Expression Of B-cell Research Articles

Expression of B-cell activating factor in pediatric patients with immune thrombocytopenia: a single institutional series and review of literature

ABSTRACT B-cell-activating factor (BAFF) is a crucial cytokine supporting survival and differentiation of B cells. Dysregulation of BAFF is involved in the pathogenesis of B-cell related autoimmune diseases including immune thrombocytopenia (ITP). The aim of this study was to evaluate the significance of BAFF expression in pediatric ITP patients. Eighty pediatric patients with ITP are subdivided in three groups. Group I included (32 patients) diagnosed with acute ITP less than 3 months, group II (48 patients) diagnosed with persistent ITP (from 3 to 12 months) and chronic ITP (more than 12 months) and group III 20 healthy controls. Complete blood picture, autoimmune profile, antiplatelet antibodies, coagulation profile, bone marrow examination, and RT-PCR were performed to detect the expression for BAF for all participants in this study. BAFF expression levels significantly increased in cases rather than in controls. BAFF Expression Value significantly increased in groups I & II (3.10 ± 1.99&3.29 ± 2.58) compared to controls (0.83 ± 0.45) as p < .001 for both. On the other hand, groups I & II were comparable in BAFF Expression Value (p = .470). BAFF expression increased in ITP patients, implying a function in the disease’s pathogenesis.

The Role of PHF6 in Hematopoiesis and Hematologic Malignancies.

Epigenetic regulation of gene expression represents an important mechanism in the maintenance of stem cell function. Alterations in epigenetic regulation contribute to the pathogenesis of hematological malignancies. Plant homeodomain finger protein 6 (PHF6) is a member of the plant homeodomain (PHD)-like zinc finger family of proteins that is involved in transcriptional regulation through the modification of the chromatin state. Germline mutation of PHF6 is the causative genetic alteration of the X-linked mental retardation Borjeson-Forssman-Lehmann syndrome (BFLS). Somatic mutations in PHF6 are identified in human leukemia, such as adult T-cell acute lymphoblastic leukemia (T-ALL, ~ 38%), pediatric T-ALL (~ 16%), acute myeloid leukemia (AML, ~ 3%), chronic myeloid leukemia (CML, ~ 2.5%), mixed phenotype acute leukemia (MPAL, ~ 20%), and high-grade B-cell lymphoma (HGBCL, ~ 3%). More recent studies imply an oncogenic effect of PHF6 in B-cell acute lymphoblastic leukemia (B-ALL) and solid tumors. These data demonstrate that PHF6 could act as a double-edged sword, either a tumor suppressor or an oncogene, in a lineage-dependent manner. However, the underlying mechanisms of PHF6 in normal hematopoiesis and leukemogenesis remain largely unknown. In this review, we summarize current knowledge of PHF6, emphasizing the role of PHF6 in hematological malignancies. Epigenetic regulation of PHF6 in B-ALL. PHF6 maintains a chromatin structure that is permissive to B-cell identity genes, but not T-cell-specific genes (left). Loss of PHF6 leads to aberrant expression of B-cell- and T-cell-specific genes resulting from lineage promiscuity and binding of T-cell transcription factors (right).

Mechanism of Velvet Antler Peptide in Preventing Mild Cognitive Impairment Based on Non-Targeted Metabolomics

Velvet antler peptide (VAP) is one of the main active ingredients in Velvet antler (VA), and its mechanism in preventing mild cognitive impairment (MCI) needs to be further studied. In this research, rats model with MCI was established, non-targeted metabonomics method was performed. Total 50 wistar rats were divided into: control, model, piracetam (PIR), high- and low-dose VAP groups, and were administered for 18 days. Scopolamine (2 mg/kg) was injected intraperitoneally to establish the MCI rat model. The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in rat serum were detected by the enzyme-linked immunosorbent assay (ELISA). The expression of B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated death promoter (Bad) and Bcl-2-associated X protein (Bax) in hippocampus of rats were detected by Western blot analysis. The brain and kidney of MCI rats were detected by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) metabolomics technology, and the differences of metabolites and the dynamic changes of metabolic pathway were analyzed based on the results of Principal Component Analysis (PCA) and Orthogonal partial least squares discriminant analysis (OPLS-DA) The correlation analysis of significantly different metabolites was then analyzed by Cluster analysis (CA), and then the differential metabolite pathway enrichment analysis was performed. According to the statistical analysis of univariate and multivariates, the models constructed between the groups are stable and reliable, the variability and separation trend are good, and there are differences in the metabolomes. In results, VAP increased the content of serum SOD, reduced the content of MDA, increased the expression level of Bcl-2 and reduced the expression level of Bad and Bax protein in MCI model rats. The relevant potential markers in the brain and kidney of MCI rats mainly included folic acid, acetyl L-carnitine (ALC), Prostaglandin E1 (PGE1), 3′,5′-cyclic-adenosine-monophosphate (cAMP) and various amino acids. Therefore, VAP could inhibit oxidative stress response, reduce hippocampal neuron apoptosis and protect neurons in MCI rats. Its mechanism may be related to regulating folate and lipid metabolism, cyclic adenosine phosphate energy cycle, amino acid biosynthesis and renal secretion of sex hormones.

Circular RNA from phosphodiesterase 4D can attenuate chondrocyte apoptosis and matrix degradation under OA milieu induced by IL-1β via circPDE4D/miR-4306/SOX9 Cascade

Background Phosphodiesterase 4D (PDE4D) is a novel molecular therapeutic agent for human diseases, including Alzheimer’s disease, ischemic stroke, asthma, and cancers. Circular RNA from PDE4D (circPDE4D; ID hsa_circ_0072568) was one of the most downregulated circRNAs in OA patients. However, its precise role in OA-related chondrocytes was largely unknown. Methods Expressions of circPDE4D, microRNA (miR)-4306, and sex-determining region Y-box 9 (SOX9) were measured by quantitative real-time PCR; protein levels of SOX9 and proteins related to apoptosis and extracellular matrix (ECM) were detected by Western blotting. Cell apoptosis was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, 5-ethynyl-2′-deoxyuridine and Annexin V-fluorescein isothiocyanate apoptosis assays. MiR-4306 response elements were predicted by bioinformatics algorithm and identified using dual-luciferase reporter, RNA immunoprecipitation, and biotin-coupled miRNA capture assays. Results CircPDE4D was markedly downregulated in OA cartilages and interleukin (IL)-1β-stressed human normal chondrocytes (HNC). Ectopic expression of circPDE4D rescued cell viability, proliferation, and expressions of B-cell lymphoma/leukemia-2 (Bcl-2) and Collagen type II α1 in IL-1β-insulted HNC, and meanwhile declined apoptosis rate and levels of Bcl-2-associated X protein, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase-1, matrix metalloproteinase-13, ADAM metallopeptidase with thrombospondin type 1 motif 5, IL-6, and IL-8. CircPDE4D and SOX9 were competing endogenous RNAs (ceRNAs) for miR-4306, and circPDE4D could positively regulate SOX9 expression via miR-4306. Conclusion CircPDE4D and miR-4306 were important regulators in regulating IL-1β-induced HNC apoptosis and matrix degradation via regulating the key transcription factor SOX9, suggesting a novel circPDE4D/miR-4306/SOX9 ceRNA pathway in OA-related chondrocyte dysfunction.

Molecular Taxonomy of Systemic Lupus Erythematosus Through Data-Driven Patient Stratification: Molecular Endotypes and Cluster-Tailored Drugs.

ObjectivesTreatment of Systemic Lupus Erythematosus (SLE) is characterized by a largely empirical approach and relative paucity of novel compound development. We sought to stratify SLE patients based on their molecular phenotype and identify putative therapeutic compounds for each molecular fingerprint.MethodsBy the use of whole blood RNA-seq data from 120 SLE patients, and in a data-driven, clinically unbiased manner, we established modules of commonly regulated genes (molecular endotypes) and re-stratified patients through hierarchical clustering. Disease activity and severity were assessed using SLEDAI-2K and Lupus Severity Index, respectively. Through an in silico drug prediction pipeline, we investigated drugs currently in use, tested in lupus clinical trials, and listed in the iLINCS prediction databases, for their ability to reverse the gene expression signatures in each molecular endotype. Drug repurposing analysis was also performed to identify perturbagens that counteract group-specific SLE signatures.ResultsMolecular taxonomy identified five lupus endotypes, each characterized by a unique gene module enrichment pattern. Neutrophilic signature group consisted primarily of patients with active lupus nephritis, while the B-cell expression group included patients with constitutional features. Patients with moderate severity and serologic activity exhibited a signature enriched for metabolic processes. Mild disease was distributed in two groups, exhibiting enhanced basic cellular functions, myelopoiesis, and autophagy. Bortezomib was predicted to reverse disturbances in the “neutrophilic” cluster, azathioprine and ixazomib in the “B-cell” cluster, and fostamatinib in the “metabolic” patient subgroup.ConclusionThe clinical spectrum of SLE encompasses distinct molecular endotypes, each defined by unique pathophysiologic aberrancies potentially reversible by distinct compounds.

Beta-thalassemia and the advent of new interventions beyond transfusion and iron chelation.

Beta-thalassaemia, including sickle cell anaemia and thalassaemia E, is most common in developing countries in tropical and subtropic regions. Because carriers have migrated there owing to demographic migration, β-thalassaemia can now be detected in areas other than malaria-endemic areas. Every year, an estimated 300 000-500 000 infants, the vast majority of whom are from developing countries, are born with a severe haemoglobin anomaly. Currently, some basic techniques, which include iron chelation therapy, hydroxyurea, blood transfusion, splenectomy and haematopoietic stem cell transplantation, are being used to manage thalassaemia patients. Despite being the backbone of treatment, traditional techniques have several drawbacks and limitations. Ineffective erythropoiesis, correction of globin chain imbalance and adjustment of iron metabolism are some of the innovative treatment methods that have been developed in the care of thalassaemia patients in recent years. Moreover, regulating the expression of B-cell lymphoma/leukaemia 11A and sex-determining region Y-box through the enhanced expression of micro RNAs can also be considered putative targets for managing haemoglobinopathies. This review focuses on the biological basis of β-globin gene production, the pathophysiology of β-thalassaemia and the treatment options that have recently been introduced.

Abstract P1-04-11: Pd-l1-expressing b-cells promote murine breast cancer development and mediate the response to anti-pd-l1 immune checkpoint inhibitor

Abstract B cells promote tumor development in murine models of breast cancer (BCa), including - as we have shown - by producing pleiotropic cytokine IL-27, which upregulates expression of the PD-L1 immune checkpoint in BCa cells and tumor-infiltrating B cells (TIL-Bs). Paradoxically, B cell activation has been shown to predict the positive response to immune checkpoint inhibitors (ICIs) by murine BCa tumors and melanoma, sarcoma and kidney cancer in patients. Here, we have addressed the role of B cell-expressed PD-L1 in BCa development and responses to ICIs by generating conditional knockout (KO) mice in which Cd274 (encoding PD-L1) was ablated only in activated B cells (AicdacreCd274fl/fl). Tumor development in AicdacreCd274fl/fl mice from engrafted syngeneic ER+ E0071 BCa cells was significantly delayed, albeit not abolished (as occurring in mice lacking B cells), in association with increased B-cell expression of CD86, a key agonistic ligand for T cell-expressed CD28, a co-stimulatory receptor whose function is inhibited by the PD-L1/PD-1 immune checkpoint in exhausted tumor-infiltrating CD8+ T cells. The heightened B-cell CD86 expression in AicdacreCd274fl/fl mice was recapitulated in AicdacreCd274fl/fl B cells stimulated in vitro by CD154, a T helper cell stimulus critical for B cell activation and differentiation as well as the antibody response. Accordingly, AicdacreCd274fl/fl mice displayed an increased antibody response to a T-dependent antigen. Likewise, treatment with the anti-PD-L1 (αPD-L1) ICI boosted wildtype B cells stimulated with CD154 in vitro to express CD86 and differentiate into potentiated plasma cells. It also effectively regressed the growth of pre-developed E0071 tumor in C57 and AicdacreCd274+/fl mice by rejuvenating tumor-infiltrating CD8+ T cells, likely through activation of previously dampened CD28 signaling. By contrast, residual tumors that had eventually developed in AicdacreCd274fl/fl mice failed to respond to αPD-L1 and continued to grow at a pace comparable to tumors treated with an isotype-matched control antibody, showing that the full anti-tumor efficacy of αPD-L1 was mediated mainly by PD-L1-expressing B cells, but not other immune cell types. Thus, PD-L1+ TIL-Bs use PD-L1 to impose a “brake” on the host anti-tumor activity, but are directly targeted by αPD-L1 to relieve the brake, “jump-start” the CD86:CD28 signaling and potentiate the anti-tumor response, thereby providing a mechanistic explanation for the opposing roles of B cells in murine BCa development and ICI responses. Our findings also provide a strong rationale to investigate the activation of PD-L1-expressing TIL-Bs by Atezolizumab® (αPD-L1) in BCa patients to underpin the patient response to this ICI. Citation Format: Mustafa Tamim Alam Khan, Christian Cervantes, Suryavathi Viswanadhapalli, Yan Hui, Ratna Vadlamudi, Zhenming Xu. Pd-l1-expressing b-cells promote murine breast cancer development and mediate the response to anti-pd-l1 immune checkpoint inhibitor [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-11.

The miRNA content of circulating exosomes in DLBCL patients and in vitro influence of DLBCL-derived exosomes on miRNA expression of healthy B-cells from peripheral blood.

Due to the heterogeneous nature of Diffuse Large B-cell Lymphoma (DLBCL), the mechanisms underlying tumor development and progression have not yet been fully elucidated. This study aimed to compare the characteristics of plasma exosomes of DLBCL patients and healthy individuals and to evaluate the exosomal interactions between DLBCL cell lines and normal B-cells. Exosome isolation was performed using an ultracentrifugation-based protocol from plasma of 20 patients with DLBCL and 20 controls. The expression of miRNAs from exosome samples was analyzed using a miRNA expression microarray. The presence of exosome-mediated communication between the lymphoma cells and normal B-cells was determined by the co-culture model. A significant increase in plasma exosome concentrations of DLBCL patients was observed. There was also a significant decrease in the expression of 33 miRNAs in plasma exosomes of DLBCL patients. It was determined that normal B-cells internalize DLBCL-derived exosomes and then miRNA expression differences observed in normal B-cells are specific to lymphoma-subtypes. MiR-3960, miR-6089 and miR-939-5p can be used as the miRNA signature in DLBCL diagnosis. We suppose that the exosomes changed the molecular signature of the target cells depending on the genomic characterization of the lymphoma cells they have originated.

Acquired Hemophilia a Relapse Is Related to Th2 Response, and Increased Expression of B-Cell Activating Factor (BAFF)

Acquired hemophilia A (AHA) is a rare autoimmune disorder caused by the development of autoantibodies against the factor (F)VIII of coagulation with higher incidence in elderly individuals. The treatment includes immunosuppressive alternatives, and the majority of patients reach complete remission of symptoms. Although the role of immune T and B cells in the physiopathology of several autoimmune diseases is widely established, few studies have explored the T and B cells immune response pathway in AHA. This study aimed to determine the role of T cells through their cytokines expression and B cells through their expression of proteins that activate the B cell receptor (BCR), such as B-cell activating factor (BAFF) and A-proliferation inducing ligand (APRIL), in a longitudinal evaluation of patients with AHA.We included in this study 11 patients, 4 (36%) male, with the diagnosis of AHA, based on the occurrence of low FVIII levels (median FVIII level at the diagnosis was 0.10 IU/dL; range 0-18.8) and anti-FVIII inhibitor titer positive (≥0.6 Bethesda units (BU)/mL) (median inhibitor titer at the diagnosis was 60BU/mL; range 7.6-1000.2). The median age at the diagnosis was 59 years (range 9-75). Idiopathic AHA was evident in 72.7% of cases, while autoimmune diseases (18.2%), and pregnancy (9.1%) were the other underlying etiologies. All patients received immunosuppressive therapy (IST). Eight (72.7%) patients used corticosteroid (CS) plus cyclophosphamide (CP), one (9.1%) received only CP, one (9.1%) used CS plus rituximab and one (9.1%) received CS, CP and three cycles of rituximab. The response to IST was defined as complete remission (CR) with anti-FVIII < 0.6BU/mL and FVIII >50IU/dL, without further IST. Relapse was defined when anti-FVIII titer became >0.6BU/mL after previous remission. In this cohort, we observed 64% of sustained CR and 4(36%) AHA patients had at least one relapse, including two AHA patients considered idiopathic and two with other autoimmune diseases associated.Peripheral blood mononuclear cells (PBMC) were collected from all AHA patients at the diagnosis previously IST (baseline), when patient achieved CR, and at the occurrence of relapse. PBMCs were maintaining frozen in liquid nitrogen. Cells were taw and cultivated with RMPI-1640 medium in 7.5x10 5 cells/well in a 48-well plate. After 24h, cells were stimulated with a 1IU/well of a full-length recombinant FVIII concentrate (rFVIII) or phorbol myristate acetate (PMA) (1µM) with ionomycin (1µM). After 24h incubation, cells were analyzed by flow cytometry at BD FACSCalibur™.To determine T CD4 + cells' cytokine production profile, we stained T cells with anti-CD4, anti-interleukins (IL)-17a, IL-4, and IL-21, anti-TGF-b, and anti-TNF-a. We observed a significant increase of IL-17a production in the rFVIII-stimulated cells isolated at baseline from all patients compared to cells isolated from healthy individuals (P=0.01). However, IL-17a expression at baseline was not different between cells from patients who sustained CR to those who relapsed. Interestingly, when we compared cells isolated at baseline from patients that relapsed after different immunosuppressive attempts, we observed a significant difference for IL-4 (P=0.009), IL-21 (P=0.02), and TGF-b (P=0.01) expression in comparison to cells from healthy individuals. However, this difference was not observed in cells from sustained CR vs. cells from healthy individuals (figure 1). Thus, suggesting that all patients present Th17 response, while only patients that relapsed also showed Th2 response at the diagnosis.We also evaluated B cells' expression of BAFF and APRIL. The cells were stained with anti-CD19, BAFF, and APRIL. Only cells isolated at baseline from AHA relapsed patients after rFVIII stimulation or without any stimuli presented increased expression of BAFF (P=0.01 with rFVIII-stimulated and P=0.007 for unstimulated cells) when compared to PBMCs isolated from healthy individuals (figure 1). BAFF is responsible for controlling B-cell maturation and is associated with autoantibody production of different autoimmune conditions.Our finds reveal an increase in the Th17 response in patients with AHA at the diagnosis. However, the presence of Th2 response and increase of BAFF expression was observed only at the diagnosis of patients with recurrence of autoimmune response to FVIII, which suggests a potential biomarker for AHA evaluation. [Display omitted] DisclosuresOzelo: BioMarin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Grifols: Other: Grant review.

Ibrutinib Treatment in CLL Interrupts CD40 Signaling Capacity and Sensitizes CLL Cells to Venetoclax

Background: For proliferation and survival, chronic lymphocytic leukemia (CLL) cells depend on interactions with cells and soluble factors present in the tumor microenvironment (TME). These interactions also increase expression of B-cell leukemia/lymphoma-2 (Bcl-2) proteins, including Bcl-XL, Mcl-1 and Bfl-1, thereby reducing drug sensitivity.In the VISION HOVON141 clinical trial, patients with relapsed or refractory CLL are treated with 2 cycles of Bruton tyrosine kinase inhibitor ibrutinib (IBR) lead-in followed by 13 cycles of IBR + Bcl-2 inhibitor venetoclax (VEN) combination before randomization. The combination of IBR + VEN may have synergistic anti-tumor effects, since IBR forces CLL cells from lymph node (LN) to the blood (PB) where they become fully dependent on Bcl-2, and succumb to VEN.To support a mechanistic basis for this premise, we investigated changes in expression of Bcl-2 proteins, effects of TME-mimicking signals, and venetoclax sensitivity before/after 2 months IBR treatment.Results: At baseline, lymph node emigrant cells (CXCR4dim/CD5high) have higher Bfl-1 expression, in addition to earlier reported Bcl-XL and Mcl-1 expression than cells immigrating back to the lymph node (CXCR4high/CD5dim)(Haselager et al., 2020). After 2 months of IBR treatment a clear reduction in all four pro-survival proteins was observed (N=17 p<0.001). Despite these changes, VEN sensitivity was not different in unstimulated PB CLL cells obtained at baseline versus at 2 months of IBR treatment (N=8; IC50=0.001µM). In contrast, CD40 stimulated CLL cells obtained at baseline are fully resistant to VEN (IC50>10µM), but unexectedly retained partial sensitivity to venetoclax after IBR treatment (IC50=0.05 µM, p-value <0.0001; Figure 1A). This suggested reduced CD40 signalling capacity and was accompanied by reduced ability to upregulate Bcl-XL, Mcl-1, and Bfl-1 expression. Indeed, cell surface expression, as well as level of CD40 activation as measured by CD95 induction, was clearly reduced after 2 months IBR. Importantly, these effects occurred in vivo, as IBR did not directly affect CD40 signaling in vitro.These data imply that under IBR treatment, when cells cannot (re-)enter LN sites, a factor is lacking that maintains or induces CD40 expression. In search of stimuli that can augment CD40 signaling capacity, it was found that BCR stimulation had no effect on CD40 expression. In contrast, TLR9 stimulation via CpG led to increased CD40 expression in CLL cells (N=7, p-value <0.0001)(Figure 1B). These findings align well with recent data which indicate a role for TLR9 signalling in vivo by unmethylated mitochondrial DNA in CLL (Kennedy et al., 2021). Taken together, these data provide a mechanistic explanation, by which a triad of signaling pathways not only involving BCR but also CD40 and TLR9 is interrupted by IBR (Figure 1C).Discussion/conclusion: IBR treatment broadly affects Bcl-2 family protein expression and CD40 signaling in vivo. The combined data indicate a novel aspect of IBR efficacy, namely its capacity to interrupt TLR9-induced CD40 upregulation, which normally primes CLL cells in the LN environment for drug resistance. This also suggests that drugs that inhibit TLR9 signaling may synergize with IBR.ReferencesHaselager, M. V., et al (2020). Changes in Bcl-2 members after ibrutinib or venetoclax uncover functional hierarchy in determining resistance to venetoclax in CLL. Blood, 136(25), 2918Kennedy, E., et al (2021). TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target. Blood, 137(22), 3064 [Display omitted] DisclosuresLevin: Roche, Janssen, Abbvie: Other: Travel Expenses, Ad-Board. Westerweel: Novartis: Research Funding; Incyte: Consultancy; BMS / Celgene: Consultancy; Pfizer: Consultancy. Niemann: CSL Behring, Genmab, Takeda, Octapharma: Consultancy; Novo Nordisk Foundation: Research Funding; Abbvie, AstraZeneca, Janssen: Consultancy, Research Funding.

Inhibition of STAT3/PD-L1 and Activation of miR193a-5p Are Critically Involved in Apoptotic Effect of Compound K in Prostate Cancer Cells.

Since the signal transducer and activator of transcription 3 (STAT3)/programmed death-ligand 1 (PD-L1) signaling plays an important role in tumor-immune microenvironments, in the present study, the role of STAT3/PD-L1 signaling in the apoptotic mechanism of an active ginseng saponin metabolite compound K (CK) was investigated in human prostate cancer cells. Here, CK exerted significant cytotoxicity without hurting RWPE1 normal prostate epithelial cells, increased sub-G1 and cleavage of Poly ADP-ribose polymerase (PARP) and attenuated the expression of pro-PARP and Pro-cysteine aspartyl-specific protease3 (pro-caspase-3) in LANCap, PC-3 and DU145 cells. Further, CK attenuated the expression of p-STAT3 and PD-L1 in DU145 cells along with disrupted the binding of STAT3 to PD-L1. Furthermore, CK effectively abrogated the expression of p-STAT3 and PD-L1 in interferon-gamma (INF-γ)-stimulated DU145cells. Additionally, CK suppressed the expression of vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), interleukin 6 (IL-6) and interleukin 10 (IL-10) as immune escape-related genes in DU145 cells. Likewise, as STAT3 targets genes, the expression of CyclinD1, c-Myc and B-cell lymphoma-extra-large (Bcl-xL) was attenuated in CK-treated DU145 cells. Notably, CK upregulated the expression of microRNA193a-5p (miR193a-5p) in DU145 cells. Consistently, miR193a-5p mimic suppressed p-STAT3, PD-L1 and pro-PARP, while miR193a-5p inhibitor reversed the ability of CK to attenuate the expression of p-STAT3, PD-L1 and pro-PARP in DU145 cells. Taken together, these findings support evidence that CK induces apoptosis via the activation of miR193a-5p and inhibition of PD-L1 and STAT3 signaling in prostate cancer cells.

Targeted inhibition of eIF4A suppresses B-cell receptor-induced translation and expression of MYC and MCL1 in chronic lymphocytic leukemia cells

Signaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

Intratumoral B cells as predictive biomarkers for response to immune check point blockade in patients with recurrent ovarian cancer

<h3>Objectives:</h3> Single agent immune check point inhibitors are largely ineffective in recurrent OC patients. A recently completed phase 2 clinical trial (NCT02853318) on recurrent ovarian cancer patients at Roswell Park combined pembrolizumab with bevacizumab and oral cyclophosphamide and showed an overall response rate of 47.5%, a median progression-free survival (PFS) of 10.0 months and disease control over 12 months in 25% of the patients. Our objective was to evaluate the transcriptomic and proteomic signatures in the tumor microenvironment (TME) to understand the factors that drive durable clinical responses. <h3>Methods:</h3> RNASeq analysis was performed on 28 patients on the clinical trial using paired baseline and on-therapy tumor biopsies. Celltype expression was deconvoluted using CIBERSORT from RNASeq results. In parallel, NanoString Digital Spatial Imaging was used to evaluate the TME in 13 of these patients for 50 immuno-oncology proteins to compare their mean expression and the changes in expression levels between responder (PFS > 12 months) and non-responder (PFS < 6 months) patients throughout the treatment. <h3>Results:</h3> RNASeq pathway enrichment analysis of baseline and on-treatment tumor biopsies revealed significant enrichment of immune-related pathways in responder patients, including B-cell signaling pathways, and a strong correlation of B-cell presence with T-cells in responding patients (p<0.001). CIBERSORT analysis showed higher levels of naïve and memory B-cells in baseline tumor samples of responders compared to non-responders, which further increased on treatment. High expression of B cell markers strongly correlated with increased PFS (CD19+ p=0.0029, CD20+ p=0.0030). Digital spatial imaging data revealed higher T-cell expression (CD4+ p=0.025, CD8+ p=0.05) and B-cell expression (CD20+ p=0.019) in baseline and on treatment tumor biopsies in responding patients. No significant changes were observed in CD20 expression during the clinical trial in non-responders. <h3>Conclusions:</h3> More recently the outcome of immune checkpoint inhibitor therapy in cancer patients has been linked to the quality and magnitude of both T cell and B cell responses within the tumor microenvironment. Similarly, we demonstrate here that B cells as crucial supportive regulators in immune tumor control.

Abstract LB160: PD-L1-expressing B cells promote murine breast cancer development and mediate the response to anti-PD-L1 immune checkpoint inhibitor to upregulate B-cell CD86 and inhibit tumor growth

Abstract B cells promote tumor development in murine models of breast cancer (BCa), including - as we have shown - by producing pleiotropic cytokine IL-27, which upregulates expression of the PD-L1 immune checkpoint in BCa cells and tumor-infiltrating B cells (TIL-Bs). Paradoxically, B cell activation has been shown to predict the positive response to immune checkpoint inhibitors (ICIs) by murine BCa tumors and melanoma, sarcoma and kidney cancer in patients. Here, we have addressed the role of B cell-expressed PD-L1 in BCa development and responses to ICIs by generating conditional knockout (KO) mice in which Cd274 (encoding PD-L1) was ablated only in activated B cells (AicdacreCd274fl/fl). Tumor development in AicdacreCd274fl/fl mice from engrafted syngeneic ER+ E0071 BCa cells was significantly delayed, albeit not abolished (as occurring in mice lacking B cells), in association with increased B-cell expression of CD86, a key agonistic ligand for T cell-expressed CD28, a co-stimulatory receptor whose function is inhibited by the PD-L1/PD-1 immune checkpoint in exhausted tumor-infiltrating CD8+ T cells. The heightened B-cell CD86 expression in AicdacreCd274fl/fl mice was recapitulated in AicdacreCd274fl/fl B cells stimulated in vitro by CD154, a T helper cell stimulus critical for B cell activation and differentiation as well as the antibody response. Accordingly, AicdacreCd274fl/fl mice displayed an increased antibody response to a T-dependent antigen. Likewise, treatment with the anti-PD-L1 (anti-PD-L1) ICI boosted wildtype B cells stimulated with CD154 in vitro to express CD86 and differentiate into potentiated plasma cells. It also effectively regressed the growth of pre-developed E0071 tumor in C57 and AicdacreCd274+/fl mice by rejuvenating tumor-infiltrating CD8+ T cells, likely through activation of previously dampened CD28 signaling. By contrast, residual tumors that had eventually developed in AicdacreCd274fl/fl mice failed to respond to αPD-L1 and continued to grow at a pace comparable to tumors treated with an isotype-matched control antibody, showing that the full anti-tumor efficacy of anti-PD-L1 was mediated mainly by PD-L1-expressing B cells, but not other immune cell types. Thus, PD-L1+ TIL-Bs use PD-L1 to impose a “brake” on the host anti-tumor activity, but are directly targeted by αPD-L1 to relieve the brake, “jump-start” the CD86:CD28 signaling and potentiate the anti-tumor response, thereby providing a mechanistic explanation for the opposing roles of B cells in murine BCa development and ICI responses. Our findings also provide a strong rationale to investigate the activation of PD-L1-expressing TIL-Bs by Atezolizumab (anti-PD-L1) in BCa patients to underpin the patient response to this ICI. Citation Format: Mustafa T. Khan, Christian Cervantes, Zexuan Liu, Suryavathi Viswanadhapalli, Harshita Gupta, Tyler Curiel, Ratna Vadlamudi, Zhenming Xu. PD-L1-expressing B cells promote murine breast cancer development and mediate the response to anti-PD-L1 immune checkpoint inhibitor to upregulate B-cell CD86 and inhibit tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB160.

TAK-243, a Small Molecule Inhibitor of Ubiquitin-Activating Enzyme (UAE), Induces ER Stress and Apoptosis in Diffuse Large B-Cell Lymphoma (DLBCL) Cells

Despite the promise of targeting B-cell receptor-associated signaling kinases in indolent lymphomas, new agents in this class show limited activity in DLBCL, which remains an unmet clinical need. Ubiquitin-proteasome systems (UPS) are altered in cancer, leading to destabilization of tumor suppressors (e.g., TP53), overexpression of proto-oncogenes (e.g., MYC), and impaired DNA repair. We and others have shown that neoplastic B-cells exhibit a state of heightened cellular stress and thereby are susceptible to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Proteasome inhibitors (e.g., bortezomib) have demonstrated clinical activity in B-cell neoplasia. Our group has previously targeted proximal components of the UPS in B-cell neoplasia by disrupting the Nedd8 pathway with pevonedistat, which inhibits the function of a subset of E3 ubiquitin ligases known as cullin-ring ligases. Here we investigated an alternative approach to targeting the UPS in DLBCL using TAK-243, a first-in-class small molecule UAE inhibitor. By inhibiting UAE, TAK-243 blocks ubiquitin conjugation, disrupting monoubiquitin signaling as well as global protein ubiquitination. Here we demonstrate that UAE inhibition effectively kills DLBCL cells via induction of ER stress.Experiments were conducted in activated B-cell (ABC)-type (OCI-LY3, U2932) and germinal center (GC)-type (OCI-LY19, SUDHL4/10/16, VAL) DLBCL cell lines, and primary neoplastic B-cells derived from peripheral blood and lymph nodes of patients with CLL and DLBCL, respectively. CD40L-expressing stromal cells were used to induce NFκB activation in primary cells. TAK-243 was obtained from Millennium Pharmaceuticals, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA).TAK-243 induced ER stress and the UPR , followed by rapid apoptosis, as evidenced by PARP cleavage within 2 h, in both DLBCL cell lines and primary neoplastic B-cells. 24 h drug treatment yielded an IC50 of ~50 nM, as estimated by Annexin V expression. TAK-243 induced apoptosis of DLBCL cell lines independent of cell-of-origin (ABC- vs. GC). By contrast, primary T cells were significantly less sensitive to TAK-243.Treatment with TAK-243 rapidly disrupted ubiquitin conjugation and subsequent degradation of proteins controlled by the UPS in neoplastic B-cells. TAK-243-induced UPR occurred within 2 h, as evidenced by oligomerization and autophosphorylation of PERK, induction of BiP/GRP78 and activation of eIF2α. After 4 h of treatment, cells exhibited late apoptotic phase of the UPR: transcriptional induction of CHOP, GADD34 (10-fold) and NOXA (4-fold), accompanied by upregulation of pro-apoptotic BH3-only proteins, stabilization of MCL-1, activation of BAK, and ultimately cleavage of caspase-3 and PARP.TAK-243 inhibited both canonical and non-canonical NFκB pathways in neoplastic B-cells, as evidenced by accumulation of IκBα, a negative pathway regulator, and diminished p100/p52 processing, respectively. We and others have shown that CD40L induces NFκB and promotes resistance to spontaneous and drug-induced apoptosis in primary neoplastic B-cells. By contrast, CD40L-stimulated cells were fully sensitive to TAK-243 (IC50=50 nM). Furthermore, TAK-243 showed lower IC50 compared to bortezomib in DLBCL cell lines and primary samples, and induced accelerated and more pronounced ER stress and NFκB inhibition.We observed increased MYC mRNA transcript and protein expression in neoplastic B-cells following treatment with TAK-243. DLBCL cells expressing high baseline MYC levels (by protein expression) showed enhanced susceptibility to TAK-243-mediated apoptosis compared with MYC-low cells. Engineered expression of MYC sensitized OCI-LY3 cells to TAK-243, while siRNA-mediated downregulation of MYC resulted in decreased apoptosis. It has been suggested that MYC may sensitize cells to apoptosis through the extrinsic pathway. Indeed, we found that TRAIL ligand sensitized DLBCL cell lines to TAK-243.Thus, the UAE inhibitor TAK-243 induces ER stress, abrogates NFκB pathway activation and promotes apoptosis in DLBCL cell lines and primary neoplastic B-cells. Its in vitro activity compares favorably with bortezomib. Targeting UAE is a novel approach to disrupt the ubiquitin-proteasome system which may hold promise in therapy of B-cell malignancies. DisclosuresBerger:Takeda Pharmaceuticals International Co.: Employment.

VP5-2021: IMpower133: Gene expression (GE) analysis in long-term survivors (LTS) with ES-SCLC treated with first-line carboplatin and etoposide (CE) ± atezolizumab (atezo)

Exploratory analyses to characterise LTS (patients [pts] who survived ≥18 mo post randomisation) in IMpower133 showed that more LTS were treated with atezo (anti–PD-L1) + CE than placebo (PBO) + CE (Liu et al. ESMO 2020). Here, we report the association of differential GE (single genes, T-effector [Teff] and B-cell signatures) and published SCLC subtypes (Gay et al. Cancer Cell 2021) in IMpower133 LTS. Treatment-naive pts with ES-SCLC were randomised 1:1 to four 21-day cycles of C (AUC 5 mg/mL/min IV, day 1) + E (100 mg/m2 IV, days 1-3) combined with atezo (1200 mg IV, day 1) or PBO, followed by maintenance atezo or PBO until disease progression or toxicity. Co-primary endpoints were PFS and OS. Differential GE was analysed using RNA-sequencing (RNA-seq) data in LTS and non-LTS. OS was assessed by Teff and B-cell gene signature expression in both arms. Distribution of recently identified neuroendocrine, non-neuroendocrine and immune-infiltrated subtypes were also assessed in LTS and non-LTS. 271 pts were in the RNA-seq biomarker-evaluable population, 253 of whom had sufficient follow-up for LTS classification (LTS RNA-seq BEP; LTS, n=64; non-LTS, n=189). Within the LTS RNA-seq BEP, significantly more LTS were in the atezo (n=39, 32%) vs PBO arm (n=25, 19%; P=0.027). In both arms within the LTS RNA-seq BEP, distributions of Teff- and B-cell−related genes and gene signatures (≥ median vs < median) were similar in LTS. Exploratory OS efficacy analyses in the LTS RNA-seq BEP suggest that benefit was observed in favour of atezo vs PBO across all Teff and B-cell expression subgroups (high [≥ median]: Teff, HR, 0.65 [95% CI, 0.43, 0.97]; B-cell, HR, 0.75 [95% CI, 0.51, 1.11], and low [< median]: Teff, HR, 0.75 [95% CI. 0.52, 1.11]; B-cell, HR, 0.66 [95% CI: 0.45, 0.97]). Among the LTS RNA-seq BEP in both arms, GE subtype distribution was broadly similar for LTS. In IMpower133, more LTS were treated with atezo + CE vs PBO + CE. Enhanced immune-related signaling was seen in LTS in both arms. Atezo and PBO had similar GE-defined subtype distribution, suggesting that subtype associations with outcome may be prognostic.

Minimalistic In Vitro Culture to Drive Human Naive B Cell Differentiation into Antibody-Secreting Cells.

High-affinity antibody-secreting cells (ASC) arise from terminal differentiation of B-cells after coordinated interactions with T follicular helper (Tfh) cells in germinal centers (GC). Elucidation of cues promoting human naive B-cells to progress into ASCs is challenging, as this process is notoriously difficult to induce in vitro while maintaining enough cell numbers to investigate the differentiation route(s). Here, we describe a minimalistic in vitro culture system that supports efficient differentiation of human naive B-cells into antibody-secreting cells. Upon initial stimulations, the interplay between level of CD40 costimulation and the Tfh cell-associated cytokines IL-21 and IL-4 determined the magnitude of B-cell expansion, immunoglobulin class-switching and expression of ASC regulator PRDM1. In contrast, the B-cell-specific transcriptional program was maintained, and efficient ASC formation was hampered. Renewed CD40 costimulation and Tfh cytokines exposure induced rapid secondary STAT3 signaling and extensive ASC differentiation, accompanied by repression of B-cell identity factors PAX5, BACH2 and IRF8 and further induction of PRDM1. Our work shows that, like in vivo, renewed CD40L costimulation also induces efficient terminal ASC differentiation after initial B-cell expansion in vitro. This culture system for efficient differentiation of human naive B-cells into ASCs, while also maintaining high cell numbers, may form an important tool in dissecting human naive B-cell differentiation, thereby enabling identification of novel transcriptional regulators and biomarkers for desired and detrimental antibody formation in humans.

The red blood cell as a novel regulator of human B-cell activation.

Non‐immune cells are increasingly recognized as important in regulating immunity, but the role of red blood cells (RBC) remains relatively unexplored, despite their abundance in the circulation and a cell surface rich in potential ligands. Here, we determine whether RBC influence the activation state of human B cells. Separation of RBC from peripheral blood mononuclear cells increased B‐cell expression of HLA‐DR/DP/DQ, whilst reconstitution reduced the levels of B‐cell activation markers HLA‐DR/DP/DQ, CD86, CD69 and CD40, as well as decreasing proliferative responses and IgM secretion. Inhibition of B cells required contact with RBC and was abrogated by either removal of sialic acids from RBC or blocking the corresponding lectin receptor CD22 on B cells. Chronic lymphocytic leukaemia B cells express low levels of CD22 and were less susceptible to inhibition by RBC, which may contribute to their activated phenotype. Taken together, the results identify a novel mechanism that may suppress inappropriate responsiveness of healthy B cells whilst circulating in the bloodstream.

PD-L1-expressing B cells promote murine breast cancer development and mediate response to anti-PD-L1 immune checkpoint inhibitor.

Abstract B cells promote tumor development in murine models of breast cancer (BCa), – as we have shown – by producing pleiotropic cytokine IL-27, which upregulates expression of PD-L1 immune checkpoint in BCa cells and tumor-infiltrating B cells (TIL-Bs). Paradoxically, B cell activation has been shown to predict a positive response to immune checkpoint inhibitors (ICIs) in murine BCa tumors and melanoma, sarcoma and kidney cancer in humans. Here, we have addressed the role of B cell-expressed PD-L1 in BCa development and responses to ICIs by generating conditional knockout mice in which Cd274 (encoding PD-L1) was ablated only in activated B cells (AicdacreCd274fl/fl). Tumor development in AicdacreCd274fl/fl mice from engrafted syngeneic ER+ E0071 BCa cells was significantly delayed, albeit not abolished (as occurring in mice lacking B cells), with a marked increase in CD86 B-cell expression, a key agonistic ligand for T cell-expressed CD28 (a co-stimulatory receptor whose function is inhibited by the PD-L1/PD-1 immune checkpoint). The heightened B-cell CD86 expression in AicdacreCd274fl/fl mice was recapitulated in vitro using CD154, a T helper cell stimulus critical for B cell activation and differentiation. Treatment with anti-PD-L1 (aPD-L1) boosted wildtype B cells, stimulated with CD154 in vitro, to express CD86 and differentiate into potentiated plasma cells. Thus, PD-L1+ TIL-Bs use PD-L1 to impose a “brake” on host anti-tumor activity but are directly targeted by aPD-L1 to relieve the brake, which “jump-starts” CD86:CD28 signaling and potentiates the anti-tumor response. Thus, providing a mechanistic explanation for the opposing roles of B cells in murine BCa development and ICI responses.

Role of B-cell endogenous interferon beta in lupus nephritis

Abstract Lupus nephritis (LN) is histologically evident yet early diagnosis can be challenging since some patients do not exhibit overt clinical manifestations until advanced stages. The objective of the present study is to determine if B-cell expression of interferon beta (IFNβ) can be a unique systemic lupus erythematosus (SLE) clinical prognostic marker and if specific histopathologic features of LN can be identified in patients with elevated B-cell expression of IFNβ. A total of 80 patients who met the ACR/EULAR classification criteria of SLE were recruited. LN was identified in 41% (N=33). Multivariate regression analysis indicated a significant positive correlation between naïve B-cell IFNβ with race (African American &amp;gt; European American; P=0.006) and LN class (P&amp;lt;0.0001). Patients with elevated serum anti-Smith (P=0.01) and anti-DNA (P=0.013) exhibited significantly higher naïve B-cell IFNβ. B-cell IFNβ was further compared with renal histopathology microscopy findings on light, electron microscopy, and immunofluorescence (IF) (N=23). Patients with a higher percentage of IFNβ in naïve B cells showed increased segmental scaring (P=0.046). These patients also exhibited aggressive LN class (P=0.14), membranous pattern (P=0.15), and global sclerosis (P=0.18), although the differences were not significant. B-cell IFNβ did not show significant correlation with IgG, IgM, IgA, and C1q, and C3 in situ staining. The present study suggests that B-cell endogenous IFNβ may be developed into a unique target-based marker for the development and severity of LN. IFNβ blockade may be developed into an individualized therapy for this subset of SLE patients.