JAK2; STAT3

The proliferation of HEL cells was greatly suppressed in a dose-dependent manner by WP10666. 2.3 μM is the IC50 value for suppressing HEL cell growth. Human HEL cells with the JAK2 V617F mutant isoform cannot proliferate when WP1066 is present [1]. When WP1066 was used to block p-STAT3, the cytotoxic effect of CTX on tumors was increased. On B16 cells, WP1066's IC50 dosage is 2.43 μM (0.865 μg/mL)[2]. At high concentrations, WP1066 suppresses the growth of normal BM progenitor cells, suppresses the proliferation of AML blast colony-forming cells, and suppresses the proliferation of AML colony-forming cells [3].

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We found that WP1066 significantly inhibited JAK2 and its downstream signal transducer and activator of transcription-3, signal transducer and activator of transcription-5, and extracellular signal-regulated kinase-1/2 pathways in a dose- and time-dependent manner. As a result, WP1066 concentrations in the low micromolar range induced time- and dose-dependent antiproliferative and proapoptotic effects in HEL cells. As expected, WP1066 inhibited the proliferation of peripheral blood hematopoietic progenitors of patients with polycythemia vera carrying the JAK2 V617F mutation in a dose-dependent manner. Conclusions: Our data suggest that WP1066 is active both in vitro and ex vivo and should be further developed for the treatment of neoplasms expressing the JAK2 V617F mutation. [1]

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Blockade of p-STAT3 with WP1066 enhances the cytotoxic effects of CTX on the tumor [2]
To determine if CTX and WP1066 exert an additive effect on direct tumor cytotoxicity, the IC50 of the agents, both individually and in combination, were evaluated on the melanoma cell line B16. The IC50 doses of WP1066 and CTX for B16 cells were 2.43 µM (0.865 µg/ml) and 4.04 mM (1.128 mg/ml), respectively. Using the IC50 dose of WP1066 (2 µM), the addition of CTX enhanced tumor cytotoxicity (Fig. 1A).
B16 has both constitutive activation of p-STAT3 (Fig. 1B) and inducible activity36. To ascertain whether CTX exerts an additive effect with WP1066 on the inhibition of p-STAT3, B16 cells were cultured in the absence or presence of WP1066, CTX, or both, and the levels of p-STAT3, total STAT3, and β-actin evaluated by Western blot analysis. Inhibition of p-STAT3 by WP1066 was enhanced with CTX in a dose-dependent manner in the B16 cells. Specifically, the combination of either 0.75 or 2.5 mg/ml of CTX with 2.5 µM of WP1066 has a similar inhibitory effect on pSTAT3 as 5 µM of WP1066 alone. This would suggest that CTX can enhance the inhibition of p-STAT3 in conjunction with WP1066 but cannot exert direct inhibition of p-STAT3 at these doses (Fig. 1B).

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Because (E)-3(6-bromopyridin-2-yl)-2-cyano-N-((S0-1-phenylethyl)acrylamide) (WP1066) is a novel analogue of the JAK2 inhibitor AG490, we tested its activity in AML cells and investigated its mechanism of action. Using clonogenic assays, we found that although WP1066 had a marginal effect on normal marrow progenitors, it inhibited the proliferation of AML colony-forming cells obtained from patients with newly diagnosed AML and that of the AML cell lines OCIM2 and K562. WP1066 inhibited OCIM2 cell multiplication by inducing accumulation of cells at the G0-G1 phase of the cell cycle. Similar to its parent compound AG490, WP1066 inhibited the phosphorylation of JAK2, but unlike AG490, WP1066 also degraded JAK2 protein, thereby blocking its downstream signal transducer and activator of transcription (STAT) and phosphoinositide-3-kinase pathways. These effects resulted in the activation of the caspase pathway. Incubation of both OCIM2 and K562 cells with WP1066 activated caspase-3, induced cleavage of poly(ADP-ribose) polymerase, and caused caspase-dependent apoptotic cell death. Thus, WP1066 is a potent JAK2 inhibitor whose effects in AML and other hematologic malignancies merit further investigation. [3]

In the tumor microenvironment, WP1066 (30 mg/kg, og) had additive effects on CTX-induced p-STAT3 pathway inhibition [2].

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WP1066 does not further enhance the therapeutic effects of cyclophosphamide on pulmonary melanoma lesions [2]
To determine whether CTX and WP1066 show additive efficacy against melanoma within the lung, metronomic CTX (o.g.), cytotoxic CTX (i.p.) or WP1066 (o.g.) were administered alone or in conjunction with one another in C57BL/6J mice who had pulmonary melanoma. In an attempt to observe an additive effect of STAT3 inhibition and CTX, a subtherapeutic dose (30 mg/kg) of WP1066 was used. The number of melanoma lesions in untreated tumor-bearing mice was 26.8 ± 11.3. Although there was no statistical difference in development of lung lesions between mice treated with the subtherapeutic WP1066 dose (33 ± 16.1) and the untreated mice (P> 0.05), administration of CTX by both metronomic and cytotoxic methods reduced the number of pulmonary lesions compared with the control (7 ± 3.5 P= 0.049 and 0.7 ± 0.6; P= 0.01, respectively). Reduction in the number of melanoma lesions was even more significant using cytotoxic CTX dosing than using metronomic CTX dosing (P= 0.03). However, no additive effect was observed in combinational therapy groups compared with groups treated alone with either metronomic (7.8 ± 3.6) or cytotoxic (0.5 ± 0.6) CTX dosing (Fig. 2).

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WP1066 does enhance the therapeutic effects of cyclophosphamide against CNS melanoma [2]
To determine whether combinational therapy of metronomic CTX dosing and WP1066 yields an additive benefit against established CNS tumors, C57BL/6J mice with intracerebral B16 were treated as previously described. The median overall survival time for tumor-bearing mice treated with the WP1066 vehicle control or PBS was 16 days (range, 13–21 days; n=10). No improvement in the median survival time was observed with the subtherapeutic WP1066 dose alone (15 days; range, 10–18 days; n= 7; P= 0.29) or metronomic (o.g.) dosing of CTX alone (18 days; range 17–36 days; n=10; P= 0.1) compared with the control. The median overall survival time of 21 days (range, 17–75 days; n=10) was slightly longer for mice with intracerebral melanoma treated with the subtherapeutic WP1066 dose in combination with metronomic CTX dosing, and this was statistically significant compared with vehicle control mice (P=0.004) and those treated with WP1066 (P<0.0001) alone but not with those treated with metronomic CTX dosing alone (P= 0.13) (Fig. 3A).

We next determined the effects of cytotoxic CTX dosing in combination with WP1066. The median overall survival time for tumor-bearing mice treated with the WP1066 vehicle control or PBS was 17 days (range, 13–17 days; n=9). No improvement in the median survival time was observed with the subtherapeutic WP1066 dose alone (17.5 days; range, 14–21 days; n= 8; P= 0.10). Survival was significantly enhanced by cytotoxic CTX monotherapy relative to the vehicle control (32 days; range, 24–108 days; P< 0.0001). The median survival time of mice with intracerebral melanoma treated with a subtherapeutic WP1066 dose and cytotoxic CTX dosing was 120 days (range, 26–127 days; n=7), which was significantly longer than for mice treated using cytotoxic CTX dosing alone (P= 0.03), WP1066 (P=0.0001), and the controls (P=0.0002) (Fig. 3B). For the mice treated with the combined therapy of WP1066 and cytotoxic CTX dosing, 57% survived long term, and there was at least a 375% increase in median survival time compared with the cytotoxic CTX alone group when the experiment was terminated to perform the tumor rechallenge experiments. At the time of death, CNS autopsy demonstrated there were no significant differences in tumor burden between treatment groups. Death was usually secondary to leptomeningeal spread of B16 tumor and secondary hydrocephalus. This would preclude measuring tumor size and harvesting for Western Blot analysis.

To determine whether mice with intracerebral tumors treated with both WP1066 and cytotoxic or metronomic dosing of CTX were able to generate long-lasting protective immune memory, mice were rechallenged with B16 cells implanted in the contra lateral hemisphere. Upon rechallenge, mice treated with the combination of the subtherapeutic WP1066 dose in conjunction with either metronomic or cytotoxic CTX dosing, had median survival times of 17 and 21 days, respectively, which did not differ significantly from the median survival time of naïve, control mice. We did not see long lasting immunity after intracerebral rechallenge experiment in any treatment groups.

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WP1066 does not further enhance immune-mediated cytotoxic effects of CTX [2]
To determine if there was an enhancement of immunological tumor cytotoxicity correlating with the therapeutic in vivo efficacy of the combinational therapy, we evaluated immune cytotoxic responses directed toward B16 melanoma cells. Splenocytes from non-tumor-bearing mice, and tumor-bearing mice treated with WP1066, metronomic (o.g.) CTX dosing, cytotoxic (i.p.) CTX dosing, or a combination of WP1066 with CTX were isolated and co-cultured with CFSE-labeled B16 target cells for 48 h to assess immune cytotoxicity toward B16 cells. Immune-mediated cytotoxicity decreased by 37% in tumor-bearing mice compared with non-tumor-bearing controls (P =0.008). The immune cells including CD3+ T cells, natural killer (NK) cells and NKT cells (splenocytes) from the tumor-bearing mice treated with WP1066, metronomic CTX dosing, and cytotoxic CTX dosing increased cytotoxic clearance of the B16 target cells by 30%, 77% and 69%, respectively, relative to tumor-bearing control mice (P = 0.05, P = 0.001, and P= 0.006, respectively). However, combinational therapy of WP1066 with either metronomic or cytotoxic CTX dosing did not significantly enhance immune-mediated cytotoxic clearance of the B16 target cells in comparison with monotherapy (Fig. 4A).

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WP1066 does not further modulate the influence of CTX on the immune cell populations [2]
To determine the in vivo effects of metronomic (o.g.) CTX dosing, cytotoxic (i.p.) CTX dosing, WP1066 administration, and combinational therapy on the number of CD8+, CD4+, and CD4+Foxp3+ Tregs, tumor-bearing mice were treated for 14 days as described above. Metronomic CTX dosing enhanced the number of CD8+ (P= 0.007) (Fig. 4B) and CD4+ T cells (P= 0.04) (Fig. 4C) and inhibited the number of Tregs within the CD4 compartment (P<0.05) (Fig. 4D) compared with the control tumor-bearing mice. The combination of metronomic CTX dosing and subtherapeutic WP1066 dosing did not further enhance the number of CD8+ (Fig. 4B) or CD4+ T cells (Fig. 4C) or further inhibit the number of CD4+FoxP3+ Tregs in the peripheral blood (P > 0.05) (Fig. 4D). In contrast, cytotoxic CTX dosing significantly suppressed CD8+ T cells (P< 0.0001) (Fig. 4B), CD4+ T cells (P= 0.0005) (Fig. 4C), and CD4+FoxP3+ Tregs (P=0.0004) (data not shown) compared with the tumor-bearing control mice and this was not further modulated with WP1066 (P> 0.05).

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WP1066 exerts an additive effect to CTX inhibition of the p-STAT3 pathway within the tumor microenvironment [2]
To clarify why there was an additive effect of CTX with WP1066 in the efficacy experiments against melanoma within the CNS but to a much lesser degree than in the lung, we investigated the levels of pSTAT3 within the local tumor microenvironments and their modulation with combination therapy. The intranuclear p-STAT3 expression within the melanomas in each group was measured using immunohistochemistry and expressed as the percentage of p-STAT3-positive cells. The presence of cytoplasmic melanin was not included in the analysis. In the CNS melanoma of control mice, p-STAT3 expression was 12.7 ± 2.3% (range 2.8–21.8%; n = 8). This was diminished to 9.2 ± 2.8% (range 2.3–18.7%; n = 9; P=0.170) in mice treated with sub-therapeutic WP1066. Metronomic CTX did not affect p-STAT3 expression at 12.7 ± 3.7% (range 4.6–37.2%; n = 8) p-STAT3 expression. In mice with CNS tumors treated with cytotoxic CTX, the p-STAT3 level was 4.5 ± 1.2% (range 1.9–10.7%; n = 7; P=0.011) and this was further reduced in combination with WP1066 to 2.3 ± 0.8% (range 0–6.5%; n = 7; P=0.001) (Fig. 5A). In marked contrast, in the control, established lung melanoma, the p-STAT3 expression was low at 2.4 ± 0.6% (range 0–3.7%; n = 5). Mice treated with sub-therapeutic WP1066 and metronomic CTX had 2.0 ± 0.4% (range 1.4–2.7%; n = 4) and 1.7 ± 0.7 % (range 0.8–3%; n =5) p-STAT3 expression, respectively. Secondary to the already low levels of p-STAT3, the addition of WP1066 did not provide any further additive effect to CTX in modulating p-STAT3 inhibition in lung tumor model (Fig. 5B). Although a significant difference could be found in the p-STAT3 expression in tumors between the brain and the lung in animals treated with the metronomic CTX, a determination of p-STAT3 levels in the cytotoxic CTX group could not be used to provide secondary validation due to the low incidence of lung tumors (i.e. marked efficacy of cytotoxic CTX in the lung). The numbers of tumor infiltrating macrophage/microglia and CD8 T cells in the tumor microenvironment between treatment groups was not significant (data not shown).

Growth inhibition assay. [1]
The 3,[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium (MTT) assay was done using an MTT-based cell proliferation/cytotoxicity assay system. Briefly, fresh low-density peripheral blood cells and various cell lines at the logarithmic phase of their growth were washed twice in RPMI 1640 containing 10% FCS and counted in a hemocytometer. Cell viability was assessed by the trypan blue (0.1%) staining method. Equal numbers of viable cells (5 × 104 per well) were incubated in a total volume of 100 μL of RPMI 1640 supplemented with 10% FCS alone or with WP1066 at increasing concentrations; the incubations were continued for up to 72 h in 96-well flat-bottomed plates at 37°C in a humidified 5% CO2 atmosphere. Experiments for each condition were done in triplicate. After incubation, 20 μL of CellTiter96 One Solution Reagent were added to each well. The plates were then incubated for an additional 60 min at 37°C in a humidified 5% CO2 atmosphere. Immediately after incubation, absorbance was read using a 96-well plate reader at a wavelength of 490 nm.

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Annexin V and propidium iodide staining. [1]
After treatment with v for 24, 48, and 72 h, cells were washed with PBS and resuspended in 100 μL of binding buffer [10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (pH 7.4), 0.15 mol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, and 1.8 mmol/L CaCl2], to which Annexin V-fluoroisothiocyanate had been added. Cells were then incubated for 15 min in the dark at room temperature. After incubation, cells were washed and then resuspended in 0.2 mL of binding buffer.

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Measurement of mitochondrial transmembrane potential. [1]
After treatment with WP1066 for up to 72 h, cells were incubated with submicromolar concentrations of MitoTracker probes to evaluate changes in the potential of the mitochondrial membrane. MitoTracker probes passively diffuse across the plasma membrane and accumulate in mitochondria. Cells were stained with MitoTracker Red CMXRos and MTGreen FM. Briefly, cells were washed in Ca2+-free PBS, stained with MitoTracker dyes, and incubated for 1 h at 37°C in the dark. CMXRos and MTGreen are incorporated into mitochondria by force of the mitochondrial membrane potential and react with thiol residues to form covalent thiol ester bonds.

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Cell cycle analysis. [3]
Cell cycle analysis was done according to standard protocols. Briefly, 5 × 106 cells were pelleted after being incubated with WP1066. The cell pellets were washed and resuspended in 2 mL of 1% paraformaldehyde in PBS. Cells were incubated with or without WP1066 for 15 min at 4°C and then washed in PBS, resuspended in 2 mL of absolute ethanol, and stored at −20°C until they were needed for staining. The stored cells were then washed twice in PBS, resuspended in 0.5 mL of propidium iodide staining buffer (50 μg/mL propidium iodide and 10 μg/mL RNase in PBS), and incubated for 1 h at room temperature in total darkness. Flow-cytometric analysis was done using a FACSCalibur flow cytometer and the CellQuest software program. Data analysis was done using the CellQuest and ModFit LT software programs.

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Annexin V assay for detection of apoptosis. [3]
To quantify the cells undergoing apoptosis, the annexin V–FITC assay was used as described previously. Briefly, WP1066-treated OCIM2 and K562 cells were washed twice with cold PBS and then resuspended in binding buffer [10 nmol/L N-(2-hydroxyethylpiperazine)-N′-2-ethanesulfonic acid, 140 nmol/L NaCl, and 5 nmol/L CaCl2 (pH, 7.4)] at a concentration of 1 × 106 cells/mL. After incubation, 100 μL of each solution was transferred to a 5-mL culture tube, to which 5 μL of annexin V–FITC was added. The tubes were gently vortexed and incubated for 15 min at room temperature in total darkness. At the end of the incubation, 400 mL of binding buffer was added to each tube, and the cells were analyzed immediately using flow cytometry with a FACSCalibur flow cytometer and the CellQuest software program. Data analysis was done using the CellQuest and ModFit LT (2.0) software programs. To determine whether WP1066-induced apoptosis was caspase dependent, OCIM2 cells were preincubated with 20 μL of the pan-caspase inhibitor Z-VAD-FMK, and apoptotic cells were detected using the annexin V assay as described above.

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Cell survival assay [2]
B16 cells were seeded at a density of 2,000 cells per well in 96-well culture plates and were treated with WP1066 at increasing concentrations of 0, 0.156, 0.313, 0.625, 1.25, 2.5, and 5.0 µM or at CTX concentrations of 0, 0.156 mg/ml (0.559 mM), 0.313 mg/ml (1.121 mM), 0.625mg/ml (2.239 mM), and 1.25 mg/ml (4.478 mM). The WP1066 diluent DMSO was used at a final concentration of 0.05% including as a control with the CTX. After 72 h of treatment, 25 µl of 5 mg/ml dimethyl thiazolyl diphenyl tetrazolium salt solution were added to each well, and the cells were cultured for 3 h at 37° C in a humidified atmosphere of 5% CO2 and 95% air. The cells were lysed with 100 µl/well of lysing buffer (50% dimethylformamide, 20% sodium dodecyl sulfate [SDS], pH 5.6) and incubated at room temperature overnight. Cell viability was evaluated by reading the O.D. at 570 nm, and the IC50 was calculated.

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Immunoblotting analysis [2]
B16 cells were seeded at a density of 2 × 106 cells/well in 6-well culture plates and incubated overnight in RPMI medium at 37° C in an atmosphere containing 5% CO2. Afterwards, B16 cells were cultured in the absence or presence of WP1066 (2.5 µM, 5 µM), CTX [0.75 mg/ml (below the IC50), 1.5 mg/ml (above the IC50], or the combination of 2.5 µM of WP1066 with CTX (0.75 mg/ml, 1.5 mg/ml) for 2 hours. Afterwards, B16 cells were pelleted and rinsed with ice-cold PBS at 1500 rpm for 5 minutes then placed for 30 minutes in ice-cold lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA) containing 1% Triton-X-100 plus phosphatase and protease inhibitors. The lysates were centrifuged at 14,000 rpm for 10 minutes at 4° C. The supernatants were collected and their protein content was quantified. Equal amounts of protein (65 µg) were electrophoretically fractionated in 8% polyacrylamide gels containing SDS, transferred to nitrocellulose membranes, and subjected to immunoblot analysis with specific antibodies against p-STAT3 (Tyr705), STAT3, and β-actin.

Metronomic dosing of CTX, delivered o.g., was at a dose of 20 mg/kg every day (weekends off) until euthanasia/death or for a maximum of 3 weeks (whichever came first) in the intracerebral model and for 2 weeks in the pulmonary model. Cytotoxic CTX treatment, delivered i.p., was administered as two weekly cycles separated by a 1-week interval for the intracerebral model and for one cycle in the pulmonary model. Each cycle consisted of a total of three doses of CTX (150 mg/kg/per dose) administered every other day (total dose of 450 mg/kg, maximal tolerated dose)16. Since greater than 80% of mice with intracerebral B16 melanoma treated with WP1066 at 40 mg/kg survive long-term20, a subtherapeutic dose of 30 mg/kg of WP1066 was used so an additive/synergistic effect with CTX could be ascertained. WP1066 was administered via o.g. in a vehicle of DMSO/polyethylene glycol (PEG) 300 (1:4 ratio) on a once every other day schedule for 9 treatments (on Mondays, Wednesdays, and Fridays). DMSO/PEG 300 vehicle alone was used for the negative control group. [2]

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Determination of inhibition of immune cells in vivo [2]
To ascertain the inhibition of the immune populations within the spleen and peripheral blood compartments, tumor-bearing mice were treated with CTX, WP1066, or CTX in combination with WP1066, for 14 days as described above. Single-cell suspensions were prepared from spleens and the peripheral blood of mice and single cells were surface-stained with FITC-conjugated anti-CD4 (L3T4) or PE-conjugated anti-CD8 (53-6.7) and were intracellularly stained with APC-conjugated-FoxP3 . The cell number of CD4+ and CD8+ T cells in the peripheral blood was counted based on positive surface staining of the respective markers relative to the total cell count of PBMCs. The percentage of FoxP3+ Tregs was calculated within the peripheral blood and within the CD4 compartment as previously described.

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Dissolved in DMSO:polyethylene glycol 300 (20:80); 40 mg/kg; Oral gavage

Caki-1 xenograft mice

[1]. WP1066, a novel JAK2 inhibitor, suppresses proliferation and induces apoptosis in erythroid human cells carrying the JAK2 V617F mutation. Clin Cancer Res, 2008, (3), 788-796.

[2]. The tumor microenvironment expression of p-STAT3 influences the efficacy of WP1066 in murine melanoma models. Int J Cancer, 2012, 131(1), 8-17.

[3]. WP1066 disrupts Janus kinase-2 and induces caspase-dependent apoptosis in acute myelogenous leukemia cells. Cancer Res, 2007, 67(23), 11291-11299.

WP1066 has been used in trials studying the treatment of Melanoma, Brain Cancer, Solid Tumors, and Central Nervous System Neoplasms.
STAT3 Inhibitor WP1066 is an orally bioavailable, small molecule inhibitor of signaling transducer and activator 3 (STAT3), with potential antineoplastic and immunomodulatory activities. Upon administration, STAT3 inhibitor WP1066 blocks the intranuclear translocation of p-STAT, thereby suppressing STAT3 signaling and decreasing the levels of downstream products including c-Myc. Additionally, WP1066 may upregulate costimulatory molecules including CD80 and CD86 on human microglia, and reverse glioma cancer stem cell (gCSC)-mediated innate and adaptive immune suppression allowing for the restoration of antitumor effector immune responses. The STAT3 pathway is overly active in many cancer types and is implicated in CSC-mediated growth, recurrence and resistance to conventional chemotherapies.

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Purpose: The discovery of an activating somatic mutation in codon 617 of the gene encoding the Janus kinase (JAK)-2 (JAK2 V617F) in patients with myeloproliferative disorders has opened new avenues for the development of targeted therapies for these malignancies. However, no effective JAK2 inhibitors are currently available for clinical use. Experimental design: We investigated the activity of (E)-3(6-bromopyridin-2-yl)-2-cyano-N-(S0-1phenylethyl)acrylamide (WP1066), a novel analogue of the JAK2 inhibitor AG490, in JAK2 V617F-positive erythroleukemia HEL cells and in blood cells from patients with polycythemia vera. Results: We found that WP1066 significantly inhibited JAK2 and its downstream signal transducer and activator of transcription-3, signal transducer and activator of transcription-5, and extracellular signal-regulated kinase-1/2 pathways in a dose- and time-dependent manner. As a result, WP1066 concentrations in the low micromolar range induced time- and dose-dependent antiproliferative and proapoptotic effects in HEL cells. As expected, WP1066 inhibited the proliferation of peripheral blood hematopoietic progenitors of patients with polycythemia vera carrying the JAK2 V617F mutation in a dose-dependent manner. Conclusions: Our data suggest that WP1066 is active both in vitro and ex vivo and should be further developed for the treatment of neoplasms expressing the JAK2 V617F mutation.[1]

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Melanoma is a common and deadly tumor that upon metastasis to the central nervous system (CNS) has median survival duration of less than 5 months. Activation of the signal transducer and activator of transcription 3 (STAT3) has been identified as a key mediator that drives the fundamental components of melanoma. We hypothesized that WP1066, a novel inhibitor of STAT3 signaling, would enhance the antitumor activity of cyclophosphamide (CTX) against melanoma, including disease within the CNS. The mechanisms of efficacy were investigated by tumor- and immune-mediated cytotoxic assays, in vivo evaluation of the reduction of regulatory T cells (Tregs) and by determining intratumoral p-STAT3 expression by immunohistochemistry. Combinational therapy of WP1066, with both metronomic and cytotoxic dosing of CTX, was investigated in a model system of systemic and intracerebral melanoma in syngeneic mice. Inhibition of p-STAT3 by WP1066 was enhanced with CTX in a dose-dependent manner. However, in mice with intracerebral melanoma, the greatest therapeutic benefit was seen in animals treated with cytotoxic CTX dosing and WP1066, whose median survival time was 120 days, an increase of 375%, with 57% long-term survivors. This treatment efficacy correlated with p-STAT3 expression levels within the tumor microenvironment. The efficacy of the combination of cytotoxic dosing of CTX with WP1066 is attributed to the direct tumor cytotoxic effects of the agents and has the greatest therapeutic potential for the treatment of CNS melanoma. [2]

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Several cytokines and growth factors that stimulate the proliferation of acute myelogenous leukemia (AML) cells transduce their signals by activating the transcription factor Janus-activated kinase 2 (JAK2). Accordingly, the inhibition of JAK2 or of its downstream signaling pathways suppresses the proliferation of AML cells. Because (E)-3(6-bromopyridin-2-yl)-2-cyano-N-((S0-1-phenylethyl)acrylamide) (WP1066) is a novel analogue of the JAK2 inhibitor AG490, we tested its activity in AML cells and investigated its mechanism of action. Using clonogenic assays, we found that although WP1066 had a marginal effect on normal marrow progenitors, it inhibited the proliferation of AML colony-forming cells obtained from patients with newly diagnosed AML and that of the AML cell lines OCIM2 and K562. WP1066 inhibited OCIM2 cell multiplication by inducing accumulation of cells at the G(0)-G(1) phase of the cell cycle. Similar to its parent compound AG490, WP1066 inhibited the phosphorylation of JAK2, but unlike AG490, WP1066 also degraded JAK2 protein, thereby blocking its downstream signal transducer and activator of transcription (STAT) and phosphoinositide-3-kinase pathways. These effects resulted in the activation of the caspase pathway. Incubation of both OCIM2 and K562 cells with WP1066 activated caspase-3, induced cleavage of poly(ADP-ribose) polymerase, and caused caspase-dependent apoptotic cell death. Thus, WP1066 is a potent JAK2 inhibitor whose effects in AML and other hematologic malignancies merit further investigation. [3]

C17H14BRN3O

356.22

355.032

C, 57.32; H, 3.96; Br, 22.43; N, 11.80; O, 4.49

857064-38-1

11210478

Off-white to light yellow solid powder

1.4±0.1 g/cm3

569.9±50.0 °C at 760 mmHg

298.5±30.1 °C

0.0±1.6 mmHg at 25°C

1.637

2.95

1

3

4

22

462

1

C[C@@H](C1=CC=CC=C1)NC(=O)/C(=C/C2=NC(=CC=C2)Br)/C#N

VFUAJMPDXIRPKO-LQELWAHVSA-N

InChI=1S/C17H14BrN3O/c1-12(13-6-3-2-4-7-13)20-17(22)14(11-19)10-15-8-5-9-16(18)21-15/h2-10,12H,1H3,(H,20,22)/b14-10+/t12-/m0/s1

(S,E)-3-(6-bromopyridin-2-yl)-2-cyano-N-(1-phenylethyl)acrylamide

WP-1066; WP 1066; WP1066; (S,E)-3-(6-Bromopyridin-2-yl)-2-cyano-N-(1-phenylethyl)acrylamide; (E)-3-(6-bromopyridin-2-yl)-2-cyano-N-[(1S)-1-phenylethyl]prop-2-enamide; UNII-63V8AIE65T; DTXSID50235007; ...; 857064-38-1; WP1066

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)