Namalwa Cells Research Articles

Chevalierlin: A spirocyclic alkaloid from a hydrothermal vent associated fungus Aspergillus chevalieri TW132-65

A previously undescribed spirodiketopiperazine-indole alkaloid, chevalierlin (1), two pairs of previously undescribed dihydroisocoumarin enantiomers eurotiumides H–I (2–3), as well as six related known compounds (4–9) were isolated from the culture of a hydrothermal vent associated fungus Aspergillus chevalieri TW132-65. Their structures were unambiguously determined by NMR, mass spectrometry, and ECD calculations. Chevalierlin (1) exhibits moderate cytotoxic activities with IC50 values of 6.20 ± 0.05 μM and 7.68 ± 0.01 μM against Namalwa and Raji cell lines.

Humanization of the antigen-recognition domain does not impinge on the antigen-binding, cytokine secretion, and antitumor reactivity of humanized nanobody-based CD19-redirected CAR-T cells

BackgroundThe immunogenicity of the antigen-recognition domains of chimeric antigen receptor (CAR)-T cells leads to immune responses that may compromise the antitumor effects of the adoptively transferred T cells. Herein, we attempt to humanize a CD19-specific VHH (named H85) using in silico techniques and investigate the impact of antigen-recognition domain humanization on CAR expression and density, cytokine secretion, and cytolytic reactivity of CAR-T cells based on the humanized VHH.MethodsH85 was humanized (named HuH85), and then HuH85 was compared with H85 in terms of conformational structure, physicochemical properties, antigenicity and immunogenicity, solubility, flexibility, stability, and CD19-binding capacity using in silico techniques. Next, H85CAR-T cells and HuH85CAR-T cells were developed and CAR expression and surface density were assessed via flow cytometry. Ultimately, the antitumor reactivity and secreted levels of IFN-γ, IL-2, and TNF-α were assessed following the co-cultivation of the CAR-T cells with Ramos, Namalwa, and K562 cells.ResultsIn silico findings demonstrated no negative impacts on HuH85 as a result of humanization. Ultimately, H85CAR and HuH85CAR could be surface-expressed on transduced T cells at comparable levels as assessed via mean fluorescence intensity. Moreover, H85CAR-T cells and HuH85CAR-T cells mediated comparable antitumor effects against Ramos and Namalwa cells and secreted comparable levels of IFN-γ, IL-2, and TNF-α following co-cultivation.ConclusionHuH85 can be used to develop immunotherapeutics against CD19-associated hematologic malignancies. Moreover, HuH85CAR-T cells must be further investigated in vitro and in preclinical xenograft models of CD19+ leukemias and lymphomas before advancing into clinical trials.

Determination of in vitro immunotoxic potencies of a series of perfluoralkylsubstances (PFASs) in human Namalwa B lymphocyte and human Jurkat T lymphocyte cells.

Exposure to PFASs is associated to several adverse health effects, such as immunotoxicity. Immunotoxic effects of PFOA and PFOS, including a reduced antibody response in both experimental animals and humans, have been reported. However, there is limited understanding of the underlying mechanisms involved. Moreover, there is only a restricted amount of immunotoxicity data available for a limited number of PFASs. In the current study the effects of 15 PFASs, including short- and long-chain perfluorinated carboxylic and sulfonic acids, fluorotelomer alcohols, and perfluoralkyl ether carboxylic acids were studied on the expression of recombinant activating gene 1 (RAG1) and RAG2 in the Namalwa human B lymphoma cell line, and on the human IL-2 promotor activity in Jurkat T-cells. Concentration-response data were subsequently used to derive in vitro relative potencies through benchmark dose analysis. In vitro relative potency factors (RPFs) were obtained for 6 and 9 PFASs based on their effect on RAG1 and RAG2 gene expression in Namalwa B-cells, respectively, and for 10 PFASs based on their inhibitory effect on IL-2 promotor activity in Jurkat T-cells. The most potent substances were HFPO-TA for the reduction of RAG1 and RAG2 gene expression in Namalwa cells (RPFs of 2.1 and 2.3 respectively), and PFDA on IL-2 promoter activity (RPF of 9.1). RAG1 and RAG2 play a crucial role in V (D)J gene recombination, a process for acquiring a varied array of antibodies crucial for antigen recognition. Hence, the effects observed in Namalwa cells might indicate a PFAS-induced impairment of generating a diverse range of B-cells essential for antigen recognition. The observed outcomes in the Jurkat T-cells suggest a possible PFAS-induced reduction of T-cell activation, which may contribute to a decline in the T-cell dependent antibody response. Altogether, the present study provides potential mechanistic insights into the reported PFAS-induced decreased antibody response. Additionally, the presented in vitro models may represent useful tools for assessing the immunotoxic potential of PFASs and prioritization for further risk assessment.

Role of Epstein-Barr Virus in Breast Cancer: Correlation with Clinical Outcome and Survival Analysis.

Background: Breast cancer is the most prevalent cancer among women worldwide. The potential involvement of Epstein-Barr virus (EBV) in breast cancer pathogenesis has been a subject of debate, but its correlation with clinical outcomes remains uncertain. Methods: In this study, we collected 276 pathologically confirmed breast cancer tissue samples from the tissue bank of MacKay Memorial Hospital and the National Health Research Institutes in Taiwan. DNA was extracted from frozen tissue using The QIAamp DNA Mini Kit. The Taqman quantitative PCR method was employed to assess the EBV copy number per cell in these samples, using NAMALWA cells as a reference. We performed statistical analyses, including 2 × 2 contingency tables, Cox regression analysis, and Kaplan-Meier survival curves, to explore the association between clinicopathologic factors and survival outcomes in breast cancer patients. We analyzed both relapse survival, which reflects the period patients remain free from cancer recurrence post-treatment, and overall survival, which encompasses all-cause mortality. Results: Our results revealed a significant association between EBV status and relapse survival (hazard ratio: 2.75, 95% CI: 1.30, 5.86; p = 0.008) in breast cancer patients. However, no significant association was found in overall survival outcomes. Additionally, we observed significant associations between ER status and tumor histologic grade with both overall and relapse survival. Patients with EBV-positive tumors exhibited higher recurrence rates compared to those with EBV-negative tumors. Furthermore, we noted significant correlations between EBV status and HER-2 (p = 0.0005) and histological grade (p = 0.02) in our cohort of breast cancer patients. Conclusions: The presence of EBV in breast cancer tumors appears to exert an impact on patient outcomes, particularly concerning recurrence rates. Our findings highlight the significance of considering EBV status as a potential prognostic marker in breast cancer patients. Nonetheless, further research is essential to elucidate the underlying molecular mechanisms and develop novel therapeutic approaches.

EXPRESSION PATTERNS OF CD90, CD117, CD123, AND CD147 IN CONTINUOUS CELL LINES OF HUMAN MATURE B-CELL TUMORS

Summary. Aim: to analyze expression patterns of CD90, CD117, CD123, and CD147 in vitro in different continuous lines of human mature B-cell neoplasms. Objects and methods: expression patterns of the surface antigens on cells of the continuous lines of Burkitt lymphoma (Namalwa and BJAB), Hodgkin lymphoma (КМ-Н2), and follicular lymphoma (Car-1) were analyzed by flow cytometry with monoclonal antibodies against CD90, CD117, CD123, and CD147. Results: BJAB and Namalwa cells did not express CD90 and CD123, while a small fraction of cells (less than 2%) was CD117-positive. КМ-Н2 and Сar-1 cells did not express any of three above mentioned antigens. The percentage of CD147-positive cells in continuous lines of human mature B-cell neoplasms amounted to 80–100%. Conclusions: CD117 expression in cell populations of Burkitt lymphoma continuous cell lines is rather rare. CD90 and CD123 are not expressed in all cell lines of human mature B-cell neoplasms under study. At the same time, ubiquitous pattern of CD147 has been evident.

Combination of Propranolol and a Novel CXCR4 Antagonist Burixafor (GPC-100) for Enhanced Hematopoietic Cell Mobilization

Burixafor (GPC-100), a novel CXCR4 antagonist, has shown safe and effective mobilization of hematopoietic stem and progenitor cells (HSPC) in a phase I clinical study. In multiple myeloma patients, GPC-100 combined with granulocyte-colony stimulating factor (G-CSF) elicited a significant increase in circulating HSPCs that was comparable with the historical results from G-CSF plus the CXCR4 antagonist plerixafor. GPC-100 is currently being investigated for HSPC mobilization in combination with the FDA-approved ß-adrenergic receptor (ß-AR) blocker propranolol in a phase II clinical trial for autologous stem cell transplant (ASCT) in multiple myeloma. Here, we present preclinical findings indicating that the addition of propranolol to GPC-100 alone or GPC-100 and G-CSF combination can address the unmet needs of patients undergoing mobilization. Additionally, we show an enhanced and unique pattern of lymphoid cell mobilization that may have applications in adoptive cell therapies. Compared to chemotherapy, ASCT significantly improves survival in multiple myeloma patients. Because HSPCs constitute a very small fraction (~ 0.1%) of circulating mononuclear cells, it is critical to mobilize a sufficient number of HSPC into peripheral blood during the ASCT process. Current therapies for HSPC mobilization include G-CSF as a monotherapy or combined with plerixafor. However, mobilization failure is experienced by 15-50% patients, leading to potential loss of ASCT as a treatment option and significant toxicity from repeated mobilization attempts. Moreover, G-CSF is contraindicated in sickle cell disease and may exacerbate autoimmune diseases. Therefore, our goal is to identify novel strategies that will not only address the mobilization failure, but also explore non-G-CSF options for adequate mobilization. G protein-coupled receptors like CXCR4 can form heteromers with other receptors and result in altered signaling pathways. Using Proximity Ligation Assay in Namalwa (lymphoma) cells, we show that endogenously expressed CXCR4 and ß 2AR form heteromers, which are absent in CXCR4 knockout cells. In cell-based assays, co-activation of both receptors with their respective ligands, CXCL12 and epinephrine, leads to a synergistic increase in calcium flux (Figure 1) as well as ß-arrestin recruitment, indicating functional consequences of the heteromerization. Complete blockade of this enhanced signaling is only achieved with co-inhibition by both antagonists. Like CXCR4, ß 2AR is also expressed by HSPCs, and in lymph nodes was shown to heteromerize with CXCR4 to increase lymphocyte retention. In multiple myeloma patients, propranolol upregulated stem-cell like gene signature. Therefore, we sought to understand the effects of propranolol on HSPC mobilization by GPC-100. In mice, single injection of GPC-100 effectively mobilized hematopoietic stem cells characterized as Lin negSca1 +cKit +(LSK) cells as well as mature Lin negCD34 + progenitor cells by 2-4-fold, respectively. Seven-day pretreatment with propranolol enhanced LSK cell mobilization by GPC-100 resulting in a 4-fold increase in circulating HSPCs (Figure 2). Addition of propranolol to G-CSF and GPC-100 led to 8-17-fold increases in mobilization of long-term repopulating HSCs (CD34 negLSK and CD150 +LSK), which were greater than the 4-7-fold increases elicited by G-CSF and G-CSF with plerixafor. Additionally, propranolol enhanced GPC-100 induced mobilization of white blood cells, neutrophils, and lymphocytes (Figure 2). Immune cell subset analyses revealed that GPC-100 induced a 3-fold increase in circulating CD8 +T cells, which was increased to 7-fold when combined with propranolol (Figure 2). For comparison, plerixafor increased CD8 + T cell mobilization by only 2-fold. GPC-100 induced mobilization of NK and B cells was also improved by propranolol. These pre-clinical data support our phase II clinical trial currently in progress (NCT05561751). In the first arm of this two-arm trial, GPC-100 is combined with orally administered propranolol, and in the second arm, G-CSF is added to this combination. We anticipate that our clinical studies can fill the gap in the current standard of care without adding a significant financial burden or co-morbidities. Furthermore, enhanced mobilization of CD8+ T cells can provide extended opportunities for improved adoptive cell therapies.

Structural Optimization of Dual-Target CD22/CD19 CAR-T and Its Anti-Tumor Effects on B Cell Malignancies

Relapse to CD19 chimeric antigen receptor-modified (CAR) T cell therapy, which associated with diminished antigen density and presence of isoforms, were emerging and precluded the widespread application of this novel therapy. As such, CARs target dual antigens, such as CD19/CD22 bispecific CAR may become a feasible and promising strategy to reduce immune escape caused by tumor clonal evolution and antigen loss. Here, we designed a novel CD19/CD22 CAR, whose scFvs were derived from HI19α and HI22 clone generated in our institution, and then screened the most efficient construct into clinical application. Considering the impact of different linkers and sequence of chains in scFvs on CAR efficiency, we constructed 4 types of TanCAR constructs and 2 types of LoopCAR constructs. Of the 6 CAR constructs, TanCAR 2 (CD22VL-CD22VH-CD19VL-CD19VH, Tan22-19 CAR) and LoopCAR 2 (CD22VL-CD19VL-CD19VH-CD22VH, Loop22×19 CAR) were validated with higher infection efficiency and killing effect on tumor cell lines. To investigate the impact of CD19/CD22 CAR on CD19 and/or CD22 positive cells, we constructed CD19KO (CD19 knock out) or CD22KO (CD22 knock out) Namalwa cell lines through CRISPR-Cas9 technology. When co-cultured with Namalwa, Namalwa-CD19KO and Namalwa-CD22KO cells, compared with CD19 CAR-T or CD22 CAR-T which could only kill its corresponding target positive cells, Tan22-19 CAR-T and Loop22×19 CAR-T had significant cytotoxicity on CD19 and/or CD22 positive cells, indicating a dual-targeting efficacy. To further explore the difference of Tan22-19 CAR-T and Loop 22×19 CAR-T under continuous exposure to antigen, re-challenge experiment was carried out. It was found that compared with Tan22-19 CAR-T, Loop22×19 CAR-T proliferated faster, eliminated tumor cells more significanly, and had a higher proportion of central memory T cells (TCM), indicating that Loop22×19 CAR-T might have a more persisting function. In the simulated tumor recurrence mouse model, single CD19 or CD22 CAR-T could only partially eliminate tumor cells, while Loop22×19 CAR-T significantly reduced the recurrence caused by antigen loss and prolonged the survival of mice compared with that of Tan22-19 CAR-T and single-target CAR-T. Then, Loop22×19 CAR-T was finally selected for evaluation of its clinical therapeutic effect. A total of 6 patients with relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) received Loop22×19 CAR-T cells therapy, in which 2 patients had undergone allogeneic hematopoietic stem cell transplantation (allo-HSCT) before receiving CAR-T therapy. The median age of the 6 patients was 18 years (range: 8-52 years), and the median percentage of blast cells in BM prior to lymphodepletion therapy was 18.1% (range, 0.03%-70.6%). Of the 6 patients, 5 received 3×106/kg CAR T-cells infusion, and the dose of the other one was 1×106/kg. Encouragingly, all 6 (100%) patients achieved MRD-negative remission within 32 days after CAR-T infusion. In addition, one BCR-ABL positive patient achieved molecular CR. These results demonstrate that Loop22×19 CAR-T therapy results in a deep and rapid response for patients with R/R B-ALL. Furthermore, after a median follow-up of 11.2 months, both the median overall survival (OS) and relapse-free survival (RFS) have not been reached. In entire cohort, 4 patients keeps remission persistantly, including 2 patients with TP53 mutation who bridged transplantation after CAR-T treatment. The remaining 2 patients experienced relapse, including 1 patient, who underwent allo-HSCT before CAR-T therapy, died 10 months after CAR-T infusion. As such, the one-year OS and RFS rate of the 6 patients were 77.8% (95% CI, 16.6%-96.5%) and 48.0% (95% CI, 0.04%-84.9%), respectively. Although CRS was detected in all 6 patients, no severe CRS (grade ≥3) or CAR-T- related neurotoxicity was observed after Loop22×19 CAR-T infusion. For the hematological adverse events, all 6 patients developed reversible grade 3-4 neutropenia. In conclusion, our study optimized bispecific CD19/22 CAR-T by constructing Tan and Loop CAR structures with different linkers and sequences of scFvs. Preclinical study demonstrated Loop structure 22×19 CAR-T exhibited more robust proliferation and anti-leukemia effect than Tan22-19. Besides, Loop22×19 CAR-T showed durable efficacy and reliable safety for B-ALL patients, and was expected to become a new strategy for reducing B-ALL recurrence.

Perfluoroalkyl substances (PFASs) decrease the expression of recombination-activating genes (RAG1 and RAG2) in human B lymphoma Namalwa cells

Per- and polyfluoroalkyl substances (PFASs) are omnipresent and have been shown to induce a wide range of adverse effects, including hepatotoxicity, developmental toxicity and immunotoxicity. So far, little information is available about the mechanisms underlying the toxicity of PFASs, including those related to their immunotoxicity. Reported immunotoxic effects of PFASs include decreased antibody responses in experimental animals and humans, indicating that PFASs may, among others, affect B cell function. In the present study, we first assessed the effects of PFOA on the transcriptome of the human Namalwa B cell line using RNA seq analysis. Gene expression changes, analyzed using Ingenuity Pathway Analysis, pointed to various cellular processes affected by PFOA, including ‘B cell development’ and ‘Primary immunodeficiency signaling’. Interestingly, PFOA decreased the expression of RAG1 and RAG2, genes involved in immunoglobulin and T cell receptor V(D)J recombination. As a next step, time- and concentration-dependent changes in the expression of RAG1 and RAG2 upon exposure to PFOA, PFNA, PFHxS and PFOS were studied through RT-qPCR analysis. Analysis with the concentration–response modeling software PROAST resulted in the following potency ranking: PFNA > PFOA > PFOS > PFHxS. Altogether, the present in vitro study provides insights into the effects of selected PFASs on B cells, identifying RAG1 and RAG2 expression as possible relevant targets that may play a role in the immunotoxicity of PFASs.

Cytotoxic shornephines and asterresins from the hydrothermal vent associated fungus Aspergillus terreus CXX-158-20

A previously unreported alkaloid, bearing an undescribed 5/7/8 tricyclic heterocyclic skeleton, shornephine D, an undescribed diketomorpholine (DKM) shornephine B, two undescribed diketomorpholine derivatives shornephine C and seco-shornephine B methyl ester, an undescribed indole-isoquinoline alkaloid asterresin C, three undescribed indole alkaloids asterresins A-B and D, together with five known compounds, were isolated from the culture of hydrothermal vent associated fungus Aspergillus terreus CXX-158-20. Their structures were unambiguously determined by nuclear magnetic resonance (NMR), mass spectrometry, Mosher's method, 13C NMR calculation in combination with DP4+, and ECD calculations. Shornephine D and asterresin C represent two undescribed heterocyclic skeletons. Asterresin D and giluterrin exhibited cytotoxicity activities with IC50 values of 3.96 μM and 7.97 μM against A549 cell line. Asterresin D exhibited cytotoxicity activities with IC50 values of 12.36 μM and 12.48 μM against Namalwa and U266 cell lines. Asterresin A and giluterrin exhibited synergistic effect with adriamycin against MCF-7 cell line.

5-Azacytidine Suppresses the Expression of Tissue-Specific Oct-1 Isoform in Namalwa Burkitt’s Lymphoma Cell Culture

Overexpression of the transcription factor POU2F1 (Oct-1) increases the malignant potential of the tumor and determines the unfavorable prognosis for both solid and hematological cases of the disease in human carcinogenesis. The Oct-1 level determines the rate of development of the disease in acute myelodysplastic leukemia (AML), and a decrease in its expression significantly delays the development of leukemia in mice; however, a complete knockout of Oct-1 leads to the death of the animals. POU2F1 (Oct-1) is expressed as several isoforms transcribed from alternative promoters. They include both ubiquitous and tissue-specific isoforms. It was shown that in Burkitt’s lymphoma Namalwa cells 5-azacytidine specifically suppresses the expression of the tissue-specific isoform Oct-1L mRNA (level of Oct-1L is abnormally increased in these cells), while not causing changes in the amount of the ubiquitous isoform Oct-1A mRNA. These results show that it is possible to selectively reduce the transcription level of the Oct-1L isoform aberrantly expressed in human tumor cells.

POU2F1(Oct-1) разнонаправленно участвует в ауторегуляции альтернативных промоторов за счет связывания с различными регуляторными участками гена POU2F1

The POU2F1 gene, which plays an important role in regulating the mammalian genome and development, has both a ubiquitous (U) and a tissue-specific (L) promoter and is subject to intricate regulation. Regions of POU2F1 gene were found to contain multiple binding sites for its product POU2F1 (Oct-1), a transcription factor. Interspecies homology in these regions was found to exceed 90% among the human, mouse, rat, pig, and dog genomes, almost all of the Oct-1 binding sites being identical. Some of the sites cluster in the vicinity of each of the two alternative promoters, while others are in the 5' noncoding region 6 kb upstream of the transcription start site. The presence of Oct-1 at the sites was demonstrated by chromatin immunoprecipitation and the electrophoretic mobility shift assay (EMSA). A POU2F1 knockdown activated the U promoter and downregulated the L promoter in Namalwa cells, while Oct-1 overexpression exerted an opposite effect. Thus, Oct-1 acts via negative feedback to autoregulate the U promoter through low-affinity Oct-1 binding sites and positive feedback to autoregulate the L promoter through high-affinity canonical (oct) sites when increasing in concentration in a natural context.

SPAG6 promotes cell proliferation and inhibits apoptosis through the PTEN/PI3K/AKT pathway in Burkitt lymphoma.

The main purpose of the present study was to elucidate the role of sperm-associated antigen 6 (SPAG6) in the occurrence and development of Burkitt lymphoma (BL) and explore the underlying molecular mechanisms. A correlation was observed between the expression of SPAG6 and the prognosis of patients with lymphoma using The Cancer Genome Atlas (TCGA) database analysis. It was demonstrated that the levels of SPAG6 in BL cells were higher compared with that in IM-9 cells by reverse transcription-PCR and western blot assays. Moreover, silencing of SPAG6 significantly decreased proliferation and increased apoptosis of Daudi and Raji cells, whereas SPAG6 overexpression exerted the opposite effects on CA46 and NAMALWA cells. When investigating the possible mechanism, it was first observed that the level of phosphatase and tensin homolog (PTEN) protein was significantly increased, while that of phosphorylated (p-)AKT protein was markedly reduced in the SPAG6-knockdown group compared with the blank control group in Daudi and Raji cells by western blot analysis. It was further ascertained whether the phosphoinositide 3-kinase (PI3K)/PTEN/protein kinase B (AKT) pathway mediates the effects of SPAG6 on cell proliferation and apoptosis, and the results demonstrated that silencing of SPAG6 suppressed the viability of Daudi and Raji cells, whereas PTEN knockdown using siRNA or SF1670 (a specific PTEN inhibitor) reversed the inhibitory effect on cell proliferation and the promoting effect on cell apoptosis induced by SPAG6 depletion in vitro as well as in vivo. These data revealed that SPAG6 may promote the proliferation and inhibit the apoptosis of BL cells via the PTEN/PI3K/AKT pathway. The results of the present study suggest that SPAG6 may play a key role in the progression of BL and may be of value as a predictive prognostic biomarker in patients with BL.

Reversine exerts cytotoxic effects through multiple cell death mechanisms in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer with limited therapeutic options for adult patients. Aurora kinases have drawn attention as potential targets in hematological neoplasms due to their high expression and biological functions. Aurora kinase A (AURKA) and AURKB are essential for a successful mitosis, acting in spindle mitotic organization and cytokinesis. Reversine is a synthetic purine analog that acts as a multi-kinase inhibitor with anti-neoplastic activity by targeting AURKA and AURKB. ALL patient gene expression data were retrieved from the Amazonia! For functional assays, Jurkat (T-ALL) and Namalwa (B-ALL) cells were exposed to increasing concentrations of reversine and submitted to various cellular and molecular assays. We found that AURKB expression was higher in ALL patient samples compared to normal lymphocytes (p < 0.0001). The ALL cell lines tested displayed aberrant AURKA and AURKB expression. In Jurkat and Namalwa cells, reversine reduced cell viability in a dose- and time-dependent manner (p < 0.05). Reversine also significantly reduced the viability of primary ALL cells. Reversine induced apoptosis and autophagy, and reduced cell proliferation in both cell lines (p < 0.05). Mitotic catastrophe markers, including cell cycle arrest at G2/M, increased cell size and DNA damage, were observed upon reversine exposure. Short- and long-term treatment with reversine inhibited autonomous clonogenicity (p < 0.05). At the molecular level, reversine reduced AURKB activity, induced SQSTM1/p62 consumption, and increased LC3BII and γ-H2AX levels. In Namalwa cells, reversine modulated 25 out of 84 autophagy-related genes, including BCL2, BAD, ULK1, ATG10, IRGM and MAP1LC3B, which indicates that reversine acts by initiating and sustaining autophagy signals in ALL cells. From our data we conclude that reversine reduces the viability of ALL cells by triggering multiple cell death mechanisms, including apoptosis, mitotic catastrophe, and autophagy. Our findings highlight reversine as a potential anticancer agent for ALL.

Knockdown of lncRNA MCM3AP-AS1 Attenuates Chemoresistance of Burkitt Lymphoma to Doxorubicin Treatment via Targeting the miR-15a/EIF4E Axis.

PurposeThe long-noncoding RNA MCM3AP-AS1 has been shown to participate in the tumorigenesis and growth of several types of cancer, but little is known about the role of MCM3AP-AS1 in the chemoresistance of lymphoma.MethodsA series of patients with Burkitt lymphoma were enrolled for clinical analysis. Daudi and Namalwa cells were used for further experiments. CCK-8 and apoptosis assays were used to assess the response to doxorubicin. Mitochondrial membrane potential assays and high-resolution respirometry were used to assess mitochondrial function. Western blotting was used to detect the expression of certain molecules. Luciferase assays and microRNA transfection were used to clarify the regulatory mechanisms of MCM3AP-AS1. An in vivo model using BALB/c nude mice was utilized to investigate the effects of MCM3AP-AS1 on cell proliferation and tumor growth.ResultsThe expression level of MCM3AP-AS1 was increased in tumors compared with normal lymph nodes, which indicated poor prognosis in patients with Burkitt lymphoma. Moreover, compared with siNC transfection, MCM3AP-AS1 knockdown decreased cell viability and increased apoptosis rates upon doxorubicin treatment compared with siNC. Further studies indicated that upregulation of several antiapoptotic factors, downstream of EIF4E, was partially responsible for MCM3AP-AS1-induced chemoresistance. Moreover, miR-15a functioned as a link between MCM3AP-AS1 and EIF4E, and was sponged by MCM3AP-AS1. Finally, we showed that the MCM3AP-AS1/miR-15a/EIF4E axis regulated the chemoresistance of lymphoma cells in vitro and in vivo.ConclusionMCM3AP-AS1/miR-15a/EIF4E axis plays a role in the chemoresistance of Burkitt lymphoma, and it might become a promising target for lymphoma therapeutics.

Heparin and heparan sulfate proteoglycans promote HIV-1 p17 matrix protein oligomerization: computational, biochemical and biological implications

p17 matrix protein released by HIV+ cells interacts with leukocytes heparan sulfate proteoglycans (HSPGs), CXCR1 and CXCR2 exerting different cytokine-like activities that contribute to AIDS pathogenesis. Since the bioactive form of several cytokines is represented by dimers/oligomers and oligomerization is promoted by binding to heparin or HSPGs, here we evaluated if heparin/HSPGs also promote p17 oligomerization. Heparin favours p17 dimer, trimer and tetramer assembly, in a time- and biphasic dose-dependent way. Heparin-induced p17 oligomerization is of electrostatic nature, being it prevented by NaCl, by removing negative sulfated groups of heparin and by neutralizing positive lysine residues in the p17 N-terminus. A new computational protocol has been implemented to study heparin chains up to 24-mer accommodating a p17 dimer. Molecular dynamics show that, in the presence of heparin, two p17 molecules undergo conformational modifications creating a continuous “electropositive channel” in which heparin sulfated groups interact with p17 basic amino acids, promoting its dimerization. At the cell surface, HSPGs induce p17 oligomerization, as demonstrated by using B-lymphoblastoid Namalwa cells overexpressing the HSPG Syndecan-1. Also, HSPGs on the surface of BJAB and Raji human B-lymphoblastoid cells are required to p17 to induce ERK1/2 activation, suggesting that HS-induced oligomerization plays a role in p17-induced lymphoid dysregulation during AIDS.

Abstract 876: CD40-induced growth inhibition of Burkitt lymphoma: A possible role for NADPH oxidase by upregulation of p67phox

Abstract CD40 stimulation together with IL4 has been shown to promote growth of normal B cells in vitro. In contrast to this, CD40 stimulation in several B cell lymphoma cell lines has been demonstrated to be growth inhibitory. Though the mechanism is not completely known, CD40-stimulated cells exhibit increased levels of reactive oxygen species (ROS). In B lymphocytes, a major source of ROS production is the NADPH oxidase (NOX) that is comprised of a membrane-bound Nox2 catalytic subunit and four cytosolic regulatory components, with the p67phox cytosolic protein being the activator factor. Upon stimulation, by either cross linking of the B-cell receptor or following treatment with PMA, the active NADPH oxidase generates superoxide. We have investigated the role of NADPH oxidase in CD40-mediated growth inhibition in Burkitt lymphoma (BL) cells. For this study, we first evaluated the effect of CD40 stimulation on the expression of Nox2 and p67phox in BL cells that demonstrate different levels of endogenous Nox2 and p67phox. Accordingly, Daudi and Raji cell lines, with endogenously high Nox2 and low p67phox, NAMALWA cells which have high Nox2 and no p67phox expression, and Ramos cells that contain no Nox2 or p67phox were analyzed. Treatment with anti-CD40 or CD40 ligand (CD40L) for 24 h induced up-regulation of p67phox in Daudi, Ramos, and NAMALWA cells, but not in the Raji line. No significant change in the expression of Nox2 was observed. Additionally, Daudi, Ramos and NAMALWA cells showed higher production of ROS after CD40 stimulation and activation with anti-IgM or PMA, in a chemiluminescence assay. To determine whether the change in ROS level was related to the upregulation of p67phox, Daudi cells that express either stable p67phox shRNA or control shRNA were generated. Treatment with CD40L for 24 h demonstrated significant ROS production only in control shRNA and not in the p67shRNA cells, confirming that the ROS production is NOX2-p67phox mediated. This enhanced ROS production was observed following treatment with CD40L alone and was enhanced further in conjunction with anti-IgM or PMA. However, cells with the p67phox shRNA showed detectable level of ROS only after activation with anti IgM, albeit to much lower levels than the control. Co-treatment of Daudi cells with anti-CD40 or CD40L and IL4 further increased the expression of p67phox. Although the basal level of ROS in the cells was unchanged upon co-treatment, the ROS generated following stimulation with anti-IgM or PMA was significantly enhanced. Preliminary MTT studies confirmed the growth inhibition of CD40-stimulated Daudi cells but not of Raji cells, suggesting a role for p67phox-dependent, ROS-related inhibition of lymphoma cell proliferation. Experiments are currently ongoing to delineate further the role of CD40 stimulation and Nox2-p67phox mediated ROS in affecting the growth of Burkitt lymphomas and signaling pathways downstream of CD40. Citation Format: Mazal Iris Dahan, Smitha Antony, Agnes Juhasz, Guojian Jiang, Mariam Konate, Jiamo Lu, Jennifer Meitzler, YongZhong Wu, Krishnendu Roy, James H. Doroshow. CD40-induced growth inhibition of Burkitt lymphoma: A possible role for NADPH oxidase by upregulation of p67phox [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 876.

ERK-dependent IL-6 positive feedback loop mediates resistance against a combined treatment using danusertib and BKM120 in Burkitt lymphoma cell lines

This study was conducted to define the synergistic effect of the PI3K inhibitor BKM120 with the pan-Aurora kinase inhibitor danusertib and the potential mechanism of resistance to the combined inhibitor treatment in Burkitt lymphoma cell lines. The combination of danusertib and BKM120 showed a synergistic effect on Namalwa cells but not on BJAB cells. The combined treatment led to ERK hyperactivation and induced IL-6 secretion in BJAB cells but not in Namalwa cells. A blockade of ERK signaling with trametinib suppressed the combination treatment-induced ERK activation, reduced IL-6 mRNA expression, and downregulated IL-6R mRNA expression, resulting in an improvement in the antitumor effect. We stepwise treated Namalwa cells with both inhibitors using on-and-off treatment cycles and found that Namalwa cells gained chemoresistance by activating the ERK/IL-6 feedback loop, suggesting that the ERK-dependent IL-6 positive feedback loop can compensate for AKT inactivation and is closely associated with adaptive resistance and relapse.

Targeting Burkitt lymphoma with a tumor cell–specific heptamethine carbocyanine‐cisplatin conjugate

BackgroundBurkitt lymphoma is a fast‐growing mature B cell malignancy, whose genetic hallmark is translocation and activation of the c‐myc gene. Prompt multiagent immunochemotherapy regimens can have favorable outcomes, but prognosis is poor in refractory or relapsed disease. We previously identified a novel family of near‐infrared heptamethine carbocyanine fluorescent dyes (HMCD or DZ) with tumor‐homing properties via organic anion‐transporting peptides. These membrane carriers have uptake in tumor cells but not normal cells in cell culture, mouse and dog tumor models, patient‐derived xenografts, and perfused kidney cancers in human patients.MethodsHere we report the cytotoxic effects of a synthesized conjugate of DZ with cisplatin (CIS) on B cell lymphoma CA46, Daudi, Namalwa, Raji, and Ramos cell lines in cell culture and in xenograft tumor formation. Impaired mitochondrial membrane permeability was examined as the mechanism of DZ‐CIS–induced lymphoma cell death.ResultsThe new conjugate, DZ‐CIS, is cytotoxic against Burkitt lymphoma cell lines and tumor models. DZ‐CIS retains tumor‐homing properties to mitochondrial and lysosomal compartments, does not accumulate in normal cells and tissues, and has no nephrotoxicity in mice. DZ‐CIS accumulated in Burkitt lymphoma cells and tumors induces apoptosis and retards tumor cell growth in culture and xenograft tumor growth in mice.ConclusionDZ‐CIS downregulated c‐myc and overcame CIS resistance in myc‐driven TP53‐mutated aggressive B cell Burkitt lymphoma. We propose that DZ‐CIS could be used to treat relapsed/refractory aggressive Burkitt lymphomas.

OSU-T315 as an Interesting Lead Molecule for Novel B Cell-Specific Therapeutics.

B cells are pathogenic in various disease processes and therefore represent an interesting target for the development of novel immunosuppressants. In the search for new therapeutic molecules, we utilized an in vitro B cell activation assay with ODN2006-stimulated Namalwa cells to screen a chemical library of small molecules for B cell modulating effects. OSU-T315, described as an inhibitor of integrin-linked kinase (ILK), was hereby identified as a hit. On human and murine primary B cells, OSU-T315 potently suppressed the proliferation and the production of antibodies and cytokines upon stimulation, suggesting that ILK could be a promising target in the modulation of B cell activity. Mice with B cell-specific knockout of ILK were generated. Surprisingly, knockout of ILK in murine B cells did not affect B cell function as assessed by several in vivo and ex vivo B cell assays and did not alter the B cell immunosuppressive activity of OSU-T315. In conclusion, OSU-T315 displays potency as B cell modulator, probably through a mechanism of action independent of ILK, and might serve as lead drug molecule for the development of novel B cell-selective drugs.

The regulatory interplay between Oct-1 isoforms contributes to hematopoiesis and the isoforms imbalance correlates with a malignant transformation of B cells.

Oct-1(POU2F1) is a DNA-binding transcription regulator and its level being highly increased in many human cancers. Oct-1 is present in the human cells as a family of functionally different isoforms which are transcribed from alternative promoters. Here, we have demonstrated that expression patterns of Oct-1 isoforms change during differentiation of hematopoetic progenitor cells (CD34+) (HPCs) to the B (CD19+) and T (CD3+) cells. While Oct-1L is expressed at a high level in the CD34+ HPCs, its expression level drops dramatically during the T-cell differentiation, although remains nearly the same in B-cells. We have described the novel human Oct-1R isoform which is conserved in mammals and is B cell-specific. Oct-1R was found in B cells, but not in HPCs. Oct-1R is transcribed from the same promoter as Oct-1L, another lymphocyte-specific isoform. Overexpression of Oct-1R and Oct-1L in the Namalwa cells leads to the repression of many genes involved in B-lymphocyte differentiation and signal transduction. Thus these isoforms may regulate the particular stages of development of normal B cells and maintain their proper differentiation status. However the extremely high level of Oct-1L isoform observed in the B-lymphoblast tumor cell lines indicated that the excess of Oct-L seem likely to considerably decrease the differentiation ability of these cells. Oct-1 may serve as a therapeutic target for many tumors, but it should be noted that in a tumor the content of a certain isoform Oct-1, rather than the total Oct-1 protein, can be increased.