Glioblastoma and glioblastoma stem cells are dependent on functional MTH1

Abstract

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro. We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease.

Keywords: DNA damage; MTH1; Nudt1; cancer stem cells; glioblastoma multiforme.

Figure 1. MTH1 is overexpressed in GBM

MTH1 mRNA is upregulated in GBM (n = 528) compared to non-tumor (NT) samples (n = 10), and high levels of MTH1 correlate with its gene copy number in GBM (TCGA dataset). Tukey’s Honest Significant Difference: ***p < 0.001 A. Inhibition of MTH1 by the small molecule inhibitors TH588 and TH1579 decreased viability of glioblastoma cell lines B.

Figure 2. MTH1 inhibitors TH588 and TH1579 target GMB cells independent of aggressiveness

The MTH1 inhibitors TH588 and TH1579 target seven patient-derived GBM cultures independent of aggressiveness (type B being more aggressive than type A) A. Dose-response curve for TH588 and TH1579 in the most aggressive (#18) and least aggressive (#7) GBM culture B. Clonogenic survival of GBM#18 exposed to TH588 and TH1579 C. Effect of TH588 and TH1579 on the cell cycle of GBM culture #7 and #18 after 24 hours of exposure D., E.

Figure 3. Inhibition of MTH1 is more potent in GBM than standard treatment

Dose-response curves for the MTH1 inhibitors TH588 and TH1579 as well as Temozolomide, AG-120, Palbocilib, Dovitinib and Ganciclovir in the GBM line #18.

Figure 4. Inhibition of MTH1 induces DNA damage in GBM cells

Inhibition of MTH1 by TH588 and TH1579 leads to an increase of yH2AX foci in GBM line #7 and #18 cells after 24 hours A. Quantification of yH2AX foci B. Comet assay reveals incorporation of 8-oxo-dGTP into DNA of GBM #7 and #18 upon treatment with MTH1 inhibitors for 24 hours C. Quantification of 8-oxo-dGTP incorporation, significance calculated with 1-way ANOVA test D.

Figure 5. Inhibition of MTH1 targets GBM stem cells

Inhibition of MTH1 with TH588 and TH1579 decreases both the CD133⁺ and CD133^- cell population of GBM culture #18 equally well A. Inhibition of MTH1 by TH588 and TH1579 impairs viability of CD133⁺ and CD133^- cells equally well B. Exposure to MTH1 inhibitors for 72 hours increases the sub-G1 population of the CD133⁺ cell population of GBM culture #18 C. Clonogenic survival of CD133⁺ cells exposed to TH588 D. and TH1579 E. Mitosis of SOX2-GFP⁺ cells is significantly prolonged upon inhibition of MTH1 F. Quantification of mitosis duration G. Comet assay reveals incorporation of 8-oxo-dGTP into DNA of the CD133⁺ cell population of GBM culture #18 H. Quantification of comet assay I.

Figure 6. MTH1 inhibitors target GBM and GBM stem cells in vivo

GBM #18-CMV-LUC cell enriched for the CD133⁺ population have been orthotopically injected into zebrafish embryos. 6 days post injection, embryos exposed to 50 µM TH1579 displayed smaller tumors A. Quantification by luminescence measurements in single embryos showed 26.4 % smaller tumors in TH1579 treated embryos (n = 43) compared to DMSO controls (n = 31; p = 0.011) B. Still images of real-time light sheet microscopy on orthotopic xenotransplants exposed to 50 µM TH1579 apoptotic cells encircled C. Determination of tumor volume of xenotransplant. Grey circle: DMSO control, black circle TH1579 treated (tumor shown in (C). The asterisks mark the time-point when the transplants started leaving the focal plane D. Immunocytochemistry on cleaved caspase as well as y-H2AX in orthotopic xenotransplants treated for 5 days with 50 µM TH1579 or DMSO E.