Sodium arsenite and arsenic trioxide differently affect the oxidative stress of lymphoblastoid cells: An intricate crosstalk between mitochondria, autophagy and cell death.

Although the toxicity of arsenic depends on its chemical forms, few studies have taken into account the ambiguous phenomenon that sodium arsenite (NaAsO2) acts as a potent carcinogen while arsenic trioxide (ATO, As2O3) serves as an effective therapeutic agent in lymphoma, suggesting that NaAsO2 and As2O3 may act via paradoxical ways to either promote or inhibit cancer pathogenesis. Here, we compared the cellular response of the two arsenical compounds, NaAsO2 and As2O3, on the Burkitt lymphoma cell model, the Epstein Barr Virus (EBV)-positive P3HR1 cells. Using flow cytometry and biochemistry analyses, we showed that a NaAsO2 treatment induces P3HR1 cell death, combined with drastic drops in ΔΨm, NAD(P)H and ATP levels. In contrast, As2O3-treated cells resist to cell death, with a moderate reduction of ΔΨm, NAD(P)H and ATP. While both compounds block cells in G2/M and affect their protein carbonylation and lipid peroxidation, As2O3 induces a milder increase in superoxide anions and H2O2 than NaAsO2, associated to a milder inhibition of antioxidant defenses. By electron microscopy, RT-qPCR and image cytometry analyses, we showed that As2O3-treated cells display an overall autophagic response, combined with mitophagy and an unfolded protein response, characteristics that were not observed following a NaAsO2 treatment. As previous works showed that As2O3 reactivates EBV in P3HR1 cells, we treated the EBV- Ramos-1 cells and showed that autophagy was not induced in these EBV- cells upon As2O3 treatment suggesting that the boost of autophagy observed in As2O3-treated P3HR1 cells could be due to the presence of EBV in these cells. Overall, our results suggest that As2O3 is an autophagic inducer which action is enhanced when EBV is present in the cells, in contrast to NaAsO2, which induces cell death. That's why As2O3 is combined with other chemicals, as all-trans retinoic acid, to better target cancer cells in therapeutic treatments.

Molecular targets of glucocorticoids that elucidate their therapeutic efficacy in aggressive lymphomas.

Glucocorticoids have been used for decades to treat lymphomas without an established mechanism of action. Using functional genomic, proteomic, and chemical screens, we discover that glucocorticoids inhibit oncogenic signaling by the B cell receptor (BCR), a recurrent feature of aggressive B cell malignancies, including diffuse large B cell lymphoma and Burkitt lymphoma. Glucocorticoids induce the glucocorticoid receptor (GR) to directly transactivate genes encoding negative regulators of BCR stability (LAPTM5; KLHL14) and the PI3 kinase pathway (INPP5D; DDIT4). GR directly represses transcription of CSK, a kinase that limits the activity of BCR-proximal Src-family kinases. CSK inhibition attenuates the constitutive BCR signaling of lymphomas by hyperactivating Src-family kinases, triggering their ubiquitination and degradation. With the knowledge that glucocorticoids disable oncogenic BCR signaling, they can now be deployed rationally to treat BCR-dependent aggressive lymphomas and used to construct mechanistically sound combination regimens with inhibitors of BTK, PI3 kinase, BCL2, and CSK.

Aflatoxin B1 and Epstein-Barr virus-induced CCL22 expression stimulates B cell infection.

Epstein-Barr Virus (EBV) infects more than 90% of the adult population worldwide. EBV infection is associated with Burkitt lymphoma (BL) though alone is not sufficient to induce carcinogenesis implying the involvement of co-factors. BL is endemic in African regions faced with mycotoxins exposure. Exposure to mycotoxins and oncogenic viruses has been shown to increase cancer risks partly through the deregulation of the immune response. A recent transcriptome profiling of B cells exposed to aflatoxin B1 (AFB1) revealed an upregulation of the Chemokine ligand 22 (CCL22) expression although the underlying mechanisms were not investigated. Here, we tested whether mycotoxins and EBV exposure may together contribute to endemic BL (eBL) carcinogenesis via immunomodulatory mechanisms involving CCL22. Our results revealed that B cells exposure to AFB1 and EBV synergistically stimulated CCL22 secretion via the activation of Nuclear Factor-kappa B pathway. By expressing EBV latent genes in B cells, we revealed that elevated levels of CCL22 result not only from the expression of the latent membrane protein LMP1 as previously reported but also from the expression of other viral latent genes. Importantly, CCL22 overexpression resulting from AFB1-exposure in vitro increased EBV infection through the activation of phosphoinositide-3-kinase pathway. Moreover, inhibiting CCL22 in vitro and in humanized mice in vivo limited EBV infection and decreased viral genes expression, supporting the notion that CCL22 overexpression plays an important role in B cell infection. These findings unravel new mechanisms that may underpin eBL development and identify novel pathways that can be targeted in drug development.

Latent Epstein-Barr virus infection collaborates with Myc over-expression in normal human B cells to induce Burkitt-like Lymphomas in mice.

Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.

7-Dehydrocholesterol is an endogenous suppressor of ferroptosis.

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

Andrographolide inhibits Burkitt's lymphoma by binding JUN and CASP3 proteins.

BACKGROUND: Burkitt's lymphoma, one of the most common subtypes of pediatric malignant lymphoma, is notorious for its swift onset, aggressive proliferation, pronounced invasiveness, and marked malignancy. The therapeutic landscape for Burkitt's lymphoma currently falls short of providing universally effective and tolerable solutions. Andrographolide, a primary active component of Andrographis paniculata, is renowned for its properties of heat-clearing, detoxification, inflammation reduction, and pain relief. It is predominantly used in treating bacterial and viral infections of the upper respiratory tract, as well as dysentery. Various reports highlight the antitumor effects of andrographolide. Yet, its specific impact and the underlying mechanism of action on Burkitt's lymphoma remain an uncharted area of research. METHOD: We employed network pharmacology to pinpoint the targets of andrographolide's action on Burkitt's lymphoma and the associated pathways. We then evaluated the impact of andrographolide on Burkitt's lymphoma using both in vitro and in vivo patient-derived xenograft (PDX) models. Concurrently, we confirmed the molecular targets of andrographolide in Burkitt's lymphoma through immunofluorescence assays. RESULT: Utilizing network pharmacology, we identified 15 relevant targets, 60 interrelationships between these targets, and numerous associated signaling pathways for andrographolide's action on Burkitt's lymphoma. In vitro efficacy tests using High-throughput Drug Sensitivity Testing and in vivo PDX model evaluations revealed that andrographolide effectively curtailed the growth of Burkitt's lymphoma. Moreover, we observed a increased in the expression of JUN (c-Jun) and CASP3 (Caspase 3) proteins in Burkitt's lymphoma cells treated with andrographolide. CONCLUSION: Andrographolide inhibits the growth of Burkitt's lymphoma by inhibiting JUN and CASP3 proteins.

Synthesis and Pro-Apoptotic Effects of Nitrovinylanthracenes and Related Compounds in Chronic Lymphocytic Leukaemia (CLL) and Burkitt's Lymphoma (BL).

Chronic lymphocytic leukaemia (CLL) is a malignancy of the immune B lymphocyte cells and is the most common leukaemia diagnosed in developed countries. In this paper, we report the synthesis and antiproliferative effects of a series of (E)-9-(2-nitrovinyl)anthracenes and related nitrostyrene compounds in CLL cell lines and also in Burkitt's lymphoma (BL) cell lines, a rare form of non-Hodgkin's immune B-cell lymphoma. The nitrostyrene scaffold was identified as a lead structure for the development of effective compounds targeting BL and CLL. The series of structurally diverse nitrostyrenes was synthesised via Henry-Knoevenagel condensation reactions. Single-crystal X-ray analysis confirmed the structure of (E)-9-chloro-10-(2-nitrobut-1-en-1-yl)anthracene (19f) and the related 4-(anthracen-9-yl)-1H-1,2,3-triazole (30a). The (E)-9-(2-nitrovinyl)anthracenes 19a, 19g and 19i-19m were found to elicit potent antiproliferative effects in both BL cell lines EBV-MUTU-1 (chemosensitive) and EBV+ DG-75 (chemoresistant) with >90% inhibition at 10 µM. Selected (E)-9-(2-nitrovinyl)anthracenes demonstrated potent antiproliferative activity in CLL cell lines, with IC50 values of 0.17 µM (HG-3) and 1.3 µM (PGA-1) for compound 19g. The pro-apoptotic effects of the most potent compounds 19a, 19g, 19i, 19l and 19m were demonstrated in both CLL cell lines HG-3 and PGA-1. The (E)-nitrostyrene and (E)-9-(2-nitrovinyl)anthracene series of compounds offer potential for further development as novel chemotherapeutics for CLL.

Network and Computational Drug Repurposing Analysis for c-Myc Inhibition in Burkitt Lymphoma.

BACKGROUND/AIM: The treatment rate of Burkitt lymphoma (BL) is still low in low-income countries and among elderly patients. The c-Myc dysregulation induced by mutations is one of the characteristics of BL. However, studies on the downstream signaling pathways of c-Myc are still lacking. This study aimed to identify the signaling pathways regulated by c-Myc. MATERIALS AND METHODS: Network and gene set analyses using c-Myc inhibition (i.e., c-Myc knock-down and c-Myc inhibitor treatment) transcriptome datasets for BL cell lines were performed to determine the pathways regulated by c-Myc. In addition, computational drug repurposing was used to identify drugs that can regulate c-Myc downstream signaling pathway. RESULTS: Computational drug repurposing revealed that the ERK/MAPK signaling pathway is regulated by c-Myc in BL and that this pathway can be modulated by vorinostat. Furthermore, in the pharmacogenomics database, vorinostat showed a cell viability half-maximal inhibitory concentration of less than 2 µM in the BL cell lines. CONCLUSION: The downstream signaling pathway regulated by c-Myc and the drug that can modulate this pathway is presented for the first time.

Chidamide Induces Epstein-Barr Virus (EBV) Lytic Infection and Acts Synergistically with Tenofovir to Eliminate EBV-Positive Burkitt Lymphoma.

Epstein-Barr virus (EBV) is a type of human γ-herpesvirus, and its reactivation plays an important role in the development of EBV-driven Burkitt lymphoma (BL). Despite intensive chemotherapy, the prognosis of relapsed/refractory BL patients remains unfavorable, and a definitive method to completely eliminate latent EBV infection is lacking. Previous studies have demonstrated that histone deacetylase (HDAC) inhibitors can induce the transition of EBV from latency to the lytic phase. The lytic activation of EBV can be inhibited by tenofovir, a potent inhibitor of DNA replication. Herein, we explored the antitumor effect and EBV clearance potential of a novel HDAC inhibitor called chidamide, combined with tenofovir, in the treatment of EBV-positive BL. In the study, chidamide exhibited inhibitory activity against HDAC. Moreover, chidamide inhibited BL cell proliferation, arrested cell cycle progression, and induced BL cell apoptosis primarily by regulating the MAPK pathways. Additionally, chidamide promoted the transcription of lytic genes, including BZLF1, BMRF1, and BMLF1 Compared with chidamide alone, the addition of tenofovir further induced growth arrest and apoptosis in EBV-positive BL cells and inhibited the transcriptions of EBV lytic genes induced by chidamide alone. Furthermore, our in vivo data demonstrated that the combination of chidamide and tenofovir had superior tumor-suppressive effects in a mouse model of BL cell tumors. The aforementioned findings confirm the synergistic effect of chidamide combined with tenofovir in inducing growth inhibition and apoptosis in EBV-positive BL cells and provide an effective strategy for eliminating EBV and EBV-associated malignancies. SIGNIFICANCE STATEMENT: High levels of Epstein-Barr virus (EBV)-DNA have consistently been associated with unfavorable progression-free survival and overall survival in EBV-associated lymphomas. Therefore, identifying novel strategies to effectively eradicate tumor cells and eliminate EBV is crucial for lymphoma patients. This study confirmed, for the first time, the synergistic effect of chidamide combined with tenofovir in the treatment of Burkitt lymphoma and the eradication of EBV virus.

c-Myc inhibits LAPTM5 expression in B-cell lymphomas.

Myc is a pivotal protooncogenic transcription factor that contributes to the development of almost all Burkitt's lymphomas and about one-third of diffuse large B-cell lymphomas. How B-cells sustain their uncontrolled proliferation due to high Myc is not yet well defined. Here, we found that Myc trans-represses the expression of murine LAPTM5, a gene coding a lysosome-associated protein, by binding to two E-boxes in the LAPTM5 promoter. While the product of intact mRNA (CDS+3'UTR) of LAPTM5 failed to suppress the growth of B-lymphomas, either the protein coded by coding sequence (CDS) itself or the non-coding 3'-untranslated region (3'UTR) mRNA was able to inhibit the growth of B-lymphomas. Moreover, Myc trans-activated miR-17-3p, which promoted tumor growth. Strikingly, LAPTM5 3'UTR contains 11 miR-17-3p-binding sites through which the LAPTM5 protein synthesis was inhibited. The functional interplay between low LAPTM5 mRNA and high miR-17-3p due to high Myc in B-lymphomas leads to further dampening of tumor-suppressive LAPTM5 protein, which promotes tumor progression. Our results indicate that Myc inhibits LAPTM5 expression in B-lymphoma cells by transcriptional and post-transcriptional modifications.

The impact of Down syndrome-specific non-malignant hematopoietic regeneration in the bone marrow on the detection of leukemic measurable residual disease.

BACKGROUND: Detection of measurable residual disease detection (MRD) by flow cytometry after the first course of chemotherapy is a standard measure of early response in patients with acute myeloid leukemia (AML). Myeloid leukemia associated with Down Syndrome (ML-DS) is a distinct form of AML. Differences in steady-state and regenerating hematopoiesis between patients with or without DS are not well understood. This understanding is essential to accurately determine the presence of residual leukemia in patients with ML-DS. METHODS: A standardized antibody panel defined quantitative antigen expression in 115 follow-up bone marrow (BM) aspirates from 45 patients following chemotherapy for ML-DS or DS precursor B-cell acute lymphoblastic leukemia (B-ALL-DS) with the "difference from normal (ΔN)" technique. When possible, FISH and SNP/CGH microarray studies were performed on sorted cell fractions. RESULTS: 93% of BM specimens submitted post chemotherapy had a clearly identifiable CD34+ CD56+ population present between 0.06% and 2.6% of total non-erythroid cells. An overlapping CD34+ HLA-DRheterogeneous population was observed among 92% of patients at a lower frequency (0.04%-0.8% of total non-erythroid cells). In B-ALL-DS patients, the same CD34+ CD56+ HLA-DRheterogeneous expression was observed. FACS-FISH/Array studies demonstrated no residual genetic clones in the DS-specific myeloid progenitor cells. CONCLUSIONS: Non-malignant myeloid progenitors in the regenerating BM of patients who have undergone chemotherapy for either ML-DS or B-ALL-DS express an immunophenotype that is different from normal BM of non-DS patients. Awareness of this DS-specific non-malignant myeloid progenitor is essential to the interpretation of MRD by flow cytometry in patients with ML-DS.

Epstein-Barr virus reactivation induces MYC-IGH spatial proximity and t(8;14) in B cells.

Burkitt lymphoma (BL) is a B cell malignancy associated with the Epstein-Barr virus (EBV). Most BL cases are characterized by a t(8;14) chromosomal translocation involving the MYC oncogene and the immunoglobulin heavy chain gene (IGH). The role of EBV in promoting this translocation remains largely unknown. Here we provide the experimental evidence that EBV reactivation from latency leads to an increase in the proximity between the MYC and IGH loci, otherwise located far away in the nuclear space both in B-lymphoblastoid cell lines and in patients' B-cells. Specific DNA damage within the MYC locus, followed by the MRE11-dependent DNA repair plays a role in this process. Using a CRISPR/Cas9-based B cell model to induce specific DNA double strand breaks in MYC and IGH loci, we have shown that the MYC-IGH proximity induced by EBV reactivation leads to an increased t(8;14) translocation frequency.

Inhibition of SUMOylation enhances DNA hypomethylating drug efficacy to reduce outgrowth of hematopoietic malignancies.

Combination therapies targeting malignancies aim to increase treatment efficacy and reduce toxicity. Hypomethylating drug 5-Aza-2'-deoxycytidine (5-Aza-2') enhances transcription of tumor suppressor genes and induces replication errors via entrapment of DNMT1, yielding DNA-protein crosslinks. Post-translational modification by SUMO plays major roles in the DNA damage response and is required for degradation of entrapped DNMT1. Here, we combine SUMOylation inhibitor TAK981 and DNA-hypomethylating agent 5-Aza-2'-deoxycytidine to improve treatment of MYC driven hematopoietic malignancies, since MYC overexpressing tumors are sensitive to SUMOylation inhibition. We studied the classical MYC driven malignancy Burkitt lymphoma, as well as diffuse large B-cell lymphoma (DLBCL) with and without MYC translocation. SUMO inhibition prolonged the entrapment of DNMT1 to DNA, resulting in DNA damage. An increase in DNA damage was observed in cells co-treated with TAK981 and 5-Aza-2'. Both drugs synergized to reduce cell proliferation in vitro in a B cell lymphoma cell panel, including Burkitt lymphoma and DLBCL. In vivo experiments combining TAK981 (25 mg/kg) and 5-Aza-2' (2.5 mg/kg) showed a significant reduction in outgrowth of Burkitt lymphoma in an orthotopic xenograft model. Our results demonstrate the potential of tailored combination of drugs, based on insight in molecular mechanisms, to improve the efficacy of cancer therapies.

Plasma membrane topography governs the 3D dynamic localization of IgM B cell antigen receptor clusters.

B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces, and are thought to perform sensory functions in screening antigen-bearing surfaces. Here, we have used lattice light-sheet microscopy in combination with tailored custom-built 4D image analysis to study the cell-surface topography of B cells of the Ramos Burkitt's Lymphoma line and the spatiotemporal organization of the IgM-BCR. Ramos B-cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A fraction of membrane-localized IgM-BCR was found in clusters, which were mainly associated with the ridges and the microvilli. The dynamic ridge-network organization and the IgM-BCR cluster mobility were linked, and both were controlled by Arp2/3 complex activity. Our results suggest that dynamic topographical features of the cell surface govern the localization and transport of IgM-BCR clusters to facilitate antigen screening by B cells.

Marine Sponge-Derived Alkaloid Induces Mitochondrial Dysfunction and Inhibits the PI3K/AKT/mTOR Signaling Pathway against Burkitt's Lymphoma.

Burkitt's lymphoma (BL) has a particularly extremely poor prognosis and the fastest growth rate among human tumors, and the development of new drugs for the treatment of BL is urgently needed. In this study, the cytotoxic properties of 3,7-bis(3,5-dimethylphenyl)-aaptamine (AP-51), a new semisynthetic alkaloid derived from the marine natural product aapatamine, were investigated using BL cell lines. Our results showed that AP-51 inhibited the proliferation of Daudi and Raji cells with IC50 values of 3.48 and 2.07 µM, respectively. Flow cytometry and Western blot analyses showed that AP-51 initiated G0/G1 phase arrest by modulating the expression of cyclin-dependent kinases (CDKs). AP-51 also induced apoptosis, as demonstrated by nuclear fragmentation, downregulation of BCL-XL and Mcl-1, and upregulation of cleaved caspase-9, cleaved caspase-3, cleaved-PARP, and cytochrome c, the markers of apoptosis regulated via the mitochondrial pathway. When it comes to mitochondria, AP-51 treatment also significantly increased the levels of intracellular mitochondrial superoxide, decreased ATP content, and reduced the expression of ATP synthase, as well as the expression of the mitochondrial respiratory chain complexes. Finally, AP-51 treatment significantly inhibited the PI3K/AKT/mTOR signaling pathway, which was shown to be associated with the induction of apoptosis. Collectively, these findings indicated that AP-51 initiated cell cycle arrest, induced apoptosis, caused mitochondrial dysfunction, and decreased the phosphorylation of PI3K/AKT/mTOR signaling pathway-related proteins and the protein levels of C-MYC, suggesting that AP-51 has therapeutic potential as a possible treatment for Burkitt's lymphoma.

Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive form of B cell lymphoma that can affect children and adults. The study of BL led to the identification of the first recurrent chromosomal aberration in lymphoma, t(8;14)(q24;q32), and subsequent discovery of the central role of MYC and Epstein-Barr virus (EBV) in tumorigenesis. Most patients with BL are cured with chemotherapy but those with relapsed or refractory disease usually die of lymphoma. Historically, endemic BL, non-endemic sporadic BL and the immunodeficiency-associated BL have been recognized, but differentiation of these epidemiological variants is confounded by the frequency of EBV positivity. Subtyping into EBV+ and EBV- BL might better describe the biological heterogeneity of the disease. Phenotypically resembling germinal centre B cells, all types of BL are characterized by dysregulation of MYC due to enhancer activation via juxtaposition with one of the three immunoglobulin loci. Additional molecular changes commonly affect B cell receptor and sphingosine-1-phosphate signalling, proliferation, survival and SWI-SNF chromatin remodelling. BL is diagnosed on the basis of morphology and high expression of MYC. BL can be effectively treated in children and adolescents with short durations of high dose-intensity multiagent chemotherapy regimens. Adults are more susceptible to toxic effects but are effectively treated with chemotherapy, including modified versions of paediatric regimens. The outcomes in patients with BL are good in high-income countries with low mortality and few late effects, but in low-income and middle-income countries, BL is diagnosed late and is usually treated with less-effective regimens affecting the overall good outcomes in patients with this lymphoma.

Novel lectin-based chimeric antigen receptors target Gb3-positive tumour cells.

The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins  that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.

[Reduced Expression of the Tissue-Specific Oct-IL Isoform Exerts an Antitumor Effect on Namalwa Burkitt's Lymphoma Cells].

Increased expression levels of the Oct-1 transcription factor is considered to be one of the key markers of poor cancer prognosis. In addition to the ubiquitous Oct-1A isoform, which is found in all cells, there also exists a tissue-specific Oct-1L isoform, which is expressed in hematopoietic cells. Oct-1L increases cell resistance to different stresses and also regulates the expression of genes controlling differentiation of hematopoietic and immune system cells. The tissue-specific Oct-1L isoform levels are significantly increased in the B-cell lymphoblastoma Namalwa and Raji lines and the T-cell lymphoblastoma Jurkat line compared to normal B and T cells. Apparently, aberrant Oct-1L overexpression not only enhances stress resistance but also leads to the disruption of developmental pathways in the cells promoting their malignant transformation. We report here that targeted suppression of the tissue-specific Oct-1L isoform expression reduces the proliferation rate of Namalwa B-lymphoblastic Burkitt's lymphoma cells, significantly increases cell death rate under hypoxic conditions, and makes cells more sensitive to chemotherapeutic agents such as docetaxel and doxorubicin. These results indicate that targeted therapy aimed at the suppression of the Oct-1 isoforms with increased expression levels in tumor cells rather than the total Oct-1, thus avoiding the traumatic effects of total Oct-1 knockdown, may be promising. Selective suppression of Oct-1 isoforms is a promising strategy in the treatment of lymphoid tumors and may contribute to mitigating the disease course and increasing survival rates in cancer patients.

The lncRNA KTN1-AS1 co-regulates a variety of Myc-target genes and enhances proliferation of Burkitt lymphoma cells.

Long non-coding RNAs (lncRNAs) are involved in many normal and oncogenic pathways through a diverse repertoire of transcriptional and posttranscriptional regulatory mechanisms. LncRNAs that are under tight regulation of well-known oncogenic transcription factors such as c-Myc (Myc) are likely to be functionally involved in their disease-promoting mechanisms. Myc is a major driver of many subsets of B cell lymphoma and to date remains an undruggable target. We identified three Myc-induced and four Myc-repressed lncRNAs by use of multiple in vitro models of Myc-driven Burkitt lymphoma and detailed analysis of Myc binding profiles. We show that the top Myc-induced lncRNA KTN1-AS1 is strongly upregulated in different types of B cell lymphoma compared with their normal counterparts. We used CRISPR-mediated genome editing to confirm that the direct induction of KTN1-AS1 by Myc is dependent on the presence of a Myc E-box-binding motif. Knockdown of KTN1-AS1 revealed a strong negative effect on the growth of three BL cell lines. Global gene expression analysis upon KTN1-AS1 depletion shows a strong enrichment of key genes in the cholesterol biosynthesis pathway as well as co-regulation of many Myc-target genes, including a moderate negative effect on the levels of Myc itself. Our study suggests a critical role for KTN1-AS1 in supporting BL cell growth by mediating co-regulation of a variety of Myc-target genes and co-activating key genes involved in cholesterol biosynthesis. Therefore, KTN1-AS1 may represent a putative novel therapeutic target in lymphoma.

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.