B-cell Lymphoma Cell Lines 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.

3D-Q-FISH/Telomere/TRF2 Nanotechnology Identifies a Progressively Disturbed Telomere/Shelterin/Lamin AC Complex as the Common Pathogenic, Molecular/Spatial Denominator of Classical Hodgkin Lymphoma.

The bi- or multinucleated Reed-Sternberg cell (RS) is the diagnostic cornerstone of Epstein-Barr Virus (EBV)-positive and EBV-negative classical Hodgkin lymphoma (cHL). cHL is a germinal center (GC)-derived B-cell disease. Hodgkin cells (H) are the mononuclear precursors of RS. An experimental model has to fulfill three conditions to qualify as common pathogenic denominator: (i) to be of GC-derived B-cell origin, (ii) to be EBV-negative to avoid EBV latency III expression and (iii) to support permanent EBV-encoded oncogenic latent membrane protein (LMP1) expression upon induction. These conditions are unified in the EBV-, diffuse large B-Cell lymphoma (DLBCL) cell line BJAB-tTA-LMP1. 3D reconstructive nanotechnology revealed spatial, quantitative and qualitative disturbance of telomere/shelterin interactions in mononuclear H-like cells, with further progression during transition to RS-like cells, including progressive complexity of the karyotype with every mitotic cycle, due to BBF (breakage/bridge/fusion) events. The findings of this model were confirmed in diagnostic patient samples and correlate with clinical outcomes. Moreover, in vitro, significant disturbance of the lamin AC/telomere interaction progressively occurred. In summary, our research over the past three decades identified cHL as the first lymphoid malignancy driven by a disturbed telomere/shelterin/lamin AC interaction, generating the diagnostic RS. Our findings may act as trailblazer for tailored therapies in refractory cHL.

Genotype from Phenotype: Using CRISPR Screens to Dissect Lymphoma Biology.

Genome-wide screens are a powerful technique to dissect the complex network of genes regulating diverse cellular phenotypes. The recent adaptation of the CRISPR-Cas9 system for genome engineering has revolutionized functional genomic screening. Here, we present protocols used to introduce Cas9 into human lymphoma cell lines, produce high-titer lentivirus of a genome-wide sgRNA library, transduce and culture cells during the screen, select cells with a specified phenotype, isolate genomic DNA, and prepare a custom library for next-generation sequencing. These protocols were tailored for loss-of-function CRISPR screens in human B-cell lymphoma cell lines but are highly amenable for other experimental purposes.

PI3Kδ activation, IL-6 overexpression, and CD37 loss cause resistance to naratuximab emtansine in lymphomas

AbstractCD37-directed antibody and cellular-based approaches have shown preclinical and promising early clinical activity. Naratuximab emtansine (Debio 1562; IMGN529) is an antibody-drug conjugate (ADC) incorporating an anti-CD37 monoclonal antibody conjugated to the maytansinoid DM1 as payload, with activity as a single agent and in combination with rituximab in patients with lymphoma. We studied naratuximab emtansine and its free payload in 54 lymphoma models, correlated its activity with CD37 expression, characterized 2 resistance mechanisms, and identified combination partners providing synergy. The activity, primarily cytotoxic, was more potent in B- than T-cell lymphoma cell lines. After prolonged exposure to the ADC, 1 diffuse large B-cell lymphoma (DLBCL) cell line developed resistance to the ADC due to the CD37 gene biallelic loss. After CD37 loss, we also observed upregulation of interleukin-6 (IL-6) and related transcripts. Recombinant IL-6 led to resistance. Anti-IL-6 antibody tocilizumab improved the ADC’s cytotoxic activity in CD37+ cells. In a second model, resistance was sustained by a PIK3CD activating mutation, with increased sensitivity to PI3Kδ inhibition and a functional dependence switch from MCL1 to BCL2. Adding idelalisib or venetoclax overcame resistance in the resistant derivative and improved cytotoxic activity in the parental cells. In conclusion, targeting B-cell lymphoma with the naratuximab emtansine showed vigorous antitumor activity as a single agent, which was also observed in models bearing genetic lesions associated with inferior outcomes, such as Myc Proto-Oncogene (MYC) translocations and TP53 inactivation or R-CHOP (rituximab, cyclophosphamide, doxorubicin, Oncovin [vincristine], and prednisone) resistance. Resistant DLBCL models identified active combinations of naratuximab emtansine with drugs targeting IL-6, PI3Kδ, and BCL2.

EZB-type diffuse large B-cell lymphoma cell lines have superior migration capabilities compared to MCD-type.

Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent aggressive B-cell lymphoma. The group is heterogeneous and the outcome is variable. A variety of approaches have been employed with the objective of improving the stratification of DLBCL patients according to their prognosis, based on the cell of origin. Recently, distinct genetic subtypes of DLBCL have been identified. Given the importance of cell migration in immune cells, the objective of this study was to ascertain whether different genetic subtypes of DLBCL exhibit disparate migration abilities. MCD- and EZB-type DLBCL cell lines were subjected to testing to ascertain their basal velocity in straight microchannels and their ability to overcome tight constrictions of 2 μm. The EZB-type cell lines showed superior basal migration velocity and constriction passage time, and a similar trend was observed in live cell imaging of native human DLBCL tissue. In addition, MCD-type DLBCL exhibited significantly elevated levels of nuclear lamin A/C, which is responsible for the stiffness of the nuclear envelope and could thus explain the disparate migration behaviours observed among these subtypes. Our study suggests that different genetic subtypes of DLBCL may not only influence the outcome after therapy but also the motility of the tumour cells.

Abstract PO-020: ZBTB24 is a novel tumor suppressor and cooperates with CDKN2A to suppress B-cell lymphomagenesis

Abstract B-cell lymphoma is the most common hematological malignancy that accounts for the sixth highest mortality among all cancers. Despite recent improvements on combined chemotherapy and immunotherapy, treatments remain challenging due to complex genetic heterogeneity and molecular pathogenesis. To explore novel mechanisms in B-cell lymphomagenesis, we performed a genome-wide CRISPR knockout screen in the murine interleukin-3 (IL-3)-dependent Ba/F3 pro-B cell line and identified Zbtb24 as a putative target whose silencing leads to elevated proliferative signaling and reduced apoptosis, conferring IL-3-independent growth in Ba/F3 cells. Silencing of ZBTB24 also accelerated growth in multiple human B-cell lymphoma cell lines. ZBTB24 is a member of the zinc-finger and BTB domain containing transcription factor that is highly expressed in spleen and bone marrow. While it has been shown that ZBTB24 may play an important role in B-cell development and activation, its involvement in B-cell lymphoma has not been described. Analysis of publicly available datasets found that ZBTB24 copy number deletion occurs in various B-cell lymphomas, which frequently co-occurs with deletion of the tumor suppressor CDKN2A. Hence, we generated B-cell specific Zbtb24 single knockout and Zbtb24/Cdkn2a double knockout mice to explore the role of Zbtb24 in vivo. While Zbtb24 and Cdkn2a single knockout mice remained tumor-free or showed low penetrance of tumorigenesis, the double knockout mice developed lymphadenopathy and splenomegaly accompanied with accelerated mortality. Flow cytometry analysis of bone marrow, spleen, and lymph nodes revealed malignant B-cells. Subsequent transplantation of the splenic cells induced identical malignant B-cell lymphomagenesis and immunophenotype in athymic nude mice, further confirming its malignant identity. These results indicate that ZBTB24 is a novel tumor suppressor in B-cell malignancy. Ongoing studies are being conducted to understand the mechanistic basis for ZBTB24 tumor suppressive function that may involve transcriptional regulation of its target gene expression. This project is supported by the NIH R01CA232246, R01CA129536, and the John Colaizzi Endowment Fund. Citation Format: Rongrong Li, Namratha Sheshadri, Jianliang Shen, Junrong Yan, Jaeyong Jung, Muhammad Usama Tariq, Y Lynn Wang, Ping Xie, Wei-Xing Zong. ZBTB24 is a novel tumor suppressor and cooperates with CDKN2A to suppress B-cell lymphomagenesis [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 PO-020.

Abstract PO-006: ABBV-319: A CD19–targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies

Abstract Glucocorticoids such as prednisone (P) and dexamethasone (D) are key components in standard-of-care immuno-chemotherapy regimens (e.g., R-CHOP, EPOCH-R, Hyper-CVAD) for the treatment of B-cell malignancy. However, prolonged systemic glucocorticoid treatment is associated with several adverse events that often result in dose reduction and reduced therapeutic efficacy. ABBV-319 is a CD19-targeting antibody-drug conjugate (ADC) with a glucocorticoid receptor modulator (GRM) payload engineered to reduce glucocorticoid-associated toxicities while possessing three distinct mechanisms of action (MOA) to increase therapeutic efficacy: (1) CD19 receptor-mediated delivery of potent glucocorticoid receptor modulator (GRM) payload to induce apoptosis, (2) inhibition of B-cell receptor (BCR) stimulated CD19 signaling, and (3) enhanced Fc-mediated effector function via afucosylation of the antibody backbone. ABBV-319 elicited potent GRM-driven anti-tumor activity against multiple malignant B-cell lines in vitro as well as in cell line-derived xenografts (CDXs) and patient-derived xenografts (PDXs) in vivo. Remarkably, a single-dose of ABBV-319 induced sustained tumor regression and enhanced anti-tumor activity compared to repeat dosing of systemic prednisolone at the maximum tolerated dose (MTD) in mice. The unconjugated CD19 monoclonal antibody (mAb) also displayed anti-proliferative activity on a subset of B-cell lymphoma cell lines through the inhibition of PI3K signaling. Moreover, afucosylation of the CD19 mAb enhanced Fc-mediated antibody-dependent cellular cytotoxicity (ADCC), and this activity was maintained after conjugation with GRM payload. Notably, ABBV-319 displayed superior efficacy compared to afucosylated CD19 mAb in human CD34+ PBMC-engrafted NSG-tg(Hu-IL15) transgenic mice, demonstrating enhanced anti-tumor activity in vivo when multiple MOAs are enabled concurrently. ABBV-319 also showed potent anti-tumor activity across multiple B-cell lymphoma models, including non-germinal center B-cell (GCB) DLBCL and relapsed lymphoma treated with R-CHOP treatment. Collectively, these data support the ongoing evaluation of ABBV-319 for the treatment of B-cell malignancy in Phase I clinical trial (NCT05512390). Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Chewei (Anderson) Chang. ABBV-319: A CD19–targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies [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 PO-006.

Targeting CD19-positive lymphomas with the antibodydrug conjugate loncastuximab tesirine: preclinical evidence of activity as a single agent and in combination therapy.

Antibody-drug conjugates (ADC) represent one of the most successful therapeutic approaches introduced into clinical practice in the last few years. Loncastuximab tesirine (ADCT-402) is a CD19-targeting ADC in which the antibody is conjugated through a protease cleavable dipeptide linker to a pyrrolobenzodiazepine dimer warhead (SG3199). Based on the results of a phase II study, loncastuximab tesirine was recently approved for adult patients with relapsed/refractory large B-cell lymphoma. We assessed the activity of loncastuximab tesirine using in vitro and in vivo models of lymphomas, correlated its activity with levels of CD19 expression, and identified combination partners providing synergy with the ADC. Loncastuximab tesirine was tested across 60 lymphoma cell lines. It had strong cytotoxic activity in B-cell lymphoma cell lines. The in vitro activity was correlated with the level of CD19 expression and intrinsic sensitivity of cell lines to the ADC's warhead. Loncastuximab tesirine was more potent than other anti-CD19 ADC (coltuximab ravtansine, huB4-DGN462), although the pattern of activity across cell lines was correlated. The activity of loncastuximab tesirine was also largely correlated with cell line sensitivity to R-CHOP. Combinatorial in vitro and in vivo experiments identified the benefit of adding loncastuximab tesirine to other agents, especially BCL2 and PI3K inhibitors. Our data support the further development of loncastuximab tesirine for use as a single agent and in combination for patients affected by mature B-cell neoplasms. The results also highlight the importance of CD19 expression and the existence of lymphoma populations characterized by resistance to multiple therapies.

ABBV-319: a CD19-targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies

AbstractGlucocorticoids are key components of the standard-of-care treatment regimens for B-cell malignancy. However, systemic glucocorticoid treatment is associated with several adverse events. ABBV-319 is a CD19-targeting antibody-drug conjugate engineered to reduce glucocorticoid-associated toxicities while possessing 3 distinct mechanisms of action (MOA) to increase therapeutic efficacy: (1) antibody-mediated delivery of a glucocorticoid receptor modulator (GRM) payload to activate apoptosis, (2) inhibition of CD19 signaling, and (3) enhanced fragment crystallizable (Fc)–mediated effector function via afucosylation of the antibody backbone. ABBV-319 elicited potent GRM-driven antitumor activity against multiple malignant B-cell lines in vitro, as well as in cell line-derived xenografts and patient-derived xenografts (PDXs) in vivo. Remarkably, a single dose of ABBV-319 induced sustained tumor regression and enhanced antitumor activity compared with repeated dosing of systemic prednisolone at the maximum tolerated dose in mice. The unconjugated CD19 monoclonal antibody (mAb) also displayed antiproliferative activity in a subset of B-cell lymphoma cell lines through the inhibition of phosphoinositide 3-kinase signaling. Moreover, afucosylation of CD19 mAb enhanced Fc-mediated antibody-dependent cellular cytotoxicity. Notably, ABBV-319 displayed superior efficacy compared with afucosylated CD19 mAb in human CD34+ peripheral blood mononuclear cell–engrafted NSG-Tg(Hu-IL15) transgenic mice, demonstrating enhanced antitumor activity when multiple MOAs are enabled. ABBV-319 also showed durable antitumor activity across multiple B-cell lymphoma PDX models, including nongerminal center B-cell diffuse large B-cell lymphoma and relapsed lymphoma after R-CHOP treatment. Collectively, these data support the ongoing evaluation of ABBV-319 in a phase 1 clinical trial.

Effects of Cannabidiol, ∆9-Tetrahydrocannabinol, and WIN 55-212-22 on the Viability of Canine and Human Non-Hodgkin Lymphoma Cell Lines.

In our previous study, we demonstrated the impact of overexpression of CB1 and CB2 cannabinoid receptors and the inhibitory effect of endocannabinoids (2-arachidonoylglycerol (2-AG) and Anandamide (AEA)) on canine (Canis lupus familiaris) and human (Homo sapiens) non-Hodgkin lymphoma (NHL) cell lines' viability compared to cells treated with a vehicle. The purpose of this study was to demonstrate the anti-cancer effects of the phytocannabinoids, cannabidiol (CBD) and ∆9-tetrahydrocannabinol (THC), and the synthetic cannabinoid WIN 55-212-22 (WIN) in canine and human lymphoma cell lines and to compare their inhibitory effect to that of endocannabinoids. We used malignant canine B-cell lymphoma (BCL) (1771 and CLB-L1) and T-cell lymphoma (TCL) (CL-1) cell lines, and human BCL cell line (RAMOS). Our cell viability assay results demonstrated, compared to the controls, a biphasic effect (concentration range from 0.5 μM to 50 μM) with a significant reduction in cancer viability for both phytocannabinoids and the synthetic cannabinoid. However, the decrease in cell viability in the TCL CL-1 line was limited to CBD. The results of the biochemical analysis using the 1771 BCL cell line revealed a significant increase in markers of oxidative stress, inflammation, and apoptosis, and a decrease in markers of mitochondrial function in cells treated with the exogenous cannabinoids compared to the control. Based on the IC50 values, CBD was the most potent phytocannabinoid in reducing lymphoma cell viability in 1771, Ramos, and CL-1. Previously, we demonstrated the endocannabinoid AEA to be more potent than 2-AG. Our study suggests that future studies should use CBD and AEA for further cannabinoid testing as they might reduce tumor burden in malignant NHL of canines and humans.

Identification of a novel form of caspase-independent cell death triggered by BH3-mimetics in diffuse large B-cell lymphoma cell lines

BH3-mimetics represent promising anti-cancer agents in tumors that rely on the anti-apoptotic function of B-Cell Lymphoma 2 (BCL2) proteins, particularly in leukemia and lymphoma cells primed for apoptosis. Mechanistically, BH3-mimetics may displace pro-apoptotic binding partners thus inducing BAX/BAK-mediated mitochondrial permeabilization followed by cytochrome c release, activation of the caspase cascade and apoptosis. Here, we describe a novel mode of caspase-independent cell death (CICD) induced by BH3-mimetics in a subset of diffuse large B-cell lymphoma (DLBCL) cells. Of note, rather than occurring via necroptosis, CICD induced immediately after mitochondrial permeabilization was associated with transcriptional reprogramming mediated by activation of c-Jun N-terminal Kinase (JNK) signaling and Activator Protein 1 (AP1). Thereby, CICD resulted in the JNK/AP1-mediated upregulation of inflammatory chemokines and increased migration of cytotoxic Natural Killer (NK) cells. Taken together, our study describes a novel mode of CICD triggered by BH3-mimetics that may alter the immune response towards dying cells.

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.

Lactate dehydrogenase A is implicated in the pathogenesis of B-cell lymphoma through regulation of the FER signaling pathway.

Lactate dehydrogenase A (LDHA) is highly expressed in various tumors. However, the role of LDHA in the pathogenesis of B-cell lymphoma remains unclear. Analysis of data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases revealed an elevated LDHA expression in diffuse large B-cell lymphoma (DLBC) tissues compared with normal tissues. Similarly, our results demonstrated a significant increase in LDHA expression in tumor tissues from the patients with B-cell lymphoma compared with those with lymphadenitis. To further elucidate potential roles of LDHA in B-cell lymphoma pathogenesis, we silenced LDHA in the Raji cells (a B-cell lymphoma cell line) using shRNA techniques. Silencing LDHA led to reduced mitochondrial membrane integrity, adenosine triphosphate (ATP) production, glycolytic activity, cell viability and invasion. Notably, LDHA knockdown substantially suppressed in vivo growth of Raji cells and extended survival in mice bearing lymphoma (Raji cells). Moreover, proteomic analysis identified feline sarcoma-related protein (FER) as a differential protein positively associated with LDHA expression. Treatment with E260, a FER inhibitor, significantly reduced the metabolism, proliferation and invasion of Raji cells. In summary, our findings highlight that LDHA plays multiple roles in B-cell lymphoma pathogenesis via FER pathways, establishing LDHA/FER may as a potential therapeutic target.

Abstract 7583: Discovery of a potent small molecule MALT1 protease inhibitor for the treatment of cancer

Abstract The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), as the core component of CBM complex (CARD9/10/11/14-BCL10-MALT1), plays an essential role in activating the NF-κB pathway and regulating the cell proliferation, differentiation and immunity. Inhibiting of MALT1 is considered a promising therapeutic approach for the treatment of several non-Hodgkin B-cell lymphomas, chronic lymphocytic leukemia (CLL), solid tumors and autoimmune diseases. Herein, we report the discovery of a novel potent MALT1 allosteric inhibitor, BPI-530616, which strongly inhibited MALT1 protease activity with subnanomolar IC50, and low off-target activity on other proteases. BPI-530616 can inhibit the proliferation of B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell line, OCI-ly3, bearing CD79b and CARD11 mutations. The cell line is intrinsically resistant to covalent and non-covalent Bruton's Tyrosine Kinase (BTK) inhibitors. Meanwhile, BPI-530616 suppressed the cellular cytokine secretion (e.g.IL-10, IL-2) or MALT1 direct substrate cleavage (e.g. CYLD). In vivo, BPI-530616 dose-dependently suppressed tumor growth and inhibited the cleavage of CYLD in OCI-ly3 xenograft mouse model. To address the role of MALT1 inhibitor in the contest of immune-oncology, a CT26 syngeneic mouse model was set up. Oral administration of BPI-530616 exhibited a good antitumor effect comparable to that of the anti-PD1 antibody, with no observable adverse effects. Additionally, BPI-530616 displayed desirable drug-like properties, including low clearance and good oral bioavailability across multiple pre-clinical species. Taken together, our research has led to the discovery of a novel, potent, selective, and orally bioavailable small molecule MALT1 inhibitor, BPI-530616. It demonstrated anti-tumor efficacy in the DLBCL animal model and showed promising effects in an immune-oncology animal model. Citation Format: Yuzhen Zhou, Bang Fu, Jie Chen, Weidong Cai, Xiao Sun, Yinlong Li, Wei Ren, Qichao Shen, Guolong Du, Xiaojun Zhou, Zhengyao Zou, Haibo Chen, Xiaoyun Liu, Cheng Yang, Xiangyong Liu, Jing Guo, Hao Wu, Lieming Ding, Jiabing Wang, Hong Lan. Discovery of a potent small molecule MALT1 protease inhibitor for the treatment of cancer [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 7583.

Abstract 1344: Improvement of the anti-tumor activity of NK cells and the efficacy of anti-CD20 monoclonal antibodies in the therapy of B cell-derived malignancies

Abstract Resistance of tumor cells to immunotherapy with monoclonal antibodies (mAbs) often relies on the reduced level of the antigen targeted by the particular mAb. We have therefore screened for the compounds able to upregulate selected antigens on the surface of lymphoma/leukemia cells, to minimize the possibility of resistance acquired by the tumor cells. We have found that members of a specific class of antibiotics, known as cation carriers, namely salinomycin (SAL), monensin (MON), narasin, and nigericin exhibit great CD20 antigen-upregulating abilities. A variety of Burkitt’s lymphoma and diffuse large B-cell lymphoma (DLBCL) cell lines as well as patients-derived primary malignant cells, such as DLBCL and chronic lymphocytic leukemia (CLL) cells exhibited upregulation of surface CD20 upon the treatment with SAL and MON at concentrations as low as 250nM and 50 nM, respectively. As a consequence, these compounds appeared to be beneficial for the therapy of lymphoma/leukemia with anti-CD20 mAbs, both in vitro and in animal models.Moreover, the proteomics analysis revealed that besides CD20, cation carriers upregulated a number of different proteins on the surface of tumor cells, including proteins implicated in the interaction with immune cells. These included ICAM1, HLA-A/B/C, CD155, CD80, CD86 and ULBP2/5/6. Particularly important appears to be the upregulation of antigens interacting with the activating receptors on NK cells, helping them to overcome the inhibitory signals and stimulate the lysis of tumor cells. Thus, the results of proteomics analysis explained why SAL- or MON-pretreated tumor cells were efficiently eliminated by NK cells, even in the absence of therapeutic mAb. In light of our discoveries, it seems reasonable to consider the cation carriers as promising drug candidates for the improvement of NK cell anti-tumor activity and potential treatment of B-cell non-Hodgkin lymphoma patients, in combination with anti-CD20 mAbs.Funding: National Science Centre (NCN, Poland), grants 2019/35/B/NZ5/01445 and 2020/39/B/NZ6/03513. Citation Format: Abdessamad Zerrouqi, Aleksandra Zdanowicz, Anna Torun, Andrzej Ciechanowicz, Bhaskar Pradhan, Grzegorz Rymkiewicz, Dimitar Efremov, Beata Pyrzynska. Improvement of the anti-tumor activity of NK cells and the efficacy of anti-CD20 monoclonal antibodies in the therapy of B cell-derived malignancies [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 1344.

Abstract 521: Anticancer mechanisms of epigallocatechin gallate revealed via cellular and molecular profiling

Abstract Introduction: Epigallocatechin gallate (EGCG) is a polyphenol present in green tea that is known for its anti-oxidant, anti-inflammatory, anti-angiogenic, pro-apoptotic, and anti-cancer properties. However, EGCG has not been systematically evaluated on a large panel of cancer cell lines. We hypothesized that EGCG exhibits varying effects on specific cancer cell genetic backgrounds and types. This study aimed to provide a comprehensive analysis of EGCG on both cell viability and gene expression profiles. Methods: 750 cancer cell lines were cultured and seeded in 1536-well plates for cell viability studies (by the CTD2 Center at the Broad Institute). After 24 h, cells were treated with 16 two-fold serial dilutions of EGCG. After 72 h, cell viability was assayed using CellTiter-Glo. For gene expression profiling (with the Broad Institute and NIH), 8 cell lines were treated with 10 µM EGCG for 6 and 24 h, and transcriptomes were assessed using Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. The CMap large-scale transcriptome dataset was used for comparisons. Results: EGCG had the highest potency in the non-Hodgkins B-cell lymphoma cell line DoHH2 (IC50=0.42 µM), the myeloid leukemia cell line Ku812 (IC50=3.76 µM), and the multiple myeloma cell line KMS-28BM (IC50=4.57 µM). Tissue-of-origin analysis showed that EGCG was most potent against lymphoid cancers, and least potent against prostate cancers. Bioinformatics-based analyses found that EGCG-treated cells resembled cells overexpressing CHEK2, DDP4, CBLC, ZNF350, and TRAF6, and cells underexpressing TRIM16. EGCG-induced gene expression resembled lapatinib and sulfasalazine treatment. Conclusions: This study provides a comprehensive examination of the differential impact of EGCG treatment on cancer cells. Subsequent research on the timing and duration of the changes, along with pathway enrichment analyses, will potentially guide the translational potential of EGCG and its chemical analogs in personalized cancer treatment approaches. Citation Format: Aria Panchal, Jacqueline H. Law, Clement Lo, Kenneth W. Yip. Anticancer mechanisms of epigallocatechin gallate revealed via cellular and molecular profiling [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 521.

Abstract 2721: FF01: a novel humanized heavy chain antibody targeting endogenous retroviruses with anti-lymphoma activity

Abstract Background: Human endogenous retroviruses (HERVs) are transposable elements (TE) derived from retrovirus infections that occurred millions of years ago. Despite constituting 8% of our genome, their biological role still needs to be explored. HERV-K (HML-2), a subgroup of HERVs, represents the most recent retroviruses integrated into the human germline DNA. Members of the HML-2 subgroup retain complete ORF with coding potential and have garnered the most attention in disease-related studies. HERVs exhibit physiological expression in embryonic stem cells but are heavily methylated and remain almost silent in normal adult tissues. Although the mechanisms underlying HERV-K contribution to tumor cell growth and survival remain poorly understood, aberrant expression of HERVs has been demonstrated in various human tumor tissues and lymphoma. These findings led us to explore HERV-K as a potential therapeutic target in B cell lymphomas .Methods: RNA data were analyzed with Telescope. Validations were performed by qPCR, Western blot, flow cytometry, and ELISA. Env structure was obtained with AlphaFold and peptides were purchased from GeneScript. Antibody-dependent cellular cytotoxicity (ADCC) was performed with the ADCC Reporter Bioassay (Promega). Internalization was assessed using the Incucyte Fabfluor-pH Antibody labeling reagents. Results: We assessed the TE expression in RNA-seq datasets of diffuse large B cell lymphoma (DLBCL) and B cells from healthy individuals. An MDS plot showed that TEs could separate neoplastic from healthy samples, demonstrating that tumoral and normal specimens have distinct TE and HERV expression profiles. Focusing on tumor samples, DLBCLs were divided into four clusters with different prognoses. Similar results were obtained in a 47 B-cell lymphoma cell line panel. Focusing on the HML-2 family in lymphoma patients, we identified many of them upregulated in tumors. We validated RNA and protein expression of the HML-2 envelope (env) in seventeen lymphoma cell lines, and in two of them, we also proved the protein surface expression. Env-specific siRNA reduced env expression determined a decrease in cell proliferation, suggesting a contribution to lymphoma cell growth. In silico examination of the env identified four linear regions with potential for antibody binding, and peptides covering them were synthesized. We screened 150 lymphoma patients' serum samples for IgG antibodies binding to the peptides, and all peptides showed positive hits. We used a phage display screen to obtain one nanobody, FF01, binding one peptide. FF01 induced ADCC in the cell line with high surface env expression and not in a negative one. FF01 was internalized in an expressing cell, proving the principle for using it as a component of an antibody-drug conjugate. Conclusions: Our data demonstrate that HERVs are in DLBCL patients and propose FF01 as a new candidate to target HERV Env+ lymphomas. Citation Format: Filippo Spriano, Luciano Cascione, Jacopo Sgrignani, Nikolai Bendik, Sara Napoli, Giulio Sartori, Eleonora Cannas, Tao Gongo, Alberto J. Arribas, Davide Rossi, Davide F. Robbiani, Andrea Cavalli, Francesco Bertoni. FF01: a novel humanized heavy chain antibody targeting endogenous retroviruses with anti-lymphoma activity [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 2721.

Abstract 6069: Discovery and preclinical development of orally bioavailable potent BTK degrader, and its early clinical development for the treatment of relapsed and refractory B-cell malignancies

Abstract Background : Bruton tyrosine kinase (BTK) is an important signaling hub that activates the B-cell receptor (BCR) signaling cascade, which is critical for growth and survival of B-cell lymphoma or leukemia. BTK inhibitors have shown anti-tumor activity in patients with B-NHL. However, nearly half of the patients on current BTK inhibitors discontinue the treatment due to adverse effects and/or mutation-induced drug resistance. A degrader is a heterobifunctional chemical compound aimed at eliminating disease-causing proteins, rather than simply inhibiting proteins’ enzymatic function, through the ubiquitin-proteasome system (UPS)-mediated protein degradation. This approach offers a unique solution for addressing relapse or refractory disease by removing overexpressed and mutated proteins, unlike traditional inhibitors. We thus designed UBX-382, a degrader targeting BTK. Results : UBX-382 showed superior degradation activity for both wild-type (WT) and mutant BTK proteins in a single-digit nanomolar range of half-maximal degradation concentration when compared to the competing degrader programs in diffuse large B-cell lymphoma cell line. UBX-382 effectively addressed 7 out of 8 previously reported resistant BTK mutants in in vitro experiments and outperformed ibrutinib, ARQ-531, and MT-802 in inhibiting tumor growth in murine xenograft models harboring WT or C481S mutant BTK-expressing TMD-8 cells. Remarkably, oral dosing of UBX-382 for &amp;lt;2 weeks led to complete tumor regression in 3 and 10 mg/kg groups in murine xenograft models. Following this, further optimization was carried out for potency and PK profile. We nominated the candidate for IND filing and are planning a phase 1 clinical trial. For the first-in-human, open-label, multicenter, phase 1 study, patients in part 1 (dose escalation with BOIN design) had R/R B-cell malignancies and had received at least 2 prior therapies. The candidate will be administered orally at 5 mg once daily followed by 2 days, for a 7-day schedule over 28-day cycles. Part 2 (dose expansion) consisted of disease-specific cohorts, including CLL/SLL, DLBCL, MCL, FL, MZL, and WM. The primary endpoints were safety and tolerability, along with the definition of the maximum tolerated dose. Secondary endpoints included pharmacokinetics/pharmacodynamics and preliminary efficacy. Conclusions : We have discovered potent BTK degrader exhibiting excellent anti-tumor activity in vitro and in vivo. In pre-clinical safety studies, the candidate of UBX demonstrated an acceptable safety profile. The candidate of UBX is currently preparing phase 1 clinical trials for hematological malignancies. Citation Format: Song Hee Lee, Sun-Mi Yoo, Ye Seul Lim, Han Wool Kim, Je Ho Ryu, Jong Hwan Lim. Discovery and preclinical development of orally bioavailable potent BTK degrader, and its early clinical development for the treatment of relapsed and refractory B-cell malignancies [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 6069.

Lysine demethylase LSD1 is associated with stemness in EBV-positive B cell lymphoma

EBV-infected lymphoma has a poor prognosis and various treatment strategies are being explored. Reports suggesting that B cell lymphoma can be induced by epigenetic regulation have piqued interest in studying mechanisms targeting epigenetic regulation. Here, we set out to identify an epigenetic regulator drug that acts synergistically with doxorubicin in EBV-positive lymphoma. We expressed the major EBV protein, LMP1, in B-cell lymphoma cell lines and used them to screen 100 epigenetic modifiers in combination with doxorubicin. The screening results identified TCP, which is an inhibitor of LSD1. Further analyses revealed that LMP1 increased the activity of LSD1 to enhance stemness ability under doxorubicin treatment, as evidenced by colony-forming and ALDEFLUOR activity assays. Quantseq 3′ mRNA sequencing analysis of potential targets regulated by LSD1 in modulating stemness revealed that the LMP1-induced upregulation of CHAC2 was decreased when LSD1 was inhibited by TCP or downregulated by siRNA. We further observed that SOX2 expression was altered in response to CHAC2 expression, suggesting that stemness is regulated. Collectively, these findings suggest that LSD1 inhibitors could serve as promising therapeutic candidates for EBV-positive lymphoma, potentially reducing stemness activity when combined with conventional drugs to offer an effective treatment approach.

Experimental model of primary intraocular lymphoma based on BALB/CaNn strain and A20 cells is optimal for investigational research.

The purpose of this project was to compare the characteristics of two experimental murine models of primary intraocular lymphoma (PIOL) and determine which experimental model is most suitable for further investigational research to elucidate the pathophysiology of PIOL and to find new therapeutical strategies. In both experimental models PIOL was induced in immunocompetent mice with intravitreal injection of syngeneic B-cell lymphoma cell lines. Murine strain C3H/HeN and cell line 38C13 were used in the first model and BALB/CaNn mice and cell line A20 in the second model. During the experiments, thorough clinical evaluation (using photo documentation, ultrasonography, and MRI) and histological evaluation were performed. In both models, the percentage of PIOL development was high, reaching nearly 80%. Disease progression was faster in C3H/HeN with exophthalmos occurring on average on day 10. Vitreous involvement was a predominant sign in the clinical presentation of this group. In BALB/CaNn mice exophthalmos occurred on average on day 22. The predominant clinical sign in the BALB/CaNn group was tumorous infiltration of the retina, optic disc, and tumorous retinal detachment. Slower progression of the disease in BALB/CaNn mice, greater possibility to examine the retina due to mild vitreous involvement, and later occurrence of exophthalmos makes this strain more suitable for further investigational research.