Diffuse Large B-cell Lymphoma Tumors Research Articles

Ethyl pyruvate attenuates cellular adhesion and proliferation of diffuse large B-cell lymphoma by targeting c-Jun.

Diffuse large B-cell lymphoma (DLBCL) stands out as the most common type of malignant cancer, representing the majority of cases of non-Hodgkin's lymphoma. Ethyl pyruvate (EP) is a derivative of pyruvic acid and found to have potent anti-tumor properties. Despite its potential benefits, the impact of EP on DLBCL remains ambiguous. Our objective is to elucidate the role of EP in modulating the development of DLBCL. Analysis of cholecystokinin-8 (CCK-8) revealed that treatment with EP significantly diminished the viability of DLBCL cells. Furthermore, EP administration suppressed colony formation and hindered cell adhesion and invasion in DLBCL cells. Examination of cell cycle progression showed that EP treatment induced arrest at the G1 phase and subsequently reduced the S phase population in DLBCL cells. EP treatment consistently exhibited apoptosis-inducing properties in Annexin-V assays, and notably downregulated the expression of Bcl-2 while increasing levels of proapoptotic cleaved caspase 3 and BAX in DLBCL cells. Additionally, EP treatment decreased the overexpression of c-Jun in c-Jun-transfected DLBCL cells. Further, EP demonstrated DNA-damaging effects in TUNEL assays. In vivo, xenograft animal models revealed that EP treatment significantly mitigated DLBCL tumor growth and suppressed DLBCL cell adhesion to bone marrow stromal cells. In summary, these findings suggest that EP mitigates DLBCL progression by inducing apoptosis, inducing cell cycle arrest, and promoting DNA damage.

Low NDRG2, regulated by the MYC/MIZ-1 complex and methylation, predicts poor outcomes in DLBCL patients.

Diffuse large B-cell lymphoma (DLBCL) represents the most common tumor in non-Hodgkin's lymphoma. N-Myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor highly expressed in healthy tissues but downregulated in many cancers. Although cell proliferation-related metabolism rewiring has been well characterized, less is known about the mechanism of metabolic changes with DLBCL. Herein, we investigated the expressions of NDRG2, MYC and Myc-interacting zinc finger protein 1 (MIZ-1) in seven human lymphoma (mostly DLBCLs) cell lines. NDRG2 expression was inversely correlated with the expressions of MYC and MIZ-1. Further, we explored the regulatory mechanism and biological functions underlying the lymphomagenesis involving NDRG2, MYC and MIZ-1. MYC and MIZ-1 promoted DLBCL cell proliferation, while NDRG2 induced apoptosis in LY8 cells. Moreover, NDRG2 methylation was reversed by the 5-Aza-2'-deoxycytidine (5-Aza-CDR) treatment, triggering the downregulation of MYC and inhibiting DLBCL cell survival. MYC interacts with NDRG2 to regulate energy metabolism associated with mTOR. Remarkably, supporting the biological significance, the converse correlation between NDRG2 and MYC was observed in human DLBCL tumor tissues (R = -0.557). Bioinformatics analysis further validated the association among NDRG2, MYC, MIZ-1, mTOR, and related metabolism genes. Additionally, NDRG2 (P = 0.001) and MYC (P < 0.001) were identified as promising prognostic biomarkers in DLBCL patients through survival analysis. Together, our data demonstrate that the MYC/MIZ-1 complex interplays with NDRG2 to influence the proliferation and apoptosis of DLBCL cells and show the characterizations of NDRG2, MYC and MIZ-1 for metabolism features and prediction prognosis in DLBCL.

Palmitic acid reduces the methylation of the FOXO1 promoter to suppress the development of diffuse large B-cell lymphoma via modulating the miR-429/DNMT3A axis

Diffuse large B-cell lymphoma (DLBCL) is characterized by significant treatment resistance. Palmitic acid (PA) has shown promising antitumor properties. This study aims to elucidate the molecular mechanisms by which PA influences DLBCL progression. We quantified the expression levels of microRNAs (miRNAs), Forkhead box protein O1 (FOXO1), and DNA methyltransferase 3A (DNMT3A) in both untreated and PA-treated DLBCL tumors and cell lines. Assessments were made of cell viability, apoptosis, and autophagy-related protein expression following PA administration. Interaction analyses among miR-429, DNMT3A, and FOXO1 were conducted using luciferase reporter assays and methylation-specific (MSP) Polymerase chain reaction (PCR). After transfecting the miR-429 inhibitor, negative control (NC) inhibitor, shRNA against DNMT3A (sh-DNMT3A), shRNA negative control (sh-NC), overexpression vector for DNMT3A (oe-DNMT3A), or overexpression negative control (oe-NC), we evaluated the effects of miR-429 and DNMT3A on cell viability, mortality, and autophagy-related protein expression in PA-treated DLBCL cell lines. The efficacy of PA was also tested in vivo using DLBCL tumor-bearing mouse models. MiR-429 and FOXO1 expression levels were downregulated, whereas DNMT3A was upregulated in DLBCL compared to the control group. PA treatment was associated with enhanced autophagy, mediated by the upregulation of miR-429 and downregulation of DNMT3A. The luciferase reporter assay and MSP confirmed that miR-429 directly inhibits DNMT3A, thereby reducing FOXO1 methylation. Subsequent experiments demonstrated that PA promotes autophagy and inhibits DLBCL progression by upregulating miR-429 and modulating the DNMT3A/FOXO1 axis. In vivo PA significantly reduced the growth of xenografted tumors through its regulatory impact on the miR-429/DNMT3A/FOXO1 axis. Palmitic acid may modulate autophagy and inhibit DLBCL progression by targeting the miR-429/DNMT3A/FOXO1 signaling pathway, suggesting a novel therapeutic target for DLBCL management.

Assessment and prognostic significance of a serum cytokine panel in diffuse large B‑cell lymphoma.

The objective of the present study was to assess the levels of circulating cytokines in patients with diffuse large B-cell lymphoma (DLBCL), and to examine the associations between the cytokine levels, clinicopathological manifestations and patient prognosis. The study enrolled 49 patients with DLBCL, 11 patients with chronic lymphocytic leukemia/small lymphocytic lymphoma and 67 healthy controls from Zhejiang Provincial People's Hospital (Hangzhou, China) between January 2017 and January 2020. The serum levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were measured using flow cytometry. The IL-6, IL-10 and IFN-γ levels were significantly raised in patients with DLBCL compared with those in the healthy controls (P<0.05). The levels of IL-10 were significantly higher in patients with raised levels of circulating lactate dehydrogenase (P<0.05), while increases in both IL-6 and IL-10 were associated with raised C-reactive protein (CRP) levels, with IL-6 levels positively associated with those of serum CRP (P<0.01; r=0.66). Additionally, International Prognostic Index (IPI) risk stratification of patients with DLBCL was strongly associated with circulating IL-6 and IL-10 levels. Raised IL-6, IL-10 and TNF-α levels were linked with worse short-term treatment efficacies (P<0.05). Moreover, the accuracy of the model predicting short-term treatment response in patients with DLBCL, obtained using the support vector machine algorithm, was 81.63%. It was also found that raised serum IL-6 and IL-10 levels, together with reduced levels of IL-17, were associated with survival of <1 year in patients with DLBCL (P<0.05), although no significant link was found between cytokine levels and long-term overall survival. In conclusion, the serum levels of IL-6, IL-10, IL-17, TNF-α and IFN-γ can potentially serve as biological indicators of DLBCL tumor immune status, and combined application with the IPI score can be a robust prognostic indicator in patients with DLBCL.

Chidamide enhances T-cell-mediated anti-tumor immune function by inhibiting NOTCH1/NFATC1 signaling pathway in ABC-type diffuse large B-cell lymphoma

Chidamide (CS055/HBI-8000, tucidinostat) has shown promising effects in the clinical treatment of various hematologic tumors. Diffuse large B-cell lymphoma (DLBCL) has shown highly heterogeneous biological characteristics. There are complex mechanisms of the role of chidamide in DLBCL for in-depth study. It is essential to probe further into the mechanism of drug-tumor interactions as a guide to clinical application and to understand the occurrence and progression of DLBCL. In vitro and in vivo models were utilized to determine the effects of chidamide on signaling pathways involved in the DLBCL tumor microenvironment. The experimental results show that chidamide inhibited the proliferation of DLBCL cell lines in a dose- and time-dependent manner, and down-regulated the expression of NOTCH1 and NFATC1 in DLBCL cells as well as decreased the concentration of IL-10 in the supernatant. In addition, chidamide significantly lowered the expression of PD1 or TIM3 on CD4+T cells and CD8+T cells and elevated the levels of IL-2, IFN-γ, and TNF-α in the serum of animal models, which augmented the function of circulating T cells and tumor-infiltrating T cells and ultimately significantly repressed the growth of tumors. These findings prove that chidamide can effectively inhibit the cell activity of DLBCL cell lines by inhibiting the activation of NOTCH1 and NFATC1 signaling pathways. It can also improve the abnormal DLBCL microenvironment in which immune escape occurs, and inhibit immune escape. This study provides a new therapeutic idea for the exploration of individualized precision therapy for patients with malignant lymphoma.

The Oncogenic Lipid Sphingosine-1-Phosphate Impedes the Phagocytosis of Tumor Cells by M1 Macrophages in Diffuse Large B Cell Lymphoma.

A total of 30-40% of diffuse large B cell lymphoma (DLBCL) patients will either not respond to the standard therapy or their disease will recur. The first-line treatment for DLBCL is rituximab and combination chemotherapy. This treatment involves the chemotherapy-induced recruitment of tumor-associated macrophages that recognize and kill rituximab-opsonized DLBCL cells. However, we lack insights into the factors responsible for the recruitment and functionality of macrophages in DLBCL tumors. We have studied the effects of the immunomodulatory lipid sphingosine-1-phosphate (S1P) on macrophage activity in DLBCL, both in vitro and in animal models. We show that tumor-derived S1P mediates the chemoattraction of both monocytes and macrophages in vitro and in animal models, an effect that is dependent upon the S1P receptor S1PR1. However, S1P inhibited M1 macrophage-mediated phagocytosis of DLBCL tumor cells opsonized with the CD20 monoclonal antibodies rituximab and ofatumumab, an effect that could be reversed by an S1PR1 inhibitor. Our data show that S1P signaling can modulate macrophage recruitment and tumor cell killing by anti-CD20 monoclonal antibodies in DLBCL. The administration of S1PR1 inhibitors could enhance the phagocytosis of tumor cells and improve outcomes for patients.

Genomic Stability, DNA Repair and the SKP1-CUL1-F-Box Complex in HIV Associated Diffuse Large B-Cell Lymphoma

Introduction: Previously we demonstrated that HIV associated diffuse large B-cell lymphomas (DLBCL) of Germinal Center B-cell subtype (GCB) are more proliferative, with enhanced genomic stability and increased expression of DNA repair genes, particularly Fanconi anemia (FA) and homologous recombination (HR) genes compared to their HIV negative counterparts (Maguire et al, 2019). It is unknown if this signature is unique to the GCB subtype or if it is also observed in Activated B-cell (ABC) and Unclassified (UNC) DLBCL. Herein we validate our HIV associated GCB DLBCL findings in a larger independent cohort and report for the first time the aberrational profile of molecularly profiled HIV associated ABC DLBCL compared to their HIV negative counterparts respectively. Methods: An independent cohort of 353 DLBCL FFPE cases was assembled, including 125 HIV-DLBCL samples. All samples underwent hematopathological review to confirm diagnosis and determine tumor content. Samples with &amp;lt;60% tumor content were macro-dissected before they were RNA/DNA extracted. Cell of origin (COO) profiles were assessed using the Lymph3Cx molecular assay which can distinguish primary mediastinal B-cell lymphoma (PMBL) like from GCB, ABC and UNC DLBCL (Mottok et al 2018). Copy number (CN) analyses were performed on samples with sufficient DNA (41 HIV neg, 32 HIV pos) using the OncoScan FFPE assay kit and the resulting data analyzed using Chromosome Analysis Suite (ChAS) v4.1 and the multisample viewer module of Reproductive Health Research Analysis Software (RHAS) v1.1. Additional digital gene expression profiling was performed using the Pan-Cancer panel (NanoString, Seattle, WA) customized to include 9 BCL2 family genes for a total of 739 genes and analyzed using the NanoString nSolver Advanced Analysis module. Results: GCB, ABC, UNC and PMBL-like proportions were 50%, 30%, 14% and 6% in HIV unassociated (HIV neg) and 50%, 23%, 25% and 2% in HIV pos tumors respectively, confirming both cohorts were GCB-enriched by molecular profiling. PMBL-like cases were excluded from further analyses. OncoScan CN analyses showed that HIV pos DLBCL were significantly less aberrant than HIV neg DLBCL (median CN per sample 0.56 vs 1.01, p&amp;lt;0.001). HIV pos GCB tumors were significantly less aberrant than both HIV neg GCB tumors (median CN per sample 0.50 vs 1.11, p&amp;lt;0.001), and HIV pos ABC tumors (median CN per sample 0.50 vs 0.81, p=0.009). HIV pos ABC tumors were also less aberrant than HIV neg ABC tumors, however, the difference was not significant overall (median CN per sample 0.81 vs 1.03, p=0.162) or for gain/amplifications (median CN per sample 0.38 vs 0.42, p=0.814), but was borderline significant for loss/deletions (median CN per sample 0.31 vs 0.47, p=0.06). GEP analyses identified Cell Cycle and DNA repair as the top two upregulated pathways in HIV pos GCB tumors compared to HIV neg GCB tumors. In contrast, Cell Cycle and DNA repair were downregulated in HIV pos ABC tumors compared to HIV neg ABC tumors. However, one gene ( RBX1) was identified as significantly upregulated in both pathways in HIV pos GCB (adjusted p-value 0.0215) and HIV pos ABC (adjusted p-value 0.0046) tumors compared to their HIV neg counterparts. A second gene ( SKP1) was also significantly upregulated in HIV pos GCB (adjusted p-value 3.94E-5) and HIV pos ABC (adjusted p-value 0.0031) tumors. Together RBX1 and SKP1 encode the two principal functional subunits of the SKP1, CUL1, F-box protein (SCF) complex which is part of the ubiquitin proteasome system (UPS) and has been reported as essential for maintaining genome and chromosome stability (Thompson et al 2021). Conclusions: These results demonstrate that HIV pos DLBCL tumors are less aberrant than their HIV neg counterparts irrespective of COO subtype. However, the degree of genomic stability differs by COO, with HIV pos GCB tumors being more genomically stable than HIV pos ABC tumors. Although both have enhanced expression of UPS related SCF genes ( RBX1, SKP1), only HIV pos GCB tumors have enhanced expression of key DNA repair genes as well including Fanconi Anemia ( BRCA2/FANCD1, FANCL) and mismatch repair ( MSH2, MSH6) genes (adjusted p-values of ≤ 0.0263). Given the reported role of SCF in genomic stability maintenance, the HIV pos COO CN differences could be due to the contributions of both DNA repair and SCF mechanisms in HIV pos GCB and mainly SCF mechanisms in HIV pos ABC tumors.

Enhanced Ferroptosis Induction Susceptibility in Diffuse Large B-Cell Lymphoma through Rocaglate-Mediated Translation Inhibition

Introduction: Diffuse Large B-cell Lymphoma (DLBCL) is a genomically complex hematologic malignancy characterized by heterogeneous pathogenesis across multiple molecular subtypes. Despite recent advances, standard frontline treatment options leave a third of patients with relapsed or refractory disease due to tumor populations resistant to therapy by a wide variety of mechanisms. Ferroptosis, an iron-dependent non-programmed cell death mechanism driven by lipid peroxidation, has emerged as a promising therapeutic opportunity in DLBCL. DLBCL demonstrates distinct sensitivity to ferroptosis activators compared to other cancer types, yet the implications in DLBCL prognosis, immune infiltration, and drug resistance remain elusive. Notably, the loss of histone deubiquitinase MYSM1 in hematopoietic stem cells significantly reduces protein synthesis, leading to reduced ferroptosis suppressors, ultimately rendering the cells more susceptible to ferroptosis. Moreover, NRF2, encoded by NFE2L, a known modulator of ferroptosis, has been linked to resistance to rocaglates (cap-dependent translation inhibitors). Intriguingly, our preliminary data indicate that rocaglates induce translation of key proteins involved in oxidative stress (i.e. SDHB) and transcription factors related to the oxidative stress response (NRF1, ATF2, NRF2). Additionally, our published findings in glioblastoma suggest the upregulation of the ferroptosis suppressor 4F2 ( SLC3A2) under translation inhibition conditions. Furthermore, our investigations uncover another potential mechanism, wherein rocaglates induce NF-κB, which was previously shown to be suppressed by dimethyl fumarate (DMF) upon ferroptosis induction in DLBCL. However, the intricate interplay between ferroptosis and translation inhibition in DLBCL remains uninvestigated, prompting our hypothesis that protein synthesis inhibitors might enhance therapeutic ferroptosis induction. Method: To test our hypothesis, we conducted a comprehensive analysis, employing TMT-pSILAC to assess the global transcriptome response to rocaglates treatment, and subsequently verified significant findings via western blot analysis. Additionally, we quantified reduced glutathione (GSH) levels, reactive oxygen species (ROS) levels, and synergy with ferroptosis inducers (e.g., erastin, RSL3) to elucidate the mechanism of rocaglates in enhancing susceptibility to the ferroptosis pathway. Moreover, we explored NF-κB, previously identified as a ferroptosis regulator in our RNAseq analysis, using ELISA and NF-κB reporter assays. Results: Our TMT-pSILAC analysis unveiled upregulation of key ferroptosis suppressors, specifically NFS1, CBS, and 4F2. Concurrent treatment with rocaglates resulted in a notable increase in GSH levels, which was attenuated when administered in combination with ferroptosis inducers (p-value &amp;lt; 0.005) concomitant with the reduction of Glutathione Peroxidase 4 (GPX4), modulator of lipid peroxidation. Although rocaglates alone did not elicit significant levels of reactive oxygen species (ROS), their combination with ferroptosis inducers resulted in dramatic elevation of ROS levels (p-value &amp;lt; 0.0005). Correspondingly, there was strong synergy between the clinical rocaglate zotatifin and different ferroptosis inducers (Bliss δ synergy score &amp;gt; 10) and antagonism with ferroptosis inhibitors (Bliss δ synergy score &amp;lt; -10). Furthermore, our investigation revealed a noteworthy upregulation of NFKB1 at the translatome level, accompanied by a significant increase in TNF-α expression at 48 hours. The NF-κB-EGFP reporter assay confirmed the enhanced expression of NF-κB in response to the treatment. Conclusion: In conclusion, rocaglate-induced stress upregulates multiple ferroptosis suppressors, suggesting that cap-dependent translation disruption triggers a protective response against ferroptosis. Strikingly, these factors did not protect DLBCL tumors from ferroptosis inducers, which synergized remarkably with rocaglates, yielding promising new combination drug strategies. The proteomic basis for ferroptosis sensitization by rocaglate therapy remains under active investigation. Understanding the intricate interplay between these pathways may unveil potential key mechanisms for DLBCL treatment, potentially offering novel therapeutic targets to improve patient outcomes.

Combination of Selinexor with BTK Inhibitor for Central Nervous System Diffuse Large B-Cell Lymphoma, Possible Mechanisms and Therapeutic Potential Exploration

Background: Primary central nervous system lymphoma (PCNSL) is a rare aggressive extra-nodal non-Hodgkin lymphoma (NHL) of which over 90% are diffuse large B-cell lymphoma (DLBCL). High-dose methotrexate (HD-MTX)-based regimens are the dominant therapy for newly diagnosed PCNSL, but the patients' complete remission rate (CRR) is merely about 50%. Bruton tyrosine kinase (BTK), a critical kinase in the B cell receptor (BCR) signaling pathway, contributes to the degradation of FOXO3a and promotes abnormal B cell proliferation. Recent studies revealed that BTK inhibitors (BTKi) play a role in various B cell lymphomas, including DLBCL, and have also shown promising efficacy in PCNSL. However, the clinical management of PCNSL is still challenging for its limited treatment options and poor prognosis, especially when patients developed acquired resistance to BTKi. Selinexor (KPT-330) is a first-ever oral small molecule Exportin-1 (nuclear output protein XPO1) inhibitor with a molecular weight of 443.31 Da, which can efficiently cross the blood-brain barrier. Currently Selinexor was found to promote the aggregation of FOXO3a and could synergistically inhibit PI3K/AKT and NF-κB pathways with BTKi to overcome acquired BTKi resistance. To investigate the therapeutic potential of Selinexor and BTKi in PCNSL, we examined the combination both in DLBCL cells and A20 transplantation mice, and further explored the clinical efficacy in PCNSL patients. Methods and Results: ABC-DLBCL cell lines (OCI-LY3 and OCI-LY10) were treated with Selinexor for 48 hours. Western blot analysis showed that Selinexor inhibited NF-κB, P-STAT3, and NFATc1 signaling pathways in DLBCL in vitro. Combining Selinexor(1μM) with ICP-022(2μM), a BTKi, resulted in more notable inhibition of the above tumor signaling pathways. A20 murine B-cell lymphoma transplantation models were divided into a control group, Selinexor monotherapy group, ICP-022 monotherapy group, and combination group. Tumor volume was measured after 30 days (Figures 1A), and the number of CD4+ T cells and CD8+ T cells and the corresponding cellular expression of PD-1 and TIM-3 in the spleen and tumor tissues of mice were detected. The results showed that the tumor volume in the combination group was minimized, and the number of CD4+ T cells and CD8+ T cells in spleens and tumors in this group was significantly increased, while PD-1 and TIM-3 expression was downregulated (Figures 1B-C). Based on these results, a prospective, open-label, single-arm, multicenter study was conducted with MSZ regimen (MTX, BTKi, Selinexor) for the treatment of PCNSL (Trial Registration Number: ChiCTR2200062154). Newly diagnosed primary and secondary central DLBCL patients (18-80 years old) received a 6-cycle MSZ regimen (HDMTX 3.5 g/m 2 3-h infusion d1, Zanubrutinib 160 mg Bid po, Selinexor 40 mg Biw po, 3 weeks per cycle) and evaluated by MRI/PET. 5 patients (2 GCB-DLBCL, 3 ABC-DLBCL) were enrolled, with a median age of 65 years (range: 54-73 years), 100% achieved metabolic remission (Figure 2A) and maintained long-term efficacy. 4 patients achieved early deep remissions of over 75% reduction with 1 cycle of treatment, and 1 patient had a complete remission after 2 cycles of treatment (Figures 2B). 3 patients chose radiation therapy as consolidation after the end of induction chemotherapy. 2 others were on maintenance therapy with Selinexor combined with Zanubrutinib, one of them has completed the two-year maintenance treatment and maintained sustained remission till now. Only 1 patient experienced several grade 3 adverse events (AEs) including anemia, decreased white blood cell and platelet counts, and elevated alanine aminotransferase.1 patient developed mild renal insufficiency, which recovered 6 months after the end of treatment. Patients were generally well-tolerated and there were no treatment-related deaths. Conclusion: This is the first report describing that Selinexor combined with BTKi can synergistically inhibit the DLBCL tumor signaling pathway and repairing the immune microenvironment. Correspondingly, in treatment of central DLBCL patients, the novel therapeutic strategy of MSZ regimen has achieved impressive results, with a CRR of 100%. Most patients attained early and rapid deep remissions with durable efficacy. This study provides theoretical support and preliminary clinical data for the combined application of Selinexor and BTK inhibitors.

Whole Exome Sequencing Analysis of Diffuse Large B-Cell Lymphoma That Progressed to Central Nervous System

Background: Recent sequencing analyses have identified distinct genetic subtypes of diffuse large B-cell lymphoma (DLBCL). The MCD subtype, characterized by MYD88 L265P and CD79B gene mutations, is frequently found in DLBCL that progresses to the central nervous system (CNS), known as secondary CNS lymphoma (sCNSL) (Ollila, Blood 2021). In addition, the MCD subtype is also frequently found in primary CNS lymphoma (PCNSL) (Radke, Nat commun 2022). Therefore, we hypothesized that specific genetic alterations that are present in the initial DLBCL tumors are associated with their progression to the CNS. We aimed to investigate i) the clinical impact of genetic subtypes of DLBCL and ii) genomic mutations that may be associated with sCNSL. To this end, genetic alterations in DLBCL samples at diagnosis that later developed into sCNSL were evaluated using whole-exome sequencing (WES). Methods: A total of 44 patients with DLBCL who developed sCNSL after receiving standard rituximab-containing chemotherapy between 2009 and 2020 were identified from five institutions. sCNSL was diagnosed on the basis of imaging and/or pathological findings. Genomic DNA (gDNA) was extracted from formalin-fixed, paraffin-embedded tumor samples obtained at the initial diagnosis of DLBCL. Finally, 26 of the 44 samples with sufficient gDNA were subjected to WES at BGI Genomic Co. Ltd. Single-nucleotide variants and short indels were detected using Mutect2 and filtered as described in Figure 1. Genetic subtypes were determined using the LymphGen algorithm (https://llmpp.nih.gov/lymphgen/index.php). The association between the clinicopathological features and genetic subtypes was also evaluated. In addition, recurrently mutated genes (&amp;gt;10% in our cohort) were identified and their alteration frequencies were compared with those in a representative general DLBCL cohort (N=574, available in the article by Schmitz, NEJM 2018) and PCNSL cohort (N=30, whole genome sequencing performed, available in the article by Radke, Nat Commun 2022) (Figure 1a). Results: The median age of the 26 patients was 69 years (range, 34-87). At the time of CNS progression, 54, 27, and 19% of the patients had parenchymal CNS disease, extraparenchymal CNS disease, and both, respectively. Immunohistological analysis showed that the non-germinal center B-cell-like (GCB) type, as defined using the Hans algorithm, and dual expression of MYC and BCL2 (DEL) were identified in 65% and 69% of patients, respectively. The median overall survival (OS) from CNS relapse was 16 months (95% confidence interval, 2-30). WES analysis showed that the median mean coverage depth of the 26 samples was 140 (range, 53-299). The median mapping rate was 94% (range, 80-99). As expected, the most frequently mutated genes in our cohort were MYD88 (50%), PIM1 (50%), BTG2 (31%), and CD79B (31%), corresponding to the MCD subtype. The LymphGen algorithm classified the 26 samples into MCD (n=12, 46%), EZB (n=5, 19%, including one with a composite feature of ST2), BN2 (n=1, 4%) and “not classified” (n=8, 31%) (Figure 1b). The CNS-IPI risk, DEL status, and CNS relapse sites did not differ among the genetic subgroups. In addition, univariate analysis showed that non-GCB status, DEL status, and genetic subtype at initial diagnosis did not influence OS. Next, we shortlisted 38 genes whose mutations were identified in three or more samples (&amp;gt;10%) of our cohort as well as in &amp;gt;10% of the PCNSL cohort (by Radke, Nat Commun 2022). We further selected genes that were less frequent (&amp;lt;3%) in the general DLBCL cohort and identified several genes, such as MUC16 and FAT4, that were more enriched in PCNSL and our sCNSL cases (Figure 1a). Metascape analysis (https://metascape.org/gp/index.html#/main/step1) revealed that the top three enriched processes associated with these genes were sensory organ development, cell adhesion via plasma membrane adhesion molecules, and morphogenesis of epithelium. Conclusion: Our study confirmed that MCD was a major genetic subtype of DLBCL that developed sCNSL. We identified recurrently mutated genes that were common in both sCSNL and PCNSL but not in the general DLBCL cohort; these genes may be associated with CNS progression. Validation of our results and investigation of the functions of these genes will help to understand the mechanisms of CNS relapse and develop more accurate markers to predict sCNSL. *Suzuki T and Yokomori R equally contributed to this study.

Oncolytic virus oHSV2 combined with PD-1/PD-L1 inhibitors exert antitumor activity by mediating CD4 + T and CD8 + T cell infiltration in the lymphoma tumor microenvironment

A novel therapeutic regimen showed that the oncolytic type II herpes simplex virus (oHSV2) was able to prevent colorectal cancer growth, recurrence, and metastasis. However, no study has yet explored whether oHSV2 has an impact on the development of diffuse large B-cell lymphoma (DLBCL). We chose the clinical chemotherapeutic drug doxorubicin (DOX) as a positive control to evaluate the effect of oHSV2 infection on the apoptotic, invasive, and proliferative capacity of DLBCL cells. We next further explored the therapeutic efficacy of oncolytic virus oHSV2 or DOX in DLBCL tumor bearing BALB/c mice, and evaluated the infiltration of CD8 + T cells and CD4 + T cells in tumor tissues. A pathological approach was used to explore the effects of oHSV2 on various organs of tumor bearing mice, including the heart, liver, and kidney. Next, SU-DHL-4 cells were co-cultured with cytotoxic T lymphocytes (CTLs) to mimic the tumor immune microenvironment (TME), to explore the impact of oHSV2 on the immune environment at the cellular level, and then analyzed the relationship between oHSV2 and the PD-1/PD-L1 immune-checkpoint. Subsequently, we further validated the efficacy of combined oHSV2 and PD-L1 treatment on transplanted tumor growth in mice at the in vivo level. DLBCL cells were sensitive to the action of the oncolytic virus oHSV2, and the decline in their proliferative activity showed a time-and dose-dependent manner. oHSV2 and DOX intervention preeminently increased the cell apoptosis, restrained cell proliferation and invasion, with the greatest changes occurring in response to oHSV2 infection. oHSV2 application effectively improved the immune status of the tumor microenvironment, favoring the invasion of CD8 + T and CD4 + T cells, thereby enhancing their antitumor effects. Besides, oHSV2 treatment has a safety profile in the organs of tumor bearing mice and indeed inhibits the PD-1/PD-L1 immune checkpoint in DLBCL. Interestingly, the combination of oHSV2 and PD-L1 antibodies results in more profound killing of DLBCL cells than oHSV2 infection alone, with a significant increase in the proportion of CD4 + T cells and CD8 + T cells. The antitumor effect was the best after combining oHSV2 and PD-L1 antibodies, suggesting that the combination therapy of oHSV2 and PD-L1 would have a better prospect for clinical application.

PAQR3 facilitates the ferroptosis of diffuse large B-cell lymphoma via the regulation of LDLR-mediated PI3K/AKT pathway.

Progesterone and adiponectin receptor 3 (PAQR3) has been found to regulate tumor progression by mediating cell ferroptosis. However, whether PAQR3 mediates ferroptosis in diffuse large B-cell lymphoma (DLBCL) needs further investigation. The mRNA and protein levels of PAQR3 and low-density lipoprotein receptor (LDLR) were assessed by qRT-PCR and WB assays. Cell proliferation was detected by MTT assay and EdU assay. Shrunken mitochondria was counted under transmission electron microscope. Cell ferroptosis was evaluated by measuring the levels of malondialdehyde, reactive oxygen species, glutathione, Fe2+ , and the protein expression of ferroptosis-related markers. PAQR3 and LDLR interaction was confirmed by RIP assay and pull-down assay. Our study showed that PAQR3 was underexpressed, while LDLR was overexpressed in DLBCL tissues and cells. Functionally, PAQR3 overexpression or LDLR knockdown restrained DLBCL cell proliferation and enhanced ferroptosis. Mechanistically, PAQR3 reduced LDLR expression by inhibiting its mRNA stability. Meanwhile, LDLR overexpression reversed PAQR3-mediated the promoting on DLBCL cell ferroptosis, and LY294002 (PI3K/AKT inhibitor) eliminated the inhibiting effects of LDLR overexpression on DLBCL cell ferroptosis. Additionally, excessive PAQR3 reduced DLBCL tumor growth by enhancing tumor cell ferroptosis through LDLR-mediated PI3K/AKT pathway. In conclusion, our data suggested that PAQR3 restrained DLBCL progression by aggravating ferroptosis, which was achieved by inhibiting LDLR expression to repress PI3K/AKT pathway.

Role of an adaptor protein human germinal center-associated lymphoma (HGAL) in cell signaling and lymphomagenesis

Human germinal center (GC)-associated lymphoma (HGAL) is a multi-domain adaptor protein expressed in GC B lymphocytes, T follicular helper (Tfh) cells and lymphomas derived from these cells. HGAL expression is an independent predictor of longer survival of diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin’s lymphoma (HL) patients. HGAL regulates B cell receptor (BCR) signaling and immunological synapse formation by binding to either the downstream effectors [e.g., spleen tyrosine kinase (Syk)] or other signaling regulators [e.g., growth factor receptor-bound protein 2 (Grb2)]. HGAL regulates the cytoskeleton that reshapes B cell morphology during BCR signaling and cell motility by at least two molecular mechanisms: enhanced Ras homolog gene family member A (RhoA) signaling and inhibition of myosin-actin translocation. These effects on the cytoskeleton decrease lymphoma dissemination in animal models and contribute to decreased lymphoma dissemination in patients. The latter may contribute to the association of HGAL protein expression with longer survival of patients with DLBCL and HL tumors. The ability to regulate multiple and distinct functions simultaneously in B cells implies that the HGAL protein level is tightly regulated. It was demonstrated that HGAL can be regulated by PR/SET domain 1 (PRDM1)/B lymphocyte-induced maturation protein-1 (BLIMP1) and interleukin-4 (IL-4) at the transcription level, by microRNA-155 (miR-155) at the post-transcriptional level, and by F-box protein 10 (FBXO10) at the post-translational level. Constitutive enforced expression of HGAL at physiological levels leads to lymphoid hyperplasia and DLBCL in mice. Future studies need to focus on identifying HGAL interactome, dissecting its interaction network, and understanding HGAL spatiotemporal signaling in live cells in physiological conditions. Further, the recent demonstration of HGAL expression in Tfh cells requires the determination of its function in these cells. These studies will contribute to new insights into the biology of these cellular subsets and how immune dysregulation contributes to lymphomagenesis.

A SINGLE‐CELL ATLAS OF DIFFUSE LARGE B CELL LYMPHOMA

Diffuse large B-cell lymphoma (DLBCL) is one of the most common yet aggressive types of B-cell lymphoma and remains incurable in 40% of patients. Herein, we profiled the transcriptomes of 94,324 cells from 17 DLBCLs and 3 control samples using single-cell RNA-sequencing technology, creating a comprehensive single-cell map for tumor and infiltrating immune cells for DLBCL. High degrees of inter- and intratumor heterogeneity were revealed, and we identified 73 gene expression programs expressed in malignant B cells. We further characterized 8 nonmalignant B-cell subclusters and 16 T-cell subclusters from the tumor microenvironment. Six myeloid subclusters were also identified, including the LAMP3+ DC subcluster enriched in ABC-DLBCL. More than 2000 likely cell-cell interactions were further predicted in DLBCL, illustrating a complex and highly dynamic DLBCL tumor microenvironment. Unique to B cell lymphomas, a strong costimulatory CD70-CD27 interaction was predicted between malignant cells and T cells. Furthermore, coinhibitory signals mediated by TIM-3 or TIGIT seemed to be the main driving force for T-cell exhaustion. Finally, we discovered that chronic hepatitis B virus infection may have a significant impact on tumor cell survival and immune evasion in DLBCL. Our results provide new insights into B-cell lymphomagenesis and may facilitate the design of targeted immunotherapy strategies. The research was funded by: Swedish Cancer Society, STINT, CIMED, Radiumhemmet, the Swedish Research Council, and the Knut and Alice Wallenberg Foundation, the Guangdong Enterprise Key Laboratory of Human Disease Genomics, the Shenzhen Key Laboratory of Single-Cell Omics Keywords: aggressive B-cell non-Hodgkin lymphoma, genomics, epigenomics, and other -omics, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

APR‐246 TRIGGERS FERRITINOPHAGY AND FERROPTOSIS OF DIFFUSE LARGE B‐CELL LYMPHOMA CELLS WITH DISTINCT TP53 MUTATIONS

Purpose: Recently, seven diffuse large B-cell lymphoma (DLBCL) genotypes were defined, among which the A53 type showed adverse outcomes. Thus, it is necessary to explore promising anticancer drugs and therapies for TP53 dysfunction patients. APR-246 is a promising compound to combat mutant p53 in solid and hematological tumors, but the effect of APR-246 on DLBCL remains unclear. Methods: A set of 2464 DLBCL cases from multiple cohorts including our center, was integrated to identify the type and localization of TP53 mutations and clinical impacts. APR-246 was applied in TP53-mutated DLBCL cells and xenograft mouse models to explore the anti-tumor effect and the underlying mechanism. Results: Of all patients, 16% contained TP53 mutations. TP53 mutations correlated with poor overall survival (OS) and progression-free survival (PFS) in all cases. Notably, TP53single mutations in the DBD (exons 5-8) resulted in poor OS and PFS, but those on the exons (3-4, 9-11) outside the DBD. DLBCL cells carrying exons 5 and 7 or intact TP53 mutations showed more sensitivity to APR-246 than those containing spliced mutations or null TP53. DLBCL cells with knocked-out TP53 showed less sensitivity to APR-246. APR-246 also significantly inhibited the DLBCL tumor growth in xenograft animal model. The decreased cell viability induced by APR-246 was almost completely rescued by either the iron chelater deferoxamine (DFO) or ferrostatin-1 (Fer-1), indicating that ferroptosis mediates cell death. APR-246-induced cell death is an action involving ROS production, which was not altered by knockout of TP53 in OCI-LY7. However, only ferroptosis was induced by APR-246 in OCI-LY3, TMD8 and TP53-knockout OCI-LY7 cells, indicating that APR-246-induced autophagy in OCI-LY7 was TP53-dependent. Further, the cells re-expressing wild-type p53 showed the similar response as OCI-LY3 harboring wild-type p53 to APR-246, while the cells with re-expressed p53 mutant showed the similar response as OCI-LY7 to APR-246. The expression of ALOX5, ALOX12, and ALOX15 were increased in DLBCL cells after APR-246 treatment for 24 h. The LC3-II level was enhanced while the autophagy substrate SQSTM1 was reduced by APR-246 treatment, confirming APR-246 induces autophagy in OCI-LY7 cells. Additionally, FTH1, NCOA4 and HERC2 were also examined in TP53 knockout DLBCL cells, and the data demonstrated that APR-246 had no effect on the level of these proteins, indicating that APR-246 restored p53 was involved in ferritinophagy in OCI-LY7 cells. These findings were further confirmed in OCI-LY7 TP53-KO cells with re-expressing mutant or wild-type p53. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Diagnostic and Prognostic Biomarkers, Genomics, Epigenomics, and Other -Omics No conflicts of interests pertinent to the abstract.

Potential role of the P2X7 receptor in the proliferation of human diffused large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of invasive non-Hodgkin lymphoma. 60-70% of patients are curable with current chemoimmunotherapy, whereas the rest are refractory or relapsed. Understanding of the interaction between DLBCL cells and tumor microenvironment raises the hope of improving overall survival of DLBCL patients. P2X7, a member of purinergic receptors P2X family, is activated by extracellular ATP and subsequently promotes the progression of various malignancies. However, its role in DLBCL has not been elucidated. In this study, the expression level of P2RX7 in DLBCL patients and cell lines was analyzed. MTS assay and EdU incorporation assay were carried out to study the effect of activated/inhibited P2X7 signaling on the proliferation of DLBCL cells. Bulk RNAseq was performed to explore potential mechanism. The results demonstrated high level expression of P2RX7 in DLBCL patients, typically in patients with relapse DLBCL. 2'(3')-O-(4-benzoylbenzoyl) adenosine 5-triphosphate (Bz-ATP), an agonist of P2X7, significantly accelerated the proliferation of DLBCL cells, whereas delayed proliferation was detected when administrated with antagonist A740003. Furthermore, a urea cycle enzyme named CPS1 (carbamoyl phosphate synthase 1), which up-regulated in P2X7-activated DLBCL cells while down-regulated in P2X7-inhibited group, was demonstrated to involve in such process. Our study reveals the role of P2X7 in the proliferation of DLBCL cells and implies that P2X7 may serve as a potential molecular target for the treatment of DLBCL.

Abstract CT129: Trial in progress: ATHENA-1 - a phase 1, open-label, first-in-human study to assess safety and tolerability of REGN5837 in combination with odronextamab in patients with relapsed/refractory aggressive B-cell non-Hodgkin lymphomas

Abstract Background: Many patients with relapsed/refractory (R/R) aggressive B-cell non-Hodgkin lymphoma (B-NHL) are unable to tolerate, access, or benefit from intensive chemo-therapeutic approaches or cellular therapies and will invariably relapse; therefore, novel approaches are urgently required. Odronextamab (REGN1979) is a hinge-stabilized, human CD20 × CD3 IgG4-based bispecific antibody that elicits T-cell-mediated cytotoxicity of malignant B cells. In a Phase 1 study, odronextamab monotherapy showed a manageable safety profile with encouraging preliminary activity in heavily pre-treated patients with R/R B-NHL (Bannerji R, et al. Lancet Haematol. 2022;9(5):e327-39). REGN5837 is a hinge-stabilized, human CD28 × CD22 IgG4-based bispecific antibody that provides a co-stimulatory signal (signal 2). When combined with odronextamab (signal 1), REGN5837 improved anti-tumor efficacy and survival in in vivo diffuse large B-cell lymphoma tumor models via enhanced T-cell expansion. We hypothesize that combining REGN5837 with odronextamab may deepen and extend anti-tumor activity in patients with aggressive lymphoma. Methods: ATHENA-1 (NCT05685173) is a Phase 1, open-label, first-in-human study of REGN5837 in combination with odronextamab in patients with R/R aggressive B-NHL. During induction, odronextamab and REGN5837 will be administered weekly over 21-day cycles. To mitigate potential CRS events, odronextamab will be introduced with step-up dosing as a monotherapy, followed by introduction of REGN5837 on C2 D15 with step-up dosing. Maintenance will consist of 28-day cycles (odronextamab and REGN5837 administration on D1, 15). Patients who achieve a sustained complete response (≥9 months) will have study drug(s) administration changed to once every 4 weeks. Patients must be aged ≥18 years, have Eastern Cooperative Oncology Group performance status ≤1, with adequate organ function, and have CD20+ aggressive B-NHL that progressed after ≥2 lines of systemic therapy containing at least an anti-CD20 antibody and an alkylating agent, with or without prior chimeric antigen receptor T-cell therapy. Exclusion criteria include prior allogeneic stem cell transplant, organ transplant, or CD20xCD3 bispecific antibodies, or mantle cell lymphoma or central nervous system lymphoma. Primary endpoints are incidence of dose-limiting toxicities and the incidence and severity of treatment-emergent adverse events. Secondary endpoints include pharmacokinetics of odronextamab and REGN5837, anti-drug antibody incidence, objective response rate, complete response rate, duration of response, progression-free survival, and overall survival. Enrolment is planned to open in early 2023. Citation Format: John Baird, Pim G. Mutsaers, Jeremy S. Abramson, Manjusha Namuduri, Jingjin Li, Nickolas A. Sophos, Min Zhu, Jurriaan Brouwer-Visser, Hesham Mohamed, Aafia Chaudhry, Andrew J. Davies. Trial in progress: ATHENA-1 - a phase 1, open-label, first-in-human study to assess safety and tolerability of REGN5837 in combination with odronextamab in patients with relapsed/refractory aggressive B-cell non-Hodgkin lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT129.

Abstract LB049: ALKBH5 emerges as a novel regulator of the BCR pathway

Abstract Global epitranscriptomic methylation of mRNA at the N6 position (m6A) is reported to be altered in B-cell and tumor development. However, the mechanistic underpinnings of its role in the pathogenesis of Diffuse large B cell lymphoma (DLBCL) are unknown. It is well established that the B-cell receptor (BCR) pathway is a primary oncogenic driver in aggressive B-cell lymphomas. We previously reported that the Ataxia Telangiectasia Mutated (ATM)/Hu Antigen R (HuR)axis, as well as Eukaryotic Translation Initiation Factor 4A (eIF4A), modulate BCR signaling. However, the impact of RNA modifications on BCR induction is still poorly understood. Addressing this burgeoning question, we noted enhanced expression of AlkB Homolog 5 (ALKBH5), an m6A demethylase, in naïve B-cells treated with BCR mimetics. Similarly, mRNA levels of ALKBH5 were reported to be modestly enhanced in IgM-treated human B-cells (GSE156195). Consistently, immunization of C57BL/6 mice with sheep red blood cells (SRBC) significantly enhanced ALKBH5 in splenic B-cells. Next, evaluating the impact of BCR induction in lymphomagenesis, we observed a robust increase in the demethylase expression in BCR mimetic-treated DLBCL cells. Notably, increased expression of ALKBH5 was observed in a murine EµMyc model and primary DLBCL tumors. In coherence, the enhanced transcript levels of ALKBH5 in TCGA-DLBCL have prognostic implications. To gain functional insight, we depleted ALKBH5, which resulted in a robust reduction of BCR pathway proteins. Unexpectedly, the mRNA levels of CD79a and BLK were minimally modified, while the SYK, BTK, and CARD11 messages were reduced. Further studies are ongoing to evaluate the potential mechanism of mRNA transport and/or stability. Citation Format: Bandish B. Kapadia, Anirban Roychowdhury, Forum Kayastha, Nahid Nanaji, Jolene Windle, Ronald B. Gartenhaus. ALKBH5 emerges as a novel regulator of the BCR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB049.

Limitations in the CAR-T therapy for diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) therapy has been the subject of much investigation for many years. CAR-T cell therapy (CAR-T therapy) is now a new treatment for DLBCL. Human T cells are genetically altered to create new CAR-T cells, which are then injected into patients' bodies to kill cancer cells via an immune reaction. This process is known as CAR-T therapy. When treating refractory/relapsing (r/r) DLBCL, CAR-T therapy has showed some fairly thrilling clinical responses in comparison to conventional therapy and has a high cure rate. A few CAR-T treatments for DLBCL have also been authorized and put on the market. CAR-T cell therapy, however, has several drawbacks and difficulties when used to treat DLBCL. Some individuals are resistant to the therapy, leading to unsuccessful treatment, for the immunosuppressive microenvironment (TME) of DLBCL tumors and the phenomena of antigen escape. Additionally, CAR-T cell treatment may have major adverse effects including CRS and ICANS. If these adverse effects are not taken into consideration, people might die. Additionally, CAR-T treatment is quite pricey and common people might not be able to afford it, which will lead to a number of ethical issues. Naturally, scientists are working to provide answers to these problems and have made some success. The fundamentals of CAR-T therapy and its drawbacks for treating DLBCL will be covered in this review (including drug resistance, side effects, ethical and financial issues). Of course, the corresponding solutions, such as developing multiple targeted CARs and inhibiting the activity of certain cytokines, will also be described in this review.

Drug-Resistance Mechanism and New Targeted Drugs and Treatments of Relapse and Refractory DLBCL.

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin's lymphoma (NHL). 30 ~ 40% of DLBCL patients were resistant to the standard R-CHOP regimen or recurrence after remission. It is currently believed that drug resistance is the main cause of the recurrence and refractory of DLBCL (R/R DLBCL). With the increased understanding of DLBCL biology, tumor microenvironment and epigenetics, some new therapies and drugs like molecular and signal pathway target therapy, chimeric antigen receptor (CAR) T-cell therapy, immune checkpoint inhibitors, antibody drug-conjugate and tafasitamab have been used for R/R DLBCL. This article will review the drug resistance mechanism and novel targeted drugs and therapies of DLBCL.