EXTH-62. COMBINATORIAL THERAPY TARGETING TRANSCIPTION AND IMMUNO-METABOLISM IN RECURRENT GLIOBLASTOMA

Recurrent glioblastomas (GBM) are notoriously difficult to treat, and in spite of aggressive chemo- and radiation therapy they inevitably recur in almost all GBM patients within 14 months following initial diagnosis. To interrogate pathways driving therapeutic resistance, we compared matched primary and recurrent IDH wild type glioblastoma samples from a cohort of patients, using RNA-Seq. Our analyses showed that pathways involved with tumor immune and metabolic reprogramming were up-regulated in recurrent GBMs compared to untreated, primary samples. Based on these findings, we tested the anti-tumor efficacy of over 20 rationally selected targeted therapeutic agents alone and in combination in a high-throughput drug screen assay designed to measure long term cell viability of primary tumorspheres from patients who underwent surgery for recurrent GBM. Top performing drug combinations from these screens were validated in vivo using patient-derived intracranial mouse models of glioma. Our data demonstrated that the combination of the transcriptional/metabolic inhibitor TG02 and the dual PI3K/mTOR inhibitor GDC-0084 significantly prolonged in vivo survival of tumor bearing mice, performing better than either drug alone. Notably, both inhibitors are undergoing human clinical trials as single agents with positive initial safety profiles and superior blood brain barrier penetrance. RNA Seq and functional assays of tumor samples from treated mice showed that only the combination treatment (TG02 + GDC-0084) significantly inhibited expression of immunomodulatory cytokines driving tumor progression, including those identified by us to be overexpressed in recurrent human GBM samples. Overall, these data suggest that combining TG02 and GDC-0084 is an effective treatment for recurrent GBMs. Furthermore, our results support the use of a translational research platform consisting of a personalized pharmaco-genomics testing, using patient-derived tumor samples towards designing more effective treatment for recurrent GBM patients.

Metabolic responses of plasma to extreme environments in overwintering Tibetan frogs Nanorana parkeri: a metabolome integrated analysis

Many animals lower their metabolic rate in response to low temperatures and scarcity of food in the winter in phenomena called hibernation or overwintering. Living at high altitude on the Tibetan Plateau where winters are very cold, the frog Nanorana parkeri, survives in one of the most hostile environments on Earth but, to date, relatively little is known about the biochemical and physiological adjustments for overwintering by this species. The present study profiled changes in plasma metabolites of N. parkeri between winter and summer using UHPLC-QE-MS non-target metabolomics in order to explore metabolic adaptations that support winter survival. The analysis showed that, in total, 11 metabolites accumulated and 95 were reduced in overwintering frogs compared with summer-active animals. Metabolites that increased included some that may have antioxidant functions (canthaxanthin, galactinol), act as a metabolic inhibitor (mono-ethylhexylphthalate), or accumulate as a product of anaerobic metabolism (lactate). Most other metabolites in plasma showed reduced levels in winter and were generally involved in energy metabolism including 11 amino acids (proline, isoleucine, leucine, valine, phenylalanine, tyrosine, arginine, tryptophan, methionine, threonine and histidine) and 4 carbohydrates (glucose, citrate, succinate, and malate). Pathway analysis indicated that aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, and nitrogen metabolism were potentially the most prominently altered pathways in overwintering frogs. Changes to these pathways are likely due to fasting and global metabolic depression in overwintering frogs. Concentrations of glucose and urea, commonly used as cryoprotectants by amphibians that winter on land, were significantly reduced during underwater hibernation in N. parkeri. In conclusion, winter survival of the high-altitude frog, N. parkeri was accompanied by substantial changes in metabolomic profiles and this study provides valuable information towards understanding the special adaptive mechanisms of N. parkeri to winter stresses.

A Warburg-like metabolic program coordinates Wnt, AMPK, and mTOR signaling pathways in epileptogenesis

Epilepsy is a complex neurological condition characterized by repeated spontaneous seizures and can be induced by initiating seizures known as status epilepticus (SE). Elaborating the critical molecular mechanisms following SE are central to understanding the establishment of chronic seizures. Here, we identify a transient program of molecular and metabolic signaling in the early epileptogenic period, centered on day five following SE in the pre-clinical kainate or pilocarpine models of temporal lobe epilepsy. Our work now elaborates a new molecular mechanism centered around Wnt signaling and a growing network comprised of metabolic reprogramming and mTOR activation. Biochemical, metabolomic, confocal microscopy and mouse genetics experiments all demonstrate coordinated activation of Wnt signaling, predominantly in neurons, and the ensuing induction of an overall aerobic glycolysis (Warburg-like phenomenon) and an altered TCA cycle in early epileptogenesis. A centerpiece of the mechanism is the regulation of pyruvate dehydrogenase (PDH) through its kinase and Wnt target genes PDK4. Intriguingly, PDH is a central gene in certain genetic epilepsies, underscoring the relevance of our elaborated mechanisms. While sharing some features with cancers, the Warburg-like metabolism in early epileptogenesis is uniquely split between neurons and astrocytes to achieve an overall novel metabolic reprogramming. This split Warburg metabolic reprogramming triggers an inhibition of AMPK and subsequent activation of mTOR, which is a signature event of epileptogenesis. Interrogation of the mechanism with the metabolic inhibitor 2-deoxyglucose surprisingly demonstrated that Wnt signaling and the resulting metabolic reprogramming lies upstream of mTOR activation in epileptogenesis. To augment the pre-clinical pilocarpine and kainate models, aspects of the proposed mechanisms were also investigated and correlated in a genetic model of constitutive Wnt signaling (deletion of the transcriptional repressor and Wnt pathway inhibitor HBP1). The results from the HBP1-/- mice provide a genetic evidence that Wnt signaling may set the threshold of acquired seizure susceptibility with a similar molecular framework. Using biochemistry and genetics, this paper outlines a new molecular framework of early epileptogenesis and advances a potential molecular platform for refining therapeutic strategies in attenuating recurrent seizures.

MitoTracker Deep Red (MTDR) Is a Metabolic Inhibitor for Targeting Mitochondria and Eradicating Cancer Stem Cells (CSCs), With Anti-Tumor and Anti-Metastatic Activity In Vivo

MitoTracker Deep Red (MTDR) is a relatively non-toxic, carbocyanine-based, far-red, fluorescent probe that is routinely used to chemically mark and visualize mitochondria in living cells. Previously, we used MTDR at low nano-molar concentrations to stain and metabolically fractionate breast cancer cells into Mito-high and Mito-low cell sub-populations, by flow-cytometry. Functionally, the Mito-high cell population was specifically enriched in cancer stem cell (CSC) activity, i) showing increased levels of ESA cell surface expression and ALDH activity, ii) elevated 3D anchorage-independent growth, iii) larger overall cell size (>12-μm) and iv) Paclitaxel-resistance. The Mito-high cell population also showed enhanced tumor-initiating activity, in an in vivo preclinical animal model. Here, we explored the hypothesis that higher nano-molar concentrations of MTDR could also be used to therapeutically target and eradicate CSCs. For this purpose, we employed an ER(+) cell line (MCF7) and two triple negative cell lines (MDA-MB-231 and MDA-MB-468), as model systems. Remarkably, MTDR inhibited 3D mammosphere formation in MCF7 and MDA-MB-468 cells, with an IC-50 between 50 to 100 nM; similar results were obtained in MDA-MB-231 cells. In addition, we now show that MTDR exhibited near complete inhibition of mitochondrial oxygen consumption rates (OCR) and ATP production, in all three breast cancer cell lines tested, at a level of 500 nM. However, basal glycolytic rates in MCF7 and MDA-MB-468 cells remained unaffected at levels of MTDR of up to 1 μM. We conclude that MTDR can be used to specifically target and eradicate CSCs, by selectively interfering with mitochondrial metabolism, by employing nano-molar concentrations of this chemical entity. In further support of this notion, MTDR significantly inhibited tumor growth and prevented metastasis in vivo, in a xenograft model employing MDA-MB-231 cells, with little or no toxicity observed. In contrast, Abemaciclib, an FDA-approved CDK4/6 inhibitor, failed to inhibit metastasis. Therefore, in the future, MTDR could be modified and optimized via medicinal chemistry, to further increase its potency and efficacy, for its ultimate clinical use in the metabolic targeting of CSCs for their eradication.

Characterization of the Domoic Acid Uptake Mechanism of the Mussel (Mytilus galloprovincialis) Digestive Gland

Cultures of the mussel Mytilus galloprovincialis are frequently affected by accumulation of the amnesic shellfish poisoning toxin domoic acid (DA). This species is characterized by a fast uptake and release of the toxin. In this work, the main characteristics of the uptake mechanism have been studied by incubation of digestive gland thin slices in media with different composition and DA concentration. DA uptake seems to follow Michaelis–Menten kinetics, with a very high estimated KM (1722 µg DA mL−1) and a Vmax of 71.9 µg DA g−1 h−1, which is similar to those found for other amino acids in invertebrates. Replacement of NaCl from the incubation media by Cl-choline (Na+-free medium) did not significantly reduce the uptake, but replacement by sorbitol (Na+-free and Cl−-depleted medium) did. A new experiment replacing all chlorides with their equivalent gluconates (Na+- and Cl−-free medium) showed an important reduction in the uptake that should be attributed to the absence of chloride, pointing to a Na+-independent, Cl− (or anion-) dependent transporter. In media with Na+ and Cl−, neither decreasing the pH nor adding cyanide (a metabolic inhibitor) had significant effect on DA uptake, suggesting that the transport mechanism is not H+- or ATP-dependent. In a chloride depleted medium, lowering pH or adding CN increased the uptake, suggesting that other anions could, at least partially, substitute chloride.