Identification of potential resistance mechanisms and therapeutic targets for the relapse of BCMA CAR-T therapy in relapsed/refractory multiple myeloma through single-cell sequencing.

BACKGROUND: BCMA CAR-T is highly effective for relapsed/refractory multiple myeloma(R/R-MM) and significantly improves the survival of patients. However, the short remission time and high relapse rate of MM patients treated with BCMA CAR-T remain bottlenecks that limit long-term survival. The immune microenvironment of the bone marrow (BM) in R/R-MM may be responsible for this. The present study aims to present an in-depth analysis of resistant mechanisms and to explore potential novel therapeutic targets for relapse of BCMA CAR-T treatment via single-cell RNA sequencing (scRNA-seq) of BM plasma cells and immune cells. METHODS: This study used 10X Genomic scRNA-seq to identify cell populations in R/R-MM CD45+ BM cells before BCMA CAR-T treatment and relapse after BCMA CAR-T treatment. Cell Ranger pipeline and CellChat were used to perform detailed analysis. RESULTS: We compared the heterogeneity of CD45+ BM cells before BCMA CAR-T treatment and relapse after BCMA CAR-T treatment. We found that the proportion of monocytes/macrophages increased, while the percentage of T cells decreased at relapse after BCMA CAR-T treatment. We then reclustered and analyzed the alterations in plasma cells, T cells, NK cells, DCs, neutrophils, and monocytes/macrophages in the BM microenvironment before BCMA CAR-T treatment and relapse after BCMA CAR-T treatment. We show here that the percentage of BCMA positive plasma cells increased at relapse after BCMA CAR-T cell therapy. Other targets such as CD38, CD24, SLAMF7, CD138, and GPRC5D were also found to be expressed in plasma cells of the R/R-MM patient at relapse after BCMA CAR-T cell therapy. Furthermore, exhausted T cells, TIGIT+NK cells, interferon-responsive DCs, and interferon-responsive neutrophils, increased in the R/R-MM patient at relapse after BCMA CAR-T cell treatment. Significantly, the proportion of IL1ßhi Mφ, S100A9hi Mφ, interferon-responsive Mφ, CD16hi Mφ, MARCO hi Mφ, and S100A11hi Mφ significantly increased in the R/R-MM patient at relapse after BCMA CAR-T cell therapy. Cell-cell communication analysis indicated that monocytes/macrophages, especially the MIF and APRIL signaling pathway are key players in R/R-MM patient at relapse after BCMA CAR-T cell therapy. CONCLUSION: Taken together, our data extend the understanding of intrinsic and extrinsic relapse of BCMA CAR-T treatment in R/R-MM patient and the potential mechanisms involved in the alterations of antigens and the induced immunosuppressive microenvironment, which may provide a basis for the optimization of BCMA CAR-T strategies. Further studies should be performed to confirm these findings.

Contributions of bone marrow monocytes/macrophages in myeloproliferative neoplasms with JAK2V617F mutation.

The classic BCR-ABL1-negative myeloproliferative neoplasm (MPN) is a highly heterogeneous hematologic tumor that includes three subtypes, namely polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). Despite having the same JAK2V617F mutation, the clinical manifestations of these three subtypes of MPN differ significantly, which suggests that the bone marrow (BM) immune microenvironment may also play an important role. In recent years, several studies have shown that peripheral blood monocytes play an important role in promoting MPN. However, to date, the role of BM monocytes/macrophages in MPN and their transcriptomic alterations remain incompletely understood. The purpose of this study was to clarify the role of BM monocytes/macrophages in MPN patients with the JAK2V617F mutation. MPN patients with the JAK2V617F mutation were enrolled in this study. We investigated the roles of monocytes/macrophages in the BM of MPN patients, using flow cytometry, monocyte/macrophage enrichment sorting, cytospins and Giemsa-Wright staining, and RNA-seq. Pearson correlation coefficient analysis was also used to detect the correlation between BM monocytes/macrophages and the MPN phenotype. In the present study, the proportion of CD163+ monocytes/macrophages increased significantly in all three subtypes of MPN. Interestingly, the percentages of CD163+ monocytes/macrophages are positively correlated with HGB in PV patients and PLT in ET patients. In contrast, the percentages of CD163+ monocytes/macrophages are negatively correlated with HGB and PLT in PMF patients. It was also found that CD14+CD16+ monocytes/macrophages increased and correlated with MPN clinical phenotypes. RNA-seq analyses demonstrated that the transcriptional expressions of monocytes/macrophages in MPN patients are relatively distinct. Gene expression profiles of BM monocytes/macrophages suggest a specialized function in support of megakaryopoiesis in ET patients. In contrast, BM monocytes/macrophages yielded a heterogeneous status in the support or inhibition of erythropoiesis. Significantly, BM monocytes/macrophages shaped an inflammatory microenvironment, which, in turn, promotes myelofibrosis. Thus, we characterized the roles of increased monocytes/macrophages in the occurrence and progression of MPNs. Our findings of the comprehensive transcriptomic characterization of BM monocytes/macrophages provide important resources to serve as a basis for future studies and future targets for the treatment of MPN patients.

Natural killer cells: a promising immunotherapy for cancer.

As a promising alternative platform for cellular immunotherapy, natural killer cells (NK) have recently gained attention as an important type of innate immune regulatory cell. NK cells can rapidly kill multiple adjacent cancer cells through non-MHC-restrictive effects. Although tumors may develop multiple resistance mechanisms to endogenous NK cell attack, in vitro activation, expansion, and genetic modification of NK cells can greatly enhance their anti-tumor activity and give them the ability to overcome drug resistance. Some of these approaches have been translated into clinical applications, and clinical trials of NK cell infusion in patients with hematological malignancies and solid tumors have thus far yielded many encouraging clinical results. CAR-T cells have exhibited great success in treating hematological malignancies, but their drawbacks include high manufacturing costs and potentially fatal toxicity, such as cytokine release syndrome. To overcome these issues, CAR-NK cells were generated through genetic engineering and demonstrated significant clinical responses and lower adverse effects compared with CAR-T cell therapy. In this review, we summarize recent advances in NK cell immunotherapy, focusing on NK cell biology and function, the types of NK cell therapy, and clinical trials and future perspectives on NK cell therapy.

GM-CSF impairs erythropoiesis by disrupting erythroblastic island formation via macrophages.

Anemia is a significant complication of chronic inflammation and may be related to dysregulated activities among erythroblastic island (EBI) macrophages. GM-CSF was reported to be upregulated and attracted as a therapeutic target in many inflammatory diseases. Among EBIs, we found that the GM-CSF receptor is preferentially and highly expressed among EBI macrophages but not among erythroblasts. GM-CSF treatment significantly decreases human EBI formation in vitro by decreasing the adhesion molecule expression of CD163. RNA-sequence analysis suggests that GM-CSF treatment impairs the supporting function of human EBI macrophages during erythropoiesis. GM-CSF treatment also polarizes human EBI macrophages from M2-like type to M1-like type. In addition, GM-CSF decreases mouse bone marrow (BM) erythroblasts as well as EBI macrophages, leading to a reduction in EBI numbers. In defining the molecular mechanism at work, we found that GM-CSF treatment significantly decreases the adhesion molecule expression of CD163 and Vcam1 in vivo. Importantly, GM-CSF treatment also decreases the phagocytosis rate of EBI macrophages in mouse BM as well as decreases the expression of the engulfment-related molecules Mertk, Axl, and Timd4. In addition, GM-CSF treatment polarizes mouse BM EBI macrophages from M2-like type to M1-like type. Thus, we document that GM-CSF impairs EBI formation in mice and humans. Our findings support that targeting GM-CSF or reprogramming EBI macrophages might be a novel strategy to treat anemia resulting from inflammatory diseases.

Single-cell map of diverse immune phenotypes in the acute myeloid leukemia microenvironment.

BACKGROUND: Knowledge of immune cell phenotypes, function, and developmental trajectory in acute myeloid leukemia (AML) microenvironment is essential for understanding mechanisms of evading immune surveillance and immunotherapy response of targeting special microenvironment components. METHODS: Using a single-cell RNA sequencing (scRNA-seq) dataset, we analyzed the immune cell phenotypes, function, and developmental trajectory of bone marrow (BM) samples from 16 AML patients and 4 healthy donors, but not AML blasts. RESULTS: We observed a significant difference between normal and AML BM immune cells. Here, we defined the diversity of dendritic cells (DC) and macrophages in different AML patients. We also identified several unique immune cell types including T helper cell 17 (TH17)-like intermediate population, cytotoxic CD4+ T subset, T cell: erythrocyte complexes, activated regulatory T cells (Treg), and CD8+ memory-like subset. Emerging AML cells remodels the BM immune microenvironment powerfully, leads to immunosuppression by accumulating exhausted/dysfunctional immune effectors, expending immune-activated types, and promoting the formation of suppressive subsets. CONCLUSION: Our results provide a comprehensive AML BM immune cell census, which can help to select pinpoint targeted drug and predict efficacy of immunotherapy.

[Proliferation and Apoptosis of Leukemia Cell Line K562 Treated with HSP90 Inhibitor 17-DMAG].

OBJECTIVE: To investigate the role of HSP90 in proliferation and apoptosis of leukemia cells K562 through detecting the effect of HSP90 inhibitors 17-[2-(Dimethylamino) ethyl] amino-17-desmethoxygeldanamycin(17-DMAG) on leukemia K562 cell lines. METHODS: The K562 cells were treated with HSP90 inhibitors 17-DMAG, the semi-quantitative PCR was used to detect HSP90 gene expression, the WST was used to detect the effect 17-DMAG on cell proliferation as well as Annexin V flow cytometry was used to detect the cell apoptosis. RESULTS: After 17-DMAG treated the K562 cells in different stage, the K562 cell growth was obviously inhibited with time dependent (48 h)(r=0.9918) and dose dependent(3.2 µmol/L) manners (r=0.9999) (P<0.01); after the K562 cells in different stage were treated with different concentrations of 17-DMAG, the K562 cells showed significant apoptosis and with dosage-dependent mauner (r=0.9903)(P<0.01); HSP90 mRNA expression decreased significantly after K562 cells were treated with different concentrations of 17-DMAG for 48 hours. 17-DAMG down-regulated the HSP90 mRNA expression in dosage-dependent mauner as well(r=0.9227) (P<0.01). CONCLUSION: HSP90 inhibitor 17-DMAG can inhibit the proliferation of K562 cells and induce their apoptosis. This study result provides laboratory basis for the treatment of leukemia patients with 17-DMAG.

[Effect of Heat Shock Protein 90 Inhibitor 17-DMAG on Proliferation and Apoptosis of Acute Lymphocytic Leukemia Cell Line Jurkat].

OBJECTIVE: To explore the effect of heat shock protein 90(HSP90) inhibitor 17-DMAG, an inhibitor specific for heat shock protein 90, on the proliferation and apoptosis of acute lymphocytic leukemia cell lines Jurkat. METHODS: Jurkat cells were collected, then were treated with 17-DMAG. The expression of HSP90 was examined by semi-quantitative RT-PCR analysis, the effect of 17-DMAG on cell proliferation were detected by using WST, and cell apoptosis were detected by using flow cytometry with Annexin V/PI double stenining. RESULTS: After Jurkat cells were treated with different concentrations of 17-DMAG for 48 hours, the HSP90 mRNA expression decreased significantly in dose dependent manner (r=0.9530, P<0.01). The IC50 was 3.17 mmol/L when the Jurkat cells were treated with 17-DMAG for 48 h; after treating Jurkat cell with 17-DMAG, the cell proliferation was inhibited(r=0.9903, P< 0.01), the cell apoptosis was increased in dose dependent manner (r=0.9876, P<0.01). CONCLUSION: 17-DMAG can inhibit the Jurkat cell proliferation and induce the Jurkat cell apoptosis.