Ancestral library identifies conserved reprogrammable liver motif on AAV capsid.

Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.

A single-cell atlas of human and mouse white adipose tissue.

White adipose tissue, once regarded as morphologically and functionally bland, is now recognized to be dynamic, plastic and heterogenous, and is involved in a wide array of biological processes including energy homeostasis, glucose and lipid handling, blood pressure control and host defence1. High-fat feeding and other metabolic stressors cause marked changes in adipose morphology, physiology and cellular composition1, and alterations in adiposity are associated with insulin resistance, dyslipidemia and type 2 diabetes2. Here we provide detailed cellular atlases of human and mouse subcutaneous and visceral white fat at single-cell resolution across a range of body weight. We identify subpopulations of adipocytes, adipose stem and progenitor cells, vascular and immune cells and demonstrate commonalities and differences across species and dietary conditions. We link specific cell types to increased risk of metabolic disease and provide an initial blueprint for a comprehensive set of interactions between individual cell types in the adipose niche in leanness and obesity. These data comprise an extensive resource for the exploration of genes, traits and cell types in the function of white adipose tissue across species, depots and nutritional conditions.

Novel Transgenic Lines to Analyze Renal Glutathione Redox Potential In Vivo.

Reactive oxygen species (ROS) are important regulators of intracellular signaling pathways in health and disease. It is implicated that ROS may play critical roles in pathogenesis of a number of kidney diseases including diabetic nephropathy. However, due to the lack of tools for in vivo detection of redox status, our knowledge of redox dynamics is still fragmentary. In this study, we present novel zebrafish UAS transgenic lines expressing mitochondrial and cytoplasmic targeted redox fluorescent biosensors, Grx1-roGFP2 and mitoGrx1-roGFP2. As the zebrafish is an ideal animal model for intravital imaging, these transgenic zebrafish provide useful tools to analyze renal redox dynamics in vivo.

Genome-Wide Interrogation of Human Cancers Identifies EGLN1 Dependency in Clear Cell Ovarian Cancers.

We hypothesized that candidate dependencies for which there are small molecules that are either approved or in advanced development for a nononcology indication may represent potential therapeutic targets. To test this hypothesis, we performed genome-scale loss-of-function screens in hundreds of cancer cell lines. We found that knockout of EGLN1, which encodes prolyl hydroxylase domain-containing protein 2 (PHD2), reduced the proliferation of a subset of clear cell ovarian cancer cell lines in vitro. EGLN1-dependent cells exhibited sensitivity to the pan-EGLN inhibitor FG-4592. The response to FG-4592 was reversed by deletion of HIF1A, demonstrating that EGLN1 dependency was related to negative regulation of HIF1A. We also found that ovarian clear cell tumors susceptible to both genetic and pharmacologic inhibition of EGLN1 required intact HIF1A. Collectively, these observations identify EGLN1 as a cancer target with therapeutic potential. SIGNIFICANCE: These findings reveal a differential dependency of clear cell ovarian cancers on EGLN1, thus identifying EGLN1 as a potential therapeutic target in clear cell ovarian cancer patients.

Defining a Cancer Dependency Map.

Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on- from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six SDs from the mean. We found predictive models for 426 dependencies (55%) by nonlinear regression modeling considering 66,646 molecular features. Many dependencies fall into a limited number of classes, and unexpectedly, in 82% of models, the top biomarkers were expression based. We demonstrated the basis behind one such predictive model linking hypermethylation of the UBB ubiquitin gene to a dependency on UBC. Together, these observations provide a foundation for a cancer dependency map that facilitates the prioritization of therapeutic targets.

The zebrafish foxj1a transcription factor regulates cilia function in response to injury and epithelial stretch.

Cilia are essential for normal organ function and developmental patterning, but their role in injury and regeneration responses is unknown. To probe the role of cilia in injury, we analyzed the function of foxj1, a transcriptional regulator of cilia genes, in response to tissue damage and renal cyst formation. Zebrafish foxj1a, but not foxj1b, was rapidly induced in response to epithelial distension and stretch, kidney cyst formation, acute kidney injury by gentamicin, and crush injury in spinal cord cells. Obstruction-induced up-regulation of foxj1a was not inhibited by cycloheximide, identifying foxj1a as a primary response gene to epithelial injury. Foxj1 was also dramatically up-regulated in murine cystic kidney disease epithelia [jck/jck (nek8) and Ift88Tg737Rpw(-/-)] as well as in response to kidney ischemia-reperfusion injury. Obstruction of the zebrafish pronephric tubule caused a rapid increase in cilia beat rate that correlated tightly with expanded tubule diameter and epithelial stretch. Zebrafish foxj1a was specifically required for cilia motility. Enhanced foxj1a expression in obstructed tubules induced cilia motility target genes efhc1, tektin-1, and dnahc9. foxj1a-deficient embryos failed to up-regulate efhc1, tektin-1, and dnahc9 and could not maintain enhanced cilia beat rates after obstruction, identifying an essential role for foxj1 in modulating cilia function after injury. These studies reveal that activation of a Foxj1 transcriptional network of ciliogenic genes is an evolutionarily conserved response to multiple forms of tissue damage and highlight enhanced cilia function as a previously uncharacterized component of organ homeostasis.

High density DNA array analysis reveals distinct genomic profiles in a subset of gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) generally harbor activating mutations in KIT or platelet-derived growth facter receptor (PDGFRA). Mutations in these receptor tyrosine kinases lead to dysregulation of downstream signaling pathways that contribute to GIST pathogenesis. GISTs with KIT or PDGFRA mutations also undergo secondary cytogenetic alterations that may indicate the involvement of additional genes important in tumor progression. Approximately 10-15% of adult and 85% of pediatric GISTs do not have mutations in KIT or in PDGFRA. Most mutant adult GISTs display large-scale genomic alterations, but little is known about the mutation-negative tumors. Using genome-wide DNA arrays, we investigated genomic imbalances in a set of 31 GISTs, including 10 KIT/PDGFRA mutation-negative tumors from nine adults and one pediatric case and 21 mutant tumors. Although all 21 mutant GISTs exhibited multiple copy number aberrations, notably losses, eight of the 10 KIT/PDGFRA mutation-negative GISTs exhibited few or no genomic alterations. One KIT/PDGFRA mutation-negative tumor exhibiting numerous genomic changes was found to harbor an alternate activating mutation, in the serine-threonine kinase BRAF. The only other mutation-negative GIST with significant chromosomal imbalances was a recurrent metastatic tumor found to harbor a homozygous deletion in chromosome arm 9p. Similar findings in several KIT-mutant GISTs identified a minimal overlapping region of deletion of approximately 0.28 Mbp in 9p21.3 that includes only the CDKN2A/2B genes, which encode inhibitors of cell-cycle kinases. These results suggest that GISTs without activating kinase mutations, whether pediatric or adult, generally exhibit a much lower level of cytogenetic progression than that observed in mutant GISTs.

Insulin-like growth factor 1 receptor is a potential therapeutic target for gastrointestinal stromal tumors.

A subset of gastrointestinal stromal tumors (GISTs) lack gain-of-function mutations in c-KIT and PDGFRalpha. These so-called wild-type (WT) GISTs tend to be less responsive to imatinib-based therapies and have a poor prognosis. We identified amplification of IGF1R in a SNP analysis of GIST and thus studied its potential as a therapeutic target in WT and mutant GIST. Expression of IGF1R and downstream effectors in clinical GIST samples was examined by using immunoblots and immunohistochemistry. The roles of IGF1R signaling in GIST and viability were analyzed by using NVP-AEW541, an inhibitor of IGF1R, alone and in combination with imatinib, or via siRNA silencing of IGF1R. IGF1R was strongly overexpressed, and IGF1R amplification was detected at a significantly higher frequency in WT GISTs, including a pediatric WT GIST, compared with mutant GISTs (P = 0.0173 and P = 0.0163, respectively). Inhibition of IGF1R activity in vitro with NVP-AEW541 or down-regulation of expression with siIGF1R led to cytotoxicity and induced apoptosis in GIST cell lines via AKT and MAPK signaling. Combination of NVP-AEW541 and imatinib in GIST cell lines induced a strong cytotoxicity response. Our results reveal that IGF1R is amplified and the protein is overexpressed in WT and pediatric GISTs. We also demonstrate that the aberrant expression of IGF1R may be associated with oncogenesis in WT GISTs and suggest an alternative and/or complementary therapeutic regimen in the clinical management of all GISTs, especially in a subset of tumors that respond less favorably to imatinib-based therapy.

Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse.

BACKGROUND: Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. RESULTS: We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. CONCLUSION: In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.

Analysis of KIT mutations in sporadic and familial gastrointestinal stromal tumors: therapeutic implications through protein modeling.

PURPOSE: Gastrointestinal stromal tumors (GIST) are characterized by expressing a gain-of-function mutation in KIT, and to a lesser extent, PDGFR. Imatinib mesylate, a tyrosine kinase inhibitor, has activity against GISTs that contain oncogenic mutations of KIT. In this study, KIT and PDGFRalpha mutation status was analyzed and protein modeling approaches were used to assess the potential effect of KIT mutations in response to imatinib therapy. EXPERIMENTAL DESIGN: Genomic DNA was isolated from GIST tumors. Exons 9, 11, 13, and 17 of c-KIT and exons 12, 14, and 18 of PDGFRalpha were evaluated for oncogenic mutations. Protein modeling was used to assess mutations within the juxtamembrane region and the kinase domain of KIT. RESULTS: Mutations in KIT exons 9, 11, and 13 were identified in GISTs with the majority of changes involving the juxtamembrane region of KIT. Molecular modeling indicates that mutations in this region result in disruption of the KIT autoinhibited conformation, and lead to gain-of-function activation of the kinase. Furthermore, a novel germ-line mutation in KIT was identified that is associated with an autosomal dominant predisposition to the development of GIST. CONCLUSIONS: We have used protein modeling and structural analyses to elucidate why patients with GIST tumors containing exon 11 mutations are the most responsive to imatinib mesylate treatment. Importantly, mutations detected in this exon and others displayed constitutive activation of KIT. Furthermore, we have found tumors that are both KIT and PDGFRalpha mutation negative, suggesting that additional, yet unidentified, abnormalities may contribute to GIST tumorigenesis.