DLBCL Cell Research Articles

Identification of miR-451 target genes as prognostic markers in diffuse large B-cell lymphoma

ABSTRACT Background B-cell lymphoma, a diverse malignancy, is intricately regulated by multiple factors. MicroRNAs (miRNAs) have been demonstrated to be important regulators of the initiation and progression of human B-cell lymphoma, but their functions need to be further explored. Research design and methods Two B-cell lymphoma cell lines, Romas and HBL-1, were engineered to overexpress miR-451, with cell proliferation assessed via cell counting assays. Flow cytometry was used to study the effects of miR-451 on cell cycle and apoptosis. Bioinformatics analyses were performed using GEO datasets (GSE181063, GSE32918, GSE56315) to identify DEGs, and potential miR-451 target genes were predicted using tools like ENCORI, TargetScan, and R packages. A risk model for DLBCL prognosis was developed using Cox and LASSO regression. qRT-PCR validated the expression of these target genes. Results This study revealed that miR-451 inhibited cell proliferation, arrested the cell cycle, and induced apoptosis in human DLBCL cell lines. Bioinformatics analysis identified 9 target genes (MMP9, AQP9, RIN2, EOMES, LCP2, SELPLG, MAL, SOCS5, S1PR3) significantly associated with DLBCL prognosis, suggesting a potential mechanism by which miR-451 suppresses DLBCL development. Conclusions Our study indicates that a specific set of miR-451 target genes may significantly influence DLBCL patient outcomes.

Increased CCL2/CCR2 axis promotes tumor progression by increasing M2 macrophages in MYC/BCL2 double-expressor DLBCL

AbstractThe pathogenesis of myelocytomatosis oncogene (MYC) and B-cell lymphoma 2 (BCL2) double-expressor diffuse large B-cell lymphoma (DE-DLBCL) remains unclear. To investigate how MYC and BCL2 contribute to tumor aggressiveness, we analyzed tumors from 14 patients each with DE-DLBCL and non–DE-DLBCL using whole transcriptome sequencing. Validation was performed using publicly available data sets, tumor tissues from 126 patients, DLBCL cell lines, and a syngeneic mouse lymphoma model. Our transcriptome analysis revealed significantly elevated messenger RNA levels of C-C motif chemokine ligand 2 (CCL2) and C-C chemokine receptor type 2 (CCR2) in DE-DLBCLs when compared with non–DE-DLBCLs (adjusted P value < .05). Transcriptomic analysis of public data sets and immunohistochemistry corroborated these findings, indicating increased levels of M2 macrophages but a reduction in T-cell infiltration in DE-DLBCLs when compared with non–DE-DLBCLs (all P < .05). CCR2 expression was observed mainly in tumor-infiltrating macrophages and not in DLBCL cells. Increased expression of CCL2 and CCR2 was significantly associated with a poor prognosis in patients with DLBCL. In the in vitro analyses, MYChigh/BCL2high DLBCL cells showed higher CCL2 expression and secretion than MYClow/BCL2low cells. MYC and BCL2 increased CCL2 expression and secretion by upregulation of nuclear factor κB p65 in DLBCL cells, and CCL2 promoted M2 polarization of macrophages. In a mouse lymphoma model, CCL2 contributed to the immunosuppressive microenvironment and tumor growth of MYChigh/BCL2high tumors. We demonstrated that the increased CCL2/CCR2 axis confers aggressiveness to DE-DLBCL by increasing M2 polarization and can be a potential therapeutic target.

A novel EZH1/2 dual inhibitor inhibits GCB DLBCL through cell cycle regulation and M2 tumor-associated macrophage polarization

The incidence of germinal center B-cell-like type diffuse large B-cell lymphoma (GCB DLBCL) is steadily increasing, with a known hereditary component. Although some molecular mechanisms in GCB DLBCL have been elucidated, understanding remains incomplete, limiting the effectiveness of targeted therapies. In GCB DLBCL patients, abnormally high expression of zeste homologs 2 (EZH2) is noted, and the compensatory effect of EZH1 following EZH2 inhibition contributes to poor prognosis. This highlights the potential of dual targeting of EZH1/2 as a promising strategy. In this study, we developed a novel inhibitor, EZH-1-P2, targeting EZH1/2, and evaluated its anti-tumor effects on DLBCL cells. Mechanistically, inhibition of EZH1/2 affects the epigenetic regulation of gene expression related to p53, impacting cell cycle progression and GCB DLBCL cell growth. Additionally, while EZH1/2 inhibition impacts NOTCH signaling, the precise mechanism by which it affects M2-type tumor-associated macrophage (M2-TAM) polarization and germinal center expansion requires further investigation. Our research introduces EZH-1-P2 as a novel inhibitor with potential as a candidate for GCB DLBCL therapy, although further studies are needed to fully elucidate its mechanisms.

A comprehensive prognostic and immune infiltration analysis of UBA1 in pan-cancer: A computational analysis and invitro experiments.

Ubiquitin like modifier activating enzyme 1 (UBA1) plays an important role in immune regulation and cellular function. However, the functional mechanism and role of UBA1 in pan-cancer have not been fully elucidated and its value in haematological tumours (diffuse large B cell lymphoma (DLBC/DLBCL) and acute myeloid leukaemia (AML/LAML)) has not been explored. We conducted a comprehensive analysis of the functional mechanism and role of UBA1 in pan-cancer using multiple databases, including differential expression analysis, clinical pathological staging analysis, prognosis analysis and immune analysis. Then, we confirmed the function of UBA1 in haematological tumours through cell experiments. The results showed that the expression of UBA1 was significantly increased in most cancers and the differential expression of UBA1 was mainly concentrated in digestive tumours, haematological tumours and brain tumours. Moreover, the high expression of UBA1 had poor prognosis in most tumours, which may be related to its involvement in various cancer-related pathways such as cell cycle, as well as its methylation level, protein phosphorylation level, immune cell infiltration and immune therapy response. Cell experiments have confirmed that UBA1 can significantly regulate the cycle progression and apoptosis of DLBCL cells and AML cells. Therefore, UBA1 may be a potential therapeutic target for haematological tumours. In summary, our study not only comprehensively analysed the functional mechanisms and clinical value of UBA1 in pan-cancer, but also validated for the first time the regulatory role of UBA1 in haematological tumours.

SNCA is a potential therapeutic target for COVID-19 infection in diffuse large B-cell lymphoma patients

Cuprotosis related genes (CRGs) have been proved to be potential therapeutic targets for coronavirus disease 2019 (COVID-19) and cancer, but their immune and molecular mechanisms in COVID-19 infection in Diffuse Large B-cell Lymphoma (DLBC/DLBCL) patients are rarely reported. Our research goal is first to screen the key CRGs in COVID-19 through univariate analysis, machine learning and clinical samples. Secondly, we determined the expression and prognostic role of key CRGs in DLBCL through pan-cancer analysis. We validated the expression levels and prognosis using multiple datasets and independent clinical samples and validated the functional role of key CRGs in DLBCL through cell experiments. Finally, we validated the expression levels of CRGs in COVID-19 infected DLBCL patients samples and analyzed their common pathways in COVID-19 and DLBCL. The results show that synuclein-alpha (SNCA) is the common key differential gene of COVID-19 and DLBCL. DLBCL cells confirm that high expression of SNCA can significantly promote cell apoptosis and significantly inhibit the cycle progression of DLBCL. High expression of SNCA can regulate the binding of major histocompatibility complexes (MHCs) and T cell receptor (TCR) by regulating immune infiltration of Dendritic cells, effectively enhancing T cell-mediated anti-tumor immunity and clearing cancer cells. In conclusion, SNCA may be a potential therapeutic target for COVID-19 infection in DLBCL patients. Our study provides a theoretical basis for improving the clinical treatment of COVID-19 infection in DLBCL patients.

Discovery of a Highly Potent and Selective HDAC8 Degrader: Advancing the Functional Understanding and Therapeutic Potential of HDAC8.

HDAC8 plays crucial roles in biological processes, from gene regulation to cell motility, making it a highly desirable target for therapeutic intervention. HDAC8 also has deacetylase-independent activity which cannot be blocked by a conventional inhibitor. In this study, we report the discovery of YX862, a highly potent and selective hydrazide-based HDAC8-proteolysis targeting chimera (PROTAC) degrader. The selectivity is achieved through rational design of the warhead to spare HDAC3 activity from the previous HDAC3/8 dual degrader YX968. We demonstrate that the degradation of HDAC8 by YX862 increases acetylation levels of its nonhistone substrates such as SMC3 without significantly triggering histone PTM, supporting HDAC8's major role in nonhistone PTM regulation. YX862 exhibits promising on-target antiproliferative activity against DLBCL cells with higher potency than the HDAC8 selective inhibitor PCI-34051. As a selective HDAC8 degrader that avoids pan-HDAC inhibition, YX862 represents a valuable tool for exploring the biological and therapeutic potential of HDAC8.

Abstract PR02: Aging-associated molecular and cellular alterations determine the biology and clinical characteristics of DLBCL in elderly patients

Abstract About 30% of DLBCLs occur in patients over 75 years old. Due to comorbidities, this potentially curable disease is treated with a reduced dose chemoimmunotherapy regimen, associated with worse response rates and outcomes than the standard approach. Our data indicates that even those able to tolerate full-dose treatments have comparatively worse outcomes. We found that DLBCLs in older patients are more frequently of the ABC-DLBCL subgroup, and their microenvironment (TME) has infiltration of inflammatory and immunosuppressed cells, both characteristics independently associated with chemoimmunotherapy resistance. These features may imply we are facing different biologically diseases. To investigate this, we developed aged DLBCL murine models by implanting three murine lymphoma cells (harboring Setd2/BCL2, Ezh2/BCL2 and Tp53 mutations) in the spleen of young (3-4 months) and aged (21-23 months) mice and monitoring tumor growth by in vivo imaging. We also performed an integrative characterization of the TME from young and aged mice by single cell RNA-sequencing (coding and BCR/TCR), immunophenotyping (flow cytometry) and (MS)-based proteomics. For these two different murine DLBCL cell lines utilized, aged mice had higher tumor burden and showed significantly worse survival than younger mice. In addition, DLBCL cells isolated from aged mice displayed higher clonality and the activation of pro-inflammatory transcriptional programs than DLBCL cells isolated from younger mice. Regarding non-malignant TME B cells, CD11c+ T-bet+ Zeb2+ age-associated B cells (AABs) were significantly more abundant in aged v young mice. AABs displayed a secretory phenotype and transcriptional up-regulation of genes associated with pro-inflammatory programs such as IL-6, Stat3 and Nfatc1. TME T cells showed profound changes including increased infiltration of Foxp3+ Tregs and Pd1+ Cd8+ T cells, as well as the appearance of Cd8+ Gzk+ aged-associated T cells (ATT). Aged mice presented alterations in the myeloid compartment with expansion of Cd45+ Cd11b+ cells. Moreover, the TME of aged mice was enriched in tumor-associated macrophages polarized towards a pro-inflammatory phenotype as well as more abundance of Ly6C+ Ly6G+ myeloid-derived suppressor cells (MDSC). Aged TMEs were also characterized by increased stiffness and extracellular matrix changes affecting collagen and proteoglycans. These TME changes, were associated with intrinsic signaling and transcriptional changes in lymphoma cells like increased activation of NF-kB pathway. Overall, these alterations were compatible with the TME of elderly DLBCL patients showing a global increase in the proportion of inflammatory and immunosuppressive (IN-LME) category (41% in 75-85 y.o. vs. 26% in 50-65 y.o.) and specifically in Tregs, MDSC and CD8 PD1HIGH cells in 75-85 y.o. This data indicates that low-grade chronic inflammation that has been associated with age-related diseases determines molecular and cellular characteristics of the DLBCL in the elderly population. Citation Format: Nahuel Zamponi, Maria Victoria Revuelta, Rossella Marullo, Nicolas Di Siervi, Nikita Kotlov, Wendy Béguelin, Leandro Cerchietti. Aging-associated molecular and cellular alterations determine the biology and clinical characteristics of DLBCL in elderly patients [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR02.

Abstract PR01: EZH2 inhibitors improve CAR-T therapy by enhancing lymphoma B cell immunogenicity and CAR-T cell functions

Abstract Although CAR-T19 therapy is widely used for relapsed/refractory B cell lymphomas, approximately half of patients experience post CAR-T relapse. The mechanisms underlying this resistance remain largely unknown and efforts to overcome this limitation are hindered by the lack of suitable pre-clinical models for study. To address these unmet needs, we generated a novel genetically engineered mouse model with conditional expression of Ezh2Y641F and BCL2 in germinal center (GC) B cells. Ezh2/BCL2 mice developed follicular lymphoma (FL) which histologically and transcriptionally recapitulates human FL. Moreover, we established a cell line derived from an Ezh2/BCL2 mouse that developed transformed FL (tFL). Injection of the cell line named “tFL-P6” in immunocompetent C57BL6 recipients resulted in rapid disease progression that histologically and transcriptionally mimics human tFL. Treatment of tFL-P6 with EZH2 inhibitors (EZH2i) didn’t affect proliferation and viability, however, the number of pretreated cells was significantly reduced when co-cultured with T cells. EZH2i pretreated tFL-P6 displayed extended interaction with T cells, as measured by live imaging. EZH2i reprogrammed tFL-P6 to restore T cell engagement genes such as ICOSL, ICAM1, OX40L, and integrins, as well as cytokines and chemokines involved in T cell recruitment, therefore enhancing immunogenicity. Notably, pretreated tFL-P6 cells were rejected by C57BL6 whereas they developed lethal disease in immunodeficient recipients. Pre-treatment of tFL-P6 as well as human DLBCL and PDX-derived cell lines significantly enhanced CAR-T19 cell killing effects in vitro. Administering CAR-T cells in mice engrafted with EZH2i-pretreated tFL-P6 significantly reduced the tumor burden and prolonged their survival (100% vs 30%, p&amp;lt;0.01). To assess the direct interactions between lymphoma cells and CAR-T cells in vivo, we performed intravital 2-photon imaging of popliteal lymph nodes using dTomato labeled CAR-T cells and GFP+ tFL-P6 cells. Pretreatment of tFL-P6 cells with EZH2i doubled the recruitment of CAR-T cells in the microenvironment and enhanced the duration of contact and surface engagement with CAR-T cells. To explore the impact of EZH2 inhibition on CAR-T cells, we manufactured CAR-T cells from splenic T cells of mice treated with EZH2i or vehicle for 14 days. Prior exposure to EZH2i didn’t affect proliferation and transduction during CAR-T production. Ex vivo assays with those CAR-T cells and tFL-P6 cells showed a superior killing effect and expansion of EZH2i-exposed CAR-T (p&amp;lt;0.001). Mice bearing tFL-P6 lymphomas displayed a longer survival when infused with EZH2i-pretreated CAR-T cells (p=0.06). Pretreatment with EZH2i resulted in an increased memory/effector ratio (p&amp;lt;0.01) and reduction of PD1+CD38+ exhausted CD8CAR-T cells (p&amp;lt;0.001). Overall, EZH2i improved CAR-T therapy by enhancing lymphoma cell immunogenicity and CAR-T cell functions. These results prompted the initiation of a clinical trial to evaluate the safety and efficacy of this combination in R/R B cell lymphomas (NCT05934838). Citation Format: Yusuke Isshiki, Xi Chen, Matt Teater, Ioannis Karagiannidis, Henna Nam, Amy Chadburn, Ari Melnick, Wendy Béguelin. EZH2 inhibitors improve CAR-T therapy by enhancing lymphoma B cell immunogenicity and CAR-T cell functions [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR01.

Single-cell and spatial transcriptomics reveal a high glycolysis B cell and tumor-associated macrophages cluster correlated with poor prognosis and exhausted immune microenvironment in diffuse large B-cell lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) is a heterogeneous malignancy characterized by varied responses to treatment and prognoses. Understanding the metabolic characteristics driving DLBCL progression is crucial for developing personalized therapies.MethodsThis study utilized multiple omics technologies including single-cell transcriptomics (n = 5), bulk transcriptomics (n = 966), spatial transcriptomics (n = 10), immunohistochemistry (n = 34), multiple immunofluorescence (n = 20) and to elucidate the metabolic features of highly malignant DLBCL cells and tumor-associated macrophages (TAMs), along with their associated tumor microenvironment. Metabolic pathway analysis facilitated by scMetabolism, and integrated analysis via hdWGCNA, identified glycolysis genes correlating with malignancy, and the prognostic value of glycolysis genes (STMN1, ENO1, PKM, and CDK1) and TAMs were verified.ResultsHigh-glycolysis malignant DLBCL tissues exhibited an immunosuppressive microenvironment characterized by abundant IFN_TAMs (CD68+CXCL10+PD-L1+) and diminished CD8+ T cell infiltration. Glycolysis genes were positively correlated with malignancy degree. IFN_TAMs exhibited high glycolysis activity and closely communicating with high-malignancy DLBCL cells identified within datasets. The glycolysis score, evaluated by seven genes, emerged as an independent prognostic factor (HR = 1.796, 95% CI: 1.077–2.995, p = 0.025 and HR = 2.631, 95% CI: 1.207–5.735, p = 0.015) along with IFN_TAMs were positively correlated with poor survival (p < 0.05) in DLBCL. Immunohistochemical validation of glycolysis markers (STMN1, ENO1, PKM, and CDK1) and multiple immunofluorescence validation of IFN_TAMs underscored their prognostic value (p < 0.05) in DLBCL.ConclusionsThis study underscores the significance of glycolysis in tumor progression and modulation of the immune microenvironment. The identified glycolysis genes and IFN_TAMs represent potential prognostic markers and therapeutic targets in DLBCL.

T Cells Spatially Regulate B Cell Receptor Signaling in Lymphomas through H3K9me3 Modifications.

Activated B cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is a subtype associated with poor survival outcomes. Despite identifying therapeutic targets through molecular characterization, targeted therapies have limited success. New strategies using immune-competent tissue models are needed to understand how DLBCL cells evade treatment. Here, synthetic hydrogel-based lymphoma organoids are used to demonstrate how signals in the lymphoid tumor microenvironment (Ly-TME) can alter B cell receptor (BCR) signaling and specific histone modifications, tri-methylation of histone 3 at lysine 9 (H3K9me3), dampening the effects of BCR pathway inhibition. Using imaging modalities, T cells increase DNA methyltransferase 3A expression and cytoskeleton formation in proximal ABC-DLBCL cells, regulated by H3K9me3. Expansion microscopy on lymphoma organoids reveals T cells increase the size and quantity of segregated H3K9me3 clusters in ABC-DLBCL cells. Findings suggest the re-organization of higher-order chromatin structures that may contribute to evasion or resistance to therapy via the emergence of novel transcriptional states. Treating ABC-DLBCL cells with a G9α histone methyltransferase inhibitor reverses T cell-mediated modulation of H3K9me3 and overcomes T cell-mediated attenuation of treatment response to BCR pathway inhibition. This study emphasizes the Ly-TME's role in altering DLBCL fate and suggests targeting aberrant signaling and microenvironmental cross-talk that can benefit high-risk patients.

Discovery of a novel BTK inhibitor S-016 and identification of a new strategy for the treatment of lymphomas including BTK inhibitor-resistant lymphomas.

Bruton's tyrosine kinase (BTK) has emerged as a therapeutic target for B-cell malignancies, which is substantiated by the efficacy of various irreversible or reversible BTK inhibitors. However, on-target BTK mutations facilitating evasion from BTK inhibition lead to resistance that limits the therapeutic efficacy of BTK inhibitors. In this study we employed structure-based drug design strategies based on established BTK inhibitors and yielded a series of BTK targeting compounds. Among them, compound S-016 bearing a unique tricyclic structure exhibited potent BTK kinase inhibitory activity with an IC50 value of 0.5 nM, comparable to a commercially available BTK inhibitor ibrutinib (IC50 = 0.4 nM). S-016, as a novel irreversible BTK inhibitor, displayed superior kinase selectivity compared to ibrutinib and significant therapeutic effects against B-cell lymphoma both in vitro and in vivo. Furthermore, we generated BTK inhibitor-resistant lymphoma cells harboring BTK C481F or A428D to explore strategies for overcoming resistance. Co-culture of these DLBCL cells with M0 macrophages led to the polarization of M0 macrophages toward the M2 phenotype, a process known to support tumor progression. Intriguingly, we demonstrated that SYHA1813, a compound targeting both VEGFR and CSF1R, effectively reshaped the tumor microenvironment (TME) and significantly overcame the acquired resistance to BTK inhibitors in both BTK-mutated and wild-type BTK DLBCL models by inhibiting angiogenesis and modulating macrophage polarization. Overall, this study not only promotes the development of new BTK inhibitors but also offers innovative treatment strategies for B-cell lymphomas, including those with BTK mutations.

Expression of alternatively spliced BCMA RNA with skipping of transmembrane domain.

7514 Background: The TNFRSF17 gene, which encodes for the B cell maturation antigen (BCMA), is a small gene expressed mainly on the surface of plasma cells and some DLBCL cells. This gene is composed of three exons. Exon 1 is responsible for the extracellular domain, exon 2 for the transmembrane domain, and exon 3 for the TRAF binding domain. The BCMA is currently targeted by various types of immunotherapies as a major therapeutic approach for the treatment of multiple myeloma (MM). This includes CAR-T cells, bi-specific antibodies, and antibody-drug conjugates (ADC). However, emerging data indicates that alternative splicing in genes is an important mechanism for expression of different isoforms that may influence antibody-based therapies. We explored the potential of the presence of alternative splicing in BCMA transcripts via sequencing of BCMA RNA in patients with lymphoma or multiple myeloma. Methods: RNA was extracted from 587 fresh bone marrow samples with lymphoid/plasma cell neoplasms or from FFPE samples with lymphoma. In addition, cfRNA was extracted from 260 peripheral blood plasma samples from patients with lymphoma or multiple myeloma. RNA was sequenced using a hybrid capture-targeted RNA panel with analysis focused on TNFRSF17 (BCMA) gene transcript. Quantification of RNA transcript was done using Salmon algorithm. Results: Of the 587 lymphoma/plasma cell samples, 161 (27%) samples showed alternative splicing involving deletion of exon 2 (BCMA∆Ex2). Of the 260 cfRNA samples, 14 (6%) showed BCMA∆Ex2. The median percentage of BCMA∆Ex2 transcripts was 0.7% of total BCMA transcripts in cellular samples as compared with 9% in cfRNA samples. In cellular samples, there was a correlation between levels of BCMA and BCMA∆Ex2 (R=0.63). Cases with higher levels of BCMA had significantly higher levels of BCMA∆Ex2 (P&lt;0.0001, Kruskal-Wallis). In contrast, cfRNA showed no correlation between levels of BCMA and levels of BCMA∆Ex2 (R=021, Spearman) and mildly higher level of BCMA∆Ex2 in cases with higher levels of BCMA (P=0.002, Kruskal-Wallis). Conclusions: BCMA exon 2 skipping is detected in a significant number of patients with multiple myeloma and lymphoma. The percentage of skipping transcripts is low in cells and relatively higher in cfRNA. Since exon 2 skipping deletes the transmembrane domain, this BCMA protein may remain in the cytoplasm or perhaps is secreted as cell-free BCMA protein. The demonstration of relatively higher levels of BCMA∆Ex2 in cfRNA is likely reflecting higher turnover of cells and raises the possibility that cells with this isoform of BCMA are more aggressive than cells without the expression of this isoform. Further studies are needed to explore the clinical relevance of the expression of such abnormal BCMA protein on treatment with the various forms of anti-body-based therapy or CAR-T.

SLC27A2 is a potential immune biomarker for hematological tumors and significantly regulates the cell cycle progression of diffuse large B-cell lymphoma

BackgroundResearch on the fatty acid metabolism related gene SLC27A2 is currently mainly focused on solid tumors, and its mechanism of action in hematological tumors has not been reported.MethodThis study aims to explore the pathological and immune mechanisms of the fatty acid metabolism related gene SLC27A2 in hematological tumors and verify its functional role in hematological tumors through cell experiments to improve treatment decisions and clinical outcomes of hematological tumors.ResultThis study identified the fatty acid metabolism related gene SLC27A2 as a common differentially expressed gene between DLBCL and AML. Immune microenvironment analysis showed that SLC27A2 was significantly positively correlated with T cell CD4 + , T cell CD8 + , endothelial cells, macrophages, and NK cells in DLBCL. In AML, there is a significant negative correlation between SLC27A2 and B cells, T cell CD8 + , and macrophages. SLC27A2 participates in the immune process of hematological tumors through T cell CD8 + and macrophages. The GESA results indicate that high expression of SLC27A2 is mainly involved in the fatty acid pathway, immune pathway, and cell cycle pathway of DLBCL. The low expression of SLC27A2 is mainly involved in the immune pathway of AML. Therefore, SLC27A2 is mainly involved in the pathological mechanisms of hematological tumors through immune pathways, and cell experiments have also confirmed that SLC27A2 is involved in the regulation of DLBCL cells.ConclusionIn summary, our research results comprehensively report for the first time the mechanism of action of SLC27A2 in the immune microenvironment of DLBCL and AML, and for the first time verify the cycle and apoptotic effects of the fatty acid related gene SLC27A2 in DLBCL cells through cell experiments. Research can help improve the treatment of AML and DLBCL patients.

Effects of polyphyllin Ⅶ on proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma cells

The aim of this study was to investigate the effects of polyphyllin Ⅶ (PP Ⅶ) on proliferation, apoptosis, and cell cycle of diffuse large B-cell lymphoma (PLBCL) cell lines U2932 and SUDHL-4. The DLBCL cell lines were divided into a control group and a PPⅦ group, and experiments were conducted using MTT assay, flow cytometry, and Western blotting.Results showed that compared with the control group, PPⅦ significantly inhibited the proliferation of U2932 and SUDHL-4 cells (P<0.05). Apoptosis assays demonstrated that treatment with 0.50 and 1.00 µmol/L PP Ⅶ significantly increased the apoptosis rates of both cell lines (P<0.05), upregulated apoptosis-related proteins, and downregulated Bcl-2 protein level (P<0.05). Cell cycle analysis revealed that PPⅦ treatment led to an increase in G0/G1-phase cells (P<0.05) and a decrease in G2/M-phase cells (P<0.05), significantly downregulated cyclin D1, CDK4, CDK6, and survivin protein expression (P<0.05). In conclusion, PPⅦ exerted anti-lymphoma effects by inhibiting proliferation, promoting apoptosis, and inducing G0/G1 phase arrest in DLBCL cells.

DLD is a potential therapeutic target for COVID-19 infection in diffuse large B-cell lymphoma patients

Since the discovery of copper induces cell death(cuprotosis) in 2022, it has been one of the biggest research hotspots. cuprotosis related genes (CRGs) has been demonstrated to be a potential therapeutic target for cancer, however, the molecular mechanism of CRGs in coronavirus disease 2019 (COVID-19) infected in DLBCL patients has not been reported yet. Therefore, our research objective is first to elucidate the mechanism and role of CRGs in COVID-19. Secondly, we conducted univariate and multivariate analysis and machine learning to screen for CRGs with common expression differences in COVID-19 and DLBCL. Finally, the functional role and immune mechanism of genes in DLBCL were confirmed through cell experiments and immune analysis. The research results show that CRGs play an important role in the occurrence and development of COVID-19. Univariate analysis and machine learning confirm that dihydrolipoamide dehydrogenase (DLD) is the common key gene of COVID-19 and DLBCL. Inhibiting the expression of DLD can significantly inhibit the cycle progression and promote cell apoptosis of DLBCL cells and can target positive regulation of Lysine-specific demethylase 1 (LSD1, also known as KDM1A) to inhibit the proliferation of DLBCL cells and promote cell apoptosis. The immune analysis results show that high-expression of DLD may reduce T cell-mediated anti-tumor immunity by regulating immune infiltration of CD8 + T cells and positively regulating immune checkpoints LAG3 and CD276. Reducing the expression of DLD can effectively enhance T cell-mediated anti-tumor immunity, thereby clearing cancer cells and preventing cancer growth. In conclusion, DLD may be a potential therapeutic target for COVID-19 infection in DLBCL patients. Our research provides a theoretical basis for improving the clinical treatment of COVID-19 infection in DLBCL.

Abstract 46: Mechanisms and impact of bystander killing by CAR T cells

Abstract While CD19-directed chimeric antigen receptor (CAR) T cell therapy has improved outcomes of patients with relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL), a majority will suffer disease relapse following CAR T cell infusion. Therefore, understanding mechanisms of CAR T cell killing is critical to enhance therapeutic efficacy. Cytolysis of CAR T cell targets presumably involves recognition of CD19 antigen by the CAR followed by activation of the granzyme/perforin axis. However, bystander elimination of CD19-negative lymphoma cells by CAR T cells has been reported, indicating that an alternative pathway may be required for optimal killing. Through in-depth phenotyping of a large panel of human DLBCLs and in vitro killing assays, we observed that CD19 expression levels on DLBCL cells and vulnerability to CAR T cell killing were weakly correlated. Conversely, cell surface expression of the death receptor, FAS, on DLBCL cells was strongly associated with susceptibility to CAR T cell killing. Although FAS expression across DLBCL cell lines varied, it could be broadly enhanced following exposure to IFNγ. Potent CAR T cell-mediated cytolysis of CD19-negative, Fas-positive murine DLBCL cells required the presence of CD19-positive lymphoma cells in the culture. CRISPR/Cas9 targeting was employed to delete FAS and/or CD19 in isogenic human DLBCL cells to generate CD19+FAS+, CD19+FAS−, CD19−FAS+, and CD19−FAS− subsets, which enabled us to define the extent to which CD19 engagement by the CAR was required to affect killing via FAS. When CAR T cells were co-cultured with equal numbers of the four gene-modified DLBCL cells described above, CD19+FAS+ DLBCL cells were killed most effectively, followed by CD19+FAS− DLBCL cells, and CD19−FAS+ DLBCL cells. Double-negative DLBCL cells were poorly killed. This result suggested that CD19 engagement by the CAR was required for optimal DLBCL cell killing via FAS, and that CD19 and FAS need not necessarily be engaged on the same target cell. Future studies will determine if Fas/FasL interactions can be exploited by overexpression of FasL in CAR T cells and examination of DLBCL killing in vitro and in vivo. Citation Format: Joanna Chorazeczewski, Lishi Xie, Xiufen Chen, Sidney Wang, Justin Kline. Mechanisms and impact of bystander killing by CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 46.

Abstract 5725: Aurkin-A, a TPX2-Aurora A inhibitor disrupts alisertib-induced polyploidy in HG-diffuse large B cell lymphoma

Abstract Chemotherapy induced polyploidy (CIP) is a mechanism of inherited drug resistance resulting in an aggressive disease course in cancer patients. Alisertib, an Aurora Kinase A (AK-A) ATP site inhibitor, induces cell cycle disruption resulting in polyaneuploidy in high-grade (MYC and BCL2 amplified or expressed) Diffuse Large B Cell Lymphoma (HG-DLBCL). Propidium Iodide cycle flow cytometry was utilized to quantify alisertib induced polyploidy in U2932 and VAL cell lines. In U2932 cells, 1µM alisertib generated 8n+ polyploidy in 48% of the total cell population after five days of treatment. Addition of Aurkin A an AK-A/TPX2 site inhibitor, disrupted alisertib induced polyploidy in a dose-dependent manner with associated increased apoptosis in DLBCL cells. Combination of Aurkin A plus alisertib significantly reduced polyploidy and sensitized DLBCL cells to alisertib. The sensitization of alisertib with Aurkin A through complete AK-A inhibition is of high clinical importance as the alisertib dose can be significantly reduced while preventing polyploidy, enhancing efficacy, and significantly reducing toxicity to normal tissue. In clinical trials of alisertib in lymphoma, a maximum tolerated dose of 1.5µM was achieved. In U2932 cells, we found the addition of 100µM Aurkin A reduced the alisertib IC50 from 983nM to 34nM. We generated a stable FUCCI U2932 cell line expressing Geminin-clover (S/G2/M) and cdt1-mKO (G1), to help monitor cell cycle progression in live cells. Using this system, we identified alisertib-induced polyploidy through endomitosis, which was eliminated with Aurkin A treatment. In a VAL cell line mouse xenograft model, we show polyploidy generation in alisertib treated mice versus vehicle control or Aurkin A. Aurkin A plus alisertib significantly reduced polyploidy and tumor growth compared to vehicle control or Aurkin A levels. Finally, we performed Data-Independent Acquisition Mass Spectrometry on alisertib, Aurkin A, and combination alisertib/Aurkin A treated U2932 cells to identify therapy induced proteomic changes. This proteomic analysis has provided insight into the mechanism of alisertib polyploidy induction and the protective mechanism of within the combination therapy. Our in vitro and in vivo studies show that Aurkin A synergizes with alisertib and significantly decreases the alisertib dose needed to disrupt polyploidy while increasing apoptosis in HG-DLBCL cells. Citation Format: Patrick J. Conway, Bárbara De la Peña Avalos, Jonathan Dao, Sebastian Montagnino, Dmytro Kovalskyy, Eloise Dray, Daruka Mahadevan. Aurkin-A, a TPX2-Aurora A inhibitor disrupts alisertib-induced polyploidy in HG-diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5725.

Abstract 3298: Unlocking the potential of OPM-383: A novel LRRK2 inhibitor in cancer therapy

Abstract Background: Leucine-rich repeat kinase 2 (LRRK2) plays a pivotal role in regulating various cellular processes, such as cell proliferation, survival, and inflammation. LRRK2 exhibits dual functionality as a serine-threonine kinase and as a GTPase. It is involved in the modulation of multiple signaling pathways, including WNT, MAPK, NF-kB and mTOR. Germline mutations in LRRK2 are associated with an increased risk of cancer, particularly hormone-related and colorectal cancers. LRRK2 also promotes tumor cell growth and survival in papillary renal and thyroid carcinomas, DLBCL and cholangiocarcinoma cells. OPM has designed and developed a novel oral LRRK2 inhibitor, OPM383, using its proprietary Nanocyclix® technology. In this study, we have evaluated the pharmacokinetic (PK) properties, efficacy and tolerability of OPM383 in a colon carcinoma model and a panel of patient-derived organoids. Materials and Methods: In vitro potency of our Type I inhibitor, OPM383, against full-length wild-type LRRK2 and the G2019S pathogenic LRRK2 mutant was assessed in in a Lanthascreen cell assay. For PK studies, we analyzed OPM383 plasma levels using UPLVC/MS-MS. Evaluation of inhibition of LRRK2 phosphorylation (Ser935) was assessed by western blot. MC-38 cells were inoculated into C57BL/6 mice. When tumor masses reached 75 mm³, mice were randomized to receive OPM383 (50 mg/kg, orally, daily), anti-PD1 antibody (10 mg/kg, ip, twice weekly), or their combination. OPM383 was also evaluated in patient-derived organoids using the SEngine-Paris® platform. Results: OPM-383 inhibited wild-type LRRK2 and LRRK2[G2019S] with an IC50 of 42 nM and 33 nM, respectively. OPM383 is highly selective among a panel of &amp;gt;400 protein kinases (only 6.7% cross-reactivity at 100 nM). The compound is permeable, metabolically stable and possesses drug-like properties. OPM-383 exhibited a favorable pharmacokinetic profile in mice (AUC of 11858 h*ng/mL and T max of 3 hours at a dose of 30 mg/kg via oral administration). Preliminary e-ADME results indicated moderate inhibition of CYP3A4BFC but no significant inhibition of CYPs 1A2, 2D6, 3A4BQ, 2C19, and 2C9 by OPM-383. After 90 minutes of oral administration in CD1 mice, we observed the inhibition of LRRK2 phosphorylation (Ser935) in the brain, kidney, and PBMCs. In assessing its potential antitumor effects, OPM383 was tested on a panel of patient-derived organoids. Notably, OPM383 inhibited the proliferation of 7 out of 29 patient-derived organoids after 6 days of exposure at various concentrations (&amp;lt;10 µM). In mice bearing subcutaneous MC38 tumors, OPM383 significantly inhibited tumor growth compared to the vehicle-treated group. Conclusion: This study characterizes a potent first-in-class LRRK2 inhibitor in cancer models. The findings suggest that OPM383 could serve as an attractive lead scaffold for designing and synthesizing a new class of kinase inhibitors for potential applications in cancer therapy. Citation Format: Maria Eugenia Riveiro, Petra Blom, Kenji Shoji, Jan Hoflack. Unlocking the potential of OPM-383: A novel LRRK2 inhibitor in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3298.

Abstract 7598: The novel eIF4A inhibitor potently synergizes with BCL2 inhibitor in high grade B-cell lymphoma with MYC and BCL2 rearrangements through regulating UPR

Abstract Double hit lymphoma(DHL) is defined to be a chromosomal break with MYC gene combined with BCL2 or BCL6. MYC is an oncogene which proliferation the cells and BCL2 inhibit cell death. These combinations could be a cause of highly aggressive clinical features and poor survival of DLBCL. The WHO-HAEM5 renames the entity DLBCL/high-grade B-cell lymphoma with MYC and BCL2 rearrangements. Despite the dismal prognosis, no widely accepted standard treatment exists to the management of DHL. Therefore, new therapeutic approaches for these patients are urgently needed. Previously, we looked at the eIf4A inhibition was shown to downregulate MYC expression in lymphoma cell lines. Venetoclax, a highly selective inhibitor of BCL2, shows clinical activity for patients with DLBCL although there are concerns about increasing toxicity. Our hypothesis in the following: If eIF4A inhibitor preferentially inhibit the translation of oncogenes such as cMyc and venetoclax inhibit the BCL2 in DHL, then the combination of eIF4A inhibitor and BCL2 inhibitor will be highly effective for DHL and potentially safe for animals and humans. We tested the anti-lymphoma effect of CR-1-31-B, in combination with Venetoclax in lymphoma cell lines using flow cytometry. We performed cell apoptosis assays in increasing concentrations of Venetoclax, CR-1-31-B and a combination of the two drugs at 48 hours. To better understand molecular mechanisms underlying the observed synergistic effect in DLBCL-DHL, RNA-seq profiling was performed on cells treated with single drugs and their combinations, respectively. Venetoclax and CR1-31-B exhibited concentration and time dependent cytotoxicity in lymphoma cell lines at low nanomolar concentrations. Overall, the combination of eIF4A inhibitor and BCL2 inhibitor showed synergy in all kinds of DLBCL cell lines. Especially DHL cell lines were more sensitive to the drug combination and showed enhanced synergy compared to the non-DHL cell lines(p&amp;lt;0.0001). We analyzed RNA-seq profiling of cells treated with single drugs and their combinations and found that the transcriptional signature of the synergistic combination was unique relative to that of either constituent monotherapy. Gene set enrichment analysis was performed in the DHL and non-DHL group. The analysis demonstrates that known Unfolded protein response(UPR) part and metabolism, are enriched in DHL groups. This suggests that the UPR and metabolism pathway are closely related to the combination effect in DHL. These findings suggest that the combination of eIF4A inhibitor and BCL2 inhibitor have potent pharmacological activity in DHL lines, it was closely related with UPR. These combination is promising and novel treatment for high-grade B-cell lymphoma with MYC and BCL2 rearrangements. Further pre-clinical investigations of this combination for these patients are needed. Citation Format: So-Young Seol, Chung Hyun Park, Soo Jeong Kim, Haerim Chung, Hyunsoo Cho, Jin Seok Kim, Yu Ri Kim. The novel eIF4A inhibitor potently synergizes with BCL2 inhibitor in high grade B-cell lymphoma with MYC and BCL2 rearrangements through regulating UPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7598.

Abstract 6044: EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases

Abstract Heterobifunctional kinase degraders (HKDs) are an important new class of therapeutic modalities, of which the first have recently entered clinical trials. HKDs are small molecules that comprise a kinase-of-interest binding moiety and an E3 ligase binding moiety connected via a spacer. HKDs are excellently suited to target kinases that have a scaffolding function, or that are resistant against conventional inhibitors. A HKD brings a kinase and an E3 ligase in proximity forming a ternary complex leading to ubiquitin-dependent catalytic degradation of the kinase of interest. This ternary complex formation is unique for each kinase which can make HKDs highly specific. To discover novel HKDs, we have developed a platform called Energetically Privileged Ligands (EPriLs), which is based on a macrocycle scaffold that binds non-covalently in the kinase ATP pocket with near-covalent affinity, and allows binding to kinases that have acquired known inhibitor resistance mutations. The macrocyclic structure allows attachment of E3 ligase ligands with multiple exit vectors. With the EPriL platform, we have developed novel HKDs for various clinically relevant kinase targets, including Bruton’s Tyrosine Kinase (BTK). BTK inhibitor drugs are used as first line treatment for chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM) and as second line treatment for mantle cell lymphoma (MCL). Despite their great success in the clinic, many patients on BTK inhibitors relapse eventually, mostly because of the emergence of resistance mutations in the BTK protein. These include BTKC481S, which abrogates binding of covalent drugs, and BTKT474I, BTKL528W and BTKV416L which preclude binding of non-covalent inhibitors like pirtobrutinib. HKDs can combat resistance by degrading such BTK variants, and by removing the scaffold function of BTK. BTK degraders such as NX-5948 and NX-2127 are currently in clinical Phase 1. To show the applicability of the EPriL platform for the discovery of novel BTK degraders, we screened a library of more than 100 EPriL HKDs for biochemical binding to BTK, and tested their propensity to induce degradation of HiBiT-tagged BTKWT, BTKC481S and BTKT474I in HEK293 cell lines. The degrading activity of hits was verified through western blot on the BTK-dependent DLBCL cell line TMD8. The best degraders - based on potency and efficacy - were tested in cell proliferation assays on a panel of TMD8 BTK mutant cell lines. In these lines, endogenous BTK was mutated by CRISPR to BTKC481S, BTKT474I, BTKL528W or BTKV416L. Several BTK degraders had potent (&amp;lt; 20 nM IC50) antiproliferative activity on most TMD8 lines, including difficult-to-target variants such as BTKV416L and BTKL528W. The unique binding mode of the EPriL scaffold thus translates to broad activity on resistance variants, while its selectivity ensures minimal degradation of off-target tyrosine kinases as observed for other degraders. Citation Format: Michelle Muller, Jos de Man, Joost C. Uitdehaag, Freek van Cauter, Sander van Gemert, Milan Hoffmann, Yvonne G. van Mil, Winfried R. Mulder, Martine B. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri de Wit, Erik Ensing, Rogier C. Buijsman. EPriL: A platform for the rapid identification of degraders of inhibitor-resistant kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6044.