Abstract 2413: Glioblastoma stem-like cells are resistant to the negative effects of increased aneuploidy on in vitro survival and radiosensitivity

Abstract

Abstract Glioblastomas (GBM) have long been classified as radioresistant tumors. Glioblastoma stem-like cells (GSCs) are a portion of GBM’s heterogenous cell population and are generally considered to be crucial drivers of tumorigenesis and radioresistance. As for most tumor cells, GSCs display a moderate level of chromosomal instability and aneuploidy. However, whether aneuploidy promotes or inhibits GSC survival and its effects on radiosensitivity remain unclear. In this study we compared the radiation response of GSC lines (NSC11 and NSC20) and the standard glioma cell line U251 after an increase in aneuploidy. This increase was induced by treatment with CFI-402257, a selective inhibitor of the mitotic kinase TTK, which plays a key role in spindle-assembly checkpoint (SAC) regulation. After an initial drug exposure of 24h, the drug was removed and cells were allowed to grow for 5 days. In both GSC lines and U251, as demonstrated by interphase chromosome FISH, this protocol resulted in approximately 150%, 50%, and 125% increases in aneuploidy of chromosomes 2, 7 and 10 respectively. After these increases in aneuploidy, the clonogenicity (in vitro survival) of U251 cells was reduced by 80%, whereas it was reduced by 50% and 30% in NSC11 and NSC20 respectively. Moreover, the increase in aneuploidy significantly enhanced the radiosensitivity of U251 cells, whereas it had no effect on the radiosensitivity of the GSCs. These changes could not be attributed to residual inhibition of TTK or deficiencies in the SAC: at 5 days after CFI inhibition no changes in nocodazole-induced SAC arrest in mitosis were detected. Additionally, after aneuploid induction no changes in the expression of the stem cell marker CD133 were detected in the GSC lines, indicating no change in differentiation status. To begin to investigate the mechanism behind these differences in radiosensitivity, γH2AX foci dispersal, a surrogate marker for DNA double strand break (DSB) repair was evaluated. After the increase in aneuploidy, cells were irradiated (2Gy) and collected 1-24h after for analysis of γH2AX. In U251 cells, the increase in aneuploidy resulted in slower foci dispersal, indicative of a decrease in DSB repair; whereas in GSCs the increased aneuploidy had no effect on foci dispersal as compared to control. Mitotic catastrophe, the primary mechanism of solid tumor cell death after irradiation, was also evaluated. The increase in aneuploidy significantly increased the frequency of radiation-induced mitotic catastrophe in U251 cells, but no changes in either GSC line were detected. Overall, these results suggest that GSCs have an enhanced ability to tolerate the negative consequences of aneuploidy on survival as well as on radiosensitivity. Such aneuploid tolerance may provide a mechanism through which GBMs exploit karyotype diversity to survive under harsh environmental conditions and after treatment. Citation Format: Ian C. Sutton, Charlotte M. Degorre, Komal Rawal, Philip J. Tofilon. Glioblastoma stem-like cells are resistant to the negative effects of increased aneuploidy on in vitro survival and radiosensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2413.