MYO1C stabilizes actin and facilitates the arrival of transport carriers at the Golgi complex

In this study, we aimed to identify the myosin motor proteins that control trafficking at the Golgi complex. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identified MYO1C as a novel player at the Golgi in a human cell line. We demonstrate that depletion of MYO1C induces Golgi complex fragmentation and decompaction. MYO1C accumulates at dynamic structures around the Golgi complex that colocalize with Golgi-associated actin dots. MYO1C depletion leads to loss of cellular F-actin, and Golgi complex decompaction is also observed after inhibition or loss of the actin-related protein 2/3 complex, Arp2/3 (also known as ARPC). We show that the functional consequence of MYO1C depletion is a delay in the arrival of incoming transport carriers, both from the anterograde and retrograde routes. We propose that MYO1C stabilizes actin at the Golgi complex, facilitating the arrival of incoming transport carriers at the Golgi.This article has an associated First Person interview with the first author of the paper.Fil: Capmany, Anahi. Institute Curie; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Medicas. Instituto de Inmunologia; ArgentinaFil: Yoshimura, Azumi. Institute Curie; FranciaFil: Kerdous, Rachid. Institute Curie; FranciaFil: Caorsi, Valentina. Abbelight; FranciaFil: Lescure, Aurianne. Institute Curie; FranciaFil: Nery, Elaine Del. Institute Curie; FranciaFil: Coudrier, Evelyne. Institute Curie; FranciaFil: Goud, Bruno. Institute Curie; FranciaFil: Schauer, Kristine. Institute Curie; Franci

Upregulation of the Mevalonate Pathway through EWSR1-FLI1/EGR2 Regulatory Axis Confers Ewing Cells Exquisite Sensitivity to Statins

Ewing sarcoma (EwS) is an aggressive primary bone cancer in children and young adults characterized by oncogenic fusions between genes encoding FET-RNA-binding proteins and ETS transcription factors, the most frequent fusion being EWSR1-FLI1. We show that EGR2, an Ewing-susceptibility gene and an essential direct target of EWSR1-FLI1, directly regulates the transcription of genes encoding key enzymes of the mevalonate (MVA) pathway. Consequently, Ewing sarcoma is one of the tumors that expresses the highest levels of mevalonate pathway genes. Moreover, genome-wide screens indicate that MVA pathway genes constitute major dependencies of Ewing cells. Accordingly, the statin inhibitors of HMG-CoA-reductase, a rate-limiting enzyme of the MVA pathway, demonstrate cytotoxicity in EwS. Statins induce increased ROS and lipid peroxidation levels, as well as decreased membrane localization of prenylated proteins, such as small GTP proteins. These metabolic effects lead to an alteration in the dynamics of S-phase progression and to apoptosis. Statin-induced effects can be rescued by downstream products of the MVA pathway. Finally, we further show that statins impair tumor growth in different Ewing PDX models. Altogether, the data show that statins, which are off-patent, well-tolerated, and inexpensive compounds, should be strongly considered in the therapeutic arsenal against this deadly childhood disease. Keywords: Ewing sarcoma; MVA pathway; new therapeutic strategy; statin

High-Throughput Drug Screening Identifies Pazopanib and Clofilium Tosylate as Promising Treatments for Malignant Rhabdoid Tumors

Summary: Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/β and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro. In vivo, reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These results strongly support testing pazopanib/CfT combination therapy in future clinical trials for RTs. : Rhabdoid tumors (RTs) are aggressive pediatric tumors characterized by SMARCB1 inactivation. Chauvin et al. identify two SMARCB1-dependent targeted therapies for RT: pazopanib, which inhibits PDGFR and FGFR2, and the potassium channel inhibitor clofilium tosylate, which induces endoplasmic reticulum stress. Combining both drugs induces cell apoptosis and reduces PDX tumor growth. Keywords: rhabdoid tumors, SMARCB1, pazopanib, clofilium tosylate, high-throughput drug screening, tyrosine kinase inhibitor

Combination Therapies Targeting ALK-aberrant Neuroblastoma in Preclinical Models

PURPOSE ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma

Screening out irrelevant cell-based models of disease

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell-and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates

Estudo da atividade cininogenásica das calicreínas tissulares, humana e porcina, utilizando substratos fluorogênicos

BV UNIFESP: Teses e dissertaçõe

Kininogenae activiy by he major cysteinyl proteinase cruzipain from Trypanosoma cruzi

Estudamos a liberacao de Lys-Bk pelas cisteino peptidases de Trypanosoma cruzi, cruzipaina e sua forma recombinante, cruzipaina 2, utilizando um substrato sintetico com sequencia analoga aquela hidrolisada no cininogenio humano, contendo as duas ligacoes que no cininogenio sao susceptiveis a clivagem para a liberacao de Lys-Bk. O estudo da simultaneidade da hidrolise das ligacoes Met-Lys e Arg-Ser, foi investigado utilizando-se um fragmento do cininogenio humano sintetizado em nosso laboratorio, contendo a sequencia completa da calidina e pelo menos 3 residuos adicionais de cada lado ( N- e C-terminal). Utilizamos tambem cininogenio humano e plasma humano como fonte de cininogenio e testamos a atividade cininogenasica em ileo isolado de cobaia. Visando estudar a especificidade da cruzipaina, utilizamos substratos fluorescentes derivados da biblioteca de peptideos. A atividade foi comparada com catepsina L. A cruzipaina foi detectada como sendo uma nova cininogenase e os ensaios de especificidade mostraram diferencas relevantes sobre sua especificidade em comparacao com a catepsina L humanaBV UNIFESP: Teses e dissertaçõe