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1.  The gamma secretase inhibitor MRK-003 attenuates pancreatic cancer growth in preclinical models 
Molecular cancer therapeutics  2012;11(9):1999-2009.
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, with most patients facing an adverse clinical outcome. Aberrant Notch pathway activation has been implicated in the initiation and progression of PDAC, specifically the aggressive phenotype of the disease. We used a panel of human PDAC cell lines, as well as patient-derived PDAC xenografts to determine whether pharmacological targeting of Notch pathway could inhibit PDAC growth and potentiate gemcitabine sensitivity. MRK-003, a potent and selective γ-secretase inhibitor, treatment is effective against PDAC as evidenced by the down-regulation of nuclear Notch1 intracellular domain (N1ICD), inhibition of anchorage independent growth, and reduction of tumor-initiating cells capable of extensive self-renewal. Pre-treatment of PDAC cells with MRK-003 in cell culture significantly inhibited the subsequent engraftment in immunocompromised mice. MRK-003 monotherapy significantly blocked tumor growth in 5 of 9 (56%) PDAC xenografts. A combination of MRK-003 and gemcitabine showed enhanced antitumor effects compared to gemcitabine in 4 of 9 (44%) PDAC xenografts, reduced tumor cell proliferation and induced both apoptosis and intra-tumoral necrosis. Gene expression analysis of untreated tumors indicated that up-regulation of nuclear factor kappa B (NFκB) pathway components were predictive of sensitivity to MRK-003, while up-regulation in B-cell receptor (BCR) signaling and nuclear factor erythroid-derived 2-like 2 (NRF2) pathway correlated with response to the combination of MRK-003 with gemcitabine. Our findings strengthen the rationale for small molecule inhibition of Notch signaling as a therapeutic strategy in PDAC.
doi:10.1158/1535-7163.MCT-12-0017
PMCID: PMC3438318  PMID: 22752426
Notch; MRK-003; gamma-secretase inhibitor; pancreatic cancer; chemotherapy
2.  Cdkn2a is an atherosclerosis modifier locus that regulates monocyte/macrophage proliferation 
Objective
Common genetic variants in a 58-kilobase region of chr 9p21, near the CDKN2A/CDKN2B tumor suppressor locus, are strongly associated with coronary artery disease. However, the underlying mechanism of action remains unknown.
Methods and Results
We previously reported a congenic mouse model harboring an atherosclerosis susceptibility locus and the region of homology with the human 9p21 locus. Microarray and transcript-specific expression analyses showed markedly decreased Cdkn2a expression, including both p16INK4a and p19ARF, but not Cdkn2b (p15INK4b), in macrophages derived from congenic mice compared to controls. Atherosclerosis studies in subcongenic strains revealed genetic complexity and narrowed one locus to a small interval including Cdkn2a/b. Bone marrow (BM) transplantation studies implicated myeloid lineage cells as the culprit cell type rather than resident vascular cells. To directly test the role of BM-derived Cdkn2a transcripts in atherogenesis and inflammatory cell proliferation, we performed a transplantation study utilizing Cdkn2a+/− cells in the Ldlr−/− mouse model. Cdkn2a-deficient BM recipients exhibited accelerated atherosclerosis, increased Ly6Chi pro-inflammatory monocytes and increased monocyte/macrophage proliferation compared to controls.
Conclusions
These data provide a plausible mechanism for accelerated atherogenesis in susceptible congenic mice, involving decreased expression of Cdkn2a and increased proliferation of monocyte/macrophages, with possible relevance to the 9p21 human locus.
doi:10.1161/ATVBAHA.111.234492
PMCID: PMC3350106  PMID: 21868699
atherosclerosis; genetics; inflammation; leukocytes
3.  Regression of Atherosclerosis Is Characterized by Broad Changes in the Plaque Macrophage Transcriptome 
PLoS ONE  2012;7(6):e39790.
We have developed a mouse model of atherosclerotic plaque regression in which an atherosclerotic aortic arch from a hyperlipidemic donor is transplanted into a normolipidemic recipient, resulting in rapid elimination of cholesterol and monocyte-derived macrophage cells (CD68+) from transplanted vessel walls. To gain a comprehensive view of the differences in gene expression patterns in macrophages associated with regressing compared with progressing atherosclerotic plaque, we compared mRNA expression patterns in CD68+ macrophages extracted from plaque in aortic aches transplanted into normolipidemic or into hyperlipidemic recipients. In CD68+ cells from regressing plaque we observed that genes associated with the contractile apparatus responsible for cellular movement (e.g. actin and myosin) were up-regulated whereas genes related to cell adhesion (e.g. cadherins, vinculin) were down-regulated. In addition, CD68+ cells from regressing plaque were characterized by enhanced expression of genes associated with an anti-inflammatory M2 macrophage phenotype, including arginase I, CD163 and the C-lectin receptor. Our analysis suggests that in regressing plaque CD68+ cells preferentially express genes that reduce cellular adhesion, enhance cellular motility, and overall act to suppress inflammation.
doi:10.1371/journal.pone.0039790
PMCID: PMC3384622  PMID: 22761902
5.  Translational Biomarkers: from Preclinical to Clinical a Report of 2009 AAPS/ACCP Biomarker Workshop 
The AAPS Journal  2011;13(2):274-283.
There have been some successes in qualifying biomarkers and applying them to drug development and clinical treatment of various diseases. A recent success is illustrated by a collaborative effort among the US Food and Drug Administration, the European Medicines Agency, and the pharmaceutical industry to provide a set of seven preclinical kidney toxicity biomarkers for drug development. Other successes include, but are not limited to, clinical biomarkers for cancer treatment and clinical management of heart transplant patients. The value of fully qualified surrogate endpoints in facilitating successful drug development is undisputed, especially for diseases in which the traditional clinical outcome can only be assessed in large, multi-year trials. Emerging biomarkers, including chemical genomic or imaging biomarkers, and measurement of circulating tumor cells hold great promise for early diagnosis of disease and as prognostic tests for managing treatment of chronic diseases such as osteoarthritis, Alzheimer disease, cardiovascular disease, and cancer. To advance the success of treating and managing these diseases, efforts are needed to establish the temporal relationship between changes in inflammatory or imaging biomarkers with the progression of the chronic disease, and in the case of cancer, between the extent of circulating cancer cells and tumor progression or remission.
doi:10.1208/s12248-011-9265-x
PMCID: PMC3085704  PMID: 21448748
biomarkers; diagnostic; diseases; gene expression; imaging
6.  Insights to transcriptional networks by using high throughput RNAi strategies 
RNA interference (RNAi) is a powerful method to unravel the role of a given gene in eukaryotic cells. The development of high throughput assay platforms such as fluorescence plate readers and high throughput microscopy has allowed the design of genome wide RNAi screens to systemically discern members of regulatory networks around various cellular processes. Here we summarize the different strategies employed in RNAi screens to reveal regulators of transcriptional networks. We focus our discussion in experimental approaches designed to uncover regulatory interactions modulating transcription factor activity.
doi:10.1093/bfgp/elp046
PMCID: PMC3097102  PMID: 19952073
RNAi screen; transcription factor; reporter; localization; post-translational modification
7.  Drosophila FoxO Regulates Organism Size and Stress Resistance through an Adenylate Cyclase▿ † 
Molecular and Cellular Biology  2009;29(19):5357-5365.
Forkhead box class O (FoxO) transcription factors are a family of conserved proteins that regulate the cellular responses to various stimuli, such as energy deprivation, stress, and developmental cues. FoxO proteins are important mediators of the insulin signaling pathway, adjusting growth and metabolism to nutrient availability. Insulin signaling acts together with the glucagon-stimulated cAMP signaling pathway to orchestrate the organism response to various nutritional conditions. In this study, we demonstrate that Drosophila melanogaster FoxO (dFoxO) regulates cAMP signaling by directly inducing the expression of an adenylate cyclase gene, ac76e. Interestingly, ac76e is expressed in a highly restricted pattern throughout fly development, limited to the corpus allatum (CA), gastric cecum, and malpighian tubules. dFoxO activation of AC76E in the CA increases starvation resistance and limits growth. Our results unravel a new role for dFoxO, integrating cAMP and insulin signaling to adapt organism growth to the existing nutritional conditions.
doi:10.1128/MCB.00302-09
PMCID: PMC2747984  PMID: 19651894
8.  FOXO-regulated transcription restricts overgrowth of Tsc mutant organs 
The Journal of Cell Biology  2008;180(4):691-696.
FOXO is thought to function as a repressor of growth that is, in turn, inhibited by insulin signaling. However, inactivating mutations in Drosophila melanogaster FOXO result in viable flies of normal size, which raises a question over the involvement of FOXO in growth regulation. Previously, a growth-suppressive role for FOXO under conditions of increased target of rapamycin (TOR) pathway activity was described. Here, we further characterize this phenomenon. We show that tuberous sclerosis complex 1 mutations cause increased FOXO levels, resulting in elevated expression of FOXO-regulated genes, some of which are known to antagonize growth-promoting pathways. Analogous transcriptional changes are observed in mammalian cells, which implies that FOXO attenuates TOR-driven growth in diverse species.
doi:10.1083/jcb.200710100
PMCID: PMC2265581  PMID: 18299344
9.  The U1 snRNP-associated factor Luc7p affects 5′ splice site selection in yeast and human 
Nucleic Acids Research  2007;35(17):5874-5885.
yLuc7p is an essential subunit of the yeast U1 snRNP and contains two putative zinc fingers. Using RNA–protein cross-linking and directed site-specific proteolysis (DSSP), we have established that the N-terminal zinc finger of yLuc7p contacts the pre-mRNA in the 5′ exon in a region close to the cap. Modifying the pre-mRNA sequence in the region contacted by yLuc7p affects splicing in a yLuc7p-dependent manner indicating that yLuc7p stabilizes U1 snRNP–pre-mRNA interaction, thus reminding of the mode of action of another U1 snRNP component, Nam8p. Database searches identified three putative human yLuc7p homologs (hLuc7A, hLuc7B1 and hLuc7B2). These proteins have an extended C-terminal tail rich in RS and RE residues, a feature characteristic of splicing factors. Consistent with a role in pre-mRNA splicing, hLuc7A localizes in the nucleus and antibodies raised against hLuc7A specifically co-precipitate U1 snRNA from human cell extracts. Interestingly, hLuc7A overexpression affects splicing of a reporter in vivo. Taken together, our data suggest that the formation of a wide network of protein–RNA interactions around the 5′ splice site by U1 snRNP-associated factors contributes to alternative splicing regulation.
doi:10.1093/nar/gkm505
PMCID: PMC2034479  PMID: 17726058

Results 1-9 (9)