Unraveling the Role of PCDH9 in Breast Cancer and Identifying Therapeutic Strategies for PCDH9-Deficient Tumors.

Introduction: Protocadherin 9 (PCDH9), a member of the cadherin superfamily of transmembrane proteins, plays a role in cell adhesion and neural development. Recent studies suggest that PCDH9 may function as a tumor suppressor in certain cancers, though its specific role in breast cancer remains unclear. Methods: UALCAN database to retrieve information on PCDH9 expression in breast cancer tissues compared with that in normal tissues. The biological effects of PCDH9 in breast cancer cells were analyzed using the DepMap database. Stable knockdown or overexpression of PCDH9 in breast cancer cell lines and subsequently assessed tumor cell proliferation and migration. Synthetic lethal screening was conducted for breast cancer cells with low PCDH9 expression or deficiency. Results: In this study, we observed significant downregulation of PCDH9 in breast cancer tissues, with its expression negatively correlated with progression-free survival. Further investigations revealed that decreased PCDH9 expression promotes breast cancer cell proliferation and migration, while overexpression of PCDH9 has the opposite effect. Subsequently, we identified the TAS-102, an approved drug for metastatic colorectal cancer, exhibited selective cytotoxicity against breast cancer cells with low PCDH9 expression. Conclusion and discussion: In summary, our study identified PCDH9 as a tumor suppressor in breast cancer and highlighted TAS-102 as a potential therapeutic option for tumors with low PCDH9 expression or deficiency. The specific interaction between TAS-102 and PCDH9 warrants further exploration, providing deeper insights into its mode of action in treating PCDH9-deficient breast cancer.

Overexpression of Igf2-derived Mir483 inhibits Igf1 expression and leads to developmental growth restriction and metabolic dysfunction in mice.

Mir483 is a conserved and highly expressed microRNA in placental mammals, embedded within the Igf2 gene. Its expression is dysregulated in a number of human diseases, including metabolic disorders and certain cancers. Here, we investigate the developmental regulation and function of Mir483 in vivo. We find that Mir483 expression is dependent on Igf2 transcription and the regulation of the Igf2/H19 imprinting control region. Transgenic Mir483 overexpression in utero causes fetal, but not placental, growth restriction through insulin-like growth factor 1 (IGF1) and IGF2 and also causes cardiovascular defects leading to fetal death. Overexpression of Mir483 post-natally results in growth stunting through IGF1 repression, increased hepatic lipid production, and excessive adiposity. IGF1 infusion rescues the post-natal growth restriction. Our findings provide insights into the function of Mir483 as a growth suppressor and metabolic regulator and suggest that it evolved within the INS-IGF2-H19 transcriptional region to limit excessive tissue growth through repression of IGF signaling.

Effect of Nitrite-Embedded Packaging Film on Growth of Listeria monocytogenes in Nitrite-free and Conventionally-Cured Bologna Sausage.

Listeria monocytogenes is a pathogen frequently associated with ready-to-eat (RTE) meat and poultry products. Nitrite is a key antimicrobial additive that can offer some degree of protection against L. monocytogenes when included in meat product formulations. The objectives of this study were to determine the potential of nitrite-embedded film to affect growth of L. monocytogenes following post-thermal processing of conventionally-cured and nitrite-free bologna. Two bologna treatment formulations, a conventionally-cured control formulation (CON) and a nitrite-free formulation (UCC), were manufactured, packaged in conventional (CF) or nitrite-embedded (NEF) film, inoculated with 3.5 log CFU/cm2 of a cocktail of L. monocytogenes strains, and stored at 10 ± 1°C. CON-NEF and UCC-NEF treatments had significantly slower (P < 0.05) growth of L. monocytogenes than CON-CF and UCC-CF, with populations in UCC-CF (which contained no nitrite) increasing by 3.4 logs after 10 d of storage in UCC-CF and 3.6 logs after 50 d in CON-CF (which had formulated nitrite only), while in the NEF-packaged samples, with or without formulated nitrite, they did not exceed the inoculum level until after day 40. Initial (day 0) residual nitrite was significantly greater (P < 0.05) in the control formulation. Packaging in NEF, however, resulted in an increase of 27-28 ppm by day 3, regardless of formulation, after which it decreased rapidly. Results suggest NEF can be used as a post-lethality antimicrobial intervention in food safety intervention strategies, in both cured and uncured processed meat products.

An update on small molecule compounds targeting synthetic lethality for cancer therapy.

Targeting cancer-specific vulnerabilities through synthetic lethality (SL) is an emerging paradigm in precision oncology. A SL strategy based on PARP inhibitors has demonstrated clinical efficacy. Advances in DNA damage response (DDR) uncover novel SL gene pairs. Beyond BRCA-PARP, emerging SL targets like ATR, ATM, DNA-PK, CHK1, WEE1, CDK12, RAD51, and RAD52 show clinical promise. Selective and bioavailable small molecule inhibitors have been developed to induce SL, but optimization for potency, specificity, and drug-like properties remains challenging. This article illuminated recent progress in the field of medicinal chemistry centered on the rational design of agents capable of eliciting SL specifically in neoplastic cells. It is envisioned that innovative strategies harnessing SL for small molecule design may unlock novel prospects for targeted cancer therapeutics going forward.

Revealing the lethal effects of Pasteurella multocida toxin on multiple organ systems.

Pasteurella multocida toxin (PMT) is one of the most important virulence factors of Pasteurella multocida type D. Pasteurella multocida infection has caused enormous economic losses in the pig farming industry. Although it is well known that this bacterial infection causes progressive atrophic rhinitis, its effects on other organ tissues in pigs are unclear. In this study, PMT was expressed and purified, and the cytotoxic effects of PMT on four types of swine cells, LLC-PK1, PAM, IPEC, and ST, were investigated. LLC-PK1 exhibited the highest sensitivity to the cytotoxic effects of PMT. Our studies revealed that a PMT concentration of 0.1 µg/kg can lead to weight loss, whereas a PMT concentration of 0.5 µg/kg can lead to death in mice. PMT causes damage to the intestines, kidneys, lungs, livers, and spleens of mice. Furthermore, PMT caused acute death in pigs at treatment concentrations greater than 5 µg/kg; at PMT concentration of 2.5 µg/kg, weight loss occurred until death. PMT mainly caused damage to the hearts, lungs, livers, spleens and kidneys of pigs. The organ coefficient showed that damage to the heart and kidneys was the most severe and caused the renal pelvis and renal pyramid to dissolve and become cavitated. Pathology revealed hemorrhage in the lungs, liver, and spleen, and the kidneys were swollen and vacuolated, which was consistent with the damaged target organs in the mice. In conclusion, these findings demonstrate that PMT is extremely toxic in vitro and in vivo, causing damage to various organs of the body, especially the kidneys and lungs. This study provides a theoretical basis for the in-depth exploration of the cytotoxic effects of PMT on target organs.

Facet-Dependent Lethality of a Contact Insecticide Crystal.

The activity of crystalline contact insecticides relies on the extraction of surface molecules by insect tarsi upon contact. Most crystals are inherently anisotropic, and surface molecules on symmetry independent faces are expected to have different free energies. The facet-dependent bioavailability and associated efficacy of insect lethality have not been investigated, however. We discriminate the bioactivity of various facets of single crystals of DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane), a well-known contact insecticide. Our findings reveal facet-dependent lethality differences of nearly 75% among four crystallographically unique facets. Furthermore, computations reveal that the respective lethalities of the facets are strongly correlated with the detachment energies of molecules from the crystal surfaces. This facet-dependent lethality suggests a pathway to enhance the efficacy of known contact insecticides through crystal habit control.

Genome-Wide CRISPR Screen Identifies KEAP1 Perturbation as a Vulnerability of ARID1A-Deficient Cells.

ARID1A is the core DNA-binding subunit of the BAF chromatin remodeling complex and is mutated in about 8% of all cancers. The frequency of ARID1A loss varies between cancer subtypes, with clear cell ovarian carcinoma (CCOC) presenting the highest incidence at > 50% of cases. Despite a growing understanding of the consequences of ARID1A loss in cancer, there remains limited targeted therapeutic options for ARID1A-deficient cancers. Using a genome-wide CRISPR screening approach, we identify KEAP1 as a genetic dependency of ARID1A in CCOC. Depletion or chemical perturbation of KEAP1 results in selective growth inhibition of ARID1A-KO cell lines and edited primary endometrial epithelial cells. While we confirm that KEAP1-NRF2 signalling is dysregulated in ARID1A-KO cells, we suggest that this synthetic lethality is not due to aberrant NRF2 signalling. Rather, we find that KEAP1 perturbation exacerbates genome instability phenotypes associated with ARID1A deficiency. Together, our findings identify a potentially novel synthetic lethal interaction of ARID1A-deficient cells.

Polθ Inhibitor (ART558) Demonstrates a Synthetic Lethal Effect with PARP and RAD52 Inhibitors in Glioblastoma Cells.

DNA repair proteins became the popular targets in research on cancer treatment. In our studies we hypothesized that inhibition of DNA polymerase theta (Polθ) and its combination with Poly (ADP-ribose) polymerase 1 (PARP1) or RAD52 inhibition and the alkylating drug temozolomide (TMZ) has an anticancer effect on glioblastoma cells (GBM21), whereas it has a low impact on normal human astrocytes (NHA). The effect of the compounds was assessed by analysis of cell viability, apoptosis, proliferation, DNA damage and cell cycle distribution, as well as gene expression. The main results show that Polθ inhibition causes a significant decrease in glioblastoma cell viability. It induces apoptosis, which is accompanied by a reduction in cell proliferation and DNA damage. Moreover, the effect was stronger when dual inhibition of Polθ with PARP1 or RAD52 was applied, and it is further enhanced by addition of TMZ. The impact on normal cells is much lower, especially when considering cell viability and DNA damage. In conclusion, we would like to highlight that Polθ inhibition used in combination with PARP1 or RAD52 inhibition has great potential to kill glioblastoma cells, and shows a synthetic lethal effect, while sparing normal astrocytes.

Potential promising of synthetic lethality in cancer research and treatment.

Cancer is a complex disease driven by multiple genetic changes, including mutations in oncogenes, tumor suppressor genes, DNA repair genes, and genes involved in cancer metabolism. Synthetic lethality (SL) is a promising approach in cancer research and treatment, where the simultaneous dysfunction of specific genes or pathways causes cell death. By targeting vulnerabilities created by these dysfunctions, SL therapies selectively kill cancer cells while sparing normal cells. SL therapies, such as PARP inhibitors, WEE1 inhibitors, ATR and ATM inhibitors, and DNA-PK inhibitors, offer a distinct approach to cancer treatment compared to conventional targeted therapies. Instead of directly inhibiting specific molecules or pathways, SL therapies exploit genetic or molecular vulnerabilities in cancer cells to induce selective cell death, offering benefits such as targeted therapy, enhanced treatment efficacy, and minimized harm to healthy tissues. SL therapies can be personalized based on each patient's unique genetic profile and combined with other treatment modalities to potentially achieve synergistic effects. They also broaden the effectiveness of treatment across different cancer types, potentially overcoming drug resistance and improving patient outcomes. This review offers an overview of the current understanding of SL mechanisms, advancements, and challenges, as well as the preclinical and clinical development of SL. It also discusses new directions and opportunities for utilizing SL in targeted therapy for anticancer treatment.

MAT2A inhibition combats metabolic and transcriptional reprogramming in cancer.

Metabolic and transcriptional reprogramming are crucial hallmarks of carcinogenesis that present exploitable vulnerabilities for the development of targeted anticancer therapies. Through controlling the balance of the cellular methionine (MET) metabolite pool, MET adenosyl transferase 2 alpha (MAT2A) regulates crucial steps during metabolism and the epigenetic control of transcription. The aberrant function of MAT2A has been shown to drive malignant transformation through metabolic addiction, transcriptional rewiring, and immune modulation of the tumor microenvironment (TME). Moreover, MAT2A sustains the survival of 5'-methylthioadenosine phosphorylase (MTAP)-deficient tumors, conferring synthetic lethality to cancers with MTAP loss, a genetic alteration that occurs in ∼15% of all cancers. Thus, the pharmacological inhibition of MAT2A is emerging as a desirable therapeutic strategy to combat tumor growth. Here, we review the latest insights into MAT2A biology, focusing on its roles in both metabolic addiction and gene expression modulation in the TME, outline the current landscape of MAT2A inhibitors, and highlight the most recent clinical developments and opportunities for MAT2A inhibition as a novel anti-tumor therapy.

First-in-human study of AMG 193, an MTA-cooperative PRMT5 inhibitor, in patients with MTAP-deleted solid tumors: results from phase 1 dose exploration.

BACKGROUND: Homozygous deletion of methylthioadenosine phosphorylase (MTAP) occurs in ∼10%-15% of solid tumors. AMG 193, a CNS-penetrant methylthioadenosine-cooperative protein arginine methyltransferase 5 (PRMT5) inhibitor, selectively induces synthetic lethality in MTAP-deleted tumors cells. Here, we report results of the completed monotherapy dose exploration evaluating AMG 193 in patients with MTAP-deleted solid tumors. PATIENTS AND METHODS: In this first-in-human, multicenter, open-label, phase 1 study, patients with advanced CDKN2A-deleted and/or MTAP-deleted solid tumors received AMG 193 orally (once [QD] or twice [BID] daily) continuously in 28-day cycle. Primary objectives were safety and tolerability assessed by dose-limiting toxicities (DLTs) and determination of the maximum-tolerated dose (MTD); secondary objectives included pharmacokinetics and preliminary antitumor activity measured by RECIST v1.1. RESULTS: As of 23 May 2024, 80 patients in dose exploration received AMG 193 at doses 40-1600 mg QD or 600 mg BID. The most common treatment-related adverse events were nausea (48.8%), fatigue (31.3%), and vomiting (30.0%). DLTs were reported in eight patients at doses ≥240 mg, including nausea, vomiting, fatigue, hypersensitivity reaction, and hypokalemia. The MTD was determined to be 1200 mg QD. Mean exposure of AMG 193 increased in a dose-proportional manner from 40 mg to 1200 mg. Among the efficacy-evaluable patients treated at the active and tolerable doses of 800 mg QD, 1200 mg QD, or 600 mg BID (n=42), objective response rate (ORR) was 21.4% (95% CI: 10.3-36.8). Responses were observed across eight different tumor types, including squamous/nonsquamous non-small cell lung cancer, pancreatic adenocarcinoma, and biliary tract cancer. At doses ≥480 mg, complete intratumoral PRMT5 inhibition was confirmed in paired MTAP-deleted tumor biopsies, and molecular responses (circulating-tumor DNA [ctDNA] clearance) were observed. CONCLUSIONS: AMG 193 demonstrated a favorable safety profile without clinically significant myelosuppression. Encouraging antitumor activity across a variety of MTAP-deleted solid tumors was observed based on ORR and ctDNA clearance.

An Additional L451G452N453 in the RpoB Protein Suppressed the Synthetic Lethality in Escherichia coli at 37 Degrees Caused by Depletion of DnaK/J and Trigger Factor.

Escherichia coli depletion of chaperone trigger factor and DnaK/J were not viable at 37°C, but viable below 30°C. Among the engineered E. coli depleted of trigger factor and DnaK/J, one strain Z625, exhibited survival at 37°C, while another strain Z629 only survived below 30°C. Comparative analysis of fatty acid profiles of Z625 and Z629 revealed absence of numerous saturated fatty acids in Z625 as compared to the wild-type E. coli BW25113. In addition, increased unsaturated fatty acids were present in Z625, whereas the fatty acids profile of Z629 closely resembled that of BW25113. Whole genome sequencing revealed a 9-bp insertion in rpoB of Z625. Combined structural analysis of simulated RpoB protein bearing the amino acid sequence L451G452N453 insertion and susceptibility analysis to rifampicin suggested that the insertion did not disturb the individual RpoB structure as beta subunit of RNA polymerase. Comparative transcriptomic analysis of Z625 and Z629 suggested that this insertion impacted transcription of the overall RNA polymerase in Z625, leading to potential repression of some genes whose overexpression was toxic to E. coli. Additionally, Z625 exhibited distinctive metabolic adaptations, likely contributing to its survival at 37°C. In summary, our study elucidated one LGN insertion in rpoB that impacts transcriptional regulation in E. coli, thereby explaining the survival of E. coli depletion of trigger factor and DnaK/J at 37°C, and these founding suggested that some simple mutations in critical genes like rpoB might play an important role in driving adaptive evolution.

Multivariate analysis of metabolic state vulnerabilities across diverse cancer contexts reveals synthetically lethal associations.

Targeting the distinct metabolic needs of tumor cells has recently emerged as a promising strategy for cancer therapy. The heterogeneous, context-dependent nature of cancer cell metabolism, however, poses challenges to identifying effective therapeutic interventions. Here, we utilize various unsupervised and supervised multivariate modeling approaches to systematically pinpoint recurrent metabolic states within hundreds of cancer cell lines, elucidate their association with tumor lineage and growth environments, and uncover vulnerabilities linked to their metabolic states across diverse genetic and tissue contexts. We validate key findings via analysis of data from patient-derived tumors and pharmacological screens and by performing genetic and pharmacological experiments. Our analysis uncovers synthetically lethal associations between the tumor metabolic state (e.g., oxidative phosphorylation), driver mutations (e.g., loss of tumor suppressor PTEN), and actionable biological targets (e.g., mitochondrial electron transport chain). Investigating the mechanisms underlying these relationships can inform the development of more precise and context-specific, metabolism-targeted cancer therapies.

GTF2H5 Identified as a crucial synthetic lethal target to counteract chemoresistance in colorectal cancer.

BACKGROUND: Synthetic lethality (SL) emerges as a novel concept being explored to combat cancer progression and resistance to conventional therapy. Despite the efficacy of chemotherapy in select cases of colorectal cancer (CRC), a substantial proportion of patients encounter challenges, leading to an adverse prognosis of CRC patients. CRC-related SL genes offer a potential avenue for identifying therapeutic targets. METHODS: CRC-related SL genes were obtained from the SynLethDB database. The bulk RNA sequencing data, mutation data, and clinical information for treated and untreated CRC patients were enrolled from the UCSC and GEO databases. The Tumor Immunology Single Cell Center database served as the repository for collecting and analyzing single-cell RNA sequencing data. The synergistic killing effect of SL genes and chemotherapeutic drugs on resistant cells was experimentally verified. RESULTS: In the present study, pivotal SL genes associated with chemoresistance identified by using WGCNA and CRC patients categorized into two groups based on these genes. Variations between the groups were most pronounced in pathways associated with extracellular matrix remodeling. Further by integrating mutation data, five potential SL genes were discerned, which were highly expressed in the presence of TP53 or KRAS mutations, leading to a severely poor prognosis. Subsequent time series analysis revealed that the expression of GTF2H5 was gradually elevated at different stages of the transition from sensitive to resistant in CRC cells. Finally, it was preliminarily verified by experiments that GTF2H5 may play a key role in driving the drug-resistant transition within CRC cells. CONCLUSIONS: The identification of SL genes that collaboratively interact with chemotherapeutic agents could provide new insights into solving the issue of chemotherapy resistance in CRC patients. And GTF2H5 wields a fundamental influence in inducing chemoresistance in CRC, which provided a potential therapeutic target for CRC.

OsIPK1 frameshift mutations disturb phosphorus homeostasis and impair starch synthesis during grain filling in rice.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) catalyzes the final step in phytic acid (InsP6) synthesis. In this study, the effects of OsIPK1 mutations on InsP6 synthesis, grain filling and their underlying mechanisms were investigated. Seven gRNAs were designed to disrupt the OsIPK1 gene via CRISPR/CAS9 system. Only 4 of them generated 29 individual insertion or deletion T0 plants, in which nine biallelic or heterozygous genotypes were identified. Segregation analysis revealed that OsIPK1 frameshift mutants are homozygous lethality. The biallelic and heterozygous frameshift mutants exhibited significant reduction in yield-related traits, particularly in the seed-setting rate and yield per plant. Despite a notable decline in pollen viability, the male and female gametes had comparable transmission rates to their progenies in the mutants. A significant number of the filling-aborted (FA) grains was observed in mature grains of these heterozygous frameshift mutants. These grains exhibited a nearly complete blockage of InsP6 synthesis, resulting in a pronounced increase in Pi content. In contrast, a slight decline in InsP6 content was observed in the plump grains. During the filling stage, owing to the excessive accumulation of Pi, starch synthesis was significantly impaired, and the endosperm development-specific gene expression was nearly abolished. Consistently, the activity of whereas AGPase, a key enzyme in starch synthesis, was significantly decreased and Pi transporter gene expression was upregulated in the FA grains. Taken together, these results demonstrate that OsIPK1 frameshift mutations result in excessive Pi accumulation, decreased starch synthesis, and ultimately leading to lower yields in rice.

The Progression of Lethality Across Multiple Suicide Attempts: A Systematic Review.

OBJECTIVE: Suicide is a major global public health concern. While some progress has been made in understanding risk factors for suicidal behavior, other relevant questions have received less attention. One such question relates to the longitudinal course of suicidal behavior amongst individuals with multiple suicide attempts. This systematic review investigated whether there is an increase in the lethality across multiple suicide attempts. METHOD: This systematic review followed PRISMA 2020 reporting guidelines. A literature search was conducted in MEDLINE, Embase and PsycINFO electronic databases from inception to August 2023 to identify studies with key terms related to multiple suicide attempts and lethality. The review included longitudinal studies with data on multiple suicide attempts, and any rating of their lethality. Covidence was used to guide the screening and extraction process. A narrative synthesis approach was used to descriptively summarize included studies. RESULTS: After identifying 828 unique abstracts for screening, 11 studies were included for narrative synthesis. Suicide attempt assessment methods and definitions were heterogenous, often indirectly inferring lethality based on suicide attempt method. Individuals with repeat attempts may be more likely to continue using the same method. CONCLUSIONS: There was no evidence to support increasing lethality across repeat suicide attempts. However, this should be interpreted along with the fact that the evidence base is scarce, heterogenous, and methodologically limited.

There was no strong evidence for an increasing pattern of lethality across repeat attempts.A higher proportion of individuals continue to use the same method across repeat attempts.The evidence base is scarce, heterogenous, and methodologically limited.

Simultaneous inhibition of ATM, ATR, and DNA-PK causes synergistic lethality.

Here we report that simultaneous inhibition of the three primary DNA damage recognition PI3 kinase-like kinases (PIKKs) -ATM, ATR, and DNA-PK- induces severe combinatorial synthetic lethality in mammalian cells. Utilizing Chinese hamster cell lines CHO and V79 and their respective PIKK mutants, we evaluated effects of inhibiting these three kinases on cell viability, DNA damage response, and chromosomal integrity. Our results demonstrate that while single or dual kinase inhibition increased cytotoxicity, inhibition of all three PIKKs results in significantly higher synergistic lethality, chromosomal aberrations, and DNA double-strand break (DSB) induction as calculated by their synergy scores. These findings suggest that the overlapping redundancy of ATM, ATR, and DNA-PK functions is critical for cell survival, and their combined inhibition greatly disrupts DNA damage signaling and repair processes, leading to cell death. This study provides insights into the potential of multi-targeted DDR kinase inhibition as an effective anticancer strategy, necessitating further research to elucidate underlying mechanisms and therapeutic applications.

Does the atrazine increase animal mortality: Unraveling through a meta-analytic study.

Atrazine is one of the most used herbicides in the world, although it is banned in several countries. Pollution of terrestrial and aquatic ecosystems represents a threat to non-target organisms, with various damages already reported in different species. However, there is controversy in studies on atrazine. The question of whether atrazine increases animal mortality is not yet clearly resolved. In this context, this study aimed to carry out a meta-analytic review, focusing on studies on environmental concentrations of the herbicide atrazine to evaluate its lethal effects on various animal species. We identified and analyzed 107 datasets through a selection process that used the Scopus, PubMed, and Web of Science (WoS) databases. A significant increase in the mortality rate of animals exposed to environmental concentrations of atrazine was observed. Nematodes, amphibians, molluscs, insects, and fish showed increased mortality after exposure to atrazine. Animals in the larval and juvenile stages showed greater susceptibility when exposed to different concentrations of atrazine. Furthermore, both commercial and pure formulations resulted in high mortality rates for exposed animals. Atrazine and other pesticides had a synergistic effect, increasing the risk of mortality in animals. There are still many gaps to be filled, and this study can serve as a basis for future regulations involving atrazine.

Age-dependent Powassan virus lethality is linked to glial cell activation and divergent neuroinflammatory cytokine responses in a murine model.

Powassan virus (POWV) is an emergent tick-borne flavivirus that causes fatal encephalitis in the elderly and long-term neurologic sequelae in survivors. How age contributes to severe POWV encephalitis remains an enigma, and no animal models have assessed age-dependent POWV neuropathology. Inoculating C57BL/6 mice with a POWV strain (LI9) currently circulating in Ixodes ticks resulted in age-dependent POWV lethality 10-20 dpi. POWV infection of 50-week-old mice was 82% fatal with lethality sequentially reduced by age to 7.1% in 10-week-old mice. POWV LI9 was neuroinvasive in mice of all ages, causing acute spongiform CNS pathology and reactive gliosis 5-15 dpi that persisted in survivors 30 dpi. High CNS viral loads were found in all mice 10 dpi. However, by 15 dpi, viral loads decreased by 2-4 logs in 10- to 40-week-old mice, while remaining at high levels in 50-week-old mice. Age-dependent differences in CNS viral loads 15 dpi occurred concomitantly with striking changes in CNS cytokine responses. In the CNS of 50-week-old mice, POWV induced Th1-type cytokines (IFNγ, IL-2, IL-12, IL-4, TNFα, IL-6), suggesting a neurodegenerative pro-inflammatory M1 microglial program. By contrast, in 10-week-old mice, POWV-induced Th2-type cytokines (IL-10, TGFß, IL-4) were consistent with a neuroprotective M2 microglial phenotype. These findings correlate age-dependent CNS cytokine responses and viral loads with POWV lethality and suggest potential neuroinflammatory therapeutic targets. Our results establish the age-dependent lethality of POWV in a murine model that mirrors human POWV severity and long-term CNS pathology in the elderly. IMPORTANCE: Powassan virus is an emerging tick-borne flavivirus causing lethal encephalitis in aged individuals. We reveal an age-dependent POWV murine model that mirrors human POWV encephalitis and long-term CNS damage in the elderly. We found that POWV is neuroinvasive and directs reactive gliosis in all age mice, but at acute stages selectively induces pro-inflammatory Th1 cytokine responses in 50-week-old mice and neuroprotective Th2 cytokine responses in 10-week-old mice. Our findings associate CNS viral loads and divergent cytokine responses with age-dependent POWV lethality and survival outcomes. Responses of young mice suggest potential therapeutic targets and approaches for preventing severe POWV encephalitis that may be broadly applicable to other neurodegenerative diseases. Our age-dependent murine POWV model permits analysis of vaccines that prevent POWV lethality, and therapeutics that resolve severe POWV encephalitis.

Inhibition of autophagy as a novel treatment for neurofibromatosis type 1 tumors.

Neurofibromatosis type 1 (NF1) is a genetic disorder caused by mutation of the NF1 gene that is associated with various symptoms, including the formation of benign tumors, called neurofibromas, within nerves. Drug treatments are currently limited. The mitogen-activated protein kinase kinase (MEK) inhibitor selumetinib is used for a subset of plexiform neurofibromas (PNs) but is not always effective and can cause side effects. Therefore, there is a clear need to discover new drugs to target NF1-deficient tumor cells. Using a Drosophila cell model of NF1, we performed synthetic lethal screens to identify novel drug targets. We identified 54 gene candidates, which were validated with variable dose analysis as a secondary screen. Pathways associated with five candidates could be targeted using existing drugs. Among these, chloroquine (CQ) and bafilomycin A1, known to target the autophagy pathway, showed the greatest potential for selectively killing NF1-deficient Drosophila cells. When further investigating autophagy-related genes, we found that 14 out of 30 genes tested had a synthetic lethal interaction with NF1. These 14 genes are involved in multiple aspects of the autophagy pathway and can be targeted with additional drugs that mediate the autophagy pathway, although CQ was the most effective. The lethal effect of autophagy inhibitors was conserved in a panel of human NF1-deficient Schwann cell lines, highlighting their translational potential. The effect of CQ was also conserved in a Drosophila NF1 in vivo model and in a xenografted NF1-deficient tumor cell line grown in mice, with CQ treatment resulting in a more significant reduction in tumor growth than selumetinib treatment. Furthermore, combined treatment with CQ and selumetinib resulted in a further reduction in NF1-deficient cell viability. In conclusion, NF1-deficient cells are vulnerable to disruption of the autophagy pathway. This pathway represents a promising target for the treatment of NF1-associated tumors, and we identified CQ as a candidate drug for the treatment of NF1 tumors.