A gain of function paradox: Targeted therapy for glioblastoma associated with abnormal NHE9 expression

Glioblastoma (GBM) is the most frequent and inevitably lethal primary brain cancer in adults. It is recognized that the overexpression of the endosomal Na+/H+ exchanger NHE9 is a potent driver of GBM progression. Patients with NHE9 overexpression have a threefold lower median survival relative to GBM patients with normal NHE9 expression, using available treatment options. New treatment strategies tailored for this GBM subset are much needed. According to the prevailing model, NHE9 overexpression leads to an increase in plasma membrane density of epidermal growth factor receptors (EGFRs) which consequently enhances GBM cell proliferation and migration. However, this increase is not specific to EGFRs. In fact, the hallmark of NHE9 overexpression is a pan‐specific increase in plasma membrane receptors. Paradoxically, we report that this gain of function in NHE9 can be exploited to effectively target GBM cells for destruction. When exposed to gold nanoparticles, NHE9 overexpressing GBM cells accumulated drastically high amounts of gold via receptor‐mediated endocytosis, relative to control. Irradiation of these cells with near‐infrared light led to apoptotic tumour cell death. A major limitation for delivering therapeutics to GBM cells is the blood‐brain barrier (BBB). Here, we demonstrate that macrophages loaded with gold nanoparticles can cross the BBB, deliver the gold nanoparticles and effect the demise of GBM cells. In combination with receptor tyrosine kinase inhibition, we show this approach holds great promise for a new GBM‐targeted therapy.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152030/1/jcmm14665.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/152030/2/jcmm14665_am.pd

MicroRNA-135a regulates NHE9 to inhibit proliferation and migration of glioblastoma cells

Abstract Background Glioblastoma multiformae (GBM) is the most aggressive type of malignant brain tumor with complex molecular profile. Overexpression of Na+/H+ Exchanger isoform 9 (NHE9) promotes tumor progression and correlates positively with insensitivity to radiochemotherapy and poor prognosis. However, molecular mechanisms responsible for increase in NHE9 levels beyond a critical threshold have not been identified. Methods Bioinformatics analysis, luciferase reporter assays, real-time PCR and western blotting were conducted to examine the expression profiles and identify microRNAs (miRNA) that target NHE9. Cell proliferation and migration assays were conducted in U87 glioblastoma cells to determine the consequence of miRNA mediated targeting of NHE9. Endosomal pH measurements, immunofluorescence microscopy and surface biotinylation experiments were conducted to characterize the mechanistic basis of regulation. Results We show that microRNA 135a (miR-135a) targets NHE9 to downregulate its expression in U87 cells. MiR-135a levels are significantly lower in glioblastoma cells compared to normal brain tissue. Downregulation of NHE9 expression by miR-135a affects proliferative and migratory capacity of U87 cells. Selectively increasing NHE9 expression in these cells restored their ability to proliferate and migrate. We demonstrate that miR-135a takes a two-pronged approach affecting epidermal growth factor receptors (EGFRs) to suppress tumor cell growth and migration. EGFR activity is a potent stimulator of oncogenic signaling. While miR-135a targets EGFR transcripts to decrease the total number of receptors made, by targeting NHE9 it routes the few EGFRs made away from the plasma membrane to dampen oncogenic signaling. NHE9 is localized to sorting endosomes in glioblastoma cells where it alkalinizes the endosome lumen by leaking protons. Downregulation of NHE9 expression by miR-135a acidifies sorting endosomes limiting EGFR trafficking to the glioblastoma cell membrane. Conclusions We propose downregulation of miR-135a as a potential mechanism underlying the high NHE9 expression observed in subset of glioblastomas. Future studies should explore miR-135a as a potential therapeutic for glioblastomas with NHE9 overexpression.https://deepblue.lib.umich.edu/bitstream/2027.42/140393/1/12964_2017_Article_209.pd

NEK1 variants confer susceptibility to amyotrophic lateral sclerosis

To identify genetic factors contributing to amyotrophic lateral sclerosis (ALS), we conducted whole-exome analyses of 1,022 index familial ALS (FALS) cases and 7,315 controls. In a new screening strategy, we performed gene-burden analyses trained with established ALS genes and identified a significant association between loss-of-function (LOF) NEK1 variants and FALS risk. Independently, autozygosity mapping for an isolated community in the Netherlands identified a NEK1 p.Arg261His variant as a candidate risk factor. Replication analyses of sporadic ALS (SALS) cases and independent control cohorts confirmed significant disease association for both p.Arg261His (10,589 samples analyzed) and NEK1 LOF variants (3,362 samples analyzed). In total, we observed NEK1 risk variants in nearly 3% of ALS cases. NEK1 has been linked to several cellular functions, including cilia formation, DNA-damage response, microtubule stability, neuronal morphology and axonal polarity. Our results provide new and important insights into ALS etiopathogenesis and genetic etiology

Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation.Objective: To identify the genetic variants associated with juvenile ALS.Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism.Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members.Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway.Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.</p

Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.Peer reviewe

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Molecular Details of the Frataxin–Scaffold Interaction during Mitochondrial Fe–S Cluster Assembly

Iron–sulfur clusters are essential to almost every life form and utilized for their unique structural and redox-targeted activities within cells during many cellular pathways. Although there are three different Fe–S cluster assembly pathways in prokaryotes (the NIF, SUF and ISC pathways) and two in eukaryotes (CIA and ISC pathways), the iron–sulfur cluster (ISC) pathway serves as the central mechanism for providing 2Fe–2S clusters, directly and indirectly, throughout the entire cell in eukaryotes. Proteins central to the eukaryotic ISC cluster assembly complex include the cysteine desulfurase, a cysteine desulfurase accessory protein, the acyl carrier protein, the scaffold protein and frataxin (in humans, NFS1, ISD11, ACP, ISCU and FXN, respectively). Recent molecular details of this complex (labeled NIAUF from the first letter from each ISC protein outlined earlier), which exists as a dimeric pentamer, have provided real structural insight into how these partner proteins arrange themselves around the cysteine desulfurase, the core dimer of the (NIAUF)2 complex. In this review, we focus on both frataxin and the scaffold within the human, fly and yeast model systems to provide a better understanding of the biophysical characteristics of each protein alone and within the FXN/ISCU complex as it exists within the larger NIAUF construct. These details support a complex dynamic interaction between the FXN and ISCU proteins when both are part of the NIAUF complex and this provides additional insight into the coordinated mechanism of Fe–S cluster assembly

Flooding and Emergency Room Visits for Gastrointestinal Illness in Massachusetts: A Case-Crossover Study

INTRODUCTION: Floods and other severe weather events are anticipated to increase as a result of global climate change. Floods can lead to outbreaks of gastroenteritis and other infectious diseases due to disruption of sewage and water infrastructure and impacts on sanitation and hygiene. Floods have also been indirectly associated with outbreaks through population displacement and crowding. METHODS: We conducted a case-crossover study to investigate the association between flooding and emergency room visits for gastrointestinal illness (ER-GI) in Massachusetts for the years 2003 through 2007. We obtained ER-GI visits from the State of Massachusetts and records of floods from the National Oceanic and Atmospheric Association's Storm Events Database. ER-GI visits were considered exposed if a flood occurred in the town of residence within three hazard periods of the visit: 0-4 days; 5-9 days; and 10-14 days. A time-stratified bi-directional design was used for control selection, matching on day of the week with two weeks lead or lag time from the ER-GI visit. Fixed effect logistic regression models were used to estimate the risk of ER-GI visits following the flood. RESULTS AND CONCLUSIONS: A total of 270,457 ER-GI visits and 129 floods occurred in Massachusetts over the study period. Across all counties, flooding was associated with an increased risk for ER-GI in the 0-4 day period after flooding (Odds Ratio: 1.08; 95% Confidence Interval: 1.03-1.12); but not the 5-9 days (Odds Ratio: 0.995; 95% Confidence Interval: 0.955-1.04) or the 10-14 days after (Odds Ratio: 0.966, 95% Confidence Interval: 0.927-1.01). Similar results were observed for different definitions of ER-GI. The effect differed across counties, suggesting local differences in the risk and impact of flooding. Statewide, across the study period, an estimated 7% of ER-GI visits in the 0-4 days after a flood event were attributable to flooding

A long-duration gamma-ray burst of dynamical origin from the nucleus of an ancient galaxy

The majority of long-duration (&gt;2 s) gamma-ray bursts (GRBs) arise from the collapse of massive stars, with a small proportion created from the merger of compact objects. Most of these systems form via standard stellar evolution pathways. However, a fraction of GRBs may result from dynamical interactions in dense environments. These channels could also contribute substantially to the samples of compact object mergers detected as gravitational wave sources. Here we report the case of GRB 191019A, a long GRB (a duration of T 90 = 64.4 ± 4.5 s), which we pinpoint close (⪅100 pc projected) to the nucleus of an ancient (&gt;1 Gyr old) host galaxy at z = 0.248. The lack of evidence for star formation and deep limits on any supernova emission disfavour a massive star origin. The most likely route for progenitor formation is via dynamical interactions in the dense nucleus of the host. The progenitor, in this case, could be a compact object merger. These may form in dense nuclear clusters or originate in a gaseous disc around the supermassive black hole. Identifying, to the best of our knowledge, a first example of a dynamically produced GRB demonstrates the role that such bursts may have in probing dense environments and constraining dynamical fractions in gravitational wave populations.</p