Adar1 deletion causes degeneration of the exocrine pancreas via Mavs-dependent interferon signaling.

Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA-binding protein that deaminates adenosine (A) to inosine (I). A-to-I editing alters post-transcriptional RNA processing, making ADAR1 a crucial regulator of gene expression. Consequently, Adar1 has been implicated in organogenesis. To determine the role of Adar1 in pancreatic development and homeostasis, we conditionally deleted Adar1 from the murine pancreas (Ptf1aCre/+; Adar1Fl/Fl). The resulting mice had stunted growth, likely due to malabsorption associated with exocrine pancreatic insufficiency. Analyses of pancreata revealed ductal cell expansion, heightened interferon-stimulated gene expression and an increased influx of immune cells. Concurrent deletion of Adar1 and Mavs, a signaling protein implicated in the innate immune pathway, rescued the degenerative phenotype and resulted in normal pancreatic development. Taken together, our work suggests that the primary function of Adar1 in the pancreas is to prevent aberrant activation of the Mavs-mediated innate immune pathway, thereby maintaining pancreatic homeostasis.

A Programmable In Vivo CRISPR Activation Model Elucidates the Oncogenic and Immunosuppressive Functions of MYC in Lung Adenocarcinoma.

Conventional genetically engineered mouse models (GEMM) are time-consuming, laborious, and offer limited spatiotemporal control. Here, we describe the development of a streamlined platform for in vivo gene activation using CRISPR activation (CRISPRa) technology. Unlike conventional GEMMs, this model system allows for flexible, sustained, and timed activation of one or more target genes using single or pooled lentiviral guides. Myc and Yap1 were used as model oncogenes to demonstrate gene activation in primary pancreatic organoid cultures in vitro and enhanced tumorigenic potential in Myc-activated organoids when transplanted orthotopically in vivo. Implementation of this model as an autochthonous lung cancer model showed that transduction-mediated activation of Myc led to accelerated tumor progression and significantly reduced overall survival relative to nontargeted tumor controls. Furthermore, Myc activation led to the acquisition of an immune suppressive, "cold" tumor microenvironment. Cross-species validation of these results using publicly available RNA/DNA-seq datasets linked MYC to a previously described immunosuppressive molecular subtype in patient tumors, thus identifying a patient cohort that may benefit from combined MYC- and immune-targeted therapies. Overall, this work demonstrates how CRISPRa can be used for rapid functional validation of putative oncogenes and may allow for the identification and evaluation of potential metastatic and oncogenic drivers through competitive screening. SIGNIFICANCE: A streamlined platform for programmable CRISPR gene activation enables rapid evaluation and functional validation of putative oncogenes in vivo.

APOBEC3A drives deaminase domain-independent chromosomal instability to promote pancreatic cancer metastasis.

Despite efforts in understanding its underlying mechanisms, the etiology of chromosomal instability (CIN) remains unclear for many tumor types. Here, we identify CIN initiation as a previously undescribed function for APOBEC3A (A3A), a cytidine deaminase upregulated across cancer types. Using genetic mouse models of pancreatic ductal adenocarcinoma (PDA) and genomics analyses in human tumor cells we show that A3A-induced CIN leads to aggressive tumors characterized by enhanced early dissemination and metastasis in a STING-dependent manner and independently of the canonical deaminase functions of A3A. We show that A3A upregulation recapitulates numerous copy number alterations commonly observed in patients with PDA, including co-deletions in DNA repair pathway genes, which in turn render these tumors susceptible to poly (ADP-ribose) polymerase inhibition. Overall, our results demonstrate that A3A plays an unexpected role in PDA as a specific driver of CIN, with significant effects on disease progression and treatment.

Multiplex Enrichment and Detection of Rare KRAS Mutations in Liquid Biopsy Samples using Digital Droplet Pre-Amplification.

Oncology research is increasingly incorporating molecular detection of circulating tumor DNA (ctDNA) as a tool for cancer surveillance and early detection. However, noninvasive monitoring of conditions with low tumor burden remains challenging, as the diagnostic sensitivity of most ctDNA assays is inversely correlated with total DNA concentration and ctDNA abundance. Here we present the Multiplex Enrichment using Droplet Pre-Amplification (MED-Amp) method, which combines single-molecule emulsification and short-round polymerase chain reaction (PCR) preamplification with digital droplet PCR detection of mutant DNA template. The MED-Amp assay increased mutant signal by over 50-fold with minimal distortion in allelic frequency. We demonstrate detection of as few as three mutant copies in wild-type DNA concentrations ranging from 5 to 50 ng. The MED-Amp assay successfully detected KRAS mutant ctDNA in 86% plasma samples obtained from patients with metastatic pancreatic ductal adenocarcinoma. This assay for high-sensitivity rare variant detection is appropriate for liquid biopsy samples or other limited clinical biospecimens.

Tracheostomy Emergencies.

Tracheostomy is a common procedure for long-term airway management. Although the overall complication rate is greater than 50%, the incidence of serious complications is low. These serious complications can, however, lead to significant morbidity and mortality and it is incumbent on the emergency provider to be prepared to deal with such tracheostomy-related emergencies. The greatest life threats to the tracheostomy patient are decannulation, obstruction, and hemorrhage. Other important but lower-acuity complications include tracheoesophageal fistula formation, tracheal stenosis, infection, and tracheocutaneous fistula formation.

Effects of exercise on circulating tumor cells among patients with resected stage I-III colon cancer.

BACKGROUND: Physical activity is associated with a lower risk of disease recurrence among colon cancer patients. Circulating tumor cells (CTC) are prognostic of disease recurrence among stage I-III colon cancer patients. The pathways through which physical activity may alter disease outcomes are unknown, but may be mediated by changes in CTCs. METHODS: Participants included 23 stage I-III colon cancer patients randomized into one of three groups: usual-care control, 150 min∙wk-1 of aerobic exercise (low-dose), and 300 min∙wk-1 of aerobic exercise (high-dose) for six months. CTCs from venous blood were quantified in a blinded fashion using an established microfluidic antibody-mediated capture device. Poisson regression models estimated the logarithmic counts of CTCs. RESULTS: At baseline, 78% (18/23) of patients had ≥1 CTC. At baseline, older age (-0.12±0.06; P = 0.04), lymphovascular invasion (0.63±0.25; P = 0.012), moderate/poor histology (1.09±0.34; P = 0.001), body mass index (0.07±0.02; P = 0.001), visceral adipose tissue (0.08±0.04; P = 0.036), insulin (0.06±0.02; P = 0.011), sICAM-1 (0.04±0.02; P = 0.037), and sVCAM-1 (0.06±0.03; P = 0.045) were associated with CTCs. Over six months, significant decreases in CTCs were observed in the low-dose (-1.34±0.34; P<0.001) and high-dose (-1.18±0.40; P = 0.004) exercise groups, whereas no significant change was observed in the control group (-0.59±0.56; P = 0.292). Over six months, reductions in body mass index (-0.07±0.02; P = 0.007), insulin (-0.08±0.03; P = 0.014), and sICAM-1 (-0.07±0.03; P = 0.005) were associated with reductions in CTCs. The main limitations of this proof-of-concept study are the small sample size, heterogenous population, and per-protocol statistical analysis. CONCLUSION: Exercise may reduce CTCs among stage I-III colon cancer patients. Changes in host factors correlated with changes in CTCs. Exercise may have a direct effect on CTCs and indirect effects through alterations in host factors. This hypothesis-generating observation derived from a small pilot study warrants further investigation and replication.

MYC regulates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma associated with poor outcome and chemoresistance.

Intratumoral phenotypic heterogeneity has been described in many tumor types, where it can contribute to drug resistance and disease recurrence. We analyzed ductal and neuroendocrine markers in pancreatic ductal adenocarcinoma, revealing heterogeneous expression of the neuroendocrine marker Synaptophysin within ductal lesions. Higher percentages of Cytokeratin-Synaptophysin dual positive tumor cells correlate with shortened disease-free survival. We observe similar lineage marker heterogeneity in mouse models of pancreatic ductal adenocarcinoma, where lineage tracing indicates that Cytokeratin-Synaptophysin dual positive cells arise from the exocrine compartment. Mechanistically, MYC binding is enriched at neuroendocrine genes in mouse tumor cells and loss of MYC reduces ductal-neuroendocrine lineage heterogeneity, while deregulated MYC expression in KRAS mutant mice increases this phenotype. Neuroendocrine marker expression is associated with chemoresistance and reducing MYC levels decreases gemcitabine-induced neuroendocrine marker expression and increases chemosensitivity. Altogether, we demonstrate that MYC facilitates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma, contributing to poor survival and chemoresistance.

Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci.

Factor V Leiden (F5L ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N-ethyl-N-nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5L (F5L/L ) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/- ). F8 deficiency enhanced the survival of F5L/LTfpi+/- mice, demonstrating that F5L/LTfpi+/- lethality is genetically suppressible. ENU-mutagenized F5L/L males and F5L/+Tfpi+/- females were crossed to generate 6,729 progeny, with 98 F5L/LTfpi+/- offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden (MF5L1-16)," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene (F3). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 (F3+/- ) suppressed F5L/LTfpi+/- lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5L/LTfpi+/- lethality (P = 1.7 × 10-6), suggesting that Actr2p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2p.R258G Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5L and also suggest a role for Actr2 in this process.

Spontaneous Irs1 passenger mutation linked to a gene-targeted SerpinB2 allele.

In characterizing mice with targeted disruption of the SerpinB2 gene, we observed animals that were small at birth with delayed growth and decreased life expectancy. Although this phenotype cosegregated with homozygosity for the inactive SerpinB2 allele, analysis of homozygous SerpinB2-deficient mice derived from two additional independent embryonic stem (ES) cell clones exhibited no growth abnormalities. Examination of additional progeny from the original SerpinB2-deficient line revealed recombination between the small phenotype (smla) and the SerpinB2 locus. The locus responsible for smla was mapped to a 2.78-Mb interval approximately 30 Mb proximal to SerpinB2, bounded by markers D1Mit382 and D1Mit216. Sequencing of Irs1 identified a nonsense mutation at serine 57 (S57X), resulting in complete loss of IRS1 protein expression. Analysis of ES cell DNA suggests that the S57X Irs1 mutation arose spontaneously in an ES cell subclone during cell culture. Although the smla phenotype is similar to previously reported Irs1 alleles, mice exhibited decreased survival, in contrast to the enhanced longevity reported for IRS1 deficiency generated by gene targeting. This discrepancy could result from differences in strain background, unintended indirect effects of the gene targeting, or the minimal genetic interference of the S57X mutation compared with the conventionally targeted Irs1-KO allele. Spontaneous mutations arising during ES cell culture may be a frequent but underappreciated occurrence. When linked to a targeted allele, such mutations could lead to incorrect assignment of phenotype and may account for a subset of markedly discordant results from experiments independently targeting the same gene.

Homozygosity for factor V Leiden leads to enhanced thrombosis and atherosclerosis in mice.

BACKGROUND: Activated protein C resistance due to factor V Leiden (FVL) is a common genetic risk factor for venous thrombosis in humans. Although the impact of FVL on the development of venous thrombosis is well established, its effect on arterial thrombosis and atherosclerosis is controversial. METHODS AND RESULTS: To determine the effect of the FVL mutation on arterial thrombosis in the mouse, wild-type (Fv+/+), heterozygous FVL (FvQ/+), and homozygous FVL (FvQ/Q) mice underwent photochemical carotid arterial injury to induce occlusive thrombosis. FvQ/Q mice formed occlusive thromboses 27+/-3 minutes (n=7) after the onset of injury, which was significantly shorter than that observed for Fv+/+ mice (56+/-7 minutes, n=9, P<0.01), whereas FvQ/+ mice (41+/-7 minutes, n=5) were intermediate (P=0.5, compared with Fv+/+). To determine the source of FVL relevant to the enhanced vascular thrombosis, bone marrow transplantation experiments were performed between Fv+/+ and FvQ/Q mice. FvQ/Q mice transplanted with Fv+/+ bone marrow formed occlusive thromboses at 35+/-5 minutes (n=7, P<0.05 compared with Fv+/+ mice), whereas Fv+/+ mice transplanted with FvQ/Q bone marrow occluded at 59+/-7 minutes (n=6, P<0.001 compared with FvQ/Q mice). To assess the effect of the FVL mutation on the development of atherosclerosis, FvQ/Q mice were crossed with the atherosclerosis-prone apolipoprotein E (ApoE)-deficient strain (ApoE-/-) to generate FvQ/Q,ApoE-/- mice. By 52 weeks of age, FvQ/Q,ApoE-/- mice (n=8) had developed more aortic atherosclerosis (40+/-6% lesion area) compared with Fv+/+,ApoE-/- mice (15+/-3% lesion area; n=12, P<0.02). CONCLUSIONS: In conclusion, homozygosity for the FVL mutation in mice leads to enhanced arterial thrombosis and atherosclerosis. The source of the FVL leading to accelerated thrombosis appears to be circulating, non-platelet-derived plasma FVL.

Lethal perinatal thrombosis in mice resulting from the interaction of tissue factor pathway inhibitor deficiency and factor V Leiden.

BACKGROUND: Factor V Leiden (FVL) is a common genetic risk factor for thrombosis in humans. The incomplete penetrance of FVL suggests important contributions from other genetic or environmental modifying factors. Variation in the expression of tissue factor pathway inhibitor (TFPI) has also been proposed as a risk factor for venous thrombosis and has been shown to enhance the prothrombotic effect of FVL in vitro. METHODS AND RESULTS: To examine the potential in vivo interaction between Tfpi and FvL, we analyzed crosses between mice carrying FvL and a deficiency of TFPI. The Fv(Q/Q),Tfpi(+/-) genotype was nearly completely fatal in the early perinatal period. Increased fibrin deposition was observed in multiple organs from the Fv(Q/Q),Tfpi(+/-) fetuses, suggesting disseminated thrombosis. CONCLUSIONS: These observations demonstrate the prothrombotic effect of modest variations in the level of TFPI expression and suggest that TFPI could be an important genetic modifier for the thrombosis associated with FVL in humans.