Multiple Myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of plasma cells. Among the different therapeutic options in this setting, chimeric antigen receptor (CAR) ...T cells recently showed unprecedent achievements in term of progression free survival. However, despite advancements in current therapies, the disease's heterogeneity remains a major challenge and all patients unavoidably relapse, rendering innovative approaches to identify precise therapeutic targets eagerly awaited. Along this line, the revolution of single-cell (sc) technologies brings new opportunities to identify precise therapeutic targets, including abundant and unique surface proteins for CAR therapy. In this study, by analyzing individual cancer cells at sc level, we aim to identify novel surface targets, facilitating the development of novel personalized cellular therapies as well as treatment sequences. To this end, we used samples collected from 9 publicly available single-cell RNA sequencing (scRNA-seq) datasets (GEO accession numbers: GSE176131, GSE189460, GSE223060, GSE210079, GSE145977, GSE124310, GSE161801, GSE163278, GSE161722) obtained from 156 patients affected by monoclonal gammopathies (19 monoclonal gammopathy of undetermined significance (MGUS), 10 smoldering multiple myeloma (SMM), 78 MM and 25 relapsed/refractory multiple myeloma (RRMM)) and 24 normal bone marrows (NBM), to conduct an unbiased search for genes showing specific expression in plasma cells (PCs), regardless of disease status. We found 15 genes showing differential expression in PCs (adj.pval<0.01 and logFC>1) and coding for surface proteins: TNFRSF17(BCMA), SDC1 (CD138), FCRL5 (GPRC5D), TNFRSF13B (TACI), CD38, SLAMF7 (CS1), CD59, FCGR2B, FGFR3, SLC44A1 (CTL1), CD320, FCRL2, IL15RA, INSR and SLAMF1. As expected, most of these molecules already represent critical therapeutic targets. After completing our initial analysis, we compared PCs transcriptomes from MM/RRMM patients with NBM, MGUS, and SMM samples. Among previously identified genes, CD320 only was significantly overexpressed in MM PCs. Next, we investigated associations between cytogenetic-related cell subclusters and previously identified surface markers. Interestingly, we found a significant association between the expression levels of TACI/CD59 and the overexpression of CCND2 and MAF, both associated with the presence of t(14:16). Next, we decided to focus on the first five genes (according to expression ranking) that do not currently have an active clinical development program in advanced phases, namely: TNFRSF13B, CD59, FCGR2B, SLC44A1, and CD320 (Fig. 1 A, values dichotomized based on their median) to uncover their (potential) impact/association with patient's outcome. In our analysis of different available expression profiling datasets (GSE4204, GSE2658, GSE57317, GSE4581, GSE4452, GSE9782, the CoMMpass study NCT01454297) involving around 2000 MM patients, we observed TNFRSF13B, CD59, and FCGR2B expression correlated with improved outcome while CD320 and SLC44A1 expression associated with worse outcome (Fig. 1 B). In conclusion, we integrated results from both scRNAseq and bulk-RNAseq/microarray data to identify new targets genes to be used in the context of personalized medicine. Interestingly, CD320 emerges as a potential candidate biomarker useful for monitoring disease evolution and predicting worse outcomes. Additionally, this approach led us to identify a series of potential new targets for MM patients, depicting a new scenario where each patient could be “screened” to identify the best molecule to be targeted. While further investigation is necessary to assess off-target toxicity and confirm clinical relevance, our analyses significantly streamline the search for tumor markers with a method potentially applicable to different malignancies, bringing us closer to identifying the best candidates for effective CAR therapy.
Introduction: Multiple myeloma (MM) is an incurable hematologic cancer characterized by abnormal plasma cell growth in the bone marrow (BM). It is preceded by two premalignant conditions: monoclonal ...gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Understanding the transition from MGUS to MM is crucial for better risk stratification, early interventions, and targeted treatments. This study aims to gain insights into immunological changes and fecal microbiota variations along the MGUS to MM transition. Methods: To comprehensively evaluate the immune composition (myeloid/lymphoid T/B/NK subpopulations and immune checkpoint distribution) in both bone marrow (BM) and peripheral blood (PB), we designed six 10-color custom and two 8-color flow cytometry panels. We analyzed samples from 13 MGUS, 12 SMM, and 63 newly diagnosed MM patients. Additionally, we evaluated cytokine/chemokine abundance in BM and PB using a 48-plex Luminex plate on 72 samples from the same MGUS, SMM, and MM patients, along with 4 healthy donors (HD). Furthermore, we performed fecal microbiome profiling on samples from 3 MGUS, 6 SMM, and 9 MM patients to investigate potential relationships between bacterial composition and MM evolution. Statistical analysis was performed in R by using specific packages (such as FlowCT). Results: Through unsupervised analysis of T cells by flow cytometry, we identified a significant increase in circulating TEMRA CD8 T cells, mostly for CD57+ cells (mean, 7.5% for MGUS vs 20.1% for SMM vs 21.6% for MM; p<0.05) and a significant reduction in naïve CD8 T TIGIT+ cells (mean, 12.4% for MGUS vs 5.98% for SMM vs 4.16% for MM; p<0.001) and naïve CD8 T TIGIT+ TIM3+ (mean, 2.18% for MGUS vs 1.24% for SMM vs 1.17% for MM; p<0.001). Regarding CD4 T subsets, we observed a reduction in BM effector memory phenotype along disease evolution (mean, 7.07% for MGUS vs 4.54% for SMM vs 3.72% for MM; p<0.05). Interestingly, we confirmed the increase of BM CD4 T cells production of IL17 (mean: 6.7% for MGUS vs 22% for MM; p<0.05) in MM patients compared to those with MGUS. In the context of myeloid cell subsets, circulating non-classical monocytes expressing HLA-DR and CD11c (mean, 1.45% for MGUS vs 0.95% for SMM vs 0.69% for MM; p<0.05) as well as granulocytes (mean, 74.18% MGUS vs 73.86% SMM vs 65.59% MM; p<0.05) were found to be reduced along MM progression. No major differences were observed for other evaluated lineages. Regarding cytokines and chemokines, a concurrent decrease in GM-CSF, IFN-a2, IFN-g, IL-1b, IL-2, IL-2Ra, IL-3, IL-10, IL-13, and MCP-1/CCL2 levels within the BM plasma throughout MM progression was observed, thus supporting a global reduction in myeloid function as well as in T cell effector activity. Moreover, PDGF-BB, IFN-a2, IL-16, and IL-8 decrease was observed in PB plasma along MM progression. Lastly, while no differences were observed in term of alpha and beta diversity, we observed an overall increase in total abundance of fecal bacteria along MM evolution (maybe related to loss of myeloid/lymphoid control according to what previously described), with several bacterial genus significantly over-represented in MM patients, including Bariatricus, CAG-83, Alistipes, Bifidobacterium, Streptococcus, Ruminococcus D, Slackia A, and Coprococcus A, while others (such as Parabacteroides B) was found to be more represented in MGUS and SMM patients. The analysis of phyla abundance indicated Firmicutes D and Lactobacillales as more abundant in MM patients, while Actinobacteriota dominated in SMM patients, and Proteobacteria and Bacteroidota were more prominent in MGUS patients. Conclusions: Overall, this study demonstrated a progressive impairment of both myeloid and lymphoid compartment in term of cellular composition, cytokine production and release and control over gut microbiome. Recovering from T cell exhaustion and IL17 production remains a major challenge for these patients.
Multiple myeloma (MM) is characterized by generalized immune suppression and increased susceptibility to infections and secondary malignancies. Malignant plasma cells (PCs) modulate the bone marrow ...microenvironment to favor their own survival and proliferation. These events lead to a severe deregulation of immune effectors. Extensive studies have been conducted to unveil the mechanisms through which MM cells negatively modulate immunity and to develop therapeutical approaches for restoring an efficient anti-MM immune response.
This review article covers both the immunosuppressive effects exerted by MM and the immunomodulatory potential of novel anti-MM agents. A brief overview on the most promising immunotherapeutic approaches in the field is also provided.
MM leads to a progressive impairment of the immune system. Different approaches have been evaluated or are currently under investigation to boost a specific anti-MM immune response. The discovery that anti-MM agents like bortezomib also retain immunomodulatory properties provides evidence to support the development of combined treatment modalities. In the next future, immunotherapy will be likely included in selective treatments in early stages or in the post-transplantation setting with non toxic modalities that control or clear the neoplastic clone.
Immunosuppressive cells have been reported to play an important role in tumor progression mainly because of their capability to promote immune-escape, angiogenesis and metastasis. Among them, myeloid ...derived suppressor cells (MDSCs) have been recently identified as immature myeloid cells, induced by tumor-associated inflammation, able to impair both innate and adaptive immunity. While murine MDSCs are usually identified by the expression of CD11b and Gr-1, human MDSCs represent a more heterogeneous population characterized by the expression of CD33 and CD11b, low or no HLA-DR and variable CD14 and CD15. In particular, the last two may alternatively identify monocyte-like or granulocyte-like MDSC subsets with different immunosuppressive properties. Recently, a substantial increase of MDSCs has been found in peripheral blood and bone marrow (BM) of multiple myeloma (MM) patients with a role in disease progression and/or drug resistance. Preclinical models recapitulating the complexity of the MM-related BM microenvironment (BMM) are major tools for the study of the interactions between MM cells and cells of the BMM (including MDSCs) and for the development of new agents targeting MM-associated immune suppressive cells.This review will focus on current strategies for human MDSCs generation and investigation of their immunosuppressive function in vitro and in vivo, taking into account the relevant relationship occurring within the MM-BMM. We will then provide trends in MDSC-associated research and suggest potential application for the treatment of MM.
Multiple myeloma (MM) is a hematologic malignancy characterized by high genomic instability. Here we provide evidence that hyper-activation of DNA ligase III (LIG3) is crucial for genomic instability ...and survival of MM cells. LIG3 mRNA expression in MM patients correlates with shorter survival and even increases with more advanced stage of disease. Knockdown of LIG3 impairs MM cells viability in vitro and in vivo, suggesting that neoplastic plasmacells are dependent on LIG3-driven repair. To investigate the mechanisms involved in LIG3 expression, we investigated the post-transcriptional regulation. We identified miR-22-3p as effective negative regulator of LIG3 in MM. Enforced expression of miR-22 in MM cells downregulated LIG3 protein, which in turn increased DNA damage inhibiting in vitro and in vivo cell growth. Taken together, our findings demonstrate that myeloma cells are addicted to LIG3, which can be effectively inhibited by miR-22, promoting a novel axis of genome stability regulation.
Proteasome inhibitor bortezomib induces apoptosis in multiple myeloma (MM) cells, and has transformed patient outcome. Using
as well as
immunodeficient and immunocompetent murine MM models, we here ...show that bortezomib also triggers immunogenic cell death (ICD) characterized by exposure of calreticulin on dying MM cells, phagocytosis of tumor cells by dendritic cells, and induction of MM specific immunity. We identify a bortezomib-triggered specific ICD-gene signature associated with better outcome in two independent MM patient cohorts. Importantly, bortezomib stimulates MM cells immunogenicity
activation of cGAS/STING pathway and production of type-I interferons; and STING agonists significantly potentiate bortezomib-induced ICD. Our studies therefore delineate mechanisms whereby bortezomib exerts immunotherapeutic activity, and provide the framework for clinical trials of STING agonists with bortezomib to induce potent tumor-specific immunity and improve patient outcome in MM.
•Loss of GABARAP abrogates the surface exposure of CRT in dying cancer cells, thus reducing anti-MM immune response after bortezomib.•Immunogenicity can be restored by combining bortezomib with an ...autophagy inducer, providing the framework for their clinical translation.
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Immunogenic cell death (ICD) is a form of cell death by which cancer treatments can induce a clinically relevant antitumor immune response in a broad range of cancers. In multiple myeloma (MM), the proteasome inhibitor bortezomib is an ICD inducer and creates durable therapeutic responses in patients. However, eventual relapse and resistance to bortezomib appear inevitable. Here, by integrating patient transcriptomic data with an analysis of calreticulin (CRT) protein interactors, we found that GABA type A receptor–associated protein (GABARAP) is a key player whose loss prevented tumor cell death from being perceived as immunogenic after bortezomib treatment. GABARAP is located on chromosome 17p, which is commonly deleted in patients with high risk MM. GABARAP deletion impaired the exposure of the eat-me signal CRT on the surface of dying MM cells in vitro and in vivo, thus reducing tumor cell phagocytosis by dendritic cells and the subsequent antitumor T-cell response. Low GABARAP was independently associated with shorter survival in patients with MM and reduced tumor immune infiltration. Mechanistically, we found that GABARAP deletion blocked ICD signaling by decreasing autophagy and altering Golgi apparatus morphology, with consequent defects in the downstream vesicular transport of CRT. Conversely, upregulating autophagy using rapamycin restored Golgi morphology, CRT exposure, and ICD signaling in GABARAPKO cells undergoing bortezomib treatment. Therefore, coupling an ICD inducer, such as bortezomib, with an autophagy inducer, such as rapamycin, may improve patient outcomes in MM, in which low GABARAP in the form of del(17p) is common and leads to worse outcomes.
Bortezomib kills multiple myeloma (MM) cells by inducing immunogenic cell death (ICD). Although it induces durable responses, most patients develop bortezomib resistance and relapse. Gulla et al propose that resistance is linked to low expression of GABA type A receptor–associated protein (GABARAP) which decreases surface expression of calreticulin, an “eat me” signal mediating ICD. GABARAP is on chromosome 17p, often deleted in high-risk MM, and low GABARAP expression predicts decreased MM survival. GABARAP deletion blocks autophagy, and inducing autophagy with rapamycin restores bortezomib responsiveness in vitro, which is a potential approach to enhance bortezomib response.
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