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General anaesthesia in the neonatal period has detrimental effects on the developing mammalian brain. The impact of underlying inflammation on anaesthesia-induced developmental neurotoxicity remains largely unknown.Postnatal day 7 (PND7) rats were randomly assigned to receive sevoflurane (3 vol% for 3 h) or carrier gas 12 h after bacterial lipopolysaccharide (LPS; 1 μg gSevoflurane or LPS treatment increased activated caspase-3 and caspase-9 expression in the hippocampal subiculum and CA1, which was greater when sevoflurane was administered in the setting of LPS-induced inflammation. Neuronal injury induced by LPS+sevoflurane treatment resulted in sex-specific behavioural outcomes when rats were tested at 5-8 weeks of age, including learning and memory deficits in males and heightened anxiety-related behaviour in females. Hippocampal caspase-1 and NLRP1 (NLR family pyrin domain containing 1), but not NLRP3, were upregulated by LPS or LPS+sevoflurane treatment, along with related proinflammatory cytokines, interleukin (IL)-1β, and IL-18. Pretreatment with Vx-765, a selective caspase-1 inhibitor, led to reduced IL-1β in LPS and LPS+sevoflurane groups. Caspase-1 inhibition by Vx-765 significantly decreased activated caspase-3 and caspase-9 immunoreactivity in the subiculum.Systemic inflammation promotes developmental neurotoxicity by worsening anaesthesia-induced neuronal damage with sex-specific behavioural outcomes. This highlights the importance of studying anaesthesia-induced neurotoxicity in more clinically relevant settings.

Inhibition of the GPX4 enzyme leads to the rapid accumulation of toxic lipid peroxides and cell death through a process known as ferroptosis. This mechanism has recently garnered significant attention in the field of oncology because many difficult-to-treat tumor types are particularly susceptible to this non-canonical form of cell death. However, robust biomarkers that predict sensitivity to ferroptosis are needed to help select well-defined tumor subtypes and patient segments that are likely to respond to GPX4-targeting. Our nascent RNA platform measures the active transcription of not only protein-coding transcripts, but also transcripts initiating from enhancer regions (enhancer RNAs) critical for defining cell type. We reasoned that these data could afford the unique opportunity to identify non-coding transcripts that could serve as novel biomarkers of sensitivity to ferroptosis. We recently discovered a potent and selective GPX4 inhibitor and profiled the compound for its ability to induce ferroptosis across a diverse group of cancer cell types. We chose several of the most sensitive and resistant cell lines for further examination using our nascent RNA platform and identified novel relationships between sensitivity to ferroptosis and cell-specific enhancer RNA profiles. Importantly, enhancer RNA profiles were more effective at stratifying sensitive and resistant cell lines than protein-coding gene expression. Using enhancer RNA expression profiles as biomarkers of drug sensitivity represents a novel strategy that could ultimately improve our ability to select patient cohorts more likely to benefit from emerging drug candidates and established therapeutics. Citation Format: Timothy Read, David Simpson, Jenna Rimel, Christy Rhine, Casey Pham, Cole Liechty, Eric Martin, Joey Azofeifa. Enhancer RNA profiles predict sensitivity to a novel GPX4 inhibitor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4913.

Induction of Ferroptosis is a promising strategy for treating therapy-resistant tumors including mesenchymal tumor types. We performed a phenotypic screen to identify small molecules that induce ferroptosis in mesenchymal cancer cells by screening for compounds whose toxicity could be rescued by the lipophilic antioxidant Ferrostatin-1. To identify compounds that induce GPX4-mediated ferroptosis, we identified several pharmacodynamic biomarkers specific to GPX4 inhibition using our nascent RNA sequencing platform, which allows for time-resolved snapshots of global transcription. We measured the transcriptional changes that occur in the hours following inhibition of GPX4 and identified HMOX1 as a robust biomarker of GPX4i-induced ferroptosis in mesenchymal cancer cell lines. Using HMOX1-induction as a guide, we uncovered a small number of compounds as potential GPX4 inhibitors. One such compound was validated as a bonafide GPX4 inhibitor through a variety of biochemical assays and selected for a hit-to-lead campaign. In addition to standard medicinal chemistry strategies, we are currently employing a newly-developed global transcriptional reporter system to stratify several novel series of GPX4 inhibitors by their transcriptional signatures. This approach represents a unique strategy for determining on- and off- target effects of a compound and for defining structure activity relationships within a chemical series. Citation Format: Timothy Read, Zoe Winigrad, Ardeshir Goliaei, Cole Liechty, Carter Grochala, Leah Damon, John Dickson, Jason Harris, Casey Pham, Jenna Rimel, Christy Rhine, Dave Simpson, Eric Martin, Joey Azofeifa. Identification of novel GPX4 inhibitors using global transcriptional reporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2755.

A central feature of diabetic wounds is the persistence of chronic inflammation, which is partly due to the prolonged presence of pro-inflammatory (M1) macrophages in diabetic wounds. Persistence of the M1 macrophage phenotype and failure to transition to the regenerative or pro-remodeling (M2) macrophage phenotype plays an indispensable role in diabetic wound impairment, however, the mechanism underlying this relationship remains unclear. Recently, microRNAs have been shown to provide an additional layer of regulation of gene expression. In particular, microRNA-21 (miR-21) is essential for an inflammatory immune response. We hypothesize that miR-21 plays a role in regulating inflammation by promoting M1 macrophage polarization and the production of reactive oxygen species (ROS). To test our hypothesis, we employed an in vivo mouse skin wound model in conjunction with an in vitro mouse model to assess miR-21 expression and macrophage polarization. First, we found that miR-21 exhibits a distinct expression pattern in each phase of healing in diabetic wounds. MiR-21 abundance was higher during early and late phases of wound repair in diabetic wounds, while it was significantly lower in the middle phase of wounding (at days 3 and 7 following wounding). In macrophage cells, M1 polarized macrophages exhibited an upregulation of miR-21, as well as the M1 and pro-inflammatory markers IL-1b, TNFa, iNos, IL-6, and IL-8. Overexpression of miR-21 in macrophage cells resulted in an upregulation of miR-21 and also increased expression of the M1 markers IL-1b, TNFa, iNos, and IL-6. Furthermore, hyperglycemia induced NOX2 expression and ROS production through the HG/miR-21/PI3K/NOX2/ROS signaling cascade. These findings provide evidence that miR-21 is involved in the regulation of inflammation. Dysregulation of miR-21 may explain the abnormal inflammation and persistent M1 macrophage polarization seen in diabetic wounds.

A central feature of diabetic (Db) wounds is the persistence of chronic inflammation, which is partly due to the prolonged presence of proinflammatory (M1) macrophages. Using in vivo and in vitro analyses, we have tested the hypothesis that long noncoding RNA GAS5 is dysregulated in Db wounds. We have assessed the contribution of GAS5 to the M1 macrophage phenotype, as well as the functional consequences of knocking down its expression. We found that expression of GAS5 is increased significantly in Db wounds and in cells isolated from Db wounds. Hyperglycemia induced GAS5 expression in macrophages in vitro. Overexpression of GAS5 in vitro promoted macrophage polarization toward an M1 phenotype by upregulating signal transducer and activator of transcription 1. Of most significance in our judgment, GAS5 loss-of-function enhanced Db wound healing. These data indicate that the relative level of long noncoding RNA GAS5 in wounds plays a key role in the wound healing response. Reductions in the levels of GAS5 in wounds appeared to enhance healing by promoting transition of M1 macrophages to M2 macrophages. Thus, our results suggest that targeting long noncoding RNA GAS5 may provide a therapeutic intervention for correcting impaired Db wound healing.