Mouse B-cell Research Articles

B-cell and plasma cell activation in a mouse model of chronic muscle pain

Fibromyalgia (FM) is a complex chronic musculoskeletal pain disorder with an elusive pathogenesis, with a strong implication of immune interactions. We recently found that IL-5 and the adaptive immune system mediates pain outcomes in fibromyalgia (FM) patients and preclinical models of FM-like chronic widespread pain (CWP). However, there is an active debate if FM/CWP has an autoimmune etiology. Preclinical models of CWP utilize a repeated insult paradigm, which resembles a primary, then secondary response similarly observed in the antibody response in which the subsequent event causes a potentiated pain response. Recent translational studies have implicated immunoglobulins (Ig) and B-cells in FM/CWP pathophysiology. To understand if these are involved in preclinical models of CWP, we performed comprehensive B-cell phenotyping in the bone marrow, circulation, and popliteal (draining) lymph nodes in the two-hit acidic saline model of CWP. We found increased MHC class II-expressing B-cells in peripheral blood, increased activated plasma cells in peripheral blood, and increased memory B-cells in the bone marrow. Interestingly, acidic pH (4.0) injected mice have reduced levels of IgG1, independent of treatment with IL-5. We have demonstrated that the acidic saline model of CWP induces T-cell mediated activation of B-cells, increased active plasma cells, and increased memory B-cells in female mice.

The rheumatoid arthritis drug auranofin exerts potent anti-lymphoma effect by stimulating TXNRD-mediated ROS generation and inhibition of energy metabolism

Since the survival of lymphoma patients who experience disease progression or relapse remains very poor, new therapeutic approaches and effective drugs are urgently needed. Here we show that auranofin (AF), an anti-rheumatoid drug thought to inhibit thioredoxin reductases (TXNRDs) as its mechanism of action, exhibited potent activity against multiple cancer types, especially effective against B cell lymphoma. Surprisingly, a knockdown of TXNRD1 and TXNRD2 did not cause significant cytotoxicity, suggesting that abrogation of TXNRD enzyme per se was insufficient to cause cancer cell death. Further mechanistic study showed that the interaction of AF with TXNRD could convert this antioxidant enzyme to a ROS-generating molecule via disrupting its electron transport, leading to a leak of electrons that interact with molecular oxygen to form superoxide. AF also suppressed energy metabolism by inhibiting both mitochondria complex II and the glycolytic enzyme GAPDH, leading to a significant depletion of ATP and inhibition of cancer growth in vitro and in vivo. Importantly, we found that the AF-mediated ROS stress could induce PD-L1 expression, revealing an unwanted effect of AF in causing immune suppression. We further showed that a combination of AF with anti-PD-1 antibody could enhance the anticancer activity in a syngeneic immune-competent mouse B-cell lymphoma model. Our study suggests that AF could be a potential drug for lymphoma treatment, and its combination with immune checkpoint inhibitors would be a logical strategy to increase the therapeutic activity.

IL-27-engineered CAR.19-NK-92 cells exhibit enhanced therapeutic efficacy

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells has shown promising results in early-phase clinical studies. However, advancing CAR-NK cell therapeutic efficacy is imperative. In this study, we investigated the impact of a fourth-generation CD19-targeted CAR (CAR.19) coexpressing IL-27 on NK-92 cells. We observed a significant improvement in NK-92 cell proliferation and cytotoxicity activity against B-cell cancer cell lines, both in vitro and in a xenograft mouse B-cell lymphoma model. Our systematic transcriptome analysis of the activated NK-92 CAR variants further supports the potential of IL-27 in fourth-generation CARs to overcome limitations of NK cell-based targeted tumor therapies by providing essential growth and activation signals. Integrating IL-27 into CAR-NK cells emerges as a promising strategy to enhance their therapeutic potential and elicit robust responses against cancer cells. These findings contribute substantially to the mounting evidence supporting the potential of fourth-generation CAR engineering in advancing NK cell-based immunotherapies.

Glycerolipid Metabolism Via the MGA-MYC-NME1-PGP Axis As a Key Regulator of Oxidative Phosphorylation in Richter's Transformation

Despite advances in targeted therapies for chronic lymphocytic leukemia (CLL), Richter's transformation (RT) remains a clinical challenge. RT is an aggressive shift from CLL to lymphoma, associated with metabolic changes and aberrations in the MYC transcriptional network. Genomic analysis of paired CLL and RT cases revealed recurrent mutations or deletions in the MYC suppressor Max-gene-associated (MGA) increase from 5% (CLL) to 30% (RT) cases, highlighting its critical yet unexplored role as a driver event. We recently developed a murine RT model using B cell-restricted in vitro editing of LSK progenitors from Cd19Cre-Cas9-del(13q)-Sf3b1 ( Sf3b1-K700E) with Mga-targeting sgRNAs. This murine model demonstrated upregulation of Mga targets Myc and Nme1 (nucleoside diphosphate kinase), a distinct OXPHOS-based transcription signature, increased oxygen consumption rate, elevated ROS levels, suggesting a significant link between the Mga-Myc-Nme1 axis and OXPHOS dysregulation in RT. Given the crucial role of metabolic reprogramming in cancer, we seek to understand the molecular mechanisms of metabolites driving OXPHOS and accelerating CLL transformation. Unraveling these processes could revolutionize therapeutic approaches for managing RT in clinical settings. To identify metabolites driving dysregulated OXPHOS in RT, we first conducted paired RNA-seq and unlabeled metabolomic profiling on normal and RT mouse splenic B cells (n=3/group). To translate our discovery from the murine RT model to human disease, we performed metabolomic profiling using MEC1 and Nalm6 cell lines with MGA deletion. Our metabolomic analysis of murine RT cells revealed 44 upregulated metabolites, including TCA cycle substrates (glutamine fold change (FC)=9.6, p=0.001;asparagine FC= 4.0, p=0.006), fatty-acid intermediates (glycerol-3P FC=11.7, p=0.003; carnitine FC=4.3, p=0.02; palmitate FC= 4.1, p= 0.08), and glycolysis intermediates (3-phosphoglycerate FC= 4.0, p=0.03; UDP-glucose FC=12.7, p=0.02; glycine FC=4.7, p=0.03). Moreover, integrated metabolite analysis (MetaboAnalyst v5.0) identified 63 common upregulated metabolites in MGA KO (knockout) human cell lines and murine RT, enriching glycerolipid, and fatty acid oxidation, associated pathways, indicating a potential role for MGA deletion in driving dysregulated fatty acid metabolism. Pathway enrichment analysis using integrated RNA-seq and metabolite analyses showed that the glycerolipid pathway was one of the most enriched pathways among mouse RT and B-cell lines (Nalm6 and MEC1) with MGA KO. Additionally, two key fatty-acid enzymes, fatty acid synthetase (FASN) and phosphoglycerate phosphatase (PGP), displayed significant mRNA and protein changes, as validated by qPCR and immunoblotting. Furthermore, elevated lipid accumulation and increased free glycerol were confirmed based on lipi-green flow cytometry and glycerol-bioluminescent assays, suggesting an important role of glycerolipid metabolism in MGA KO-associated OXPHOS. PGP, a glycerol-3-phosphatase, regulates glycerol-3-P levels, a key intermediate in glucose, lipid, and energy metabolism. Overexpression of MYC and NME1 increases PGP levels in B-cell lines (MEC1, Nalm6, and Jeko), confirming PGP as a direct target of the MGA-MYC-NME1 axis. To determine the role of PGP in MGA KO (knock-out) cells, we used CRISPR/Cas9 to KO PGP in MEC1, Nalm6, and JeKo cell lines. KO of PGP decreased cell growth and reduced OXPHOS compared to controls based on CCK8 absorbance and Seahorse Mitostress assays respectively. Moreover, PGP KO downregulated the mTOR (p4E-BP1) pathway, emphasizing the significance of dysregulated glycerol and fatty acid metabolism in CLL to RT-driven OXPHOS. Our study reveals key metabolites driving RT and highlights the crucial role of the MGA-MYC-NME1-PGP axis in regulating OXPHOS in Mga KO RT cells. Our results thus suggest targeting glycerolipid metabolism and related pathways may offer effective strategies to conquer RT

A Multimodal Imaging-Supported Down Syndrome Mouse Model of RSV Infection.

Individuals with Down syndrome (DS) are more prone to develop severe respiratory tract infections. Although a RSV infection has a high clinical impact and severe outcome in individuals with DS, no vaccine nor effective therapeutics are available. Any research into infection pathophysiology or prophylactic and therapeutic antiviral strategies in the specific context of DS would greatly benefit this patient population, but currently such relevant animal models are lacking. This study aimed to develop and characterize the first mouse model of RSV infection in a DS-specific context. Ts65Dn mice and wild type littermates were inoculated with a bioluminescence imaging-enabled recombinant human RSV to longitudinally track viral replication in host cells throughout infection progression. This resulted in an active infection in the upper airways and lungs with similar viral load in Ts65Dn mice and euploid mice. Flow cytometric analysis of leukocytes in lungs and spleen demonstrated immune alterations with lower CD8+ T cells and B-cells in Ts65Dn mice. Overall, our study presents a novel DS-specific mouse model of hRSV infection and shows that potential in using the Ts65Dn preclinical model to study immune-specific responses of RSV in the context of DS and supports the need for models representing the pathological development.

Abstract 1757: Characterizing novel immunomodulating interactions for potentiating HDAC3 inhibition in non-Hodgkin lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and represents a highly heterogeneous and aggressive disease. Up to 40% of all DLBCL patients experience treatment failure after the standard immunochemotherapy R-CHOP. Defining novel therapeutic strategies is therefore a pressing need. Reduced acetylation at histone 3 lysine 27 (H3K27ac) represents a pathogenetic hallmark of DLBCL and selective histone deacetylase 3 inhibition (HDAC3i) has recently emerged as a viable strategy to reverse abnormal epigenetic signatures and induce strong anti-tumor effects by increasing H3K27ac. Despite this compelling rationale, the overall response remains suboptimal, suggesting unrecognized mechanisms of resistance. To unbiasedly uncover sensitizers, we performed a genome-wide CRISPR screen and identified knockout (KO) of the top candidate GNAS (encoding G-protein α subunit, Gαs) for sensitizing resistant DLBCL cells to HDAC3i. The sensitizing effects on reduced viable cell number were only observed in GNAS KO cells treated with HDAC3i, but not in the control cells (“synthetic lethality”). We have validated this sensitizing phenotype in multiple resistant cell lines including three human DLBCL cell lines and one mouse B-cell lymphoma cell line. Interestingly, we showed that the sensitization was not related to the canonical G-protein functions but to novel regulatory mechanisms in viral mimicry activation and chromatin modification. Compared with the control conditions, the interaction between GNAS KO and HDAC3i has been linked with differential chromatin accessibility and endogenous retrovirus expression, upregulated double-stranded RNA expression, and increased phosphorylation of TBK1 whose activation plays a vital role in antiviral immune responses. Strikingly, a selective TBK1 degrader completely and specifically rescued the sensitizing phenotype in GNAS KO cells treated with HDAC3i, confirming the critical role of TBK1 in inducing sensitization. Furthermore, we identified potential immune interactions by exploring the clinical data from tumor samples of 309 DLBCL patients that low GNAS expression was correlated with cytokine and inflammatory signaling as well as stromal cell interactions in the tumor microenvironment, altogether associated with longer overall survival. These observations were aligned with our results in the mouse xenograft and allograft tumor experiments that GNAS KO with/without HDAC3i led to differential phenotypes. Overall, our research uncovers novel immunomodulating activities for potentiating the efficacy of epigenetic agents in non-Hodgkin lymphoma. This expands our understanding of novel interactions between immune and epigenetic regulation in lymphoma biology and provides new therapeutic opportunities for patients with resistant lymphoma who have poor prognoses and limited treatment options. Citation Format: Michael Y. He, Kit I. Tong, Mehran Bakhtiari, Ryder Whittaker Hawkins, Ting Liu, Yong Zeng, Noorhan Ghanem, Housheng Hansen He, Robert Kridel. Characterizing novel immunomodulating interactions for potentiating HDAC3 inhibition in non-Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1757.

Abstract 2586: OncogenicPIK3CA promotes hematopoietic reprogramming and cell transdifferentiation developing hematopoietic malignancy

Abstract PIK3CA mutation is one of the most common, recurrent genetic abbrations identified across multiple types of cancers. Such mutations sustain activation of PI3K/AKT/mTOR pathway, being considered reliable therapeutic targets; yet, the functional consequences still remain unclear. Angiosarcomas are a heterogenous group of soft-tissue sarcomas that form malignant endothelium with disorganized, irregular blood-filled vascular spaces, and a subset of the vascular tumors harbor PIK3CA mutations. In this study, we found that one of 11 canine angiosarcoma cells gave rise to vascular tumors in 3 of 3 mice in a series of xenograft experiments using a total of 86 immunodeficient BNX mice. Intriguingly, we also observed that xenografts developed lymphoproliferative tumors indicating a mouse B-cell origin in 3 of 4 mice from one case of canine patient-derived tumor xenografts: four tumor cases surgically implanted in a total of 16 BNX mice. Additionally, canine angiosarcoma cells induced splenomegaly with expansion of Ter-119+ erythroid progenitors in NSG mice. Canine angiosarcoma cells (two cell lines; DHSA-1426 and EFB) were capable of hematopoietic expansion and cell lineage differentiaton in vitro. Since PIK3CA mutations appear to promote cellular stemness and impairment of cell lineage differentiation, we determined if the mutations play a role in hematopoietic regulations of angiosarcoma cells. DHSA-1426 angiosarcoma cells were used to induce PIK3CA H1047R mutations using CRISPR/Cas9. We found that PIK3CA mutant angiosarcoma cells dysregulated AKT, ERK, and mTOR pathway with enrichement of inflammatory cytokines such as IL-6, IL-8, and MCP-1. Single cell RNA-seq data showed that PIK3CA mutant angiosarcoma cells established distinct single cell clusters representing immune reactions, cellular stemness, and lineage differentiation. Furthermore, PIK3CA mutant angiosarcoma cells developed malignant hematopoietic tumor in one of five BNX mice, while no evidence of hematopoietic tumor formation was found in mice transplanted with non-mutant cells. Altogether, our data suggest that angiosarcoma cells have the capacity to promote hematopoietic imbalance impacting on cellular ontogeny and lineage differentiation, potentially mediated by oncogenic PIK3CA. Our ongoing work is determining the mechanism of the cell fate decisions using bi-potential hematoendothelial progenitors derived from induced pluripotent stem cells harboring oncogenic PIK3CA. Citation Format: Emma Kozurek, Erin B. Dickerson, Jong Hyuk Kim. OncogenicPIK3CA promotes hematopoietic reprogramming and cell transdifferentiation developing hematopoietic malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2586.

Identification of functional gene modules by integrating multi-omics data and known molecular interactions.

Multi-omics data integration has emerged as a promising approach to identify patient subgroups. However, in terms of grouping genes (or gene products) into co-expression modules, data integration methods suffer from two main drawbacks. First, most existing methods only consider genes or samples measured in all different datasets. Second, known molecular interactions (e.g., transcriptional regulatory interactions, protein-protein interactions and biological pathways) cannot be utilized to assist in module detection. Herein, we present a novel data integration framework, Correlation-based Local Approximation of Membership (CLAM), which provides two methodological innovations to address these limitations: 1) constructing a trans-omics neighborhood matrix by integrating multi-omics datasets and known molecular interactions, and 2) using a local approximation procedure to define gene modules from the matrix. Applying Correlation-based Local Approximation of Membership to human colorectal cancer (CRC) and mouse B-cell differentiation multi-omics data obtained from The Cancer Genome Atlas (TCGA), Clinical Proteomics Tumor Analysis Consortium (CPTAC), Gene Expression Omnibus (GEO) and ProteomeXchange database, we demonstrated its superior ability to recover biologically relevant modules and gene ontology (GO) terms. Further investigation of the colorectal cancer modules revealed numerous transcription factors and KEGG pathways that played crucial roles in colorectal cancer progression. Module-based survival analysis constructed four survival-related networks in which pairwise gene correlations were significantly correlated with colorectal cancer patient survival. Overall, the series of evaluations demonstrated the great potential of Correlation-based Local Approximation of Membership for identifying modular biomarkers for complex diseases. We implemented Correlation-based Local Approximation of Membership as a user-friendly application available at https://github.com/free1234hm/CLAM.

BCL6-Mediated Escape from Negative Selection Enables RAS-Driven B-Cell Transformation

Background: In B-cell acute lymphoblastic leukemia (B-ALL), RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) are found in ~40% of cases (Irving et al., 2014; Jerchel et al., 2018; Zhang et al., 2011). Evidence suggests that RAS-pathway mutations in B-ALL subclones are selected for during therapy and are associated with relapse, which has a poor prognosis and remains one of the leading causes of death in children with malignancy (Irving et al., 2014; Jerchel et al., 2018). Beyond its role as a driver of B-ALL, ERK-activation can induce expression of PRDM1, which functions as a tumor suppressor and triggers negative B-cell selection and cell death (Yasuda et al., 2011; Hug et al., 2014; Setz et al., 2018). During B-cell development, PRDM1 and the proto-oncogene BCL6 are reciprocal antagonists (Shaffer et al., 2002). Here, we examined the mechanism by which oncogenic RAS-ERK signaling in B-ALL avoids negative selection and cell death to promote leukemogenesis. Results: Mimicking survival signals from pre-B cell receptor (pre-BCR), constitutive Erk activation upon oncogenic RAS (NRASG12D) expression in mouse B-cell precursors massively induced the mRNA (~390-fold) and protein (~50-fold) levels of the proto-oncogene Bcl6. Highlighting an essential role of Erk-signaling in upregulation of Bcl6 expression downstream of oncogenic RAS, pharmacological Erk-inhibition markedly reduced NRASG12D-mediated Bcl6 induction. Both the pre-BCR linker molecule Blnk and the surrogate light chains (SLCs) of the pre-BCR are required for Erk-mediated induction of Bcl6. Genetic ablation of Blnk or the SLC component λ5 abrogated RAS-mediated Bcl6-induction. Hence, oncogenic expression of Bcl6 in RAS-driven B-ALL cells depended on structural elements of the pre-BCR. Additionally, ERK-activation in patient-derived B-ALL cells had the same effect on BCL6 expression. Pharmacological reactivation of ERK (BCI-215) markedly induced BCL6 expression, whereas small molecule inhibition of ERK-activity (trametinib) reduced BCL6 levels in patient-derived B-ALL cells. Consistent with BCL6-transactivation downstream of ERK-activation, ChIP-seq analysis revealed binding of ERK-dependent transcription factors (CREB1, ELK1, EGR1, JUND and C-JUN) to the BCL6 promoter in human B cells. Altogether, our findings illuminated a critical role of ERK-signaling in positive regulation of BCL6 expression. Using a genetic model for Cre-mediated deletion of Bcl6 in NRASG12D B-ALL cells, we found that Bcl6 was required for leukemia-initiation in transplant recipient mice (P=0.007). Patient-derived RAS-driven B-ALL cells were highly sensitive to pharmacological inhibition of BCL6 using peptide (RI-BPI) and small molecule (FX1) inhibitors. Furthermore, pharmacological inhibition of BCL6 delayed onset of fatal disease and prolonged survival of transplant recipient mice bearing patient-derived RAS-driven B-ALL cells (P=0.009). Taken together, our findings demonstrated that suppressing BCL6 function is a promising option for targeted therapeutics in RAS-driven B-ALL. Mechanistically, genetic ablation or pharmacological inhibition of BCL6 increased PRDM1 expression. Gene expression analysis revealed that ectopic expression of Prdm1 in B-cell precursors promoted gene expression programs of apoptosis and cell cycle arrest (GSE111692). While Bcl6-deletion resulted in cell death of B-ALL cells, this was largely reversed by shRNA-mediated silencing of Prdm1. Altogether, our results showed that compromised leukemogenesis was a result of aberrant PRDM1 expression in BCL6-deficient RAS-driven B-ALL cells. Conclusions: We previously investigated targeted engagement of negative selection in B-ALL and showed that hyperactivation of SYK or AKT functionally mimics autoreactive pre-BCR signaling and results in negative selection and cell death (Chen et al., 2015; Shojaee et al., 2016). Here, we reinforced this concept by providing a mechanistic basis on how negative selection is downregulated in RAS-driven B-ALL and how it can be reactivated for therapeutic benefit - namely, BCL6 curbs ERK-mediated induction of PRDM1 expression to promote leukemogenesis and this mechanism can be exploited as synthetic lethality in the treatment of this disease.

The oxidative phosphorylation inhibitor IM156 suppresses B-cell activation by regulating mitochondrial membrane potential and contributes to the mitigation of systemic lupus erythematosus

Current treatment strategies for autoimmune diseases may not sufficiently control aberrant metabolism in B-cells. To address this concern, we investigated a biguanide derivative, IM156, as a potential regulator for B-cell metabolism invitro and invivo on overactive B-cells stimulated by the pro-inflammatory receptor TLR-9 agonist CpG oligodeoxynucleotide, a mimic of viral/bacterial DNA. Using RNA sequencing, we analyzed the B-cell transcriptome expression, identifying the major molecular pathways affected by IM156 invivo. We also evaluated the anti-inflammatory effects of IM156 in lupus-prone NZB/W F1 mice. CD19+B-cells exhibited higher mitochondrial mass and mitochondrial membrane potential compared to T-cells and were more susceptible to IM156-mediated oxidative phosphorylation inhibition. Invivo, IM156 inhibited mitochondrial oxidative phosphorylation, cell cycle progression, plasmablast differentiation, and activation marker levels in CpG oligodeoxynucleotide-stimulated mouse spleen B-cells. Interestingly, IM156 treatment significantly increased overall survival, reduced glomerulonephritis and inhibited B-cell activation in the NZB/W F1 mice. Thus, our data indicated that IM156 suppressed the mitochondrial membrane potentials of activated B-cells in mice, contributing to the mitigation of lupus activity. Hence, IM156 may represent a therapeutic alternative for autoimmune disease mediated by B-cell hyperactivity.

Neutralizing monoclonal antibodies elicited by mosaic RBD nanoparticles bind conserved sarbecovirus epitopes

Increased immune evasion by SARS-CoV-2 variants of concern highlights the need for new therapeutic neutralizing antibodies. Immunization with nanoparticles co-displaying spike receptor-binding domains (RBDs) from eight sarbecoviruses (mosaic-8 RBD-nanoparticles) efficiently elicits cross-reactive polyclonal antibodies against conserved sarbecovirus RBD epitopes. Here, we identified monoclonal antibodies (mAbs) capable of cross-reactive binding and neutralization of animal sarbecoviruses and SARS-CoV-2 variants by screening single mouse B-cells secreting IgGs that bind two or more sarbecovirus RBDs. Single-particle cryo-EM structures of antibody–spike complexes, including a Fab-Omicron complex, mapped neutralizing mAbs to conserved class 1/4 RBD epitopes. Structural analyses revealed neutralization mechanisms, potentials for intra-spike trimer crosslinking by IgGs, and induced changes in trimer upon Fab binding. In addition, we identified a mAb resembling Bebtelovimab, an EUA-approved human class 3 anti-RBD mAb. These results support using mosaic RBD-nanoparticle vaccination to generate and identify therapeutic pan-sarbecovirus and pan-variant mAbs.

BCL-2 isoform β promotes angiogenesis by TRiC-mediated upregulation of VEGF-A in lymphoma.

Bcl-2 (B-cell lymphoma 2), the first identified anti-apoptosis factor, encodes two transcripts, the long isoform α and the short isoform β. The current understanding of the Bcl-2 function mainly focuses on Bcl-2α, while little is known about the function of Bcl-2β, which lacks the transmembrane domain and contains 10 unique amino acids at the C-terminus instead. Here, we analyzed the expressions of BCL-2 two isoforms in diffused large B-cell lymphoma (DLBCL) and found a significant positive correlation between them. Then, with the CRISPR/Cas9-based transcriptional activator (CRISPRa), we generated mouse B-cell lymphomas with Bcl-2 upregulation from the endogenous locus, in which both Bcl-2α and Bcl-2β levels were increased. Bcl-2β itself promoted angiogenesis both in vitro and in vivo through increased vascular endothelial growth factor A (VEGF-A). Inhibiting VEGF receptors with Axitinib reduced angiogenesis induced by Bcl-2β overexpression. Co-immunoprecipitation and mass spectrometry analysis revealed that Bcl-2β interacted with the T-complex protein ring complex (TRiC). Disruption of TRiC significantly impaired the angiogenesis-promoting activity of Bcl-2β, indicated by reduced VEGF-A protein level and HUVEC tube formation. Thus, our study suggests that Bcl-2 isoform β plays a role in promoting tumor angiogenesis through the Bcl-2β-TRiC-VEGF-A axis.

Design and Optimization of Thienopyrimidine Derivatives as Potent and Selective PI3Kδ Inhibitors for the Treatment of B-Cell Malignancies.

Phosphoinositide 3-kinase δ (PI3Kδ) plays a critical role in B lymphocyte (B-cell) development and activation and has been a validated target for the treatment of B-cell malignancies. Herein, we report a series of thienopyrimidine derivatives as novel potent and selective PI3Kδ inhibitors based on a scaffold hopping design strategy. Among them, compound 6 exhibited nanomolar PI3Kδ potency and a favorable selectivity profile compared to other class I PI3K isoforms. In cellular assays, compound 6 showed antiproliferative activity against a panel of B-cell lymphoma cell lines in a low micromolar range, caused cell cycle arrest, and induced apoptosis in Pfeiffer and SU-DHL-6 cells. Further, compound 6 inhibited the activation of mouse B-cells. With support from in vivo pharmacokinetic studies, compound 6 demonstrated significant anticancer efficacy in a Pfeiffer xenograft mouse model. Overall, compound 6 is a promising PI3Kδ inhibitor worthy of further preclinical investigation for the treatment of B-cell malignancies.

Genomic profile of radiation-induced early-onset mouse B-cell lymphoma recapitulates features of Philadelphia chromosome-like acute lymphoblastic leukemia in humans.

Epidemiological studies have revealed a radiation-related increase in the risk of developing acute lymphoblastic leukemia (ALL). Our recent study revealed early induction and increased risk of precursor B-cell (pB) lymphomas in mice after radiation exposure. However, the genomic landscape of radiation-induced B-cell lymphomas remains unclear. To identify the relevant genetic alterations in mice, whole-exome sequencing was performed on both early-onset and late-onset B-cell lymphomas that developed spontaneously or after gamma-irradiation. In addition to multiple driver mutations, the data revealed that interstitial deletion of chromosome 4, including Pax5, and missense mutations in Jak3 are unique genomic alterations in radiation-induced, early-onset B-cell lymphomas. RNA sequencing revealed a pB-cell-type gene-expression profile with no involvement of known fusion genes for human ALLs in the early-onset B-cell lymphomas. Activation of Jak3/Stat5 signaling in early-onset B-cell lymphomas was validated using western capillary electrophoresis. Those features were similar to those of Philadelphia chromosome-like ALL. Our data suggest a critical role for Pax5 loss-of-function mutations in initiating B-cell leukemogenesis coupled with activation of Jak3/Stat5 signaling as a basis for the rapid development of radiation-induced pB-ALL. These molecular signatures for radiation-induced cancers will inform both risk assessment and potential targeted therapies for pB-ALL.

Effect of the combination of caffeine and taurine of different concentrations on activated B-cells

Caffeinated drinks are popular because of their mood- and performance-enhancing effects. Energy drinks in particular are advertised with these properties, with the content of caffeine and taurine being a central component of these products. However, little is known about the influence of taurine and caffeine on immune cells. This article answers the following research question: “How high is the effect of the combination of caffeine and taurine of different concentrations on activated B-cells”.Immunoglobulin production is a measure of B-cell activity. This property is determined in mouse B-cells. For this purpose, these cells are taken from the spleen of mice, induced with CD40/IL4 and the activity is checked with the aid of an immunoglobulin ELISA. Low doses of taurine and caffeine in combination do not cause any significant change in immunoglobulin production, whereas high doses (4 or 40 mg / ml) lead to cell death.

AVM0703 Tumor Debulking Enhances Cy/Flu Efficacy

AVM0703 is a high concentration (24 mg/mL) dexamethasone phosphate drug product that permits the safe administration of the doses necessary to mobilize gamma delta+, bi-specific natural killer T (NKT) cells. AVM0703 is in clinical trials as a stand-alone treatment for relapsed/refractory (R/R) Non-Hodgkin's Lymphoma (NHL). Even more exciting is the potential combination of AVM0703 with standard chemotherapy to reduce the number of treatment cycles while maintaining efficacy. A therapeutic solution that reduces the number of chemotherapy cycles could lower the risk of secondary malignancies, decrease long-term toxicities, and limit costs associated with management of chemotherapy toxicities. AVM0703 has been well tolerated to date, with mild to moderate and self-limiting side effects. This indicates the use of AVM0703 in combination therapies may greatly improve quality of life for patients during treatment.AVM0703 has been tested in an aggressive mouse B-cell lymphoma model (A20) alone and in combination with cyclophosphamide/fludarabine (CyFlu). The tumor killing effect of AVM0703 on A20 cells in Matrigel TM embedded tumors is evident as early as 3 hours after dosing, assessed by flow cytometry. Maximum A20 cell death is observed 24 hours after treatment in blood (Figure 1) and between 3- and 24-hours in tumors (Figure 2). Treatment with AVM0703 alone was compared with Cy/Flu alone and with a combination of single dose AVM0703 followed by one Cy/Flu cycle. Tumor growth was monitored by caliper measurements and blood and tumors were analyzed by flow cytometry at the end point, 7-days after Cy/Flu dosing. AVM0703 alone was superior to one cycle of Cy/Flu and the combination of a dose of AVM0703 followed by Cy/Flu was the most effective (Figure 3). Of the mice treated with the combination, 33% had no visible tumor 7-days post-dose. We are currently testing AVM0703 administered between 6- and 24-hours before a single Cy/Flu dose and predict that A20 eradication should be more complete in all compartments. Acute 7 day studies will be followed by long-term studies to evaluate A20 tumor escape or recurrence. In previous studies with long term monitoring of tumor growth, AVM0703 administered 3 days before one cycle of Cy/Flu induced long-term stable disease up to 50 days in tumor bearing mice (Figure 4a) compared to two cycles of Cy/Flu where 33% of the tumors escaped (Figure 4b). Additional studies in combination with R-CHOP are underway.AVM0703 has the ability to debulk the tumor by mobilizing gamma delta+, bi-specific natural killer T (NKT) cells. When added to a chemotherapeutic regimen, the chemotherapy may be more effective due to the lower tumor burden in the body. As cancer survivors live longer, the late term consequences of chemotherapy have become apparent, which include secondary malignancies. The challenge for new treatments is to reduce toxicities without sacrificing efficacy. AVM0703 has the potential to be one solution when used in combination with chemotherapy. Future clinical trials will determine if debulking with AVM0703 results in a reduction of the number of necessary R-CHOP cycles while maintaining or enhancing the efficacy rate. [Display omitted] DisclosuresDeisher: AVM Biotechnology, LLC: Current Employment. Sawas: AVM Biotechnology, LLC: Current Employment. Suwito: AVM Biotechnology, LLC: Current Employment. Rylatt: AVM Biotechnology, LLC: Current Employment. Jarzyna: AVM Biotechnology, LLC: Current Employment. Zahid: AVM Biotechnology, LLC: Ended employment in the past 24 months. Parthasarathy: AVM Biotechnology, LLC: Consultancy, Ended employment in the past 24 months. Poulin: AVM Biotechnology, LLC: Current Employment. Lee-Diaz: AVM Biotechnology, LLC: Current Employment.

Surface-associated antigen induces permeabilization of primary mouse B-cells and lysosome exocytosis facilitating antigen uptake and presentation to T-cells.

B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here, we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.

Radiation Therapy Activates Interferon-Responsive Genes in Follicular B-Cells in the Tumor-Draining Lymph Node

Radiation therapy (RT) constitutes a fundamental treatment modality for head and neck squamous cell carcinoma. As the use of checkpoint blockade immunotherapy becomes more prevalent and combinatorial therapies are explored, it is imperative to understand how RT alters specific immune cell subsets. Prior studies have investigated the effect of RT on T-cells and myeloid cells; more recent studies have begun to study B-cells and tertiary lymphoid structures, yet much remains to be uncovered as B-cells are critical in facilitating an antigen-specific adaptive anti-tumor response.Wild type C3H mice were injected subcutaneously with 5 × 105 cells of AT-84-E7-OVA into the right flank. Mice were then treated with 12 Gy of focused RT direct at the tumor. Tumor-draining lymph nodes were dissected and FACS was used to isolate B-cells. Cells were then counted and loaded onto a 10x-Genomics Chromium controller. 10x-Genomics v3 libraries were then prepared per manufacturer recommendations and then sequenced with a target of a minimum of 20000 reads per cell using an Illumina Hiseq 4000. Single-cell RNA sequencing data were then initially processed using Cell Ranger, converted into matrix files, and subsequently analyzed using Seurat v3. Pathway analysis was performed using Metascape.Single-cell RNA-sequencing data of mice that were untreated and treated with RT alone revealed 7 distinct clusters of B-cells in the tumor-draining lymph node that could be broadly distinguished as being follicular vs germinal center B cells. Amongst the follicular B-cells, there was one cluster that constituted 1.7% of B-cells in untreated mice and 5.1% in mice treated with RT. Compared to naïve follicular B-cells, this cluster had 88 upregulated genes; pathway analysis demonstrated that these genes were involved in: "response to interferon-alpha" (7 genes, log(P) = -11.3), and "positive regulation of interferon-beta production" (5 genes, log(P) = -6.5). Upon closer analysis, cells in this cluster derived from mice treated with RT had 77 genes upregulated compared to those from untreated mice. Successive pathway analysis exhibited that these genes were related to: "generation of precursor metabolites and energy" (10 genes, log(P) = -5.9), "positive regulation of cell cycle" (6 genes, log(P) = -2.7), amongst others.RT significantly increased the proportion of B-cells that expressed genes involved in interferon-related pathways and additionally altered the intrinsic phenotype of this B-cell subset. Continued investigation into how B-cells react to conventional therapies may reveal further therapeutic targets to successively polarize B-cell activation and exploit B-cell-mediated anti-tumor responses.

Cohesin Core Complex Gene Dosage Contributes to Germinal Center Derived Lymphoma Phenotypes and Outcomes.

The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit Smc3 was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of Smc3 deficiency in GC lymphomagenesis. Transcriptional profiling of Smc3 haploinsufficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors Tet2 and Kmt2d. Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like Tet2, which was aberrantly downregulated in Smc3 deficient lymphomas. Tet2 plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in Smc3 mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although Smc3 deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in Smc3 haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the Smc3 haploinsufficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical outcome and low expression of cohesin core subunits. Reciprocally, reduced expression of cohesin subunits was an independent risk factor for worse survival int DLBCL patient cohorts. Collectively, the data suggest that Smc3 functions as a bona fide tumor suppressor for lymphomas through non-genetic mechanisms, and drives disease by disrupting the commitment of GC B-cells to the plasma cell fate.

Thymoquinone ameliorates age-related hearing loss in C57BL/6J mice by modulating Sirt1 activity and Bak1 expression

Age-related hearing loss (AHL) is the most common sensory disorder of aged population. Currently, one of the most important sources of experimental medicine for AHL is medicinal plants. This study performed the first investigation of the effect of thymoquinone (TQ), a potent antioxidant, on AHL. Here, we used inbred C57BL/6J mice (B6 mice) as a successful experimental model of the early onset of AHL. The behavioral assessment of hearing revealed that the injection of a high dose of TQ (40 mg/kg; TQ40) significantly improved the auditory sensitivity of B6 mice at all tested frequencies (8, 16 and 22 kHz). Histological sections of cochlea from B6 mice injected with a low dose (20 mg/kg; TQ20) and high dose showed relatively less degenerative signs in the modiolus, hair cells and spiral ligaments, the main constituents of the cochlea. In addition, TQ40 completely restored the normal pattern of hair cells in B6 mice, as shown in scanning electron micrographs. Our data indicated that TQ20 and TQ40 reduced levels of Bak1-mediated apoptosis in the cochlea of B6 mice. Interestingly, the level of Sirt1, a positive regulator of autophagy, was significantly increased in B6 mice administered TQ40. In conclusion, TQ relieves the symptoms of AHL by downregulating Bak1 and activating Sirt1 in the cochlea of B6 mice.