Burkitt Lymphoma Tumors Research Articles

Detection of anti-EBV TCR CDR3s associated with better outcomes for EBV-positive, Ugandan cases of Burkitt lymphoma.

Burkitt lymphoma (BL) has a tight association with Epstein-Barr virus (EBV), especially in sub-Saharan Africa. While the relationship between BL and EBV is well documented, the relationship between the anti-EBV adaptive immune response, particularly in sub-Saharan African cases, and disease course, has not been substantially investigated. An analysis of T-cell receptor (TCR) complementarity determining region-3 (CDR3) sequences, reported here, from EBV-positive, Ugandan BL tumor samples revealed a correlation between the presence of anti-EBV CDR3s and improved overall survival probabilities. Furthermore, chemical complementarity assessments demonstrated higher complementarity for TCR CDR3s and EBV epitopes in the cases where there had been a detection of the anti-EBV CDR3 AA sequence matches in the BL tumor samples. Overall, the results reported here raise the question of whether EBV targeted immunotherapy would lead to better BL outcomes?

G protein-coupled receptor 183 mediates the sensitization of Burkitt lymphoma tumors to CD47 immune checkpoint blockade by anti-CD20/PI3Kδi dual therapy.

Immunotherapy-based regimens have considerably improved the survival rate of B-cell non-Hodgkin lymphoma (B-NHL) patients in the last decades; however, most disease subtypes remain almost incurable. TG-1801, a bispecific antibody that targets CD47 selectively on CD19+ B-cells, is under clinical evaluation in relapsed/refractory (R/R) B-NHL patients either as a single-agent or in combination with ublituximab, a new generation CD20 antibody. A set of eight B-NHL cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells, M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 alone or combined with the U2 regimen associating ublituximab to the PI3Kδ inhibitor umbralisib, was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and/or quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). CRISPR-Cas9 gene edition was used to selectively abrogate GPR183 gene expression in B-NHL cells. In vivo, drug efficacy was determined in immunodeficient (NSG mice) or immune-competent (chicken embryo chorioallantoic membrane (CAM)) B-NHL xenograft models. Using a panel of B-NHL co-cultures, we show that TG-1801, by disrupting the CD47-SIRPα axis, potentiates anti-CD20-mediated ADCC and ADCP. This led to a remarkable and durable antitumor effect of the triplet therapy composed by TG-1801 and U2 regimen, in vitro, as well as in mice and CAM xenograft models of B-NHL. Transcriptomic analysis also uncovered the upregulation of the G protein-coupled and inflammatory receptor, GPR183, as a crucial event associated with the efficacy of the triplet combination. Genetic depletion and pharmacological inhibition of GPR183 impaired ADCP initiation, cytoskeleton remodeling and cell migration in 2D and 3D spheroid B-NHL co-cultures, and disrupted macrophage-mediated control of tumor growth in B-NHL CAM xenografts. Altogether, our results support a crucial role for GPR183 in the recognition and elimination of malignant B cells upon concomitant targeting of CD20, CD47 and PI3Kδ, and warrant further clinical evaluation of this triplet regimen in B-NHL.

Endemic Burkitt lymphoma avatar mouse models for exploring inter-patient tumor variation and testing targeted therapies.

Endemic Burkitt lymphoma (BL) is a childhood cancer in sub-Saharan Africa characterized by Epstein-Barr virus and malaria-associated aberrant B-cell activation and MYC chromosomal translocation. Survival rates hover at 50% after conventional chemotherapies; therefore, clinically relevant models are necessary to test additional therapies. Hence, we established five patient-derived BL tumor cell lines and corresponding NSG-BL avatar mouse models. Transcriptomics confirmed that our BL lines maintained fidelity from patient tumors to NSG-BL tumors. However, we found significant variation in tumor growth and survival among NSG-BL avatars and in Epstein-Barr virus protein expression patterns. We tested rituximab responsiveness and found one NSG-BL model exhibiting direct sensitivity, characterized by apoptotic gene expression counterbalanced by unfolded protein response and mTOR pro-survival pathways. In rituximab-unresponsive tumors, we observed an IFN-α signature confirmed by the expression of IRF7 and ISG15. Our results demonstrate significant inter-patient tumor variation and heterogeneity, and that contemporary patient-derived BL cell lines and NSG-BL avatars are feasible tools to guide new therapeutic strategies and improve outcomes for these children.

DNA Methylation-Based Burkitt Lymphoma Epitypes Have Distinct Molecular and Clinical Features

Introduction: Genome-wide hypomethylation coupled with hypermethylation of tumor suppressor genes is a hallmark of many cancers. Consistent changes in DNA methylation patterns are also a feature of immune cells as they undergo terminal differentiation and maturation. In previous studies, it has been shown that malignant B cells harbor a combination of de novo methylation while retaining some epigenetic features characteristic of their cell of origin. Although information on the genetic features of Burkitt Lymphoma (BL) is expanding, limited information exists on the methylation landscape and its role in pathogenesis. We sought to identify consistent DNA methylation changes and their interplay between BL pathogenesis and other features such as EBV status and somatic mutations. We performed an integrative analysis using comprehensive genome-wide CpG methylation profiling, whole genome sequencing, and gene expression analysis. Methods: Samples were collected as part of the Burkitt Lymphoma Genome Sequencing Project (BLGSP). DNA from 218 primary BL tumors (154 pediatric (111 EBV+), 64 adult (19 EBV+)), 13 BL cell lines (6 EBV+), and 6 normal centroblast (CB) samples were profiled using the Illumina EPIC array. DNA and RNA from biopsies and cell lines underwent whole genome and RNA sequencing to enable the correlation of DNA methylation status with gene expression and genomic alterations. Nine of the BL samples were separately subjected to whole genome sequencing using the PromethION (Oxford Nanopore) to infer genome-wide methylation landscape. Publicly available data from 1595 B cell samples representing both normal subpopulations and tumor subtypes were used for further comparison. To search for methylation patterns within BL, beta values from the 10000 most variable CpGs were used as features for non-negative matrix factorization (NMF) clustering. Results: Clustering revealed two distinct epigenetic subgroups (epitypes) within BL, which we have named hyperBL (cluster1) and hypoBL (cluster2). We found pediatric and adult tumors to be proportionally split across the two epitypes; however, we noted an over-representation of EBV+ tumors in hyperBL. PCA analysis comparing BL with the additional B-cell samples revealed a separation between the epitypes, with hyperBL projecting farther along the trajectory of B cell differentiation, suggestive of a more terminally differentiated B-cell. Investigating the differential methylation between epitypes revealed hyperBL to be associated with greater hypermethylation compared to hypoBL and CBs. The hypermethylated CpGs in hyperBL mainly affected promoter regions within CpG islands while hypomethylated CpGs were mostly intergenic or in open seas. Using PromethION data to allow for genome-wide analyses, we found hypermethylated regions of hyperBL were enriched for binding motifs for several transcription factors involved in B cell development including: IRF4, PAX5, YY1, BCL6, SP1, STAT3, and KLF9. We used mitotic clock estimates based on DNA methylation (epiCMIT) and found hyperBL had significantly elevated values, suggesting a greater proliferative history of these tumors. To further establish a molecular basis for these epitypes, we searched for gene expression differences and distinct driver mutation patterns. The genomes of hyperBL tumors harbored an overall greater mutation burden and significantly higher mutation burden within regions affected by aberrant somatic hypermutation (aSHM), which was associated with a higher frequency of mutations attributed to the SBS9 mutational signature. Gene expression differences indicate hypoBL tumors have upregulation of IRF4 induced pathways. To determine the clinical relevance of the unique epigenetic clusters we conducted survival analyses and found hyperBL samples to have significantly inferior progression free survival. Conclusion: This work identifies novel epigenetic subgroups within BL with characteristic genetic differences between them. HyperBL is associated with hypermethylation, a higher mutation burden, elevated rates of aSHM, and inferior PFS. These results establish a subset of patients with distinct epigenetic, molecular and clinical features and further elucidates key mechanisms underlying BL pathogenesis. Together these data highlight the need and potential for strategies that exploit these epigenetic changes as a therapeutic target. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phosphorylation of PBK/TOPK Tyr74 by JAK2 promotes Burkitt lymphoma tumor growth

Burkitt lymphoma (BL), which is characterized by high invasiveness, is a subgroup of non-Hodgkin lymphoma. Although BL is regarded as a highly curable disease, especially for children, some patients unfortunately still do not respond adequately. The understanding of the etiology and molecular mechanisms of BL is still limited, and targeted therapies are still lacking. Here, we found that T-LAK cell-derived protein kinase (TOPK) and phosphorylated Janus kinase 2 (p-JAK2) are highly expressed in the tissues of BL patients. We report that TOPK directly binds to and is phosphorylated at Tyr74 by JAK2. Histone H3, one of the downstream targets of TOPK, is also phosphorylated in vivo and in vitro. Furthermore, we report that the phosphorylation of TOPK at Tyr74 by JAK2 plays a vital role in the proliferation of BL cells and promotes BL tumorigenesis in vivo. Phosphorylation of TOPK at Tyr74 by JAK2 enhances the stability of TOPK. Collectively, our results suggest that the JAK2/TOPK/histone H3 axis plays a key role in the proliferation of BL cells and BL tumorigenesis in vivo.

L-Asparaginase synergizes with etoposide via the PI3K/Akt/mTOR pathway in Epstein-Barr virus-positive Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive Epstein-Barr virus (EBV)-driven B-cell lymphoma characterized by the translocation and rearrangement of the c-Myc proto-oncogene. High-intensity multidrug chemotherapy regimens have a limited effect on the survival of refractory or relapsed BL patients, mainly owing to the high EBV load and drug resistance. l-asparaginase ( l-Asp) and etoposide (VP-16) play a beneficial role in EBV-related lymphoproliferative diseases; however, their roles and mechanisms in BL remain unclear. In this study, we found that VP-16 inhibited BL cell proliferation and arrested the cell cycle at the G2 /M phase. It also induced autophagy and activated the extrinsic and intrinsic apoptotic signaling pathways in BL cells. Mechanistically, VP-16 inhibited c-Myc expression and regulated the PI3K/Akt/mTOR signaling pathway. Notably, VP-16 also showed a specific synergistic effect with l-Asp to induce apoptosis in EBV-positive BL cells but not in EBV-negative BL cells. VP-16 combined with l-Asp further inhibited c-Myc expression and downregulated the PI3K/Akt/mTOR signaling pathway. Additionally, we found that VP-16 inhibited the expression of latent membrane protein 1 (LMP1), and in combination with l-Asp further decreased LMP1 expression in Raji cells. Our in vivo data also showed that the dual-drug combination significantly inhibited the growth of BL tumors and prolonged the survival of mice compared to VP-16 alone. In conclusion, this study provides new evidence that l-Asp may enhance the antitumor effect of VP-16 by inhibiting the PI3K/Akt/mTOR signaling pathway in EBV-positive BL cells.

RNA helicase DHX15 decreases cell apoptosis by NF-\u03baB signaling pathway in Burkitt lymphoma

BackgroundDHX15 is one of the RNA helicase family members involving in several biological processes. Studies have reported that overexpression of DHX15 is related to cancer progression. However, the role of DHX15 in Burkitt lymphoma (BL) and latent Epstein-Barr virus (EBV) infection remains to be elucidated.MethodsExpression of DHX15 was measured in BL patient by immunohistochemical staining. In vitro study, a CCK-8 assay was used to analyze cell proliferation and flow cytometry was performed to assess cell cycle, apoptosis and mitochondria membrane potential. Members of NF-κB signaling pathway and apoptotic-related proteins expression were measured by western-blot. EBV latent infection products and RNA polymerase III transcripts expression were determined by quantitative real-time PCR and western-blot. In vivo study, HE, IHC, TUNEL and ISH assays were used to analyze the effect of DHX15 on subcutaneous tumor nodes formation.ResultsDHX15 was overexpressed in Burkitt lymphoma patients and tends to be associated with poor progression-free survival and poor overall survival. Knockdown of DHX15 significantly inhibited BL tumor growth, reduced cell proliferation, induced cell cycle arrest and increased cell apoptosis. Further analysis showed that canonical NF-κB signaling and its downstream targets, mitochondria and Caspase were involved in the increased cell apoptosis after DHX15 gene knockdown. Furthermore, knockdown of DHX15 reduced EBV latent infection products expression and inhibited RNA polymerase III activity.ConclusionDHX15 may be an oncogene in the development of BL and a potential therapeutic target for the treatment of BL and latent EBV infection.

Polatuzumab Vedotin Alone or in Combination with Obinutuzumab Synergistically Enhances In-Vitro Cytotoxicity and Cytokine Release Against CD20+/ CD79b+ Burkitt Lymphoma (BL) /Primary Mediastinal Large B Cell Lymphoma (PMBL)

Background: Pediatric Burkitt Lymphoma (PBL) represents the most common malignancy in childhood and adolescent non-Hodgkin Lymphoma (NHL) but occurs in less than 5% of adult NHL cases (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012). Mature B-cell NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis with rituximab based immunochemotherapy (Cairo et al Blood. 2007,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). The prognosis of PBL has significantly improved over the last 40 years through the use of short and intense multi-agent immunochemotherapy, however, a subset of patients with relapsed/refractory disease has immunochemotherapy resistant disease and a dismal prognosis (≤ 20% 5 yr. EFS),(Cairo et al.Blood. 2007; Cairo et al. JCO.2012). It is therefore critical to investigate and to develop targeted translational strategies in BL/PMBL in order to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease.The hu-anti-CD79b-vc-MMAE (Polatuzumab Vedotin, PV) an antibody drug conjugate (ADC) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). More recently PV has well tolerated in adults with CD79b refractory CLL (Palanca-Wessels et al. Lancet Oncol, 2014). Obinutuzumab, a glycoengineered type II CD20 antibody, has been shown to enhance cell death and antibody dependent cytotoxicity assay (ADCC) vs. rituximab (RTX) (Herter et al, Clinc Can Res, 2013). We previously observed that PV or obinutuzumab alone (Awasthi/Cairo et al., AACR 2017, Awasthi/Cairo et al., BJH 2015) significantly enhanced tumor cell death and increased overall survival against BL and PMBL xenografted mice. However, additive/synergistic effects of PV with obinutuzumab against mature PMBL/BL are unknown.Objective: To determine the efficacy of the PV, obinutuzumab or RTX alone or in combination with PV+ obinutuzumab vs. PV+RTX against PMBL and RTX sensitive/resistant BL tumor cell lines.Methods: Raji/Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and PMBL: Karpas1106P (ATCC, USA) tumor cells were cultured in RPMI with 10 or 20% FBS.Tumor cells were incubated with PV (generously supplied by Genentech Inc.) with obinutuzumab (generously provided by Roche) or rituximab (100ug/ml) for 4 hrs with NK cells.PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells (Lee et al, PLoS One, 2012).CD56+/CD3- isolated and expanded NK cells were used for cytotoxicity assay. Cytotoxicity was determined by DELFIA cytotoxicity assay at 10:1 E: T ratio and cytokine secretion was measured by multiplex ELISA assay kit.Results: Obinutuzumab+NK, RTX+NK (100µg/ml) compared to PV+NK cells (20ug/ml, 10:1E:T ratio) significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1%, (p=0.001 and p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 and p=NS), respectively and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 and NS), respectively. PV+ obinutuzumab+NK, further significantly improved in-vitro cytotoxicity compared to PV+ rituximab+NK, Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007, Figure 1A), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004, Figure1B) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003, Figure1C), respectively. Furthermore, IFN-g level from supernatant of PV+obinutuzumab+NK was significantly increased compared to obinutuzumab+NK alone, Raji, 22.14±2.1 vs. 9.41±6.5 ug/ml (p=0.01), Raji4RH, 20.30±9.8vs.17.02±2.5 ug/ml (p=0.05) and Karpas1106P, 18.10±1.5vs.9.56±5.5 ug/ml (p=0.03), respectively.Conclusion: Our preliminary data indicates thatPV in combination with obinutuzumab significantly enhances cell death in RTX sensitive/resistant CD79b+/CD20+ BL and PMBL compared to obinutuzumab or PV alone. Furthermore, PV+ obinutuzumab significantly enhanced cytokine secretion in BL (RTX sensitive/resistant) and PMBL compared to obinutuzumab alone. [Display omitted] DisclosuresKlein:Roche: Employment, Equity Ownership, Other: patents. Lee:Miltenyi: Speakers Bureau; Cyto-Sen Therapeutics, Inc: Other: Founder, Vice President, Medical Director; Intellia Therapeutics, Inc: Consultancy; Courier Therapeutics, Inc: Membership on an entity's Board of Directors or advisory committees. Cairo:Jazz Pharmaceuticals: Speakers Bureau.

Transcription factor 3 (TCF3) combined with histone deacetylase 3 (HDAC3) down-regulates microRNA-101 to promote Burkitt lymphoma cell proliferation and inhibit apoptosis

ABSTRACT To explore the function of transcription factor 3 (TCF3) on the proliferation and apoptosis of Burkitt lymphoma cells and its mechanism. qRT-PCR was performed to determine the expression of TCF3, histone deacetylase 3 (HDAC3), and microRNA-101 (miR-101) in the Burkitt lymphoma (BL) tumor tissues and lymph node tissues with reactive lymph node hyperplasia (RLNH). We found that the expression of TCF3 and HDAC3 was up-regulated in BL tumor tissues and lymphoma cells, and the miR-101 expression was down-regulated. And TCF3 and HDAC3 were negatively correlated with the expression of miR-101, respectively. In addition, knockdown of TCF3 can inhibit BL cell proliferation, reduce cell viability and promote cell apoptosis, retain the cell cycle in the G0/G1 phase, and inhibit the expression of Akt/mTOR pathway-related proteins (p-Akt and p-mTOR). When miR-101 was overexpressed, the results were the same as when TCF3 was knocked down. Moreover, we used Co-immunoprecipitation (Co-IP) to detect the interaction between TCF3 and HDAC3, and performed the Chromatin immunoprecipitation (ChIP) experiment to detect the enrichment of TCF3 and HDAC3 in the promoter region of miR-101. We found that TCF3 can interact with HDAC3 and is enriched in the miR-101 promoter region. In conclusion, TCF3 combined with HDAC3 down-regulates the expression of miR-101, thereby promoting the proliferation of BL cells and inhibiting their apoptosis.

The Association between the Comprehensive Epstein-Barr Virus Serologic Profile and Endemic Burkitt Lymphoma.

The discovery of Epstein-Barr virus (EBV) in Burkitt lymphoma tumors represented the first link between a virus and cancer in humans, but the underlying role of this virus in endemic Burkitt lymphoma remains unclear. Nearly all children in Burkitt lymphoma-endemic areas are seropositive for EBV, but only a small percentage develop disease. Variation in EBV-directed immunity could be an explanatory cofactor. We examined serum from 150 Burkitt lymphoma cases and 150 controls using a protein microarray that measured IgG and IgA antibodies against 202 sequences across the entire EBV proteome. Variation in the EBV-directed antibody repertoire between Burkitt lymphoma cases and controls was assessed using unpaired t tests. ORs quantifying the association between anti-EBV IgG response tertiles and Burkitt lymphoma status were adjusted for age, sex, and study year. Thirty-three anti-EBV IgG responses were elevated in Burkitt lymphoma cases compared with controls (P ≤ 0.0003). Burkitt lymphoma-associated IgG elevations were strongest for EBV proteins involved in viral replication and antiapoptotic signaling. Specifically, we observed ORs ≥4 for BMRF1 (early antigen), BBLF1 (tegument protein), BHRF1 (Bcl-2 homolog), BZLF1 (Zebra), BILF2 (glycoprotein), BLRF2 [viral capsid antigen (VCA)p23], BDLF4, and BFRF3 (VCAp18). Adjustment for malaria exposure and inheritance of the sickle cell variant did not alter associations. Our data suggest that the anti-EBV serologic profile in patients with Burkitt lymphoma is altered, with strong elevations in 33 of the measured anti-EBV IgG antibodies relative to disease-free children. The Burkitt lymphoma-specific signature included EBV-based markers relevant for viral replication and antiapoptotic activity, providing clues for future Burkitt lymphoma pathogenesis research.

Epstein barr virus IgG and EBER-1 in Burkitt's lymphoma children at a referral hospital in western Kenya.

Burkitt's lymphoma (BL) is a frequent childhood B cell non-Hodgkin's lymphoma (NHL) in equatorial Africa associated with infections. Chronic Epstein Barr virus (EBV) infections can lead to host immune stimulation that may trigger genetic translocation(s), neoplastic transformation and proliferation of B cells. We determined EBV immunoglobulin G (IgG) in sera from participants and EBER-1 in tumour sections in confirmed BL cases at Moi Teaching and Referral Hospital (MTRH). A cross sectional study of children with clinical and histology diagnosis of NHL from whom BL status were confirmed by immunohistochemistry (IHC) was carried out. Epstein Barr virus IgG in sera was determine using Enzyme-linked immunosorbant assay, IHC for EBER-1 and MYC protein in tumour sections. Demographic and clinical information were obtained from questionnaires and hospital files respectively. Ninety three percent of sera were EBV IgG positive of which 31.7% were confirmed as BL. All jaw BL tumours and 86.7% of BL tumours carried EBER-1 antigen. Odds ratio of EBER-1 positive was 1.39, 95% CI: 0.16-12.19 in BL tumours regardless of age or gender. EBV infection among the study participants may be associated with BL, however, EBER-1 and MYC negative in BL tumours suggest alternative BL pathogenesis or variant.

MCL-1 Inhibition By the Selective MCL-1 Inhibitor AMG-176 Induces in Vitro Activity Against Burkitt Lymphoma Cell Lines and Synergistically Enhances the Cytotoxic Effect of Chemotherapy and BH3 Mimetics

Background: Although >90% of Burkitt Lymphoma (BL) patients will be cured by intensive multi-agent immunochemotherapy, relapsed and refractory BL patients have poor prognoses demonstrating the need to identify novel therapies. Myeloid cell leukemia-1 (MCL-1) is a pro-survival protein within the BCL-2 family that has been investigated as a therapeutic target in several leukemia and lymphoma pre-clinical models and clinical studies. Analysis of pediatric BL tumors has implicated MCL-1 in BL pathogenesis (Giulino-Roth et al., Blood 2012) and targeting MCL-1 may be effective in MYC-driven lymphomas (Kelly et al., Genes Dev 2014). High MCL-1 expression has also been associated with therapy resistance and can confer resistance to BH3 mimetic agents (Carrington et al., Cell Death Differ 2017). AMG176, a selective MCL-1 inhibitor, is an effective promoter of apoptosis and is currently under investigation in clinical trials for relapsed and refractory multiple myeloma, lymphoma, and acute myeloid leukemia. Hypothesis: We hypothesized that MCL-1 inhibition with AMG176 will promote apoptosis and increase sensitivity of BL therapy-sensitive (TSCL) and therapy-resistant (TRCL) cell lines to other chemotherapy drugs. Methods: A panel of TSCLs (Raji, Ramos and Daudi) and one TRCL (Raji 4RH) BL cell lines were exposed to increasing concentrations of AMG176 as a single agent and in combination with Doxorubicin or Venetoclax at different time points. Cell viability was analyzed using PrestoBlue. Synergy in drug combination exposures was determined by Calcusyn software and reported as a combination index (CI) value with values increasingly <1 indicating increasing synergistic activity. Apoptosis induction was analyzed by flow cytometry for Annexin V (AV) and Sytox-7AAD and by western blotting for PARP. Effects of MCL-1 inhibition on the expression of other pro- and anti-apoptotic BCL-2 family member proteins was assessed by western blotting. AMG176 mechanism(s) of action was evaluated by co-immunoprecipitation followed by western blotting for pro-apoptotic BCL-2 family members known to be bound by MCL-1. Results: Single agent AMG176 exposure of BL cell lines exhibited a dose and time-dependent decrease in viable cells. 48hr IC50 values of Ramos (0.3097uM) and Raji (1.652uM) showed an increased sensitivity to AMG176 as compared to Daudi (9.616uM) and Raji 4RH (12.45uM). Exposure of cells to AMG176 also resulted in altered expression levels of pro-survival and pro-apoptotic proteins of the BCL-2 family proteins. Co-immunoprecipitation performed after 48hrs of AMG176 exposure exhibited a decrease in MCL-1:BIM and MCL-1:BAK complexes in Raji and Ramos cells promoting a pro-apoptotic environment. After 48hr exposure to AMG176, induction of apoptosis was observed in a dose-dependent manner in all cell lines as evidenced by increased PARP cleavage on western blot and AV-Sytox positivity on flow cytometry. To investigate the ability of MCL-1 inhibition to enhance the activity of cytotoxic chemotherapy, BL cells were exposed to AMG176 in combination with Doxorubicin. Daudi and Raji exhibited strong synergy with peak synergistic activity observed at 72hrs (Daudi CI values <0.1 at all AGM176 concentrations with 0.1-0.2uM Doxorubicin; Raji CI ranged from 0.233-0.295 at multiple AMG176 concentrations with <0.4uM Doxorubicin) while a minimal effect was observed in Ramos and Raji 4RH. As high MCL-1 is associated with resistance to Venetoclax by sequestering free BIM following Venetoclax exposure, the combination of AMG176 and Venetoclax was investigated and also showed significant synergy in BL cells. A minimal effect was observed in Daudi and Raji 4RH in Venetoclax combination but Ramos and Raji, the more AMG176 sensitive cell lines, showed peak synergy at 24hrs at high doses of Venetoclax (Ramos CI values ranged from 0.3-0.478; Raji CI ranged from 0.511 to 0.67). Conclusion: MCL-1 inhibition with AMG176 demonstrates dose- and time-dependent anti-lymphoma activity impairing in vitro proliferation and inducing apoptosis in BL cells. AMG176 exhibits synergistic activity in combination with cytotoxic chemotherapy in both therapy-sensitive and therapy-resistant BL cell lines and synergistically enhances response to the BH3 mimetic agent Venetoclax. These findings highlight the potential of MCL-1 inhibition as a therapeutic option in BL. This work was supported by T35 NIH training grant (T35AI089693). Disclosures No relevant conflicts of interest to declare.

68Ga]Ga/[177Lu]Lu-BL01, a Novel Theranostic Pair for Targeting C-X-C Chemokine Receptor 4.

C-X-C chemokine receptor type 4 (CXCR4) is overexpressed in hematological and solid malignancies. LY2510924 is a potent peptide antagonist of CXCR4. A derivative of LY2510924, BL01, was evaluated for theranostic applications targeting CXCR4. Methods: BL01 was synthesized by solid phase approach. A Lys(ivDde) residue was added at the C-terminus of LY2510924 (cyclo[Phe-Tyr-Lys(iPr)-d-Arg-2-Nal-Gly-d-Glu]-Lys(iPr)-NH2). A DOTA chelator was conjugated to the side chain of the deprotected exogenous Lys residue. The binding affinity of Ga/Lu-BL01 was determined by competitive radioligand binding assays. BL01 was radiolabeled with 68GaCl3 or 177LuCl3. Biodistribution studies were performed in mice bearing Daudi Burkitt's lymphoma tumor xenografts at selected time points. PET imaging studies were performed with [68Ga]Ga-BL01, with blocking experiments performed with preinjection of LY2510924. The stability of [68Ga]Ga/[177Lu]Lu-BL01 was assessed in mouse plasma. Results: Ga-BL01 and Lu-BL01 have nanomolar affinity for CXCR4. [68Ga]Ga-BL01 was obtained in 58 ± 5% decay-corrected radiochemical yields and >99% radiochemical purity with a molar activity of 40 ± 11 GBq/μmol, while [177Lu]Lu-BL01 was obtained in 65 ± 6% decay-corrected radiochemical yields and >99% radiochemical purity with a molar activity of 120 ± 21 GBq/μmol. [68Ga]Ga-BL01 and [177Lu]Lu-BL01 were excreted primarily through the renal pathway. Daudi xenografts were clearly delineated in PET images with good contrast. On the basis of biodistribution data, tumor uptake of [68Ga]Ga-BL01 was 10.2 ± 2.56% injected dose per gram (%ID/g) at 1 h postinjection (p.i.). Spleen (12.6 ± 2.36 %ID/g) and lungs (13.2 ± 2.98 %ID/g), organs that express CXCR4, had high uptake as well. Preinjection of LY2510924 reduced average uptake of [68Ga]Ga-BL01 in tumors by 88%, demonstrating target specificity. The uptake of [68Ga]Ga-BL01 in tumor increased to 15.3 ± 1.86 %ID/g at 2 h p.i., with improved contrast. [177Lu]Lu-BL01 has similar pharmacokinetics as [68Ga]Ga-BL01 at 1 h p.i. The highest uptake was observed in tumor (14.0 ± 1.11 %ID/g), followed by the lungs (13.0 ± 1.27 %ID/g) and spleen (11.6 ± 1.78 %ID/g). The tumor uptake increased to 16.2 ± 2.69 %ID/g at 4 h p.i., before declining slightly to 10.1 ± 1.41 %ID/g at 24 h p.i. Both compounds were stable in vivo, as no metabolites were observed at 5 min p.i. Conclusions: [68Ga]Ga-BL01 and [177Lu]Lu-BL01 are a promising theranostic pair for imaging and endoradiotherapy of CXCR4-expressing malignancies.

Abstract 2698: eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of lymphoma

Abstract Dysregulated messenger RNA (mRNA) translation drives the pathogenesis of multiple hematological malignancies. In lymphoma this includes the upregulation of key driver oncogenes and anti-apoptotic proteins (e.g., MYC, CCND1/3, BCL2 and MCL1) that contain a highly structured 5’-untranslated region (UTR) in their mRNA requiring enhanced eIF4A helicase activity for translation. eIF4A is a component of the eIF4F translation initiation complex and catalyzes the ATP-dependent unwinding of RNA duplexes and facilitates 43S ribosome scanning within the 5’-UTR. The activation of oncogenic signaling pathways, including RAS and PI3K, enhance eIF4A activity through phosphorylation of eIF4B, eIF4G and PDCD4 which facilitates formation of eIF4F and full activation of eIF4A. The PI3K/AKT/mTOR pathway is frequently activated in lymphoma, promoting the translation of oncogenes with complex 5’-UTRs that are required for tumor cell proliferation, survival and metastasis. eFT226 is a potent and sequence selective eIF4A1 inhibitor that promotes eIF4A1 binding to specific 5’-UTR polypurine and/or G-quadraplex recognition motifs leading to a selective block in ribosome mRNA scanning. The sequence dependency of eFT226 translational inhibition was evaluated in cell-based reporter assays demonstrating >100-fold greater sensitivity for reporter constructs containing a polypurine motif in the 5’-UTR (IC50 ~2 nM). Direct binding studies also confirmed the formation of a stable ternary complex with increased drug residence time between eFT226, eIF4A1 and RNA oligonucleotides containing polypurine motifs. The ability of eFT226 to inhibit MYC or MCL1 expression was found to be dependent on the presence of their respective 5’-UTR supporting a translational regulation mechanism dependent on recognition elements within the 5’-UTR. eFT226 shows potent anti-proliferative activity (GI50 < 15 nM) against a panel of B-cell lymphoma cell lines. Treatment with eFT226 leads to coordinated inhibition of MYC, CCND1/3, BCL2 or MCL1 protein expression resulting in significant anti-tumor activity. eFT226 has good pharmacokinetic properties and exhibits significant in vivo activity across a panel of diffuse large B cell lymphoma (DLBCL), and Burkitt lymphoma tumor models with ≤1 mg/kg/week IV administration. Further evaluation of predictive markers of sensitivity or resistance has shown that tumors with mTOR mediated activation of eIF4A are most sensitive to eFT226. In addition, tumors with PTEN mutations do not exhibit activated eIF4A and are generally resistant to induction of apoptosis by eFT226, resulting in reduced in vivo efficacy. The association of eFT226 activity with PI3K/mTOR pathway activation and mutational status provides a means to identify patient subsets during clinical development. Clinical trials in patients with lymphoma and other malignancies are planned. Citation Format: Peggy A. Thompson, Boreth Eam, Nathan P. Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Eric Sung, Ana Parra, Jocelyn Staunton, Gary G. Chiang, Christopher J. Wegerski, Andres Nevarez, Jeff Clarine, Samuel Sperry, Alan Xiang, Chinh Tran, Christian Nilewski, Garrick K. Packard, Theodore Michels, Paul A. Sprengeler, Justin T. Ernst, Siegfried H. Reich, Kevin R. Webster. eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of 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 2698.

MiR‑29 promoter and enhancer methylation identified by pyrosequencing in Burkitt lymhoma cells: Interplay between MYC and miR‑29 regulation.

Deregulation of microRNA expression plays a significant role in several cancer types including Burkitt lymphoma (BL). MicroRNA genes may be regulated through epigenetic mechanisms, such as specific histone modifications and/or DNA methylation of CpG islands in promoter regions, or by regions that are located next to microRNA genes. Given the regulatory role of MYC in miR‑29 expression, methylation as an additional mechanism for miR‑29 silencing was investigated. Methylation of miR‑29a/b/c in BL tumour samples and BL cell lines (BL41 and Raji) was assessed by pyrosequencing assay. BL cells were treated with 5‑aza‑2'‑deoxicitidine (decitabine) and evaluated for miR‑29a/b/c expression and methylation status. MYC, DNMT1 and DNMT3B protein expression were accessed by western blotting. For Epstein‑Barr virus (EBV) microRNA (miR)‑BART6 inhibition, the cells were transiently transfected with anti‑BART6‑5p. BL tumour samples and BL cell lines presented miR‑29a/b1 and miR‑29b2/c genes methylated in CpG sites located in both the promoter and enhancer regions. The treatment of BL cells with decitabine reduced methylation, induced miR‑29s expression and downregulated MYC protein levels in a dose‑dependent manner. Notably, inhibition of EBV miR‑BART6‑5p combined with decitabine enhanced miR‑29 expression in an EBV‑BL cell line. In conclusion, the miR‑29a/b1 and miR‑29b2/c genes have methylated CpG sequences at promoter and enhancer regions that may contribute to the regulation of miR‑29 expression in BL tumours. The present findings indicated interplay between MYC and miR‑29 regulation, highlighting the potential role of EBV‑miRNAs in miR‑29 regulation for BL pathogenesis.

Two Pathways of p27Kip1 Degradation Are Required for Murine Lymphoma Driven by Myc and EBV Latent Membrane Protein 2A.

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A), expressed in EBV latency, contributes to Burkitt lymphoma (BL) development in a murine model by acting as a constitutively active B cell receptor (BCR) mimic. Mice expressing both LMP2A and MYC transgenes (LMP2A/λ-MYC) develop tumors significantly faster than mice only expressing MYC (λ-MYC). Previously, we demonstrated the cell cycle inhibitor p27Kip1 is present at significantly lower levels in LMP2A/λ-MYC mice due to increased posttranslational degradation. P27Kip1 degradation can occur in the cytoplasm following phosphorylation on serine 10 (S10) or in the nucleus via the SCFSkp2 complex, which depends on Cks1. We previously demonstrated an S10A knock-in of p27Kip1 (p27S10A/S10A) that prevented S10 phosphorylation failed to significantly delay tumor onset in LMP2A/λ-MYC mice. We also previously demonstrated that a Cks1 knockout partially delayed tumor onset in LMP2A/λ-MYC mice, but onset was still significantly faster than that in λ-MYC mice. Here, we have combined both genetic manipulations in what we call p27Super mice. LMP2A/λ-MYC/p27Super mice and λ-MYC/p27Super mice both displayed dramatic delays in tumor onset. Strikingly, tumor development in LMP2A/λ-MYC/p27Super mice was later than that in λ-MYC mice and not significantly different from that in λ-MYC/p27Super mice. The p27Super genotype also normalized G1-S-phase cell cycle progression, spleen size, and splenic architecture in LMP2A/λ-MYC mice. Our results reveal both major pathways of p27Kip1 degradation are required for the accelerated BL development driven by LMP2A in our BL model and that blocking both degradation pathways is sufficient to delay Myc-driven tumor development with or without LMP2A.IMPORTANCE BL is a cancer that primarily affects children. The side effects of chemotherapy highlight the need for better BL treatments. Many BL tumors contain EBV, and our goal is to determine what makes EBV-positive BL different from EBV-negative BL. This may lead to more specific treatments for both types. All cases of BL require overexpression of MYC Mice engineered to express EBV LMP2A along with MYC (LMP2A/λ-MYC mice) develop tumors much more quickly than mice only expressing MYC (λ-MYC mice). Blocking degradation of the cell cycle inhibitor protein p27Kip1 in LMP2A/λ-MYC mice causes tumors to develop later than in λ-MYC mice, showing that p27Kip1 degradation may play a larger role in EBV-positive BL than EBV-negative BL. Furthermore, our studies suggest the cell cycle is an attractive target as a treatment option for LMP2A-positive cancers in humans.

Regulation of Cytokines/Chemokines Release and Anti-Tumor Effect of Expanded Natural Killer (NK) Cells By a Novel Fusion of N-820 (2B8T2M), an IL-15 Superagonist with 4 Single-Chain Anti-CD20 Antibody Domains, Against Rituximab Resistant Burkitt Lymphoma (BL)

Background: The prognosis of children with relapsed/refractory B-cell (CD20+) non-Hodgkin lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemo-radiotherapy resistance (Cairo et al, JCO, 2012, Cairo et al, BJH, 2018). Rituximab has been widely used in frontline treatment of B-NHL, however, some patients retreated with rituximab will relapse which limits patient treatment options (Goldman/Cairo, Leukemia, 2013). Several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering of NK cells with chimeric antigen receptors (Chu/Cairo et al, Can Imm Res 2015), as well as second-generation engineered anti-CD20 antibodies (Tiwari/Cairo et al BJH 2015). To enhance NK based therapy, our group has successfully expanded the functionally activated peripheral blood NK cells (exPBNK) with irradiated feeder cells (Chu/Cairo, et al, Can Imm Res 2015). To enhance NK cell-based targeted therapy, we had successfully engineered expanded NK cells with chimeric antigen receptors (Chu/Cairo, et al, Can Imm Res 2015) and combined expanded NK with an anti-CD20 targeted IL-15 fusion protein N-820 (2B8T2M) targeting rituximab-sensitive and -resistant BL (Chu/Cairo, et al, ASH 2017). N-820 was generated by fusing ALT-803 to four single-chain antibody domains of rituximab (Liu/Wong, et al, JBC, 2016). ALT-803 is a superagonist of an IL-15 variant bound to an IL-15RαSu-Fc fusion with enhanced IL-15 biological activity (Zhu et al. 2009 J Immunol), longer half-life and increased potency (Han, et al. Cytokine. 2011). N-820 displayed tri-specific binding activity through its recognition of CD20 on tumor cells, activated NK cells to enhance antibody-dependent cellular cytotoxicity (ADCC), and induced apoptosis of B-lymphoma cells (Liu/Wong, et al, JBC, 2016).Objective: To investigate how N-820 modulates the crosstalk between BL tumor cells and expanded NK cells by monitoring cytokines, chemokines and growth factors released and the anti-tumor effects of N-820 on NK cells in BL xenografts.Method: PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells and isolated as we have previously described with K562-mbIL15-41BBL (Chu/Cairo, et al, Can Imm Res 2015). ALT-803 and N-820 were generously provided by Altor Bioscience. Equal doses of N-820, Rituximab (R), ALT-803, R+ALT-803, obinutuzumab (obinu, generously provided by Christian Klein, PhDfrom Roche) were used for comparison. IgG was used as control. Rituximab-sensitive Raji and -resistant BL cells Raji-2R and Raji-4RH were used as target cells. NK cells were sorted by Beckman Coulter MoFlo XDP high-speed sorter and NK purity was confirmed by flow cytometry. Cytokines, chemokines and growth factors mRNA levels in NK cells were monitored by real-time PCR. Secreted cytokines, chemokines and growth factors were examined by standard ELISAs. Raji-2R-Luc cells were injected into NSG mice. ExPBNK+N-820 and controls were injected to the xenografted NSG mice. The tumor burden was measured with the IVIS-200 system.Results: We found that ALT-803 and N-820 at equal doses significantly enhanced the expression of NK activating receptors such as NKG2D, NKp30, NKp44, and CD16 on exPBNK compared to IgG controls. Additionally, N-820 significantly enhanced exPBNK cytotoxicity against Raji, Raji-2R and Raji-4RH cells compared to the controls IgG, R, ALT-803, R+ALT-803, or obinu (p<0.001). We further examined 84 genes of cytokines, chemokines and growth factors by real time PCR. Among these 84 genes, N-820 up-regulated 26 genes and down-regulated 22 genes in exPBNK when co-cultured with Raji-2R compared to exPBNK alone. Using ELISAs, we confirmed that N-820 significantly enhanced IFN-g, granzyme B, and GM-CSF (Fig.1A) release from exPBNK against Raji-2R and Raji-4RH compared to IgG and R+ALT-803 (p<0.001). We also found that N-820 significantly reduced MDC (CCL22) (Fig.1B) release from Raji-2R cells compared to IgG (p<0.01) and R+ALT-803 (p<0.05). In NSG mice bearing Raji-2R tumor xenografts, we found that N-820 added to exPBNK significantly reduced tumor burden (p<0.05) and expanded mice survival (Fig.1C) (p<0.05) compared to control groups.Conclusions: N-820 significantly enhanced exPBNK cytotoxicity, IFN-g, granzyme B, GM-CSF release from exPBNK in-vitro, inhibited MDC release from BL and increased anti-tumor activity against BL xenografts in NSG mice. [Display omitted] DisclosuresJeng:Altor: Employment. Alter:NANTCell / Altor Bioscience: Employment. Rhode:NANTCell / Altor Bioscience: Employment, Patents & Royalties: Altor Bioscience. Lee:NantKwest: Employment. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Wong:Altor: Employment, Equity Ownership, Patents & Royalties: Altor Bioscience. Cairo:Janssen: Research Funding.

Abstract 1788: Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with obinutuzumab

Abstract Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD20+/CD79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. EFS, Cairo et al. Blood. 2007; Gerrard/Cairo et al., Blood, 2013, Goldman/Cairo et al. Leukemia, 2013). PV has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res.2009). We previously observed that obinutuzumab (Anti-CD20 mAb) significantly enhanced cell death and increased overall survival against BL (Awasthi/Cairo et al., BJH 2015) in xenografted NSG mice. However, additive/synergistic effects of PV with obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV or obinutuzumab/RTX alone or in combination against PMBL and rituximab (RTX) sensitive/resistant BL cell lines. Methods: Raji4RH (provided by M. Barth, MD, Roswell Park Cancer Institute) and Raji/ Karpas1106P (ATCC, USA) were cultured in RPMI. Tumor cells were incubated with PV, and/or anti-CD79b, MMAE (generously supplied by Genentech Inc.) with obinutuzumab /rituximab (100ug/ml) for 4 hr with NK cells at 10:1 E: T ratio and cytotoxicity was determined by DELFIA cytotoxicity assay. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS, isotype control, PV, antiCD79B mAb and MMAE (5mg/kg). Mice were xenografted with intravenous injections of Luc+ BL and PMBL cells and tumor burden was monitored by IVIS spectrum system. Results: OS of mice receiving PV alone was significantly increased compared to antiCD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Obinutuzumab+NK, rituximab+NK compared to PV+NK cells significantly enhanced cell lysis in Raji, 65.9±2.4% vs. 38.9±5.4% vs. 44.24±8.1% (p=0.001 &amp; p=0.001), Raji4RH, 52.8±9.4% vs. 16.04±7.2% vs.47.0±8.2% (p=0.03 &amp; p=NS) and Karpas1106P, 66.10±5.3% vs.48.2±3.9% vs. 61.6±10.06% (p=0.004 &amp; NS), respectively. PV+ obinutuzumab+NK, significantly improved cytotoxicity compared to PV+ rituximab+NK in Raji, 93.6±6.1% vs 79.9±5.3% (p=0.007), Raji4RH, 78.07±2.05% vs 63.5±0.16% (p=0.004) and Karpas1106P, 88.3±6.3 %vs.73.03±3.03% (p=0.003), respectively. Conclusion: Our preliminary data indicates that PV significantly increased survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with obinutuzumab significantly enhances cytotoxicity in BL and PMBL compared to obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Christeen Azmy, Janet Ayello, Christian Klein, Mitchell S. Cairo. Enhanced in vitro/in vivo cytotoxicity against Burkitt lymphoma/primary mediastinal large B cell lymphoma by polatuzumab vedotin (hu- anti-CD79b-vc-MMAE, PV) alone or in combination with obinutuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1788.

Argonaute 2 RNA Immunoprecipitation Reveals Distinct miRNA Targetomes of Primary Burkitt Lymphoma Tumors and Normal B Cells

miRNAs are small noncoding RNAs involved in the posttranscriptional regulation of gene expression. Deregulated miRNA levels have been linked to Burkitt lymphoma (BL) pathogenesis. To date, the number of known pathogenesis-related miRNA-target gene interactions is limited. Here, we determined for the first time the miRNA targetomes of primary BL tumors and normal B cells. AGO2-RNA immunoprecipitation of two frozen diagnostic BL tissue samples and three CD19+ B-cell samples isolated from routinely removed tonsils showed distinct miRNA targetomes of BL and normal B cells. In contrast to normal B cells, miRNA target genes in BL were enriched for targets of the oncogenic miR-17 to 92 cluster, and were involved mainly in cell cycle and cell death. Immunohistochemistry on BL and tonsil tissues confirmed altered protein levels for two of six selected miRNA targets, in line with the differential AGO2-IP enrichment between BL and normal B cells. A comparison of AGO2-IP-enriched genes in primary BL cases with BL cell lines indicated that despite a considerable overlap, the miRNA targetomes of BL cell lines show substantial differences with the targetomes of primary BL tumors. In summary, we identified distinct miRNA targetomes of BL and normal B cells, and showed both the necessity and feasibility of studying miRNA-target gene interactions in primary tumors.

MiR-29 silencing modulates the expression of target genes related to proliferation, apoptosis and methylation in Burkitt lymphoma cells.

Burkitt lymphoma (BL) is a B-cell lymphoma frequently diagnosed in children. It is characterized by MYC translocations, which lead to the constitutive expression of the MYC oncogene. MYC contributes to miR-29 repression through an E-box MYC binding site on the miR-29b-1/miR-29a promoter region. We evaluated the role of miR-29a/b/c and their predicted targets in BL pathogenesis. Mature sequences of miR-29a/b/c were transfected to the BL cell lines BL41 and Raji, and evaluated for DNMT3B, MCL1, BIM, CDK6, AKT and TCL1 protein expression as well as for MCL-1 and CDK6 mRNA expression. BL cells were treated with 5-aza-2'-deoxycytidine (decitabine) and evaluated for miR29 expressions and methylation status. DNMT3B inhibition was performed by DNMT3B siRNA. Ectopic expression of miR-29s in BL cells decreased CDK6, DNMT3B, TCL1 and MCL-1 protein levels, but CDK6 and MCL-1 mRNA expression was unaffected by miR-29. Decitabine enhanced miR-29 expression levels and decreased CDK6 protein expression. Additionally, inhibition of DNMT3B by siRNA increased miR-29a/b expression. Notably, the miR-29a/b1 and miR-29b2/c promoter genes showed methylated CpG sequences that were demethylated after decitabine treatments. Furthermore, MYC-negative tumours had higher levels of miR-29 expression compared with MYC-translocated cases, suggesting that MYC regulates miR-29 in BL tumours. Our results suggest a significant role for miR-29s in BL pathogenesis in altering the expression of targets involved in critical cancer pathways, such as cell cycle control, apoptosis inhibition and DNA methylation. Moreover, methylation-mediated miR-29 epigenetic silencing may occur during BL development.