role in multiple myeloma. However, large-scale cohort studies investigating residual NPCs, especially at the minimal residual disease (MRD) phase, are currently lacking. In this study, we conducted a comprehensive investigation into the clinical significance of residual NPCs throughout the entire disease course in 1363 myeloma patients from the NICHE cohort (NCT04645199). Our results revealed that myeloma patients with high baseline NPCs ratio (≥5%) exhibited distinct indolent features, characterized by lower tumor burden, reduced frequencies of cytopenia, immunoparesis, and high-risk cytogenetics. Importantly, high residual NPCs ratio at diagnosis or relapse was independently associated with favorable survival. High absolute percentages of NPCs at undetectable MRD were related with superior clinical benefit and immune reconstitution. At MRD-positive phases, grouping based on NPCs ratio (<50%, 50-90%, ≥90%) demonstrated better risk stratification compared to residual tumor log levels. Based on the time-dependent NPCs ratio trend, we developed a dynamic MRD model that classifies patients into three groups with diverse longitudinal trends, leading to distinct prognoses. Collectively, residual NPCs serves not only as a valuable complementary biomarker for risk stratification but also provides valuable insights on reclassifications and kinetics of MRD.

Integrating palliative care into multiple myeloma management : Optimizing quality of life across the disease continuum.

Multiple myeloma (MM) poses significant challenges due to its complex symptomatology and evolving treatment landscape. While therapeutic advances have improved survival outcomes, holistic management of MM requires addressing the numerous physical and psychosocial needs of patients. Palliative care (PC) offers acomprehensive approach to symptom management and supportive care on aphysical, psychosocial and spiritual level; however, its role in MM remains underexplored. This retrospective single-center study examines the outcome of 22 MM patients admitted to the Division of Palliative Medicine at the Medical University of Vienna. We investigated reasons for admission, symptom severity, functional status, length of stay and overall survival. Most common reasons for palliative care unit (PCU) admission were nutritional problems (82%), fatigue (77%) and pain (68%). Median ECOG score at PCU admission was 3. The timepoint within the timeline of myeloma disease at which hospitalization took place varied greatly. Some patients were hospitalized shortly after diagnosis, other patients after many years of active disease and therapy. Median time from MM initial diagnosis to first PCU stay was 4.3years (range 0.6-23.8 years). The median length of hospital (PCU) stay was 11days (range 1-127days) and 45% of patients died during PCU hospitalization. The reduction in symptom burden as aresult of the inpatient stay in the PCU is reflected in the PERS2ON score, which was measured on the day of admission (median23 days, range 6-32days) and on the day of discharge (median16 days, range 7-20days). PC interventions effectively addressed the complex symptom burden experienced by patients with MM. Multidimensional approaches encompassing physical, psychological and social domains proved instrumental in optimizing quality of life. Integrating PC principles into MM management paradigms is essential to prioritize patient-centered care across the disease continuum.

PDCD4 interacting with PIK3CB and CTSZ promotes the apoptosis of multiple myeloma cells.

The role of programmed cell death 4 (PDCD4) in multiple myeloma (MM) development remains unknown. Here, we investigated its role and action mechanism in MM. Bioinformatic analysis indicated that patients with MM and high PDCD4 expression had higher overall survival than those with low PDCD4 expression. PDCD4 expression promoted MM cell apoptosis and inhibited their viability invitro and tumor growth invivo. RNA-binding protein immunoprecipitation sequencing analysis showed that PDCD4 is bound to the 5' UTR of the apoptosis-related genes PIK3CB, Cathepsin Z (CTSZ), and X-chromosome-linked apoptosis inhibitor (XIAP). PDCD4 knockdown reduced the cell apoptosis rate, which was rescued by adding PIK3CB, CTSZ, or XIAP inhibitors. Dual luciferase reporter assays confirmed the internal ribosome entry site (IRES) activity of the 5' UTRs of PIK3CB and CTSZ. An RNA pull-down assay confirmed binding of the 5' UTR of PIK3CB and CTSZ to PDCD4, identifying the specific binding fragments. PDCD4 is expected to promote MM cell apoptosis by binding to the IRES domain in the 5' UTR of PIK3CB and CTSZ and inhibiting their translation. Our findings suggest that PDCD4 plays an important role in MM development by regulating the expression of PIK3CB, CTSZ, and XIAP, and highlight new potential molecular targets for MM treatment.

TAZ downregulated ANXA1 expression to modulate myeloma cell interactions with bone marrow mesenchymal stromal cells

We and others have previously shown that TAZ plays a tumor suppressive role in multiple myeloma. However, recent reports suggest that molecular crosstalk between the myeloma cells and bone marrow stromal components contributes to the myeloma cell survival and drug resistance. These reports further point to reciprocal interaction via adhesion molecules as the most prominent mechanism of intercellular crosstalk between myeloma cells and bone marrow mesenchymal stromal cells (BM-MSCs). YAP/TAZ silencing/expression has been shown to correlate across all cancers with a set of adhesion/extracellular matrix proteins. Therefore, we hypothesized that TAZ may regulate myeloma cell interaction with BM stromal cells by influencing the expression of distinct cell adhesion signatures. We used previously established TAZ myeloma cell line models, including DELTA47-pLENTI or TAZ knockout DELTA47 cells cocultured with or without BM-MSCs, as our study models. Using RNA sequencing analysis, we performed the first comprehensive screen for cell adhesion-related transcriptional targets of TAZ in multiple myeloma (MM). In doing so, we uncovered an enrichment of cell adhesion-related genes in TAZ knockout DELTA47 cells relatively to pLENTI-DELTA47 cells, including 11 genes with log2 fold change > 2 (p < 0.05), namely, ANXA1, ADGRL2, NCAM1, NCAM2, ADGRL3, CXADR, ALCAM, JAM2, KIRREL1, KIRREL2, and ADGRG7, suggesting possible relationship with TAZ. We validated ANXA1 as a bona fide target of TAZ in MM. We show that TAZ represses myeloma cell migration and interaction with BM-MSCs by transcriptionally downregulating ANXA1 expression via TEAD-dependent mechanism. Our data provide new insights into the understanding of the role of TAZ in the intercellular communication signals between myeloma cells and BM-MSCs. Our findings also suggest that ANXA1 represents a putative cell adhesion target to attenuate BM-MSC driven, tumor-promoting interaction with myeloma cells.

The role of immune checkpoint inhibitors: Variable number of tandem repeat (VNTR) polymorphism in the second exon of the P-selectin glycoprotein ligand-1 (PSGL-1) gene polymorphism in multiple myeloma.

Somatic mutations and polymorphisms may play a role in multiple myeloma (MM) susceptibility and survival. One of the immune checkpoint inhibitors is P-selectin glycoprotein ligand-1 (PSGL-1); the majority of tumor-infiltrating leukocytes express PSGL-1, causing T cell and immune inhibition via PSGL-1 mediator molecules. We aimed to investigate the effect of variable number of tandem repeat (VNTR) polymorphism in the second exon of the PSGL-1 gene on MM susceptibility, response to treatment and survival in our patient group. A total of 238 patients diagnosed with MM between January 2010 and January 2021 and 162 healthy individuals as a control group were included in this cross-sectional study. The genotypes of the VNTR polymorphism in the second exon of the PSGL-1 gene were statistically compared between patients and healthy controls; the statistically significant effects of the genotypes on response to first-line treatment and survival were examined. The AC genotype was significantly higher in healthy controls compared to patients diagnosed with MM (p < 0.001). The median PFS in patients with AA/AB/AC was 56 months, while it was 100 months in patients with BB/CC. The hazard ratio of 1.34 for PFS was found to be clinically significant and having the BB/CC genotype could provide a longer PFS compared to others, but it was not statistically significant due to the sample size. Our study results will shed light on new study plans in terms of immune checkpoint target therapies among conventional treatment preferences in MM.

Determination of Serum Cathepsin G Level in Patients with Multiple Myeloma

Multiple myeloma is a hematological malignancy identified by bone marrow infiltration of clonal plasma cells. It is still not a curable disease under current conditions. Cathepsin G is a serine protease playing a role in inflammation that is present in the azurophilic granules of neutrophils. It is known that there is a relationship between Cathepsin G and chronic inflammatory diseases and tumors. The goal of the study is to define its role in multiple myeloma. In the study, 33 patients newly diagnosed with MM who were never received treatment and 33 control subjects were included. Basic laboratory parameters and Cathepsin G levels were examined both in the myeloma patient group and control group. While the serum CathG level in the control group is 22.84 ng/mL, the serum CathG level of the MM patient group is 10.77 ng/mL. Serum CathG level in the control group is statistically significantly higher than the MM patient group. The aim of the present study is to contribute to the literature in hematological malignancies, to figure out the role of Cathepsin G in multiple myeloma, and to open a door to new treatment options for multiple myeloma, which is an incurable disease, yet.

Identification of lipid metabolism-related gene signature in the bone marrow microenvironment of multiple myelomas through deep analysis of transcriptomic data

Dysregulated lipid metabolism in the bone marrow microenvironment (BMM) plays a vital role in multiple myeloma (MM) development, progression, and drug resistance. However, the exact mechanism by which lipid metabolism impacts the BMM, promotes tumorigenesis, and triggers drug resistance remains to be fully elucidated.By analyzing the bulk sequencing and single-cell sequencing data of MM patients, we identified lipid metabolism-related genes differential expression significantly associated with MM prognosis, referred to as LMRPgenes. Using a cohort of ten machine learning algorithms and 117 combinations, LMRPgenes predictive models were constructed. Further exploration of the effects of the model risk score (RS) on the survival status, immune status of patients with BMM, and response to immunotherapy was conducted. The study also facilitated the identification of personalized therapeutic strategies targeting specified risk categories within patient cohorts.Analysis of the scRNA-seq data revealed increased lipid metabolism-related gene enrichment scores (LMESs) in erythroblasts and progenitor, malignant, and Tprolif cells but decreased LMESs in lymphocytes. LMESs were also strongly correlated with most of the 50 hallmark pathways within these cell populations. An elevated malignant cell ratio and reduced lymphocytes were observed in the high LMES group. Moreover, the LMRPgenes predictive model, consisting of 14 genes, showed great predictive power. The risk score emerged as an independent indicator of poor outcomes. Inverse relationships between the RS and immune status were noted, and a high RS was associated with impaired immunotherapy responses. Drug sensitivity assays indicated the effectiveness of bortezomib, buparlisib, dinaciclib, staurosporine, rapamycin, and MST-312 in the high-RS group, suggesting their potential for treating patients with high-RS values and poor response to immunotherapy. Ultimately, upon verification via qRT-PCR, we observed a significant upregulation of ACBD6 in NDMM group compared to the control group.Our research enhances the knowledge base regarding the association between lipid metabolism-related genes (LMRGs) and the BMM in MM patients, offering substantive insights into the mechanistic effects of the BMM mediated by LMRGs.Graphical abstract

Cytidine triphosphate synthase 1-mediated metabolic reprogramming promotes proliferation and drug resistance in multiple myeloma

Upregulation of metabolism-related gene cytidine triphosphate synthase 1 (CTPS1) is associated with poor prognosis in multiple myeloma (MM). However, its role in MM remains unclear. In this study, bioinformatics analysis revealed significant differences in CTPS1 expression levels among various plasma cell malignancies. The patients with high CTPS1 expression had poor overall survival, progression-free survival, and event-free survival. CTPS1 was significantly correlated with sex, albumin, β2 microglobulin, lactate dehydrogenase, and advanced disease. In vitro experiments demonstrated that CTPS1-overexpressing (CTPS1-OE) cells proliferated faster than CTPS1-short hairpin RNA (CTPS1-sh) cells. NRG-SGM3 mice showed significantly accelerated tumor growth in the CTPS1-OE group. CTPS1-OE decreased sensitivity to bortezomib, whereas CTPS1-sh increased sensitivity to bortezomib in MM cell lines. Mechanistically, CTPS1 was primarily involved in metabolism processes. Additionally, CTPS1 was closely related to several co-expressed genes such as MYC and the bone marrow immune microenvironment. In conclusion, CTPS1 is a significant prognostic biomarker for patients with MM, suggesting a potential therapeutic target.

MicroRNA-138 promotes the progression of multiple myeloma through targeting paired PAX5

BackgroundMultiple myeloma cancer stem cells (MMSC) have been considered as the leading cause of multiple myeloma (MM) drug resistance and eventual relapse, microRNAs (miRNAs) collectively participate in the progression of MM. However, the pathogenesis of miR-138 in MMSC is still not fully understood. ObjectiveThe intention of this study was to investigate the mechanism and role of miR-138 in multiple myeloma. MethodBone marrow samples and peripheral blood from patients and normal controls were collected. Use Magnet-based Cancer Stem Cell Isolation Kit to separate and extract MMSC. Real-time quantitative PCR (RT-qPCR) was carried out to determine mRNA level. Western blot was applied to detect protein levels. MTT and flow cytometry were conducted to examine the proliferation and apoptosis of MMSC. Finally, dual-luciferase reporter gene assays were performed to confirm that paired box 5 (PAX5) is a direct target for miR-138. ResultsCompared with normal group, the expression of miR-138 in patients was significantly up-regulated, and the expression of miR-138 was in a negative correlation with PAX5. Additionally, downregulated miR-138 facilitated the apoptosis and inhibited the proliferation of MMSC in vitro and in vivo. Downregulated miR-138 moderated the expression of PAX5, Bcl-2, Bax, and Caspase-3. PAX5 was a direct target of miR-138. ConclusionTaken together, miR-138 plays a carcinogenic role in MM, and miR-138 adjusted the proliferation and apoptosis of MMSC by targeting PAX5. miR-138 has the probability of becoming a new medicinal target for the treatment of MM.

Circulating miR-16 and miR-21 Levels in Multiple Myeloma: Prognostic Significance of Survival and Response to Lenalidomide Treatment.

MicroRNAs (miRNAs), particularly miR-16 and miR-21, play a crucial role in multiple myeloma (MM) pathogenesis by regulating gene expression. This study evaluated the prognostic significance of circulating miR-16 and miR-21 expression levels in 48 patients with MM at diagnosis treated with lenalidomide-dexamethasone (LD) compared with 15 healthy individuals (HI). All patients were treated with LD, 13 at first line and 35 at relapse, of whom 21 were tested twice at diagnosis and before LD initiation. The results revealed significantly lower levels of miR-16 and miR-21 in patients than in HIs, both at diagnosis and relapse, with decreased miR-16 levels at diagnosis, indicating improved overall survival (OS) (p value 0.024). Furthermore, miR-16 and miR-21 levels were associated with disease markers, while both correlated with the depth of response and mir-16 with sustained response to LD treatment. Ratios of both miR-16 and miR-21 expression levels (prior to LD treatment/diagnosis) below two predicted a shorter time to response (p = 0.027) and a longer time to next treatment (p = 0.042), respectively. These findings suggested a prognostic value for serum miR-16 and miR-21 levels in MM, as their expression levels correlated with disease variables and treatment outcomes.

Do Alarmins Have a Role in Multiple Myeloma?

Calprotectin (CLP), S100A6, and high mobility group nucleosome-binding protein 1 (HMGN1), known as alarmins, are involved in the pathogenesis of many tumors. In this study, we aimed to investigate the relationships of serum CLP, S100A6, and HMGN1 levels with the clinical and laboratory findings of patients with multiple myeloma (MM) and their roles in the pathogenesis of MM. We measured the serum CLP, S100A6, and HMGN1 levels of 55 newly diagnosed patients and 32 healthy controls using the sandwich enzyme-linked immunosorbent assay method. The medical records of the patients were also reviewed. Serum CLP, S100A6, and HMGN1 levels were significantly decreased in MM patients compared to the control group (p=0.012, p=0.001, and p=0.030, respectively). Receiver operating characteristic analysis was used to determine diagnostic cut-off values for serum CLP, S100A6, and HMGN1 of <98 ng/mL (area under the curve [AUC]: 0.663, 95% confidence interval [CI]: 0.554-0.761, p=0.009), <1174.5 pg/mL (AUC: 0.706, 95% CI: 0.598-0.799, p=0.001), and <440.18 pg/mL (AUC: 0.640, 95% CI: 0.530-0.740, p=0.03), respectively. CLP levels were found to be statistically significantly higher in patients with light chain MM (91.58±22.57 ng/mL) compared to heavy chain MM (79.42±15.83 ng/mL) (p=0.03). A negative correlation was observed between CLP and M protein, immunoglobulin G, globulin, and beta-2 microglobulin (correlation coefficients: -0.361, -0.370, -0.279, -0.300, respectively; p=0.024, p=0.06, p=0.04, p=0.0033). In this study, we found that serum CLP, S100A6, and HMGN1 levels were statistically lower in patients with newly diagnosed MM compared to the control group. These results suggest that CLP may bind to the paraprotein produced by heavy chain MM in the blood, causing its blood levels to be low. Additionally, low levels of HMGN1, which is involved in DNA repair, suggest that HMGN1 may contribute to the complex genetic abnormalities found in cases of MM.

Antibody targeting of surface P-selectin glycoprotein ligand 1 leads to lymphoma apoptosis and tumorigenesis inhibition.

Lymphomas are a heterogeneous group of diseases that originate from T, B or natural killer cells. Lymphoma treatment is based on chemotherapy, radiotherapy, and monoclonal antibody (mAb) or other immunotherapies. The P-selectin glycoprotein ligand 1 (PSGL-1) is expressed at the surface of hematological malignant cells and has been shown to have a pro-oncogenic role in multiple myeloma and lymphoma. Here, we investigated the expression and therapeutic potential of PSGL-1 in T and B cell lymphomas. By flow cytometry analysis, we found that PSGL-1 was expressed in both T and B cell-derived lymphoma cell lines but generally at higher levels in T cell lymphoma cell lines. For most T and B cell-derived lymphoma cell lines, in vitro targeting with the PL1 mAb, which recognizes the PSGL-1 N-terminal extracellular region and blocks functional interactions with selectins, resulted in reduced cell viability. The PL1 mAb pro-apoptotic activity was shown to be dose-dependent, to be linked to increased ERK kinase phosphorylation, and to be dependent on the MAP kinase signaling pathway. Importantly, anti-PSGL-1 treatment of mice xenografted with the HUT-78 cutaneous T-cell lymphoma cell line resulted in decreased tumor growth, had no effect on in vivo proliferation, but increased the levels of apoptosis in tumors. Anti-PSGL-1 treatment of mice xenografted with a Burkitt lymphoma cell line that was resistant to anti-PSGL-1 treatment in vitro, had no impact on tumorigenesis. These findings show that PSGL-1 antibody targeting triggers lymphoma cell apoptosis and substantiates PSGL-1 as a potential target for lymphoma therapy.

ALKHB5-demethylated lncRNA SNHG15 promotes myeloma tumorigenicity by increasing chromatin accessibility and recruiting H3K36me3 modifier SETD2.

Chromatin instability plays a crucial role in multiple myeloma (MM) relapse and progression, but its mechanism remains obscure. Here, we uncovered that m6A-demethylase ALKBH5 upregulated and stabilized long noncoding RNA (lncRNA) small nucleolar RNA host gene 15 (SNHG15), which was elevated in MM and positively correlated with unfavorable clinical prognosis factors. ALKBH5-SNHG15 axis participated in viability and migration/invasion of myeloma cell lines and MM-xenografted SCID/NOD mice. Mechanically, ALKBH5 promoted the expression of trimethylated histone H3 at lysine 36 (H3K36me3) methyltransferase SETD2 through lncRNA SNHG15-mediated protein stability. ALKBH5-SNHG15 axis increased chromatin accessibility and altered the H3K36me3 enrichment at the gene body, which is responsible for transcription elongation. Our study suggested a novel epigenetically interaction of N6-methyladenosine (m6A) methylation, lncRNA SNHG15, and histone SETD2/H3K36me3 modifications in myeloma progression, indicating that ALKBH5 and lncRNA SNHG15 could serve as potential novel therapeutic targets for MM treatment.NEW & NOTEWORTHY To our knowledge, this study first demonstrated the prognostic significance and biological function of long noncoding RNA (lncRNA) small nucleolar RNA host gene 15 (SNHG15) in multiple myeloma (MM), and indicated a novel revelation on the effect of N6-methyladenosine (m6A)-regulated lncRNA on MM tumorigenicity. Moreover, the novel chromatin-regulatory mechanism of lncRNA by interacting with epigenetic modifiers including m6A demethylase ALKBH5 and H3K36me3 methyltransferase SETD2 in myeloma progression elucidated intricate mechanism of tumor pathogenesis.

Short-Chain Fatty Acid Production by Gut Microbiota Predicts Treatment Response in Multiple Myeloma.

The gut microbiota plays important roles in health and disease. We questioned whether the gut microbiota and related metabolites are altered in monoclonal gammopathies and evaluated their potential role in multiple myeloma and its response to treatment. We used 16S rRNA sequencing to characterize and compare the gut microbiota of patients with monoclonal gammopathy of undetermined significance (n = 11), smoldering multiple myeloma (n = 9), newly diagnosed multiple myeloma (n = 11), relapsed/refractory multiple myeloma (n = 6), or with complete remission (n = 9). Short-chain fatty acids (SCFA) were quantified in serum and tested in cell lines. Relevant metabolites were validated in a second cohort of 62 patients. Significant differences in alpha- and beta diversity were present across the groups and both were lower in patients with relapse/refractory disease and higher in patients with complete remission after treatment. Differences were found in the abundance of several microbiota taxa across disease progression and in response to treatment. Bacteria involved in SCFA production, including Prevotella, Blautia, Weissella, and Agathobacter, were more represented in the premalignant or complete remission samples, and patients with higher levels of Agathobacter showed better overall survival. Serum levels of butyrate and propionate decreased across disease progression and butyrate was positively associated with a better response. Both metabolites had antiproliferative effects in multiple myeloma cell lines. We demonstrate that SCFAs metabolites and the gut microbiota associated with their production might have beneficial effects in disease evolution and response to treatment, underscoring its therapeutic potential and value as a predictor.

Acute kidney injury in multiple myeloma patients undergoing autologous hematopoietic stem cell transplant: a cohort study

BackgroundAutologous hematopoietic stem cell transplant plays an important role in multiple myeloma (MM) treatment. Increasing incidence of MM and growing awareness of acute kidney injury (AKI) as a complication of hematopoietic stem cell transplant results in the need to better understand AKI in these patients. We aimed to evaluate incidence, risk factors and 5-year prognostic impact of AKI in MM patients undergoing autologous hematopoietic stem cell transplant.MethodsRetrospective cohort study. AKI was defined by the KDIGO classification using creatinine and urinary output criteria. We used survival analysis methods considering competing events for risk factors and disease-free survival, Cox proportional regression for overall survival and stepwise regression methods for multivariable models.ResultsWe analyzed data regarding 143 patients. The cumulative incidence of AKI and moderate-to-severe AKI was 49.7% and 14.1%, respectively. Factors with independent impact on AKI were obesity (HR: 1.83, 95% CI 1.07–3.11; p = 0.026), Hematopoietic cell transplantation—specific comorbidity index (HCT-CI) ≥ 2 (HR: 1.85, 95% CI 1.08–3.17), chronic kidney disease (CKD) (HR: 2.06, 95% CI 1.05–4.04), amyloidosis (HR: 2.25, 95% CI 1.25–4.06), mucositis grade 3–4 (HR: 2.19, 95% CI 1.25–3.86) and exposure to nephrotoxic drugs (HR: 2.0856, 95% CI 1.04–4.19). Moderate-to-severe AKI had an impact (HR: 1.62, 95% CI 1.15–2.31) on 5-year overall survival.ConclusionAcute kidney injury affects almost half of MM patients undergoing autologous hematopoietic stem cell transplantation, and reduction in urinary output allows early diagnosis in almost a quarter of the patients. Obesity, HCT-CI ≥ 2, CKD, amyloidosis, mucositis grade 3–4 and exposure to nephrotoxic drugs are significant risk factors. Moderate-to-severe AKI is associated with lower 5-year overall survival.Graphical abstract

TAS0612, a Triple Inhibitor for RSK, AKT, and S6K, Is a Promising Anti-Myeloma Agent Regardless of Types of Cytogenetic/Genetic Abnormality

Introduction: Despite advancements in therapeutic approaches, treatment outcome for multiple myeloma (MM) remains unsatisfactory. One critical factor of the poor outcomes is the complex inter-patient and intra-clonal heterogeneity of cytogenetic, genetic, and molecular abnormalities. To overcome this, the exploration of therapeutic target molecules that play a universal and essential role in MM regardless of the types of molecular profile is crucial even in the post-genomic era. We have previously demonstrated that RSK2 Ser227 at the N-terminal kinase domain (NTKD), as the downstream signaling hub of the RAS/ERK pathway, plays essential roles in the proliferation and survival of myeloma cells (Shimura Y, Mol Cancer Ther 2012). We also identified that RSK2-NTKD is constitutively activated by constitutive active PDPK1, the master serine-threonine kinase of &amp;gt;20 AGC kinases, in myeloma cells of approximately 90% of MM patients regardless of cytogenetic abnormalities (Chinen Y, Cancer Res 2014, Tatekawa S, Br J Haematol 2017). Moreover, the combinatory blockade of RSK2 and AKT, one of the other important molecules regulated by PDPK1, exhibited synergistic anti-myeloma efficacy through the modulation of various critical gene sets involved in myeloma pathophysiology, such as MYC (Isa R, Int J Mol Sci 2022). Based upon these, this study investigates the anti-myeloma potency of TAS0612 (Taiho Oncology Inc., Princeton, NJ) that targets RSK, including RSK2, AKT, and S6K, and inhibits the bindings to their respective substrate molecules (Shibata T, Mol Cancer Ther 2020). Methods and Results: Firstly, we examined the activation status of RSK, AKT, and S6K in eight human myeloma-derived cell lines (HMCLs) with diverse genetic and molecular profiles at the protein level. RSK2 Ser227 was found to be commonly activated through phosphorylation in all HMCLs examined. In contrast, the activation patterns of p-AKT Ser473 and p-S6K Thr389 were inconsistent among eight HMCLs, and depending on cell types. Despite these cell type-dependent effects, the exposure to TAS0612 resulted in the accumulation of p-total RSK Ser380, p-AKT Ser473, and p-S6K Thr389 in the majority of HMCLs examined, and this led to the subsequent inactivation of their respective target substrates, such as p-YB1 Ser102, p-PRAS Thr246, and p-S6 Ser235/236, through dephosphorylation. In accompany with these molecular effects, the modified MTT assay showed the dose-dependent growth inhibitory effects of TAS0612 in all cell lines with various types of cytogenetic/genetic abnormalities with IC50 values mostly below 0.5 μM (48 hours). In addition, flow cytometry analyses for the cell cycle analysis and the apoptosis assay by the counterstaining of propidium iodide and Annexin-V showed that TAS0612 induced G1/S cell cycle arrest and apoptotic cell death. Furthermore, patient-derived primary myeloma cells were labeled with anti-CD138 MicroBeads, and were positively isolated from bone marrow mononuclear cells from patients with newly diagnosed MM (n=7) and relapsed/refractory MM (n=11), and, then, were treated by TAS0612. As the results, the ex vivo treatment of 2.5-5.0 μM TAS0612 for 72 hours showed robust cell reductive effects on primary-myeloma cells from all 18 patients examined irrespective of cytogenetic profile. To clarify the precise molecular effects of TAS0612 treatment in myeloma cells, we conducted a comprehensive molecular analysis with TAS0612 treatment by gene expression profiling. The results showed that TAS0612 treatment significantly modulated the gene sets involved in Myc signaling, mTOR signaling, and the core serum response (CSR). Additionally, TAS0612 caused the concomitant upregulation of several bona-fide tumor suppressor genes, including TP53INP, HBP1, FBOX32, and YPEL3, while the downregulation of oncogenes LYAR and HSPA1. Intriguingly, according to MMRF CoMMpass Study, 8 of 18 upregulated genes by TAS0612 in HMCLs were reported to be associated with favorable survival outcomes when highly expressed in myeloma cells. Conclusion: Our findings strongly support the potential of TAS0612 as an effective anti-myeloma agent. Further preclinical investigations are necessary to substantiate these results and facilitate its future clinical development.

Network Modeling of Apoptosis-Related Genes Reveal a High-Risk Cluster in Multiple Myeloma

The plasma cell cancer multiple myeloma (MM) varies significantly in genomic characteristics, response to therapy, and long-term prognosis. Recent studies have shown the complexity of biological systems associated with poor outcomes in MM. One hallmark of cancer pathogenesis, applicable to MM, is the dysregulation of apoptotic cell death. Understanding the relationship between known drivers of MM and critical pathways in the apoptosis gene network is key towards identifying novel therapeutic targets. Recently, there has been a surge of interest in developing network-based approaches to study gene interactions and use machine learning tools to further elucidate useful biological factors. It is well-known that genes operate as network systems; however, interpretable computational methods are still limited. In this work, we combine a set of network analysis tools to provide novel biological insights into how the apoptosis network is dysregulated in MM. We identify a set of genes associated with poor outcomes in MM using both univariate and network-based approaches. Genomic and clinical data, including RNA-sequencing (RNA-seq) and copy number alteration (CNA), for 659 subjects were obtained from the Multiple Myeloma Research Foundation's CoMMpass (IA19) database. The apoptosis gene set is defined using the apoptosis hallmark pathway provided by the gene set enrichment analysis tool. The interactions between the given list of genes are provided via MetaCore. Univariate gene modeling is done using a Cox proportional hazards model. To determine the relationship between genes, we used Ollivier-Ricci curvature (ORC), a measure of network robustness. The ORC value between two genes incorporates information about both the two genes in question and their local neighborhoods, a concept critical for understanding the role a neighborhood of genes has on a given connection. Once edge information is computed, we used a uniform manifold approximation and projection (UMAP) to reduce the complexity of the dataset. The resulting projection is clustered using k-means clustering, and survival analysis of the given clusters is done using the Kaplan-Meier (KM) method. To understand which features are most informative, we used a random forest to predict the cluster labels using the RNA-seq data. After five-fold cross-validation, feature importance was computed using random forest's permutation importance methodology. All statistical significance testing was corrected for multiple comparisons via the Benjamini-Hochberg false discovery rate method with an alpha set to 0.05. Using RNA-seq data of genes associated with apoptosis, we identified nineteen apoptosis genes associated with poor prognosis in MM. Some of these genes, such as ANXA1 and WEE1 are known to have an effect in MM, while others, such as AVPR1A have yet to be studied extensively for their role in MM. When examining CNA data associated with apoptosis, a similar pattern emerged - nineteen genes were associated with poor prognosis. However, between the two key gene sets found by RNA-seq and CNA, only FAS appeared in both lists. FAS, Fas Cell Surface Death Receptor, is critical for triggering apoptosis. Half of the 37 genes identified using RNA-seq and CNA data are directly connected to the TP53 gene. While TP53 mutations are not a marker of the presence of MM itself, TP53 mutations have been shown to be associated with poorer outcomes. After reducing the complexity of the Ollivier-Ricci curvature analysis of the RNA-seq apoptosis network using UMAPs, the data revealed five clusters with KM curves for progression-free survival were significant (p&amp;lt;0.001). To identify the genes most impactful for separating these clusters, we trained a random forest model on the RNA-seq - note, it was the edge robustness values that were clustered - and evaluated its performance for predicted class membership. The model had an overall accuracy of approximately 78% with F1 scores for each class ranging from 61% to 87%. The top three features associated with the model include CTNNB1, RELA, and LEF1. Of note, CTNNB1 and RELA are directly interacted with the TP53 gene. Because these three genes discriminate between all five clusters, their expression levels may play a mediating effect on MM outcomes. Further investigation with external datasets is needed to validate these results.

Tumor Suppressive Mir-7 Targeting PSME3 Improves the Efficacy of Histone Deacetylases Inhibitors in Multiple Myeloma

Background: Histone deacetylases (HDACs) play a tumor-promoting role in multiple myeloma (MM), contributing to disease progression and treatment resistance. Panobinostat, an HDAC inhibitor, is clinicallyrelapsed or refractory MM. However, HDAC inhibitors have several limitations, such as drug resistance, poor tolerance, and limited use of combination drugs. Our previous comprehensive analysis of microRNAs modulated by HDAC inhibitors revealed new molecular pathogenesis and potential therapeutic targets in malignant lymphomas. However, the relationship between HDAC inhibitors and microRNAs in MM remains largely unknown. Aim and methods: We treated the MM cell lines (MM.1S and KMS-11) with HDAC inhibitors (10 nM panobinostat and 1 µM vorinostat), collected RNA 48 h later, and conducted a comprehensive microarray analysisFurthermore, we explored the novel clinical impact of HDAC inhibitors via functional analysis of microRNAs. Results: Microarray analysis showed that no microRNAs were commonly upregulated; however, only hsa-miR-7-5p (miR-7) was commonly downregulated in MM.1S and KMS-11 cells treated with both HDAC inhibitors. After exposing MM cell lines (MM.1S, KMS-11, RPMI-8226, H929, and U266) and patient samples to 10 nM panobinostat and 1 µM vorinostat for 48 h, we performed quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and observed significant downregulation in the expression of miR-7. Next, we examined the function of miR-7 in MM by introducing miR-7 into MM cell lines. Introducing miR-7 significantly suppressed the proliferation of MM.1S and KMS-11 cells. We hypothesized that the introduction of miR-7 enhances the effects of HDAC inhibitors. We introduced miR-7 into MM.1S and cultured them for 48 h with panobinostat, followed by an apoptosis assay. The introduction of miR-7 significantly enhanced the apoptosis-inducing effect of panobinostat, suggesting a tumor-suppressive role of miR-7 in MM. Furthermore, we examined the regulatory mechanisms of miR-7 in MM cells. Because it has been reported that MYC promotes miR-7 transcription, we focused on the role of MYC in miR-7 expression in MM. After exposing MM.1S and KMS-11 cells to HDAC inhibitors for 24 h, western blot analysis revealed that MYC was downregulated. After exposing MM.1S and KMS-11 cells to the BRD4 inhibitor JQ1, which suppresses MYC, at concentrations of 50 and 100 nM for 48 h, qRT-PCR showed that the expression of miR-7 was significantly downregulated in these cell lines. Similarly, introducing siRNA for MYC into MM.1S cells downregulated the expression of miR-7. These results suggest that HDAC inhibitors downregulate miR-7 expression by suppressing MYC expression. In addition, we examined the effect of bortezomib, a proteasome inhibitor, in combination with MM on the expression of miR-7. After exposing MM.1S, KMS-11, and H929 cells to 5 nM and 10 nM bortezomib for 48 h, we performed qRT-PCR and observed a significant upregulation in the expression of miR-7. Therefore, anti-myeloma effects of panobinostat by restoring miR-7 expression. Finally, we investigated miR-7 targets that mediate the anti-myeloma effects. We introduced miR-7 into MM.1S cells via electroporation, collected RNA 48 h later, and performed microarray analysis. As a result, 26 genes were identified, and their expression was downregulated by less than one-third. Among these, four genes ( SLC6A9, LRRC59, EXOSC2, and PSME3) possess conserved binding sites for miR-7. PSME3 (proteasome activator subunit 3：PA28ɤ) has been reported as an oncogene in MM, and we focused on PSME3. Introducing miR-7 into MM.1S cells via electroporation significantly suppressed PSME3 expression. Furthermore, when MM.1S cells were exposed to HDAC inhibitors, PSME3 expression was significantly upregulated. It has been reported that the inhibition of PSME3 increases sensitivity to proteasome inhibitors in MM. The favorable combination effect of bortezomib with HDAC inhibitors could be due to the modulation of the miR-7- PSME3 axis. Conclusion: We revealed that miR-7 exerts anti-myeloma effects and that PSME3 is one of the targets of miR-7. miR-7 is an interesting microRNA because it is positively regulated by MYC, even though it is tumor-suppressive. Elucidating the role of miR-7 may lead to the re-discovery of HDAC inhibitors in MM therapeutic strategies.

Chimeric Antigen Receptor T Cells Targeting LILRB4, an Immunoreceptor Mediating T-Cell Suppression, Are Potently Effective in Multiple Myeloma

Significant progress has been made in the treatment of multiple myeloma (MM) however, no strategy to date has proven curative and relapse invariably occurs. We recently developed an integrated pipeline to identify biologically and therapeutically relevant targets for MM immunotherapy by using Mass-Spectrometry analyses from seven MM cell lines and RNA-seq data from 900+ patients. Starting from 4,000+ candidates, ILT3 aka LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor emerged as a promising candidate target: it has absent/low expression in most normal tissues and high and frequent expression in many primary patient samples we used for validation, similarly to BCMA (Di Meo et al., Cell Reports Medicine 2023). We sought to examine its functional role in MM and develop Chimeric Antigen Receptor (CAR) T-cells targeting LILRB4. Interrogating RNA-seq data from MM patients, LILRB4 expression results higher in patients with high-risk cytogenetics such as t:(14;16), t:(14;20), and t:(8;14) translocations compared to patients with no genomic abnormalities. Investigating function, we find no difference between cell proliferation of U266/KMS11/OPM2 genetically depleted of LILRB4 (KO) compared to controls by measuring cell growth in vitro for 120 hours and MTS assays. Similarly, when KO and control cells are seeded in plastic chambers containing a porous membrane and serum-free medium, both KO and control cells are attracted to the lower chambers containing FBS, suggesting that LILRB4 is also not required for MM cell migration. Despite these initial observations, RNAseq data from LILRB4 KO and overexpressing (OE) U266 MM cells identify ~3,000 differentially expressed genes compared to controls. Thus, we next investigate potential extrinsic effects on T-cell proliferation by co-culturing T-cells from healthy donors with LILRB4 KO or OE U266 MM cells (T:E ratio 1:2). T cells are previously activated with anti-CD3/CD28-coated beads and IL-2. 5 days after activation, T cells and LILRB4 KO U266 MM are seeded in the same well plate. After 48h of co-culture, T-cell proliferation is decreased (-30%) in the presence of LILRB4 U266 OE MM cells, whereas it is increased (+50%) when the co-culture includes LILRB4 U266 KO cells. CD25 expression is also reduced by 20% on T cells co-cultured with LILRB4 OE cells and increased by the same extent when T cells are co-cultured with LILRB4 KO cells, suggesting a role as a negative immune receptor. This effect seems dependent on the direct cell-cell interaction given that when T and MM cells are separated by a transwell in co-culture, no effect on the T cell proliferation is observed. Finally, we develop multiple Chimeric Antigen Receptor (CAR) constructs targeting LILRB4, by using different single-chain variable fragments (scFVs) and rearranging the variable regions of the heavy (VH) and light (VL) regions. We used lentiviral LILRB4 scFV-41BB-CD3zeta vectors. LILRB4 CAR T-cells specifically kill U266, H929 and KMS11 MM cell lines in vitro with cytotoxicity ranging from 30 to 80% depending on the construct (T:E ratio 1:10). Equal efficacy is observed in U266 cells resistant to Bortezomib and BCMA KO U266cells. No effect on LILRB4 null cells is noted. These data suggest that LILRB4 is a negative immune receptor that is active on malignant plasma cells mediating T-cell suppression, similar to previously described data in leukemia (Deng et al., Nature 2018). Our work supports the rationale development of ILT3 CAR-T cell therapy as a viable therapeutic approach for MM patients especially high-risk patients. Ongoing studies are investigating the functional role of LILRB4 in mediating the cross-talk between MM ad immune cells.