Dynamic Single-Cell RNA-Seq reveals mechanism of Selinexor-Resistance in Chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a type of hematologic malignancies caused by BCR-ABL chimeric oncogene. Resistance to tyrosine kinase inhibitors (TKIs) leads to the progression of CML into advanced stages. Selinexor is a small molecule inhibitor that targets a nuclear transporter called Exportin 1. Combined with imatinib, selinexor has been shown to disrupt nuclear-cytoplasmic transport signal of leukemia stem cells, resulting in cell death. The objective of this study was to investigate the mechanism of drug resistance to selinexor in CML. We established K562 cell line resistant to selinexor and conducted single cell dynamic transcriptome sequencing to analyze the heterogeneity within the parental and selinexor resistant cell populations. We identified specific gene expression changes associated with resistance to selinexor. Our results revealed differential expression patterns in genes such as MT2A, TFPI, MTND3, and HMGCS1 in the total RNA, as well as MT-TW, DNAJB1, and HSPB1 in the newly synthesized RNA, between the parental and drug-resistant groups. By applying pseudo-time analysis, we discovered that a specific cluster of cells exhibited characteristics of tumor stem cells. Furthermore, we observed a gradual decrease in the expression of ferroptosis-related molecules as drug resistance developed. In vitro experiments confirmed that the combination of a ferroptosis inducer called RSL3 effectively overcame drug resistance. In conclusion, this study revealed the resistance mechanism of selinexor in CML. In conclusion, we identified a subgroup of CML cells with tumor stem cell properties and demonstrated that ferroptosis inducer improved the efficacy of selinexor in overcoming drug resistance.

Liquid extramedullary disease in multiple myeloma strongly predicts a poor prognosis and is associated with bortezomib resistance gene upregulation.

BACKGROUND-AIM: Patients with multiple myeloma (MM) relapse with extramedullary disease (EMD) exhibits an aggressive disease course and poor prognostic features. Myelomatous effusion (ME) is a rare subtype of EMD. METHODS: In this retrospective study, we analyzed the baseline characteristics and therapies of 14 EMD patients relapse with ME and 21 EMD patients relapse without ME. RESULTS: Patients with ME relapse demonstrated higher concentrations of serum lactate dehydrogenase, a higher fraction in the International Staging System stage III, and poorer event-free survival (EFS) (9.3 vs. 36.57 months; P = 0.0013) and overall survival (OS) (12.06 vs. 42.64 months; P < 0.001). The multivariate analysis showed that the presence of ME (hazard ratio [HR] 12.57; P = 0.003) and lack of autologous hematopoietic stem cell transplantation therapy (HR 4.382; P = 0.014) were predictive factors for poor OS. Using single-cell RNA sequencing, we discovered several bortezomib resistance genes were highly expressed in extramedullary malignant plasma cells. CONCLUSIONS: The presence of ME strongly predicts a poor prognosis in patients with MM relapse with EMD, and bortezomib resistance genes are highly expressed in extramedullary malignant plasma cells.

Identification of evolutionary mechanisms of myelomatous effusion by single-cell RNA sequencing.

Myelomatous effusion (ME) is a rare manifestation of extramedullary multiple myeloma (MM) with limited therapeutic options and poor outcomes. The molecular mechanisms underlying ME are incompletely understood. We profiled transcriptomes of bone marrow, peripheral blood (PB), and pleural effusion/ascites from 3 patients with ME using single-cell RNA sequencing analysis. We found that ME contained a higher percentage of cytotoxic T cells, whereas PB contained a higher proportion of naive T cells. Malignant cells varied within and between sites and patients in their expression of signatures. We identified a gene module highly expressed in intramedullary and extramedullary plasma cell clusters and defined cell clusters expressing this gene set as extramedullary-initiating cells (EMICs). This gene set was associated with increased cellular proliferation, involved in p53 signaling, and related to poor prognosis in MM. The transcriptional regulators E2F1, YY1, and SMAD1 were activated in EMICs. Leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4) was upregulated in extramedullary EMICs. We confirmed that LILRB4 promoted MM cell migration in vitro. This study provided insight into the evolutionary mechanisms of ME and defined EMICs and LILRB4 associated with extramedullary development.

Identification of long noncoding RNA NEAT1 as a key gene involved in the extramedullary disease of multiple myeloma by bioinformatics analysis.

OBJECTIVE: Long non-coding RNAs (lncRNAs) are involved in tumorigenesis and play a key role in cancer progression. To determine whether lncRNAs are involved in extramedullary disease of multiple myeloma (EMD), we analyzed the expression profile of lncRNAs in EMD. METHODS: Three pairs of EMD patients and their intramedullary MM cells were screened by microarray first. We extracted data from gene chips and made an identification of lncRNAs and mRNAs with significant differences between EMD group and non EMD group. WGCNA confirmed the EMD related gene module and drew a heat map to further determine the key gene lncRNA-NEAT1. In the meantime, bone marrow and extramedullary samples (hydrothorax and ascites) were collected from 2 MM patients and subjected to single-cell RNA-seq. Single cell Transcriptome analysis was conducted to verify the gene expression difference of malignant plasma cells derived from intramedullary and extramedullary. Then we verified high expression level of lncRNA-NEAT1 in EMD patients by using quantitative real-time PCR (qRT-PCR) and analyzed the correlation between expression patterns and survival and molecular genetics analysis of the LncRNA (NEAT1) involved in MM patients. At last, cell experiments were conducted to observe the effects of down-regulation of NEAT1on the proliferation, cell cycle and PTEN pathway related proteins of multiple myeloma cell lines U266 and RPMI8226. RESULTS: We identified one of the EMD related key gene is lncRNA-NEAT1. Compared with patients without extramedullary lesions, intramedullary MM cells in EMD patients expressed NEAT1 highly. The outcome of parallel single-cell RNA sequencing (RNA-seq) revealed NEAT1 level of plasma cells came from pleural effusion /ascites increased significantly compared with myeloma-stricken bone marrow. By survival and molecular genetic analysis, NEAT1 gene expression was not associated with OS and PFS in MM patients. However, the expression of NEAT1 is related to adverse therapeutic reactions and the progression of MM. We found that the expressions of NEAT1 were negatively associated with albumin levels and were positively associated with gain of chromosome 1q, IGH-CCND1, IGH@-FGFR3/WHSC1,and IGH-MAF gene fusion, respectively. At the level of cell experiment, CCK-8, soft agar clone formation experiment and CFSE staining showed that down regulating NEAT1 could inhibit the proliferation of U266 and RPMI8226 cells; Cell cycle detection showed that down-regulation of NEAT1 would interfere with the cell cycle process, and RPMI 8226 cells were blocked in G1 phase. Western blot analysis showed that when the expression of NEAT1 was down regulated in U266 and RPMI 8226 cells, the expression of PTEN and p-PTEN (phosphorylated phosphatase and tensin homologue) was up-regulated, and the expression of PI3K, p-PI3K (human phosphorylated inositol 3 kinase), Akt, p-Akt (phosphorylated protein kinase B). DISCUCCION AND CONCLUSION: This study provides novel insights into the lncRNA-NEAT1 and reveals that NEAT1 maybe a potential lncRNA biomarkers in EMD.

Monocytic myeloid-derived suppressive cells mitigate over-adipogenesis of bone marrow microenvironment in aplastic anemia by inhibiting CD8+ T cells.

Aplastic anemia (AA) is a blood disorder resulted from over-activated T-cell related hematopoietic failure, with the characterization of hypocellularity and enhanced adipogenic differentiation of mesenchymal stroma cells (MSCs) in bone marrow (BM). However, little is known about the relationship between immune imbalance and polarized adipogenic abnormity of BM microenvironment in this disease entity. In the present study, we differentiated BM-MSCs into osteoblastic or adipogenic lineages to mimic the osteo-adipogenic differentiation. Activated CD8+ T cells and interferon-γ (IFN-γ) were found to stimulate adipogenesis of BM-MSCs either in vitro or in vivo of AA mouse model. Interestingly, myeloid-derived suppressive cells (MDSCs), one of the immune-regulating populations, were decreased within BM of AA mice. We found that it was not CD11b+Ly6G+Ly6C- granulocytic-MDSCs (gMDSCs) but CD11b+Ly6G-Ly6C+ monocytic-MDSCs (mMDSCs) inhibiting both T cell proliferation and IFN-γ production via inducible nitric oxide synthetase (iNOS) pathway. Single-cell RNA-sequencing (scRNA-seq) of AA- and mMDSCs-treated murine BM cells revealed that mMDSCs transfusion could reconstitute BM hematopoietic progenitors by inhibiting T cells population and signature cytokines and decreasing immature Adipo-Cxcl12-abundant reticular cells within BM. Multi-injection of mMDSCs into AA mice reduced intra-BM T cells infiltration and suppressed BM adipogenesis, which subsequently restored the intra-BM immune balance and eventually prevented pancytopenia and hypo-hematopoiesis. In conclusion, adoptive transfusion of mMDSCs might be a novel immune-regulating strategy to treat AA, accounting for not only restoring the intra-BM immune balance but also improving stroma's multi-differentiating microenvironment.

Advances in the treatment of extramedullary disease in multiple myeloma.

Extramedullary disease (EMD) is characterized by plasma cells outside of bone marrow in multiple myeloma (MM) patients, which results in an adverse prognosis. The cornerstone of treatment consists of combination therapy including proteasome inhibitors, immunomodulatory agents, steroids, followed by consolidative autologous hematopoietic stem cell transplantation in eligible patients. This review summarized the recent advances in the treatment of EMD. Bortezomib based therapy showed efficacy and was recommended to treat EMD. Marizomib had advantages in the treatment of central nervous system-multiple myeloma (CNS-MM) because of its good central nervous system penetrability. Immunomodulatory drugs such as lenalidomide and pomalidomide have been reported to be effective. Isatuximab and selinexor were also active. Based on the treatment experience of EMD in our department, we summarized treatment approach for EMD. However, the benefits of patients with EMD from the new era of novel drugs were limited. Novel drugs combination, monoclonal antibody, molecular targeted therapy, cellular immunotherapy and autologous stem cell transplantation (ASCT) are under investigation. Therapeutic studies and clinical trials specifically target EMD should be conducted. Hopefully, these treatment options for EMD will be demonstrated efficacy in the future.

The gut microbial metabolite trimethylamine N-oxide aggravates GVHD by inducing M1 macrophage polarization in mice.

The diversity of the human microbiome heralds the difference of the impact that gut microbial metabolites exert on allogenic graft-versus-host (GVH) disease (GVHD), even though short-chain fatty acids and indole were demonstrated to reduce its severity. In this study, we dissected the role of choline-metabolized trimethylamine N-oxide (TMAO) in the GVHD process. Either TMAO or a high-choline diet enhanced the allogenic GVH reaction, whereas the analog of choline, 3,3-dimethyl-1-butanol reversed TMAO-induced GVHD severity. Interestingly, TMAO-induced alloreactive T-cell proliferation and differentiation into T-helper (Th) subtypes was seen in GVHD mice but not in in vitro cultures. We thus investigated the role of macrophage polarization, which was absent from the in vitro culture system. F4/80+CD11b+CD16/32+ M1 macrophage and signature genes, IL-1ß, IL-6, TNF-α, CXCL9, and CXCL10, were increased in TMAO-induced GVHD tissues and in TMAO-cultured bone marrow-derived macrophages (BMDMs). Inhibition of the NLRP3 inflammasome reversed TMAO-stimulated M1 features, indicating that NLRP3 is the key proteolytic activator involved in the macrophage's response to TMAO stimulation. Consistently, mitochondrial reactive oxygen species and enhanced NF-κB nuclear relocalization were investigated in TMAO-stimulated BMDMs. In vivo depletion of NLRP3 in GVHD recipients not only blocked M1 polarization but also reversed GVHD severity in the presence of TMAO treatment. In conclusion, our data revealed that TMAO-induced GVHD progression resulted from Th1 and Th17 differentiation, which is mediated by the polarized M1 macrophage requiring NLRP3 inflammasome activation. It provides the link among the host choline diet, microbial metabolites, and GVH reaction, shedding light on alleviating GVHD by controlling choline intake.

Toll-like receptor 4 protects against irradiation-induced hematopoietic injury by promoting granulopoiesis and alleviating marrow adipogenesis.

Irradiation induces severe damage in the hematopoietic system, which leads to bone marrow hyperplasia, pancytopenia, and aggravated tissue formation in bone marrow. Studies have shown that Toll-like receptor 4 (TLR4) has a protective effect against irradiation, but the underlying mechanism remains unclear. In this study, we used a TLR4 knockout (TLR4-/-) mouse irradiation model and found that the white blood cell and platelet counts in the peripheral blood of TLR4-/- mice recovered slowly after irradiation, with bone marrow hyperplasia and increased mortality. Additionally, we found that the proportion of CD11b+Gr1+ granulocytes in the peripheral blood and bone marrow of TLR4-/- mice was lower than that of wild-type mice after irradiation. Further, we found that the expression of NADPH Oxidases (NOXs) in the bone marrow was down-regulated after irradiation of TLR4-/- mice, and administration of the NOXs inhibitor VAS2870 reduced the proportion of CD11b+Gr1+ cells in the bone marrow and peripheral blood of wild-type mice after irradiation. Irradiation induced severe marrow adipocytes accumulation in TLR4-/- mice, TLR4 ligand lipopolysaccharide promoted proliferation and inhibited adipogenic differentiation of mesenchymal stromal cells. In summary, our data suggest that TLR4 promotes myeloid hyperplasia by up-regulating the expression of NOXs after irradiation, prohibits marrow adipogensis and increases the tolerance of mice to irradiation.

Cyclosporin A inhibits adipogenic differentiation and regulates immunomodulatory functions of murine mesenchymal stem cells.

Aplastic anemia (AA) is generally considered as an immune-mediated bone marrow failure syndrome. Several studies show that bone marrow mesenchymal stem cells (BM-MSCs), as key cellular components of the bone marrow microenvironment, are also involved in the pathogenic mechanism of AA. Cyclosporin A (CsA) is a classic immunosuppressive drug for AA, and it specifically inhibits mammalian T cells by preventing activation of transcription factors involved in cytokine gene expression. However, little is known about the effect of CsA on the BM-MSCs. In this study, murine BM-MSCs were stimulated in the presence of CsA. Further, we found that CsA could inhibit murine BM-MSC proliferation and promote BM-MSC apoptosis, what's more CsA could inhibit adipogenic differentiation. Our study also showed that CsA could inhibit interleukin-6 expression in BM-MSCs, while promoting programmed death-ligand 2 expression. In conclusion, our results proposed that CsA may exert an effect on regulating the bone marrow environment by influencing BM-MSCs, which have a beneficial effect on treating AA.