Gabriel J. Sandoval

The Scaffolding Protein Synapse-Associated Protein 97 Is Required for Enhanced Signaling Through Isotype-Switched IgG Memory B Cell Receptors

A scaffolding protein clusters B cell receptors to enable the rapid, high-titer antibody responses of memory B cells. Boosting Antibody Production The initial exposure of naïve B cells that have IgM B cell receptors (BCRs) on their surface to a foreign antigen produces a primary antibody response and generates memory B cells that have IgG BCRs, which respond to subsequent encounters with the same antigen by rapidly producing large amounts of antibodies. Liu et al. investigated differences in the signaling capacities of IgG and IgM BCRs and found that the scaffold protein SAP97 bound to IgG, but not IgM, BCRs at the immunological synapse, enabling BCR clustering and enhanced signaling. These findings may provide therapeutic targets to block enhanced BCR activation in autoimmune disease and in some B cell tumors. After their first encounter with a foreign antigen, naïve B cells that have immunoglobulin M (IgM) B cell receptors (BCRs) trigger the primary antibody response and the generation of memory B cells with IgG BCRs. When these memory B cells reencounter the same antigen, the cell surface IgG BCRs stimulate their rapid differentiation into plasma cells that release large amounts of IgG antibodies. We showed that the conserved cytoplasmic tail of the IgG BCR, which contains a putative PDZ (postsynaptic density 95/disc large/zona occludens 1)–binding motif, associated with synapse-associated protein 97 (SAP97), a PDZ domain–containing scaffolding molecule that is involved in controlling receptor density and signal strength at neuronal synapses. SAP97 accumulated and bound to IgG BCRs in the immunological synapses that formed in response to B cell engagement with antigen. Knocking down SAP97 in IgG+ B cells or mutating the putative PDZ-binding motif in the BCR tail impaired formation of the immunological synapse, initiation of IgG BCR signaling, and downstream activation of the mitogen-activated protein kinase p38. Thus, heightened B cell memory responses are encoded, in part, by a mechanism that involves SAP97 serving as a scaffolding protein in the IgG BCR immunological synapse.

Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

RAG-mediated DNA double-strand breaks activate a cell type–specific checkpoint to inhibit pre–B cell receptor signals

B-lineage cells reconcile the competing needs of proliferation and genome stability.

Polarity gene discs large homolog 1 regulates the generation of memory T cells

Mammalian ortholog of Drosophila cell polarity protein, Dlg1, plays a critical role in neural synapse formation, epithelial cell homeostasis, and urogenital development. More recently, it has been proposed that Dlg1 may also be involved in the regulation of T‐cell proliferation, migration, and Ag‐receptor signaling. However, a requirement for Dlg1 in development and function of T lineage cells remains to be established. In this study, we investigated a role for Dlg1 during T‐cell development and function using a combination of conditional Dlg1 KO and two different Cre expression systems where Dlg1 deficiency is restricted to the T‐cell lineage only, or all hematopoietic cells. Here, using three different TCR models, we show that Dlg1 is not required during development and selection of thymocytes bearing functionally rearranged TCR transgenes. Moreover, Dlg1 is dispensable in the activation and proliferative expansion of Ag‐specific TCR‐transgenic CD4+ and CD8+ T cells in vitro and in vivo. Surprisingly, however, we show that Dlg1 is required for normal generation of memory T cells during endogenous response to cognate Ag. Thus, Dlg1 is not required for the thymocyte selection or the activation of primary T cells, however it is involved in the generation of memory T cells.

Novel Mechanism of Tumor Suppression by Polarity Gene Discs Large 1 (DLG1) Revealed in a Murine Model of Pediatric B-ALL

Using two different murine models of pre–B-cell leukemia, Sandoval and colleagues delineate the mechanism of tumor suppression by the PDZ domain polarity gene DLG1, which interacts with and stabilizes the PTEN protein in early-stage B cells. Drosophila melanogaster discs large (dlg) is an essential tumor suppressor gene (TSG) controlling epithelial cell growth and polarity of the fly imaginal discs in pupal development. A mammalian ortholog, Dlg1, is involved in embryonic urogenital morphogenesis, postsynaptic densities in neurons, and immune synapses in lymphocytes. However, a potential role for Dlg1 as a mammalian TSG is unknown. Here, we present evidence that loss of Dlg1 confers strong predisposition to the development of malignancies in a murine model of pediatric B-cell acute lymphoblastic leukemia (B-ALL). Using mice with conditionally deleted Dlg1 alleles, we identify a novel “pre-leukemic” stage of developmentally arrested early B-lineage cells marked by preeminent c-Myc expression. Mechanistically, we show that in B-lineage progenitors Dlg1 interacts with and stabilizes the PTEN protein, regulating its half-life and steady-state abundance. The loss of Dlg1 does not affect the level of PTEN mRNAs but results in a dramatic decrease in PTEN protein, leading to excessive phosphoinositide 3-kinase signaling and proliferation. Our data suggest a novel model of tumor suppression by a PDZ domain-containing polarity gene in hematopoietic cancers. Cancer Immunol Res; 1(6); 426–37. ©2013 AACR.

ITAM signaling in dendritic cells controls T helper cell priming by regulating MHC class II recycling

Immature dendritic cells (DCs) specialize in antigen capture and maintain a highly dynamic pool of intracellular major histocompatibility complex class II (MHCII) that continuously recycles from peptide loading compartments to the plasma membrane and back again. This process facilitates sampling of environmental antigens for presentation to T helper cells. Here, we show that a signaling pathway mediated by the DC immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptors (DAP12 and FcRγ) and Vav family guanine nucleotide exchange factors controls the half-life of surface peptide-MHCII (pMHCII) complexes and is critical for CD4 T-cell triggering in vitro. Strikingly, mice with disrupted DC ITAMs show defective T helper cell priming in vivo and are protected from experimental autoimmune encephalitis. Mechanistically, we show that deficiency in ITAM signaling results in increased pMHCII internalization, impaired recycling, and an accumulation of ubiquitinated MHCII species that are prematurely degraded in lysosomes. We propose a novel mechanism for control of T helper cell priming.

Cutting Edge: Cell-Autonomous Control of IL-7 Response Revealed in a Novel Stage of Precursor B Cells

During early stages of B-lineage differentiation in bone marrow, signals emanating from IL-7R and pre-BCR are thought to synergistically induce proliferative expansion of progenitor cells. Paradoxically, loss of pre-BCR–signaling components is associated with leukemia in both mice and humans. Exactly how progenitor B cells perform the task of balancing proliferative burst dependent on IL-7 with the termination of IL-7 signals and the initiation of L chain gene rearrangement remains to be elucidated. In this article, we provide genetic and functional evidence that the cessation of the IL-7 response of pre-B cells is controlled via a cell-autonomous mechanism that operates at a discrete developmental transition inside Fraction C′ (large pre-BII) marked by transient expression of c-Myc. Our data indicate that pre-BCR cooperates with IL-7R in expanding the pre-B cell pool, but it is also critical to control the differentiation program shutting off the c-Myc gene in large pre-B cells.

Loss of DAP12 and FcRγ drives exaggerated IL-12 production and CD8(+) T cell response by CCR2(+) Mo-DCs.

Dap12 and FcRγ, the two transmembrane ITAM-containing signaling adaptors expressed in dendritic cells (DC), are implicated in the regulation of DC function. Several activating and adhesion receptors including integrins require these chains for their function in triggering downstream signaling and effector pathways, however the exact role(s) for Dap12 and FcRγ remains elusive as their loss can lead to both attenuating and enhancing effects. Here, we report that mice congenitally lacking both Dap12 and FcRγ chains (DF) show a massively enhanced effector CD8+ T cell response to protein antigen immunization or West Nile Virus (WNV) infection. Thus, immunization of DF mice with MHCI-restricted OVA peptide leads to accumulation of IL-12-producing monocyte-derived dendritic cells (Mo-DC) in draining lymph nodes, followed by vastly enhanced generation of antigen-specific IFNγ-producing CD8+ T cells. Moreover, DF mice show increased viral clearance in the WNV infection model. Depletion of CCR2+ monocytes/macrophages in vivo by administration anti-CCR2 antibodies or clodronate liposomes completely prevents the exaggerated CD8+ T cell response in DF mice. Mechanistically, we show that the loss of Dap12 and FcRγ-mediated signals in Mo-DC leads to a disruption of GM-CSF receptor-induced STAT5 activation resulting in upregulation of expression of IRF8, a transcription factor. Consequently, Dap12- and FcRγ-deficiency exacerbates GM-CSF-driven monocyte differentiation and production of inflammatory Mo-DC. Our data suggest a novel cross-talk between DC-ITAM and GM-CSF signaling pathways, which controls Mo-DC differentiation, IL-12 production, and CD8+ T cell responses.

Going beyond genetics to discover cancer targets

Two recent studies demonstrate the power of integrating tumor genotype information with epigenetic and proteomic studies to discover potential therapeutic targets in breast cancer.

Binding of TMPRSS2-ERG to BAF Chromatin Remodeling Complexes Mediates Prostate Oncogenesis

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.