Tumor-vascular interactions promote STING-driven inflammation in the tumor microenvironment

The recruitment of T cells following intratumoral administration of Stimulation of Interferon Genes (STING) agonists in the tumor microenvironment (TME) is a critical event in the STING-driven antitumor immune response, a pathway with great relevance in the context of cancer immunotherapy. We have previously demonstrated that LKB1 mutation is associated with suppression of tumor cell STING levels and reduced production of T-cell chemoattractants such as CXCL10 in KRAS-driven non-small cell lung cancer (NSCLC). Consistent with this, immunohistochemical staining of patient samples showed poor infiltration of CD3, CD4, and CD8 T cells into LKB1 negative versus LKB1 intact cancer epithelium, and instead, retention of T-cells in stroma. To examine how LKB1 alters immune cell recruitment in a STING-dependent manner, we used a 3-D microfluidic co-culture system to study interactions between vasculature and tumor spheroids derived from a KRAS/LKB1 mutated (KL) cell line with LKB1 reconstitution +/- STING deletion. To form the vasculature, we co-cultured tumor spheroids with fibroblasts and endothelial cells for 7 days, and identified changes in morphology, cytokine production, and gene expression that occur in co-culture. We first observed that co-culture induced synergistic production of multiple immune cell chemo-attractants such as CXCL10, CCL2, CCL5, and G-CSF. Interestingly, this more physiologic ex vivo tumor model of LKB1 reconstitution revealed particularly strong cooperative production of STING-dependent cytokines such as CXCL10 in the vasculature. Moreover, STING depletion in LKB1 reconstituted tumor cells did not significantly attenuate production of CXCL10 and other cytokines in co-culture, suggesting that tumor/vessel interaction may promote STING activation in the vasculature regardless of cancer cell-intrinsic STING function. Furthermore, although there was no appreciable response after treatment of KL cancer cells with cGAMP based STING agonists, treatment of isolated 3-D vascular networks with cGAMP enhanced vascular permeability and increased production of CXCL10 and CCL5, possibly contributing to defective chemokine gradients that retain T cells near the vasculature. Thus, developing these more complex models that incorporate the vasculature may elucidate important aspects of STING biology and may ultimately aid further development of effective immunotherapies targeting this signaling axi

Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses.

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy

Estimation of genetic diversity and relatedness in a mango germplasm collection using SNP markers and a simplified visual analysis method

Mango is a globally important tropical fruit but lacks genomic tools to support cultivar identification and to enable breeding efforts. Assessing the genetic diversity and relatedness of mango germplasm is essential for identifying genetically distant parents with favorable agronomic traits to produce hybrid populations enabling selection of improved cultivars. We thus genotyped 1915 mango accessions from the United States, Senegal, Thailand, and Australia with 272 single nucleotide polymorphism (SNP) markers identifying over 520,000 genotypes. These accessions represent the available diversity from both public and private germplasm collections in these countries, as well as accessions from smaller international collections. The study included Mangifera indica, other Mangifera species, and accessions from half sibling populations. Genotype data were analyzed using an affinity propagation method to define 258 groups. Using a simple visual method, no more than 30 SNPs are needed to distinguish a single cultivar of interest from all other cultivars in the dataset enabling the accurate identification of important commercial cultivars. As these SNP markers provided accurate genotype data for accessions from different genera as well as half siblings, the majority of the genetic diversity of the mango germplasm and related species that were genotyped has been captured. The dataset contains a large collection of open-pollinated half siblings from known maternal parents. A simple visual method can also be used to identify self-pollinated individuals among the half siblings of known maternal parents and, in some cases, to infer likely candidates for the paternal parent. Identification of self-pollinated individuals is particularly important in terms of selection of improved cultivars, as due to high levels of heterozygosity, self-pollinated progeny are likely to uncover deleterious recessive alleles. Genotyping of progeny at the seedling stage and removal of self-pollinated progeny can increase the efficiency and decrease the costs of selection of improved cultivars from open-pollinated populations

Synthetic Lethal Interaction between Oncogenic KRAS Dependency and STK33 Suppression in Human Cancer Cells

An alternative to therapeutic targeting of oncogenes is to perform “synthetic lethality” screens for genes that are essential only in the context of specific cancer-causing mutations. We used high-throughput RNA interference (RNAi) to identify synthetic lethal interactions in cancer cells harboring mutant KRAS, the most commonly mutated human oncogene. We find that cells that are dependent on mutant KRAS exhibit sensitivity to suppression of the serine/threonine kinase STK33 irrespective of tissue origin, whereas STK33 is not required by KRAS-independent cells. STK33 promotes cancer cell viability in a kinase activity-dependent manner by regulating the suppression of mitochondrial apoptosis mediated through S6K1-induced inactivation of the death agonist BAD selectively in mutant KRAS-dependent cells. These observations identify STK33 as a target for treatment of mutant KRAS-driven cancers and demonstrate the potential of RNAi screens for discovering functional dependencies created by oncogenic mutations that may enable therapeutic intervention for cancers with “undruggable” genetic alterations.National Institutes of Health (U.S.) (grant R33 CA128625)National Institutes of Health (U.S.) (grant NIH U54 CA112962)National Institutes of Health (U.S.) (grant P01 CA095616)National Institutes of Health (U.S.) (grant P01 CA66996)Starr Cancer ConsortiumDoris Duke Charitable FoundationMPN Research FoundationDeutsche Forschungsgemeinschaft (grant SCHO 1215/1-1)Deutsche Forschungsgemeinschaft (grant FR 2113/1-1)Brain Science FoundationLeukemia & Lymphoma Society of Americ

Characterizing genomic alterations in cancer by complementary functional associations.

Systematic efforts to sequence the cancer genome have identified large numbers of mutations and copy number alterations in human cancers. However, elucidating the functional consequences of these variants, and their interactions to drive or maintain oncogenic states, remains a challenge in cancer research. We developed REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene dependency of oncogenic pathways or sensitivity to a drug treatment. We used REVEALER to uncover complementary genomic alterations associated with the transcriptional activation of β-catenin and NRF2, MEK-inhibitor sensitivity, and KRAS dependency. REVEALER successfully identified both known and new associations, demonstrating the power of combining functional profiles with extensive characterization of genomic alterations in cancer genomes

Creation of an avocado unambiguous genotype SNP database for germplasm curation and as an aid to breeders

Avocado (Persea americana) is an important tropical and subtropical fruit tree crop. Traditional tree breeding programs face the challenges of long generation times and significant expense in land and personnel resources. Avocado selection and breeding can be more efficient and less expensive through the development and application of molecular markers. A total of 1524 individuals were genotyped with 384 SNPs creating the largest SNP genotype database for avocado. These individuals correspond to four extensive germplasm collections including two housed in Florida and two in California. In addition, hybrids and selections from two rootstock breeding programs have been genotyped. Genotype data were analyzed using an affinity propagation method to define 155 groups. The 384 SNP markers provided accurate genotype data for individuals from different Persea species as well as half-siblings. Therefore, the majority of the genetic diversity of the avocado germplasm and related species that were genotyped has been captured. A simple visual method can also be used to identify self-pollinated individuals among the half-siblings of known maternal parents and, in some cases, to infer likely candidates for the paternal parent. Finally, this dataset is unambiguous so breeders can determine the genetic diversity of their breeding stock to optimize avocado breeding and selection programs by identifying outcrossed individuals at the seedling stage, thus increasing the efficiency of avocado genetic improvement.Supplementary Table 1. The sequences of the 384 SNP assays with the associated linkage group (LG), map position in centimorgans (cM), and annotation where available.Supplementary Table 2. Colored data file with affinity propagation groups, silhouette scores, and related data for biallelic SNP markers (384) used to genotype 1,524 germplasm individuals. Data were curated to remove markers with greater than 5% missing data and individuals with greater than 5% missing data in a recursive fashion, resulting in a dataset of genotypes of 377 markers for 1,461 individuals.Supplementary Table 3. Examples of SNP markers that distinguish between landraces.Data archiving statement. All SNP data from this study can be found in Supplementary Table 2.Trust Fund Cooperative Agreement 58-6038-7-006 with the California Avocado Commission and USDA-ARS CRIS 58-6038-21000-022-00D.http://link.springer.com/journal/11295hj2020BiochemistryGeneticsMicrobiology and Plant Patholog

Virtual Reality, 360⁰ Video, and Journalism Studies: Conceptual Approaches to Immersive Technologies

A growing number of newsrooms are experimenting with Virtual Reality (VR) and other immersive storytelling techniques, typically supported by technology companies that see journalism as a potential vehicle for taking VR mainstream. The resulting pieces have been wide-ranging in topic, style, and scope, but all introduce new complexities to journalistic norms and practices. To date, however, journalism studies scholars have conducted relatively little research into these immersive technologies. This essay proposes three conceptual approaches to examining VR journalism: Actor-Network Theory, normative theory, and a sociological perspective on journalistic work

Effects of parental origins and length of residency on adiposity measures and nutrition in urban middle school students: a cross-sectional study

Background: The prevalence of obesity in U.S. has been rising at an alarming rate, particularly among Hispanic, African, and Asian minority groups. This trend is due in part to excessive calorie consumption and sedentary lifestyle. We sought to investigate whether parental origins influence eating behaviors in healthy urban middle school students. Methods: A multiethnic/racial population of students (N = 182) enrolled in the ROAD (Reduce Obesity and Diabetes) Study, a school-based trial to assess clinical, behavioral, and biochemical risk factors for adiposity and its co-morbidities completed questionnaires regarding parental origins, length of US residency, and food behaviors and preferences. The primary behavioral questionnaire outcome variables were nutrition knowledge, attitude, intention and behavior, which were then related to anthropometric measures of waist circumference, BMI z-scores, and percent body fat. Two-way analysis of variance was used to evaluate the joint effects of number of parents born in the U.S. and ethnicity on food preference and knowledge score. The Tukey-Kramer method was used to compute pairwise comparisons to determine where differences lie. Analysis of covariance (ANCOVA) was used to analyze the joint effects of number of parents born in the US and student ethnicity, along with the interaction term, on each adiposity measure outcome. Pearson correlation coefficients were used to examine the relationships between maternal and paternal length of residency in the US with measures of adiposity, food preference and food knowledge. Results: African Americans had significantly higher BMI, waist circumference and body fat percentage compared to other racial and ethnic groups. Neither ethnicity/race nor parental origins had an impact on nutrition behavior. Mothers’ length of US residency positively correlated with students’ nutrition knowledge, but not food attitude, intention or behavior. Conclusions: Adiposity measures in children differ according to ethnicity and race. In contrast, food behaviors in this middle school sample were not influenced by parental origins. Longer maternal US residency benefited offspring in terms of nutrition knowledge only. We suggest that interventions to prevent obesity begin in early childhood

Whole-exome sequencing and clinical interpretation of FFPE tumor samples to guide precision cancer medicine

Translating whole exome sequencing (WES) for prospective clinical use may impact the care of cancer patients; however, multiple innovations are necessary for clinical implementation. These include: (1) rapid and robust WES from formalin-fixed paraffin embedded (FFPE) tumor tissue, (2) analytical output similar to data from frozen samples, and (3) clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival FFPE tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a “long tail” of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15/16 patients. In one patient, previously undetected findings guided clinical trial enrollment leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine