B-cell Lines Research Articles

Separation of B- and T-Cell-Specific Signaling Molecules Prevents Oncogenic Transformation in Lymphoid Malignancies

Background and significance: Despite the highly homologous structure and function of proteins controlling signal transduction from the BCR and TCR, B- and T-cells have evolved separate sets of linker and kinase molecules and mechanisms to ensure that their expression is strictly separated between B- and T-lymphoid lineages. While this is the case among normal B- and T-cells, we and others showed that the separation of B- and T-cell-specific signaling molecules is often blurred in lymphoid malignancies. For instance, B-ALL, CLL, and MCL often aberrantly express the T-cell kinase ZAP70, whereas PTCLs and γδTCR CTCLs often express SYK or the ITK-SYK fusion. Here we investigated the mechanisms driving the aberrant cross-lineage expression of BCR- and TCR-downstream signaling molecules and how the promiscuous expression of these molecules contributes to the malignant transformation of lymphoid malignancies. Results: We have previously shown that the B-cell-specific kinase SYK and the T-cell-specific kinase ZAP70 are segregated to prevent autoimmunity and malignant transformation of pre-germinal center B-cells. In pre-malignant B-cells, SYK drives negative selection through Ca 2+-signaling via BTK-BLNK phosphorylation and downstream activation of NFAT. The aberrant expression of ZAP70 diverts SYK signaling towards CD19-PI3K, weakening negative selection, and consequently permitting the expansion of pre-malignant cells. This represents a novel key mechanism of aberrantly expressed signaling molecules in the etiology and persistence of B-lymphoid malignancy. We previously showed that co-expression of the T-cell kinase ZAP70 with the B-cell kinase SYK dramatically accelerated the development of fatal disease in a mouse model of CLL, consistent with poor clinical outcomes of ZAP70 + CLL in patients. To elucidate the role of ZAP70 co-expression in the initiation of B-ALL, pre-B-cells were isolated from Zap70 -/- mice and transformed ex vivo with BCR-ABL1 and NRAS G12D to model B-ALL. These cells showed significantly impaired colony-forming capacity compared to transformed wild-type cells. To corroborate these results, we crossed BCR-ABL1-transgenic with Zap70 -/- mice. Next, patient-derived B-ALL and MCL xenografts (PDX) were engineered to carry a homozygous knock-in of dTAG at the C-terminus of ZAP70. Upon degradation of ZAP70-dTAG, consequences on cellular fitness and proliferation were assessed, as well as the downstream activation of Ca 2+-signaling and NFAT activation ( Figure 1). Additionally, a mouse strain carrying a bulky ATP-analog-sensitive variant of Zap70 (M413A) was crossed with the p190 strain to assess the effect of ZAP70 inhibition after the manifestation of B-cell leukemia. Since the expression of ZAP70 increases upon malignant transformation of pre-germinal center B-cells, we used Genomic Locus Proteomics (GLoPro) to identify transcription factors driving this transcriptional upregulation ( Figure 1). By comparing malignant B- and T-cell lines, which either express only SYK or ZAP70 or co-express them, we were able to isolate specific mediators of aberrant transcription of ZAP70, including β-catenin/WNT signaling. ChIP-qPCR, CRISPR/Cas9-mediated knockout, as well as dTAG, were used to validate the binding of transcription factors identified by GLoPro to the ZAP70 enhancers and their role in ZAP70 expression. Apart from ZAP70, we identified multiple aberrantly co-expressed T-cell signaling proteins by comparative proteomic analysis of malignant B- and T-cell lines. These included LCK, PLCγ1, and SLP-76, while the BTK-homolog ITK is completely absent in B-cell lines. In future work, we aim to elucidate the transcriptional regulation and consequences of the loss of these aberrantly co-expressed proteins. Further, we will perform a CRISPR/Cas9-based switch of BTK and ITK to explore the effects of forced co-expression of these segregated proteins. Conclusions: We conclude that malignant pre-germinal center B-cells increase the expression of ZAP70 through the formation of aberrant transcription machinery. Furthermore, our work elucidates the role of ZAP70 not only for the initiation but also for the maintenance of B-cell leukemia. Other T-cell signaling molecules are also aberrantly expressed in B-cell malignancies and further investigation will highlight their transcriptional regulation and role during and after malignant transformation.

T-Cell Signaling Mediates the Epigenetic Priming of Germinal Center B-Cell Plasticity and Stem Functionality

Chemo-immunotherapy can prolong life expectancy of patients with Diffuse Large B-cell Lymphomas (DLBCL). However, the emergence of donor-derived lymphoma cases after stem cell transplantation and the relapse of a third of DLBCL patients, strongly suggests the existence of persisting lymphoma repopulating cells (LRC) with stem-like functionality. This apparently controversial concept arises from the fact that DLBCLs originate from mature rather than progenitor cells. Identifying the biological and epigenetic features of these elusive LRC, and how they arise from normal germinal center (GC) B cells, is therefore critical and remains underexplored. Although fully mature, GC B cells naturally manifest remarkable phenotypic plasticity as compared to other cell types. We hypothesized that this plasticity reflects stem-like programming and functionality. Along these lines, we found that GC B cells are uniquely enriched for pluripotent stem cell transcriptional signatures, and increased chromatin accessibility at stem cell enhancers. We reasoned that the capacity to undergo induced Pluripotent Stem Cells (iPSC) reprogramming could serve as a surrogate assay for stem-like plasticity. Therefore, we investigated the potential of GC B cells to form iPSCs using a doxycycline-dependent mouse strain that enables the inducible expression of Yamanaka transcription factors (TF) in any given cell. Strikingly, GC B cells isolated from our mice manifested a 10-fold (p<0.0001) increase in iPSC reprogramming as compared to other mature B cells. GC B cells are a heterogenous mix of functionally distinct subpopulations. Using single cell RNA-seq and iPSC assays in sorted cells, we noted that the rare population (~3%) of GC B cells selected by T-cell help manifested the stem-like transcriptional profile and iPSC reprogramming phenotype. Moreover, blocking T-cell help during the GC reaction in vivo using CD40 blockade abrogated iPSC forming capacity. Lymphoma-associated EZH2 Y641F mutations impair GC B-cell interactions with T FH cells due to aberrant repression of immune synapse genes. Accordingly, EZH2 Y641F GC B cells manifested loss of the iPSC phenotype. T-cell help induces MYC in GC B cells. However, induction of MYC in mature naïve B cells did not confer them with iPSC potential indicating that other mechanisms must be at play. Lymphoma-associated Btg1 Q36Hmutations confer fitness in GC B cells by enhancing their response to T-cell help. Reciprocal to EZH2 Y641F, Btg1 Q36Hmutant GC B cells manifested further significant increase in iPSC formation. Performing multi-ome (simultaneous single-cell RNAseq and ATACseq) studies in GC B cells revealed massive gain in chromatin accessibility of bona fide stem cell super-enhancers in GC B cells that received T-cell help. There was also a reduced accessibility at genes that maintain B-cell lineage (e.g. Pax5) and GC phenotypes (e.g. Foxo1). Finally, lymphoma-associated Histone 1 mutations induce upregulation of stem cell programs in GCs and could enhance iPSC reprogramming in fibroblasts. We therefore hypothesized that H1 deficiency would overcome the GC dependency on T-cells for their plasticity phenotype. Indeed, increased iPSC formation occurred across all GC subpopulations in H1 C/E-/- (HIST1C/HIST1E knockout) mice. We speculate that restricting plasticity to B cells under selection by T cells, limits the potential for GC B cells to acquire high levels of plasticity, hence reducing the potential for these cells to initiate lymphomas. Consistent with this notion, we found that DLBCL patients enriched for these stem cell signatures manifested inferior clinical outcomes.

Effect of Antigen Valency on Autoreactive B-Cell Targeting.

Many autoimmune diseases are characterized by B cells that mistakenly recognize autoantigens and produce antibodies toward self-proteins. Current therapies aim to suppress the immune system, which is associated with adverse effects. An attractive and more specific approach is to target the autoreactive B cells selectively through their unique B-cell receptor (BCR) using an autoantigen coupled to an effector molecule able to modulate the B-cell activity. The cellular response upon antigen binding, such as receptor internalization, impacts the choice of effector molecule. In this study, we systematically investigated how a panel of well-defined mono-, di-, tetra-, and octavalent peptide antigens affects the binding, activation, and internalization of the BCR. To test our constructs, we used a B-cell line expressing a BCR against citrullinated antigens, the main autoimmune epitope in rheumatoid arthritis. We found that the dimeric antigen construct has superior targeting properties compared to those of its monomeric and multimeric counterparts, indicating that it can serve as a basis for future antigen-specific targeting studies for the treatment of RA.

Targeting GD2-Positive Tumor Cells by Pegylated scFv Fragment-Drug Conjugates Carrying Maytansinoids DM1 and DM4.

Oligomerization of antibody fragments via modification with polyethylene glycol (pegylation) may alter their function and properties, leading to a multivalent interaction of the resulting constructs with the target antigen. In a recent study, we generated pegylated monomers and multimers of scFv fragments of GD2-specific antibodies using maleimide-thiol chemistry. Multimerization enhanced the antigen-binding properties and demonstrated a more efficient tumor uptake in a syngeneic GD2-positive mouse cancer model compared to monomeric antibody fragments, thereby providing a rationale for improving the therapeutic characteristics of GD2-specific antibody fragments. In this work, we obtained pegylated conjugates of scFv fragments of GD2-specific antibodies with maytansinoids DM1 or DM4 using tetravalent PEG-maleimide (PEG4). The protein products from the two-stage thiol-maleimide reaction resolved by gel electrophoresis indicated that pegylated scFv fragments constituted the predominant part of the protein bands, and most of the scFv formed pegylated monomers and dimers. The conjugates retained the ability to bind ganglioside GD2 comparable to that of the parental scFv fragment and to specifically interact with GD2-positive cells. Both induced significant inhibitory effects in the GD2-positive B78-D14 cell line, in contrast to the GD2-negative B16 cell line. The decrease in the B78-D14 cell viability when treated with scFv-PEG4-DM4 was more prominent than that for scFv-PEG4-DM1, and was characterized by a twofold lower half-maximal inhibitory concentration (IC50). Unlike the parental scFv fragment, the product of scFv and PEG4 conjugation (scFv-PEG4), consisting predominantly of pegylated scFv multimers and monomers, induced direct cell death in the GD2-positive B78-D14 cells. However, the potency of scFv-PEG4 was low in the selected concentration range, thus demonstrating that the cytotoxic effect of DM1 and DM4 within the antibody fragment-drug conjugates was primary. The suggested approach may contribute to development of novel configurations of antibody fragment-drug conjugates for cancer treatment.

About the blood characteristics and adaptation status variability in intact Balb/c mice of different sex

Purpose of the study. Was to study the values and variability of traditionally determined blood parameters and index of the adaption status in intact mature Balb/c mice of different sex.Materials and methods. Studies were carried out on 20 animals of mature age with spf-status, males (10) and females (10). Cytological and biochemical parameters of blood and weight characteristics of the organs of the immune system and adrenal glands were studied (considering the constitutional features of mice of this line). Statistica 10.0 software package was used for statistical analysis. The coefficient of variation (CV) was determined. The significance of statistical differences was assessed using the Mann-Whitney test.Results. Sex differences were found in the ratio of albumins and globulins, the content of urea and alkaline phosphatase, the relative proportion of lymphocytes and monocytes in the blood count, the erythrocytes’ count and the level of hemoglobin in erythrocytes. There were also indicators which had values of severe variability (CV up to 51 %), the number of those in males was higher than in females. The information content of the weight characteristics of the studied organs is reduced due to the constitutional features of Balb/c females. The identified sex differences indicated a higher adaptive status of female rodents compared to males and let to propose the prevalence of different activity of T-cell, B-cell and myeloid line in animals of different sexes.Conclusion. The study of weak and moderate shifts in traditional laboratory parameters reflecting the adaptive status and characteristics of the blood of sexually mature males and females of the Balb/c line under physiological conditions made it possible to identify the sex characteristics of systemic regulatory processes that may be important for the body's resistance to malignant growth and the effectiveness of antitumor treatment. The current areas of further research are also indicated.

African American patients with Multiple Sclerosis (MS) have higher proportions of CD19+ and CD20+ B-cell lineage cells in their cerebrospinal fluid than White MS patients

ObjectivesTo compare proportions of B-cell lineage CD19+ and CD20+ cells in CSF of African-American (AA) and White (W) patients with MS. BackgroundAA MS patients are more likely to have oligoclonal bands in CSF, higher IgG index in CSF, and higher circulating plasmablasts in blood than W MS patients. It is unknown whether the proportion of B-cells in CSF differs between AA and W patients in MS. MethodsDemographics, disease-related information, treatment history were retrospectively collected on patients with MS who self-identified as AA or W and underwent flow cytometry of CSF during diagnostic work-up. Proportion of B-lymphocytes, T-lymphocytes, NK cells, monocytes, and plasma cells were analyzed with flow cytometry. Results20 AA and 56 W MS patients fulfilled our inclusion criteria. The groups had similar demographics, CSF cell counts, protein and glucose CSF concentrations, and oligoclonal band number. IgG index was higher in AA compared to W (1.08 vs. 0.85, p = 0.031). AA had higher proportions of CD19+ (5.46 % AA vs. 2.26 % W, p = 0.006) and CD20+ (4.64 % AA vs. 1.91 % W, p = 0.004) cells but did not significantly differ in proportion of CD4+, CD8+, CD38+ bright B-cells, NK cells and monocytes. ConclusionsB-cells are overrepresented in the CSF of African American patients with MS relative to Whites.

Abstract PO-025: The effect of tissue of origin on oncogenesis in HPV-related malignancies: Comparative analysis of molecular and immune signatures in HPV-positive head and neck cancer and cervical cancer

Abstract Background: Despite sharing a common causal agent and viral oncogenes, HPV-positive head and neck cancer (HPV+ HNC) and cervical cancer (HPV+ CC) have markedly distinct natural histories and therapy responses. Here we sought to elucidate how the tissue of origin and oncogenic etiology drive molecular and microenvironmental differences between these two malignancies. Methods: Somatic mutation and RNA expression data for HPV+ HNC, HPV+ CC and HPV-negative head and neck cancer (HPV- HNC) were obtained from The Cancer Genome Atlas (TCGA). Mutation data were used to define repertoires of somatic mutations and mutational signatures. Single-sample gene set enrichment analysis (ssGSEA) was used to identify enriched immune signatures. Immune cell infiltration levels were estimated using xCell. Comparisons were primarily made between HPV+ HNC and HPV+ CC samples, with comparisons between HPV+ HNC vs. HPV- HNC tumors serving as a control to distinguish molecular/immune signatures driven by tissue of origin versus viral etiology. Results: Exome and RNA-sequencing data were available for 72 HPV+ HNC, 139 HPV+ CC, and 415 HPV- HNC tumors, and 70 HPV+ HNC, 139 HPV+ CC, and 413 HPV- HNC cases, respectively. Recurrently altered genes in head and neck and cervical cancer were not statistically significantly different between HPV+ HNC and HPV+ CC, although as previously reported HPV+ HNC and HPV- HNC differed by the higher frequency of TP53, CDKN2A, FAT1, and NOTCH1 mutations in the later. An evaluation of mutational signatures revealed that although both HPV+ HNC and CC had evidence of APOBEC mutagenesis (Signatures 2 and 13), Signature 2 and 13 exposures were both significantly higher in HPV+ CC relative to HPV+ HNC. APOBEC enrichment scores confirmed that HPV+ CC had higher APOBEC signal than HPV+ HNC despite both being virally driven. While both cancers were commonly affected by mutagenesis from aging and showed no differences in Signature 1, HPV+ HNC exhibited a higher correlation with Signature 5. Immune de-convolution evaluating 36 cell populations identified 13 cell types with different frequencies between two cancers (q < 0.1). The abundance of B-cell lineages, CD4+ naïve T-cells, and Tregs significantly differed in HPV+ HNC and HPV+ CC (p<0.001). These findings led us to investigate expression signatures of tertiary lymphoid structures, which we identified as markedly higher in HPV+ HNC versus HPV+ CC or HPV- HNC (p < 0.001, for both comparisons). Activated T-cells, however, did not appear to differ between HPV+ HNC or CC as determined by the previously defined cytolytic score. Conclusion: We find that while tissue of origin seems not to influence recurrently mutated oncogenes in HPV-related malignancies, it has significant effects on phenotypic consequences in these tumors including influencing both mutational signature exposure and the tumor microenvironment. These findings may inform further investigations into the unique molecular and microenvironmental characteristics of these virally driven tumors. Citation Format: Reith R. Sarkar, Xin Pei, Andrea Gazzo, Yingjie Zhu, Nancy Lee, Jorge Reis-Filho, Dmitriy Zamarin, Britta Weigelt, Nadeem Riaz. The effect of tissue of origin on oncogenesis in HPV-related malignancies: Comparative analysis of molecular and immune signatures in HPV-positive head and neck cancer and cervical cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-025.

Alternative splicing of its 5′-UTR limits CD20 mRNA translation and enables resistance to CD20-directed immunotherapies

AbstractAberrant skipping of coding exons in CD19 and CD22 compromises the response to immunotherapy in B-cell malignancies. Here, we showed that the MS4A1 gene encoding human CD20 also produces several messenger RNA (mRNA) isoforms with distinct 5′ untranslated regions. Four variants (V1-4) were detected using RNA sequencing (RNA-seq) at distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma, only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform contained upstream open reading frames and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, whereas V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed chimeric antigen receptor T cells were able to kill both V3- and V1-expressing cells, but the bispecific T-cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on 4 postmosunetuzumab follicular lymphoma relapses and discovered that in 2 of them, the downregulation of CD20 was accompanied by a V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.

Abstract 24 Development of an In Vitro B-Cell Differentiation Model for the Characterization of the Origin and Causation of ETV6::RUNX1+ Preleukemic Cells

Abstract Introduction Pregnant women are exposed to various carcinogenic substances that can subsequently affect their progeny. Recurrent preleukemic chromosomal translocations characteristic for childhood acute lymphoblastic leukemia (ALL) frequently emerge before birth in utero. The most common translocation t (12;21) occurs in 25% of B-cell precursor ALL and results in the formation of the chimeric oncogenic transcription factor ETV6::RUNX1. Objectives We aim to analyze factors promoting the generation of preleukemic ETV6::RUNX1 translocations, investigate the biological origin of these preleukemic cells, and develop an in vitro model of early B-cell differentiation to analyze the mechanisms underlying the persistence of ETV6::RUNX1+ preleukemic cells and leukemia evolution in children. Methods Genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) (Schafer et. al., 2018) was applied to identify ETV6::RUNX1+ cord blood units (CBU). Screening results were validated by Sanger sequencing and quantified by real-time PCR (ipsogen ETV6-RUNX1 Kit, Qiagen). ETV6::RUNX1+ CBUs were thawed, sorted for CD34+ and CD19+ cells, and analyzed by qPCR. CD34+ were differentiated towards B-cell lineage on MSC feeder lines according to a modified protocol (Ichii et. al., 2008). Results We screened a cohort of 1405 cord bloods collected in 2004 and discovered that 103 (7.3%) carried the ETV6::RUNX1 fusion gene. The preleukemic translocation was detected in CD34+ stem and early B progenitor cells. For the establishment of an in vitro B-cell differentiation model, bone marrow (BM-MSCs) and CB (USSCs, CB-MSCs) stromal feeder cells were compared and the effect of different media (SCGM, DMEM/5 % FCS, MSCBM/10 % FCS) in the presence of stem cell factor (SCF), Flt-3, IL3, +/- IL7 was assessed. BM-MSCs demonstrated the highest ability to support B-lymphopoieses towards CD19+ cell differentiation in MSCBM media (~30%), primarily due to the release of IL-7 by the feeder layer. Currently, factors that may promote generation of preleukemic translocations in utero e.g. nitrosamines contained in food products, are being tested in this model. Discussion We identified the HSC/early B progenitor state as the origin of ETV6::RUNX1 translocations in CB. The establishment of an in vitro B-cell differentiation system will allow to test the impact of environmental carcinogens on the generation of ETV6::RUNX1+ pre-leukemic cells and their leukemic evolution.

Secondary Mutations of the EGFR Gene That Confer Resistance to Mobocertinib in EGFR Exon 20 Insertion

IntroductionApproximately 10% of mutations in the EGFR gene in NSCLC are in-frame insertions in exon 20 (X20ins). These tumors usually do not respond to conventional EGFR tyrosine kinase inhibitors (TKIs). Several novel EGFR TKIs active for X20ins are in clinical development, including mobocertinib, which was recently approved by the U.S. Food and Drug Administration. However, acquired resistance during treatment with these TKIs still occurs as in the case of EGFR TKIs of earlier generations. MethodsWe chronically exposed murine pro–B-cell line cells transduced with the five most common X20ins (A763_Y764insFQEA, V769_D770insASV, D770_N771insSVD, H773_V774insNPH and H773_V774insH) to mobocertinib in the presence of N-ethyl-N-nitrosourea and searched for secondary EGFR mutations. We evaluated the efficacies of several EGFR X20ins inhibitors, including zipalertinib and sunvozertinib, against cells with acquired resistant mutations. ResultsAll secondary mutations resulting in acquired resistance to mobocertinib were exclusively C797S in insFQEA and insSVD. However, in the case of other X20ins (insASV, insNPH, and insH), T790M or C797S secondary mutations contributed to acquired resistance to mobocertinib. The emergence of T790M was more frequent in cells treated with lower drug concentrations. Sunvozertinib exhibited good activity against resistant cells with T790M. Cells with C797S were refractory to all EGFR TKIs, except for erlotinib, which was active for insFQEA with C797S. ConclusionsT790M or C797S, depending on the original X20ins mutations, conferred acquired resistance to mobocertinib. Sunvozertinib may be the treatment of choice for patients with tumors resistant to mobocertinib because of T790M.

HLA DQ protein changes the cell surface distribution pattern of HLA proteins as monitored by Förster resonance energy transfer and high-resolution electron microscopy.

Peptide presentation by MHC class I and MHC class II molecules plays important roles in the regulation of the immune response. One factor in these displays is the density of antigen, which must exceed a critical threshold for the effective activation of T cells. Nonrandom distribution of MHC class I and class II has already been detected at the nanometer level and at higher hierarchical levels. It is not clear how the absence and reappearance of some protein molecules can influence the nonrandom distribution. Therefore, we performed experiments on HLA II-deficient bare lymphocyte syndrome (BLS1) cells: we created a stable transfected cell line, tDQ6-BLS-1, and were able to detect the effect of the appearance of HLA-DQ6 molecules on the homo and heteroassociation of different cell surface molecules by comparing Förster resonance energy transfer (FRET) efficiency on transfected cells to that on nontransfected BLS-1 and JY human B-cell lines. Our FRET results show a decrease in homoassociation FRET between HLA I chains in HLA-DQ6-transfected tDQ6-BLS-1 cells compared with the parent BLS-1 cell line and an increase in heteroassociation FRET between HLA I and HLA II (compared with JY cells), suggesting a similar pattern of antigen presentation by the HLA-DQ6 allele. Transmission electron microscopy (TEM) revealed that both HLA class I and class II molecules formed clusters at higher hierarchical levels on the tDQ6-BLS-1 cells, and the de novo synthesized HLA DQ molecules did not intersperse with HLA class I islands. These observations could be important in understanding the fine tuning of the immune response.

Hemin treatment drives viral reactivation and plasma cell differentiation of EBV latently infected B cells.

Epstein-Barr virus (EBV) and Plasmodium falciparum have a well described role in the development of endemic Burkitt lymphoma (BL), yet the mechanisms involved remain unknown. A major hallmark of malarial disease is hemolysis and bystander eryptosis of red blood cells, which causes release of free heme in large quantities into peripheral blood. We hypothesized that heme released during malaria infection drives differentiation of latently infected EBV-positive B cells, resulting in viral reactivation and release of infectious virus. To test this hypothesis, we used the EBV-positive Mutu I B-cell line and treated with hemin (the oxidized form of heme) and evaluated evidence of EBV reactivation. Hemin treatment resulted in the expression of EBV immediate early, early and late lytic gene transcripts. In addition, expression of CD138, a marker of plasma cells was co-expressed with the late lytic protein gp350 on hemin treated Mutu I cells. Finally, DNase-resistant EBV DNA indicative of virion production was detected in supernatant. To assess the transcriptional changes induced by hemin treatment, RNA sequencing was performed on mock- and hemin-treated Mutu I cells, and a shift from mature B cell transcripts to plasma cell transcripts was identified. To identify the mechanism of hemin-induced B cell differentiation, we measured levels of the plasma cell transcriptional repressor, BACH2, that contains specific heme binding sites. Hemin treatment caused significant degradation of BACH2 by 24 hours post-treatment in four BL cell lines (two EBV positive, two EBV negative). Knockdown of BACH2 in Mutu I cells using siRNAs significantly increased CD138+gp350+ cells to levels similar to treatment with hemin. This suggested that hemin induced BACH2 degradation was responsible for plasma cell differentiation and viral reactivation. Together, these data support a model where EBV reactivation can occur during malaria infection via heme modulation, providing a mechanistic link between malaria and EBV.

Comparison of Ki67 Proliferation Index in Gastrointestinal Non-Hodgkin Large B-Cell Lymphomas: The Conventional Method of Evaluation or AI Evaluation?

The most common NHL subtype in SEEU is DLBCL (39%), and it manifests with a variety of cellular morphologies and a high proliferation index. Also, the GI tract is the most common site of extranodal NHLs, and most NHLs involving the GI tract are of B-cell lineage, of which diffuse large B-cell lymphoma is the most common subtype, irrespective of location. The last few years have seen digital pathology as a vital technology that has a positive impact on diagnostics, but studies on the use of DP for lymphoma identification, however, are still restricted to only determining whether a tumor is present or absent. Using the example of cases of malignant NHL, we aim to investigate the diagnostic utility of DP using QuPath software in evaluating the proliferation index and the prognostic significance and to show that improved visualization and analysis contribute to the convergence of these complementary diagnostic modalities for lymphomas. The average proliferation index (Ki67) was 58.33% with values between 10% and 85%. After the stratification of the cases, an increased proliferation index was observed in the majority of cases (53.33%), and this aspect was associated with the advanced age of the patients (p = 0.045). Visual assessment provides lower Ki67 values than automated digital image analysis. However, the agreement coefficient between the conventional method and the AI method indicates an excellent level of reliability (ICC1-0.970, ICC2-0.990). The multivariate analysis revealed that in the cases where the proliferation index Ki67 is high (˃70%), the IPI score represents an important risk factor predicting mortality (HR = 10.597, p = 0.033).

Circulating Biomarkers Associated with the Diagnosis and Prognosis of B-Cell Progenitor Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is a hematological disease characterized by the dysfunction of the hematopoietic system that leads to arrest at a specific stage of stem cells development, suppressing the average production of cellular hematologic components. BCP-ALL is a neoplasm of the B-cell lineage progenitor. BCP-ALL is caused and perpetuated by several mechanisms that provide the disease with its tumor potential and genetic and cytological characteristics. These pathological features are used for diagnosis and the prognostication of BCP-ALL. However, most of these paraclinical tools can only be obtained by bone marrow aspiration, which, as it is an invasive study, can delay the diagnosis and follow-up of the disease, in addition to the anesthetic risk it entails for pediatric patients. For this reason, it is crucial to find noninvasive and accessible ways to supply information concerning diagnosis, prognosis, and the monitoring of the disease, such as circulating biomarkers. In oncology, a biomarker is any measurable indicator that demonstrates the presence of malignancy, tumoral behavior, prognosis, or responses to treatments. This review summarizes circulating molecules associated with BCP-ALL with potential diagnostic value, classificatory capacity during monitoring specific clinic features of the disease, and/or capacity to identify each BCP-ALL stage regarding its evolution and outcome of the patients with BCP-ALL. In the same way, we provide and classify biomarkers that may be used in further studies focused on clinical approaches or therapeutic target identification for BCP-ALL.

Targeting FoxO proteins induces lytic reactivation of KSHV for treating herpesviral primary effusion lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus consisting of both latent and lytic life cycles. Primary effusion lymphoma (PEL) is an aggressive B-cell lineage lymphoma, dominantly latently infected by KSHV. The latent infection of KSHV is persistent and poses an obstacle to killing tumor cells. Like the "shock and kill" strategy designed to eliminate latent HIV reservoir, methods that induce viral lytic reactivation in tumor latently infected by viruses represent a unique antineoplastic strategy, as it could potentially increase the specificity of cytotoxicity in cancer. Inspired by this conception, we proposed that the induction of KSHV lytic reactivation from latency could be a potential therapeutic stratagem for KSHV-associated cancers. Oxidative stress, the clinical hallmark of PEL, is one of the most prominent inducers for KSHV reactivation. Paradoxically, we found that hydrogen peroxide (H2O2) triggers robust cytotoxic effects on KSHV-negative rather than KSHV-positive B lymphoma cells in a dose-dependent manner. Mechanistically, we identified forkhead box protein O1 (FoxO1) and FoxO3 as irrevocable antioxidant defense genes and both of them are upregulated by KSHV latent infection, which is essential for the promoted ROS scavenging in KSHV-positive B lymphoma cells. Pharmacological inhibition or functional knockdown of either FoxO1 or FoxO3 is sufficient to ablate the antioxidant ability and therefore increases the intracellular ROS level that further reverses KSHV from latency to active lytic replication in PEL cells, resulting in tremendous cell death both in vitro and in vivo. Additionally, the elevated level of ROS by inhibiting FoxO proteins further sensitizes PEL cells to ROS-induced apoptosis. Our study therefore demonstrated that the lytic reactivation of KSHV by inhibiting FoxO proteins is a promising therapeutic approach for PEL, which could be further extended to other virus-associated diseases.

Usefulness and Limitations of Current Diagnostic Strategies for Pulmonary Mucosa-Associated Lymphoid Tissue Lymphoma: Lessons Learned From a Large Cohort.

The pathologic diagnosis of pulmonary extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT) is challenging. To evaluate the diagnostic usefulness and limitations of current diagnostic strategies for pulmonary MALT lymphoma. A retrospective review of 120 cases of pulmonary MALT lymphoma from 2014 through 2021 was performed. Clinicoradiologic presentations overlapped with previous observations in patients with MALT lymphoma, such as a wide age range, female predominance, frequent association with autoimmune disease or immunodeficiency, and broad imaging findings. The histopathologic diagnosis was based on a combination of morphology, immunohistochemistry, and demonstration of B-cell lineage clonality. Two-thirds (76 of 113) of MALT lymphomas had lymphoplasmacytoid cytomorphology. Occasionally, MALT lymphomas were associated with granulomas/giant cells (29%, 35 of 120) or immunoglobulin deposition disease (21%, 25 of 120), including light chain/heavy chain deposition disease, amyloidosis, and/or crystal storing histiocytosis. While CD5, CD10, Bcl-2, and Bcl-6 rarely revealed aberrancies, aberrant CD43 expression either on B-cells or on plasma cells was detected in 42% (27 of 64) of cases, including cases for which proof of clonality could not be obtained. κ/λ in situ hybridization was particularly useful for tumors with lymphoplasmacytoid morphology but performed poorly in lymphomas having no plasmacytic differentiation. κ/λ immunohistochemistry showed no additional usefulness when applied together with κ/λ in situ hybridization. Immunoglobulin gene rearrangement studies by polymerase chain reaction achieved high detection rates of clonality in all cytomorphologic subgroups. Our study offers a practical evaluation of common diagnostic tests in pulmonary MALT lymphoma. We offer recommendations for a diagnostic workup that takes into consideration the usefulness and the specific limitations of the various diagnostic strategies.

A dual function for the chromatin organizer Special A-T rich Binding Protein 1 in B-lineage cells.

SATB1 (Special A-T rich Binding protein 1) is a cell type-specific factor that regulates the genetic network in developing T cells and neurons. In T cells, SATB1 is required for lineage commitment, VDJ recombination, development and maturation. Considering that its expression varies during B-cell differentiation, the involvement of SATB1 needs to be clarified in this lineage. Using a KO mouse model in which SATB1 was deleted from the pro-B-cell stage, we examined the consequences of SATB1 deletion in naive and activated B-cell subsets. Our model indicates first, unlike its essential function in T cells, that SATB1 is dispensable for B-cell development and the establishment of a broad IgH repertoire. Second, we show that SATB1 exhibits an ambivalent function in mature B cells, acting sequentially as a positive and negative regulator of Ig gene transcription in naive and activated cells, respectively. Third, our study indicates that the negative regulatory function of SATB1 in B cells extends to the germinal center response, in which this factor limits somatic hypermutation of Ig genes.

The off-target effects of AID in carcinogenesis.

Activation-induced cytidine deaminase (AID) plays a crucial role in promoting B cell diversification through somatic hypermutation (SHM) and class switch recombination (CSR). While AID is primarily associated with the physiological function of humoral immune response, it has also been linked to the initiation and progression of lymphomas. Abnormalities in AID have been shown to disrupt gene networks and signaling pathways in both B-cell and T-cell lineage lymphoblastic leukemia, although the full extent of its role in carcinogenesis remains unclear. This review proposes an alternative role for AID and explores its off-target effects in regulating tumorigenesis. In this review, we first provide an overview of the physiological function of AID and its regulation. AID plays a crucial role in promoting B cell diversification through SHM and CSR. We then discuss the off-target effects of AID, which includes inducing mutations of non-Igs, epigenetic modification, and the alternative role as a cofactor. We also explore the networks that keep AID in line. Furthermore, we summarize the off-target effects of AID in autoimmune diseases and hematological neoplasms. Finally, we assess the off-target effects of AID in solid tumors. The primary focus of this review is to understand how and when AID targets specific gene loci and how this affects carcinogenesis. Overall, this review aims to provide a comprehensive understanding of the physiological and off-target effects of AID, which will contribute to the development of novel therapeutic strategies for autoimmune diseases, hematological neoplasms, and solid tumors.

Intravascular Large B-Cell Lymphoma Detected by Cutaneous Biopsy with Pancytopenia and Negative Bone Marrow Biopsy

Intravascular large B-cell lymphoma is a B-cell lineage neoplasm, which is typically recognized as a high-grade neoplasm with poor outcomes. Though rare, the cutaneous variant can present with multiple lesions of the skin and negative systemic staging. We present a case of intravascular large B-cell lymphoma first identified on cutaneous biopsy, with pancytopenia, in addition to negative bone marrow biopsy and serum protein electrophoresis. The patient is a 71-year-old male who presented with upper body pruritis and pancytopenia, as well as weight loss and fatigue. A full body skin exam incidentally revealed a well-demarcated, firm, bound down atrophic lichenified plaque on the right anterior thigh. A punch biopsy revealed large, atypical lymphoid cells exclusively localized within the lumina of variably sized blood vessels, immunoreactive for CD20, BCL6, MUM1, and CD5, but negative for CD3, CD10, cyclin D1, and TdT. A diagnosis of intravascular large B-cell lymphoma was made, and he was referred to oncology for further treatment. Full body skin exam is a mainstay of dermatologic practice, and it is essential to pursue biopsy with uncharacteristic lesions, regardless of the presenting concern.

LC-MS/MS-Guided Molecular Networking for Targeted Discovery of Undescribed and Bioactive Ophiobolins from Bipolaris eleusines.

An integrated purification procedure through the LC-MS/MS-based molecular networking strategy combined with bioactive evaluation was first ushered for discovering bioactive ophiobolins from Bipolaris eleusines. Ophiobolins were mainly dispersed in five clusters, which were classified based on different ring systems and functional groups. Nine undescribed ophiobolins (1-6 and 9-11) and an undescribed natural product (8) along with two known analogs (7 and 12) were isolated in target. The undescribed structures were characterized by HR-ESI-MS, NMR spectra, and X-ray diffraction experiments. Compounds 3-12 exhibited strong phytotoxic effects on green foxtails by producing visible lesions, and compounds 1-10 and 12 displayed different levels of cytotoxic activities against cancer cell lines B16, Hep G2, and MCF-7, from which the possible structure-activity relationships were then suggested. The results have supported that bioactivity-guided molecular networking is an efficient strategy to expedite the discovery of undescribed bioactive natural products.