Discovery of JSI-124 (Cucurbitacin I), a Selective Janus Kinase/Signal

[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 Mice1

Michelle A. Blaskovich,2 Jiazhi Sun,2 Alan Cantor, James Turkson, Richard Jove, and Saõ¬d M. Sebti3 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 Gene knockout and other experiments implicated STATs in many important physiological functions such as immune modulation, in- Constitutively activated, tyrosine-phosphorylated signal transducer flammation, proliferation, differentiation, development, cell survival, and activator of transcription (STAT) 3 plays a pivotal role in human and apoptosis (1–4). tumor malignancy. To discover disrupters of aberrant STAT3 signaling pathways as novel anticancer drugs, we developed a phosphotyrosine STAT tyrosine phosphorylation is required for the biological func- STAT3 cytoblot. Using this high throughput 96-well plate assay, we tion of STATs. This occurs when cytokines such as interleukin 6 and identified JSI-124 (cucurbitacin I) from the National Cancer Institute IFN or growth factors such as platelet-derived growth factor and Diversity Set. JSI-124 suppressed the levels of phosphotyrosine STAT3 in epidermal growth factor bind their respective receptors, which results v-Src-transformed NIH 3T3 cells and human cancer cells potently (IC50 in STAT protein recruitment to the inner surface of the plasma value of 500 nM in the human lung adenocarcinoma A549) and rapidly membrane in the vicinity of the cytoplasmic portion of the receptors (complete inhibition within 1Ð2 h). The suppression of phosphotyrosine (5, 6). Tyrosine kinases that are known to phosphorylate STATs are STAT3 levels resulted in the inhibition of STAT3 DNA binding and non-RTKs such as Src and the JAKs, JAK1 and JAK2. Other possible STAT3-mediated but not serum response element-mediated gene tran- tyrosine kinases that can phosphorylate STATs are peptide growth scription. JSI-124 also decreased the levels of tyrosine-phosphorylated Janus kinase (JAK) but not those of Src. JSI-124 was highly selective for factor receptors such as platelet-derived growth factor receptor and JAK/STAT3 and did not inhibit other oncogenic and tumor survival EGFR. The cellular levels of STATs that are tyrosine phosphorylated pathways such as those mediated by Akt, extracellular signal-regulated could also be regulated by phosphotyrosine STAT phosphatases such kinase 1/2, or c-Jun NH2-terminal kinase. Finally, JSI-124 (1 mg/kg/day) as SHP-1 and SHP-2 (7–9). Once tyrosine phosphorylated, STAT potently inhibited the growth in nude mice of A549 tumors, v-Src-trans- monomers dimerize via reciprocal phosphotyrosine-SH2 interactions formed NIH 3T3 tumors, and the human breast carcinoma MDA-MB-468, and translocate to the nucleus, where they bind DNA and regulate all of which express high levels of constitutively activated STAT3, but it gene transcription (3, 10). Whereas tyrosine phosphorylation of did not affect the growth of oncogenic Ras-transformed NIH 3T3 tumors STATs regulates dimerization, nuclear translocation and DNA bind- that are STAT3 independent or of the human lung adenocarcinoma ing, serine/threonine phosphorylation is believed to regulate the tran- Calu-1, which has barely detectable levels of phosphotyrosine STAT3. JSI-124 also inhibited tumor growth and significantly increased survival scriptional activity of STATs (11). of immunologically competent mice bearing murine melanoma with con- Several lines of evidence have implicated some STAT family stitutively activated STAT3. These results give strong support for phar- members in malignant transformation and tumor cell survival (12, 13). macologically targeting the JAK/STAT3 signaling pathway for anticancer STAT3 involvement in oncogenesis is the most thoroughly charac- drug discovery. terized. First, STAT3 is found constitutively tyrosine phosphorylated and activated in many human cancers (12–14). This abnormal activa- INTRODUCTION tion of STAT3 is prevalent in breast, pancreatic, ovarian, head and neck, brain, and prostate carcinomas, as well as in melanomas, leu- STATs4 are key signal transduction proteins that play a dual role of kemias, and lymphomas. In those tumors investigated, aberrant transducing biological information from cell surface receptors to the STAT3 activation is required for growth and survival (12–14). Sec- cytoplasm and translocating to the nucleus, where, as transcription ond, many known oncogenes, especially those belonging to the non- factors, they regulate gene expression (reviewed in Refs. 1–4). Mam- RTK family such as src, induce constitutive activation of STAT3 (15). malian cells express seven different STATs (1, 2, 3, 4, 5a, 5b, and 6). Third, expression of a constitutively activated mutant of STAT3, for which stable dimerization was forced through disulfide covalent link- Received 6/13/02; accepted 1/9/03. age, was shown to be sufficient to induce cell transformation and The costs of publication of this article were defrayed in part by the payment of page tumor growth in nude mice (16). Finally, perhaps the most compelling charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. evidence for the requirement of STAT3 for oncogenesis and its 1 Supported in part by National Cancer Institute Drug Discovery Program Project validation as an anticancer drug target comes from experiments in Grant CA78038. 2 These authors contributed equally to this work. which a dominant negative form of STAT3 was used in cultured cells 3 To whom requests for reprints should be addressed, at Drug Discovery Program, H. and in gene therapy animal experiments to show that blocking aber- Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL rant activation of STAT3 results in inhibition of tumor growth and 33612. Phone: (813) 979-6734; E-mail: [email protected]. 4 The abbreviations used are: JAK, Janus kinase; STAT, signal transducer and activator survival and induction of apoptosis with few side effects to normal of transcription; EGFR, epidermal growth factor receptor; NCI, National Cancer Institute; cells (17, 18). TBS, Tris-buffered saline [10 mM Tris (pH 7.4), 150 mM NaCl]; PMSF, phenylmethyl- On the basis of the above observations, we and others have pro- sulfonyl fluoride; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; JNK, c-Jun NH2-terminal kinase; SRE, serum response element; RIPA, radioim- posed to target STAT3 for the development of novel anticancer drugs munoprecipitation assay; TUNEL, terminal deoxynucleotidyl transferase-mediated nick (reviewed in Refs. 12–14). Depending on the aberrant genetic alter- end labeling; EMSA, electrophoretic mobility shift analysis; ERK, extracellular signal- regulated kinase; PI3k, phosphatidylinositol 3Ј-kinase; RTK, receptor tyrosine kinase; ations that result in constitutively tyrosine-phosphorylated, activated SHP, SH2-containing phosphatase; DAP1, 4Ј-6-diamidino-2-phenylindole. STAT3, several approaches could be undertaken including blocking 1270

ligand-receptor interactions, inhibiting receptor and non-RTKs, acti- 20 at a 1:1000 dilution for1hatroom temperature. Western blots were vating phosphotyrosine STAT3 phosphatases, and blocking STAT3 visualized using enhanced chemiluminescence as described previously (21). dimerization, nuclear translocation, DNA binding, and gene transcrip- Immunoprecipitation of STAT3. A549 cells were treated for 4 h with tion. In addition, gene therapy, antisense, or RNA interference ap- vehicle or JSI-124 and then lysed in 150 mM HEPES (pH 7.5), 150 mM NaCl, proaches can also be attempted. One approach that we have taken, the 1mM EDTA, 0.5% NP40, 10% glycerol, 5 mM NaF, 1 mM DTT, 1 mM PMSF, ␮ results of which are described in this study, is to identify small 2mM sodium orthovanadate, and 5 g/ml leupeptin. Sample lysates were collected and cleared, and then 500 ␮g of lysate were immunoprecipitated with molecules capable of interfering with the signaling events leading to 50 ng of STAT3 antibody overnight at 4°C and rocked with 25 ␮l of protein the abnormally elevated levels of tyrosine-phosphorylated STAT3 in A/G PLUS-agarose (Santa Cruz Biotechnology) for1hat4°C. Samples were many human cancer cells. washed four times with lysis buffer and then boiled in 2ϫ SDS-PAGE sample buffer and run on 10% SDS-PAGE gel. Protein was transferred to nitrocellu- MATERIALS AND METHODS lose and then blotted as described above for both phospho-specific STAT3 and STAT3. Cell Lines. All human and murine tumor cell lines used were obtained DNA Binding and Transcription. The STAT3 reporter, pLucTKS3, driv- from the American Type Culture Collection (Manassas, VA). Stably trans- ing expression of firefly luciferase has been described previously (11). The fected v-Src, oncogenic H-Ras, and vector NIH 3T3 cell lines have been pLucTKS3 plasmid harbors seven copies of a sequence corresponding to the described previously (11, 19). STAT3-specific binding site in the promoter of the human C-reactive protein NCI Diversity Set. The NCI Structural Diversity Set is a library of 1,992 gene (22). The STAT3-independent plasmid, pRLSRE, contains two copies of compounds selected from the approximately 140,000-compound NCI drug the SRE from the c-fos promoter (23), subcloned into Renilla luciferase depository. These compounds were selected based on various criteria including reporter, pRL-null (Promega Corp., Madison, WI). availability, drug-like structure, uniqueness of pharmacophore, and anticancer Transfection and Generation of Stable Clones. NIH 3T3/v-Src/ activity as determined by cell growth inhibition assays against a panel of pLucTKS3 and NIH 3T3/v-Src/pRLSRE are stable clones that were generated human tumor cell lines. In-depth data on the selection, structures, and activities by transfecting NIH 3T3/v-Src fibroblasts with pLucTKS3 or pRLSRE and of these diversity set compounds can be found on the NCI Developmental selecting for G418-resistant and zeocin clones, respectively (11, 24). In the Therapeutics Program web site.5 There are additional NCI web sites that have case of NIH 3T3/v-Src/pLucTKS3/pRLSRE, pRLSRE was transfected into links to more specific information about the antiproliferative properties of NIH 3T3/v-Src/pLucTKS3 cells, and stable G418-resistant clones were se- 6 these Diversity Set compounds. lected. Transfections were carried out with LipofectAMINE Plus (Invitrogen Cytoblot Screening for Phospho-STAT3 Inhibition. NIH 3T3 cells sta- Corp., Carlsbad, CA), according to the manufacturer’s protocol. Concerning bly transfected with v-Src or NIH 3T3 vector control cells (11) were plated into treatment of cells with inhibitors, Src-transformed NIH 3T3 cells stably ex- sterile, opaque, 96-well tissue culture plates at 25,000 cells/well. After over- pressing reporter constructs pLucTKS3, pRLSRE, or both were treated with night growth at 37°C, the cells were treated for4hinthepresence of either JSI-124 (10 ␮M) for 24–48 h before harvesting cells for cytosolic and nuclear ␮ vehicle control or 10 M of NCI Diversity Set compounds. After treatment, extract preparation and luciferase assay. ␮ cells were washed in 100 l of cold TBS and then fixed for1hat4°C with Preparation of Cytosolic Extracts. Cytosolic extracts were prepared from ␮ cold 3.7% formaldehyde in TBS (190 l/well) as described previously for a fibroblasts as described previously (11). Briefly, after two washes with PBS similar cytoblot for phospho-nucleolin (20). Membranes were permeabilized and equilibration for 5 min with 0.5 ml of PBS-0.5 mM EDTA, cells were during a 5-min incubation in ice-cold methanol at 4°C. Cells were washed with scraped off the dishes, and the cell pellet was obtained by centrifugation ␮ 3% milk in TBS (180 l/well) and then rocked overnight at 4°C with 3% milk (4,500 ϫ g for 2 min at 4°C). Cells were resuspended in 0.4 ml of low-salt in TBS (50 ␮l/well) containing a 1:1,000 dilution of anti-phospho-STAT3 HEPES buffer [10 mM HEPES (pH 7.8), 10 mM KCl, 0.1 mM EGTA, 0.1 mM (P-Tyr 705; Cell Signaling Technology, Beverly, MA) and a 1:2,000 dilution EDTA, 1 mM PMSF, and 1 mM DTT] for 15 min, lysed by the addition of 20 of horseradish peroxidase-conjugated goat antirabbit IgG (Jackson Immuno- ␮l of 10% NP40, and centrifuged (10,000 ϫ g for30sat4°C) to obtain the Research, West Grove, PA). Antibodies were aspirated, and then plates were cytosolic supernatant, which was used for luciferase assays (Promega Corp.) washed twice with TBS (180 ␮l/well). Results were visualized by adding measured with a luminometer. Western blot chemiluminescence reagent directly to the wells of the plates, Nuclear Extract Preparation and Gel Shift Assay. Nuclear extract prep- incubating at room temperature for 5 min, and then placing X-ray film directly aration and EMSA were carried out as described previously (11). The 32P- on top of the plate in a dark room for 1–5 min. Quantification of results radiolabeled oligonucleotide probe is hSIE (high affinity sis-inducible element, was done using a GS-700 scanning densitometer (Bio-Rad Laboratories, m67 variant, 5Ј-AGCTTCATTTCCCGTAAATCCCTA-3Ј) that binds STAT1 Hercules, CA). and STAT3. Western Blotting. Treated cell samples were lysed in 30 mM HEPES (pH JAK Kinase Assays. A549, MDA-MB-468, and v-Src-transformed NIH 7.5), 10 mM NaCl, 5 mM MgCl2,25mM NaF, 1 mM EGTA, 1% Triton X-100, 3T3 cells were harvested, washed three times in PBS [10 mM sodium phos- 10% glycerol, 2 mM sodium orthovanadate, 10 ␮g/ml aprotinin, 10 ␮g/ml phate (pH 7.4), 137 mM NaCl, and 1 mM sodium orthovanadate], and then soybean trypsin inhibitor, 25 ␮g/ml leupeptin, 2 mM PMSF, and 6.4 mg/ml p-nitrophenyl phosphate. Phospho-STAT3, phospho-Akt, phospho-MEK, and lysed for 30 min on ice in JAK kinase lysis buffer [25 mM HEPES (pH 7.4), phospho-p42/p44 mitogen-activated protein kinase antibodies were obtained 0.1% Triton X-100, 0.5 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF, ␮ ␮ from Cell Signaling Technologies (Cambridge, MA). Antibodies to STAT3, 10 g/ml aprotinin, and 10 g/ml leupeptin]. Samples were spun at high speed ␮ JAK2, and phospho-JNK were purchased from Santa Cruz Biotechnology to clear, and 800-1000 g of protein were immunoprecipitated per treatment (Santa Cruz, CA). Phospho-JAK2 antibody came from Upstate Biotechnology condition with 50 ng of either JAK1 or JAK2 antibody (Santa Cruz Biotech- ␮ (Lake Placid, NY). Membranes were blocked in either 5% milk in PBS (pH nology) with rocking overnight at 4°C. Twenty-five l of protein A/G PLUS- 7.4) containing 0.1% Tween 20 or 1% BSA in TBS (pH 7.5) containing 0.1% agarose were then added, and rocking continued for1hat4°C. Samples were Tween 20. Phospho-specific antibodies (except phospho-mitogen-activated spun to collect agarose pellet, and pellet was washed twice in wash buffer [50 protein kinase and phospho-JNK) were incubated in 1% BSA in TBS (pH 7.5) mM HEPES (pH 7.4), 0.1% Triton X-100, 0.5 mM DTT, and 150 mM NaCl] containing 0.1% Tween 20, whereas all other antibodies were diluted in 5% and once in phosphorylation buffer [50 mM HEPES (pH 7.4), 0.1% Triton milk in PBS (pH 7.4) containing 0.1% Tween 20 for either2hatroom X-100, 0.5 mM DTT, 6.25 mM manganese chloride, and 100 mM NaCl]. Kinase temperature or overnight at 4°C. Horseradish peroxidase-conjugated secondary reactions were performed at 30°C for 15 min in a final volume of 100 ␮lof antibodies (Jackson ImmunoResearch) were diluted in 5% milk in either PBS phosphorylation buffer. Samples were pretreated with DMSO control, JSI-124, (pH 7.4) containing 0.1% Tween 20 or TBS (pH 7.5) containing 0.1% Tween and control compounds [AG490 (100 ␮M) and PD180970 (2 ␮M)] before addition of 20 ␮Ci/sample [␥-32P]ATP. The reaction was halted using stop buffer (wash buffer ϩ 10 mM EDTA), samples were spun to collect pellet, and 5 http://dtp.nci.nih.gov/. 6 http://dtp.nci.nih.gov/branches/dscb/diversity_explanation.html and http://dtp.nci. then pellet was washed once with stop buffer and twice with wash buffer. nih.gov/docs/dtp_search.html. Samples were then placed in 2ϫ SDS-PAGE sample buffer, boiled at 100°C, 1271

and run on 8% SDS-PAGE gels to separate proteins. Autophosphorylation RESULTS results were visualized by autoradiography. Src Kinase Assay. A549, MDA-MB-468, and v-Src cells were harvested Development of Phosphotyrosine STAT3-specific Cytoblot and lysed for 30 min on ice in RIPA150 buffer [10 mM Tris (pH 7.5), 150 mM High Throughput Assay and Identification of JSI-124. STAT3 is NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 100 ␮M found tyrosine phosphorylated and constitutively activated in many sodium orthovanadate, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 1 ␮g/ml human cancer types. Blockade of this aberrant activation using dom- antipain]. Samples were spun at high speed to clear, and then 1000 ␮gof inant negative STAT3 was shown previously to result in inhibition of protein per treatment condition were immunoprecipitated with 2 ␮g of v-Src tumor growth and induction of tumor cell apoptosis, giving strong antibody (Ab-1; Oncogene Research Products, San Diego, CA) with rocking support to the validation of STAT3 as a cancer drug discovery target ␮ overnight at 4°C. Twenty-five l of protein A/G PLUS-agarose were then (reviewed in Refs. 12–14). In an attempt to identify novel anticancer added, and rocking continued for4hat4°C. Samples were spun to collect drugs based on interference with the aberrant activation of STAT3, we agarose pellet, and pellet was washed three times in RIPA150 buffer, twice in RIPA10 buffer [10 mM Tris (pH 7.5), 10 mM NaCl, 10% glycerol, 5 mM have developed a 96-well plate high throughput cytoblot assay in EDTA, 1% Triton X-100, 0.1% SDS, 100 ␮M sodium orthovanadate, 10 ␮g/ml which the levels of activated tyrosine-phosphorylated STAT3 are aprotinin, 10 ␮g/ml leupeptin, and 1 ␮g/ml antipain], and three times in 40 mM determined by an antibody specific to tyrosine-phosphorylated Tris (pH 7.4). Pellet was then resuspended in 30 ␮l of kinase reaction buffer STAT3. Using this cytoblot assay, we have evaluated a 1992- ␮ ␥ 32 [20 mM Tris (pH 7.4) and 5 mM MgCl2] containing 10 Ci of [ - P]ATP. compound library from the NCI (the NCI Diversity Set) for agents Inhibitors were preincubated for several minutes before the addition of ATP. capable of blocking v-Src activation of STAT3 in NIH 3T3 cells as Kinase reactions were carried out at room temperature for 15 min. Reaction described in “Materials and Methods.” Analysis of the cytoblot results ϫ was stopped with the addition of 2 SDS-PAGE sample buffer. Samples were indicated that several compounds inhibited activation of STAT3 to boiled and run on 10% SDS-PAGE gels. Autophosphorylation results were various degrees. The most potent of these compounds, JSI-124 (NCI visualized by autoradiography. identifier: NSC 521777), suppressed v-Src-activated STAT3 at a In Vitro Cellular Proliferation and TUNEL Assays. Subconfluent A549, ␮ MDA-MB-468, Calu-1, v-Src-transformed NIH 3T3, H-Ras-transformed NIH concentration of 10 M. Fig. 1a shows the structure of JSI-124, which 3T3, and vector NIH 3T3 cells were grown in the presence of 10 ␮M JSI-124 is also known as cucurbitacin I (26–28). Fig. 1b shows an example of or DMSO vehicle control. After 24 h, cells were harvested by trypsinization a 96-well plate cytoblot where the effects of 88 compounds from the and counted via trypan blue exclusion assay to determine cellular viability. NCI Diversity Set on phosphotyrosine STAT3 levels were evaluated. Cells (75,000–150,000, depending on cell line) were then spun onto glass JSI-124 (10 ␮M) at position D6 on the plate reduced phosphotyrosine slides using a Cytospin 3 centrifuge (Thermo Shandon Inc., Pittsburgh, PA). STAT3 to barely detectable levels. After fixing cells to the slides with 4% paraformaldehyde in PBS (pH 7.5) for JSI-124 Suppresses Phosphotyrosine STAT3 Levels in Human 1 h at room temperature, cells were labeled for apoptotic DNA strand breaks Cancer Cell Lines. Fig. 1 identifies JSI-124 as an inhibitor of v-Src by TUNEL reaction using an in situ cell death detection kit (Roche Applied activation of STAT3 in NIH 3T3 cells. To determine whether JSI-124 Science, Indianapolis, IN), according to the manufacturer’s instructions, and then mounted in Vectashield mounting medium (Vector Laboratories, Burl- ingame, CA) containing 4Ј,6-diamidino-2-phenylindole (DAP1) to counter- stain DNA. Fluorescein-labeled DNA strand breaks (TUNEL-positive cells) were then visualized using a fluorescence microscope (Leica Microsystems Inc., Bannockburn, IL), and pictures were taken with a digital camera (Diag- nostic Instruments, Inc., Sterling Heights, MI). TUNEL-positive nuclei were counted and compared with DAP1-stained nuclei to determine the percentage induction of apoptosis by 10 ␮M JSI-124. Antitumor Activity in the Nude Mouse Tumor Xenograft Model. Nude mice and C57 BL-6 mice (NCI, Bethesda, MD) were maintained in accordance with the Institutional Animal Care and Use Committee procedures and guide- lines. v-Src-transformed and oncogenic H-Ras-transformed NIH 3T3, A549, MDA-MB-468, and Calu-1 cells were harvested, resuspended in PBS, and injected s.c. into the right and left flank (10 ϫ 106 cells/flank) of 8-week-old female nude mice as reported previously (25). Similarly, murine B16-F10 melanoma cells were injected s.c. into the right and left flank (106 cells/flank) of C57 black mice. When tumors reached about 150 mm3, animals were randomized (5 animals/group; 2 tumors/animal) and dosed i.p. with 0.2 ml vehicle of drug once daily. Control animals received DMSO (20%) vehicle, whereas treated animals were injected with JSI-124 (1 mg/kg/day) in 20% DMSO in water. The tumor volumes were determined by measuring the length (l) and the width (w) and calculating the volume (V ϭ lw2/2) as described previously (25). Statistical significance between control and treated animals was evaluated by using Student’s t test. Antitumor Activity in Immuno-Competent Mouse Model. For the mouse survival experiments, C57 BL-6 mice were given s.c. injections of B16-F10 cells (105-106 cells/flank). On day 16 after implantation, the mice were randomized (6 animals/group) and treated with either vehicle or JSI-124 (1 mg/kg/day) for 25 days. The percentage of surviving mice was determined by monitoring the death of mice until all mice died. Two experiments of 12 animals each (6 control mice Fig. 1. Identification of JSI-124 (cucurbitacin I) from the NCI Diversity Set using a phosphotyrosine STAT3 high throughput cytoblot assay. a, chemical structure of JSI-124 and 6 mice treated with JSI-124) were carried out. For each of the two experi- (cucurbitacin I). b, v-Src-transformed NIH 3T3 cells containing constitutively activated ments, mice receiving JSI-124 were compared with those receiving vehicle control tyrosine-phosphorylated STAT3 were plated in all wells of the 96-well plate, except for with respect to survival using the permutation log-rank test as implemented in the wells 1A, 1B, 1C, and 1D, where NIH 3T3 cells transfected with empty vector were plated. statistical software package, ProcStatXact (Ps are two sided and exact). The results Wells A–H of Lane 1 were treated for 4 h with vehicle, whereas all other wells were treated with compounds from the NCI Diversity Set (each well received a different of both experiments were then pooled in a stratified analysis and resulted in a P compound). The cells were then permeabilized and cytoblotted with antiphosphotyrosine of 0.01. STAT3 antibody as described in “Materials and Methods.” 1272

Fig. 2. JSI-124 suppresses phosphotyrosine STAT3 levels in v-src-transformed NIH 3T3 cells and human cancer cell lines. a, phosphotyrosine STAT3 levels in various human cell lines. Lysates from a variety of human cancer cell lines were processed for Western blotting using antiphosphotyrosine STAT3 antibody as described in “Materi- als and Methods.” b, JSI-124 suppresses phosphotyrosine STAT3 levels. v-Src-transformed NIH 3T3 cells, A549, MDA-MB-231, MDA-MB-468, and Panc-1 cells were treated for 4 h with either vehicle or JSI-124 (10 ␮M) and then harvested and processed for antiphosphotyrosine STAT3 Western blotting as described for a. c, JSI-124 suppresses phosphotyrosine STAT3 levels without affecting STAT3 protein levels. A549 and MDA-MB-468 cells were treated as described in b, except that the lysates were first immunoprecipitated with anti- STAT3 antibody and immunoblotted with either antiphosphotyrosine STAT3 antibody or anti- STAT3 antibody. Data are representative of three independent experiments for a–c.

suppresses phosphotyrosine STAT3 levels in human cancer cell lines, gene expression. To this end, we first evaluated the effect of JSI-124 we first evaluated several human cancer cell lines and identified those on STAT3 DNA-binding activity by EMSA. v-Src-transformed NIH with high levels of tyrosine-phosphorylated STAT3 (Fig. 2a). Among 3T3 cells and A549 cells were treated with vehicle or JSI-124, and the human cancer cell lines evaluated, A549 (a lung adenocarcinoma), nuclear extracts containing activated STAT3 were incubated with MDA-MB-468 and MDA-MB-231 (two breast carcinomas), and 32P-labeled hSIE oligonucleotide probe for EMSA as described in Panc-1 (a pancreatic carcinoma) contained high levels of tyrosine- phosphorylated STAT3. These human cancer cell lines, along with the positive control cell line (v-Src-transformed NIH 3T3 cells), were treated with either vehicle or JSI-124 (10 ␮M) for 4 h, and the cell lysates were processed for Western blotting with antiphosphotyrosine STAT3 antibody as described in “Materials and Methods.” Fig. 2b shows that JSI-124 was very effective at reducing the levels of tyrosine-phosphorylated STAT3. These results confirm those of the cytoblot and demonstrate the ability of JSI-124 to suppress the levels of constitutively activated, tyrosine-phosphorylated STAT3 not only in v-Src-transformed NIH 3T3 murine fibroblasts but also in human cancer cells of epithelial origin. Because the phosphotyrosine STAT3 antibody could possibly cross-react with other tyrosine-phosphorylated proteins, we confirmed that JSI-124 suppresses phosphotyrosine STAT3 levels by first im- munoprecipitating STAT3 with an anti-STAT3 antibody and then Western blotting with antiphosphotyrosine STAT3 antibody. Fig. 2c shows that treatment of A549 and MDA-MB-468 cells with JSI-124 (10 ␮M) for 4 h reduced phosphotyrosine STAT3 to barely detectable levels. To determine whether JSI-124 affected the STAT3 protein levels, we reblotted with anti-STAT3 antibody. Fig. 2c also shows that JSI-124 has no effect on the protein levels of STAT3. Thus, JSI-124 suppresses the phosphotyrosine levels of STAT3 without affecting its Fig. 3. JSI-124 inhibits STAT3 DNA-binding activity and STAT3-mediated transcrip- protein levels. tion. a, v-Src-transformed NIH 3T3 cells and A549 cells were treated with vehicle or JSI-124 Inhibits STAT3 Signaling by Disrupting STAT3 DNA- JSI-124 and then harvested and processed for EMSA as described in “Materials and binding Activity and STAT3-mediated Gene Expression. Tyrosine Methods.” Samples in Lanes 1–4 and 6–9 are from cells that were treated with vehicle control, whereas samples from Lanes 5 and 10 are from cells treated with JSI-124. Lanes phosphorylation of STAT3 is required for its biological activity (re- 2, 3, 7, and 8 are from samples supershifted with anti-STAT1 (Lanes 2 and 3)or viewed in Refs. 1–4). We therefore reasoned that the suppression by anti-STAT3 antibodies (Lanes 7 and 8). b, v-Src-transformed NIH 3T3 cells transfected with either pLucTKS3- or pRLSRE-dependent luciferase reporters were treated with JSI-124 of the phosphotyrosine levels of STAT3 should lead to either vehicle or JSI-124 and processed for luciferase assays as described in “Materials disruption of STAT3 DNA-binding activity and STAT3-mediated and Methods.” Data are representative of two independent experiments. 1273

“Materials and Methods.” Fig. 3a shows that STAT3 DNA-binding out affecting the pRLSRE reporter. Because v-Src activates pLucTKS3 in activity was greatly reduced in nuclear extracts from v-Src/NIH 3T3 a STAT3-dependent manner and activates pRLSRE in a STAT3-inde- and A549 cells treated with JSI-124 compared with extracts from pendent manner in v-Src-transformed NIH 3T3 cells, the results shown in vehicle-treated cells (Fig. 3, Lanes 4 versus 5 and Lanes 9 versus 10). Fig. 3b demonstrate that JSI-124 is specific to STAT3-mediated tran- To confirm that the band seen in the gel contains STAT3-DNA scription. Thus, JSI-124 inhibits STAT3 signaling by suppressing phos- complexes, the nuclear extracts were preincubated with anti-STAT3 photyrosine levels of STAT3, inhibiting STAT3 DNA binding and or anti-STAT1 antibodies. The anti-STAT3 antibody, but not the STAT3-mediated gene expression. anti-STAT1 antibody, supershifted or blocked the complex, demon- JSI-124 Suppresses Phosphotyrosine Levels of STAT3 and strating that the protein-DNA complex contains STAT3, not STAT1 JAK2 but not Src in A549 and MDA-MB-468 Cells. The ability of (Fig. 3a, Lanes 1, 2, 3, 6, 7, and 8). These results demonstrate that JSI-124 to suppress phosphotyrosine levels of STAT3 suggests that JSI-124, by reducing the levels of tyrosine-phosphorylated STAT3, this agent may interfere with the function of the upstream tyrosine inhibits STAT3 signaling, resulting in disruption of STAT3 DNA kinases JAK and Src that are known to phosphorylate STAT3. We binding. therefore evaluated the effects of JSI-124 on the phosphotyrosine We next determined whether this suppression of STAT3 activation levels of JAK2 and Src in whole cells and the ability of JSI-124 to results in inhibition of STAT3-mediated gene expression. To this end, inhibit Src, JAK1, and JAK2 kinase activities in vitro. Fig. 4a shows v-Src/NIH 3T3 fibroblasts that stably express pLucTKS3 or that stably that treatment of A549 and MDA-MB-468 cells with JSI-124 results express pRLSRE were treated with either vehicle or JSI-124, and cyto- in reduction of the levels of tyrosine-phosphorylated STAT3, with solic extracts were prepared for luciferase assays as described in “Mate- A549 cells being more sensitive than MDA-MB-468 cells. Further- rials and Methods.” Fig. 3b shows that JSI-124 significantly suppresses more, JSI-124 was also effective at suppressing the levels of tyrosine- induction of the STAT3-dependent pLucTKS3 luciferase reporter with- phosphorylated JAK2, but not the levels of tyrosine-phosphorylated

Fig. 4. Effects of JSI-124 on phosphotyrosine levels and kinase activities of JAK and Src kinases. a, JSI-124 suppresses phosphotyrosine levels of STAT3 and JAK2 but not Src. A549 and MDA-MB-468 cells were treated with various concentrations of JSI-124 and processed for immunoblotting with antibodies specific for either phosphotyrosine STAT3, phosphotyrosine JAK2, or phosphotyrosine Src as described in “Materials and Methods.” The membranes were also reblotted with antibodies to STAT3 and JAK2. b, suppression by JSI- 124 of phosphotyrosine STAT3 and JAK2 is rapid. A549 and MDA-MB-468 cells were treated with JSI- 124 (10 ␮M) for various lengths of time (0–240 min) and processed as described above. c, JSI-124 does not inhibit JAK1, JAK2, and Src kinase activities. Lysates from v-Src-transformed cells, A549 cells, and MDA- MB-468 cells were immunoprecipitated with antibodies against JAK1, JAK2, and Src. Autophosphorylation kinase assays were then performed as described in “Ma- terials and Methods.” Immunoprecipitates were incubated either with vehicle control (C), JSI-124 (J), the JAK kinase inhibitor AG490 (A), or the Src kinase inhibitor PD180970 (P). Data are representative of three independent experiments.

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Src. JSI-124 has no effect on the protein levels of STAT3 and JAK2 JSI-124 Inhibits Cellular Proliferation and Induces Apoptosis. in both cell lines (Fig. 4a). The ability of JSI-124 to selectively inhibit the JAK/STAT3 pathway The effects of JSI-124 on JAK/STAT3 signaling described above were suggested that it may inhibit proliferation and/or induce apoptosis determined after4hofJSI-124 treatment. To ascertain the length of preferentially in those tumors that express constitutively activated treatment time required for JSI-124 to suppress phosphotyrosine STAT3 STAT3. To this end, we treated cells that express constitutively and JAK levels, we carried out a time course experiment. Fig. 4b shows activated STAT3 (A549, MDA-MB-468, and v-Src-transformed NIH that treatment of A549 and MDA-MB-468 cells with JSI-124 for as little 3T3) and cells that do not (Calu-1, H-Ras-transformed NIH 3T3, and as 60 min was effective, and in both cell lines the suppression was vector NIH 3T3) with JSI-124 as described in “Materials and Meth- complete after 2 h. Thus, the suppression by JSI-124 of the levels of ods.” Fig. 6 shows that 10 ␮M JSI-124 inhibited cellular proliferation tyrosine-phosphorylated STAT3 and JAK2 is rapid. by 73% (A549), 80% (MDA-MB-468), 90% (v-Src/3T3), 68% (H- We next evaluated the ability of JSI-124 to inhibit the kinase Ras/3T3), 71% (vector/3T3), and 70% (Calu-1). Fig. 6 also shows that activities of Src, JAK1, and JAK2 in vitro. To this end, we JSI-124 induced apoptosis by 18-fold (A549), 15-fold (MDA-MB- immunoprecipitated Src, JAK1, and JAK2 from either A549, 468), 11-fold (v-Src/3T3), 1.4-fold (H-Ras/3T3), 1.2-fold (vector/ MDA-MB-468, or v-Src/NIH 3T3 cells and incubated the immu- 3T3), and 1.3-fold (Calu-1). Thus, JSI-124 induces apoptosis only in noprecipitates with either vehicle control, JSI-124, the JAK tyro- cells that express constitutively activated tyrosine-phosphorylated sine kinase inhibitor AG490, or the Src kinase inhibitor PD180970 STAT3. In contrast, the ability of JSI-124 to inhibit cellular prolifer- and followed autophosphorylation of Src, JAK1, and JAK2 as ation is independent of STAT3 activation status. described in “Materials and Methods.” Fig. 4c shows that, as JSI-124 Inhibits Growth in Mice of Tumors with High Levels of expected, in all three cell lines PD180970 inhibits Src but not Constitutively Activated STAT3. Previous studies have shown that JAK1 or JAK2 activities (A549 did not have JAK1 kinase activity). interfering with STAT3 signaling using a gene therapy approach with Similarly, AG490 inhibited JAK1 and JAK2 but not Src kinase a dominant negative variant of STAT3 (STAT3-␤) resulted in induc- activities. In contrast, all three kinase activities were not affected tion of apoptosis and inhibition of the growth of melanoma cells in by JSI-124 (Fig. 4c). Therefore, although in whole cells JSI-124 is mice (17, 18). Because JSI-124 inhibits aberrantly activated STAT3 very effective at suppressing the levels of tyrosine-phosphorylated signaling, DNA binding, and STAT3-mediated gene expression, and STAT3 and JAK2, it is unable to directly inhibit Src, JAK1, or because it induced apoptosis only in cancer cells with constitutively JAK2 kinase activities in vitro. activated STAT3, we reasoned that the growth in nude mice of tumors JSI-124 Is Highly Selective for the JAK/STAT3 Signaling Path- with constitutively activated STAT3 should be more sensitive to way Over the Akt, ERK, and JNK Signaling Pathways. To deter- JSI-124 than that of tumors with low or without constitutively acti- mine whether the effects of JSI-124 were selective to the JAK/STAT3 vated STAT3. To this end, we s.c. implanted A549, MDA-MB-468, pathway over other oncogenic and survival pathways, we treated A549 and Calu-1 cells in nude mice. When the tumors reached an average and MDA-MB-468 cells with various concentrations of JSI-124 and size of about 150 mm3, the animals were randomized and treated i.p. processed the lysates for Western blotting with antibodies specific for with either vehicle or JSI-124 (1 mg/kg/day) as described in “Mate- phospho-STAT3, phospho-ERK1/2, phospho-JNK, and phospho-Akt as rials and Methods.” Fig. 7 shows that A549 and Calu-1 tumors from described in “Materials and Methods.” Fig. 5 shows that A549 and animals treated with vehicle grew to about 500 mm3 26 days after MDA-MB-468 have constitutively phosphorylated ERK1/ERK2, JNK1, tumor implantation. MDA-MB-468 tumors treated with vehicle con- and Akt in addition to phospho-STAT3. Treatment with JSI-124 resulted trol grew to about 300 mm3 60 days after tumor implantation. JSI-124 in suppression of phospho-STAT3 levels in both cell lines. In contrast, inhibited A549 and MDA-MB-468 tumor growth by 76% and 86%, treatment with JSI-124 had no inhibitory effect on phospho-Akt, phos- respectively. In contrast, JSI-124 had little effect on the growth of pho-ERK1/2, or phospho-JNK with a concentration as high as 10 ␮M. Calu-1 cells in nude mice. Treatment of mice bearing A549 cells with With ERK1/2, not only did JSI-124 not inhibit the levels of phosphoryl- a reduced dose of JSI-124 (0.5 mg/kg/day) for 23 days also inhibited ation, it actually increased the levels of phosphorylation. Thus, these tumor growth by 52% (data not shown). At both doses, 1 and 0.5 results demonstrate that JSI-124 suppressive effects are highly selective mg/kg/day, JSI-124 had no effects on body weight, activity, or food for the JAK/STAT3 pathway over the ERK, JNK, and Akt tumor survival intake of mice. However, at the local site of drug injection, the and oncogenic signaling pathways. peritoneal cavity, JSI-124 at the 1 mg/kg/day dose caused edema. A

Fig. 5. JSI-124 suppresses phosphorylation levels of STAT3 but not ERK1/ERK2, JNK, and Akt. A549 and MDA- MB-468 were treated with various concentrations of JSI-124 (0–10 ␮M) and processed for immunoblotting with phospho- specific antibody against either STAT3, ERK1, ERK2, JNK, or Akt as described in “Materials and Methods.” Data are representative of three independent experiments.

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Fig. 6. JSI-124 induces apoptosis and inhibits proliferation in cultured cells. A549, MDA-MB-468, Calu-1, v-Src/NIH 3T3, H-Ras/NIH 3T3, and vector/NIH 3T3 cells were treated for 24 h with 10 ␮M JSI-124. Cells were harvested and counted to determine inhibition of proliferation. The percentage of inhibition of proliferation was determined by dividing the number of viable cells of JSI-124-treated samples by those of the control. Cells were then spun onto slides for apoptosis determination by TUNEL reaction as described in “Materials and Methods.” The percentage of TUNEL-positive values corresponds to average and SE of nine determinations of slides from three independent experiments with 3 fields/slide. Apoptosis fold increase was obtained by dividing the percentage of TUNEL-positive values of JSI-124-treated samples by that of control samples. similar observation was made by the NCI Developmental Therapeu- deficient nude mice. Furthermore, the studies did not investigate the tics Program, where edema was observed at the s.c. site of injection.7 effects of JSI-124 on the survival of mice bearing death-inducing The results from A549, Calu-1, and MDA-MB-468 xenograft studies tumors. We therefore evaluated the ability of JSI-124 to inhibit tumor suggest that human cancer cells that express constitutively activated growth and increase survival of immunologically competent C57 STAT3 should be sensitive to JSI-124. We further reasoned that if the BL-6 mice that had been s.c. implanted with murine B16-F10 mela- ability of JSI-124 to inhibit tumor growth in nude mice depends on noma that expresses constitutively activated STAT3 (17). Fig. 7 constitutively activated STAT3, v-Src-transformed NIH 3T3 cells that shows that B16-F10 tumors from control mice given injections with require constitutively activated STAT3 for malignant transformation (15) vehicle grew to an average size of 1194 Ϯ 141 mm3, whereas those should be sensitive to JSI-124, whereas oncogenic Ras-transformed NIH treated with JSI-124 (1 mg/kg/day) grew only to an average size of 3T3 cells, in which STAT3 is not constitutively activated (see Fig. 2a), 588 Ϯ 94 mm3. Thus, JSI-124 treatment inhibited tumor growth by should be resistant. Fig. 7 shows that, in the absence of JSI-124, the 56%. To determine the effect of JSI-124 on mouse survival, we s.c. growth of both v-Src- and Ras-transformed NIH 3T3 tumors was highly implanted B16-F10 melanoma and followed mouse survival over aggressive, and tumors reached average sizes of about 2500 and 1000 time. Fig. 7 shows that mice treated with vehicle begin to die on day mm3, respectively, within 9 days of tumor cell implantation. Fig. 7 also 19 after B16-F10 implantation. By day 21, half of the mice were dead, shows that JSI-124 (1 mg/kg/day) inhibited the growth of v-Src/NIH 3T3 and by day 35, all six mice were dead. In contrast, none of the tumors by 64%. In contrast, the growth of Ras/NIH 3T3 tumors was JSI-124-treated mice were dead by day 23, half of the mice died on resistant to JSI-124. These results, coupled with those from the human day 34, and all of the mice died by day 42. Fig. 7 also shows that 50% ϭ tumor xenografts, strongly suggest that JSI-124 selectively targets tumors of vehicle-treated mice survived up until day 21 (T50 21), whereas with constitutively activated STAT3 signaling. the JSI-124-treated group of mice had a longer T50 of 34 days. Thus,

The above results were obtained from experiments with immune- treatment with JSI-124 significantly increased the life span (T50 increase of 13 days) of immunologically competent mice that were 7 J. Johnson, personal communication. implanted with B16-F10 melanoma. 1276

Fig. 7. JSI-124 increases mouse survival and inhibits tumor growth in mice of MDA-MB-468, A549, B16-F10, and v-Src/NIH 3T3 cells but does not inhibit tumor growth in Calu-1 and Ras/NIH 3T3 cells. MDA-MB-468, A549, Calu-1, v-Src/NIH 3T3, and Ras/NIH 3T3 cells were implanted s.c. in nude mice, whereas B16-F10 cells were implanted s.c. in C57 BL-6 mice. When the tumors reached an average size of about 100–150 mm3, animals were randomized (5 animals/group; 2 tumors/animal) and treated with either vehicle (F) or 1 mg/kg/day JSI-124 ()asde- scribed in “Materials and Methods.” Each measure- ment represents an average of 10 tumors. Data are representative of four (B16-F10), three (A549, v-Src/ NIH 3T3, and Ras/NIH 3T3), and two (MDA- MB-468 and Calu-1) independent experiments. †, P ϭ 0.08. For the mouse ,ء ;P Ͻ 0.05 ,ءء ;P Ͻ 0.01 survival studies, B16-F10 cells were implanted s.c. in C57 BL-6 mice, and on day 16 after implantation, the mice were randomized (6 animals/group) and treated with either vehicle (F) or JSI-124 (Œ; 1 mg/kg/day) for 25 days. The percentage of surviving mice was determined by monitoring the death of mice over a period of 42 days until all mice died. Data are representative of two independent experiments carried out with 12 mice each (6 vehicle control-treated mice and 6 JSI-124-treated mice). For statistical analysis, in each of the two experiments, control animals were compared with JSI-124-treated animals with respect to survival using the permutation log-rank test as implemented in the statistical software package, Proc- StatXact. The results of both experiments were pooled in a stratified analysis resulting in P ϭ 0.01.

DISCUSSION shown to suppress skin carcinogenesis (33), inhibit cell adhesion (34), and disrupt the actin and vimentin cytoskeleton in prostate carcinoma Many modern anticancer drug discovery approaches have focused cells (27, 31). Although the reports on cucurbitacin biological activity on targeting signal transduction pathways involving RTKs (e.g., suggest antiproliferative activity and possible antitumor activity, no ErbB2 and EGFR), farnesylated proteins (e.g., Ras), and nonreceptor data are available concerning the oncogenic and tumor survival path- cytosolic kinases (e.g., Raf, MEK, PI3k, and Akt; Ref. 29). These ways that are targeted by cucurbitacins. important efforts resulted in several novel agents such as RTK mono- In this report, we demonstrate that cucurbitacin I (JSI-124) reduced clonal antibodies; RTK, farnesyltransferase, Raf, and MEK inhibitors the levels of phosphotyrosine of constitutively activated STAT3 in that are presently in clinical trials; and some such as the Bcr-Abl many human cancer cell lines including pancreatic, lung, and breast tyrosine kinase inhibitor STI-571 (Gleevec), which has recently been carcinomas. This suppression in the levels of constitutively activated approved by the Food and Drug Administration for chronic myelog- STAT3 resulted in blockade of STAT3 DNA-binding activity and enous leukemia. In contrast to this heavily exploited area, little has STAT3-mediated gene transcription. JSI-124 was highly selective for been done to target the STAT3 signaling pathway. However, exper- disrupting STAT3 signaling over other pivotal oncogenic and tumor iments in animal models using gene therapy with a dominant negative form of STAT3 and a constitutively active mutant of STAT3, as well survival pathways. For example, in two cell lines, the human lung as the prevalence of constitutively activated STAT3 in many human adenocarcinoma A549 and the human breast carcinoma MDA-MB- cancers, strongly suggest STAT3 as having a causal role in oncogen- 468, JSI-124 did not inhibit the constitutive activation of the Ser/Thr esis (12–14). Furthermore, the fact that constitutive activation of protein kinase B, PKB/Akt, indicating that the PI3k/Akt survival STAT3 induces genes such as cyclin D1, c-myc, and bcl-xl that are pathway is not a target for JSI-124. Similarly, the Ras/Raf/MEK/ERK intimately involved in oncogenesis and tumor survival, coupled with oncogenic signaling pathway was not inhibited by JSI-124 in these the fact that constitutively activated STAT3 is required for survival of two cell lines. Finally, the constitutive activation of JNK in A549 and some human cancer cells, further validates the STAT3 signaling MDA-MB-468 was not affected by JSI-124, indicating that the stress- pathway as a selective cancer drug discovery target (12–14). activated protein kinase signaling pathway is not targeted by JSI-124. In this study, we have identified JSI-124, a selective JAK/STAT3 Many human cancers rely on the PI3k/Akt and Ras/Raf/MEK/ERK signaling pathway inhibitor with potent antitumor activity against pathways to induce malignant transformation and tumor survival. For human tumors in nude mice, from the NCI Diversity Set of 1,992 example, the great majority of tumors overexpress the ErbB family of compounds. JSI-124 is a plant natural product identified previously as receptors such as EGFR and ErbB2 and contain mutant forms of Ras. cucurbitacin I, a member of the cucurbitacin family of compounds that These RTK and Ras genetic alterations result in constitutive activation are isolated from various plant families such as the Cucurbitaceae and of the PI3k/Akt and Ras/Raf/MEK/ERK pathways. The fact that Cruciferae and have been used as folk medicines for centuries in JSI-124 inhibits tumor growth and blocks STAT3 signaling without countries such as China and India. However, little was known about inhibiting the constitutive activation of Akt and ERK1/ERK2 suggests the biological activities of the various cucurbitacins until recently. that its ability to inhibit the growth of A549 and MDA-MB-468 in Some cucurbitacins have been shown to have anti-inflammatory and nude mice does not depend on blocking Akt and ERK activation. This analgesic as well as cytotoxic effects. Furthermore, cucurbitacins also suggests that JSI-124 may be more selective toward inhibiting the were also found to inhibit DNA, RNA, and protein synthesis in HeLa growth of tumors with constitutively activated STAT3. Consistent cells (30) and to inhibit proliferation of HeLa cells (30), endothelial with this, JSI-124 induces apoptosis in A549, MDA-MB-468, and cells (31), and T lymphocytes (32). Finally, some cucurbitacins were v-Src/NIH 3T3 cells that express constitutively activated STAT3, but 1277

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2003 American Association for Cancer Research. DISCOVERY OF JSI-124, A JAK-STAT3 PATHWAY INHIBITOR it does not induce apoptosis in Calu-1, H-Ras/NIH 3T3, and vector/ levels of activated STAT3 further validates interference with STAT3 NIH 3T3 cells that do not express constitutively activated STAT3. signaling as a sound approach to cancer chemotherapy. Present studies Interestingly, the ability of JSI-124 to inhibit proliferation in vitro did are geared toward evaluating the antitumor efficacy of JSI-124 in a not depend on STAT3 activation status. However, inhibition of tumor broader spectrum of human cancer cell lines and identifying the growth in animals did depend on STAT3 activation status. Indeed, biochemical target of JSI-124. consistent with the apoptosis data, but not the proliferation data, we found v-Src-transformed NIH 3T3 tumors that depend on constitu- ACKNOWLEDGMENTS tively activated STAT3 for malignant transformation to be sensitive to JSI-124 antitumor activity in nude mice. In contrast, oncogenic Ras- We thank Drs. Edward Sausville, Jill Johnson, George Johnson, Daniel transformed NIH 3T3 tumors, in which STAT3 is not constitutively Zaharevitz, and Robert Schultz from the NCI Developmental Therapeutics activated, were resistant to JSI-124. Furthermore, the fact that JSI-124 Program for providing us with the Diversity Set. We also thank Dr. Alex Levitzki for AG490, Dr. Alan Kraker for PD180970, and the H. Lee Moffitt inhibited the growth in mice of the human lung adenocarcinoma Cancer Center Biostatistics and Informatics Core for statistical analysis. (A549), human breast carcinoma (MDA-MB-468), and murine melanoma (B16-F10), all of which express constitutively activated REFERENCES STAT3, but did not inhibit growth of the human lung adenocarcinoma (Calu-1) that has very low levels of tyrosine-phosphorylated STAT3 1. Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H., and Schreiber, R. D. How cells respond to interferons. Annu. Rev. Biochem., 67: 227–264, 1998. gives further support to the notion that the ability of JSI-124 to inhibit 2. Horvath, C. M., and Darnell, J. E. The state of the STATs: recent developments in the tumor growth depends on an aberrantly activated STAT3 signaling study of signal transduction to the nucleus. Curr. Opin. Cell Biol., 9: 233–239, 1997. pathway. Importantly, JSI-124 also significantly increased the sur- 3. Ihle, J. N., and Kerr, I. M. JAKs and STATs in signaling by the cytokine receptor superfamily. Trends Genet., 11: 69–74, 1995. vival of immunologically competent mice in which B16-F10 murine 4. Schindler, C., and Darnell, J. E., Jr. Transcriptional responses to polypeptide ligands: melanoma was implanted. the JAK-STAT pathway. Annu. Rev. Biochem., 64: 621–651, 1995. 5. Ihle, J. N., Witthuhn, B. A., Quelle, F. W., Yamamoto, K., and Silvennoinen, O. The ability of JSI-124 to suppress the cellular levels of phosphoty- Signaling through the hematopoietic cytokine receptors. Annu. Rev. Immunol., 13: rosine-STAT3 but not phospho-ERK1/2, phospho-JNK, and phospho- 369–398, 1995. Akt suggested that a STAT3 tyrosine kinase is a possible molecular target 6. Leaman, D. W., Leung, S., Li, X., and Stark, G. R. Regulation of STAT-dependent pathways by growth factors and cytokines. FASEB J., 10: 1578–1588, 1996. for JSI-124. Consistent with a direct inhibition of the enzymatic activity 7. Schaper, F., Gendo, C., Eck, M., Schmitz, J., Grimm, C., Anhuf, D., Kerr, I. M., and of a tyrosine kinase is the fact that suppression of the STAT3 phospho- Heinrich, P. C. Activation of the protein tyrosine phosphatase SHP2 via the interleu- tyrosine levels was rapid (observed as early as 30 min and complete after kin-6 signal transducing receptor protein gp130 requires tyrosine kinase JAK1 and limits acute-phase protein expression. Biochem. J., 335: 557–565, 1998. only2hoftreatment). There are two well-characterized STAT3 tyrosine 8. Stofega, M. R., Wang, H., Ullrich, A., and Carter-Su, C. Growth hormone regulation kinases: JAK and Src kinase. Because phosphotyrosine JAK2 levels were of SIRP and SHP-2 tyrosyl phosphorylation and association. J. Biol. Chem., 273: 7112–7117, 1998. also reduced by JSI-124, this suggested that JAK2 is likely not the target. 9. Yu, C. L., Jin, Y. J., and Burakoff, S. J. Cytosolic tyrosine dephosphorylation of STAT5. This was confirmed by in vitro kinase assays where JAK2 and JAK1 Potential role of SHP-2 in STAT5 regulation. J. Biol. Chem., 275: 599–604, 2000. enzymatic activities were inhibited by AG490, a known JAK inhibitor, 10. Seidel, H. M., Milocco, L. H., Lamb, P., Darnell, J. E., Jr., Stein, R. B., and Rosen, J. Spacing of palindromic half sites as a determinant of selective STAT (signal but were not inhibited by JSI-124. Similarly, Src kinase activity was transducers and activators of transcription) DNA binding and transcriptional activity. inhibited in vitro by the known Src kinase inhibitor PD180970 but was Proc. Natl. Acad. Sci. USA, 92: 3041–3045, 1995. not inhibited by JSI-124, indicating that Src kinase is not a target. The 11. Turkson, J., Bowman, T., Adnane, J., Zhang, Y., Djeu, J. Y., Sekharam, M., Frank, D. A., Holzman, L. B., Wu, J., Sebti, S., and Jove, R. Requirement for Ras/Rac1- RTK EGFR, which is also believed to phosphorylate STAT3, is most mediated p38 and c-Jun N-terminal kinase signaling in STAT3 transcriptional activity likely not a target either because epidermal growth factor stimulation of induced by the Src oncoprotein. Mol. Cell. Biol., 19: 7519–7528, 1999. 12. Bowman, T., Yu, H., Sebti, S., Dalton, W., and Jove, R. Signal transducers and EGFR tyrosine phosphorylation in breast cell line MCF-10A and EGFR- activators of transcription: novel targets for anticancer therapeutics. Cancer Control, overexpressing NIH 3T3 cells was inhibited only minimally by JSI-124 6: 427–435, 1999. (data not shown). 13. Turkson, J., and Jove, R. STAT proteins: novel molecular targets for cancer drug discovery. Oncogene, 19: 6613–6626, 2000. Reduction in phosphotyrosine levels could be a result of either 14. Bowman, T., Garcia, R., Turkson, J., and Jove, R. STATs in oncogenesis. Oncogene, inhibition of protein tyrosine kinases or activation of protein phos- 19: 2474–2488, 2000. photyrosine phosphatases. STAT3 is known to be phosphotyrosine 15. Yu, C. L., Meyer, D. J., Campbell, G. S., Larner, A. C., Carter-Su, C., Schwartz, J., and Jove, R. Enhanced DNA-binding activity of a STAT3-related protein in cells dephosphorylated by two protein phosphotyrosine phosphatases, transformed by the Src oncoprotein. Science (Wash. DC), 269: 81–83, 1995. SHP-1 and SHP-2 (7–9), and JSI-124 could down-regulate phospho- 16. Bromberg, J. F., Wrzeszczynska, M. H., Devgan, G., Zhao, Y., Pestell, R. G., Albanese, C., and Darnell, J. E., Jr. STAT3 as an oncogene. Cell, 98: 295–303, 1999. tyrosine-STAT3 levels by promoting the protein phosphatase activi- 17. Niu, G., Heller, R., Catlett-Falcone, R., Coppola, D., Jaroszeski, M., Dalton, W., Jove, ties of SHP-1 and SHP-2. Alternatively, JSI-124 could also activate R., and Yu, H. Gene therapy with dominant-negative STAT3 suppresses growth of the physiological inhibitors that are known to directly or indirectly down- murine melanoma B16 tumor in vivo. Cancer Res., 59: 5059–5063, 1999. 18. Catlett-Falcone, R., Landowski, T. H., Oshiro, M. M., Turkson, J., Levitzki, A., regulate STAT3 activation. These include suppressors of cytokine Savino, R., Ciliberto, G., Moscinski, L., Fernandez-Luna, J. L., Nunez, G., Dalton, signaling, STAT-induced STAT inhibitor, JAK-binding protein, and W. S., and Jove, R. Constitutive activation of STAT3 signaling confers resistance to STAT3-interacting protein (13). apoptosis in human U266 myeloma cells. Immunity, 10: 105–115, 1999. 19. Lerner, E. C., Qian, Y., Blaskovich, M. A., Fossum, R. D., Vogt, A., Sun, J., Cox, In summary, we have developed a phosphotyrosine STAT3-specific A. D., Der, C. J., Hamilton, A. D., and Sebti, S. M. Ras CAAX peptidomimetic cytoblot that led us to the discovery of a JAK/STAT3 signaling FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accu- mulation of inactive Ras-Raf complexes. J. Biol. Chem., 270: 26802–26806, 1995. inhibitor with potent antitumor activity. JSI-124 blocked activation of 20. Stockwell, B. R., Haggarty, S. J., and Schreiber, S. L. High-throughput screening of STAT3 in several human cancer cell lines that contain high levels of small molecules in miniaturized mammalian cell-based assays involving posttransla- constitutively activated tyrosine-phosphorylated STAT3 and subse- tional modifications. Chem. Biol., 6: 71–83, 1999. 21. Blaskovich, M. A., Lin, Q., Delarue, F. L., Sun, J., Park, H. S., Coppola, D., quently inhibited STAT3 DNA-binding activity and STAT3- Hamilton, A. D., and Sebti, S. M. Design of GFB-111, a platelet-derived growth dependent gene expression. This JAK/STAT3 signaling disrupter is factor binding molecule with antiangiogenic and anticancer activity against human highly selective in that other oncogenic and tumor survival pathways tumors in mice. Nat. Biotechnol., 18: 1065–1070, 2000. 22. Zhang, D., Sun, M., Samols, D., and Kushner, I. STAT3 participates in transcriptional were not affected. The ability of JSI-124 to increase mouse survival, activation of the C-reactive protein gene by interleukin-6. J. Biol. Chem., 271: to inhibit growth in mice of human and murine tumors and oncogene- 9503–9509, 1996. 23. Yamauchi, K., Holt, K., and Pessin, J. E. Phosphatidylinositol 3-kinase functions transformed NIH 3T3 tumors with high levels of constitutively acti- upstream of Ras and Raf in mediating insulin stimulation of c-fos transcription. vated STAT3, and to not inhibit the growth of those tumors with low J. Biol. Chem., 268: 14597–14600, 1993. 1278

24. Turkson, J., Ryan, D., Kim, J. S., Zhang, Y., Chen, Z., Haura, E., Laudano, A., Sebti, 29. Sebti, S. New drug targets and therapies for cancer. Oncog. Rev., 19: 6549–6692, S., Hamilton, A. D., and Jove, R. Phosphotyrosyl peptides block STAT3-mediated 2000. DNA-binding activity, gene regulation, and cell transformation. J. Biol. Chem., 276: 30. Witkowski, A., Woynarowska, B., and Konopa, J. Inhibition of the biosynthesis of 45443–45455, 2001. deoxyribonucleic acid, ribonucleic acid, and protein in HeLa S3 cells by cucurb- 25. Sun, J., Blaskovich, M. A., Knowles, D., Qian, Y., Ohkanda, J., Bailey, R. D., itacins, glucocorticoid-like cytotoxic triterpenes. Biochem. Pharmacol., 33: 995– Hamilton, A. D., and Sebti, S. M. Antitumor efficacy of a novel class of non-thiol- 1004, 1984. containing peptidomimetic inhibitors of farnesyltransferase and geranylgeranyltrans- 31. Duncan, M. D., and Duncan, K. L. Cucurbitacin E targets proliferating endothelia. ferase I: combination therapy with the cytotoxic agents cisplatin, Taxol, and gemcit- J. Surg. Res., 69: 55–60, 1997. abine. Cancer Res., 59: 4919–4926, 1999. 32. Smit, H. F., van den Berg, A. J., Kroes, B. H., Beukelman, C. J., Quarles van Ufford, 26. Witkowski, A., and Konopa, J. Binding of the cytotoxic and antitumor triterpenes, H. C., van Dijk, H., and Labadie, R. P. Inhibition of T-lymphocyte proliferation by cucurbitacins, to glucocorticoid receptors of HeLa cells. Biochim. Biophys. Acta, cucurbitacins from Picrorhiza scrophulariaeflora. J. Nat. Prod. (Lloydia), 63: 1300– 674: 246–255, 1981. 1302, 2000. 27. Duncan, K. L., Duncan, M. D., Alley, M. C., and Sausville, E. A. Cucurbitacin 33. Konoshima, T., Takasaki, M., Kozuka, M., Nagao, T., Okabe, H., Irino, N., E-induced disruption of the actin and vimentin cytoskeleton in prostate carcinoma Nakasumi, T., Tokuda, H., and Nishino, H. Inhibitory effects of cucurbitane triter- cells. Biochem. Pharmacol., 52: 1553–1560, 1996. penoids on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumor. 28. Dinan, L., Whiting, P., Girault, J. P., Lafont, R., Dhadialla, T. S., Cress, D. E., II. Biol. Pharm. Bull., 18: 284–287, 1995. Mugat, B., Antoniewski, C., and Lepesant, J. A. Cucurbitacins are insect steroid 34. Musza, L. L., Speight, P., McElhiney, S., Barrow, C. J., Gillum, A. M., Cooper, R., hormone antagonists acting at the ecdysteroid receptor. Biochem. J., 327: 643– and Killar, L. M. Cucurbitacins, cell adhesion inhibitors from Conobea scoparioides. 650, 1997. J. Nat. Prod. (Lloydia), 57: 1498–1502, 1994.

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Michelle A. Blaskovich, Jiazhi Sun, Alan Cantor, et al.

Cancer Res 2003;63:1270-1279.

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