Progression Of Diffuse Large B-cell Lymphoma Research Articles

FOXO3 suppresses lymphoma progression through promoting miR-34b/HSPG2 axis.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma, which caused many patients to lose their precious lives. FOXO3 was a suppressor in various cancers, however, the role and mechanism of FOXO3 in DLBCL remain unclear. Bioinformatics analysis was used to offer information FOXO3 expression and its expression for prognosis of DLBCL patients. The abundance of genes and proteins was evaluated using RT-qPCR and western blot. Cell proliferation and apoptosis was detected by CCK-8 and flow cytometry. The interactions among FOXO3, miR-34b, and HSPG2 were predicted by TransmiR and Starbase and validated using dual luciferase reporter assay, ChIP assay, and RIP assay. Our findings revealed that FOXO3 expression was abnormally declined in DLBCL cells. FOXO3 upregulation restrained cell proliferation and promoted cell apoptosis of DLBCL cells, while miR-34b inhibitor eliminated these influences. Similarly, miR-34b mimic suppressed malignant behaviors of DLBCL cells, which were abolished by HSPG2 overexpression. Mechanically, FOXO3 induced miR-34b expression through interacting with miR-34b promoter and HSPG2 was a targeted gene of miR-34b. FOXO3 attenuated the capability of cell proliferation and promoted cell apoptosis rate of DLBCL cells through affecting miR-34b/HSPG2 axis, therefore inhibiting DLBCL progression.

Arsenic disulfide promoted the demethylation of PTPL1 in diffuse large B cell lymphoma cells.

Promoter hypermethylation of the tumor suppressor gene is one of the well-studied causes of cancer development. The drugs that reverse the process by driving demethylation could be a candidate for anticancer therapy. This study was designed to investigate the effects of arsenic disulfide on PTPL1 methylation in diffuse large B cell lymphoma (DLBCL). We knocked down the expression of PTPL1 in two DLBCL cell lines (i.e., DB and SU-DHL-4 cells) using siRNA. Then the DLBCL proliferation was determined in the presence of PTPL1 knockdown. The methylation of PTPL1 in DLBCL cells was analyzed by methylation specific PCR (MSPCR). The effect of arsenic disulfide on the PTPL1 methylation was determined in DLBCL cell lines in the presence of different concentrations of arsenic disulfide (5 µM, 10 µM and 20 µM), respectively. To investigate the potential mechanism on the arsenic disulfide-mediated methylation, the mRNA expression of DNMT1, DNMT3B and MBD2 was determined. PTPL1 functioned as a tumor suppressor gene in DLBCL cells, which was featured by the fact that PTPL1 knockdown promoted the proliferation of DLBCL cells. PTPL1 was found hypermethylated in DLBCL cells. Arsenic disulfide promoted the PTPL1 demethylation in a dose-dependent manner, which was related to the inhibition of DNMTs and the increase of MBD2. Experimental evidence shows that PTPL1 functions as a tumor suppressor gene in DLBCL progression. PTPL1 hyper-methylation could be reversed by arsenic disulfide in a dose-dependent manner.

SENP1 knockdown potentiates the apoptosis, cell cycle arrest, and reduces cisplatin resistance of diffuse large B cell lymphoma cells via inducing ferroptosis.

Ferroptosis has been regarded as a critical event in the process of diffuse large B cell lymphoma (DLBCL). Sentrin-specific protease 1 (SENP1) has emerged as an oncogene in multiple human malignancies. The present work was to investigate the effects of SENP1 on the progression of DLBCL and the possible regulatory mechanism involving ferroptosis. SENP1 expression in DLBCL tissues, parental and cisplatin-resistant DLBCL cells were, respectively, tested by GEPIA database, RT-qPCR, and Western blot. Cell viability was estimated via CCK-8 assay. Flow cytometry analysis estimated cell apoptosis and cycle. Western blot examined the expression of apoptosis-, cell cycle-, and ferroptosis-associated proteins. TBARS assay and BODIPY 581/591 C11 probe measured lipid peroxidation. Related assay kit assessed total iron levels. CCK-8 and flow cytometry evaluated cisplatin resistance. SENP1 expression was raised in DLBCL tissues and cells. SENP1 knockdown reduced cell viability, boosted cell apoptosis, cell cycle arrest, and elevated cisplatin sensitivity in DLBCL. SENP1 depletion drove the ferroptosis of both parental and cisplatin-resistant DLBCL cells and ferroptosis inhibitor Fer-1 reversed the influences of SENP1 inhibition on cell viability, apoptosis, cell cycle, and cisplatin resistance in DLBCL. Anyway, SENP1 absence might facilitate ferroptosis to obstruct the development of DLBCL and cisplatin resistance.

Chidamide enhances T-cell-mediated anti-tumor immune function by inhibiting NOTCH1/NFATC1 signaling pathway in ABC-type diffuse large B-cell lymphoma

Chidamide (CS055/HBI-8000, tucidinostat) has shown promising effects in the clinical treatment of various hematologic tumors. Diffuse large B-cell lymphoma (DLBCL) has shown highly heterogeneous biological characteristics. There are complex mechanisms of the role of chidamide in DLBCL for in-depth study. It is essential to probe further into the mechanism of drug-tumor interactions as a guide to clinical application and to understand the occurrence and progression of DLBCL. In vitro and in vivo models were utilized to determine the effects of chidamide on signaling pathways involved in the DLBCL tumor microenvironment. The experimental results show that chidamide inhibited the proliferation of DLBCL cell lines in a dose- and time-dependent manner, and down-regulated the expression of NOTCH1 and NFATC1 in DLBCL cells as well as decreased the concentration of IL-10 in the supernatant. In addition, chidamide significantly lowered the expression of PD1 or TIM3 on CD4+T cells and CD8+T cells and elevated the levels of IL-2, IFN-γ, and TNF-α in the serum of animal models, which augmented the function of circulating T cells and tumor-infiltrating T cells and ultimately significantly repressed the growth of tumors. These findings prove that chidamide can effectively inhibit the cell activity of DLBCL cell lines by inhibiting the activation of NOTCH1 and NFATC1 signaling pathways. It can also improve the abnormal DLBCL microenvironment in which immune escape occurs, and inhibit immune escape. This study provides a new therapeutic idea for the exploration of individualized precision therapy for patients with malignant lymphoma.

NCAPD3 promotes diffuse large B-cell lymphoma progression through modulating SIRT1 expression in an H3K9 monomethylation-dependent manner

IntroductionCondensin, a family of structural maintenance of chromosome complexes, has been shown to regulate chromosome compaction and segregation during mitosis. NCAPD3, a HEAT-repeat subunit of condensin II, plays a dominant role in condensin-mediated chromosome dynamics but remains unexplored in lymphoma. ObjectivesThe study aims to unravel the molecular function and mechanism of NCAPD3 in diffuse large B-cell lymphoma (DLBCL). MethodsThe expression and clinical significance of NCAPD3 were assessed in public database and clinical specimens. Chromosome spreads, co-immunoprecipitation (co-IP), mass spectrometry (MS), and chromatin immunoprecipitation (ChIP) assays were conducted to untangle the role and mechanism of NCAPD3 in DLBCL. ResultsNCAPD3 was highly expressed in DLBCL, correlated with poor prognosis. NCAPD3 deficiency impeded cell proliferation, induced apoptosis and increased the chemosensitivity. Instead, NCAPD3 overexpression facilitated cell proliferation. In vivo experiments further indicated targeting NCAPD3 suppressed tumor growth. Noteworthily, NCAPD3 deficiency disturbed the mitosis, triggering the formation of aneuploids. To reveal the function of NCAPD3 in DLBCL, chromosome spreads were conducted, presenting that chromosomes became compact upon NCAPD3 overexpression, instead, loose, twisted and lacking axial rigidity upon NCAPD3 absence. Meanwhile, the classical transcription-activated marker, H3K4 trimethylation, was found globally upregulated after NCAPD3 knockout, suggesting that NCAPD3 might participate in chromatin remodeling and transcription regulation. MS revealed NCAPD3 could interact with transcription factor, TFII I. Further co-IP and ChIP assays verified NCAPD3 could be anchored at the promoter of SIRT1 by TFII I and then supported the transcription of SIRT1 via recognizing H3K9 monomethylation (H3K9me1) on SIRT1 promoter. Function reversion assay verified the oncogenic role of NCAPD3 in DLBCL was partially mediated by SIRT1. ConclusionThis study demonstrated that dysregulation of NCAPD3 could disturb chromosome compaction and segregation and regulate the transcription activity of SIRT1 in an H3K9me1-dependent manner, which provided novel insights into targeted strategy for DLBCL.

MYC-dependent MiR-7-5p regulated apoptosis and autophagy in diffuse large B cell lymphoma by targeting AMBRA1.

Diffuse large B-cell lymphoma (DLBCL) is the leading cause of mortality from invasive hematological malignancies worldwide. MicroRNA-7-5p (miR-7-5p) has been shown to be a tumor suppressor in several types of tumors. However, its role in DLBCL is not fully understood. This study explored the role of miR-7-5p in the progression of DLBCL and pursued the underlying mechanism. Quantitative real-time PCR and transfection of miRNA mimic and inhibitors were used to assess the effects of miR-7-5p on autophagy and apoptosis in SU-DHL-4 and SU-DHL-10 cells. Dual-luciferase reporter assay was used to identify target genes of miR-7-5p. Immunofluorescence, flow cytometry, and western blotting (WB) were performed to explore the underlying mechanism and downstream pathways of miR-7-5p and AMBRA1 in DLBCL cells. MiR-7-5p was upregulated in DLBCL cells. Luciferase reporter assays implicated AMBRA1 as a downstream target of miR-7-5p in DLBCL. WB and flow cytometry showed that an increase in miR-7-5p level and a decrease in AMBRA1 expression led to a decrease in autophagy and apoptosis-related protein expression. Furthermore, miR-7-5p prevented c-MYC dephosphorylation through AMBRA1 downregulation. On the contrary, c-MYC increased the expression of miR-7-5p, thereby establishing positive feedback on miR-7-5p transcription. The addition of hydroxychloroquine, an autophagy inhibitor, reduced autophagy and increased apoptosis in DLBCL cells. In vivo experiments further proved that the increase of miR-7-5p played a regulatory role in the expression of downstream AMBRA1 and c-MYC. These results demonstrate that c-MYC-dependent MiR-7-5p suppressed autophagy and apoptosis by targeting AMBRA1 in DLBCL cells. MiR-7-5p also suppressed autophagy and apoptosis by targeting AMBRA1 in DLBCL cells. Therefore, these data suggest that targeting miR-7-5p may be a promising strategy in DLBCL therapy.

Statement of Retraction: Knockdown of zinc finger protein 267 suppresses diffuse large B-cell lymphoma progression, metastasis, and cancer stem cell properties

Statement of Retraction: Knockdown of zinc finger protein 267 suppresses diffuse large B-cell lymphoma progression, metastasis, and cancer stem cell properties

Treatment of Diffuse Large B-Cell Lymphoma at the Moscow City Clinical Hospital No. 52: A Retrospective Clinical Trial Based on the Single-Center Experience and Analysis of Data from Its In-House Registry

Background. The term diffuse large B-cell lymphoma (DLBCL) defines a heterogeneous group of lymphatic tumors. DLBCL is the most frequent immunomorphological variant among aggressive non-Hodgkin lymphomas (NHLs) in adults. It accounts for 30–40 % of all NHLs. Long-term results of treating newly diagnosed DLBCL have not been reliably evidenced in healthcare practice and, therefore, require further study.
 Aim. To assess the efficacy of chemotherapy for newly diagnosed DLBCL in terms of the 5-year progression-free survival (PFS) based on the analysis of data from the in-house hematology service registry at the Moscow City Clinical Hospital No. 52.
 Materials & Methods. The study enrolled 156 patients with newly diagnosed DLBCL in the period from 2015 to 2022. The patients were 35–85 years of age (median 65 years).
 Results. First-line R-CHOP/R-miniCHOP chemotherapy was administered to 70 % of patients, 28 % of patients received R-DA-EPOCH, and 2 % were treated either with R-B or R-CVP. Complete response was achieved in 100 (65 %) patients: 75 out of them (75 %) received R-CHOP/R-miniCHOP, whereas 25 (25 %) received R-DA-EPOCH. Induction mortality was below 2.5 %. The 5-year PFS was 32 % with the survival median of 20 months. As confirmed by the multivariate analysis, the age over 60 years (p = 0.003), high IPI risk group (p = 0.015), advanced stage of the disease (p = 0.002), and non-GCB subtype of tumor (p = 0.045) can be regarded as independent predictors of early DLBCL progression.
 Conclusion. DLBCL is an aggressive B-cell lymphoma and one of the most frequent immunomorphological NHL variants in the clinical practice of the Moscow City Clinical Hospital No. 52. Despite the use of generally accepted standard immunochemotherapy regimens, the results attained by the present study illustrate unresolved challenges in chemotherapy for newly diagnosed DLBCL patients. By now, more effective first-line DLBCL therapy methods already exist, which are confirmed by the results of clinical trials. As it is sometimes impossible to further escalate immunochemotherapy for obvious reasons (age restrictions, health status, co-morbidities, etc.), a new promising strategy appears to be the personalized chemotherapy based on the study of genetic DLBCL profile of each particular patient.

Tissue-Matched IgH Gene Rearrangement of Circulating Tumor DNA Shows Significant Value in Predicting the Progression of Diffuse Large B Cell Lymphoma.

Evidence has demonstrated that monitoring of the variable, diversity, and joining gene segments (VDJ) rearrangement of the immunoglobulin (Ig) genes in the circulating tumor DNA (ctDNA) is of value in predicting the outcomes of diffuse large B cell lymphoma (DLBCL). In this study, we investigated the role of VDJ rearrangement proportion in ctDNA for predicting DLBCL progression. Patients diagnosed with newly diagnosed DLBCL were included in this study. The VDJ sequences of IgH were detected using next-generation sequencing (NGS) in formalin-fixed paraffin-embedded tissue and/or peripheral blood. The clonotype of the highest proportion in the peripheral blood was defined as the "dominant circulating clonotype," whilst the clonotype of the highest proportion in matched tissue that is detected in peripheral blood was defined as the "dominant tissue-matched clonotype." The decision tree, a machine learning-based methodology, was used to establish a progression-predicting model through a combination of "dominant tissue-matched clonotype" proportion or "dominant circulating clonotype" proportion, and the clinicopathological information, including age, sex, cell of origin, stage, international prognostic index, lactate dehydrogenase, number of extranodal involvements and β2-microglobulin. A total of 55 patients with eligible sequencing data were used for prognosis analysis, among which 36 patients had matched tissue samples. The concordance rate of "dominant circulating clonotype" and "dominant tissue-matched clonotype" was 19.44% (7/36). The decision tree model showed that the combination of extranodal involvement event and "dominant circulating clonotype" proportion (≥37%) had a clinical value in predicting the prognosis of DLBCL following combined chemotherapy (sensitivity, 0.63; specificity, 0.81; positive prediction value (PPV), 0.59; negative prediction value, 0.83; kappa value, 0.42). Noticeably, the combination of the "dominant tissue-matched clonotype" and extranodal involvement event showed a higher value in predicting the progression (sensitivity, 0.85; specificity, 0.78; PPV, 0.69; kappa value, 0.64). IgH proportion detected in the ctDNA samples traced from tissue samples has a high clinical value in predicting the progression of DLBCL.

Tumor-derived Exosomal ENO2 Modulates Polarization of Tumor-associated Macrophages through Reprogramming Glycolysis to Promote Progression of Diffuse Large B-cell Lymphoma.

Macrophages can be polarized into functional classically activated (M1) or alternatively activated (M2) phenotype. Tumor-associated macrophages (TAMs) mainly exhibit M2 phenotype. Previous works determined that up-regulation of enolase 2 (ENO2) in diffuse large B-cell lymphoma (DLBCL) cells can promote macrophages to an M2-like phenotype, thereby consequently promoting the progression of DLBCL. Exosomes are a subset of extracellular vesicles, carrying various bioactive molecules, mediate signals transduction and regulate immune cells. In our study, we investigated the role and related mechanisms of DLBCL-derived exosomal ENO2 in regulating macrophage polarization during DLBCL progression via bioinformatics analysis and a series of experiments. The results of bioinformatics analysis indicated that high expression of ENO2 was positively correlated with DLBCL progression and macrophages M2/M1 ratio. ENO2 protein levels were increased in the exosomes of the sera of DLBCL patients and DLBCL cells. Moreover, the DLBCL-derived exosomes were assimilated by macrophages and then regulated macrophage polarization. The results of in vitro and in vivo experiments showed that DLBCL-derived exosomal ENO2 modulated macrophages polarization (increased M2 phenotype and decreased M1 phenotype), thereby promoting DLBCL proliferation, migration, and invasion. We then revealed that the modulation of macrophages polarization by DLBCL-derived exosomal ENO2 depended on glycolysis and was promoted through GSK3β/β-catenin/c-Myc signaling pathway. These findings suggested that DLBCL-derived exosomal ENO2 accelerated glycolysis via GSK3β/β-catenin/c-Myc signaling pathway to ultimately promote macrophages to an M2-like phenotype, which can promote the proliferation, migration and invasion of DLBCL, suggesting that exosomal ENO2 may be a promising therapeutic target and prognostic biomarker for DLBCL.

M6A-related lncRNAs as potential biomarkers and the lncRNA ELFN1-AS1/miR-182-5p/BCL-2 regulatory axis in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid subtype. However, unsatisfactory survival outcomes remain a major challenge, and the underlying mechanisms are poorly understood. N6-methyladenosine (m6A), the most common internal modification of eukaryotic mRNA, participates in cancer pathogenesis. In this study, m6A-associated long non-coding RNAs (lncRNA) were retrieved from publicly available databases. Univariate, LASSO, and multivariate Cox regression analyses were performed to establish an m6A-associated lncRNA model specific to DLBCL. Kaplan-Meier curves, principal component analysis, functional enrichment analyses and nomographs were used to study the risk model. The underlying clinicopathological characteristics and drug sensitivity predictions against the model were identified. Risk modelling based on the three m6A-associated lncRNAs was an independent prognostic factor. By regrouping patients using our model-based method, we could differentiate patients more accurately for their response to immunotherapy. In addition, prospective compounds that can target DLBCL subtypes have been identified. The m6A-associated lncRNA risk-scoring model developed herein holds implications for DLBCL prognosis and clinical response prediction to immunotherapy. In addition, we used bioinformatic tools to identify and verify the ceRNA of the m6A-associated lncRNA ELFN1-AS1/miR-182-5p/BCL-2 regulatory axis. ELFN1-AS1 was highly expressed in DLBCL and DLBCL cell lines. ELFN1-AS1 inhibition significantly reduced the proliferation of DLBCL cells and promoted apoptosis. ABT-263 inhibits proliferation and promotes apoptosis in DLBCL cells. Invitro and invivo studies have shown that ABT-263 combined with si-ELFN1-AS1 can inhibit DLBCL progression.

Aberrant Translation Regulated By METTL1/WDR4-Mediated tRNA N 7-Methylguanosine Modification Drives DLBCL Progression

Cancer cells selectively promote translation of specific oncogenic transcripts to facilitate cancer survival and progression, but the underlying mechanisms are poorly understood. Here, we find that N 7 - methylguanosine (m 7G) tRNA modification and its methyltransferase complex components, METTL1 and WDR4, are significantly upregulated in diffuse large B-cell lymphoma (DLBCL) and associated with poor prognosis. We further reveal the critical role of METTL1/WDR4 in promoting DLBCL cell survival and progression using loss- and gain-of-function assays in vivo. Mechanistically, m 7G tRNA modification selectively regulates the translation of oncogenic transcripts, in m 7G-tRNA-decoded codon-frequency-dependent mechanisms. Moreover, using overexpression and knockout mouse models, we demonstrate the crucial oncogenic function of Mettl1-mediated m 7G tRNA modification in promoting DLBCL tumorigenesis and progression in vivo. Our study uncovers the important physiological function and mechanism of METTL1-mediated m 7G tRNA modification in the regulation of oncogenic mRNA translation and cancer progression.

Lymphoma-Derived IL-10 Is a Key Immunomodulatory Factor at the Tumor Microenvironment of Activated B-Cell Diffuse Large B-Cell Lymphoma and Influences In Vivo Responses to Immunotherapy

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of aggressive non-Hodgkin's lymphoma with ~40% of patients experiencing refractory or relapsed disease to R-CHOP immunochemotherapy. These higher risk patients often exhibit gene expression profiles that resemble a late germinal center-derived activated B-cell (ABC) phenotype, where diverse specific mutations in genes along BCR and TLR signaling pathways converge into constitutive activation of the NF-κB pathway. Identification of factors that contribute to the survival of malignant ABC-DLBCL cells is crucial for designing specific interventions. In this setting, previous in vitro studies in DLBCL cell lines have evidenced that IL-10 is a direct target of NF-κB signaling that sustains lymphoma cell survival by an auto-stimulatory loop via STAT3 signaling. However, the action of IL-10 could be more complex within the lymphoma microenvironment, as IL-10 can have both stimulatory and inhibitory effects depending on the cellular source of IL-10, the timing of its secretion, and the type of immune cells that receives signals via IL-10 receptor (IL-10R). To explore in vivo the relevance of lymphoma-derived IL-10, we have generated a novel quintuple transgenic mouse model that we call pBIC10, after crossing our previous ABC-DLBCL-like pBIC mice carrying constitutive NF-κB signaling (Pascual et al., Blood 2019) to mice with a floxed Il10 gene for conditional knock-out of IL-10 specifically in malignant ABC-DLBCL cells. As expected, increased intracellular IL-10 signaling was detected in primary murine DLBCL cells from pBIC mice but not in IL-10 deficient pBIC10 mice, which promoted lymphoma cell survival ex vivo as revealed by perturbation studies of the IL-10/JAK1/STAT3 autocrine loop with blocking monoclonal antibodies or selected inhibitors. However, in vivo genetic deletion of lymphoma-derived IL-10 did not offer improved overall survival but, on the contrary, notably accelerated DLBCL progression in pBIC10 mice (Figure 1A, left). A comprehensive integration of multiparametric flow cytometry and transcriptomic analyses using bulk and single-cell RNA-seq of lymphoma cells and the tumor microenvironment coupled with BCR- and TCR-seq, identified lymphoma-derived IL-10 as a key immunomodulator of the DLBCL microenvironment, including unexpected protective paracrine functions in the progression of DLBCL. By comparing IL-10-proficient (pBIC) and -deficient (pBIC10) murine DLBCL models, we observed a multifaceted role of IL-10 produced by DLBCL cells, enabling immune-effector processes (facilitating immune chemotaxis, antigen presentation, and IFNγ responses) that yielded higher percentages of long-lived stem-like CD8 +TCF-1 +PD-1 lo/int T cells and dendritic cells, while restraining PD-1/LAG3-driven T-cell exhaustion and reducing immunosuppression by CD4 +CD25 +FOXP3 + regulatory T (Treg) cells. Thus, depletion of lymphoma-derived IL-10 decreased T-cell cytotoxicity and increased Treg-driven immunosuppression, which collectively accelerated lymphoma development. Consequently, T-cell targeted immune-checkpoint therapy (with anti-PD-1 antibodies) was effective only in IL-10-proficient DLBCL but not in IL-10-deficient pBIC10 mice (Figure 1A, right). Further comparative studies between pBIC and pBIC10 mice showed that tumor vascularization and expression of B-cell calcium channels, which have been previously associated with sensitivity to rituximab, were counteracted by lymphoma-derived IL-10. Accordingly, anti-CD20-based targeting of lymphoma cells (with rituximab surrogate antibodies) showed best in vivo responses in IL-10-deficient DLBCL mice with respect to pBIC mice (Figure 1A, right). Consistent with this preclinical data, analyses of transcriptional IL-10-associated hallmarks to stratify DLBCL patients (Schmitz et al., NEJM 2018) identified better clinical responses to R-CHOP in patients with pBIC10-like DLBCL phenotype (Figure 1B). Collectively, our study provides new functional and mechanistic insights into the role of lymphoma-derived IL-10 in the pathogenesis of DLBCL and the restraint of an immunosuppressed tumor microenvironment, which holds predictive potential as a biomarker for immunotherapy responses in DLBCL.

NCAPD3 Prompts Diffuse Large B-Cell Lymphoma Progression through Modulating SIRT1 Expression in H3K9 Monomethylation-Dependent Manner

Chromosomal instability, including number and structure abnormality, is the critical molecular basis in lymphoid oncogenesis. Condensin, a family of structural maintenance of chromosome complexes, has been shown to regulate chromosome compaction and segregation during mitosis. NCAPD3, a HEAT-repeat subunit of condensin II, plays a dominant role in condensin-mediated chromosome dynamics, whose abnormalities are related to tumorigenesis and developmental disorders but remain unexplored in lymphoma. This study aims to unravel the expression, molecular function and mechanism of NCAPD3 in diffuse large B-cell lymphoma (DLBCL). We first elucidated the expression of NCAPD3 in DLBCL. Lymph node tissues from 70 DLBCL patients and 35 reactive hyperplasia cases were collected with informed consent approved by the Medical Ethical Committee of Shandong Provincial Hospital. Higher NCAPD3 expression was identified in DLBCL tissues and correlated with advanced Ann Arbor stage and higher IPI score. The survival analysis presented patients with higher NCAPD3 suffered shorter survival (P=0.045). Meanwhile, upregulation of NCAPD3 was validated in DLBCL cells compared with normal CD19+ B-cell. We further explored the biological functions of NCAPD3 in DLBCL and constructed NCAPD3-knockdown, knockout and overexpression models. Deficiency of NCAPD3 impeded cell proliferation, induced apoptosis and increased the sensitivity to doxorubicin and ibrutinib. Instead, NCAPD3 overexpression facilitated cell proliferation and therapeutic resistance. In vivo experiments further indicated that xenograft DLBCL mice with deficient NCAPD3 presented delayed tumor growth. These suggested NCAPD3 promoted DLBCL progression. Strikingly, we found knocking down NCAPD3 disturbed the mitosis, triggering the formation of aneuploidy, due to its function in chromatin structure and dynamics. Hence, we further portrayed the transformation of chromosome architecture under different NCAPD3 expression. Chromosome spreads exhibited chromosomes became slim and elongated upon NCAPD3 overexpression, instead, NCAPD3 knockout triggered short and highly-curled transformation with the global upregulation of H3K4me3, an indication of transcriptional activation and chromatin decompaction (Figure 1). This suggested NCAPD3 could modulate transcriptional activity. Therewith, we investigated how NCAPD3 regulated gene transcription. We examined the interacting proteins of NCAPD3 by mass spectrometry and Clusters of Orthologous Groups analysis revealed they were mainly enriched in RNA processing and modification, chromatin dynamics and cell cycle control. Of them, transcription factor, GTF2I, may be responsible for NCAPD3-mediated transcription regulation. Coimmunoprecipitation (co-IP) verified their interaction and chromatin immunoprecipitation (ChIP) presented they were both located on the promoter of target gene, SIRT1. We further examined SIRT1 expression after regulating NCAPD3 expression, showing a significantly positive correlation. This suggested NCAPD3 could support SIRT1 transcription via being anchored by GTF2I on the promoter of SIRT1. We further elucidated the potential mechanism how NCAPD3 regulated SIRT1 expression. Previous study found that NCAPD3 could interact with histone methylation markers in mammalian genome, thus, we explored whether NCAPD3 could modulate the SIRT1 expression via recognizing the histone methylation marker on SIRT1 promoter. Co-IP assay identified the interaction between NCAPD3 and monomethylation of lysine residue K9 of histone H3 (H3K9me1), an active promoter tag, in DLBCL and ChIP assay verified the location of H3k9me1 on SIRT1 promoter. When NCAPD3 downregulated, H3K9me1 on SIRT promoter diminished, ruining the transcription of SIRT. To clarify whether the oncogenic effects of NCAPD3 was mediated by SIRT1, we conducted functional reversion assays, showing regulating SIRT1 partially reversed the effects of NCAPD3. The above suggested NCAPD3 prompted DLBCL progression through modulating SIRT expression in H3k9me1-mediated manner. In conclusion, this study unraveled that dysregulation of NCAPD3 could disturb chromosome compaction and segregation, induce genome instability and alter the transcription of SIRT1 via modulating H3k9 monomethylation, which provided novel insights into NCAPD3-based targeting strategy for DLBCL.

Extrachromosomal Circular DNA Drives the Progression of DLBCL through Activating the Sting Pathway

Objective Chromosome instability (CIN) as a hallmark of cancer is correlated with the prognosis of patients with diffuse large B cell lymphoma (DLBCL). However, little is known about how it drives the tumorigenesis and progression. DNA damage affects CIN and accumulated cytosolic DNA which activates the DNA sensing machinery cGAS-STING pathway in a sequence-independent manner. Extrachromosomal circular DNA (eccDNA), a type of circular DNA that derived from genomic DNA is independent of chromosomes. Existing widely in nature, eccDNA could be involved in tumorigenesis, heterogeneity and drug resistance. The purpose of this study is to reveal the expression profile of eccDNA in DLBCL, and to explore how eccDNA promotes the occurrence and development of the disease through the cGAS-independent STING pathway. Methods The expression profile of eccDNA in DLBCL was detected through multi-omics sequencing analysis including circular DNA sequencing (circle-seq), single-cell sequencing (scRNA-seq), whole exome sequencing (WES) and atomic force microscopy. CCK8, scRNA-seq, etc. were used to reveal the activation of eccDNA on the STING pathway to promote cell proliferation. In vivo and in vitro models were treated with chemotherapeutic drugs to verify the hypothesis that DNA damage induces the production of eccDNA, thereby activating the cGAS-independent STING pathway. GEO databases were applied to verify the prognosis of eccDNA-related gene sets, animal models to explore the anti-tumor effect of DNA damage therapy combined with STING inhibitors. Results Through the integration of multi-omics analysis results of 18 DLBCL cell lines, we divided DLBCL cells into two groups with high (H group) or low (L group) eccDNA abundance separately and confirmed that cells with high wGII were accompanied by abundant eccDNA. It suggested the potential roles of eccDNA in prognostic evaluation. Subsequent research revealed that eccDNA was upregulated in patients with DLBCL especially in R/R DLBCL. Pseudo-time analysis indicated that cells developed in two different differentiation directions. GSEA analysis demonstrated that high abundance of eccDNA contributed to the cell proliferative potential. To verify the role of eccDNA in cell proliferation, eccDNA extracted from H group cells were transfected into L group cells. As a result, cells transfected with eccDNA showed a significantly higher rate of propagation. These results confirmed that eccDNA had the promotional effects on proliferation DLBCL cells. GEO databases (GSE31312, GSE10846 and GSE87371) were applied to verify the prognosis of eccDNA-related gene sets. Gens that upregulated in H group was selected to construct a panel and overall survival analysis showed that patients with high levels of the panel had a worse prognosis. DNA damage repair mechanism is a key component to maintain the stability of genome. Numerous studies have suggested that DNA damage repair is conducive to the generation of eccDNA. Here, we found that cells with serious DNA damage degree were accompanied with increased γH2AX levels. EccDNA was induced with the treatment of DNA damage agents (cisplatin, doxorubicin, irinotecan and olaparib). Meanwhile, the levels of p-STING and downstream proteins were significantly upregulated. This phenomenon was also confirmed with immunodeficient and immunocompetent mice. H-151, the inhibitor of STING showed effective inhibitory effect on DLBCL through in vitro and in vivo assay. cells with high eccDNA abundance, and the results were further confirmed with scRNA-seq analysis. The combinational effects of STING inhibitor with DNA damage agents were observed. These results suggested that cells with high eccDNA abundance were more sensitive to the inhibition of STING pathway and the combined use of chemotherapy drugs and STING inhibitors can significantly enhance the anti-tumor effect. Conclusion DLBCL is a highly heterogeneous aggressive non-Hodgkin's lymphoma. STING plays a double-edged role in tumor development and immune regulation. However, its role in DLBCL and related mechanisms have not been reported so far. Our study reveals the expression, function and clinical significance of eccDNA in DLBCL for the first time, clarifies the mechanism of eccDNA-mediated STING pathway activation in disease progression and drug resistance, and provides a new theoretical basis for clinical treatment and prognosis evaluation of DLBCL.

Investigating the Drug Resistance Mechanisms of Refractory Diffuse Large B-Cell Lymphoma Based on Proteomic Research

Diffuse large B-cell lymphoma (DLBCL) is the most common histological subtype of non-Hodgkin's lymphoma, more than half of the patients of which can achieve complete remission or even be cured with multidrug immunochemotherapy regimen(R-CHOP). However, for patients with relapsed or refractory DLBCL, there is no effective treatment, while the unfavorable prognosis is related to the primary and acquired resistance to existing therapies. Proteomics is the study of the presence and activity of all proteins in cells. In exploring the mechanisms of DLBCL progression, proteomic technology can identify and quantify changed proteins in DLBCL, reveal the relationship between changes in protein expression levels and different stages of lymphoma occurrence and development, and thus provide information on variation in related proteins and protein-related signaling molecules. Based on proteomic technology research, this study is expected to discover new molecular mechanisms, further clarify resistance mechanisms, and find new therapeutic targets for DLBCL, providing more powerful weapons for better prognosis. We first collected paired clinical patient samples that met the experimental requirements and selected biopsy specimens from 3 patients who relapsed within 1 year after systemic treatment, including biopsy specimens at initial diagnosis and relapse. Next, paraffin sections and HE-stains were performed on the samples, and target areas were micro dissected using a laser microscope. The dissected samples were collected in specific EP tubes, lysed ultrasonically by adding lysis buffer to each group of samples, and the protein concentration of the supernatant was determined after centrifugation. Equal amounts of protein from each sample were digested with trypsin and the final concentration was controlled. Analysis was then performed using the EASY-nLC 1200 ultra-high performance liquid phase system and the Q Exactive TM HF-X mass spectrometer. Finally, bioinformatic analysis was performed to analyze differential proteins and find relevant drug targets or markers. The number of identified peptides was 36,107, the number of identified proteins was 6,338, and the number of quantifiable proteins was 6,165. The top five differentially upregulated proteins in the volcano plot of differential proteins were HOPX, TSNAX, PTMA, FTH1, and IFI44, respectively; and the top five downregulated differential proteins were KRT9, KRT1, KRT10, KRT2, and PIP, respectively. Through enrichment analysis of differential proteins, we noticed the ferroptosis pathway as a key pathway, and found that FTH1, TF, and GSH were involved in iron death-related processes, while FTH1 was one of the top five upregulated differential proteins. Iron death is an iron-dependent, non-apoptotic form of cell death discovered in recent years, activation of which can lead to tumor cell death and inhibit tumor growth. Increased expression of the iron-binding subunit FTH1 can suppress activation of iron death and reduce iron death in tumor cells. This suggests that by developing targeted drugs that act on increasing FTH1 expression or iron death inducers which are capable of slowing down the process, it may be possible to address the problem of traditional chemo-resistance in DLBCL and provide new directions for overcoming DLBCL resistance.

Long non-coding RNA SNHG17 may function as a competitive endogenous RNA in diffuse large B-cell lymphoma progression by sponging miR-34a-5p.

We investigated the functional mechanism of long non-coding small nucleolar host gene 17 (SNHG17) in diffuse large B-cell lymphoma (DLBCL). lncRNAs related to the prognosis of patients with DLBCL were screened to analyze long non-coding small nucleolar host gene 17 (SNHG17) expression in DLBCL and normal tissues, and a nomogram established for predicting DLBCL prognosis. SNHG17 expression in B-cell lymphoma cells was detected using qPCR. The effects of SNHG17 with/without doxorubicin on the proliferation and apoptosis of DoHH2 and Daudi were detected. The effects of combined SNHG17 and doxorubicin were analyzed. The regulatory function of SNHG17 in DLBCL was investigated using a mouse tumor xenotransplantation model. RNA sequencing was used to analyze the signaling pathways involved in SNHG17 knockdown in B-cell lymphoma cell lines. The target relationships among SNHG17, microRNA, and downstream mRNA biomolecules were detected. A higher SNHG17 level predicted a lower survival rate. SNHG17 was highly expressed in DLBCL patient tissues and cell lines. We established a prognostic model containing SNHG17 expression, which could effectively predict the overall survival rate of DLBCL patients. SNHG17 knockdown inhibited the proliferation and induced the apoptosis of B-cell lymphoma cells, and the combination of SNHG17 and doxorubicin had a synergistic effect. SNHG17, miR-34a-5p, and ZESTE gene enhancer homolog 2 (EZH2) had common hypothetical binding sites, and the luciferase reporter assay verified that miR-34a-5p was the direct target of SNHG17, and EZH2 was the direct target of miR-34a-5p. The carcinogenic function of SNHG17 in the proliferation and apoptosis of DLBCL cells was partially reversed by a miR-34a-5p inhibitor. SNHG17 increases EZH2 levels by inhibiting miR-34a-5p. Our findings indicate SNHG17 as critical for promoting DLBCL progression by regulating the EZH2 signaling pathway and sponging miR-34a-5p. These findings provide a new prognostic marker and therapeutic target for the prognosis and treatment of DLBCL.

Sex differences in abdominal visceral fat composition and metabolism as predictors of DLBCL prognosis: A retrospective cohort study

PurposeWe aimed to determine if sex differences in abdominal visceral fat composition and metabolism can help predict the prognosis of diffuse large B-cell lymphoma (DLBCL) patients. MethodsThis retrospective cohort study included 117 DLBCL patients. The area and metabolic activity of subcutaneous adipose tissue and visceral adipose tissue were measured using CT and PET imaging. Kaplan–Meier survival analysis was employed to evaluate the effect of these parameters on progression-free survival. Multivariate Cox proportional hazard regression models were used to determine the effects of relative visceral fat area (rVFA) on sex-specific survival. ResultsFemales with an rVFA greater than the optimal threshold of 35 % and a visceral-to-subcutaneous adipose tissue ratio (V/S) >3.24 had worse progression-free survival (p = 0.01, 0.001, respectively). No rVFA or V/S were identified in significantly stratified males with DLBCL (p = 0.249 and 0.895, respectively). Combining the changes in rVFA and V/S identified a subgroup of females with high rVFA and V/S values and exceptionally poor outcomes. The rVFA was a significant predictor of DLBCL progression in females alone. ConclusionOnce female DLBCL patients accumulate fat over the tolerable range in the visceral area, they might be at an increased risk of progression (hazard ratio, 3.87; 95 % CI, 1.81–12.69, p = 0.02).Sex differences in visceral fat composition and metabolism may provide a new risk stratification system for patients with DLBCL.

MiR-525-5p inhibits diffuse large B cell lymphoma progression via the Myd88/NF-κB signaling pathway.

Diffuse large B-cell lymphoma (DLBCL) is a B-cell lymphoma with a high degree of aggressiveness. Recently, evidence has shown that miR-525-5p is decreased in DLBCL, suggesting its possible involvement in tumor progression. In this study, miR-525-5p suppressed proliferation, invasion and clonogenicity, and increased apoptosis of U2932 cells, whereas miR-525-5p silencing enhanced tumor cell growth. Next, miR-525-5p targets the 3'-UTR of Myd88, and Myd88 protein was increased in lymphoma tissues. Similar to the miR-525-5p mimic, Myd88 siRNA suppressed proliferation, invasion, and clonogenicity, and enhanced apoptosis of U2932 cells. We observed that Myd88 reversed the inhibitory effect of miR-525-5p on tumor cell growth by transfecting cells with miR-525-5p mimics alone or together with Myd88 overexpression vector. In addition, in vivo studies have shown that compared to the control group, U2932 cells with upregulated miR-525-5p expression have a reduced ability to induce tumor formation. In conclusion, our results demonstrate that miR-525-5p inhibits the progression of DLBCL through the Myd88/NF-κB pathway, which largely fills the gap of previous studies, and our results may provide a new reference for the targeted treatment of DLBCL.

Circ_SPEF2 Regulates the Balance of Treg Cells by Regulating miR-16-5p/BACH2 in Lymphoma and Participates in the Immune Response.

This study aims to explore the potential mechanism of action of the newly discovered hsa_circ_0128899 (circSPEF2) in diffuse large B-cell lymphoma (DLBCL). circSPEF2, miR-16-5p and BTB and CNC homologue 2 (BACH2) expression patterns in DLBCL patients and cell lines were studied by RT-qPCR. The biological function of circSPEF2 in vitro and in vivo was investigated by function acquisition experiments. The proliferation activity of lymphoma cells was detected by MTT. Bax, Caspase-3, and Bcl-2 were determined by Western Blot. Apoptosis and the ratio of CD4 to Treg of immune cells in the co-culture system were analyzed by flow cytometry. The mechanism of action of circSPEF2 in DLBCL progression was further investigated by RIP and dual luciferase reporter experiments. circSPEF2 was a circRNA with abnormally down-regulated expression in DLBCL. Increasing circSPEF2 expression inhibited the proliferative activity and induced apoptosis of lymphoma cells in vitro and in vivo, as well as increased CD4+T cells and decreased Treg cell proportion of immune cells in the tumor microenvironment. Mechanically, circSPEF2 was bound to miR-16-5p expression, while BACH2 was targeted by miR-16-5p. circSPEF2 overexpression-mediated effects on lymphoma progression were reversible by upregulating miR-16-5p or downregulating BACH2. circSPEF2 can influence DLBCL progression by managing cellular proliferation and apoptosis and the proportion of immune cells Treg and CD4 through the miR-16-5p/BACH2 axis.