Localized Store-Operated Calcium Influx Represses CD95-Dependent Apoptotic Effects of Rituximab in Non-Hodgkin B Lymphomas

Ren

et al. 2018

Cell Physiol Biochem

Background/Aims: Multiple myeloma (MM) is a plasma cell neoplasm which constitutes about 10% of all hematologic malignancies. Despite the development and application of novel agents, MM still undergoes an aggressive and incurable course in the vast majority of patients. Ca²⁺ is one of the critical regulators of cell migration. Ca²⁺ influx is essential for the migration of various types of cells including tumor cells. However, the role of store-operated calcium entry (SOC) channels, the only Ca²⁺ channels of non-excitable cells, has not yet been reported in MM cell survival. Methods: We evaluated the expression of Stim1 and Orai1 (two key regulators of SOC) in MM tissues and cell lines by immunohistochemical assay, quantitative real-time PCR assay and western blot. MM cell lines were pretreated with pharmacological blockers and siRNAs, and then MM cell proliferation, cell cycle arrest, and apoptosis were examined by FACS (flow cytometry) assay, and Annexin V-FITC/PI staining. The correlation between the expression of Stim1 (or Orai1) level and outcome in MM were assessed by using Progress Free Survival (PFS). Results: Stim1 and Orai1 were both abundantly expressed in MM tissue and MM cell lines. Inhibition of SOCE reduced MM cell viability, and induced cell cycle arrest and apoptosis. Stim1 or Orai1 silencing also reduced cell viability, caused cell apoptosis and cell cycle arrest in MM cell lines. Over-expression of Stim1/Orai1 in MM patients was closely associated with the clinical outcome of MM. Conclusion: The Stim1/Orai1-mediated signaling participates in the pathogenesis of MM, which represents an attractive target for future therapeutic intervention.

Section: Discussionmentioning

confidence: 56%

Orai1 and Stim1 Mediate the Majority of Store-Operated Calcium Entry in Multiple Myeloma and Have Strong Implications for Adverse Prognosis

Ren

et al. 2018

Cell Physiol Biochem

Clinical and Experimental Immunology

“…The basic molecular mechanism including the interactions between CD20 and its antibody are not well understood, while many studies have examined the clinical efficacy of CD20 antibodies. In particular, although previous studies have shown that CD20 over‐expressing cells exhibit store‐operated Ca 2+ entry activity , the molecular mechanism of the channel activity of CD20 and its effect on antibody function remain unclear. In our study, we found that the store‐operated mode of Ca 2+ influx of CD20 is mediated by Orai1 and STIM1.…”

Section: Discussionmentioning

confidence: 99%

“…Rituximab binding to CD20 causes CD20 aggregation in the raft region, which is one of the typical characteristics of Type I categorized CD20 antibodies . This enhances the efficient establishment of a mega pore by activating the complement cascade which, in turn, causes complement‐dependent cytotoxicity .…”

Section: Introductionmentioning

confidence: 99%

“…This is where ion channels such as Orai1 form puncta in the membrane, which is more fluid than the neighbouring region, but the puncta form a rigid membrane to anchor the membrane protein . The contribution of STIM1 and Orai1 to rituximab‐induced Ca 2+ influx through CD20 has been reported . However, their roles in obinutuzumab‐related events remain unclear, particularly the role of STIM1 in binding‐induced cell death.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

STIM1 knock-down decreases the affinity of obinutuzumab for CD20 by altering CD20 localization to Triton-soluble membrane

Heo

Jin

Park

et al. 2020

Obinutuzumab is thought to exert its effects through its high antibodydependent cellular cytotoxicity (ADCC) via glyco-engineering of the Fc region. In addition, obinutuzumab causes direct binding-induced cell death (DCD) only by specifically binding to its target CD20, a Ca 2+ channel. However, the specific features of CD20 related to obinutuzumab bindinginduction of cell death are not clearly understood. In this study, we evaluated the relationship between the Ca 2+ channel features of CD20 as a store-operated Ca 2+ channel (SOC) and obinutuzumab binding-induced cell death. Ca 2+ channel function and biochemical analysis revealed that CD20 is an Orai1-and stromal interaction molecule (STIM1)-dependent Ca 2+ pore. However, binding of obinutuzumab on CD20 did not have any effect on Ca 2+ influx activity of CD20; the direct cell death rate mediated by obinutuzumab binding was almost equivalent with or without the extracellular Ca 2+ condition. Given the apparent interaction between STIM1 and CD20, we observed Triton-X solubilized obinutuzumab-bound CD20 accompanied by STIM1. Subsequently, obinutuzumab binding and cell death were decreased by STIM1 knock-down in Ramos B cells. Thus, STIM1 directly contributes to cell death by increasing the affinity of cells for obinutuzumab by transferring CD20 to the Triton-soluble membrane region.STIM1 increases obinutuzumab affinity to CD20 © 2020 British Society for Immunology, Clinical and Experimental Immunology, 200: 260-271 261Ca 2+ via B cell receptor activation [19,20]. Additionally, Ca 2+ influx of CD20 is related to resistance to rituximab treatment [21]. The relationship between Ca 2+ and obinutuzumab binding is not clearly understood. The fact that cell death occurs only by obinutuzumab binding leads us to expect that excessive Ca 2+ influx through CD20 is the underlying cell death mechanism. However, reports of intracellular calcium responses after binding of obinutuzumab remain controversial, and there are no reports supporting the hypothesis that Ca 2+ influx is a key cell death mechanism. Therefore, in-depth understanding of the CD20 channel features, its exact biological role and interactions with obinutuzumab binding and cell death are essential to unveil the pharmacological mechanism of obinutuzumab.Rituximab binding to CD20 causes CD20 aggregation in the raft region, which is one of the typical characteristics of Type I categorized CD20 antibodies [16]. This enhances the efficient establishment of a mega pore by activating the complement cascade which, in turn, causes complement-dependent cytotoxicity [1,22,23]. In contrast, Type II antibodies such as obinutuzumab do not cause CD20 aggregation in the raft region, and thus show low complement-dependent cell death and instead exhibit high ADCC [3,24]. The epitope region is bound by rituximab and obinutuzumab overlap [25]; however, only obinutuzumab causes binding-induced cell death [3,8,9]. Structural analysis has revealed that the binding mode and plasma membrane localization of rituximab and obinutuzumab diff...

“…Binding of RTX to CD20 induces complement-dependent cytotoxicity (CDC) via the classic pathway [12] and/or recruitment of immune effector cells, leading to antibody-dependent cellular cytotoxicity [13,14]. Moreover, recognition of CD20 by RTX leads to the aggregation in lipid rafts [15], which initiates a cascade of intracellular signaling events involving calcium influx [16,17] and phosphorylation by SRC family kinases [18,19], leading to apoptosis and/or cell cycle arrest [18,[20][21][22].…”

Section: Introductionmentioning

confidence: 99%

Identification of BLNK and BTK as mediators of rituximab‐induced programmed cell death by CRISPR screens in GCB‐subtype diffuse large B‐cell lymphoma

et al. 2020

Diffuse large B‐cell lymphoma (DLBCL) is characterized by extensive genetic heterogeneity, and this results in unpredictable responses to the current treatment, R‐CHOP, which consists of a cancer drug combination supplemented with the humanized CD20‐targeting monoclonal antibody rituximab. Despite improvements in the patient response rate through rituximab addition to the treatment plan, up to 40% of DLBCL patients end in a relapsed or refractory state due to inherent or acquired resistance to the regimen. Here, we employ a lentiviral genome‐wide clustered regularly interspaced short palindromic repeats library screening approach to identify genes involved in facilitating the rituximab response in cancerous B cells. Along with the CD20‐encoding MS4A1 gene, we identify genes related to B‐cell receptor (BCR) signaling as mediators of the intracellular signaling response to rituximab. More specifically, the B‐cell linker protein (BLNK) and Bruton's tyrosine kinase (BTK) genes stand out as pivotal genes in facilitating direct rituximab‐induced apoptosis through mechanisms that occur alongside complement‐dependent cytotoxicity (CDC). Our findings demonstrate that rituximab triggers BCR signaling in a BLNK‐ and BTK‐dependent manner and support the existing notion that intertwined CD20 and BCR signaling pathways in germinal center B‐cell‐like‐subtype DLBCL lead to programmed cell death.