Patient Multiple Myeloma Cells Research Articles

Original vs. Generic Plerixafor for the Mobilization of Stem Cells in Multiple Myeloma Patients

Original vs. Generic Plerixafor for the Mobilization of Stem Cells in Multiple Myeloma Patients

CiRS-7 circular RNA overexpression in plasma cells is a promising molecular biomarker of unfavorable prognosis in multiple myeloma.

Several non-coding RNAs are known to be associated with the pathobiology and progression of multiple myeloma (MM). ciRS-7 (also known as CDR1-AS), a key oncogenic circular RNA (circRNA) that sponges miR-7-5p and other cancer-related microRNAs, was recently found to be downregulated in malignant plasma cells resistant to immunomodulatory drugs. Considering that various circRNAs have a strong potential as molecular biomarkers, we aimed to investigate the expression of ciRS-7 in plasma cell disorders, assess its prognostic importance in MM, and compare these findings with those of individuals with smoldering MM (SMM) and monoclonal gammopathy of unknown significance (MGUS). This study included 171 patients (110 newly diagnosed MM, 34 SMM, and 27 MGUS cases), from which bone marrow aspirate samples were collected for CD138+ plasma cell selection. Total RNA was reversely transcribed using random hexamer primers, and the expression levels of ciRS-7 were quantified using an in-house-developed protocol that includes pre-amplification and real-time quantitative polymerase chain reaction. ciRS-7 levels were found to significantly differ among CD138+ plasma cells of MM, SMM, and MGUS patients. ROC analysis indicated that ciRS-7 expression effectively distinguishes between MM and SMM patients. Moreover, high levels of ciRS-7 were associated with unfavorable prognosis in MM, independently of MM patients' age and Revised International Staging System stage. Additionally, in silico analysis predicted the binding of 85 microRNAs to ciRS-7. In conclusion, this study provides novel insights into the role of ciRS-7 as a promising molecular marker able to distinguish MM from SMM and predict prognosis in MM.

Dynamic changes in γδT cells and bone resorption markers after zoledronic acid in multiple myeloma: A prospective study

We conducted a prospective evaluation for the dynamic change of γδT cells in peripheral blood (PB) and N-telopeptide of type I collagen in urine (uNTX) of patients diagnosed with multiple myeloma (MM) who underwent their initial treatment with zoledronic acid (ZOA; Zobonic®, TTY, Taiwan). Between March 2012 and November 2015, a total of 35 patients were enrolled, including 25 newly diagnosed MM (NDMM) patients. The percentage of γδT cells in PB was assessed at 20 days prior to the first ZOA infusion, then at day 8, day 64, and day 85 after the infusion. Simultaneously, uNTX levels were measured as well. Thirty-three patients who had received at least one dose of ZOA were included in subsequent analysis. We identified three dynamic change patterns for γδT cells: fluctuated pattern, continuously increasing pattern, and continuously decreasing pattern. Among NDMM patients, those exhibiting a continuously increasing pattern showed a significantly shorter overall survival compared to those with the other two patterns combined (4.7 months vs. 92.9 months, p = 0.037). For uNTX, which levels significantly decreased following ZOA treatment. In conclusion, our findings reveal three distinct dynamic change patterns for γδT cells after ZOA initiation, with continuously increasing pattern being associated with a poor prognosis. These findings prompt further inquiry into the role of γδT cells in MM patients and support the suppressive nature of γδT cells and their associated tumor microenvironment.

G-Quadruplex Forming DNA Sequence Context Is Enriched around Points of Somatic Mutations in a Subset of Multiple Myeloma Patients.

Multiple myeloma (MM) is the second most common hematological malignancy, which remains incurable despite recent advances in treatment strategies. Like other forms of cancer, MM is characterized by genomic instability, caused by defects in DNA repair. Along with mutations in DNA repair genes and genotoxic drugs used to treat MM, non-canonical secondary DNA structures (four-stranded G-quadruplex structures) can affect accumulation of somatic mutations and chromosomal abnormalities in the tumor cells of MM patients. Here, we tested the hypothesis that G-quadruplex structures may influence the distribution of somatic mutations in the tumor cells of MM patients. We sequenced exomes of normal and tumor cells of 11 MM patients and analyzed the data for the presence of G4 context around points of somatic mutations. To identify molecular mechanisms that could affect mutational profile of tumors, we also analyzed mutational signatures in tumor cells as well as germline mutations for the presence of specific SNPs in DNA repair genes or in genes regulating G-quadruplex unwinding. In several patients, we found that sites of somatic mutations are frequently located in regions with G4 context. This pattern correlated with specific germline variants found in these patients. We discuss the possible implications of these variants for mutation accumulation and specificity in MM and propose that the extent of G4 context enrichment around somatic mutation sites may be a novel metric characterizing mutational processes in tumors.

HDAC Inhibition Induces CD26 Expression on Multiple Myeloma Cells via the c-Myc/Sp1-mediated Promoter Activation.

There is a desire to induce and sustain CD26 expression on multiple myeloma cells to elicit superior anti-myeloma response by humanized anti-CD26 mAb therapy. HDACi upregulates the expression levels of CD26 on myeloma cells via the increased acetylation of c-MycK323 on the CD26 promoter, leading to initiation of CD26 transcription, thereby synergistically augments the efficacy of CD26mAb against CD26neg myeloma cells.

Analysis of Peripheral Blood T Lymphocyte Subsets in Multiple Myeloma Patients.

The aim of this study was to investigate the changes of T lymphocyte subsets (Th1, Th2, Tc1, Tc2, and Th17) and memory T lymphocyte subsets (Tcm and Tem) in patients with multiple myeloma (MM) at different stages of the disease. In total, 25 newly diagnosed patients with MM were selected as the study subjects and 30 healthy people were selected as the control group. The subsets of T lymphocytes such as Th1, Th2, Tc1, Tc2, Th17, Tcm, and Tem in the peripheral blood were detected by flow cytometry at the time of initial diagnosis, infection, and remission. Th1, Tem, and Tcm cells in MM patients showed a significant decrease compared to the control group. Th2 and Th17 cells in MM patients showed a significant increase compared to the control group. Total Th1 cells and memory Th1 cells in MM patients with bacterial infection were significantly higher than at initial diagnosis (p < 0.05). The Tcm of Th2 cells in the remission stage were significantly higher than those in MM patients with no remission. MM patients have decreased Th1 cells and increased Th2 and Th17 cells. The changes in memory Th1 cells were related to bacterial infection in MM patients. The increase of Tcm of Th2 cells may be associated with disease remission. The balance of T lymphocyte subsets plays an important role in the pathogenic course of MM.

Research and Analysis of Molecules such as CD28, CD45RA, CD45RO, CD38, HLA-DR, and CD57 on T Cells in Multiple Myeloma.

For many years it has been postulated that the immune system controls the progress of multiple myeloma (MM). However, the phenotypes of T cells in MM remain to be elucidated. In this study, we compared the phenotypes of T cells, which were obtained from the peripheral blood, in MM patients with those in healthy donors (HD). The expression of CCR7, CD57, CD28, HLA-DR, CD38, CD45RA, and CD45RO were assessed on T cells from MM patients and HDs using multicolor flow cytometry (MFC). For this study, 17 newly diagnosed MM patients were selected, and 20 healthy people were selected as a control group. MFC was used to detect the markers on T cells. We detected significant increases in the expression levels of HLA-DR, CD38, and CD57on CD8+ T cells, significant decreases in the expression levels of CD28 and CD45RA on CD8+ T cells, and a decrease of CD4+ effec-tor T cells in MM patients, compared to the HD group. Our study shows that the accumulation of peripheral CD8+CD57+T cells, CD8+CD38high T cells, and CD8+HLA-DR+CD38high T cells is reflective of an ongoing antitumor T cell response and a progressive immune dysfunction in MM. During chemotherapy, the recovery of immune function can be monitored by detecting the proportion of activated molecules of T lymphocytes.

Multidimensional role of adapalene in regulating cell death in multiple myeloma.

Multiple myeloma (MM) remains a challenging condition to cure, with persistent drug resistance negating the benefits of treatment advancements. The unraveling complexities in programmed cell death (PCD), inclusive of apoptosis, autophagy, and ferroptosis, have highlighted novel therapeutic avenues. Our study focuses on deciphering how adapalene (ADA), a small molecule compound, accelerates the demise of MM cells via targeting their compensatory survival mechanisms. To assess the impact of ADA on MM, we employed flow cytometry and trypan blue exclusion assays to determine cell viabilities across MM cell lines and primary patient samples post-treatment. To delineate ADA's therapeutic targets and mechanisms, we conducted RNA sequencing (RNA-seq), gene set enrichment analysis (GSEA), molecular docking, and molecular dynamics simulations. We further designed pre-clinical trials emphasizing MM, exploring the efficacy of ADA as a standalone and in combination with bortezomib (BTZ). ADA elicited a dose-responsive induction of MM cell death. Building upon ADA's anti-MM capabilities as a single agent, we proposed that ADA-BTZ co-treatment might amplify this lethality. Indeed, ADA and BTZ together greatly potentiated MM cell death. ADA proved beneficial in restoring BTZ susceptibility in BTZ-resistant relapsed or refractory MM (RRMM) patient cells. Molecular simulations highlighted ADA's high affinity (-9.17kcal/mol) for CD138, with MM-GBSA revealing a binding free energy of -27.39kcal/mol. Detailed interaction analyses indicated hydrogen-bonding of ADA with CD138 at the Asp35 and Gln34 residues. Additionally, ADA emerged as a versatile instigator of both ferroptosis and apoptosis in MM cells. Furthermore, ADA disrupted activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway triggered by BTZ, fostering cell death in BTZ-resistant MM subsets. ADA demonstrates a comprehensive capability to orchestrate MM cell death, exerting pronounced anti-MM activity while disrupting NF-κB-related drug resistance. ADA sensitization of MM cells to BTZ unravels its potential as a novel therapeutic drug for MM management.

BR109, a Novel Fully Humanized T-Cell-Engaging Bispecific Antibody with GPRC5D Binding, Has Potent Antitumor Activities in Multiple Myeloma.

At present, multiple myeloma (MM) is still an essentially incurable hematologic malignancy. Although BCMA-targeted therapies have achieved remarkable results, BCMA levels were found to be downregulated in patients with MM who relapsed after these treatments. Therefore, the search for other antigens specific to MM has become a priority. Independently of BCMA expression, G-protein-coupled receptor family C group 5 member D (GPRC5D) is mainly expressed in the plasma cells of MM patients, while it is expressed in a limited number of normal tissues. Combining MM-specific antigen GPRC5D and T-cell-mediated therapies would be a promising therapeutic strategy for MM. Recently, we constructed a new anti-GPRC5D × anti-CD3 T-cell-engaging bispecific antibody (TCB), BR109, which was capable of binding to human GPRC5D and human CD3ε. Moreover, BR109 was proven to have relatively good stability and antitumor activity. BR109 could specifically trigger T-cell-mediated cytotoxicity against many GPRC5D-positive MM cells in vitro. Meanwhile, antitumor activity was demonstrated in MM cell line xenograft mouse models with human immune cell reconstitution. These preclinical studies have formed a solid foundation for the evaluation of MM treatment efficacy in clinical trials.

Lipid-Mediated Modulation of DNA Damage Signaling As a Prognostic and Therapeutic Strategy Against Multiple Myeloma

Multiple myeloma (MM), characterized by the accumulation of tumor plasma cells in the bone marrow, is the second most common hematologic cancer. Occurrence of chemoresistance invariably leads patients to relapse and results in loss of clinical control over the disease. Cancer cells show a deregulation of different pathways, including lipid metabolism alterations and high genomic instability. Yet, no functional relationship between these two features has been identified. We have shown in a previous work that the metabolism of lipids impacts in how DNA damage is detected, signaled and repaired (Ovejero et al 2023 EMBO J). We reported that breaks in the DNA trigger the birth of lipid droplets (LD), a central lipid-storage organelle, and that this event is key for cells to tolerate this specific DNA damage. Given that MM cells are particularly dependent on DNA repair pathways for survival, we postulated that new therapeutic perspectives would emerge by targeting the formation of LD. Analysis of the LD formation gene expression profile of a cohort of newly diagnosed MM patients (UAMS-TT2 MM cohort, n = 345) allowed the establishment of a LD score comprising 13 genes associated with a significant prognostic value after multiple testing correction. The LD score gene expression-based risk score enabled the identification of MM patients with a poor outcome. The prognostic value of the LD score was validated in two other independent cohorts of newly diagnosed patients (HM cohort, n = 206; UAMS-TT3 MM cohort, n = 158) and in a cohort of patients at relapse treated with anti-CD38 MoAb (Mtp-Dara, n = 49). We subsequently studied the presence of LD in primary MM cells obtained from 34 patients. Importantly, we observed a significant positive correlation between the percentage of LD-positive cells and the value of the LD score (p = 0,02). This prompted us to investigate the therapeutic interest of interfering with LD formation to target MM cells. To this end, we treated 26 Human Myeloma Cell Lines (HMCLs) with drugs selected on an educated-guess basis and identified positive hits. These results were validated employing a shRNA strategy in selected HCMLs. Flow cytometry analyses revealed that this regime triggered a cell cycle halt, where cells accumulated in the G 1 phase at the expense of S and G 2. This was accompanied by a decrease in c-MYC expression, as well as an induction of cell proliferation arrest markers such as the G 1-checkpoint CDK inhibitors p21 and p27. Mechanistically, we observed a significant increase in Chk1 and Chk2 phosphorylations, two downstream events of DNA damage signaling despite that basal DNA damage levels remained unchanged. Together, these data convey the notion that limiting LD formation in MM cell lines boosts the detection of endogenously present DNA damage, thus enforcing a more robust DNA damage signaling which culminates in a drastic loss of proliferative capacity and viability. Of major importance, we validated the therapeutic interest of this strategy in primary MM cells from patients (n = 19). A significant higher toxicity in MM cells was found compared to normal cells from the microenvironment (tumoral: ****, p &amp;lt; 0.0001; normal: ***, p = 0,0006). Moreover, we tested the therapeutic interest of combining this approach with conventional treatments used in MM. Interestingly, we identified a synergistic effect when combined with melphalan, a DNA-alkylating agent used in MM treatment. This synergy was observed in HCMLs and in primary MM cells of patients (n = 7). Furthermore, significant synergistic effects were found in HMCLs by combining with the proteasome inhibitors bortezomib and carfilzomib. Altogether, these data unveil both the LD score and LD-bearing cell quantification as new prognostic factors in MM, and open new therapeutic perspectives to improve the treatment of MM patients by targeting LD metabolism.

Regulation of BCMA By the Ubiquitin Proteasome System Enables Optimization of BCMA-Targeting Therapies in Multiple Myeloma

Background: The exclusive expression of the B-cell maturation antigen (BCMA), in normal and malignant plasma cells, currently makes it a prominent target for immune cell therapies in multiple myeloma (MM) including chimeric antigen receptor T-cell (CART) and bi-specific T cell engaging antibody (BiTE) therapies. Despite initial high response rates, most patients eventually relapse highlighting the need to enhance therapy efficacy. Although loss of BCMA-antigen expression marks one reason for therapy failure, little is known about the mechanistic means of antigen escape. The ubiquitin proteasome system (UPS) controls abundance and protein degradation of about 80-90% of all cellular proteins. Protein degradation is initiated by substrate ubiquitination, which is orchestrated by the sequential action of enzymes whereby only E3-ligases possesses substrate specificity. Deubiquitinating enzymes (DUBs) can reverse substrate ubiquitination. Against this background, we raised the question whether BCMA antigen presentation, expression, and activity is regulated by the UPS and whether the UPS can be utilized to enhance BCMA directed CART therapies in MM. Methods: Protein turnover and regulation by the UPS was assayed using cycloheximide and proteasomal inhibitors (PI) followed by immunoblotting. Direct substrate ubiquitylation was confirmed by ubiquitylation assays based on immunoprecipitation and image-based analysis. Changes in antigen surface presentation were determined using FACS analysis. Changes in cellular signaling and downstream effects were assessed by immunoblotting and quantitative PCR (qPCR). Primary CD138 + cells were obtained from bone marrow aspirations followed by MACS ® cell separation. To assess the effect of PI on CART killing capacity, a classic co-culture was set up in vitro. Using a proximity-based protein purification method (TurboID), UPS-related interactors of BCMA were identified. Results: For the first time, we show that BCMA is a short-lived protein with a half-life of approximately 30 min in MM cells. Degradation of BCMA is abrogated upon PI treatment. Inhibition of γ-secretase, an established protease of the extracellular domain of BCMA does not impair BCMA turnover. Furthermore, we show that BCMA is indeed ubiquitylated in MM cells and that ubiquitylation is induced by the ligand APRIL. These results support the hypothesis that the UPS regulates BCMA abundance. In both MM cell line models and primary CD138 + MM cells of PI refractory/ relapsed MM (RRMM) patients, inhibition of the proteasome leads to a significant increase of BCMA on the cell surface without promoting oncogenic NF-κB signaling in MM cells. Subsequently, we show that the increase in BCMA surface expression upon PI treatment substantially increases the efficacy of BCMA directed CART cells in co-cultures with MM cells and primary CD138 + MM patient cells. Specific members of the UPS (E3 ligases and DUBs) identified from our proteome wide BCMA interactome analysis are currently being validated. Conclusion: Here we show, for the first time, that BCMA is ubiquitylated and degraded by the ubiquitin proteasome system in multiple myeloma. The increase in BCMA expression upon proteasomal inhibition enhanced efficacy of BCMA-targeting CART cells. These insights provide a rationale for combining BCMA CART cells with PI in patients with BCMA CART relapsed/refractory disease and persistence. The identification of BCMA-specific E3-ligases and DUBs may represent novel therapeutic targets to specifically prevent BCMA degradation and antigen escape.

Development of Next Generation Antibody-Polymer-Drug Conjugates for Treatment of Multiple Myeloma

Background: Antibody-drug conjugates (ADCs) are an effective class of cancer therapeutics, but current ADCs face stability challenges, resulting in premature payload release and off-target toxicity. To overcome this, we developed a novel strategy using antibody-polymer-based drug conjugates (pADCs). Our pADCs have a number of advantages compared to currently FDA approved ADCs, including: 1) Increased drug payload without altering binding site numbers, ensuring maximum drug loading without affecting antigen and Fc binding; 2) Potential for synergistic immunotherapy and chemotherapy; 3) Improved pharmacokinetics and reduced immunogenicity with modification using water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, providing significant stealth effect; and 4) Enhanced stability under physiological conditions, reducing premature payload release and off-target toxicity. Panobinostat, a pan-HDAC (histone deacetylase) inhibitor, enhances CD38 expression and improves pADC efficiency against MM cells. Our hypothesis is that our pADC system induces strong cytotoxicity, further enhanced by panobinostat-induced CD38 upregulation, leading to more efficient treatment outcomes. Methods:mAbs decorated with cyclooctyne (DBCO) moieties in the hinge region were prepared in two steps: mAbs were reduced with tris(2-carboxyethyl)phosphine (TCEP), then thiol groups were capped with DBCO-PEG4-Mal. Azide-functionalized semitelechelic (ST) polymer-drug conjugates (ST-P-drug-N3) were synthesized via RAFT polymerization. Antibody-polymer-drug conjugates were prepared by CLICK chemistry. MM.1S and RPMI 8226 cell lines (ATCC) were used as the MM cell model. The cell internalization was revealed by confocal imaging. Primary MM cells were collected from the aspirate of eight patients with primary plasma cell leukemia and isolated via Ficoll centrifugation. CD38 upregulation was done by incubating with 10 or 25 nM panobinostat for 24 h and the apoptosis detection employed Annexin V and Propidium Iodide staining followed by flow cytometry analysis. Results: A series of CLICK-able mAbs and semitelechelic polymer-drug conjugates were synthesized. Among them, we firstly prepared Epirubicin (EPI)-based pADCs, such as DARA-P-EPI, ISA-P-EPI and DRO-P-EPI (Fig.1A). Unlike naked mAbs, pADCs can induce EPI concentration-dependent cytotoxicity. Surface calreticulin (CRT) was assessed and demonstrated the induction of EPI-dependent immunogenic cell death on MM.1S cells (Fig.1B). The pADC mechanism of action involves binding to cell surface antigens, internalization via endosomes, and subsequent cleavage of drugs at lysosomes by cathepsin B. This minimizes premature drug release and reduces off-target toxicity (Fig.1C). Ex vivo studies on patient MM cells revealed that pADCs induced significantly higher apoptosis compared to naked mAbs and free EPI, highlighting their efficacy (Fig.1D). CD38-based pADC's efficacy was enhanced by pretreatment with panobinostat (Fig.1F), which increased CD38 expression (Fig.1E). When incubating with NK cells and pADC, the number of target cells decreased significantly indicating the pADC preserve the original mAb ADCC functionality. Notably, co-treatment of RPMI8226 cells with anti-CD38 and GDC0980, a PI3k/mTOR inhibitor, significantly improved cytotoxicity, suggesting DARA's potential for chemo sensitization (Fig.1G). Conclusion: pADC's innovative design offers a high likelihood of successful clinical development and holds significant promise for MM therapy. Moreover, the unique mechanism provides potential to effectively overcome mAb and multiple drug resistance. Its facile nature allows for easy incorporation of multiple types of payloads into the ADC, making its synthesis highly scalable .

Immunological Subgroups Predicting Efficacy of Elotuzumab in Multiple Myeloma Patients: Insights from the GMMG-HD6 Clinical Trial

Introduction: Elotuzumab, a monoclonal antibody targeting SLAM family member 7 protein (SLAMF7) on multiple myeloma (MM) cells, has shown promise in relapsed/refractory MM when combined with immunomodulatory agents. However, its effectiveness in newly diagnosed patients did not show improved progression-free survival (PFS) nor overall survival. In a previous study we demonstrated that elotuzumab specifically depleted high SLAMF7 expressing regulatory CD8 + T cells by macrophage induced antibody dependent phagocytosis. This study aimed to identify immunological predictive factors for elotuzumab efficacy, enabling improved risk stratification and personalized treatment decisions based on the detection and quantification of SLAMF7-positive T cell subsets. Patients and Methods: This pre-planned accompanying immunological research analyzed samples from the German-Speaking Myeloma Multicenter Group (GMMG) HD6 clinical trial (NCT02495922). The trial investigated elotuzumab in combination with lenalidomide/bortezomib/dexamethasone induction and consolidation, followed by lenalidomide maintenance in transplant-eligible newly diagnosed MM patients and found no progression-free or overall survival advantage with the addition of elotuzumab in any treatment sequence. Peripheral blood samples were collected at three time points: trial inclusion (T1), after induction therapy (T2), and during consolidation or maintenance therapy (T3). Flow cytometry was used to assess T cell subpopulations. Results: Elotuzumab during induction therapy significantly reduced the percentage of regulatory SLAMF7 high expressing CD8 + CD28 - ( p &amp;lt; 0.001) and effector CD8 + T cells ( p &amp;lt; 0.001) in MM patients after induction therapy and during maintenance therapy. A T2 landmark multivariate analysis (after induction therapy) revealed that a higher percentage of effector CD8 + T cells at T1 showed a borderline advantageous effect on the progression-free survival (PFS) ( HR = 0.98 [CI 95% 0.97; 1.00], p = 0.010) regardless of the study arm. Further, in a multivariate analysis a high regulatory CD8 + T cell level at T2 was associated with an adverse PFS in patients receiving elotuzumab in addition to lenalidomide during consolidation and maintenance (PFS from T2 for high vs. low regulatory CD8 + T cell levels at T2, HR = 3.64 [CI 95% 1.58; 8.36], p = 0.002 for patients that received elotuzumab during consolidation and maintenance only; regulatory CD8 + T cells at T2 [high versus low] HR = 5.93 [CI 95% 2.30; 15.26], p &amp;lt; 0.001 for patients that received elotuzumab during induction, consolidation and maintenance). Conclusion: The efficacy of elotuzumab in MM patients may be influenced by the frequency of regulatory CD8 + T cells after induction therapy. As high levels of regulatory CD8 + T cells showed adverse progression-free survival in MM patients, the assessment of the level of regulatory CD8 + SLAMF7 + T cells could be a prerequisite for MM patients' response to elotuzumab. This study highlights the importance of T cell level-driven immunological treatment approaches when utilizing elotuzumab.

Dysregulation of Cellular Immunity in High-Risk Multiple Myeloma

Introduction: Multiple myeloma (MM) is the second most prevalent hematological malignancy, characterized by the abnormal proliferation of plasma cells in the bone marrow. Approximately 20% of MM patients present with high-risk (HR) clinical and genetic features that are associated with aggressive disease course and poor prognosis. Novel immune therapies are changing the paradigm of MM treatment. A comprehensive understanding of immune dysregulation within the tumor microenvironment (TME) of HR vs standard risk (SR) MM patients could inform the development of targeted immunotherapeutic strategies for HR MM. Methods: 55 HR and 25 SR patients were selected from the Mt. Sinai Hematologic Malignancies Tissue Bank (IRB# STUDY-11-01978). HR included one or more cytogenic abnormalities (HRCA) such as translocations t(4;14), deletion of chromosome 17 (del17p), and gain/amplification of 1q (1q+/++) based on the Second Revised International Staging System (R2-ISS). We also included nine patients with t(14;16) translocation alone or combined with one or two of the other HRCA. We performed comprehensive immune phenotyping of the bone marrow micro-environment on specimens from HR and SR patients using mass cytometry (CyTOF) and single cell RNA sequencing (scRNASeq) of bone marrow mononuclear cells (BMMC) depleted of CD138 positive MM cells. Results: ScRNAseq of BMMC from HR MM patients revealed a robust cytotoxic and cytolytic signature on CD4+ T cells relative to SR, characterized by the upregulation of genes such as GNLY, NKG7, PRF1, and IFNG, and a higher frequency of Granzyme K-positive (GZMK+) CD8+ T cells, previously described as progenitors of exhausted cells. Both CD4 and CD8 T cells, along with monocytes, exhibited high expression of a group of Interferon-stimulated genes (ISGs) including IFI27, IFIT3, IFI44L, IFITM1/3, and OASL. Notably, high expression of this group of ISGs was associated with T cell exhaustion and poor prognosis in the setting of immune checkpoint inhibitor therapy for solid tumors. Mass cytometry analysis confirmed the activated state of T cells in HR MM patients, with increased expression of immune activation markers HLA-DR, CD38, and the senescence marker CD57. Furthermore, HR patients exhibited higher frequencies of mast cells, CD14+ Monocytes and plasmacytoid dendritic cells compared to SR, suggesting an inflammatory and immunosuppressive environment that may dampen effective antitumor immune responses. Conclusions: These results indicate distinct bone marrow microenvironments of HR and SR MM. CD4 and CD8 T cells appear to be more activated in HR patients compared to SR. However, the higher frequencies of mast cells and plasmacytoid dendritic cells, and the prominent expression of ISGs associated with inhibition of anti-tumor immunity suggest dysregulation and exhaustion of T cell activity in HR patients. Collectively, these findings highlight the complex immune dynamics in HR MM and provide insights into the underlying immune dysfunction which may contribute to disease progression and therapy resistance in HR MM. Such insights have the potential to inform the design of targeted therapeutic interventions aimed at modulating the immune response and improving treatment outcomes for HR MM patients.

Synergistic Effects of Type I PRMT1 and Type II PRMT5 Inhibitors Against Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by the extensive expansion of monoclonal aberrant plasma cells in the bone marrow and the second most prevalent hematological malignancies worldwide. Despite the promising outcomes of advanced therapies, relapsed/refractory (RR) diseases remain incurable, highlighting an unmet clinical need to identify novel therapeutic targets. An emerging field of investigation is the role of aberrant post-translational modifications in multiple myeloma pathology. Arginine methylation is one of the most predominant post-translational modifications in cells, catalyzed by protein arginine methyltransferases (PRMTs) that plays a crucial role in multiple cellular processes. Type I PRMTs catalyze mono- (MMA) and asymmetric demethylation (ADMA) of arginine; Type II PRMTs catalyze mono- and symmetric demethylation (SDMA) of arginine on proteins; Type III PRMT mediates MMA only. Aberrant expression of PRMTs is strongly associated with poor prognosis of solid cancers and hematologic malignancies. Recently, we identified PRMT1 as a novel therapeutic vulnerability in MM cells. Among PRMTs, PRMT1 is the highest expressed gene in MM cell lines and patient cells. The elevation of PRMT1 expression is strongly associated with relapsed/refractory patients and poor clinical outcomes. Deletion of PRMT1 and suppression of the PRMT1's enzymatic activity exhibited a strong anti-MM in vitro via suppressing cell cycle and elevating the apoptosis. We also found that inhibition of PRMT1 boosts the T cell activation in the co-culture setup, indicating a potential enhancement of immunotherapy. Using a subcutaneous xenograft model, we showed that suppressing PRTM1 in vivo by either genetic or pharmaceutical approaches robustly inhibits MM tumor growth and extends survival of tumor-bearing mice. Our findings demonstrated that PRMT1 is indispensable in MM. PRMT5, a PRMT type II, is prognostically relevant and a known druggable target in MM. PRMT1 and PRMT5 inversely regulate post-transcription and have been implicated in human cancers. Recent studies in solid tumor showed a strong anti-tumor effect in vivo using the combinational inhibition of PRMT1 and PRMT5. We hypothesize that PRMT1 and PRMT5 might have a complementary function in MM cells. The combinational inhibition PRMT1 and PRTM5 will exhibit a synergistic anti-MM effect. To address this hypothesis, we treated MM cell lines with two clinical trial inhibitors: GSK3368715 (GSK) and EPZ 015666 (EPZ), suppressing PRMT type I and type II inhibitor, respectively. We first performed dose-dependent cell viability inhibition and selected effective doses for drug combination treatments in three different MM cell lines. The combination of GSK and EPZ demonstrated a strong synergistic effect in suppressing the viability of MM cells. Using the SynergyFinder algorithm, we found that the synergistic indexes (&amp;gt;10, synergism) for 3 MM cell lines JJN3, MM1S, and KMS11 were 33.5, 32.8, and 14.3, respectively. We next assessed the effects of combined treatment on the cell cycle and apoptosis of MM cells. We found that combination of GSK and EPZ strikingly elevated the apoptosis and suppressed cell cycle of MM cell lines compared single treatment and untreated control. Intriguingly, the combination treatment of two inhibitors significantly decreased MMA, SDMA, and ADMA, suggesting a robust suppression of global arginine methylation. Of note, we found a similar anti-MM effect when treating bortezomib-resistant MM cells with GSK and EPZ, indicating that PRMT type I and type II inhibitors potentially have a substantial clinical impact on relapsed MM. Of note, the same dose of GSK and EPZ that exhibits the synergistic anti-MM effect, didn't suppress the viability of healthy peripheral blood cells. In summary, our preliminary data suggest that suppression of both PRMT type I and type II might provide a potential effective combination treatment for both newly diagnosed and relapsed MM patients. We're currently investigating the underling mechanisms of how PRMTs regulate MM pathology and validating the in vivo efficacy of combination treatment using patient-derived xenograft.

Anti-BCMA Dual Epitope-Binding CAR-Marrow Infiltrating Lymphocytes (MILs) Could Offer a Potent Innovative Immunotherapeutic Tool Against Multiple Myeloma

Introduction: Chimeric antigen receptor (CAR) T cells are a promising new treatment for multiple myeloma (MM). Marrow-infiltrating lymphocytes (MILs) derived from MM patient's bone marrow are another potential innovative T cell source for CAR-based cell therapy, capable of robustly and specifically targeting myeloma cells through polyclonal targets. MILs possess polyclonal central memory T cell populations, which could be as effective MM killings via polyantigenic cytotoxic T cells with greater recognitions of broad range of tumor antigens via the polyclonal memory TCR as well as the CAR pathway with strong recognition of tumor antigens. Nanobodies are currently the smallest stable antibody molecules having high affinity for antigen, allowing them to strongly penetrate tumor tissue and even directly to the blood-brain barrier while remaining low toxicity and immunogenicity. With the peculiar properties, nanobodies have been widely used as the antigen biding domain of CAR-T cell. In this study, we investigated anti-B-cell maturation antigen (BCMA) dual epitope-binding CAR-MILs against MM. Methods: CD3 + T cells isolated from bone marrow mononuclear cells of MM patients were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2. The nanobodies VHH01 and VHH02 were provided as dual epitope-binding for BCMA (Senlang, China). After expansion and activation for 2 days, anti-BCMA dual epitope-binding CAR was transduced to the activated MILs and, they were stimulated with Ex Vivo CD3/CD28 in the presence of recombinant human IL-2, IL-7, IL-15, and IL-21. Expanded and activated BCMA dual epitope-binding CAR-MILs (BCMA CAR-MILs) were harvested on days 7, 14, and 21 for further experiments. Results: The transduction efficiency of BCMA CAR-MILs was approximately 34%, and the expansion of BCMA CAR-MILs was &amp;gt; 100 folds over 21-day culture period. Additionally, BCMA CAR-MILs showed a dramatically increased proportion of CD8 + T cells, while the proportion of CD4 + T cells decreased. Moreover, BCMA CAR-MILs were enriched with a higher proportion of central memory T cells (Tcm) (80%), while the proportions of regulatory T cells (Tregs) and checkpoint molecules (TIGIT, TIM3, and CD73) were not so high. The BCMA CAR-MILs showed potent cytotoxicity, particularly high levels of CD107a degranulation against BCMA-expressing target cancer cell lines, such as Daudi, Raji, RPMI8226, and U266B1 cells, and autologous CD138 + primary myeloma cells. Conclusion: This study suggested that BCMA dual epitope-binding CAR-MILs were easily expanded and activated using bone marrow mononuclear cells from MM patients with high transduction efficiency and induced a potent anti-myeloma immune response, which may offer an alternative innovative immunotherapeutic approach against MM patients in the future.

Outcomes after CAR T Cells in Multiple Myeloma Patients with Extramedullary and Paramedullary Disease

Background: Extramedullary disease (EMD) is a poor prognostic factor for multiple myeloma patients even in the era of novel therapies, while patients with paramedullary disease (PMD) have historically fared better. We sought to characterize EMD and PMD in the peri-CAR T period, assess their effects on outcomes after CAR T, and evaluate the ability of CAR T-cell therapy to clear EMD. Methods: We retrospectively analyzed patients with relapsed/refractory multiple myeloma (RRMM) treated with CAR T-cell therapy in a clinical trial or with a commercially approved product at The Mount Sinai Hospital who had at least 3 months of follow-up. EMD was defined as the presence of soft tissue lesions not contiguous with bone and was quantified by measuring the sum of the product of the two longest perpendicular diameters (SPD) of each lesion. PMD was defined as the presence of soft tissue lesions contiguous with bone. Results: A total of 134 patients received CAR T-cell therapy for RRMM between April 2017 and February 2023 and were followed for a median of 18.6 months, with 59 patients treated with ciltacabtagene autoleucel, 52 with idecabtagene vicleucel, and 23 with other products on clinical trials. Thirty-four patients (25.4%) had EMD at baseline (EMD+), while patients without EMD were further subdivided into those with PMD (PMD-only, 25 patients, 18.7%) and those with neither EMD nor PMD (75 patients, 56.0%). Among patients with measurable EMD, median SPD was 11.15 cm 2 (range 0.25-200.10 cm 2). Patients were heavily pretreated with triple-class refractory status present in 94.1% of EMD+ patients, 96.0% of PMD-only patients, and 74.7% of patients with neither. Compared to patients with neither EMD nor PMD, PMD-only patients had similar PFS (HR 0.98, 95% CI 0.51-1.87) and OS (HR 1.34, 95% CI 0.48-3.76), while EMD+ patients exhibited significantly worse PFS (HR 1.87, 95% CI 1.13-3.09) and OS (HR 3.78, 95% CI 1.81-7.92). Overall response rates (ORR) were 76.5% in EMD+, 92.0% in PMD-only, and 88.0% in patients with neither. Among EMD+ patients, those with the highest tumor burdens (SPD ≥50 cm 2, n = 7) had ORR of 57.1% with no complete responses (3 PD, 3 PR, 1 VGPR). Conversely, EMD+ patients with SPD &amp;lt;50 cm 2 (n = 27) had 81.5% ORR and deeper responses (66.7% complete response). Of the 134 patients, 69 patients relapsed including 34 with EMD at relapse. Among the 34 patients with baseline EMD, 23 (67.6%) relapsed including 21 (61.8%) who relapsed with EMD. EMD was present in its prior location for 13 patients, while EMD cleared in prior locations and was present in new locations for 8 patients. Of 100 patients who had no EMD at baseline, only 13 (13.0%) relapsed with EMD. All patients with bulky EMD (SPD ≥50 cm 2) relapsed with EMD. Among the 34 patients with EMD at relapse, 28 were assessed for MRD status in the bone marrow at the time of relapse, of which 12 (42.9%) remained MRD negative by next generation flow (sensitivity 10 -5). EMD post-BCMA CAR T was biopsied and stained for BCMA in 7 relapsing patients, of which 6 were BCMA positive and 1 was BCMA negative. Conclusion: EMD is a well-recognized poor prognostic factor in multiple myeloma. In our single-center retrospective analysis, the presence of EMD but not PMD was associated with significantly worse PFS and OS following CAR T-cell therapy. EMD+ patients with SPD &amp;lt;50 cm 2 experienced high response rates and deep responses following CAR T-cell therapy (with two-thirds of patients clearing EMD), while patients with the highest extramedullary tumor burdens (SPD ≥50 cm 2) did not. This shows the ability of CAR T-cell therapy to benefit patients in this high-risk group when extramedullary tumor burden is lower. We contend that controlling extramedullary sites of disease prior to CAR T-cell therapy with optimal bridging chemotherapy and/or radiation therapy may be an effective strategy for improving outcomes. Half of patients relapsing after CAR T-cell therapy had EMD, and the majority of patients with baseline EMD ultimately relapsed with EMD despite having been radiographically EMD negative following CAR T treatment. Finally, 42.9% of patients relapsing with EMD after CAR T-cell therapy remained MRD negative in the marrow, showing bone marrow MRD does not adequately represent overall disease control post-CAR T.

The Potential of JAG Ligands as Therapeutic Targets and Predictive Biomarkers in Multiple Myeloma.

The NOTCH ligands JAG1 and JAG2 have been correlated in vitro with multiple myeloma (MM) cell proliferation, drug resistance, self-renewal and a pathological crosstalk with the tumor microenvironment resulting in angiogenesis and osteoclastogenesis. These findings suggest that a therapeutic approach targeting JAG ligands might be helpful for the care of MM patients and lead us to explore the role of JAG1 and JAG2 in a MM in vivo model and primary patient samples. JAG1 and JAG2 protein expression represents a common feature in MM cell lines; therefore, we assessed their function through JAG1/2 conditional silencing in a MM xenograft model. We observed that JAG1 and JAG2 showed potential as therapeutic targets in MM, as their silencing resulted in a reduction in the tumor burden. Moreover, JAG1 and JAG2 protein expression in MM patients was positively correlated with the presence of MM cells in patients' bone marrow biopsies. Finally, taking advantage of the Multiple Myeloma Research Foundation (MMRF) CoMMpass global dataset, we showed that JAG2 gene expression level was a predictive biomarker associated with patients' overall survival and progression-free survival, independently from other main molecular or clinical features. Overall, these results strengthened the rationale for the development of a JAG1/2-tailored approach and the use of JAG2 as a predictive biomarker in MM.

Extracellular vesicles derived from immortalized human natural killer cell line NK3.3 as a novel therapeutic for multiple myeloma.

Over the last decade, there have been many advancements in the therapeutic treatment of multiple myeloma (MM), including the use of natural killer (NK) cells. However, despite promising results from clinical trials, there are concerns over the use of NK cell-based therapy. Cells often undergo growth arrest, limiting their experimental utility; donor cells are extremely heterogeneous, resulting in content variability; and patients receiving allogeneic cells are at risk for graft-versus-host disease and/or cytokine release syndrome. Extracellular vesicles (EVs) have emerged as a new natural therapeutic tool. EVs are known to carry cargo derived from the parent cell from which they originate. NK cells play an important role in the innate immune system, targeting and killing tumor cells. This has led many researchers to isolate EVs from NK cells for their cytotoxic potential. In this study, we isolated EVs from the NK cell line, NK3.3, which was derived from the peripheral blood of a healthy donor. Currently, it is the only normal human NK cell line reported with all the functional characteristics of healthy NK cells. To address the issue of growth arrest, we immortalized NK3.3 cells with lentivirus encoding the catalytic subunit of human telomerase htert (NK3.3-LTV). EVs from these cells were isolated using a modified polyethylene glycol (PEG)-acetate precipitation protocol to simplify processing and increase EV yield. We demonstrated that NK3.3-LTV EVs target both sensitive and drug-resistant MM cell lines as well as primary patient MM cells in vitro, decreasing proliferation and inducing apoptotic cell death as well as or better than EVs from non-immortalized cells with no toxicity towards normal cells. This study is the first step towards developing an immunotherapeutic product designed to treat patients with relapsed/refractory MM.

High expression of VARS promotes the growth of multiple myeloma cells by causing imbalance in valine metabolism.

Multiple myeloma (MM) is a plasma cell malignancy occurring in middle and old age. MM is still an incurable disease due to its frequent recurrence and drug resistance. However, its pathogenesis is still unclear. Abnormal amino acid metabolism is one of the important characteristics of MM, and the important metabolic pathway of amino acids participates in protein synthesis as basic raw materials. Aminoacyl transfer ribonucleic acid synthetase (ARS) gene is a key regulatory gene in protein synthesis. This study aims to explore the molecular mechanism for ARS, a key factor of amino acid metabolism, in regulating amino acid metabolism in MM and affecting MM growth. The corresponding gene number was combined with the gene expression profile GSE5900 dataset and GSE2658 dataset in Gene Expression Omnibus (GEO) database to standardize the gene expression data of ARS. GSEA_4.2.0 software was used to analyze the difference of gene enrichment between healthy donors (HD) and MM patients in GEO database. GraphPad Prism 7 was used to draw heat maps and perform data analysis. Kaplan-Meier and Cox regression model were used to analyze the expression of ARS gene and the prognosis of MM patients, respectively. Bone marrow samples from 7 newly diagnosed MM patients were collected, CD138+ and CD138- cells were obtained by using CD138 antibody magnetic beads, and the expression of ARS in MM clinical samples was analyzed by real-time RT-PCR. Human B lymphocyte GM12878 cells and human MM cell lines ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11, MM1.s cells were selected as the study objects. The expression of ARS in MM cell lines was analyzed by real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) lentiviruses were used to construct gene knock-out plasmids (VARS-sh group). No-load plasmids (scramble group) and gene knock-out plasmids (VARS-sh group) were transfected into HEK 293T cells with for virus packaging, respectively. Stable expression cell lines were established by infecting ARP1 and OCI-MY5 cells, and the effects of knockout valyl-tRNA synthetase (VARS) gene on proliferation and apoptosis of MM cells were detected by cell counting and flow cytometry, respectively. GEO data were divided into a high expression group and a low expression group according to the expression of VARS. Bioinformatics analysis was performed to explore the downstream pathways affected by VARS. Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) and high performance liquid chromatography (HPLC) were used to detect the valine content in CD138+ cells and ARP1, OCI-MY5 cells and supernatant of knockdown VARS gene in bone marrow samples from patients, respectively. Gene enrichment analysis showed that tRNA processing related genes were significantly enriched in MM compared with HD (P<0.0001). Further screening of tRNA processing-pathway related subsets revealed that cytoplasmic aminoacyl tRNA synthetase family genes were significantly enriched in MM (P<0.0001). The results of gene expression heat map showed that the ARS family genes except alanyl-tRNA synthetase (AARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase (SARS) in GEO data were highly expressed in MM (all P<0.01). With the development of monoclonal gammopathy of undetermined significance (MGUS) to MM, the gene expression level was increased gradually. Kaplan-Meier univariate analysis of survival results showed that there were significant differences in the prognosis of MM patients in methionyl-tRNA synthetase (MARS), asparaginyl-tRNA synthetase (NARS) and VARS between the high expression group and the low expression group (all P<0.05). Cox regression model multivariate analysis showed that the high expression of VARS was associated with abnormal overall survival time of MM (HR=1.83, 95% CI 1.10 to 3.06, P=0.021). The high expression of NARS (HR=0.90, 95% CI 0.34 to 2.38) and MARS (HR=1.59, 95% CI 0.73 to 3.50) had no effect on the overall survival time of MM patients (both P>0.05). Real-time RT-PCR and Western blotting showed that VARS, MARS and NARS were highly expressed in CD138+ MM cells and MM cell lines of clinical patients (all P<0.05). Cell counting and flow cytometry results showed that the proliferation of MM cells by knockout VARS was significantly inhibited (P<0.01), the proportion of apoptosis was significantly increased (P<0.05). Bioinformatics analysis showed that in addition to several pathways including the cell cycle regulated by VARS, the valine, leucine and isoleucine catabolic pathways were upregulated. Non-targeted metabolomics data showed reduced valine content in CD138+ tumor cells in MM patients compared to HD (P<0.05). HPLC results showed that compared with the scramble group, the intracellular and medium supernatant content of ARP1 cells and the medium supernatant of OCI-MY5 in the VARS-shRNA group was increased (all P<0.05). MM patients with abnormal high expression of VARS have a poor prognosis. VARS promotes the malignant growth of MM cells by affecting the regulation of valine metabolism.