SUMMARY PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches.

SummaryCell surface metalloproteases coordinate signaling during development, tissue homeostasis, and disease. TACE (TNF-α-converting enzyme), is responsible for cleavage (“shedding”) of membrane-tethered signaling molecules, including the cytokine TNF, and activating ligands of the EGFR. The trafficking of TACE within the secretory pathway requires its binding to iRhom2, which mediates the exit of TACE from the endoplasmic reticulum. An important, but mechanistically unclear, feature of TACE biology is its ability to be stimulated rapidly on the cell surface by numerous inflammatory and growth-promoting agents. Here, we report a role for iRhom2 in TACE stimulation on the cell surface. TACE shedding stimuli trigger MAP kinase-dependent phosphorylation of iRhom2 N-terminal cytoplasmic tail. This recruits 14-3-3 proteins, enforcing the dissociation of TACE from complexes with iRhom2, promoting the cleavage of TACE substrates. Our data reveal that iRhom2 controls multiple aspects of TACE biology, including stimulated shedding on the cell surface.

The apical inflammatory cytokine TNF regulates numerous important biological processes including inflammation and cell death, and drives inflammatory diseases. TNF secretion requires TACE (also called ADAM17), which cleaves TNF from its transmembrane tether. The trafficking of TACE to the cell surface, and stimulation of its proteolytic activity, depends on membrane proteins, called iRhoms. To delineate how the TNF/TACE/iRhom axis is regulated, we performed an immunoprecipitation/mass spectrometry screen to identify iRhom-binding proteins. This identified a novel protein, that we name iTAP (iRhom Tail-Associated Protein) that binds to iRhoms, enhancing the cell surface stability of iRhoms and TACE, preventing their degradation in lysosomes. Depleting iTAP in primary human macrophages profoundly impaired TNF production and tissues from iTAP KO mice exhibit a pronounced depletion in active TACE levels. Our work identifies iTAP as a physiological regulator of TNF signalling and a novel target for the control of inflammation.

There are two well characterized modes of action of drugs acting against eukaryotic topoisomerase II. Anti-cancer topoisomerase II poisons such as etoposide, amsacrine, and doxorubicin stabilize an intermediate in the topoisomerase II reaction in which the two topoisomerase II subunits are covalently bound to DNA via a phosphotyrosine linkage. This covalent intermediate, termed the covalent complex plays a critical role in cell killing by anti-topoisomerase II agents (reviewed in Refs. 1-3). The second class of agents do not stabilize the covalent intermediate of the topoisomerase II reaction, but inhibit the enzyme at other points of the reaction cycle (1, 4). Since blocking the enzyme at other points of the reaction cycle does not result in DNA damage, this second class of agents is thought to kill cells by depriving them of the essential enzyme activity of topoisomerase II. This second class of inhibitors has been termed catalytic inhibitors to distinguish them from agents that act by stabilizing covalent complexes.A major class of catalytic inhibitors of prokaryotic topoisomerase II inhibits topoisomerase activity by preventing ATP binding (5). These inhibitors include novobiocin and the coumermycins. Most of these inhibitors have relatively low potency against eukaryotic topoisomerases (5). Other, more potent catalytic inhibitors of eukaryotic topoisomerases have been described, these include anthracyclines such as aclarubicin that intercalate in DNA and prevent the binding of the enzyme to DNA (6, 7), and merbarone, which inhibits DNA cleavage by the enzyme (8 -10).Wang and colleagues (11) have demonstrated that during the course of the topoisomerase II reaction, the enzyme forms a closed clamp around DNA. ATP binding is required to generate a closed clamp with wild type topoisomerase II, and ATP hydrolysis generates a conformational change that leads to reopening of the clamp (11,12). Subsequently, Roca et al. (13) showed that bisdioxopiperazines inhibit the re-opening of the closed clamp, and also blocks ATP hydrolysis. Therefore, bisdioxopiperazines would sequester topoisomerase II in the closed clamp conformation, and inhibit enzyme activity inside the cell.Support for this mode of action of bisdioxopiperazines in vivo has been obtained from both yeast and mammalian cells. Overexpression of topoisomerase II in yeast leads to resistance to 1 while reducing the activity of the enzyme leads to increased cell killing, suggesting that cell death arises from a lack of topoisomerase II activity. Studies by Andoh and colleagues have shown that ICRF-187 or ICRF-193 exposure results in a failure to complete a normal mitosis, and can generate polyploid cells (4). In vitro, bisdioxopiperazines prevent decatenation of replicated chromosomes by topoisomerase II (4,14). These results are consistent with the hypothesis that a

We describe a rapid, simple, and efficient method for recovering glutathione S-transferase (GST)- and His6-tagged maltose binding protein (MBP) fusion proteins from inclusion bodies. Incubation of inclusion bodies with 10% sarkosyl effectively solubilized >95% of proteins, while high-yield recovery of sarkosyl-solubilized fusion proteins was obtained with a specific ratio of Triton X-100 and CHAPS. We demonstrate for the first time that this combination of three detergents significantly improves binding efficiency of GST and GST fusion proteins to gluthathione (GSH) Sepharose.

An immature state of cellular differentiation-characterized by stem cell-like tendencies and impaired differentiation-is a hallmark of cancer. Using glioblastoma multiforme (GBM) as a model system, we sought to determine whether molecular determinants that drive cells toward terminal differentiation are also genetically targeted in carcinogenesis and whether neutralizing such genes also plays an active role to reinforce the impaired differentiation state and promote malignancy. To that end, we screened 71 genes with known roles in promoting nervous system development that also sustain copy number loss in GBM through antineoplastic assay and identified A2BP1 (ataxin 2 binding protein 1, Rbfox1), an RNAbinding and splicing regulator that is deleted in 10% of GBM cases. Integrated in silico analysis of GBM profiles to elucidate the A2BP1 pathway and its role in glioma identified myelin transcription factor 1-like (Myt1L) as a direct transcriptional regulator of A2BP1. Reintroduction of A2BP1 or Myt1L in GBM cell lines and glioma stem cells profoundly inhibited tumorigenesis in multiple assays, and conversely, shRNA-mediated knockdown of A2BP1 or Myt1L in premalignant neural stem cells compromised neuronal lineage differentiation and promoted orthotopic tumor formation. On the mechanistic level, with the top-represented downstream target TPM1 as an illustrative example, we demonstrated that, among its multiple functions, A2BP1 serves to regulate TPM1's alternative splicing to promote cytoskeletal organization and terminal differentiation and suppress malignancy. Thus, in addition to the activation of self-renewal pathways, the neutralization of genetic programs that drive cells toward terminal differentiation may also promote immature and highly plastic developmental states that contribute to the aggressive malignant properties of GBM.oncogenomics | cancer stem cells

Expression of the Gria1-targeting miRNA miR-501-3p is increased locally in dendrites after NMDAR activation and is required for NMDAR-dependent inhibition of GluA1 expression and long-lasting spine shrinkage and elimination.

DNA topoisomerases are enzymes that catalyze changes in the topology of DNA via a mechanism involving the transient breakage and rejoining of phosphodiester bonds in the DNA backbone (for reviews, see Refs. 1-7). Their biological functions include the removal of DNA supercoils generated during various cellular processes, such as DNA replication and transcription, decatenation of the intertwined DNA duplexes for the segregation of chromosomes, and regulating the cellular level of DNA supercoiling.DNA topoisomerases can be divided into three subfamilies, types IA, IB, and II, on the basis of sequence homology and mechanism of action. Type II enzymes (topo II) 1 catalyze the ATP-dependent passage of a DNA segment through a transient double-strand break in another segment. In such a reaction, the enzyme-mediated reversible cleavage is generated by the transesterification reactions between a pair of active site tyrosines and two DNA phosphodiester bonds staggered 4 base pairs apart. Eukaryotic topo II has a dimer structure, whereas bacterial topo IIs, including gyrase and topo IV, are A 2 B 2 heterotetramers. However, the N-terminal and the central part of eukaryotic topo II are homologous to the bacterial gyrB and gyrA subunits, respectively.Recent biochemical and structural studies have provided further understandings on the mechanism of topo II-catalyzed reaction (Refs. 8 -11; also reviewed in Ref. 12). The reaction starts with the enzyme binding to a segment (G-segment) of DNA and generating a double strand break to serve as a protein-mediated DNA gate. The ATPase domain of the enzyme, which is located at the N-terminal part of each subunit, can dimerize and close the N-terminal protein clamp (N-gate) upon the binding of ATP. This closure can capture another DNA segment (T-segment) and trigger a cascade of conformational changes in the enzyme-DNA complex, which results in the passage of T-segment through the double-strand DNA break in the G-segment, the resealing of the DNA gate, and the exit of transported T-segment through the C-terminal protein clamp (C-gate).Results from the mapping of proteolytic cleavage sites revealed at least three domains within each subunit of eukaryotic topo II (Refs. 13-15; outlined in Fig. 1). The N-terminal domain is homologous to the N-terminal half of gyrB subunit, which possesses the ATPase activity (16,17). The core domain includes the regions homologous to the C-terminal one-third of the gyrB subunit (BЈ) and N-terminal two-thirds of the gyrA subunit (AЈ). This part of the gyrA subunit contains the active site tyrosine and is involved in breaking and rejoining the DNA backbone (reviewed in Ref. 5). Recently, the crystal structure of the core domain of yeast topo II has been solved, showing a heart-shaped profile and two dimer contacts at the BЈ-BЈ and AЈ-AЈ interfaces (10). The hydrophilic C-terminal domain of eukaryotic topo II is less conserved between different species and has been shown to be dispensable for the biochemical activities of topo .Some of the antibiotics and an...