RESUMEN
Germinal centers (GCs) that form in mucosal sites are exposed to gut-derived factors that have the potential to influence homeostasis independent of antigen receptor-driven selective processes. The G-protein Gα13 confines B cells to the GC and limits the development of GC-derived lymphoma. We discovered that Gα13-deficiency fuels the GC reaction via increased mTORC1 signaling and Myc protein expression specifically in the mesenteric lymph node (mLN). The competitive advantage of Gα13-deficient GC B cells (GCBs) in mLN was not dependent on T cell help or gut microbiota. Instead, Gα13-deficient GCBs were selectively dependent on dietary nutrients likely due to greater access to gut lymphatics. Specifically, we found that diet-derived glutamine supported proliferation and Myc expression in Gα13-deficient GCBs in the mLN. Thus, GC confinement limits the effects of dietary glutamine on GC dynamics in mucosal tissues. Gα13 pathway mutations coopt these processes to promote the gut tropism of aggressive lymphoma.
Asunto(s)
Linfocitos B , Proliferación Celular , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Centro Germinal , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Noqueados , Centro Germinal/inmunología , Centro Germinal/metabolismo , Animales , Ratones , Linfocitos B/inmunología , Linfocitos B/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/inmunología , Nutrientes/metabolismo , Transducción de Señal , Glutamina/metabolismo , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/inmunología , Membrana Mucosa/metabolismo , Membrana Mucosa/inmunologíaRESUMEN
Immunoglobulin A (IgA) maintains commensal communities in the intestine while preventing dysbiosis. IgA generated against intestinal microbes assures the simultaneous binding to multiple, diverse commensal-derived antigens. However, the exact mechanisms by which B cells mount broadly reactive IgA to the gut microbiome remains elusive. Here, we have shown that IgA B cell receptor (BCR) is required for B cell fitness during the germinal center (GC) reaction in Peyer's patches (PPs) and for generation of gut-homing plasma cells (PCs). We demonstrate that IgA BCR drove heightened intracellular signaling in mouse and human B cells, and as a consequence, IgA+ B cells received stronger positive selection cues. Mechanistically, IgA BCR signaling offset Fas-mediated death, possibly rescuing low-affinity B cells to promote a broad humoral response to commensals. Our findings reveal an additional mechanism linking BCR signaling, B cell fate, and antibody production location, which have implications for how intestinal antigen recognition shapes humoral immunity.
Asunto(s)
Linfocitos B , Ganglios Linfáticos Agregados , Ratones , Humanos , Animales , Antígenos/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Inmunoglobulina A , Mucosa IntestinalRESUMEN
Diets high in cholesterol alter intestinal immunity. Here, we examined how the cholesterol metabolite 25-hydroxycholesterol (25-HC) impacts the intestinal B cell response. Mice lacking cholesterol 25-hydroxylase (CH25H), the enzyme generating 25-HC, had higher frequencies of immunoglobulin A (IgA)-secreting antigen-specific B cells upon immunization or infection. 25-HC did not affect class-switch recombination but rather restrained plasma cell (PC) differentiation. 25-HC was produced by follicular dendritic cells and increased in response to dietary cholesterol. Mechanistically, 25-HC restricted activation of the sterol-sensing transcription factor SREBP2, thereby regulating B cell cholesterol biosynthesis. Ectopic expression of SREBP2 in germinal center B cells induced rapid PC differentiation, whereas SREBP2 deficiency reduced PC output in vitro and in vivo. High-cholesterol diet impaired, whereas Ch25h deficiency enhanced, the IgA response against Salmonella and the resulting protection from systemic bacterial dissemination. Thus, a 25-HC-SREBP2 axis shapes the humoral response at the intestinal barrier, providing insight into the effect of high dietary cholesterol in intestinal immunity.
Asunto(s)
Diferenciación Celular/inmunología , Hidroxicolesteroles/metabolismo , Inmunoglobulina A/inmunología , Células Plasmáticas/inmunología , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Animales , Colesterol en la Dieta/inmunología , Colesterol en la Dieta/metabolismo , Hidroxicolesteroles/inmunología , Inmunoglobulina A/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Ratones , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/metabolismo , Células Plasmáticas/metabolismoRESUMEN
BACKGROUND: The identification of oncogenic mutations in diffuse large B-cell lymphoma (DLBCL) has led to the development of drugs that target essential survival pathways, but whether targeting multiple survival pathways may be curative in DLBCL is unknown. METHODS: We performed a single-center, phase 1b-2 study of a regimen of venetoclax, ibrutinib, prednisone, obinutuzumab, and lenalidomide (ViPOR) in relapsed or refractory DLBCL. In phase 1b, which included patients with DLBCL and indolent lymphomas, four dose levels of venetoclax were evaluated to identify the recommended phase 2 dose, with fixed doses of the other four drugs. A phase 2 expansion in patients with germinal-center B-cell (GCB) and non-GCB DLBCL was performed. ViPOR was administered every 21 days for six cycles. RESULTS: In phase 1b of the study, involving 20 patients (10 with DLBCL), a single dose-limiting toxic effect of grade 3 intracranial hemorrhage occurred, a result that established venetoclax at a dose of 800 mg as the recommended phase 2 dose. Phase 2 included 40 patients with DLBCL. Toxic effects that were observed among all the patients included grade 3 or 4 neutropenia (in 24% of the cycles), thrombocytopenia (in 23%), anemia (in 7%), and febrile neutropenia (in 1%). Objective responses occurred in 54% of 48 evaluable patients with DLBCL, and complete responses occurred in 38%; complete responses were exclusively in patients with non-GCB DLBCL and high-grade B-cell lymphoma with rearrangements of MYC and BCL2 or BCL6 (or both). Circulating tumor DNA was undetectable in 33% of the patients at the end of ViPOR therapy. With a median follow-up of 40 months, 2-year progression-free survival and overall survival were 34% (95% confidence interval [CI], 21 to 47) and 36% (95% CI, 23 to 49), respectively. CONCLUSIONS: Treatment with ViPOR was associated with durable remissions in patients with specific molecular DLBCL subtypes and was associated with mainly reversible adverse events. (Funded by the Intramural Research Program of the National Cancer Institute and the National Center for Advancing Translational Sciences of the National Institutes of Health and others; ClinicalTrials.gov number, NCT03223610.).
Asunto(s)
Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica , Lenalidomida , Linfoma de Células B Grandes Difuso , Piperidinas , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adenina/análogos & derivados , Adenina/efectos adversos , Adenina/uso terapéutico , Adenina/administración & dosificación , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Compuestos Bicíclicos Heterocíclicos con Puentes/efectos adversos , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Lenalidomida/efectos adversos , Lenalidomida/administración & dosificación , Lenalidomida/uso terapéutico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/mortalidad , Terapia Molecular Dirigida , Piperidinas/efectos adversos , Piperidinas/uso terapéutico , Piperidinas/administración & dosificación , Prednisona/efectos adversos , Prednisona/administración & dosificación , Prednisona/uso terapéutico , Supervivencia sin Progresión , Pirazoles/efectos adversos , Pirazoles/uso terapéutico , Pirazoles/administración & dosificación , Pirimidinas/efectos adversos , Pirimidinas/uso terapéutico , Pirimidinas/administración & dosificación , Recurrencia , Sulfonamidas/efectos adversos , Sulfonamidas/administración & dosificación , Sulfonamidas/uso terapéuticoRESUMEN
Germinal centres are important sites for antibody diversification and affinity maturation, and are also a common origin of B cell malignancies. Despite being made up of motile cells, germinal centres are tightly confined within B cell follicles. The cues that promote this confinement are incompletely understood. P2RY8 is a Gα13-coupled receptor that mediates the inhibition of migration and regulates the growth of B cells in lymphoid tissues1,2. P2RY8 is frequently mutated in germinal-centre B cell-like diffuse large B cell lymphoma (GCB-DLBCL) and Burkitt lymphoma1,3-6, and the ligand for this receptor has not yet been identified. Here we perform a search for P2RY8 ligands and find P2RY8 bioactivity in bile and in culture supernatants of several mouse and human cell lines. Using a seven-step biochemical fractionation procedure and a drop-out mass spectrometry approach, we show that a previously undescribed biomolecule, S-geranylgeranyl-L-glutathione (GGG), is a potent P2RY8 ligand that is detectable in lymphoid tissues at the nanomolar level. GGG inhibited the chemokine-mediated migration of human germinal-centre B cells and T follicular helper cells, and antagonized the induction of phosphorylated AKT in germinal-centre B cells. We also found that the enzyme gamma-glutamyltransferase-5 (GGT5), which was highly expressed by follicular dendritic cells, metabolized GGG to a form that did not activate the receptor. Overexpression of GGT5 disrupted the ability of P2RY8 to promote B cell confinement to germinal centres, which indicates that GGT5 establishes a GGG gradient in lymphoid tissues. This work defines GGG as an intercellular signalling molecule that is involved in organizing and controlling germinal-centre responses. As the P2RY8 locus is modified in several other types of cancer in addition to GCB-DLBCL and Burkitt lymphoma, we speculate that GGG might have organizing and growth-regulatory roles in multiple human tissues.
Asunto(s)
Linfocitos B/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Línea Celular , Movimiento Celular/efectos de los fármacos , Quimiocinas/inmunología , Femenino , Centro Germinal/citología , Centro Germinal/inmunología , Humanos , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Tonsila Palatina/citología , Tonsila Palatina/inmunología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Purinérgicos P2Y/genética , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , gamma-Glutamiltransferasa/metabolismoRESUMEN
Unique molecular vulnerabilities have been identified in the aggressive MCD/C5 genetic subclass of diffuse large B-cell lymphoma (DLBCL). However, the premalignant cell-of-origin exhibiting MCD-like dependencies remains elusive. In this study, we examined animals carrying up to 4 hallmark genetic lesions found in MCD consisting of gain-of-function mutations in Myd88 and Cd79b, loss of Prdm1, and overexpression of BCL2. We discovered that expression of combinations of these alleles in vivo promoted a cell-intrinsic accumulation of B cells in spontaneous splenic germinal centers (GCs). As with MCD, these premalignant B cells were enriched for B-cell receptors (BCRs) with evidence of self-reactivity, displayed a de novo dependence on Tlr9, and were more sensitive to inhibition of Bruton's tyrosine kinase. Mutant spontaneous splenic GC B cells (GCB) showed increased proliferation and IRF4 expression. Mice carrying all 4 genetic lesions showed a >50-fold expansion of spontaneous splenic GCs exhibiting aberrant histologic features with a dark zone immunophenotype and went on to develop DLBCL in the spleen with age. Thus, by combining multiple hallmark genetic alterations associated with MCD, our study identifies aberrant spontaneous splenic GCBs as a likely cell-of-origin for this aggressive genetic subtype of lymphoma.
Asunto(s)
Linfoma de Células B Grandes Difuso , Bazo , Animales , Linfocitos B/patología , Centro Germinal/patología , Linfoma de Células B Grandes Difuso/patología , Ratones , Mutación , Bazo/patologíaRESUMEN
Epstein-Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.
Asunto(s)
Herpesvirus Humano 4/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/fisiología , Linfocitos B/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , Fosforilación , Transducción de Señal , Quinasa Syk/metabolismoRESUMEN
Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.
Asunto(s)
Linfocitos B/metabolismo , Linfocitos B/patología , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Centro Germinal/patología , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Transducción de Señal , Animales , Sangre/inmunología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Línea Celular Tumoral , Movimiento Celular/genética , Humanos , Linfa/citología , Linfoma de Células B Grandes Difuso/genética , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Proteína Oncogénica v-akt/genética , Proteína Oncogénica v-akt/metabolismo , Receptores de Lisoesfingolípidos/deficiencia , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Receptores Purinérgicos P2Y/genética , Receptores Purinérgicos P2Y/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/deficiencia , Factores de Intercambio de Guanina Nucleótido Rho/genética , Receptores de Esfingosina-1-FosfatoRESUMEN
Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor ß receptor 2 (TGFßR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the ß2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules.
Asunto(s)
Linfocitos B/inmunología , Cadenas Ligeras de Clatrina/genética , Endocitosis/inmunología , Eliminación de Gen , Cambio de Clase de Inmunoglobulina , Animales , Linfocitos B/patología , Corteza Cerebral/citología , Corteza Cerebral/inmunología , Cadenas Ligeras de Clatrina/inmunología , Regulación de la Expresión Génica , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina A/genética , Hígado/citología , Hígado/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/citología , Miocardio/inmunología , Especificidad de Órganos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/inmunología , Receptores CXCR4/genética , Receptores CXCR4/inmunología , Receptores Opioides delta/genética , Receptores Opioides delta/inmunología , Receptores de Factores de Crecimiento Transformadores beta/agonistas , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Multiple myeloma (MM) is an incurable plasma cell malignancy that exploits transcriptional networks driven by IRF4. We employ a multi-omics approach to discover IRF4 vulnerabilities, integrating functional genomics screening, spatial proteomics, and global chromatin mapping. ARID1A, a member of the SWI/SNF chromatin remodeling complex, is required for IRF4 expression and functionally associates with IRF4 protein on chromatin. Deleting Arid1a in activated murine B cells disrupts IRF4-dependent transcriptional networks and blocks plasma cell differentiation. Targeting SWI/SNF activity leads to rapid loss of IRF4-target gene expression and quenches global amplification of oncogenic gene expression by MYC, resulting in profound toxicity to MM cells. Notably, MM patients with aggressive disease bear the signature of SWI/SNF activity, and SMARCA2/4 inhibitors remain effective in immunomodulatory drug (IMiD)-resistant MM cells. Moreover, combinations of SWI/SNF and MEK inhibitors demonstrate synergistic toxicity to MM cells, providing a promising strategy for relapsed/refractory disease.
Asunto(s)
Proteínas de Unión al ADN , Factores Reguladores del Interferón , Mieloma Múltiple , Células Plasmáticas , Factores de Transcripción , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Factores Reguladores del Interferón/metabolismo , Factores Reguladores del Interferón/genética , Animales , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Humanos , Ratones , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Línea Celular Tumoral , Diferenciación Celular/efectos de los fármacosRESUMEN
Glucocorticoids have been used for decades to treat lymphomas without an established mechanism of action. Using functional genomic, proteomic, and chemical screens, we discover that glucocorticoids inhibit oncogenic signaling by the B cell receptor (BCR), a recurrent feature of aggressive B cell malignancies, including diffuse large B cell lymphoma and Burkitt lymphoma. Glucocorticoids induce the glucocorticoid receptor (GR) to directly transactivate genes encoding negative regulators of BCR stability (LAPTM5; KLHL14) and the PI3 kinase pathway (INPP5D; DDIT4). GR directly represses transcription of CSK, a kinase that limits the activity of BCR-proximal Src-family kinases. CSK inhibition attenuates the constitutive BCR signaling of lymphomas by hyperactivating Src-family kinases, triggering their ubiquitination and degradation. With the knowledge that glucocorticoids disable oncogenic BCR signaling, they can now be deployed rationally to treat BCR-dependent aggressive lymphomas and used to construct mechanistically sound combination regimens with inhibitors of BTK, PI3 kinase, BCL2, and CSK.
Asunto(s)
Glucocorticoides , Receptores de Antígenos de Linfocitos B , Humanos , Glucocorticoides/farmacología , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Transducción de Señal/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Ratones , Línea Celular Tumoral , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , Terapia Molecular Dirigida/métodos , Fosfatidilinositol 3-Quinasas/metabolismo , Familia-src Quinasas/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the CD79B subunit of the B cell receptor (BCR). When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL. To identify determinants of Pola-V sensitivity, we used CRISPR-Cas9 screening for genes that modulated Pola-V toxicity for lymphomas or the surface expression of its target, CD79B. Our results reveal the striking impact of CD79B glycosylation on Pola-V epitope availability on the lymphoma cell surface and on Pola-V toxicity. Genetic, pharmacological, and enzymatic approaches that remove sialic acid from N-linked glycans enhanced lymphoma killing by Pola-V. Pola-V toxicity was also modulated by KLHL6, an E3 ubiquitin ligase that is recurrently inactivated in germinal center derived lymphomas. We reveal how KLHL6 targets CD79B for degradation in normal and malignant germinal center B cells, thereby determining expression of the surface BCR complex. Our findings suggest precision medicine strategies to optimize Pola-V as a lymphoma therapeutic.
RESUMEN
Follicular lymphoma (FL) is a generally incurable malignancy that evolves from developmentally blocked germinal center (GC) B cells. To promote survival and immune escape, tumor B cells undergo significant genetic changes and extensively remodel the lymphoid microenvironment. Dynamic interactions between tumor B cells and the tumor microenvironment (TME) are hypothesized to contribute to the broad spectrum of clinical behaviors observed among FL patients. Despite the urgent need, existing clinical tools do not reliably predict disease behavior. Using a multi-modal strategy, we examined cell-intrinsic and -extrinsic factors governing progression and therapeutic outcomes in FL patients enrolled onto a prospective clinical trial. By leveraging the strengths of each platform, we identify several tumor-specific features and microenvironmental patterns enriched in individuals who experience early relapse, the most high-risk FL patients. These features include stromal desmoplasia and changes to the follicular growth pattern present 20 months before first progression and first relapse.
Asunto(s)
Linfoma Folicular , Humanos , Linfocitos B , Linfoma Folicular/genética , Multiómica , Estudios Prospectivos , Recurrencia , Microambiente Tumoral , Ensayos Clínicos como AsuntoRESUMEN
Diffuse large B-cell lymphoma (DLBCL) can be subdivided into the activated B-cell (ABC) and germinal center B cell-like (GCB) subtypes. Self-antigen engagement of B-cell receptors (BCR) in ABC tumors induces their clustering, thereby initiating chronic active signaling and activation of NF-κB and PI3 kinase. Constitutive BCR signaling is essential in some GCB tumors but primarily activates PI3 kinase. We devised genome-wide CRISPR-Cas9 screens to identify regulators of IRF4, a direct transcriptional target of NF-κB and an indicator of proximal BCR signaling in ABC DLBCL. Unexpectedly, inactivation of N-linked protein glycosylation by the oligosaccharyltransferase-B (OST-B) complex reduced IRF4 expression. OST-B inhibition of BCR glycosylation reduced BCR clustering and internalization while promoting its association with CD22, which attenuated PI3 kinase and NF-κB activation. By directly interfering with proximal BCR signaling, OST-B inactivation killed models of ABC and GCB DLBCL, supporting the development of selective OST-B inhibitors for the treatment of these aggressive cancers. SIGNIFICANCE: DLBCL depends on constitutive BCR activation and signaling. There are currently no therapeutics that target the BCR directly and attenuate its pathologic signaling. Here, we unraveled a therapeutically exploitable, OST-B-dependent glycosylation pathway that drives BCR organization and proximal BCR signaling. This article is highlighted in the In This Issue feature, p. 1749.
Asunto(s)
Linfoma de Células B Grandes Difuso , FN-kappa B , Humanos , FN-kappa B/metabolismo , Glicosilación , Transducción de Señal , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Línea Celular TumoralRESUMEN
Fas is highly expressed on germinal center (GC) B cells, and mutations of FAS have been reported in diffuse large B cell lymphoma (DLBCL). Although GC-derived DLBCL has better overall outcomes than other DLBCL types, some cases are refractory, and the molecular basis for this is often unknown. We show that Fas is a strong cell-intrinsic regulator of GC B cells that promotes cell death in the light zone, likely via T follicular helper (Tfh) cell-derived Fas ligand. In the absence of Fas, GCs were more clonally diverse due to an accumulation of cells that did not demonstrably bind antigen. FAS alterations occurred most commonly in GC-derived DLBCL, were associated with inferior outcomes and an enrichment of Tfh cells, and co-occurred with deficiency in HVEM and PD-L1 that regulate the Tfh-B cell interaction. This work shows that Fas is critically required for GC homeostasis and suggests that loss of Tfh-mediated counterselection in the GC contributes to lethality in GC-derived lymphoma.
Asunto(s)
Centro Germinal/patología , Linfoma/metabolismo , Linfoma/patología , Receptor fas/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Linfocitos B/inmunología , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular , Proteína Ligando Fas/metabolismo , Eliminación de Gen , Centro Germinal/metabolismo , Humanos , Inmunización , Ganglios Linfáticos/metabolismo , Linfoma/inmunología , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/patología , Ratones Endogámicos C57BL , Modelos Biológicos , Invasividad Neoplásica , Especificidad de Órganos , Unión Proteica , Linfocitos T Colaboradores-Inductores/inmunología , Regulación hacia Arriba , Receptor fas/deficienciaRESUMEN
Fas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.
Asunto(s)
Apoptosis/fisiología , Estructuras de la Membrana Celular/metabolismo , Membrana Celular/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Receptor fas/metabolismo , Animales , Proteínas de Arabidopsis/metabolismo , Células COS , Caspasas/metabolismo , Línea Celular , Ácido Graso Desaturasas/metabolismo , Humanos , Células Jurkat , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/metabolismo , Mutación/genética , Estructura Terciaria de Proteína/genética , Transducción de Señal/genética , Receptor fas/genéticaRESUMEN
B cells in germinal centers (GCs) cycle between light zone (LZ) and dark zone (DZ). The cues in the GC microenvironment that regulate the transition from LZ to DZ have not been well characterized. In Peyer's patches (PPs), transforming growth factor-ß (TGFß) promotes IgA induction in activated B cells that can then differentiate into GC B cells. We show here that TGFß signaling occurs in B cells in GCs and is distinct from signaling that occurs in activated B cells in PPs. Whereas in activated B cells TGFß signaling is required for IgA induction, in the GC it was instead required for the transition from LZ to DZ. In the absence of TGFß signaling, there was an accumulation of LZ GC B cells and reduced antibody affinity maturation likely due to reduced activation of Foxo1. This work identifies TGFß as a microenvironmental cue that is critical for GC homeostasis and function.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Ganglios Linfáticos Agregados/inmunología , Transducción de Señal/inmunología , Factor de Crecimiento Transformador beta/inmunología , Animales , Linfocitos B/metabolismo , Proteína Forkhead Box O1/inmunología , Proteína Forkhead Box O1/metabolismo , Centro Germinal/citología , Centro Germinal/metabolismo , Inmunoglobulina A/inmunología , Inmunoglobulina A/metabolismo , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ganglios Linfáticos Agregados/citología , Ganglios Linfáticos Agregados/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/inmunología , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Receptores CCR6/genética , Receptores CCR6/inmunología , Receptores CCR6/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The orphan Gα13-coupled receptor P2RY8 is mutated in human germinal center (GC)-derived lymphomas and was recently found to promote B cell association with GCs in a mouse model. Here we establish that P2RY8 promotes clustering of activated B cells within follicles in a follicular dendritic cell (FDC)-dependent manner. Although mice lack a P2RY8 orthologue, we show that mouse GC B cell clustering is also dependent on FDCs acting to support the function of a Gα13-coupled receptor. Mutations in GNA13 and its downstream effector ARHGEF1 are associated with the development of disseminated GC-derived lymphomas. We find that egress of Gna13 mutant GC B cells from lymph nodes in the mouse depends on sphingosine-1-phosphate receptor-3. These findings provide evidence that FDCs promote GC confinement of both human and mouse GC B cells via Gα13-dependent pathways, and they show that dissemination of Gα13-deficient GC B cells additionally requires an egress-promoting receptor.