Diffuse Large B-cell Lymphoma Cell Lines Research Articles

Design, synthesis and pharmacological evaluation of 2,3-dihydrobenzofuran IRAK4 inhibitors for the treatment of diffuse large B-cell lymphoma

Interleukin-1 receptor associated kinase 4 (IRAK4) is a critical mediator of MYD88 L265P-induced NF-κB activation, indicating it is a promising therapeutic target for diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of 2,3-dihydrobenzofuran IRAK4 inhibitors through structure-based drug design. The representative compound 22 exhibited strong IRAK4 inhibitory potency (IRAK4 IC50 = 8.7 nM), favorable kinase selectivity and high antiproliferative activity against the MYD88 L265P DLBCL cell line (OCI-LY10 IC50 = 0.248 μM). Compound 22 also exhibited the ability to inhibit the activation of IRAK4 signaling pathway and induce apoptosis in MYD88 L265P DLBCL cell line. In combination with Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, 22 showed enhanced apoptosis-inducing effect and antiproliferative potency. The most advanced compound 22 in this inhibitor series holds promise for further development into efficacious and selective IRAK4 inhibitors for the treatment of DLBCL.

Platycodin D induces proliferation inhibition and mitochondrial apoptosis in diffuse large B-cell lymphoma

Patients with diffuse large B-cell lymphoma (DLBCL) have unsatisfactory outcomes, especially when relapse occurs after initial chemotherapy. Platycodin D (PD), a triterpenoid saponin isolated from the root of Platycodon grandiflorum (Jacq.) A. DC., has demonstrated potent anticancer activities. However, information regarding the effect of PD on malignant lymphoma remains unavailable. In the present study, we showed that PD dose dependently inhibited the viability of a serial of established DLBCL cell lines representing different molecular subtypes, and their sensitivities to PD were comparable. Mitochondrial dysfunction and subsequent intrinsic apoptosis were induced by PD, as indicated by the loss of mitochondrial membrane potential (MMP) and the increase in the percentage of Annexin Ⅴ-positive cells. Mechanistically, PD treatment downregulated the expression levels of antiapoptotic proteins including MCL-1, BCL-2, and BCL-XL, whereas the expression level of proapoptotic protein BAK was upregulated, followed by the cleavage of the DNA repair enzyme PARP. Moreover, PD synergistically enhanced the cytotoxicity of BCL-2 inhibitor venetoclax. In a SUDHL-4-derived xenograft mouse model, the PD administration significantly constrained the tumor growth without obvious side effects. Therefore, our results provide new insights into the role of PD in lymphoma therapy.

Abstract 3451: XS-04: A first-in-class dual IRAK4/BTK inhibitor for the treatment of hematological malignancies

Abstract Bruton's tyrosine kinase (BTK), a critical mediator of B-cell receptor (BCR) signaling, is a validated therapeutic target found across multiple B-cell cancers. Though initially responding to treatment with a BTK inhibitor, many patients with chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), or Waldenström macroglobulinemia (WM) eventually progressed and clinical responses to BTK inhibitors are often poor in diffuse large B-cell lymphoma (DLBCL) patients. These highlight the need to understand the resistance mechanisms and improve responses to BTK inhibitors. The serine/threonine protein kinase IRAK4 is a key modulator of MYD88, which is found to be mutated in various aggressive B-cell lymphomas, including DLBCL. As BCR signaling and MYD88 signaling converge downstream on NF-kB activation, which plays an essential role in proliferation and survival of lymphoma cells, blockade of the two signaling pathways presents a potential therapeutic strategy. Here, we investigated the pharmacokinetic properties and anti-tumor activity of XS-04, a first-in-class dual inhibitor targeting both IRAK4 and BTK, using preclinical models of DLBCL subtypes, including tumors resistant to BTK inhibitors. XS-04 showed potent inhibition of IRAK4 and BTK kinases, with IC50 values of 0.2 and 2.9 nM, respectively. Favorable pharmacokinetic properties were also seen across multiple non-clinical species. Using in vitro anti-proliferative assays, we found that XS-04 inhibited proliferation of various DLBCL cell lines with IC50s of 30 to 40 nM. Mechanistically, XS-04 suppressed NF-kB activity in DLBCL cells and in vivo xenograft tumors, as indicated by decreased phosphorylation of IkBa. Oral dosing of XS-04 in TMD8 xenograft model led to a dose-dependent inhibition of tumor growth, which was comparable to the inhibition shown by a combination of BTK and IRAK4 inhibitors. In addition, XS-04 treatment resulted in tumor regression in a patient-derived ibrutinib-resistant MCL xenograft model, without significant changes in body weight. XS-04, a first-in-class IRAK4/BTK dual inhibitor, shows broad anti-tumor activity across preclinical models of DLBCL subtypes, including tumors resistant to BTK inhibitors. These results support XS-04 as a therapeutic candidate for the treatment of hematological malignancies. An IND submission is planned for 2023. Citation Format: Chunyan Zhao, Jianfei Wang, Aiguo Liu, Xin Gao, Yanfen Teng, Lei Liu, Yang Zhang, Xiaohong Yu. XS-04: A first-in-class dual IRAK4/BTK inhibitor for the treatment of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3451.

Abstract 1771: Characterization of RHOA inactivation as a driver of CAR-T therapy resistance in diffuse large B-cell lymphoma

Abstract Background: Front-line chemoimmunotherapy for diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin lymphoma, is effective, however at least 40% of patients relapse or are refractory (r/r). Of this r/r DLBCL cohort, approximately 10% will be cured with conventional second-line treatments consisting of salvage chemotherapy and autologous stem cell transplant. CD19-directed chimeric antigen receptor T-cell (CAR-19) therapies are promising new second-line options in diffuse large B-cell lymphoma (DLBCL) and show exciting 30-40% durable complete responses in heavily pretreated patients. Recent whole-genome sequencing data published by our lab revealed that deletion of Ras homolog family member A (RHOA) is strongly associated with poor response to CAR-19 therapy in r/r DLBCL patients (p = 0.0013). In newly diagnosed DLBCL, 20% of patients harbor a RHOA deletion and another 5% have loss-of-function mutations. To date, no work has comprehensively identified the role of RHOA in DLBCL or how its inactivation may protect CAR-T resistant tumors. Methods: We created a novel in vivo model of RHOA deficient lymphoma by transplanting Eμ-Myc transgenic hematopoietic stem cells (HSCs) transduced with a shRhoa vector. We also created a syngeneic model of RHOA-deficient lymphoma with MYC-driven BL3750 cells. In vitro flow cytometry and Xcelligence real-time cell analysis experiments were utilized to assess cytotoxicity of CAR-19 against RHOA-deficient lymphoma cell lines. RNAseq analysis was conducted on Rhoa knockdown cell lines to elucidate cell-intrinsic mechanisms of lymphomagenesis. Cell proliferation and migration assays were performed to characterize the phenotype of RHOA-deficient DLBCL cell lines. Results: 18 mice were transplanted (11 shRhoa, 7 shRenilla control) and are being monitored for lymphoma development. In vitro CAR-T experiments demonstrated that while RHOA-deficient cells demonstrate a similar degree of killing initially, a population of RHOA-deficient cells persists and enrich at later time points. RNA-seq analysis of A20 shRhoa murine lymphoma cells uncovered enrichment of gene sets associated will cell-cycle progression and interferon (INF) signaling pathways. Cell proliferation assay and Western blot of RHOA inactivated cell lines revealed increased proliferation in shRhoa cells through the AKT/mTOR pathway compared to controls. Conclusions: RHOA-deficient lymphomas, while not entirely evading CAR-19 cells in vitro, demonstrate resistance mechanisms that allows a small population to persist. RNA-seq analysis reveals upregulated oncogenic and immunoinhibitory pathways, consistent with in vitro cell proliferation and Western blot assays. Further research will challenge in vivo RHOA-deficient tumors with CAR-19 cells and employ scRNA-seq to reveal transcriptomic changes within RHOA-deficient lymphoma responsible for CAR-19 resistance. Citation Format: Austin Newsam, Yitzhar Goretsky, Evan Roberts, Daniel Bilbao, Jonathan Schatz. Characterization of RHOA inactivation as a driver of CAR-T therapy resistance in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1771.

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.

HIF-1α mediates CXCR4 transcription to activate the AKT/mTOR signaling pathway and augment the viability and migration of activated B cell-like diffuse large B-cell lymphoma cells.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy with a high relapse rate. We previously found that C-X-C motif chemokine receptor 4 (CXCR4) was highly expressed in DLBCL and associated with poor prognosis. This study focused on the effect of hypoxia-inducible factor-1α (HIF-1α) on CXCR4 expression and the DLBCL progression. Two activated B cell-like DLBCL cell lines Ly-3 and SUDHL2 were transfected with overexpression and knockdown plasmids or HIF-1α. The viability and migration of DLBCL cells were significantly increased under hypoxic conditions, or upon HIF-1α overexpression under normoxic conditions, but the HIF-1α downregulation led to inverse trends. However, the promoting effects of HIF-1α overexpression on DLBCL cells were suppressed by Plerixafor (a CXCR4 inhibitor). The luciferase and chromatin immunoprecipitation assays revealed that HIF-1α bound to the functional site HRE1 on CXCR4 promoter to activate its transcription. HIF-1α-mediated CXCR4 activation further led to increased phosphorylation of AKT/mTOR under hypoxic conditions. Taken together, this work reports that HIF-1α promotes viability and migration of activated B cell-like cells under hypoxia, which might involve the transcription of CXCR4 and the activation of the AKT/mTOR pathway. The finding may provide novel lights in the management of DCBCL.

In Silico Identification and Functional Characterization of Genetic Variations across DLBCL Cell Lines.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma and frequently develops through the accumulation of several genetic variations. With the advancement in high-throughput techniques, in addition to mutations and copy number variations, structural variations have gained importance for their role in genome instability leading to tumorigenesis. In this study, in order to understand the genetics of DLBCL pathogenesis, we carried out a whole-genome mutation profile analysis of eleven human cell lines from germinal-center B-cell-like (GCB-7) and activated B-cell-like (ABC-4) subtypes of DLBCL. Analysis of genetic variations including small sequence variants and large structural variations across the cell lines revealed distinct variation profiles indicating the heterogeneous nature of DLBCL and the need for novel patient stratification methods to design potential intervention strategies. Validation and prognostic significance of the variants was assessed using annotations provided for DLBCL samples in cBioPortal for Cancer Genomics. Combining genetic variations revealed new subgroups between the subtypes and associated enriched pathways, viz., PI3K-AKT signaling, cell cycle, TGF-beta signaling, and WNT signaling. Mutation landscape analysis also revealed drug-variant associations and possible effectiveness of known and novel DLBCL treatments. From the whole-genome-based mutation analysis, our findings suggest putative molecular genetics of DLBCL lymphomagenesis and potential genomics-driven precision treatments.

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.

E3 ubiquitin ligase ASB8 promotes selinexor-induced proteasomal degradation of XPO1

Selinexor (KPT-330), a small-molecule inhibitor of exportin-1 (XPO1, CRM1) with potent anticancer activity, has recently been granted FDA approval for treatment of relapsed/refractory multiple myeloma and diffuse large B-cell lymphoma (DLBCL), with a number of additional indications currently under clinical investigation. Since selinexor has often demonstrated synergy when used in combination with other drugs, notably bortezomib and dexamethasone, a more comprehensive approach to uncover new beneficial interactions would be of great value. Moreover, stratifying patients, personalizing therapeutics and improving clinical outcomes requires a better understanding of the genetic vulnerabilities and resistance mechanisms underlying drug response. Here, we used CRISPR-Cas9 loss-of-function chemogenetic screening to identify drug-gene interactions with selinexor in chronic myeloid leukemia, multiple myeloma and DLBCL cell lines. We identified the TGFβ-SMAD4 pathway as an important mediator of resistance to selinexor in multiple myeloma cells. Moreover, higher activity of this pathway correlated with prolonged progression-free survival in multiple myeloma patients treated with selinexor, indicating that the TGFβ-SMAD4 pathway is a potential biomarker predictive of therapeutic outcome. In addition, we identified ASB8 (ankyrin repeat and SOCS box containing 8) as a shared modulator of selinexor sensitivity across all tested cancer types, with both ASB8 knockout and overexpression resulting in selinexor hypersensitivity. Mechanistically, we showed that ASB8 promotes selinexor-induced proteasomal degradation of XPO1. This study provides insight into the genetic factors that influence response to selinexor treatment and could support both the development of predictive biomarkers as well as new drug combinations.

Effects of the omega-3 fatty acid DHA on histone and p53 acetylation in diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) often develops resistance and/or relapses in response to immunochemotherapy. Epigenetic modifiers are frequently mutated in DLBCL, i.e., the lysine (histone) acetyltransferases CREBBP and EP300. Mutations in CBP/p300 can prevent the proper acetylation and activation of (i) enhancer sequences of genes required for essential functions (e.g., germinal center exit and differentiation) and (ii) the tumor suppressor p53. Based on evidence that omega-3 fatty acids (ω-3 FAs) affect histone acetylation in various cancers, we investigated whether ω-3 FA docosahexaenoic acid (DHA) could modify levels of histone and p53 acetylation in three DLBCL cell lines (at different CREBBP/EP300 mutational status) versus normal B cells. Exposure to DHA at clinically attainable doses was shown to significantly alter the genome-wide levels of histone posttranslational modifications in a cell-line-dependent and dose-dependent manner. Although histone acetylation did not increase uniformly, as initially expected, levels of p53 acetylation increased consistently. Quantitative reverse transcription polymerase chain reaction results revealed significant changes in expression of multiple genes, including increased expression of CREBBP and of PRDM1 (required for differentiation into plasma cells or memory B cells). Taken together, our results provide (to our knowledge) the first characterization of the epigenetic effects of ω-3 FAs in DLBCL.

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.

Prognostic Significance of Ribosome-related Genes Signature in Diffuse Large B Cell Lymphoma.

Background: The diffuse large B-cell lymphoma (DLBCL) is a heterogeneous lymphoma with a dismal outcome, due to approximately 40% patients will be relapsed or refractory to the standard therapy of rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Therefore, we need urgently to explore the approach to classify the risk of DLBCL patients accurately and accurately targeting therapy. The ribosome is a vital cellular organelle that is mainly responsible for translation mRNA into protein, moreover, more and more reports revealed that ribosome was associated with cellular proliferation and tumorigenesis. Therefore, our study aimed to construct a prognostic model of DLBCL patients using ribosome-related genes (RibGs). Method: We screened differentially expressed RibGs between healthy donors' B cells and DLBCL patients' malignant B cells in GSE56315 dataset. Next, we performed analyses of univariate Cox regression, the least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox regression analyses to establish the prognostic model consisting of 15 RibGs in GSE10846 training set. Then, we validated the model by a range of analyses including Cox regression, Kaplan-Meier survival, ROC curve, and nomogram in training and validation cohorts. Results: The RibGs model showed a reliably predictive capability. We found the upregulated pathways in high-risk group most associated with innate immune reaction such as interferon response, complement and inflammatory responses. In addition, a nomogram including age, gender, IPI score and risk score was constructed to help explain the prognostic model. We also discovered the high-risk patients were more sensitive to some certain drugs. Finally, knocking out the NLE1 could inhibit the proliferation of DLBCL cell lines. Conclusion: As far as we know, it is the first time to predict the prognosis of DLBCL using the RibGs and give a new sight for DLBCL treatment. Importantly, the RibGs model could be acted as a supplementary to the IPI in classifying the risk of DLBCL patients.

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.

In Vitro Diffuse Large B-Cell Lymphoma Cell Line Models as Tools to Investigate Novel Immunotherapeutic Strategies

Despite the high incidence of diffuse large B-cell lymphoma (DLBCL), its management constitutes an ongoing challenge. The most common DLBCL variants include activated B-cell (ABC) and germinal center B-cell-like (GCB) subtypes including DLBCL with MYC and BCL2/BCL6 rearrangements which vary among each other with sensitivity to standard rituximab (RTX)-based chemoimmunotherapy regimens and lead to distinct clinical outcomes. However, as first line therapies lead to resistance/relapse (r/r) in about half of treated patients, there is an unmet clinical need to identify novel therapeutic strategies tailored for these patients. In particular, immunotherapy constitutes an attractive option largely explored in preclinical and clinical studies. Patient-derived cell lines that model primary tumor are indispensable tools that facilitate preclinical research. The current review provides an overview of available DLBCL cell line models and their utility in designing novel immunotherapeutic strategies.

Pharmacological Inhibitory Effect of Imetelstat, a Novel Human Telomerase Inhibitor in Diffuse Large B-Cell Lymphoma and Peripheral T-Cell Lymphoma

Telomerase activity is present in some non-Hodgkin lymphomas (NHL), including diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell lymphomas (PTCL), and correlated with proliferation index, suggesting anti-telomerase drugs may be efficacious in some types of NHL (Ely et al. 2000). Imetelstat, a telomerase inhibitor that specifically targets the RNA template of human telomerase, is a potent, first-in-class, competitive inhibitor of telomerase enzymatic activity and has demonstrated clinical activity in myeloid malignancies (Steensma et al. 2021; Mascarenhas et al. 2021). No studies have been conducted to demonstrate the effects of imetelstat in DLBCL and PTCL. In our study, we characterized telomerase activity and telomere length, as well as the in-vitro effects of imetelstat on cell viability and apoptosis of a panel of DLBCL and PTCL cell lines. Six DLBCL cell lines, including germinal B cells (GCB: DOHH2, HT, SUDHL4), non-germinal B cells (HBL-1, TMD8), and one high-grade B cell lymphoma line RC with MYC and BCL2 gene rearrangements (double hit lymphomas; ), and five PTCL cell lines (Karpas 299, H9, MAC2A, SUDHL1, and SUPM2) were tested for imetelstat single agent activity as assessed by change of telomerase activity using ScienCell's Telomerase Activity Quantification qPCR Assay Kit, cell viability by Cell Titer-Glo Luminescent Cell Viability Assay and apoptosis by Annexin V/Propidium Iodide flow assay. Baseline telomerase activity and telomere length were measured for each cell line to correlate with imetelstat activity. In addition, PTCL cell lines were treated with Suberoylanilide hydroxamic acid (SAHA) and imetelstat to determine if the combination had an impact on cell viability over either of the single agents. All DLBCL and PTCL cell lines have higher baseline telomerase activity with Ct 20.6-23.2 cycles than the telomerase-positive cell lysate control with Ct 30.6 cycles used in the assay. The dose and time-dependent effects of imetelstat were examined through cell viability and apoptosis assays. Imetelstat sensitivity was higher in DLBCL than in PTCL cell lines used in this study. Preliminary experiments determined that IC50 3-17.5 μM imetelstat markedly reduced cell viability and induced apoptosis in DLBCL cell lines. Specifically, non-GCB DLBCL cell lines (IC50 = 3-4.1 μM) were more sensitive than the GCB-DLBCL (IC50 = 6-8.2 μM). While the level of effects in cell viability and apoptosis by imetelstat varied in the DLBCL cell lines, the double hit DLBCL cell line RC was the least sensitive to imetelstat (IC50 = 17.5 μM) after treatment for 8 days. In contrast, compared to DLBCL cell lines, imetelstat single-agent activity on cell viability was limited in PTCL cell lines, although a time and dose-dependent reduction of telomerase activity were noted. Additionally, the greater inhibitory effect in DLBCL than in PTCL cell lines by imetelstat treatment may be attributed to higher telomerase activity observed in DLBCL compared to PTCL cell lines. Furthermore, the PTCL cell lines had a ~7.3 fold longer telomere length than DLBCL cell lines, which was associated with less response to imetelstat than DLBCL lines. A combination of SAHA with imetelstat was more effective than a single agent alone, synergistic activity was observed in Karpas 299, while other PTCL lines had an additive effect. Our findings indicate that imetelstat was more sensitive in non-GCB subtypes than in GCB-DLBCL. While in PTCL cell lines, the activity of imetelstat was enhanced when combined with SAHA. In the lymphoma landscape, telomerase may be a therapeutic target for ABC-type DLBCL; the novel telomerase inhibitor Imetelstat may present one of the potential treatment options.

Inhibition of PRMT5 Increases Sensitivity to BH3 Mimetics in Aggressive B Cell Malignancies

Introduction: Protein Arginine Methyltransferase 5 (PRMT5) is a type II methyltransferase that catalyzes symmetric dimethylation of arginine. PRMT5 is highly expressed in several types of aggressive B cell malignancies, and increased PRMT5 expression has been associated with poor disease prognosis. Several small molecule inhibitors targeting PRMT5 have been developed and showed promising anti-tumor effect in pre-clinical studies including in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). JNJ-64619178, a potent and specific PRMT5 inhibitor, exhibited broad antiproliferative activity in cancer cell lines from multiple cancer types and cell lines derived xenograft mouse model (Brehmer et al., 2021). In a phase 1 clinical trial of JNJ-64619178 (NCT03573310), patients with advanced solid tumors and non-Hodgkin lymphoma demonstrated manageable toxicity and preliminary evidence of antitumor activity at selected dose levels (Villar et al., 2020). In addition, in patients with lower-risk myelodysplastic syndromes, JNJ-64619178 had manageable hematologic toxicity at the selected expansion dose, but did not have significant clinical activity (Haque et al., 2021). Inhibition of PRMT5 alone could induce cell cycle arrest instead of apoptosis depending on the types of cancer (Zhu et al., 2019), therefore we hypothesized that the combination of PRMT5i and pro-apoptotic BH3 mimetics therapies could be synergistic in aggressive B cell malignancies. Methods: We used dynamic BH3 profiling (DBP) to measure the net pro-apoptotic signaling induced by ex vivo drug treatments (as per Montero et al., 2015). Other standard techniques included shRNA mediated gene knockdown, Western blot, cell viability assays including PI/Annexin V and CellTiter-Glo, and reactive oxygen species (ROS) measurement by flow cytometry with dihydroethidium (DHE) staining. The DLBCL cell lines (HBL1, TMD8, RI-1, OCI-Ly1, Karpas 422, SUDHL4, Toledo), double hit lymphoma (DHL) patient-derived xenograft (PDX) cell lines (DW19), mantle cell lymphoma (MCL) cell lines (Mino, Jeko-1), T cell acute leukemia (T-ALL) cell line (Jurkat) and Burkitt lymphoma (BL) cell line (Raji) were used to investigate the in vitro anti-cancer activity of JNJ-64619178 (Janssen Pharmaceuticals) and BH3 mimetics (venetoclax [BCL2i], S63845 [MCL-1i] and A1331852 [BCL-xLi], Selleckchem). SynergyFinder was used to calculate drug synergism. Results: In all 7 DLBCL cell lines and the DHL-PDX cell line, we found synergistic induction of apoptosis with JNJ-64619178 plus venetoclax (synergy score, 2.82~51.42). The lowest level of synergy was observed in OCI-Ly1 cells, which are intrinsically highly sensitive to venetoclax monotherapy. The combination of JNJ-64619178 with S63845 synergistically induced apoptosis in 5 out of 7 cell lines (synergy score, -1.91~35.64). While the combination of JNJ-64619178 with A1331852 synergistically induced apoptosis in all 7 cell lines (synergy score 1.88~46.73), 4 of them had weak synergy score (1.88~9.4). To confirm the effects we observed with pharmacologic inhibition of PRMT5, we performed shRNA mediated knockdown of PRMT5, which similarly revealed increased sensitivity to venetoclax. In MCL cell lines, combining JNJ-64619178 with venetoclax or A1331852 was more synergistic in inducing cell death than combining with S63845. The T-ALL Jurkat cell line was resistant to JNJ-64619178 and single BH3 mimetic-induced apoptosis. Combining JNJ-64619178 with A1331852 but not venetoclax or S63845 strongly induced apoptosis. The Burkitt cell line Raji was resistant to JNJ-64619178, venetoclax and A1331852, but was sensitive to S63845. The addition of JNJ-64619178 and a single BH3 mimetic in these Raji cells had only a minor synergistic effect. The synergy scores are summarized in Figure 1. Using DBP, JNJ-64619178 was found to increase overall apoptotic priming of DLBCL and MCL cell lines (> 15% increase in cytochrome c loss). By measuring ROS levels, we found increased ROS production (> 2-fold increase in median fluorescent intensity) in response to JNJ-64619178 before the onset of apoptosis, suggesting a possible mechanism by which JNJ-64619178 increases apoptotic priming. Conclusions: The combination of a PRMT5 inhibitor with BH3 mimetics, especially venetoclax, is worthy of further exploration as a potential therapeutic strategy for DLBCL and MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Pyroptosis-Related Signature and Development of a Novel Prognostic Model in Diffuse Large B-Cell Lymphoma

Introduction Pyroptosis is a programmed cell death triggered by inflammatory vesicles, which is manifested by cellular distension until the cell membrane ruptures, leading to the release of cellular contents and thus an intense inflammatory response. Recently, emerging evidence suggests that pyroptosis plays an essential role in the development and progression of multiple cancers. However, the role of pyroptosis in diffuse large B-cell lymphoma (DLBCL) remains elusive. This study aimed to identify the pyroptosis-related signature in DLBCL, construct a novel prognostic model, and further analyze the involved immune infiltration features. Methods The expression profile data of DLBCL and normal samples of pyroptosis-related genes (PRGs) were analyzed, and the clinical characteristics of 533 DLBCL patients were further investigated. A prognostic model based on six PRGs was established using Cox univariate and LASSO Cox regression analysis. The expression of differential expressed PRGs was validated by qRT-PCR in DLBCL cell lines. Single-sample gene set enrichment analysis (ssGSEA) was applied to compare immune infiltration characteristics in subgroups. Results: To explore the expression profile characteristics of PRGs in DLBCL, the mRNA expression levels of PRGs were evaluated in DLBCL and normal samples. The results revealed that most PRGs were dysregulated in DLBCL samples. Subsequently, 8 hub genes, including PJVK, TNF, CASP1, CASP3, CASP5, CASP8, NLRP3, and IL18, were screened. The clinical significance of the PRGs in DLBCL was further evaluated. 19 genes were significantly associated with overall survival (OS) of DLBCL patients in the univariate Cox regression. SCAF11, CASP8, CASP9, NLRP1, NLRP6, and TIRAP were selected for the risk model according to the minimum criteria of the LASSO Cox regression, and DLBCL patients were divided into groups of high and low. The low-risk group exhibited lower mortality rates and longer survival times than the high-risk group (Figure 1A). Significantly shorter OS was observed in DLBCL patients with high risks (Figure 1B). Additionally, the ROC curves indicated the excellent prediction performance of this model. To further verify the expression levels of PRGs in DLBCL, we assessed the mRNA expression levels of the above six PRGs by qRT-PCR in DLBCL cell lines. Compared with normal samples, CASP8, CASP9, NLRP1, NLRP6, and TIRAP were significantly down-regulated in DLBCL cell lines, while SCAF11 was significantly up-regulated. These results revealed the outstanding predictive efficacy and superiority of this model. The potential biological role and the mechanism of PRGs causing different risks were further explored. GO and KEGG enrichment illuminated that these PRGs may be involved in cellular protein modification processes and regulation of JAK-STAT signaling pathway. Given the strongly correlated JAK-STAT signaling pathway and tumor immune environment, we analyzed the immune status of different risk groups by applying ssGSEA. The low-risk group showed higher activity immune infiltration and immune checkpoint pathways than the high-risk group. We found that the risk scores corresponded with the immune profile, and the elevated immune activity might contribute to the antitumor effect of DLBCL. Conclusion In summary, our study identified for the first time that pyroptosis is closely associated with the development and progression of DLBCL. A comprehensive prognostic model was developed based on the characteristics of PRGs in DLBCL, which accurately predicts the prognosis of DLBCL patients. Pyroptosis-targeting coupled with immunotherapies would be a promising alternative treatment approach for DLBCL. Keywords: Pyroptosis, DLBCL, Prognostic model, Immune infiltration features Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification of Histone Acetylation Regulator KAT2A As a Potential Prognostic Biomarker in Diffuse Large B-Cell Lymphoma

Introduction Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous lymphoid neoplasm with variations in the expression of genes and genetic alterations. Epigenetic alterations are associated with lymphoma pathogenesis, one of which is histone acetylation. Ferroptosis is a novel type of cell death with distinct properties and recognizing functions involved in physical conditions or various cancers. However, the functions of histone acetylation and ferroptosis in DLBCL remain unclear. Our study assesses the significance and mechanisms of ferroptosis-related genes (FRG) and histone acetylation regulators (HAG). Methods Using consensus clustering, we categorized DLBCL patients based on histone acetylation regulators and ferroptosis-related genes (HFGs) expression. The WGCNA identified genes and mechanisms that affect HF clusters. LASSO Cox regression was used to calculate risk scores. To assess the accuracy of this model, Kaplan-Meier survival analysis, univariate and multivariate Cox regression analysis, and ROC curve analysis were performed. qRT-PCR was used to validate the expression of four HFGs in DLBCL cell lines. Results A total of 38 HA regulators were evaluated for mutations. According to the study, CREBBP had the highest mutation frequency. Based on CREBBP mutations, patients were divided into mutant and wild groups, and several histone acetylation regulators were overexpressed in the mutant group. There was a high rate of copy number variation (CNV) in some genes. To demonstrate the potential malignant mechanisms of HFGs in DLBCL development and their prognostic value. A significant positive self-correlation was observed in HAGs and FRGs. A negative correlation was found between KAT2A, the HA writer, and the majority of FRGs (Fig. 1A). 36 genes were selected (P < 0.05) by univariate regression correlations with clinical prognosis. Consensus clustering was used to categorize the expression of DLBCL patients. K-M survival analysis showed that HFcluster B had a worse prognosis. To investigate the underlying mechanisms, WGCNA was used to divide genes into different modules. Afterwards, we selected the blue and green modules to analyze their functions. KEGG enrichment emphasized oncological pathways like PI3K-Akt, focal adhesion, Hippo, and MAPK signaling pathway. Based on HFGs and their corresponding signatures, we developed a high-quality and high-accuracy gene signature prediction model. Following LASSO Cox regression, a four-gene signature model was identified. Patients with DLBCL in the high-risk group had a significantly shorter OS (P < 0.001, Fig. 1B). ROC curves, univariate and multivariate Cox regression analyses also revealed that riskScore served as an independent indicator for patients with DLBCL (P < 0.001). GSEA enrichment analysis identified JAK-STAT, P53, and APOPTOSIS as the most differentially enriched genes for malignant mechanisms. Analyses revealed that molecular mechanisms mediate histone acetylation and ferroptosis. The results of PCR in CD19+ B cells and DLBCL cell lines also confirmed the accuracy of the model. Due to its close relationship to ferroptosis-related genes, KAT2A was explored in greater depth. Further study revealed that KAT2A was significantly correlated with age (P = 0.028) and IPI (P = 0.016). In univariate analysis, KAT2A was an independent indicator of DLBCL patients (P < 0.001). The expression level of KAT2A was negatively correlated with OS in DLBCL patients (P = 0.003). Conclusions There were significant correlations among these HFGs, indicating their potential role in DLBCL development and predictive value for prognosis. Additionally, our study shows that KAT2A is associated with prognosis in patients with DLBCL. Further study of the mechanisms of KAT2A in DLBCL may help clinicians to individualize the treatment of this patient population. Keywords: histone acetylation, ferroptosis, mechanism, DLBCL Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Translatome Reprogramming By Rocaglates in Lymphoma Induces Drug Resistance through up-Regulation of CD98hc, Activating Downstream AKT Survival Signaling

Background: Up to 40% of diffuse large B-cell lymphoma (DLBCL) patients do not achieve a cure in response to standard immunochemotherapy R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). In addition, related aggressive lymphomas such as high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (a.k.a. double and triple-hit lymphomas) have persistently inferior outcomes in response to frontline therapy, and no widely accepted alternative exists yet for these high-risk patients. Therefore, there is a strong unmet need to identify novel, effective therapeutics. B-cell lymphomas are driven by deregulation of several oncoproteins that rely on cap-dependent translation, including MYC, MCL1, Cyclin D1, and BCL2. Among strategies to bypass drug-resistance driven by redundant signaling pathways, targeting cap-dependent translation has been shown to be effective. Several studies highlight various types of lymphoma, including the "undruggable” MYC-driven B cell lymphoma, that respond to rocaglates - natural and synthetic translation inhibitors. Among these compounds, eFT226 (zotatifin), is currently in phase I/II evaluation for solid tumors. Despite advancement of rocaglates to clinical evaluation, their mechanisms of action on target molecules remains incompletely defined to advance clinical use for B-cell lymphoma. Methods and Results: Using unbiased proteomic strategies, previous findings from our laboratory in glioblastoma in vitro models have shown that rocaglates induce a far more complex, dynamic process than simple translation inhibition. Indeed, despite translational repression, we discovered significant adaptation that caused active translational synthesis of many targets in the translation machinery (i.e eEF1ε1) and the translatome. We have also shown that some upregulated proteins counteract drug effects (i.e., CD98 heavy chain-CD98hc). Our previous findings and additional data confirmed that rocaglates also induced reprogramming in a DLBCL patient-derived xenograft (PDX) mouse model, and in several DLBCL cell lines. Intriguingly, in DLBCL cell lines we found upregulation of the survival mediators CD98hc and AKT-signaling in persistent cells at 48 hours upon zotatifin treatment via Western Blot and flow cytometry, possibly representing a novel mechanism of resistance driven by CD98hc in response to rocaglate treatment. To further explore the role of the CD98hc counteracting mechanism, we established a doxycycline-inducible (TET-on) CD98hc-overexpressing DLBCL cell line (SU-DHL10), and we consistently found that overexpression of CD98hc was responsible for decreased sensitivity to zotatifin treatment (48h, 72h) by ATP-dependent viability assay. This data was further corroborated by Annexin-V/PI staining via flow cytometry, depicting a significant decrease in cell death upon 12.5 nM zotatifin treatment for 24h (P=0.0074) and 48h (P=0.0010) in induced versus uninduced CD98hc-overexpressing SU-DHL10 cells. For statistical analysis, repeated measures two-way Anova with Sidaks multiple comparison test were used. Conclusions: We define for the first time CD98hc as a new putative driver of rocaglate resistance in B-cell lymphoma. Paradoxically, CD98hc is upregulated as part of an overall cellular translational response to rocaglate exposure, but it provides insights and new potential targets for further drug combinations. This study lays the foundation for new treatment strategies to optimize the use of zotatifin to overcome rocaglate resistance in lymphoma patients.