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Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control 1 . ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS 1 . Although several ALS-associated genes have been shown to affect immune functions 2 , whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression 3 . Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene ( SETX ). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (T EMRA ) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded T EMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state. An immune signature characterized by activated antigen-specific CD8 T cells is identified in the brain and blood of mice with amyotrophic lateral sclerosis-4 (ALS4), suggesting that the immune system is involved in ALS4 neurodegeneration.

Proper developmental progression during embryogenesis depends on a precise, stepwise, sculpting of transcriptional networks during cell fate transitions. During pre-implantation development, waves of epigenetic reprogramming aid in the reorganization of the gene regulatory landscape to allow for the induction of cell-type specific gene programs. Chromatin modifying enzymes play a key role in the repression and activation of transcriptional networks during cell fate transitions. The derivation of mouse embryonic stem cells has yielded a powerful in vitro model that allowed for the elucidation of molecular mechanisms required for proper establishment of gene regulatory networks during embryogenesis. Naïve mESC are representative of the epiblast found in a E3.5 embryo and can be induced to transition to a primed state that represents the pre-streak post-implantation epiblast [epiblast-like cell (EpiLC)]. Although these two share similar characteristics, their epigenome and transcriptional networks differ. Particularly, naïve pluripotent stem cells exabit transposable elements (TEs) activity compared to their primed counterparts. TE are remnants of ancient retroviruses that have stably integrated into out genomes and can be passed to progeny in a mendelian fashion. Moreover, TEs have become domesticated to contain gene regulatory function, however, their mis-regulation can be detrimental to cellular homeostasis. In pluripotent stem cells TEs are repressed through the histone modifications whereas in differentiated progeny are highly methylated. However, approximately 20% of TE are found within open chromatin regions throughout the course of development. How the transcriptional activity of these TEs is control remains unknown.As TEs comprise the bulk of the noncoding RNA compendium in mouse and human genes, they make for an ideal target for the RNA exosome. The RNA exosome is a high conserved multisubunit RNA degradation machine that targets transcripts that are derived from all three RNA polymerases. However, the exosome preferentially degrades noncoding RNA that is produced from gene regulatory regions, such as promoter and enhancers. As TE have the potential to regulate host genes, we hypothesized the RNA exosome co-transcriptional degrades TE found in open chromatin regions. We show that in the absence of the RNA exosome, various classes of TE are upregulated in mESC and EpiLC. Of note, we find MERVL to be amongst the top upregulated TE. MERVL expression is confined in the totipotent 2-cell embryo and becomes subsequently silenced as development progresses. Inability to degrade MERVL results in a reversion of pluripotent stem cells to a 2-cell like cell (2CLC) state. Additionally, we observed the exonization of MERVL LTR into host genes creating chimeric transcripts. Depression of MERVL in the absence of the results in increase RNAPII recruitment to the long terminal repeats (LTRs) and enrichment of H3K27ac, with global changes in methylation. Lastly, we use these data to inform about the etiology of pontocellular hypoplasia (PCH1b) in which mutations in the exosome subunit Exosc3 result in neurological disease. Together, our results indicate that the RNA exosome functions to maintain cellular states to prevent reversion to a previous cell state through co-transcriptional degradation of regulatory RNA.

Metabolic reprogramming promotes cancer aggressiveness and an immune-suppressive tumor microenvironment. Loss of the Y chromosome (LOY) drives both phenotypes in bladder cancer (BC). We investigated the hypothesis that LOY leads to metabolic reprogramming using untargeted metabolomic profiling of human BC cells and analysis of pan-cancer transcriptomic datasets. This revealed that aerobic glycolysis is activated in LOY BC cells. Since prior work showed that expression of collagen receptor DDR2 drives BC progression and DDR2 is a regulator of tumor metabolism, we investigated if DDR2 is implicated in metabolic reprogramming of LOY-tumors. Analysis of scRNAseq data from 251 patients with 12 tumor types found that LOY and DDR2 expression promote aerobic glycolysis, and this was confirmed by metabolomics. Deletion of DDR2 in LOY BC cells reduced glycolytic flux, inhibited cell proliferation, reduced EMT and stemness features, and promoted apoptosis. Our data provide a rationale for using LOY as a tumor selection biomarker for DDR2 targeted therapeutics.

Metabolic reprogramming promotes cancer aggressiveness and an immune-suppressive tumor microenvironment. Loss of the Y chromosome (LOY) drives both phenotypes in bladder cancer (BC). We investigated the hypothesis that LOY leads to metabolic reprogramming using untargeted metabolomic profiling of human BC cells and analysis of pan-cancer transcriptomic datasets. This revealed that aerobic glycolysis is activated in LOY BC cells. Since prior work showed that expression of collagen receptor DDR2 drives BC progression and DDR2 is a regulator of tumor metabolism, we investigated if DDR2 is implicated in metabolic reprogramming of LOY-tumors. Analysis of scRNAseq data from 251 patients with 12 tumor types found that LOY and DDR2 expression promote aerobic glycolysis, and this was confirmed by metabolomics. Deletion of DDR2 in LOY BC cells reduced glycolytic flux, inhibited cell proliferation, reduced EMT and stemness features, and promoted apoptosis. Our data provide a rationale for using LOY as a tumor selection biomarker for DDR2 targeted therapeutics. Loss of the Y chromosome augments glycolytic metabolism in bladder cancer cells, and this is in part dependent on the collagen receptor DDR2.

The lack of physiologically relevant in vitro prostate models has impeded studies of organ development and prostate tumorigenesis. We reprogrammed peripheral blood mononuclear cells (PBMCs) from individuals with and without pathogenic-germline BRCA2 mutation (MUT_BRCA2, CON_BRCA2) into induced pluripotent stem cells (iPSCs), which showed no differences in morphology, proliferation, or pluripotency markers. Differentiation of MUT_BRCA2 iPSCs into prostate organoids (iPROS) using defined growth factors and signaling molecules resulted in disrupted morphology, impaired polarity, increased proliferation, and elevated prostate-specific antigen (PSA) secretion compared to CON_BRCA2 iPROS. Transcriptomic profiling revealed early prostate cancer (PCa) signatures. Upon exposure to dietary carcinogens, MUT_BRCA2 iPROS showed further PSA elevation, enhanced proliferation, AMACR upregulation, p63 reducetion are markers of aggressive PCa. In vivo, MUT_BRCA2 iPROS formed tumors in immunodeficient mice. This patient-derived iPROS-platform recapitulates human-prostate mopphology and function, models early tumorigenesis events, and provides a valuable tool for studying PCa biology and enabling personalized drug discovery. In this study, we developed patients’ iPSC-derived prostate organoids (iPROS) with or without a pathogenic BRCA2 germline mutation that display human-prostate like morphology and function. MUT_BRCA2 iPROS displayed disrupted morphology, early tumorigenic changes, and formed tumors in mice. Upon carcinogen exposure, they showed markers of aggressive prostate cancer. This platform models early prostate tumorigenesis and enables personalized studies of cancer initiation and therapeutic response.

Human pluripotent stem cells are a tremendous tool to model early human development and disease including their use in the in vitro generation of blood cell fates. Hematopoietic progenitors and stem cells are the primary source of blood and the immune system from early development to adulthood and arise through successive waves of hemogenic mesoderm either in the yolk sac or embryo proper. Researchers have long sought a tractable human model for observing and distinguishing these waves of hematopoiesis in the dish for human developmental and disease modeling. Here we report a high-efficiency method for differentiating human pluripotent stem cells into an aorta-gonad-mesonephros-like definitive hemogenic mesoderm capable of giving rise to definitive hematopoietic progenitor and stem cells. The hematopoietic progenitor and stem cells exhibit robust multilineage in vitro colony forming potential. Gene expression analysis and single cell sequencing strongly support the developmental timing and notion that the pluripotent stem cell derived hematopoietic stem and progenitors are strikingly like bone fide hematopoietic stem cells. The hematopoietic progenitors can be subsequently differentiated into polarized macrophage and T-cells in vitro. Minimal silencing was observed upon differentiation of the pluripotent stem cells to hematopoietic lineages when conducting gene editing. Finally, upon engraftment into immunodeficient animals the hematopoietic progenitors and stem cells differentiate into multiple lineages including B-cells, T-cells, NK-cells, and monocytes.

in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans. Competing Interest Statement The Garcia-Sastre Laboratory has received research support from Pfizer, Senhwa Biosciences, 7Hills Pharma, Pharmamar, Blade Therapuetics, Avimex, Johnson & Johnson, Dynavax, Kenall Manufacturing and ImmunityBio. Adolfo Garcia-Sastre has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Accurius and Esperovax. M.J.T. is an employee, and M.S. is a co-founder of Enhanc3D Genomics Ltd. I.M. is an inventor in the patent, Serial Number: 16/063,009