Precision delivery of RAS-inhibiting siRNA to KRAS driven cancer via peptide-based nanoparticles

Over 95% of pancreatic adenocarcinomas (PDACs), as well as a large fraction of other tumor types, such as colorectal adenocarcinoma, are driven by KRAS activation. However, no direct RAS inhibitors exist for cancer therapy. Furthermore, the delivery of therapeutic agents of any kind to PDAC in particular has been hindered by the extensive desmoplasia and resultant drug delivery challenges that accompanies these tumors. Small interfering RNA (siRNA) is a promising modality for anti-neoplastic therapy due to its precision and wide range of potential therapeutic targets. Unfortunately, siRNA therapy is limited by low serum half-life, vulnerability to intracellular digestion, and transient therapeutic effect. We assessed the ability of a peptide based, oligonucleotide condensing, endosomolytic nanoparticle (NP) system to deliver siRNA to KRAS-driven cancers. We show that this peptide-based NP is avidly taken up by cancer cell

TMEM97 and PGRMC1 do not mediate sigma-2 ligand-induced cell death

Abstract Sigma-2 receptors have been implicated in both tumor proliferation and neurodegenerative diseases. Recently the sigma-2 receptor was identified as transmembrane protein 97 (TMEM97). Progesterone receptor membrane component 1 (PGRMC1) was also recently reported to form a complex with TMEM97 and the low density lipoprotein (LDL) receptor, and this trimeric complex is responsible for the rapid internalization of LDL. Sigma-2 receptor ligands with various structures have been shown to induce cell death in cancer cells. In the current study, we examined the role of TMEM97 and PGRMC1 in mediating sigma-2 ligand-induced cell death. Cell viability and caspase-3 assays were performed in control, TMEM97 knockout (KO), PGRMC1 KO, and TMEM97/PGRMC1 double KO cell lines treated with several sigma-2 ligands. The data showed that knockout of TMEM97, PGRMC1, or both did not affect the concentrations of sigma-2 ligands that induced 50% of cell death (EC50), suggesting that cytotoxic effects of these compounds are not mediated by TMEM97 or PGRMC1. Sigma-1 receptor ligands, (+)-pentazocine and NE-100, did not block sigma-2 ligand cytotoxicity, suggesting that sigma-1 receptor was not responsible for sigma-2 ligand cytotoxicity. We also examined whether the alternative, residual binding site (RBS) of 1,3-Di-o-tolylguanidine (DTG) could be responsible for sigma-2 ligand cytotoxicity. Our data showed that the binding affinities (K i) of sigma-2 ligands on the DTG RBS did not correlate with the cytotoxicity potency (EC50) of these ligands, suggesting that the DTG RBS was not fully responsible for sigma-2 ligand cytotoxicity. In addition, we showed that knocking out TMEM97, PGRMC1, or both reduced the initial internalization rate of a sigma-2 fluorescent ligand, SW120. However, concentrations of internalized SW120 became identical later in the control and knockout cells. These data suggest that the initial internalization process of sigma-2 ligands does not appear to mediate the cell-killing effect of sigma-2 ligands. In summary, we have provided evidence that sigma-2 receptor/TMEM97 and PGRMC1 do not mediate sigma-2 ligand cytotoxicity. Our work will facilitate elucidating mechanisms of sigma-2 ligand cytotoxicity

Testing asteroseismology with Gaia DR2: Hierarchical models of the Red Clump

Asteroseismology provides fundamental stellar parameters independent of distance, but subject to systematics under calibration. Gaia DR2 has provided parallaxes for a billion stars, which are offset by a parallax zero-point. Red Clump (RC) stars have a narrow spread in luminosity, thus functioning as standard candles to calibrate these systematics. This work measures how the magnitude and spread of the RC in the Kepler field are affected by changes to temperature and scaling relations for seismology, and changes to the parallax zero-point for Gaia. We use a sample of 5576 RC stars classified through asteroseismology. We apply hierarchical Bayesian latent variable models, finding the population level properties of the RC with seismology, and use those as priors on Gaia parallaxes to find the parallax zero-point offset. We then find the position of the RC using published values for the zero-point. We find a seismic temperature insensitive spread of the RC of ~0.03 mag in the 2MASS K band and a larger and slightly temperature-dependent spread of ~0.13 mag in the Gaia G band. This intrinsic dispersion in the K band provides a distance precision of ~1% for RC stars. Using Gaia data alone, we find a mean zero-point of -41 $\pm$ 10 $\mu$as. This offset yields RC absolute magnitudes of -1.634 $\pm$ 0.018 in K and 0.546 $\pm$ 0.016 in G. Obtaining these same values through seismology would require a global temperature shift of ~-70 K, which is compatible with known systematics in spectroscopy.Comment: Accepted for publication in MNRA

The role of supplier performance evaluations in mitigating risk: assessing evaluation processes and behaviors

Given the level of outsourcing, supplier performance evaluation (SPE) is a critical supply chain process. SPEs are used to record supplier performance levels to inform future supplier selections, and thus mitigate the risk of adverse selection. Numerous weaknesses associated with industrial buyers\u27 collection and use of supplier performance information call SPE effectiveness into question. The risk-related factors affecting SPE effectiveness have not been empirically explored, including misuses of the tool. This research identifies the factors affecting SPE risk mitigation effectiveness. It employs a mixed method of qualitative interviews of buyers and suppliers in order to develop a model of SPE risk mitigation effectiveness using structural equations modeling of survey data from a rare sample of 131 performance assessors. Findings implicate the importance of a thoroughly defined scope of work, an accurate SPE, and documented rating justifications. Additionally, dissonance among several performance evaluators and the fear of a supplier\u27s dispute detract from SPE risk mitigation effectiveness. Finally, this research unveils how SPEs are weaponized, pursuing short-term gains and clouding the view of the supplier\u27s performance thereby hindering the long-term, risk-mitigating purpose of SPEs. Two separate forms of opportunism - threat and debt - are discovered and have differing effects

Lysosomal membrane permeabilization is an early event in sigma-2 receptor ligand mediated cell death in pancreatic cancer

BACKGROUND: Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. RESULTS: Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. CONCLUSIONS: Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death following LMP protected by DEVD-FMK and α-toco, which is also known to stabilize the mitochondrial membrane during apoptotic stimuli. These differences in mechanism are likely dependent on the structural class of the compounds versus the inherent sigma-2 binding affinity. As resistance of pancreatic cancers to specific apoptotic stimuli from chemotherapy is better appreciated, and patient-tailored treatments become more available, ligands with high sigma-2 receptor affinity should be chosen based on sensitivities to apoptotic pathways

Mesothelin\u27s minimal MUC16 binding moiety converts TR3 into a potent cancer therapeutic via hierarchical binding events at the plasma membrane

TRAIL has been extensively explored as a cancer drug based on its tumor-selective activity profile but it is incapable per se of discriminating between death receptors expressed by normal host cells and transformed cancer cells. Furthermore, it is well documented that surface tethering substantially increases its biologic activity. We have previously reported on Meso-TR3, a constitutive TRAIL trimer targeted to the biomarker MUC16 (CA125), in which the entire ectodomain of human mesothelin was genetically fused to the TR3 platform, facilitating attachment to the cancer cells via the MUC16 receptor. Here, we designed a truncation variant, in which the minimal 64 amino acid MUC16 binding domain of mesothelin was incorporated into TR3. It turned out that the dual-domain biologic Meso64-TR3 retained its high MUC16 affinity and bound to the cancer cells quickly, independent of the TR3/death receptor interaction. Furthermore, it was substantially more potent than Meso-TR3 and TR3 in vitro and in a preclinical xenograft model of MUC16-dependent ovarian cancer. Phenotypically, Meso64-TR3 is more closely related to non-targeted TR3, evident by indistinguishable activity profiles on MUC16-deficient cancers and similar thermal stability characteristics. Overall, Meso64-TR3 represents a fully human, MUC16-targetd TRAIL-based biologic, ideally suited for exploring preclinical and clinical evaluation studies in MUC16-dependent malignancies

Evidence for an extensive hydrothermal plume in the Tonga-Fiji region of the South Pacific

Author Posting. © American Geophysical Union, 2004. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Geochemistry Geophysics Geosystems 5 (2004): Q01003, doi:10.1029/2003GC000607.Several hydrographic stations in the vicinity of the Samoa Islands have 3He/4He above the regional background in the depth range of 1500–1800 m, indicating injection of mantle helium from a local hydrothermal source. The highest δ(3He) = 43.4% was detected at 1726-m depth at 15.0°S, 173.1°W in the bathymetric gap between the Samoa Islands and the northern end of the Tonga-Kermadec Arc. The δ(3He) profile at this station decreases to δ(3He) = 26% at 2500-m depth. The relatively shallow depth of the maximum hydrothermal signal suggests a source different from the conventional Pacific basin helium plume centered at 2500 m that is carried westward from the East Pacific Rise. Stations to the west of this locality show a progressive decrease in the maximum δ(3He) values in the depth range of 1480–1790 m out to 169°E. Stations east of the Tonga-Fiji region show lower 3He values (<26%) at 1700 m and the profiles are dominated by a deeper maximum at 2500 m, presumably the distal traces of hydrothermal input from East Pacific Rise. This pattern in the 3He distribution suggests that the 1700-m deep helium plume is carried in a northwesterly direction some 2000 km from its source near the northern end of the Tonga-Kermadec Arc. At this time very little is known about the source of this hydrothermal plume or the details of its areal extent. Numerous seamounts and rift zones in the region are possible hydrothermal sources for the plume. The summit crater of Vailulu'u, a young seamount at the eastern end of the Samoa chain, was recently discovered to be hydrothermally active at ∼600 m depth [Hart et al., 2000]. However this shallow hydrothermal field on Vailulu'u is an unlikely source for the deeper 1700-m signal. The most likely source would appear to be the extensional zones of the northern Lau Basin system, such as the Mangatolo Triple Junction. Just as the helium plume emanating from Lo'ihi has helped our understanding of the circulation near the Hawaiian Islands [Lupton, 1996], this helium plume in the Tonga-Fiji region has great potential for delineating circulation in this area of the south Pacific.This work was supported by the NOAA Vents Program and by Grants OCE91-05884, OCE92-96237, OCE92-96169, and OCE98-20132 of the Ocean Sciences Division of the National Science Foundation

Novel treatment option for MUC16-positive malignancies with the targeted TRAIL-based fusion protein Meso-TR3

BACKGROUND: The targeted delivery of cancer therapeutics represents an ongoing challenge in the field of drug development. TRAIL is a promising cancer drug but its activity profile could benefit from a cancer-selective delivery mechanism, which would reduce potential side effects and increase treatment efficiencies. We recently developed the novel TRAIL-based drug platform TR3, a genetically fused trimer with the capacity for further molecular modifications such as the addition of tumor-directed targeting moieties. MUC16 (CA125) is a well characterized biomarker in several human malignancies including ovarian, pancreatic and breast cancer. Mesothelin is known to interact with MUC16 with high affinity. In order to deliver TR3 selectively to MUC16-expressing cancers, we investigated the possibility of targeted TR3 delivery employing the high affinity mesothelin/MUC16 ligand/receptor interaction. METHODS: Using genetic engineering, we designed the novel cancer drug Meso-TR3, a fusion protein between native mesothelin and TR3. The recombinant proteins were produced with mammalian HEK293T cells. Meso-TR3 was characterized for binding selectivity and killing efficacy against MUC16-positive cancer cells and controls that lack MUC16 expression. Drug efficacy experiments were performed in vitro and in vivo employing an intraperitoneal xenograft mouse model of ovarian cancer. RESULTS: Similar to soluble mesothelin itself, the strong MUC16 binding property was retained in the Meso-TR3 fusion protein. The high affinity ligand/receptor interaction was associated with a selective accumulation of the cancer drug on MUC16-expressing cancer targets and directly correlated with increased killing activity in vitro and in a xenograft mouse model of ovarian cancer. The relevance of the mesothelin/MUC16 interaction for attaching Meso-TR3 to the cancer cells was verified by competitive blocking experiments using soluble mesothelin. Mechanistic studies using soluble DR5-Fc and caspase blocking assays confirmed engagement of the extrinsic death receptor pathway. Compared to non-targeted TR3, Meso-TR3 displayed a much reduced killing potency on cells that lack MUC16. CONCLUSIONS: Soluble Meso-TR3 targets the cancer biomarker MUC16 in vitro and in vivo. Following attachment to the tumor via surface bound MUC16, Meso-TR3 acquires full activation with superior killing profiles compared to non-targeted TR3, while its bioactivity is substantially reduced on cells that lack the tumor marker. This prodrug phenomenon represents a highly desirable property because it has the potential to enhance cancer killing with fewer side-effects than non-targeted TRAIL-based therapeutics. Thus, further exploration of this novel fusion protein is warranted as a possible therapeutic for patients with MUC16-positive malignancies