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Wnt-modulator in surface ectoderm (WISE) is a secreted modulator of Wnt signaling expressed in the adult kidney. Activation of Wnt signaling has been observed in renal transplants developing interstitial fibrosis and tubular atrophy; however, whether WISE contributes to chronic changes is not well understood. Here, we found moderate to high expression of WISE mRNA in a rat model of renal transplantation and in kidneys from normal rats. Treatment with a neutralizing antibody against WISE improved proteinuria and graft function, which correlated with higher levels of β-catenin protein in kidney allografts. In addition, treatment with the anti-WISE antibody reduced infiltration of CD68(+) macrophages and CD8(+) T cells, attenuated glomerular and interstitial injury, and decreased biomarkers of renal injury. This treatment reduced expression of genes involved in immune responses and in fibrogenic pathways. In summary, WISE contributes to renal dysfunction by promoting tubular atrophy and interstitial fibrosis.

O1 Combinatorial CD8+ and PD-L1+ cell densities correlate with response and improved survival in non-small cell lung cancer (NSCLC) patients treated with durvalumab #### Sonja Althammer1, Keith Steele2, Marlon Rebelatto2, Tze Heng Tan1, Tobias Wiestler1, Guenter Schmidt1, Brandon Higgs2, Xia

We conducted a genome-wide screen using the mutagen N-ethyl-N-nitrosourea to identify recessive mutations in genes that lead to altered lipid traits in mice. We screened 7,546 G3 mice that were of mixed C57BL/6J (B6) x C3.SW-H2(b)/SnJ (C3) genomes and identified three pedigrees with differences in plasma HDL-cholesterol. Genome scan analyses mapped three distinct loci to chromosomes 3, 4, and 7. An S1748L missense mutation was identified in ABCA1 in one pedigree with undetectable levels of HDL-cholesterol and resulted in reduced protein levels. This phenotype was completely penetrant, semi-dominant, and cosegregated with high plasma triglycerides. Mice in a second pedigree had very high levels of plasma total cholesterol and HDL-cholesterol (up to 800 mg/dl total cholesterol). Despite a high degree of phenotype lability and reduced penetrance, an I68N missense mutation was identified in the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). Finally, a second high HDL-cholesterol pedigree of mice, again with a highly labile phenotype and reduced penetrance, was mapped to a 7 Mb locus on chromosome 3. These results illustrate the use of a hybrid background for simultaneous screening and mapping of mutagenized pedigrees of mice and identification of three novel alleles of HDL-cholesterol phenotypes.

Insulin resistance in mice typically does not manifest as diabetes due to multiple compensatory mechanisms. Here, we present a novel digenic model of type 2 diabetes in mice heterozygous for a null allele of the insulin receptor and an N-ethyl-N-nitrosourea–induced alternative splice mutation in the regulatory protein phosphatase 2A (PP2A) subunit PPP2R2A. Inheritance of either allele independently results in insulin resistance but not overt diabetes. Doubly heterozygous mice exhibit progressive hyperglycemia, hyperinsulinemia, and impaired glucose tolerance from 12 weeks of age without significant increase in body weight. Alternative splicing of Ppp2r2a decreased PPP2R2A protein levels. This reduction in PPP2R2A containing PP2A phosphatase holoenzyme was associated with decreased serine/threonine protein kinase AKT protein levels. Ultimately, reduced insulin-stimulated phosphorylated AKT levels were observed, a result that was confirmed in Hepa1-6, C2C12, and differentiated 3T3-L1 cells knocked down using Ppp2r2a small interfering RNAs. Altered AKT signaling and expression of gluconeogenic genes in the fed state contributed to an insulin resistance and hyperglycemia phenotype. This model demonstrates how genetic changes with individually small phenotypic effects interact to cause diabetes and how differences in expression of hypomorphic alleles of PPP2R2A and potentially other regulatory proteins have deleterious effects and may therefore be relevant in determining diabetes risk.

Air was injected through a well in a thin transparent tank filled with saturated glass beads to study how the size and air saturation of air sparging plumes are affected by particle size and gradation; operational parameters such as injection pressure, well depth, injection pressure pulsing; and well outlet configuration. V-shaped air plumes with an apex between 40° and 60° were obtained for all tests. The air pressure required to initiate sparging agreed closely with the sum of the air entry pressure and the hydrostatic pressure, with higher initiation pressures required in the fine and well-graded beads. Higher air flow rates and air saturations were obtained in coarser beads at a given pressure, and the variation in flow rate was consistent with estimated air permeabilities. Peak average air saturations were 28–56% for the coarse-medium beads, 10% for the well-graded beads, and 8% for the fine beads. Air saturation and the radius of influence increased modestly (

The MET receptor tyrosine kinase is involved in cell growth, survival, and invasion. Clinical studies with small molecule MET inhibitors have shown the role of biomarkers in identifying patients most likely to benefit from MET-targeted therapy. AMG 337 is an oral, small molecule, ATP-competitive, highly selective inhibitor of the MET receptor. Herein, we describe AMG 337 preclinical activity and mechanism of action in MET-dependent tumor models. These studies suggest MET is the only therapeutic target for AMG 337. In an unbiased tumor cell line proliferation screen (260 cell lines), a closely related analogue of AMG 337, Compound 5, exhibited activity in 2 of 260 cell lines; both were MET-amplified. Additional studies examining the effects of AMG 337 on the proliferation of a limited panel of cell lines with varying MET copy numbers revealed that high-level focal MET amplification (>12 copies) was required to confer MET oncogene addiction and AMG 337 sensitivity. One MET-amplified cell line, H1573 (>12 copies), was AMG 337 insensitive, possibly because of a downstream G12A KRAS mutation. Mechanism-of-action studies in sensitive MET-amplified cell lines demonstrated that AMG 337 inhibited MET and adaptor protein Gab-1 phosphorylation, subsequently blocking the downstream PI3K and MAPK pathways. AMG 337 exhibited potency in pharmacodynamic assays evaluating MET signaling in tumor xenograft models; >90% inhibition of Gab-1 phosphorylation was observed at 0.75 mg/kg. These findings describe the preclinical activity and mechanism of action of AMG 337 in MET-dependent tumor models and indicate its potential as a novel therapeutic for the treatment of MET-dependent tumors. Mol Cancer Ther; 15(7); 1568–79. ©2016 AACR.

Seeds of soybean (Glycine max (L.) Merr.) were harvested from greenhouse-grown plants and fractures of the seed coat were examined with a scanning electron microscope. The seed coat was well differentiated from the outer integument when the seed had reached approximately 30% maximum seed size. At this time, the osteosclereids began to separate, becoming fully detached along their radial walls by 50% maximum seed size. Macrosclereid secondary wall development occurred during growth of the seed from 50 to 100% maximum seed size. Near R6 (100% maximum seed size) the endothelium began differentiation from the integumentary tapetum (inner integument) and was fully differentiated by physiological maturity (R7). From R7 to harvest maturity (R8) the seed lost moisture content and decreased in size. The parenchyma of the seed coat collapsed in response to this dehydration.

Acquired drug resistance is a long-standing problem of cancer therapeutics. The issue has become even more vexing with the development of highly selective agents; resistance to gefitinib or erlotinib is acquired frequently in lung adenocarcinomas. Thus, anticipating acquired resistance and understanding its basis may help us develop strategies to prevent or circumvent occurrence. Through its receptor tyrosine kinase Met, hepatocyte growth factor (HGF) regulates mitogenesis, motogenesis, and morphogenesis during development and adulthood. HGF/Met signaling also contributes to cancer progression in many malignancies, including glioblastoma. Rilotumumab is a fully human neutralizing monoclonal antibody against HGF tested in multiple Phase 2 clinical trials, including mono therapy in renal cell carcinoma and glioblastoma, as well as combination trials in gastric, colorectal, small cell lung cancers and castrate resistant prostate cancer. To generate a cellular model of acquired resistance to rilotumumab, an HGF/Met dependent human glioblastoma-derived cell line (U87-MG) was grown in rilotumumab (600 nM) for 120 days. Growth rate, HGF secretion, Met content and Met activation state were 10-fold, 10,000-fold, 10-fold and 80-fold higher than the parental cell values, respectively. The HGF and MET coding sequences were normal in both parental and resistant cells. Quantitative PCR studies to determine mRNA levels of all HGF isoforms revealed a dramatic increase in full-length HGF transcript. CGH array studies indicated amplification within both HGF and MET genes. Xenograft studies confirmed that tumor growth was resistant to rilotumumab, however the resistant cell line and tumors remained sensitive to a highly selective Met tyrosine kinase inhibitor, suggesting that resistance was achieved via increased HGF/Met signaling rather than mutation or activation of alternate pathways. Microarray expression analysis demonstrated transcript profiles that were consistent with HGF/Met pathway activation. Thus the molecular basis of acquired resistance in this model differs from those prevalent in: [1] lung cancers treated with EGFR inhibitors and medulloblastomas treated with hedgehog inhibitors, where most cases acquire secondary mutations in the targeted kinase; [2] breast cancers treated with HER2 inhibitors, where PTEN loss, p27 downregulation, and activation of other receptors are primary causes; or [3] malignant melanoma treated with BRAF inhibitors, where increased signaling via multiple pathways lead to PI3K- and/or MEK-mediated reactivation of the MAPK pathway. In addition to the importance of HGF/Met pathway activity in selecting glioblastoma patients for HGF-targeted therapeutics, our results suggest that monitoring Met pathway activity could provide early indications of acquired resistance to these agents, and that Met kinase inhibitors may still be efficacious when resistance occurs. Citation Format: Fabiola Cecchi, Karen Rex, Joanna Schmidt, Daniel Baker, Michael A. Damore, Angela Coxon, Teresa L. Burgess, Donald P. Bottaro. A cellular model of acquired resistance to rilotumumab (AMG 102) in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5637. doi:10.1158/1538-7445.AM2013-5637

Acquired drug resistance is a long-standing problem of cancer therapeutics, for example chemoresistance to temozolomide occurs in >90% of recurrent gliomas. The issue has become even more vexing with the development of highly selective targeted agents; e.g. agents targeting the epidermal and hepatocyte growth factor (EGF and HGF, respectively) pathways; resistance to gefitinib and erlotinib has already been frequently demonstrated in lung adenocarcinomas. Thus, anticipating acquired resistance and understanding its basis may help us develop clinical strategies to prevent or circumvent its occurrence. HGF, through its receptor tyrosine kinase Met, regulates mitogenesis, motogenesis, and morphogenesis in a range of cellular targets during development and homeostasis. HGF/Met signaling also contributes to oncogenesis and tumor progression in many prevalent human malignancies, including glioblastoma. Rilotumumab (AMG102) is a fully human neutralizing monoclonal antibody against HGF tested in multiple Phase 2 clinical trials, including mono therapy in renal cell carcinoma and Glioblastoma, as well as combination trials in gastric, colorectal and small cell lung cancers and castrate resistant prostate cancer. To generate a cellular model of acquired resistance to rilotumumab, the HGF/Met dependent human glioblastoma-derived cell line U87-MG, was grown in continuous exposure to 600 nM rilotumumab for 120 days. Growth rate, HGF secretion, Met content and Met activation state were 10-fold, 10,000-fold, 10-fold and 80-fold higher than the parental cell values, respectively. The HGF and MET coding sequences and the HGF promoter DATE region, where truncation reportedly increases HGF expression level, were found to have normal sequence and length in both parental and resistant cell lines. Quantitative PCR studies to determine mRNA levels of all HGF isoforms revealed a dramatic increase in full-length HGF transcript. CGH array studies indicated amplification within both HGF and MET genes. Xenograft studies confirmed that tumor growth was resistant to rilotumumab, however the resistant cell line and tumors remained sensitive to a highly selective Met tyrosine kinase inhibitor suggesting that resistance was achieved via increased HGF/Met signaling rather than mutation or activation of alternate pathways. Microarray expression analysis demonstrated transcript profiles that were consistent with HGF/Met pathway activation. Thus the molecular basis of acquired resistance in this model differs from those prevalent in: [1] lung cancers treated with EGFR inhibitors and medulloblastomas treated with hedgehog inhibitors, where nearly all cases acquire secondary mutations in the targeted kinase; [2] breast cancers treated with HER2 inhibitors, where PTEN loss, p27 downregulation, and activation of other receptors are primary causes; or [3] malignant melanoma treated with BRAF inhibitors, where increased signaling by ARAF, CRAF, IGFR1, PDGFR, and other sources lead to PI3K- and/or MEK-mediated reactivation of the MAPK pathway. In addition to the importance of HGF/Met pathway activity in selecting glioblastoma patients for HGF-targeted therapeutics, our results suggest that monitoring Met pathway activity could provide early indications of acquired resistance to these agents, and that Met kinase inhibitors may still be efficacious when resistance occurs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A204.

Magnetic resonance imaging (MRI) is steadily becoming recognized as a premier imaging modality for evaluating the thoracic aorta. Its noninvasive, nonimaging approach and refined resolution capabilities effectively combined the advantages of echocardiography, angiography, and computed tomography (CT) under the auspices of a single imaging device. On static spin-echo MRI, blood flow appears dark, allowing superb depiction of both intra- and extra vascular anatomy. With dynamic MR studies, gradient echo (cine) and phase velocity mapping techniques have proven effective in assessing the physiological consequences of anatomic vascular abnormalities in a qualitative and quantitative manner, respectively. Magnetic resonance angiography (MRA) is fast becoming a prominent vascular imaging method. Although still evolving, MRA bolsters the vascular imaging capabilities of MRI by enhancing the imaging resolution of the major branch vessels and collateral vessels. With continued advances in this area, MRI may adopt the role as the primary imaging method for assessing the thoracic aorta.

Seeds of soybean (Glycine max (L.) Merr.) were harvested from greenhouse-grown plants and fractures of the seed coat were examined with a scanning electron microscope. The seed coat was well differentiated from the outer integument when the seed had reached approximately 30% maximum seed size. At this time, the osteosclereids began to separate, becoming fully detached along their radial walls by 50% maximum seed size. Macrosclereid secondary wall development occurred during growth of the seed from 50 to 100% maximum seed size. Near R6 (100% maximum seed size) the endothelium began differentiation from the integumentary tapetum (inner integument) and was fully differentiated by physiological maturity (R7). From R7 to harvest maturity (R8) the seed lost moisture content and decreased in size. The parenchyma of the seed coat collapsed in response to this dehydration.

Phomopsis longicolla Hobbs, a causal agent of Phomopsis seed decay, colonizes the seed coats and embryos of mature soybean (Glycine max (L.) Merr.) seeds. The mode of infection of immature soybean pods and seeds by P. longicolla was examined using scanning electron microscopy. Phomopsis longicolla penetrated directly through pod walls by appressoria. Penetration through stomata in the pod surface was not observed. Immature seeds were also penetrated directly. Heavily infected immature seeds showed extensive colonization of the seed coats and embryos.

Background C57BLKS/J (BLKS) mice are susceptible to islet exhaustion in insulin-resistant states as compared with C57BL6/J (B6) mice, as observed by the presence of the leptin receptor (Lepr) allele, Leprdb/db. Furthermore, DBA2/J (DBA) mice are also susceptible to β-cell failure and share 25% of their genome with BLKS; thus the DBA genome may contribute to β-cell dysfunction in BLKS mice. Results Here we show that BLKS mice exhibit elevated insulin secretion, as evidenced by improved glucose tolerance and increased islet insulin secretion compared with B6 mice, and describe interstrain transcriptional differences in glucose response. Transcriptional differences between BLKS and B6 mice were identified by expression profiling of isolated islets from both strains. Genomic mapping of gene expression differences demonstrated a significant association of expression differences with DBA loci in BLKS mice (P = 4×10-27). Conclusion Two genes, Nicotinamide nucleotide transhydrogenase (Nnt) and Pleiomorphic adenoma gene like 1 (Plagl1), were 4 and 7.2-fold higher respectively in BLKS islets, and may be major contributors to increased insulin secretion by BLKS islets. Contrary to reports for B6 mice, BLKS mice do not harbor a mutant Nnt gene. We detected 16 synonymous polymorphisms and a two-amino acid deletion in the Plagl1 gene in BLKS mice. Several inflammatory glucose-responsive genes are expressed at a higher level in BLKS, suggesting an inflammatory component to BLKS islet dysfunction. This study describes physiological differences between BLKS and B6 mice, and provides evidence for a causative role of the DBA genome in β-cell dysfunction in BLKS mice.