Tumorigenesis Of Diffuse Large B-cell Lymphoma Research Articles

LncRNA CHROMR/miR-27b-3p/MET axis promotes the proliferation, invasion, and contributes to rituximab resistance in diffuse large B-cell lymphoma

Long non-coding RNAs (LncRNAs) could regulate chemoresistance through sponging microRNAs (miRNAs) and sequestering RNA binding proteins. However, the mechanism of lncRNAs in rituximab resistance in diffuse large B-cell lymphoma (DLBCL) is largely unknown. Here, we investigated the functions and molecular mechanisms of lncRNA CHROMR in DLBCL tumorigenesis and chemoresistance. LncRNA CHROMR is highly expressed in DLBCL tissues and cells. We examined the oncogenic functions of lncRNA CHROMR in DLBCL by a panel of gain-or-loss-of-function assays and in vitro experiments. LncRNA CHROMR suppression promotes CD20 transcription in DLBCL cells and inhibits rituximab resistance. RNA immunoprecipitation, RNA pull-down and dual luciferase reporter assay reveal that lncRNA CHROMR sponges with miR-27b-3p to regulate mesenchymal-epithelial transition factor (MET) levels and Akt signaling in DLBCL cells. Targeting the lncRNA CHROMR/miR-27b-3p/MET axis reduces DLBCL tumorigenesis. Altogether, these findings provide a new regulatory model, lncRNA CHROMR/miR-27b-3p/MET, which can serve as a potential therapeutic target for DLBCL.

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.

Targeting YTHDF2 inhibits tumorigenesis of diffuse large B-cell lymphoma through ACER2-mediated ceramide catabolism

IntroductionEpigenetic alterations play crucial roles in diffuse large B-cell lymphoma (DLBCL). Disturbances in lipid metabolism contribute to tumor progression. However, studies in epigenetics, especially its critical regulator YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), on lipid metabolism regulation in DLBCL are unidentified. ObjectivesElucidate the prognostic value and biological functions of YTHDF2 in DLBCL and illuminate the underlying epigenetic regulation mechanism of lipid metabolism by YTHDF2 in DLBCL development. MethodsThe expression and clinical value of YTHDF2 in DLBCL were performed in public databases and clinical specimens. The biological functions of YTHDF2 in DLBCL were determined in vivo and in vitro through overexpression and CRISPR/Cas9-mediated knockout of YTHDF2. RNA sequencing, lipidomics, methylated RNA immunoprecipitation sequencing, RNA immunoprecipitation-qPCR, luciferase activity assay, and RNA stability experiments were used to explore the potential mechanism by which YTHDF2 contributed to DLBCL progression. ResultsYTHDF2 was highly expressed in DLBCL, and related to poor prognosis. YTHDF2 overexpression exerted a tumor-promoting effect in DLBCL, and knockdown of YTHDF2 restricted DLBCL cell proliferation, arrested cell cycle in the G2/M phase, facilitated apoptosis, and enhanced drug sensitivity to ibrutinib and venetoclax. In addition, YTHDF2 knockout drastically suppressed tumor growth in xenograft DLBCL models. Furthermore, a regulatory role of YTHDF2 in ceramide metabolism was identified in DLBCL cells. Exogenous ceramide effectively inhibited the malignant phenotype of DLBCL cells in vitro. The binding of YTHDF2 to m6A sites on alkaline ceramidase 2 (ACER2) mRNA promoted its stability and expression. Enhanced ACER2 expression hydrolyzed ceramides, disrupting the balance between ceramide and sphingosine-1-phosphate (S1P), activating the ERK and PI3K/AKT pathways, and leading to DLBCL tumorigenesis. ConclusionThis study demonstrated that YTHDF2 contributed to the progression of DLBCL by regulating ACER2-mediated ceramide metabolism in an m6A-dependent manner, providing novel insights into targeted therapies.

Alpha‐ketoglutarate Suppresses Tumor Growth of Diffuse Large B‐cell Lymphoma by Inducing Ferroptosis

Background: Metabolic reprogramming is one of the vital characteristics of cancers. Glutamine metabolism is an essential metabolic pathway in tumorigenesis, which provides a ready carbon and nitrogen source to support tumor biosynthesis, energy metabolism, and intracellular homeostasis. As a type of lymphoma with heterogeneity, diffuse large B-cell lymphoma (DLBCL) is characterized by severe metabolic vulnerability represents. Given the importance of glutamine metabolism in human cancer, revealing the characteristics of glutamine metabolism in DLBCL is expected to provide new pathogenesis and effective treatment strategies for patients. Methods: Peripheral blood serum of 120 DLBCL patients and 60 healthy donors were collected for untargeted metabolomics sequencing, followed by metabolic characteristics analysis. Subsequently, the biological functions and mechanisms of α-ketoglutarate (α-KG) were explored by RNA sequencing. Finally, ROS detection, ATP detection, and lipid peroxidation were used to verify the mechanism of α-KG-induced ferroptosis. Results: Untargeted metabolomics profiling revealed that the metabolic characteristics of DLBCL patients were significantly different from healthy controls. Among the differentially expressed metabolic pathways, glutamine metabolism accounted for the highest weight, suggesting the importance of glutamine metabolism in the tumorigenesis of DLBCL (Figure 1A). Notably, glutamate, glutamine, and α-KG were the critical metabolites in glutamine metabolism. Clinical data analysis identified that high glutamine concentrations and low decreased α-KG were associated with poor prognosis in DLBCL patients. Subsequently, dimethyl α-ketoglutarate (DM-αKG) was used to reverse glutamine metabolism and increase α-KG concentration. In vitro studies showed that DM-αKG treatment significantly inhibited cell proliferation of DLBCL cells both in vitro and in vivo (Figure 1B). In addition, DM-αKG induced non-apoptotic cell death phenotypes, represented by cell membrane swelling and LDH release. To further explore the functional mechanisms of α-KG in DLBCL, we performed RNA-sequencing in DM-αKG-treated DLBCL cells. As shown in enrichment analysis, DM-αKG treatment showed apparent dysfunction in the hypoxia-inducible factor pathway, oxidative stress response, and ferroptosis (Figure 1C). In particular, DM-αKG treatment promoted ROS release and lipid peroxidation, followed by impaired ATP production and decreased mitochondrial pathway expression. Moreover, differentially expressed genes analysis identified an increased expression of TP53 in the ferroptosis pathway, indicating the importance of TP53 in α-KG-induced ferroptosis (Figure 1D). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Metabolism No conflicts of interests pertinent to the abstract.

Upregulation of USP25 promotes progression of human diffuse large B-cell lymphoma through blocking the ubiquitinated degradation of MDM2

Diffuse large B cell lymphoma (DLBCL) is a type of cancer that originates from abnormal B cells in the lymph nodes or other lymphoid tissues. Dysfunction of deubiquitinases is frequently implicated in malignant progression. This study planned to uncover the biological roles of deubiquitinase USP25 during DLBCL tumorigenesis. In this study we identified USP25 as a novel oncogene which is frequently upregulated in DLBCL and associated with dismal prognosis of patients. Moreover, USP25 silencing was found to inhibit DLBCL growth, migration, while induced an obvious increase in apoptosis in vitro. Meanwhile, USP25 could promote DLBCL tumour growth and lung metastasis in vivo. Mechanistically, the co-immunoprecipitation test provided a mechanistic explanation, showing that USP25 directly interacted with murine double minute 2 (MDM2) and MDM2 protein stability was maintained by USP25 mediated deubiquitination. In addition, overexpression of USP25 with C178A mutation failed to decrease its modification on MDM2 stability. Further mechanism-of-action studies demonstrated that USP25 promoted DLBCL progression via stabilizing MDM2 and consequently decreasing p53 expression. In addition, further analysis showed that the oncogenic effect of USP25 was relied on MDM2-p53 signaling pathway-mediated cell-cycle accelerating. Collective, USP25 was shown to be an important upstream regulator of the MDM2-p53 signaling pathway in DLBCL, and it has the potential to be employed as a novel target gene in the development of new therapeutic applications.

NAT10 Promotes Tumorigenesis of Diffuse Large B-Cell Lymphoma By ac4C-Dependent Acetylation of SLC30A9

Introduction: N4-acetylcytidine (ac4C) is a chemical and conserved modification, which plays an essential role in the stability and regulation of mRNA translation. However, the role of RNA ac4C modification in diffuse large B-cell lymphoma (DLBCL) remains unclear. N-acetyltransferase 10 (NAT10), the key enzyme of ac4C modification, participates in various cellular processes. Herein, we explored the functional significance and regulatory mechanism of NAT10-mediated mRNA ac4C modification in the tumorigenesis of DLBCL, which is expected to propose novel therapeutic strategy. Methods: Lymph node biopsies from 104 newly diagnosed DLBCL patients and 20 reactive lymphoid hyperplasia were collected with informed consents. The biological function of NAT10 was explored through NAT10 knockdown (NAT10 KD) and CRISPR/Cas9 mediated genomic deletion (NAT10 KO). RNA sequencing and acRIP-seq were performed to detect the dysregulated RNA in DLBCL. LC-MS/MS, RIP assay and RNA stability assay were performed to investigate the functional mechanism of NAT10. SCID beige mice were subcutaneously injected into LY1 cells to establish DLBCL xenograft model. Animal experiments were performed in accordance with the principles of the Institutional Animal Care. Results: We first evaluated the expression level of NAT10 in DLBCL as well as analyzed the correlation between NAT10 expression and clinicopathologic characteristics. Upregulation of NAT10 mRNA were identified in DLBCL. Survival analysis revealed that high NAT10 expression was associated with poor prognosis of DLBCL patients. In addition, high expression levels of NAT10 protein were validated in DLBCL cell lines and newly diagnosed DLBCL patients. To verify the biological functions of NAT10, we deleted NAT10 expression by CRISPR/Cas9 mediated genomic-editing system. Deletion of NAT10 induced cell cycle attest in G0/G1 phase. Knockdown of NAT10 resulted in similar anti-lymphoma effects. We further investigated the activities of NAT10 inhibition in DLBCL xenograft mice model. The results revealed that deletion of NAT10 significantly suppressed proliferation of DLBCL cells. RNA sequencing and acRIP-seq of LY1 cells with NAT10 knockout were performed to explore the mechanism of NAT10 in DLBCL. Knockout of NAT10 reduced the enrichment of ac4C peaks and significantly down-regulated the expression of Solute Carrier Family 30 Member 9 (SLC30A9) mRNA levels through integrative analysis of the sequencing results (Figure 1). RIP-qPCR further verified the combination of SLC30A9 and NAT10. Notably, SLC30A9 was downregulated upon NAT10 knockout, and positively correlated with NAT10 expression, suggesting that SLC30A9 may act as a feasible downstream target of NAT10 in DLBCL. Given that NAT10 is an RNA acetyltransferase, we next explored whether NAT10-mediated expression of SLC30A9 was dependent on ac4C modification. Lower global ac4C level was showed in DLBCL cells with NAT10 knockout. Analysis of SLC30A9 mRNA sequence found that the vast majority of ac4C peaks were distributed in the protein-coding region. Moreover, the NAT10 mut plasmid without a functional RNA helicase domain (K290A) or acetyltransferase domain (G641E) was constructed to explore the regulatory mechanism of NAT10 on SLC30A9 expression. Overexpression of NAT10 wild-type (WT) but not NAT10 mutant induced a significant increase in SLC30A9 expression levels. Furthermore, overexpression of NAT10 WT significantly increased the half-life of SLC30A9 mRNA (Figure 2). The above results suggested that SLC30A9 was regulated by NAT10 in an ac4C-dependent manner. Conclusions: Taken together, this study provides preclinical evidence for the significance of NAT10 in lymphomagenesis and highlights the regulatory mechanism of NAT10-mediated the expression of SLC30A9 in an ac4C-dependent manner in DLBCL. Further investigations on the specific NAT10 inhibitors in DLBCL will outline a promising treatment approach to target this newly identified oncogene in DLBCL therapy. Keywords: Diffuse large B-cell lymphoma (DLBCL), N-acetyltransferase 10 (NAT10), Solute Carrier Family 30 Member 9 (SLC30A9), CRISPR/Cas9 Disclosure: No relevant conflicts of interest to declare. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Viral Agents as Potential Drivers of Diffuse Large B-Cell Lymphoma Tumorigenesis.

Among numerous causative agents recognized as oncogenic drivers, 13% of total cancer cases occur as a result of viral infections. The intricacy and diversity of carcinogenic processes, however, raise significant concerns about the mechanistic function of viruses in cancer. All tumor-associated viruses have been shown to encode viral oncogenes with a potential for cell transformation and the development of malignancies, including diffuse large B-cell lymphoma (DLBCL). Given the difficulties in identifying single mechanistic explanations, it is necessary to combine ideas from systems biology and viral evolution to comprehend the processes driving viral cancer. The potential for more efficient and acceptable therapies lies in targeted medicines that aim at viral proteins or trigger immune responses to either avoid infection or eliminate infected or cancerous cells. In this review, we aim to describe the role of viral infections and their mechanistic approaches in DLBCL tumorigenesis. To the best of our knowledge, this is the first review summarizing the oncogenic potential of numerous viral agents in DLBCL development.

PHOSPHO1 Serves as a Key Metabolism-Related Biomarker in the Tumorigenesis of Diffuse Large B-cell Lymphoma.

ObjectiveDiffuse large B-cell lymphoma (DLBCL) is an aggressive type of non-Hodgkin lymphoma. Due to its genetic heterogeneity and abnormal metabolism, many DLBCL patients have a poor prognosis. This study investigated the key metabolism-related genes and potential mechanisms.MethodsDifferentially expressed genes, differentially expressed transcription factors (TFs), and differentially expressed metabolism-related genes (DEMRGs) of glucose and lipid metabolic processes were identified using the edgeR package. Key DEMRGs were screened by Lasso regression, and a prediction model was constructed. The cell type identification by estimating relative subsets of RNA transcripts algorithm was utilized to assess the fraction of immune cells, and Gene Set Enrichment Analysis was used to determine immune-related pathways. A regulatory network was constructed with significant co-expression interactions among TFs, DEMRGs, immune cells/pathways, and hallmark pathways.ResultsA total of 1551 DEMRGs were identified. A prognostic model with a high applicability (area under the curve=0.921) was constructed with 13 DEMRGs. Tumorigenesis of DLBCL was highly related to the neutrophil count. Four DEMRGs (PRXL2AB, CCN1, DECR2 and PHOSPHO1) with 32 TF—DEMRG, 36 DEMRG—pathway, 14 DEMRG—immune-cell, 9 DEMRG—immune-gene-set, and 67 DEMRG—protein-chip interactions were used to construct the regulatory network.ConclusionWe provided a prognostic prediction model based on 13 DEMRGs for DLBCL. We found that phosphatase, orphan 1 (PHOSPHO1) is positively regulated by regulatory factor X5 (RFX5) and mediates MYC proto-oncogene (MYC) targeting the V2 pathway and neutrophils.

Glycoprotein PTGDS promotes tumorigenesis of diffuse large B-cell lymphoma by MYH9-mediated regulation of Wnt\u2013\u03b2-catenin\u2013STAT3 signaling

Glycoprotein prostaglandin D2 synthase (PTGDS) is a member of the lipocalin superfamily and plays dual roles in prostaglandins metabolism and lipid transport. PTGDS has been involved in various cellular processes including the tumorigenesis of solid tumors, yet its role in carcinogenesis is contradictory and the significance of PTGDS in hematological malignancies is ill-defined. Here, we aimed to explore the expression and function of PTGDS in diffuse large B-cell lymphoma (DLBCL), especially the potential role of PTGDS inhibitor, AT56, in lymphoma therapy. Remarkable high expression of PTGDS was found in DLBCL, which was significantly correlated with poor prognosis. PTGDS overexpression and rhPTGDS were found to promote cell proliferation. Besides, in vitro and in vivo studies indicated that PTGDS knockdown and AT56 treatment exerted an anti-tumor effect by regulating cell viability, proliferation, apoptosis, cell cycle, and invasion, and enhanced the drug sensitivity to adriamycin and bendamustine through promoting DNA damage. Moreover, the co-immunoprecipitation-based mass spectrum identified the interaction between PTGDS and MYH9, which was found to promote DLBCL progression. PTGDS inhibition led to reduced expression of MYH9, and then declined activation of the Wnt-β-catenin-STAT3 pathway through influencing the ubiquitination and degradation of GSK3-β in DLBCL. The rescue experiment demonstrated that PTGDS exerted an oncogenic role through regulating MYH9 and then the Wnt-β-catenin-STAT3 pathway. Based on point mutation of glycosylation sites, we confirmed the N-glycosylation of PTGDS in Asn51 and Asn78 and found that abnormal glycosylation of PTGDS resulted in its nuclear translocation, prolonged half-life, and enhanced cell proliferation. Collectively, our findings identified for the first time that glycoprotein PTGDS promoted tumorigenesis of DLBCL through MYH9-mediated regulation of Wnt-β-catenin-STAT3 signaling, and highlighted the potential role of AT56 as a novel therapeutic strategy for DLBCL treatment.

GPNMB promotes the progression of diffuse large B cell lymphoma via YAP1-mediated activation of the Wnt/β-catenin signaling pathway

Glycoprotein non-metastatic melanoma protein B (GPNMB) has been confirmed to be related to the pathogenesis of tumors. However, the potential impact of GPNMB on the progression of diffuse large B-cell lymphoma (DLBCL) is unclear. In this study, the expression levels of GPNMB and Yes-associated protein (YAP) were analyzed using qRT-PCT and Western blot assay. Cell counting kit-8, EdU, and flow cytometry assays were used to detect the proliferation and apoptosis of DLBCL cells. A nude mice xenograft model was established for in vivo research. Results showed that GPNMB and YAP1 were upregulated in DLBCL cell lines. Knockdown of GPNMB inhibited cell proliferation and promoted apoptosis in DLBCL cells. Additionally, the expression levels of YAP1 and the downstream effector of Hippo pathway (c-myc) were markedly decreased when GPNMB was knocked down. Moreover, knockdown of GPNMB inhibited the nuclear translocation of β-catenin protein, which could be abolished by YAP1 overexpression. Simultaneously, the anti-proliferative and pro-apoptotic effects of GPNMB knockdown could be reversed by YAP1 overexpression or LiCl (the activator of Wnt/β-catenin pathway). Furthermore, the mice xenograft model confirmed that inhibition of GPNMB restrained the tumorigenesis of DLBCL in vivo. In conclusion, GPNMB could partly activate the Wnt/β-catenin signaling pathway by targeting YAP1, so as to participate in tumorigenesis of DLBCL.

Methylation silencing CDH23 is a poor prognostic marker in diffuse large B-cell lymphoma.

Cadherin-23(CDH23) mediates homotypic and heterotypic cell-cell adhesions in cancer cells. However, the epigenetic regulation, the biological functions, the mechanisms and the prognostic value of CDH23 in diffuse large B-cell lymphoma (DLBCL) are still unclear. The Gene Expression Profiling Interactive Analysis (GEPIA) and the Gene Expression Omnibus (GEO) database were employed to analyze the CDH23 expression level in DLBCL. The correlation of CDH23 expression and methylation was analyzed by LinkedOmics database. The prognostic value was analyzed via GEPIA. Correlated genes, target kinase, target miRNA, target transcription factor and biological functions were identified by LinkedOmics and GeneMANIA database. The relationship between CDH23 and the immune cell infiltration was explored by the Tumor Immune Estimation Resource (TIMER). The expression of CDH23 was reduced by DNA methylation significantly in DLBCL tissue. Reduction of CDH23 represented poor outcome of DLBCL patients. Functional enrichment analysis showed that CDH23 mainly enriched in cancer cell growth, cell metastasis, cell adhesion, cell cycle, drug catabolic process, leukocyte mediated immunity and DNA repair by some cancer related kinases, miRNAs and transcription factors. These results indicated that methylated reduction of CDH23 represented poor outcome of DLBCL. CDH23 is associated with essential biological functions and key molecules in DLBCL. CDH23 may play crucial roles in DLBCL tumorigenesis. Our results lay a foundation for further investigation of the role of CDH23 in DLBCL tumorigenesis.

MicroRNA‑196a‑3p inhibits cell proliferation and promotes cell apoptosis by targeting ADP ribosylation factor 4 in diffuse large B‑cell lymphoma.

Diffuse large B‑cell lymphoma (DLBCL) is the most prevalent type of non‑Hodgkin's lymphoma with a heterogeneous molecular pathogenesis and aggressive clinical manifestations. The aim of the present study was to investigate the role of miR‑196a‑3p and its target gene in the development and progression of DLBCL. RT‑qPCR was used to detect the miR‑196a‑3p expression level in human DLBCL cell lines and DLBCL pathological tissues and compare them with the normal control. The clinical significance of the miR‑196a‑3p expression was also analyzed in DLBCL patients. Next, the effect of miR‑196a‑3p overexpression on the cell cycle, apoptosis, and proliferation of DLBCL cells was evaluated. To explore its underlying mechanism, the target gene of miR‑196a‑3p was predicted and validated using bioinformatics and molecular biological approaches. Finally, the expression of this target gene in clinical specimens and its correlation with clinicopathological characteristics were determined. The decreased expression of miR‑196a‑3p was validated in DLBCL, with further analysis proving that it was correlated with poor prognosis. It was shown that the overexpression of miR‑196a‑3p was associated with cell cycle arrest, enhanced apoptosis, and inhibited proliferation in DLBCL cells. Furthermore, ADP ribosylation factor 4 (ARF4) was verified as the downstream target gene of miR‑196a‑3p. Similar to miR‑196a‑3p restoration invitro, endogenous ARF4‑knockdown was proven to inhibit cell proliferation through cell cycle arrest and elevate apoptosis in DLBCL. The present results indicated that miR‑196a‑3p downregulation contributed to the tumorigenesis of DLBCL by targeting ARF4 expression, which may be used as a novel prognostic marker or potential molecular therapeutic target for DLBCL management in the future.

CD20-Modified Exosomes Delivered E260 Inhibits Progression of Diffuse Large B-Cell Lymphoma By Restoring Hippo Activity

Introduction: Fer tyrosine kinase (Fer) is ubiquitously expressed in various cancer types and has been proved to play a pivotal role in tumorigenesis and metastasis. E260, as an effective inhibitor of Fer, is selective lethal to cancer cells. However, the effect of Fer and E260 in diffuse large B-cell lymphoma (DLBCL) remain elusive. Thus, we set out to investigate the function and mechanisms of E260 targeting Fer in DLBCL for a treatment purpose. Methods: The impact of Fer expression to predict clinical outcome was evaluated in 50 newly diagnosed DLBCL and 30 healthy donors between 2014 and 2019 by immunohistochemistry. This study was approved by the ethics board of Shandong Provincial Hospital, and informed consent was obtained. Next, we assessed the oncogenic effect of Fer by using gain-and loss-of-function assays. Fer inhibitor E260 was used in the treatment of DLBCL cells. CD20 targeted exosomes were isolated from CD20 transfected HEK293T cells and then incubated with E260 in order to generate CD20-EXO-E260. The anti-tumor efficacy of E260 and CD20-EXO-E260 were assessed by cell proliferation, apoptosis, migration and drug sensitivity assays. To elucidate the regulatory mechanism, we test the expression of Hippo-YAP pathway and YAP location after E260 treated by western blotting and immunofluorescent staining. Interaction of the Fer and AJUBA was further validated by coimmunoprecipitation (Co-IP) assay. To further explore the clinical value of Fer, we isolated and test Fer expression in exosomes from the plasma of DLBCL patients. ROC curve was performed to test the diagnostic value of exosomal Fer in DLBCL. Results: To explore the association of Fer expression with DLBCL tumorigenesis, we examined Fer expression using immunohistochemical staining. Fer was upregulated in DLBCL patients (Fig.1A) and correlated with adverse clinical parameters (Fig.1B) and shorter overall survival time (Fig.1C). Since Fer was also increased in the DLBCL cell lines, we then set out to explore the biological function of Fer in DLBCL cells (Fig.1D). Transfected efficacy was verified by western blot (Fig2.A). In vitro gain-and loss-of-function assays demonstrated the stimulatory role of Fer on cell viability, migration and chemoresistance (Fig2.B-D). Recently, exosomes have been emerged as promising drug delivery carriers. Thus, we generated CD20-modified exosomes as an alternative to targeting deliver E260 to DLBCL cells. This event not only facilitated the targeting efficacy of E260 (Fig.3A) but also showed higher efficacy than E260 in eliminating DLBCL progression through inhibiting cell proliferation and chemotaxis, enhancing cell apoptosis and drug sensitivity both in vitro and in vivo (Fig.3B-F). In the view of GO and KEGG analysis, Fer mainly enrichment in regulating cell proliferation, adhesion, response to drug and Hippo pathway in cancer (Fig.4A). Consistent with this, we demonstrated that E260 activated Hippo pathway and leading to YAP cytoplasmic sequestration (Fig.4B-C). Co-IP assay confirmed the physical interaction between Fer and AJUBA. Furthermore, upregulated AJUBA reversed E260 mediated cell proliferation inhibition (Fig.4D). Thus, we suggest that E260 inhibits DLBCL progression by downregulating AJUBA which restores Hippo tumor-suppressor activity. In the mice model, the levels of plasma exosomal Fer could reflex the tumor burden (Fig.5A). We next detected that exosomal Fer and AJUBA expression increased in the plasma of DLBCL patients (Fig.5B-C) and released after R-CHOP therapy (Fig.5D). Besides, Fer expression was positively related with AJUBA (Fig.5E). As shown in the Figure 5F-G, exosomal Fer could act as a promising indicator in early diagnosis and disease progression for DLBCL patients. Conclusion: We demonstrated for the first time the oncogenic role of Fer in DLBCL progression and therefore provided an appealing therapeutic target. CD20-targeted exosomes delivery E260 are more effective in eliminating DLBCL progression through diminishing AJUBA and activating Hippo pathway. Furthermore, we provided circulating exosomal Fer as a novel non-invasive biomarker for DLBCL diagnosis. Disclosures No relevant conflicts of interest to declare.

Bufalin inhibits human diffuse large B-cell lymphoma tumorigenesis by inducing cell death through the Ca2+/NFATC1/cMYC pathway

The 5-year survival rate of diffuse large B-cell lymphoma (DLBCL) can reach 60%. However, nearly half of patients undergo relapse/refractory issues with a survival period of less than 2 years. New therapeutic approaches are therefore needed to improve chemotherapy efficacy and patient survival. Bufalin (BF), isolated from the traditional Chinese medicine Chansu, has been reported to play an anticancer role in multiple cancer cell types. However, there are few reports of the effects of BF on the growth of DLBCL. In the present study, we demonstrated that BF exerts antitumor activity in DLBCL cells, both in vitro and in vivo. Treatment of DLBCL cells with BF resulted in increased proliferation and apoptosis in a dose- and time-dependent manner. Daily intraperitoneal injection of 1.5 mg/kg BF significantly delayed DLBCL xenograft growth in NOD/SCID mice without affecting body weight. Bioinformatics analysis showed that BF may regulate NFATC1 protein and affect expression of its downstream gene, cMYC. Our results suggest that BF can attenuate NFATC1 translocation by reducing the intracellular calcium concentration; BF may also have a low synergistic effect with cyclosporin A. In conclusion, we demonstrated that BF exerts antitumor activity that is mediated at least in part by the Ca2+/NFATC1/cMYC pathway. Our findings suggest that BF can be effectively applied as a novel potential therapeutic agent for DLBCL.

MDM2-P53 Signaling Pathway-Mediated Upregulation of CDC20 Promotes Progression of Human Diffuse Large B-Cell Lymphoma.

BackgroundCell-division cycle 20 (CDC20) is overexpressed in a variety of tumor cells and is negatively regulated by wild-type p53 (wtp53). Our previous study uncovered that CDC20 was upregulated and associated with poor outcome in diffuse large B-cell lymphoma (DLBCL) based on bioinformatics analysis. Dysregulation of the MDM2-p53 is a major mechanism to promote DLBCL. Thus, we hypothesized that CDC20 could be a downstream gene of the MDM2-p53 signaling pathway. However, the clinical significance and mechanistic role of a novel MDM2-p53-CDC20 signaling pathway in DLBCL have still remained unclear.Materials and MethodsRT-qPCR was performed in MDM2 knocked down (KD) and control (Ctrl) OCI-Ly3/OCI-Ly10 cells to investigate whether CDC20 was a downstream gene of the MDM2-p53 pathway. The effects of CDC20 on cell proliferation, cell cycle and apoptosis were assessed, as well as the role of CDC20 in suppressing tumorigenicity in vivo. Furthermore, we also investigated the roles of CDC20 and MDM2 in progression of DLBCL and the underlying mechanisms.ResultsThe results of RT-qPCR revealed that CDC20 was downregulated while TP53 was upregulated in MDM2 KD OCI-Ly3 and OCI-Ly10 cells. It was unveiled that the expression levels of CDC20 and MDM2 were upregulated in DLBCL tissues and cells, and high CDC20 expression was correlated with adverse clinical features and poor outcome. Functional assays showed that downregulation of CDC20 could inhibit proliferation, induce apoptosis and cell cycle arrest in vitro. In addition, inactivation of the MDM2-p53 pathway by downregulation of MDM2 restored wtp53 expression level and reduced CDC20 protein level in OCI-Ly3 and OCI-Ly10 cells. Besides, targeting CDC20 was found to suppress tumorigenesis of DLBCL in vivo.ConclusionCDC20 was identified as a key downstream gene of the MDM2-p53 signaling pathway in DLBCL and may be used as a novel target gene to guide therapeutic applications.

NCAPD3 Regulates Tumor Growth and Predicts Prognosis in Diffuse Large B-Cell Lymphoma

Introduction NCAPD3 (Non-SMC Condensin II Complex Subunit D3) is a regulatory subunit of the condensing-2 complex. Recent studies have shown that somatic mutations in genes encoding subunits of condensins associated with microcephaly, primary autosomal recessive and several cancers. NCAPD3 was highlighted as an outcome predictor in pancreatic ductal adenocarcinoma (PDAC) and a new biomarker for subtype-1 prostate cancer that improves prognostication. Yet, no literature has been reported regarding the expression and biological role of NCAPD3 in diffuse large B cell lymphoma (DLBCL). Hence, the aim of our study is to evaluate the functional significance and mechanism of NCAPD3 in DLBCL. Methods Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers with informed consents. Expression levels of NCAPD3 mRNA and protein in DLBCL cell lines and PBMCs were detected by quantitative RT-PCR and western blotting. Immunohistochemistry (IHC) was conducted to assess the expression of NCAPD3 on paraffin-embedded tissues from 70 de novo DLBCL patients (study group) and 35 reactive hyperplasia patients (control group) with informed contents. Microarray datasets GSE32918 and GSE83632 were obtained from Gene Expression Omnibus. Survival analysis, protein-protein interaction (PPI) and gene set enrichment analysis (GSEA) in gene expression profiles were performed. Lentivirus vectors either targeting NCAPD3 (shNCAPD3) or empty lentiviral vector (shControl) were stably transfected into DLBCL cells. Cell proliferation was analyzed by cell counting kit (CCK-8). The apoptosis and cell cycle assays were carried out by flow cytometry. p < 0.05 was considered statistically significant. Results Markedly increased expression of NCAPD3 was detected in DLBCL cell lines at mRNA and protein level compared to those in healthy volunteers' PBMCs (Fig. 1a-b). We also observed higher NCAPD3 expression levels in DLBCL tissues than in reactive hyperplasia upon IHC staining (Fig. 1c). Expression of NCAPD3 protein was revealed in significant positive correlation with advanced Ann Arbor stage (p=0.046) and IPI score (p=0.017, Fig. 1d). Bioinformatics analysis showed that high NCAPD3 expression in DLBCL was turned up to be correlate with shorter overall survival according to GSE32918 (p=0.013, Fig. 2a). PPI network and GSEA indicated that NCAPD3 was functional enriched in chromatin regulation, cell cycle, histone methylation, NF-κB signaling and Toll-like receptor signaling pathway (Fig. 2b-c). Relevant mechanism is now the focus of ongoing experiments. Lentivirus mediated loss-of-function assays were performed to further investigate the biological role of NCAPD3 in DLBCL. Effective knockdown (shNCAPD3) was confirmed by qRT-PCR and western blot (Fig. 3a-b). Stable expression of shNCAPD3 in DLBCL cells exhibited growth suppression, increased fast-onset apoptosis, and induced GO/G1 phase arrest when compared to the control group (Fig. 3c-e). Conclusion Our investigations identified for the first time the oncogenic role of NCAPD3 in DLBCL tumorigenesis by bioinformatics analysis and in vitro evaluation. Expression of NCAPD3 was upregulated, and associated with adverse outcome of DLBCL patients. NCAPD3 inhibition by RNAi exerted anti-tumor efficacy in inhibiting cell growth, promoting apoptosis and blocking cell cycle. This study suggests that Sirt6 could be a potential molecular target for the treatment of DLBCL. Further study on it is under way. Disclosures No relevant conflicts of interest to declare.

Blockade of HMGB1 signaling pathway by ethyl pyruvate inhibits tumor growth in diffuse large B-cell lymphoma

High mobility group box 1 (HMGB1) protein in the tumor microenvironment actively contributes to tumor progression but its role in diffuse large B-cell lymphoma (DLBCL) is unknown. The aim of this study was to determine the mechanism by which HMGB1 promotes tumor growth in DLBCL and whether blockade of HMGB1 signaling pathway could inhibit tumorigenesis. We report that HMGB1 promotes proliferation of DLBCL cells by activation of AKT, extracellular signal-regulated kinases 1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3) and SRC Proto-Oncogene, Non-Receptor Tyrosine Kinase (Src). Ethyl pyruvate (EP), an anti-inflammatory agent, inhibits HMGB1 active release from DLBCL cells and significantly inhibited proliferation of DLBCL cells in vitro. Treatment with EP significantly prevented and inhibited tumor growth in vivo and prolonged DLBCL-bearing mice survival. EP significantly downregulated HMGB1 expression and phosphorylation of Src and ERK1/2 in mice lymphoma tissue. EP induced accumulation of the cell cycle inhibitor p27 but downregulated expression of cyclin-dependent kinase 2 (CDK2). Increased nuclear translocation of p27 interacted with CDK2 and cyclin A, which led to blockade of cell cycle progression at the G1 to S phase transition. In conclusion, we demonstrated for the first time that blockade of HMGB1-mediated signaling pathway by EP effectively inhibited DLBCL tumorigenesis and disease progression.

ROR1 associates unfavorable prognosis and promotes lymphoma growth in DLBCL by affecting PI3K/Akt/mTOR signaling pathway

The receptor-tyrosine-kinase (RTK)-like orphan receptor 1 (ROR1) is a transmembrane glycoprotein regarded as a tumor-associated antigen. ROR1 plays an important role in cancer development, but the detailed function of ROR1 in diffuse large B-cell lymphoma (DLBCL) remains unclear. In this study, we first detected ROR1 expression and evaluated the relationship between ROR1 expression and the clinicopathological characteristics of DLBCL patients. Next we employed shRNA-mediated knockdown of ROR1 in DLBCL cell line to explore the characteristics of ROR1 in DLBCL development both in vitro and in vivo. The results showed a significantly higher level of ROR1 in DLBCL tissues than in lymphatic hyperplasia tissues. High ROR1 expression was correlated with unfavorable prognosis in DLBCL patients. Furthermore, ROR1 knockdown inhibited the growth and induced the apoptosis in DLBCL cells and xenografts. In addition, shROR1 inhibited activation of the PI3K/Akt/mTOR signaling pathway, both in vitro and in vivo. Taken together, our results suggest that ROR1 is a novel prognostic marker for DLBCL survival and ROR1 significantly promotes DLBCL tumorigenesis by regulating the PI3K/Akt/mTOR signaling pathway. Targeting ROR1 may provide a promising strategy for DLBCL treatment. © 2019 BioFactors, 45(3):416-426, 2019.

MicroRNA-155 Downregulation Promotes Cell Cycle Arrest and Apoptosis in Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma in the adult population, and treatment of DLBCL is still unfavorable. Therefore, there is an urgent requirement to investigate the molecular mechanisms underlying DLBCL tumorigenesis. To study the potential function of microRNA-155 (miR-155) involved in the regulation of lymphoma, we monitored lymphoma cell behavior including proliferation, cell cycle, and apoptosis using CCK-8 and flow cytometry analysis. Real-time PCR was used to detect the expression levels of miR-155 in 118 lymphoma patients’ tissues, and Western blot was also used to analyze the expression level of proteins correlated with cell cycle and apoptosis in lymphoma cells. miR-155 expression levels were higher in lymphoma tissues compared with adjacent tissues. Downregulation of miR-155 inhibited lymphoma cell progress by arresting cell cycle in the G0/G1 phase and promoting apoptosis. Cell cycle-correlated proteins (cyclin B1, cyclin D1, and CDK4) were inhibited by downregulation of miR-155. Apoptosis-correlated proteins level (Bax/Bcl-2 and caspase 3 activity) were increased by downregulation of miR-155. In addition, a significant inverse correlation between the level of miR-155 and transforming growth factor-β receptor 2 (TGFBR2) was observed, which has been demonstrated to be a novel tumor suppressor gene. A further in vivo tumor formation study in nude mice indicated that downregulation of miR-155 in lymphoma cells delayed the progress of tumor formation. These findings indicate that miR-155 may serve as a useful potential target for the treatment of lymphoma.

Differential expression of viral agents in lymphoma tissues of patients with ABC diffuse large B-cell lymphoma from high and low endemic infectious disease regions.

Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin's lymphoma (NHL) in adults, accounts for approximately 30-40% of newly diagnosed lymphomas worldwide. Environmental factors, such as viruses and bacteria, may contribute to cancer development through chronic inflammation and the integration of oncogenes, and have previously been indicated in cervical cancer, hepatocellular carcinoma, gastric cancer and lymphoproliferative disorders. In the present study, the presence of microbial agents was analyzed in the lymphoma tissue of patients with activated B-cell like (ABC) DLBCL. The present study compared two groups of patients from geographically varied regions that possess a difference in the prevalence of viral and other microbial agents. The patient populations were from Sweden (a low endemic infectious disease region) and Egypt (a high endemic infectious disease region). A differential expression of several viruses in lymphoma tissues was noted when comparing Swedish and Egyptian patients. JC polyomavirus (JCV) was detected in Swedish and Egyptian patients and, uniquely, the complete hepatitis B virus (HBV) genome was detected only in Egyptian lymphoma patients. None of these viruses were detected in control lymph tissues from Sweden or Egypt. In total, 38% of the Egyptian patients were found to have HBV surface antigens (HBsAgs) in their serum; however, HBsAgs were not found in any of the Swedish patients. The percentage of serum HBsAgs in Egyptian patients with ABC DLBCL was significantly increased compared with the general Egyptian population (P<0.05). The present study may support a notion that viral agents, including JCV and HBV, may be involved in the tumorigenesis of DLBCL in regions of high infectious disease.