In Vivo Effects of Notch Signaling Inhibitor on Tumor Growth and Its Mechanism of Action.

[Background] The Notch signaling pathway plays crucial roles in a variety of cellular functions, including cell proliferation, differentiation and apoptosis. When constitutively activated, Notch signaling has been proved to be oncogenic. It has been reported that human Notch1 activating mutations are observed in approximately 50% of human T-cell acute lymphoblastic leukemia (T-ALL) patients, and that the gamma-secretase inhibitor (GSI), which is known to block Notch signaling, induces cell cycle arrest in vitro in T-ALL cell lines with Notch1 activating mutations. Notch signaling has also been suggested to play a critical role in tumor angiogenesis. The purpose of this study is to examine whether GSI is potent in vivo in eradicating established tumors made of human T-ALL cell lines in xenograft models, and to elucidate the mechanism of action.

[Methods] Human T-ALL cell lines (ALL-SIL, DND-41, TALL-1, HPB-ALL), which were sensitive to GSI in vitro, were implanted subcutaneously to SCID mice. After 3 to 6 weeks, when the tumor diameter reached to approximately 1 cm, the treatment with GSI, YO01027, a derivative of LY411575, by oral administration at 0.1, 1, 10 mg/kg/day was initiated and continued for at least 30 days. The tumor volume was measured to evaluate efficacy of treatment. Tumor vascular density was assessed sequentially until tumors began to shrink, by immunohistochemical staining with a vascular endothelial cell marker, CD31. In addition, we performed in vivo angiogenesis assay using Matrigel, either with or without GSI treatment of mice for 6 days. Hemoglobin content was measured by Drabkin’s method to evaluate neovascularization in Matrigel.

[Results & Discussion] We found that the treatment with GSI resulted in partial (in HPB-ALL) or complete (in ALL-SIL, DND-41, and TALL-1) regression of subcutaneous tumors. When treated with YO01027 at 10 mg/kg/day, the growth of tumors made of HPB-ALL was suppressed to less than 50% compared to the growth without treatment. Tumors made of ALL-SIL, DND-41, and TALL-1 completely regressed by the treatment with YO01027 at 1 mg/kg/day in 2 to 3 weeks. Notably, it was demonstrated that tumor vascular density was significantly decreased before the tumors regress. In tumors made of ALL-SIL and DND-41, vascular density decreased more than 40% at the treatment days 5 to 8, suggesting that the inhibition of tumor angiogenesis was one of the mechanisms how GSI acts as a strong anti-tumor agent in vivo. In vivo angiogenesis assay showed significant (about 40% compared to the control) decrease in neovascularization in Matrigel by the treatment with YO01027 at 1 to 10 mg/kg/day.

[Conclusion] Our observations indicate that the Notch signaling inhibitor, GSI, is highly effective in treating T-ALL with Notch1 activating mutations in our xenograft model, the efficacy of which might be attributable to (1) direct apoptosis of T-ALL cells and (2) inhibition of tumor angiogenesis.