Burkitt's Lymphoma Cell Research Articles

Nonbiological Factors Affecting Outcomes in Adolescent and Young Adults with Lymphoma

Background: Lymphoma is one of the most common cancers in adolescent and young adults (AYA). Although histology, stage, age, and gender are known to affect outcome, little is known about nonbiological factors (NBF) that may affect access to care on survival.Objectives: To determine the impact of NBF in the survival of AYA with classical Hodgkin lymphoma (cHL) or non-Hodgkin lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), lymphoblastic lymphoma (LL), and anaplastic large cell lymphoma (ALCL).Design/Methods: The impact of NBF (insurance status, median household income, and educational achievement in the county of residence) along with biological factors (histology, stage, gender, age, and race/ethnicity) on survival of AYA lymphoma patients was analyzed using a SEER-18 (Surveillance, Epidemiology, and End Results) cohort diagnosed between 2007 and 2014.Results: There were 8,173 cases of cHL and 4,973 cases of NHL with a median follow-up of 44 months and 32 months, respectively. Five-year overall survival (OS) for AYA patients with localized cHL according to insurance status was 98% vs. 91% vs. 92% for insured, Medicaid, and uninsured patients, respectively (P < 0.001). The 5-year OS for advanced stage cHL was 91.5% vs. 85% vs. 85% for insured, Medicaid, and uninsured patients, respectively (P < 0.001). In multivariate analysis, the increase risk of death was associated with Medicaid (HR 2.23, 95% C.I. 1.76-2.81), and uninsured (HR 1.88, 95% C.I. 1.38-2.55), after adjustment for age, gender, histology and stage (P<0.001). Five-year OS for AYA patients with localized NHL according to insurance status was 90% vs. 76.5% vs. 84.5% for insured, Medicaid, and uninsured patients, respectively (P < 0.001). The 5-year OS for advanced stage NHL was 76% vs. 57%, and 62% for insured, Medicaid, and uninsured patients, respectively (P < 0.001). In multivariate analysis, the increased risk of death in AYA NHL was associated with Medicaid (HR 1.95, 95% CI 1.67-2.27), and uninsured (HR 1.48, 95% CI 1.18-1.84) (P<0.001) and county level of income in the second (HR 1.33, 95% C.I. 1.07-2.64), third (HR 1.54, 95% C.I. 1.25-1.90), and forth (HR 1.57, 95% C.I. 1.27-1.93) (P< 0.001) lower quartiles (P<0.001), after adjustment for age, gender, histology, and stage.Conclusion: Lack of insurance or Medicaid insured status and lower income are associated with increased mortality in AYA lymphomas, indicating an opportunity to increase outcomes by addressing NBF. DisclosuresCosta:Karyopharm: Research Funding; Amgen: Honoraria, Research Funding; Janssen: Research Funding; Celgene: Honoraria, Research Funding; Abbvie: Research Funding; Sanofi: Honoraria; BMS: Research Funding.

Targeting the Glyco-Antigen CD75s with the Tetravalent, Fc-Engineered Antibody ‘Ebu-141 Tetra’ Induces Potent Killing of B Cell Lymphoma and Plasma Cell Tumors

While monoclonal antibodies (MoAb) are already well established for the treatment of B cell-derived malignancies and usually show a good safety profile, not all patients benefit and relapses may be a problem. In order to identify novel surface structures suitable for antibody-based therapies and to improve killing mechanisms, ‘EBU-141 Tetra’ was developed.The parental MoAb EBU-141 is of mouse IgMk isotype, was generated in our laboratory and recognizes the glyco-antigen CD75s (previously CDw75), which are α-2,6-sialylated lactosamines on cell surface glycoproteins and glycolipids. CD75s was found on normal mature B cells and subsets of T cells, but is also expressed on certain lymphatic and solid tumors, e.g. mature B cell lymphoma, pancreatic and prostate cancer cells. EBU-141 specifically binds to CD75s on most B cell lymphoma, including Burkitt's lymphoma, FL, DLBCL, MCL, CLL, and interestingly plasma cell tumors. In addition, EBU-141 showed reactivity on a few cases of peripheral T-cell lymphoma, whereas classical Hodgkin lymphomas were consistently negative. Previously a chimeric IgG1k antibody, chEBU-141, was derived from EBU-141. Compared to the parental IgM antibody, chEBU-141 showed strongly reduced binding avidity, but was moderately effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) of mature B cell lymphoma and malignant plasma cells via recruitment of NK cells. However, chEBU-141 lacked the potent complement-dependent cytotoxicity (CDC) observed with the parental EBU-141 antibody. The aim of this study was to generate a tetravalent binding, Fc-engineered chEBU-141 IgG1 antibody with enhanced binding avidity for CD75s and potent effector functions for antibody-based therapy of mature B cell lymphomas and multiple myeloma.Using the variable regions of EBU-141, the chimeric IgG1κ antibody with a protein-engineered Fc and tetravalent binding properties, named ‘EBU-141 Tetra’, was generated. This MoAb and relevant controls were produced by transient transfection of 293T cells and purified from cell culture supernatants by affinity chromatography. Direct anti-tumor effects and Fc-mediated modes of action were investigated in cell proliferation assays and chromium release experiments using lymphoma and myeloma cell lines. Peripheral blood mononuclear cells and serum of healthy donors were used as source of human effector cells and complement in the cytotoxicity experiments.The ‘EBU-141 Tetra’ showed improved binding to CD75s on cell surface of mature B cell lymphoma as well as myeloma plasma cells compared to the bivalent binding chEBU-141 IgG1. The higher avidity for CD75s resulted in markedly improved ADCC activity of the ‘EBU-141 Tetra’ against Daudi Burkitt's lymphoma and U266 plasma cells with EC50 values in the picomolar range and higher maximum lysis rates. In addition, the ‘EBU-141 Tetra’ regained CDC activity of the parental EBU-141 and demonstrated efficient killing of Burkitt's lymphoma and myeloma cell lines with human serum as complement source. Thus, recruitment of immune effector cells and activation of the complement system are the main modes of action of the novel, tetravalent, chimeric, Fc-engineered antibody ‘EBU-141 Tetra’ antibody.Our findings further demonstrate that highly potent IgG-like antibodies against glycan-structures can be generated from mouse IgM antibodies and may open a new therapeutic window for therapy of patients with mature B cell lymphomas and multiple myeloma. DisclosuresKlausz:Affimed: Research Funding. Otte:Affimed: Research Funding. Klapper:HTG Molecular Diagnostics, Inc.: Research Funding; Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Peipp:Affimed: Research Funding. Gramatzki:Affimed: Research Funding.

Identification of Posttranvslationally Modified Neoantigens As Targets of B Cell Receptors of Burkitt Lymphoma

Introduction:Burkitt lymphoma represents the most aggressive neoplasm of mature sIg+ B cells. Besides the characterisitc translocation of the MYC gene with an immunoglobulin heavy or light chain gene locus, activating mutations in the TCF3 gene and inactivating mutations in the ID3 gene represent the key events in the pathogenesis of Burkitt lymphoma. These TCF3/ID3 mutations result in tonic and antigen-independent B cell receptor (BCR) pathway activation. Additionally, chronic BCR activation by antigens might play a role in Burkitt lymphoma pathogenesis and we set out to identify such potential target antigens of BCRs of Burkitt lymphoma.Methods:BCRs were expressed as recombinant Fabs in TG1 E.coli based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen sporadic Burkitt lymphoma specimens. Additionally, natural Fabs and recombinant Fabs were produced of 8 established Burkitt lymphoma cell lines by Papain digestion and BCR expression cloning. The purified pooled Fabs were screened for reactivities against non-modified and differently posttranslationally modified human protein macroarrays. Reactivities were verified by ELISA with coated N-terminally FLAG-tagged candidate antigens, each separately for the posttranslationally modified and non-modified isoforms. Recombinant Fabs derived of mantle cell lymphoma, diffuse large B cell lymphoma and primary central nervous system lymphoma served as controls. Moreover the functional effects on proliferation and BCR pathway activation after addition of the identified target antigens to Burkitt lymphoma cell lines with and without reactive BCRs were analyzed by proliferation assays and western blots. Finally, mutation status, methylation status and expression level of identified target antigens were analyzed in ICGC MMML-Seq lymphoma databases for differences in Burkitt lymphoma versus distinct lymphoma entities.Results:The Burkitt lymphoma derived Fabs were tested on posttranslationally modified protein arrays. The BCR of CA46 line showed specific reactivity against sumoylated Bystin, the Fab of the BL41 line reacted specifically against acetylated HSP40. Recombinant Fabs of diffuse large B cell lymphoma, primary central nervous system lymphoma and mantle cell lymphoma did neither bind sumoylated Bystin nor acetylated HSP40. Addition of the posttranslationally modified cognate antigens to respective Burkitt lymphoma cell line with the reactive BCR induced proliferaton. The analysis of the ICGC MMML-Seq lymphoma databases (representing different cohorts) showed a higher expression of Bystin in Burkitt lymphoma compared to other aggressive B-cell lymphomas. However, the mutation and methylation status of Bystin and HSP40 in the ICGC MMML-seq cohort did not provide any direct indication of the origin of their immunogenic post-translational modifications.Conclusions:A subgroup of sporadic Burkitt lymphoma has autoreactive BCRs with specific affinity against posttranslationally modified self antigens, demonstrating a new aspect in the pathogenesis of Burkitt lymphoma. Specific secondary modifications, as sumolytation of Bystin or acetylation of HSP40 appear to evoke the immunogenicity. Future studies will focus on the functional consequences of the antigen/BCR interaction on Burkitt cells. Furthermore, the causes of these posttranslationally modified neoantigens will be investigated in more detail. DisclosuresStilgenbauer:Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding.

Inhibition of Pro-Survival Pathways in DLBCL Cells By Functional Lipoprotein-like Nanoparticles

Introduction: Diffuse large B cell lymphoma (DLBCL) cells have an increased demand for cholesterol and cholesteryl esters to maintain membrane-anchored pro-survival signaling pathways, such as B cell receptor (BCR) signaling. In addition to BCR-mediated stimulation of de novo cholesterol synthesis, another mechanism employed by lymphoma cells to maintain cholesterol and cholesteryl ester homeostasis is by binding cholesterol-rich HDLs via the high-affinity receptor, scavenger receptor type B-1 (SCARB1). We previously reported that, unlike normal B cells, DLBCL cells highly express SCARB1, and that synthetic, cholesterol-poor high-density lipoprotein (HDL)-like nanoparticles (HDL NPs) exquisitely target SCARB1, deplete cellular cholesterol, and induce lymphoma cell death in vitro and in vivo. Given the critical role of cholesterol in maintaining proper membrane organization for BCR-mediated intracellular signaling, we hypothesized that HDL NP binding to SCARB1 and subsequent cellular cholesterol depletion modulates the organization of the cell membrane, reduces intracellular signaling downstream of membrane-anchored pathways like the BCR, and modulates the expression of genes relevant to cholesterol synthesis. Collectively, these mechanisms potently induce lymphoma cell death.Methods: We focused on germinal center (GC) DLBCL and Burkitt's Lymphoma (BL), due to the previously reported sensitivity of the SUDHL4 (GC DLBCL) and Ramos (BL) cell lines to HDL NP-induced cell death. In addition to SUDHL4 and Ramos, we tested the GC DLBCL cell line SUDHL6 and the BL cell lines Raji, Daudi and Namalwa. HDL NPs were synthesized, purified and characterized using standard protocols. Cell viability was quantified using the MTS assay, and total cholesterol levels were measured using the Amplex Red cholesterol assay. A blocking antibody against SCARB1 was used to inhibit binding of HDL NPs to the receptor. Confocal microscopy was used to visualize changes in membrane organization of SUDHL4 and Ramos cells following HDL NP treatment. Changes in the phosphorylation of signaling kinases (e.g. AKT, ERK1/2) following HDL NP treatment was measured using phospho-kinase arrays, phos-flow analysis, and western blot assays. RNA was harvested from cells treated with the HDL NPs for 48 hours and analyzed using Illumina's HT-12 microarray and RT-qPCR. Protein expression changes were measured using western blot assays.Results: HDL NPs potently induced cell death in all of the GC DBLCL and BL cell lines, all of which expressed SCARB1, with HDL NP IC50 values between 1 ~ 5 nM. As expected, HDL NP-induced cell death correlated with reduced total cellular cholesterol levels. Inhibition of HDL NP binding to SCARB1 by antibody blockade protected cells from HDL NP-induced cell death. HDL NP treatment induced a reorganization of the plasma membrane in SUDHL4 and Ramos cells, resulting in clustering of SCARB1, a phenomenon not seen in cholesterol-rich human HDL or saline controls. HDL NP treatment led to a decrease in the phosphorylation of a number of kinases downstream of BCR signaling, including AKT, ERK1/2, LCK and LYN over time. HDL NPs demonstrated synergy with small molecule inhibitors of various signaling kinases, such as ATK (inhibitor = GDC-0068) and SYK (inhibitor = R406), in Ramos and SUDHL4 cells. By microarray analysis and RT-qPCR, HDL NPs up-regulated a number of cholesterol biosynthesis genes, as well as the cell cycle inhibitor p21 and the pro-apoptotic protein APOPT1.Conclusions: These data demonstrate that HDL NPs bind SCARB1, altering the organization of the plasma membrane and reducing cellular cholesterol levels. Collectively, this results in decreased membrane-anchored pro-survival signaling, changes in gene expression pathways, and, ultimately, lymphoma cell death. Enhancing cholesterol depletion strategies, such as the combination of the targeted cholesterol depletion agent HDL NP with small molecule inhibitors of signaling kinases (e.g. AKT and SYK inhibitors), represents a novel and targeted therapeutic strategy for DLBCL and may be broadly applicable to other malignancies dependent on cholesterol homeostasis and membrane-anchored signaling pathways. DisclosuresKarmali:Gilead: Speakers Bureau; AstraZeneca: Speakers Bureau. Thaxton:AuraSense: Other: Co-founder of the biotech company AuraSense.

Sp140 is a multi-SUMO-1 target and its PHD finger promotes SUMOylation of the adjacent Bromodomain

BackgroundHuman Sp140 protein is a leukocyte-specific member of the speckled protein (Sp) family (Sp100, Sp110, Sp140, Sp140L), a class of multi-domain nuclear proteins involved in intrinsic immunity and transcriptional regulation. Sp140 regulates macrophage transcriptional program and is implicated in several haematologic malignancies. Little is known about Sp140 structural domains and its post-translational modifications. MethodsWe used mass spectrometry and biochemical experiments to investigate endogenous Sp140 SUMOylation in Burkitt's Lymphoma cells and Sp140 SUMOylation sites in HEK293T cells, FLAG-Sp140 transfected and His6-SUMO-1T95K infected. NMR spectroscopy and in vitro SUMOylation reactions were applied to investigate the role of Sp140 PHD finger in the SUMOylation of the adjacent BRD. ResultsEndogenous Sp140 is a SUMO-1 target, whereby FLAG-Sp140 harbors at least 13 SUMOylation sites distributed along the protein sequence, including the BRD. NMR experiments prove direct binding of the SUMO E2 ligase Ubc9 and SUMO-1 to PHD-BRDSp140. In vitro SUMOylation reactions show that the PHDSp140 behaves as SUMO E3 ligase, assisting intramolecular SUMOylation of the adjacent BRD. ConclusionsSp140 is multi-SUMOylated and its PHD finger works as versatile protein-protein interaction platform promoting intramolecular SUMOylation of the adjacent BRD. Thus, combinatorial association of Sp140 chromatin binding domains generates a multifaceted interaction scaffold, whose function goes beyond the canonical histone recognition. General significanceThe addition of Sp140 to the increasing lists of multi-SUMOylated proteins opens new perspectives for molecular studies on Sp140 transcriptional activity, where SUMOylation could represent a regulatory route and a docking surface for the recruitment and assembly of leukocyte-specific transcription regulators.

CD38-Specific Biparatopic Heavy Chain Antibodies Display Potent Complement-Dependent Cytotoxicity Against Multiple Myeloma Cells.

CD38 is overexpressed by multiple myeloma cells and has emerged as a target for therapeutic antibodies. Nanobodies are soluble single domain antibody fragments derived from the VHH variable domain of heavy chain antibodies naturally occurring in camelids. We previously identified distinct llama nanobodies that recognize three non-overlapping epitopes of the extracellular domain of CD38. Here, we fused these VHH domains to the hinge, CH2, and CH3 domains of human IgG1, yielding highly soluble chimeric llama/human heavy chain antibodies (hcAbs). We analyzed the capacity of these hcAbs to mediate complement-dependent cytotoxicity (CDC) to CD38-expressing human multiple myeloma and Burkitt lymphoma cell lines. Combinations of two hcAbs that recognize distinct, non-overlapping epitopes of CD38 mediated potent CDC, in contrast to the hcAb monotherapy with only weak CDC capacity. Similarly, combining daratumumab with a hcAb that recognizes a non-overlapping epitope resulted in dramatically enhanced CDC. Further, introducing the E345R HexaBody mutation into the CH3 domain strongly enhanced the CDC potency of hcAbs to CD38-expressing cells. Exploiting their high solubility, we genetically fused two distinct nanobodies into heteromeric dimers via a flexible peptide linker and then fused these nanobody dimers to the hinge, CH2 and CH3 domains of human IgG1, yielding highly soluble, biparatopic hcAbs. These biparatopic hcAbs elicited CDC toward CD38-expressing myeloma cells more effectively than daratumumab. Our results underscore the advantage of nanobodies vs. pairs of VH and VL domains for constructing bispecific antibodies. Moreover, the CD38-specific biparatopic heavy chain antibodies described here represent potential new powerful therapeutics for treatment of multiple myeloma.

Targeting the MYC Oncogene in Burkitt Lymphoma through HSP90 Inhibition.

Overexpression of the MYC oncogene is a key feature of many human malignancies including Burkitt lymphoma. While MYC is widely regarded to be a promising therapeutic target, a clinically effective MYC inhibitor is still elusive. Here, we report an alternative strategy, targeting MYC indirectly through inhibition of the HSP90 machinery. We found that inhibition of HSP90 function reduces MYC expression in human Burkitt lymphoma through suppression of MYC transcription and destabilization of MYC protein, thereby diminishing the proliferation of tumor cells. Consistently, treatment of Burkitt lymphoma cell lines with HSP90 inhibitors (17-AAG or 17-DMAG) was accompanied by downregulation of canonical MYC target genes. Combination treatment with 17-DMAG and the proteasome inhibitor, MG-132, led to accumulation of MYC protein, indicating that upon HSP90 inhibition, MYC is degraded by the proteasome. Using co-immunoprecipitation, we furthermore demonstrated a direct interaction between MYC and HSP90, indicating that MYC is an HSP90 client protein in Burkitt lymphoma. Together, we report here the use of HSP90 inhibitors as an alternative approach to target the MYC oncogene and its network in Burkitt lymphoma.

MiR-150 might inhibit cell proliferation and promote cell apoptosis by targeting LMO4 in Burkitt lymphoma.

This study aimed at investigating the effect of microRNA-150 (miR-150) on cell proliferation of Burkitt lymphoma and its molecular mechanism. Gene expression analysis was applied to identify target genes of miR-150 in Burkitt lymphoma cell line ST486 based on the dataset from the Gene Expression Omnibus (GEO) datasets GSE86432. miRNA mimics, inhibitor and small interfering RNA (siRNA) were fluorescently labeled by Cy3, whereas plasmid vector was labeled by EGFP. Cells were viewed by fluorescence microscope and transfection efficiency was evaluated through fluorescent cell percentage. Quantitative real-time polymerase chain reaction analysis (qRT-PCR) and western blot were applied to detect the expression level of miR-150 and LMO4. Cell proliferation, cell cycle, and apoptosis were explored by CCK-8, flow cytometry. Targeting relationship was validated by the Luciferase reporter assay. Tumor xenograft and immunohistochemical analysis were conducted in nude mice model. In Burkitt lymphoma cells, miR-150 expression was significantly lower than normal ones, whereas the expression of LMO4 was upregulated. miR-150 might inhibit cell proliferation and promoted apoptosis in Burkitt lymphoma deterioration by downregulating LMO4. The results of tumor xenograft further confirmed the role of miR-150 in Burkitt lymphoma. Targeting LMO4 is a significant mechanism by which miR-150 suppresses cell growth and promotes apoptosis in Burkitt lymphoma cells, thus may provide a novel target for Burkitt lymphoma therapy in the future.

Two-Step Co-Immunoprecipitation (TIP).

In the past few decades, numerous approaches have been developed to investigate protein-protein and protein-nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co-immunoprecipitation (co-IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two-step co-immunoprecipitation (TIP) technique. As compared to standard co-IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes. Here, we report a detailed TIP procedure that we used to purify a protein-protein complex from Burkitt lymphoma cells and from primary human CD4+ T cells. In addition, this unit describes an application of TIP for the isolation of transcription-factor-bound chromatin. © 2018 by John Wiley & Sons, Inc.

Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.

Ibrutinib significantly inhibited Bruton’s tyrosine kinase (BTK) phosphorylation,in-vitro proliferation and enhanced overall survival in a preclinical Burkitt lymphoma (BL) model

ABSTRACT Pediatric and adult patients with recurrent/refractory Burkitt lymphoma (BL) continue to have poor outcomes, emphasizing the need for newer therapeutic agents. Bruton’s tyrosine kinase (BTK) is activated following B-cell receptor stimulation and in part regulates normal B-cell development. Ibrutinib, a selective and irreversible BTK inhibitor, has been efficacious in chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), Waldenström’s macroglobulinemia, and marginal zone lymphoma. In this study, we investigated the efficacy of ibrutinib alone and in selective adjuvant combinations against BL in-vitro and in a human BL xenografted immune-deficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mouse model. Our data demonstrated that phospho-BTK level was significantly reduced in BL cells treated with ibrutinib (p < 0.001). Moreover, we observed a significant decrease in cell proliferation as well as significant decrease in IC50 of ibrutinib in combination with dexamethasone, rituximab, obinutuzumab, carfilzomib, and doxorubicin (p < 0.001). In-vivo studies demonstrated ibrutinib treated mice had a significantly prolonged survival with median survival of mice following ibrutinib treatment (32 days) (24 days) (p < 0.02). In conclusion, our findings demonstrate the significant in-vitro and preclinical in-vivo effects of ibrutinib in BL. Based on our preclinical results in this investigation, there is an on-going clinical trial comparing overall survival in children and adolescents with relapsed/refractory BL treated with chemoimmunotherapy with or without ibrutinib (NCT02703272).

Tuberous sclerosis complex is required for tumor maintenance in MYC‐driven Burkitt's lymphoma

The tuberous sclerosis complex (TSC) 1/2 is a negative regulator of the nutrient‐sensing kinase mechanistic target of rapamycin complex (mTORC1), and its function is generally associated with tumor suppression. Nevertheless, biallelic loss of function of TSC1 or TSC2 is rarely found in malignant tumors. Here, we show that TSC1/2 is highly expressed in Burkitt's lymphoma cell lines and patient samples of human Burkitt's lymphoma, a prototypical MYC‐driven cancer. Mechanistically, we show that MYC induces TSC1 expression by transcriptional activation of the TSC1 promoter and repression of miR‐15a. TSC1 knockdown results in elevated mTORC1‐dependent mitochondrial respiration enhanced ROS production and apoptosis. Moreover, TSC1 deficiency attenuates tumor growth in a xenograft mouse model. Our study reveals a novel role for TSC1 in securing homeostasis between MYC and mTORC1 that is required for cell survival and tumor maintenance in Burkitt's lymphoma. The study identifies TSC1/2 inhibition and/or mTORC1 hyperactivation as a novel therapeutic strategy for MYC‐driven cancers.

Spatial and temporal variations of childhood cancers: Literature review and contribution of the French national registry.

BackgroundSignificant increases in childhood cancer incidence since the 1970s have been consistently reported worldwide, but the persistence of the increase on recent periods is discussed. No conclusion can be drawn concerning the spatial variations of childhood cancer, either. This study is an in‐depth investigation of the spatial and temporal variations of childhood cancer in France. An extensive review of all the studies published since 2000 on those issues is provided.MethodsThe study included 25 877 cases of childhood cancer registered nationwide over 2000‐2014. The spatial heterogeneity (overdispersion, autocorrelation, overall heterogeneity) was tested, on two geographic scales, and two spatial scan methods were used to detect clusters of cases. The annual average percent change (AAPC) in incidence rate was estimated with Poisson regression models, and joinpoint analyses were considered.ResultsGlioma and non‐Hodgkin lymphoma cases exhibited some spatial heterogeneity and two large clusters were detected. Overall, the incidence rate of childhood cancer was stable over 2000‐2014 (AAPC = −0.1% [−0.3%; 0.2%]). A log‐linear positive trend was significantly evidenced for gliomas other than pilocytic astrocytomas (AAPC = 1.8% [0.9%; 2.7%]), with some suggestion of a leveling‐off at the end of the period, while Burkitt lymphoma and germ cell tumor incidence rates decreased (AAPC = −2.2% [−3.8%; −0.5%] and AAPC = −1.9% [−3.4%; −0.3%], respectively). No spatial heterogeneity or significant time variation was evidenced for other cancers.ConclusionSeveral types of childhood cancer displayed some spatial heterogeneity and two large clusters were detected, the origins of which are to be investigated and might include differences in case ascertainment. Overall, the results do not support a sustained increase in incidence rates of childhood cancer in recent years.

Evaluation of curcumin, a natural product in turmeric, on Burkitt lymphoma and acute myeloid leukemia cancer stem cell markers.

Curcumin, a bioactive compound from turmeric, eliminates cancer stem cells (CSCs) in some cancers. This study evaluates the effects of curcumin on CSC markers in Burkitt lymphoma and acute myeloid leukemia cells. Cells were treated with increasing concentrations of curcumin, followed by an ALDEFLUOR assay, colony formation assay and western blot analysis for the CSC-associated proteins, Gli-1, Notch-1 and Cyclin D1. Markers associated with CSCs were decreased in cells treated with curcumin. This included a decrease in the percentage of ALDH-positive cells, a decrease in colony formation and the downregulation of Gli-1, Notch-1 and Cyclin D1. These results indicate that curcumin decreased CSC markers in lymphoma/leukemia cells, potentially through inhibiting self-renewal.

MiR-129 targets CDK1 and iASPP to modulate Burkitt lymphoma cell proliferation in a TAp63-dependent manner.

Burkitt lymphoma is one of the most common lymphatic system cancers with poor outcome in adult patients. p53-induced apoptosis is a critical signaling for preventing tumor development. Cyclin B/cyclin-dependent kinase 1 (CDK1) phosphorylates inhibitor of apoptosis stimulating protein of P53 (iASPP) to promote iASPP nucleus localization and its inhibitory effect on p53. However, p53 is frequently mutated in Burkitt lymphoma, which gains novel oncogenic properties. Recently, the p53 family member, p63, became an attractive gene for the therapeutic strategies for patients with cancer. Therefore, we investigated the role of iASPP in the transactivation domain p63 (TAp63)-dependent cell proliferation inhibition in Burkitt lymphoma. We verified that the oncogenic effect of iASPP on Burkitt lymphoma is TAp63 dependent rather than p53 and confirmed that the interaction between CDK1 and iASPP enhanced the inhibitory effect of iASPP on p53 and TAp63. An online tool predicated that miR-129 might bind to 3'-untranslated region of iASPP and CDK1. We revealed that miR-129 acted as a tumor suppressor by inhibiting cancer cell proliferation and inhibiting CDK1 and iASPP via direct binding. An miR-129 inhibitor increased nucleus iASPP and decreased nucleus p53 and TAp63 levels, which could be reversed by the CDK1 knockdown, indicating that miR-129 might target CDK1 to inhibit iASPP phosphorylation, thus hindering iASPP nucleus localization and its inhibitory effect on p53 and TAp63 protein levels. Taken together, miR-129 could targetedly inhibit the expression of CDK1 and iASPP. CDK1 knockdown inhibits iASPP S84/S113 phosphorylation, thus blocking iASPP nucleus localization, suppressing the inhibitory effect of iASPP on p53 and TAp63, and restoring TAp63-induced proliferation inhibition in Burkitt lymphoma cells.

Drug-based perturbation screen uncovers synergistic drug combinations in Burkitt lymphoma

Burkitt lymphoma (BL) is a highly aggressive B-cell lymphoma associated with MYC translocation. Here, we describe drug response profiling of 42 blood cancer cell lines including 17 BL to 32 drugs targeting key cancer pathways and provide a systematic study of drug combinations in BL cell lines. Based on drug response, we identified cell line specific sensitivities, i.e. to venetoclax driven by BCL2 overexpression and partitioned subsets of BL driven by response to kinase inhibitors. In the combination screen, including BET, BTK and PI3K inhibitors, we identified synergistic combinations of PI3K and BTK inhibition with drugs targeting Akt, mTOR, BET and doxorubicin. A detailed comparison of PI3K and BTKi combinations identified subtle differences, in line with convergent pathway activity. Most synergistic combinations were identified for the BET inhibitor OTX015, which showed synergistic effects for 41% of combinations including inhibitors of PI3K/AKT/mTOR signalling. The strongest synergy was observed for the combination of the CDK 2/7/9 inhibitor SNS032 and OTX015. Our data provide a landscape of drug combination effects in BL and suggest that targeting CDK and BET could provide a novel vulnerability of BL.

BET Inhibition-Induced GSK3β Feedback Enhances Lymphoma Vulnerability to PI3K Inhibitors

The phosphatidylinositol 3 kinase (PI3K)-glycogen synthase kinase β (GSK3β) axis plays a central role in MYC-driven lymphomagenesis, and MYC targeting with bromodomain and extraterminal protein family inhibitors (BETi) is a promising treatment strategy in lymphoma. In a high-throughput combinatorial drug screening experiment, BETi enhance the antiproliferative effects of PI3K inhibitors in a panel of diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma cell lines. BETi or MYC silencing upregulates several PI3K pathway genes and induces GSK3β S9 inhibitory phosphorylation, resulting in increased β-catenin protein abundance. Furthermore, BETi or MYC silencing increases GSK3β S9 phosphorylation levels and β-catenin protein abundance through downregulating the E2 ubiquitin conjugating enzymes UBE2C and UBE2T. In a mouse xenograft DLBCL model, BETi decrease MYC, UBE2C, and UBE2T and increase phospho-GSK3β S9 levels, enhancing the anti-proliferative effect of PI3K inhibitors. Our study reveals prosurvival feedbacks induced by BETi involving GSK3β regulation, providing a mechanistic rationale for combination strategies.

Infection of Epstein⁻Barr Virus in Type III Latency Modulates Biogenesis of Exosomes and the Expression Profile of Exosomal miRNAs in the Burkitt Lymphoma Mutu Cell Lines.

Infection of Epstein–Barr virus (EBV), a ubiquitous human gamma herpesvirus, is associated with various malignancies in B lymphocytes and epithelial cells. EBV encodes 49 microRNAs in two separated regions, termed the BART and BHRF1 loci. Although accumulating evidence demonstrates that EBV infection regulates the profile of microRNAs in the cells, little is known about the microRNAs in exosomes released from infected cells. Here, we characterized the expression profile of intracellular and exosomal microRNAs in EBV-negative, and two related EBV-infected Burkitt lymphoma cell lines having type I and type III latency by next-generation sequencing. We found that the biogenesis of exosomes is upregulated in type III latently infected cells compared with EBV-negative and type I latently infected cells. We also observed that viral and several specific host microRNAs were predominantly incorporated in the exosomes released from the cells in type III latency. We confirmed that multiple viral microRNAs were transferred to the epithelial cells cocultured with EBV-infected B cells. Our findings indicate that EBV infection, in particular in type III latency, modulates the biogenesis of exosomes and the profile of exosomal microRNAs, potentially contributing to phenotypic changes in cells receiving these exosomes.

MYC-related microRNAs signatures in non-Hodgkin B-cell lymphomas and their relationships with core cellular pathways.

In order to investigate the role of microRNAs in the pathogenesis of different B-cell lymhoma subtypes, we have applied an array-based assay to a series of 76 mixed non-Hodgkin B-cell lymphomas, including Burkitt’s lymphoma (BL), diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, mantle cell lymphoma (MCL) and follicular lymphoma. Lymphomas clustered according to histological subtypes, driven by two miRNA clusters (the miR-29 family and the miR-17-92 cluster). Since the two miRNA clusters are known to be MYC-regulated, we investigated whether this would be supported in MYC-driven experimental models, and found that this signature separated BL cell lines and a MYC-translocated MCL cell lines from normal germinal center B-cells and other B-cell populations. Similar results were also reproduced in tissue samples comparing BL and reactive lymph node samples. The same series was then quantitatively analyzed for MYC expression by immunohistochemistry and MYC protein levels were compared with corresponding miRNA signatures. A specific metric was developed to summarize the levels of MYC-related microRNAs and the corresponding protein levels. We found that MYC-related signatures are directly related to MYC protein expression across the whole spectrum of B-cells and B-cell lymphoma, suggesting that the MYC-responsive machinery shows predominantly quantitative, rather than qualitative, modifications in B-cell lymphoma. Novel MYC-related miRNAs were also discovered by this approach. Finally, network analysis found that in BL MYC-related differentially expressed miRNAs could control, either positively or negatively, a limited number of hub proteins, including BCL2, CDK6, MYB, ZEB1, CTNNB1, BAX and XBP1.

Abstract LB-269: IDH2 inhibition enhances proteasome inhibitor responsiveness in hematological malignancies

Abstract The introduction of proteasome inhibitors (PIs) into the clinic has transformed the treatment of patients affected by multiple myeloma (MM) and mantle-cell lymphoma (MCL) establishing new standards of care. Despite these improvements, patients continuously relapse or are intrinsically resistant to PIs. Here, to identify druggable targets that synergize with PIs, we carried out a functional screening in MM cell lines using a short hairpin RNA library targeting 152 cancer driver genes, highly representative of all signaling pathways. The Isocitrate Dehydrogenase 2 (IDH2) gene was identified as a top candidate, showing a synthetic lethal activity with the PI Carfilzomib (CFZ). IDH2 is a NADP(+) dependent mitochondrial enzyme which catalyzes the oxidative decarboxylation of isocitrate to 2-oxoglutarate in the tricarboxylic acid (TCA) cycle. We demonstrated that combinations of the pharmacological IDH2 inhibitor AGI-6780 with FDA approved PIs significantly increased apoptotic cell death in ten MM cell lines, both sensitive and resistant to PIs. Combined treatments triggered synergistic cytotoxicity also in four MCL and in two Burkitt's lymphoma cell lines. Importantly, CFZ/AGI-6780 treatment increased death of primary CD138-positive cells from nine MM patients and exhibited a favorable cytotoxicity profile towards peripheral blood mononucleated cells and bone marrow-derived stromal cells. Mechanistically, CFZ/AGI-6780 combination significantly decreased TCA cycle activity and ATP levels, as a consequence of enhanced IDH2 enzymatic inhibition. In contrast, only a slight increase of mitochondrial reactive oxygen species (ROS) was observed. CFZ treatment reduced the expression of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme required for IDH2 activation through the NAD(+)-dependent deacetylase SIRT3. Consistently, combination of CFZ with either NAMPT (FK866) or SIRT3 (AGK7) inhibitors impaired IDH2 activity and increased MM cell death, thus phenocopying CFZ/AGI-6780 effects and putting the proteasome in a direct link with IDH2 inhibition. Finally, inducible IDH2 knock-down enhanced the therapeutic efficacy of CFZ in a subcutaneous xenograft model of MM, resulting in inhibition of tumor progression and extended survival. In conclusion, our data demonstrate that IDH2 inhibition increases the therapeutic efficacy of PIs, thus providing compelling evidence for treatments with lower and less toxic doses, and broadening the application of PIs to other malignancies. Citation Format: Elisa Bergaggio, Chiara Riganti, Giulia Garaffo, Elisabetta Mereu, Nicoletta Vitale, Cecilia Bandini, Elisa Pellegrino, Paola Omedè, Katia Todoerti, Valentina Audrito, Antonio Rossi, Francesco Bertoni, Silvia Deaglio, Antonino Neri, Antonio Palumbo, Roberto Piva. IDH2 inhibition enhances proteasome inhibitor responsiveness in hematological malignancies [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 LB-269.