We have identified a critical role for amplified FGFR2 in gastric cancer cell proliferation and survival. In a panel of gastric cancer cell lines, fibroblast growth factor receptor 2 (FGFR2) was overexpressed and tyrosine phosphorylated selectively in FGFR2-amplified cell lines KatoIII, Snu16, and OCUM-2M. FGFR2 kinase inhibition by a specific smallmolecule inhibitor resulted in selective and potent growth inhibition in FGFR2-amplified cell lines, resulting in growth arrest in KatoIII cells and prominent induction of apoptosis in both Snu16 and OCUM-2M cells. FGFR2-amplified cell lines also contained elevated phosphotyrosine in EGFR, Her2, and Erbb3, but the elevated phosphorylation in EGFR could not be inhibited by gefitinib or erlotinib. We show that the elevated EGFR, Her2, and Erbb3 phosphotyrosine is dependent on FGFR2, revealing EGFR family kinases to be downstream targets of amplified FGFR2. Moreover, shRNA to Erbb3 resulted in a loss of proliferation, confirming a functional role for the activated EGFR signaling pathway. These results reveal that both the FGFR2 and EGFR family signaling pathways are activated in FGFR2-amplified gastric cancer cell lines to drive cell proliferation and survival. Inhibitors of FGFR2 or Erbb3 signaling may have therapeutic efficacy in the subset of gastric cancers containing FGFR2 amplification.

Background and purpose:  γ‐Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T‐cell acute lymphoblastic leukaemia (T‐ALL). The ability of GSIs to decrease T‐ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti‐tumour efficacy. Experimental approach:  Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Aβ40 and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours. Key results:  Three days of >70% Notch pathway inhibition was sufficient to provide an anti‐tumour effect and was well tolerated. GSI‐induced conversion of mouse epithelial cells to a secretory lineage was time‐ and dose‐dependent. Anti‐tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch‐dependent regulation of mitochondrial homeostasis. Conclusions and implications:  Intermittent but potent inhibition of Notch signalling is sufficient for anti‐tumour efficacy in these T‐ALL models. These findings provide support for the use of GSI in Notch‐dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.

Although retinoic acid (RA) has been shown to be critical for lung development, little is known about when RA is required and the role of individual RA receptors (RAR) in this process. Previously reported data from an RA responsive element RARE-lacZ reporter mouse show that when epithelial tubules are branching and differentiating RA signaling becomes markedly down-regulated in the epithelium. It is unclear why this down-regulation occurs and what role it might play in the developing lung. Here we analyze the effects of preventing potential progenitors of the distal lung from turning off RA signaling by locally expressing constitutively activated RAR␣ or RAR␤ chimeric receptors (RARVP16) in branching airways of transgenic mice. Continued RA activation resulted in lung immaturity in both cases, but the phenotypes were remarkably different. RAR␣VP16 lungs did not expand to form saccules or morphologically identifiable type I cells. High levels of surfactant protein C (Sp-C), thyroid transcription factor-1 (Ttf1), and Gata6, but not Sp-A or Sp-B in the epithelium at birth suggested that in these lungs differentiation was arrested at an early stage. These alterations were not observed in RAR␤VP16 lungs, which showed relatively less severe changes. Our data suggest a model in which activation of RAR signaling at the onset of lung development establishes an initial program that assigns distal cell fate to the prospective lung epithelium. Downregulation of RA signaling, however, is required to allow completion of later steps of this differentiation program that ultimately form mature type I and II cells.

The amount of genomic information about leukemia cells currently far exceeds our overall understanding of the precise genetic events that ultimately drive disease development and progression. Effective implementation of personalized medicine will require tools to distinguish actionable genetic alterations within the complex genetic landscape of leukemia. In this study, we performed kinase inhibitor screens to predict functional gene targets in primary specimens from patients with acute myeloid leukemia (AML) and chronic myelomonocytic leukemia (CMML). Deep sequencing of the same patient specimens identified genetic alterations that were then integrated with the functionally important targets using the HitWalker algorithm to prioritize the mutant genes that most likely explain the observed drug sensitivity patterns. Through this process, we identified Tyrosine Kinase Non-receptor 2 (TNK2) point mutations that exhibited oncogenic capacity. Importantly, the integration of functional and genomic data using HitWalker allowed for prioritization of rare oncogenic mutations that may have been missed through genomic analysis alone. These mutations were sensitive to the multi-kinase inhibitor dasatinib, which antagonizes TNK2 kinase activity, as well as novel TNK2 inhibitors, XMD8-87 and XMD16-5, with greater target specificity. We also identified activating truncation mutations in other tumor types that were sensitive to XMD8-87 and XMD16-5, exemplifying the potential utility of these compounds across tumor types dependent on TNK2. Collectively, our findings highlight a more sensitive approach for identifying actionable genomic lesions that may be infrequently mutated or overlooked, and provide a new method for the prioritization of candidate genetic mutations.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, adult-onset motor neuron disease that arises as a dominantly inherited trait in approximately 10% of ALS cases. Mutations in one gene, cytosolic Cu/Zn superoxide dismutase (SOD1), account for approximately 25% of familial ALS (FALS) cases. We have performed a genetic linkage screen in 16 pedigrees with FALS with no evidence for mutations in the SOD1 gene and have identified novel ALS loci on chromosomes 16 and 20. The analysis of these genes will delineate pathways implicated as determinants of motor-neuron viability and provide insights into possible therapies for ALS.

IGF binding protein (IGFBP)-3 can induce apoptosis in human prostate cancer cells directly without sequestering IGF-I and -II. The molecular mechanisms responsible for the IGF-independent actions of IGFBP-3 remain unclear. IGFBP-3, a secreted protein, can be internalized and translocate to the nucleus. It binds to the nuclear retinoid X receptor (RXR)-alpha. Binding to RXR-alpha has been proposed to be required for IGFBP-3 to induce apoptosis. The present study tests this hypothesis in the PC-3 human prostate cancer cell line. PC-3 cells express RXR-alpha, and apoptosis is induced by incubation with RXR-specific ligand. A COOH-terminal region in IGFBP-3 (residues 215-232) contains a nuclear localization signal, and binding domains for RXR-alpha and heparin (HBD). Different combinations of the 11 amino acids in this region that differ from IGFBP-1, a related IGFBP, which does not localize to the nucleus or bind RXR-alpha, were mutated to the IGFBP-1 sequence. By confocal imaging, mutation of residues 228-KGRKR-232 in nonsecreted IGFBP-3 diminished its nuclear localization. IGFBP-3 binding to glutathione S-transferase-RXR-alpha only was lost when all 11 sites were mutated (HBD-11m-IGFBP-3). Expressed nuclear RXR-alpha did not transport cytoplasmic IGFBP-3 nuclear localization signal mutants that can bind RXR-alpha to the nucleus even after treatment with RXR ligand. Expressed HBD-11m-IGFBP-3 still induced apoptosis in PC-3 cells in an IGF-independent manner as determined by flow cytometric analysis of Annexin V staining. We conclude that in PC-3 cells, RXR-alpha is not required for the nuclear translocation of IGFBP-3 and that IGFBP-3 can induce apoptosis in human prostate cancer cells without binding RXR-alpha.

We report a new approach for target quantification directly within DNA duplex. Our assay is based on the formation of a new biomolecular structure, the PD-loop. The approach takes advantage of a selective hybridization of a probe to double-stranded DNA (dsDNA), which is locally opened by a pair of bis-PNA oligomers. To optimize the technique, several experimental formats are tested with the use of PNA and oligonucleotide probes. The highest sensitivity is achieved when the hybridized probe is extended and multiply labeled with 125I-dCTP by DNA polymerase via strand displacement in the presence of single-strand binding (SSB) protein. In this case, the PNA-assisted probe hybridization combined with the method of multiphoton detection (MPD) allows to monitor sub-attomolar amounts of the HIV-1 target on the background of unrelated DNA at sub-nCi level of radioactivity. The developed robust methodology is highly discriminative to single mutations, thus being of practical use for DNA analysis.

High-content image analysis captures many cellular parameters, but current methods of interpretation of acquired multiple dimensions assume a normal distribution, which is rarely seen in biological data sets. We describe a novel statistically based approach that collapses a set of cellular measurements into a single value, permitting a simplified and unbiased comparison of heterogeneous cellular populations. Differences in multiple cellular responses across two populations are measured using nonparametric Kolmogorov-Smirnov (KS) statistics. This method can be used to study cellular functions, to identify novel target genes and pharmacodynamic biomarkers, and to characterize drug mechanisms of action.