Inhibition of autoantigen-induced B-cell receptor (BCR) internalization as a therapeutic strategy in Diffuse Large B Cell Lymphoma (DLBCL)

Inhibition of autoantigen-induced B-cell receptor (BCR) internalization as a therapeutic strategy in Diffuse Large B Cell Lymphoma (DLBCL) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Inhibition of autoantigen-induced B-cell receptor (BCR) internalization as a therapeutic strategy in Diffuse Large B Cell Lymphoma (DLBCL) Patryk Górniak, Anna Polak, Anna Rams, Kristina Kupcova, Eliza Glodkowska-Mrowka, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7260543/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Feb, 2026 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract BCR signal dependency is a hallmark of diffuse large B-cell lymphoma (DLBCL) and other B-cell lymphoid malignancies originating from germinal centers. Chronic-active BCR signaling, typical for the more aggressive activated B-cell subtype (ABC) of DLBCLs, is often attributed to activating mutations within the BCR signaling cascade and continuous stimulation of the BCR by autoantigens. In certain ABC-DLBCLs, the BCR forms an intracellular multiprotein supercomplex with TLR9 and MYD88, which generates signals from endolysosomes. However, it is not clear whether the internalization of BCR is required for sustained signaling, nor have the mechanisms responsible for BCR trafficking been defined. A detailed and mechanistic characterization of receptor trafficking and its consequences is crucial for elucidating new therapeutic targets. To address these questions, we developed DLBCL cell models with modified ovalbumin (OVA)-specific hypervariable regions (HVRs) in the BCRs using CRISPR-Cas9 technology. Modified BCRs were incapable of binding self-antigens, while still responding in a controlled fashion to stimulation with ovalbumin. Using these genetic models, we demonstrated that autoantigens drive a complex BCR-dependent signaling program and facilitate the assembly of the intracellular BCR-TLR9-IκB complex, promoting NFκB pathway activation. Furthermore, we showed that the binding of autoantigens to the BCR leads to the internalization of the BCR-autoantigen complex via clathrin-mediated endocytosis (CME). Using genetic models with inducible inhibition of this endocytic pathway, we found that BCR internalization is essential for the oncogenic activation of BCR-dependent signaling pathways and the formation of the BCR-TLR9-IκB complex in autoantigen-dependent ABC-DLBCL cells. Finally, CME inhibition with dynamin-2 antagonists, such as phenothiazine derivatives, reduces BCR signaling, cell viability, and synergizes with SYK and PI3Kδ inhibitors. Since phenothiazines have well-defined safety and pharmacokinetic profiles, our data provide a framework for the rational design of clinical trials employing these drugs in the autoantigen-dependent subset of DLBCL. Health sciences/Diseases/Haematological diseases/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/B-cell lymphoma Biological sciences/Cancer/Cancer therapy/Targeted therapies Health sciences/Medical research/Translational research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Diffuse large B cell lymphoma (DLBCL) is the most common aggressive lymphoid malignancy in adults, accounting for up to 35% of non-Hodgkin lymphomas [1,2]. DLBCLs arise from B-cells that have encountered antigens and undergone the germinal center (GC) reaction, a process particularly conducive to somatic mutations [3]. DLBCLs are subdivided into the more aggressive, activated B-cell (ABC)-type, and the less aggressive, germinal center B-cell (GCB)-type [4]. While this classification provides fundamental insights into the molecular characteristics of DLBCL and has prognostic value, recent genomic profiling studies have revealed a more nuanced substructure within DLBCL, identifying at least five distinct subtypes with different mutational spectra [1,5]. Although transformed B cells acquire new pro-survival mutations during the GC reaction, they frequently remain dependent on B-cell receptor (BCR) signaling. Similar to their normal counterparts, DLBCL cells exhibit two different patterns of signaling emanating from the BCR: tonic and chronic-active, characteristic of GCB and ABC subtypes, respectively [6,7]. Tonic BCR signaling is antigen-independent, with phosphatidylinositide 3-kinase (PI3K) and AKT being the key mediators [6,8–10]. In contrast, chronic-active BCR signaling engages multiple pathways and transcriptional networks, with a crucial role of NFκB activation - a hallmark of ABC-type DLBCLs. This mode of signaling resembles antigen-dependent BCR activation in normal B cells [7, 11] While activating mutations in CARD11 can explain the constitutive BCR signal in a fraction of cases, other mutations (e.g., in CD79A/B or MYD88 , present in ~20-25% and up to 40% of ABC-DLBCLs, respectively) cannot initiate the signal by themselves [2, 7, 12]. Consistent with this, certain lymphomas exhibit non-random IGHV segment usage, suggesting antigen-dependent selection and signaling [12]. In line with these findings, BCR engagement by autoantigens or homotypic BCR interactions have been identified in selected DLBCL cases [13–16]. Numerous molecules and epitopes acting as BCR autoantigens have been characterized in ABC-type DLBCL cell lines, including N-acetyl-lactosamine glycans on cell surface proteins in HBL1, an epitope within the FR2 fragment of the BCR in TMD8, or molecules in apoptotic debris stimulating BCRs in U2932 and OCI-LY10 cells [13]. The engagement of autoantigens has also been demonstrated in DLBCL patients. Screening for potential BCR antigens identified hypo-phosphorylated arsenite resistance protein 2 (Ars2) as a candidate autoantigen in primary ABC-type DLBCL [16, 17]. Moreover, in primary central nervous system lymphoma (PCNSL)—a specific extranodal subtype of DLBCL—hyper-N-glycosylated SAMD14 and neurabin-I were identified as self-antigens [18]. Importantly, BCR-engaging autoantigens have been also identified in other lymphoid malignancies, such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) [14, 15]. Consistent with the pro-survival role of activated BCR signaling, targeting intracellular BCR-associated pathways with small-molecule inhibitors emerged as a new, promising therapeutic strategy. Inhibitors of BCR signaling have been registered for clinical use in CLL and MCL, but not in DLBCL – most likely due to molecular heterogeneity and the type of BCR signaling [11, 12, 19]. Importantly, subsequent studies clearly demonstrated that certain subgroups of DLBCL patients can benefit from this strategy, paving the way to further studies on strategies augmenting the activity of the BCR signal inhibitors [12, 19–22]. While the pro-survival role of autoantigens in DLBCL and other lymphomas is well-established, the proximal events triggering signaling pathways induced by BCR engagement are less well-defined. In certain ABC-DLBCLs, the BCR forms an intracellular multiprotein supercomplex with MYD88 and TLR9, generating signals from endolysosomes [23]. These observations indicate that BCRs in these ABC-DLBCLs are internalized, similarly to BCRs in normal B-cells following antigen engagement. However, it is not clear whether the internalization of the BCR is required for sustained signaling in DLBCL cells, nor have the mechanisms responsible for BCR trafficking been defined. Likewise, the role of autoantigens in this process remains undefined. To fill this gap, we generated DLBCL cell models with ovalbumin (OVA)-recognizing BCR hypervariable regions (HVRs). Cells with modified BCRs can no longer bind their original target autoantigens, but are responsive to stimulation with OVA under strictly controlled conditions. Our results show that BCR (auto)antigen engagement in DLBCL cells leads to the internalization of the BCR-antigen complex. This process is crucial for the formation of a previously reported signalosome complex containing the BCR and TLR9 as well as phosphorylated IκB, [23], which triggers the canonical NFκB pathway and enhances the survival of DLBCL cells. Furthermore, we demonstrate that disruption of clathrin-mediated endocytosis (CME) using dominant-negative dynamin-2 mutants decreases BCR internalization and DLBCL cell viability. Importantly, phenothiazine derivatives, acting as pharmacologic dynamin-2 antagonists, recapitulate these effects both in vitro and in vivo DLBCL xenograft models. Taken together, BCR internalization represents a novel therapeutic vulnerability in DLBCL and, potentially, also for other types of autoantigen-dependent lymphomas. Our data suggest that phenothiazines can be further evaluated and repurposed as candidates for DLBCL treatment, particularly in combination with BCR signaling pathway blockers, including SYK or PI3Kδ inhibitors. Results Autoantigen binding is required for sustained BCR signaling in ABC-DLBCL cells To investigate the proximal mechanisms of autoantigen-mediated BCR signal induction in DLBCL, we employed CRISPR/Cas9 knock-in technology to modify BCR specificity in two autoantigen-dependent ABC-type cell lines (U2932 and HBL1) and one autoantigen-independent GCB-type cell line (OCI-LY19). Specifically, we replaced the hypervariable regions (HVRs) in the immunoglobulin heavy (IgH) and light chains (IgL) with ovalbumin (OVA)-recognizing HVRs derived from isolated OVA-reactive B cells [24]. With this approach, cells with fully modified HVR express GFP (IgH-derived) and mTurquoise (IgL-derived) and can be isolated using FACS (Supplementary Fig. 1).Endogenous HVRs from the cell lines being targeted were used to generate appropriate controls (Fig. 1A). This approach successfully produced DLBCL cell models with OVA-recognizing BCRs that no longer bind cognate autoantigens (Fig. 1B), enabling studies on the mechanisms of autoantigen-induced signal generation in DLBCL cells. In cell lines that naturally express autoantigen-specific BCRs, replacement with OVA-reactive HVRs exhibited caused an increase in cell-surface BCR levels. In contrast, OVA-reactive BCR surface levels in the autoantigen-independent OCI-LY19 cells were not increased when compared to the control cells (Fig. 1C). Both the native and the dual HVR-replaced BCRs were capable of signal generation, as demonstrated by calcium flux analysis following anti-IgM-mediated receptor cross-linking (Fig. 1D). However, OVA stimulation induced calcium fluxes only in cells with OVA-reactive HVRs (Fig. 1D), confirming the functionality and antigen specificity of the engineered BCRs and the utility of the generated cell models for further studies. To characterize the autoantigen-dependent signaling, we first assessed tyrosine-phosphorylated proteins in ABC-DLBCL cells with OVA-specific and endogenous HVRs. In both HBL1 and U2932, cells expressing the OVA-specific BCR exhibited decreased tyrosine phosphorylation of numerous proteins when compared to controls (Fig. 2A). Incubation of these cells with OVA upregulated tyrosine phosphorylation in these proteins (Fig. 2B). Next, we compared global tyrosine and serine/threonine kinase activities in OVA-specific and control cells using the chip-based phosphoproteomic PamGene platform. This functional kinase assay measures protein kinase activity directly in cellular lysates by quantifying phosphorylation of peptides printed on a chip (Supplementary Fig. 2). Cells with OVA-specific BCRs (incapable of autoantigen binding), exhibited significantly reduced activity of proximal BCR signaling mediators, including SRC family kinases (e.g., LYN, FYN, BLK), SYK tyrosine kinase, Bruton's tyrosine kinase (BTK), and protein kinase C. Furthermore, these OVA-specific cells also showed decreased activity of kinases essential for the regulation of cell proliferation (cyclin-dependent kinases, CDKs), protein biosynthesis (ribosomal S6 kinases, RSKs), and kinases engaged in oncogenic signaling (JAKs, PIMs, FLT1/3/4, PDGFRs, FGFRs, TRKA/B/C) (Fig. 2C and Supplementary File 1). NFκB activation is a hallmark of ABC-type DLBCLs. Since altering BCR specificity and eliminating autoantigen binding decreased the activity of multiple BCR-related kinases and global tyrosine phosphorylation, we evaluated the changes in NFκB activity in engineered cells. For this purpose, we evaluated the phosphorylation levels of the NFκB inhibitory protein IκBα and the expression of NFκB-dependent genes as markers of NFκB activity. These analyses revealed significantly higher levels of IκBα phosphorylation and NFκB-dependent transcripts in the autoantigen-reactive cells, highlighting the crucial role of sustained BCR autoantigen engagement for continuous NFκB activation (Fig. 3A and 3B). In a subset of ABC-DLBCLs (referred to as MCD or C5 clusters) [1, 5], NFκB pathway activation is mediated by the intracellular BCR-TLR9 complex [23]. Using Proximity Ligation Assay (PLA) analysis, we found that in OVA-reactive cells, the assembly of the endolysosomal BCR-TLR9 complex (as indicated by interaction with the LAMP1 marker) was significantly inhibited. Furthermore, the interaction between this complex and the phosphorylated form of IκBα, which is critical for NFκB activation, was also diminished in OVA-reactive models (Fig. 3C). Collectively, these findings suggest that autoantigen binding by the BCR in ABC-DLBCL cells facilitates the recruitment of BCR and TLR9 into a functional endolysosomal BCR-TLR9 complex, contributing to NFκB activation. Antigenic stimulation in DLBCL cells leads to BCR internalization In normal B cells, antigen binding to the BCR triggers rapid clustering within membrane lipid rafts and subsequently leads to the internalization of the BCR-antigen complex. Following internalization, this complex is sorted into early endosomes and eventually into late endosomes [25]. DLBCL cells with OVA-specific BCRs exhibited increased BCR surface density and reduced formation of the endolysosomal BCR-TLR9 complex (Fig. 1C and 3C), indicating that the loss of autoantigenic stimulation results in BCR membrane retention. To confirm this scenario, we incubated OVA-specific cell models with anti-IgM (a positive control), OVA, and 17mer-OVA peptide conjugated with biotin and complexed with avidin. Anti-IgM triggered BCR endocytosis in both HVR types, but OVA or 17mer-OVA exhibited this effect only in cells with OVA-specific HVRs (Fig. 4A). Time-course analysis demonstrated that BCR internalization is a dynamic process, resulting in a significant decrease in BCR surface levels within minutes and progressing up to 3h (Fig. 4B). Since BCR-engaging DLBCL autoantigens can be either extracellular or expressed on the plasma membrane [13], we asked whether membrane-anchored autoantigens could also initiate BCR endocytosis. To address this question, we generated a model derived from the autoantigen-independent OCI-LY19 GCB-DLBCL cell line with OVA-specific BCRs. This cell line was modified to express a doxycycline (Dox)-inducible construct including the truncated murine CD8a (mCD8) fused to the OVA peptide (Fig. 4C). In this model, Dox-induced expression of membrane-anchored mCD8-OVA resulted in a significant decrease of the surface BCR levels, demonstrating that membrane-expressed autoantigens can initiate BCR internalization in DLBCL cells (Fig. 4D). Inhibition of BCR internalization blocks BCR-TLR9 complex formation and pro-survival signaling in ABC-DLBCL cells Given the observations that altered BCR specificity and loss of autoantigen binding led to BCR membrane retention, decreased formation of intracellular BCR-TLR9 supercomplexes, and reduced NFκB activity, we hypothesized that blocking BCR internalization might represent a novel DLBCL therapeutic strategy. Since elimination of autoantigens inducing BCR internalization is not feasible, we hypothesized that blocking BCR endocytosis would be a more general approach to inhibit autoantigen–dependent DLBCL cells. To evaluate the consequences of inhibition of endocytosis for DLBCL cell survival, we first utilized available datasets from CRISPR-Cas9 screening studies [2] and the KEGG Endocytosis Human Gene Set. With this approach, we found that knockout of multiple genes in the clathrin-mediated endocytosis (CME) pathway resulted in reduced DLBCL cell viability (Fig. 5A). CME is responsible for the internalization of multiple surface receptors, including BCR-antigen complexes [26,27]. DNM2 (dynamin-2) GTP-ase plays an essential role in CME. DNM2 forms ring-like structures around the neck of the invaginating vesicle and facilitates the scission of endocytic vesicles from the cell membrane. Importantly, DNM2 depletion exhibited a stronger effect on DLBCL survival than depletion of other genes coding for proteins targeted by available small molecule inhibitors (Supplementary Fig. 3). To assess the role of DNM2 and CME in autoantigen-induced BCR internalization and the subsequent activation of BCR signaling in DLBCL cells, we generated cellular models with inducible expression of a dynamin-2 K44A mutant lacking GTPase activity and shown to inhibit CME in a dominant-negative manner (DN-DNM2) (Fig. 5B) [28, 29]. Overexpression of DN-DNM2 markedly decreased internalization of the transferrin receptor, confirming the inhibition of CME (Supplementary Fig. 4). DN-DNM2 expression increased BCR surface levels in untreated, autoantigen-dependent cells. Likewise, DN-DNM2 blocked BCR internalization after anti-IgM cross-linking (Fig. 5C). Consistent with the data from the CRISPR-Cas9 screen, DN-DNM2 expression resulted in growth inhibition in the generated cell models, particularly in U2932 cells (Fig. 5D). BCR membrane retention after CME inhibition suggested reduced endolysosomal BCR-TLR9 complex formation. Indeed, CME inhibition in HBL1 cells led to a marked decrease in BCR-TLR9-pIκB complexes (Fig. 6A), which subsequently resulted in the downregulation of NFκB-dependent gene expression (Fig. 6B). To further characterize the consequences of CME inhibition, we evaluated the changes in tyrosine and serine-threonine kinase activity in HBL1 cells after DN-DNM2 induction using the PamGene platform. Inhibition of CME dampened the activation of several signaling pathways (mTOR, CDKs, PKCs, and RSKs), similar to the effects of switching endogenous HVRs for OVA-recognizing HVRs. However, unlike the genetic change in BCR specificity, CME inhibition led to increased activity of proximal BCR signaling kinases, including SRC family kinases (including LYN) and SYK - a critical tyrosine kinase involved in tonic BCR signaling [30]. Consistent with this, DNM2 inhibition increased the activity of SRC family kinases (including LYN) and AKT, the key kinase mediating tonic BCR signaling [6, 10] (Fig. 6C and 6D and Supplementary File 2). These observations suggest that blocking endocytosis increases surface BCR density, which may enhance tonic BCR signaling and represent a cellular attempt to compensate for the loss of the chronic-active signal. Phenothiazine derivatives inhibit antigen-induced BCR internalization Our studies indicate that clathrin- and dynamin-2 (DNM2)-dependent endocytosis is essential for antigen-induced BCR internalization and the survival of ABC-DLBCL cells (Fig. 5D). This led us to hypothesize that inhibition of BCR endocytosis could serve as a therapeutic strategy for DLBCL treatment. The clathrin- and DNM2-dependent endocytosis pathway can be inhibited by various chemical inhibitors, including phenothiazine derivatives, used for decades as anti-psychotic or anti-emetic agents [31]. To investigate the effect of phenothiazines on CME and BCR internalization, we first confirmed the inhibition of transferrin receptor internalization in prochloroperazine (PCH) or chlorpromazine (CPZ) -treated cells (Supplementary Fig. 5). Next, we evaluated the impact of these compounds on BCR endocytosis using previously described OVA-reactive DLBCL models and unmodified DLBCL cells. PCH and CPZ increased surface BCR levels in untreated cells. More importantly, receptor cross-linking or OVA treatment -induced BCR internalization was markedly attenuated by PCH or CPZ (Fig. 7A and 7B). These results indicate that autoantigen-induced BCR internalization can be pharmacologically inhibited by phenothiazine derivatives, leading us to hypothesize that these compounds could inhibit DLBCL growth. Phenothiazine derivatives inhibit DLBCL cell growth and synergize with BCR pathway inhibitors To address this question,we assessed the toxicity of PCH and CPZ in a panel of ABC-DLBCL cell lines: U2932, RIVA, HBL1 and TMD8. While all cell lines were sensitive to high PCH and CPZ doses (10 μM), U2932 and RIVA (CD79B-WT lines) exhibited markedly higher sensitivity to phenothiazine derivatives than HBL1 and TMD8 (CD79B-mutants) (Fig. 7C). To test the activity of phenothiazine derivatives against a DLBCL model in vivo , we generated U2932 xenografts in NSG mice. Animals with established disease were divided into two cohorts, with one group receiving vehicle alone and the other treated with PCH every three days starting on day 13 (4 or 8 mg/kg). Treatment with PCH significantly inhibited tumor growth in this model (Fig. 7D). Given the observed switching to tonic BCR signaling following DNM2/CME inhibition in kinome studies (Fig. 6C), we hypothesized that blocking kinases mediating tonic signaling would synergize with CME inhibition. For this reason, we examined the cytotoxic effects of combined inhibition of BCR internalization with SYK/PI3K blockade in DLBCL cell lines. For these studies, we chose HBL1 and TMD8 cell lines. HBL1 was shown to reprogram its BCR signaling to tonic mode after DNM2 inhibition, and both cell lines were moderately sensitive to resistance to phenothiazine derivatives. The combination of PCH with SYK inhibitors (Fostamatinib and Entospletinib) or a PI3K inhibitor (Idelalisib) exhibited strong synergistic effects in the assessed DLBCL cell lines (Fig. 7E). Discussion In normal B-cells, antigen engagement triggers rapid clustering of the BCR within membrane lipid rafts, initiates signaling, and subsequently leads to the internalization of the BCR–antigen complex. The internalized BCR–antigen complex is sorted into early endosomes and subsequently into major histocompatibility complex class II (MHC II) containing late endosomes. Notably, the BCR in the endosomal compartment is capable of continuous signaling [27]. Moreover, in a murine model of SLE, pathological B-cell proliferation depends on binding of TLR7 and/or TLR9 ligands, which are components of self-antigens internalized by the BCR and delivered to endosomes [32]. These observations underscore the role of autoantigens in facilitating the efficient transport of BCR and TLR9 to endolysosomes, where they form the signaling supercomplexes. In this study, we employed precise genomic editing to modify BCR specificity and control BCR membrane-cytosol trafficking and signaling in DLBCL cells. We demonstrate that similar to normal B-lymphocytes, DLBCL cells internalize the BCR complex rapidly after antigen engagement. The complex is delivered to the endolysosomal compartment, where it forms active BCR signalosomes. When the antigen specificity is altered and BCRs are unable to bind native autoantigens, BCRs are no longer internalized. As a consequence, receptor density in ABC-DLBCL cells increases and the formation of intracellular signaling complexes is decreased. These observations suggested that blocking endocytosis might have similar consequences as blocking BCR antigen engagement, while being therapeutically achievable. As expected, blocking endocytosis in HBL1 cells with a dominant-negative, catalytically inactive DNM2 mutant decreased the formation of endolysosomal BCR-TLR9 signaling supercomplexes and reduced NFκB activity. Surprisingly, DNM2 inhibition simultaneously led to activation of multiple kinases involved in tonic BCR signal generation. These observations suggest that when the autoantigen is present and available, but endocytosis is blocked, BCRs are cross-linked on the surface and the signaling is rewired to mimic a tonic signal. Surprisingly, blockade of DNM2 triggered activation of additional kinases with potentially compensatory functions. For example, CME inhibition was associated with the activation of ephrin (Eph) receptor tyrosine kinases. Eph receptors mediate bidirectional tumor-microenvironment communication, regulating tumor cell proliferation, migration, invasion, angiogenesis, and metastasis in vivo [33, 34]. These observations identify potential resistance mechanisms that may limit the efficacy of therapeutic strategies based on DNM2 inhibition. Consistent with this, the combination of PI3Kδ or SYK inhibitors (tonic signal antagonists) synergized with DNM2 inhibition. Since the role of Eph receptors in DLBCL has not been defined, these findings prompt further mechanistic studies. Phenothiazines are antipsychotic and anti-emetic drugs [35] acting as antagonists of dopamine receptors, but are also potent inhibitors of dynamin, making them effective at blocking clathrin-mediated endocytosis and receptor internalization [36–40]. Phenothiazines administered at anti-emetic doses demonstrate an acceptable safety profile, supporting their potential repurposing as a therapeutic option for DLBCL. According to published calculations [41], a dose of 5 mg prochlorperazine/kg/day in mice corresponds to approximately 0.405 mg/kg/day for humans (24.3 mg/day for a 60 kg person). The recommended clinical dose of prochlorperazine for preventing nausea and vomiting is 30 mg daily [42]. Consistent with this, prochlorperazine in our experiments was used at the therapeutically available dose range and exhibited a significant tumor growth-inhibitory effect in mice. Therefore, achieving an anti-tumor effect in human subjects is likely feasible. In our in vitro analyses, phenothiazines effectively dampened BCR internalization and markedly decreased proliferation of the CD79B wild-type U2932 and RIVA cells, whereas CD79B-mutant HBL1 and TMD8 cells were less sensitive. These observations suggest that the BCRs with wild-type BCRs are more prone to internalization and thus, more susceptible to endocytosis inhibitors. Mechanistically, mutations in the CD79B subunit of BCR are associated with increased BCR surface expression [7]. Moreover, the membrane-proximal ITAM YxxØ motif in CD79B, a common mutation target in ABC DLBCL, is crucial for the binding of adaptor protein 2 (AP2), the primary mediator of receptor endocytosis via clathrin-coated pits, suggesting that CD79B may regulate BCR internalization [43]. Genetic analyses indicate that 94.2% of CD79A/B mutations are heterozygous, meaning that some CD79A/B heterodimers forming the BCR will consist of the wild-type subunits. We therefore hypothesize that ABC-DLBCL cells with CD79A/B mutations possess two pools of BCRs: a wild-type BCR that can undergo self-antigen-induced internalization and trigger signaling with TLR9 from the endolysosomal compartment, and a BCR with mutated CD79A/B, less susceptible to CME – mediated internalization and remaining on the surface, initiating tonic BCR signaling (Supplementary Fig. 6). These findings suggest a potential shift to tonic BCR signaling when BCR internalization is blocked, particularly in the CD79B-mutant cells. Therefore, inhibiting both BCR internalization and tonic BCR signaling could represent an effective strategy for autoantigen-dependent cells with CD79A/B mutations. Indeed, the combination of phenothiazines with proximal inhibitors of BCR signaling demonstrated significantly greater cytotoxicity than monotherapy. In summary, we provide genetic, proteomic, and functional evidence that autoantigen-induced endocytosis of the BCR receptor is a key mechanism supporting BCR signaling and the survival of DLBCL cells with autoantigen-specific BCRs. Furthermore, we demonstrate that blocking endocytosis should be considered a rational therapeutic strategy in this group of lymphoid malignancies. The well-defined toxicity, pharmacokinetics, and pharmacodynamics of phenothiazines may facilitate design of clinical trials aimed at repurposing these drugs. Materials And Methods Cell lines and culture conditions For our study, we used the following DLBCL cell lines: OCI-Ly19 (RRID: CVCL_1878), HBL-1 (RRID: CVCL_4213), TMD8 (RRID: CVCL_A442), U-2932 (RRID: CVCL_1896), and Riva (RRID: CVCL_1885). Cells were cultured at 37°C with 5% CO 2 in RPMI media with L-glutamine (Sigma Aldrich, St. Louis, MO, USA) supplemented with: 10% fetal bovine serum (FBS) (Sigma Aldrich), 20 mM HEPES buffer (Lonza, Basel, Switzerland), 1 mM sodium pyruvate (Sigma Aldrich), and penicillin/streptomycin (final concentration 50 U/mL and 50 U/mL, respectively; Sigma Aldrich). Chemicals and inhibitors The following chemicals and inhibitors were used in the study:Prochlorperazine (Selleckchem, Houston, TX, USA, #S4631); Prochlorperazine Malate CRS - for in vivo studies (EDQM, Strasbourg, France, #P3200000); Chlorpromazine (Selleckchem, #S2456); Fostamatinib (Selleckchem, #S2206); Entospletinib (Selleckchem, #S7523); Idelalisib (MedChemExpress, Monmouth Junction, NJ, USA, #HY-13026). Knock-in experiments CRISPR/Cas9-mediated homologous recombination (HR) was used to generate knock-in (KI) modifications. DNA double-strand breaks (DSBs) at the desired genomic loci were introduced using pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330; Addgene, Watertown, MA, USA, #42230), which encodes both Cas9 and a guide RNA (gRNA) [44]. For KI, pX330 plasmids were co-electroporated with an HR template plasmid containing a left homology arm (LHA, 200–400 bp), the insert sequence, and a right homology arm (RHA, 200–400 bp). All homology arms and inserts were designed with silent mutations to prevent re-targeting by Cas9/gRNA. Homology arms and KI sequences were synthesized as gBlocks Gene Fragments, cloned into pSC-B-amp/kan (StrataClone Ultra Blunt PCR Cloning Kit, Agilent, Santa Clara, CA, USA), and sequence-verified. The HR template plasmids were constructed by combining the LHA and RHA as a single gBlock, separated by a cassette with two type IIS restriction enzyme sites for seamless KI sequence insertion. Specific details and sequences for all HR templates can be found in the Sequences section in the Supplement. To modify BCR specificity, the same KI approach was used to replace the BCR hypervariable region (HVR). Two pX330 plasmids induced DSBs flanking the original HVR, and a repair template plasmid provided the new ovalbumin (OVA)-specific HVR and a fluorescent protein (FP) marker. OVA-specific HVRs were based on published sequences from OBI Rag1−/− mice with OVA-reactive B cells [24]. To recreate the complete OVA HVR, we incorporated the 5’ portions of the full HVRs (including the leader sequence and V intron) based on the mouse reference sequence predicted by IMGT/V-QUEST. The repair template plasmid, from 5’ to 3’, included: LHA, FP, a 58-amino acid F2A sequence, HVR, and RHA. The FP cDNA was positioned with a Kozak sequence for in-frame translation (emGFP for H-HVR, mTurquoise2 for L-HVR). The F2A sequence ensured optimal separation of FP and HVR. The inserted HVR included its leader sequence and V intron. Following HR-mediated KI, the modified genomic sequence spanned from the V region translation initiation site to the 3’ end of the J segment, with the endogenous IgH or IgL promoter driving the expression of the FP (as a modification marker) alongside the separate IgH or IgL containing the replaced HVRs. In all experiments, we used control HVR replacements in which the original (endogenous) HVR was inserted along with the fluorescent marker. BCR HVR sequences for each cell line were previously published (Havranek et al., 2017) [6] . The CRISPR/Cas9 target sequences used for HVR replacement, and the basic characteristics of the DLBCL cell lines used, are provided in Supplementary Table 1 and Supplementary Table 2, respectively. HA fragments with FPs and F2A, and individual H-HVR and L-HVR fragments, are also listed in the Sequences section in the Supplement. For dual HVR replacement, both IgH and IgL HVRs were targeted simultaneously by electroporating six plasmids (upstream and downstream CRISPR/Cas9 and HR template for each Ig chain), 4 μg each, into 1.2 million cells in 120 μL R buffer. Double HVR-replaced (GFP- and mTurquoise2-positive) cells were monitored by flow cytometry. Inducible expression of DNM2 and mCD8-OVA To enable doxycycline-inducible expression of wild-type or K44A mutant DNM2, synthesized coding sequences for both variants (ATG Biosynthetics) were PCR-amplified and cloned into the transposon-based pSBtet-Bla vector (Addgene #60510) using the NEBuilder® HiFi DNA Assembly Kit. For stable genomic integration, 6 μg of the resulting transposon plasmid and 4 μg of the transposase plasmid pCMV(CAT)T7-SB100 (Addgene #34879) were electroporated into 1.2 million cells using the Neon Transfection System (Thermo Fisher Scientific, Waltham, MA, USA). Transfected cells were selected with blasticidin (10 μg/mL) (Thermo Fisher Scientific). For surface expression of the OVA peptide, a fusion construct comprising the transmembrane region of mouse CD8a and the OVA peptide (mCD8a-OVA) was cloned into the pSBtet-Bla vector. Cells were co-electroporated with this construct and the transposase plasmid using the Neon device. All nucleotide sequences are provided in the Supplement. Transfection Cells were transfected with plasmid DNA using the Neon electroporation system (Thermo Fisher Scientific) in 100 μL volumes. Log-phase cells were cultured with daily medium changes for three days prior to electroporation and washed once with PBS. For each electroporation, 1.2 million cells were resuspended in 120 μL of buffer R, mixed with maxiprep-purified plasmid DNA (PureLink HiPure Kit, Thermo Fisher Scientific), and electroporated under cell line–specific conditions (Supplementary Table 3). Afterward, cells were transferred to 3 mL of pre-warmed, antibiotic-free medium. Cell viability and growth assay Growth rates and viability after inhibitor treatment were assessed by bead-based flow cytometry. Cells were stained with SYTOX® Red (Thermo Fisher, 1:1000). Before analysis, 10 μL of 1:10 diluted 6.0–8.0 μm polystyrene beads (Spherotech, Lake Forest, IL, USA) were added. Beads and cells were distinguished by scatter on a CytoFLEX cytometer (Beckman Coulter, Indianapolis, IN, USA). Absolute cell numbers were determined by comparing bead and cell counts. If the culture was maintained over time through passaging at known dilutions, a growth curve reflecting the exponential increase in absolute cell number (logarithmic) over time (linear) was constructed. IC₅₀ values were calculated using Quest Graph™ IC50 Calculator (AAT Bioquest, Pleasanton, CA, USA), and drug synergy was analyzed with SynergyFinder. Proximity Ligation Assay (PLA) Cells were centrifuged, resuspended in PBS, and plated on precision coverslips for 20 min at 37°C. After adhesion, cells were fixed with 4% paraformaldehyde (Sigma Aldrich) for 20 min, washed, and membranes labeled with 5 μg/ml WGA-Alexa Fluor 488 (Thermo Fisher Scientific) for 10 min. Cells were permeabilized with 0.5% Triton X-100 (Sigma) in PBS for 10 min, washed, and blocked in Duolink Blocking Buffer (Sigma Aldrich) for 30 min. Primary antibodies (see Supplementary Table 5) were diluted in Duolink Antibody Diluent and incubated overnight at 4°C. After washing, Duolink Probes (Sigma Aldrich) were added and incubated for 1 h at 37°C, followed by washes. Ligation and amplification were performed with Duolink In Situ Detection Reagents Red kit (Sigma Aldrich) per manufacturer’s instructions. Cells were mounted in Prolong Gold with DAPI (Invitrogen, Carlsbad, CA, USA). Images were acquired on a Zeiss Axio Imager.Z2 fluorescence microscope and analyzed in ImageJ/FIJI. The antibodies we used for PLA analysis were previously validated by Phelan et al. [23] and are listed in the Supplementary Table 5. Flow cytometry-based BCR internalization assay DLBCL cell lines (HBL1, U2932, OCI-Ly19) were stimulated with 5 μg/mL anti-human IgM F(ab')₂ fragments (Jackson ImmunoResearch, West Grove, PA, USA). To induce OVA-specific BCR internalization, cells were treated with either 1 μM full-length ovalbumin (Sigma Aldrich) or a biotinylated OVA 17-mer peptide (FDKLPGFGDSIEAQGGK; GenScript, Nanjing, China) pre-complexed with avidin at a 4:1 molar ratio. For stimulation, 20 μL of a 10 μM peptide-avidin complex (calculated based on avidin concentration) was added to 1 mL of cell suspension. Following stimulation, cells were placed on ice to halt internalization, stained with APC-conjugated anti-human kappa light chain antibody (Invitrogen), and analyzed by flow cytometry (CytoFLEX, Beckman Coulter). Surface BCR levels were quantified using FlowJo software. Transferrin Internalization Assay Cells were incubated with Alexa Fluor 488–labeled transferrin (20 µg/mL; Invitrogen) for 1 hour at 37°C to allow internalization. Following incubation, cells were placed on ice to halt further uptake, washed, and incubated with or without anti–Alexa 488 quenching antibody (Invitrogen) to distinguish internalized from surface-bound transferrin. Samples were analyzed by flow cytometry (CytoFLEX, Beckman Coulter), and data were processed using FlowJo software (FlowJo, LLC). Flow cytometry For surface antigen detection (mCD8-OVA and BCR), 0.5 × 10⁶ cells were washed, stained with the appropriate antibody in FACS buffer (PBS with 1% FBS) for 30 min on ice, washed, and resuspended in FACS buffer. Data were acquired on a CytoFLEX cytometer and analyzed with FlowJo. Western-Blot Cells were washed in PBS and lysed in RIPA buffer with protease and phosphatase inhibitors (Roche) as described [45]. Proteins were separated via SDS–PAGE on 4–15% gradient gels (Bio-Rad), transferred to PVDF membranes (Millipore, Burlington, MA, USA), and blocked with 5% BSA/TBST. Membranes were incubated with primary antibodies (1:1000; Supplementary Table 1) overnight at 4°C, then with HRP-conjugated secondary antibodies. Signals were visualized using ECL (Perkin Elmer, Waltham, MA, USA) and captured with the G:Box system (Syngene, Bengaluru, India). Real-time PCR RNA was extracted using the GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland), and cDNA was synthesized with the Transcriptor Universal cDNA Master (Roche, Basel, Switzerland). qPCR was performed using SYBR Green Master Mix on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). Expression levels were normalized to GAPDH using the ΔΔCT method. Primer sequences are listed in Supplementary Table 4. Tyrosine and Serine/threonine kinase profiling For tyrosine and serine/threonine kinase profiling, the PamStation12 with PTK and STK PamChip peptide arrays (PamGene, Wolvenhoek, Netherland) was utilized. Analysis was performed according to the manufacturer’s instructions. In brief, chips were blocked with 2% BSA (Sigma-Aldrich). Proteins were extracted from fresh-frozen cell pellets using T-PER Buffer (Thermo Fisher Scientific), supplemented with 1:100 Phosphatase Inhibitor Cocktail and 1:100 Halt Protease Inhibitor Cocktail (EDTA-free, Thermo Fisher Scientific). 1 μg of protein per sample was applied to the chips with kinase buffer, ATP, and FITC-labeled antibodies. Signal intensities were quantified using BioNavigator 6.1.42 (PamGene), expressed per 100 ms exposure, and log-transformed. A mean value of 2,000 was established to ensure quality standards. Normalization was applied, and two replicated quantifications were combined using a false discovery rate (FDR) < 1%. A P value 10% fold change were considered significant. Calcium flux For calcium flux analysis, cells were loaded with Calbryte 630AM by resuspending in RPMI with 2% FBS and 25 mM HEPES containing 10 µM Calbryte 630AM (AAT Bioquest) and 0.05% Pluronic® F-127 (Thermo Fisher Scientific). Cells were incubated for 45 min at 37°C in the dark, washed twice with DMEM containing 2% FBS, and resuspended in loading medium. For stimulation, cells were treated with 6 µg/mL F(ab')₂ anti-human IgM goat antibody (Jackson ImmunoResearch) or 1 µM ovalbumin (Sigma Aldrich). Calcium responses were measured in the red channel using a CytoFLEX Flow Cytometer (Beckman Coulter). In vivo experiments For in vivo assessment of prochlorperazine (PCH) activity, 5 × 10⁶ U2932 cells mixed with 30% Matrigel Matrix (Corning, Corning, NY, USA, #354230) were injected subcutaneously into 8–12 week old female NOD.Cg- Prkdc scid Il2rg tm1Wjl (NSG) mice (Animalab, Poznan, Poland). All procedures were approved by the II Local Ethical Committee for Experiments on Animals in Warsaw, Poland (approval No WAW2/129/2023) and conducted in accordance with Directive 2010/63/EU. Mice were housed in specific pathogen-free conditions in individually ventilated cages under a 12-h light/dark cycle with ad libitum access to food and water. When tumors reached ≥100 mm³, 15 mice were randomized into three groups (n=5 per group) with similar mean tumor volumes and treated with 4 mg/kg PCH, 8 mg/kg PCH, or vehicle (H₂O) intraperitoneally for 15 consecutive days. Tumor growth was measured with digital calipers. After the final dose, mice were euthanized. Statistical Analysis. Statistical analyses were performed using GraphPad Prism 9.5.1 (GraphPad, Inc., La Jolla, CA, USA). Statistical tests are described in the figure legends and were nonparametric and two-sided unless otherwise indicated. P values < 0.05 were considered statistically significant, with significance denoted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. Declarations Acknowledgments This work has been supported by the research grants from the Polish National Science Centre (2019/35/D/NZ5/03354) and the Polish National Agency for Academic Exchange (PPN/BEK/2020/1/00173). Conflict of Interest The authors declare no potential conflicts of interest. Authors’ Contributions P. Górniak: Conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, writing–original draft, writing–review and editing. O. Havranek : Resources, investigation and methodology, writing–review and editing. A. Polak : Investigation and methodology, writing–review and editing . A. Rams : Investigation and methodology, writing–review and editing . K. Kupcova: Investigation and methodology, writing–review and editing E. Głodkowska-Mrówka : Resources and methodology, writing–review and editing Z. Pilch : Investigation and methodology, writing–review and editing. M. Miączyńska : Resources and methodology, writing–review and editing. D. Nowis : Investigation and methodology, writing–review and editing. J. Gołąb : Investigation and methodology, writing–review and editing R. E. Davis :Resources, investigation and methodology, writing–review and editing. P. Juszczyński : Supervision, writing–original draft, writing–review and editing. Data Availability The data generated in this study are available within the article and its supplementary data files. Other data that support this study and script to reproduce the analyses are available from the corresponding author upon reasonable request. References Chapuy B, Stewart C, Dunford AJ, Kim J, Kamburov A, Redd RA, et al. Molecular subtypes of diffuse large B cell lymphoma are associated with distinct pathogenic mechanisms and outcomes. Nat Med. 2018;24:679–90. 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Additional Declarations (Not answered) Supplementary Files SupplemantaryFile1.txt Supplementary File 1. Comparison of tyrosine and serine/threonine kinase activities in HBL1 OVA-specific vs. HBL1 control cells using the chip-based phosphoproteomic PamGene platform. SupplementaryFile2.txt Supplementary File 2. Comparison of tyrosine and serine/threonine kinase activities in K44A-DNM2 mutant vs. wild-type DNM2-expressing HBL1 cells using the chip-based phosphoproteomic PamGene platform. SupplementaryFile3.pdf Supplementary File 3. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7260543","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":504541536,"identity":"9f1bb8b9-486d-4be5-8510-c9f385657cad","order_by":0,"name":"Patryk 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13:51:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":256965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStrategy for modifying the hypervariable region (HVR) to alter BCR specificity and validation of cellular genetic models. (A)\u003c/strong\u003e Schematic representation of the experimental strategy to replace the immunoglobulin heavy (IgH) and light (IgL) chain HVR fragments in DLBCL cells. Complementary DNA encoding a fluorescent protein (GFP for IgH, and the CFP variant mTurquoise2 for IgL) is followed by sequences encoding a 2A peptide, which creates a break during translation, a signal peptide, and either an OVA-specific HVR or an endogenous HVR. This construct was knocked into the start of the IgH and IgL translation sites using CRISPR-Cas9 methodology; \u003cstrong\u003e(B)\u003c/strong\u003e Scheme illustrating the predicted consequences of HVR replacement, leading to changes in BCR specificity and capabilities of autoantigen binding in autoantigen-dependent (ABC-type) and autoantigen-independent (GCB-type) cells; \u003cstrong\u003e(C)\u003c/strong\u003eSurface BCR levels in DLBCL cells with OVA-recognizing and endogenous HVRs. BCR levels were assessed by flow cytometry. The results are presented as histograms and mean fluorescence intensity (MFI) plots (means ± standard deviations (SD) of two independent replicates); \u003cstrong\u003e(D)\u003c/strong\u003eCalcium fluxes in response to BCR stimulation in DLBCL cell models. Cells were incubated with anti-IgM (6 μg/mL) or OVA (1 μM). Arrows indicate the moments of OVA or anti-IgM addition.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/ec544a5724dafedd7102af11.png"},{"id":90335020,"identity":"1bb6af84-0292-4926-92bd-8af2c1fa560d","added_by":"auto","created_at":"2025-09-01 13:59:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":940193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAutoantigen binding activates oncogenic BCR signaling in DLBCL cells\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003eWestern blot analysis of tyrosine-phosphorylated proteins in cells with OVA-recognizing and endogenous HVRs. GAPDH was used as a loading control; \u003cstrong\u003e(B)\u003c/strong\u003e Western blot analysis of tyrosine-phosphorylated proteins in cells with OVA-recognizing HVRs following stimulation with OVA (1 μM) for the indicated times. GAPDH was used as a loading control; \u003cstrong\u003e(C)\u003c/strong\u003e Differential analysis of kinase activity levels in HBL1 cells with OVA-specific HVRs compared to those with endogenous HVRs, presented as a kinome tree (values \u0026gt;0 indicate higher activity in OVA-reactive cells). Each cell model was evaluated in two replicates, data presents the average values. \u003cstrong\u003eAGC\u003c/strong\u003e – \u003cem\u003eProtein Kinase A, G, and C family; \u003c/em\u003e\u003cstrong\u003eCAMK\u003c/strong\u003e – \u003cem\u003eCalcium/Calmodulin-dependent Protein Kinase; \u003c/em\u003e\u003cstrong\u003eCK1\u003c/strong\u003e– \u003cem\u003eCasein Kinase 1 family; \u003c/em\u003e\u003cstrong\u003eCMGC\u003c/strong\u003e – \u003cem\u003eCDK, MAPK, GSK3, and CLK kinases; \u003c/em\u003e\u003cstrong\u003eTK\u003c/strong\u003e– \u003cem\u003eTyrosine Kinases and \u003c/em\u003e\u003cstrong\u003eTKL\u003c/strong\u003e – \u003cem\u003eTyrosine Kinase-Like.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/893b0da3303409cf689cefcb.png"},{"id":90333940,"identity":"49841ca3-bf88-4f2b-ac73-179a6e2cab68","added_by":"auto","created_at":"2025-09-01 13:51:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":348634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAutoantigen binding induces the assembly of the BCR-TLR9 complex in the endosomal compartment and activates NFκB pathway in DLBCL cells\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e NFκB-dependent gene expression in cells with OVA-recognizing and endogenous HVRs. Relative mRNA expression was assessed by real-time PCR, with GAPDH used as a reference gene. Data from three independent experiments are presented, with error bars representing standard deviation (SD). \u003cem\u003eP\u003c/em\u003e values were calculated by using two-sided unpaired \u003cem\u003et\u003c/em\u003e test; \u003cstrong\u003e(B)\u003c/strong\u003e Western blot analysis of phospho-IκBα in U2932 and HBL1 cells with OVA-recognizing and endogenous HVRs; \u003cstrong\u003e(C)\u003c/strong\u003e Proximity ligation assays showing co-localization (red puncta) of IgM-LAMP1, IgM-TLR9, and IgM-pIκBα in genetic models with OVA-recognizing and endogenous HVRs. Nuclei were stained with DAPI (blue), and membranes were visualized using WGA (green). To determine the Proximity Ligation Assay (PLA) score, the number of PLA spots per cell was counted in three OVA-specific samples and three control samples. The results from the OVA-specific samples were then normalized to the average value from the control samples, which was arbitrarily set to 100. Box and whisker plots display the mean PLA score, with whiskers depicting the range of all observed data. \u003cem\u003eP \u003c/em\u003evalues were determined using a two-sided t-test; ****\u003cem\u003eP\u003c/em\u003e ≤ 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/3e2d08b085b6b92acd27559f.png"},{"id":90333943,"identity":"c41fb6cc-6335-4fa3-93c1-db74377ea98d","added_by":"auto","created_at":"2025-09-01 13:51:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":320471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntigenic stimulation in DLBCL cells leads to the BCR internalization. (A)\u003c/strong\u003e BCR internalization is observed as a decrease in surface BCR levels in response to BCR stimulation in DLBCL cell models. Cells were incubated with anti-IgM (6 μg/mL), OVA (1 μM), or an OVA-17mer-biotin complexed with avidin for 3 hours. Surface BCR levels were assessed by flow cytometry; \u003cstrong\u003e(B)\u003c/strong\u003eTime course of surface BCR level decrease in response to OVA treatment (1 μM) in cells with OVA-specific HVRs. BCR levels were assessed by flow cytometry, with results presented as histograms and mean fluorescence intensity (MFI) plots; \u003cstrong\u003e(C)\u003c/strong\u003e Schematic representation of BCR internalization in DLBCL genetic models, mimicking the co-expression of BCRs and autoantigens on the plasma membrane. The cell model was derived from the autoantigen-independent OCI-LY19 GCB-DLBCL cell line, showing stable expression of OVA-specific BCRs and doxycycline (Dox)-inducible expression of a transmembrane protein, a truncated murine CD8a fused to the cognate OVA peptide (mCD8-OVA); \u003cstrong\u003e(D)\u003c/strong\u003e Surface levels of OVA-specific BCR and mCD8-OVA in the OCI-LY19-based genetic model. Cells were treated with DOX (50 or 100 ng/mL) for 24 hours and assessed by flow cytometry. Results are presented as dot plots, histograms, and MFI plots.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/75239eaad73f590dfbeb7938.png"},{"id":90333947,"identity":"fd1c4541-b78b-43c9-bb12-f7568ef5422c","added_by":"auto","created_at":"2025-09-01 13:51:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":381622,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBCR internalization is clathrin- and dynamin 2-dependent and is crucial for sustaining the growth of DLBCL cells\u003c/strong\u003e.\u003cstrong\u003e (A)\u003c/strong\u003e CRISPR-Cas9 screening data from Reddy et al. (2017) were analyzed using the KEGG Endocytosis Human Gene Set (hsa04144). A CRISPR score ≤ 0 indicates a growth-inhibitory effect of the depletion of a particular gene; \u003cstrong\u003e(B)\u003c/strong\u003e Western blot analysis showing the induction of expression of the wild-type Dynamin-2 (DNM2wt) and the dominant-negative K44A mutant (DNM2mut) in U2932- and HBL1-derived genetic models after treatment with doxycycline (DOX) (100 ng/mL for 24 hours). GAPDH was used as a loading control; \u003cstrong\u003e(C)\u003c/strong\u003e Surface levels of BCR in cells expressing DNM2wt or DNM2mut. Cells were pretreated with DOX (100 ng/mL) for 24 hours and stimulated with anti-IgM (6 μg/mL) for 3 hours. Analysis was performed using flow cytometry. Results are presented as histograms and mean fluorescence intensity (MFI) plots (means ± SD of three independent replicates). \u003cem\u003eP\u003c/em\u003e values were calculated by using two-sided unpaired \u003cem\u003et\u003c/em\u003e test. The line colors on the histograms correspond to the bar colors on the MFI chart; \u003cstrong\u003e(D)\u003c/strong\u003e Absolute growth curves of U2932 and HBL1 cells expressing DNM2wt or DNM2mut. To induce DNM2wt/mut, cells were treated with DOX (100 ng/mL). The chart displays the mean results from three replicates ± SD. \u003cem\u003eP \u003c/em\u003evalues were calculated using a two-sided t-test.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/25156f938db9daf7075b2b31.png"},{"id":90333945,"identity":"b7461229-7714-44a5-a09d-7224246892e5","added_by":"auto","created_at":"2025-09-01 13:51:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":859280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of\u003c/strong\u003e \u003cstrong\u003eBCR internalization blocks BCR-TLR9 complex formation and pro-survival signaling in ABC-DLBCL cells. (A)\u003c/strong\u003e Proximity ligation assays showing co-localization (red puncta) of IgM-LAMP1, IgM-TLR9, and IgM-pIκBα in HBL1 cells expressing DNM2wt or DNM2mut. Nuclei were stained with DAPI (blue), and membranes were visualized with WGA (green). To determine the Proximity Ligation Assay (PLA) score, the number of PLA spots per cell was counted in three DNM2mut samples and three control samples. The results from the DNM2mut samples were then normalized to the average value from the control samples, which was arbitrarily set to 100. Box and whisker plots display the mean PLA score, with whiskers depicting the range of all observed data. \u003cem\u003eP \u003c/em\u003e\u0026nbsp;values were determined using a two-sided t-test; ****\u003cem\u003eP\u003c/em\u003e ≤ 0.0001; \u003cstrong\u003e(B)\u003c/strong\u003e Expression of NFκB-dependent genes in HBL1 cells expressing DNM2mut or DNM2wt. To induce DNM2wt/mut, cells were incubated with DOX (100 ng/mL) for 24 hours. Relative mRNA expression was assessed by real-time PCR, using GAPDH as a reference gene. Data from two independent experiments are presented, with error bars representing the SD. \u003cem\u003eP\u003c/em\u003e values were calculated by using two-sided unpaired \u003cem\u003et\u003c/em\u003e test;\u003cstrong\u003e (C)\u003c/strong\u003eDifferential analysis of kinase activity in HBL1 cells expressing DNM2mut versus DNM2wt, presented as a kinome tree (\u0026gt;0 indicates higher activity in OVA-reactive cells). To induce DNM2wt/mut, cells were incubated with DOX (100 ng/mL) for 24 hours. Two replicate quantifications were performed for each cell model; \u003cstrong\u003e(D)\u003c/strong\u003e Western blot analysis of phosphorylated proximal BCR signaling mediators in HBL1 cells expressing DNM2mut and DNM2wt variants after treatment with doxycycline (DOX) (100 ng/mL for 24 hours). The α-SRC antibody cross-reacts with LYN, FYN, LCK, YES and HCK. GAPDH was used as a loading control.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/3092a9e7f1e2512951450a74.png"},{"id":90335022,"identity":"e54f6624-e747-491e-86a8-940fae6cdec4","added_by":"auto","created_at":"2025-09-01 13:59:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":406518,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenothiazine derivatives inhibit autoantigen-induced BCR internalization and DLBCL cell growth.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eBCR internalization in phenothiazine derivative-treated DLBCL cells. U2932 and HBL1 cells were treated with DMSO, prochlorperazine (PCH) (5 and 10 μM), or chlorpromazine (CPZ) (10 and 20 μM) for 16 hours and then stimulated with anti-IgM (6 μg/mL for 1 hour) to induce BCR internalization. Surface BCR levels were assessed by flow cytometry, and the results are presented as histograms and mean fluorescence intensity (MFI) plots (means ± SD of two independent replicates). \u003cem\u003eP\u003c/em\u003e values were calculated by using two-sided unpaired \u003cem\u003et\u003c/em\u003e test; \u003cstrong\u003e(B)\u003c/strong\u003e BCR internalization in phenothiazine derivative-treated OVA-specific DLBCL cells. Genetic models expressing OVA-specific BCRs were treated with DMSO, prochlorperazine (PCH) (5 and 10 μM), or chlorpromazine (CPZ) (10 and 20 μM) for 16 hours and then stimulated with OVA (1 μM for 1 hour) to induce BCR internalization. Surface BCR levels were assessed by flow cytometry, and the results are presented as histograms and MFI plots (mean ± SD of two independent replicates). \u003cem\u003eP\u003c/em\u003e values were calculated by using two-sided unpaired \u003cem\u003et\u003c/em\u003e test; \u003cstrong\u003e(C)\u003c/strong\u003e Toxicity of prochlorperazine (PCH) and chlorpromazine (CPZ) in DLBCL cell lines. Cells were incubated for 72 hours with DMSO or the indicated concentrations of drugs. Afterward, cells were stained with SYTOX Red, and live cells were counted by flow cytometry. Graphs represent averaged data from three experiments; CD79B mutation status in cell lines is indicated above the bar plots; \u003cstrong\u003e(D)\u003c/strong\u003e Tumor growth kinetics in prochlorperazine or vehicle-treated mice. U2932 cells were suspended in 30% Matrigel and injected into NSG mice. When tumor volume reached ≥100 mm³, mice were treated with intraperitoneal injections of prochlorperazine (4 mg/kg or 8 mg/kg) or vehicle (H₂O) as indicated. Each experimental group consisted of five mice. Asterisks indicate \u003cem\u003eP \u003c/em\u003evalues \u0026lt; 0.05 in a one-sided Student's t-test; \u003cstrong\u003e(E)\u003c/strong\u003eDrug synergy in TMD8 and HBL1 cells treated with prochlorperazine and the indicated drugs. Cells were incubated for 72 hours with DMSO or drug combinations. Afterward, cells were stained with SYTOX Red, and live cells were counted by flow cytometry. The highest single agent (HSA) synergy model was used for evaluation.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/bbc8bbea920f0de44af3a922.png"},{"id":104397239,"identity":"a7e3ddf7-ed0d-4b7f-90e8-e0d86c06ebf3","added_by":"auto","created_at":"2026-03-11 11:45:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4448057,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/21ddbc47-4d10-4967-a6a1-6ad535fd424c.pdf"},{"id":90335019,"identity":"0bc69440-fcc5-4feb-ae3b-1dda185fb104","added_by":"auto","created_at":"2025-09-01 13:59:33","extension":"txt","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary File 1\u003c/strong\u003e. Comparison of tyrosine and serine/threonine kinase activities in HBL1 OVA-specific vs. HBL1 control cells using the chip-based phosphoproteomic PamGene platform.\u003c/p\u003e","description":"","filename":"SupplemantaryFile1.txt","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/4ea00d78972e9260a9507c05.txt"},{"id":90333938,"identity":"cdbd7035-dbbc-4229-af37-b93ea9b97e8b","added_by":"auto","created_at":"2025-09-01 13:51:33","extension":"txt","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary File 2\u003c/strong\u003e. Comparison of tyrosine and serine/threonine kinase activities in K44A-DNM2 mutant vs. wild-type DNM2-expressing HBL1 cells using the chip-based phosphoproteomic PamGene platform.\u003c/p\u003e","description":"","filename":"SupplementaryFile2.txt","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/5218417b42c2d33f9c8b2109.txt"},{"id":90333946,"identity":"61d7ff72-db8a-470a-8e46-2e508b3702c9","added_by":"auto","created_at":"2025-09-01 13:51:33","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":857474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary File 3. \u003c/strong\u003eOriginal WB\u003c/p\u003e","description":"","filename":"SupplementaryFile3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/c23f16cc74e43a00ecca1802.pdf"},{"id":90335330,"identity":"aec3d4a9-6edb-4e5f-a55b-faffc24f52f1","added_by":"auto","created_at":"2025-09-01 14:07:33","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1376459,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Information\u003c/p\u003e","description":"","filename":"SupplementPGPJFINAL.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7260543/v1/61faa59741c79c9bed8c67c0.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Inhibition of autoantigen-induced B-cell receptor (BCR) internalization as a therapeutic strategy in Diffuse Large B Cell Lymphoma (DLBCL)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiffuse large B cell lymphoma (DLBCL) is the most common aggressive lymphoid malignancy in adults, accounting for up to 35% of non-Hodgkin lymphomas [1,2]. DLBCLs arise from B-cells that have encountered antigens and undergone the germinal center (GC) reaction, a process particularly conducive to somatic mutations [3]. DLBCLs are subdivided into the more aggressive, activated B-cell (ABC)-type, and the less aggressive, germinal center B-cell (GCB)-type [4]. While this classification provides fundamental insights into the molecular characteristics of DLBCL and has prognostic value, recent genomic profiling studies have revealed a more nuanced substructure within DLBCL, identifying at least five distinct subtypes with different mutational spectra [1,5].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Although transformed B cells acquire new pro-survival mutations during the GC reaction, they frequently remain dependent on B-cell receptor (BCR) signaling. Similar to their normal counterparts, DLBCL cells exhibit two different patterns of signaling emanating from the BCR: tonic and chronic-active, characteristic of GCB and ABC subtypes, respectively [6,7]. Tonic BCR signaling is antigen-independent, with phosphatidylinositide 3-kinase (PI3K) and AKT being the key mediators\u0026nbsp;[6,8\u0026ndash;10]. In contrast, chronic-active BCR signaling engages multiple pathways and transcriptional networks, with a crucial role of NF\u0026kappa;B activation - a hallmark of ABC-type DLBCLs. This mode of signaling resembles antigen-dependent BCR activation in normal B cells\u0026nbsp;[7, 11]\u0026nbsp;While activating mutations in \u003cem\u003eCARD11\u003c/em\u003e can explain the constitutive BCR signal in a fraction of cases, other mutations (e.g., in \u003cem\u003eCD79A/B\u003c/em\u003e or \u003cem\u003eMYD88\u003c/em\u003e, present in ~20-25% and up to 40% of ABC-DLBCLs, respectively) cannot initiate the signal by themselves\u0026nbsp;[2, 7, 12]. Consistent with this, certain lymphomas exhibit non-random IGHV segment usage, suggesting antigen-dependent selection and signaling\u0026nbsp;[12]. In line with these findings, BCR engagement by autoantigens or homotypic BCR interactions have been identified in selected DLBCL cases\u0026nbsp;[13\u0026ndash;16].\u0026nbsp;Numerous molecules and epitopes acting as BCR autoantigens have been characterized in ABC-type DLBCL cell lines, including N-acetyl-lactosamine glycans on cell surface proteins in HBL1, an epitope within the FR2 fragment of the BCR in TMD8, or molecules in apoptotic debris stimulating BCRs in U2932 and OCI-LY10 cells\u0026nbsp;[13]. The engagement of autoantigens has also been demonstrated in DLBCL patients. Screening for potential BCR antigens identified hypo-phosphorylated arsenite resistance protein 2 (Ars2) as a candidate autoantigen in primary ABC-type DLBCL\u0026nbsp;[16, 17]. Moreover, in primary central nervous system lymphoma (PCNSL)\u0026mdash;a specific extranodal subtype of DLBCL\u0026mdash;hyper-N-glycosylated SAMD14 and neurabin-I were identified as self-antigens\u0026nbsp;[18]. Importantly, BCR-engaging autoantigens have been also identified in other lymphoid malignancies, such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL)\u0026nbsp;[14, 15].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsistent with the pro-survival role of activated BCR signaling, targeting intracellular BCR-associated pathways with small-molecule inhibitors emerged as a new, promising therapeutic strategy. Inhibitors of BCR signaling have been registered for clinical use in CLL and MCL, but not in DLBCL \u0026ndash; most likely due to molecular heterogeneity and the type of BCR signaling [11, 12, 19]. Importantly, subsequent studies clearly demonstrated that certain subgroups of DLBCL patients can benefit from this strategy, paving the way to further studies on strategies augmenting the activity of the BCR signal inhibitors\u0026nbsp;[12, 19\u0026ndash;22].\u003c/p\u003e\n\u003cp\u003eWhile the pro-survival role of autoantigens in DLBCL and other lymphomas is well-established, the proximal events triggering signaling pathways induced by BCR engagement are less well-defined. In certain ABC-DLBCLs, the BCR forms an intracellular multiprotein supercomplex with MYD88 and TLR9, generating signals from endolysosomes [23]. These observations indicate that BCRs in these ABC-DLBCLs are internalized, similarly to BCRs in normal B-cells following antigen engagement. However, it is not clear whether the internalization of the BCR is required for sustained signaling in DLBCL cells, nor have the mechanisms responsible for BCR trafficking been defined. Likewise, the role of autoantigens in this process remains undefined. To fill this gap, we generated DLBCL cell models with ovalbumin (OVA)-recognizing BCR hypervariable regions (HVRs). Cells with modified BCRs can no longer bind their original target autoantigens, but are responsive to stimulation with OVA under strictly controlled conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur results show that BCR (auto)antigen engagement in DLBCL cells leads to the internalization of the BCR-antigen complex. This process is crucial for the formation of a previously reported signalosome complex containing the BCR and TLR9 as well as phosphorylated I\u0026kappa;B, [23], \u0026nbsp;which triggers the \u0026nbsp;canonical NF\u0026kappa;B pathway and enhances the survival of DLBCL cells. Furthermore, we demonstrate that disruption of clathrin-mediated endocytosis (CME) using dominant-negative dynamin-2 mutants decreases BCR internalization and DLBCL cell viability. Importantly, phenothiazine derivatives, acting as pharmacologic dynamin-2 antagonists, recapitulate these effects both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eDLBCL xenograft models.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTaken together, BCR internalization represents a novel therapeutic vulnerability in DLBCL and, potentially, also for other types of autoantigen-dependent lymphomas. Our data suggest that phenothiazines can be further evaluated and repurposed as candidates for DLBCL treatment, particularly in combination with BCR signaling pathway blockers, including SYK or PI3K\u0026delta; inhibitors.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eAutoantigen binding is required for sustained BCR signaling in ABC-DLBCL cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the proximal mechanisms of autoantigen-mediated BCR signal induction in DLBCL, we employed CRISPR/Cas9 knock-in technology to modify BCR specificity in two autoantigen-dependent ABC-type cell lines (U2932 and HBL1) and one autoantigen-independent GCB-type cell line (OCI-LY19). Specifically, we replaced the hypervariable regions (HVRs) in the immunoglobulin heavy (IgH) and light chains (IgL) with ovalbumin (OVA)-recognizing HVRs derived from isolated OVA-reactive B cells [24]. With this approach, cells with fully modified HVR express GFP (IgH-derived) and mTurquoise (IgL-derived) and can be isolated using FACS (Supplementary Fig. 1).Endogenous HVRs from the cell lines being targeted were used to generate appropriate controls (Fig. 1A). This approach successfully produced DLBCL cell models with OVA-recognizing BCRs that no longer bind cognate autoantigens (Fig. 1B), enabling studies on the mechanisms of autoantigen-induced signal generation in DLBCL cells.\u003c/p\u003e\n\u003cp\u003eIn cell lines that naturally express autoantigen-specific BCRs, replacement with OVA-reactive HVRs exhibited caused an increase in cell-surface \u0026nbsp;BCR levels. In contrast, OVA-reactive BCR surface levels in the autoantigen-independent OCI-LY19 cells were not increased when compared to the control cells (Fig. 1C). Both the native and the dual HVR-replaced BCRs were capable of signal \u0026nbsp;generation, as demonstrated by calcium flux analysis following anti-IgM-mediated receptor cross-linking (Fig. 1D). However, OVA stimulation induced calcium fluxes only in cells with OVA-reactive HVRs (Fig. 1D), confirming the functionality and antigen specificity of the engineered BCRs and the utility of the generated cell models for further studies.\u003c/p\u003e\n\u003cp\u003eTo characterize the autoantigen-dependent signaling, we first assessed tyrosine-phosphorylated proteins in ABC-DLBCL cells with OVA-specific and endogenous HVRs. In both HBL1 and U2932, cells expressing the OVA-specific BCR exhibited decreased tyrosine phosphorylation of numerous proteins when compared to controls (Fig. 2A). Incubation of these cells with OVA upregulated tyrosine phosphorylation in these proteins (Fig. 2B). Next, we compared global tyrosine and serine/threonine kinase activities in OVA-specific and control cells using the chip-based phosphoproteomic PamGene platform. This functional kinase assay measures protein kinase activity directly in cellular lysates by quantifying phosphorylation of peptides printed on a chip (Supplementary Fig. 2). Cells with OVA-specific BCRs (incapable of autoantigen binding), exhibited significantly reduced activity of proximal BCR signaling mediators, including SRC family kinases (e.g., LYN, FYN, BLK), SYK tyrosine kinase, Bruton\u0026apos;s tyrosine kinase (BTK), and protein kinase C. Furthermore, these OVA-specific cells also showed decreased activity of kinases essential for the regulation of cell proliferation (cyclin-dependent kinases, CDKs), protein biosynthesis (ribosomal S6 kinases, RSKs), and kinases engaged in oncogenic signaling (JAKs, PIMs, FLT1/3/4, PDGFRs, FGFRs, TRKA/B/C) (Fig. 2C and Supplementary File 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNF\u0026kappa;B activation is a hallmark of ABC-type DLBCLs. Since altering BCR specificity and eliminating autoantigen binding decreased the activity of multiple BCR-related kinases and global tyrosine phosphorylation, we evaluated the changes in NF\u0026kappa;B activity in engineered cells. For this purpose, we evaluated the phosphorylation levels of the NF\u0026kappa;B inhibitory protein I\u0026kappa;B\u0026alpha; and the expression of NF\u0026kappa;B-dependent genes as markers of NF\u0026kappa;B activity. These analyses revealed significantly higher levels of I\u0026kappa;B\u0026alpha; phosphorylation and NF\u0026kappa;B-dependent transcripts in the autoantigen-reactive cells, highlighting the crucial role of sustained BCR autoantigen engagement for continuous NF\u0026kappa;B activation (Fig. 3A and 3B).\u003c/p\u003e\n\u003cp\u003eIn a subset of ABC-DLBCLs (referred to as MCD or C5 clusters) [1, 5], NF\u0026kappa;B pathway activation is mediated by the intracellular BCR-TLR9 complex [23]. Using Proximity Ligation Assay (PLA) analysis, we found that in OVA-reactive cells, the assembly of the endolysosomal BCR-TLR9 complex (as indicated by interaction with the LAMP1 marker) was significantly inhibited. Furthermore, the interaction between this complex and the phosphorylated form of I\u0026kappa;B\u0026alpha;, which is critical for NF\u0026kappa;B activation, was also diminished in OVA-reactive models (Fig. 3C).\u003c/p\u003e\n\u003cp\u003eCollectively, these findings suggest that autoantigen binding by the BCR in ABC-DLBCL cells facilitates the recruitment of BCR and TLR9 into a functional endolysosomal BCR-TLR9 complex, contributing to NF\u0026kappa;B activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntigenic stimulation in DLBCL cells leads to BCR internalization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn normal B cells, antigen binding to the BCR triggers rapid clustering within membrane lipid rafts and subsequently leads to the internalization of the BCR-antigen complex. Following internalization, this complex is sorted into early endosomes and eventually into late endosomes [25]. DLBCL cells with OVA-specific BCRs exhibited increased BCR surface density and reduced formation of the endolysosomal BCR-TLR9 complex (Fig. 1C and 3C), indicating that the loss of autoantigenic stimulation results in BCR membrane retention.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo confirm this scenario, we incubated OVA-specific cell models with anti-IgM (a positive control), OVA, and 17mer-OVA peptide conjugated\u0026nbsp;with biotin and complexed with avidin. Anti-IgM triggered BCR endocytosis in both HVR types, but OVA or 17mer-OVA exhibited this effect only in cells with OVA-specific HVRs (Fig. 4A). Time-course analysis demonstrated that BCR internalization is a dynamic process, resulting in a significant decrease in BCR surface levels within minutes and progressing up to 3h (Fig. 4B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince BCR-engaging DLBCL autoantigens can be either extracellular or expressed on the plasma membrane [13], we\u0026nbsp;asked whether membrane-anchored autoantigens could also initiate BCR endocytosis. To address this question, we generated a model derived from the autoantigen-independent OCI-LY19 GCB-DLBCL cell line with OVA-specific BCRs. This cell line was modified to express a doxycycline (Dox)-inducible construct including the truncated murine CD8a (mCD8) fused to the OVA peptide (Fig. 4C). In this model, Dox-induced expression of membrane-anchored mCD8-OVA resulted in a significant decrease of the surface BCR levels, demonstrating that membrane-expressed autoantigens can initiate BCR internalization in DLBCL cells (Fig. 4D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of\u003c/strong\u003e \u003cstrong\u003eBCR internalization blocks BCR-TLR9 complex formation and pro-survival signaling in ABC-DLBCL cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the observations that altered BCR specificity and loss of autoantigen binding led to BCR membrane retention, decreased formation of intracellular BCR-TLR9 supercomplexes, and reduced NF\u0026kappa;B activity, we hypothesized that blocking BCR internalization might represent a novel DLBCL therapeutic strategy. Since elimination of autoantigens inducing BCR internalization is not feasible, we hypothesized that blocking BCR endocytosis would be a more general approach to inhibit autoantigen\u0026ndash;dependent DLBCL cells. To evaluate the consequences of inhibition of endocytosis for DLBCL cell survival, we first utilized available datasets from CRISPR-Cas9 screening studies [2]\u0026nbsp;and the KEGG Endocytosis Human Gene Set. With this approach, we found that knockout of multiple genes in the clathrin-mediated endocytosis (CME) pathway resulted in reduced DLBCL cell viability (Fig. 5A). CME is responsible for the internalization of multiple surface receptors, including BCR-antigen complexes\u0026nbsp;[26,27]. DNM2 (dynamin-2) GTP-ase plays an essential role in CME. DNM2 forms ring-like structures around the neck of the invaginating vesicle and facilitates the scission of endocytic vesicles from the cell membrane. Importantly, DNM2 depletion exhibited a stronger effect on DLBCL survival than depletion of other genes coding for proteins targeted by available small molecule inhibitors (Supplementary Fig. 3).\u003c/p\u003e\n\u003cp\u003eTo assess the role of DNM2 and CME in autoantigen-induced BCR internalization and the subsequent activation of BCR signaling in DLBCL cells, we generated cellular models with inducible expression of a dynamin-2 K44A mutant lacking GTPase activity and shown to inhibit CME in a dominant-negative manner (DN-DNM2) (Fig. 5B) [28, \u0026nbsp;29]. Overexpression of DN-DNM2 markedly decreased internalization of the transferrin receptor, confirming the inhibition of CME (Supplementary Fig. 4). DN-DNM2 expression increased BCR surface levels in untreated, autoantigen-dependent cells. Likewise, DN-DNM2 blocked BCR internalization after anti-IgM cross-linking (Fig. 5C). Consistent with the data from the CRISPR-Cas9 screen, DN-DNM2 expression resulted in growth inhibition in the generated cell models, particularly in U2932 cells (Fig. 5D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBCR membrane retention after CME inhibition suggested reduced endolysosomal BCR-TLR9 complex formation. Indeed, CME inhibition in HBL1 cells led to a marked decrease in BCR-TLR9-pI\u0026kappa;B complexes (Fig. 6A), which subsequently resulted in the downregulation of NF\u0026kappa;B-dependent gene expression (Fig. 6B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further characterize the consequences of CME inhibition, we evaluated the changes in tyrosine and serine-threonine kinase activity in HBL1 cells after DN-DNM2 induction using the PamGene platform. Inhibition of CME dampened the activation of several signaling pathways (mTOR, CDKs, PKCs, and RSKs), similar to the effects of switching endogenous HVRs for OVA-recognizing HVRs. However, unlike the genetic change in BCR specificity, CME inhibition led to increased activity of proximal BCR signaling kinases, including SRC family kinases (including LYN) and SYK - a critical tyrosine kinase involved in tonic BCR signaling [30]. Consistent with this, DNM2 inhibition increased the activity of SRC family kinases (including LYN) and AKT, the key kinase mediating tonic BCR signaling\u0026nbsp;[6, 10]\u0026nbsp;(Fig. 6C and 6D and Supplementary File 2). These observations suggest that blocking endocytosis increases surface BCR density, which may enhance tonic BCR signaling and represent a cellular attempt to compensate for the loss of the chronic-active signal.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenothiazine derivatives inhibit antigen-induced BCR internalization\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur studies indicate that clathrin- and dynamin-2 (DNM2)-dependent endocytosis is essential for antigen-induced BCR internalization and the survival of ABC-DLBCL cells (Fig. 5D). This led us to hypothesize that inhibition of BCR endocytosis could serve as a therapeutic strategy for DLBCL treatment. The clathrin- and DNM2-dependent endocytosis pathway can be inhibited by various chemical inhibitors, including phenothiazine derivatives, used for decades as anti-psychotic or anti-emetic agents [31]. To investigate the effect of phenothiazines on CME and BCR internalization, we first confirmed the inhibition of transferrin receptor internalization in prochloroperazine (PCH) or chlorpromazine (CPZ) -treated cells (Supplementary Fig. 5). Next, we evaluated the impact of these compounds on BCR endocytosis using previously described OVA-reactive DLBCL models and unmodified DLBCL cells. PCH and CPZ increased surface BCR levels in untreated cells. More importantly, receptor cross-linking or OVA treatment -induced BCR internalization was markedly attenuated by PCH or CPZ (Fig. 7A and 7B). These results indicate that autoantigen-induced BCR internalization can be pharmacologically inhibited by phenothiazine derivatives, leading us to hypothesize that these compounds could inhibit DLBCL growth.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenothiazine derivatives inhibit DLBCL cell growth and synergize with BCR pathway inhibitors\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo address this question,we assessed the toxicity of PCH and CPZ in a panel of ABC-DLBCL cell lines: U2932, RIVA, HBL1 and TMD8. While all cell lines were sensitive to high PCH and CPZ doses (10 \u0026mu;M), U2932 and RIVA (CD79B-WT lines) exhibited markedly higher sensitivity to phenothiazine derivatives than HBL1 and TMD8 (CD79B-mutants) (Fig. 7C). To test the activity of phenothiazine derivatives against a DLBCL model \u003cem\u003ein vivo ,\u0026nbsp;\u003c/em\u003ewe generated U2932 xenografts in NSG mice. Animals with established disease were divided into two cohorts, with one group receiving vehicle alone and the other treated with PCH every three days starting on day 13 (4 or 8 mg/kg). Treatment with PCH significantly inhibited tumor growth in this model (Fig. 7D).\u003c/p\u003e\n\u003cp\u003eGiven the observed switching to tonic BCR signaling following DNM2/CME inhibition in kinome studies (Fig. 6C), we hypothesized that blocking kinases mediating tonic signaling would synergize with CME inhibition. For this reason, we examined the cytotoxic effects of combined inhibition of BCR internalization with SYK/PI3K blockade in DLBCL cell lines. For these studies, we chose HBL1 and TMD8 cell lines. HBL1 was shown to reprogram its BCR signaling to tonic mode after DNM2 inhibition, and both cell lines were moderately sensitive to resistance to phenothiazine derivatives. The combination of PCH with SYK inhibitors (Fostamatinib and Entospletinib) or a PI3K inhibitor (Idelalisib) exhibited strong synergistic effects in the assessed DLBCL cell lines (Fig. 7E).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn normal B-cells, antigen engagement triggers rapid clustering of the BCR within membrane lipid rafts, initiates signaling, and subsequently leads to the internalization of the BCR\u0026ndash;antigen complex. The internalized BCR\u0026ndash;antigen complex is sorted into early endosomes and subsequently into major histocompatibility complex class II (MHC II) containing late endosomes. Notably, the BCR in the endosomal compartment is capable of continuous signaling [27]. Moreover, in a murine model of SLE, pathological B-cell proliferation depends on binding of TLR7 and/or TLR9 ligands, which are components of self-antigens internalized by the BCR and delivered to endosomes [32]. These observations underscore the role of autoantigens in facilitating the efficient transport of BCR and TLR9 to endolysosomes, where they form the signaling supercomplexes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we employed precise genomic editing to modify BCR specificity and control BCR membrane-cytosol trafficking and signaling in DLBCL cells. We demonstrate that similar to normal B-lymphocytes, DLBCL cells internalize the BCR complex rapidly after antigen engagement. The complex is delivered to the endolysosomal compartment, where it forms active BCR signalosomes. When the antigen specificity is altered and BCRs are unable to bind native autoantigens, BCRs are no longer internalized. As a consequence, receptor density in ABC-DLBCL cells increases and the formation of intracellular signaling complexes is decreased.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese observations suggested that blocking endocytosis might have similar consequences as blocking BCR antigen engagement, while being therapeutically achievable. As expected, blocking endocytosis in HBL1 cells with a dominant-negative, catalytically inactive DNM2 mutant decreased the formation of endolysosomal BCR-TLR9 signaling supercomplexes and reduced NF\u0026kappa;B activity. Surprisingly, DNM2 inhibition simultaneously led to activation of multiple kinases involved in tonic BCR signal generation. These observations suggest that when the autoantigen is present and available, but endocytosis is blocked, BCRs are cross-linked on the surface and the signaling is rewired to mimic a tonic signal. Surprisingly, blockade of DNM2 triggered activation of additional kinases with potentially compensatory functions. For example, CME inhibition was associated with the activation of ephrin (Eph) receptor tyrosine kinases. Eph receptors mediate bidirectional tumor-microenvironment communication, regulating tumor cell proliferation, migration, invasion, angiogenesis, and metastasis \u003cem\u003ein vivo\u003c/em\u003e [33, 34]. These observations identify potential resistance mechanisms that may limit the efficacy of therapeutic strategies based on DNM2 inhibition. Consistent with this, the combination of PI3K\u0026delta; or SYK inhibitors (tonic signal antagonists) synergized with DNM2 inhibition. Since the role of Eph receptors in DLBCL has not been defined, these findings prompt further mechanistic studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Phenothiazines are antipsychotic and anti-emetic drugs [35] acting as antagonists of dopamine receptors, but are also potent inhibitors of dynamin, making them effective at blocking clathrin-mediated endocytosis and receptor internalization [36\u0026ndash;40]. Phenothiazines administered at anti-emetic doses demonstrate an acceptable safety profile, supporting their potential repurposing as a therapeutic option for DLBCL. According to published calculations [41], a dose of 5 mg prochlorperazine/kg/day in mice corresponds to approximately 0.405 mg/kg/day for humans (24.3 mg/day for a 60 kg person). The recommended clinical dose of prochlorperazine for preventing nausea and vomiting is 30 mg daily [42]. Consistent with this, prochlorperazine in our experiments was used at the therapeutically available dose range and exhibited a significant tumor growth-inhibitory effect in mice. Therefore, achieving an anti-tumor effect in human subjects is likely feasible.\u003c/p\u003e\n\u003cp\u003eIn our \u003cem\u003ein vitro\u003c/em\u003e analyses, phenothiazines effectively dampened BCR internalization and markedly decreased proliferation of the CD79B wild-type U2932 and RIVA cells, whereas CD79B-mutant HBL1 and TMD8 cells were less sensitive. These observations suggest that the BCRs with wild-type BCRs are more prone to internalization and thus, more susceptible to endocytosis inhibitors. Mechanistically, mutations in the CD79B subunit of BCR are associated with increased BCR surface expression [7]. Moreover, the membrane-proximal ITAM Yxx\u0026Oslash; motif in CD79B, a common mutation target in ABC DLBCL, is crucial for the binding of adaptor protein 2 (AP2), the primary mediator of receptor endocytosis via clathrin-coated pits, suggesting that CD79B may regulate BCR internalization [43]. Genetic analyses indicate that 94.2% of CD79A/B mutations are heterozygous, meaning that some CD79A/B heterodimers forming the BCR will consist of the wild-type subunits. We therefore hypothesize that ABC-DLBCL cells with CD79A/B mutations possess two pools of BCRs: a wild-type BCR that can undergo self-antigen-induced internalization and trigger signaling with TLR9 from the endolysosomal compartment, and a BCR with mutated CD79A/B, less susceptible to CME \u0026ndash; mediated internalization and remaining on the surface, initiating tonic BCR signaling (Supplementary Fig. 6). These findings suggest a potential shift to tonic BCR signaling when BCR internalization is blocked, particularly in the CD79B-mutant cells. Therefore, inhibiting both BCR internalization and tonic BCR signaling could represent an effective strategy for autoantigen-dependent cells with CD79A/B mutations. Indeed, the combination of phenothiazines with proximal inhibitors of BCR signaling demonstrated significantly greater cytotoxicity than monotherapy.\u003c/p\u003e\n\u003cp\u003eIn summary, we provide genetic, proteomic, and functional evidence that autoantigen-induced endocytosis of the BCR receptor is a key mechanism supporting BCR signaling and the survival of DLBCL cells with autoantigen-specific BCRs. Furthermore, we demonstrate that blocking endocytosis should be considered a rational therapeutic strategy in this group of lymphoid malignancies. The well-defined toxicity, pharmacokinetics, and pharmacodynamics of phenothiazines may facilitate design of clinical trials aimed at repurposing these drugs.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell lines and culture conditions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor our study, we used the following DLBCL cell lines: OCI-Ly19 (RRID: CVCL_1878), HBL-1 (RRID: CVCL_4213), TMD8 (RRID: CVCL_A442), U-2932 (RRID: CVCL_1896), and Riva (RRID: CVCL_1885). Cells were cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in RPMI media with L-glutamine (Sigma Aldrich, St. Louis, MO, USA) supplemented with: 10% fetal bovine serum (FBS) (Sigma Aldrich), 20 mM HEPES buffer (Lonza, Basel, Switzerland), 1 mM sodium pyruvate (Sigma Aldrich), and penicillin/streptomycin (final concentration 50 U/mL and 50 U/mL, respectively; Sigma Aldrich).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemicals and inhibitors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following chemicals and inhibitors were used in the study:Prochlorperazine (Selleckchem, Houston, TX, USA, #S4631); Prochlorperazine Malate CRS - for \u003cem\u003ein vivo\u003c/em\u003e studies (EDQM, Strasbourg, France, #P3200000); Chlorpromazine (Selleckchem, #S2456); Fostamatinib (Selleckchem, #S2206); Entospletinib (Selleckchem, #S7523); Idelalisib (MedChemExpress, Monmouth Junction, NJ, USA, #HY-13026).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnock-in experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCRISPR/Cas9-mediated homologous recombination (HR) was used to generate knock-in (KI) modifications. DNA double-strand breaks (DSBs) at the desired genomic loci were introduced using pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330; Addgene, Watertown, MA, USA,\u0026nbsp;#42230), which encodes both Cas9 and a guide RNA (gRNA) [44]. For KI, pX330 plasmids were co-electroporated with an HR template plasmid containing a left homology arm (LHA, 200\u0026ndash;400 bp), the insert sequence, and a right homology arm (RHA, 200\u0026ndash;400 bp). All homology arms and inserts were designed with silent mutations to prevent re-targeting by Cas9/gRNA. Homology arms and KI sequences were synthesized as gBlocks Gene Fragments, cloned into pSC-B-amp/kan (StrataClone Ultra Blunt PCR Cloning Kit, Agilent, Santa Clara, CA, USA), and sequence-verified. The HR template plasmids were constructed by combining the LHA and RHA as a single gBlock, separated by a cassette with two type IIS restriction enzyme sites for seamless KI sequence insertion.\u0026nbsp;Specific details and sequences for all HR templates can be found in the Sequences section in the Supplement.\u003c/p\u003e\n\u003cp\u003eTo modify BCR specificity, the same KI approach was used to replace the BCR hypervariable region (HVR). Two pX330 plasmids induced DSBs flanking the original HVR, and a repair template plasmid provided the new ovalbumin (OVA)-specific HVR and a fluorescent protein (FP) marker. OVA-specific HVRs were based on published sequences from OBI Rag1\u0026minus;/\u0026minus; mice with OVA-reactive B cells [24]. To recreate the complete OVA HVR, we incorporated the 5\u0026rsquo; portions of the full HVRs (including the leader sequence and V intron) based on the mouse reference sequence predicted by IMGT/V-QUEST.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe repair template plasmid, from 5\u0026rsquo; to 3\u0026rsquo;, included: LHA, FP, a 58-amino acid F2A sequence, HVR, and RHA. The FP cDNA was positioned with a Kozak sequence for in-frame translation (emGFP for H-HVR, mTurquoise2 for L-HVR). The F2A sequence ensured optimal separation of FP and HVR. The inserted HVR included its leader sequence and V intron. Following HR-mediated KI, the modified genomic sequence spanned from the V region translation initiation site to the 3\u0026rsquo; end of the J segment, with the endogenous IgH or IgL promoter driving the expression of the FP (as a modification marker) alongside the separate IgH or IgL containing the replaced HVRs. In all experiments, we used control HVR replacements in which the original (endogenous) HVR was inserted along with the fluorescent marker.\u003c/p\u003e\n\u003cp\u003eBCR HVR sequences for each cell line were previously published (Havranek et al., 2017) [6]\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eThe CRISPR/Cas9 target sequences used for HVR replacement, and the basic characteristics of the DLBCL cell lines used, are provided in Supplementary Table 1 and Supplementary Table 2, respectively.\u0026nbsp;HA fragments with FPs and F2A, and individual H-HVR and L-HVR fragments,\u0026nbsp;are also listed in the Sequences section in the Supplement.\u003c/p\u003e\n\u003cp\u003eFor dual HVR replacement, both IgH and IgL HVRs were targeted simultaneously by electroporating six plasmids (upstream and downstream CRISPR/Cas9 and HR template for each Ig chain), 4 \u0026mu;g each, into 1.2 million cells in 120 \u0026mu;L R buffer. Double HVR-replaced (GFP- and mTurquoise2-positive) cells were monitored by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInducible expression of DNM2 and mCD8-OVA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo enable doxycycline-inducible expression of wild-type or K44A mutant DNM2, synthesized coding sequences for both variants (ATG Biosynthetics) were PCR-amplified and cloned into the transposon-based pSBtet-Bla vector (Addgene #60510) using the NEBuilder\u0026reg; HiFi DNA Assembly Kit. For stable genomic integration, 6 \u0026mu;g of the resulting transposon plasmid and 4 \u0026mu;g of the transposase plasmid pCMV(CAT)T7-SB100 (Addgene #34879) were electroporated into 1.2 million cells using the Neon Transfection System (Thermo Fisher Scientific,\u0026nbsp;Waltham, MA, USA). Transfected cells were selected with blasticidin (10 \u0026mu;g/mL) (Thermo Fisher Scientific).\u003c/p\u003e\n\u003cp\u003eFor surface expression of the OVA peptide, a fusion construct comprising the transmembrane region of mouse CD8a and the OVA peptide (mCD8a-OVA) was cloned into the pSBtet-Bla vector. Cells were co-electroporated with this construct and the transposase plasmid using the Neon device. All nucleotide sequences are provided in the Supplement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were transfected with plasmid DNA using the Neon electroporation system (Thermo Fisher Scientific) in 100 \u0026mu;L volumes. Log-phase cells were cultured with daily medium changes for three days prior to electroporation and washed once with PBS. For each electroporation, 1.2 million cells were resuspended in 120 \u0026mu;L of buffer R, mixed with maxiprep-purified plasmid DNA (PureLink HiPure Kit, Thermo Fisher Scientific), and electroporated under cell line\u0026ndash;specific conditions (Supplementary Table 3). Afterward, cells were transferred to 3 mL of pre-warmed, antibiotic-free medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability and growth assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGrowth rates and viability after inhibitor treatment were assessed by bead-based flow cytometry. Cells were stained with SYTOX\u0026reg; Red (Thermo Fisher, 1:1000). Before analysis, 10 \u0026mu;L of 1:10 diluted 6.0\u0026ndash;8.0 \u0026mu;m polystyrene beads (Spherotech,\u0026nbsp;Lake Forest, IL, USA) were added. Beads and cells were distinguished by scatter on a CytoFLEX cytometer (Beckman Coulter,\u0026nbsp;Indianapolis, IN, USA). Absolute cell numbers were determined by comparing bead and cell counts. If the culture was maintained over time through passaging at known dilutions, a growth curve reflecting the exponential increase in absolute cell number (logarithmic) over time (linear) was constructed. IC₅₀ values were calculated using Quest Graph\u0026trade; IC50 Calculator (AAT Bioquest, Pleasanton, CA, USA), and drug synergy was analyzed with SynergyFinder.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProximity Ligation Assay (PLA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were centrifuged, resuspended in PBS, and plated on precision coverslips for 20 min at 37\u0026deg;C. After adhesion, cells were fixed with 4% paraformaldehyde (Sigma Aldrich) for 20 min, washed, and membranes labeled with 5 \u0026mu;g/ml WGA-Alexa Fluor 488 (Thermo Fisher Scientific) for 10 min. Cells were permeabilized with 0.5% Triton X-100 (Sigma) in PBS for 10 min, washed, and blocked in Duolink Blocking Buffer (Sigma Aldrich) for 30 min. Primary antibodies (see Supplementary Table 5) were diluted in Duolink Antibody Diluent and incubated overnight at 4\u0026deg;C. After washing, Duolink Probes (Sigma Aldrich) were added and incubated for 1 h at 37\u0026deg;C, followed by washes. Ligation and amplification were performed with Duolink In Situ Detection Reagents Red kit (Sigma Aldrich) per manufacturer\u0026rsquo;s instructions. Cells were mounted in Prolong Gold with DAPI (Invitrogen,\u0026nbsp;Carlsbad, CA, USA). Images were acquired on a Zeiss Axio Imager.Z2 fluorescence microscope and analyzed in ImageJ/FIJI.\u003c/p\u003e\n\u003cp\u003eThe antibodies we used for PLA analysis were previously validated by Phelan et al. [23] and are listed in the Supplementary Table 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry-based BCR internalization assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDLBCL cell lines (HBL1, U2932, OCI-Ly19) were stimulated with 5 \u0026mu;g/mL anti-human IgM F(ab\u0026apos;)₂ fragments (Jackson ImmunoResearch,\u0026nbsp;West Grove, PA, USA). To induce OVA-specific BCR internalization, cells were treated with either 1 \u0026mu;M full-length ovalbumin (Sigma Aldrich) or a biotinylated OVA 17-mer peptide (FDKLPGFGDSIEAQGGK; GenScript,\u0026nbsp;Nanjing, China) pre-complexed with avidin at a 4:1 molar ratio. For stimulation, 20 \u0026mu;L of a 10 \u0026mu;M peptide-avidin complex (calculated based on avidin concentration) was added to 1 mL of cell suspension. Following stimulation, cells were placed on ice to halt internalization, stained with APC-conjugated anti-human kappa light chain antibody (Invitrogen), and analyzed by flow cytometry (CytoFLEX, Beckman Coulter). Surface BCR levels were quantified using FlowJo software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransferrin Internalization Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were incubated with Alexa Fluor 488\u0026ndash;labeled transferrin (20 \u0026micro;g/mL; Invitrogen) for 1 hour at 37\u0026deg;C to allow internalization. Following incubation, cells were placed on ice to halt further uptake, washed, and incubated with or without anti\u0026ndash;Alexa 488 quenching antibody (Invitrogen) to distinguish internalized from surface-bound transferrin. Samples were analyzed by flow cytometry (CytoFLEX, Beckman Coulter), and data were processed using FlowJo software (FlowJo, LLC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor surface antigen detection (mCD8-OVA and BCR), 0.5 \u0026times; 10⁶ cells were washed, stained with the appropriate antibody in FACS buffer (PBS with 1% FBS) for 30 min on ice, washed, and resuspended in FACS buffer. Data were acquired on a CytoFLEX cytometer and analyzed with FlowJo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern-Blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were washed in PBS and lysed in RIPA buffer with protease and phosphatase inhibitors (Roche)\u0026nbsp;as described\u0026nbsp;[45].\u0026nbsp;Proteins were separated via SDS\u0026ndash;PAGE on 4\u0026ndash;15% gradient gels (Bio-Rad), transferred to PVDF membranes (Millipore,\u0026nbsp;Burlington, MA, USA), and blocked with 5% BSA/TBST. Membranes were incubated with primary antibodies (1:1000; Supplementary Table 1) overnight at 4\u0026deg;C, then with HRP-conjugated secondary antibodies. Signals were visualized using ECL (Perkin Elmer,\u0026nbsp;Waltham, MA, USA) and captured with the G:Box system (Syngene,\u0026nbsp;Bengaluru, India).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReal-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was extracted using the GeneMATRIX Universal RNA Purification Kit (EURx, Gdansk, Poland), and cDNA was synthesized with the Transcriptor Universal cDNA Master (Roche, Basel, Switzerland). qPCR was performed using SYBR Green Master Mix on a CFX96 Real-Time System (Bio-Rad,\u0026nbsp;Hercules, CA, USA). Expression levels were normalized to GAPDH using the \u0026Delta;\u0026Delta;CT method. Primer sequences are listed in Supplementary Table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTyrosine and Serine/threonine kinase profiling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor tyrosine and serine/threonine kinase profiling, the PamStation12 with PTK and STK PamChip peptide arrays (PamGene,\u0026nbsp;Wolvenhoek, Netherland) was utilized. Analysis was performed according to the manufacturer\u0026rsquo;s instructions. In brief, chips were blocked with 2% BSA (Sigma-Aldrich). Proteins were extracted from fresh-frozen cell pellets using T-PER Buffer (Thermo Fisher Scientific), supplemented with 1:100 Phosphatase Inhibitor Cocktail and 1:100 Halt Protease Inhibitor Cocktail (EDTA-free, Thermo Fisher Scientific). 1 \u0026mu;g of protein per sample was applied to the chips with kinase buffer, ATP, and FITC-labeled antibodies.\u003c/p\u003e\n\u003cp\u003eSignal intensities were quantified using BioNavigator 6.1.42 (PamGene), expressed per 100 ms exposure, and log-transformed. A mean value of \u0026lt;20% for peptides with a signal \u0026gt;2,000 was established to ensure quality standards. Normalization was applied, and two replicated quantifications were combined using a false discovery rate (FDR) \u0026lt; 1%. A \u003cem\u003eP\u0026nbsp;\u003c/em\u003evalue \u0026lt; 0.05 and a \u0026gt;10% fold change were considered significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCalcium flux\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor calcium flux analysis, cells were loaded with Calbryte 630AM by resuspending in RPMI with 2% FBS and 25 mM HEPES containing 10 \u0026micro;M Calbryte 630AM (AAT Bioquest) and 0.05% Pluronic\u0026reg; F-127 (Thermo Fisher Scientific). Cells were incubated for 45 min at 37\u0026deg;C in the dark, washed twice with DMEM containing 2% FBS, and resuspended in loading medium. For stimulation, cells were treated with 6 \u0026micro;g/mL F(ab\u0026apos;)₂ anti-human IgM goat antibody (Jackson ImmunoResearch) or 1 \u0026micro;M ovalbumin (Sigma Aldrich). Calcium responses were measured in the red channel using a CytoFLEX Flow Cytometer (Beckman Coulter).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor in vivo assessment of prochlorperazine (PCH) activity, 5 \u0026times; 10⁶ U2932 cells mixed with 30% Matrigel Matrix (Corning, Corning, NY, USA, #354230) were injected subcutaneously into 8\u0026ndash;12 week old female NOD.Cg- \u003cem\u003ePrkdc\u003csup\u003escid\u003c/sup\u003eIl2rg\u003csup\u003etm1Wjl\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e(NSG) mice (Animalab, Poznan, Poland). All procedures were approved by the II Local Ethical Committee for Experiments on Animals in Warsaw, Poland (approval No WAW2/129/2023) and conducted in accordance with Directive 2010/63/EU. Mice were housed in specific pathogen-free conditions in individually ventilated cages under a 12-h light/dark cycle with ad libitum access to food and water. When tumors reached \u0026ge;100 mm\u0026sup3;, 15 mice were randomized into three groups (n=5 per group) with similar mean tumor volumes and treated with 4 mg/kg PCH, 8 mg/kg PCH, or vehicle (H₂O) intraperitoneally for 15 consecutive days. Tumor growth was measured with digital calipers. After the final dose, mice were euthanized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism 9.5.1 (GraphPad, Inc., La Jolla, CA, USA). Statistical tests are described in the figure legends and were nonparametric and two-sided unless otherwise indicated. P values \u0026lt; 0.05 were considered statistically significant, with significance denoted as follows: *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; and ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has been supported by the research grants from the Polish National Science Centre (2019/35/D/NZ5/03354) and the Polish National Agency for Academic Exchange (PPN/BEK/2020/1/00173).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP. G\u0026oacute;rniak:\u0026nbsp;\u003c/strong\u003eConceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, writing\u0026ndash;original draft, writing\u0026ndash;review and editing. \u003cstrong\u003eO. Havranek\u003c/strong\u003e: Resources, investigation and methodology, writing\u0026ndash;review and editing. \u003cstrong\u003eA. Polak\u003c/strong\u003e: Investigation and methodology, writing\u0026ndash;review and editing . \u003cstrong\u003eA. Rams\u003c/strong\u003e: Investigation and methodology, writing\u0026ndash;review and editing\u003cstrong\u003e. K. Kupcova:\u003c/strong\u003e Investigation and methodology, writing\u0026ndash;review and editing\u0026nbsp;\u003cstrong\u003eE. Głodkowska-Mr\u0026oacute;wka\u003c/strong\u003e:\u0026nbsp;Resources and methodology, writing\u0026ndash;review and editing \u003cstrong\u003eZ. Pilch\u003c/strong\u003e: Investigation and methodology, writing\u0026ndash;review and editing. \u003cstrong\u003eM. Miączyńska\u003c/strong\u003e: Resources and methodology, writing\u0026ndash;review and editing. \u003cstrong\u003eD. Nowis\u003c/strong\u003e: Investigation and methodology, writing\u0026ndash;review and editing. \u0026nbsp; \u003cstrong\u003eJ. Gołąb\u003c/strong\u003e: Investigation and methodology, writing\u0026ndash;review and editing \u003cstrong\u003eR. E. Davis\u003c/strong\u003e:Resources, investigation and methodology, writing\u0026ndash;review and editing. \u003cstrong\u003eP. Juszczyński\u003c/strong\u003e: Supervision, writing\u0026ndash;original draft, writing\u0026ndash;review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated in this study are available within the article and its supplementary data files.\u0026nbsp;\u0026nbsp;Other data that support this study and script to reproduce the analyses are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChapuy B, Stewart C, Dunford AJ, Kim J, Kamburov A, Redd RA, et al. Molecular subtypes of diffuse large B cell lymphoma are associated with distinct pathogenic mechanisms and outcomes. Nat Med. 2018;24:679\u0026ndash;90. \u003c/li\u003e\n\u003cli\u003eReddy A, Zhang J, Davis NS, Moffitt AB, Love CL, Waldrop A, et al. Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma. 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FASEB J Off Publ Fed Am Soc Exp Biol. 2008;22:659\u0026ndash;61. \u003c/li\u003e\n\u003cli\u003eFallon R, Fraser C, Moriarty K. Recommended management of nausea and vomiting. Prescriber. 2007;18:50\u0026ndash;61. \u003c/li\u003e\n\u003cli\u003eBusman-Sahay K, Drake L, Sitaram A, Marks M, Drake JR. Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs. PloS One. 2013;8:e54938. \u003c/li\u003e\n\u003cli\u003eCong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 2013;339:819\u0026ndash;23. \u003c/li\u003e\n\u003cli\u003eJuszczynski P, Chen L, O\u0026rsquo;Donnell E, Polo JM, Ranuncolo SM, Dalla-Favera R, et al. BCL6 modulates tonic BCR signaling in diffuse large B-cell lymphomas by repressing the SYK phosphatase, PTPROt. Blood. 2009;114:5315-21.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7260543/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7260543/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"BCR signal dependency is a hallmark of diffuse large B-cell lymphoma (DLBCL) and other B-cell lymphoid malignancies originating from germinal centers. Chronic-active BCR signaling, typical for the more aggressive activated B-cell subtype (ABC) of DLBCLs, is often attributed to activating mutations within the BCR signaling cascade and continuous stimulation of the BCR by autoantigens. In certain ABC-DLBCLs, the BCR forms an intracellular multiprotein supercomplex with TLR9 and MYD88, which generates signals from endolysosomes. However, it is not clear whether the internalization of BCR is required for sustained signaling, nor have the mechanisms responsible for BCR trafficking been defined. A detailed and mechanistic characterization of receptor trafficking and its consequences is crucial for elucidating new therapeutic targets. To address these questions, we developed DLBCL cell models with modified ovalbumin (OVA)-specific hypervariable regions (HVRs) in the BCRs using CRISPR-Cas9 technology. Modified BCRs were incapable of binding self-antigens, while still responding in a controlled fashion to stimulation with ovalbumin. Using these genetic models, we demonstrated that autoantigens drive a complex BCR-dependent signaling program and facilitate the assembly of the intracellular BCR-TLR9-IκB complex, promoting NFκB pathway activation. Furthermore, we showed that the binding of autoantigens to the BCR leads to the internalization of the BCR-autoantigen complex via clathrin-mediated endocytosis (CME). Using genetic models with inducible inhibition of this endocytic pathway, we found that BCR internalization is essential for the oncogenic activation of BCR-dependent signaling pathways and the formation of the BCR-TLR9-IκB complex in autoantigen-dependent ABC-DLBCL cells. Finally, CME inhibition with dynamin-2 antagonists, such as phenothiazine derivatives, reduces BCR signaling, cell viability, and synergizes with SYK and PI3Kδ inhibitors. Since phenothiazines have well-defined safety and pharmacokinetic profiles, our data provide a framework for the rational design of clinical trials employing these drugs in the autoantigen-dependent subset of DLBCL.","manuscriptTitle":"Inhibition of autoantigen-induced B-cell receptor (BCR) internalization as a therapeutic strategy in Diffuse Large B Cell Lymphoma (DLBCL)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 13:51:28","doi":"10.21203/rs.3.rs-7260543/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-09-10T10:33:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-09T14:33:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-07T13:25:40+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-26T10:34:40+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-25T03:25:30+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-08-23T10:07:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-01T09:24:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-31T09:31:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2025-07-31T09:31:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f94de022-956b-47b7-8b06-3d9b8571ac44","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53605212,"name":"Health sciences/Diseases/Haematological diseases/Haematological cancer/Lymphoma/Non-hodgkin lymphoma/B-cell lymphoma"},{"id":53605213,"name":"Biological sciences/Cancer/Cancer therapy/Targeted therapies"},{"id":53605214,"name":"Health sciences/Medical research/Translational research"}],"tags":[],"updatedAt":"2026-02-20T08:11:51+00:00","versionOfRecord":{"articleIdentity":"rs-7260543","link":"https://doi.org/10.1038/s41419-026-08446-1","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2026-02-11 05:00:00","publishedOnDateReadable":"February 11th, 2026"},"versionCreatedAt":"2025-09-01 13:51:28","video":"","vorDoi":"10.1038/s41419-026-08446-1","vorDoiUrl":"https://doi.org/10.1038/s41419-026-08446-1","workflowStages":[]},"version":"v1","identity":"rs-7260543","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7260543","identity":"rs-7260543","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}