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.

Circular ZDHHC11 supports Burkitt lymphoma growth independent of its miR-150 binding capacity.

We previously showed that MYC promoted Burkitt lymphoma (BL) growth by inhibiting the tumor suppressor miR-150, resulting in release of miR-150 targets MYB and ZDHHC11. The ZDHHC11 gene encodes three different transcripts including a mRNA (pcZDHHC11), a linear long non-coding RNA (lncZDHHC11) and a circular RNA (circZDHHC11). All transcripts contain the same region with 18 miR-150 binding sites. Here we studied the relevance of circZDHHC11, including this miR-150 binding site region, for growth of BL cells. CircZDHHC11 was mainly present in the cytoplasmic fraction in BL cells and its localization was not altered upon miR-150 overexpression. Knockdown of circZDHHC11 caused a strong inhibition of BL growth without affecting the expression levels of MYC, MYB, miR-150 and other genes. Overexpression of circZDHHC11 neither affected cell growth, nor rescued the phenotype induced by miR-150 overexpression. Genomic deletion of the miR-150 binding site region did not affect growth, nor did it change the effect of circZDHHC11 knockdown. This indicated that the miR-150 binding site region is dispensable for the growth promoting role of circZDHHC11. To conclude, our results show that circZDHHC11 is a crucial factor supporting BL cell growth independent of its ability to sponge miR-150.

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.

Targeting latent viral infection in EBV-associated lymphomas.

Epstein-Barr virus (EBV) contributes to the development of a significant subset of human lymphomas. As a herpes virus, EBV can transition between a lytic state which is required to establish infection and a latent state where a limited number of viral antigens are expressed which allows infected cells to escape immune surveillance. Three broad latency programs have been described which are defined by the expression of viral proteins RNA, with latency I being the most restrictive expressing only EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) and latency III expressing the full panel of latent viral genes including the latent membrane proteins 1 and 2 (LMP1/2), and EBNA 2, 3, and leader protein (LP) which induce a robust T-cell response. The therapeutic use of EBV-specific T-cells has advanced the treatment of EBV-associated lymphoma, however this approach is only effective against EBV-associated lymphomas that express the latency II or III program. Latency I tumors such as Burkitt lymphoma (BL) and a subset of diffuse large B-cell lymphomas (DLBCL) evade the host immune response to EBV and are resistant to EBV-specific T-cell therapies. Thus, strategies for inducing a switch from the latency I to the latency II or III program in EBV+ tumors are being investigated as mechanisms to sensitize tumors to T-cell mediated killing. Here, we review what is known about the establishment and regulation of latency in EBV infected B-cells, the role of EBV-specific T-cells in lymphoma, and strategies to convert latency I tumors to latency II/III.

Burkitt lymphoma: The effect of age, sex and delay to diagnosis on treatment completion and outcome of treatment in 934 Patients in Cameroon.

INTRODUCTION: The role of age and sex in the presentation and outcome of endemic Burkitt lymphoma (BL) has not been studied recently. This study analysed these factors in 934 patients with BL who had received cyclophosphamide and intrathecal methotrexate as treatment. METHODS: Records of 934 children diagnosed with BL from 2004 to 2015 were obtained from our Paediatric Oncology Networked Database (POND) cancer registry. Age at diagnosis, sex, disease stage, time to diagnosis, delay in diagnosis, completion of treatment, rate of abandonment, and one-year survival rates were recorded and statistically analysed. RESULTS: The male to female ratio of 1.41 for the study population of 934. The median delay from onset of symptoms to diagnosis was 31 days. The St Jude stage distribution was I = 6.4%, II = 5.9%, III = 71.5% and IV = 16.2%. Significantly more patients presented with stage III disease in age groups 5-9 and 10-14 years than 0-4 years. The overall 1-year survival rate was 53.45%, respectively 77.1% for stage I, 67.9% for stage II, 55.1% for stage III and 32.4% for stage IV disease (p<0.001). There was no significant difference in survival by sex and age group. CONCLUSION: Patients aged under 5 years presented with less-advanced disease, but survival was not affected by age. Sex did not influence delay to diagnosis and overall survival. The long delay between the onset of symptoms and diagnosis emphasises the need for interventions to achieve an earlier diagnosis and a better survival rate.

From the Total Synthesis of Semi-Viriditoxin, Semi-Viriditoxic Acid and Dimeric Naphthopyranones to their Biological Activities in Burkitt B Cell Lymphoma.

Dimeric naphthopyranones are known to be biologically active, however, for the corresponding monomeric naphthopyranones this information is still elusive. Here the first enantioselective total synthesis of semi-viriditoxic acid as well as the synthesis of semi-viriditoxin and derivatives is reported. The key intermediate in the synthesis of naphthopyranones is an α,ß-unsaturated δ-lactone, which we synthesized in two different ways (Ghosez-cyclization and Grubbs ring-closing metathesis), while the domino-Michael-Dieckmann reaction of the α,ß-unsaturated δ-lactone with an orsellinic acid derivative is the key reaction. A structure-activity relationship study was performed measuring the cytotoxicity in Burkitt B lymphoma cells (Ramos). The dimeric structure was found to be crucial for biological activity: Only the dimeric naphthopyranones showed cytotoxic and apoptotic activity, whereas the monomers did not display any activity at all.

Deciphering stage 0 hematogones by flow cytometry in follow-up bone marrow samples of pediatric B-Acute lymphoblastic leukemia cases: A potential mimicker of residual disease after anti CD19 therapy.

CD19 is frequently targeted for immunotherapy in B cell malignancies, which may result in loss of CD19 expression in leukemic cells as an escape mechanism. Stage 0 hematogones (Hgs) are normal CD19-negative very early B cell precursors that can be potentially mistaken for CD19 negative residual leukemic cells by flow cytometry (FCM) in B cell acute lymphoblastic leukemia (BCP-ALL) cases treated with anti CD19 therapy. Our main objective was to characterize and study the incidence of stage 0 hematogones in follow-up bone marrow samples of pediatric BCP-ALL cases. We analyzed the flow cytometry standard files of 61 pediatric BCP-ALL cases treated with conventional chemotherapy and targeted anti-CD19 therapy, for identifying the residual disease and normal B cell precursors including stage 0 Hgs. A non-CD19 alternate gating strategy was used to isolate the B cells for detecting the residual disease and stage 0 Hgs. The stage 0 Hgs were seen in 95% of marrow samples containing CD19+ Hgs. When compared with controls and posttransplant marrow samples, the fraction of stage 0 Hgs was higher in patients receiving anti CD19 therapy (p = 0.0048), but it was not significant when compared with patients receiving chemotherapy (p = 0.1788). Isolated stage 0 Hgs are found in samples treated with anti-CD19 therapy simulating CD19 negative residual illness. Our findings aid in understanding the stage 0 Hgs and its association with CD19+ Hgs in anti CD19 therapy and conventional chemotherapy. This is crucial as it can be potentially mistaken for residual disease in patients treated with anti CD19 therapy.

Primary manifestation of HIV-related Burkitt lymphoma in the oral and maxillofacial regions.

BACKGROUND: Burkitt lymphoma (BL) is a subtype of non-Hodgkin lymphoma. It is strongly associated with HIV infection and has a highly aggressive clinical course. The involvement of the maxillofacial region in BL has rarely been reported. CASE DESCRIPTION: A 36-year-old woman with HIV-positive status had painless bilateral swelling of the oral mucosa and middle and lower thirds of the face. Microscopic analysis of the oral lesion revealed an atypical lymphoid infiltrate with a starry sky pattern. The lymphoid cells expressed cluster of differentiation 20, cluster of differentiation 10, B-cell lymphoma 6, and c-Myc; the Ki-67 proliferative index was high. The tumor cells were positive for Epstein-Barr virus. These results led to the diagnosis of HIV-related BL. PRACTICAL IMPLICATIONS: BL and other immunodeficiency-related lymphoproliferative malignancies may affect the oral and maxillofacial regions and should be included in the differential diagnosis of rapidly expanding swelling in young patients.

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.

The urgency of Burkitt lymphoma diagnosis in fluid cytology-A tertiary care experience.

BACKGROUND: Burkitt lymphoma (BL) is an aggressive high-grade B-cell non-Hodgkin lymphoma commonly diagnosed in young age and is known to involve extra nodal sites. But the involvement of body fluids by BL is an uncommon presentation. Rapid diagnosis of BL is vital to prevent complications like tumour lysis syndrome. Cytological examination of body fluids continues to be an indispensable tool for rapid diagnosis of BL. OBJECTIVES: In this study, we aim to study the clinical, cytomorphological and immunophenotypic characteristics of BL involving serous effusions and other fluids. MATERIALS AND METHODS: In this retrospective study, 17 cases reported as BL in fluid cytology from 2016 to 2022 were collected and reviewed. We performed a comprehensive analysis of the clinical data, cytomorphological features, immunophenotyping data along with the haematological workup of these cases. We have also compared with the histopathological diagnosis for those cases where biopsy was available. RESULTS: BL more commonly involved ascitic fluid (52%), followed by pleural fluid (4 cases) and cerebrospinal fluid (CSF; 4 cases). Primary diagnosis of BL in fluid was done in 88% of the cases. Bone marrow involvement was noted in two cases. Cytological smears showed discrete monomorphous population of medium-sized atypical lymphoid cells with frequent apoptotic bodies. Classic cytoplasmic punched out vacuoles were observed in 88% of the cases. Immunophenotyping data was available for 12 cases in which tumour cells showed positivity for CD20 (100%), CD10 (4 of 7 cases), BCL6 (3 of 5 cases) and cMYC (7 of 7 cases-100%) and were negative for Terminal deoxynucleotidyl transferase (TdT) (11 of 11 cases). Mean Ki67 labelling index was 95%. Histopathological diagnosis was available for 9 cases, and there was 100% agreement between cytological and histopathological diagnosis in 7 cases. CONCLUSION: Precise diagnosis of BL can be rendered in body fluids by identification of classic cytomorphological features and by performing supportive ancillary tests in fluids for immunophenotyping.

Optimal dosage of rituximab for children with Burkitt lymphoma.

The current chemotherapy treatments have led to an improvement in survival rates for pediatric Burkitt's lymphoma (BL). Survival in children with high-grade, mature B-cell non-Hodgkin's lymphoma (B-NHL) has been prolonged by six rituximab doses combined with chemotherapy, whereas the efficacy of four doses has not been reported. This study aimed to explore optimal therapeutic strategies-the number of doses of rituximab based on different risk groups-and also aim to investigate the clinical characteristics of Chinese pediatric BL. This study consecutively enrolled children with BL in Beijing Children's Hospital who received French-American-British mature B-cell lymphoma 96 (FAB/LMB96). The patients were divided into three groups: R0 group (chemotherapy alone), R6 group (chemotherapy combined with six rituximab doses), and R4 group (chemotherapy combined with four rituximab doses). The clinical characteristics and outcomes were evaluated. Univariate and multivariate analyses and prognostic nomogram were used to assess prognostic factors. A nomogram was developed that predicted overall survival based on the Cox proportional hazards model, and the concordance index (C-index) and a calibration curve were used to determine its predictive and discriminatory capacity. We enrolled 385 boys and 71 girls, with a median age of 6 years (1-14 years). Of these, 296 patients (65%) had initial abdominal symptoms, 182 (40%) had bulky disease, 46 (10%) had B symptoms, 77 (16.9%) had BL-ALL (blasts ≥ 25% in bone marrow (BM)), 96 (21%) had central nervous system (CNS) disease, 406 (89%) were in stages III-IV, 378 (83%) were in group C, 170 (37.2%) had lactate dehydrogenase (LDH) levels ≥ 1000 U/L at initial diagnosis, and 137 (30%) had tumor lysis syndrome. The R0, R6, and R4 groups included 79, 144, and 227 patients, respectively. Six patients were excluded due to treatment withdrawal for various reasons. The 3-year overall survival (OS) and event-free survival (EFS) percentages were 92% ± 1.3% and 91.3% ± 1.3%, respectively, in all cohorts, whereas the 3-year EFS percentage was 83.5% ± 4.2%, 93% ± 2.1%, and 92.9% ± 1.8% in the R0, R6, and R4 groups, respectively (P = 0.025). The nomogram included four important variables based on a multivariate analysis of the primary cohort: course of disease ≤ 20 days, presence of bulky disease at the beginning of diagnosis, central nervous system(CNS) invasion, and dosage of rituximab. The calibration curve showed that the nomogram was able to predict 3-year OS accurately. The C-index of the nomogram for OS prediction was 0.79 for both cohorts. In our hospital, pediatric BL was more commonly observed in school-age boys with an abdominal mass and mostly in advanced stages at initial diagnosis. The FAB/LMB96 regimen combined with rituximab significantly increased survival outcomes. We observed no significant differences between four and six doses of rituximab in terms of treatment outcomes. The proposed nomogram provides an individualized risk estimate of OS in patients with BL and may assist treatment decision-making or rituximab dose design.

Feasibility and outcomes after dose reduction of immunochemotherapy in young adults with Burkitt lymphoma and leukemia: results of the BURKIMAB14 trial.

High dose-intensive or infusional intermediate-dose immunochemotherapy is highly effective treatment for Burkitt lymphoma irrespective of human immunodeficiency virus (HIV) infection. However, toxicities of these regimens are relevant, especially in older adults and elderly patients. The prospective multicenter BURKIMAB14 trial included four to six blocks of immunochemotherapy according to stage (localized: 1 and 2 non-bulky; advanced: 2 bulky, 3, 4) and age, with dose reduction in patients >55 years old. Dose-intensity of chemotherapy was reduced in patients ≤55 years old after achieving complete metabolic response (CMR). Their outcomes were compared with those of similar patients included in the former BURKIMAB08 trial, in which there was no dose reduction. CMR was attained in 86 of 107 (80%) patients (17/19 in localized stages and 69/88 in advanced stages). Patients from the BURKIMAB14 trial ≤55 years old showed similar overall survival (OS), fewer infections and cytopenias than patients from the BURKIMAB08 trial. Patients >55 years old had a significantly higher treatment- related mortality despite dose reduction of chemotherapy. With a median follow-up of 3.61 years the 4-year OS probability was 73% (range, 63-81%). Age (≤55 vs. >55 years) and stage (localized vs. advanced) had prognostic significance. No significant differences in OS were observed in HIV-positive versus HIV-negative patients. The results of BURKIMAB14 are similar to those of other dose-intensive immunochemotherapy trials. Age >55 years and advanced stage, but not HIV infection, were associated with poor survival. Dose reduction of chemotherapy in young adults in CMR is safe and does not impact outcomes (clinicaltrials gov. Identifier: NCT05049473).

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.

Mosaic chromosomal alterations in peripheral blood leukocytes of children in sub-Saharan Africa.

In high-income countries, mosaic chromosomal alterations in peripheral blood leukocytes are associated with an elevated risk of adverse health outcomes, including hematologic malignancies. We investigate mosaic chromosomal alterations in sub-Saharan Africa among 931 children with Burkitt lymphoma, an aggressive lymphoma commonly characterized by immunoglobulin-MYC chromosomal rearrangements, 3822 Burkitt lymphoma-free children, and 674 cancer-free men from Ghana. We find autosomal and X chromosome mosaic chromosomal alterations in 3.4% and 1.7% of Burkitt lymphoma-free children, and 8.4% and 3.7% of children with Burkitt lymphoma (P-values = 5.7×10-11 and 3.74×10-2, respectively). Autosomal mosaic chromosomal alterations are detected in 14.0% of Ghanaian men and increase with age. Mosaic chromosomal alterations in Burkitt lymphoma cases include gains on chromosomes 1q and 8, the latter spanning MYC, while mosaic chromosomal alterations in Burkitt lymphoma-free children include copy-neutral loss of heterozygosity on chromosomes 10, 14, and 16. Our results highlight mosaic chromosomal alterations in sub-Saharan African populations as a promising area of research.

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.

Evaluation of potential role of R-loop and G-quadruplex DNA in the fragility of c-MYC during chromosomal translocation associated with Burkitt's lymphoma.

t(8;14) translocation is the hallmark of Burkitt's lymphoma and results in c-MYC deregulation. During the translocation, c-MYC gene on chromosome 8 gets juxtaposed to the Ig switch regions on chromosome 14. Although the promoter of c-MYC has been investigated for its mechanism of fragility, little is known about other c-MYC breakpoint regions. We have analyzed the translocation break points at the exon 1/intron 1 of c-MYC locus from patients with Burkitt's lymphoma. Results showed that the breakpoint region, when present on a plasmid, could fold into an R-loop confirmation in a transcription-dependent manner. Sodium bisulfite modification assay revealed significant single-strandedness on chromosomal DNA of Burkitt's lymphoma cell line, Raji, and normal lymphocytes, revealing distinct R-loops covering up to 100 bp region. Besides, ChIP-DRIP analysis reveals that the R-loop antibody can bind to the breakpoint region. Further, we show the formation of stable parallel intramolecular G-quadruplex on non-template strand of the genome. Finally, incubation of purified AID in vitro or overexpression of AID within the cells led to enhanced mutation frequency at the c-MYC breakpoint region. Interestingly, anti-γH2AX can bind to DSBs generated at the c-MYC breakpoint region within the cells. The formation of R-loop and G-quadruplex was found to be mutually exclusive. Therefore, our results suggest that AID can bind to the single-stranded region of the R-loop and G4 DNA, leading to the deamination of cytosines to uracil and induction of DNA breaks in one of the DNA strands, leading to double-strand break, which could culminate in t(8;14) chromosomal translocation.