The Therapeutic and Diagnostic Potential of Amyloid ß Oligomers Selective Antibodies to Treat Alzheimer's Disease.

Improvements have been made in the diagnosis of Alzheimer's disease (AD), manifesting mostly in the development of in vivo imaging methods that allow for the detection of pathological changes in AD by magnetic resonance imaging (MRI) and positron emission tomography (PET) scans. Many of these imaging methods, however, use agents that probe amyloid fibrils and plaques-species that do not correlate well with disease progression and are not present at the earliest stages of the disease. Amyloid ß oligomers (AßOs), rather, are now widely accepted as the Aß species most germane to AD onset and progression. Here we report evidence further supporting the role of AßOs as pathological instigators of AD and introduce promising anti-AßO diagnostic probes capable of distinguishing the 5xFAD mouse model from wild type mice by PET and MRI. In a developmental study, Aß oligomers in 5xFAD mice were found to appear at 3 months of age, just prior to the onset of memory dysfunction, and spread as memory worsened. The increase of AßOs is prominent in the subiculum and correlates with concomitant development of reactive astrocytosis. The impact of these AßOs on memory is in harmony with findings that intraventricular injection of synthetic AßOs into wild type mice induced hippocampal dependent memory dysfunction within 24 h. Compelling support for the conclusion that endogenous AßOs cause memory loss was found in experiments showing that intranasal inoculation of AßO-selective antibodies into 5xFAD mice completely restored memory function, measured 30-40 days post-inoculation. These antibodies, which were modified to give MRI and PET imaging probes, were able to distinguish 5xFAD mice from wild type littermates. These results provide strong support for the role of AßOs in instigating memory loss and salient AD neuropathology, and they demonstrate that AßO selective antibodies have potential both for therapeutics and for diagnostics.

Lgr5-positive endothelial progenitor cells occupy a tumor and injury prone niche in the kidney vasa recta.

The Wnt pathway co-receptor, Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5), labels tumor-prone stem cell populations in certain types of tissue. In this study, we show that ARID1A and PIK3CA mutations in LGR5+ cells result in renal angiosarcomas in adult mice. The tumors originate in the renal medulla. We further show that LGR5 labels SOX17+/CD31+/CD34+/CD133+/AQP1+/CD146+ endothelial progenitor cells within the descending vasa recta or straight arterioles of the kidney, which are specialized capillaries that maintain medullary osmotic gradients necessary for water reabsorption and the production of concentrated urine. LGR5+ endothelial progenitor cells are tightly associated with contractile pericytes within the descending vasa recta. Long-term in vivo lineage tracing revealed that LGR5+ cells give rise to renal medullary vasculature. We further show that LGR5+ cells are activated in response to ischemic kidney injury. Our findings uncover a physiologically relevant endothelial progenitor cell population within the kidney vasa recta.

Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF from Chromatin in a Schedule-dependent Manner.

PURPOSE: The successful clinical translation of compounds that target specific oncogenic transcription factors will require an understanding of the mechanism of target suppression to optimize the dose and schedule of administration. We have previously shown trabectedin reverses the gene signature of the EWS-FLI1 transcription factor. In this report, we establish the mechanism of suppression and use it to justify the reevaluation of this drug in the clinic in patients with Ewing sarcoma.Experimental Design: We demonstrate a novel epigenetic mechanism of trabectedin using biochemical fractionation and chromatin immunoprecipitation sequencing. We link the effect to drug schedule and EWS-FLI1 downstream target expression using confocal microscopy, qPCR, Western blot analysis, and cell viability assays. Finally, we quantitate target suppression within the three-dimensional architecture of the tumor in vivo using 18F-FLT imaging. RESULTS: Trabectedin evicts the SWI/SNF chromatin-remodeling complex from chromatin and redistributes EWS-FLI1 in the nucleus leading to a marked increase in H3K27me3 and H3K9me3 at EWS-FLI1 target genes. These effects only occur at high concentrations of trabectedin leading to suppression of EWS-FLI1 target genes and a loss of cell viability. In vivo, low-dose irinotecan is required to improve the magnitude, penetrance, and duration of target suppression in the three-dimensional architecture of the tumor leading to differentiation of the Ewing sarcoma xenograft into benign mesenchymal tissue. CONCLUSIONS: These data provide the justification to evaluate trabectedin in the clinic on a short infusion schedule in combination with low-dose irinotecan with 18F-FLT PET imaging in patients with Ewing sarcoma.

Phosphorylation of TXNIP by AKT Mediates Acute Influx of Glucose in Response to Insulin.

Growth factors, such as insulin, can induce both acute and long-term glucose uptake into cells. Apart from the rapid, insulin-induced fusion of glucose transporter (GLUT)4 storage vesicles with the cell surface that occurs in muscle and adipose tissues, the mechanism behind acute induction has been unclear in other systems. Thioredoxin interacting protein (TXNIP) has been shown to be a negative regulator of cellular glucose uptake. TXNIP is transcriptionally induced by glucose and reduces glucose influx by promoting GLUT1 endocytosis. Here, we report that TXNIP is a direct substrate of protein kinase B (AKT) and is responsible for mediating AKT-dependent acute glucose influx after growth factor stimulation. Furthermore, TXNIP functions as an adaptor for the basal endocytosis of GLUT4 in vivo, its absence allows excess glucose uptake in muscle and adipose tissues, causing hypoglycemia during fasting. Altogether, TXNIP serves as a key node of signal regulation and response for modulating glucose influx through GLUT1 and GLUT4.

Near infrared fluorescent imaging of brain tumor with IR780 dye incorporated phospholipid nanoparticles.

BACKGROUND: Near-IR fluorescence (NIRF) imaging is becoming a promising approach in preclinical tumor detection and clinical image-guided oncological surgery. While heptamethine cyanine dye IR780 has excellent tumor targeting and imaging potential, its hydrophobic property limits its clinical use. In this study, we developed nanoparticle formulations to facilitate the use of IR780 for fluorescent imaging of malignant brain tumor. METHODS: Self-assembled IR780-liposomes and IR780-phospholipid micelles were prepared and their NIRF properties were characterized. The intracellular accumulation of IR780-nanoparticles in glioma cells were determined using confocal microscopy. The in vivo brain tumor targeting and NIRF imaging capacity of IR780-nanoparticles were evaluated using U87MG glioma ectopic and orthotopic xenograft models and a spontaneous glioma mouse model driven by RAS/RTK activation. RESULTS: The loading of IR780 into liposomes or phospholipid micelles was efficient. The particle diameter of IR780-liposomes and IR780-phospholipid micelles were 95 and 26 nm, respectively. While stock solutions of each preparation were maintained at ready-to-use condition, the IR780-phospholipid micelles were more stable. In tissue culture cells, IR780-nanoparticles prepared by either method accumulated in mitochondria, however, in animals the IR780-phospholipid micelles showed enhanced intra-tumoral accumulation in U87MG ectopic tumors. Moreover, IR780-phospholipid micelles also showed preferred intracranial tumor accumulation and potent NIRF signal intensity in glioma orthotopic models at a real-time, non-invasive manner. CONCLUSION: The IR780-phospholipid micelles demonstrated tumor-specific NIRF imaging capacity in glioma preclinical mouse models, providing great potential for clinical imaging and image-guided surgery of brain tumors.

18F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma.

Ewing sarcoma is a bone and soft-tissue tumor that depends on the activity of the EWS-FLI1 transcription factor for cell survival. Although a number of compounds have been shown to inhibit EWS-FLI1 in vitro, a clinical EWS-FLI1-directed therapy has not been achieved. One problem plaguing drug development efforts is the lack of a suitable, non-invasive, pharmacodynamic marker of EWS-FLI1 activity. Here we show that 18F-FLT PET (18F- 3'-deoxy-3'-fluorothymidine positron emission tomography) reflects EWS-FLI1 activity in Ewing sarcoma cells both in vitro and in vivo. 18F-FLT is transported into the cell by ENT1 and ENT2, where it is phosphorylated by TK1 and trapped intracellularly. In this report, we show that silencing of EWS-FLI1 with either siRNA or small-molecule EWS-FLI1 inhibitors suppressed the expression of ENT1, ENT2, and TK1 and thus decreased 18F-FLT PET activity. This effect was not through a generalized loss in viability or metabolic suppression, as there was no suppression of 18F-FDG PET activity and no suppression with chemotherapy. These results provide the basis for the clinical translation of 18F-FLT as a companion biomarker of EWS-FLI1 activity and a novel diagnostic imaging approach for Ewing sarcoma.

(99m)TC-Methylene diphosphonate uptake at injury site correlates with osteoblast differentiation and mineralization during bone healing in mice.

(99m)Tc-Methylene diphosphonate ((99m)Tc-MDP) is widely used in clinical settings to detect bone abnormalities. However, the mechanism of (99m)Tc-MDP uptake in bone is not well elucidated. In this study, we utilized a mouse tibia injury model, single-photon emission computed tomography (gamma scintigraphy or SPECT), ex vivo micro-computed tomography, and histology to monitor (99m)Tc-MDP uptake in injury sites during skeletal healing. In an ex vivo culture system, calvarial cells were differentiated into osteoblasts with osteogenic medium, pulsed with (99m)Tc-MDP at different time points, and quantitated for (99m)Tc-MDP uptake with a gamma counter. We demonstrated that (99m)Tc-MDP uptake in the injury sites corresponded to osteoblast generation in those sites throughout the healing process. The (99m)Tc-MDP uptake within the injury sites peaked on day 7 post-injury, while the injury sites were occupied by mature osteoblasts also starting from day 7. (99m)Tc-MDP uptake started to decrease 14 days post-surgery, when we observed the highest level of bony tissue in the injury sites. We also found that (99m)Tc-MDP uptake was associated with osteoblast maturation and mineralization in vitro. This study provides direct and biological evidence for (99m)Tc-MDP uptake in osteoblasts during bone healing in vivo and in vitro.

Targeting ornithine decarboxylase reverses the LIN28/Let-7 axis and inhibits glycolytic metabolism in neuroblastoma.

LIN28 has emerged as an oncogenic driver in a number of cancers, including neuroblastoma (NB). Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that impact the LIN28/Let-7 pathway could be beneficial in treating NB patients. The LIN28/Let-7 pathway affects many cellular processes including the regulation of cancer stem cells and glycolytic metabolism. Polyamines, regulated by ornithine decarboxylase (ODC) modulate eIF-5A which is a direct regulator of the LIN28/Let-7 axis. We propose that therapy inhibiting ODC will restore balance to the LIN28/Let-7 axis, suppress glycolytic metabolism, and decrease MYCN protein expression in NB. Difluoromethylornithine (DFMO) is an inhibitor of ODC in clinical trials for children with NB. In vitro experiments using NB cell lines, BE(2)-C, SMS-KCNR, and CHLA90 show that DFMO treatment reduced LIN28B and MYCN protein levels and increased Let-7 miRNA and decreased neurosphere formation. Glycolytic metabolic activity decreased with DFMO treatment in vivo. Additionally, sensitivity to DFMO treatment correlated with LIN28B overexpression (BE(2)-C>SMS-KCNR>CHLA90). This is the first study to demonstrate that DFMO treatment restores balance to the LIN28/Let-7 axis and inhibits glycolytic metabolism and neurosphere formation in NB and that PET scans may be a meaningful imaging tool to evaluate the therapeutic effects of DFMO treatment.