[CANCER RESEARCH 63, 1270 –1279, March 15, 2003] Discovery of JSI-124 (Cucurbitacin I), a Selective Janus Kinase/Signal Transducer and Activator of Transcription 3 Signaling Pathway Inhibitor with Potent Antitumor Activity against Human and Murine Cancer Cells in Mice 1 Michelle A. Blaskovich, 2 Jiazhi Sun, 2 Alan Cantor, James Turkson, Richard Jove, and Saı ¨d M. Sebti 3 Drug Discovery Program [M. A. B., J. S., S. M. S.] and Molecular Oncology Program [J. T., R. J.], Biostatistics and Informatics Core [A. C.], H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, and Departments of Oncology and Biochemistry and Molecular Biology, University of South Florida, Tampa, Florida 33612 [M. A. B., J. S., J. T., R. J., S. M. S.] ABSTRACT Constitutively activated, tyrosine-phosphorylated signal transducer and activator of transcription (STAT) 3 plays a pivotal role in human tumor malignancy. To discover disrupters of aberrant STAT3 signaling pathways as novel anticancer drugs, we developed a phosphotyrosine STAT3 cytoblot. Using this high throughput 96-well plate assay, we identified JSI-124 (cucurbitacin I) from the National Cancer Institute Diversity Set. JSI-124 suppressed the levels of phosphotyrosine STAT3 in v-Src-transformed NIH 3T3 cells and human cancer cells potently (IC 50 value of 500 nM in the human lung adenocarcinoma A549) and rapidly (complete inhibition within 1–2 h). The suppression of phosphotyrosine STAT3 levels resulted in the inhibition of STAT3 DNA binding and STAT3-mediated but not serum response element-mediated gene tran- scription. JSI-124 also decreased the levels of tyrosine-phosphorylated Janus kinase (JAK) but not those of Src. JSI-124 was highly selective for JAK/STAT3 and did not inhibit other oncogenic and tumor survival pathways such as those mediated by Akt, extracellular signal-regulated kinase 1/2, or c-Jun NH 2 -terminal kinase. Finally, JSI-124 (1 mg/kg/day) potently inhibited the growth in nude mice of A549 tumors, v-Src-trans- formed NIH 3T3 tumors, and the human breast carcinoma MDA-MB-468, all of which express high levels of constitutively activated STAT3, but it did not affect the growth of oncogenic Ras-transformed NIH 3T3 tumors that are STAT3 independent or of the human lung adenocarcinoma Calu-1, which has barely detectable levels of phosphotyrosine STAT3. JSI-124 also inhibited tumor growth and significantly increased survival of immunologically competent mice bearing murine melanoma with con- stitutively activated STAT3. These results give strong support for phar- macologically targeting the JAK/STAT3 signaling pathway for anticancer drug discovery. INTRODUCTION STATs 4 are key signal transduction proteins that play a dual role of transducing biological information from cell surface receptors to the cytoplasm and translocating to the nucleus, where, as transcription factors, they regulate gene expression (reviewed in Refs. 1– 4). Mam- malian cells express seven different STATs (1, 2, 3, 4, 5a, 5b, and 6). Gene knockout and other experiments implicated STATs in many important physiological functions such as immune modulation, in- flammation, proliferation, differentiation, development, cell survival, and apoptosis (1– 4). STAT tyrosine phosphorylation is required for the biological func- tion of STATs. This occurs when cytokines such as interleukin 6 and IFN or growth factors such as platelet-derived growth factor and epidermal growth factor bind their respective receptors, which results in STAT protein recruitment to the inner surface of the plasma membrane in the vicinity of the cytoplasmic portion of the receptors (5, 6). Tyrosine kinases that are known to phosphorylate STATs are non-RTKs such as Src and the JAKs, JAK1 and JAK2. Other possible tyrosine kinases that can phosphorylate STATs are peptide growth factor receptors such as platelet-derived growth factor receptor and EGFR. The cellular levels of STATs that are tyrosine phosphorylated could also be regulated by phosphotyrosine STAT phosphatases such as SHP-1 and SHP-2 (7–9). Once tyrosine phosphorylated, STAT monomers dimerize via reciprocal phosphotyrosine-SH2 interactions and translocate to the nucleus, where they bind DNA and regulate gene transcription (3, 10). Whereas tyrosine phosphorylation of STATs regulates dimerization, nuclear translocation and DNA bind- ing, serine/threonine phosphorylation is believed to regulate the tran- scriptional activity of STATs (11). Several lines of evidence have implicated some STAT family members in malignant transformation and tumor cell survival (12, 13). STAT3 involvement in oncogenesis is the most thoroughly charac- terized. First, STAT3 is found constitutively tyrosine phosphorylated and activated in many human cancers (12–14). This abnormal activa- tion of STAT3 is prevalent in breast, pancreatic, ovarian, head and neck, brain, and prostate carcinomas, as well as in melanomas, leu- kemias, and lymphomas. In those tumors investigated, aberrant STAT3 activation is required for growth and survival (12–14). Sec- ond, many known oncogenes, especially those belonging to the non- RTK family such as src, induce constitutive activation of STAT3 (15). Third, expression of a constitutively activated mutant of STAT3, for which stable dimerization was forced through disulfide covalent link- age, was shown to be sufficient to induce cell transformation and tumor growth in nude mice (16). Finally, perhaps the most compelling evidence for the requirement of STAT3 for oncogenesis and its validation as an anticancer drug target comes from experiments in which a dominant negative form of STAT3 was used in cultured cells and in gene therapy animal experiments to show that blocking aber- rant activation of STAT3 results in inhibition of tumor growth and survival and induction of apoptosis with few side effects to normal cells (17, 18). On the basis of the above observations, we and others have pro- posed to target STAT3 for the development of novel anticancer drugs (reviewed in Refs. 12–14). Depending on the aberrant genetic alter- ations that result in constitutively tyrosine-phosphorylated, activated STAT3, several approaches could be undertaken including blocking Received 6/13/02; accepted 1/9/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by National Cancer Institute Drug Discovery Program Project Grant CA78038. 2 These authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612. Phone: (813) 979-6734; E-mail: sebti@moffitt.usf.edu. 4 The abbreviations used are: JAK, Janus kinase; STAT, signal transducer and activator of transcription; EGFR, epidermal growth factor receptor; NCI, National Cancer Institute; TBS, Tris-buffered saline [10 mM Tris (pH 7.4), 150 mM NaCl]; PMSF, phenylmethyl- sulfonyl fluoride; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; JNK, c-Jun NH 2 -terminal kinase; SRE, serum response element; RIPA, radioim- munoprecipitation assay; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; EMSA, electrophoretic mobility shift analysis; ERK, extracellular signal- regulated kinase; PI3k, phosphatidylinositol 3'-kinase; RTK, receptor tyrosine kinase; SHP, SH2-containing phosphatase; DAP1, 4'-6-diamidino-2-phenylindole. 1270