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Background: PARP inhibitors are currently approved for the treatment of germline BRCA1/2 mutant metastatic breast cancer (MBC), which accounts for 5-10% of breast cancer. We hypothesize that a PARP inhibitor may also have efficacy in somatic BRCA1/2 mutant MBC, expanding the potential clinical applicability of PARP inhibitors. We previously demonstrated that somatic BRCA1/2 mutations can be identified by both cell-free DNA and tumor tissue genotyping in a subset of patients with MBC who are not germline BRCA1/2 carriers. Furthermore, a PARP inhibitor was demonstrated to induce cell growth inhibition in a circulating tumor cell culture model generated from a patient with pathogenic somatic BRCA1 mutant MBC (Vidula, Dubash, CCR, 2020). In this trial, we are evaluating the efficacy of a PARP inhibitor in somatic BRCA1/2 mutant MBC. Trial Design: This phase II investigator-initiated clinical trial is enrolling 30 patients with somatic BRCA1/2 mutant MBC identified via cell-free DNA or tumor tissue genotyping. Patients are treated with talazoparib, a PARP inhibitor, until disease progression. At baseline and every 3 months, patients undergo CT chest, abdomen, and pelvis, and a bone scan for disease assessment. Patients undergo blood collection at baseline for the Cancer Risk B (CR-B) assay, a novel flow variant assay to assess double-strand break repair mutations in circulating blood cells (Syeda, 2017) and monthly blood collection for cell-free DNA analysis to evaluate changes in the genomic environment. Eligibility Criteria: Patients with MBC with a pathogenic somatic BRCA1/2 mutation identified by cell-free DNA or tumor tissue genotyping are eligible. Both patients with triple-negative breast cancer (≥ 1 prior chemotherapy) or hormone receptor positive/HER2- MBC (≥ 1 prior hormone therapy) are eligible. Patients should not be known germline BRCA1/2 carriers. There is no limit on prior therapies including receipt of a prior platinum (in the absence of disease progression on prior platinum). A prior PARP inhibitor is not allowed. Adequate performance status and organ function are needed. Specific Aims: Primary aim is progression-free survival (PFS) assessed by RECIST 1.1. Secondary aims include objective response rate and toxicity assessed by NCI CTCAE v 5.0. Exploratory aims include assessing impact of BRCA1/2 reversion mutations in cell-free DNA, studying serial changes in BRCA1/2 mutant allelic frequency in cell-free DNA, comparing pre- and post-treatment cell-free DNA results to identify changes in the genomic environment, assessing the CR-B assay positivity rate, and correlating these biomarker analyses with patient response. Statistical Methods: This study uses a two-stage design with 80% power to demonstrate that talazoparib is associated with “success” (PFS > 12 weeks) in 53% patients (4% alpha). Accrual: This study (NCT03990896) is open at Massachusetts General Hospital, MD Anderson, University of California San Francisco, and Emory, with pending activation at Northwestern, Cornell, and Vanderbilt. Five patients are enrolled as of 7/2022. Funding: This study is supported by a Pfizer ASPIRE award and Conquer Cancer Foundation of ASCO–Breast Cancer Research Foundation- Career Development Award. Contact information: Neelima Vidula, MD, Massachusetts General Hospital, nvidula@mgh.harvard.edu Citation Format: Neelima Vidula, Senthil Damodaran, Erica L. Blouch, Nora Horick, Nathan Royce Ruffle-Deignan, Manali Bhave, Ami N. Shah, Leticia Varella, Vandana Abramson, Joseph Sparano, Leif Ellisen, Ishraq Alim, Harry Ostrer, Hope Rugo, Aditya Bardia. Phase II study of talazoparib, a PARP inhibitor, in somatic BRCA1/2 mutant metastatic breast cancer identified by cell-free DNA or tumor tissue genotyping [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-11-01.

PURPOSE: Heritable pathogenic variants in the DNA mismatch repair (MMR) pathway cause Lynch syndrome, a condition that significantly increases risk of colorectal and other cancers. At least half of individuals tested using gene panel sequencing have a variant of uncertain significance or no variant identified leading to no diagnosis. To fill this diagnostic gap, we developed Cancer Risk C (CR-C), a flow variant assay test. METHODS: In response to treatment with an alkylating agent, individual assays of the nuclear translocation of MLH1, MSH2, BARD1, PMS2, and BRCA2 proteins and the nuclear phosphorylation of the ATM and ATR proteins distinguished pathogenic/likely pathogenic (P/LP) from benign/likely benign variants in MMR genes. RESULTS: A risk classification score based on MLH1, MSH2, and ATR assays was 100% sensitive and 98% specific. Causality of MMR P/LP variants was shown through gene editing and rescue. In individuals with suspected Lynch syndrome but no P/LP, CR-C identified most (73%) as having germline MMR defects. Direct comparison of CR-C on matched blood samples and lymphoblastoid cell lines yielded comparable results (r(2) > 0.9). CONCLUSION: For identifying germline MMR defects, CR-C provides augmentation to traditional panel sequencing through greater accuracy, shorter turnaround time (48 hours), and performance on blood with minimal sample handling.

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.

Modern definitions of epigenetics incorporate models for transient but biologically important changes in gene expression that are unrelated to DNA code but responsive to environmental changes such as injury-induced stress. In this scheme, changes in oxygen levels (hypoxia) and/or metabolic co-factors (iron deficiency or diminished 2-oxoglutarate levels) are transduced into broad genetic programs that return the cell and the organism to a homeostatic set point. Over the past two decades, exciting studies have identified a superfamily of iron-, oxygen-, and 2-oxoglutarate-dependent dioxygenases that sit in the nucleus as modulators of transcription factor stability, co-activator function, histone demethylases, and DNA demethylases. These studies have provided a concrete molecular scheme for how changes in metabolism observed in a host of neurological conditions, including stroke, traumatic brain injury, and Alzheimer's disease, could be transduced into adaptive gene expression to protect the nervous system. We will discuss these enzymes in this short review, focusing primarily on the ten eleven translocation (TET) DNA demethylases, the jumonji (JmJc) histone demethylases, and the oxygen-sensing prolyl hydroxylase domain enzymes (HIF PHDs). This article is part of a Special Issue entitled SI: Neuroprotection.

Neuronal vulnerability to ischemia is dependent on the balance between prosurvival and prodeath cellular signaling. In the latter, it is increasingly appreciated that toxic Ca2+influx can occur not only via postsynaptic glutamate receptors, but also through other cation conductances. One such conductance, the Transient receptor potential melastatin type-2 (TRPM2) channel, is a nonspecific cation channel having homology to TRPM7, a conductance reported to play a key role in anoxic neuronal death. The role of TRPM2 conductances in ischemic Ca2+influx has been difficult to study because of the lack of specific modulators. Here we used TRPM2-null mice (TRPM2−/−) to study how TRPM2 may modulate neuronal vulnerability to ischemia. TRPM2−/−mice subjected to transient middle cerebral artery occlusion exhibited smaller infarcts when compared with wild-type animals, suggesting that the absence of TRPM2 is neuroprotective. Surprisingly, field potentials (fEPSPs) recorded during redox modulation in brain slices taken from TRPM2−/−mice revealed increased excitability, a phenomenon normally associated with ischemic vulnerability, whereas wild-type fEPSPs were unaffected. The upregulation in fEPSP in TRPM2−/−neurons was blocked selectively by a GluN2A antagonist. This increase in excitability of TRPM2−/−fEPSPs during redox modulation depended on the upregulation and downregulation of GluN2A- and GluN2B-containing NMDARs, respectively, and on augmented prosurvival signaling via Akt and ERK pathways culminating in the inhibition of the proapoptotic factor GSK3β. Our results suggest that TRPM2 plays a role in downregulating prosurvival signals in central neurons and that TRPM2 channels may comprise a therapeutic target for preventing ischemic damage.

During a stroke, the brain integrates stress and damage signals to ensure neuronal death or survival to optimize survival of the whole organism. Stresses relevant to stroke include hypoxia, glucose deprivation, and oxidative stress. Our laboratory has focused on understanding how these stresses are transduced into changes in gene expression by focusing on transcription factors activated by stress, and whether these transcription factors mediate prodeath or prosurvival responses. Prior data from our laboratory have shown that when transcriptional changes that integrate cell survival responses fail and stress or damage is persistent, programmed cell death is activated. Specifically, we discuss the transcription factors activating transcription factor 4 and hypoxia inducible factor and the role they play during stroke. We also explore the development of therapeutic strategies to target the activity of these transcription factors to provide neuroprotection during stroke.

Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier–permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models.

Mitochondrial impairment induced by oxidative stress is a main characteristic of intrinsic cell death pathways in neurons underlying the pathology of neurodegenerative diseases. Therefore, protection of mitochondrial integrity and function is emerging as a promising strategy to prevent neuronal damage. Here, we show that pharmacological inhibition of hypoxia-inducible factor prolyl-4-hydroxylases (HIF-PHDs) by adaptaquin inhibits lipid peroxidation and fully maintains mitochondrial function as indicated by restored mitochondrial membrane potential and ATP production, reduced formation of mitochondrial reactive oxygen species (ROS) and preserved mitochondrial respiration, thereby protecting neuronal HT-22 cells in a model of glutamate-induced oxytosis. Selective reduction of PHD1 protein using CRISPR/Cas9 technology also reduced both lipid peroxidation and mitochondrial impairment, and attenuated glutamate toxicity in the HT-22 cells. Regulation of activating transcription factor 4 (ATF4) expression levels and related target genes may mediate these beneficial effects. Overall, these results expose HIF-PHDs as promising targets to protect mitochondria and, thereby, neurons from oxidative cell death.

Hydrogen peroxide (H2O2) is produced endogenously in a number of cellular compartments, including the mitochondria, the endoplasmic reticulum, peroxisomes, and at the plasma membrane, and can play divergent roles as a second messenger or a pathological toxin. It is assumed that the tuned production of H2O2 within neuronal and non-neuronal cells regulates a discreet balance between survival and death. However, a major challenge in understanding the physiological versus pathological role of H2O2 in cells has been the lack of validated methods that can spatially, temporally, and quantitatively modulate H2O2 production. A promising means of regulating endogenous H2O2 is through the expression of peroxide-producing enzyme D-amino acid oxidase (DAAO from Rhodotorula gracilis lacking a peroxisomal targeting sequence). Using viral vectors to express DAAO in distinct cell types and using targeting sequences to target DAAO to distinct subcellular sites, we can manipulate H2O2 production by applying the substrate D-alanine or permeable analogs of D-alanine. In this chapter, we describe the use of DAAO to produce H2O2 in culture models and the real-time visual validation of this technique using two-photon microscopy and chemoselective fluorescent probes.

Article history: Accepted 25 February 2010 Available online 4 March 2010 Adiponectin (ADP) is a peptide produced by adipose tissue, which acts as an insulin sensitizing hormone. Recent studies have shown that adiponectin receptors (AdipoR1 and AdipoR2) are present in the CNS, and although adiponectin does appear in both circulation and the cerebrospinal fluid there is still some debate as to whether or not ADP crosses the blood brain barrier (BBB). Circumventricular organs (CVO) are CNS sites which lack normal BBB, and thus represent sites at which circulating adiponectin may act to directly influence the CNS. The subfornical organ (SFO) is a CVO that has been implicated in the regulation of energy balance as a consequence of the ability of SFO neurons to respond to a number of different circulating satiety signals including amylin, CCK, PYY and ghrelin. Our recent microarray analysis suggested the presence of adiponectin receptors in the SFO. We report here that the SFO shows a high density of mRNA for both adiponectin receptors (AdipoR1 and AdipoR2), and that ADP influences the excitability of dissociated SFO neurons. Separate subpopulations of SFO neurons were either depolarized (8.9±0.9 mV, 21 of 97 cells), or hyperpolarized (�8.0±0.5 mV, 34 of 97 cells), by bath application of 10 nM ADP, effects which were concentration dependent and reversible. Our microarray analysis also suggested that 48 h of food deprivation resulted in specific increases in AdipoR2 mRNA expression (no effect on AdipoR1 mRNA), observations which we confirm here using real-time PCR techniques. The effects of food deprivation also resulted in a change in the responsiveness of SFO neurons to adiponectin with 77% (8/11) of cells tested responding to adiponectin with depolarization, while no hyperpolarizations were observed. These observations support the concept that the SFO may be a key player in sensing circulating ADP and transmitting such information to critical CNS sites involved in the regulation of energy balance.

As interest in the gut microbiome has grown in recent years, attention has turned to the impact of our diet on our brain. The benefits of a high fiber diet in the colon have been well documented in epidemiological studies, but its potential impact on the brain has largely been understudied. Here, we will review evidence that butyrate, a short-chain fatty acid (SCFA) produced by bacterial fermentation of fiber in the colon, can improve brain health. Butyrate has been extensively studied as a histone deacetylase (HDAC) inhibitor but also functions as a ligand for a subset of G protein-coupled receptors and as an energy metabolite. These diverse modes of action make it well suited for solving the wide array of imbalances frequently encountered in neurological disorders. In this review, we will integrate evidence from the disparate fields of gastroenterology and neuroscience to hypothesize that the metabolism of a high fiber diet in the gut can alter gene expression in the brain to prevent neurodegeneration and promote regeneration.