Acute Influenza Infection Promotes Lung Tumor Growth by Reprogramming the Tumor Microenvironment.

One billion people worldwide get flu every year, including patients with non-small cell lung cancer (NSCLC). However, the impact of acute influenza A virus (IAV) infection on the composition of the tumor microenvironment (TME) and the clinical outcome of patients with NSCLC is largely unknown. We set out to understand how IAV load impacts cancer growth and modifies cellular and molecular players in the TME. Herein, we report that IAV can infect both tumor and immune cells, resulting in a long-term protumoral effect in tumor-bearing mice. Mechanistically, IAV impaired tumor-specific T-cell responses, led to the exhaustion of memory CD8+ T cells and induced PD-L1 expression on tumor cells. IAV infection modulated the transcriptomic profile of the TME, fine-tuning it toward immunosuppression, carcinogenesis, and lipid and drug metabolism. Consistent with these data, the transcriptional module induced by IAV infection in tumor cells in tumor-bearing mice was also found in human patients with lung adenocarcinoma and correlated with poor overall survival. In conclusion, we found that IAV infection worsened lung tumor progression by reprogramming the TME toward a more aggressive state.

Clonal hematopoiesis driven by chromosome 1q/MDM4 trisomy defines a canonical route toward leukemia in Fanconi anemia.

Fanconi anemia (FA) patients experience chromosome instability, yielding hematopoietic stem/progenitor cell (HSPC) exhaustion and predisposition to poor-prognosis myeloid leukemia. Based on a longitudinal cohort of 335 patients, we performed clinical, genomic, and functional studies in 62 patients with clonal evolution. We found a unique pattern of somatic structural variants and mutations that shares features of BRCA-related cancers, the FA-hallmark being unbalanced, microhomology-mediated translocations driving copy-number alterations. Half the patients developed chromosome 1q gain, driving clonal hematopoiesis through MDM4 trisomy downmodulating p53 signaling later followed by secondary acute myeloid lukemia genomic alterations. Functionally, MDM4 triplication conferred greater fitness to murine and human primary FA HSPCs, rescued inflammation-mediated bone marrow failure, and drove clonal dominance in FA mouse models, while targeting MDM4 impaired leukemia cells in vitro and in vivo. Our results identify a linear route toward secondary leukemogenesis whereby early MDM4-driven downregulation of basal p53 activation plays a pivotal role, opening monitoring and therapeutic prospects.

Assessing and enhancing migration of human myogenic progenitors using directed iPS cell differentiation and advanced tissue modelling.

Muscle satellite stem cells (MuSCs) are responsible for skeletal muscle growth and regeneration. Despite their differentiation potential, human MuSCs have limited in vitro expansion and in vivo migration capacity, limiting their use in cell therapies for diseases affecting multiple skeletal muscles. Several protocols have been developed to derive MuSC-like progenitors from human induced pluripotent stem (iPS) cells (hiPSCs) to establish a source of myogenic cells with controllable proliferation and differentiation. However, current hiPSC myogenic derivatives also suffer from limitations of cell migration, ultimately delaying their clinical translation. Here we use a multi-disciplinary approach including bioinformatics and tissue engineering to show that DLL4 and PDGF-BB improve migration of hiPSC-derived myogenic progenitors. Transcriptomic analyses demonstrate that this property is conserved across species and multiple hiPSC lines, consistent with results from single cell motility profiling. Treated cells showed enhanced trans-endothelial migration in transwell assays. Finally, increased motility was detected in a novel humanised assay to study cell migration using 3D artificial muscles, harnessing advanced tissue modelling to move hiPSCs closer to future muscle gene and cell therapies.

Reversal of RNA toxicity in myotonic dystrophy via a decoy RNA-binding protein with high affinity for expanded CUG repeats.

Myotonic dystrophy type 1 (DM1) is an RNA-dominant disease whose pathogenesis stems from the functional loss of muscleblind-like RNA-binding proteins (RBPs), which causes the formation of alternative-splicing defects. The loss of functional muscleblind-like protein 1 (MBNL1) results from its nuclear sequestration by mutant transcripts containing pathogenic expanded CUG repeats (CUGexp). Here we show that an RBP engineered to act as a decoy for CUGexp reverses the toxicity of the mutant transcripts. In vitro, the binding of the RBP decoy to CUGexp in immortalized muscle cells derived from a patient with DM1 released sequestered endogenous MBNL1 from nuclear RNA foci, restored MBNL1 activity, and corrected the transcriptomic signature of DM1. In mice with DM1, the local or systemic delivery of the RBP decoy via an adeno-associated virus into the animals' skeletal muscle led to the long-lasting correction of the splicing defects and to ameliorated disease pathology. Our findings support the development of decoy RBPs with high binding affinities for expanded RNA repeats as a therapeutic strategy for myotonic dystrophies.

The non-muscle ADF/cofilin-1 controls sarcomeric actin filament integrity and force production in striated muscle laminopathies.

Cofilins are important for the regulation of the actin cytoskeleton, sarcomere organization, and force production. The role of cofilin-1, the non-muscle-specific isoform, in muscle function remains unclear. Mutations in LMNA encoding A-type lamins, intermediate filament proteins of the nuclear envelope, cause autosomal Emery-Dreifuss muscular dystrophy (EDMD). Here, we report increased cofilin-1 expression in LMNA mutant muscle cells caused by the inability of proteasome degradation, suggesting a protective role by ERK1/2. It is known that phosphorylated ERK1/2 directly binds to and catalyzes phosphorylation of the actin-depolymerizing factor cofilin-1 on Thr25. In vivo ectopic expression of cofilin-1, as well as its phosphorylated form on Thr25, impairs sarcomere structure and force generation. These findings present a mechanism that provides insight into the molecular pathogenesis of muscular dystrophies caused by LMNA mutations.

Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice.

Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nucleus-retained mutant DMPK (DM1 protein kinase) transcripts containing CUG expansions (CUGexps). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Thus, low-dose treatment with Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease transcriptome. Moreover, treated DM1 patient-derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1.

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations.

AtWRKY15 perturbation abolishes the mitochondrial stress response that steers osmotic stress tolerance in Arabidopsis.

Environmental stresses adversely affect plant growth and development. A common theme within these adverse conditions is the perturbation of reactive oxygen species (ROS) homeostasis. Here, we demonstrate that the ROS-inducible Arabidopsis thaliana WRKY15 transcription factor (AtWRKY15) modulates plant growth and salt/osmotic stress responses. By transcriptome profiling, a divergent stress response was identified in transgenic WRKY15-overexpressing plants that linked a stimulated endoplasmic reticulum-to-nucleus communication to a disrupted mitochondrial stress response under salt-stress conditions. We show that mitochondrial calcium-flux sensing might be important for regulating an active mitochondrial retrograde signaling and launching an appropriate defense response to confer salt-stress tolerance.

AGRONOMICS1: a new resource for Arabidopsis transcriptome profiling.

Transcriptome profiling has become a routine tool in biology. For Arabidopsis (Arabidopsis thaliana), the Affymetrix ATH1 expression array is most commonly used, but it lacks about one-third of all annotated genes present in the reference strain. An alternative are tiling arrays, but previous designs have not allowed the simultaneous analysis of both strands on a single array. We introduce AGRONOMICS1, a new Affymetrix Arabidopsis microarray that contains the complete paths of both genome strands, with on average one 25mer probe per 35-bp genome sequence window. In addition, the new AGRONOMICS1 array contains all perfect match probes from the original ATH1 array, allowing for seamless integration of the very large existing ATH1 knowledge base. The AGRONOMICS1 array can be used for diverse functional genomics applications such as reliable expression profiling of more than 30,000 genes, detection of alternative splicing, and chromatin immunoprecipitation coupled to microarrays (ChIP-chip). Here, we describe the design of the array and compare its performance with that of the ATH1 array. We find results from both microarrays to be of similar quality, but AGRONOMICS1 arrays yield robust expression information for many more genes, as expected. Analysis of the ATH1 probes on AGRONOMICS1 arrays produces results that closely mirror those of ATH1 arrays. Finally, the AGRONOMICS1 array is shown to be useful for ChIP-chip experiments. We show that heterochromatic H3K9me2 is strongly confined to the gene body of target genes in euchromatic chromosome regions, suggesting that spreading of heterochromatin is limited outside of pericentromeric regions.

Quantitative RNA expression analysis with Affymetrix Tiling 1.0R arrays identifies new E2F target genes.

The Affymetrix ATH1 array provides a robust standard tool for transcriptome analysis, but unfortunately does not represent all of the transcribed genes in Arabidopsis thaliana. Recently, Affymetrix has introduced its Arabidopsis Tiling 1.0R array, which offers whole-genome coverage of the sequenced Col-0 reference strain. Here, we present an approach to exploit this platform for quantitative mRNA expression analysis, and compare the results with those obtained using ATH1 arrays. We also propose a method for selecting unique tiling probes for each annotated gene or transcript in the most current genome annotation, TAIR7, generating Chip Definition Files for the Tiling 1.0R array. As a test case, we compared the transcriptome of wild-type plants with that of transgenic plants overproducing the heterodimeric E2Fa-DPa transcription factor. We show that with the appropriate data pre-processing, the estimated changes per gene for those with significantly different expression levels is very similar for the two array types. With the tiling arrays we could identify 368 new E2F-regulated genes, with a large fraction including an E2F motif in the promoter. The latter groups increase the number of excellent candidates for new, direct E2F targets by almost twofold, from 181 to 334.

At-TAX: a whole genome tiling array resource for developmental expression analysis and transcript identification in Arabidopsis thaliana.

Gene expression maps for model organisms, including Arabidopsis thaliana, have typically been created using gene-centric expression arrays. Here, we describe a comprehensive expression atlas, Arabidopsis thaliana Tiling Array Express (At-TAX), which is based on whole-genome tiling arrays. We demonstrate that tiling arrays are accurate tools for gene expression analysis and identified more than 1,000 unannotated transcribed regions. Visualizations of gene expression estimates, transcribed regions, and tiling probe measurements are accessible online at the At-TAX homepage.