Human B-cell Lymphoma Cell Lines Research Articles

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.

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.

The Ublituximab-Umbralisib (U2) Drug Regimen Potentiates the Activity of the Novel CD47-CD19 Bispecific Antibody, TG-1801, through the Activation of the G Protein-Coupled Receptor EBI2/GPR183

Introduction: TG-1801 is a novel, bispecific anti-CD47 and anti-CD19 fully human IgG1 antibody that targets CD47 selectively on CD19+ B-cells, sparing red blood cells or platelets and blocking the CD47-SIRPα macrophage checkpoint on mature B cells. TG-1801 is currently in clinical trial as a single agent or in combination with ublituximab, a glyco-engineered CD20 antibody, in B-cell non-Hodgkin lymphoma (B-NHL). The doublet therapy of ublituximab with the dual PI3Kδ/CK1e inhibitor umbralisib (“U2” regimen), provides a non-chemotherapy backbone regimen on which several novel multidrug combinations are being explored clinically. Here we explored in vitro and in vivo potential synergies between TG-1801 and ublituximab, umbralisib, and the U2 combination, in preclinical models of B-NHL.Methods: A panel of n=12 human B-cell lymphoma cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells (BMSCs), M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 +/- U2 treatments was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). In vivo, drug efficacy was determined in a Raji xenograft model, by dosing tumor-bearing mice for 17 days with ublituximab (5mg/kg, qw) +/- umbralisib (150mg/kg, bid), the combination of both and/or TG-1801 (5mg/kg, qw).Results: Here we show on a panel of lymphoma cells lines that the number of receptors per cell and the ratio CD47/CD19 do not impact TG-1801-mediated ADCC or phagocytosis (ADCP). In addition, we show that TG-1801 potentiated ublituximab-mediated ADCC and ADCP and exhibited a similar additive effect when added to U2 combination. In vivo, ublituximab alone displayed a tumor growth inhibition (TGI) of 88%, with 3/8 mice harboring a barely palpable tumor, while the umbralisib alone treatment arm showed a TGI of 50%, with 2/8 mice lacking detectable tumors. TG-1801 exhibited a 76% TGI with 1/8 tumor free-mouse. Most importantly, the combination of TG-1801 with umbralisib alone, ublituximab alone, and U2 achieved TGI of 85%, 93% and 93% respectively, with more tumor-free mice 35 days after the last dose in these three groups. Interestingly, this superior anti-tumor effect of the different TG-1801 combinations was associated with a higher infiltration of mouse macrophages within the tumors as assessed by F4/80 IHC labeling. RNA-seq analysis of the Raji xenografts and of n=4 representative in vitro B-NHL co-cultures treated with TG-1801, U2 or the triple combination uncovered the upregulation of the G-protein coupled receptor EBI2/GRP183 as a common event associated with the synergistic antitumor effects of TG-1801 and U2 in vitro and in vivo. A critical role of EBI2 in the regulation of macrophage activity, B cell migration and in the promotion of a pro-inflammatory phenotype was demonstrated upon exposure of the co-cultures with the EBI2 small molecule inhibitor NIBR189, which impaired the U2/TG-1801-evoked ADCP, B-cell cytoskeleton remodeling and inflammatory cytokine production.Conclusion: The data presented here set the preclinical rationale and support a combination strategy of the novel CD47-CD19 bispecific antibody TG-1801 in B-NHL with other B-cell targeted mechanisms, including umbralisib and ublituximab. DisclosuresNormant: TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Miskin: TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Roue: TG Therapeutics, Inc.: Research Funding.

Impaired ribosome biogenesis checkpoint activation induces p53-dependent MCL-1 degradation and MYC-driven lymphoma death

AbstractMYC-driven B-cell lymphomas are addicted to increased levels of ribosome biogenesis (RiBi), offering the potential for therapeutic intervention. However, it is unclear whether inhibition of RiBi suppresses lymphomagenesis by decreasing translational capacity and/or by p53 activation mediated by the impaired RiBi checkpoint (IRBC). Here we generated Eμ-Myc lymphoma cells expressing inducible short hairpin RNAs to either ribosomal protein L7a (RPL7a) or RPL11, the latter an essential component of the IRBC. The loss of either protein reduced RiBi, protein synthesis, and cell proliferation to similar extents. However, only RPL7a depletion induced p53-mediated apoptosis through the selective proteasomal degradation of antiapoptotic MCL-1, indicating the critical role of the IRBC in this mechanism. Strikingly, low concentrations of the US Food and Drug Administration–approved anticancer RNA polymerase I inhibitor Actinomycin D (ActD) dramatically prolonged the survival of mice harboring Trp53+/+;Eμ-Myc but not Trp53–/–;Eμ-Myc lymphomas, which provides a rationale for treating MYC-driven B-cell lymphomas with ActD. Importantly, the molecular effects of ActD on Eμ-Myc cells were recapitulated in human B-cell lymphoma cell lines, highlighting the potential for ActD as a therapeutic avenue for p53 wild-type lymphoma.

Abstract 2497: FCN-338, a novel and selective Bcl-2 inhibitor, exhibits potent anti-tumor activity in B-cell lymphoma

Abstract B-cell lymphoma-2 (Bcl-2) family proteins are key regulators of apoptosis, which is vital for proper tissue development and cellular homeostasis. Apoptosis occurs via activation of two different pathways: the extrinsic pathway, triggered by a death ligand binding to a death receptor, such as TNF-α to TNFR1; and the intrinsic pathway, regulated by Bcl-2 family proteins and their complex protein-protein interactions. Bcl-2 family proteins are functionally classified as either anti-apoptotic, such as Bcl-2, Bcl-xL and Mcl-1, or pro-apoptotic, including Bid, Bim, Bad, Bak and Bax. Anti-apoptotic Bcl-2 family members are often found to be up-regulated in many types of human cancers, such as different subtypes of B-cell lymphoma and diverse solid tumors, and are frequently been correlated with decreased susceptibility to chemotherapeutics and to increased radio-resistance. Overexpression of Bcl-2 proteins is an independent indicator of poor prognosis in tumors including chronic lymphocytic leukemia (CLL), prostate cancer, and small cell lung cancer (SCLC). Therefore, targeting Bcl-2 and its pro-survival relatives that form the core anti-apoptotic machinery, has emerged as a promising therapeutic approach in cancer. To that end, we have developed a novel and selective Bcl-2 inhibitor, FCN-338, with nanomolar affinity to Bcl-2 and physicochemical properties suitable for oral administration. FCN-338 exhibits much greater affinity to Bcl-2 than Bcl-xL, suggesting its good potential to avoid thrombocytopenia caused by the inhibition of Bcl-xL. FCN-338 has shown remarkable anti-proliferation potency against a panel of Bcl-2-addicted human B-cell lymphoma cell lines, including DOHH2 follicular lymphoma (FL) cell line (IC50 2 nM), Mino diffuse large B-cell lymphoma (DLBCL) cell line (IC50 23 nM), and RS4;11 acute lymphoblastic leukemia (ALL) cell line (IC50 35 nM), but not in Bcl-xL-dependent H146 SCLC cell line (IC50 >4,500 nM). Correspondingly, in vivo efficacy of FCN-338 was demonstrated in a variety of xenograft models derived from FL, DLBCL and ALL tumors. FCN-338 dramatically caused tumor regression in a dose-dependent manner. In the non-clinical studies, FCN-338 exhibits much improved pharmacokinetic properties with higher T1/2, dose-normalized AUC and bioavailability in dogs compared with the FDA-approved Bcl-2 inhibitor ABT-199. Preferable safety profiles of FCN-338 were shown with no potential hERG inhibitory effect and less drug-drug interaction potential, as evidenced by no inhibitory effect (IC50 >50 μM) on CYP2C9 enzyme compared with ABT-199 (IC50 1.05 μM). Together, our findings highlight the therapeutic potential of FCN-338 to be used as effective therapy across a broad range of Bcl-2-addicted B-cell malignancies. Citation Format: Shu Lin, Xingdong Zhao, Hongbin Liu, Huajie Zhang, Zhifang Chen, Lihua Jiang, Yanxin Liu, Min Lin, Yue Rong, Rui Tan, Zuwen Zhou, Zongyao Zou, Yuwei Gao, Weibo Wang. FCN-338, a novel and selective Bcl-2 inhibitor, exhibits potent anti-tumor activity in B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2497.

Targeting MYC-Driven B-Cell Lymphoma By Inhibition of the Histone Methyltransferase DOT1L

Aberrant activation of the MYC proto-oncogene is a recurrent feature in human B-cell lymphomas of diverse sub-types, correlating with adverse prognosis and therapy resistance. Direct pharmacological MYC-targeting has proved difficult, but recent studies have shown that targeting chromatin regulators critical for MYC-driven oncogenesis may provide alternative avenues for therapeutic intervention. Recently, it has been demonstrated that MYC-driven oncogenesis in certain solid tumors is dependent on the histone 3 lysine 79 (H3K79) methyltransferase DOT1L. We hypothesized that B-cell lymphomas with hyperactive MYC-signaling might be responsive to DOT1L inhibition. In order to test this hypothesis, we tested the effect of the DOT1L inhibitor Pinometostat (EPZ-5676) on a panel of human B-cell lymphoma cell lines featuring elevated MYC. Pinometostat treatment reduced global H3K79 methylation levels, accompanied by a time and dose-dependent decrease in proliferation of several Burkitt's lymphoma cell lines including P493-6, Daudi and Raji. We observed that key MYC-target genes including CDK4, PPAT and NPM1 were significantly downregulated upon Pinometostat treatment, suggesting that DOT1L is required for the transcriptional activation of MYC-target genes in these cells. Pinometostat-treated B-lymphoma cells showed a significant decrease of cells in S-phase compared to controls as assessed by BrdU-labeling assays. Similar results were also obtained in a panel of B-cell lymphoma cell lines with MYC-rearrangements including mantle cell lymphoma (MCL) cell lines Jeko-1, JVM2, Mino-1 and Maver-1 and the diffused large B-cell lymphoma (DLBCL) cell line Karpas 422.Next, we sought to investigate whether the DOT1L-dependence of MYC-driven B-cell lymphoma could be reproduced in a well-defined model of MYC-driven B-cell lymphoma. Towards this end, we utilized a mouse model in which expression of the Cre recombinase from a B cell specific promoter leads to ectopic expression of a transgenic human MYC allele and concomitant deletion of the tumor suppressor Pten in B cells. Similar to our in vitro studies, Pinometostat treatment led to a significant reduction in proliferation of B-cell lymphoma cells from these mice with an IC50 of 0.5 µM.Furthermore, we sought to ascertain whether these findings reflected on-target effects related to DOT1L inhibition. Therefore, we deleted DOT1L using CRISPR/Cas9 in B-cell lymphoma cell lines and assessed the effect on proliferation using competitive-proliferation assays. We observed that DOT1L-deletion progressively diminished the relative growth of anti-DOT1L sgRNA-expressing P493-6 and Jeko1 cells compared to non-targeted cells invitro. In order to test the requirement for DOT1L in lymphoma propagation in vivo, we performed intravenous injections of equal number of Jeko-1 cells with either anti-DOT1L or anti-Renilla control sgRNAs into sub-lethally irradiated non-obese diabetic/severe combined immunodeficiency mice (NOD/SCID) mice. Mice injected with control anti-Renilla sgRNAs succumbed to disease with a median latency of 34 days while the latency of disease in the anti-DOT1L sgRNA cohort was 45 days. In summary, DOT1L depletion significantly delayed disease latency in this invivo disseminated model of B-cell lymphoma (P=0.02).We then performed transcriptomic analyses of Pinometostat-treated B-cell lymphoma cell lines compared to DMSO-treated counterparts using RNA-seq. Gene-set enrichment analysis (GSEA) of RNA-seq data from three different B-cell lymphoma cell lines demonstrated that Pinometostat treatment significantly decreased the expression of MYC-target genes. In order to investigate the intriguing role of DOT1L in regulating MYC-target gene expression, we used ChIP-seq to assess the genome-wide occupancy of MYC following DOT1L inhibitor treatment. Strikingly, our studies demonstrated that DOT1L inhibition significantly reduced the chromatin occupancy of MYC.Taken together, our experiments demonstrate the role of DOT1L in MYC-driven B-cell lymphoma pathogenesis invitro and invivo. Furthermore, our genome-wide studies demonstrate the importance of DOT1L for genomic MYC occupancy. Based on these findings, we propose that therapeutic DOT1L targeting may be a viable strategy in MYC-driven B-cell lymphoma. DisclosuresWeigert:Roche: Research Funding; Novartis: Research Funding. Rickert:Pfizer: Employment. Ren:Elli Lilly: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arima Genomics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Deshpande:Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees; A2A Pharma: Membership on an entity's Board of Directors or advisory committees.

CD3-Activating Bi-Specific Antibody Targeting CD19 on B Cells in Mono- and Bi-Valent Format

Immunotherapies targeting B-lineage-specific surface marker CD19 had demonstrated promising clinical results. Two CD19 CAR-T therapies (Kymriah® and Yescarta®) have been approved by FDA to treat patients with B cell malignancies, however, the complicated manufacturing process and low throughput limit its accessibility to more patients, especially in developing countries. The first CD3-activating bi-specific antibody targeting CD19, Blincyto, or CD19 BiTE, was approved to treat relapsed and refractory acute lymphoblastic lymphoma (r/r ALL). The relatively short half-life of Blincyto requires continuous IV infusion for weeks to maintain a steady levels of drug exposure, not to mention the high risk of developing severe cytokine release syndrome in patients.We had established a bispecific antibody platform ITabTM (immunotherapy antibody) for the generation of CD3-activating bi-specific antibodies that could potentially overcome the shortcomings of BiTEs. A CH1 domain was introduced into the ITabTM construct design with the intent to increase the molecular weight thus led to extend the serum half-life of the bispecific antibody. A novel CD3-activating and monkey cross-reactive antibody was generated with a less degree of T cell activation and cytokine release compared to BiTEs. A bi-valent binding to tumor associated antigen (TAA) format was established to target tumor cells and/or stem cells expressing very low levels of TAA. We report here the biological properties of the mono-valent/bi-valent binding of CD19 bi-specific antibody with CD3-activating activity (A-319/A-329).A series of studies were conducted to evaluate the bioactivities of A-319/A-329 in vitro and in vivo including binding to CD3 and CD19 antigens, T-cell and B-cell binding activities, T cell activation and proliferation and B cell killing activities in vitro as well as in vivo efficacy using human PBMC engrafted mouse xenograft models. The in vitro data showed that the mono-valent and bi-valent CD19 binding had little effect on the CD3-associated activities including CD3 antigen binding affinity, T cell binding and T cell activation. In contrast, the bi-valent binding format A-329 showed better potency compared to the mono-valent format A-319 in CD19 binding (KD 0.89 nM vs 19.4 nM); B cell binding (EC50 at 2.3 pM vs 462 pM); in vitro human B cell killing (EC50 0.2 pM vs 3.4 pM). Both A-319 and A-329 were capable of mediating tumor cell lysis with EC50 at 0.03~4 pM. A-329 demonstrated a greater killing activity on Pfeiffer, a human diffuse large B-cell lymphoma (DLBCL) cell line with a low expression of CD19 antigen. In human PBMC engrafted NOG mouse xenograft model, a dose-dependent tumor growth inhibition was observed at 0.5~100 µg/kg in both A-319 and A-329. In monkey studies, when A-319 and A-329 was dosed at 3, 10, 30 µg/kg, twice or three times weekly via IV infusion for A-329 or A-319. Dose-dependent elimination of peripheral blood B cells were observed with both ITabTM. The CD19 bi-valent format of A-329 revealed more complete B cell killing in monkeys. No significant difference of cytokine induction or liver injuries were observed between A-319 and A-329.These results demonstrated that both A-319 and A-329 may benefit patients with B cell malignancies with less dosing frequency and lower cytokine inductions especially, A-329 may have the potential to targeting the low CD19 expressing tumor stem cells. DisclosuresNo relevant conflicts of interest to declare.

Construction of lung tumor cells using RFP and Fluc dual-labeled system

Objective To establish a human lung cancer cell line that can stably express firefly luciferase (Fluc) and red fluorescent protein (RFP) gene so as to lay the foundation for the further establishment of a live-imaging lung cancer xenograft model in nude mice and therapeutic research. Methods The lentiviral vector pHBLV-Fluc-RFP containing luciferase and red fluorescent protein was constructed and then transfected into 293T cells for virus packaging. The complete virus was used to infect human lung cancer cell lines A549, H1975 and human B-cell lymphoma cell line K562. The stable cell lines were obtained by puromycin selection. Fluorescence microscopy and quantitative PCR were used to confirm the RFP and Fluc expression. Results The lentiviral vector pHBLV-Fluc-RFP was successfully constructed. Cancer cell line A549, H1975 and K562 stably expressing Fluc and RFP was obtained. The real-time quantitative PCR results showed that the relative expression of Fluc gene in the three stable infected cells was much higher than that in the corresponding wild-type cells, and the differences were statistically significant(all P<0.05). Conclusion The human lung cancer cell line A549, H1975 and human B-cell lymphoma cell line K562 with dual expression of RFP and Fluc were obtained, which provided a new model of fluorescent cells for in vivo imaging of immunodeficient mouse models such as nude mice. Key words: Red fluorescent protein; Firefly luciferase; Lentivirus; Lung cancer cell fluorescence model

DEAD-Box Protein RNA-Helicase DDX6 Regulates the Expression of HER2 and FGFR2 at the Post-Transcriptional Step in Gastric Cancer Cells.

The human DEAD/H-box RNA helicase DDX6 (RCK/p54) is a protein encoded by the fusion gene from the t(11;14)(q23;q32) chromosomal translocation observed in human B-cell lymphoma cell line RC-K8. DDX6 has a variety of functions such as translation initiation, pre-mRNA splicing, and ribosome assembly. However, details of the regulatory mechanism governing DDX6 and the functions of DDX6 are largely unknown. Previously, we reported that DDX6 is overexpressed in most malignant cell lines and clinical colorectal tumor samples and that DDX6 positively contributes to the pathogenesis of various cancers. In the current study, we aimed at revealing the function of DDX6 in HER2 and FGFR2 related human gastric cancer (GC) by using clinical samples and GC cell lines. DDX6 protein was overexpressed in about 60% of the clinical samples; HER2, in 35%; and FGFR2, in 30%, (n = 20). Interestingly, the DDX6 protein was overexpressed in all HER2-positive samples (n = 7), and in 83% (5 of 6) of the FGFR2-positive samples, which could reflect the contribution of DDX6 to the expression of HER2 and FGFR2. In the GC cell line MKN7, which has HER2 amplification, the knockdown of DDX6 by siR-DDX6 led to the decreased expression of the HER2 protein. On the other hand, the knockdown of HER2 did not influence the DDX6 expression. Similar results were also obtained for the KATO-III and HSC39 cell lines having amplified FGFR2 expression. The increased expression of DDX6 induced a significantly increased expression of the HER2 protein without increasing the mRNA expression. The results of an RNP Immunoprecipitation (RIP)-assay using GC cells indicated that the DDX6 protein acted as an RNA-binding protein for HER2 and FGFR2 mRNAs and positively regulated their post-transcriptional processes. These findings demonstrated that DDX6 was an upstream molecule that positively regulated the expression of HER2 and FGFR2 at the post-transcriptional step in GC cells.

Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma.

T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG) mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP) in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.

1-Thiazol-2-yl-N-3-methyl-1H-pyrozole-5-carboxylic acid derivatives as antitumor agents

A class of substituted 1-thiazol-2-yl-N-3-methyl-1H-pyrozole-5-carboxylic acid derivatives was found to have potent anti-proliferative activity against a broad range of tumor cell lines. A compound from this class (14) was profiled across a broad panel of hematologic and solid tumor cancer cell lines demonstrating cell cycle arrest at the G0/G1 interphase and has potent anti-proliferative activity against a distinct and select set of cancer cell types with no observed effects on normal human cells. An example is the selective inhibition of human B-cell lymphoma cell line (BJAB). Compound 14 was orally bioavailable and tolerated well in mice. Synthesis and structure activity relationships (SAR) in this series of compounds are discussed.

Abstract 2655: Drug resistant B-cell tumors eliminated by novel therapeutic antibodies in vivo

Abstract B-cell malignancies have been successfully targeted in the clinic by therapies such as anti-CD20 antibody rituximab or Bruton’s tyrosine kinase inhibitor ibrutinib. However, leukemias and lymphomas remain incurable due to primary or acquired resistance, ultimately leaving patients without an effective treatment option. We sought to circumvent this drug resistance by pursuing an alternative target known as B cell activating factor receptor (BAFF-R). Despite past limited success, BAFF-R remains a prime target for B-cell lymphoma and leukemia therapeutic antibody development due to its key role in B-cell proliferation and development. We report the development of two novel monoclonal antibodies (mAbs) against human (h) BAFF-R. The mAbs were generated by immunizing mice with (h)BAFF-R-expressing mouse fibroblast cells presenting a natively folded, cell-surface immunogen. The two mAbs presented unique complementarity determining regions that specifically bound (h)BAFF-R-expressing mouse fibroblast cells but not the parental counterpart. Furthermore, the antibodies were specific to B-cell containing organs such as tonsil and spleen, by immunohistochemical staining and without detectable reactivity in heart, lung, brain, liver, and kidney tissues. To tailor the antibodies for clinical application, a human IgG1 Fc capable of eliciting an immune response was substituted, creating chimeric versions. We showed that both chimeric mAbs bound with high affinity to human B-cell lymphoma cell lines including JeKo-1 (mantle cell lymphoma; MCL), SU-DHL-6 (diffuse large B cell lymphoma; DLBCL), Raji (Burkitt lymphoma), and RL (follicular lymphoma). The chimeric antibodies also elicited antibody-dependent cell-mediated cytotoxicity (ADCC) with primary human natural killer (NK) cells in vitro against these tumor lines as well as primary lymphoma samples (n=5) from patients who progressed after rituximab exposure. Most notably, the antibodies demonstrated efficacy in two in vivo drug resistant lymphoma models we developed, a rituximab-resistant CD20 genomic knockout variant of JeKo-1 and the naturally ibrutinib-resistant Z-138. Using these lymphomas lines for xenogeneic tumor models in NOD scid gamma (NSG) mice, we found our antibodies significantly inhibited tumor growth, conferring long-term and tumor free survival on the mice. Our in vitro and in vivo results robustly demonstrate the high specificity and significant anti-tumor effects of our anti-BAFF-R antibodies against a broad variety of B-cell malignancies, especially against cases of rituximab and ibrutinib resistance. This successful development of novel anti-BAFF-R therapeutic antibodies warrants support for further translational development for clinical use in light of current resistance cases. Citation Format: Hong Qin, Guowei Wei, Ippei Sakamaki, Zhenyuan Dong, Wesley A. Cheng, Diane L. Smith, Feng Wen, Han Sun, Soung-chul Cha, Sattva S. Neelapu, Larry W. Kwak. Drug resistant B-cell tumors eliminated by novel therapeutic antibodies in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2655. doi:10.1158/1538-7445.AM2017-2655

FOXO4 expression is related to stem cell-like properties and resistance to treatment in diffuse large B-cell lymphoma.

Cancer stem cells are proposed to be responsible for resistance to chemotherapeutic agents, including doxorubicin. As phenylbutyrate enhances cancer stem cell properties, we analyzed surviving lymphoma cells after treatment with doxorubicin and phenylbutyrate. Human B-cell lymphoma cell lines, including Toledo, BJAB, Daudi, and Raji were incubated with IC90 concentrations of doxorubicin (300 nM) or phenylbutyrate (8 mM). After 48 h, live cells were sorted and analyzed for their resistance to treatment by examining gene expression profiles using cDNA microarray and biological characteristics. A small fraction of lymphoma cells that survived after drug application showed higher expression of stem cell markers (NANOG, and SOX2) and superior ability of self-renewal and sphere formation, compared to untreated control cells (P < 0.05). Gene expression analysis disclosed elevated expression of 41 genes, including FOXO4, in the four lymphoma cell lines that survived drug treatment. Overexpression of FOXO4 was evident in lymphoma cells surviving after phenylbutyrate treatment and refractory patient-derived lymphoma cells. Induction of FOXO4 expression promoted self-renewal whereas its knockdown led to diminished expression of stem cell markers and colony-forming ability of lymphoma cells. Immunohistochemical staining for FOXO4 in tumor tissue of diffuse large B-cell lymphoma revealed nuclear localization and significant association with poor prognosis. In conclusion, lymphoma cells resistant to treatment exhibit stem cell-like properties and enhanced FOXO4 expression. The presence of FOXO4-expressing cells in tumor tissue and their association with poor survival supports a role of FOXO4 in promoting stem cell properties resulting in poor outcomes.

Treatment of Human B-Cell Lymphomas Using Minicircle DNA Vector Expressing Anti-CD3/CD20 in a Mouse Model.

Bispecific antibodies (BsAbs), capable of directing T cells to kill specific cancer cells by transiently binding the two cell types, have emerged as one class of promising cancer immunotherapies. However, their wide clinical application might be hampered by two deficiencies: high cost and inconvenience in drug administration. This study presents concept-proving data that these problems could be bypassed by using an enhanced nonviral DNA vector minicircle (MC) to produce BsAb in vivo. It was found that the anti-CD3/CD20 produced from the minicircle (MC.CD20) could effectively mediate the T-cell killing of multiple CD20-positive human B-cell lymphoma cell lines in vitro. More importantly, it was demonstrated that delivery of 5 μg of MC.CD20 to mouse liver via hydrodynamic injection resulted in both the expression of a therapeutic level of anti-CD3/CD20 throughout the 32-day experiment and effective anticancer activity in a B-cell lymphoma xenograft mouse model. The data suggest that MC encoding the BsAbs may become an attractive cancer immunotherapy modality based on its excellent features of safety, efficacy, and convenience in both preparation and use, and its affordability once the delivery technology matures.

Newcastle Disease Virus: Potential Therapeutic Application for Human and Canine Lymphoma.

Research on oncolytic viruses has mostly been directed towards the treatment of solid tumors, which has yielded limited information regarding their activity in hematological cancer. It has also been directed towards the treatment of humans, yet veterinary medicine may also benefit. Several strains of the Newcastle disease virus (NDV) have been used as oncolytics in vitro and in a number of in vivo experiments. We studied the cytolytic effect of NDV-MLS, a low virulence attenuated lentogenic strain, on a human large B-cell lymphoma cell line (SU-DHL-4), as well as on primary canine-derived B-cell lymphoma cells, and compared them to healthy peripheral blood mononuclear cells (PBMC) from both humans and dogs. NDV-MLS reduced cell survival in both human (42% ± 5%) and dog (34% ± 12%) lymphoma cells as compared to untreated controls. No significant effect on PBMC was seen. Cell death involved apoptosis as documented by flow-cytometry. NDV-MLS infections of malignant lymphoma tumors in vivo in dogs were confirmed by electron microscopy. Early (24 h) biodistribution of intravenous injection of 1 × 1012 TCID50 (tissue culture infective dose) in a dog with T-cell lymphoma showed viral localization only in the kidney, the salivary gland, the lung and the stomach by immunohistochemistry and/or endpoint PCR. We conclude that NDV-MLS may be a promising agent for the treatment of lymphomas. Future research is needed to elucidate the optimal therapeutic regimen and establish appropriate biosafety measures.

Antibody-Interferon-Alpha Fusion Protein Therapy for the Treatment of B-Cell Non-Hodgkin Lymphoma: Enhanced ADCC, Inhibition of Proliferation, and In Vivo Eradication of CD20+ Human Lymphomas

Background: Interferon-alpha (IFNα) is a pleiotrophic cytokine with direct anti-tumor and immunostimulatory effects. Currently IFNα is approved for the treatment of multiple hematologic malignancies, including non-Hodgkin lymphoma (NHL). However, its clinical utility has been hindered by dose-limiting toxicitiy due to systemic activation of the interferon receptor. To overcome this limitation, we engineered anti-tumor antibody-IFNα fusion proteins to selectively increase delivery of IFN to the tumor site and reduce systemic toxicity. We previously reported that IGN002, an anti-CD20-IFNα fusion protein, exhibits enhanced complement-dependent cytotoxicity (CDC) compared to rituximab, and inhibits proliferation and induces apoptosis of human B-cell NHL (Yamada et al, ASCO 2013). We now extend these previous findings and show that IGN002 possesses enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function and superior in vivo anti-tumor activity against B-cell NHL, compared to rituximab.Methods: IGN002 was evaluated against a panel of human Burkitt and diffuse large B-cell lymphoma (DLBCL) cell lines. Proliferation was measured by [3 H]-thymidine incorporation, STAT1 activation by flow cytometry, ADCC by lactate dehydrogenase release using human PBMC effectors, and IFN bioactivity by encephalomyocarditis (EMC) viral protection assay. NHL xenografts were grown in SCID mice.Results: IGN002 more potently inhibited the growth of NHL cell lines expressing CD20 than rituximab or unfused IFNα. Intrinsic IFNα activity of IGN002 was reduced in viral protection and anti-proliferation assays using cells lacking CD20 expression. STAT1 activation by IGN002 was enhanced on cells expressing the target antigen, whereas a control antibody-IFNα fusion protein showed reduced STAT activation activity compared to unfused IFNα. Together, these results indicate that fusion of IFNα to the antibody results in reduced IFN effects on cells not bearing the tumor antigen target. IGN002 exhibited enhanced ADCC activity compared to rituximab against Daudi, Ramos, and Raji NHL cells in long-term (overnight incubation) assays, demonstrating both higher potency and higher maximal cytotoxicity. This result is possibly due to activation of the effector cell populations by the fused IFNα moiety, as IFN is known to activate both NK cells and monocytes. The in vivo anti-tumor efficacy of IGN002 was compared to rituximab and a control antibody-IFNα fusion protein against 10-day established Raji NHL xenografts. IGN002 was superior to both rituximab and the control fusion protein, achieving a longer median survival and higher long-term survival rate (p = 0.0015 and < 0.0001 vs. rituximab and control fusion protein, respectively). The in vivo anti-tumor efficacy of IGN002 was also compared to rituximab at three equimolar dose levels (5 mg/kg, 1 mg/kg, and 0.2 mg/kg antibody) against 10-day established Daudi NHL xenografts. IGN002 showed superior efficacy compared to rituximab at all doses (p < 0.001), achieving tumor eradication (100% long-term survival) in all mice treated at all three dose levels, whereas rituximab only delayed tumor progression.Conclusions: IGN002 demonstrated more robust direct anti-proliferative and antibody effector functions than rituximab against human NHL cells in vitro, and also showed the ability to eradicate established NHL xenografts in vivo. Against cells expressing the CD20 target antigen, IGN002 exhibited greater anti-proliferative potency than unfused IFNα. In contrast, the anti-proliferative and anti-viral potency of IGN002 was reduced against cells lacking CD20, compared to unfused IFNα. These findings support the hypothesis that tumor antigen-targeted IFN therapeutics may possess a broader therapeutic index than unfused IFNα, inhibiting tumor growth by multiple mechanisms while reducing systemic toxicity. These results support the further development of IGN002 for the treatment of B-cell NHL, and a first-in-human phase I clinical study will begin later this year in the United States. DisclosuresTimmerman:Janssen: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Valor Biotherapeutics: Research Funding. Steward:ImmunGene, Inc.: Employment. Minning:Valor Biotherapeutics, LLC: Consultancy. Sachdev:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Gresser:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Khare:Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morrison:ImmunGene, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Caspase-dependent drug-induced apoptosis is regulated by cell surface sialylation in human B-cell lymphoma.

The important role of sialic acid in various biological phenomena is well-established. In order to further clarify the role of sialic acid in cell death induced by various stimuli, the present study compared the cell survival of the HBL-2 human diffuse large B-cell lymphoma cell line upon anticancer drug-induced cell death, with or without neuraminidase pretreatment: Cell survival was assessed using flow cytometry. Upon treatment with doxorubicin or etoposide, the HBL-2 cell viability decreased. In etoposide-induced cell death, the HBL-2 cells demonstrated nuclear fragmentation, which was consistent with morphologically apoptotic cells. In addition, a higher decrease in the cell viability of etoposide-treated HBL-2 cells was observed in cells pretreated with neuraminidase compared with cells that were not pretreated. Furthermore, the caspase-3, caspase-8 and caspase-9 activities in etoposide-induced apoptosis demonstrated a greater increase upon neuraminidase pretreatment compared with no neuraminidase pretreatment. In conclusion, cell surface sialylation appears to protect lymphoma cells from anticancer drug-induced apoptosis.

Anti-CD20 antibody induces the improvement of cytokine-induced killer cell activity via the STAT and MAPK/ERK signaling pathways.

There is a current requirement for novel therapeutic strategies for the treatment of hematopoietic tumors. Residual tumor cells are the main origin of tumor relapse. The aim of this study was to eliminate the residual tumor cells of hematopoietic tumors. Cytokine-induced killer (CIK) cells are used in immunotherapy to deplete the residual cells. However, it is necessary to increase the antitumor activity and clinical applicability of CIK cells. The present study investigated the antitumor activity of CIK cells to the SU-DHL2 human B-cell lymphoma and K562 human chronic myelogenous leukemia cell lines. CD3+CD56+ cells from healthy donors were expanded in culture with cytokines and anti-CD20 monoclonal antibody (mAb; rituximab) to generate CIK cells. A preliminary investigation of their mechanism was then performed. The increase in the cytotoxicity of the CIK cells induced by the anti-CD20 mAb was associated with an increase in the expression of cytotoxic factors. The expression of components of the signal transducer and activator of transcription (STAT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways was found to increase. Upregulation of the expression of STAT1, STAT3 and STAT5 is important as these co-stimulatory molecules enhance T-cell proliferation. Activation of the MAPK signaling pathway is a possible mechanism for the anti-apoptosis effect on the proliferation of CIK cells. In conclusion, anti-CD20 mAb may play an important role in the improvement of CIK-mediated cytotoxicity to tumor cells. These observations may aid in the improvement of the effects of immunotherapy in depleting the residual cells of hematopoietic tumors. Thus, the use of CIK cells cultured with anti-CD20 mAb could be a novel therapeutic strategy for the depletion of chemotherapy-resistant or residual cells in anaplastic large and B-cell lymphoma.

T Cells Expressing CD19-Specific Chimeric Antigen Receptors Are Inhibited By Indoleamine 2,3-Dioxygenase in Tumors

Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme that mediates the metabolism of tryptophan to kynurenines, which have an inhibitory activity on immune cells. IDO-positive tumors thus establish a microenvironment in which NK and T cells are inactivated, and high IDO expression by tumor cells and high serum kynurenine levels correlate with poor prognosis in diffuse large B-cell lymphoma patients.CD19-specific chimeric antigen receptor T cell (CART) therapy is a promising new approach against B-cell malignancies but not every tumor responds. To determine whether the presence of IDO in tumors affects CART activity, we first investigated the expression of IDO by the human B-cell lymphoma cell lines Raji, Daudi, BJAB and Jeko-1. We found that only Jeko-1 expresses IDO and produces kynurenine natively. IDO was not expressed by the other cell lines, even after exposure to IFNγ, a known IDO inducer. Based on these results, we chose Raji as a baseline IDO-negative cell line and made an IDO-positive Raji clone by retroviral transduction with human IDO cDNA (Raji-IDO); a clone transduced with an empty vector served as control (Raji-control). We injected SCID mice subcutaneously in opposite flanks with luciferase-transduced Raji-control and Raji-IDO cells. Seven days later, we injected human non-transduced T cells (NTs) or CARTs intravenously. In the NT group, tumors had similar growth on both sides. In contrast, in the CART group, Raji-control tumors had significantly slower growth than Raji-IDO tumors (3.1 ± 1.1×108 and 20 ± 7.3×108 bioluminescence units [BU] at day 7, respectively, P = 0.03). We also found that CARTs significantly inhibited Raji-control tumor growth (NT: 27 ± 6.8×108 vs CART: 3.1 ± 1.1×108 BU at day 7, P = 0.02), but did not affect Raji-IDO tumors (NT: 24 ± 5.4×108 vs CART: 20 ± 7.3×108 BU at day 7, P = 0.35). In another experiment, Raji-IDO cells were injected subcutaneously, and mice were treated with an IDO inhibitor (1-methyl-tryptophan, 1-MT), CARTs, or both. The combination treatment produced significantly better tumor control than either single therapy (1-MT: 45 ± 6.8×108 and CART: 22 ± 4.6×108 vs both: 8.2 ± 3×108 BU at day 7, P = 0.001 and 0.04, respectively). Thus, the IDO inhibitor protects CARTs against the deleterious effects of IDO-positive tumors.To investigate potential mechanisms for CART inhibition by IDO, we assessed the effect of kynurenine and found that even low concentrations (12.5 µM) inhibited CART proliferation in response to IL-2, IL-7, IL-15 or CD19-positive targets, although there was no effect on proliferation of B-cell lymphoma cell lines at this kynurenine concentration. CART apoptosis was also increased by kynurenine (8.6 ± 0.6%, 13.9 ± 2.2%, and 33.5 ± 10.6% annexin V-positive cells with 0, 12.5, or 25 µM kynurenine). In coculture of CARTs with wild-type Raji cells, the latter were eliminated by day 6 in the absence of kynurenine, but increased in numbers (in parallel with a decrease in CARTs) in its presence. Kynurenine also inhibited the release of IFNγ (13,143 ± 848 pg/mL vs 2,663 ± 1,873 pg/mL, P = 0.02) and IL-2 (718 ± 355 pg/mL and 199 ± 165 pg/mL, P = 0.03) by CARTs. Expression of granzyme B, PD-1 and CTLA-4 on CARTs was not significantly affected.Fludarabine has been reported to downregulate IDO expression in tumors and this drug is used in many lymphodepleting regimens that are administered before CART infusion in an effort to augment the efficacy of these therapies. However, the beneficial mechanism of lymphodepleting chemotherapy drugs is not fully understood. Therefore, we also measured the effect of fludarabine and mafosfamide (a cyclophosphamide analog) on IDO expression by Jeko-1 cells. We found that both drugs downregulate IDO expression by Jeko-1, even in the presence of IFNγ.In summary, expression of IDO by tumor cells inhibits CART activity, likely because kynurenine is produced and has negative effects on proliferation and cytokine secretion by CARTs. Fludarabine and cyclophosphamide downregulate IDO expression in tumors and this effect may contribute to the benefits of lymphodepletion before CART therapy. Direct IDO inhibitors may further improve clinical CART activity against IDO-positive tumors. DisclosuresNo relevant conflicts of interest to declare.

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.