Human Diffuse Large B-cell Lymphoma Cell Research Articles

Abstract LB081: Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma

Abstract Telomerase (TERT) is an enzyme critical for carcinogenesis expressed in about 90% of human cancers, including aggressive diffuse large B-cell lymphoma (DLBCL). Targeting TERT proven difficult due to on-target toxicities to non-malignant hematopoietic stem cells and potentially to activated immune effector cells. To overcome these challenges, we developed novel TLR9+ cell-selective CpG-oligodeoxynucleotides (ODNs) to deliver telomere-damaging nucleoside analogue, 6-thio-2’-deoxy-guanosine (6tdG), into target B cell lymphoma cells in vitro and in vivo. Human and mouse TLR9+/TERT+ lymphoma cells rapidly internalized CpG(6tdG)-oligonucleotides through active endocytosis. 6tdG molecules were released from the 3’ end of the oligonucleotide into cytosol and cell nucleus. TERT-mediated 6tdG incorporation into telomeres, led to DNA damage and lymphoma cell death. In vitro, CpG(6tdG)-oligonucleotides showed potency in targeting variety of human DLBCL cells in nanomolar range without detectable toxicity to non-malignant TERT— cells or human TERT+ hematopoietic stem cells and activated T cells. Importantly, the repeated systemic administration of CpG(6tdG)-oligonucleotides was well-tolerated by humanized hCD34/NOG mice, without detectable decrease in the percentage of hematopoietic stem cells, myeloid cells or T cells although the numbers of B cells were partly reduced as expected. Intravenously injected CpG(6tdG)-oligonucleotides, but not 6tdG nucleoside, had potent and dose-dependent antitumor effects against xenotransplanted models of human GC- or ABC-subtypes of DLBCL, such as OCI-Ly18 or OCI-Ly3, respectively. Furthermore, we assessed CpG(6tdG)-oligonucleotide activity against aggressive, syngeneic TERT+ Eμ-myc/15A lymphoma in immunocompetent mice. CpG(6tdG)-oligonucleotide, but not the CpG-oligonucleotide alone, 6tdG alone or co-injection of thereof, was effective in triggering regression of established 15A tumors with long-term survival of the majority of mice. The effect of CpG(6tdG)-oligonucleotide correlated with the activation of tumor antigen-specific T cell-mediated antitumor immune responses. In contrast, when tested in immunodeficient mice, CpG(6tdG)-oligonucleotide induced significant but transient regression of 15A lymphoma. These results suggest that the oligonucleotide elicits two-pronged activity triggering direct lymphoma cell cytotoxicity and also immune-mediated antitumor effects. Overall, this cell-selective strategy could provide an effective and safer strategy against aggressive TERT+ DLBCL cells with potential to engage and support T cell-based antitumor immunity. Citation Format: Chunsong Yu, Yong Liang, Piotr Swiderski, Larry Kwak, Marcin Kortylewski. Novel targeted oligonucleotide-based telomere disruptor for immunotherapy of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB081.

Abstract 605: ABBV-101, a highly potent and selective clinical stage Bruton tyrosine kinase degrader, overcomes BTK mutation-induced resistance to BTK inhibitors

Abstract Bruton tyrosine kinase (BTK), a clinically validated target for various B-cell malignancies, plays a critical role in regulating cell growth, adhesion, and homing to lymphoid tissues that provide a microenvironment favorable to cancer cells. Despite the success of approved covalent BTK inhibitors in diseases such as CLL, most patients do not achieve complete response with monotherapy and eventually relapse, often via the emergence of the BTK-C481S mutation. Although second-generation reversible BTK inhibitors may temporarily overcome the C481S mutation, novel BTK mutations associated with resistance emerged in the clinic recently. Additionally, covalent BTK inhibitors have not shown sufficient differentiation from standard-of-care (SoC) in B-cell malignancies, such as diffuse large B cell lymphoma (DLBCL) to warrant approval to date. To address these unmet needs, a next-generation orally bioavailable BTK degrader ABBV-101 has been developed. Unlike BTK inhibitors that solely bind to and impede the catalytic domain of BTK, ABBV-101 eliminates the protein in a highly selective fashion. This degradation mechanism targets both the catalytic and scaffolding functions of BTK, and thus may enable deeper and more durable responses in patients with B-cell malignancies. Cellular activity of ABBV-101 was determined in a panel of DLBCL cell lines. We show that most non-germinal center B cell DLBCL (non-GCB-DLBCL) cell lines are sensitive to ABBV-101. To determine whether ABBV-101 could overcome BTK mutations-induced resistance, we engineered human DLBCL cell line TMD8 using CRISPR technology to express these BTK mutations and tested for sensitivity to ABBV-101 and BTK inhibitors. We show that ABBV-101 demonstrates similar potent activity against all BTK mutations associated with resistance to both covalent and reversible BTK inhibitors. The modulation of downstream pathway by ABBV-101 and how it differentiates from other BTK inhibitors will be presented. Further, ABBV-101 induces complete tumor regression in the BTK-C481S TMD8 DLBCL xenograft model. A spontaneous mouse CLL model carrying BTK-C481S mutation was developed through crossing BTK-C481S knock-in mice with the Emu-TCL1 transgenic mice in the C57BL/6 background. ABBV-101 completely inhibits BTK-C481S Emu-TCL1 CLL burden increase in the blood compartment and reduces CLL burden in the spleen and lymph nodes, whereas covalent BTK inhibitors show no activity. Finally, combination with a BCL-2 inhibitor enhances the efficacy of ABBV-101 in CLL and DLBCL models. Taken together, our results suggest that ABBV-101 has potent in vitro and in vivo efficacy. ABBV-101 is currently in phase 1 clinical trial for a variety of B-cell malignancies. ClinicalTrials.gov identifier: NCT05753501 Citation Format: Chin Pan, Jacob Riehm, Tarikere L. Gururaja, Haiyan Li, Henry Nguyen, Yongjiao Zhai, Xiaoping Xie, Rong-Xian Ding, Rebecca Matthew, Hana Y. Hoh, Zhaozhong J. Jia, Bo Liu, Cassandra P. Shu, Claudina A. Stevenson, Christine Will, Federico Innocenti, Wissam Assaily, Lloyd T. Lam, Alexey Rivkin. ABBV-101, a highly potent and selective clinical stage Bruton tyrosine kinase degrader, overcomes BTK mutation-induced resistance to BTK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 605.

Kaempferol inhibits the proliferation and migration of Epstein-Barr virus-positive diffuse large B-cell lymphoma cells

Purpose: To assess the effect of kaempferol on the growth, apoptosis, and motility of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) cells, and to elucidate its mechanism of action. 
 Methods: Human DLBCL cells were used to generate EBV-positive (EBV +) cell line Pfeiffer. CCK-8, colony formation, flow cytometry (FCM), and immunoblot assays were employed to determine the effects of kaempferol on the growth and apoptosis of EBV + DLBCL cells. Cell motility was evaluated by Transwell and immunoblot assays. Immunoblot assay was further conducted to unravel the mechanism of action. 
 Results: Kaempferol inhibited the growth of EBV + DLBCL cells. It also enhanced the apoptosis of EBV + DLBCL cells, but inhibited the motility of the cells. Furthermore, kaempferol inhibited PI3K/AKT axis, thereby suppressing DLBCL. 
 Conclusion: Kaempferol inhibits the growth and motility of EBV + DLBCL cells via PI3K/AKT axis. It is therefore a potential drug for the treatment of DLBCL.

High Level of IL-16 Secreted By Non-GCB DLBCL Facilitates Tumor Growth Though the Tumor-Promoting and Immunosuppressive Tumor Microenvironment

Introduction: Diffuse Large B-Cell Lymphoma (DLBCL) is a highly heterogeneous non-Hodgkin's lymphoma. Based on cell origin, DLBCL is classified into two main molecular subtypes: the germinal center B-cell-like (GCB) subtype and the activated B-cell-like (ABC) subtype. ABC subtype is more aggressive and associated with a worse prognosis than GCB subtype. Previous studies have shown that cytokines play an important role in the growth of ABC subtypes, and IL-16 was supposed to be highly expressed in ABC-DLBCL, however it has not been explored in depth in DLBCL. In this study, we broadly assessed the expression and role of IL-16 on DLBCL. Method: Serum and tissues samples from patients with DLBCL and whole blood from healthy donors were collected. The characterization of protein expression, mRNA expression, and levels of cytokines in cells and tissues was detected by Western blotting, IHC/IHF, PCR, ELISA, RNA-seq, and Flow cytometry. Cells experiments such as cell proliferation, cell apoptosis, cell cycle, cell migration, and ADPC were detected by CCK-8, Flow cytometry, and Transwells. Gene knockdown or overexpression was conducted by lentiviruses and then verified by Western blotting and ELISA. We established subcutaneous tumors (human DLBCL cells and murine DLBCL cells) in BALB/c mice, BALC/c nude mice, and NOD SCID mice for the tumor size or the therapeutic evaluation. Results: First, we found that IL-16, secreted by the cleavage of active caspase-3 from the lymphoma cells under tumor burden, was highly expressed in ABC-DLBCL compared to GCB-DLBCL. The serum IL-16 levels were positively correlated with unfavorable prognosis in DLBCL patients. The dynamic change of serum IL-16 levels accurately predicted the treatment response in patients with DLBCL who received R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone) or anti-CD19 Chimeric Antigen Receptor (CAR) T cells. Interestingly, bioactive IL-16 showed rare effect on DLBCL cells in vitro, but significantly promoted their growth in vivo. Mechanically, tumor-derived bioactive IL-16 promoted the migration of monocytes via CD4+ receptor, thereby inducing the enrichment of monocytes-derived tumor-associated macrophages in the tumor microenvironment (TME) of DLBCL, which accelerated the tumor growth primarily by expressing cytokines such as IL-6, inducing angiogenesis, and exerting immunosuppressive effects through the expression of cytokines like IL-10; thus, conferring growth advantage to the ABC- DLBCL. To translate the findings into clinical application, we design a novel treatment strategy to overcome the poor outcomes in ABC-DLBCL patients with high IL-16 expression by combination of high-dose cyclophosphamide (CTX) and the bispecific antibody targeting CD20 and CD47 (IMM0306). IMM0306 effectively reversed the tumor-promoting effects of macrophages in subsets of ABC-DLBCL, while CTX induced IL-16 and increased the infiltration of macrophages in the TME. The increasing effector cells/tumor cells ratio further improved the antitumor effect of IMM0306. The combination of high-dose CTX and IMM0306 was confirmed to significantly inhibit the growth of ABC-DLBCL with high IL-16 expression. A clinical trial is being designed to evaluate the potential for clinical applications. Conclusion: In conclusion, this study provides insight into the expression and role of IL-16 in DLBCL. IL-16 is proposed to be highly expressed in ABC-DLBCL, and serum IL-16 indicate the tumor burden, treatment response, and clinical prognosis of DLBCL patients. IL-16 may be a key driver of the TME of DLBCL, which can confer growth advantage to the ABC-DLBCL. Our data provides a theoretical basis for future clinical treatment. A novel treatment regimen (IMM0306 and CTX) could attenuate the pro-oncogenic roles of IL-16 to prevent ABC-DLBCL progression.

Abbv-101, a Highly Potent and Selective Clinical Stage Bruton Tyrosine Kinase Degrader for the Treatment of B-Cell Malignancies

Bruton tyrosine kinase (BTK), a clinically validated target for various B-cell malignancies, plays a critical role in regulating cell growth, adhesion and homing to key lymphoid tissues that provide a microenvironment favorable to cancer cells. Despite the success of approved covalent BTK inhibitors in diseases such as CLL, most patients do not achieve complete response with monotherapy and eventually relapse, often via the emergence of the BTK-C481S mutation. Additionally, covalent BTK inhibitors have not shown sufficient differentiation from standard-of-care (SoC) in B-cell malignancies such as diffuse large B cell lymphoma (DLBCL) to warrant approval to date. To address these unmet needs, a next-generation orally bioavailable BTK degrader ABBV-101 has been developed. Unlike BTK inhibitors that solely bind to and impede the catalytic domain of BTK, ABBV-101 eliminates the protein in a highly selective fashion. This degradation mechanism targets both the catalytic and scaffolding functions of BTK, and thus may enable deeper and more durable responses in patients with B-cell malignancies. In preclinical studies, the BTK degrader ABBV-101 has shown potent in vitro and in vivo efficacy in BCR pathway-dependent leukemia/lymphoma models, including covalent BTK inhibitor resistant BTK-C481S systemic mouse CLL and human DLBCL models. ABBV-101 degrades BTK-WT and BTK-C481S with similar sub-nanomolar potency in the human DLBCL cell line TMD8 which translates to potent cellular growth inhibition. In addition to the BTK-C481S mutation, ABBV-101 demonstrates similar potent activity against novel BTK mutations associated with resistance to reversible BTK inhibitors in the clinic, an emerging unmet need. ABBV-101 induces complete tumor regression in the BTK-C481S TMD8 DLBCL xenograft model. A spontaneous mouse CLL model carrying BTK-C481S mutation was developed through crossing BTK-C481S knock-in mice with the Emu-TCL1 transgenic mice in the C57BL/6 background. ABBV-101 completely inhibits BTK-C481S Emu-TCL1 CLL burden increase in the blood compartment and reduces CLL burden in the spleen and lymph nodes, whereas covalent BTK inhibitors show no activity. Further, ABBV-101 induces complete tumor regressions in multiple non-GCB DLBCL PDX models, demonstrating deeper and more durable response than covalent and reversible inhibitors. Combination with a BCL-2 inhibitor enhances the efficacy of ABBV-101 in CLL and DLBCL models. Kinome and global proteomics profiling showed ABBV-101 to be highly selective, a designed feature that may enable favorable tolerability in patients. ABBV-101 is currently in phase 1 clinical trial for a variety of B-cell malignancies. ClinicalTrials.gov identifier: NCT05753501

ONO-7018, a First-in-Class MALT1 Inhibitor, Provides Novel Therapeutic Strategies for B Cell Malignancies: Overcoming BTK Inhibitor Acquired Resistance and Enhancing the Antitumor Effect of BTK Inhibitors

Introduction: B cell receptor (BCR) signaling plays a crucial role in promoting survival and growth in B cell malignancies, and several compounds targeting the signaling pathways have been approved. Bruton's tyrosine kinase (BTK) is a key molecule in BCR signaling pathways and BTK inhibitors are one of the essential treatment options for chronic lymphocytic leukemia (CLL) and certain subtypes of B cell Non-Hodgkin Lymphoma. However, there are issues that acquired resistance mutations in the BTK kinase domain and gain of function mutation of its downstream molecules, which lead to BTK inhibitor resistance and limit its efficacy. For example, BTK C481S mutation is well known as a resistant mutation to covalent BTK inhibitors and several mutations of BTK (such as T474I and L528W mutation) have recently been reported in relapsed or refractory CLL patients with acquired resistance to pirtobrutinib, a non-covalent BTK inhibitor. In addition, there are unmet medical needs to improve the duration of response of current standard therapies including BTK inhibitors for B cell malignancies. Therefore, therapeutic strategies to overcome the BTK inhibitor resistance and novel combination therapies to enhance the efficacy of BTK inhibitors are required. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is known as a key growth and survival regulator in BCR signaling and an emerging therapeutic target. ONO-7018 (formerly known as CTX-177), a potent and selective MALT1 inhibitor, demonstrated preclinical efficacy in several lymphoma models. Aim: Firstly, to explore the potential of ONO-7018 to overcome BTK inhibitor acquired resistance, we tested the antitumor effects of ONO-7018 on cell lines harboring BTK inhibitor-resistant mutations. Secondly, we examined the combination antitumor efficacy of ONO-7018 and tirabrutinib, a second generation BTK inhibitor in vitro and in vivo. Methods: Diffuse large B-cell lymphoma (DLBCL) cell lines harboring BTK inhibitor-resistant mutations (BTK T474I, C481S, or L528: PLCG2 R665W) were generated by continuous passage with BTK inhibitor or CRISPR-Cas9 gene editing followed by single cell cloning. In in vivo study, human DLBCL cell line, TMD8 cells were subcutaneously implanted in immune deficient mice. ONO-7018 and tirabrutinib were orally administered to the mice twice a day. Results: Anti-proliferative activities of ONO-7018 on these BTK inhibitor resistant cell lines were evaluated in vitro. ONO-7018 inhibited the proliferation of these cells with comparable IC 50 values to the parent cell line. Anti-proliferative activities of ONO-7018 alone or in combination with tirabrutinib on DLBCL and mantle cell lymphoma (MCL) cell lines were evaluated in vitro. ONO-7018 exhibited synergistic effects with tirabrutinib in both DLBCL and MCL cell lines. In addition, combination treatment of ONO-7018 with tirabrutinib strongly inhibited the expression of interferon regulatory factor 4, which is a downstream signal molecule in BCR signaling pathways, compared to their single treatment. We then evaluated the antitumor effects of ONO-7018 in combination with tirabrutinib in the xenograft model. Combination treatment of ONO-7018 with tirabrutinib showed dramatic antitumor effect with tumor regression. These results suggest that ONO-7018 could demonstrate antitumor effect on patients with B cell malignancies harboring BTK inhibitor acquired resistant mutations and synergistic antitumor effect with BTK inhibitors in clinic. Conclusion: ONO-7018 would provide novel therapeutic strategies to overcome the BTK inhibitor acquired resistance and enhance the antitumor effect of BTK inhibitors in clinic. Phase 1 study of ONO-7018 (NCT05515406) is currently ongoing.

A Combination of CPI-0610 and SAHA Induces Apoptosis through STAT3 and p38 Signalling Pathways in Diffuse Large B-cell Lymphoma Cells.

Although immunotherapies have greatly improved diffuse large B-cell lymphoma (DLBCL) prognosis, a proportion of patients remain to be relapsed or refractory. Therefore, the identification of novel therapeutic targets and drugs is urgently required. Inhibition of the bromodomain and extra-terminal (BET) proteins has been a promising therapeutic strategy for various haematologic cancers. CPI-0610 is a potent and selective BET inhibitor. The effects of CPI-0610 in DLBCL cells have not been reported yet. The aim of this study was to assess the effects of CPI-0610 in DLBCL and its underlying mechanisms. DLBCL cells were treated with CPI-0610, followed by measuring cell viability, cell cycle, apoptosis, autophagy, and specific cell signaling pathways. Moreover, immunodeficient mice were engrafted with SUDHL2 cells and then treated with CPI-0610 for analysis of tumor burden. We also analyzed the synergistic effect of CPI-0610 with histone deacetylase inhibitor suberoylanilide hydroxamic acid. The present study demonstrated that CPI-0610 displayed cell cytotoxicity by arresting the G1 cell cycle and inducing endogenous and exogenous apoptotic pathways. Additionally, CPI-0610 decreased BRD4 and c-Myc expressions and affected MAPK, JAK/STAT, and AKT signalling pathways in human DLBCL cells. An in vivo experiment exhibited that CPI-0610 decreased the primary tumour growth of the DLBCL xenograft model. Furthermore, the use of CPI-0610 in combination with suberoylanilide hydroxamic acid exhibited a specific synergistic effect in inducing apoptosis through the regulation of STAT3 and p38. Targeting BET may be an effective therapeutic strategy and potentiated by a combination with histone deacetylase inhibition in DLBCL.

Effect of MiR-424-5p on the Drug Resistance of Diffuse Large B-Cell Lymphoma Cells by Regulating PD-1/PD-L1 Signaling Pathway

To explore the effect of microRNA-424-5p (miR-424-5p) on the drug resistance of diffuse large B-cell lymphoma (DLBCL) cells by regulating the programmed death receptor-1 (PD-1)/programmed death ligand-1 (PD-L1) signaling pathway. Human DLBCL cell line CRL2631 cells were induced to construct CRL2631-CHOP resistant cell line. RT-qPCR and Western blot were used to detect the expression levels of MiR-424-5p, PD-L1 mRNA and protein, and multidrug resistance gene-1 (MDR-1) protein in CRL2631 cells and CRL2631-CHOP cells, respectively. The target genes of MiR-424-5p was verified by dual luciferase reporter assay. The miRNA simulation/interference technology and thiazole blue (MTT) method were used to detect the resistance of CRL2631 cells and CRL2631-CHOP cells to CHOP. Compared with CRL2631 cells, the drug resistance of CRL2631-CHOP cells to CHOP and the levels of MDR-1 protein (P<0.05), PD-L1 mRNA and protein in the cells were significantly increased (both P<0.001), while the relative level of MiR-424-5p was significantly reduced (P<0.001). The result of the dual luciferase reporter assay showed that PD-L1 was the direct downstream target gene of MiR-424-5p (P<0.001). After transfection of MiR-424-5p inhibitor, the resistance of CRL2631 cells to CHOP drugs increased, and the expression level of MDR-1 protein (P<0.01), PD-L1 mRNA and protein also increased significantly (both P<0.01). After transfection of MiR-424-5p mimics, the resistance of CRL2631-CHOP cells to CHOP drugs decreased, and the expression level of MDR-1 protein (P<0.001), PD-L1 mRNA and protein also decreased significantly (both P<0.001). Overexpression of PD-L1 could reverse the inhibitory effect of upregulating MiR-424-5p on PD-L1 (P<0.001). Down-regulation of MiR-424-5p enhances the drug resistance of DLBCL cells by regulating the PD-1/PD-L1 signaling pathway.

FOXP1-GINS1 axis promotes DLBCL proliferation and directs doxorubicin resistance.

GINS1 is overexpressed in several types of cancers including leukemia and linked to poor outcomes. However, GINS1 remains poorly investigated in DLBCL (diffuse large B-cell lymphoma). This project aimed to explore the expression, functions and regulation of GINS1 in DLBCL. In this study, through analysis of clinical specimens from DLBCL patients, we uncovered that GINS1 was upregulated in DLBCL. By EMSA, ChIP and luciferase reporter assays, it was found that FOXP1 transcriptionally activated GINS1 expression by directly binding to the promoter region of the GINS1 gene. Western blotting and RT-PCR also revealed that GINS1 expression positively correlated with FOXP1 in human DLBCL specimens and cell lines. In an in vivo xenograft lymphoma mouse model, the FOXP1/GINS1 regulatory axis was also validated. Moreover, with CCK8 cell proliferation assays and colony formation assay, elevated GINS1 expression was found to be associated with doxorubicin resistance in lymphoma cells. Our findings showed that the FOXP1-GINS1 axis played a critical role in DLBCL development and doxorubicin resistance, and targeting the FOXP1-GINS1 axis could be a potential therapeutic approach for DLBCL treatment.

Serine Protease 3 Promotes Progression of Diffuse Large B-Cell Lymphoma and Serves as a Novel Prognostic Predictor.

Diffuse large B-cell lymphoma (DLBCL) ranks among the most prevalent malignancies of the lymphohematopoietic system in adults. The PRSS (Serine Protease) protein family members had been reported to be involved in carcinogenesis as well as tumor progression. Here, we aimed to explore the expression profile of PRSS3 in DLBCL and investigate its clinical significance as well as detailed functions. We retrospectively enrolled 155 DLBCL patients from our hospital and tested protein expression level of PRSS3 through immunohistochemical staining. Accordingly, PRSS3 was highly expressed in certain DLBCL tissues. Chi-square test revealed that higher PRSS3 expression was correlated with advanced Ann Arbor stage, elevated serum LDH level, and higher International Prognostic Index. Moreover, univariate and multivariate analyses confirmed that higher PRSS3 can act as an independent unfavorable prognostic predictor for DLBCL. Two human DLBCL cell lines, SUDHL10 and OCI-LY3, were subjected for knockdown assays, followed by phonotype tests including proliferation and invasion. According to the cellular experiments, PRSS3-knockdown resulted in impaired DLBCL proliferation in the two cell lines above. Taken together, PRSS3 is a novel prognostic factor for DLBCL, which functions by multiple signaling pathways.

Combined multiomics analysis reveals the mechanism of CENPF overexpression-mediated immune dysfunction in diffuse large B-cell lymphoma in vitro.

Diffuse large B-cell lymphoma (DLBCL) is one of the most common aggressive B-cell lymphomas with significant heterogeneity. More than half of patients are cured, but 40%-45% still face relapse or develop drug resistance, and the mechanism is not yet known. In this study, Centrimeric protein F (CENPF) overexpression was found in several DLBCL patients with relapsed or refractory disease compared to patients with complete remission. Thus, the human DLBCL cell line SU-DHL-4 was chosen for this study, and CENPF was upregulated in that cell line by using an adenovirus in vitro. Mass spectrometry-based quantitative proteome analysis was first performed, and the results showed that the expression levels of various proteins were increased when CENPF was upregulated, and these proteins are mainly involved in cellular processes, biological regulation, immune system processes and transcriptional regulator activity. Bioinformatics data analysis revealed that the main enriched proteins, including UBE2A, UBE2C, UBE2S, TRIP12, HERC2, PIRH2, and PIAS, were involved in various ubiquitin-related kinase activities and ubiquitination processes. Thus, ubiquitinome analysis was further performed, and the results demonstrated that proteins in many immune-related cellular pathways, such as natural killer cell-mediated cytotoxicity, the T-cell receptor signaling pathway and the B-cell receptor signaling pathway, were significantly deubiquitinated after CENPF was upregulated in DLBCL cells. Furthermore, TIMER2.0 was also used to reveal the association between CENPF and immune infiltration in DLBCL. The results showed that CENPF expression was positively correlated with CD8+ T cells, NK cells and B lymphocytes in DLBCL samples but negatively correlated with regulatory T cells. Aberrant activation of CENPF may induce immune dysregulation in DLBCL cells by mediating protein deubiquitination in various immune signaling pathways, which leads to tumor escape of DLBCL, but further experimental validation is still needed.

Abstract A24: BTG1 mutations confer a fitness advantage and promote aggressive B cell lymphoma development by lowering the threshold for MYC induction

Abstract BTG1 somatic mutations are exclusively found in mature B cell malignancies, ~12% diffuse large B cell lymphoma (DLBCL) and are particularly enriched in the MCD/cluster 5 subtype of ABC-DLBCL, characterized by extranodal dissemination and poor clinical outcome. However, the mechanism of action and clinical relevance of BTG1 mutations remain unknown. We find that BTG1 mutations score among the top mutations with DLBCL driver potential, using a rigorous genomic and epigenomic covariates analysis. Most notably, BTG1 mutant patients presented with inferior clinical outcome (p=0.0011) in ABC-DLBCL cases from 5 cohorts and BTG1 mutation was independently associated with lower overall survival in a multivariable Cox regression analysis (p=0.0190) DLBCL originates from mature B cells having experienced the germinal center (GC) reaction. We therefore generated a conditional knockin mouse model to express the most frequent Btg1 Q36H in B cells. Btg1 Q36H GC B cells showed a dramatic fitness advantage in in vivo competitive assays. This effect was specific to the GC compartment and was dependent on T cells. RNAseq showed that Btg1Q36H GC B cells were markedly enriched for genes normally transiently induced upon positive selection by T cells, including MYC targets. The same signatures were enriched in BTG1 mutant DLBCL patients and isogenic BTG1Q36H vs BTG1WT human DLBCL cell lines. We observed a higher proportion of MYC-expressing cells in Btg1Q36H GCs without an increase in maximal MYC levels per cell, also confirmed in human DLBCL lines and primary human tonsillar B cells, suggesting a lowered threshold for MYC induction in mutant cells. Mechanistically, ~800 transcripts associated with BTG1WT, but not BTG1Q36H. These belong to the same gene sets that characterize positively selected GC B cell, including MYC itself. BTG1Q36H therefore showed reduced association with the MYC mRNA. We found that this associated with facilitated MYC protein synthesis. Polysome profiling also showed higher translation capacity in BTG1Q36H DLBCL cells. Crossing our Btg1 Q36H mice to the VavP-Bcl2 model showed that VavP-Bcl2+Btg1Q36H mice displayed shorter survival, earlier onset of lymphoma and dysplastic B cell infiltration into non lymphoid organs. Immunoglobulin genes sequencing showed that VavP-Bcl2+Btg1Q36H lymphoma B cells were highly clonal, extensively mutated and selected. Importantly, the lack of BTG1 deletions suggested that BTG1 missense mutations do not cause a full loss-of-function of the protein. Indeed, we observed that shRNA-mediated knockdown of BTG1 resulted in apoptosis. Collectively, BTG1 mutations contribute to the formation of aggressive lymphomas through an entirely novel mechanism, by lowering the threshold to MYC induction in response to T cell selection signals, conferring dramatic fitness. This effect appears to correspond to a partial loss of function disrupting a novel GC context-specific check point, whereby BTG1 normally attenuates spurious MYC translation to tightly restrict fitness potential. Citation Format: Coraline Mlynarczyk, Matt Teater, Juhee Pae, Ling Wang, Jonatan Ersching, Antonin Papin, Darko Barisic, Ersilia Barin, Kenneth B. Hoehn, Zhengming Chen, Diu T. T. Nguyen, Chiara Evans, Ashley S. Doane, Michael G. Kharas, David W. Scott, Gabriel Victora, Ari Melnick. BTG1 mutations confer a fitness advantage and promote aggressive B cell lymphoma development by lowering the threshold for MYC induction [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A24.

Transcriptomics-Based Investigation of Molecular Mechanisms Underlying Apoptosis Induced by ZnO Nanoparticles in Human Diffuse Large B-Cell Lymphoma.

IntroductionZinc oxide nanoparticles (ZnO NPs) show anti-cancer activity. Diffuse Large B-cell Lymphoma (DLBCL) is a type of B-cell malignancies with unsatisfying treatment outcomes. This study was set to assess the potential of ZnO NPs to selectively induce apoptosis in human DLBCL cells (OCI-LY3), and to describe possible molecular mechanisms of action.MethodsThe impact of ZnO NPs on DLBCL cells and normal peripheral blood mononuclear cells (PBMCs) was studied using cytotoxicity assay and flow-cytometry. Transcriptomics analysis was conducted to identify ZnO NPs-dependent changes in the transcriptomic profiles of DLBCL cells.ResultsZnO NPs selectively induced apoptosis in DLBCL cells, and caused changes in their transcriptomes. Deferential gene expression (DGE) with fold change (FC) ≥3 and p ≤ 0.008 with corrected p ≤ 0.05 was identified for 528 genes; 125 genes were over-expressed and 403 genes were under-expressed in ZnO NPs-treated DLBCL cells. The over-expressed genes involved in biological processes and pathways like stress response to metal ion, cellular response to zinc ion, metallothioneins bind metals, oxidative stress, and negative regulation of growth. In contrast, the under-expressed genes were implicated in DNA packaging complex, signaling by NOTCH, negative regulation of gene expression by epigenetic, signaling by WNT, M phase of cell cycle, and telomere maintenance. Setting the FC to ≥1.5 with p ≤ 0.05 and corrected p ≤ 0.1 showed ZnO NPs to induce over-expression of anti-oxidant genes and under-expression of oncogenes; target B-cell receptor (BCR) signaling pathway and NF-κB pathway; and promote apoptosis by intrinsic and extrinsic pathways.DiscussionOverall, ZnO NPs selectively induced apoptosis in DLBCL cells, and possible molecular mechanisms of action were described.

LncRNA NORAD Mediates the Proliferation and Apoptosis of Diffuse Large-B-Cell Lymphoma via Regulation of miR-345-3p/TRAF6 Axis

Diffuse large-B-cell lymphoma (DLBCL), as the most common subtype of B Cell Non-Hodgkin lymphoma (B-NHL), is one of the lymphoid malignancies with poor prognosis worldwide. Non-coding RNA activated by the DNA damage (NORAD), a novel identified lncRNA involved in the DNA repairment process, is reportedly to participate in carcinogenesis, and it is predicted to sponge miR-345-3p. However, LncRNA NORAD has never been investigated in DLBCL. To investigate the role of LncRNA NORAD and miR-345-3p in DLBCL cells and explore the underlying mechanisms. LncRNA NORAD and miR-345-3p levels were determined in the blood samples from patients with B-NHL. Human DLBCL cell lines DB and SU-DHL-4 were transfected with LncRNA NORAD small interfering RNA, miRNA-345-3p mimics, or miRNA-345-3p inhibitor using Lipofectamine RNAiMAX Reagent. Cell cycle, proliferation, and apoptosis were assessed in the transfected cells. Silencing of lncRNA NORAD and overexpression of miR-345-3p both inhibited cell proliferation, induced cell cycle arrest, and triggered apoptosis in DLBCL cells. Inhibition of miR-345-3p counteracted the suppression effects of LncRNA NORAD silencing on DLBCL progression. In addition, LncRNA NORAD shared the regulatory binding sites of miR-345-3p with TNF receptor associated factor 6 (TRAF6). Knockdown of LncRNA NORAD decreased the levels of TRAF6, simultaneously, resulted in deactivation of PI3K/Akt pathway in DLBCL cells. LncRNA NORAD regulated DLBCL cell growth and apoptosis via miR-345-3p/TRAF6/PI3K/Akt axis.

LncRNA PVT1 facilitates DLBCL development via miR-34b-5p/Foxp1 pathway.

Diffuse large B-cell lymphoma (DLBCL) is the most prevalent subtype of non-Hodgkin lymphoma and is a very aggressive malignancy with tumor growing rapidly in organs like lymph nodes. The pathogenesis of DLBCL is not clear and the prognosis of DLBCL requires improvement. Here, we investigated the mechanisms of DLBCL, with the focus on lncRNA PVT1/miR-34b-5p/Foxp1 axis. Human DLBCL tissues from diagnosed DLBCL patients and four human DLBCL cell lines, one normal human B lymphoblastoid cell line were used. qRT-PCR and western blotting were employed to measure expression levels of lncRNA PVT1, Foxp1, miR-34b-5p, β-catenin, and proliferation-related proteins. MTT assay and colony formation assay were performed to determine cell proliferation. Flow cytometry was used to examine cell apoptosis. ChIP and Dual-luciferase assay were utilized to validate interactions of Foxp1/promoters, PVT1/miR-34b-5p and miR-34b-5p/Foxp1. Mouse tumor xenograft model was used to determine the effect of sh-PVT1 on tumor growth in vivo. In this study, we found PVT1 and Foxp1 were elevated in DLBCL tissues and cells while miR-34b-5p was decreased. Knockdown of PVT1, overexpression of miR-34b-5p, or Foxp1 knockdown repressed DLBCL cell proliferation but enhanced cell apoptosis. PVT1 directly bound miR-34b-5p to disinhibit Foxp1/β-catenin signaling. Foxp1 regulated CDK4, CyclinD1, and p53 expression via binding with their promoters. Knockdown of Foxp1 partially reversed the effects of miR-34b-5p inhibitor on DLBCL cell proliferation and apoptosis. Inhibition of PVT1 through shRNA suppressed DLBCL tumor growth in vivo. All in all, lncRNA PVT1 promotes DLBCL progression via acting as a miR-34b-5p sponge to disinhibit Foxp1/β-catenin signaling.

BTK and PI3K Inhibitors Reveal Synergistic Inhibitory Anti-Tumoral Effects in Canine Diffuse Large B-Cell Lymphoma Cells.

Bruton’s tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) in the B-cell receptor (BCR) signaling pathway are considered potential therapeutic targets for the treatment of B-cell lymphomas, among which, diffuse large B-cell lymphoma (DLBCL) is the most common type. Herein, we comparatively evaluated the single and combined application of the BTK inhibitor ibrutinib and the selective PI3Kγ inhibitor AS-605240 in the canine DLBCL cell line CLBL-1. For further comparison, key findings were additionally analyzed in canine B-cell leukemia GL-1 and human DLBCL cell line SU-DHL-4. While ibrutinib alone induced significant anti-proliferative effects on all cell lines in a dose-dependent manner, AS-605240 only induced anti-proliferative effects at high concentrations. Interestingly, ibrutinib and AS-605240 acted synergistically, reducing cell proliferation and increasing apoptosis/necrosis in all cell lines and inducing morphological changes in CLBL-1. Moreover, the combined application of ibrutinib and AS-605240 reduced relative phosphorylation and, in some instances, the levels of the BTK, AKT, GSK3β, and ERK proteins. Comparative variant analysis of RNA-seq data among canine B- and T-lymphoid cell lines and primary B-cell lymphoma samples revealed potentially high-impact somatic variants in the genes that encode PI3K, which may explain why AS-605240 does not singly inhibit the proliferation of cell lines. The combination of ibrutinib and AS-605240 represents a promising approach that warrants further in vivo evaluation in dogs, potentially bearing significant value for the treatment of human DLBCL.

Lw-213 Synergizes with Rituximab to Inhibit Diffuse Large B-Cell Lymphomas By Upregulating CD20

Background：Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens.Up to one-third of diffuse large B cell lymphoma (DLBCL) patients eventually develop resistance to R-CHOP regimen, since the remaining therapeutic options are limited. LW-213, a derivative of wogonin, is reported to possess antineoplastic properties in a variety of cancers, but whether it has effects on DLBCL is n-ot known. Studies have reported that upregulation of CD20 expression b-y either HDACi or silenced SOX2 expression showed sensitizing potential in Rituximab-induced cell death in malignant B cells. Our study was to explore whether LW-213 could sensitize DLBCL to Rixutimab thus improve therapeutic efficacy.Methods： Two DLBCL cell lines, RI-1 (ABC subtype) and Su-DHL -8 (GCB subtype), were used in our study. RI-1 and Su-DHL-8 cells were treated with LW-213 at different doses and for different times, and their proliferation and viability were detected by Cell counting kit-8 (CCK8).Flow cytometry was used to determine surface CD20 expression. Western blotting and q-PCR were applied to examine the protein and mRNA levels of CD20, SOX2, Ace-H3 and Ace-H3K27. CDC assay was used to evaluate the synergistic effects of LW-213 and Rixutimab.Results：We showed that LW-213 inhibited the proliferation of human DLBCL cell lines (Su-DHL-8、RI-1 ) in dose-and time-dependent manners with IC 50 values at the low μmol/L levels, meanwhile it potently inhibited primary lymphoma cells derived from peripheral blood of B-cel-l lymphoma patients. Furthermore, LW-213 significantly increased CD20 surface expression and the acetylation level of histone in DLBCL cell li-nes. Inversely，the SOX2 expression level remarkably decreased. Finally,Combination with LW-213 significantly synergized Rituximab-induced cell death in vitro.Conclusion： The results demonstrate that LW-213 sensitizes DLBCL cells to Rituximab in vitro by upregulating CD20 expression and the SOX2/ace-H3K27/ace-H3 axis may plays a critical role in CD20 upregulation processing. Even though this strategy is important in vitro models,the upregulating CD20 expression therapy against DLBCL proposed in this study warrants further study in vivo and clinical trials .Keywords：CD20 DLBCL Rituximab SOX2 Histone Deacetylation DisclosuresNo relevant conflicts of interest to declare.

CCR2 Expression Promotes Diffuse Large B Lymphoma Cell Survival and Invasion

Objective: Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma world wide. It is a phenotypically and genetically heterogeneous disease, accounting for 30-40% of all cases. 50%-70% of patients can be cured by the R-CHOP regimen, but nearly one-third of patients develop relapsed or refractory disease. CC chemokine receptor 2 (CCR2), the high affinity receptor of CC-Chemokine Ligand 2 (CCL2), which is the most representative of the CC chemokine family members, has be regarded to involve in tumor growth, angiogenesis, epithelial mesenchymal transition, metastasis and immune escape etc.. In recent years, the role and mechanism in DLBCL has not been reported yet. Our preliminary study showed that high expression of CCR2 was correlated with clinicopathological characteristics, and an adverse prognostic factor for overall survival (OS) and progression-free survival (PFS) of DLBCL patients. The purpose of this study is to investigate the role of CCR2 expression in DLBCL cells proliferation and migration by in vitro and in vivo.Methods: CCR2 expression were analyzed in human DLBCL cell lines (SUDHL-2, SUDHL-4, SUDHL-6, OCI-Ly8 and OCI-Ly10) by Western blot (WB). SUDHL-2 and OCI-Ly8 cells were incubated with CCR2 antagonist SC-202525 (Santa Crutz Biotechnology), and control cells were left untreated. The proliferation, migration, apoptosis and signaling pathway were detected by CCK8, transwell, flow cytometry (FC) and WB, respectively in vitro. The engraftment, tumor growth, dissemination and survival time were observed in BALB/c nude mice.Results: CCR2 were expressed in all human DLBCL cell lines (relative CCR2 expression was higher in SUDHL-2, SUDHL-4 and OCI-Ly8 than in SUDHL-6 and OCI-Ly10 cell lines). Blockade of CCR2 expression signaling with CCR2 antagonist inhibited tumor cell proliferation, migration and anti-apoptosis ability. The signaling involved in the proliferation and migration of DLBCL cells by activating PI3K/Akt signaling pathway, and induced apoptosis through activation of P38MARK signaling pathway. Expression of CCR2 was also associated with increased engraftment, tumor growth and dissemination, and decreased survival time in xenograft mice. Furthermore, administration of CCR2 antagonist decreased tumor growth and dissemination of DLBCL cells, and increased survival time in the xenograft model.Conclusions: Our study demonstrates that CCR2 plays an important role in the development of DLBCL by stimulating cell proliferation, migration and anti-apoptosis. The inhibition of CCR2 may, therefore, be a potential target for anticancer therapy in DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Effects of Arsenic Disulfide Combined with Itraconazole on Proli- feration and Apoptosis and Hedgehog Pathway of Diffuse Large B-Cell Lymphoma Cells

To investigate the effect of arsenic disulfide (AS2S2) combined with itraconazole on the proliferation, apoptosis and hedgehog pathway of diffuse large B-cell lymphoma (DLBCL) cells. The human DLBCL cell OCI-LY3 was treated with different concentrations of AS2S2 and itraconazole. Cell proliferation inhibition was detected by CCK-8, cell apoptosis rate was determined by flow cytometry. The expression levels of BCL-2, BAX, SMO and GLi1 were detected by Western blot. The DLBCL cell viability was decreased significantly at 24, 48 or 72 h as cultured with itraconazole. Along with the increasing of itraconazole concentration, the DLBCL cell viability was significantly reduced as compared with that in control group, and the results showed statistically significant(r=-0.690，r=-0.639, r=-0.833, r=-0.808, r=-0.578). The inhibitory and apoptosis rates of the cells were significantly increased as compared with those of the single drug-treated group after treated by the combination of itraconazole and AS2S2(P<0.05). The protein levels of SMO and Glil were significantly down-regulated after treated by arsenic disulfide and itraconazole alone(P<0.01). The protein expression levels of SMO and Glil was down-regulated in the combined-treatment group(P<0.01). Itraconazole can inhibit proliferation of DLBCL cells in a concentration-and time-dependent manner. In addition, the combination of AS2S2 and itraconazole show a synergistic effects, which may be related with the down-regulated protein expression of SMO and Glil of Hedgehog signaling pathway.

Viability of diffuse large B-cell lymphoma cells is regulated by kynurenine 3-monooxygenase activity.

Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogeneous lymphoid malignancy that is the most common type of lymphoma in Japan. Previous studies have demonstrated that patients with DLBCL have a poor prognosis due to increased levels of indoleamine 2,3-dioxygnase and kynurenine (KYN). However, the roles of metabolites acting downstream of KYN and associated enzymes are not fully understood. The present study investigated the role of kynurenine 3-monooxygenase (KMO), which catalyzes the conversion of KYN to 3-hydroxykynurenine (3-HK), using serum samples from patients with DLBCL and human DLBCL cell lines with different KMO expression [STR-428 cells with high levels of KMO expression (KMOhigh) and KML-1 cells with low levels of KMO expression (KMOlow)]. Serum samples from 28 patients with DLBCL and 34 healthy volunteers were used to investigate the association between prognosis and KMO activity or 3-HK levels. Furthermore, to investigate the roles of KMO and its related metabolites, STR-428 and KML-1 cell lines, and the lymph nodes of patients with DLBCL were analyzed by reverse transcription-quantitative PCR for KMO, KYNU, 3-hydroxyanthranilate-3,4-dioxygenase and quinolinate phosphoribosyltransferase, by western blotting, and immunohistochemical or immunofluorescence staining for KMO, and by cell viability and NAD+/NADH assays. KYN pathway metabolites in serum samples were measured by HPLC. Serum 3-HK levels were regulated independently of serum KYN levels, and increased serum 3-HK levels and KMO activity were found to be associated with worse disease progression. Notably, the addition of KMO inhibitors and 3-HK negatively and positively regulated the viability of DLBCL cells, respectively. Furthermore, NAD+ levels in KMOhigh STR-428 cells were significantly higher than those in KMOlow KML-1 cells. These results suggested that 3-HK generated by KMO activity may be involved in the regulation of DLBCL cell viability via NAD+ synthesis.