Abstract LB-32: Glioma treatment with temozolomide and Notch inhibition suppresses neurosphere formation and xenograft formation
Authors: Gilbert Candace A. , Hermance Nicole , Daou Marie-Claire , Moser Richard P. , Kelliher Michelle , Ross Alonzo H. 
Published: February 2011

Abstract
Malignant gliomas are treated with a combination of surgery, radiation and temozolomide (TMZ), however these therapies do not result in a long term cure. Our lab has found a population of human glioma cells that undergo a transient cell cycle arrest in response to chemotherapy. Treatment with TMZ decreases sphere formation; however, after a short recovery period, a small number of cells resume sphere formation. This neurosphere recovery assay allows us to analyze drugs that target pathways involved in chemoprotection. One promising target is the Notch pathway, which is over-expressed in gliomas and can be suppressed using gamma-secretase inhibitors (GSIs). If Notch facilitates escape from cell chemotoxicity, the combination of TMZ and GSI treatment would be expected to inhibit recovery following chemotherapy. We demonstrate that TMZ + GSI treatment resulted in a decrease in initial sphere formation similar to TMZ-only treatment; however, TMZ + DAPT treated cells did not recover. Low doses of GSI-only treatment had no apparent effect on sphere formation. To determine if the treated cultures self-renew, the remaining spheres were analyzed for secondary sphere formation. TMZ-only cultures have high rates of secondary sphere formation, while cells from the TMZ + GSI-treated cultures have minimal self-renewal capabilities. To establish that the GSI synergizes with TMZ by targeting a Notch family member, cultures were infected with an intracellular form of NOTCH1 (NICD) and treated with TMZ + GSI. NICD expressing glioma cells were not sensitive to TMZ + GSI treatment and were capable of recovery and secondary sphere formation. Interestingly, we also found that the TMZ + GSI synergy was dependent on the order in which the drugs were administered. Recovery of sphere formation was inhibited when TMZ was administered first, followed by GSI treatment 24 hrs later. If the GSI was administered prior to, or concurrently with TMZ, the culture was able to recover and form secondary spheres. To analyze the mechanism of TMZ + GSI treatment, we found that there was an increase in the number of cells expressing the senescence-associated ß-galactosidase. This demonstrates that TMZ + GSI treatment induces senescence in human glioma cultures. Finally, when human glioma cell lines were treated in vitro and implanted in immunodeficient mice, TMZ + GSI treatment resulted in extended latency and greatly increased survival. In addition, in vivo TMZ + GSI treatment completely blocked tumor progression in 50% of mice, while none of the mice with in vivo TMZ-only treatment survived. This data demonstrates the importance of the Notch pathway in chemoprotection and maintenance of TMZ-treated gliomas. The addition of GSIs to current treatments may be a promising new target-directed therapy to decrease the rate of brain tumor recurrence.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-32.