Therapeutic Target In Diffuse Large B-cell Lymphoma Research Articles

BCL11A expression worsens the prognosis of DLBCL and its co-expression with C-MYC predicts poor survival

Non-Hodgkin's lymphoma (NHL) is a significant global malignancy, with diffuse large B cell lymphoma (DLBCL) being the most prevalent subtype, accounting for 25–50 % of newly diagnosed cases in China. Despite a 60 % survival rate achieved with R-CHOP regiment for DLBCL, approximately 40 % of patients experience relapse or develop resistance to treatment. While the oncogenic transcription factor B-cell chronic lymphocytic leukaemia/lymphoma 11 A (BCL11A) has been implicated in various tumors, its specific role in DLBCL remains unclear. In this study, we conducted retrospective histomorphological and immunophenotypic analyses on paraffin sample tissues and collected fresh tissue samples for protein and mRNA analyses to investigate the relationship between BCL11A and DLBCL. Additionally, we classified DLBCL into subtypes based on cells of origin (COO) and examined the expressions of BCL11A, C-MYC, P53 and other protein expressions to better understand the factors contributing to poor clinical outcomes in DLBCL. Our findings revealed elevated BCL11A expression in DLBCL, with increased expression associated with worse prognosis and higher C-MYC expression. Patients exhibiting co-expression of C-MYC and BCL11A had significantly lower survival rates compared to those with singular expression. Furthermore, BCL11A protein expression levels demonstrated significant associations with P53 and C-MYC protein expression levels in the Germinal Center B-cell-like (GCB) subtype. These findings suggest that BCL11A may serve as a potential prognostic marker and therapeutic target for DLBCL.

Combinatorial Therapy of CDK9 Inhibitor with CD19 CAR-T to Reciprocally Overcome Therapy Resistance and Enhance Treatment Efficacies Against Aggressive B-Cell Lymphomas

Diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are subgroups of aggressive B-cell lymphoma associated with unacceptably high rates of refractoriness to intensive treatment and significantly shortened survival in this setting. In addition to standard therapies, modern lymphoma care has incorporated targeted agents including Bruton's tyrosine kinase inhibitors and BCL-2 antagonists. Despite achieving initial clinical responses, many patients eventually succumb to disease relapse following treatment with these novel agents. Meanwhile, chimeric-antigen receptor (CAR) T cell therapies have produced unprecedented initial responses in patients with relapsed or refractory DLBCL and MCL, yet at follow-up, only 40% of patients are able to sustain durable remissions. Thus, there is an urgent need to improve the duration of response and delay or prevent CAR-T therapy resistance. To this end, we previously performed mechanistic and translational studies aimed at developing CDK9 as a novel therapeutic target in DLBCL and MCL. This work involved the interrogation of gene expression and functional screening of a large panel of primary patient samples and lymphoma cell lines, ultimately revealing an essential transcription state characterized by increased RNA polymerase II-mediated transcription as the primary driver of therapy resistance evolution and fatal disease progression. Although clinical trials of CDK9 inhibitors (CDK9i) in B-cell lymphomas have shown promise in drug and CAR-T resistant lymphoma patients, mono-targeting of CDK9 has encountered clinical challenges, particularly the development of resistance in initial responders. Thus, understanding CDK9i resistance evolution mechanisms and identifying ways to target the key resistance-associated molecular determinants and subpopulation(s) of cells capable of giving rise to resistant disease holds great potential for advancing lymphoma therapy. Here, we extend our previous work with CDK9i to identify the underlying molecular determinants of CDK9i resistance and, ultimately, to design rational combination therapies to enhance and sustain CDK9i efficacy. In doing so, we are able to demonstrate that: 1) DLBCL and MCL are exquisitely sensitive to CDK9 inhibition (NVP2/AZD4573) regardless of genetic background or resistance status, 2) populations of CDK9i (NVP2/AZD4573) treatment-induced drug tolerant persister (DTP) cells are responsible for driving therapy resistance evolution, 3) CDK9i (NVP2/AZD4573) treatment induces an immunogenic response via activation of proinflammatory and IFN pathways ex vivo and in vivo, and 4) CDK9i and CD19 CAR-T therapies exhibit reciprocally enhanced efficacy in a manner that can overcome therapy resistance in DLBCL and MCL. The outcomes of this study have broad applicability across B-cell malignancies for eradicating both targeted and CAR-T therapy resistance, which we anticipate can be readily translated to the clinic and have immediate impact on DLBCL/MCL patient care.

Eukaryotic initiation factor 3a promotes the development of diffuse large B-cell lymphoma through regulating cell proliferation

BackgroundOne-third of diffuse large B-cell lymphoma (DLBCL) patients suffer relapse after standard treatment. Eukaryotic initiation factor 3a (eIF3a) is a key player in the initial stage of translation, which has been widely reported to be correlated with tumorigenesis and therapeutic response. This study aimed to explore the biological role of eIF3a, evaluate its prognostic and therapeutic potential in DLBCL.MethodsRNA-seq datasets from GEO database were utilized to detect the expression and prognostic role of eIF3a in DLBCL patients. Protein level of eIF3a was estimated by western blot and immunohistochemical. Next, DLBCL cells were transfected with lentiviral vector either eIF3a-knockdown or empty to assess the biological role of eIF3a. Then, samples were divided into 2 clusters based on eIF3a expression and differentially expressed genes (DEGs) were identified. Function enrichment and mutation analysis of DEGs were employed to detect potential biological roles. Moreover, we also applied pan-cancer and chemosensitivity analysis for deep exploration.ResultseIF3a expression was found to be higher in DLBCL than healthy controls, which was associated with worse prognosis. The expression of eIF3a protein was significantly increased in DLBCL cell lines compared with peripheral blood mononuclear cells (PBMCs) from healthy donors. eIF3a knockdown inhibited the proliferation of DLBCL cells and the expression of proliferation-related proteins and increase cell apoptosis rate. Besides, 114 DEGs were identified which had a close linkage to cell cycle and tumor immune. eIF3a and DEGs mutations were found to be correlated to chemosensitivity and vital signal pathways. Pan-cancer analysis demonstrated that high eIF3a expression was associated with worse prognosis in several tumors. Moreover, eIF3a expression was found to be related to chemosensitivity of several anti-tumor drugs in DLBCL, including Vincristine and Wee1 inhibitor.ConclusionsWe firstly revealed the high expression and prognostic role of eIF3a in DLBCL, and eIF3a might promote the development of DLBCL through regulating cell proliferation and apoptosis. eIF3a expression was related to immune profile and chemosensitivity in DLBCL. These results suggest that eIF3a could serve as a potential prognostic biomarker and therapeutic target in 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.

Ras interacting protein 1 facilitated proliferation and invasion of diffuse large B-cell lymphoma cells

ABSTRACT A GTPase binding protein, Ras interacting protein 1 (RASIP1), has been reported with a tumor-promoting role in lung cancer cells, and its role in lymphoma remains unknown. The analysis of medical databank shows that RASIP1 is upregulated in diffuse large B-cell lymphoma (DLBCL) specimens. In this article, we demonstrated that RASIP1 is highly expressed in DLBCL cell lines, compared with primary B cells. The gain- and loss-of-function experiments were performed to investigate the effects of RASIP1 on DLBCL cells. CCK-8, flow cytometry, western blot, and transwell assays demonstrated that silence of RASIP1 inhibited proliferation, cell cycle transition, and invasion and induced significant apoptosis in DLBCL cells, and ectopic expression of RASIP1 played opposite roles. Xenograft results revealed that RASIP1 facilitated the growth of DLBCL cells in vivo. These findings suggest that RASIP1 may be required for malignancy of DLBCL cells. In addition, we also found that the expression of RASIP1 was negatively regulated by forkhead box O3 (FOXO3), which has been reported to suppress the proliferation of DLBCL cells. Our results indicate that FOXO3 is bound to the promoter sequence of RASIP1 and inhibits its transcription. The suppressive effects of FOXO3 on proliferation and invasion of DLBCL cells were neutralized by RASIP1. In conclusion, we demonstrate that FOXO3 negatively regulated RASIP1 facilitates growth and invasion of DLBCL cells, provides novel diagnostic markers and therapeutic targets for DLBCL in clinic.

The Cyclin-G Associated Kinase (GAK) Is a Critical Dependency for Cell-Cycle Progression in Diffuse Large B-Cell Lymphoma Associated with Deregulation of the Retinoblastoma Tumor Suppressor

Background: Recurrent genomic alterations in specific cancers reveal tumor drivers that inform the development of rational therapeutic approaches. In diffuse large B-cell lymphoma (DLBCL), such oncoprotein-focused drug development has achieved only limited success. Phenotypic screening, less explored in DLBCL drug discovery, is an emerging alternative that can be leveraged especially through the application of machine-learning analyses to raw screening data. Under this paradigm, we reveal strong therapeutic potential in DLBCL of inhibiting the cyclin-G associated kinase (GAK). GAK participates in clathrin-mediated endocytosis and regulates mitotic-spindle alignment during cell division. It has been studied as an antiviral target that interrupts internalization at cell membranes but is minimally explored in cancer therapeutics and is a novel target in DLBCL. Methods and Results: We performed a first-of-its-kind screen of kinase inhibitors in DLBCL followed by machine learning-based analysis using our in-house target identification platform, idTRAX. The screen revealed GAK as a strong target for both activated B-cell (ABC) and germinal center B-cell (GCB) derived cell lines with complete sparing of peripheral-blood mononuclear cells (PBMCs), included as non-malignant controls. We analyzed gene-expression data available for 414 previously untreated DLBCL patients (Lenz cohort) and found tumors with high GAK expression had dramatically worse overall survival (OS) compared to those with low expression (hazard ratio=1.84 (95% conf. int. 1.33-2.53), p=0.0002). Mechanistically, we explored GAK's role in endocytosis but found no change in cell-surface B-cell receptor (BCR) expression after treatment with the GAK tool compound SGC-GAK-1. In addition, there was no evident perturbation of signaling pathways downstream of BCR by immunoblotting. We found instead that GAK inhibition in DLBCL results in profound disruption of progression through mitosis, potently triggering the spindle assembly checkpoint (SAC) and causing accumulation in G2 before onset of apoptosis. We explored whether GAK dependency is induced due to cell cycle deregulation. Analysis of gene-expression data in the Lenz cohort showed a striking correlation between high GAK expression and low expression of RB1, encoding the tumor suppressive master cell-cycle regulator retinoblastoma-associated protein (RB, p<0.0001). The ABC-DLBCL cell lines RIVA and U2932 completely lack RB expression and show high sensitivity and accumulation in G2 in response to SGC-GAK-1 treatment. Immunofluorescent confocal microscopy revealed profound disruption of mitotic spindles and shattering of chromosomes in these cells and, to a lesser extent, in RB-competent lines. SGC-GAK-1 is not optimized for use as a chemical probe in vivo, but target validation through shRNA knockdown in DLBCL patient-derived xenograft (PDX) models is underway and expected at time of presentation. Conclusions: GAK is a novel kinase therapeutic target in DLBCL revealed by our target identification platform, idTRAX. GAK's role in mitotic-spindle alignment represents a critical kinase dependency for DLBCL tumors, in particular those with cell-cycle deregulation due to loss of RB function. Our ongoing efforts to synthesize novel GAK inhibitors and reveal synergistic targets may reveal new therapeutic strategies for translation to clinical trials for DLBCL patients with unmet clinical needs.

Knockdown of the long non-coding RNA CACNA1G-AS1 enhances cytotoxicity and apoptosis of human diffuse large B cell lymphoma by regulating miR-3160-5p.

: Long non-coding RNAs (lncRNAs) have been confirmed to be connected with tumor proliferation, apoptosis, metastasis and recurrence. Previous studies have indicated that lncRNA calcium voltage-gated channel subunit α1 G (CACNA1G)-antisense 1 (AS1) can function as a pro-oncogene in several types of cancer. However, the specific role and mechanism of CACNA1G-AS1 have not been fully elucidated in human diffuse large B cell lymphoma (DLBCL). In the present study, CACNA1G-AS1 expression was verified in DLBCL tissues and cells by reverse transcription-quantitative PCR, and the relationship between CACNA1G-AS1 and microRNA (miR)-3160-5p was confirmed using luciferase reporter assays. After CACNA1G-AS1-knockdown and miR-3160-5p-overexpression, MTT, colony formation and flow cytometry assays were conducted to assess the changes in the cytotoxicity and apoptosis of OCI-Ly10 and SUDHL-4 cells. In addition, in vivo experiments were performed to determine the impact of CACNA1G-AS1-knockdown on tumor growth and apoptosis. It was revealed that CACNA1G-AS1 was highly expressed in DLBCL tissues and cells and that expression of CACNA1G-AS1 was associated with the clinical stage of DLBCL. Functionally, CACNA1G-AS1-knockdown was demonstrated to increase cytotoxicity and expedite apoptosis in DLBCL cells in vitro and in vivo. In addition, CACNA1G-AS1 could downregulate miR-3160-5p by targeting binding in DLBCL cells. Overexpression of miR-3160-5p had the same effects on the cytotoxicity and apoptosis of DLBCL cells as CACNA1G-AS1-knockdown. Overall, the present study revealed that CACNA1G-AS1-knockdown and miR-3160-5p-overexpression could prevent DLBCL carcinogenesis, which might provide novel therapeutic targets for DLBCL.

Exploration of the molecular mechanisms, shared gene signatures, and MicroRNAs between systemic lupus erythematosus and diffuse large B cell lymphoma by bioinformatics analysis.

Systemic lupus erythematosus (SLE) is a complex heterogeneous systemic autoimmune disease. Previous studies have shown that SLE may be related to diffuse large B cell lymphoma (DLBCL), but the mechanism of their relationship is still unclear. The present study aimed to explore the common genetic molecular mechanisms, core shared genes, and miRNAs between SLE and DLBCL as well as to investigate the diagnostic markers of DLBCL. The SLE and DLBCL microarray data were downloaded from the comprehensive Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) was used to identify co-expression modules. Four core shared genes were screened out by various algorithms and validated in other cohorts. Finally, we constructed a common core gene-miRNA network using the human microRNA disease database (HMDD) and TarBase. Using WGCNA, four modules were identified as important modules for SLE and DLBCL. Enrichment analysis of the shared genes showed that the highly activated NF-κB pathway was a common feature of the pathophysiology. Four core shared genes, namely, PSMB10, PSMB4, TAF10, and NFΚBIA, were screened out. These core shared genes were significantly upregulated in both diseases, and they may be potential diagnostic markers of DLBCL. The core gene-miRNA network showed that miR-155-5p, regulating the shared NF-κB pathway, may play an important role in the susceptibility of SLE patients to DLBCL. The present study revealed that NF-κB pathway in SLE may be a crucial susceptible factor for DLBCL. In addition, we identified PSMB10, PSMB4, TAF10, NFΚBIA and miR-155 involved in the common pathogenesis as potential biomarkers and therapeutic targets for DLBCL.

CircPCBP2 promotes the stemness and chemoresistance of DLBCL via targeting miR-33a/b to disinhibit PD-L1.

Diffuse large B‐cell lymphoma (DLBCL) is the most common lymphoid malignancy with a high relapse rate of up to 40%. The prognosis of the disease needs improvement and requires a understanding of its molecular mechanism. We investigated the mechanisms of DLBCL development and its sensitivity to chemotherapy by focusing on circPCBP2/miR‐33a/b/PD‐L1 axis. Human DLBCL specimens and cultured cancer cell lines were used. Features of circPCBP2 were systematically characterized through Sanger sequencing, Actinomycin D, RNase R treatment, and FISH. The expression levels of circPCBP2, miR‐33a/b, PD‐L1, stemness‐related markers, ERK/AKT and JAK2/STAT3 signaling were measured using qRT‐PCR, western blotting, and immunohistochemistry. Stemness of DLBCL cells was assessed through spheroid formation assay and flow cytometry. Cell viability and apoptosis upon cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) treatment were determined using MTT assay and flow cytometry, respectively. Interactions of circPCBP2‐miR‐33a/b and miR‐33a/b‐PD‐L1 were validated using dual luciferase activity assay and RNA‐RIP. Nude mouse xenograft model was used to assess the function of circPCBP2 in DLBCL growth in vivo. circPCBP2 was upregulated in human DLBCL specimens and cultured DLBCL cells while miR‐33a/b was reduced. Knockdown of circPCBP2 or miR‐33a/b overexpression inhibited the stemness of DLBCL cells and promoted cancer cell apoptosis upon CHOP treatment. circPCBP2 directly bound with miR‐33a/b while miR‐33a/b targeted PD‐L1 3’‐UTR. circPCBP2 disinhibited PD‐L1 signaling via sponging miR‐33a/b. miR‐33a/b inhibitor and activating PD‐L1 reversed the effects of circPCBP2 knockdown and miR‐33a/b mimics, respectively. circPBCP2 knockdown restrained DLBCL growth in vivo and potentiated the anti‐tumor effects of CHOP. In conclusion, circPCBP2 enhances DLBCL cell stemness but suppresses its sensitivity to CHOP via sponging miR‐33a/b to disinhibit PD‐L1 expression. circPCBP2/miR‐33a/b/PD‐L1 axis could serve as a diagnosis marker or therapeutic target for DLBCL.

A Novel Autochthonous Mouse Model Serves As a Preclinical Evaluation Platform and Explores Dual BTK and BCL2 Inhibition for Activated B Cell-like Diffuse Large B Cell Lymphoma

A Novel Autochthonous Mouse Model Serves As a Preclinical Evaluation Platform and Explores Dual BTK and BCL2 Inhibition for Activated B Cell-like Diffuse Large B Cell Lymphoma

Fascin Inhibition As a Novel Therapeeutic Strategy in Diffuse Large B-Cell Lymphoma

Fascin is an actin bundling protein used to form cellular projections (e.g. filipodia) involved in cell motility and invasion. Cancer cells often upregulate fascin, and in many cancer types this is associated with poor prognosis, likely due to increased invasive and metastatic potential. We have developed fascin inhibitors that prevent actin bundling and block breast cancer metastasis in mice. Here we explored the potential role of fascin inhibition in lymphoma. We have shown that fascin is upregulated in 44% of canine B-cell (n=62) and 57% of T-cell lymphoma cases (n=28), and that increased fascin expression is correlated with worse overall survival (p=0.03). B-cell lymphoma cases had a median survival time of 388 days while that of T-cell cases was 263 days. We have further shown that a fascin inhibitor, NP-G2-044, kills human and canine diffuse large B-cell lymphoma (DLBCL) cells in vitro, and in vivo in mice bearing canine DLBCL xenografts, with GI50s ranging from 5 to 200uM. Animal toxicology studies (performed by Charles River Laboratories) show no hematologic or other toxicity in dogs and rats at concentrations up to 1,000 mg/kg/day for 28 days; these doses are much higher than the doses needed to achieve concentrations that kill lymphoma in vivo . Therefore, we are planning clinical studies in pet dogs with lymphoma to establish proof of principle prior to a human clinical trial. Collectively, our results demonstrate that fascin is a potential new therapeutic target in DLBCL. DisclosuresHuang:Novita: Equity Ownership, Patents & Royalties: Cornell University and Novita Pharmaceutical has patient on the fascin described here. .

Expression and Prognostic Significance of PD-L2 in Diffuse Large B-Cell Lymphoma.

Recent studies suggest that programmed death ligand-2 (PD-L2) constitutes an important antitumor immune response. Here, we investigated the relationship between PD-L2 expression and clinicopathological features in diffuse large B-cell lymphoma (DLBCL). Immunohistochemistry showed that positive expression of PD-L2 was observed in 45 of 181 newly diagnosed patients, including 14 cases with expression exclusively on tumor cells (TCs) and 31 cases with the expression on both TCs and immune cells (ICs) in the tumor microenvironment (TME). In 21 recurrent patients, positive expression of PD-L2 was present in six cases, including two cases with expression exclusively on TCs, and four cases with the expression on both TCs and ICs in the TME. Patients with PD-L2 tumor proportion score (TPS) ≥1% exhibited a better ECOG performance status (PS) (ECOG PS score <2, P = 0.041), lower international prognostic index (IPI) score (P < 0.001), and early Ann Arbor stage (Ann Arbor stage I or II, P = 0.010). Similarly, patients with PD-L2 immune proportion score (IPS) ≥1% also exhibited a better ECOG PS (ECOG PS score < 2, P = 0.006) and lower IPI score (P = 0.001). Survival analysis showed that patients with PD-L2 TPS ≥1% exhibited prolonged overall survival (OS) and progression-free survival (PFS). However, survival analysis showed no prognostic significance based on expression of PD-L2 on ICs in the TME. TC PD-L2 expression was significantly associated with OS (P = 0.041) and PFS (P = 0.001). In the multivariate analysis, TC PD-L2 expression was an independent prognostic risk factor for PFS (P = 0.013), but not for OS (P = 0.249). Furthermore, we found that higher TC and IC PD-L2 expression was associated with higher objective response rate (ORR). Moreover, we demonstrated that the expression level of PD-L2 was positively correlated with the expression status of M1 macrophage markers CD86. Our findings highlight PD-L2 as a promising therapeutic target in DLBCL.

LncRNA SNHG14 promotes oncogenesis and immune evasion in diffuse large-B-cell lymphoma by sequestering miR-152-3p

This study aimed to explore the role of small nucleolar RNA host gene 14 (SNHG14) in the pathogenesis of diffuse large-B-cell lymphoma (DLBCL). DLBCL cell lines (OCI-Ly7 and OCI-Ly3) and specimens from patients were collected to evaluate the roles of SNHG14 in DLBCL pathogenesis. The results showed that SNHG14 expression increased and miR-152-3p expression decreased in DLBCL tissues and cell lines, indicating a negative correlation between miR-152-3p and SNHG14 expression. Moreover, SNHG14 was found to promote DLBCL growth, migration, and EMT-like processes in vitro, and directly inhibits miR-152-3p gene expression via sequestration of the miR-152-3p transcripts in DLBCL. Additionally, SNHG14/miR-152-3p inhibits apoptosis and promotes cell proliferation on cytotoxic T lymphocytes (CTLs) in DLBCL via the PD-1/PD-L1 checkpoint. Furthermore, both the immune escape and progression of DLBCL are advanced by SNHG14 expression via its interactions with miR-152-3p. Collective, this suggests that SNHG14 is a potential diagnostic, prognostic, and therapeutic target for DLBCL.

Targeting Glycosylated Ptgds Displays Anti-Tumor Activities in Diffuse Large B-Cell Lymphoma through Down-Regulation of Wnt Pathway

Introduction: Prostaglandin D2 Synthase (PTGDS), a member of lipocalin superfamily, plays a dual role in catalyzing the conversion of PGH2 to PGD2 and transporting lipophilic substances. PTGDS protein is with different degrees of glycosylation. AT56 is a selective, competitive, and highly bioavailable inhibitor of PTGDS. However, the function and mechanism of PTGDS in diffuse large B-cell lymphoma (DLBCL) remains ill defined. Herein, we aimed to investigate the functional significance of PTGDS and proposed a novel therapeutic strategy for DLBCL. Methods: Lymph node biopsies from 53 de novo DLBCL patients and 28 reactive hyperplasia cases, and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were collected with informed consents. CD19+ B cells were purified by CD19+ magnetic microbeads. The expression levels of PTGDS in DLBCL cell lines and serum were detected by western blotting and ELISA, respectively. Lentivirus vectors were transfected to stably knockdown or overexpress PTGDS. After the treatment of Tunicamycin (Tun), an N-glycosylation inhibitor, western blotting and immunofluorescence were performed to validate the molecular weight and location of PTGDS protein. Results: We first evaluated the expression of PTGDS in DLBCL. Upregulation of PTGDS mRNA in DLBCL cells was identified based on Oncomine database (Fig.1A). Then, the expression level of PTGDS protein in tumor tissue (n=53) was validated to be higher in comparison with control group (Fig.1B). Furthermore, survival analysis revealed that PTGDS high expression was associated with reduced overall survival of DLBCL patients (Fig.1C). Moreover, high level of soluble PTGDS protein was detected in the serum of DLBCL patients (n=53, Fig.1D). High expression of PTGDS was also confirmed in DLBCL cells by western blotting (Fig.2A). The biological function of PTGDS in progression of DLBCL was further verified. Gene ontology and KEGG analysis revealed that PTGDS was enriched in cellular process and biological regulation (Fig.2B). After the treatment with rhPTGDS, increased proliferation of DLBCL cell was observed in a dose-dependent manner (Fig.2D), and overexpression of PTGDS also promoted cell growth (Fig.2E). Furthermore, knockdown of PTGDS (shPTGDS) significantly restrained cell proliferation (Fig.2F), promoted cell cycle arrest (Fig.2G) and cell apoptosis (Fig.2H). AT56 suppressed the proliferation of DLBCL cells in a dose- and time-dependent manner (Fig. 3A). Additionally, with the treatment of AT56, DLBCL cells exhibited induced G0/G1 phase arrest (Fig. 3B) and increased cell apoptosis (Fig. 3C). As Bendamustine and Adriamycin were found to decrease the mRNA level of PTGDS (Fig. 3D), we further observed that AT56 enhanced sensitivity to them in cell proliferation (Fig. 3E) and cell apoptosis (Fig. 3F). Next, we explored the underlying mechanism of PTGDS in DLBCL progression. The expression of Wnt pathway molecules, such as p-LRP6, β-catenin, p-GSK3-β, Lef-1, p-STAT3, were decreased with treatment of AT56 in dose-dependent manner (Fig. 4A). Besides, STAT3 inhibitor WP1066 was found to restore the proliferation promotion (Fig. 4B) caused by PTGDS overexpression. Moreover, we validated that Wnt pathway activator Wnt3a could restore the phenotype changes caused by AT56, including cell proliferation (Fig. 4C), cell apoptosis (Fig. 4D) and cell cycle (Fig. 4E). Glycosylation, a kind of post-translational modification, could significantly alter protein function and then cellular characteristics. Protein Blast analysis indicated the potential glycosylation sites (Fig. 5A) on PTGDS protein (Ser29, Asn51 and Asn78). Furthermore, Tunicamycin was used to inhibit the N-glycosylation of protein and molecular weight of PTGDS changed from 37kD to 21kD (Fig.5B). Besides, the deglycosylation could promote the translocation of PTGDS into the nucleus (Fig.5C-D), indicating the potential role of glycosylated PTGDS in DLBCL. Conclusions : Our investigations identified for the first time the aberrant overexpression of PTGDS in DLBCL, which was associated with poor prognosis. AT56 exerted anti-tumor effect via inhibiting Wnt pathway. Besides, PTGDS protein in DLBCL cells were highly glycosylated and deglycosylation promoted its translocation into nucleus, indicating the mechanism of PTGDS in DLBCL. Further investigation is warranted to substantiate PTGDS as a promising therapeutic target in DLBCL. Disclosures No relevant conflicts of interest to declare.

Systematic Analysis of Competing Endogenous RNA Networks in Diffuse Large B-Cell Lymphoma and Hodgkin's Lymphoma.

Lymphoma is a systemic malignancy, originating from the lymphatic system, which accounts for 3 to 4% of all tumors. There are two major subtypes of lymphoma, namely, diffuse large B-cell lymphoma (DLBCL) and Hodgkin’s lymphoma (HL). Elucidation of the pathogenesis of these two lymphoma types is crucial for the identification of potential therapeutic targets. Compared with the corresponding knowledge of other diseases, the understanding of the regulatory networks involved in DLBCL and HL is relatively deficient. To address this, we comprehensively analyzed the mRNAs, lncRNAs, and miRNAs that were differentially expressed between normal and tumor samples of DLBCL and HL. In addition, functional enrichment analysis of the differentially expressed mRNAs was performed. We constructed two specific ceRNA networks of DLBCL and HL. The pathways enriched by dysregulated mRNAs in DLBCL and HL were mainly involved in immune responses, transcription process, and metabolism process. The ceRNA network analysis revealed that 45 ceRNAs were shared between the two ceRNA networks, including five pivotal lncRNAs (MALAT1, CTBP1-AS, THUMPD3-AS, PSMA3-AS1, and NUTM2A-AS1). In addition, we proposed a DLBCL survival risk model based on a DLBCL-specific network constructed by Lasso regression analysis. The model, which is based on eight mRNAs, exhibited excellent performance in regard to predicting outcomes in DLBCL patients, with a p value of 0.0017 and AUC of 0.9783. In summary, although the molecular mechanisms underlying tumorigenesis in DLBCL and HL were quite different, the same pivotal lncRNAs acted as key regulators. Our findings identify novel potential prognostic and therapeutic targets for DLBCL and HL.

MYC-regulated lncRNA NEAT1 promotes B cell proliferation and lymphomagenesis via the miR-34b-5p-GLI1 pathway in diffuse large B-cell lymphoma

BackgroundLncRNA NEAT1 has been identified as a tumour driver in many human cancers. However, the underlying mechanism of lncRNA NEAT1 in diffuse large B-cell lymphoma (DLBCL) progression is unclear.MethodsThe expression levels of NEAT1, GLI1 and miR-34b-5p were detected by RT-qPCR and Western blotting in DLBCL tissues and cell lines. MTT and colony formation assays were performed to examine cell proliferation, while annexin-V staining and TUNEL assays were performed to measure cell apoptosis. The effect of NEAT1, GLI1 and miR-34b-5p on cell cycle-associated proteins was evaluated by Western blotting. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were employed to investigate the interaction between NEAT1 and miR-34b-5p or GLI1 and miR-34b-5p. Moreover, chromatin immunoprecipitation (ChIP) was performed to demonstrate the interaction between MYC and NEAT1.ResultsNEAT1 and GLI1 were upregulated while miR-34b-5p was downregulated in DLBCL tissues and cell lines compared to normal controls. Knockdown of NEAT1 or overexpression of miR-34b-5p inhibited cell proliferation but promoted cell apoptosis. Overexpression of NEAT1 reversed GLI1-knockdown induced attenuation of cell proliferation. In other words, NEAT1 acted as a competing endogenous RNA (ceRNA), regulating the miR-34b-5p-GLI1 axis, further affecting the proliferation of DLBCL. Moreover, MYC modulated NEAT1 transcription by directly binding to the NEAT1 promoter.ConclusionWe revealed that MYC-regulated NEAT1 promoted DLBCL proliferation via the miR-34b-5p-GLI1 pathway, which could provide a novel therapeutic target for DLBCL.

Targeting Polo-like Kinase 4 for Therapeutic Benefit in the Patients with Diffuse Large B-Cell Lymphoma

Abstract: Polo-like kinase 4 (PLK4) is a serine/threonine kinase that plays a critical role in regulating centriole duplication. PLK4 overexpression causes centriole amplification and genomic instability, which is the hallmark of cancer. Thus, PLK4 has been suggested as a tumor promoter role in solid malignancies. CFI-400945 is a highly selective small-molecule inhibitor of PLK4. CFI-400945 treatment results in centriole duplication defects, which leads to mitotic catastrophe and cell death. The antineoplastic effects of CFI-400945 have been reported in many cancers and this inhibitor has entered Phase I clinical trials for solid tumors. However, the role of PLK4 and cellular effects of CFI-40045 in hematological malignancies remains elusive. In the present study, we found that PLK4 expression is up-regulated at both transcriptional and translational levels in diffuse large B-cell lymphoma (DLBCL) cell lines (Fig. 1a, Fig. 1b). DLBCL patients displayed high protein levels of PLK4 as visualized by immunohistochemistry (Fig. 1c). In vitro, treatment of DLBCL cell lines with CFI-400945 induced a decrease in cell proliferation and an increase in cell apoptosis and a block at the G2-M transition in a dose-dependent manner (Fig. 1d, Fig. 1e, Fig. 1f). Furthermore, we found that the antineoplastic effects of CFI-400945 in DLBCL cell lines were due to the increased DNA damage, as observed by increased accumulation of γH2AX through phosphorylation of CHK1/CHK2 (Fig. 1g). Since PLK4 inhibition affects DNA damage, we next aimed to explore the effects of PLK4 expression on sensitivity of DNA damage-inducing drugs (e.g., doxorubicin). We found that silencing of PLK4 via shRNA improve the anti-tumor effects of doxorubicin in DLBCL cell lines (Fig. 1h, Fig. 1i). PLK4 inhibition in combination with doxorubicin significantly increased phosphorylation of H2AX compared to cells treated with doxorubicin alone (Fig. 1j). In summary, our data indicates that PLK4 is aberrantly expressed in DLBCL cell lines and tissues. Targeting PLK4 with the selective inhibitor CFI-400945 suppresses cell proliferation and induces apoptotic deaths and causes DNA damage in vitro. Our findings established PLK4 as a potential therapeutic target in DLBCL. Silencing of PLK4 expression induces accumulation of DNA damage and enhances the cytotoxic effects of doxorubicin. Thus, the levels of PLK4 may serve as a determinant of doxorubicin sensitivity and a predictive biomarker of the patients with DLBCL treated with R-CHOP based therapy. Figure 1 Disclosures No relevant conflicts of interest to declare.

Abstract 2055: Differential expression of TP73 isoforms modulates cell survival and therapeutic response in diffuse large B-cell lymphoma

Abstract Rearrangements of the chromosome locus 1p36 with ensuing deletion or disruption of TP73, one of the most distally located putative tumor suppressor genes, is frequent in non-Hodgkin lymphoma (NHL) and confers inferior prognosis. TP73 shares homology to TP53 and is capable of transactivating p53 target genes. Earlier reports in normal B-cells and lymphoma animal models suggest an important role of p73 in B-cell lymphomas, however, the biological significance of p73 isoforms in Diffuse large B-cell lymphoma (DLBCL) is not clear. Diffuse large B-cell lymphoma is the most common form of NHL, and although long-term remission is achieved with current therapies, relapse occurs in almost one-third of the patients. Previously, we have demonstrated that the differential expression of p73 isoforms (TAp73 and ΔNp73) correlate with proliferation and apoptosis in DLBCL. In the present study, we examined whether modulating the expression of these p73 isoforms in DLBCL cells regulates cell survival, proliferation, and response to the chemotherapeutic agent, doxorubicin. We performed in vitro studies in DLBCL cells and modulated the expression of the two opposing p73 isoforms (TAp73 and ΔNp73) using expression vectors and siRNA in DHL16 (GCB subtype) and OCI-Ly3 (ABC subtype) cells. Under normal conditions TAp73-transfected cells showed comparable growth to control vector-transfected cells; however, the TAp73-transfected cells were more susceptible to serum deprivation as well as doxorubicin. In contrast, ΔNp73-transfected cells had accelerated growth when compared with control vector-transfected cells under normal conditions and were more resistant to serum deprivation and doxorubicin treatment. Subsequently, we analyzed the p73 direct transcriptional pro-apoptotic targets in cells transfected with TAp73 and ΔNp73 and vector control. Both the common p53 and p73 targets (PUMA and NOXA) as well as the exclusively p73 targetsGRAMD4 and BIM, were up-regulated in the TAp73-transfected cells compared to control cells. A reduction of PUMA, BIM, and GRAMD4 was observed in the ΔNp73-transfected cells as compared to control cells. Furthermore, we observed that p73 knockdown using p73 siRNA altered the growth and the response to doxorubicin in both DLBCL cell lines. Together, these data demonstrate the significant biological role of p73 isoforms in DLBCL pathogenesis and therapeutic response. Our findings illustrate that modulation of TP73 isoforms induces apoptosis and expression of pro-apoptotic targets, thus establishing p73 as a potential therapeutic target in DLBCL. Citation Format: Hesham M. Hassan, Michelle L. Varney, Sugandha Saxena, Rakesh K. Singh, Bhavana J. Dave. Differential expression of TP73 isoforms modulates cell survival and therapeutic response in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2055.

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.

Lnc SMAD5-AS1 as ceRNA inhibit proliferation of diffuse large B cell lymphoma via Wnt/\u03b2-catenin pathway by sponging miR-135b-5p to elevate expression of APC

Diffuse large B cell lymphoma (DLBCL) is a common and fatal hematological malignancy. Long noncoding RNAs (lncRNAs) have emerged as crucial biomarkers and regulators in many cancers. Novel lncRNA biomarker in DLBCL needs to be investigated badly, as well as its function and molecular mechanism. To further explore, microarray analysis was performed to identify the differentially expressed lncRNAs in DLBCL tissues. To investigate the biological functions of SMAD5-AS1, we performed gain- and loss-of-function experiments in vitro and in vivo. Furthermore, bioinformatics analysis, dual-luciferase reporter assays, Argonaute 2-RNA immunoprecipitation (AGO2-RIP), RNA pull-down assay, quantitative PCR arrays, western blot assay, TOPFlash/FOPFlash reporter assay, and rescue experiments were conducted to explore the underlying mechanisms of competitive endogenous RNAs (ceRNAs). We found that SMAD5-AS1 was down-regulated in DLBCL tissues and cell lines. Functionally, SMAD5-AS1 downregulation promoted cell proliferation in vitro and in vivo, whereas SMAD5-AS1 overexpression could lead to the opposite effects in vitro and in vivo. Bioinformatics analysis and luciferase assays revealed that miR-135b-5p was a direct target of SMAD5-AS1, which was validated by dual-luciferase reporter assays, AGO2-RIP, RNA pull-down assay, and rescue experiments. Also, dual-luciferase reporter assays and rescue experiments demonstrated that miR-135b-5p targeted the adenomatous polyposis coli (APC) gene directly. SMAD5-AS1/miR-135b-5p inhibits the cell proliferation via inactivating the classic Wnt/β-catenin pathway in the form of APC dependency. Our results indicated that SMAD5-AS1 inhibits DLBCL proliferation by sponging miR-135b-5p to up-regulate APC expression and inactivate classic Wnt/β-catenin pathway, suggesting that SMAD5-AS1 may act as a potential biomarker and therapeutic target for DLBCL.