Presented by:Presented by:Khuram AzizKhuram Aziz

• Discovery of JSI-124, a selective Discovery of JSI-124, a selective JAK/STAT3 inhibitor with antitumor JAK/STAT3 inhibitor with antitumor activity against human and murine activity against human and murine cancer cells in MICEcancer cells in MICE

introductionintroduction

• Discovery of disrupter in abberant Discovery of disrupter in abberant activation of STATactivation of STAT

• Supression of levels of phosphotyrosine Supression of levels of phosphotyrosine STAT3 will inhibit proliferation and STAT3 will inhibit proliferation and induce apoptosisinduce apoptosis

• Use of JSI-124Use of JSI-124• Which are inhibited by it;Which are inhibited by it;• A549 tumors, v-src-transformed NIH A549 tumors, v-src-transformed NIH

3T3 tumors, MDA-MB-4683T3 tumors, MDA-MB-468

Which are not affectedWhich are not affected

• Ras transformed tumors, akt Ras transformed tumors, akt pathways, c-jun nh2 terminal kinase, pathways, c-jun nh2 terminal kinase, extracellular signal regulated extracellular signal regulated kinases ½, calu-1kinases ½, calu-1

• STATS are of diferent types.STATS are of diferent types.• They have dual roleThey have dual role• They are involved in many They are involved in many

physiological functionsphysiological functions

• For the biological functioning, these For the biological functioning, these must be tyrosine phosphorylatedmust be tyrosine phosphorylated

• Tyrosine kinases that are involved to Tyrosine kinases that are involved to phosphorylate are non-RTKs, JAKs phosphorylate are non-RTKs, JAKs and peptide growth factorsand peptide growth factors

• Regulated by SHP-1 and SHP-2Regulated by SHP-1 and SHP-2

STAT3 invovlment in STAT3 invovlment in oncogenesis is most oncogenesis is most

thoroughly..thoroughly..• First, STAT3 is constitutively First, STAT3 is constitutively

tyrosine phosphorylatedtyrosine phosphorylated• Second, for expression of activated Second, for expression of activated

mutant STAT3, stable dimerization mutant STAT3, stable dimerization was forcedwas forced

• Its validation as an anticancer drug Its validation as an anticancer drug targettarget

Materials and MethodsMaterials and Methods

• Cell Lines. Cell Lines. All human and murine All human and murine tumor cell lines used were obtainedtumor cell lines used were obtained

• from the American Type Culture from the American Type Culture Collection (Manassas, VA). Stably Collection (Manassas, VA). Stably transfectedtransfected

v-Src, oncogenic H-Ras, and vector v-Src, oncogenic H-Ras, and vector NIH 3T3 cell lines have been NIH 3T3 cell lines have been described previously .described previously .

NCI Diversity Set.NCI Diversity Set.

• The NCI Structural Diversity Set is a library of 1,992The NCI Structural Diversity Set is a library of 1,992• compounds selected from the approximately 140,000-compounds selected from the approximately 140,000-

compound NCI drugcompound NCI drug• depository. These compounds were selected based on depository. These compounds were selected based on

various criteria includingvarious criteria including• availability, drug-like structure, uniqueness of availability, drug-like structure, uniqueness of

pharmacophore, and anticancerpharmacophore, and anticancer

activity as determined by cell growth inhibition assays activity as determined by cell growth inhibition assays against a panel ofagainst a panel of

human tumor cell lines. In-depth data on the selection, human tumor cell lines. In-depth data on the selection, structures, and activitiesstructures, and activities

of these diversity set compounds can be found on the NCI of these diversity set compounds can be found on the NCI DevelopmentalDevelopmental

Cytoblot Screening for Cytoblot Screening for Phospho-STAT3 Phospho-STAT3

Inhibition.Inhibition.• NIH 3T3 cells stably transfected with v-Src or NIH NIH 3T3 cells stably transfected with v-Src or NIH

3T3 vector control cells (11) were plated into 3T3 vector control cells (11) were plated into sterile, opaque, 96-well tissue culture plates at sterile, opaque, 96-well tissue culture plates at 25,000 cells/well.25,000 cells/well.

• After overnight growth at 37°C, the cells were After overnight growth at 37°C, the cells were treated for 4 h in the presence of either vehicle treated for 4 h in the presence of either vehicle control or 10 _M of NCI Diversity Set compounds. control or 10 _M of NCI Diversity Set compounds.

• After treatment,cells were washed in 100 _l of cold After treatment,cells were washed in 100 _l of cold TBS and TBS and

• then fixed for 1 h at 4°C with cold 3.7% then fixed for 1 h at 4°C with cold 3.7% formaldehyde in TBS (190 _l/well) as described formaldehyde in TBS (190 _l/well) as described previously for a similar cytoblot for phospho-nucleolin (20).previously for a similar cytoblot for phospho-nucleolin (20).

..• Membranes were permeabilized during a 5-Membranes were permeabilized during a 5-min incubation in ice-cold methanol at 4°C.min incubation in ice-cold methanol at 4°C.

• Cells were washed with 3% milk in TBS (180 Cells were washed with 3% milk in TBS (180 _l/well) and _l/well) and

• then rocked overnight at 4°C with 3% milk in then rocked overnight at 4°C with 3% milk in TBS (50 _l/well) containing a 1:1,000 dilution TBS (50 _l/well) containing a 1:1,000 dilution of anti-phospho-STAT3 (P-Tyr 705; Cell of anti-phospho-STAT3 (P-Tyr 705; Cell Signaling Technology, Beverly, MA) and a Signaling Technology, Beverly, MA) and a 1:2,000 dilution of horseradish peroxidase-1:2,000 dilution of horseradish peroxidase-conjugated goat antirabbit IgG (Jackson conjugated goat antirabbit IgG (Jackson Immuno Research, West Grove, PA). Immuno Research, West Grove, PA).

• Antibodies were aspirated, and then plates Antibodies were aspirated, and then plates were washed twice with TBS (180 _l/well).were washed twice with TBS (180 _l/well).

..• Results were visualized by adding Results were visualized by adding

Western blot chemiluminescence Western blot chemiluminescence reagent directly to the wells of the reagent directly to the wells of the plates, incubating at room plates, incubating at room temperature for 5 min, and temperature for 5 min, and

• then placing X-ray film directly on top then placing X-ray film directly on top of the plate in a dark room for 1–5 of the plate in a dark room for 1–5 min. Quantification of results was min. Quantification of results was done using a GS-700 scanning done using a GS-700 scanning densitometer.densitometer.

Western BlottingWestern Blotting

• Treated cell samples were lysed in 30 mM HEPES Treated cell samples were lysed in 30 mM HEPES (pH 7.5), 10 mM NaCl, 5 mM MgCl2, 25 mM NaF, (pH 7.5), 10 mM NaCl, 5 mM MgCl2, 25 mM NaF, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 2 mM 1 mM EGTA, 1% Triton X-100, 10% glycerol, 2 mM sodium orthovanadate, 10 _g/ml aprotinin, 10 _g/ml sodium orthovanadate, 10 _g/ml aprotinin, 10 _g/ml soybean trypsin inhibitor, 25 _g/ml leupeptin, 2 mM soybean trypsin inhibitor, 25 _g/ml leupeptin, 2 mM PMSF, and 6.4 mg/ml PMSF, and 6.4 mg/ml pp-nitrophenyl phosphate. -nitrophenyl phosphate. Phospho-STAT3, phospho-Akt, phospho-MEK, andPhospho-STAT3, phospho-Akt, phospho-MEK, and

• phospho-p42/p44 mitogen-activated protein kinase phospho-p42/p44 mitogen-activated protein kinase antibodies were obtained from Cell Signaling antibodies were obtained from Cell Signaling Technologies.Technologies.

• Antibodies to STAT3, JAK2, and phospho-JNK were Antibodies to STAT3, JAK2, and phospho-JNK were purchased.purchased.

• Phospho-JAK2 antibody came from Upstate Phospho-JAK2 antibody came from Upstate Biotechnology. Membranes were blocked in either 5% Biotechnology. Membranes were blocked in either 5% milk in PBS (pH 7.4) containing 0.1% Tween 20 or 1% milk in PBS (pH 7.4) containing 0.1% Tween 20 or 1% BSA in TBS (pH 7.5) containing 0.1% Tween 20. BSA in TBS (pH 7.5) containing 0.1% Tween 20.

• Phospho-specific antibodies (except phospho-Phospho-specific antibodies (except phospho-mitogen-activatedmitogen-activated

protein kinase and phospho-JNK) were incubated in 1% protein kinase and phospho-JNK) were incubated in 1% BSA in TBS (pH 7.5) containing 0.1% Tween 20, BSA in TBS (pH 7.5) containing 0.1% Tween 20,

whereas all other antibodies were diluted in 5% milk in whereas all other antibodies were diluted in 5% milk in PBS (pH 7.4) containing 0.1% Tween 20 for either 2 h PBS (pH 7.4) containing 0.1% Tween 20 for either 2 h at room temperature or overnight at 4°C.at room temperature or overnight at 4°C.

• Horseradish peroxidase-conjugated Horseradish peroxidase-conjugated secondarysecondary

antibodies (Jackson ImmunoResearch) antibodies (Jackson ImmunoResearch) were diluted in 5% milk in either PBS (pH were diluted in 5% milk in either PBS (pH 7.4) containing 0.1% Tween 20 or TBS 7.4) containing 0.1% Tween 20 or TBS (pH 7.5) containing 0.1% Tween 20 at a (pH 7.5) containing 0.1% Tween 20 at a 1:1000 dilution for 1 h at room 1:1000 dilution for 1 h at room temperature. temperature.

• Western blots were visualized using Western blots were visualized using enhanced chemiluminescenceenhanced chemiluminescence

Immunoprecipitation of Immunoprecipitation of STAT3STAT3

• A549 cells were treated for 4 h with A549 cells were treated for 4 h with vehicle or JSI-124 and then lysed in vehicle or JSI-124 and then lysed in 150 mM HEPES (pH 7.5), 150 mM 150 mM HEPES (pH 7.5), 150 mM NaCl,NaCl,

• 1 mM EDTA, 0.5% NP40, 10% 1 mM EDTA, 0.5% NP40, 10% glycerol, 5 mM NaF, 1 mM DTT, 1 glycerol, 5 mM NaF, 1 mM DTT, 1 mM PMSF, 2 mM sodium mM PMSF, 2 mM sodium orthovanadate, and 5 _g/ml orthovanadate, and 5 _g/ml leupeptin.leupeptin.

• Sample lysates were collected and cleared, and Sample lysates were collected and cleared, and then 500 _g of lysate were immunoprecipitated then 500 _g of lysate were immunoprecipitated with50 ng of STAT3 antibody overnight at 4°C with50 ng of STAT3 antibody overnight at 4°C and rocked with 25 _l of protein A/G PLUS-and rocked with 25 _l of protein A/G PLUS-agarose (Santa Cruz Biotechnology) for 1 h at agarose (Santa Cruz Biotechnology) for 1 h at 4°C. 4°C.

• Samples were washed four times with lysis buffer Samples were washed four times with lysis buffer and then boiled in 2_ SDS-PAGE sample buffer and then boiled in 2_ SDS-PAGE sample buffer and run on 10% SDS-PAGE gel. Protein was and run on 10% SDS-PAGE gel. Protein was transferred to nitrocellulosetransferred to nitrocellulose

• blotted as described above for both phospho-blotted as described above for both phospho-specific STAT3 and STAT3.specific STAT3 and STAT3.

• DNA Binding and TranscriptionDNA Binding and Transcription• Transfection and Generation of Transfection and Generation of

Stable ClonesStable Clones• Preparation of Cytosolic ExtractsPreparation of Cytosolic Extracts• Nuclear Extract Preparation and Nuclear Extract Preparation and

Gel Shift AssayGel Shift Assay

JAK Kinase AssaysJAK Kinase Assays

• A549, MDA-MB-468, and v-Src-A549, MDA-MB-468, and v-Src-transformed NIH3T3 cells were harvested,transformed NIH3T3 cells were harvested,

• washed three times in PBS [10 mM washed three times in PBS [10 mM sodium phosphate (pH 7.4), 137 mM NaCl, sodium phosphate (pH 7.4), 137 mM NaCl, and 1 mM sodium orthovanadate]and 1 mM sodium orthovanadate]

• lysed for 30 min on ice in JAK kinase lysis lysed for 30 min on ice in JAK kinase lysis buffer [25 mM HEPES (pH 7.4), 0.1% buffer [25 mM HEPES (pH 7.4), 0.1% Triton X-100, 0.5 mM DTT, 1 mM sodium Triton X-100, 0.5 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF, 10 _g/ml orthovanadate, 1 mM PMSF, 10 _g/ml aprotinin, and 10 _g/ml leupeptin].aprotinin, and 10 _g/ml leupeptin].

• Samples were spun at high speed to clear, and 800-1000 _g Samples were spun at high speed to clear, and 800-1000 _g of protein were immunoprecipitated per treatment condition of protein were immunoprecipitated per treatment condition with 50 ng of either JAK1 or JAK2 antibody with 50 ng of either JAK1 or JAK2 antibody

• with rocking overnight at 4°C. Twenty-five _l of protein A/G with rocking overnight at 4°C. Twenty-five _l of protein A/G PLUSagarose were then added, and rocking continued for 1 PLUSagarose were then added, and rocking continued for 1 h at 4°C. h at 4°C.

• Samples were spun to collect agarose pellet, and pellet was Samples were spun to collect agarose pellet, and pellet was washed twice in wash buffer [50 mM HEPES (pH 7.4), 0.1% washed twice in wash buffer [50 mM HEPES (pH 7.4), 0.1% Triton X-100, 0.5 mM DTT, and 150 mM NaCl]Triton X-100, 0.5 mM DTT, and 150 mM NaCl]

• and once in phosphorylation buffer [50 mM HEPES (pH 7.4), and once in phosphorylation buffer [50 mM HEPES (pH 7.4), 0.1% Triton X-100, 0.5 mM DTT, 6.25 mM manganese 0.1% Triton X-100, 0.5 mM DTT, 6.25 mM manganese chloride, and 100 mM NaCl]. Kinase reactions were chloride, and 100 mM NaCl]. Kinase reactions were performed at 30°C for 15 min in a final volume of 100 _l of performed at 30°C for 15 min in a final volume of 100 _l of phosphorylation buffer.phosphorylation buffer.

• Samples were pretreated with DMSO control, Samples were pretreated with DMSO control, JSI-124, and control compounds [AG490 (100 JSI-124, and control compounds [AG490 (100 _M) and PD180970 (2 _M)] before addition of _M) and PD180970 (2 _M)] before addition of 20 _Ci/sample [_-32P]ATP. 20 _Ci/sample [_-32P]ATP.

• The reaction was halted using stop buffer The reaction was halted using stop buffer (wash buffer _ 10 mM EDTA), samples were (wash buffer _ 10 mM EDTA), samples were spun to collect pellet, spun to collect pellet,

• then pellet was washed once with stop buffer then pellet was washed once with stop buffer and twice with wash buffer.and twice with wash buffer.

• Samples were then placed in 2_ SDS-PAGE Samples were then placed in 2_ SDS-PAGE sample buffer, boiled at 100°Csample buffer, boiled at 100°C

• run on 8% SDS-PAGE gels to run on 8% SDS-PAGE gels to separate proteins. separate proteins. AutophosphorylationAutophosphorylation

• results were visualized by results were visualized by autoradiography.autoradiography.

• Src Kinase AssaySrc Kinase Assay• In Vitro In Vitro Cellular Proliferation Cellular Proliferation

and TUNEL Assaysand TUNEL Assays

Antitumor Activity in the Antitumor Activity in the Nude Mouse Tumor Nude Mouse Tumor Xenograft Model.Xenograft Model.

• Nude mice and C57 BL-6 mice (NCI, Bethesda, Nude mice and C57 BL-6 mice (NCI, Bethesda, MD) were maintained in accordance with the MD) were maintained in accordance with the Institutional Animal Care and Use Committee Institutional Animal Care and Use Committee procedures and guidelines.procedures and guidelines.

• v-Src-transformed and oncogenic H-Ras-v-Src-transformed and oncogenic H-Ras-transformed NIH 3T3, A549, MDA-MB-468, and transformed NIH 3T3, A549, MDA-MB-468, and Calu-1 cells were harvested, resuspended in Calu-1 cells were harvested, resuspended in PBS, and injected s.c. into the right and left PBS, and injected s.c. into the right and left flank (10 _ 106 cells/flank) of 8-week-old female flank (10 _ 106 cells/flank) of 8-week-old female nude mice as reported previously (25).nude mice as reported previously (25).

• Similarly, murine B16-F10 melanoma cells were Similarly, murine B16-F10 melanoma cells were injected s.c. into the right and left flank (106 injected s.c. into the right and left flank (106 cells/flank) of C57 black mice. cells/flank) of C57 black mice.

• When tumors reached about 150 mm3, animals were When tumors reached about 150 mm3, animals were randomized (5 animals/group; 2 tumors/animal) and randomized (5 animals/group; 2 tumors/animal) and dosed i.p. with 0.2 ml vehicle of drug once daily. dosed i.p. with 0.2 ml vehicle of drug once daily. Control animals received DMSO (20%) vehicle,Control animals received DMSO (20%) vehicle,

• whereas treated animals were injected with JSI-124 whereas treated animals were injected with JSI-124 (1 mg/kg/day) in 20% DMSO in water. The tumor (1 mg/kg/day) in 20% DMSO in water. The tumor volumes were determined by measuring the length volumes were determined by measuring the length ((ll) and the width () and the width (ww) and calculating the volume () and calculating the volume (V V _ _ lwlw2/2)2/2)

RESULTSRESULTS

• Development of Phosphotyrosine STAT3-Development of Phosphotyrosine STAT3-specific Cytoblotspecific Cytoblot

• High Throughput Assay and High Throughput Assay and Identification of JSI-124.Identification of JSI-124.

• Experiments in animal models using gene Experiments in animal models using gene therapy with a dominant negative form of therapy with a dominant negative form of STAT3 and a constitutively active mutant of STAT3 and a constitutively active mutant of STAT3, as well as the prevalence of STAT3, as well as the prevalence of constitutively activated STAT3 in many constitutively activated STAT3 in many human cancers, strongly suggest STAT3 as human cancers, strongly suggest STAT3 as having a causal role in oncogenesishaving a causal role in oncogenesis

• constitutive activation of STAT3 constitutive activation of STAT3 induces genes such as cyclin D1, c-myc, induces genes such as cyclin D1, c-myc, and bcl-xl that are intimately involved and bcl-xl that are intimately involved in oncogenesis and tumor survival, in oncogenesis and tumor survival, coupled with the fact that constitutively coupled with the fact that constitutively activated STAT3 is required for survival activated STAT3 is required for survival of some human cancer cells, further of some human cancer cells, further validates the STAT3 signaling pathway validates the STAT3 signaling pathway as a selective cancer drug discovery as a selective cancer drug discovery targettarget

• we have identified JSI-124, a we have identified JSI-124, a selective JAK/STAT3 signaling selective JAK/STAT3 signaling pathway inhibitor with potent pathway inhibitor with potent antitumor activity against human antitumor activity against human tumors in nude mice, from the NCI tumors in nude mice, from the NCI Diversity Set of 1,992 compounds.Diversity Set of 1,992 compounds.

• cucurbitacin I (JSI-124) reduced the levels of cucurbitacin I (JSI-124) reduced the levels of phosphotyrosine of constitutively activated phosphotyrosine of constitutively activated STAT3 in many human cancer cell lines STAT3 in many human cancer cell lines including pancreatic, lung, and breast including pancreatic, lung, and breast carcinomas. This suppression in the levels of carcinomas. This suppression in the levels of constitutively activated STAT3 resulted in constitutively activated STAT3 resulted in blockade of STAT3 DNA-binding activity and blockade of STAT3 DNA-binding activity and STAT3-mediated gene transcription. JSI-124 STAT3-mediated gene transcription. JSI-124 was highly selective for disrupting STAT3 was highly selective for disrupting STAT3 signaling over other pivotal oncogenic and signaling over other pivotal oncogenic and tumor survival pathways.tumor survival pathways.

• JSI-124 may be more selective toward JSI-124 may be more selective toward inhibiting the growth of tumors with inhibiting the growth of tumors with constitutively activated STAT3.constitutively activated STAT3.

• Reduction in phosphotyrosine levels could be a Reduction in phosphotyrosine levels could be a result of either inhibition of protein tyrosine result of either inhibition of protein tyrosine kinases or activation of protein kinases or activation of protein phosphotyrosine phosphatases. STAT3 is phosphotyrosine phosphatases. STAT3 is known to be phosphotyrosine known to be phosphotyrosine dephosphorylated by two protein dephosphorylated by two protein phosphotyrosine phosphatases,phosphotyrosine phosphatases,

• SHP-1 and SHP-2SHP-1 and SHP-2