Effect of the JAK2/STAT3 inhibitor SAR317461 on human glioblastoma tumorspheres.
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.
Translational Neuro-Oncology Laboratories, Moores Cancer Center, UC San Diego, 3855 Health Sciences Drive, MC#0819, La Jolla, CA, 92093-0819, USA. rmukthavaram@ucsd.edu.|Department of Neurosciences, UC San Diego, La Jolla, CA, USA. rmukthavaram@ucsd.edu.
Rajesh
R
Mukthavaram
Department of Orthopedic Surgery, Xuzhou 3rd Hospital, Affiliated Hospital of Jiangsu University, No. 131 Huancheng Road, 221005, Xuzhou, China. 15162100079@163.com.
Xiao
X
Ouyang
Translational Neuro-Oncology Laboratories, Moores Cancer Center, UC San Diego, 3855 Health Sciences Drive, MC#0819, La Jolla, CA, 92093-0819, USA. Rsaklech@gmail.com.
Rohit
R
Saklecha
Translational Neuro-Oncology Laboratories, Moores Cancer Center, UC San Diego, 3855 Health Sciences Drive, MC#0819, La Jolla, CA, 92093-0819, USA. pejiang@ucsd.edu.
Pengfei
P
Jiang
Translational Neuro-Oncology Laboratories, Moores Cancer Center, UC San Diego, 3855 Health Sciences Drive, MC#0819, La Jolla, CA, 92093-0819, USA. nanomura@ucsd.edu.
Natsuko
N
Nomura
Translational Neuro-Oncology Laboratories, Moores Cancer Center, UC San Diego, 3855 Health Sciences Drive, MC#0819, La Jolla, CA, 92093-0819, USA. spingle@ucsd.edu.
Sandeep C
SC
Pingle
Laboratory of Tumor Targeted Therapy, Shanghai Advanced Research Institute, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 99 Haike Road, 201210, Shanghai, China. guof@sari.ac.cn.
Fang
F
Guo
Translational Neuro-Oncology Laboratories, Moores Cancer Center, UC San Diego, 3855 Health Sciences Drive, MC#0819, La Jolla, CA, 92093-0819, USA. mmakale@ucsd.edu.
Milan
M
Makale
Translational Neuro-Oncology Laboratories, Moores Cancer Center, UC San Diego, 3855 Health Sciences Drive, MC#0819, La Jolla, CA, 92093-0819, USA. skesari@ucsd.edu.|Department of Neurosciences, UC San Diego, La Jolla, CA, USA. skesari@ucsd.edu.|Department of Radiation Medicine and Applied Sciences, UC San Diego, La Jolla, CA, USA. skesari@ucsd.edu.
Santosh
S
Kesari
D000293:Adolescent; D000328:Adult; D000368:Aged; D001343:Autophagy; D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor / Q000187:drug effects; D002469:Cell Separation; D002470:Cell Survival; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D020128:Inhibitory Concentration 50; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors*; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D055728:Primary Myelofibrosis / Q000378:metabolism*; D016016:Proportional Hazards Models; D011743:Pyrimidines / Q000737:chemistry*; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors*; D013449:Sulfonamides / Q000737:chemistry*; D055815:Young Adult; D000096926:Benzenesulfonamides
D004791:Enzyme Inhibitors; D053614:Janus Kinase 2; D011743:Pyrimidines; D050796:STAT3 Transcription Factor; D013449:Sulfonamides; C494086:STAT3 protein, human; C522865:TG101209; D000096926:Benzenesulfonamides; C507924:JAK2 protein, human