Effect of the JAK2/STAT3 inhibitor SAR317461 on human glioblastoma tumorspheres

Abstract

Background: The STAT3 transcription factor is a major intracellular signaling protein and is frequently dysregulated in the most common and lethal brain malignancy in adults, glioblastoma multiforme (GBM). Activation of STAT3 in GBM correlates with malignancy and poor prognosis. The phosphorylating signal transducer JAK2 activates STAT3 in response to cytokines and growth factors. Currently there are no JAK-STAT pathway inhibitors in clinical trials for GBM, so we sought to examine the anti-GBM activity of SAR317461 (Sanofi-Aventis), a newer generation, highly potent JAK2 inhibitor that exhibits low toxicity and good pharmacokinetics. SAR317461 was initially approved for patient testing in the treatment of primary myelofibrosis (PMF), and has shown activity in preclinical models of melanoma and pulmonary cancer, but has not been tested in GBM.

Methods: We hypothesized that a potent small molecule JAK2 inhibitor could overcome the heterogeneous nature of GBM, and suppress a range of patient derived GBM tumorsphere lines and immortalized GBM cell lines. We treated with SAR317461 to determine IC50 values, and using Western blot analysis we asked whether the response was linked to STAT3 expression. Western blot analysis, FACS, and cell viability studies were used to identify the mechanism of SAR317461 induced cell death.

Results: We report for the first time that the JAK2 inhibitor SAR317461 clearly inhibited STAT3 phosphorylation and had substantial activity against cells (IC50 1-10 µM) from 6 of 7 different patient GSC derived GBM tumorsphere lines and three immortalized GBM lines. One patient GSC derived line did not constitutively express STAT3 and was more resistant to SAR317461 (IC50 ≈25 µM). In terms of mechanism we found cleaved PARP and clear apoptosis following SAR317461. SAR317461 also induced autophagy and the addition of an autophagy inhibitor markedly enhanced cell killing by SAR317461.

Conclusions: We conclude that SAR317461 potently inhibits STAT3 phosphorylation and that it has significant activity against those GBM cells which express activated STAT3. Further studies are warranted in terms of the potential of SAR317461 as single and combined therapy for selectively treating human patients afflicted with GBMs expressing activation of the JAK2-STAT3 signaling axis.

Fig. 1

TCGA data show that STAT3 expression in GBM correlates with lower patient survival. a Relative STAT3 expression levels according to GBM patient risk group based on TCGA genetic profiling. Higher risk individuals had higher STAT3 expression and their expression levels are denoted by red symbols, while lower STAT3 expression that was associated with lower survival (b) and lower risk is shown in green. b Survival curves, high risk in red indicates reduced survival; the lower risk group survival is denoted by green curve. P values calculated using log rank indicate significance at the 95 % confidence level (p < 0.05). Survival analysis was censored by survival months which means even though patients may have been lost to follow-up, partial data was acquired and incorporated in the survival curves according to study month.

Fig. 2

STAT3 phosphorylation status in GBM cell lines. a Representative images of untreated tumorspheres derived from GSCs acquired from GBM tumors. The images simply illustrate the rounded conformation of healthy tumorspheres. b STAT3 phosphorylation in patient derived tumorsphere cell lines. c Immortalized adherent cell lines.

Fig. 3

Inhibitory effect of SAR317461 on the proliferation of GBM lines. GSCs and established cells from established GBM lines were seeded into each well of a 96-well micro-culture plate and SAR317461 (40–0.075 μM) was added. Each point in every curve shows the mean for three samples and the bars are standard error of the mean. The standard deviation of IC₅₀ values for 7 tumorsphere lines treated with SAR317461 IC₅₀ value is approximately 8.1 with a mean of 4.88 (4.88 ± 8.1). Hence the IC₅₀ of 25 for SK892, which does not express pSTAT3, lies more than 2 standard deviations outside this interval. The IC₅₀s for U87, A172 and U251 are 7-8 μM. a (SK1035, SK987 and SK892), b (GBM4 and GBM8), c (SK262, SK429) are IC₅₀ curves for patient derived cell lines. d IC₅₀ curve of the U87, A172 and U251 immortalized GBM cell lines. e Shows representative images of GBM8 cells treated with 10 µM SAR317461 and control solution (DMSO).

Fig. 4

SAR317461 inactivated STAT3, induced PARP cleavage to mediate apoptosis. a Western blots of phosphorylated STAT3 at Tyr705 and total STAT3, Bcl-Xl and Parp in GBM4 and GBM8 cells treated with different concentrations of SAR317461 and harvested at 16 h. GADPH loading control. b Cells were treated with the 2 µM concentrations of SAR317461 for 16 h and then subjected to flow cytometry for cell cycle analysis. i GBM4 cells treated with DMSO. ii GBM4 cells treated with 2 uM of SAR317461. iii GBM8 cells treated with DMSO. iv GBM8 cells treated with 2uM of SAR317461. Percent of cells in sub G1 phase is indicated.

Fig. 5

JAK2 inhibitor SAR317461 induced cell autophagy and cell autophagy inhibitors enhanced cell death induced by SAR317461. a Stable U251 cell line expressed GFP-LC3 induced GFP clusters with 1 µM SAR317461 treatment. b Cell viability decreased dramatically after SAR317461 and cell autophagy inhibitors. There were three replicates per condition and for the starvation control cells were cultured in media without FBS. Statistical differences between cell viability obtained in cells treated with SAR317461 with or without autophagy inhibitors were determined using a 2-tailed, paired t test. Bars indicate standard error of the mean and p values of less than 0.05 indicated statistical significance and are denoted by asterisks above the columns. c Photomicrographs showing cell morphology and density with various treatments.