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We have previously identified the natural product obtusaquinone (OBT) as a potent antineoplastic agent with promising in vivo activity in glioblastoma and breast cancer through the activation of oxidative stress; however, the molecular properties of this compound remained elusive. We used a multidisciplinary approach comprising medicinal chemistry, quantitative mass spectrometry-based proteomics, functional studies in cancer cells, and pharmacokinetic analysis, as well as mouse xenograft models to develop and validate novel OBT analogs and characterize the molecular mechanism of action of OBT. We show here that OBT binds to cysteine residues with a particular affinity to cysteine-rich Keap1, a member of the CUL3 ubiquitin ligase complex. This binding promotes an overall stress response and results in ubiquitination and proteasomal degradation of Keap1 and downstream activation of the Nrf2 pathway. Using positron emission tomography (PET) imaging with the PET-tracer 2-[(18)F]fluoro-2-deoxy-D-glucose (FDG), we confirm that OBT is able to penetrate the brain and functionally target brain tumors. Finally, we show that an OBT analog with improved pharmacological properties, including enhanced potency, stability, and solubility, retains the antineoplastic properties in a xenograft mouse model.

Summary Inherent plasticity and various survival cues allow glioblastoma stem-like cells (GSCs) to survive and proliferate under intrinsic and extrinsic stress conditions. Here, we report that GSCs depend on the adaptive activation of ER stress and subsequent activation of lipogenesis and particularly stearoyl CoA desaturase (SCD1), which promotes ER homeostasis, cytoprotection, and tumor initiation. Pharmacological targeting of SCD1 is particularly toxic due to the accumulation of saturated fatty acids, which exacerbates ER stress, triggers apoptosis, impairs RAD51-mediated DNA repair, and achieves a remarkable therapeutic outcome with 25%–100% cure rate in xenograft mouse models. Mechanistically, divergent cell fates under varying levels of ER stress are primarily controlled by the ER sensor IRE1, which either promotes SCD1 transcriptional activation or converts to apoptotic signaling when SCD1 activity is impaired. Taken together, the dependence of GSCs on fatty acid desaturation presents an exploitable vulnerability to target glioblastoma., Graphical Abstract, Highlights • SCD1 is essential for GSC maintenance and confers tumor growth advantage in vivo • Therapeutic targeting of SCD1 is highly effective in GBM mouse models • SCD1 exerts a cytoprotective function in GSCs by regulating ER homeostasis • ER stress promotes lipogenesis through the activation of IRE1-SREBP1-SCD1 axis, In this article, Pinkham and colleagues demonstrate that the fatty acid desaturase stearoyl CoA desaturase (SCD1) is essential for the maintenance of glioblastoma cancer stem cells. SCD1 is activated by ER stress and exerts a cytoprotective function by regulating ER homeostasis, thus favoring survival and tumor growth. Pharmacological targeting of SCD1 exhibits potent therapeutic efficacy in brain tumor mouse models.

Neurologic manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in pediatric patients have been reported in the acute and postinfectious stages of coronavirus disease 2019. Acute disseminated encephalomyelitis (ADEM) typically presents in children after a viral illness at a mean age of 3 to 7 years. A total of 60% to 90% of literature-reported pediatric patients with ADEM have minimal to no neurologic deficits at long-term follow-up. We present a 17-month-old developmentally typical girl with parental complaints of irritability, upper extremity weakness, and gait disturbance. She presented to the hospital afebrile and irritable with right-sided nasolabial fold flattening, neck stiffness, left upper extremity rigidity, right upper extremity paresis, bilateral lower extremity hyperreflexia, and truncal ataxia. During her hospital course, she became somnolent with autonomic instability and was transferred to intensive care. Contrasted brain MRI revealed diffuse patchy T2 hyperintensities without contrast enhancement. Nasopharyngeal SARS-CoV-2 polymerase chain reaction and serum antibody testing results were positive. Cerebral spinal fluid analysis was unremarkable. Respiratory viral panel and autoimmune encephalitis and demyelinating disorders panel results were negative. She was started on high-dose methylprednisolone and intravenous immunoglobulin, with improvement in mental status, focal deficits, and ambulation. After hospital discharge, she received inpatient rehabilitation for 2 weeks and at 2 month follow-up had a full neurologic recovery. We report the youngest case of postinfectious ADEM due to SARS-CoV-2 in a toddler. Early recognition of autoimmune and inflammatory complications of SARS-CoV-2 is vital for early aggressive immunomodulatory treatment and, consequently, improved morbidity in these patients.

Adeno-associated virus (AAV)-based gene therapy is gaining popularity owing to its excellent safety profile and effective therapeutic outcomes in a number of diseases. Intravenous (IV) injection of AAV into the tail vein, facial vein and retro-orbital (RO) venous sinus have all been useful strategies to infuse the viral vector systemically. However, tail vein injection is technically challenging in juvenile mice, and injection at young ages (≤ postnatal day-(P)21) is essentially impossible. The temporal or facial vein is localized anterior to the ear bud and is markedly visible in the first couple of days postnatally. However, this method is age-dependent and requires a dissecting microscope. Retro-orbital injection (ROI), on the other hand, is suitable for all murine ages, including newborn and older mice, and is relatively less stressful to animals compared to tail vein injection. Although many reports have shown ROI as an effective route of AAV delivery, herein we aim to highlight and summarize the methods and benefits of ROI. To capture the full spectrum of transduction efficiency mediated by ROI, we transduced the editing-dependent reporter mice (Ai9 Cre reporter mice) with the AAV9 vector, which targets a wide range of peripheral tissues with exceptional brain tropism. We also provide a comprehensive description of the ROI technique to facilitate viral vector administration without complications.

We have previously identified the natural product Obtusaquinone (OBT) as a potent antineoplastic agent with promising in vivo activity in glioblastoma and breast cancer through the activation of oxidative stress; however, the molecular properties of this compound remained elusive. We used a multidisciplinary approach comprising medicinal chemistry, quantitative mass spectrometry-based proteomics, functional studies in cancer cells, pharmacokinetic analysis, as well as mouse xenograft models to develop and validate novel OBT analogs and charaterize the molecular mechanism of action of OBT. We here show that OBT and analogs, which have improved pharmacological properties, bind to cysteine residues with particular affinity to cysteine-rich Keap1, a member of the CUL3 ubiquitin ligase complex. This binding promotes an overall stress response and results in ubiquitination and proteasomal degradation of Keap1 and downstream activation of the Nrf2 pathway.

Tracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR (R2 = 0.9722) and NHEJ (R2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo. Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1-10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.

After publication of this article, the authors realized that the Supplementary Figure S5B had an error in it. Specifically, the labels for BIR and AXD1480 on the x-axis were in the wrong place. All text referring to the figure, including the legend, are correct and this does not impact the findings of the study. This error has not been corrected in the original version of the Article.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Glioblastoma is an incurable and highly aggressive brain tumor. Understanding therapeutic resistance and survival mechanisms driving this tumor type is key to finding effective therapies. Smac mimetics (SM) emerged as attractive cancer therapeutics particularly for tumor populations that are highly resistant to conventional apoptosis-inducing therapies. We evaluated the therapeutic efficacy of SM on Glioma stem-like cells (GSCs) and showed that this family of compounds stimulates an adaptive response triggered by TNFα. Increased expression of TNFα results in a prolonged and sustained activation of NF-κB and STAT3 signaling thus activating several tumor cell resistance mechanisms in GSCs. We show that STAT3 activation is contingent on EZH2 activation and uncover a synergistic lethality between SM and EZH2 inhibitors. Therapeutic inhibition of EZH2 impaired the viability of SM-treated GSCs. Our study outlines the molecular underpinnings of SM resistance in glioblastoma and provides mechanistic insight to overcome this resistance and increase therapeutic efficacy.

Tracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR (R2 = 0.9722) and NHEJ (R2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo. Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1–10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.

Proper migration of neurons is essential for the formation and normal functioning of the nervous system. Defects in neuronal migration underlie a number of neurologic diseases in humans. Although cell migration is crucial for neural development, molecular mechanisms guiding neuronal migration remain to be elucidated fully. Newborn neurons from the embryonic medial ganglionic eminence (MGE) migrate a long distance dorsally in the developing brain, giving rise to several types of interneurons in the neocortex.In this study, we developed an immunocytochemistry (ICC) protocol to stain neurons migrating out of the MGE explant embedded in Matrigel. We also established a protocol to efficiently transfect cells in MGE explants, achieving a transduction efficiency of more than 30%.In addition, we developed microfluidic chambers for explants that allow visualization of the vectorial migration of individual neurons from mouse embryonic MGE explants. Our microfluidic system allows monitoring of the distribution of cellular organelles (e.g. Golgi) within migrating neurons which have been stained with commercial molecular dyes or transfected with adeno-associated virus (AAV) expressing reporter proteins.These methods provide new paradigms to study neuronal migration in real-time.

In a previous study, we described the antineoplastic properties of the natural compound Obtusaquinone (OBT) against glioblastoma, breast cancer and other cancer types. We have shown that OBT targets cancer cells by increasing the production of reactive oxygen species (ROS) leading to DNA damage and apoptotic cell death. However, the mechanism behind the tumor targeting properties of OBT is not fully elucidated, hampering further preclinical development. In our current study we combined medical chemistry, quantitative mass-spectrometry-based proteomics, functional cancer cell studies, pharmokinetics and in vivo xenograft models to understand the mechanism of action of this compound and develop new OBT analogues with enhanced pharmacokinetic properties. Mechanistically, we now show that OBT binds cysteine residues, and in particular cysteine residues on Keap1. Our data also demonstrates that binding of OBT to Keap1 leads to its ubiquitination and degradation by the ubiquitin proteasome pathway (UPP). Since Keap1 acts as a negative regulator of Nrf2, its degradation following treatment with OBT results in the activation of Nrf2 signaling. Further, our pharmacokinetic studies demonstrate that OBT is able to cross the blood-brain barrier. In a GBM cancer stem cell xenograft model, OBT treatment results in 50% decrease in glucose uptake as detected using FDG-PET imaging. In conclusion, we show here that OBT binding to Keap1 is crucial for its anti-cancer properties, and that this binding leads to ubiquitination and proteasomal degradation of Keap1 and the activation of Nrf2 pathway. Citation Format: Cintia Carla da Hora, Elie Tabet, Christian Elias Badr, Bakhos Tannous. Obtusaquinone is a cysteine-modifying compound that targets Keap1 for degradation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B112. doi:10.1158/1535-7163.TARG-19-B112

Early-onset dystonia is associated with the deletion of one of a pair of glutamic acid residues (c.904_906delGAG/c.907_909delGAG; p.Glu302del/Glu303del; ΔE 302/303) near the carboxyl-terminus of torsinA, a member of the AAA+ protein family that localizes to the endoplasmic reticulum (ER) lumen and nuclear envelope (NE). This deletion commonly underlies early-onset DYT1 dystonia. While the role of the disease-causing mutation, torsinAΔE, has been established through genetic association studies, it is much less clear whether other rare human variants of torsinA are pathogenic. Two missense variations have been described in single patients; R288Q (c.863G>A; p.Arg288Gln; R288Q) identified in a patient with onset of severe generalized dystonia and myoclonus since infancy, and F205I (c.613T>A, p.Phe205Ile; F205I) in a psychiatric patient with late-onset focal dystonia. In this study, we have undertaken a series of analyses comparing the biochemical and cellular effects of these rare variants to torsinAΔE and wild-type (wt) torsinA in order to reveal whether there are common dysfunctional features. The results revealed that the variants, R288Q and F205I, are more similar in their properties to torsinAΔE protein than to torsinAwt. These findings provide functional evidence for the potential pathogenic nature of these rare sequence variants in the TOR1A gene, thus implicating these pathologies in the development of dystonia.

Background Molecular profile of glioblastoma multiforme (GBM) revealed 4 subtypes, 2 of which, proneural and mesenchymal, have been predominantly observed, with the latter displaying a more aggressive phenotype and increased therapeutic resistance. Single-cell RNA sequencing revealed that multiple subtypes actually reside within the same tumor, suggesting cellular heterogeneity in GBM. Further, plasticity between these 2 subtypes is observed during tumor recurrence and in response to radiation therapy. Methods Patient-derived GBM stemlike cells were cultured as neurospheres. These cells were differentiated in serum by attaching to the culture dishes. The "floating" cells that were not attached/differentiated were harvested from the conditioned medium. The characteristics of these cells were studied with limiting dilution assays and immunofluorescence staining. Cell growth and nuclear factor-kappaB (NFkB) activation were monitored using bioluminescent assays as well as quantitative polymerase chain reaction and western blotting. In vivo tumorigenesis was evaluated in orthotopic xenograft models using bioluminescence imaging. Results Patient-derived GBM stemlike cells undergo differentiation by attaching to the culture dish in serum-containing medium. We observed that a small subset of these cells escape this adhesion/differentiation and grow as floating cells. These cells displayed enhanced cancer stem cell properties with a molecular and phenotypic mesenchymal signature, including resistance to radiation and targeted therapies, a more aggressive tumor formation, and NFkB activation. Conclusion Our results endorse inherent intratumor molecular subtype heterogeneity in glioblastoma and provide a valuable approach to study phenotypic plasticity, which could be applied to find novel therapeutic strategies to eradicate this aggressive tumor and can be extended to other cancer types.

Rapid tumor growth is often accompanied by increased glycolytic metabolism and production of lactic acid creating an acidic tumor environment. Under these conditions, only tumor cells resistant to low pH are able to survive. Glioma stem cells tend to be localized to tumor niches where limited oxygen availability and low pH are predominant. Two glioma stem cells subtypes, proneural and mesenchymal, have been described with the latter displaying a more aggressive phenotype and increased therapeutic resistance. Plasticity between these two subtypes has been observed during tumor recurrence or in response to radiation therapy. Modeling glioblastoma mesenchymal plasticity could have many clinical implications. Here, we show that certain culture conditions reproducibly trigger mesenchymal transition of primary glioblastoma stem cells neurospheres. We identified a subpopulation of glioblastoma stem cells growing as floating cells even under differentiation conditions in the presence of serum. These floating cells displayed stem-like properties with a molecular and phenotypic mesenchymal signature, including resistance to radiation and targeted therapies along with a more aggressive tumor formation. Our results suggest that acidic pH in the cell culture medium promotes this mesenchymal transition. These findings provide a valuable approach to study phenotypic plasticity of glioblastoma, which could be applied to find novel therapeutic strategies to eradicate this aggressive tumor type and could be extended to other cancer types.

Microfluidic platforms for quantitative evaluation of cell biologic processes allow low cost and time efficient research studies of biological and pathological events, such as monitoring cell migration by real-time imaging. In healthy and disease states, cell migration is crucial in development and wound healing, as well as to maintain the body's homeostasis. The microfluidic chambers allow precise measurements to investigate whether fibroblasts carrying a mutation in the TOR1A gene, underlying the hereditary neurologic disease--DYT1 dystonia, have decreased migration properties when compared to control cells. We observed that fibroblasts from DYT1 patients showed abnormalities in basic features of cell migration, such as reduced velocity and persistence of movement. The microfluidic method enabled us to demonstrate reduced polarization of the nucleus and abnormal orientation of nuclei and Golgi inside the moving DYT1 patient cells compared to control cells, as well as vectorial movement of single cells. We report here different assays useful in determining various parameters of cell migration in DYT1 patient cells as a consequence of the TOR1A gene mutation, including a microfluidic platform, which provides a means to evaluate real-time vectorial movement with single cell resolution in a three-dimensional environment

TorsinA is an AAA + ATPase located within the lumen of the endoplasmic reticulum and nuclear envelope, with a mutant form causing early onset torsion dystonia (DYT1). Here we report a new function for torsinA in endoplasmic reticulum-associated degradation (ERAD). Retro-translocation and proteosomal degradation of a mutant cystic fibrosis transmembrane conductance regulator (CFTR ∆F508) was inhibited by downregulation of torsinA or overexpression of mutant torsinA, and facilitated by increased torsinA. Retro-translocation of cholera toxin was also decreased by downregulation of torsinA. TorsinA associates with proteins implicated in ERAD, including Derlin-1, VImP and p97. Further, torsinA reduces endoplasmic reticulum stress in nematodes overexpressing CFTR ∆F508, and fibroblasts from DYT1 dystonia patients are more sensitive than controls to endoplasmic reticulum stress and less able to degrade mutant CFTR. Therefore, compromised ERAD function in the cells of DYT1 patients may increase sensitivity to endoplasmic reticulum stress with consequent alterations in neuronal function contributing to the disease state.