P roteins of the STAT (signal transducer and activator of transcription) family are activated in response to cytokines and growth factors and promote proliferation, survival, and other biological processes (1-3). STATs are activated by phosphorylation of a critical tyrosine residue, which is mediated by growth factor receptor tyrosine kinases, Janus kinases, or the Src family kinases. Upon tyrosine phosphorylation, dimers of STATs formed between two phosphorylated monomers translocate to the nucleus, bind to specific DNA-response elements in the promoters of target genes, and induce gene expression. Aberrant activity of one of the family members, Stat3, contributes to carcinogenesis and tumor progression by up-regulating gene expression and promoting dysregulated growth, survival, and angiogenesis and modulating immune responses (2-9).As a critical step in STAT activation (10), the dimerization between two STAT monomers presents an attractive target to abolish Stat3 DNA-binding and transcriptional activity and to inhibit Stat3 biological functions (11, 12). Stat3 dimerization relies on the reciprocal binding of the SH2 domain of one monomer to the Pro-pTyr-Leu-Lys-Thr-Lys sequence of the other Stat3 monomer. To pursue the development of inhibitors of Stat3 signaling, key structural information gleaned from the x-ray crystal structure of the Stat3␤ homodimer (13) was used in the computational modeling and automated docking of small molecules into the SH2 domain of a Stat3 monomer, relative to the bound native pTyr peptide, to identify binders of the Stat3 SH2 domain, and potentially disruptors of Stat3⅐Stat3 dimers (14, 15).Structure-based high-throughput virtual screening of the National Cancer Institute (NCI) chemical libraries identified the high-scoring compound NSC 74859 (resynthesized as a pure sample and named S3I-201), which selectively inhibits Stat3 DNA-binding activity in vitro with an IC 50 value of 86 Ϯ 33 M. Furthermore, S3I-201 induces growth inhibition and apoptosis of malignant cells in part by constitutively inhibiting active Stat3 and induces human breast tumor regression in xenograft models. ResultsComputational Modeling and Virtual Screening. Our computational modeling and virtual screening study used the GLIDE (Grid-based Ligand Docking from Energetics) software (16, 17) (available from Schrödinger, Portland, OR) for the docking simulations and relied on the x-ray crystal structure of the Stat3␤ homodimer bound to DNA (13) determined at 2.25-Å resolution (1BG1 in the Protein Data Bank). For the virtual screening, DNA was removed and only one of the two monomers was used (see Fig. 1). To validate the docking approach, the native pTyr (pY) peptide, APpYLKT, was extracted from the crystal structure of one of the monomers and docked to the other monomer, whereby GLIDE produced a docking mode that closely resembled the x-ray crystal structure (data not shown). Three-dimensional structures of compounds from the NCI's chemical libraries were downloaded from the NCI Developmental Therapeutics Progra...

Ras-induced malignant transformation requiresRas proteins are plasma membrane-associated GTPases that function as relay switches transducing biological information from extracellular signals to the nucleus (for review, see Refs.

Signaling via Jak2/STAT3 is critically important for normal dendritic cell (DC) differentiation. In addition, we have previously demonstrated that hyperactivation of the Jak2/STAT3 pathway induced by tumor-derived factors (TDF) may be responsible for abnormal DC differentiation in cancer. In this study, using a novel selective inhibitor of Jak2/STAT3, JSI-124, we investigated the mechanism of the Jak2/STAT3 effect on DCs and the possibility of pharmacological regulation of DC differentiation in cancer. Our experiments have demonstrated that JSI-124 overcomes the differentiation block induced by TDF and promotes the differentiation of mature DCs and macrophages. Surprisingly, inhibition of Jak2/STAT3 signaling resulted in dramatic activation of immature DCs generated in the presence of TDF as well as in control medium. This activation manifested in up-regulation of MHC class II, costimulatory molecules, and a dramatic increase in the ability to stimulate allogeneic or Ag-specific T cells. Inhibition of Jak2/STAT3 signaling resulted in activation of the transcription factor NF-κB. This up-regulation was not due to a conventional pathway involving IκBα, but was probably due to a block of the dominant negative effect of STAT3. This indicates that Jak2/STAT3 play an important role in negative regulation of DC activation, and pharmacological inhibition of the Jak2/STAT3 pathway can be used to enhance DC function.

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