scholarly journals The Novel HDAC Inhibitor Chidamide Synergizes with Rituximab to Inhibit DLBCL Tumor Growth in Vitro and In Vivo By up-Regulating CD20 Expression

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2947-2947
Author(s):  
Xu-Wen Guan ◽  
Wang Hua-Qing ◽  
Li Jia ◽  
Feng-Ting Liu
Keyword(s):  
Cell Death ◽  
Tumor Growth ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Combined Treatment ◽  
B Cell Lymphoma ◽  
Surface Expression ◽  
Cd20 Expression

Abstract Background: Histone deacetylases (HDACs) are crucial proteins for supporting tumorigenesis. HDACs reverse chromatin acetylation and alter transcription of oncogenes and tumor suppressor genes by removing acetyl groups from histones. HDAC inhibitors are considered as promising anti-cancer drugs, particularly in combination with other standard treatment regimens. Chidamide is the world first oral HDAC inhibitor which selectively inhibits class I HDAC1, HDAC2, and HDAC3 as well as class IIb HDAC10. Chidamide has been approved by China FDA in 2015 for the treatment of relapsed or refractory peripheral T-cell lymphoma. Diffuse large B-cell lymphoma (DLBCL) is the most aggressive form of B-cell lymphoma. Treatment with R-CHOP i.e. Rituximab (the anti-CD20 monoclonal antibody) plus CHOP (Cyclophosphamide, doxorubicin, vincristine, and prednisone) has significantly improved clinical outcome for DLBCL patients. However, treatment-induced deacetylation of CD20 gene and consequently down-regulation of CD20 protein expression causes an acquired resistance to further treatment with R-CHOP. We hypothesize that inhibition of HDACs by Chidamide could overcome Rituximab-mediated down-regulation of CD20 and facilitate Rituximab-induced DLBCL tumor growth inhibition. The aim of this study is to determine the synergistic effect of Chidamide and Rituximab in the treatment of DLBCL in vitro and in vivo. Methods: The levels of CD20 (MS4A1) mRNA expression and clinical outcomes in patients with DLBCL treated either with R-CHOP or CHOP were obtained from the Gene Expression Omnibus (GEO) repository (NCBI GSE 10846). The association of CD20 expression with overall survival (OS) was analyzed by Cox regression analysis and the cut-off point was calculated by the X-tile software. CD20 protein surface expression and Rituximab-induced cell death were analyzed by flow cytometry. The IC50s of Chidamide and the synergisms with Rituximab (10 µg/ml) on five DLBCB cell lines (OCI-LY3, OCI-LY7, Su-DHL6, Su-DHL8, and Su-DLH10) were determined by MTT test after cells were treated with a range of concentrations of Chidamide with or without Rituximab for 24 hours. The synergism was calculated using ComboSyn software to obtain the combination index (CI). For in vivo experiments, the human DLBCL cell line OCI-LY7 were injected to 6 weeks BALB/C nude mice to develop xenograft DLBCL mice models. After tumors were palpable, mice were divided into four groups and injected with NaCl (control), Rituximab, Chidamide and Rituximab plus Chidamide daily for three weeks. The tumor volumes were monitored frequently during the treatment. Results: In R-CHOP treated cohort (n=233), higher expression of CD20 expression (n=137) is significantly associated with superior clinical outcomes compared with lower CD20 expression (n=96) with P=0.0038, HR=0.4753, 95% CI=0.274-0.779. However, the levels of CD20 have no effect on clinical outcome in DLBCL patients treated with CHOP (n=183). The levels of CD20 protein surface expression on five DLBCL cell lines were significantly and positively correlated with the sensitivities of cells to Rituximab-induced cell death (P=0.0018, R=0.88). HDAC1, HDCA2 and HDCA3 proteins were detected in these DLBCL cell lines. Treatment with Rituximab significantly reduced CD20 surface expression but treatment with Chidamide significantly increased CD20 surface expression in DLBCL cells. The CI numbers for combined treatment with Chidamide and Rituximab were either <0.01 (very strong synergism) or <0.3 (strong synergism), indicating that Chidamide significantly synergized Rituximab-induced cell death. For in vivo assay, treatment with either Rituximab or Chidamide alone slightly but not significantly reduced tumor volume. Combination with Chidamide and Rituximab significantly inhibited tumor growth in DLBCL xenograft mice (P<0.0001). Mice with combined treatment showed significantly prolonged survival compared with other groups. Conclusions: our data demonstrate for the first time that inhibition of HDACs by Chidamide significantly synergized Rituximab-induced tumor growth inhibition in vitro and in vivo. We propose that CD20 surface expression should be used clinically to evaluate treatment response in patients with DLBCL. Chidamide is a promising sensitizer for the treatment of DLBCL with R-CHOP. Disclosures No relevant conflicts of interest to declare.

Targeting B-Cell Malignancies With Anti-CD20-Interleukin-21 Fusokine

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 377-377 ◽  
Author(s):  
Shruti Bhatt ◽  
Daxing Zhu ◽  
Xiaoyu Jiang ◽  
Seung-uon Shin ◽  
John M Timmerman ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract The anti-CD20 antibody rituximab has revolutionized the treatment for B cell non-Hodgkin lymphomas (NHLs). However, rituximab has limited effectiveness as a single agent in some NHL subtypes and its clinical efficacy is compromised by acquired drug resistance. As a result, many patients still succumb to NHLs. Hence, strategies that enhance the activity of anti-CD20 antibody may improve patient outcome. Interleukin-21 (IL21), a member of the IL2 cytokine family, exerts diverse regulatory effects on natural killer (NK), T and B cells. IL21 has been reported to possess potent anti-tumor activity against a variety of cancers not expressing IL21 receptor (IL21R) through activation of the immune system and is in clinical trials for renal cell carcinoma and metastatic melanoma. We have recently reported that apart from immuno-stimulatory effects, IL21 exerts direct cytotoxicity on IL21R expressing diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines and primary tumors both in vitro as well in vivo (Sarosiek et al Blood 2010; Bhatt et al AACR 2013). Herein we designed a fusion protein comprising IL21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL21 fusokine) to improve efficacy of its individual components and prolong IL21 half-life. We have verified the expression of full length fusion protein and demonstrated that αCD20-IL21 fusokine retained binding ability to its individual components; CD20 and IL21R, as analyzed by immunofluorescence and flow-cytometry analyses. Similar to our previous study of IL21 in DLBCL, treatment of B cell lymphoma cell lines with fusokine lead to phosphorylation of STAT1 and STAT3, upregulation of cMYC and BAX and downregulation of BCL-2 and BCL-XL, implying the activation of IL21R dependent signaling to trigger cytotoxic effects. In vitro, direct cell death induced by αCD20-IL21 fusokine in DLBCL (RCK8, WSU and Farage) and MCL (Mino, HBL2 and SP53) cell lines was markedly increased compared to its individual components (IL21 and parent αCD20-IgG1 antibody). More importantly, fusokine treatment resulted in cell death of MCL cell lines (L128, G519 and UPN1) that were found to be resistant to IL21 alone treatment. Furthermore, treatment of freshly isolated primary NHL cells with the αCD20-IL21 fusokine also exhibited a 40-50% increase in direct cell death compared to its individual components. Previous studies reported that IL21 enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by activation of NK cells. ADCC assays using chromium release with purified human NK cells demonstrated that ADCC induced by the parent antibody was enhanced in the presence of IL21 while IL21 alone had minimal effect on the lysis of Raji, Daudi, and Jeko1 target cells. Notably, αCD20-IL21 fusokine demonstrated increased ADCC activity in comparison to parent antibody plus IL21 in Raji, Daudi and Jeko-1 cells (p<0.001, p<0.005 and p<0.001, respectively). Similar results were obtained in primary MCL tumor cells. Consistent with this finding, fusokine treatment resulted in enhanced activation of the NK cells as assessed by CD69 upregulation and CD16 downregulation using flow-cytometry. Complement dependent cytotoxicity (CDC) of the fusokine was similar to the parent antibody and rituximab in Raji cells. Studies analyzing in vivo effects of the fusokine are in progress and will be presented at the meeting. These data strongly suggest that together with direct apoptotic potential, an anti-CD20 IL21 fusokine retains the ability to trigger indirect cell killing mediated via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, our study demonstrates that anti-tumor effects of IL21 and anti-CD20 antibodies can be enhanced by conjugation of IL21 with anti-CD20 antibody that may serve as a novel anti-lymphoma therapy. Disclosures: Rosenblatt: Seattle Genetics, Inc.: Research Funding.

scholarly journals Novel HDAC inhibitor Chidamide synergizes with Rituximab to inhibit diffuse large B-cell lymphoma tumour growth by upregulating CD20

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xu-Wen Guan ◽  
Hua-Qing Wang ◽  
Wei-Wei Ban ◽  
Zhi Chang ◽  
Hai-Zhu Chen ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Tumour Growth ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Surface Expression ◽  
Large B Cell Lymphoma ◽  
Large B Cell

AbstractLoss of CD20 is a major obstacle for the retreatment of relapsed/refractory diffuse large B cell lymphoma (DLBCL) with Rituximab-associated regimens. Histone deacetylation causes gene silencing and inhibits CD20 expression. Chidamide is a novel inhibitor for histone deacetylases (HDACs). We hypothesize that Chidamide could overcome Rituximab-mediated down-regulation of CD20 and facilitate Rituximab-induced killing. In this study, we determine the mechanism of synergy of Chidamide with Rituximab in DLBCL using in vitro and in vivo models. We found that the levels of CD20 protein surface expression on five DLBCL cell lines were significantly and positively correlated with the sensitivities of cells to Rituximab. Treatment with Rituximab significantly reduced CD20 surface expression at the protein levels. RNA sequencing showed that Chidamide significantly increased expression of more than 2000 transcriptomes in DLBCL cells, around 1000 transcriptomes belong to the cell membrane and cell periphery pathways, including MS4A1. Chidamide significantly increased CD20 surface expression in DLBCL cell lines. Combination with Chidamide significantly synergized Rituximab-induced cell death in vitro and significantly inhibited tumour growth in DLBCL-bearing xenograft mice. A patient with relapsed/refractory DLBCL achieved a complete response after three cycles combined treatment with Chidamide and Rituximab. In conclusion, our data demonstrate for the first time that inhibition of HDACs by Chidamide significantly enhanced Rituximab-induced tumour growth inhibition in vitro and in vivo. We propose that CD20 surface expression should be used clinically to evaluate treatment response in patients with DLBCL. Chidamide is a promising sensitizer for the retreatment of DLBCL with Rituximab.

scholarly journals Bio-Inspired Functional Lipoprotein-like Nanoparticles (Flip-NPs) Cause Cholesterol Starvation and Ferroptosis in B-Cell Lymphomas: Studies in Cell Lines, Xenograft Models and Primary Patient Samples

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2857-2857
Author(s):  
Jonathan Rink ◽  
Adam Yuh Lin ◽  
Shuo Yang ◽  
Amir Behdad ◽  
Reem Karmali ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cholesterol Reduction ◽  
Xenograft Models

Introduction: Hematologic malignancies, including B cell lymphomas such as diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), have increased demands for cholesterol and cholesteryl esters to maintain membrane anchored pro-proliferative and pro-survival signaling pathways, including B cell receptor signaling. Recent evidence suggests that certain cancer cell lines, including several anaplastic large T cell lymphoma (ALCL) cell lines, are auxotrophic for cholesterol and are sensitive to cholesterol reduction-induced ferroptosis (Garcia-Bermudez, Nature 2019), an iron dependent form of programmed cell death characterized by accumulation of lipid peroxides. We have developed a cholesterol depleting functional lipoprotein-like nanoparticle (Flip-NP) that specifically targets the high-affinity HDL receptor, scavenger receptor type B1 (SCARB1), which maintains cellular and cell membrane cholesterol homeostasis. Our prior data demonstrated that Flip-NPs induce B cell lymphoma cell death in vitro and in in vivo xenograft models. Accordingly, we hypothesized that the mechanism of cell death by Flip-NPs in B cell lymphomas is ferroptosis, and that Flip-NPs would be potent therapy for an expanded number of cholesterol-addicted malignancies, including ALCL. Methods: After informed consent, primary B cell lymphoma cells were isolated from excisional biopsies from patients with FL or DLBCL. The SUDHL4 [germinal center (GC) DLBCL], Ramos [Burkitt's lymphoma], SUDHL1 [ALCL] and SR-786 [ALCL] cell lines were used for in vitro experiments. SCARB1 expression was quantified using flow cytometry and western blot analysis. Cell viability was quantified using the MTS assay and flow cytometry. Ferroptosis was measured using the lipophilic antioxidant ferrostatin-1 or the iron chelator deferoxamine. Gene expression changes were quantified using RT-qPCR. Lipid peroxidation was measured using C11-BODIPY and flow cytometry. SUDHL1 and SUDHL4 flank tumor xenografts were initiated in SCID-beige mice, with Flip-NPs administered 3 times per week IV. Results: Primary B cell lymphoma cells were isolated from patients with FL (n=4) or DLBCL (n=2), and all samples expressed some level of SCARB1 by flow cytometry. Flip-NPs increased cell death in 3 of the 4 FL samples and 1 of 2 DLBCL samples. In Ramos and SUDHL4 cells, RT-qPCR data showed that Flip-NP-mediated cholesterol reduction led to up-regulation of cholesterol biosynthesis genes and down-regulation of glutathione peroxidase-4 (GPX4), a critical protein responsible for degradation of lipid peroxides. Correspondingly, as shown with C11-BODIPY, Flip-NP treatment increased lipid peroxide accumulation in Ramos and SUDHL4 cells. Addition of ferrostatin-1 or deferoxamine reduced Flip-NP induced cell death, demonstrating that the mechanism-of-action of Flip-NPs involves, at least in part, ferroptosis. Given the sensitivity of cholesterol auxotrophic cell lines to cholesterol reduction-induced ferroptosis, we tested the efficacy of the Flip-NPs against cholesterol auxotrophic ALK+ ALCL cell lines SUDHL1 and SR-786. SCARB1 was expressed in both cell lines. Flip-NPs potently induced cell death in both SUDHL1 and SR-786 cells in vitro. In vivo, systemic administration of Flip-NPs reduced tumor volumes in both SUDHL4 and SUDHL1 tumor xenograft models. Conclusions: Our data show that Flip-NPs reduce GPX4 expression and increase lipid peroxide accumulation in B cell lymphoma cell lines, resulting in ferroptosis. Expanding on these results, Flip-NP efficacy was also demonstrated in cholesterol auxotrophic ALK+ ALCL cell lines and primary patient-derived B cell lymphoma cells. These in vitro results translated to in vivo murine models, as systemic administration of Flip-NPs potently reduced DLBCL and ALK+ ALCL tumor xenograft burden. Flip-NPs are a molecularly targeted, first-in-class therapy that may be effective for malignancies reliant upon cellular cholesterol. Disclosures Behdad: Pfizer: Other: Speaker; Thermo Fisher: Membership on an entity's Board of Directors or advisory committees; Loxo-Bayer: Membership on an entity's Board of Directors or advisory committees. Karmali:Astrazeneca: Speakers Bureau; Takeda, BMS: Other: Research Funding to Institution; Gilead/Kite; Juno/Celgene: Consultancy, Speakers Bureau. Thaxton:Zylem: Other: Co-founder of the biotech company Zylem. Gordon:Juno/Celgene: Other: Advisory Board, Research Funding; Gilead: Other: Advisory Board; Bayer: Other: Advisory Board; Zylem LLC: Other: co-founder; research in nanoparticles in cancer.

scholarly journals Repurposing dasatinib for diffuse large B cell lymphoma

2019 ◽  
Vol 116 (34) ◽  
pp. 16981-16986 ◽  
Author(s):  
Claudio Scuoppo ◽  
Jiguang Wang ◽  
Mirjana Persaud ◽  
Sandeep K. Mittan ◽  
Katia Basso ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Multikinase Inhibitor ◽  
Large B Cell Lymphoma ◽  
Large B Cell

To repurpose compounds for diffuse large B cell lymphoma (DLBCL), we screened a library of drugs and other targeted compounds approved by the US Food and Drug Administration on 9 cell lines and validated the results on a panel of 32 genetically characterized DLBCL cell lines. Dasatinib, a multikinase inhibitor, was effective against 50% of DLBCL cell lines, as well as against in vivo xenografts. Dasatinib was more broadly active than the Bruton kinase inhibitor ibrutinib and overcame ibrutinib resistance. Tumors exhibiting dasatinib resistance were commonly characterized by activation of the PI3K pathway and loss of PTEN expression as a specific biomarker. PI3K suppression by mTORC2 inhibition synergized with dasatinib and abolished resistance in vitro and in vivo. These results provide a proof of concept for the repurposing approach in DLBCL, and point to dasatinib as an attractive strategy for further clinical development in lymphomas.

scholarly journals Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

2003 ◽  
Vol 77 (3) ◽  
pp. 2134-2146 ◽  
Author(s):  
Vicky M.-H. Sung ◽  
Shigetaka Shimodaira ◽  
Alison L. Doughty ◽  
Gaston R. Picchio ◽  
Huong Can ◽  
...  
Keyword(s):  
Hepatitis C ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Culture System ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Hcv Rna

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

HCD122, an Antagonist Human Anti-CD40 Monoclonal Antibody, Enhances Efficacy of CHOP in Tumor Xenograft Model of Human Diffuse Large B-Cell Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 528-528 ◽  
Author(s):  
Mohammad Luqman ◽  
Ssucheng J. Hsu ◽  
Matthew Ericson ◽  
Sha Klabunde ◽  
Seema Kantak
Keyword(s):  
Monoclonal Antibody ◽  
Clinical Trials ◽  
Tumor Growth ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract HCD122 (formerly known as CHIR-12.12), is a fully human anti-CD40 monoclonal antibody (mAb) currently in Phase I clinical trials for treatment of chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). An IgG1 antibody selected for its potency as an antagonist of the CD40 signaling pathway, HCD122 both inhibits CD40/CD40L-stimulated growth of lymphoma cells ex vivo, and mediates highly effective Antibody Dependent Cell-mediated Cytotoxicity (ADCC) in vitro. As a single agent, HCD122 exhibits potent anti-tumor activity in vivo, in preclinical models of MM, Hodgkin’s lymphoma, Burkitt’s lymphoma, mantle cell lymphoma and diffused large B-cell lymphoma (DLBCL). Although several therapeutic antibodies approved for treatment of Non-Hodgkin’s Lymphoma have clinical activity as single agents, combining these antibodies with standard-of-care chemotherapeutic regimens such as CHOP (cytoxan, vincristine, doxorubicin and prednisone) is proving optimal for both increasing response rates and extending survival, and antibodies currently in clinical development are likely to be used in combination therapies in the future. Therefore the studies reported here examine the effects of combining HCD122 with CHOP, the standard for treatment of high grade NHL, in in vitro and in vivo models of DLBCL. In the xenograft RL model of DLBCL, HCD122 administered intraperitoneally weekly at 1 mg/kg as a single agent, or in combination with CHOP (H-CHOP), and CHOP alone all significantly reduced tumor growth at day 25 when compared to treatment with huIgG1 control antibody (P<0.001). However, tumor growth delay (time to reach tumor size of 500 mm3) was significantly longer for H-CHOP (17.5 days), than for CHOP (8 days) or HCD122 (6 days) (p < 0.001). No toxicity was observed with the H-CHOP combination. Interestingly, at the end of the study (day 35), reduction in tumor growth was significantly greater in the treatment group that received H-CHOP than the groups that received either 10 mg/kg Rituxan plus CHOP (R-CHOP) (p < 0.05) or CHOP alone (p < 0.001). These data show that in this model, treatment with the combination H-CHOP results in greater anti-tumor efficacy than with either modality alone or R-CHOP. We have observed that in vitro, exposure to CD40 Ligand (CD40L) results in aggregation of DLBCL cells, and postulate that interfering with the ability of cancer cells to adhere and interact with each other and their microenvironment may potentiate the effect of chemotherapeutics. To elucidate the mechanism by which the combination of HCD122 and CHOP enhanced efficacy in vivo, we developed an in vitro system to examine the effects of HCD122 on the expression of adhesion molecules in the RL and SU-DHL-4 cell lines. In these studies, HCD122 inhibited CD40L-induced expression of CD54, CD86 and CD95 in both cell lines, as well as aggregation of SU-DHL-4 cells. The combined effect of each of the components of CHOP with HCD122 in three-dimensional spheroid cultures is currently under investigation. These data provide a therapeutic rationale for combination of HCD122 with CHOP in DLBCL clinical trials.

Constitutively Active STAT6 Represses BCL6 in Primary Mediastinal B Cell Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2417-2417
Author(s):  
Olga Ritz ◽  
Jochen K Lennerz ◽  
Karolin Rommel ◽  
Karola Dorsch ◽  
Elena Kelsch ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Ectopic Expression ◽  
B Cell Lymphoma ◽  
Proximal Promoter ◽  
Constitutively Active

Abstract Abstract 2417 Primary mediastinal B-cell lymphoma (PMBL) is a subtype of diffuse large B-cell lymphoma (DLBCL) that affects predominantly young women (Swerdlow et al. 2008). Despite improvements due to addition of rituximab, which has become state of the art treatment, 20% of PMBL patients succumb to disease progression or relapse. Notably, here are currently no registered trials that are actively recruiting PMBL-patients and a better understanding of the underlying pathobiology may identify novel therapeutic targets and provide an alternative to dose escalation (Steidl and Gascoyne 2011). BCL6 is a key germinal center B-cell transcription factor that suppresses genes involved in lymphocyte activation, differentiation, cell cycle arrest and DNA damage response gene. BCL6 is aberrantly expressed in certain DLBCL subgroups and BCL6 overexpression is sufficient for lymphomagenesis in mice (Cattoretti et al. 2005). In cellular- and murine DLBCL models, targeting of BCL6 via retroinverted BCL6 peptid inhibitor (RI-BPI) appears effective (Polo et al. 2004; Cerchietti et al. 2010). In conjunction with the relatively restricted expression pattern of BCL6, these data collectively suggest BCL6 as a candidate for targeted therapy in BCL6-positive lymphomas. Despite substantial work on BCL6 in lymphomas, the function of BCL6 in PMBL is unknown. To address the BCL6 function in PMBL, we performed BCL6 depletion by siRNA in all three available PMBL cell lines: K1106, U-2940 and MedB-1. We found that BCL6 acts pro-proliferative and anti-apoptotic; however, PMBL models were only partially dependent on and not addicted to BCL6. Given that BCL6 expression in all PMBL cell lines is variable with a notable fraction of BCL6-negative cells, we argued that increasing the fraction of BCL6-positive cells might increase the level of BCL6-dependence. Since IL-4/STAT6 signaling upregulates BCL6 in mouse lymphocytes (Schroder et al. 2002), we treated PMBL cell lines with IL-4 (or IL-13) and, as expected, observed increased phosphorylated (p)STAT6 levels. Surprisingly, the pSTAT6 increase was not associated with higher – but with drastically lower BCL6 protein levels. Moreover, in untreated cells, co-localization studies for pSTAT6- and BCL6 demonstrated staining in mutually exclusive subsets of cells (Figure 1A), suggesting negative interaction between BCL6 and pSTAT6. Other STAT family members were already shown to participate in the transcriptional regulation of BCL6. Thus, we examined binding of STAT6 to the proximal promoter of BCL6 in all PMBL cell lines using shift assay and chromatin immunoprecipitation. We found that STAT6 can bind all five GAS binding sites within the BCL6 promoter in vitro and in all PMBL cell lines STAT6 was bound to proximal BCL6 promoter in vivo. Furthermore, transient STAT6 depletion by siRNA and/or ectopic expression of constitutively active STAT6 confirms that pSTAT6 is sufficient for transcriptional repression of BCL6. Co-localization studies in primary patient samples demonstrated mutually exclusive BCL6/pSTAT6 distribution as a visual hallmark of the repression mechanism (Figure 1B, C). Thus, our data demonstrate for the first time that constitutively active STAT6 transcriptionally represses BCL6 in PMBL. In conjunction with functional data, the delineated repression mechanism may prevent addiction to one single oncogenic pathway (i.e. BCL6) in PMBL. Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Figure 1. Mutually exclusive distribution of BCL6 and pSTAT6 in PMBL Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lw-213 Synergizes with Rituximab to Inhibit Diffuse Large B-Cell Lymphomas By Upregulating CD20

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4366-4366
Author(s):  
Yuchen Li ◽  
Hui Li ◽  
Hui Hui ◽  
Jingyan Xu
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Cell Counting ◽  
Mrna Levels ◽  
Sox2 Expression ◽  
Cd20 Expression ◽  
Large B Cell

Abstract Background：Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens.Up to one-third of diffuse large B cell lymphoma (DLBCL) patients eventually develop resistance to R-CHOP regimen, since the remaining therapeutic options are limited. LW-213, a derivative of wogonin, is reported to possess antineoplastic properties in a variety of cancers, but whether it has effects on DLBCL is n-ot known. Studies have reported that upregulation of CD20 expression b-y either HDACi or silenced SOX2 expression showed sensitizing potential in Rituximab-induced cell death in malignant B cells. Our study was to explore whether LW-213 could sensitize DLBCL to Rixutimab thus improve therapeutic efficacy. Methods： Two DLBCL cell lines, RI-1 (ABC subtype) and Su-DHL -8 (GCB subtype), were used in our study. RI-1 and Su-DHL-8 cells were treated with LW-213 at different doses and for different times, and their proliferation and viability were detected by Cell counting kit-8 (CCK8).Flow cytometry was used to determine surface CD20 expression. Western blotting and q-PCR were applied to examine the protein and mRNA levels of CD20, SOX2, Ace-H3 and Ace-H3K27. CDC assay was used to evaluate the synergistic effects of LW-213 and Rixutimab. Results：We showed that LW-213 inhibited the proliferation of human DLBCL cell lines (Su-DHL-8、RI-1 ) in dose-and time-dependent manners with IC 50 values at the low μmol/L levels, meanwhile it potently inhibited primary lymphoma cells derived from peripheral blood of B-cel-l lymphoma patients. Furthermore, LW-213 significantly increased CD20 surface expression and the acetylation level of histone in DLBCL cell li-nes. Inversely，the SOX2 expression level remarkably decreased. Finally,Combination with LW-213 significantly synergized Rituximab-induced cell death in vitro. Conclusion： The results demonstrate that LW-213 sensitizes DLBCL cells to Rituximab in vitro by upregulating CD20 expression and the SOX2/ace-H3K27/ace-H3 axis may plays a critical role in CD20 upregulation processing. Even though this strategy is important in vitro models,the upregulating CD20 expression therapy against DLBCL proposed in this study warrants further study in vivo and clinical trials . Keywords：CD20 DLBCL Rituximab SOX2 Histone Deacetylation Disclosures No relevant conflicts of interest to declare.

scholarly journals Combining the IAP Antagonist Tolinapant with a DNA Hypomethylating Agent Enhances Immunogenic Cell Death in Preclinical Models of T-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3986-3986
Author(s):  
George A. Ward ◽  
Simone Jueliger ◽  
Martin Sims ◽  
Matthew Davis ◽  
Adam Boxall ◽  
...  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Cell Lines ◽  
Western Blotting ◽  
Inflammatory Mediators ◽  
Cell Lymphoma ◽  
T Cell Lymphoma ◽  
Immunogenic Cell Death

Abstract Introduction: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. A Phase 1 clinical study investigating the combination of tolinapant and ASTX727 (oral decitabine) in AML is currently in progress (NCT04155580). Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death (ICD), such as necroptosis, by rational combination of our clinical compounds in pre-clinical models of T-cell lymphoma (TCL). Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key apoptosis, necroptosis or pyroptosis biomarkers were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. RIPK3- or MLKL-knockout cell lines were generated by CRISPR to demonstrate involvement of necroptosis in drug-induced cell death in a T-cell lymphoma cell line (BW5147.G.1.4) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key inflammatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma by Luminex assay (Ampersand). Results: Combined treatment of tolinapant and decitabine led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating on-target activity of tolinapant and decitabine, respectively. The combination of tolinapant and decitabine acted synergistically in mouse and human T-cell lymphoma cell lines to reduce viability in proliferation assays. Necroptosis was induced by decitabine or tolinapant alone in mouse TCL cell lines with robust activation of the RIPK1/RIPK3/MLKL necroptosis pathway when caspase activity was inhibited, and the combination of both agents enhanced loss of viability. Furthermore, we demonstrated decitabine treatment led to re-expression of both RIPK3 and MLKL in mouse cell lines, supporting published evidence that methylation can silence these key biomarkers (Koo et al., Cell Research, 2015; Koch et al., Neoplasia, 2021). Enhanced release of chemokine, cytokine and DAMPs was demonstrated with the combination of agents in vitro and in vivo. By removal of key necroptosis pathway components using CRISPR, we confirmed the importance of this lytic cell death pathway by demonstrating that RIPK3 -/- and MLKL -/- T-cell lymphoma (BW5147.G.1.4) cell lines had reduced necroptosis potential after treatment with tolinapant or decitabine alone or in combination; and demonstrate reduced release of inflammatory mediators in vitro. Finally, our in vivo evaluation of the combination of agents in mouse syngeneic models suggested that increased anti-tumour activity and immune-potentiating systemic biomarker modulation can be achieved with a tolerated dosing regimen of both compounds. Conclusion: These data demonstrate that decitabine enhances immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. This finding provides strong rationale to explore this combination clinically. Disclosures Sims: Astex Pharmaceuticals: Current Employment. Davis: Astex Pharmacueticals: Current Employment. Smyth: Astex Pharmaceuticals: Current Employment.

scholarly journals Targeting Positive Cofactor 4 induces autophagic cell death in MYC-expressing diffuse large B cell lymphoma

2021 ◽  
Author(s):  
Le Ma ◽  
Qiang Gong ◽  
Zelin Chen ◽  
Yu Wang ◽  
Xu Tan ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Autophagic Cell Death ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background: The MYC-expressing diffuse large B-cell lymphoma (DLBCL) is one of the refractory lymphomas. The pathogenesis of MYC-expressing DLBCL is still unclear, and there is a lack of effective therapy. In this study, we have explored the clinical significance and the molecular mechanisms of transcription co-activator 4 (PC4) in MYC-expressing DLBCL.Methods: We investigated PC4 expression in 54 cases of DLBCL patients’ tissues and matched normal specimens, and studied the molecular mechanisms of PC4 in MYC-expressing DLBCL both in vitro and in vivo.Results: We reported for the first time that targeting c-Myc could induce autophagic cell death in MYC-expressing DLBCL cell lines. We next characterized that PC4 was an upstream regulator of c-Myc, and PC4 was overexpressed in DLBCL and was closely related to clinical staging, prognosis and c-Myc expression. Further, our in vivo and in vitro studies revealed that PC4 knockdown could induce autophagic cell death of MYC-expressing DLBCL. And inhibition of c-Myc mediated aerobic glycolysis and activation of AMPK / mTOR signaling pathway were responsible for the autophagic cell death induced by PC4 knockdown in MYC-expressing DLBCL. Through the CHIP, DLRTM and EMSA assay, we also found that PC4 exerted its oncogenic functions by directly binding to c-Myc promoters.Conclusions: PC4 exerts its oncogenic functions by directly binding to c-Myc promoters. Inhibition of PC4 can induce autophagic cell death of MYC-expressing DLBCL. Our study provides novel insights into the functions and mechanisms of PC4 in MYC-expressing DLBCL, and suggests that PC4 might be a promising therapeutic target for MYC-expressing DLBCL.

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