Diffuse Large B-cell Lymphoma Cell Proliferation Research Articles

Ironomycin Induces Diffuse Large B-Cell Lymphoma Cell Death By Targeting Iron Metabolism Addiction

Diffuse large B cell lymphoma (DLBCL) is the most common hematologic malignancy. Although more than half of these patients may achieve long-term remission, the majority of the remaining patients succumb to DLBCL.These patients relapse after conventional Rituximab (R)-based chemotherapy regimen, such as CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) in association with drug resistance. Iron is essential for many fundamental cellular functions, including proliferation and DNA synthesis. Accumulating evidence reveals that abnormal iron metabolism plays an important role in carcinogenesis and in the progression of many tumors. In cancer cells, the demand for iron increases in response to sustained deregulation of cell proliferation and DNA synthesis. Based on these data, we searched to identify if iron metabolism pathway is deregulated in DLBCL and could be exploited to develop novel therapeutic strategies. A list of 63 genes related to iron metabolism in cancer was defined. Using Maxstat R function, we showed that 12 genes out of the 63 investigated have a prognostic value in two independent cohorts of DLBCL patients treated by R-CHOP (Melnick cohort, n=69 and Lenz cohort, n=233). Based on these prognostic genes, we created a gene expression profile (GEP)-based risk score as the sum of the beta coefficients weighted by ±1 according to the patient signal above or below the probe set Maxstat value as previously reported (Herviou L et al. Clinical epigenetics 2018). The iron score was significantly associated with high-risk DLBCL in 3 independent cohorts of patients. GSEA analysis revealed that high-risk DLBCL patients defined by iron score are significantly enriched in genes involved in MYC amplification and MYC targets (p< 0.01). These data demonstrated that high iron score allows to identify DLBCL patients with a poor outcome and that could benefit from targeted therapy. We analyzed the therapeutic interest of Ironomycin, a new promising iron depleting molecule. Ironomycin is known to sequester iron in the lysosome and to induce ferroptosis (Mai TT et al. Nature Chemistry 2017). Ironomycin inhibits DLBCL cell proliferation in a panel of 16 DLBCL cell lines, at nanomolar concentrations (Median IC50: 30nM; range: 7.2 - 91.5 nM) compared to other iron chelators. Ironomycin induces significant cell growth inhibition, apoptosis of DLBCL cells and DNA double strand break accumulation. Furthermore, apoptosis induced by Ironomycin was not reversed by Iron supplementation. Caspase activation and apoptosis induction mediated by Ironomycin could be partially reversed by pan-caspase inhibitor QVD (p<0.001). Ferroptosis inhibitor, Ferrostatin-1, also partially reversed Ironomycin-induced apoptosis (p<0.001). According to ferroptosis induction, Ironomycin induced ROS (CM-H2DCFDA staining), lipid peroxidation (BODIPY-C11 staining) and autophagy (LC3B puncta formation), at 48H, in DLBCL cells. We also identified a delayed progression of replication forks upon Ironomycin treatment in DLBCL cell lines (p<0.0001). Ironomycin induces significant phosphorylation of RPA2 that is a marker of DNA-replication stress and DNA damage response. Interestingly, we identified a significant correlation between basal replication stress monitored by ATR and CHK2 phosphorylation and sensitivity to Ironomycin in DLBCL cell lines. With major importance, we validated the therapeutic interest of Ironomycin in primary DLBCL cells of patients (n=5) without major toxicity for non-tumor cells from the microenvironment. Ironomycin significantly reduces the median number of viable primary DLBCL cells by 69.3% and 77.8%, at respectively 50 and 100nM concentrations (p<0.01 and p<0.001). Furthermore, Ironomycin presented a low toxicity on hematopoietic progenitors (CFU assays, n=5) compared to conventional treatment (p<0.001). We tested the therapeutic interest to combine Ironomycin with conventional chemotherapy used in DLBCL including cyclophosphamide, doxorubicin and etoposide. Interestingly, we identified a significant synergistic effect when Ironomycin is combined with Doxorubicin. Altogether, these data demonstrated that a subgroup of high-risk DLBCL patients could be identified with the iron score and could benefit from Iron metabolism inhibitor treatment. Disclosures Cartron: Roche, Celgene: Consultancy; Sanofi, Gilead, Janssen, Roche, Celgene: Honoraria. Moreaux:Diag2Tec: Other: Co-founder of Diag2Tec company.

Blockade of Yes-Associated Protein As a Novel Therapeutic Approach in Diffuse Large B-Cell Lymphoma

Introduction Yes-associated protein (YAP) is an important transcriptional regulator and effector of the Hippo signaling that has emerged as a novel determinant of malignancy in several human tumors. Verteporfin (VP), a clinically used photosensitizer for treatment of macular degeneration, is recently identified as a potential inhibitor of YAP expression independent of light activation. However, the function and mechanism of Hippo-YAP in diffuse large B-cell lymphoma (DLBCL) remains ill defined. Herein, we investigated the functional significance of YAP and proposed a novel therapeutic strategy for DLBCL. Methods Lymph node biopsies from 60 de novo DLBCL patients and 30 reactive hyperplasia cases were collected with informed consents. The biological function of YAP was evaluated via RNAi-mediated knockdown and CRISPR/Cas9 mediated genomic deletion. RNA-sequencing was conducted to detect the dysregulated RNAs in DLBCL cell with YAP knockout. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses were used to explore the function of these differentially expressed RNAs. SCID-Beige mice were subcutaneously injected with DLBCL cells to establish xenograft model. Animal experiments were performed in accordance with the principles of the Institutional Animal Care. Results We first examined the expression of YAP in Oncomine database and discovered the upregulation of YAP mRNA in DLBCL cells (Fig.1A). High expression of YAP protein was validated in a cohort of newly diagnosed DLBCL patients (n=60). Survival analysis revealed that YAP expression was associated with aggressive disease process (p=0.014, Fig.1B). Functional enrichment analyses of YAP in DLBCL microarray profiles revealed that YAP was enriched in cellular process and biological regulation (Fig. 1C). Knockdown of YAP (shYAP) significantly suppressed cell proliferation and promoted cell cycle arrest (Fig.2A-C). VP restrained proliferation of DLBCL cells in a dose- and time-dependent manner (Fig. 2D). Treatment of VP significantly promoted cell apoptosis and cell cycle arrest in DLBCL cells (Fig. 2E). We next validated the involved mechanism of the anti-tumor effect of VP in DLBCL cells. Protein expression levels of YAP and TEAD were significantly inhibited by VP in dose-dependent manner (Fig.2F). In addition, treatment of VP remarkably restrained the mRNA expression of YAP targeted genes, including CTGF, CYR61 and NF2 (Fig. 2G). Genomic network enrichment of YAP interactions was established in STRING database (Fig. 2H). To validate the involvement of YAP in DLBCL pathogenesis, we deleted YAP expression by CRISPR/Cas9 genomic-editing system. YAP deletion (sgYAP) resulted in significantly reduction in cell proliferation and arrest of cell cycle (Fig.3A). To explore the activities of YAP inhibition in vivo, xenograft DLBCL mice model was established by LY1 cells with YAP-deletion. Compared to the control group, tumors with YAP-deletion displayed reduced growth and decreased expression levels of Ki67 and C-myc (Fig. 3B-C). To further explore the translational regulation mechanism of YAP in DLBCL, integrated analysis of mRNA and non-coding RNA (ncRNA) expression profiles were conducted by whole-transcriptome sequencing (RNA-seq). 158 miRNAs, 29 lncRNAs, 74 circRNAs and 528 mRNAs were shown to be significantly differentially expressed (DE) in DLBCL cells with YAP knockout (Fig. 4A-C). The subsequent GO and KEGG pathway analysis revealed that DE molecules mainly related to cellular metabolic process and pathways. In addition, the miRNA-circRNA network was established (Fig. 4D). Conclusion Taken together, these findings provide in vitro and in vivo pre-clinical evidence for the crucial role of YAP in lymphomagenesis and highlight that blockade of YAP represents an attractive approach in DLBCL. The clinical used photosensitizer Verteporfin exerted anti-tumor effect via disrupting YAP-TEAD complex. Further interrogation on the regulatory mechanism of YAP in DLBCL will outline a promising therapeutic option to utilize this newly identified oncogene in DLBCL therapy. Disclosures No relevant conflicts of interest to declare.

A circular RNA from APC inhibits the proliferation of diffuse large B-cell lymphoma by inactivating Wnt/β-catenin signaling via interacting with TET1 and miR-888.

Circular RNA (circRNA), a type of non-coding RNA, can promote or suppress tumorigenesis. To investigate the involvement of circRNA in diffuse large B-cell lymphoma (DLBCL), we performed a circRNA microarray analysis on paired DLBCL and normal tissues. We identified a novel and highly stable circRNA originating from the back-splicing of APC exon 7 to exon 14, circ-APC (hsa_circ_0127621), which was downregulated in DLBCL tissues, cell lines and plasma. In gain-of-function experiments, ectopic expression of circ-APC inhibited DLBCL cell proliferation in vitro and tumor growth in vivo. Cytoplasmic circ-APC functioned as a sponge for miR-888, thus post-transcriptionally upregulating APC by alleviating the repressive effects of miR-888 on this gene. Further, nuclear circ-APC bound to the APC promoter and recruited the DNA demethylase TET1, thereby transcriptionally upregulating APC. Upon its upregulation, APC dampened the canonical Wnt/β-catenin signaling pathway by reducing the accumulation of β-catenin in the nucleus, thereby retarding DLBCL growth. Clinically, circ-APC was found to be an effective diagnostic and prognostic biomarker for patients with DLBCL. Our study suggests that circ-APC is a novel proliferation inhibitor, and that restoring circ-APC expression may be a promising therapeutic approach for DLBCL patients.

Downregulation of long non-coding RNA TUG1 suppresses tumor growth by promoting ubiquitination of MET in diffuse large B-cell lymphoma.

Long non-coding RNAs (lncRNAs) can modulate gene expression through different mechanisms, but the fundamental molecular mechanism between lncRNAs and MET protein in diffuse large B-cell lymphoma (DLBCL) was poorly understood. The expression of lncRNA TUG1 and MET in DLBCL tissues and cell lines was determined by quantitative real-time PCR and western blotting. Cell proliferation, invasion and apoptosis were determined by cell counting kit-8 assay, transwell assay and flow cytometer. The animal xenograft model was established by the injection of DLBCL cells carrying si-TUG1. The expression of TUG1 and MET was upregulated in DLBCL tissues and cells. We demonstrated that MET was altered in the TUG1 knockdown DLBCL cells, and confirmed the interaction between TUG1 and MET by RNA pull-down and RNA immunoprecipitation. Furthermore, knockdown of TUG1 reduced MET protein level by promoting ubiquitination, and suppressed tumor growth in vitro and in vivo. Our findings demonstrated that TUG1 exerted its oncogenic function in DLBCL by inhibiting the ubiquitination and the subsequent degradation of MET. Knockdown of TUG1 through MET downregulation suppressed DLBCL cell proliferation and tumor growth.

Stress-Induced Premature Senescence Promotes Proliferation by Activating the SENEX and p16INK4a/Retinoblastoma (Rb) Pathway in Diffuse Large B-Cell Lymphoma

Objective:Cellular senescence has been thought to be an important barrier to tumor formation. Recent studies have shown that stress-induced premature senescence (SIPS) can promote partial tumor invasion, but how SIPS affects diffuse large B-cell lymphoma (DLBCL) remains inconclusive. This study aimed to address that issue.Materials and Methods:The immunophenotype of the LY8 cell line was measured with flow cytometry. SIPS induced by tert-butyl hydroperoxide (tBHP) was detected by senescence β-galactosidase staining. Cell proliferation was analyzed with CCK8 and expression levels of ARHGAP18 (SENEX gene-encoding protein), p16/p21, and Rb/pRb were measured with western blot. LY8 cells were transfected with SENEX-SiRNA/NC and verified by western blot.Results:Our results suggested that the immunophenotype of the LY8 cell line is CD19-, CD20-, and CD10-positive and the immunoglobulin light chain is the kappa type. The cellular senescence model of DLBCL could be successfully induced by 30 μM tBHP. ARHGAP18, p21, p16, and Rb protein levels were significantly increased but the level of pRb expression was decreased in the SIPS group compared with other groups. Meanwhile, the proliferation rate was increased in the SIPS group more than other tBHP groups. Furthermore, the expressions of p21 and p16 were significantly decreased in the SENEX-SiRNA group compared with the negative control group.Conclusion:SIPS formation activates ARHGAP18 and the p16/Rb pathway and promotes DLBCL cell proliferation. Furthermore, SENEX activates the p16 pathway in DLBCL. SIPS promotes proliferation by activating SENEX and the p16/Rb pathway in DLBCL. SENEX-related SIPS may serve as an important target for relapsed/refractory DLBCL therapy.

Soluble CXCL16 promotes TNF-α-induced apoptosis in DLBCL via the AMAD10-NF-κB regulatory feedback loop.

We had previously identified that the co-expression of transmembrane CXCL16 (TM-CXCL16) and itsreceptor CXCR6 is an independent risk factor for poor survival in patients with diffuse large B-cell lymphoma (DLBCL). However, the impact of the soluble form of CXCL16 (sCXCL16) on the pathogenesis of DLBCL remains unknown. In the present study, the synergistic effect of sCXCL16 and tumor necrosis factor α (TNF-α) on apoptosis in DLBCL cell lines (OCI-LY8 and OCI-LY10) was investigated in vitro. sCXCL16 reinforced TNF-α-mediated inhibition of DLBCL cell proliferation, as determined by the cell counting kit-8 assay. The results of annexin V staining showed that sCXCL16 enhanced TNF-α-induced apoptosis in OCI-LY8 and OCI-LY10 cells through a death receptor-caspase signaling pathway. The results of gene microarray suggested a significant upregulation of differentially expressed genes in the TNF signaling pathway. sCXCL16 increased the concentration of extracellular TNF-α by binding to CXCR6 to activate the nuclear factor-κB (NF-κB) signaling pathway. TNF-α also induced the secretion of sCXCL16 by increasing the expression of ADAM10, which is known to cleave TM-CXCL16 to yield sCXCL16. Moreover, bioinformatics analysis revealed that elevated TNF-α and ADAM10 expression levels in tumor tissues predicted better survival in patients with DLBCL. Thus, our study suggests that sCXCL16 enhances TNF-α-induced apoptosis of DLBCL cells, which may involve a positive feedback loop consisting of TNF-α, ADAM10, sCXCL16, and members of the NF-κB pathway. sCXCL16 and TNF-α may be used as prognostic markers in the clinic, and their combinational use is a promising approach in the context of DLBCL therapy.

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.

The significance of chemokines in diffuse large B-cell lymphoma: a systematic review and future insights.

Despite the progress made in molecular and clinical research, patients with diffuse large B-cell lymphoma (DLBCL) still have a bad prognosis. Recently, chemokines/chemokine receptors have become the subject of interest in relation to DLBCL. Studies have demonstrated the important role of chemokines/chemokine receptors in the communication between DLBCL cells and tumor microenvironment. Studies have also reported the ability of chemokines/chemokine receptors in promoting the proliferation and invasion of DLBCL cells. Here, we summarize the data on mechanisms of DLBCL supporting the involvement of chemokine/chemokine receptor changes. We focus on the available evidence regarding chemokines/chemokine receptors as biomarkers and therapeutic targets for DLBCL.

Novel Epigenetic Vulnerabilities for Diffuse Large B-Cell Lymphoma

Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of B-cell Non-Hodgkin Lymphoma (NHL), representing a third of all new cases. DLBCL is further sub-divided into various molecular sub-types based on gene expression and co-occurring genetic alterations. Gene expression based- subtypes include the germinal center B-cell (GCB) and the activated B-cell type (ABC) subtypes, with the ABC sub-type having a poorer prognosis than the GCB sub-type. Interestingly, 30-40% of all DLBCL patients harbor mutations in key epigenetic regulators, EZH2, KMT2D, CREBBP, EP300, and mutations in histone proteins themselves.Through an unbiased cell-based phenotypic screen, we discovered that inhibition of lysine demethylases, specifically KDM4C and KDM4A, represents a vulnerability across 15 different DLBCL cell lines including germinal center B-cell (GCB) and activated B-cell (ABC) type lines, with a GI50 value between 75nM to ~200nM, while sparing leukemia lines. Consistently, treatment of xenograft-based animal models of DLBCL with a low dose of KDM4A/KDM4C inhibitor delivered intra-peritoneally three times a week, results in a drastic reduction of tumor burden. Both KDM4A and KDM4C catalyze the removal of histone H3 K9 di- and tri-methylation (H3K9me2/3) and H3K36 di- and tri-methylation (H3K36me2/3). H3K9me2/3 is associated with promoter and enhancer repression, while H3K36me2/3 is present in gene bodies during transcription but also functions as a chromatin repressor Consistently, we have identified key enhancers, including those associated with IKZF1, that are “decommissioned” after inhibition of KDM4A/KDM4C. Repressed enhancer activity, through loss of H3K27 acetylation and H3K4me1, and gain of H3K9me2/3, results in a rapid transcriptional downregulation of IKZF1 and its partners including IKZF3. Given the role of IKZF1 in the transcriptional regulation of key B Cell Receptor (BCR) signaling components, we show that KDM4A/KDM4C inhibition leads to a downregulation of SYK, a proximal BCR-signaling component, which likely precedes DLBCL cell apoptosis. In addition, we observed an activation of extra-lineage transcription factors such as CEBPA and CEBPB, which are normally repressed by IKZF1 in the lymphoid lineage. A concomitant downregulation of the B-cell gene expression program and an upregulation of the myeloid (CD14+ monocytic) gene expression program is also observed, implying a “trans-differentiation” of DLBCL cells into the monocyte lineage. This lineage-switch correlates with an increased population of CD14+ expressing cells. Finally, using DLBCL patient data sets, we can show that over-expression of either KDM4A or KDM4C is associated with poor prognosis in DLBCL patients. In summary, we have discovered that KDM4A/KDM4C inhibition results in an increase of repressive histone modifications at several intra-genic “enhancers” of genes that are responsible for the survival and proliferation of DLBCL cells. The elucidation of this unique epigenetic mechanism provides a strong rationale for the development of novel targeted therapies against both multiple subtypes of DLBCL. [Display omitted] DisclosuresShipp:Bayer: Research Funding; AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

SIRT6 As a Potential Therapeutic Target in Diffuse Large B-Cell Lymphoma

IntroductionSirt6 is a member of the mammalian sirtuins (SIRTs) family, involved in diverse biological processes. It is dysregulated in various solid tumors and multiple myeloma. Sirt6 can be considered a double-edged sword in cancer, as its dual role of both an oncogene and a tumor suppressor. However, no literature has been reported regarding the functional significance of Sirt6 in diffuse large B-cell lymphoma (DLBCL) to date. Thus we evaluated the expression and function of Sirt6 in both DLBCL tissues and cell lines in this present study, as well as assessed the proliferation of DLBCL cells when treated with a pan-sirtuin inhibitor Nicotinamide (NAM) and a selected Sirt6 inhibitor, OSS_128167.MethodsMicroarray datasets GSE83632 and GSE32918 were obtained from Gene Expression Omnibus. Differential expression analysis in gene expression profiles GSE83632 were performed through classical Bayesian method provided by the Limma package and then extracted the SIRT6 gene expression value. GSE32918 was annotated with the R-package illumineHumanWGDASLv3.db data probe, converted into gene symbol, and extracted expression data of Sirt6 for survival analysis. Immunohistochemistry (IHC) was conducted to assess the expression of Sirt6 on araffin-embedded tissues from 60 de novo DLBCL patients (study group) and 30 reactive hyperplasia patients (control group) with informed contents. Then further analysis was performed to find the correlations between Sirt6 expression and the clinical characteristics of DLBCL patients. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers with informed consents. Expression levels of Sirt6 mRNA and protein in DLBCL cell lines (LY1, LY8, LY3 and Val) and PBMCs (control) were detected by quantitative RT-PCR and western blotting. Lentivirus vectors transfected DLBCL cells to stably knockdown or overexpress Sirt6. Apoptosis was assessed by PE/7AAD. DLBCL cell lines were treated with NAM and OSS_128167, cell proliferation was analyzed by cell counting kit (CCK-8).ResultsBioinformatics analysis showed that SIRT6 was significantly up-regulated in DLBCL than in normal samples. High Sirt6 expression in DLBCL was turned up to be correlate with shorter overall survival according to GSE32918 (Figure 1A-B). These data indicated a relevant role for Sirt6 in the biology of DLBCL. We observed markedly increased level of Sirt6 in DLBCL lymph nodes by IHC. Expression of Sirt6 was positively correlated with Ann Arbor stage and international prognostic index (IPI) score of DLBCL patients. Aberrantly overexpression of Sirt6 was also confirmed in DLBCL cell lines at mRNA and protein level (Figure 2A-C). Our findings on Sirt6 were consistent with those from publicly available data sets.Sirt6 knockdown caused increased apoptosis rate, and on the contrary, Sirt6 over-expression had brought about decreased apoptosis (Figure 3A-C). We then investigated the function of Sirt inhibitors on the proliferation of DLBCL cells. LY1 and Val cells were treated with different doses of NAM at 24-72 hours. Cell proliferation was inhibited in a dose-dependent manner. LY1 and LY8 cells were treated with different doses of OSS_128167 at 24-72 hours and triggered similar effect, cell proliferation was inhibited by 60% at the concentration of 100µM for 48 hours (Figure 4A-B).ConclusionOur investigations identified for the first time that expression levels of Sirt6 were up-regulated in DLBCL and associated with adverse outcome by bioinformatics analysis and in vitro experiments. Knockdown of Sirt6 lead to increased apoptosis and over-expression of Sirt6 had the opposite effect. DLBCL cells treated with pan-Sirt inhibitor and selective Sirt6 inhibitor, revealed reduced proliferation in a dose-dependent manner. This study suggests that Sirt6 could be a potential molecular target for the treatment of DLBCL. Further research on it is under way. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Avadomide (CC-122) Alters T Cell Repertoire and Enhances Infiltration of Lymphocytes into Tumor Microenvironment in DLBCL Patients

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, constituting 30-40% of all new cases. Avadomide, a small molecule cereblon modulator currently being developed in DLBCL, binds to cereblon in the CRL4CRBN E3 ligase, leading to ubiquitination and subsequent proteasomal degradation of transcription factors Aiolos and Ikaros. This results in decreased proliferation and increased apoptosis of DLBCL cells, independent of cell-of-origin, and immunostimulatory effects in T and NK cells, as measured by increased cytokine production, cell surface activation markers, and enhanced antibody-dependent cellular cytotoxicity. A novel gene expression-based classifier, which detects DLBCL patients with T cell and macrophage infiltration within the tumor microenvironment, has been shown to enrich for responders to avadomide. Avadomide, as a single agent and in combination with rituximab, is currently being investigated in relapsed/refractory DLBCL (NCT01421524 and NCT02031419).Methods: Eighty-one DLBCL patients were enrolled in the expansion phase of the CC-122-ST-001 study (NCT01421524). Peripheral blood T cell subsets were enumerated at screening (baseline), cycle 1 day 15 (C1D15) and cycle 2 day 15 (C2D15) by flow cytometric immunophenotyping. Ex vivo production of IL-2 and IFNγ, as a measure of T cell activation, was determined using the α-CD3 TruCulture Assay. Changes from baseline were evaluated using the t-test with P<0.05 considered significant. T cell receptor (TCR) repertoire analysis through TCRB CDR3 region sequencing was done to derive metrics of population diversity and composition. RNAseq was performed on screening and on-treatment (C1D10/15) biopsies; gene expression deconvolution analyses were used to identify immune cell populations within the tumor microenvironment.Results: Avadomide treatment results in decreased peripheral CD4+ and CD8+ naïve (CD45RA+/CD45RO-) T cells and increased memory (CD45RA-/CD45RO+) and activated (HLA-DR+) T cells, without significantly affecting the absolute numbers of total CD3+, CD4+ or CD8+ populations (Table). High-dimensional single-cell mass cytometry of longitudinally collected peripheral blood samples confirmed the significant increase in CD8+ memory T cells and identified an increase in Treg populations and decreases in CD16+ monocytes and dendritic cells (adj. P<0.02). A single dose of avadomide on C1D1 significantly activated T cells, as indicated by a 300% increase in IL-2 (P=0.018) and 185% increase in IFNγ (P=0.003) secretion. Assessment of TCR B clonotypes revealed that avadomide increases the TCRB repertoire breadth, while reducing its clonality. To understand the influence of avadomide treatment on the tumor microenvironment, we performed RNA sequencing on tumor biopsies collected at screening and two weeks after initiating avadomide treatment (n=18 patients). Deconvolution analyses identified an increase in the expression of genes indicative of various T cell populations, dendritic cells and macrophages, while B cell associated gene expression decreased in on-treatment biopsies compared to screening biopsies. Gene set enrichment analysis (GSEA) revealed significantly increased expression of genes associated with “HALLMARK Interferon Alpha Response” (adj. P=0.04), indicative of an increase in Type I/II interferon production by cells such as T and NK cells. Buttressing the in vitro observations of avadomide-mediated inhibition of DLBCL cell proliferation, GSEA identified a decrease in “E2F targets” (adj. P=0.007) consistent with decreased proliferation of malignant B cells.Conclusion: Avadomide is a potent immunomodulating agent with multiple immune activating properties, including positive effects on T cell activation, as well as a broad expansion of T cell populations as defined by an increase in the richness of the T cell repertoire in blood. In addition, our data demonstrate decreased proliferation of malignant B cells in the tumor, with concomitant increased trafficking of immune cells, such as dendritic cells and macrophages, to the tumor microenvironment. These data further delineate the immune enhancing activity of avadomide in DLBCL patients beyond T-cell activation and provide rational combination strategies. [Display omitted] DisclosuresHagner:Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership.

Combination of Enzastaurin and Ibrutinib Synergistically Induces Anti-Tumor Effects in Diffuse Large B Cell Lymphoma

Background:Diffuse large B cell lymphoma(DLBCL), the most common form of lymphoma, is characterized by a heterogeneous tumor entity. Although durable remissions can be achieved in more than half of these patients, DLBCL remains a significant clinical challenge, with approximately 30% of patients not being cured. BCR-associated kinases (SYK, BTK, PI3K)inhibitors have exhibited encouraging pre-clinical and clinical effects, as reported by many researchers.Early studies demonstrated that PKCβ inhibitors alter phosphorylation level of BTK, which enhances activation of BTK signaling pathway.Here we aim to investigate whether the combination of PKCβ inhibitor enzastaurin and BTK inhibitor ibrutinib has synergistic anti-tumor effects in DLBCL.Methods:In vitro cell proliferation was analyzed using Cell Titer-Glo Luminescent Cell Viability Assay. Induction of apoptosis and cell cycle arrest were measured by flow cytometry. Western Blotting analysis was used to detect the essential regulatory enzymes in related signaling pathways. RNA-seq was conducted to evaluate the whole-transcriptome changes brought by co-treatment with low doses of enzastaurin and ibrutinib. The synergistic anti-tumor effect of enzastaurin and ibrutinib was also evaluated in vivo.Results: Combination of enzastaurin and ibrutinib produced a lasting synergistic effect on the survival and proliferation of ABC (HBL-1,TMD8,SU-DHL-2)and GCB (SU-DHL-6, OCI-LY7)DLBCL cells lines. Exposure of these DLBCL cells to minimally toxic concentration of enzastaurin and ibrutinib resulted in more significant inhibitory activity than each agent alone. In addition, the combination of these two agents led to an enhanced apoptosis and induced G1 phase arrest compared with monotherapy, which were accompanied by marked regulation of apoptosis-related and cell cycle-related proteins. Combination treatment with enzastaurin and ibrutinib, to some extent, also functioned synergistically to inhibit cell migration and invasion compared with single agent. Moreover, combination of enzastaurin and ibrutinib seemed to be more effective in inhibition p-ERK, p-AKT and p-P38 in DLBCL cell lines. In addition, RNA-seq results showed that co-treatment with low doses of enzastaurin and ibrutinib could effectively downregulate BCR, NF-κB, JAK and MAPK related signaling pathways, and the pathway analysis results were consistent with the immunoblotting analysis. Furthermore, the mRNA expression analysis using real-time PCR further indicated that treatment with enzastaurin and ibrutinib significantly decreased the mRNA levels of NOTCH1 which has been recognized as an important oncogenic regulator of hematological malignancy. The combination effect of enzastaurin and ibrutinib in inhibiting proliferation of DLBCL cells probably was realized through suppression of NOTCH1 expression. Finally, the anti-tumor activity of co-treatment with enzastaurin and ibrutinib was also demonstrated using xenograft mice models.Conclusion:We investigated the combination of enzastaurin and ibrutinib in ABC and GCB DLBCL, demonstrating the co-treatment had synergistic anti-tumor effects in DLBCL. These results provided a sound foundation for an attractive therapeutic treatment, and the simultaneous suppression of BTK and PKCβ might be an new treatment strategy for DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Heat Shock Factor 1 Reprograms the DLBCL Microenvironment to Evade Immune Surveillance and Support Tumor Growth

The primary components of the stromal lymphoma microenvironment (LME) are the cancer-associated fibroblasts (CAF) and the extracellular matrix (ECM) they produce. CAFs are derived from healthy fibroblasts that have been hijacked and transcriptionally reprogrammed by cancer cells into a novel biological entity to promote tumorigenesis. While the key effectors of these programs are largely unknown, recent data indicates that in a variety of solid tumors and T-acute lymphoblastic leukemias an important transcriptional programmer of the microenvironment is the Heat Shock Factor 1 (HSF1). Hence, we sought to determine the contribution of HSF1 within the stromal LME to the acquisition of lymphomagenic features in diffuse large B-cell lymphoma (DLBCL).We first analyzed the activity of HSF1 in 80 DLBCL patients by RNA-sequencing. Patients were segregated into having “high” and “low” HSF1 activity based on the expression of the canonical stress response target genes. We found distinct patterns of stromal-associated genes between DLBCL having “low” vs. “high” HSF1 activity. Furthermore, ChIP-sequencing data of lymphoma cells and CAFs showed the emergence of distinct transcriptional programs that are differentially orchestrated by HSF1. To delve deeper into the mechanism of these effects, we co-cultured lymphoma cells with fibroblasts having either HSF1 WT or HSF1 null in 3D collagen-containing lymphoma organoids. We found that only organoids harboring HSF1 WT fibroblasts were supportive of DLBCL cell proliferation. Moreover, DLBCL cells responsive and non-responsive to HSF1 downregulation in a cell-autonomous manner, failed to proliferate when cultured in HSF1-deficient LME organoids.To further elucidate this mechanism, we implanted a murine HSF1-positive DLBCL cell line (A20) into HSF1 WT (n=13) and HSF1 null (n=11) syngeneic mice and monitored lymphoma development for up to 14 days. We observed a continuous growth of HSF1-positive murine DLBCL in HSF1 WT mice; however, DLBCL in HSF1 null mice underwent spontaneous collapse after day 7, leading to almost complete tumor eradication. We further investigated the lymphoma microarchitecture, ECM biomechanics and composition in relation to the cellular components in serial tumor sections at several time points. At day 7, when no significant difference in tumor sizes exists between HSF1 WTs and nulls, we found significant differences in tissue stiffness (p=0.017), collagen microarchitectureby fiber alignment (p<0.001) and elastic fiber area (p<0.001). ECM proteomics by HPLC-MS/MS of the ECM-enriched fractions of these murine DLBCLs at days 5, 7 and 13 followed by network analysis demonstrated highly dynamic changes in ECM composition. Specifically, comparing the “matrisomes” of HSF1 WT and null tumors at day 7, we identified differentially regulated ECM modules that facilitate collagen functionalization and abundance of small leucine-rich proteoglycans; suggesting a failure of the HSF1 null stroma in efficiently remodeling the ECM. We identified dynamic changes in specific matrisome components with known roles in tumor proliferation and immune activity. These biochemical and biophysical changeswere in turn capable of modulating the cellular composition of DLBCL tumors. By day 7, the LME in HSF1 null was less supportive of lymphoma growth as measured by lower cellularity (p=4.5e-18) and proliferation of lymphoma cells by Ki67 (p=7.6e-6); and increased apoptosis by TUNEL (p=5.6e-5). In regards to CD31 positivity based on endothelial cells and vascularization, we observed no significant differences between HSF1 WT and HSF1 null tumors, however, there were significant changes in the repertoire of immune infiltrating cells. Specifically, HSF1 null LME harbored higher fractions of effector cd3+ T-cells (p=3.0e-6) and mac2+macrophages (p=9.4e-11); and a decrease in the suppressive cells cd3+foxp3+Tregs (p=6.6e-19) and cd11b+ gr1+myeloid-derived suppressor cells (p=0.0467).Taken together, our data in DLBCL specimens and cell lines, lymphoma-fibroblasts organoids, and a murine DLBCL model, all demonstrate that HSF1 activity in DLBCL drives an ECM program that confers microarchitectural and biophysical properties to the LME that are supportive of lymphoma cell proliferation and infiltration by immune suppressive populations. DisclosuresCerchietti:Celgene: Research Funding; Weill Cornell Medicine: Employment.

MiR‑101 regulates the cell proliferation and apoptosis in diffuse large B‑cell lymphoma by targeting MEK1 via regulation of the ERK/MAPK signaling pathway

MAPK kinase1 (MEK1) is an upstream protein kinase of extracellular signal regulated kinase (ERK), which activates the ERK/MAPK (mitogen activated protein kinase) pathway. Importantly, bioinformatic analysis has shown that there is a target complementary binding site between miR‑101 and MEK1. The present study aimed to ascertain whether or not miR‑101 plays a role in regulating MEK1 expression, ERK/MAPK pathway activity, and the proliferation and apoptosis in a diffuse large Bcell lymphoma (DLBCL) cell line. DLBCL tumor samples were collected from patients in our hospital, and lymphatic tissues with reactive lymphoid hyperplasia were selected as controls. The patients were divided into high and low expression groups, and then the survival rate of the two groups was compared using Kaplan‑Meier method, as well the effect of miR‑101 and MEK1 mRNA expression on survival and prognosis was analyzed. The expression of miR‑101, MEK1 and p‑MEK1 between normal lymphoblastic cell lines (HCC1954 BL and NCI‑BL2009) and lymphoma cell lines (SU‑DHL‑4 and Farage) was compared. Lymphoma SU‑DHL‑4 and Farage cells were cultured invitro, and then divided into the following groups: miR‑NC group; miR‑101 mimic group; siRNA‑NC group; and siRNA‑MEK1 group. The expression of miR‑101, MEK1, p‑MEK1, p‑ERK1/2 and Bcl‑2 was compared. Cell apoptosis was detected by flow cytometry, and cell proliferation was detected by EdU staining. The results showed that targeted regulation existed between miR‑101 and MEK1, and the decreased expression of miR‑101 was related to the pathogenesis and prognosis of DLBCL. Upregulation of miR‑101 inhibited DLBCL cell proliferation and facilitated apoptosis by inhibiting the expression of MEK1.

Wilms\u2019 tumor 1-associating protein plays an aggressive role in diffuse large B-cell lymphoma and forms a complex with BCL6 via Hsp90

BackgroundWilms’ tumor 1-associating protein (WTAP) is a nuclear protein, which is ubiquitously expressed in many tissues. Furthermore, in various types of malignancies WTAP is overexpressed and plays a role as an oncogene. The function of WTAP in diffuse large B-cell lymphoma (DLBCL), however, remains unclear.MethodsImmunohistochemistry was applied to evaluate the levels of WTAP expression in DLBCL tissues and normal lymphoid tissues. Overexpression and knock-down of WTAP in DLBCL cell lines, verified on mRNA and protein level served to analyze cell proliferation and apoptosis in DLBCL cell lines by flow cytometry. Finally, co-immunoprecipitation (Co-IP), IP, and GST-pull down assessed the interaction of WTAP with Heat shock protein 90 (Hsp90) and B-cell lymphoma 6 (BCL6) as well as determined the extend of its ubiquitinylation.ResultsWTAP protein levels were consistently upregulated in DLBCL tissues. WTAP promoted DLBCL cell proliferation and improved the ability to confront apoptosis, while knockdown of WTAP in DLBCL cell lines allowed a significant higher apoptosis rate after treatment with Etoposide, an anti-tumor drug. The stable expression of WTAP was depended on Hsp90. In line, we demonstrated that WTAP could form a complex with BCL6 via Hsp90 in vivo and in vitro.ConclusionWTAP is highly expressed in DLBCL, promoting growth and anti-apoptosis in DLBCL cell lines. WTAP is a client protein of Hsp90 and can appear in a complex with BCL6 and Hsp90 in DLBCL. Down-regulation of WTAP could improve the chemotherapeutic treatments in DLBCL.

Expression of MiR-296-5p in Diffuse Large B-Cell Lymphoma and Its Influence on Biological Behavior of Tumor Cells

To detect the expression of miRNA-296-5p in diffuse large B cell lymphoma (DLBCL) cells and study the proliferation, migration and apoptosis of DLBCL cells after interfering the expression of miRNA-296-5p. The expression of miRNA-296-5p in DLBCL cells line DB cells was detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The possible roles of miRNA-296-5p in the biological and behavioral properties of DLBCL-DB cells were explored by transfection of miRNA-296-5p inhibitor for miRNA-296-5p knockdown, and detected by using CCK-8 method, Transwell method and Annexin V-FITC/PI. miRNA-296-5p was highly expressed in DLBCL-DB cells, and the inhibition of miRNA-296-5p could induce suppression of cell proliferation and migration, but the cell apoptosis was not changed significantly. The high expression of miRNA-296-5p may relate with the occurence and development of DLBCL, which may be a new therapeutic target for DLBCL.

Increased interleukin-17A levels promote rituximab resistance by suppressing p53 expression and predict an unfavorable prognosis in patients with diffuse large B cell lymphoma.

Rituximab resistance has become increasingly common in patients with diffuse large B cell lymphoma (DLBCL). However, the mechanisms involved remain unclear. In this study, we aimed to examine the effect of rituximab on interleukin (IL)-17A and to investigate the role of IL-17A in rituximab resistance and its prognostic value in patients with DLBCL. Our retrospective analysis revealed that rituximab increased IL-6 expression levels in patients with DLBCL, and the increased IL-6 levels in turn induced the differentiation of Th17 and IL-17+Foxp3+ Treg cells, which secreted IL-17A both in vivo and in vitro. We then examined the effects of IL-17A on the apoptosis and proliferation of, and p53 expression in DLBCL cells, and found that IL-17A prevented rituximab-induced apoptosis and promoted the proliferation of DLBCL cells by suppressing p53 expression in vitro. The survival data of 73 patients with DLBCL suggested that high peripheral blood levels of IL-17A predicted an unfavorable survival. On the whole, our data indicate that rituximab promotes Th17 and IL-17+Foxp3+ Treg cells to secrete IL-17A, which in turn promotes rituximab resistance, partially by suppressing p53 expression and inhibiting rituximab-induced DLBCL cell apoptosis. IL-17A may thus prove to be a useful prognostic marker in patients with DLBCL.

MiR-155 regulates lymphoma cell proliferation and apoptosis through targeting SOCS3/JAK-STAT3 signaling pathway.

Janus kinase (JAK)- signal transducer and activator of transcription (STAT) signaling pathway participates in regulating cell proliferation, differentiation, and apoptosis, and related to lymphoma. Suppressors of cytokine signaling 3 (SOCS3) is a negative regulator of the JAK-STAT signaling pathway. SOCS3 reduction and miR-155 up-regulation are associated with lymphoma pathogenesis. Bioinformatics analysis showed the complementary binding site between miR-155 and SOCS3. This study aimed to investigate the role of miR-155 in regulating SOCS3/JAK-STAT signaling pathway and affecting diffuse large B cell lymphoma (DLBCL) cell proliferation and apoptosis. DLBCL tumor sample was collected from the patients in our hospital. Lymphatic tissue derived from reactive lymphoid hyperplasia patients were selected as control. MicroRNA-155 (MiR-155) and SOCS3 expressions were detected. Dual luciferase assay was used to verify the targeted relationship between miR-155 and SOCS3. OCI-LY10 cells were cultured in vitro and divided into five groups, including miR-NC, miR-155 inhibitor, pIRES2-Blank, pIRES2-SOCS3, and miR-155 + pIRES2-SOCS3 groups. SOCS3, p-JAK1, p-JAK2, p-STAT3, and Survivin expressions were tested. Cell apoptosis and proliferation were detected by flow cytometry. MiR-155 expression significantly increased, while SOCS3 level declined in DLBCL tissue compared with control. MiR-155 targeted regulated SOCS3 expression. MiR-155 inhibitor and/or pIRES2-SOCS3 transfection markedly up-regulated SOCS3 expression, reduced p-JAK1, p-JAK2, p-STAT3, and Survivin levels, attenuated cell proliferation, and enhanced cell apoptosis in OCI-LY10 cells. Down-regulation of miR-155 inhibited DLBCL cell proliferation and facilitated apoptosis through up-regulating SOCS3 expression to suppress JAK-STAT3 signaling pathway.

HDAC6 regulates microRNA-27b that suppresses proliferation, promotes apoptosis and target MET in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. Histone deacetylase 6 (HDAC6) is frequently altered in DLBCL and inhibition of HDAC6 has potent anti-tumor effects in vitro and in vivo. We profiled miRNAs that altered in the HDAC6 knockdown DLBCL cells with NanoString nCounter assay and identified microRNA-27b (miR-27b) as the most significantly increased miRNA. We validated decreased expression of miR-27b in DLBCL tissues, and we found that low expression of miR-27b was associated with poor overall survival of patients with DLBCL. In addition, forced expression of miR-27b suppressed DLBCL cell viability and proliferation in vitro, and inhibited tumor growth in vivo. Mechanistically, Rel A/p65 is found to negatively regulate miR-27b expression, and its acetylation and block of nuclear translocalization caused by HDAC6 inhibition significantly elevates miR-27b expression. Furthermore, miR-27b targets MET and thus represses the MET/PI3K/AKT pathway. These findings highlight an important role of miR-27b in the development of DLBCL and uncover a HDAC6-Rel A/p65-miR-27b-MET signaling pathway. Elevating miR-27b through HDAC6 inhibition would be a promising strategy for DLBCL treatment.

ICT1 predicts a poor survival and correlated with cell proliferation in diffuse large B-cell lymphoma

Immature colon carcinoma transcript-1 (ICT1) is a crucial member of the large mitoribosomal subunit in mitochondrial ribosome, which has been shown to be closely related to tumorigenesis. Its expression and function in human diffuse large B-cell lymphoma (DLBCL), however, remained elusive. In this study, analysis of public available Oncomine database suggested that the expression levels of ICT1 mRNA was significantly upregulated in DLBCL tissues. Consistently, we described ICT1 was remarkably upregulated in fresh DLBCL samples compared with the corresponding normal tissues using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting. Moreover, ICT1 overexpression was associated with the poor overall survival (OS) of DLBCL patients. Finally, we used DLBCL cell lines to further probe the potential mechanisms, and found shRNA-mediated knockdown of ICT1 significantly suppressed DLBCL cell proliferation, induced cell cycle arrest at G0/G1 phase and apoptosis in vitro. Further verification showed that inhibition of ICT1 gene expression caused the upregulation of the p21, Bad and caspase-3, and downregulation of PCNA, Survivin, CDK4, CDK6 and Cyclin D1. Taken together, this study suggested that ICT1 may play an oncogenic role in human DLBCL by promoting cell proliferation and it might be a biomarker of unfavorable prognosis in DLBCL patients.