Activating Transcription Factor 3 Stimulates Follicle-Stimulating Hormone-ß Expression In Vitro But Is Dispensable for Follicle-Stimulating Hormone Production in Murine Gonadotropes In Vivo.

Follicle-stimulating hormone (FSH), a dimeric glycoprotein produced by pituitary gonadotrope cells, regulates spermatogenesis in males and ovarian follicle growth in females. Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates FSHß subunit gene (Fshb) transcription, though the underlying mechanisms are poorly understood. To address this gap in knowledge, we examined changes in pituitary gene expression in GnRH-deficient mice (hpg) treated with a regimen of exogenous GnRH that increases pituitary Fshb but not luteinizing hormone ß (Lhb) messenger RNA levels. Activating transcription factor 3 (Atf3) was among the most upregulated genes. Activating transcription factor 3 (ATF3) can heterodimerize with members of the activator protein 1 family to regulate gene transcription. Co-expression of ATF3 with JunB stimulated murine Fshb, but not Lhb, promoter-reporter activity in homologous LßT2b cells. ATF3 also synergized with a constitutively active activin type I receptor to increase endogenous Fshb expression in these cells. Nevertheless, FSH production was intact in gonadotrope-specific Atf3 knockout [conditional knockout (cKO)] mice. Ovarian follicle development, ovulation, and litter sizes were equivalent between cKOs and controls. Testis weights and sperm counts did not differ between genotypes. Following gonadectomy, increases in LH secretion were enhanced in cKO animals. Though FSH levels did not differ between genotypes, post-gonadectomy increases in pituitary Fshb and gonadotropin α subunit expression were more pronounced in cKO than control mice. These data indicate that ATF3 can selectively stimulate Fshb expression in vitro but is not required for FSH production in vivo.

Skeletal muscle transcriptome response to a bout of endurance exercise in physically active and sedentary older adults.

Age-related declines in cardiorespiratory fitness and physical function are mitigated by regular endurance exercise in older adults. This may be due, in part, to changes in the transcriptional program of skeletal muscle following repeated bouts of exercise. However, the impact of chronic exercise training on the transcriptional response to an acute bout of endurance exercise has not been clearly determined. Here, we characterized baseline differences in muscle transcriptome and exercise-induced response in older adults who were active/endurance trained or sedentary. RNA-sequencing was performed on vastus lateralis biopsy specimens obtained before, immediately after, and 3 h following a bout of endurance exercise (40 min of cycling at 60%-70% of heart rate reserve). Using a recently developed bioinformatics approach, we found that transcript signatures related to type I myofibers, mitochondria, and endothelial cells were higher in active/endurance-trained adults and were associated with key phenotypic features including V̇o2peak, ATPmax, and muscle fiber proportion. Immune cell signatures were elevated in the sedentary group and linked to visceral and intermuscular adipose tissue mass. Following acute exercise, we observed distinct temporal transcriptional signatures that were largely similar among groups. Enrichment analysis revealed catabolic processes were uniquely enriched in the sedentary group at the 3-h postexercise timepoint. In summary, this study revealed key transcriptional signatures that distinguished active and sedentary adults, which were associated with difference in oxidative capacity and depot-specific adiposity. The acute response signatures were consistent with beneficial effects of endurance exercise to improve muscle health in older adults irrespective of exercise history and adiposity.NEW & NOTEWORTHY Muscle transcript signatures associated with oxidative capacity and immune cells underlie important phenotypic and clinical characteristics of older adults who are endurance trained or sedentary. Despite divergent phenotypes, the temporal transcriptional signatures in response to an acute bout of endurance exercise were largely similar among groups. These data provide new insight into the transcriptional programs of aging muscle and the beneficial effects of endurance exercise to promote healthy aging in older adults.

Anti-invasive efficacy and survival benefit of the YAP-TEAD inhibitor verteporfin in preclinical glioblastoma models.

BACKGROUND: Glioblastoma (GBM) remains a largely incurable disease as current therapy fails to target the invasive nature of glioma growth in disease progression and recurrence. Here, we use the FDA-approved drug and small molecule Hippo inhibitor Verteporfin (VP) to target YAP-TEAD activity, known to mediate convergent aspects of tumor invasion/metastasis, and assess the drug's efficacy and survival benefit in GBM models. METHODS: Up to 8 low-passage patient-derived GBM cell lines with distinct genomic drivers, including 3 primary/recurrent pairs, were treated with VP or vehicle (VEH) to assess in vitro effects on proliferation, migration, invasion, YAP-TEAD activity, and transcriptomics. Patient-derived orthotopic xenograft (PDX) models were used to assess VP's brain penetrance and effects on tumor burden and survival. RESULTS: VP treatment disturbed YAP/TAZ-TEAD activity; disrupted transcriptome signatures related to invasion, epithelial-to-mesenchymal, and proneural-to-mesenchymal transition, phenocopying TEAD1-knockout effects; and impaired tumor migration/invasion dynamics across primary and recurrent GBM lines. In an aggressive orthotopic PDX GBM model, short-term VP treatment consistently diminished core and infiltrative tumor burden, which was associated with decreased tumor expression of Ki67, nuclear YAP, TEAD1, and TEAD-associated targets EGFR, CDH2, and ITGB1. Finally, long-term VP treatment appeared nontoxic and conferred survival benefit compared to VEH in 2 PDX models: as monotherapy in primary (de novo) GBM and in combination with Temozolomide chemoradiation in recurrent GBM, where VP treatment associated with increased MGMT methylation. CONCLUSIONS: We demonstrate combined anti-invasive and anti-proliferative efficacy for VP with survival benefit in preclinical GBM models, indicating potential therapeutic value of this already FDA-approved drug if repurposed for GBM patients.

Prototypical oncogene family Myc defines unappreciated distinct lineage states of small cell lung cancer.

Comprehensive genomic analyses of small cell lung cancer (SCLC) have revealed frequent mutually exclusive genomic amplification of MYC family members. Hence, it has been long suggested that they are functionally equivalent; however, more recently, their expression has been associated with specific neuroendocrine markers and distinct histopathology. Here, we explored a previously undescribed role of L-Myc and c-Myc as lineage-determining factors contributing to SCLC molecular subtypes and histology. Integrated transcriptomic and epigenomic analyses showed that L-Myc and c-Myc impart neuronal and non-neuroendocrine-associated transcriptional programs, respectively, both associated with distinct SCLC lineage. Genetic replacement of c-Myc with L-Myc in c-Myc-SCLC induced a neuronal state but was insufficient to induce ASCL1-SCLC. In contrast, c-Myc induced transition from ASCL1-SCLC to NEUROD1-SCLC characterized by distinct large-cell neuroendocrine carcinoma-like histopathology. Collectively, we characterize a role of historically defined general oncogenes, c-Myc and L-Myc, for regulating lineage plasticity across molecular and histological subtypes.

Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma.

The intrinsic drivers of migration in glioblastoma (GBM) are poorly understood. To better capture the native molecular imprint of GBM and its developmental context, here we isolate human stem cell populations from GBM (GSC) and germinal matrix tissues and map their chromatin accessibility via ATAC-seq. We uncover two distinct regulatory GSC signatures, a developmentally shared/proliferative and a tumor-specific/migratory one in which TEAD1/4 motifs are uniquely overrepresented. Using ChIP-PCR, we validate TEAD1 trans occupancy at accessibility sites within AQP4, EGFR, and CDH4. To further characterize TEAD's functional role in GBM, we knockout TEAD1 or TEAD4 in patient-derived GBM lines using CRISPR-Cas9. TEAD1 ablation robustly diminishes migration, both in vitro and in vivo, and alters migratory and EMT transcriptome signatures with consistent downregulation of its target AQP4. TEAD1 overexpression restores AQP4 expression, and both TEAD1 and AQP4 overexpression rescue migratory deficits in TEAD1-knockout cells, implicating a direct regulatory role for TEAD1-AQP4 in GBM migration.

DNA polymerase η mutational signatures are found in a variety of different types of cancer.

DNA polymerase (pol) η is a specialized error-prone polymerase with at least two quite different and contrasting cellular roles: to mitigate the genetic consequences of solar UV irradiation, and promote somatic hypermutation in the variable regions of immunoglobulin genes. Misregulation and mistargeting of pol η can compromise genome integrity. We explored whether the mutational signature of pol η could be found in datasets of human somatic mutations derived from normal and cancer cells. A substantial excess of single and tandem somatic mutations within known pol η mutable motifs was noted in skin cancer as well as in many other types of human cancer, suggesting that somatic mutations in A:T bases generated by DNA polymerase η are a common feature of tumorigenesis. Another peculiarity of pol ηmutational signatures, mutations in YCG motifs, led us to speculate that error-prone DNA synthesis opposite methylated CpG dinucleotides by misregulated pol η in tumors might constitute an additional mechanism of cytosine demethylation in this hypermutable dinucleotide.

Prospective Isolation and Comparison of Human Germinal Matrix and Glioblastoma EGFR+ Populations with Stem Cell Properties.

Characterization of non-neoplastic and malignant human stem cell populations in their native state can provide new insights into gliomagenesis. Here we developed a purification strategy to directly isolate EGFR+/- populations from human germinal matrix (GM) and adult subventricular zone autopsy tissues, and from de novo glioblastoma (GBM) resections, enriching for cells capable of binding EGF ligand (LBEGFR+), and uniquely compared their functional and molecular properties. LBEGFR+ populations in both GM and GBM encompassed all sphere-forming cells and displayed proliferative stem cell properties in vitro. In xenografts, LBEGFR+ GBM cells showed robust tumor initiation and progression to high-grade, infiltrative gliomas. Whole-transcriptome sequencing analysis confirmed enrichment of proliferative pathways in both developing and neoplastic freshly isolated EGFR+ populations, and identified both unique and shared sets of genes. The ability to prospectively isolate stem cell populations using native ligand-binding capacity opens new doors onto understanding both normal human development and tumor cell biology.

Activation induced deaminase mutational signature overlaps with CpG methylation sites in follicular lymphoma and other cancers.

Follicular lymphoma (FL) is an uncurable cancer characterized by progressive severity of relapses. We analyzed sequence context specificity of mutations in the B cells from a large cohort of FL patients. We revealed substantial excess of mutations within a novel hybrid nucleotide motif: the signature of somatic hypermutation (SHM) enzyme, Activation Induced Deaminase (AID), which overlaps the CpG methylation site. This finding implies that in FL the SHM machinery acts at genomic sites containing methylated cytosine. We identified the prevalence of this hybrid mutational signature in many other types of human cancer, suggesting that AID-mediated, CpG-methylation dependent mutagenesis is a common feature of tumorigenesis.

The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination.

Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination.

Astrocytic TYMP and VEGFA drive blood-brain barrier opening in inflammatory central nervous system lesions.

In inflammatory central nervous system conditions such as multiple sclerosis, breakdown of the blood-brain barrier is a key event in lesion pathogenesis, predisposing to oedema, excitotoxicity, and ingress of plasma proteins and inflammatory cells. Recently, we showed that reactive astrocytes drive blood-brain barrier opening, via production of vascular endothelial growth factor A (VEGFA). Here, we now identify thymidine phosphorylase (TYMP; previously known as endothelial cell growth factor 1, ECGF1) as a second key astrocyte-derived permeability factor, which interacts with VEGFA to induce blood-brain barrier disruption. The two are co-induced NFκB1-dependently in human astrocytes by the cytokine interleukin 1 beta (IL1B), and inactivation of Vegfa in vivo potentiates TYMP induction. In human central nervous system microvascular endothelial cells, VEGFA and the TYMP product 2-deoxy-d-ribose cooperatively repress tight junction proteins, driving permeability. Notably, this response represents part of a wider pattern of endothelial plasticity: 2-deoxy-d-ribose and VEGFA produce transcriptional programs encompassing angiogenic and permeability genes, and together regulate a third unique cohort. Functionally, each promotes proliferation and viability, and they cooperatively drive motility and angiogenesis. Importantly, introduction of either into mouse cortex promotes blood-brain barrier breakdown, and together they induce severe barrier disruption. In the multiple sclerosis model experimental autoimmune encephalitis, TYMP and VEGFA co-localize to reactive astrocytes, and correlate with blood-brain barrier permeability. Critically, blockade of either reduces neurologic deficit, blood-brain barrier disruption and pathology, and inhibiting both in combination enhances tissue preservation. Suggesting importance in human disease, TYMP and VEGFA both localize to reactive astrocytes in multiple sclerosis lesion samples. Collectively, these data identify TYMP as an astrocyte-derived permeability factor, and suggest TYMP and VEGFA together promote blood-brain barrier breakdown.

Combinatorial cytokine code generates anti-viral state in dendritic cells.

The physiological function of the immune system and the response to therapeutic immunomodulators may be sensitive to combinatorial cytokine micro-environments that shape the responses of specific immune cells. Previous work shows that paracrine cytokines released by virus-infected human dendritic cells (DC) can dictate the maturation state of naïve DCs. To understand the effects of paracrine signaling, we systematically studied the effects of combinations cytokines in this complex mixture in generating an anti-viral state. After naïve DCs were exposed to either IFNß or to paracrine signaling released by DCs infected by Newcastle disease virus (NDV), microarray analysis revealed a large number of genes that were differently regulated by the DC-secreted paracrine signaling. In order to identify the cytokine mechanisms involved, we identified 20 cytokines secreted by NDV infected DCs for which the corresponding receptor gene is expressed in naïve DCs. By exposing cells to all combinations of 19 cytokines (leave-one-out studies), we identified five cytokines (IFNß, TNFα, IL-1ß, TNFSF15, and IL28) as candidates for regulating DC maturation markers. Subsequent experiments identified IFNß, TNFα, and IL1ß as the major contributors to this anti-viral state. This finding was supported by infection studies in vitro, by T-cell activation studies and by in vivo infection studies in mouse. Combination of cytokines can cause response states in DCs that differ from those achieved by the individual cytokines alone. These results suggest that the cytokine microenvironment may act via a combinatorial code to direct the response state of specific immune cells. Further elucidation of this code may provide insight into responses to infection and neoplasia as well as guide the development of combinatorial cytokine immunomodulation for infectious, autoimmune, and immunosurveillance-related diseases.

Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters.

Key synaptic proteins from the soluble SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters, and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and nonclustered molecules at the membrane of PC12 cells with single-molecule precision. Direct stochastic optical reconstruction microscopy images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The nonclustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Although syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical, and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule-based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.

Role of cell-to-cell variability in activating a positive feedback antiviral response in human dendritic cells.

In the first few hours following Newcastle disease viral infection of human monocyte-derived dendritic cells, the induction of IFNB1 is extremely low and the secreted type I interferon response is below the limits of ELISA assay. However, many interferon-induced genes are activated at this time, for example DDX58 (RIGI), which in response to viral RNA induces IFNB1. We investigated whether the early induction of IFNBI in only a small percentage of infected cells leads to low level IFN secretion that then induces IFN-responsive genes in all cells. We developed an agent-based mathematical model to explore the IFNBI and DDX58 temporal dynamics. Simulations showed that a small number of early responder cells provide a mechanism for efficient and controlled activation of the DDX58-IFNBI positive feedback loop. The model predicted distributions of single cell responses that were confirmed by single cell mRNA measurements. The results suggest that large cell-to-cell variation plays an important role in the early innate immune response, and that the variability is essential for the efficient activation of the IFNB1 based feedback loop.