Palila were listed as a federal endangered species in 1967, primarily due to two centuries of habitat destruction by non-native ungulates. The United States Fish and Wildlife Service designated Critical Habitat for palila in 1977, and federal court rul-ings in 1979, 1987, and 1998 mandated Hawai‘i Department of Land and Natural Resources (DLNR) to remove all sheep and goats from Critical Habitat. In 2010, over 30 years since the original ruling, DLNR initiated construction of a 6-foot-tall ungulate- proof fence around Mauna Kea to eliminate ingress of feral sheep, hybrid-mouflon sheep, goats, and cattle. Twenty-four miles of fence were completed around the southern portion of Mauna Kea by 2013. DLNR also developed a strategic plan for the permanent removal of non-native ungulates from palila critical habitat (PCH) and received a Competi-tive State Wildlife Grant to fund eradication efforts. Implementation of the plan and dispersal of the funds began in April 2013. A population estimate was conducted in May 2013 using distance sampling that produced a point estimate of 2,046 hybrid-mouflon sheep with a 95% confidence interval of 482- 3,614 sheep. From January 1 2003 to February 6 2014, 3,270 sheep and 26 goats were removed from PCH by aerial shooting (2,150), aerial drives (228), and public hunting (792). Of the 2,147 sheep shot during aerial shooting, 2,081 were shot when not tracking Judas sheep (sheep fitted with radio collars that facilitate locating herds from the helicopter), and 66 were shot when tracking Judas sheep. With the population reduction, aerial shoot efforts are now dependent upon tracking the 17 Judas sheep currently equipped with radio-collars to find herds that have learned to avoid aerial hunting by hiding under the forest canopy. Future aerial shoots will be conducted on a monthly basis and missions will likely to be 2 days. To reach the goal of sheep eradication in PCH prior to the depletion of remaining funds, it is essential to track all Judas sheep every mission to increase removal rates. Otherwise, the sheep popula-tion will likely recover and continue to damage the remaining forest in PCH that palila depend upon.

Hawai`i Volcanoes National Park acquired the 115,000-acre Kahuku Ranch Unit in 2003. The Kahuku Unit contains numerous exceptional natural resources including endemic plants and birds, many of which are federally listed threatened and endangered species. Eleven European mouflon sheep were introduced to Kahuku from 1968-1974 for private trophy hunting. Because Hawaiian plants did not evolve with mammalian herbivores, managers began to control the large mouflon population with a closely directed volunteer program. Since 2004, more than 1,900 mouflon have been removed through this program. We estimated that there were 2,586 ± 705 (90% CI) mouflon at Kahuku in November 2004, but by December 2006 the population decreased 30%. Although the ram:ewe ratio did not change significantly after this population decrease, the mean (±95% CI) overall ratio in 2004 was 1:2.4 (1:2.1-1:2.7) and 1:2.7 (1:2.4-1:3.1) in 2006. We found that 82.6% of adult ewes (n = 26) were pregnant with a single fetus in early 2007, and there was a significant increase in the number of lambs per ewe from 2005 to 2007 consistent with a density-dependent response. The maximum (±95% CI) number of lambs per ewe was 0.484 (0.412-0.558) in 2005 and 0.667 (0.587-0.750) in 2007. The directed volunteer program has been more successful in reducing mouflon abundance at Kahuku than species such as feral pigs elsewhere on Hawai`i Island, but some population-level responses such as increased reproduction could result from density decreases.

For nearly a decade, the difficulties associated with both the determination and reproducibility of Ras-dependency indexes (RDIs) have limited their application and further delineation of the biology underlying Ras dependency. In this report, we describe the application of a computational single sample gene set enrichment analysis (ssGSEA) method to derive RDIs with gene expression data. The computationally derived RDIs across the Cancer Cell Line Encyclopedia (CCLE) cell lines show excellent agreement with the experimentally derived values and high correlation with a previous in-house siRNA effector node (siREN) study and external studies. Using EMT signature-derived RDIs and data from cell lines representing the extremes in RAS dependency, we identified enriched pathways distinguishing these classes, including the Fas signaling pathway and a putative Ras-independent pathway first identified in NK cells. Importantly, extension of the method to patient samples from The Cancer Genome Atlas (TCGA) showed the same consensus differential expression patterns for these two pathways across multiple tissue types. Last, the computational RDIs displayed a significant association with TCGA cancer patients' survival outcomes. Together, these lines of evidence confirm that our computationally derived RDIs faithfully represent a measure of Ras dependency in both cancer cell lines and patient samples. The application of such computational RDIs can provide insights into Ras biology and potential clinical applications.

The 3 human RAS genes play pivotal roles regulating proliferation, differentiation, and survival in normal cells and become mutated in 15% to 20% of all human tumors and amplified in many others. In this report, we examined data from The Cancer Genome Atlas to investigate the relationship between RAS gene mutational status and messenger RNA expression. We show that all 3 RAS genes exhibit increased expression when they are mutated in a context-dependent manner. In the case of KRAS, this increase is manifested by a larger proportional increase in KRAS4A than KRAS4B, although both increase significantly. In addition, the mutational status of RAS genes can be associated with expression changes in other RAS genes, with most of these cases showing decreased expression. The mutational status associations with expression are recapitulated in cancer cell lines. Increases in expression are mediated by both copy number variation and contextual differences, including mutational status of epidermal growth factor receptor (EGFR) and BRAF. These findings potentially reveal an adaptive response during tumor evolution that is dependent on the mutational status of proximal genes in the RAS pathway and cellular context. Cell contextual differences in these adaptations may influence therapeutic responsiveness and alternative resistance mechanisms.

KRAS mutations occur in ∼35% of colorectal cancers and promote tumor growth by constitutively activating the mitogen-activated protein kinase (MAPK) pathway. KRAS mutations at codons 12, 13, or 61 are thought to prevent GAP protein-stimulated GTP hydrolysis and render KRAS-mutated colorectal cancers unresponsive to epidermal growth factor receptor (EGFR) inhibitors. We report here that KRAS G13-mutated cancer cells are frequently comutated with NF1 GAP but NF1 is rarely mutated in cancers with KRAS codon 12 or 61 mutations. Neurofibromin protein (encoded by the NF1 gene) hydrolyzes GTP directly in complex with KRAS G13D, and KRAS G13D-mutated cells can respond to EGFR inhibitors in a neurofibromin-dependent manner. Structures of the wild type and G13D mutant of KRAS in complex with neurofibromin (RasGAP domain) provide the structural basis for neurofibromin-mediated GTP hydrolysis. These results reveal that KRAS G13D is responsive to neurofibromin-stimulated hydrolysis and suggest that a subset of KRAS G13-mutated colorectal cancers that are neurofibromin-competent may respond to EGFR therapies.

Oncogenic Ras mutants play a major role in the etiology of most aggressive and deadly carcinomas in humans. In spite of continuous efforts, effective pharmacological treatments targeting oncogenic Ras isoforms have not been developed. Cell-surface proteins represent top therapeutic targets primarily due to their accessibility and susceptibility to different modes of cancer therapy. To expand the treatment options of cancers driven by oncogenic Ras, new targets need to be identified and characterized at the surface of cancer cells expressing oncogenic Ras mutants. Here, we describe a mass spectrometry-based method for molecular profiling of the cell surface using KRasG12V transfected MCF10A (MCF10A-KRasG12V) as a model cell line of constitutively activated KRas and native MCF10A cells transduced with an empty vector (EV) as control. An extensive molecular map of the KRas surface was achieved by applying, in parallel, targeted hydrazide-based cell-surface capturing technology and global shotgun membrane proteomics to identify the proteins on the KRasG12V surface. This method allowed for integrated proteomic analysis that identified more than 500 cell-surface proteins found unique or upregulated on the surface of MCF10A-KRasG12V cells. Multistep bioinformatic processing was employed to elucidate and prioritize targets for cross-validation. Scanning electron microscopy and phenotypic cancer cell assays revealed changes at the cell surface consistent with malignant epithelial-to-mesenchymal transformation secondary to KRasG12V activation. Taken together, this dataset significantly expands the map of the KRasG12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.