The JAK2 inhibitor AZD1480 potently blocks Stat3 signaling and oncogenesis in solid tumors

Abstract

Persistent activation of Stat3 is oncogenic and is prevalent in a wide variety of human cancers. Chronic cytokine stimulation is associated with Stat3 activation in some tumors, implicating cytokine receptor-associated Jak family kinases. Using Jak2 inhibitors, we demonstrate a central role of Jaks in modulating basal and cytokine-induced Stat3 activation in human solid tumor cell lines. Inhibition of Jak2 activity is associated with abrogation of Stat3 nuclear translocation and tumorigenesis. The Jak2 inhibitor AZD1480 suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity. We demonstrate the essential role of Stat3 downstream of Jaks by inhibition of tumor growth using short hairpin RNA targeting Stat3. Our data support a key role of Jak kinase activity in Stat3-dependent tumorigenesis.

Fig.1. Janus kinase family selectivity of AZD1480

Jak family kinase selectivity was determined using both enzymatic assays and Ba/F3 cells engineered to express constitutively active Jak kinases by fusing the kinase domain of Jak1, Jak2, Jak3 and Tyk2 with the dimerization domain of TEL. A, Chemical structure of AZD1480. B, Jak1, 2 and 3 enzymatic assays were carried out in triplicate at K_m levels of ATP and 5 mM ATP. Ranges depicted represent +/− SD. C, Inhibition of Stat5 phosphorylation in Ba/F3 TEL-Jak2, TEL-Jak3, TEL-Jak1 and TEL-Tyk2 cells. TEL-Jak cells were treated with the indicated concentrations of AZD1480 for 1 h and the levels of phospho-Stat5 were determined by western immunoblotting. Signal intensity was quantified using Licor Odyssey software. IC₅₀ values were calculated from a minimum of three independent experiments. D, Inhibition of Jak1, 2, 3 and Tyk2 kinase driven cellular proliferation in engineered Ba/F3 cell lines. TEL-Jak cells were plated in 96-well plates, treated 24h later with AZD1480, and incubated for 48h. Cell proliferation was determined using the Alamar Blue assay. GI₅₀ values were calculated from a minimum of four independent experiments. Ranges depicted represent +/− SD.

Fig. 2. Jak kinase inhibition blocks Stat3 phosphorylation in a panel of human solid tumor cell lines

Cells were treated with either DMSO (V - vehicle control, 0.01%), Jak2 inhibitor AZ960 (0.3 µM), Src inhibitor Dasatinib (0.3 µM), EGFR inhibitor Gefitinib (1.0 µM) or Met inhibitor PF-2341066 (0.3 µM) for 1 h and cell lysates were probed for pStat3^Tyr705 and Stat3 by western blot. A, Representative western immunoblots showing decreased pStat3^Tyr705 in cells treated with a Jak2 inhibitor. Hep3B cells were either stimulated with IL-6 (10 ng/ml) for 30 min or pretreated with the indicated inhibitor for 30 min and then co-treated with IL-6 for an additional 30 min. B, Color-coded table of cell lines screened for Stat3 phosphorylation. Drug treatments that resulted in a decrease in Stat3 phosphorylation are shown in green, those which did not modulate pStat3 are shown in red.

Fig. 3. Inhibition of Jak2 blocks nuclear translocation of Stat3

A, Inhibition of OSM-stimulated Stat3 phosphorylation. Western blot of whole cell lysates prepared from OSM-stimulated MEF-Stat3-YFP cells treated with the indicated concentrations of AZD1480. Cells were pretreated with AZD1480 for 2 h, and were then stimulated with 25 ng/mL OSM for 30 min. B, Inhibition of Stat3 nuclear translocation. OSM stimulated MEF-Stat3-YFP cells were treated with the indicated concentrations of AZD1480, then stained with DAPI and visualized by confocal microscopy. Blue depicts the nucleus and yellow depicts localization of Stat3 protein. The bar represents a distance of 10µm C, Quantification of nuclear translocation of Stat3 of the images in panel B, method described in text. Error bars represent SD amongst the data obtained from five cells in each cohort of the experiment outlined in panel B.

Fig. 4. Jak2 inhibition suppresses Stat3 mediated tumorigenesis

A, Expression of Stat3 promotes formation of MEF tumors in mice. Tumor growth of MEF cells (grey line), and MEF-Stat3-YFP cells (black line). Results presented as mean tumor volume (n=8), error bars represent SE and P (t-test) at day 15. B, Treatment of MEF-Stat3-YFP tumor bearing mice, once daily with 50 mg/kg of AZD1480 (p.o.) (black line) or vehicle-treated control (grey line). Mice were dosed on a repeating schedule of 5 continuous d of once daily dosing, followed by 2 d of rest, for 24 d. Arrow indicates initiation of treatment. Results are presented as mean tumor volume (n=6), SE, and P (t-test) at day 33 is indicated. C, AZD1480 inhibits pStat3 activity in MEF-Stat3-YFP tumors. Western blot of tumors following the efficacy study outlined in Panel B. Tumors were harvested 2 h post treatment. D, Intravital multiphoton laser microscopy of Stat3 subcellular localization in a tumor. Two photon imaging of intact MEF-Stat3-YFP tumors treated with AZD1480 at 50 mg/kg for 2 weeks, as described above or vehicle alone. Tumors were visualized 2 h after treatment. Stat3 was visualized real time through exciting the YFP fluorophore tethered to the Stat3 protein. Individual fluorescence channels are presented in the vehicle-treated and AZD1480 treated tumors. Merged images in the control tumor display nuclear localization of Stat3 as depicted by the white dots in the field, created by an overlap of the nuclei and Stat3 emissions. Merged images of the treated tumor depict areas of Stat3 fluorescence that do not overlap with nuclei fluorescence, indicating cytoplasmic localization of Stat3. The bar represents a distance of 100 µM.

Fig. 5. Jak2 mediates IL-6 dependent survival in human prostate cancer cells

A, Stat3 activity was assayed by western blot in LN-17 cells treated for 24 h with the indicated concentrations of AZD1480. B, EMSA to visualize Stat3 DNA binding post treatment with a Jak2 inhibitor. LN-17 cells were treated as in panel A and EMSA was performed to monitor Stat3 homodimer DNA binding activity. The first lane is the control treated sample incubated with a Stat3 blocking antibody to confirm Stat3 binding. C, Cell viability was assayed with an MTS assay 72 h post treatment with the indicated concentrations of AZD1480. Error bars represent the SD of three experiments conducted independently (~ indicates P < 0.02, * indicates P < 0.01). D, Flow cytometric analysis of LN-17 cells stained with Annexin V 72 h post treatment with the indicated concentrations of AZD1480. Error bars represent the SD amongst triplicate samples (* indicates P < 0.01). E, Apoptosis was assayed by western blot with an antibody against the cleaved 85kD PARP fragment in LN-17 cells treated with the indicated concentrations of AZD1480 for 72 h. F, Two siRNAs (300 nM) targeting Jak2 were transfected using the Amaxa Nucleofector system and were harvested 48 h later. Cell lysates were immunoblotted with indicated antibodies. A non-silencing (NS) siRNA was used as a negative control.

Fig. 6. Jak kinase inhibition abrogates IL-6 induced and constitutive Stat3 activity of human cancer cells

A, MDAH2774 cells express IL-6 and IL-6R. Culture medium and cell lysates were collected from MDAH2774 cells following 1, 2, and 3 d of culture, and tumor lysates were collected from mice bearing MDAH2774 xenografts (n=7). Human IL-6 was measured by Luminex Immunoassay using the Milliplex cytokine kit (Millipore). For culture medium and cell lysate, error bars denote the SD for each sample measured in duplicate. For tumor lysate, samples were measured in triplicate and the error bar denotes the SD of the mean measurements. IL-6R was assayed in three individual tumor lysates by western blot. B, AZD1480 inhibits constitutive Stat3 activity in human cancer cells. Western blot of whole cell lysates of DU145, MDA-MB-468, and MDAH2774 cells prepared 2 h post treatment with the indicated concentrations of AZD1480. C, Western blot analysis of whole cell lysates prepared from DU145, MDA-MB-468, and MDAH2774 cells which were starved in 5% CS-FBS for 18 h (DU145 and MDA-MB-468), or serum-starved for 3 h (MDAH2774), and then treated with the indicated concentrations of AZD1480 for 2 h. Cells were then stimulated with 10 ng/mL IL-6 for 15 min following the pretreatment with AZD1480. D, Jak1 siRNA blocks phosphorylation of Stat3 in MDAH2774 cells. Jak1-siRNAs, Jak2-siRNAs or Tyk2-siRNAs (100 nM) were transfected into MDAH2774 cells using the Amaxa Nucleofector system and were harvested 24 h and 48 h later. Cell lysates were immunoblotted with indicated antibodies. GAPDH siRNA was used as a negative control.

Fig. 7. AZD1480 suppresses the growth of human xenograft tumors harboring constitutive Stat3 activity

A, DU145 and MDA-MB-468 tumor-bearing mice were treated once daily with AZD1480 (black line) at 50 mg/kg (p.o.), or vehicle (grey line), for 5 d, followed by 2 d of rest, over a total course of treatment of 26 d (DU145) or 30 d (MDA-MB-468). Arrow indicates initiation of treatment. Results are presented as mean tumor volume (n=7), SE and P (t-test) at day 37 (DU145) or day 57 (MDA-MB-468) is indicated. MDAH2774-bearing mice were treated twice daily with 1, 10 and 30 mg/kg (p.o.) for 26 consecutive days. Arrow indicates initiation of therapy. Results presented as mean tumor volume, bars indicate SE and P value (t-test) at day 44 is indicated. B, Western blot analysis of tumors 2 h post treatment with AZD1480. DU145 tumors were obtained after 26 d of treatment with 50 mg/kg AZD1480, MDA-MB-468 tumors after 30 d of treatment with 50 mg/kg AZD1480, and MDAH2774 tumors were exposed to only a single dose of 30 mg/kg AZD1480.

Fig. 8. Expression of Stat3 shRNA inhibits the growth of human breast cancer cells

A, Western blot of whole-cell lysates derived from MDA-MB-468 cells grown in culture expressing Stat3 shRNA1 or vector alone. B, Reduction of Stat3 expression results in inhibition of MDA-MB-468 tumor growth. The growth of MDA-MB-468 subcutaneous xenografts expressing either Stat3 shRNA1 or vector alone control were monitored for 58 d. Results are presented as mean tumor volume (n=6) with SE, and P (t-test) at day 58 is indicated. C, Western blot of whole-cell lysates derived from MDA-MB-468 cells grown in culture expressing Stat3 shRNA2 or non-silencing (NS) control shRNA. D, The growth of MDA-MB-468 subcutaneous xenografts expressing either Stat3 shRNA2 or NS shRNA were monitored for 56 d. Results are presented as mean tumor volume (n=6) with SE, and P (t-test) at day 56 is indicated.