Sodium stibogluconate and CD47-SIRPα blockade overcome resistance of anti-CD20-opsonized B cells to neutrophil killing

Anti-CD20 antibodies, like rituximab, are broadly used to treat B cell malignancies. These antibodies can induce various effector functions, including immune cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Neutrophils can induce ADCC towards solid cancer cells by trogoptosis, a cytotoxic mechanism known to be dependent on trogocytosis. However, neutrophils appear incapable of killing rituximab-opsonized B lymphoma cells. Nevertheless, neutrophils do trogocytose rituximab-opsonized B lymphoma cells, yet this only reduces CD20 surface expression, and is thought to render tumor cells therapeutically resistant to further rituximab-dependent destruction. Here, we demonstrate that resistance of B lymphoma cells towards neutrophil killing can be overcome by a combination of CD47-SIRPα checkpoint blockade and sodium stibogluconate (SSG), an anti-leishmanial drug and documented inhibitor of the tyrosine phosphatase SHP-1. SSG enhanced neutrophil-mediated ADCC of solid tumor cells, but enabled B lymphoma cell trogoptotic killing, by turning trogocytosis from a resistance-contributing mechanism into a cytotoxic anti-cancer one. The killing in the presence of SSG required both antibody opsonization of the target cells, as well as disruption of CD47-SIRPα interactions. These results provide a more detailed understanding of the role of neutrophil trogocytosis in antibody-mediated destruction of B cells and clues on how to further optimize antibody therapy of B cell malignancies.

CircEAF2 counteracts Epstein-Barr virus-positive diffuse large B-cell lymphoma progression via miR-BART19-3p/APC/β-catenin axis

Background Epstein-Barr virus (EBV) represents an important pathogenic factor of lymphoma and is significantly associated with poor clinical outcome of diffuse large B-cell lymphoma (DLBCL). Circular RNAs (circRNAs) play an essential role in lymphoma progression. However, the underlying mechanism of circRNA on DLBCL progression related to EBV remains largely unknown. Methods CircRNA was screened by high-throughput sequencing in tumor samples of 12 patients with DLBCL according to EBV infection status. Expression of circEAF2, as well as the relationship with clinical characteristics and prognosis, were further analyzed in tumor samples of 100 DLBCL patients using quantitative real-time PCR. Gain- and loss-of-function experiments were conducted to investigate the biological functions of circEAF2 both in vitro and in vivo. The underlying mechanism of circRNA on DLBCL progression were further determined by RNA sequencing, RNA pull down assay, dual-luciferase reporter assay, rescue experiments and western blotting. Results We identified a novel circRNA circEAF2, which was downregulated in EBV + DLBCL and negatively correlated with EBV infection and DLBCL progression. In EBV-positive B lymphoma cells, circEAF2 overexpression induced lymphoma cell apoptosis and sensitized lymphoma cells to epirubicin. As mechanism of action, circEAF2 specifically targeted EBV-encoded miR-BART19-3p, upregulated APC, and suppressed downstream β-catenin expression, resulting in inactivation of Wnt signaling pathway and inhibition of EBV + DLBCL cell proliferation. In EBV-positive B-lymphoma murine models, xenografted tumors with circEAF2 overexpression presented decreased Ki-67 positivity, increased cell apoptosis and retarded tumor growth. Conclusions CircEAF2 counteracted EBV + DLBCL progression via miR-BART19-3p/APC/β-catenin axis, referring circEAF2 as a potential prognostic biomarker. Therapeutic targeting EBV-encoded miRNA may be a promising strategy in treating EBV-associated lymphoid malignancies.

Vitreous and Optic Nerve Infiltration of an Adrenal B Lymphoma

<b><i>Aim:</i></b> This study aimed to report a case of vitreous and optic nerve infiltration of a primary adrenal B lymphoma. <b><i>Case Report:</i></b> An 81-year-old patient consulted for decrease in vision in the left eye and pain with ocular movements during the last week. The patient was being treated with chemotherapy for an adrenal gland diffuse large B-cell lymphoma. On examination, vitritis was seen in both eyes and optic disc edema in his left eye. After an extensive study based on orbital and brain magnetic resonance imaging and vitreous cytology, an ocular infiltration by systemic B lymphoma was confirmed. A treatment based on intravitreal methotrexate was carried out, achieving the regression of the vitreous infiltration and resolution of the optic disc edema. Systemic B lymphoma metastasizing ocular structures is extremely infrequent. The manifestations may mimic an inflammatory disease. Ophthalmologists should be aware of these manifestations and consider among the masquerade syndromes.

Anticancer Activity of Continentalic Acid in B-Cell Lymphoma

Aralia continentalis has been used in Korea as a folk remedy for arthralgia, rheumatism, and inflammation. However, its anti-lymphoma effect remains uncharacterized. Here, we demonstrate that A. continentalis extract and its three diterpenes efficiently kill B-lymphoma cells. Our in vitro and in vivo results suggest that the cytotoxic activities of continentalic acid, a major diterpene from A. continentalis extract, are specific towards cancer cells while leaving normal murine cells and tissues unharmed. Mechanistically, continentalic acid represses the expression of pro-survival Bcl-2 family members, such as Mcl-1 and Bcl-xL. It dissociates the mitochondrial membrane potential, leading to the stimulation of effector caspase 3/7 activities and, ultimately, cell death. Intriguingly, this agent therapeutically synergizes with roflumilast, a pan-PDE4 inhibitor that has been successfully repurposed for the treatment of aggressive B-cell malignancies in recent clinical tests. Our findings unveiled that A. continentalis extract and three of the plant’s diterpenes exhibit anti-cancer activities. We also demonstrate the synergistic inhibitory effect of continentalic acid on the survival of B-lymphoma cells when combined with roflumilast. Taken in conjunction, continentalic acid may hold significant potential for the treatment of B-cell lymphoma.

A Metabolomics Investigation of the Metabolic Changes of Raji B Lymphoma Cells Undergoing Apoptosis Induced by Zinc Ions

Zinc plays a pivotal role in the function of cells and can induce apoptosis in various cancer cells, including Raji B lymphoma. However, the metabolic mechanism of Zn-induced apoptosis in Raji cells has not been explored. In this study, we performed global metabolic profiling using UPLC−Orbitrap−MS to assess the apoptosis of Raji cells induced by Zn ions released from ZnO nanorods. Multivariate analysis and database searches identified altered metabolites. Furthermore, the differences in the phosphorylation of 1380 proteins were also evaluated by Full Moon kinase array to discover the protein associated Zn−induced apoptosis. From the results, a prominent increase in glycerophosphocholine and fatty acids was observed after Zn ion treatment, but only arachidonic acid was shown to induce apoptosis. The kinase array revealed that the phosphorylation of p53, GTPase activation protein, CaMK2a, PPAR−γ, and PLA−2 was changed. From the pathway analysis, metabolic changes showed earlier onset than protein signaling, which were related to choline metabolism. LC−MS analysis was used to quantify the intracellular choline concentration, which decreased after Zn treatment, which may be related to the choline consumption required to produce choline-containing metabolites. Overall, we found that choline metabolism plays an important role in Zn-induced Raji cell apoptosis.

IgG surface mobility promotes antibody-dependent cellular phagocytosis by Syk and Arp2/3 mediated reorganization of Fcγ receptors in macrophages.

By visualizing the movements of Rituximab during Antibody dependent cellular phagocytosis (ADCP) of B lymphoma cells by macrophages, we found that Fcγ receptors (FcγR) on the macrophage surface microcluster, recruit Syk and undergro large-scale reorganization at the phagocytic synapse prior to and during engulfment of the target cell. Given these dramatic rearrangements, we analyzed how the surface mobility of Rituximab contributes to FcγR signal amplification and ADCP efficiency. Depolymerization of the target cell actin cytoskeleton resulted in free diffusion of Rituximab docked to CD20, enhanced microcluster reorganization, Syk recruitment and ADCP. Conversely, immobilization of Rituximab by chemical fixation impaired microcluster formation and diminished Syk recruitment and ADCP. In macrophages lacking Syk, Rituximab accumulated at the base of the phagosome and were trogocytosed, consistent with Syk kinase activity being necessary to trigger the redistribution of Rituximab-FcγR during engulfment and to prevent antigenic modulation of the target. Total internal reflection fluorescence analysis of FcγR-IgG on fluid supported lipid bilayers revealed a membrane topography displaying inward reaching leading edges and protruding contact sites reminiscent of podosomes. This topography was distinct from the closely apposed macrophage/target membranes observed during engagement of IgG displayed on immobile supported lipid bilayers. The organization of this contact, pseudopod extension and the rearrangement of microclusters depended critically on Arp 2/3. Thus, Syk and Arp2/3 coordinate actin rearrangements and FcγR-IgG complexes that were of previously unrecognized complexity for the clearance of cells displaying surface-mobile antigens. ADCP is an important effector mechanism for the removal of malignant, immunologically aberrant, and infected cells during treatment with therapeutic antibodies or adaptive immune responses. Most transmembrane protein antigens are mobile with transient confinement from the actin of the target cell. This work demonstrates that macrophage forces overcome these confinements to rearrange FcγR-IgG-antigen complexes before and during ADCP. Thus, new paradigms are needed as ADCP has largely been studied using model target particles that display immobile antigens. Moreover, we found that the mobility of the therapeutic antibody, Rituximab, on the surface of B lymphoma cells foretells ADCP efficacy, with lower densities of IgG mediating ADCP on increasingly mobile antigens.

The Clinical and Biological Effects of PD-1 Expression on Tumor Cells in Diffuse Large B-Cell Lymphoma

The clinical and biological significance of programmed death-1 (PD-1) expression by B-lymphoma cells is largely unknown. Here, using multicolor immunofluorescent staining (MC-IF), we investigated PD-1 and PD-L1 expression in PAX5+ (B-lymphoma), CD68+ (macrophage), or CD3+ (T-cell) cells in formalin-fixed, paraffin-embedded samples of 32 consecutive patients with de novo diffuse large B-cell lymphoma (DLBCL) treated with rituximab plus chemotherapy. PD-1- and PD-L1-expressing PAX5+ cells were observed in 59% and 3% of the patients, respectively. PD-1-expressing CD3+ lymphocytes and PD-L1-expressing CD68+ macrophages were observed in 89% and 86% of the patients, respectively. PD-L1 expression on PAX5+ lymphoma cells or CD68+ macrophages and PD-1 expression on CD3+ lymphocytes were not correlated with prognosis. However, patients with PD-1 expression on lymphoma cells showed shorter progression-free survival than those lacking PD-1-expressing lymphoma cells (p = 0.033). Furthermore, genetically modified PD-1-knockout human B-lymphoma VAL cells showed reduced cell growth and migration, and decreased S6 kinase phosphorylation than VAL/mock cells. Our data suggest that PD-1 expression on DLBCL cells detected by MC-IF was associated with poor prognosis and cell-intrinsic PD-1 signaling was related with cell growth and migration in a subpopulation of B-cell lymphoma. These findings may allow the development of distinct DLBCL subtypes affecting prognosis.