Human Burkitt Lymphoma Cell Research Articles

Sodium diethyldithiocarbamate trihydrate: an effective and selective compound for hematological malignancies.

Myeloid leukemias and lymphomas are among the most common and well-studied hematological malignancies. However, due to the aggressiveness and rapid progression of certain subtypes, treating these diseases remains a challenge. Considering the promising results of diethyldithiocarbamates in preclinical and clinical oncology trials, this study aimed to investigate the potential of sodium diethyldithiocarbamate trihydrate (DETC) as a prototype for developing new drugs to treat hematological malignancies. In silico analysis using SwissADME was conducted to evaluate the physicochemical characteristics and pharmacokinetic properties of DETC. An in vitro investigation utilizing the MTT assay assessed the cytotoxic effects of DETC on neoplastic and non-neoplastic cell lines. Selectivity was determined using a selectivity index and a hemolysis assay, while the mechanism of cell death in neoplastic cell lines was examined through flow cytometry analysis of pro-apoptotic and anti-apoptotic protein levels. The results demonstrated that the physicochemical characteristics of DETC are suitable for oral administration. Furthermore, the compound showed promising cytotoxic activity against human myeloid leukemia (K562) and Burkitt's lymphoma (Daudi) cell lines, with high selectivity for neoplastic cells over non-neoplastic cells of the bone marrow microenvironment (HS-5 cell line). Moreover, hemolysis was observed only at very high concentrations. The cytotoxicity mechanism of DETC against both neoplastic cell lines involved cell cycle arrest and the production of reactive oxygen species. In K562 cells, cell death was induced via apoptosis. Additional experiments are needed to confirm the exact mechanism of cell death in Daudi Burkitt's lymphoma cells.

Comparable efficacy of oral bendamustine versus intravenous administration in treating hematologic malignancies.

The purpose of this study was to analyze potential differences in antitumor efficacy and pharmacokinetics between intravenous (IV) bendamustine and a novel orally administered (PO) bendamustine agent that is utilizing the beneficial properties of superstaturated solid dispersions formulated in nanoparticles. Pharmacokinetics of IV versus PO bendamustine were determined by analysis of plasma samples collected from NSG mice treated with either IV or PO bendamustine. Plasma samples were analyzed using liquid chromatography-mass spectrometry following a liquid-liquid extraction to determine peak bendamustine concentration, area under the concentration-time curve, and the half-life in-vivo. In-vitro cytotoxicity of bendamustine against human non-Hodgkin Burkitt's Lymphoma (Raji), multiple myeloma (MM.1s), and B-cell acute lymphoblastic leukemia (RS4;11) cell lines was determined over time using MTS assays. Luciferase-tagged versions of the aforementioned cell lines were used to determine in-vivo bendamustine cytotoxicity of IV versus PO bendamustine at two different doses. Bendamustine at a high dose in-vitro causes cell death. There was no significant difference in antitumor activity between IV and novel PO bendamustine at a physiologically relevant concentration in all three xenograft models. In-vivo pharmacokinetics showed the oral bioavailability of bendamustine in mice to be 51.4%. The novel oral bendamustine agent tested exhibits good oral bioavailability and systemic exposure for in-vivo antitumor efficacy comparable to IV bendamustine. An oral bendamustine formulation offers exciting clinical potential as an additional method of administration for bendamustine and warrants further evaluation in clinical studies.

Effect of Celastrus Orbiculatus Extract on proliferation and apoptosis of human Burkitt lymphoma cells.

The lymphoma incidence rate is on the rise, with invasive forms particularly prone to relapse following conventional treatment, posing a significant threat to human life and wellbeing. Numerous studies have shown that traditional Chinese botanical drug medicine offers promising therapeutic benefits for various malignancies, with previous experimental findings indicating that Celastrus orbiculatus extract effectively combats digestive tract tumors. However, its impact on lymphoma remains unexplored. This study aims to investigate the impact and underlying mechanisms of COE on the proliferation and apoptosis of Burkitt lymphoma cells. We diluted COE in RPMI-1640 medium to create various working concentrations and introduced it to human Burkitt lymphoma Raji and Ramos cells. To evaluate cell viability, we used the CCK-8 assay, and we observed morphological changes using HE staining. We also conducted Annexin V-PI and JC-1 staining experiments to assess apoptosis. By combining the cell cycle experiment with the EDU assay, we gained insights into the effects of COE on DNA replication in lymphoma cells. Using Western blotting, we detected alterations in apoptosis-related proteins. In vivo experiments revealed that following COE intervention, tumor volume decreased, survival time was prolonged, spleen size reduced, and the expression of tumor apoptosis-related proteins changed. Our findings indicate that COE effectively inhibits lymphoma cell proliferation and promotes apoptosis by regulating these apoptosis-related proteins.

SMARCA4 is a haploinsufficient B cell lymphoma tumor suppressor that fine-tunes centrocyte cell fate decisions

SMARCA4 encodes one of two mutually exclusive ATPase subunits in the BRG/BRM associated factor (BAF) complex that is recruited by transcription factors (TFs) to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ∼30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperated with MYC over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of TFs that are activated in centrocytes to drive GC-exit, including SPI1 (PU.1), IRF family, and NF-κB. Loss of activity for these factors phenocopied aberrant BCL6 activity within murine centrocytes and human Burkitt lymphoma cells. SMARCA4 therefore facilitates chromatin accessibility for TFs that shape centrocyte trajectories, and loss of fine-control of these programs biases toward centroblast cell-fate, GC hyperplasia and lymphoma.

PICH deficiency limits the progression of MYC-induced B-cell lymphoma

Plk1-interacting checkpoint helicase (PICH) is a DNA translocase involved in resolving ultrafine anaphase DNA bridges and, therefore, is important to safeguard chromosome segregation and stability. PICH is overexpressed in various human cancers, particularly in lymphomas such as Burkitt lymphoma, which is caused by MYC translocations. To investigate the relevance of PICH in cancer development and progression, we have combined novel PICH-deficient mouse models with the Eμ-Myc transgenic mouse model, which recapitulates B-cell lymphoma development. We have observed that PICH deficiency delays the onset of MYC-induced lymphomas in Pich heterozygous females. Moreover, using a Pich conditional knockout mouse model, we have found that Pich deletion in adult mice improves the survival of Eμ-Myc transgenic mice. Notably, we show that Pich deletion in healthy adult mice is well tolerated, supporting PICH as a suitable target for anticancer therapies. Finally, we have corroborated these findings in two human Burkitt lymphoma cell lines and we have found that the death of cancer cells was accompanied by chromosomal instability. Based on these findings, we propose PICH as a potential therapeutic target for Burkitt lymphoma and for other cancers where PICH is overexpressed.

TGF-β Inhibitor SD-208 Enhances the Anti-Tumor Efficacy of CD19 CAR-T Cells through a Reduction of CAR-Treg in Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is an incurable hematological malignancy with poor prognosis. Chimeric antigen receptor T cells (CAR-T) therapy is a milestone in the immunotherapy of relapsed or refractory B cells ALL (r/r B-ALL), but most cases are inevitably relapsed after CAR-T therapy and the causes are complex. Our preliminary work, a phase II clinical trial (#NCT02735291) involving pediatrics and adults with r/r B-ALL who were treated with CD19 CAR-T, exhibited that the overall survival (OS) and relapse-free survival (RFS) in patients with higher Treg are lower, and higher Treg may preclude the efficacy of CAR T therapy in r/r B-ALL. Further analysis of the frequencies of Treg within CD4 + cells at CD19 CAR-T therapy in r/r B-ALL patients investigated that higher circulating Treg, especially 1 week after CD19 CAR-T cells infusion, was a poor predict indicator for CD19 CAR-T therapy in r/r B-ALL patients. Methods: In the present study, we investigated the significance of CAR-Treg in CD19 CAR-T cells in clinical response and prognosis, and explored an inhibitive effect of TGF-β inhibitor SD-208 on the proliferation and lethality of CAR-Treg. Regulatory T cells (Treg) in CD19 CAR-T cells were tested in 34 patients with r/r B-ALL received CD19 CAR-T cells therapy, and then its significance on the complete remission (CR) rate, relapse-free survival (RFS) and overall survival (OS) was validated respectively. Next, TGF-β inhibitor SD-208 was added or not added in the culturing protocols of CD19 CAR-T cells, then the proportion of Treg and CAR-Treg as well as the anti-tumor ability of CD19 CAR-T cells were analyzed and compared in vitro. Results: 1) Higher proportion of Treg (≥7.2%) in CAR-T cells was a negative factor affecting RFS (p=0.0120) and OS (p=0.0162) in the r/r B-ALL patients with CAR-T therapy. 2) CD4 +CD25 +CD127 low/- Treg proliferated from 7.01% to 25.26% after anti-CD3 and anti-CD28 Abs activation. PBMCs cultured with SD-208 exhibited lower Treg levels than those of control groups (15.73% vs. 29.08%). 3) CD4 +CD25 +CD127 low/- Treg levels stimulated by SD-208 in CD19 CAR-T cells were lower than those in control groups both 24 and 48 hours (38.68% vs. 40.47% in 24 hours, 23.58% vs. 36.19% in 48 hours), and the treatment for 48 hours was most obvious. CAR +CD4 +CD25 +CD127 low/- CAR-Treg levels in CD19 CAR-T cells decreased by SD-208 stimulating compared with controls especially 48 hours (35.07% vs. 37.31% in 24 hours, 20.94% vs. 34.41% in 48 hours). The results exhibit that about 90% Treg in CD19 CAR-T cells are CAR-Treg, and inhibiting TGF-β by TGF-β/SMAD inhibitor SD-208 reduce the proliferation of Treg especially CAR-Treg in CD19 CAR-T cells. 4) To determine the function to CD19 CAR-T cell, LDH activity was utilized for tumor cell damage assay. Human Burkitt's lymphoma cell line Raji (CD19 +) was co-cultured with CD19 CAR-T cell for 24 hours before LDH assay. The percent of cytotoxicity calculated depending on the LDH activities showed that the inhibition rate of SD-208 treated CD19 CAR-T cell was significantly higher than that of control. To further determine the function of CD19 CAR-T cell Granzyme B and Perforin were tested. The results exhibited that SD-208 treated CD19 CAR-T cell had higher rates of Granzyme B and Perforin in CD3 +, CD3 +CD4 +, CD3 +CD8 + cells than controls, and achieved more functional. Conclusions: Inhibition of TGF-β by TGF-β/SMAD inhibitor SD-208 inhibit the proliferation of Treg in PBMCs of r/r B-ALL patients. TGF-β inhibitor SD-208 might improve the anti-tumor ability of CD19 CAR-T cells via decreasing Treg and CAR-Treg in r/r B-ALL.

DIAGNOSTIC YIELD OF IN VITRO VITREOUS BIOPSY FOR INTRAOCULAR LYMPHOMA AT VARIABLE VITREOUS CUTTER SPEEDS USING 25-GAUGE VITRECTOMY.

Primary vitreoretinal lymphoma is the most common intraocular lymphoproliferative disorder. We evaluated the diagnostic yield of pars plana vitrectomy, specifically using modern high cut rate dual-cycle cutters, on in vitro cell viability and diagnostic yield. Human Burkitt lymphoma cell line Namalwa at 2 x 10^5 cells/mL was aspirated by 25-gauge dual-blade guillotine-type vitrectomy at five speeds (500, 1,000, 4,000, 7,500, or 15,000 cuts per minute). Cell viability and diagnostic yield in each subtype group were determined using hemocytometry, viable cell count using Cell Counting Kit-8, and pathologist-guided manual count. No significant deviation in cell count was identified in any cut rate by ANOVA ( P = 0.61), and no trends in the number of viable cells were identified across cut rates (R 2 = 0.188, P = 0.47). Among histologic cell counts per cut-rate, neither linear regression (R = 0.531, P = 0.16) nor ANOVA ( P = 0.096) were statistically significant. There was no significant degradation in the number of viable cells with increasing cut speed. These results suggest that in contrast to previous findings using 20g or 23g vitrectomy for diagnostic vitrectomy, modern vitrectomy systems may be used at up to 15,000 cpm without compromising the viability of lymphoma cells.

Efficacy of Antibodies Targeting TfR1 in Xenograft Mouse Models of AIDS-Related Non-Hodgkin Lymphoma.

Transferrin receptor 1 (TfR1), also known as CD71, is a transmembrane protein involved in the cellular uptake of iron and the regulation of cell growth. This receptor is expressed at low levels on a variety of normal cells, but is upregulated on cells with a high rate of proliferation, including malignant cells and activated immune cells. Infection with the human immunodeficiency virus (HIV) leads to the chronic activation of B cells, resulting in high expression of TfR1, B-cell dysfunction, and ultimately the development of acquired immunodeficiency syndrome-related B-cell non-Hodgkin lymphoma (AIDS-NHL). Importantly, TfR1 expression is correlated with the stage and prognosis of NHL. Thus, it is a meaningful target for antibody-based NHL therapy. We previously developed a mouse/human chimeric IgG3 specific for TfR1 (ch128.1/IgG3) and showed that this antibody exhibits antitumor activity in an in vivo model of AIDS-NHL using NOD-SCID mice challenged intraperitoneally with 2F7 human Burkitt lymphoma (BL) cells that harbor the Epstein-Barr virus (EBV). We have also developed an IgG1 version of ch128.1 that shows significant antitumor activity in SCID-Beige mouse models of disseminated multiple myeloma, another B-cell malignancy. Here, we aim to explore the utility of ch128.1/IgG1 and its humanized version (hu128.1) in mouse models of AIDS-NHL. To accomplish this goal, we used the 2F7 cell line variant 2F7-BR44, which is more aggressive than the parental cell line and forms metastases in the brain of mice after systemic (intravenous) administration. We also used the human BL cell line JB, which in contrast to 2F7, is EBV-negative, allowing us to study both EBV-infected and non-infected NHL tumors. Treatment with ch128.1/IgG1 or hu128.1 of SCID-Beige mice challenged locally (subcutaneously) with 2F7-BR44 or JB cells results in significant antitumor activity against different stages of disease. Treatment of mice challenged systemically (intravenously) with either 2F7-BR44 or JB cells also showed significant antitumor activity, including long-term survival. Taken together, our results suggest that targeting TfR1 with antibodies, such as ch128.1/IgG1 or hu128.1, has potential as an effective therapy for AIDS-NHL.

Physicochemical Characterization and Antitumor Activity of Fucoidan and Its Degraded Products from Sargassum hemiphyllum (Turner) C. Agardh.

Fucoidan has many biological functions, including anti-tumor activity. Additionally, it has been suggested that low-molecular-weight fucoidans have greater bioactivities. This study aimed to examine the degradation, purification, physicochemical characterization and in vitro antitumor activity of fucoidan from Sargassum hemiphyllum (Turner) C. Agardh. Fucoidan was isolated using DEAE-cellulose-52 (F1, F2), Vc-H2O2 degration, and Sepharose CL-6B gel (DF1, DF2) from crude Sargassum fucoidans. Physicochemical characteristics of four isolated fucoidans were examined using chemical and monosaccharide composition, average molecular weight (Mw), and FTIR. Furthermore, the anti-proliferative effects of purified fucoidans on human hepatocellular carcinoma cells (HepG2), human Burkitt Lymphoma cells (MCF-7), human uterine carcinoma cells (Hela) and human lung cancer cells (A549) were analyzed by MTT method. The apoptosis of HepG2 cells was detected by flow cytometry. Our data suggest that the contents of polysaccharide, L-fucose and sulfate of DF2 were the highest, which were 73.93%, 23.02% and 29.88%, respectively. DF1 has the smallest molecular weight (14,893 Da) followed by DF2 (21,292 Da). The four fractions are mainly composed of fucose, mannose and rhamnose, and the infrared spectra are similar, all of which contain polysaccharide and sulfate characteristic absorption peaks. The results of MTT assay showed that the four fractions had inhibitory effects on HepG2 and A549 in the range of 0.5-8 mg/mL, and the four fractions had strong cytotoxic effects on HepG2 cells. DF2 had the best inhibitory effect on HepG2 (IC50 = 2.2 mg/mL). In general, the antitumor activity of Sargassum fucoidans is related to the content of L-fucose, sulfate and molecular weight, and Sargassum fucoidan has the best inhibitory effect on HepG2 hepatocellular carcinoma cells. Furthermore, when compared to MCF-7, Hela, and A549 cells, Sargassum fucoidans had the best capacity to reduce the viability of human hepatocellular carcinoma cells (HepG2) and to induce cell apoptosis, proving itself to have a good potential in anti-liver cancer therapy.

Evaluation of potential cytotoxic and apoptotic effects of paclitaxel and docetaxel in human Burkitt lymphoma cell lines

Purpose: To study the cytotoxicity of paclitaxel and docetaxel on cell viability and apoptosis in Burkitt lymphoma cell lines.
 Methods: The cytotoxic activity of Paclitaxel and Docetaxel against Burkitt lymphoma cells was evaluated using the XTT cell viability test. The cells were exposed to increasing concentrations of paclitaxel and docetaxel ranging from 0.0001 to 10 μM for 48 h. Cell cycle analysis and apoptosis were also assessed using flow cytometry-based experiments.
 Results: Both paclitaxel and docetaxel exhibited a concentration-dependent cytotoxic effect with IC50 of 5.32 and 6.58 μM, respectively. Furthermore, paclitaxel and docetaxel were shown to have a significant apoptotic effect on Burkitt lymphoma cells (p < 0.01). Furthermore, paclitaxel and docetaxel arrested Burkitt lymphoma cells at the G2/M phase.
 Conclusion: Paclitaxel and docetaxel are potential candidates for the treatment of Burkitt lymphoma. Since both drugs are already licensed for many indications, the addition of Burkitt lymphoma to the list of indications of these drugs would be a much easier and faster process. However, there is a need for further in vitro and in vivo research to fully elucidate their mechanisms of action.

Lymphoma cell-derived extracellular vesicles inhibit autophagy and apoptosis to promote lymphoma cell growth via the microRNA-106a/Beclin1 axis

ABSTRACT Lymphoma is a common malignant tumor globally. Tumor-derived extracellular vesicles (Evs) participate in genetic information exchange between tumor cells. We investigated the role and mechanism of human Burkitt lymphoma cells Raji-derived Evs (Raji-Evs) in lymphoma cells. Effects of Evs on lymphoma cell proliferation, invasion, autophagy, and apoptosis were assessed using Cell Counting Kit-8 method, Transwell assay, laser confocal microscopy, Western blotting, and flow cytometry. microRNA (miR)-106a expression in lymphoma cells was determined using reverse transcription-quantitative polymerase chain reaction and then downregulated in Raji cells and then Evs were isolated (Evs-in-miR-106a) to evaluate its role in lymphoma cell growth. The binding relationship between miR-106a and Beclin1 was verified using RNA pull-down and dual-luciferase assays. Beclin1 was overexpressed in SU-DHL-4 and Farage cells and SU-DHL-4 cell autophagy and apoptosis were detected. The levels of miR-106a and Beclin1 in SU-DHL-4 cells were detected after adding autophagy inhibitors. The tumorigenicity assay in nude mice was performed to validate the effects of Raji-Evs in vivo. Raji-Evs promoted lymphoma cell proliferation and invasion and increased miR-106a. miR-106a knockdown reversed Evs-promoted lymphoma cell proliferation and invasion. miR-106a carried by Raji-Evs targeted Beclin1 expression. Beclin1 overexpression or miR-106a inhibitor reversed the effects of Evs on lymphoma cell autophagy and apoptosis. Autophagy inhibitors elevated miR-106a expression and lowered Beclin1 expression. Raji-Evs-carried miR-106a inhibited Beclin1-dependent autophagy and apoptosis in lymphoma cells, which were further verified in vivo, together with promoted tumor growth. We proved that Raji-Evs inhibited lymphoma cell autophagy and apoptosis and promoted cell growth via the miR-106a/Beclin1 axis.

Phosphoglycerate Dehydrogenase Is Required for Germinal Center Formation and Is a Therapeutic Target in MYC-driven Lymphoma

The fields of cancer- and immuno-metabolism have re-emerged as areas of significant translational potential. Even though the upregulation of glycolysis by proliferating lymphocytes is the basis for widely used clinical tests such as FDG-PET, little is known about which metabolic pathways are involved in the utilization of glucose to support B-cell proliferation. The synthesis of serine from glucose has been demonstrated to be a key metabolic pathway supporting cellular proliferation in some healthy and malignant cell types. Importantly, this pathway is regulated by MYC, which is known to be essential for germinal centre formation and is commonly dysregulated in lymphoma. Despite this, the role that the serine synthesis pathway (SSP) plays in germinal center biology and pathology has not been previously investigated. We performed a comprehensive characterization of the role of the SSP in germinal center B cells and lymphomas derived from these cells.We demonstrate that upregulation of a functional SSP is a metabolic hallmark of B-cell activation and the germinal center reaction. We show that both human and murine resting naïve B cells lack expression of the first two enzymes in this pathway, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) enzymes. However, B-cell activation, predominantly through the B-cell receptor, robustly induces the expression of these enzymes in vitro, resulting in an acquired ability to synthesize serine, glycine and the purine nucleotides adenosine and guanosine from glucose. This is reflected in striking expression of PHGDH and PSAT1 within germinal centers but not in marginal zones confirming that this upregulation is occurring in germinal B cells activated in vivo. We then proceeded to investigate the impact of inhibiting PHGDH on germinal center formation and high-affinity antibody production in vivo. This was done both genetically, using a conditional B-cell knockout mouse model, and pharmacologically using a specific inhibitor of PHGDH, PH-755. Importantly, we show that PHGDH inhibition impairs germinal center formation with a resultant reduction in high-affinity antibody production. Mechanistic experiments demonstrate that PHGDH inhibition effectively blocks cells from synthesising serine and glycine from glucose, making them unable to proliferate in environments that lack these amino acids.We then investigated role of PHDGH and PSAT1 in Burkitt Lymphoma (BL), Diffuse Large B Cell Lymphoma (DLBCL) and Chronic Lymphocytic Leukemia (CLL). Notably, very high expression of these two proteins was observed in BL, with intermediate-to-high expression in DLBCL and relatively low expression in CLL, where expression was restricted to proliferation centers. Given the heterogeneity of expression in DLBCL patients, we next interrogated a published GSE database (Lenz et al. NEJM 2008) to investigate the impact on outcome. Notably high expression of PSAT1 was significantly associated with a poorer overall survival rate in DLBCL.We then investigated whether the SSP could be a therapeutic target in lymphoma. We demonstrate that PHGDH inhibition effectively inhibits de novo serine, glycine and purine nucleotide synthesis from glucose, resulting in impaired proliferation and increased apoptosis in a panel of human BL cell lines in vitro. We then analyzed the impact of PHGDH inhibition on lymphoma development in vivo using Eµ-myc mice, which harbour Myc coupled to the IgH enhancer characteristic of BL. Importantly we confirm the role of MYC by demonstrating that Eµ-Myc B-cells show significantly higher expression of PHGDH and PSAT1 expression resulting in increased serine and glycine synthesis when compared to control cells. We demonstrate that pharmacological inhibition of PHGDH using PH-755 impairs lymphoma progression in this model. We confirm the importance of PHGDH by showing that genetic ablation of Phgdh in Eµ-myc cells in a tamoxifen inducible system (using Eµ-myc/+;Rosa26-CreER T2/+;Phgdh fl/fl mice) also results in a significant reduction in lymphoma progression.Taken together, this work represents the first report of the role of the SSP in the biology of the germinal centre response and lymphomas derived from these cells. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in MYC-driven lymphoma, which is an area of significant unmet need. DisclosuresGribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria. Calado: Myricx Pharma: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties: Cancer Treatments. WO patent WO 2020/128475 A1 (2020).

Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma

AbstractThe expression of BCL6 in B-cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions, or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase that is involved in the degradation of BCL6, and it is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of patients with Burkitt lymphoma (BL). FBXO11 mutations impaired BCL6 degradation, and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of 1 or 2 copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B-cell lymphoma onset, providing experimental evidence that FBXO11 is a haploinsufficient oncosuppressor in B-cell lymphoma. In wild-type and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degraders or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL, we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches, such as BCL6 degraders and direct MYC inhibition, could be exploited as a targeted therapy for BL.

Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt's lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sμ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.

The Effect of the MK0339 Neutrophil Elastase Inhibitor on Proliferation and Maturation of iPSC Derived from Patients with ELANE, TCIRG1 and Whim Syndrome Associated Neutropenia

Genetic neutropenias are heterogeneous group of hematological diseases characterized by impaired survival and maturation of myeloid precursors in bone marrow. G-CSF and HSCT are currently the only effective treatment options. MK-0339 is a potent, cell permeable, orally absorbed inhibitor of neutrophil elastase (NE), previously investigated in preclinical and clinical studies by Merck/DuPont as a potential anti-inflammatory drug. We have previously reported that MK-0339 increases neutrophil proliferation in cellular models of ELANE associated neutropenia (2016 [ASH Annual Meeting Abstracts] Blood. 2016;128:406; 2017 J Leuk Biol [in press]). To examine the specificity of these responses, we investigated the effects of MK-0339 on iPSCs from healthy volunteers and compared its effects on differentiation of iPSC derived from patients with ELANE, TCIRG-1 and CXCR4 associated neutropenia.We generated induced pluripotent stem cells (iPSC) from dermal fibroblasts from healthy volunteers and neutropenic patients harboring ELANE, TCIRG1 and CXCR4 mutations and developed a feeder free, reproducible and efficient protocol for myeloid differentiation.In our initial experiments, we observed that iPSC lines derived from patients with ELANE mutations (P139L and G214R) consistently grew much slower and formed much smaller colonies than the controls using both mTeSR1/matrigel and DEF-CS feeder free culture medium systems. We observed more than 3-fold growth reduction of patient derived lines compared to healthy volunteers.We next tested the effects of MK-0339 under the same culture conditions. Growth of control and ELANE patient cell lines was enhanced, and the cells appeared more robust, after addition of 1uM MK-0339. We were surprised to see a similar proliferative effect (~40% increase) with Ramos (human Burkitt's lymphoma cell line) cells cultured in the presence of 1uM MK-0339, although neither the Ramos cells nor the ELANE iPSCs had detectable levels of ELANE expression. The enhanced growth of control and patient iPSCs and the results with Ramos cells suggested that the effects of MK-0339 are not solely mediated via inhibition of NE.We next compared myeloid differentiation and CD34+ cell proliferation of iPSC from patients with ELANE, TCIRG1 and CXCR4 associated neutropenia in HPC expansion medium, using granulocytic differentiation markers and flow cytometry. Myeloid maturation was impaired and inefficient for both ELANE (P139L and G214R) patient derived cell lines. Myeloid differentiation of the TCIRG1 patient derived was also impaired. Introduction of MK-0339 restored the impaired myeloid differentiation capacity of both ELANE and TCIRG1 positive patient derived cell lines. An iPSC line derived from a WHIM (CXCR4 mutation) patient appeared to have normal maturation and proliferation. Western blot analysis revealed elevated levels of endoplasmic reticulum chaperone GRP78/BiP in ELANE and TCIRG1 mutant cell lines which was corrected by MK-0339 treatment.These studies demonstrate heretofore unreported proliferative effects of MK-0339 on cell lines not expressing neutrophil elastase. They suggest important mechanisms beyond enzyme inhibition to account for these findings. Although preliminary, we believe that this work suggests that MK-0339 may be a novel, oral therapy for both ELANE and non-ELANE associated neutropenia. DisclosuresDale:University of Washington: Employment, Research Funding; National Institutes of Health: Research Funding; WedMD/Medscape: Membership on an entity's Board of Directors or advisory committees; Wolter Kluwer: Other: Editor, Current Opinions in Hematology; Philips: Research Funding; X4Pharma: Research Funding; Shire: Other: Independent Review Board; Omeros: Other: DSMB; Coherus: Consultancy; Boheringer-Ingelheim: Consultancy; Prolong: Consultancy; Hospira: Consultancy; Genkyotex: Other: DSMB (work completed 6/2015); Genzyme (now owned by Sanofi-Aventis): Consultancy, Patents & Royalties, Research Funding; Cellerant: Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Consultancy, Editor, Current Opinions in Hematology, Honoraria; Amgen: Consultancy, Research Funding; American College of Physicians: Other: Editor and author; GlaxoSmithKline: Equity Ownership; Johnson&Johnson: Equity Ownership.

RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production.

Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.

Enhanced electrochemiluminescence ratiometric cytosensing based on surface plasmon resonance of Au nanoparticles and nanosucculent films

Ramos cells are human Burkitt's lymphoma cells, which are a kind of cancer cells to facilitate the monitoring of the relevant biological processes of cancers. Sensitive and accurate detection of Ramos cells using emerging ratiometric ECL biosensing technology shows increasing importance, however, the target analytes of current ratiometric ECL biosensors are mainly limited to DNA/RNA or proteins. In this study, we proposed a dual-potential ratiometric sensing strategy for the electrochemiluminescence detection of Ramos cells based on two types of electrochemiluminescence (ECL)-responding molecular. Au nanosucculent films (AuNFs) were electrodeposited on the fluorine doped tin oxide (FTO) electrode to increase the effective area of the electrode for more efficient assembly of DNA and effectively improving the conductivity of the sensing interfaces. In the presence of Ramos cells, aptamers capped with Au@luminol would conjugate with Ramos cells and then remove from AuNFs, accompanying the decrease of ECL signal from Au@luminol. Then, Au–DNA was captured and alternately hybridized with DNA-modified CdS nanocrystals (NCs) on the surface of AuNFs with the formation of a super reticulate structure. The reticulate structure not only raised another identified ECL signal from CdS NCs but also greatly promoted its ECL intensity from the surface plasmon resonance originating from Au NPs. The value of log (ECLCdS/ECLluminol) and the logarithm of the number of cells exhibit considerable linear relation ranging from 80 to 8 × 105 cells mL−1 with a low detection limit of 20 cells mL−1 (S/N = 3). The selectivity and specificity of this dual-potential ECL sensor showed good performance and indicated considerable promise in avoiding false-positive results in detection.

Effect of Autophagy to Drug Resistance of B-cell Lymphoma Cells

To investigate the effect of autophagy on the drug resistance of different human lymphoma cells. Human Burkitt's lymphoma cell Daudi, human B lymphoma cell SUDHL-4, and human mantle cell lymphoma cell JeKo-1 were taken as the research subjects. The expression of Atg5 was inhibited by the treatments of autophagy inhibitors or stable interference via lentivirus infection. The autophagy activity of B lymphoma cell was changed, and the changes of lymphoma cells to the drug resistance of ADR and VCR was observed. JeKo-1 cells showed the strongest resistance to ADR and VCR, followed by SUDHL-4, and Daudi cells showed the weakest resistance to ADR and VCR. At the same time, JeKo-1 cells showed the strongest autophagy activity, followed by SUDHL-4, and Daudi cells showed the weakest autophagy activity. After the treatments of autophagy inhibitors or stable Atg5 interference, the resistance of lymphoma cells to ADR and VCR was significantly weakened, and there was the positive correlation at the drug resistance and the autophagy activity of B lymphoma cell. The higher autophagy activity in lymphoma cells, the lower chemotherapy resistance of the lymphoma cells after autophagy was inhibited.

Nanoencapsulated rituximab mediates superior cellular immunity against metastatic B-cell lymphoma in a complement competent humanized mouse model

BackgroundDespite the numerous applications of monoclonal antibodies (mAbs) in cancer therapeutics, animal models available to test the therapeutic efficacy of new mAbs are limited. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice are one...

The Antimicrobial Peptide, Nisin, Synergistically Enhances the Cytotoxic and Apoptotic Effects of Rituximab Treatment on Human Burkitt's Lymphoma Cell Lines.

Non-Hodgkin's lymphomas comprise the most common hematological cancers worldwide and consist of a heterogenous group of malignancies affecting the lymphoid system. Treatment of non-Hodgkin's lymphoma has been significantly enhanced with the addition of Rituximab to the standard chemotherapy regimen. However, even with the advancement of treatment patients continue to relapse and develop resistance to Rituximab, rendering subsequent treatments unsuccessful. The use of novel drugs with unique antitumor mechanisms has gained considerable attention. In this study, we explored the in vitro anti-cancer effects of the combined therapy of Rituximab and Nisin on human Burkitt's lymphoma cells. The human Burkitt's lymphoma cells lines, Raji and Daudi, were treated with Nisin, Rituximab, or a combination of the two agents at various concentrations. Cytotoxicity following treatment was determined using cell viability assay. The degree of apoptosis was verified via flow cytometric analysis using FITC annexin V/PI staining. Our findings show that the combined treatment of Rituximab and Nisin results in a more significant reduction in the survival of Raji and Daudi Burkitt's lymphoma cells, compared to Nisin or Rituximab treatment alone. Additionally, our results indicate that Nisin can induce a significant degree of apoptosis in the Burkitt's lymphoma cells compared to the negative controls. However, the addition of Nisin to the Rituximab treatment synergistically enhances the apoptotic antitumor effect. This study demonstrates the synergistic antitumor effect of Nisin treatment in vitro to enhance tumor cell apoptosis and the potential value of Nisin as an adjunct therapy in the treatment of lymphoma.