The mammalian target of rapamycin regulates cholesterol biosynthetic gene expression and exhibits a rapamycin-resistant transcriptional profile.

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation in response to growth factor and nutrient signaling. Consequently, this kinase is implicated in metabolic diseases including cancer and diabetes, so there is great interest in understanding the complete spectrum of mTOR-regulated networks. mTOR exists in two functionally distinct complexes, mTORC1 and mTORC2, and whereas the natural product rapamycin inhibits only a subset of mTORC1 functions, recently developed ATP-competitive mTOR inhibitors have revealed new roles for both complexes. A number of studies have highlighted mTORC1 as a regulator of lipid homeostasis. We show that the ATP-competitive inhibitor PP242, but not rapamycin, significantly down-regulates cholesterol biosynthesis genes in a 4E-BP1-dependent manner in NIH 3T3 cells, whereas S6 kinase 1 is the dominant regulator in hepatocellular carcinoma cells. To identify other rapamycin-resistant transcriptional outputs of mTOR, we compared the expression profiles of NIH 3T3 cells treated with rapamycin versus PP242. PP242 caused 1,666 genes to be differentially expressed whereas rapamycin affected only 88 genes. Our analysis provides a genomewide view of the transcriptional outputs of mTOR signaling that are insensitive to rapamycin.

Multimeric Anti-DR5 IgM Agonist Antibody IGM-8444 Is a Potent Inducer of Cancer Cell Apoptosis and Synergizes with Chemotherapy and BCL-2 Inhibitor ABT-199.

Death receptor 5 (DR5) is an attractive target for cancer therapy due to its broad upregulated expression in multiple cancers and ability to directly induce apoptosis. Though anti-DR5 IgG antibodies have been evaluated in clinical trials, limited efficacy has been attributed to insufficient receptor crosslinking. IGM-8444 is an engineered, multivalent agonistic IgM antibody with 10 binding sites to DR5 that induces cancer cell apoptosis through efficient DR5 multimerization. IGM-8444 bound to DR5 with high avidity and was substantially more potent than an IgG with the same binding domains. IGM-8444 induced cytotoxicity in a broad panel of solid and hematologic cancer cell lines but did not kill primary human hepatocytes in vitro, a potential toxicity of DR5 agonists. In multiple xenograft tumor models, IGM-8444 monotherapy inhibited tumor growth, with strong and sustained tumor regression observed in a gastric PDX model. When combined with chemotherapy or the BCL-2 inhibitor ABT-199, IGM-8444 exhibited synergistic in vitro tumor cytotoxicity and enhanced in vivo efficacy, without augmenting in vitro hepatotoxicity. These results support the clinical development of IGM-8444 in solid and hematologic malignancies as a monotherapy and in combination with chemotherapy or BCL-2 inhibition.

Chemical genetic approach for kinase-substrate mapping by covalent capture of thiophosphopeptides and analysis by mass spectrometry.

Mapping kinase-substrate interactions demands robust methods to rapidly and unequivocally identify substrates from complex protein mixtures. Toward this goal, we present a method in which a kinase, engineered to utilize synthetic ATPγS analogs, specifically thiophosphorylates its substrates in a complex lysate. The thiophosphate label provides a bio-orthogonal tag that can be used to affinity purify and identify labeled proteins. Following the labeling reaction, proteins are digested with trypsin; thiol-containing peptides are then covalently captured and non-thiol-containing peptides are washed from the resin. Oxidation-promoted hydrolysis, at sites of thiophosphorylation, releases phosphopeptides for analysis by tandem mass spectrometry. By incorporating two specificity gates-kinase engineering and peptide affinity purification-this method yields high-confidence substrate identifications. This method gives both the identity of the substrates and phosphorylation-site localization. With this information, investigators can analyze the biological significance of the phosphorylation mark immediately following confirmation of the kinase-substrate relationship. Here, we provide an optimized version of this technique to further enable widespread utilization of this technology. Curr. Protoc. Chem Biol. 2:15-36. © 2010 by John Wiley & Sons, Inc.