Unknown,Transcriptomics,Genomics,Proteomics

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Comparing global transcriptional changes that resulted from loss of Ypk1 or Ypk2 activity

ABSTRACT: To identify genes regulated by the AGC kinases Ypk1 and Ypk2 Experiment is designed to identify genes regulated by the kinases Ypk1 and Ypk2. These are functionally redundant AGC kinases such that yeast cells can grow in the presence of either one but the absence of both is lethal. We used a chemical genetic approach to selectively inhibit engineered ATP analog sensitive alleles (AS alleles) of each kinase. Experimental design is such that wild type (WT) Ypk1 or Ypk2 or (AS) Ypk1 or Ypk2 genes are expressed individually on plasmids in a strain background that is deleted for both Ypk1 and Ypk2 (ypk1 delta ypk2 delta). Cultures expressing these plasmids are treated with an ATP analog that is specific for AS Ypk1 or AS Ypk2. Thus, cells expressing wild type Ypk1 or Ypk2 are not sensitive but cells expressing their AS counterparts are sensitive. Arrays typically compare expression patterns in WT versus AS expressing cells at the same time point following inhibitor treatment.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Ted Powers

PROVIDER: E-GEOD-33185 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase, is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer and PTEN-deficient MES-SA uterine tumor xenografts was demonstrated. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 due to compensatory feedback loops was observed. Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which demonstrates a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human Phase I trial. The PTEN-deficient U87MG glioblastoma cell line was treated for 6 hours with vehicle control (DMSO) or to different concentrations of AT13148 and CCT128930 (0.1uM, 1xGI50 and 3XGI50).

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Comparing global transcriptional changes that resulted from loss of Ypk1 or Ypk2 activity

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