Bone Marrow Of Multiple Myeloma Patients Research Articles

B-Cell Maturation Antigen (BCMA)-Specific, CentyrinTM-Based, PiggyBacTM-Transposed CAR-T Memory Stem Cells Are Effective Against p53-/- and Patient-Derived Multiple Myeloma Tumors

B-Cell Maturation Antigen (BCMA)-Specific, CentyrinTM-Based, PiggyBacTM-Transposed CAR-T Memory Stem Cells Are Effective Against p53-/- and Patient-Derived Multiple Myeloma Tumors

Immune Microenvironment Contributes to Dysregulation of Aid/Apobec Expression and Genomic Instability in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy with marked genomic instability. Using whole genome sequencing, we have previously demonstrated that myeloma cells display heterogeneity in mutational spectrum and diverse patterns of clonal evolution. We have also identified AID/APOBEC-driven signature as one of 2 biologically distinct mutational signatures responsible for the majority of observed mutations. Our data show that several members of APOBEC family (including 3A, B, C, and G) are up regulated in patient MM cells and cell lines. Using SNP array and whole genome sequencing, we have also confirmed that elevated expression of APOBECs (3B, C, and G) contributes to genomic instability in MM cells. Since genomic instability and associated clonal evolution could also be driven by pressures from microenvironment, in this study we investigated the role of microenvironment in dysregulation of AID/APOBECs in MM cells.In normal cellular environment, AID and APOBECs are expressed by B and T cells and play a key role in adaptive and innate immunity. However, their expression is tightly regulated in order to prevent nonspecific damage to cellular DNA. We hypothesized that abnormal immune components and soluble factors in the bone marrow microenvironment can induce APOBEC expression in MM cells and can contribute to genomic instability. As we have previously shown that pDCs, MDSCs, Th17cells and B1/B2 cell ratio were significantly elevated in peripheral blood and bone marrow of MM patients, we evaluated the role of these immune cells (iDC, pDC, B, B1, and T cells), and soluble components (IL6, IL-17, IL-27, IFNa, and IFNb) on APOBEC expression and activity in myeloma cells. We used purified immune cells from healthy donors and co-cultured them with MM cells. Following a 3 day co-culture, MM cells were purified and analyzed. APOBEC expression by RT-qPCR. Co-culture with immune cells caused increased expression (range: 2-100 fold) of several APOBEC family genes in myeloma cells. Among different immune cells, iDC and pDC induced the expression of AID, A3A, A3G, and A3H; TLR9 activated B cells induced the expression of AID and A3G; B1 cells induced expression of AID, A3A and A3G; and, IL-17 activated T cells induced the expression of AID, A3A and A3G. Among soluble components of immune microenvironment, interferon a and β induced APOBEC expression (A3A, A3G, and A3H; range: 4-15 fold) in myeloma cells after treatment. Interestingly, none of the interleukins tested showed any impact on APOBEC gene expression in these cells. We then tested if IRF9, a downstream effector of type-1 interferons is involved in the interferon-mediated induction of APOBECs. IRF9-siRNA significantly inhibited interferon-mediated induction of APOBECs (by 50%) in myeloma cells suggesting that interferon mediated up-regulation of APOBECs in MM cells is mediated at least in part by IRF9. We also used a functional assay and demonstrated that increased expression of APOBEC genes was associated with increased APOBEC deaminase activity in MM cells. To investigate the genomic impact of interferon induced APOBEC activity, we treated MM cells with IFN-β and evaluated its impact on abasic sites (that could directly result from APOBEC deaminase activity) and micronuclei assay (a marker of genomic instability). The number of abasic sites and micronuclei were increased by 2 and 4 fold respectively, in cells treated with IFN-β. Overall, these results suggest that BM microenvironment can induce APOBEC expression/activity and associated genomic instability in MM cells. DisclosuresNo relevant conflicts of interest to declare.

Targeting CD47 As a Novel Therapeutic Strategy in Multiple Myeloma

Introduction: CD47 is an immune checkpoint coordinating the innate and adaptive immune systems which allows cancer cells to escape the innate immune response by sending inhibitory signals to macrophages and regulates the adaptive immune anti-tumor response. Hence, CD47 has been identified as a novel therapeutic target in cancers, including multiple myeloma (MM) which is localized mainly in the bone marrow (BM). MM cells rely heavily on the interactions with tumor microenvironment (TME) and immune system. Macrophages are enriched in the BM of MM patients compared to healthy controls, and were demonstrated to support MM growth, in opposition to their intrinsic phagocytic properties. We study the new class of immune checkpoint inhibitor such as anti-CD47 monoclonal antibody (mAb) which enhance MM killing. We tested two anti-CD47 mAbs provided; Vx1000R (blocking only) - blocking CD47/SIRPα binding and Vx1004R (dual function) - blocking CD47/SIRPα binding and inducing cell death activity.The objectives of this study were: (1) to test the expression of CD47 (Vx1000R and Vx1004R binding) to MM cells, stromal cells (MSP-1 and HS5) and peripheral blood mononuclear cell (PBMC) sub-populations; (2) to study anti-CD47 mAbs binding in hypoxic conditions; (3) to investigate the effect of anti-CD47 mAbs on MM cell apoptosis in 2D and the 3D tissue engineered BM (3DTEBM; a scaffold incorporating BM plasma from MM patients, MM cells, and accessory cells) to reproduce the TME; (4) to test the cytotoxicity of Vx1004R on PBMC sub-populations in 3D culture; and (5) to examine the effect of Vx1004R on MM cell phagocytosis ex vivo by macrophages.Methods: We performed meta-analysis of CD47 mRNA expression in dataset available online in healthy, MGUS, smoldering MM and MM subjects. We tested the binding of Vx1000R and Vx1004R mAbs on MM.1S, H929, RPMI8226 and U266 cells and stromal cells (MSP-1 and HS5) in normoxia (21% O2) and hypoxia (1% O2), and in the 3DTEBM at 24h and 72h. We also tested Vx1000R binding to PBMC sub-populations (from 12 MM patients) including CD3 (T cells), CD14 (monocytes/macrophages), CD16 (NK cells, eosinophils, and neutrophils), CD19 (B cells), CD123 (pDC and basophils) and CD138 (MM cells).Next, we examined the cytotoxic activity of CD47 mAbs on MM cells alone and in co-culture with the BM negative fraction (CD138-depleted) and stromal cells (MSP-1) in 2D cultures by performing MTT survival assay, and in 3DTEBM cultures by flow cytometry. The cytotoxicity of Vx1004R was studied on PBMCs incorporated into a similar 3D model and treated with Vx1004R; proliferation and apoptosis of different sub-populations of PBMC were assessed via flow cytometry. Lastly, the effect of Vx1004R on MM cell phagocytosis by macrophages (CD14+; green) was tested after 2h of co-culture with MM cells (pre-labeled with DiD+; red) in the presence or absence of Vx1004R. Phagocytosis was determined as double positive (green+ red+) cells by flow cytometry.Results: The meta-analysis results demonstrated that CD47 mRNA expression increases with tumor progression (compared to early stage disease and healthy subjects). We demonstrated that MM.1S, H929, U266 and RPMI8226 cells and stromal cells (MSP-1 and HS5) express high levels of CD47, which does not alter in hypoxia, and that MM cells increase CD47 expression in 3DTEBM at 72h. CD47 (Vx1000R) is expressed in different sub-populations of PBMCs with the highest expression in MM cells. In addition, we have shown that anti-CD47 dual function mAb inhibits proliferation of MM cells, especially when co-cultured with negative fraction and MSP-1 stroma in 3DTEBM cultures. Moreover, we showed that anti-CD47 mAb induces phagocytosis of MM cells ex vivo by macrophages.Conclusions: CD47 is a promising target candidate for MM treatment, since CD47 mRNA increases with MM progression, and CD47 protein increases in MM cells in the presence of TME in the 3DTEBM, suggesting involvement in pathophysiology of MM. Moreover, anti-CD47 mAb enhances killing of MM cells when co-cultured with TME; however, it does not affect apoptosis of PBMC sub-populations in the 3DTEBM. Finally, anti-CD47 mAb induces phagocytosis of MM cells ex vivo by macrophages. These results suggest that anti-CD47 mAbs are applicable for MM treatment. Further studies are warranted to examine the effect of anti-CD47 mAbs as a novel strategy to inhibit the progression and dissemination of MM in vivo, especially when combined with chemotherapy. DisclosuresVij:Takeda: Honoraria, Research Funding; Jazz: Honoraria; Abbvie: Honoraria; Celgene: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria; Bristol-Meyers-Squibb: Honoraria; Konypharma: Honoraria. Capoccia:Tioma: Employment. Manning:Tioma: Employment. Azab:Abbvie: Research Funding; Verastem: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner; Selexys: Research Funding; Karyopharm: Research Funding; Cell Works: Research Funding; Cleave Bioscience: Research Funding; Cellatrix LLC: Other: Founder and owner; Glycomimetics: Research Funding; Tioma: Research Funding.

Quantitative and Functional Alterations of 6-Sulfo Lacnac Dendritic Cells in Multiple Myeloma

Multiple myeloma (MM) is characterized by expansion of abnormal plasma cells in the bone marrow (BM). Monocytes and macrophages have been shown to accumulate in the microenvironment / BM of MM patients while promoting MM cells survival and proliferation. Recently, another population of myeloid cells expressing the M-DC8 antigen containing a 6-sulfo LacNAc structure, called 6-sulfo LacNac dendritic cells (Slan-DCs) has been described. Functionally, Slan-DCs have potent pro-inflammatory properties based on their capacity to produce large amounts of Tumor Necrosis Factor-α and Interleukin (IL) 12p70 upon stimulation with Toll-like receptor ligands such as R848. They also express C-X-C chemokine receptor type 4 which is important in homing to the BM. Nevertheless, little is known about microenvironmental interactions between MM cells and Slan-DCs.To gain insights on the Slan-DCs' potential role in MM pathophysiology, we evaluated their phenotype and functions in the BM and blood of patients with newly diagnosed symptomatic MM. Using multicolor flow cytometry, we performed quantitative and functional analyses. Compared to healthy donors (n=11), the percentages and absolute numbers of circulating HLA-DR+ M-DC8+ Slan-DCs were significantly decreased in the blood of MM patients (n=23) (for absolute number: 20.64 vs 7.61 Slan-DCs/μL, p=0.0015 and for percentage: 1.06 vs 0.49%, p=0.029). In contrast, there were no significant difference in the frequencies of Slan-DCs in the patients BM.We next addressed their functional properties, in particular cytokine production. We observed a significant decrease in IL-12-producing Slan-DCs from MM patients as compared to healthy donors both in blood (25.88% versus 56.86% respectively, p<0.0001) and BM (25.11% versus 48.83% respectively, p=0.016). In order to investigate whether the decrease in IL-12 secretion was due to the MM cells, circulating Slan-DCs from healthy controls were stimulated with R848 in the presence of 4 different MM cell lines. The capacity of Slan-DCs from healthy controls to secrete IL-12 under this stimulation was measured by ELISA. We observed that the RPMI-8226, KMS-12-PE and LP-1 MM cell lines inhibited R848-induced IL-12 secretion by Slan-DCs. Furthermore, we could show that primary MM cells from patients at diagnosis (magnetically sorted CD138+ cells from patients) were able to inhibit IL-12 secretion by Slan-DCs from healthy donors (n=5).Since MM cells inhibited Slan-DCs secretion of IL-12 and given the pivotal role of IL-12 in Th1 polarization, we investigated whether MM cells could modulate the capacity of Slan-DCs to induce T cell proliferation and Th1 differentiation. To this aim, we co-cultured T cells and Slan-DCs (n=5). Here, we found that normal Slan-DCs can efficiently induce the proliferation of T cells. In addition, their ability to promote T cell proliferation was more pronounced after R848 priming. However, after co-culture with RPMI-8226 MM cell line, the fraction of proliferating T cells at day 7 was significantly decreased (58.14% versus 28.37% ; p=0.03). Moreover, RPMI-8226 MM cell line markedly impaired the capacity of Slan-DCs to induce the differentiation of naïve CD4+ T cells into IFNγ-producing Th1 cells (34.59% versus 7.89% ; p=0.04).Based on the above results, we hypothesized that Slan-DCs' pro-inflammatory phenotype could also be impacted by MM cells. Thus, freshly-sorted Slan-DCs were cultured with or without MM cell lines and their phenotype was analyzed by flow cytometry (n=9). When Slan-DCs were cultured in the presence of MM cells, expression of CD14 and CD16, but not CD11b, was induced, in line with a phenotype of an “intermediate” monocytes population. Moreover, in the presence of MM cells, the expression of the costimulatory molecule CD86 was weakly induced on Slan-DCs (Mean of Fluorescence Intensity = 102049 versus 199470, p=0.004).In conclusion, these results provide evidence that MM cells are able to significantly impair the capacity of Slan-DCs to secrete IL-12 and stimulate CD3+ T cell proliferation and to polarize naïve CD4+ T cell into Th1 cells. The latter suggests that MM cells are able to strongly suppress the pro-inflammatory function of Slan-DCs, directing them towards a more tolerogenic profile. These findings reveal a novel mechanism of immune escape in MM, and may pave the way for newer immune-intervention strategies in combination with other currently available therapies. DisclosuresGarderet:Amgen: Honoraria; Takeda: Honoraria. Mohty:Sanofi: Honoraria, Speakers Bureau.

Preclinical Evaluation of MEDI2228, a BCMA-Targeting Pyrrolobenzodiazepine-Linked Antibody Drug Conjugate for the Treatment of Multiple Myeloma

Despite remarkable progress in treatment over the past two decades, multiple myeloma (MM) remains an incurable disease. B-cell maturation antigen (BCMA, TNFRSF17) is a suitable therapeutic target for the treatment of MM due to its restricted expression on normal plasma cells and universal expression in myeloma cells. Targeting BCMA with an antibody-drug conjugate (ADC) is an attractive therapeutic option, combining the specificity of a monoclonal antibody with a potent cytotoxic drug to preferentially eliminate antigen-positive cells for the treatment of cancer.Here we describe MEDI2228, a novel ADC that targets BCMA. MEDI2228 is composed of a fully human antibody site-specifically conjugated to a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker. Flow cytometry studies show that MEDI2228 is rapidly internalized and trafficked to lysosomes. Upon release, the warhead binds to the minor-groove and cross-links DNA, leading to DNA damage and apoptotic cell death.The anti-tumor activity of MEDI2228 was assessed in vitro in 10 MM and plasma cell leukemia (PCL) cell lines encompassing several cytogenetic subtypes. MEDI2228 was highly active in 8 of 10 MM cell lines tested (IC50 range 6 to 210 ng/mL) including cell lines with high (~19,000 receptors/cell) or low (~930 receptors/cell) expression of BCMA. In addition, MEDI2228 is active in the presence of bone marrow stromal cells, which have been shown to play a role in chemotherapy resistance, and in cell models resistant to Lenalidomide. In vivo antitumor activity was evaluated in 4 separate MM and PCL subcutaneous xenograft models and 2 PCL disseminated xenograft models, where a single injection of MEDI2228 induced tumor regression at doses as low as 0.1 mg/kg.A soluble form of BCMA (sBCMA) has been detected in the serum of MM patients (Sanchez et. al. Br J Haematol 2012) and is comprised of the entire extracellular domain of the molecule (Laurent et. al. Nat Commun 2015). Therefore, sBCMA could diminish the effects of antibody-based therapies. We found that levels of sBCMA are elevated in the sera of MM patients compared with healthy donor controls, with levels ranging from 23 ng/mL to 728 ng/mL. The functional features of sBCMA and recombinant monomeric human BCMA are similar (Laurent et. al. Nat Commun 2015). Therefore, to mitigate the potential effects of sBCMA on the efficacy of our drug, the antibody component of MEDI2228 was selected because it possessed weak binding to recombinant monomeric human BCMA and strong binding to membrane-bound BCMA. The in vitro cytotoxicity of MEDI2228 in the presence of clinically-relevant levels of sBCMA was evaluated using NCI-H929 and MM.1S cells. MEDI2228 was cytotoxic to both MM.1S and NCI-H929 in vitro, killing an average of 95% of tumor cells in the presence of sBCMA at levels up to 720 ng/mL with little impact on IC50. ADCs developed from antibodies that possessed a similar affinity between monomeric BCMA and membrane-bound BCMA exhibited a sBCMA-dose dependent drop in potency, with a 20-fold shift in IC50 in the presence of 720 ng/mL sBCMA.MM is a clonally heterogeneous disease, and previously it has been reported that a small population of CD19+CD138- clonogenic cells can be identified in the bone marrow (BM) of MM patients (Matsui et. al. Blood 2004). We used flow cytometric sorting of CD19 and CD138 populations and outgrowth in methocult to confirm that CD19+CD138- cells are the clonogenic cells in MM BM samples. Furthermore, we found that this population contains the same light chain restriction, genetic translocation, and aberrant phenotype as the MM plasma cells. These cells express BCMA and can be killed by MEDI2228 in methocult outgrowth assays.These data demonstrate that MEDI2228 exhibits potent antitumor activity in preclinical models of MM. Importantly, in vitro experiments suggest that this activity is maintained in the presence of sBCMA. MEDI2228, bearing a potent PBD payload, may effectively target both the bulk myeloma plasma cells as well as the more quiescent, CD19+CD138- clonogenic cells, which may offer an opportunity for more durable clinical response in this genetically heterogeneous disease. DisclosuresKinneer:MedImmune: Employment. Meekin:MedImmune: Employment. Varkey:MedImmune: Employment. Xiao:MedImmune: Employment; Bristol-Myers Squibb: Employment. Zhong:MedImmune: Employment. Breen:MedImmune: Employment. Hurt:MedImmune: Employment. Thomas:MedImmune: Employment. Flynn:MedImmune: Employment. Hynes:MedImmune: Employment. Bezabeh:Salubris Biotherapeutics Inc: Employment; MedImmune: Employment. Chen:MedImmune: Employment. Wetzel:MedImmune: Employment. Chen:MedImmune: Employment. Anderson:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Herbst:MedImmune: Employment. Tice:MedImmune: Employment.

Identification and monitoring of Copy Number Variants (CNV) in monoclonal gammopathy

ABSTRACT Monoclonal gammopathy of undetermined significance (MGUS) represents the pre-clinical stage of Multiple Myeloma (MM) with the 5% of MGUS progresses to MM. Although the progression from MGUS to MM has not been completely characterized, it is possible to monitor the DNA modifications of patients diagnosed with MGUS to detect early specific genomic abnormalities, including copy number variations (CNV). The CNVs of chromosome 1q and chromosome 13q are associated with a worse prognosis in MM. In the present study, we showed that it is possible to monitor the 1q21 gain and 13q deletion frequencies in gDNA using digital PCR. The CNV analysis of three cell lines with a well-characterized cytogenetic profile were compared with measures performed by a real-time PCR approach and with a digital PCR approach. Then, we analyzed CNVs in CD138+ plasma cells isolated from bone marrow of MGUS and MM patients. Our results show that digital PCR and targeted DNA monitoring represent a specific and accurate technique for the early detection of specific genomic abnormalities both in MM and in MGUS patients. Our results could represent a remarkable advancement in MM and MGUS diagnosis and in CNV analysis for the evaluation of the risk of progression from MGUS to MM.

Activated Natural Killer Cells Withstand the Relatively Low Glucose Concentrations Found in the Bone Marrow of Multiple Myeloma Patients.

Infusion of ex vivo expanded and cytokine-activated natural killer (NK) cells is a promising alternative way to treat multiple myeloma (MM). However, the tumor microenvironment (TME) may suppress their function. While reduced glucose availability is a TME hallmark of many solid tumors, glucose levels within the TME of hematological malignancies residing in the bone marrow (BM) remain unknown. Here, we measured glucose levels in the BM of MM patients and tested the effect of different glucose levels on NK cells. BM glucose levels were measured using a biochemical analyzer. Compared to the normal range of blood glucose, BM glucose levels were lower in 6 of 9 patients (479-1231 mg/L; mean=731.8 mg/L). The effect of different glucose levels on NK cell cytotoxicity was tested in 4-hour cytotoxicity assays with tumor cells. 500 mg/L glucose (representing low range of MM BM) during the 4-hour cytotoxicity assay did not negatively affect cytotoxicity of activated NK cells, while higher glucose concentrations (4000 mg/L) diminished NK cell cytotoxicity. Since clinical application of NK cell therapy might require ex vivo expansion, expanded NK cells were exposed to a range of glucose concentrations from 500-4000 mg/L for a longer period (4 days). This did not reduce cytotoxicity or IFN-γ secretion nor affected their phenotypic profile. In summary, low glucose concentrations, as found in BM of MM patients, by itself did not compromise the anti-tumor potential of IL-2 activated NK cells in vitro. Although follow up studies in models with a more complex TME would be relevant, our data suggest that highly activated NK cells could be used to target tumors with a reduced glucose environment.

Expression of activated integrin β7 in multiple myeloma patients.

Multiple myeloma (MM) is still extremely difficult to cure, and new therapeutic drugs are needed. We recently found that integrin β7 is constitutively activated in MM cells, and chimeric antigen receptor (CAR) T cells targeting activated integrin β7 have a significant anti-MM effect. In this study, we performed flow cytometry analysis of the expression of activated integrin β7 in bone marrow cells from 137 symptomatic MM patients. In 60/137 (44%) MM patients, activated integrin β7 was detected in most MM cells (> 80% of MM cells were in the positive gate). Activated integrin β7 was highly expressed in MM cells even in heavily treated patients. It also showed high expression in many CD38lo/-CD138-CD19+B cells, which reportedly include clonotypic B cells, in the bone marrow of MM patients. Taken together, these results suggest that CAR T-cell therapy targeting activated integrin β7 has the potential to benefit many patients with relapsed or refractory MM.

Tumor Microenvironment Proteomics: Lessons From Multiple Myeloma.

Although the “seed and soil” hypothesis was proposed by Stephen Paget at the end of the 19th century, where he postulated that tumor cells (seeds) need a propitious medium (soil) to be able to establish metastases, only recently the tumor microenvironment started to be more studied in the field of Oncology. Multiple myeloma (MM), a malignancy of plasma cells, can be considered one of the types of cancers where there is more evidence in the literature of the central role that the bone marrow (BM) microenvironment plays, contributing to proliferation, survival, migration, and drug resistance of tumor cells. Despite all advances in the therapeutic arsenal for MM treatment in the last years, the disease remains incurable. Thus, studies aiming a better understanding of the pathophysiology of the disease, as well as searching for new therapeutic targets are necessary and welcome. Therefore, the present study aimed to evaluate the protein expression profiling of mononuclear cells derived from BM of MM patients in comparison with these same cell types derived from healthy individuals, in order to fill this gap in MM treatment. Proteomic analysis was performed using the mass spectrometry technique and further analyses were done using bioinformatics tools, to identify dysregulated biological pathways and/or processes in the BM microenvironment of patients with MM as a result of the disease. Among the pathways identified in this study, we can highlight an upregulation of proteins related to protein biosynthesis, especially chaperone proteins, in patients with MM. Additionally, we also found an upregulation of several proteins involved in energy metabolism, which is one of the cancer hallmarks. Finally, with regard to the downregulated proteins, we can highlight mainly those involved in different pathways of the immune response, corroborating the data that has demonstrated that the immune system of MM is impaired and, therefore, the immunotherapies that have been studied recently for the treatment of the disease are extremely necessary in the search for a control and a cure for these patients who live with the disease.

Monocyte Subsets and Serum Inflammatory and Bone-Associated Markers in Monoclonal Gammopathy of Undetermined Significance and Multiple Myeloma.

Simple SummaryWe investigated the distribution of different subsets of monocytes (Mo) in blood and bone marrow (BM) of newly-diagnosed untreated monoclonal gammopathy of undetermined significance (MGUS), smoldering (SMM) and active multiple myeloma (MM), and its relationship with immune/bone serum-marker profiles. Our results showed decreased production of BM Mo with decreased counts of classical Mo (cMo) in BM and blood of SMM and MM, but not MGUS. Conversely, intermediate and non-classical Mo were significantly increased in MGUS, SMM and MM BM. In parallel, increased levels of interleukin (IL)1β were observed in a fraction of MGUS and SMM, while increased serum IL8 was characteristic of SMM and MM, and higher serum IL6, RANKL and bone alkaline phosphatase concentrations, together with decreased counts of FcεRI+cMo, were restricted to MM presenting with bone lesions. These results provide new insights in the pathogenesis of plasma cell neoplasms and the potential role of FcεRI+cMo in normal bone homeostasis.Background. Monocyte/macrophages have been shown to be altered in monoclonal gammopathy of undetermined significance (MGUS), smoldering (SMM) and active multiple myeloma (MM), with an impact on the disruption of the homeostasis of the normal bone marrow (BM) microenvironment. Methods: We investigated the distribution of different subsets of monocytes (Mo) in blood and BM of newly-diagnosed untreated MGUS (n = 23), SMM (n = 14) and MM (n = 99) patients vs. healthy donors (HD; n = 107), in parallel to a large panel of cytokines and bone-associated serum biomarkers. Results: Our results showed normal production of monocyte precursors and classical Mo (cMo) in MGUS, while decreased in SMM and MM (p ≤ 0.02), in association with lower blood counts of recently-produced CD62L+ cMo in SMM (p = 0.004) and of all subsets of (CD62L+, CD62L− and FcεRI+) cMo in MM (p ≤ 0.02). In contrast, intermediate and end-stage non-classical Mo were increased in BM of MGUS (p ≤ 0.03), SMM (p ≤ 0.03) and MM (p ≤ 0.002), while normal (MGUS and SMM) or decreased (MM; p = 0.01) in blood. In parallel, increased serum levels of interleukin (IL)1β were observed in MGUS (p = 0.007) and SMM (p = 0.01), higher concentrations of serum IL8 were found in SMM (p = 0.01) and MM (p = 0.002), and higher serum IL6 (p = 0.002), RANKL (p = 0.01) and bone alkaline phosphatase (BALP) levels (p = 0.01) with decreased counts of FcεRI+ cMo, were restricted to MM presenting with osteolytic lesions. This translated into three distinct immune/bone profiles: (1) normal (typical of HD and most MGUS cases); (2) senescent-like (increased IL1β and/or IL8, found in a minority of MGUS, most SMM and few MM cases with no bone lesions); and (3) pro-inflammatory-high serum IL6, RANKL and BALP with significantly (p = 0.01) decreased blood counts of immunomodulatory FcεRI+ cMo-, typical of MM presenting with bone lesions. Conclusions: These results provide new insight into the pathogenesis of plasma cell neoplasms and the potential role of FcεRI+ cMo in normal bone homeostasis.

The role of regulatory T cells in multiple myeloma progression

Regulatory T cells (Tregs) are a subset of CD4+ T lymphocytes that suppress the functions of antigen-presenting cells and effector T cells, characterized by the expression of transcription factor forkhead box P3 (FOXP3). Recent studies have reported an increase in the number of Tregs in the bone marrow (BM) of multiple myeloma (MM) patients. However, the role and mechanisms of Treg accumulation in the BM of MM patients remain debatable. Here, we present our data demonstrating the significance of Tregs in the context of MM disease progression. Using the transplantable MM mouse model, we observed a significant increase in Tregs in the BM of MM-injected mice from the early disease stage. We observed extended survival in MM-injected mice with Treg depletion than in mice without Treg depletion, demonstrating direct in vivo evidence that Tregs enhance disease progression in MM. It is noteworthy that type 1 interferon (IFN) signaling is activated in MM-associated Tregs. By using type 1 IFN receptor blocking antibody treatment and type 1 IFN receptor knockout Tregs, we demonstrated a significant decrease in MM-associated Treg proliferation, which was associated with longer survival in MM-injected mice. Thus, we have demonstrated that Tregs play a significant role in MM progression; the function and homeostasis of Tregs are regulated by type 1 IFN secreted in the BM microenvironment.

Metabolic markers for diagnosis and risk-prediction of multiple myeloma

AimsTo discriminate metabolic biomarkers for diagnosis and risk prediction of multiple myeloma (MM) on a basis of metabolic characteristics in systemic circulation and local pathogenic niche. Main methodsA gas chromatography mass spectrometry-based untargeted metabolomics analysis was performed within the bone marrow (BM) supernatants and peripheral plasma from healthy donors and patients with MM. Key findingsDistinct metabolic features between MM patients and healthy volunteers were profiled in both BM and plasma. Metabolic profiles of subgroups in which MM patients undergo high/medium/low risk displayed risk-dependent metabolic shift especially in BM. In MM patients, up-regulated glutamate level and down-regulated glutamine level in BM indicated enhanced glutamate metabolism which provided NH4+ for ammonia utilization. This resulted in increased level of urea and creatinine produced from urea cycle, arginine and proline metabolism in both BM and plasma collected from MM patients. The disorders of tricarboxylic acid cycle and carnitine synthesis were unique in BM of MM patients. Receiver operating characteristic curve analysis indicated that aspartate was a candidate plasma biomarker for diagnosis with the highest sensitivity and specificity in both BM and plasma. Threonine was identified as a preferential plasma biomarker for risk prediction due to significant relation with various risk indexes of MM in both BM and plasma. SignificanceThe perturbed glutamate metabolism and carnitine synthesis in BM of MM patients provided a new sight on pathogenesis of MM. The plasma level of aspartate and threonine may become a preferential metabolic marker for diagnosis and risk prediction of MM, respectively.

Myeloma Cells Deplete Bone Marrow Glutamine and Inhibit Osteoblast Differentiation Limiting Asparagine Availability.

Simple SummaryOsteolytic bone lesions represent an important clinical feature of multiple myeloma (MM). MM cells metabolize very high amounts of glutamine (Gln) and significantly lower Gln in the bone marrow. In this contribution we demonstrate that MM-dependent Gln depletion impairs the differentiation of bone marrow mesenchymal stromal cells into osteoblasts, the cells that form new bone tissue. We also found that osteoblast differentiation is associated with increased expression of glutaminase, the main enzyme that metabolizes Gln, SNAT2, a transporter able to accumulate Gln into the cells, and asparagine synthetase, the enzyme that uses Gln to obtain asparagine (Asn). Asn rescued osteoblast differentiation of Gln-starved mesenchymal stromal cells. These results demonstrate that MM cells impair osteoblast differentiation, hindering mesenchymal Asn synthesis through Gln depletion. Besides providing a metabolic mechanism underlying osteolytic lesions in MM, these results suggest that Asn supplementation may prevent bone disease in MM patients.Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.

RORC overexpression as a sign of Th17 lymphocytes accumulation in multiple myeloma bone marrow

The role of the bone marrow microenvironment in supporting the proliferation and survival of the abnormal plasma cells in multiple myeloma (MM) is well established. Such microenvironment is rich of cytokines like IL-6, TGF-β, IL-1 and IL-23 which are known to promote the differentiation of Th17 lymphocytes, a T helper subpopulation. Th17 cells secrete IL-17, a cytokine involved in the pathophysiology of several auto-immune diseases. Yet, its involvement in cancers remains unclear. Herein, we aimed to try to understand the role of Th17 lymphocytes in multiple myeloma.Bone marrow samples were prospectively collected from 29 MM patients and 23 healthy bone marrow donors for allograft. Mononuclear bone marrow cells were isolated by Ficoll-Hypaque gradient and CD138+ plasma cells were depleted using magnetic beads. The quantification of Th17 cells was performed by flow cytometry in the CD138 negative cells. The mRNA expression of IL17 and RORc was quantified using real time PCR in the same subset. The mRNA expression of IL17R was analyzed in plasma cells (CD138+ cells). Data obtained from patients and healthy donors were compared by both non-parametric Mann-Whitney U test and Spearman test.A significant increase of IL17 and RORC mRNA expression was found in the bone marrow microenvironment of MM patients compared to healthy donors. Th17 cells were also increased in the bone marrow of MM patients compared to healthy donors. Interestingly, the mRNA expression of IL17R was significantly decreased in MM patients. Yet, no correlation was found between the gene expression IL17, RORC and IL17R and the bone marrow infiltration or the stage of the disease. Collectively, our results suggest the involvement of Th17 cells in the pathophysiology of MM. Such data further support the use of anti-IL-17 antibodies as a therapeutic approach in MM.

Long non-coding RNA CCAT2 as a potential serum biomarker for diagnosis and prognosis of multiple myeloma.

Increasing knowledge of long non-coding RNAs (lncRNAs) has shown that they can be used as circulating tumor markers. Also, considerable evidences have revealed that lncRNAs have important roles in tumor diagnosis and prognosis. The lncRNA CCAT2 has manifested its carcinogenic effect in a variety of tumors, but the serum expression level and clinical value in multiple myeloma (MM) remain to be explored. In our study, the expression of lncRNA CCAT2 is upregulated in the serum and bone marrow of MM patients by using quantitative real-time polymerase chain reaction (qRT-PCR). The high expression level of CCAT2 in the serum of MM patients correlated with International Scoring System (ISS) stages, renal dysfunction, serum β2-microglobulin (β2-MG) concentration, and light chain (κ and λ) concentrations. Area under the curve (AUC) of CCAT2 in serum is 0.899. Besides, the sensitivity and specificity were 85.80% and 83%, respectively. Furthermore, combination of CCAT2, IgA, HGB, and β2-MG significantly improved the MM diagnostic sensitivity and AUC. Here, our present investigation indicates that serum circulating CCAT2 may serve as a potential tumor marker for diagnosis and prognosis of MM.

PD-1 Blockade Reinvigorates Bone Marrow CD8+ T Cells from Patients with Multiple Myeloma in the Presence of TGFβ Inhibitors

Immune-checkpoint inhibitors have shown therapeutic efficacy in various malignant diseases. However, anti-programmed death (PD)-1 therapy has not shown clinical efficacy in multiple myeloma. Bone marrow (BM) mononuclear cells were obtained from 77 newly diagnosed multiple myeloma patients. We examined the expression of immune-checkpoint receptors in BM CD8+ T cells and their functional restoration by ex vivo treatment with anti-PD-1 and TGFβ inhibitors. We confirmed the upregulation of PD-1 and PD-L1 expression in CD8+ T cells and myeloma cells, respectively, from the BM of multiple myeloma patients. PD-1-expressing CD8+ T cells from the BM of multiple myeloma patients coexpressed other checkpoint inhibitory receptors and exhibited a terminally differentiated phenotype. These results were also observed in BM CD8+ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. BM CD8+ T cells from multiple myeloma patients exhibited reduced proliferation and cytokine production upon T-cell receptor stimulation. However, anti-PD-1 did not increase the proliferation of BM CD8+ T cells from multiple myeloma patients, indicating that T-cell exhaustion in multiple myeloma is hardly reversed by PD-1 blockade alone. Intriguingly, anti-PD-1 significantly increased the proliferation of BM CD8+ T cells from multiple myeloma patients in the presence of inhibitors of TGFβ, which was overexpressed by myeloma cells. Our findings indicate that combined blockade of PD-1 and TGFβ may be useful for the treatment of multiple myeloma.

Targeting Cancer Associated Fibroblasts in the Bone Marrow Prevents Resistance to Chimeric Antigen Receptor T Cell Therapy in Multiple Myeloma

Introduction Cellular immunotherapy is a rapidly progressing field in multiple myeloma (MM). Multiple clinical trials have reported impressive efficacy of B cell maturation antigen (BCMA) directed chimeric antigen receptor cell therapy (BCMA CART) in MM. While trials demonstrated a high response rate, the durable response rate is around 30%. Most patients lose their CART cells, and the disease relapses within the first year, suggesting an inhibition by the MM tumor microenvironment (TME). Therefore strategies to overcome this inhibition would represent a major advance in this field. Cancer associated fibroblasts (CAFs) within the TME play a critical role in promoting tumor growth and immunosuppression. Objective CAFs from MM patients lead BCMA CART cell dysfunction and targeting both MM cells and CAFs can overcome this resistance. Methods CAFs were isolated from the bone marrow (BM) of MM patients and purified using anti fibroblast beads. MM-TME mouse model was established by injecting a 1:1 ratio of BCMA+ OPM2 (MM cell line) to CAFs. Five different CART cells were tested in this study. For single transduced CAR: BCMA, CS1, and fibroblast activation protein (FAP). For dual transduced CAR: BCMA-CS1 and BCMA-FAP CART. These were generated through the double transduction of two lentiviral vectors. Results BCMA CART cell proliferation assay showed inhibition of CART expansion in the presence of CAFs, and this was mediated by TGF-β (Fig A). MM-TME mouse model revealed a significant acceleration of MM progression (Fig B). To identify targets, we verified FAP expression on CAFs. Interestingly, there was a low level of CS1, but not BCMA expressed on CAFs (Fig C). Then, we evaluated the impact of CAFs on functions of BCMA CART cells compared to dual CART cells. When CART cells were stimulated with the BCMA+ MM cell line MM1S, in the presence of CAFs, the proliferation of BCMA CART cells, but not the dual CART cells was significantly inhibited (Fig A). Similarly, in the presence of CAFs, the production of key effector cytokines by BCMA CART cells, but not the dual CART cells was reduced (Fig D). Finally, we studied the impact of CAFs on CART cell functions in vivo. First, using OPM2 xenografts, treatment with BCMA CART cells was able to eradicate MM (Fig E). Then, the MM-TME model was used to assess the effect of targeting CAFs. Mice were treated with 1) control T cells, 2) BCMA CART cells, 3) BCMA-FAP CART cells, or 4) BCMA-CS1 CART cells. A low dose (1 × 106 iv) of CART cell was used to induce relapse. BCMA CART cells led to a transient antitumor activity in this model (mice died within 2 weeks), while dual CART cells resulted in durable remissions, a higher number of circulating T cells, and long term survival (Fig F). Conclusion We demonstrate for the first time that dual targeting both malignant plasma cells and the CAFs within the TME is a novel strategy to overcome resistance to CART cell therapy in MM.

NUPR1 Silencing Induces Autophagy-Mediated Apoptosis in Multiple Myeloma Cells Through the PI3K/AKT/mTOR Pathway.

Nuclear protein 1 (NUPR1) is a stress-related small molecule and plays important roles in various tumors, including multiple myeloma (MM). Autophagy is essential for maintaining cellular homoeostasis in response to stress and, together with apoptosis, determines cell fate. Previous studies indicate that NUPR1 is involved in cancer progression of MM, but the underlying mechanisms have not been elucidated. In this study, we confirmed that NUPR1 and basal autophagy markers were highly expressed in the bone marrow of MM patients. The overexpression of NUPR1 was correlated with staging (both by Revised International Staging System [RISS] and Durie-Salmon [D-S] Staging System), levels of hemoglobin and calcium, and bone marrow plasma cell ratio in the MM patients. NUPR1 silencing reduced autophagy activities and induced apoptosis in U266 and RPMI 8226. We further observed a decrease in NUPR1 silencing-induced apoptosis in the presence of rapamycin, while an increase in apoptosis after chloroquine and 3-methyladenine treatment. Analysis of the mechanism indicated that PI3K/AKT/mTOR pathway was involved in autophagy-mediated apoptosis upon NUPR1 knockdown. In summary, our results demonstrate that NUPR1 silencing suppresses autophagy activities and induces autophagy-mediated apoptosis in MM cells through the PI3K/AKT/mTOR pathway, which exhibits potential as a treatment strategy for MM.

Metabolic approaches to rescue antitumor Vγ9Vδ2 T-cell functions in myeloma.

Vγ9Vδ2 T cells are immune effector cells very well-suited for immunotherapy, but clinical results have been disappointing in multiple myeloma (MM) and other cancers. We have shown that Vg9Vd2 T cells are victimized prematurely by the immune suppressive tumor microenvironment (TME) established by myeloma and neighbouring cells in the bone marrow (BM) of MM patients. One major mechanism is the highly redundant expression of multiple immunecheckpoints/immune checkpoint-ligands (ICP/ICP-L) in the TME impairing antimyeloma Vg9Vd2 T-cell immune responses. Another major immune suppressive mechanism is the metabolic reset driven by myeloma cells in the TME to satisfy their energetic needs to the detriment of effector cells. Recently, it has become clear that the ICP/ICP-L circuitry and metabolic checkpoints (MCP) jointly operate in the TME of cancer patients to promote tumor cell growth and suppress antitumor immune responses. In this review, we discuss the possible interactions between ICP/ICP-L and MCP in the TME of MM patients that may compromise the immune competence of BM Vγ9Vδ2 T cells, envisaging novel combination therapies to improve the outcome of immune-based interventions.

PD-1 Blockade Reinvigorates Bone Marrow CD8+ T Cells from Patients with Multiple Myeloma in the Presence of TGF-β Inhibitors

Programmed cell death (PD)-1/PD-Ligand 1(PD-L1) blockade that reinvigorates exhausted T cells has been approved for the treatment of various solid tumors and hematological malignancies. However, in a clinical trial of multiple myeloma (MM) patients, anti-PD-1 monotherapy did not result in a clinical response. Furthermore, clinical trials of combining PD-1 blockade with immunomodulatory drugs or anti-CD38 monoclonal antibody failed to demonstrate clinical benefits in MM patients. To overcome the limitation of anti-PD-1 therapy in MM, the phenotype and differentiation of CD8+ T cells need to be characterized in the bone marrow (BM) of MM patients, particularly by analyzing myeloma antigen-specific CD8+ T cells. In addition, the role of immunosuppressive factors abundant in the MM microenvironment should be considered, including TGF-β. First, we confirmed the upregulation of PD-1 and PD-L1 expression in CD8+ T cells and myeloma cells, respectively, from the BM of MM patients. PD-1-expressing CD8+ T cells from the BM of MM patients co-expressed other checkpoint inhibitory receptors including Tim-3, LAG-3, and TIGIT. We also investigated the expression of T-cell transcription factors, such as T-bet, and EOMES, which are related to T-cell differentiation. In BM from MM patients, PD-1+CD8+ T cells had a higher percentage of EomeshiT-betlo cells than PD-1-CD8+ T cells. These data demonstrate that PD-1-expressing CD8+ T cells from the BM of MM patients exhibit a terminally differentiated phenotype with co-expression of multiple immune checkpoint inhibitory receptors. These results were also observed in BM CD8+ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. Next, we investigated proliferation and cytokine production of BM CD8+ T cells from MM patients. BM CD8+ T cells from MM patients exhibited reduced proliferation and cytokine production upon T cell receptor (TCR) stimulation, compared to BM CD8+ T cells from other control group such as of undetermined significance. However, both anti-PD-1 alone and combined blockade of PD-1 with other immune checkpoint receptors, such as Tim-3, Lag-3, or TIGIT, did not increase the proliferation of BM CD8+ T cells from MM patients. Likewise, anti-PD-1 treatment failed to induce reinvigoration of BM CD8+ T cells stimulated with HLA-A*0201-restricted myeloma antigen peptides, including NY-ESO-1157-165 and HM1.2422-30 peptides. These data demonstrate that blocking PD-1 is not sufficient to restore the function of BM CD8+ T cells from MM patients. It has been known that TGF-β, which is actively secreted by malignant plasma cells and BM stromal cells, can inhibit T-cell responses. We confirmed that the major source of TGF- β1 is plasma cells including myeloma cells among BMMCs from MM patients, and the number of TGF- β1-producing plasma cells, including myeloma cells, is increased in the BM of MM patients. We investigated whether blocking TGF-β signaling enhances reinvigoration of BM CD8+ T cells from MM patients. The combined blockade of PD-1 and TGF- β significantly increased the proliferation of BM CD8+ T cells from MM patients in the presence of TCR stimulation. The production of IFN-γ and TNF by BM CD8+ T cells was also rescued by combined blockade of PD-1 and TGF-β. Moreover, combination of anti-PD-1 antibody and TGF-β inhibitors increased proliferative responses of BM CD8+ T cells from HLA-A2+ MM patients stimulated with a mixture of HLA-A*0201-restricted myeloma antigen peptides (NY-ESO-1157-165 and HM1.2422-30 peptides). Thus, PD-1 blockade reinvigorates BM CD8+ T cells from MM patients in the presence of TGF-β inhibitors. Taken together, BM CD8+ T cells and myeloma antigen-specific CD8+ T cells express increased levels of PD-1 and have a terminally exhausted phenotype in MM patients. Under TGF-β inhibition, anti-PD-1 reinvigorates BM CD8+ T cells from MM patients, but PD-1 blockade alone does not restore the function of BM CD8+ T cells. Blocking both TGF-β and PD-1 can be a promising therapeutic strategy for the treatment of MM. Disclosures No relevant conflicts of interest to declare.