Lisa Wodicka

A Genome-Wide Transcriptional Analysis of the Mitotic Cell Cycle

Progression through the eukaryotic cell cycle is known to be both regulated and accompanied by periodic fluctuation in the expression levels of numerous genes. We report here the genome-wide characterization of mRNA transcript levels during the cell cycle of the budding yeast S. cerevisiae. Cell cycle-dependent periodicity was found for 416 of the 6220 monitored transcripts. More than 25% of the 416 genes were found directly adjacent to other genes in the genome that displayed induction in the same cell cycle phase, suggesting a mechanism for local chromosomal organization in global mRNA regulation. More than 60% of the characterized genes that displayed mRNA fluctuation have already been implicated in cell cycle period-specific biological roles. Because more than 20% of human proteins display significant homology to yeast proteins, these results also link a range of human genes to cell cycle period-specific biological functions.

Functional and Genomic Analyses Reveal an Essential Coordination between the Unfolded Protein Response and ER-Associated Degradation

The unfolded protein response (UPR) regulates gene expression in response to stress in the endoplasmic reticulum (ER). We determined the transcriptional scope of the UPR using DNA microarrays. Rather than regulating only ER-resident chaperones and phospholipid biosynthesis, as anticipated from earlier work, the UPR affects multiple ER and secretory pathway functions. Studies of UPR targets engaged in ER-associated protein degradation (ERAD) reveal an intimate coordination between these responses: efficient ERAD requires an intact UPR, and UPR induction increases ERAD capacity. Conversely, loss of ERAD leads to constitutive UPR induction. Finally, simultaneous loss of ERAD and the UPR greatly decreases cell viability. Thus, the UPR and ERAD are dynamic responses required for the coordinated disposal of misfolded proteins even in the absence of acute stress.

Genome-wide expression monitoring in Saccharomyces cerevisiae

The genomic sequence of the budding yeast Saccharomyces cerevisiae has been used to design and synthesize high-density oligonucleotide arrays for monitoring the expression levels of nearly all yeast genes. This direct and highly parallel approach involves the hybridization of total mRNA populations to a set of four arrays that contain a total of more than 260,000 specifically chosen oligonucleotides synthesized in situ using light-directed combinatorial chemistry. The measurements are quantitative, sensitive, specific, and reproducible. Expression levels ranging from less than 0.1 copies to several hundred copies per cell have been measured for cells grown in rich and minimal media. Nearly 90% of all yeast mRNAs are observed to be present under both conditions, with approximately 50% present at levels between 0.1 and 1 copy per cell. Many of the genes observed to be differentially expressed under these conditions are expected, but large differences are also observed for many previously uncharacterized genes.

Genomic analysis of the host response to hepatitis C virus infection

We have examined the progression of hepatitis C virus (HCV) infections by gene expression analysis of liver biopsies in acutely infected chimpanzees that developed persistent infection, transient viral clearance, or sustained clearance. Both common responses and outcome-specific changes in expression were observed. All chimpanzees showed gene expression patterns consistent with an IFN-α response that correlated with the magnitude and duration of infection. Transient and sustained viral clearance were uniquely associated with induction of IFN-γ-induced genes and other genes involved in antigen processing and presentation and the adaptive immune response. During the early stages of infection, host genes involved in lipid metabolism were also differentially regulated. We also show that drugs that affect these biosynthetic pathways can regulate HCV replication in HCV replicon systems. Our results reveal genome-wide transcriptional changes that reflect the establishment, spread, and control of infection, and they reveal potentially unique antiviral programs associated with clearance of HCV infection.

Dual kinase-bromodomain inhibitors for rationally designed polypharmacology.

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multi-target profile has, however, necessitated the application of combination therapies, which can pose significant clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as novel targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase/bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, clinical PLK1 and JAK2/FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a novel strategy for rational single agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase/bromodomain inhibitors.

Abstract 4238: BROMOscan - a high throughput, quantitative ligand binding platform identifies best-in-class bromodomain inhibitors from a screen of mature compounds targeting other protein classes.

Post-translationally appended acetyllysine marks on histone tails are key regulatory features of the epigenetic code. Bromodomains are “readers” of this specific lysine acetylation code, playing an important role in chromatin remodeling and regulation of gene expression. Bromodomains have emerged as an important new druggable target class in small-molecule inhibitor drug discovery, and several bromodomain-containing proteins have been associated with disease. There are 57 bromodomains contained in 41 different proteins; however, few small molecule bromodomain inhibitors have been reported. One primary factor limiting the discovery of new inhibitors is the absence of a comprehensive biochemical bromodomain screening platform. Here we describe the application of DiscoveRx Corporation9s proven ligand binding assay technology (KINOMEscan) to the development of quantitative ligand binding assays for human bromodomains (BROMOscan). We have developed a carefully validated assay panel that covers >30 percent of the human bromodomain family, and this panel is suitable for HTS, selectivity profiling, and quantitative affinity (Kd) assessment. We have used this panel internally to identify novel bromodomain inhibitors and, remarkably, have demonstrated that known, mature inhibitors thought to be selective for targets from other protein families have best in class affinity for bromodomains as well. These data shall be presented, as will a description of the BROMOscan panel replete with extensive assay validation data. Citation Format: Elizabeth Quinn, Lisa Wodicka, Pietro Ciceri, Gabriel Pallares, Elyssa Pickle, Adam Torrey, Mark Floyd, Jeremy Hunt, Daniel Treiber. BROMOscan - a high throughput, quantitative ligand binding platform identifies best-in-class bromodomain inhibitors from a screen of mature compounds targeting other protein classes. [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 4238. doi:10.1158/1538-7445.AM2013-4238

Abstract LB-390: High throughput, quantitative ligand binding assays for human bromodomains

Post translationally appended acetyl-lysine marks on histone tails are key regulatory features of the epigenetic code. Bromodomains are “readers” of this specific lysine acetylation code, playing an important role in chromatin remodeling and regulation of gene expression. Bromodomains have emerged as an important new druggable target class in small-molecule inhibitor drug discovery, and several bromodomain-containing proteins have been associated with disease. There are 57 bromodomains contained in 41 different proteins, however, few small molecule inhibitors of bromodomains have been identified to date. The primary factor limiting the discovery of new inhibitors is the absence of a comprehensive, biochemical screening platform for bromodomains. Here we describe the application of DiscoveRx Corporation9s novel competitive binding assay technology (used to build the KINOMEscan panel of >450 kinase assays) to the development of quantitative ligand binding assays for human bromodomains. By applying our established methodologies, we have rapidly developed an assay panel that covers >15% of the bromodomain family, with the ultimate goal of developing a comprehensive panel, comparable to the KINOMEscan kinase assay panel. A robust bromodomain assay panel suitable for high throughput screening that delivers quantitative ligand binding data will facilitate the identification and optimization of potent and selective small molecule bromodomain inhibitors suitable for both pharmaceutical use and as tool compounds to further elucidate the roles of bromodomains in human disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-390. doi:1538-7445.AM2012-LB-390