Scott R. Floyd

Aims: Ferroptosis, an iron-dependent form of regulated cell death, contributes to neuronal injury after ischemic stroke. Because our previous cancer-cell screens identified ataxia-telangiectasia mutated (ATM) kinase, as a ferroptosis regulator, we investigated whether ATM inhibition protects neural tissue after ischemic stroke and examined determinants of therapeutic response. Methods: ATM inhibitors, including the brain-penetrant clinical compound AZD1390, were evaluated in cultured cancer and neuronal cells, oxygen-glucose-deprived rat and mouse brain slices, and permanent middle cerebral artery occlusion models using multiple inbred and Rabep2-genotype mouse strains. Cell viability, cytotoxicity, lipid peroxidation, ferroptosis-associated markers, infarct volume, and collateral vessel density were assessed. Topical AZD1390 was also tested in mice with impaired collateral networks. Results: ATM inhibition reduced ferroptosis-associated lipid oxidative injury and protected neurons in multiple preclinical stroke models. AZD1390 decreased lipid peroxidation and neuronal death, and reduced infarct volume in B6/J and B6/NJ mice, but not in BALB mice. Treatment efficacy varied among strains and was correlated with collateral vascular density. Systemic AZD1390 was likewise ineffective in Rabep2-knockout mice with deficient collateral networks. Early topical administration also reduced infarct volume in mice with limited collateral networks; however, differences in treatment timing and route preclude direct attribution to improved local drug delivery. Conclusion: ATM inhibition may attenuate ferroptosis-associated neuronal injury after ischemic stroke, while collateral vascular anatomy may influence therapeutic response. These findings support further mechanistic and translational evaluation of ferroptosis-targeting strategies in stroke.

Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation and contributes to diverse pathologies including ischemia-reperfusion injury and neurodegenerative disorders. Current ferroptosis inhibitors largely function as nonspecific radical-trapping antioxidants, limiting their clinical utility. We previously identified MESH1 as a key regulator of ferroptosis through its NADPH phosphatase activity. Here, we identify 4,5,6,7-tetrabromo-1 H -benzotriazole (TBB) as a small molecule inhibitor of MESH1 with an IC ₅₀ value of 4.7 ± 0.3 µM. X-ray crystallography revealed the molecular determinants of TBB recognition which are corroborated through structure-activity relationships of TBB analogs. TBB protected multiple cell lines against ferroptosis in vitro , and this effect was mitigated by MESH1 knockdown, consistent with on-target activity. Furthermore, TBB reduced neuronal death in an ex vivo brain slice model of Alzheimer’s disease. Collectively, these findings establish TBB as a bona fide small-molecule MESH1 inhibitor that suppresses ferroptosis and establishes MESH1 as a promising therapeutic target. Graphical Depicting mechanism of TBB suppressing ferroptosis through the inhibition of MESH1. Figure Created with Biorender.com

Purpose: Ultra-high dose rate (FLASH) irradiation is reported to reduce normal tissue toxicity while maintaining tumor control, however mechanism(s) remain obscure. To study FLASH mechanisms in brain tissue, we developed a novel experimental platform featuring a unique high-energy electron linear accelerator (High Intensity Gamma Source, HIGS) paired with an organotypic ex vivo brain metastasis model. Methods: We varied inter-pulse spacing to modulate the mean dose rate (MDR) of our unique 35 MeV electron beam, while maintaining extremely high instantaneous dose rate (IDR), and used film dosimetry to characterize dosimetry and targeting accuracy. We combined this HIGS-FLASH beam with an organotypic rat brain slice/breast carcinoma co-culture model of brain metastasis to assess effects on normal and neoplastic tissues. Live cell and bioluminescence imaging demonstrated cancer cell growth effects, while normal tissue responses and immune activation were assessed with live cell imaging, cytokine profiles, and confocal microscopy. We performed comparison experiments with 20 MeV electrons from a Varian clinical linear accelerator (VCLA) operating at conventional dose rate. Results: The highest IDR of the HIGS-FLASH beam to-date was 20.7 ± 0.6 MGy/s, with maximum MDR of 20.7 MGy/s (1 μs pulse of 20.7 Gy). Beam targeting was accurate to < 1 mm and reproducible. HIGS-FLASH and VCLA dose rates equivalently decreased cancer cell growth. HIGS-FLASH irradiation significantly increased TNFα and fractalkine levels and confocal microscopy revealed distinct changes in microglial morphology in normal brain slices, suggesting microglia activation following HIGS-FLASH irradiation. Conclusions: Our novel experimental platform produces extremely high dose rates and rapid normal/neoplastic tissue readouts for mechanistic research into the effects of FLASH radiation on the brain. HIGS-FLASH irradiation induces comparable cancer cell growth inhibition but differential effects on cytokines and microglial morphology, suggesting that acute innate immune responses may be involved in FLASH normal tissue effects in the brain.

The cystine-xCT transporter/glutathione/GPX4 axis is the canonical pathway protecting cells from ferroptosis. Whereas GPX4-targeting ferroptosis-inducing compounds (FINs) act independently of mitochondria, xCT-targeting FINs require mitochondrial lipid peroxidation, though the mechanism remains unclear. Because cysteine is also a precursor for coenzyme A (CoA) biosynthesis, here, we demonstrated that CoA supplementation selectively prevented ferroptosis triggered by xCT inhibition by regulating the mitochondrial thioredoxin system. Our data showed that CoA regulated the in vitro enzymatic activity of mitochondrial thioredoxin reductase-2 (TXNRD2) by covalently modifying the thiol group of cysteine (CoAlation) on Cys-483. Replacing Cys-483 with alanine on TXNRD2 abolished its enzymatic activity and ability to protect cells against ferroptosis. Targeting xCT to limit cysteine import and, therefore, CoA biosynthesis reduced CoAlation on TXNRD2. Furthermore, the fibroblasts from patients with disrupted CoA metabolism had increased mitochondrial lipid peroxidation. In organotypic brain slice cultures, inhibition of CoA biosynthesis led to an oxidized thioredoxin system, increased mitochondrial lipid peroxidation, and loss of cell viability, which were all rescued by ferrostatin-1. These findings identified CoA-mediated posttranslational modification to regulate the thioredoxin system as an alternative ferroptosis protection pathway with potential clinical relevance for patients with disrupted CoA metabolism.

Background: Ferroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation. This process has been implicated in various diseases, including ischemic stroke. Ischemic stroke leads to oxidative stress, iron overload, and reactive oxygen species (ROS) accumulation, which collectively may trigger ferroptotic neuronal cell death. However, the regulatory mechanisms of ferroptosis in stroke remain poorly understood. Previous studies have identified ataxia telangiectasia mutated (ATM), a DNA damage kinase, as a critical regulator of ferroptosis. However, the therapeutic potential of this discovery remains unknown. Methods: We investigated the effect of ATM inhibitors, including the brain-penetrant AZD1390, on ferroptosis using in vitro, ex vivo , and in vivo models of ischemic stroke. Our analysis included assessments of cell viability, lipid peroxidation, ferroptosis marker expression, and infarct volume. Result: ATM inhibitors significantly alleviated ferroptosis-induced cell death in cultured cells and ex vivo murine brain slice cultures. In the oxygen-glucose deprivation (OGD) stroke model, treatment with AZD1390 reduced the expression of ferroptosis markers (xCT and PTGS2) and diminished neuronal cell death in rat and mouse brain slices. Furthermore, in a mouse model of ischemic stroke, AZD1390 decreased infarct volume confirming its therapeutic efficacy in vivo . Conclusions: This study identifies ferroptosis as a critical mechanism in ischemic stroke-induced neuronal cell death and highlights ATM inhibition, particularly with AZD1390, as a promising therapeutic candidate for mitigating stroke-associated damage. Targeting ferroptosis may provide a translationally relevant strategy to mitigate neuronal injury and improve clinical outcomes for stroke patients.

TodevelopanovelUncertaintyQuantification (UQ) framework to estimate the uncertainty of patient survival models in the absence of ground truth, we developed and evaluated our approach based on a dataset of 1383 patients treated with stereotactic radiosurgery (SRS) for brain metastases between January 2015 and December 2020. Our motivating hypothesis is that a time-to-event prediction of a test patient on inference is more certain given a higher feature-space-similarity to patients in the training set. Therefore, the uncertainty for a particular patient-of-interest is represented by the concordance index between a patient similarity rank and a prediction similarity rank. Model uncertainty was defined as the increased percentage of the max uncertainty-constrained-AUC compared to the model AUC. We evaluated our method on multiple clinically-relevant endpoints, including time to intracranial progression (ICP), progression-free survival (PFS) after SRS, overall survival (OS), and time to ICP and/or death (ICPD), on a variety of both statistical and non-statistical models, including CoxPH, conditional survival forest (CSF), and neural multi-task linear regression (NMTLR). Our results show that all models had the lowest uncertainty on ICP (2.21%) and the highest uncertainty (17.28%) on ICPD. OS models demonstrated high variation in uncertainty performance, where NMTLR had the lowest uncertainty(1.96%)and CSF had the highest uncertainty (14.29%). In conclusion, our method can estimate the uncertainty of individual patient survival modeling results. As expected, our data empirically demonstrate that as model uncertainty measured via our technique increases, the similarity between a feature-space and its predicted outcome decreases.

Background: Genetically engineered mouse models (GEMMs) of cancer are powerful tools to study mechanisms of disease progression and therapy response, yet little is known about how these models respond to multimodality therapy used in patients. Radiation therapy (RT) is frequently used to treat localized cancers with curative intent, delay progression of oligometastases, and palliate symptoms of metastatic disease. Methods: Here we report the development, testing, and validation of a platform to immobilize and target tumors in mice with stereotactic ablative RT (SART). Xenograft and autochthonous tumor models were treated with hypofractionated ablative doses of radiotherapy. Results: We demonstrate that hypofractionated regimens used in clinical practice can be effectively delivered in mouse models. SART alters tumor stroma and the immune environment, improves survival in GEMMs of primary prostate and colorectal cancer, and synergizes with androgen deprivation in prostate cancer. Complete pathologic responses were achieved in xenograft models, but not in GEMMs. Conclusions: While SART is capable of fully ablating xenografts, it is unable to completely eradicate disease in GEMMs, arguing that resistance to potentially curative therapy can be modeled in GEMMs.

Radiation necrosis, also known as treatment-induced necrosis, has emerged as an important adverse effect following stereotactic radiotherapy (SRS) for brain metastases. The improved survival of patients with brain metastases and increased use of combined systemic therapy and SRS have contributed to a growing incidence of necrosis. The cyclic GMP-AMP (cGAMP) synthase (cGAS) and stimulator of interferon genes (STING) pathway (cGAS-STING) represents a key biological mechanism linking radiation-induced DNA damage to pro-inflammatory effects and innate immunity. By recognizing cytosolic double-stranded DNA, cGAS induces a signaling cascade that results in the upregulation of type 1 interferons and dendritic cell activation. This pathway could play a key role in the pathogenesis of necrosis and provides attractive targets for therapeutic development. Immunotherapy and other novel systemic agents may potentiate activation of cGAS-STING signaling following radiotherapy and increase necrosis risk. Advancements in dosimetric strategies, novel imaging modalities, artificial intelligence, and circulating biomarkers could improve the management of necrosis. This review provides new insights into the pathophysiology of necrosis and synthesizes our current understanding regarding the diagnosis, risk factors, and management options of necrosis while highlighting novel avenues for discovery.