Specific Inhibition of the Nuclear Exporter Exportin-1 Attenuates Kidney Cancer Growth

Purpose

Despite the advent of FDA-approved therapeutics to a limited number of available targets (kinases and mTOR), PFS of kidney cancer (RCC) has been extended only one to two years due to the development of drug resistance. Here, we evaluate a novel therapeutic for RCC which targets the exportin-1 (XPO1) inhibitor.

Materials and Methods

RCC cells were treated with the orally available XPO1 inhibitor, KPT-330, and cell viability and Annexin V (apoptosis) assays, and cell cycle analyses were performed to evaluate the efficacy of KPT-330 in two RCC cell lines. Immunoblotting and immunofluorescence analysis were performed to validate mechanisms of XPO1 inhibition. The efficacy and on-target effects of KPT-330 were further analyzed in vivo in RCC xenograft mice, and KPT-330-resistant cells were established to evaluate potential mechanisms of KPT-330 resistance.

Results

KPT-330 attenuated RCC viability through growth inhibition and apoptosis induction both in vitro and in vivo, a process in which increased nuclear localization of p21 by XPO1 inhibition played a major role. In addition, KPT-330 resistant cells remained sensitive to the currently approved for RCC multi-kinase inhibitors (sunitinib, sorafenib) and mTOR inhibitors (everolimus, temsirolimus), suggesting that these targeted therapeutics would remain useful as second line therapeutics following KPT-330 treatment.

Conclusion

The orally-available XPO1 inhibitor, KPT-330, represents a novel target for RCC whose in vivo efficacy approaches that of sunitinib. In addition, cells resistant to KPT-330 retain their ability to respond to available RCC therapeutics suggesting a novel approach for treatment in KPT-330-naïve as well as -resistant RCC patients.     

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.     

Allometric Models for Predicting Aboveground Biomass in Two Widespread Woody Plants in Hawaii

Allometric models are important for quantifying biomass and carbon storage in terrestrial ecosystems. Generalized allometry exists for tropical trees, but species‐ and site‐specific models are more accurate. We developed species‐specific models to predict aboveground biomass in two of the most ubiquitous natives in Hawaiian forests and shrublands, Metrosideros polymorpha and Dodonaea viscosa. The utility of the M. polymorpha allometry for predicting biomass across a range of sites was explored by comparing size structure (diameter at breast height vs. tree height) of the trees used to develop the models against trees from four M. polymorpha‐dominated forests along a precipitation gradient (1630–2380 mm). We also compared individual tree biomass estimated with the M. polymorpha model against existing generalized equations, and the D. viscosa model with an existing species‐specific model. Our models were highly significant and displayed minimal bias. Metrosideros polymorpha size structures from the three highest precipitation sites fell well within the 95% confidence intervals for the harvested trees, indicating that the models are applicable at these sites. However, size structure in the area with the lowest precipitation differed from those in the higher rainfall sites, emphasizing that care should be taken in applying the models too widely. Existing generalized allometry differed from the M. polymorpha model by up to 88 percent, particularly at the extremes of the data range examined, underestimating biomass in small trees and overestimating in large trees. The existing D. viscosa model underestimated biomass across all sizes by a mean of 43 percent compared to our model. The species‐specific models presented here should enable more accurate estimates of biomass and carbon sequestration in Hawaiian forests and shrublands.     

Indonesia’s blue carbon: a globally significant and vulnerable sink for seagrass and mangrove carbon

The global significance of carbon storage in Indonesia’s coastal wetlands was assessed based on published and unpublished measurements of the organic carbon content of living seagrass and mangrove biomass and soil pools. For seagrasses, median above- and below-ground biomass was 0.29 and 1.13 Mg C ha^?1 respectively; the median soil pool was 118.1 Mg C ha^?1. Combining plant biomass and soil, median carbon storage in an Indonesian seagrass meadow is 119.5 Mg C ha^?1. Extrapolated to the estimated total seagrass area of 30,000 km², the national storage value is 368.5 Tg C. For mangroves, median above- and below-ground biomass was 159.1 and 16.7 Mg C ha^?1, respectively; the median soil pool was 774.7 Mg C ha^?1. The median carbon storage in an Indonesian mangrove forest is 950.5 Mg C ha^?1. Extrapolated to the total estimated mangrove area of 31,894 km², the national storage value is 3.0 Pg C, a likely underestimate if these habitats sequester carbon at soil depths >1 m and/or sequester inorganic carbon. Together, Indonesia’s seagrasses and mangroves conservatively account for 3.4 Pg C, roughly 17 % of the world’s blue carbon reservoir. Continued degradation and destruction of these wetlands has important consequences for CO₂ emissions and dissolved carbon exchange with adjacent coastal waters. We estimate that roughly 29,040 Gg CO₂ (eq.) is returned annually to the atmosphere–ocean pool. This amount is equivalent to about 3.2 % of Indonesia’s annual emissions associated with forest and peat land conversion. These results highlight the urgent need for blue carbon and REDD+ projects as a means to stem the decline in wetland area and to mitigate the release of a significant fraction of the world’s coastal carbon stores.     

Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage

The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1-Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1-Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1-Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1-Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.     

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na⁺] and [K⁺] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na⁺‐selective class I‐HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single‐nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near‐isogenic lines (NILs): 157‐14 (double homozygote for the cheesmaniae alleles) and 157‐17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157‐14 in comparison with 157‐17. A significant difference between NILs was also found for [K⁺] and the [Na⁺]/[K⁺] ratio in leaf and stem but not for [Na⁺] arising a disagreement with the corresponding RIL population. Their association with leaf [Na⁺] and salt tolerance in tomato is also discussed.     

Phenolic antioxidants: potent inhibitors of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

Bis(2-hydroxy-3-tert-butyl-5-methylphenyl)methane (bis-phenol) is the most potent inhibitor of the (Ca2+ + Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum yet identified. The compound behaves as a reversible, tight-binding inhibitor with apparent Ki = 0.3 microM. Butylated hydroxytoluene, butylated hydroxyanisole, and 4-nonylphenol are also effective inhibitors. These observations are of particular interest in light of the widespread use of such phenolic antioxidants and stabilizers in the food industry and in the manufacture of rubbers and plastics and the ease with which the compounds are extracted into organic solvents.     

AUTORADIOGRAPHIC STUDY OF TYPE II-CELL HYPERPLASIA IN LUNGS OF MICE CHRONICALL EXPOSED TO URETHANE

Hyperplasia of pulmonary alveolar epithelium was induced by exposure of adult mice to 0–1% urethane in drinking water, and their lungs were analysed using combined autoradiographic and morphometric methods. The response of pulmonary epithelium showed three phases: an initial one of 3 weeks in which the number of type II cells was consistently low and, after a transient decrease in ³H-thymidine labeling index, the latter steadily increased. Degenerating or dead type II cells were found, consequently this period was considered one in which cell death was disproportionately increased over cell production. Between the 3rd and 6th weeks the number of type II cells doubled, and thereafter increased at a slower rate. The ³H-thymidine labeling index reached its peak at 6 weeks. The third phase was marked by a decline in labeling index which returned to near-normal levels by the 16th week. The number of type II cells declined slowly after the 10th week and was still elevated at the end of the observation period. Tumors consisting of type II cells continued to arise even during the period of declining labeling index. The dissociation between the proliferative response which was reversible, and the neoplastic response which was not, supports the assumption that the major part of the observed population growth or hyperplasia induced by urethane was a form of regeneration repair secondary to cell death and may be unrelated to tumor growth.