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Antibody-drug conjugates (ADCs) are increasingly used in clinic for multiple indications and may improve upon the activity of parental antibodies by delivering cytotoxic payloads into target cells. This activity is predicated upon internalization to release the cytotoxic payloads intracellularly. Since binding of ADCs to their cell surface targets does not guarantee their internalization, we hypothesize that proteolysis targeting chimeras (PROTACs) could improve the activity of ADCs through forced internalization. We show that PROTACs improve internalization of antibodies or their derivative antibody drug conjugates when both agents target the same oncogenic cell surface proteins (EGFR, HER2 or MET) by 1.4-1.9 fold in most models. PROTACs also significantly enhance cytotoxicity with HER2-targeting ADCs. These effects depend on dynamin and proteolysis. This application of PROTACs may impact the use of ADCs and provides a rationale to combine these agents in clinical trials., Competing Interests: Competing interests: Mayo Clinic submitted patent 63/560,270 related to this work for the institution on 01 March 2024 listing the inventors Aaron S. Mansfield M.D., Ezequiel J. Tolosa Ph.D., Jennifer R. Ayers-Ringler Ph.D., Lin Yang Ph.D., and Farhad Kosari Ph.D. that is under review at the time of publication. Mitesh Borad reports grants to Mayo Clinic from Senhwa Pharmaceuticals, Adaptimmune, Agios Pharmaceuticals, Halozyme Pharmaceuticals, Celgene Pharmaceuticals, EMD Merck Serono, Toray, Dicerna, Taiho Pharmaceuticals, Sun Biopharma, Isis Pharmaceuticals, Redhill Pharmaceuticals, Boston Biomed, Basilea, Incyte Pharmaceuticals, Mirna Pharmaceuticals, Medimmune, Bioline, Sillajen, ARIAD Pharmaceuticals, PUMA Pharmaceuticals, Novartis Pharmaceuticals, QED Pharmaceuticals, Pieris Pharmaceuticals; and consulting to self from ADC Therapeutics, Exelixis Pharmaceuticals, Inspyr Therapeutics, G1 Therapeutics, Immunovative Therapies, OncBioMune Pharmaceuticals, Western Oncolytics, Lynx Group, Genentech, Merck, and Huya. Aaron Mansfield reports consulting fees to his institution from Genentech, Bristol Myers Squibb, AbbVie, AstraZeneca, Janssen, BeiGene, Takeda, Genzyme, Gilead Sciences, and Johnson & Johnson Global Services., (© 2024. The Author(s).)

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screen them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver. MET exon 14 skipping mutations (METex14Δ) are found in lung cancers and result in the loss of a degron that is required for E3-ligase recognition and subsequent ubiquitination, prolonging the half-life and oncogenicity of MET. Since proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that promote target degradation by the proteosome, we sought to restore degradation of MET lost with METex14Δ using a MET-targeting PROTAC. We generated a library of sixty PROTACs of which 37 used the MET inhibitor capmatinib as the protein of interest targeting ligand. We screened this PROTAC library for targeted degradation of METex14Δ-GFP using live cell imaging. We benchmarked out MET-targeting PROTACs to that of a previously reported MET-targeting PROTAC, SJF8240. Curve fitting live cell imaging data affords determination of time required to degrade 50% of the target protein (DT50), which was used in determining structure activity relationships. A promising candidate, 48-284, identified from the screen, exhibited classic PROTAC characteristics, was > 15-fold more potent than SJF8240, had fewer off targets compared to SJF8240, and degraded MET in multiple cell lines.

Background: Aneurysm treatments are crucial to minimize the rupture risk. The underlying molecular processes mediating cellular remodeling, endothelialization, and aneurysm healing following endovascular treatment are poorly understood. The current study aims to explore circulating miRNA as a treatment and outcome-associated biomarkers in patients undergoing endovascular treatment., Methods: Patients undergoing endovascular interventions for unruptured intracranial aneurysms, using either flow diverter placement or coil embolization, were enrolled. Blood samples were collected before the intervention and during a follow-up period between 6 and 18 months. Total mRNA/miRNA was isolated from plasma, followed by RNA-seq analysis. Gene Ontology analysis was used to identify pathways linked to altered miRNA expression., Results: Twenty-three patients participated, with 13 (56.5%) undergoing flow diversion and 10 (43.5%) coil embolization. The median follow-up sample collection time was 10.70 months (SEM ± 1.32). No significant differences in angiographic occlusion were noted between intervention groups. Differentially expressed miRNAs were not identified between groups at baseline. However, at follow-up, 39 miRNAs were upregulated and 41 were downregulated, independent of intervention. Notably, three miRNAs (miR-4746-5p, miR-4685-3p, and miR-490-3p) were downregulated in the flow diversion group compared to the coil embolization group. Bioinformatics analysis revealed associations with upregulated fluid shear stress, p53, adherens junction pathways, along with downregulated apoptosis pathways., Conclusions: This study suggests that fluid shear stress and apoptosis may influence aneurysm healing or thromboembolic events in flow diverter-treated patients. Further research is warranted to elucidate the functional significance of these findings in treatment outcomes, providing valuable insights for improved patient care in intracranial aneurysm management., Competing Interests: Declaration of conflicting interestsThe authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: DFK holds equity in Nested Knowledge, Superior Medical Editors, Conway Medical, Marblehead Medical, and Piraeus Medical. He receives grant support from MicroVention, Medtronic, Balt, and Insera Therapeutics; has served on the Data Safety Monitoring Board for Vesalio; and received royalties from Medtronic. WB holds equity in Nested Knowledge, Superior Medical Editors, Piraeus Medical, Sonoris Medical, and MIVI Neurovascular. He receives royalties from Medtronic and Balloon Guide Catheter Technology. He receives consulting fees from Medtronic, Stryker, Imperative Care, Microvention, MIVI Neurovascular, Cerenovus, Asahi, and Balt. He serves in a leadership or fiduciary role for MIVI Neurovascular, Marblehead Medical LLC, Interventional Neuroradiology (Editor in Chief), Piraeus Medical, and WFITN. RK is contracted/consultant for Cerenovus Inc., Medtronic, Endovascular Engineering, Frontier Bio, Sensome Inc., Endomimetics, Ancure LLC, Neurogami Medical, MIVI Biosciences, Monarch Biosciences, Stryker Inc., Conway Medical, Pireus Medical, and Bionaut Labs. All remaining authors have declared no conflicts of interest.

Background: Flow diverters (FDs) are an effective treatment for intracranial aneurysms, though not free from hemorrhagic complications. A previous study demonstrated increased vascular contractility after FD-implantation as a potential mechanism of distal complications. Our study aimed to investigate whether L-arginine medication affects vascular contractility following FD deployment in a rabbit model., Methods: FDs were implanted in the aorta of normal rabbits (+FD, n = 10), with sham-operated aorta as controls (n = 5). L-Arginine was given in the drinking water (2.25% L-arginine hydrochloride) of half of the +FD animals (+FD/+Arg). Force contraction vascular contractility studies were performed on the aortic rings proximal and distal to the FD using an organ bath. Total eNOS, eNOS(pS1177), eNOS(pT495), COX-2, and S100A4 were quantified by western analysis on total protein lysates from aortic segments, normalizing to GAPDH., Results: Mean vascular contractility was 53% higher in distal relative to proximal aortic segments (P = 0.0038) in +FD animals, but were not significantly different in +FD/+Arg animals, or in sham-operated controls. The +FD animals expressed significantly reduced levels of eNOS(pS1177) than sham-operated controls (P = 0.0335), while both the +FD and +FD/+Arg groups had reduced levels of eNOS(pT495) relative to sham-operated controls (P = 0.0331 and P = 0.0311, respectively)., Conclusion: These results suggest that L-arginine medication reduces distal vascular contractility after FD treatment via nitric oxide production and thus might mitigate risk for downstream complications.

Background Concerns over the neurotoxic potential of retained gadolinium in brain tissues after intravenous gadolinium-based contrast agent (GBCA) administration have led to pronounced worldwide use changes, yet the clinical sequelae of gadolinium retention remain undefined. Purpose To assess clinical and neurologic effects and potential neurotoxicity of gadolinium retention in rats after administration of various GBCAs. Materials and Methods From March 2017 through July 2018, 183 male Wistar rats received 20 intravenous injections of 2.5 mmol per kilogram of body weight (80 human equivalent doses) of various GBCAs (gadodiamide, gadobenate, gadopentetate, gadoxetate, gadobutrol, gadoterate, and gadoteridol) or saline over 4 weeks. Rats were evaluated 6 and 34 weeks after injection with five behavioral tests, and inductively coupled plasma mass spectrometry, transmission electron microscopy, and histopathology were performed on urine, serum, cerebrospinal fluid (CSF), basal ganglia, dentate nucleus, and kidney samples. Dunnett post hoc test and Wilcoxon rank sum test were used to compare differences between treatment groups. Results No evidence of differences in any behavioral test was observed between GBCA-exposed rats and control animals at either 6 or 34 weeks ( P = .08 to P = .99). Gadolinium concentrations in both neuroanatomic locations were higher in linear GBCA-exposed rats than macrocyclic GBCA-exposed rats at 6 and 34 weeks ( P < .001). Gadolinium clearance over time varied among GBCAs, with gadobutrol having the largest clearance (median: 62% for basal ganglia, 70% for dentate) and gadodiamide having no substantial clearance. At 34 weeks, gadolinium was largely cleared from the CSF and serum of gadodiamide-, gadobenate-, gadoterate-, and gadobutrol-exposed rats, especially for the macrocyclic agents (range: 70%-98% removal for CSF, 34%-94% removal for serum), and was nearly completely removed from urine (range: 96%-99% removal). Transmission electron microscopy was used to detect gadolinium foci in linear GBCA-exposed brain tissue, but no histopathologic differences were observed for any GBCA. Conclusion In this rat model, no clinical evidence of neurotoxicity was observed after exposure to linear and macrocyclic gadolinium-based contrast agents at supradiagnostic doses. © RSNA, 2022 Online supplemental material is available for this article.

Thrombus characteristics are dependent on clot composition, but identification of the etiology based on histological analysis has proved inconclusive. Identification of proteomic signatures may help to differentiate between clots of different etiologies such as cardioembolic, large artery atherosclerotic, and other known etiologies, information that could enhance an individualized medicine approach to secondary stroke prevention. In this study, total protein extracts from cardioembolic (n=25) and large artery atherosclerotic (n=23) thrombus specimens were arrayed in quadruplicate on nitrocellulose slides and immunostained for 31 proteins using a Dako Autostainer (Agilent Technologies, Inc., Santa Clara, USA). We quantified 31 proteins involved in platelet and/or endothelial function, inflammation, oxidative stress, and metabolism. Pathway analysis showed more heterogeneity and protein network interactions in the cardioembolic clots but no specific correlations with clot etiology. Reverse-phase protein arrays are a powerful tool for assessing cellular interactions within the clot microenvironment and may enhance understanding of clot formation and origination. This tool could be further explored to help in identifying stroke etiology in large vessel occlusion patients with embolic stroke of an undetermined source., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2021, Abbasi et al.)

The poorly understood mechanisms of aneurysm healing contribute substantially to the pressing medical problem of coiled aneurysm recanalization. Using an established saccular aneurysm model, we developed an animal model system in rabbits to study aneurysm and skin wound healing concurrently in the same animal. We found treated aneurysm healing to be similar to skin wound healing both histologically and in biomarker gene and protein expression, but in a delayed fashion., (© 2019 by American Journal of Neuroradiology.)

Although neurotensin (NT) analogs are known to produce antipsychotic-like effects, the therapeutic possibility of a brain penetrant NTS1 agonist in treating psychiatric disorders has not been well studied. Here, we examined whether PD149163, a brain-penetrant NTS1-specific agonist, displays antipsychotic-like effects in C57BL/6J mice by investigating the effect of PD149163 on amphetamine-mediated hyperactivity and amphetamine-induced disruption of prepulse inhibition. In addition, we assessed the effect of PD149163 on glycogen synthase kinase-3 (GSK-3) activity, a downstream molecular target of antipsychotics and mood stabilizers, using phospho-specific antibodies. PD149163 (0.1 and 0.5mg/kg) inhibited amphetamine-induced hyperactivity in mice, indicating that NTS1 activation inhibits psychomotor agitation. PD149163 (0.5mg/kg) also increased prepulse inhibition, suggesting that NTS1 activation reduces prepulse inhibition deficits which often co-occur with psychosis in humans. Interestingly, PD149163 increased the inhibitory serine phosphorylation on both GSK-3α and GSK-3β in a dose- and time-dependent manner in the nucleus accumbens and medial prefrontal cortex of the mice. Moreover, PD149163 inhibited GSK-3 activity in the nucleus accumbens and medial prefrontal cortex in the presence of amphetamine. Thus, like most current antipsychotics and mood stabilizers, PD149163 inhibited GSK-3 activity in cortico-striatal circuitry. Together, our findings indicate that PD149163 may be a novel antipsychotic., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recent studies have demonstrated that the function of glia is not restricted to the support of neuronal function. In fact, astrocytes are essential for neuronal activity in the brain and play an important role in the regulation of complex behavior. Astrocytes actively participate in synapse formation and brain information processing by releasing and uptaking glutamate, D-serine, adenosine 5'-triphosphate (ATP), and adenosine. In the central nervous system, adenosine-mediated neuronal activity modulates the actions of other neurotransmitter systems. Adenosinergic fine-tuning of the glutamate system in particular has been shown to regulate circadian rhythmicity and sleep, as well as alcohol-related behavior and drinking. Adenosine gates both photic (light-induced) glutamatergic and nonphotic (alerting) input to the circadian clock located in the suprachiasmatic nucleus of the hypothalamus. Astrocytic, SNARE-mediated ATP release provides the extracellular adenosine that drives homeostatic sleep. Acute ethanol increases extracellular adenosine, which mediates the ataxic and hypnotic/sedative effects of alcohol, while chronic ethanol leads to downregulated adenosine signaling that underlies insomnia, a major predictor of relapse. Studies using mice lacking the equilibrative nucleoside transporter 1 have illuminated how adenosine functions through neuroglial interactions involving glutamate uptake transporter GLT-1 [referred to as excitatory amino acid transporter 2 (EAAT2) in human] and possibly water channel aquaporin 4 to regulate ethanol sensitivity, reward-related motivational processes, and alcohol intake.

Postnatal oligodendrocyte progenitor cells (OPC) self-renew, generate mature oligodendrocytes, and are a cellular origin of oligodendrogliomas. We show that the proteoglycan NG2 segregates asymmetrically during mitosis to generate OPC cells of distinct fate. NG2 is required for asymmetric segregation of EGFR to the NG2(+) progeny, which consequently activates EGFR and undergoes EGF-dependent proliferation and self-renewal. In contrast, the NG2(-) progeny differentiates. In a mouse model, decreased NG2 asymmetry coincides with premalignant, abnormal self-renewal rather than differentiation and with tumor-initiating potential. Asymmetric division of human NG2(+) cells is prevalent in non-neoplastic tissue but is decreased in oligodendrogliomas. Regulators of asymmetric cell division are misexpressed in low-grade oligodendrogliomas. Our results identify loss of asymmetric division associated with the neoplastic transformation of OPC., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Malignant astrocytic brain tumors are among the most lethal cancers. Quiescent and therapy-resistant neural stem cell (NSC)-like cells in astrocytomas are likely to contribute to poor outcome. Malignant oligodendroglial brain tumors, in contrast, are therapy sensitive. Using magnetic resonance imaging (MRI) and detailed developmental analyses, we demonstrated that murine oligodendroglioma cells show characteristics of oligodendrocyte progenitor cells (OPCs) and are therapy sensitive, and that OPC rather than NSC markers enriched for tumor formation. MRI of human oligodendroglioma also suggested a white matter (WM) origin, with markers for OPCs rather than NSCs similarly enriching for tumor formation. Our results suggest that oligodendroglioma cells show hallmarks of OPCs, and that a progenitor rather than a NSC origin underlies improved prognosis in patients with this tumor., (Copyright © 2010 Elsevier Inc. All rights reserved.)