Characterization of two new high-grade B-cell lymphoma cell lines with MYC and BCL2 rearrangements that are suitable for in vitro drug sensitivity studies

Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

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Abstract 3702: Establishment and characterization of a novelMYC/BCL2double-hit high-grade B-cell lymphoma cell line HLA-3

10.1158/1538-7445.sabcs18-3702 ◽

2019 ◽

Author(s):

Hui Guo ◽

Hui Zhang ◽

Krystle Nomie ◽

Liang Zhang ◽

Kelley P. Murfin ◽

...

Keyword(s):

B Cell ◽

Cell Line ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

High Grade ◽

Lymphoma Cell Line

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Analysis of Adct-602 Pre-Clinical Activity in B-Cell Lymphoma Models and Identification of Potential Biomarkers for Its Activity

Blood ◽

10.1182/blood-2020-134664 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 10-11

Author(s):

Eugenio Gaudio ◽

Chiara Tarantelli ◽

Luciano Cascione ◽

Filippo Spriano ◽

Gaetanina Golino ◽

...

Keyword(s):

B Cell ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Advisory Committees ◽

Travel Grant

Introduction. CD22 is a cell surface marker expressed by the vast majority of normal and neoplastic B-cells. ADCT-602 is an antibody drug conjugate (ADC) composed of Emab-C220, an engineered version of the anti-CD22 humanized IgG1 antibody epratuzumab, site-specifically conjugated to SG3249, which includes the DNA minor groove crosslinking pyrrolobenzodiazepine (PBD) dimer SG3199 linked to the antibody via a protease-cleavable linker (Zammarchi et al, ASH 2016). ADCT-602 is currently being tested in a phase I/II clinical trial (NCT03698552) in recurrent or refractory B-cell acute lymphoblastic leukemia (B-ALL) patients. Here, we assessed its in vitro anti-lymphoma activity, also exploring for potential biomarkers and mechanisms of resistance. Methods. Fifty-seven human lymphoma cell lines were exposed to ADCT-602, an isotype-control ADC and the PBD dimer SG3199 as single agents for 96h, followed by MTT proliferation assay and IC50 calculation. Quantum Simply Cellular (QSC) microspheres were used for the quantitative determination of cellular CD22 antigen expression (Bangs Laboratories. Inc). Differences in IC50 values among lymphoma subtypes were calculated using the Wilcoxon rank-sum test. Statistical significance was defined by P values of 0.05 or less. Sensitivity analysis to ADCT-602 was performed by integrating different omics data, such as Illumina HT-12 microarray data (GSE94669), HTG EdgeSeq Oncology Biomarker Panel data (GSE103934) and DNA copy number variations. Results. The median IC50 for ADCT-602 was 200 pM (95%C.I, 90-400 pM) in 48 B-cell lymphoma lines (including three Hodgkin lymphoma cell lines), and 1850 pM in nine T-cell lymphoma lines (95%C.I, 700-15000 pM). ADCT-602 was more active in B- than in T-cell lymphomas, as expected based on the pattern of CD22 expression (P < 0.005). Focusing on B-cell lymphomas, ADCT-602 in vitro activity was not correlated with its target expression measured both at the cell surface protein level (absolute quantitation, n=48, r=0.06 P=ns) and at the RNA level (Illumina HT-12 arrays, n=42, r=0.28, P=ns; HTG EdgeSeq Oncology Biomarker Panel, n=36, r=0.24, P=ns). In vitro activity was stronger in marginal zone lymphoma (MZL) cell lines than other B-cell lymphoma models (median IC50s 62.5 vs 312.5 pM; P = 0.03) as well as in diffuse large B-cell lymphoma (DLBCL) cell lines with BCL2 and MYC translocations (DHT DLBCL) versus DLBCL with none or a single translocation (median IC50s 25 vs 400 pM, P = 0.03). No associations were seen with TP53 status or other histology (mantle cell lymphoma, DLBCL, DLBCL cell of origin). We then exploited the gene expression profiling data using the Illumina HT-12 microarray gene expression technology. Within all the B-cell lymphoma cell lines (sensitive, n= 25; resistant, n= 23) we identified 1.207 genes down-regulated (FC-) and 1,104 genes up-regulated (FC+) in resistant cell lines. To delineate the pathways associated with the different degrees of sensitivity to ADCT-602, we performed a gene set enrichment analysis (GSEA; GSEA hallmarks and c2.common pathways) on the pre-ranked limma data. Transcripts up-regulated in resistant cell lines were enriched of genes coding for proteins involved in respiratory electron transport, oxidative phosphorylation and proteasome. Conversely, transcripts up-regulated in the sensitive cell lines were enriched of genes coding for proteins involved in inflammation, chemokine signaling, p53 response, IL2/STAT5 signaling, hypoxia, TGF-beta and interferon response. Similar gene signatures were picked up using the HTG platform, which can be applied to formalin-fixed paraffin-embedded clinical specimens, despite the smaller number of investigated genes. Conclusion. ADCT-602 showed in vitro anti-tumor activity across a large panel of B-cell lymphoma models of various histology. Expression signatures and other features (MZL or DHT DLBCL histology), but not the expression levels of its target, were associated with different sensitivity to the ADC. Our data supports the clinical evaluation of ADCT-602 in patients with B-cell lymphoma in addition to B-ALL. Disclosures Zucca: Kite: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Other: Travel Grants; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Research Funding; Celltrion Healthcare: Membership on an entity's Board of Directors or advisory committees. Stathis:PharmaMar: Other: Travel Grant; Member of the steering committee of the trial of this abstract: Other; Loxo: Honoraria, Other, Research Funding; Cellestia: Research Funding; Roche: Other, Research Funding; Novartis: Other, Research Funding; Bayer: Other, Research Funding; Merck: Other, Research Funding; Pfizer: Other, Research Funding; MEI Pharma: Other, Research Funding; ADC Therapeutcis: Other, Research Funding; Abbvie: Other: Travel Grant. Van Berkel:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zammarchi:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bertoni:ADC-Therapeutics: Research Funding; Bayer AG: Research Funding; Helsinn: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Nordic Nanovector ASA: Research Funding; Astra Zeneca: Other: travel grant; Amgen: Other: travel grant.

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Combination of Enzastaurin, a PKC Inhibitor, and Revlimid ® has Synergistic Activity In Non-Hodgkin Lymphoma

Blood ◽

10.1182/blood.v116.21.4905.4905 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4905-4905 ◽

Cited By ~ 1

Author(s):

Stefano Sacchi ◽

Maria Cosenza ◽

Monica Civallero ◽

Giulia Grisendi ◽

Erika Road ◽

...

Keyword(s):

B Cell ◽

Cell Line ◽

Hodgkin Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Indolent Lymphoma ◽

Non Hodgkin Lymphoma

Abstract Abstract 4905 A curative treatment does not exist for indolent lymphoma and eventually patients die for progression and complications related to their disease. Thus, there is a need of new less toxic and more active treatment. Enzastaurin, a novel targeted agent, inhibits PKC-β by interacting competitively as its ATP-binding site. Several studies have shown that enzastaurin exhibits growth inhibiting effects on a wide array of cultured human tumour cells. Revlimid ® (lenalidomide), an oral immunomodulatory drug, have shown antineoplastic activity in various tumours, including multiple myeloma (MM), myelodysplastic syndrome (MDS), B-CLL, renal-cell carcinoma and prostate cancer and it is approved for the treatment of patients with MM and MDS bearing a deletion 5q. In the present research, we demonstrate that Revlimid ® alone induce G0/G1 arrest in WSU-NHL cell line, but not apoptosis. This would suggest that, in vitro, Revlimid ® has more a cytostatic than a cytotoxic effect in this cell line. Further, we have demonstrated that the combination of doses as low as 1 mM of Enzastaurin and Revlimid ® exerts, in vitro, a strong synergistic anti lymphoma activity. We also have showed that the combination decreases viability and induce apoptosis in B-cell lymphoma cell lines and peripheral blood mononuclear cells (PBMCs) from follicular lymphoma (FL) patients. The combination has no effect on normal PBMC and suppresses cell proliferation of B-cell lymphoma cell lines when co-cultured with bone marrow stromal cells (BMSCs) in a system that mimics the BM microenvironment. The combination induces a significantly higher rate of apoptosis in comparison with these caused by each agents utilized alone, as showed by flow cytometry. The combination activates both the extrinsic and intrinsic pathways of apoptosis resulting in caspase-8, caspase-9, caspase-3 and PARP cleavage. Furthermore, we have evaluated whether the combination has the ability to trigger apoptosis through BAD and we have showed its ability to activate BAD. Further, we have demonstrated that the combination decreases the expression of phosphorylated AKT and of some AKT downstream targets such as GSK-3β, m-TOR and p70S6. In addition, we have found that the combination reduces the activation of phosphorylated MAPK and of the downstream effector p90RSK. The MAPK signaling pathways have a multiple roles in natural processes such as cell growth, differentiation, and apoptosis. Taken together, these observations suggest that interrupting the PI3K/AKT and MAPK pathways is a promising therapeutic strategy against B-cell lymphoma cell lines. Therefore, these preclinical data, together with promising results obtained with Revlimid ® in the treatment of non-Hodgkin lymphoma, provide the rationale for evaluating the combination of Enzastaurin and Revlimid ® in the treatment of indolent lymphoma. These compounds, with a favourable toxicity profile, are not classic chemotherapeutic agents causing severe side effects and could be considered an example of a new innovative attempt of an anti-cancer “soft treatment”. Disclosures: No relevant conflicts of interest to declare.

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Novel BAFF-Receptor Antibody to Natively Folded and Glycosylated Recombinant Protein Eliminates Drug Resistant B Cell Malignancies In Vivo

Blood ◽

10.1182/blood.v128.22.468.468 ◽

2016 ◽

Vol 128 (22) ◽

pp. 468-468

Author(s):

Hong Qin ◽

Guowei Wei ◽

Ippei Sakamaki ◽

Zhenyuan Dong ◽

Diane Lynne Smith ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

Acquired Resistance ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Drug Resistant ◽

L Cells

Abstract Background: Targeted monoclonal antibodies (mAbs) such as the anti-CD20 rituximab, are proven therapies in lymphoma, yet these diseases remain incurable because of primary or acquired resistance. Using a eukaryotic expression system to produce antigen closely representing endogenous protein, we developed a new therapeutic antibody against an alternative lymphoma target. B cell activating factor receptor (BAFF-R/TNFRSF13C) is a tumor-necrosis factor receptor superfamily member specifically involved in B lymphocyte development and mature B cell survival. Although earlier attempts to target the BAFF/BAFF-R axis therapeutically for B cell tumors yielded limited success, BAFF-R remains an attractive target for B cell lymphoma therapeutic antibody development, particularly for rituximab-resistant tumors. Methods and Results: We generated 2 mAbs to human BAFF-R expressed as a natively folded, eukaryotically glycosylated cell-surface immunogen on engineered mouse fibroblast (L) cells. Both mAbs specifically bound BAFF-R-expressing L cells, but not the parental counterparts. Each of the complementarity determining regions (CDRs) of the 2 mAbs are unique, suggesting different binding epitopes. Both mAbs bound with high affinity to the human B cell lymphoma cell lines JeKo-1 (mantle cell lymphoma; MCL), SU-DHL6 (diffuse large B cell lymphoma; DLBCL), Raji (Burkitt's lymphoma) and RL (follicular lymphoma). Because our goal is to develop antibodies for clinical use, we substituted in human IgG1 Fc to generate the chimeric mAbs C55 and C90. The chimeric mAbs retained the binding specificity and affinity of the mouse antibodies to their target cells. By immunohistochemistry C55 and C90 staining was specific to the B cell-containing organs tonsil and spleen. No detectable staining was observed in heart, lung, brain, liver, and kidney. Using primary human natural killer (NK) cells as effectors, we demonstrated the chimeric antibodies induced potent antibody-dependent cellular cytotoxicity (ADCC) against BAFF-R-expressing L cells and the JeKo-1, SU-DHL6, Raji and RL human lymphoma cell lines. C55 and C90 were also able to elicit ADCC in primary human lymphomas; they efficiently killed tumor cells from patients with MCL, DLBCL, follicular lymphoma and chronic lymphocytic leukemia (CLL) (n=8). Notably, 5 of these primary lymphomas were from patients who had relapsed after rituximab treatment (2 MCL, 3 CLL). We next determined the activity of C55 and C90 in models of drug-resistant lymphoma. Both ibrutinib and rituximab are effective anti-lymphoma agents, however, primary or acquired resistance to these drugs is common. We derived a rituximab-resistant JeKo-1 variant (JeKo-1 CD20KO) using the CRISPR/HDR system to knock-out CD20 gene expression, and also used the naturally ibrutinib-resistant (Z-138) lymphoma cell line. We confirmed that the C55 and C90 anti-BAFF-R antibodies induced ADCC in both drug-resistant cell lines in vitro. Using xenogenic tumor models in NOD scid gamma (NSG) mice we observed remarkable in vivo anti-tumor effects of both the C55 and C90 chimeric antibodies. We found our antibodies significantly inhibited growth of implanted Z-138 and JeKo-1 CD20KO lymphomas (P<0.001; Figure). Conclusion: In contrast to previously reported BAFF-R antibodies, our in vitro and in vivo results strongly support the translational development of our novel BAFF-R-specific monoclonal antibodies, especially as an alternative immunotherapy against ribuximab- or ibrunitib-resisitant B cell maglinancies. Other preliminary data also suggest BAFF-R may be an effective target of CAR T cells. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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Abstract 3702: Establishment and characterization of a novelMYC/BCL2double-hit high-grade B-cell lymphoma cell line HLA-3

10.1158/1538-7445.am2019-3702 ◽

2019 ◽

Author(s):

Hui Guo ◽

Hui Zhang ◽

Krystle Nomie ◽

Liang Zhang ◽

Kelley P. Murfin ◽

...

Keyword(s):

B Cell ◽

Cell Line ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

High Grade ◽

Lymphoma Cell Line

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974 Additive cytotoxic and proapoptotic effects of external radiation and Rituximab on B cell lymphoma cell lines in vitro

European Journal of Cancer Supplements ◽

10.1016/s1359-6349(03)91001-9 ◽

2003 ◽

Vol 1 (5) ◽

pp. S292

Author(s):

H. Schmidberger ◽

S. Dingeldein ◽

M. Rave-Fraenk ◽

J. Hasenkamp ◽

W. Jung ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

External Radiation

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Epratuzumab (Humanized Anti-CD22 MAb) Conjugated with SN-38, a New Antibody-Drug Conjugate (ADC) for the Treatment of Hematologic Tumors: Preclinical Studies Alone and In Combination with Veltuzumab, a Humanized Anti-CD20 MAb

Blood ◽

10.1182/blood.v116.21.3941.3941 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3941-3941

Author(s):

David M Goldenberg ◽

Serengulam Govindan ◽

Tom M Cardillo ◽

Robert M Sharkey

Keyword(s):

B Cell ◽

Cell Lines ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

In Vivo Studies ◽

Anti Cd20 ◽

Anti Tumor Activity

Abstract Abstract 3941 Background: Monoclonal antibody (MAb) therapy has had a significant impact on the management of B-cell malignancies, but is most often used in combination with chemotherapy. We developed an ADC that combines SN-38, the active component of irinotecan, a topoisomerase I inhibitor, with the internalizing, humanized, anti-CD22 IgG, epratuzumab, and determined its activity alone and in combination with an anti-CD20 antibody therapy (veltuzumab). Methods: Epratuzumab was conjugated with SN-38 (E-SN-38) at a mole ratio of ∼6:1. The conjugate is designed specifically to be released slowly in the presence of serum (50% released over ∼1.5 days), allowing liberation of the drug when internalized, but also being released locally after being bound to the tumor. In vitro and in vivo studies were performed to assess the activity of the conjugate against several subcutaneously- or intravenously-inoculated B-cell lymphoma cell lines. In vivo studies also examined combination therapy using E-SN-38 and the veltuzumab (V). Results: In vitro studies in 4 B-cell lymphoma cells lines (Daudi, Raji, Ramos, WSU-FSCCL) and 4 acute lymphoblastic lymphoma cell lines (697, REH, MN-60, and RS4;11) expressing varying amounts of CD22 showed an IC50 for E-SN-38 in the nanomolar range, confirming potent activity. Nude mice bearing SC Ramos human lymphoma had significant selective anti-tumor activity compared to a control, non-targeting, IgG-SN-38 conjugate, at a dosing regimen of 75 to 250 μg of the conjugates given twice-weekly for 4 weeks. Significant anti-tumor activity was also found in several other cell lines. When combined with veltuzumab, significant improvement in therapeutic activity was observed. For example, median survival in a WSU-FSCCL human follicular B-cell lymphoma IV model with treatment initiated 5 days after implantation was 42 d (0/10 surviving at 160 d) and 91 d (2/10 surviving) for untreated and veltuzumab-treated animals, respectively; 63d (0/10 surviving after 160 d) and >160 d (with 6/10 surviving) for E-SN-38 and E-SN-38 + V, respectively; and 63 d (0/10) and 91 d (2/10) for non-targeting IgG-SN-38 conjugate alone and combined with V). The E-SN-38 conjugate combined with V was significantly better than all treatment or control groups (P ≤ 0.05). Conclusion: E-SN-38 ADC is a potent therapeutic, even at non-toxic dose levels, and shows significantly enhanced efficacy when combined with anti-CD20 immunotherapy, representing an important new ADC treatment regimen. Disclosures: Goldenberg: Immunomedics, Inc.: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Govindan:Immunomedics, Inc.: Employment. Cardillo:Immunomedics, Inc.: Employment.

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