AstA Signaling Functions as an Evolutionary Conserved Mechanism Timing Juvenile to Adult

The onset of sexual maturation is the result of a hormonal cascade peaking with the production of steroid hormones. In animals undergoing a program of determinate growth, sexual maturation also coincides with the attainment of adult size. The exact signals that time the onset of maturation and the mechanisms coupling growth and maturation remain elusive. Here, we show that the Drosophila neuropeptide AstA and its receptor AstAR1 act as a brain trigger for maturation and juvenile growth. We first identified AstAR1 in an RNAi-based genetic screen as a key regulator of sexual maturation. Its specific knockdown in prothoracicotropic hormone (PTTH)-producing neurons delays the onset of maturation by impairing PTTH secretion. In addition to its role in PTTH neurons, AstAR1 is required in the brain insulin-producing cells (IPCs) to promote insulin secretion and systemic growth. AstAR1 function is mediated by the AstA neuropeptide that is expressed in two bilateral neurons contacting the PTTH neurons and the IPCs. Silencing brain AstA expression delays the onset of maturation, therefore extending the growth period. However, no pupal overgrowth is observed, indicating that, in these conditions, the growth-promoting function of AstAR1 is also impaired. These data suggest that AstA/AstAR1 acts to coordinate juvenile growth with maturation. Interesting, AstA/AstAR1 is homologous to KISS/GPR54, a ligand-receptor signal required for human puberty, suggesting that an evolutionary conserved neural circuitry controls the onset of maturation.

anti-cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) monoclonal antibody ipilimumab (Ipi) in patients (pts) with metastatic melanoma

Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint

Hotspot mutations in the spliceosome gene SF3B1 are reported in ∼20% of uveal melanomas. SF3B1 is involved in 3′-splice site (3’ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1(R625/K666) mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3’ss. Modelling the differential junctions in SF3B1(WT) and SF3B1(R625/K666) cell lines demonstrates that the deregulated splice pattern strictly depends on SF3B1 status and on the 3’ss-sequence context. SF3B1(WT) knockdown or overexpression do not reproduce the SF3B1(R625/K666) splice pattern, qualifying SF3B1(R625/K666) as change-of-function mutants. Mutagenesis of predicted branchpoints reveals that the SF3B1(R625/K666)-promoted splice pattern is a direct result of alternative branchpoint usage. Altogether, this study provides a better understanding of the mechanisms underlying splicing alterations induced by mutant SF3B1 in cancer, and reveals a role for alternative branchpoints in disease.

SHH medulloblastoma in a young adult with a TCF4 germline pathogenic variation.

A large collection of integrated genomically characterized patient-derived xenografts highlighting

Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-Derived Xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n=61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of SMGs (Significantly Mutated Gene) and SCNAs (Somatic Copy Number Alteration) in PDX and TCGA TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes.TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research. KEYS WORD: Triple-negative breast cancer, targeted therapies, patient-derived xenograft (PDX), integrated genomic analysis. This article is protected by copyright. All rights reserved.

Cécile Boutonnet

Immunization with antigen-pulsed dendritic cells significantly improves the immune response to weak

Dendritic cells (DCs) are the only professional antigen-presenting cells endowed with the ability to stimulate naïve T cells and initiate a primary immune response. For this reason, DC-based immunization has been shown to be highly effective in eliciting CTL responses to viruses and tumor-associated antigens. Here we report on the use of DC immunization to enhance the B cell-mediated humoral immune response to highly conserved proteins and the application of this approach to the generation of monoclonal antibodies (mAbs) against these proteins. To illustrate the technique we describe the production of mAbs to class II transactivator (CIITA), the major histocompatibility complex (MHC) CIITA, a difficult immunogen owing to its high degree of identity among species. We show that mice immunized with a combination of an intravenous injection of DCs pulsed with recombinant fragments of CIITA followed by intraperitoneal injection of the antigen in incomplete Freund’s adjuvant induced a detectable antibody response against CIITA, while sera from mice immunized using the traditional method (i.e. intraperitoneal immunization with 50mug of protein in complete Freund’s adjuvant) gave an almost undetectable response. Furthermore, a total of four fusion experiments demonstrate that immunization with Ag-pulsed DCs is necessary for the efficient generation of hybridomas and a good yield of mAbs specific for the recombinant and the native endogenous CIITA protein. Conversely, four independent fusions carried out with splenocytes from mice immunized using the traditional method failed to produce anti-CIITA hybridomas. We propose that immunization with antigen-loaded DCs should be the method of preference when attempting to raise mAbs against weak self-immunogens.

Arc/Arg3.1 governs inflammatory dendritic cell migration from the skin and thereby controls T cell

Skin-migratory dendritic cells (migDCs) are pivotal antigen-presenting cells that continuously transport antigens to draining lymph nodes and regulate immune responses. However, identification of migDCs is complicated by the lack of distinguishing markers, and it remains unclear which molecules determine their migratory capacity during inflammation. We show that, in the skin, the neuronal plasticity molecule activity-regulated cytoskeleton-associated protein/activity-regulated gene 3.1 (Arc/Arg3.1) was strictly confined to migDCs. Mechanistically, Arc/Arg3.1 was required for accelerated DC migration during inflammation because it regulated actin dynamics through nonmuscle myosin II. Accordingly, Arc/Arg3.1-dependent DC migration was critical for mounting T cell responses in experimental autoimmune encephalomyelitis and allergic contact dermatitis. Thus, Arc/Arg3.1 was restricted to migDCs in the skin and drove fast DC migration by exclusively coordinating cytoskeletal changes in response to inflammatory challenges. These findings commend Arc/Arg3.1 as a universal switch in migDCs that may be exploited to selectively modify immune responses.

Kerstin Wendland

UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8 T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs.

Anton Tekhneriadov

Khanh My Pham

Pierre Conan

Sara Chabi

ATP promotes the fast migration of dendritic cells through the activity of pannexin 1 channels and

Upon its release from injured cells, such as infected, transformed, inflamed, or necrotic cells, extracellular adenosine-5′-triphosphate (ATP) acts as a danger signal that recruits phagocytes, such as neutrophils, macrophages, and dendritic cells (DCs), to the site of injury. The sensing of extracellular ATP occurs through purinergic (P2) receptors. We investigated the cellular mechanisms linking purinergic signaling to DC motility. We found that ATP stimulated fast DC motility through an autocrine signaling loop, which was initiated by the activation of P2X receptors and further amplified by pannexin 1 (Panx1) channels. Upon stimulation of the P2X receptor by ATP, Panx1 contributed to fast DC motility by increasing the permeability of the plasma membrane, which resulted in supplementary ATP release. In the absence of Panx1, DCs failed to increase their speed of migration in response to ATP, despite exhibiting a normal P2X receptor-mediated Ca response. In addition to DC migration, Panx1 channel- and P2X receptor-dependent signaling was further required to stimulate the reorganization of the actin cytoskeleton. In vivo, functional Panx1 channels were required for the homing of DCs to lymph nodes, although they were dispensable for DC maturation. These data suggest that P2X receptors and Panx1 channels are crucial players in the regulation of DC migration to endogenous danger signals.

Caveolin-1 Expression Increases upon Maturation in Dendritic Cells and Promotes Their Migration to

Dendritic cell (DC) trafficking from peripheral tissues to lymph nodes (LNs) is a key step required to initiate T cell responses against pathogens as well as tumors. In this context, cellular membrane protrusions and the actin cytoskeleton are essential to guide DC migration towards chemotactic signals. Caveolin-1 (CAV1) is a scaffolding protein that modulates signaling pathways leading to remodeling of the actin cytoskeleton and enhanced migration of cancer cells. However, whether CAV1 is relevant for DC function and specifically for DC migration to LNs is unknown. Here, we show that CAV1 expression is upregulated in DCs upon LPS- and TNF-α-induced maturation. CAV1 deficiency did not affect differentiation, maturation, or the ability of DCs to activate CD8 T cells . However, CAV1-deficient (CAV1) DCs displayed reduced trafficking to draining LNs in control and inflammatory conditions. , CAV1 DCs showed reduced directional migration in CCL21 gradients in transwell assays without affecting migration velocity in confined microchannels or three-dimensional collagen matrices. In addition, CAV1 DCs displayed reduced activation of the small GTPase Rac1, a regulator of actin cytoskeletal remodeling, and lower numbers of F-actin-forming protrusions. Furthermore, mice adoptively transferred with peptide-pulsed CAV1 DCs showed reduced CD8 T cell responses and antitumor protection. Our results suggest that CAV1 promotes the activation of Rac1 and the formation of membrane protrusions that favor DC chemotactic trafficking toward LNs where they can initiate cytotoxic T cell responses.

Reconstitution of cell migration at a glance.

Single cells migrate in a myriad of physiological contexts, such as tissue patrolling by immune cells, and during neurogenesis and tissue remodeling, as well as in metastasis, the spread of cancer cells. To understand the basic principles of single-cell migration, a reductionist approach can be taken. This aims to control and deconstruct the complexity of different cellular microenvironments into simpler elementary constrains that can be recombined together. This approach is the cell microenvironment equivalent of reconstituted systems that combine elementary molecular players to understand cellular functions. In this Cell Science at a Glance article and accompanying poster, we present selected experimental setups that mimic different events that cells undergo during migration These include polydimethylsiloxane (PDMS) devices to deform whole cells or organelles, micro patterning, nano-fabricated structures like grooves, and compartmentalized collagen chambers with chemical gradients. We also outline the main contribution of each technique to the understanding of different aspects of single-cell migration.

Probing Ligand and Cation Binding Sites in G-Quadruplex Nucleic Acids by Mass Spectrometry and

Mass spectrometry provides exquisite detail on ligand and cation binding stoichiometries with a DNA target. The next important step is to develop reliable methods to determine the cation and ligand binding sites in each complex separated by the mass spectrometer. To circumvent the caveat of ligand derivatization for cross-linking, which may alter the ligand binding mode, we explored a tandem mass spectrometry (MS/MS) method that does not require ligand derivatization, and is therefore also applicable to localize metal cations. By obtaining more negative charge states for the complexes using supercharging agents, and by creating radical ions by electron photodetachment, oligonucleotide bonds become weaker than the DNA-cation or DNA-ligand noncovalent bonds upon collision-induced dissociation of the radicals. This electron photodetachment (EPD) method allows to locate the binding regions of cations and ligands by top-down sequencing of the oligonucleotide target. The very potent G-quadruplex ligands 360A and PhenDC3 were found to replace a potassium cation and bind close to the central loop of 4-repeat human telomeric sequences.

LRP8 is overexpressed in estrogen-negative breast cancers and a potential target for these tumors.

Triple-negative breast cancer (TNBC) is the breast cancer subtype with the worst prognosis. New treatments improving the survival of TNBC patients are, therefore, urgently required. We performed a transcriptome microarray analysis to identify new treatment targets for TNBC. We found that low-density lipoprotein receptor-related protein 8 (LRP8) was more strongly expressed in estrogen receptor-negative breast tumors, including TNBCs and those overexpressing HER2, than in luminal breast tumors and normal breast tissues. LRP8 depletion decreased cell proliferation more efficiently in estrogen receptor-negative breast cancer cell lines: TNBC and HER2 overexpressing cell lines. We next focused on TNBC cells for which targeted therapies are not available. LRP8 depletion induced an arrest of the cell cycle progression in G1 phase and programmed cell death. We also found that LRP8 is required for anchorage-independent growth in vitro, and that its depletion in vivo slowed tumor growth in a xenograft model. Our findings suggest that new approaches targeting LRP8 may constitute promising treatments for hormone-negative breast cancers, those overexpressing HER2 and TNBCs.

Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) promotes the aggressiveness of human uveal melanoma

Uveal melanoma (UM) is an aggressive tumor in which approximately 50% of patients develop metastasis. Expression of the PTP4A3 gene, encoding a phosphatase, is predictive of poor patient survival. PTP4A3 expression in UM cells increases their migration in vitro and invasiveness in vivo. Here, we show that CRMP2 is mostly dephosphorylated on T514 in PTP4A3 expressing cells. We also demonstrate that inhibition of CRMP2 expression in UM cells expressing PTP4A3 increases their migration in vitro and invasiveness in vivo. This phenotype is accompanied by modifications of the actin microfilament network, with shortened filaments, whereas cells with a inactive mutant of the phosphatase do not show the same behavior. In addition, we showed that the cell cytoplasm becomes stiffer when CRMP2 is downregulated or PTP4A3 is expressed. Our results suggest that PTP4A3 acts upstream of CRMP2 in UM cells to enhance their migration and invasiveness and that a low level of CRMP2 in tumors is predictive of poor patient survival.