Effects of Clinically Relevant MPL Mutations in the Transmembrane Domain Revealed at the Atomic Level through Computational Modeling

BACKGROUND: Mutations in the thrombopoietin receptor (MPL) may activate relevant pathways and lead to chronic myeloproliferative neoplasms (MPNs). The mechanisms of MPL activation remain elusive because of a lack of experimental structures. Modern computational biology techniques were utilized to explore the mechanisms of MPL protein activation due to various mutations. RESULTS: Transmembrane (TM) domain predictions, homology modeling, ab initio protein structure prediction, and molecular dynamics (MD) simulations were used to build structural dynamic models of wild-type and four clinically observed mutants of MPL. The simulation results suggest that S505 and W515 are important in keeping the TM domain in its correct position within the membrane. Mutations at either of these two positions cause movement of the TM domain, altering the conformation of the nearby intracellular domain in unexpected ways, and may cause the unwanted constitutive activation of MPL's kinase partner, JAK2. CONCLUSIONS: Our findings represent the first full-scale molecular dynamics simulations of the wild-type and clinically observed mutants of the MPL protein, a critical element of the MPL-JAK2-STAT signaling pathway. In contrast to usual explanations for the activation mechanism that are based on the relative translational movement between rigid domains of MPL, our results suggest that mutations within the TM region could result in conformational changes including tilt and rotation (azimuthal) angles along the membrane axis. Such changes may significantly alter the conformation of the adjacent and intrinsically flexible intracellular domain. Hence, caution should be exercised when interpreting experimental evidence based on rigid models of cytokine receptors or similar systems

Efficient and versatile CRISPR engineering of human neurons in culture to model neurological disorders

The recent identification of multiple new genetic causes of neurological disorders highlights the need for model systems that give experimental access to the underlying biology. In particular, the ability to couple disease-causing mutations with human neuronal differentiation systems would be beneficial. Gene targeting is a well-known approach for dissecting gene function, but low rates of homologous recombination in somatic cells (including neuronal cells) have traditionally impeded the development of robust cellular models of neurological disorders. Recently, however, CRISPR/Cas9 gene editing technologies have expanded the number of systems within which gene targeting is possible. Here we adopt as a model system LUHMES cells, a commercially available diploid human female mesencephalic cell line that differentiates into homogeneous mature neurons in 1-2 weeks. We describe optimised methods for transfection and selection of neuronal progenitor cells carrying targeted genomic alterations using CRISPR/Cas9 technology. By targeting the endogenous X-linked MECP2 locus, we introduced four independent missense mutations that cause the autism spectrum disorder Rett syndrome and observed the desired genetic structure in 3-26% of selected clones, including gene targeting of the inactive X chromosome. Similar efficiencies were achieved by introducing neurodevelopmental disorder-causing mutations at the autosomal EEF1A2 locus on chromosome 20. Our results indicate that efficiency of genetic “knock-in” is determined by the location of the mutation within the donor DNA molecule. Furthermore, we successfully introduced an mCherry tag at the MECP2 locus to yield a fusion protein, demonstrating that larger insertions are also straightforward in this system. We suggest that our optimised methods for altering the genome of LUHMES cells make them an attractive model for the study of neurogenetic disorders

Efficient delivery of angiostatin K1-5 into tumors following insertion of an NGR peptide into adenovirus capsid.

International audienceAdenovirus (Ad)-mediated delivery of anti-angiogenic molecules into tumors constitutes an appealing approach for growth inhibition. However, lack of expression on tumors of Ad receptors leads to weak tumor transduction. Therefore, to provide Ad with a new entry pathway into tumors, an NGR peptide was inserted into either fiber (AdFNGR) or hexon (AdHNGR) capsid proteins. This strategy provided Ad with a very efficient entry pathway in both endothelial cells and tumor cells, with the highest efficacy observed for AdHNGR. Using pharmacological, biochemical and genetic approaches, AdHNGR and AdFNGR were shown to bind not only to CD13 receptor, but also to alphavbeta3 integrins. Both vectors were efficient tools to deliver angiostatin K1-5 cDNA into endothelial cells, thus leading to a dramatic inhibition of their proliferation and increased cell death. Although AdHNGR and Adwt were found to display similar gene transduction efficacy in Lewis lung carcinoma (LLC), pseudotyping AdHNGR with an Ad3-fiber unmasked the ability of NGR-peptide to target these tumors. As a result, delivery of angiostatin K1-5 cDNA into highly aggressive tumors translated into a stronger inhibition of their growth. Altogether, our results suggest that NGR-bearing Ad are valuable tools to realize the potential of this anti-angiogenic approach to anti-tumor therapy.Gene Therapy advance online publication, 30 July 2009; doi:10.1038/gt.2009.97

SOCS3 inhibits TPO-stimulated, but not spontaneous, megakaryocytic growth in primary myelofibrosis.

International audienc

p63/MT1-MMP axis is required for in situ to invasive transition in basal-like breast cancer

The transition of ductal carcinoma in situ (DCIS) to invasive breast carcinoma requires tumor cells to cross the basement membrane (BM). However, mechanisms underlying BM transmigration are poorly understood. Here, we report that expression of membrane-type 1 (MT1)-matrix metalloproteinase (MMP), a key component of the BM invasion program, increases during breast cancer progression at the in situ to invasive breast carcinoma transition. In the intraductal xenograft model, MT1-MMP is required for BM transmigration of MCF10DCIS.com breast adenocarcinoma cells and is overexpressed in cell clusters overlying focal BM disruptions and at the invasive tumor front. Mirrored upregulation of p63 and MT1-MMP is observed at the edge of MCF10DCIS.com xenograft tumors and p63 is required for induction of MT1-MMP-dependent invasive program in response to microenvironmental signals. Immunohistochemistry and analysis of public database reveal that p63 and MT1-MMP are upregulated in human basal-like breast tumors suggesting that p63/MT1-MMP axis contributes to progression of basal-like breast cancers with elevated p63 and MT1-MMP levels.Fil: Lodillinsky, Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Centre National de la Recherche Scientifique; FranciaFil: Infante, E.. Centre National de la Recherche Scientifique; FranciaFil: Guichard, A.. Centre National de la Recherche Scientifique; FranciaFil: Chaligné, R.. Génétique Et Biologie Du Développement; FranciaFil: Fuhrmann, L.. Centre National de la Recherche Scientifique; FranciaFil: Cyrta, J.. Centre National de la Recherche Scientifique; FranciaFil: Irondelle, Marie. Centre National de la Recherche Scientifique; FranciaFil: Lagoutte, Emilie. Centre National de la Recherche Scientifique; FranciaFil: Vacher, Sophie. Institut Curie; FranciaFil: Bonsang-Kitzis, H.. Institut Curie; FranciaFil: Glukhova, M.. Centre National de la Recherche Scientifique; FranciaFil: Reyal, F.. Institut Curie; FranciaFil: Bièche, I.. Institut Curie; FranciaFil: Vincent Salomon, Anne. Institut Curie; Francia. Génétique Et Biologie Du Développement; FranciaFil: Chavrier, Philippe. Centre National de la Recherche Scientifique; Franci

ROQUIN/RC3H1 alterations are not found in angioimmunoblastic T-cell lymphoma.

Angioimmunoblastic T-cell Lymphoma (AITL) is one of the most frequent T-cell lymphoma entities. Follicular helper T lymphocytes (TFH) are recognized as the normal cellular counterpart of the neoplastic component. Despite a clonal T-cell feature and few described recurrent cytogenetic abnormalities, a driving oncogenic event has not been identified so far. It has been recently reported that in mice, heterozygous inactivation of Roquin/Rc3h1, a RING type E3 ubiquitine ligase, recapitulates many of the clinical, histological, and cellular features associated with human AITL. In this study we explored whether ROQUIN alterations could be an initial event in the human AITL oncogenic process. Using microarray and RT-PCR analyses, we investigated the levels of ROQUIN transcripts in TFH tumor cells purified from AITL (n = 8) and reactive tonsils (n = 12) and found similar levels of ROQUIN expression in both. Moreover, we also demonstrated that ROQUIN protein was expressed by AITL TFH (PD1+) cells. We then analysed ROQUIN coding sequence in 12 tumor cell-rich AITL samples and found no mutation in any of the samples. Finally, we analysed the expression of MiR101, a putative partner of ROQUIN involved in the modulation of ICOS expression and found similar levels of expression in tumor and reactive TFH. Altogether, this study shows that neither alteration of ROQUIN gene nor deregulation of miR101 expression is likely to be a frequent recurrent event in AITL