Recombinant Newcastle Disease Virus Immunotherapy Drives Oncolytic Effects and Durable Systemic Antitumor Immunity.

A recombinant Newcastle Disease Virus (NDV), encoding either a human (NDVhuGM-CSF, MEDI5395) or murine (NDVmuGM-CSF) GM-CSF transgene, combined broad oncolytic activity with the ability to significantly modulate genes related to immune functionality in human tumor cells. Replication in murine tumor lines was significantly diminished relative to human tumor cells. Nonetheless, intratumoral injection of NDVmuGM-CSF conferred antitumor effects in three syngeneic models in vivo; with efficacy further augmented by concomitant treatment with anti-PD-1/PD-L1 or T-cell agonists. Ex vivo immune profiling, including T-cell receptor sequencing, revealed profound immune-contexture changes consistent with priming and potentiation of adaptive immunity and tumor microenvironment (TME) reprogramming toward an immune-permissive state. CRISPR modifications rendered CT26 tumors significantly more permissive to NDV replication, and in this setting, NDVmuGM-CSF confers immune-mediated effects in the noninjected tumor in vivo Taken together, the data support the thesis that MEDI5395 primes and augments cell-mediated antitumor immunity and has significant utility as a combination partner with other immunomodulatory cancer treatments.

Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo.

Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. Here, we report that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd's Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo.

c-Abl, Lamellipodin, and Ena/VASP proteins cooperate in dorsal ruffling of fibroblasts and axonal morphogenesis.

BACKGROUND: Tight regulation of cell motility is essential for many physiological processes, such as formation of a functional nervous system and wound healing. Drosophila Abl negatively regulates the actin cytoskeleton effector protein Ena during neuronal development in flies, and it has been postulated that this may occur through an unknown intermediary. Lamellipodin (Lpd) regulates cell motility and recruits Ena/VASP proteins (Ena, Mena, VASP, EVL) to the leading edge of cells. However, the regulation of this recruitment has remained unsolved. RESULTS: Here we show that Lpd is a substrate of Abl kinases and binds to the Abl SH2 domain. Phosphorylation of Lpd positively regulates the interaction between Lpd and Ena/VASP proteins. Consistently, efficient recruitment of Mena and EVL to Lpd at the leading edge requires Abl kinases. Furthermore, transient Lpd phosphorylation by Abl kinases upon netrin-1 stimulation of primary cortical neurons positively correlates with an increase in Lpd-Mena coprecipitation. Lpd is also transiently phosphorylated by Abl kinases upon platelet-derived growth factor (PDGF) stimulation, regulates PDGF-induced dorsal ruffling of fibroblasts and axonal morphogenesis, and cooperates with c-Abl in an Ena/VASP-dependent manner. CONCLUSIONS: Our findings suggest that Abl kinases positively regulate Lpd-Ena/VASP interaction, Ena/VASP recruitment to Lpd at the leading edge, and Lpd-Ena/VASP function in axonal morphogenesis and in PDGF-induced dorsal ruffling. Our data do not support the suggested negative regulatory role of Abl for Ena. Instead, we propose that Lpd is the hitherto unknown intermediary between Abl and Ena/VASP proteins.

Scrib regulates PAK activity during the cell migration process.

Genetic studies have highlighted the key role of Scrib in the development of Metazoans. Deficiency in Scrib impairs many aspects of cell polarity and cell movement although the mechanisms involved remain unclear. In mammals, Scrib belongs to a protein complex containing betaPIX, an exchange factor for Rac/Cdc42, and GIT1, a GTPase activating protein for ARF6 implicated in receptor recycling and exocytosis. Here we show that the Scrib complex associates with PAK, a serine-threonine kinase family crucial for cell migration. PAK colocalizes with members of the Scrib complex at the leading edge of heregulin-treated T47D breast cancer cells. We demonstrate that the Scrib complex is required for epithelial cells and primary mouse embryonic fibroblasts to efficiently respond to chemoattractant cues. In Scrib-deficient cells, the pool of cortical PAK is decreased, thereby precluding its proper activation by Rac. Loss of Scrib also impairs the polarized distribution of active Rac at the leading edge and compromises the regulated activation of the GTPase in T47D cells and mouse embryonic fibroblasts. These data underscore the role of Scrib in cell migration and show the strong impact of Scrib in the function of PAK and Rac, two key molecules implicated in this process.

The hScrib/Dlg apico-basal control complex is differentially targeted by HPV-16 and HPV-18 E6 proteins.

The E6 proteins of the high-risk Human papillomaviruses (HPV) types have a well-documented ability to target certain cellular proteins for ubiquitin-mediated degradation via the proteasome. Previous studies have shown that E6 proteins interact differently with different target proteins, and that the viral proteins, depending upon the target, may recruit diverse cellular ubiquitin-protein ligases. In this study, we have examined the abilities of E6 proteins from HPV-16 and HPV-18 to interact with and induce the degradation of two PDZ domain-containing targets, Dlg and hScrib. We have also mapped the binding site of E6 on hScrib and shown that the interaction of E6 with hScrib is distinct from its interactions with other PDZ domain-containing targets. This is reflected in the efficiency with which the two viral E6 proteins can inhibit hScrib's suppression of cell transformation.Dlg and hScrib have complementary activities in the control of epithelial cell polarity and the fact that both are targeted by high-risk HPV E6 proteins underlines their importance. Our finding that they are each targeted differently by HPV-16 and HPV-18 E 6 s suggests that the two viruses are subjected to somewhat different constraints and provides a possible explanation for the apparent redundancy in targeting both parts of this important control mechanism.

hScrib interacts with ZO-2 at the cell-cell junctions of epithelial cells.

In Drosophila, the tumor suppressor Scribble is localized at the septate junctions of epithelial cells. Its mammalian homologue, hScrib, is a basolateral protein likely associated to proteins of the cell-cell junctions. We report the direct interaction between hScrib and ZO-2, a junction-associated protein. This interaction relies on two PDZ domains of hScrib and on the C-terminal motif of ZO-2. Both proteins localise at cell-cell junctions of epithelial cells. A point mutation in the LRR of hScrib delocalises the protein from the plasma membrane and abrogates the interaction with ZO-2 but not with betaPIX. Tyrosine phosphorylation of hScrib does not impair the interaction with ZO-2. We show a direct link between two junctional proteins that are down-regulated during cancer progression.

Junctional recruitment of mammalian Scribble relies on E-cadherin engagement.

Members of the LAP protein family, LET-413 in Caenorhabditis elegans, Scribble in Drosophila melanogaster, and Erbin, Lano, Densin-180 and hScrib in mammals, have conserved structural features. LET-413 and Scribble are junctional proteins involved in establishing and maintaining epithelial cell polarity. scribble also behaves as a neoplastic tumor suppressor gene. We show here that, in epithelial cells, hScrib is recruited at cell-cell junctions in an E-cadherin-dependent manner as shown by calcium switch assays in MDCK cells, re-expression of E-cadherin in MDA-231 cells treated by 5-Aza-2'-deoxycytidine (5Aza), and siRNA experiments. hScrib is restricted at the basolateral membrane of epithelial cells by its LRR domain, and is enriched in Triton X-100-insoluble fractions. In breast cancers, most lobular tumors did not express hScrib and E-cadherin while ductal tumors had a less frequent downregulation of hScrib. Our data provide additional insights on the modalities of recruitment of hScrib at the cell-cell junctions, and establish a potential link between the E-cadherin and hScrib tumor suppressors.

Thyrotropin receptor trafficking relies on the hScrib-betaPIX-GIT1-ARF6 pathway.

G protein-coupled receptors are regulated by ligand stimulation, endocytosis, degradation of recycling to the cell surface. Little information is available on the molecular mechanisms underlying G protein-coupled receptors recycling. We have investigated recycling of the G protein-coupled thyroid stimulating hormone receptor (TSHR) and found that it relies on hScrib, a membrane-associated PDZ protein. hScrib directly binds to TSHR, inhibits basal receptor endocytosis and promotes recycling, and thus TSHR signalling, at the cell membrane. We previously demonstrated that hScrib is associated with a betaPIX-GIT1 complex comprised of a guanine nucleotide exchange factor and a GTPase-activating protein for ADP ribosylation factors that is involved in vesicle trafficking. We used dominant-negative constructs and small interfering RNA to show that TSHR recycling is regulated by the interaction between hScrib and betaPIX, and by the activity of GIT1. In addition, ARF6, a major target for GIT1, is activated during TSH stimulation of HEK293 and FRTL-5 thyroid cells, and plays a key role in TSHR recycling. Thus, we have uncovered an hScrib-betaPIX-GIT1-ARF6 pathway devoted to TSHR trafficking and function.

Mammalian Scribble forms a tight complex with the betaPIX exchange factor.

Drosophila Scribble is implicated in the development of normal synapse structure and epithelial tissues, but it remains unclear how it plays a role and which process it controls. The mammalian homolog of Scribble, hScrib, has a primary structure and subcellular localization similar to that of its fly homolog, but its function remains unknown. Here we have used tandem mass spectrometry to identify major components of the hScrib network. We show that it includes betaPIX (also called Cool-1), a guanine nucleotide exchange factor (GEF), and its partner GIT1 (also called p95-APP1), a GTPase activating protein (GAP). betaPIX directly binds to the hScrib PDZ domains, and the hScrib/betaPIX complex is efficiently recovered in epithelial and neuronal cells and tissues. In cerebellar granule cell cultures, hScrib and betaPIX are both partially localized at neuronal presynaptic compartments. Furthermore, we show that hScrib is required to anchor betaPIX at the cell cortex and that dominant-negative betaPIX or hScrib proteins can each inhibit Ca2+-dependent exocytosis in neuroendocrine PC12 cells, demonstrating a functional relationship between these proteins. These data reveal the existence of a tight hScrib/betaPIX interaction and suggest that this complex potentially plays a role in neuronal transmission.

Basolateral targeting by leucine-rich repeat domains in epithelial cells.

The asymmetric distribution of proteins to basolateral and apical membranes is an important feature of epithelial cell polarity. To investigate how basolateral LAP proteins (LRR (for leucine-rich repeats) and PDZ (for PSD-95/Discs-large/ZO-1), which play key roles in cell polarity, reach their target membrane, we carried out a structure-function study of three LAP proteins: Caenorhabditis elegans LET-413, human Erbin and human Scribble (hScrib). Deletion and point mutation analyses establish that their LRR domain is crucial for basolateral membrane targeting. This property is specific to the LRR domain of LAP proteins, as the non-LAP protein SUR-8 does not localize at the basolateral membrane of epithelial cells, despite having a closely related LRR domain. Importantly, functional studies of LET-413 in C. elegans show that although its PDZ domain is dispensable during embryogenesis, its LRR domain is essential. Our data establish a novel paradigm for protein localization by showing that a subset of LRR domains direct subcellular localization in polarized cells.

Wdr12, a mouse gene encoding a novel WD-Repeat Protein with a notchless-like amino-terminal domain.

The WD-repeat protein family consists of a large group of structurally related yet functionally diverse proteins found predominantly in eukaryotic cells. These factors contain several (4-16) copies of a recognizable amino-acid sequence motif (the WD unit) thought to be organized into a "propeller-like" structure involved in protein-protein regulatory interactions. Here, we report the cloning of a mouse cDNA, referred to as Wdr12, which encodes a novel WD-repeat protein of 423 amino acids. The WDR12 protein was predicted to contain seven WD units and a nuclear localization signal located within a protruding peptide between the third and fourth WD domains. The amino-terminal region shows similarity to that of the Notchless WD repeat protein. Sequence comparisons revealed WDR12 orthologs in various eukaryotic species. Wdr12 seems to correspond to a single-copy gene in the mouse genome, located within the C1-C2 bands of chromosome 1. These data, together with the results of Wdr12 gene expression studies and evidence of in vitro binding of WDR12 to the cytoplasmic domain of Notch1, led us to postulate a function for the WDR12 protein in the modulation of Notch signaling activity.

Interaction between Erbin and a Catenin-related protein in epithelial cells.

Integrity of epithelial tissues relies on the proper apical-basolateral polarity of epithelial cells. Members of the LAP (LRR and PDZ) protein family such as LET-413 and Scribble are involved in maintaining epithelial cell polarity in Caenorhabditis elegans and Drosophila melanogaster, respectively. We previously described Erbin as a mammalian LET-413 homologue interacting with ERBB2/HER2, an epidermal growth factor receptor family member. Erbin and ERBB2/HER2 are located in the basolateral membranes of epithelial cells. We show here that Erbin interacts with p0071 (also called plakophilin-4), an armadillo repeat protein linked to the cytoskeleton. Erbin binds to p0071 in vitro and in vivo in a PDZ domain-dependent manner, and both proteins colocalized in desmosomes of epithelial cells. Using a dominant negative approach, we found that integrity of epithelial cell monolayer is impaired when interaction between Erbin and p0071 is disrupted. We propose that Erbin is connected by p0071 to cytoskeletal networks in an interaction crucial for epithelial homeostasis.