A fundamental component of cellular radioresponse is the translational control of gene expression. As a critical regulator of translational control is the eIF4F cap binding complex, we investigated whether eIF4A, the RNA helicase component of eIF4F, can serve as a target for radiosensitization. Knockdown of eIF4A using siRNA reduced translational efficiency, as determined from polysome profiles, and enhanced tumor cell radiosensitivity as determined by clonogenic survival. The increased radiosensitivity was accompanied by a delayed dispersion of radiation-induced γH2AX foci, suggestive of an inhibition of DNA double strand break repair. Studies were then extended to (-)-SDS-1-021, a pharmacological inhibitor of eIF4A. Treatment of cells with the rocaglate (-)-SDS-1-021 resulted in a decrease in translational efficiency as well as protein synthesis. (-)-SDS-1-021 treatment also enhanced the radiosensitivity of tumor cell lines. This (-)-SDS-1-021-induced radiosensitization was accompanied by a delay in radiation-induced γH2AX foci dispersal, consistent with a causative role for the inhibition of double strand break repair. In contrast, while (-)-SDS-1-021 inhibited translation and protein synthesis in a normal fibroblast cell line, it had no effect on radiosensitivity of normal cells. Subcutaneous xenografts were then used to evaluate the in vivo response to (-)-SDS-1-021 and radiation. Treatment of mice bearing subcutaneous xenografts with (-)-SDS-1-021 decreased tumor translational efficiency as determined by polysome profiles. While (-)-SDS-1-021 treatment alone had no effect on tumor growth, it significantly enhanced the radiation-induced growth delay. These results suggest that eIF4A is a tumor selective target for radiosensitization.

Supplementary Data from Inhibition of the Translation Initiation Factor eIF4A Enhances Tumor Cell Radiosensitivity

Supplementary Figure from Inhibition of the Translation Initiation Factor eIF4A Enhances Tumor Cell Radiosensitivity

Supplementary Figure from Inhibition of the Translation Initiation Factor eIF4A Enhances Tumor Cell Radiosensitivity

The cellular radioresponse is regulated by constitutively expressed proteins and by new gene expression. We have previously shown that radiation induces changes in gene expression primarily via translational control. Regulation of gene translation largely occurs at the initiation stage, and many factors that regulate initiation impact the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4F is comprised of the cap-binding protein eIF4E, the RNA helicase eIF4A, and the scaffold protein eIF4G. Because eIF4A is considered to regulate the translation of mRNAs involved in cell survival and proliferation, we investigated it as a target for tumor cell radiosensitization. Knockdown of eIF4A1 in the U251 glioblastoma and the HeLa cervical carcinoma cell lines resulted in a decrease in translational efficiency as determined by polysome profiling and an increase in cellular radiosensitivity as determined by clonogenic survival analysis. There was also an increase in γH2AX foci remaining at 24h after irradiation, consistent with an inhibition of DNA double strand break repair. These results suggest that eIF4A serves as a determinant of cellular radiosensitivity. As an additional approach to inhibiting eIF4A, we utilized the rocaglate hydroxamate (-)-SDS-1-021, which prevents eIF4A inclusion into eIF4F. Treatment of U251, HeLa and PSN1 pancreatic carcinoma cells with 10 nM (-)-SDS-1-021 resulted in decreased translational efficiency, which was accompanied by a decrease in protein synthesis. Treatment of the tumor cell lines with (-)-SDS-1-021 immediately before radiation resulted in an increase in radiosensitivity as well as an increase in γH2AX foci remaining at 24h, similar to eIF4A1 knockdown. Importantly, whereas treatment of the normal human fibroblast cell line MRC9 with (-)-SDS-1-021 also inhibited translation and protein synthesis, there was no effect on radiosensitivity. To gain a better understanding of the mechanism of radiosensitization by (-)-SDS-1-021, RNAseq was performed using polysome-bound mRNAs isolated from U251 and MRC9 cells. Cells were treated with 10 nM (-)-SDS-1-021, immediately irradiated (2Gy), and then harvested six hours post-irradiation. In U251 cells treated with 2Gy alone, 192 genes were translationally upregulated. The upregulation of 160 of these genes was inhibited by (-)-SDS-1-021 in the combination treatment group. Similarly, in MRC9 cells, 322 genes were translationally upregulated in response to radiation, and the upregulation of 173 of these was inhibited in the combination treatment group. By Ingenuity Pathway Analysis, the radiation-induced, (-)-SDS-1-021-sensitive genes in U251 were significantly enriched for proteins involved in DNA replication, recombination, and repair, which is in contrast to the MRC9 (-)-SDS-1-021 genes. Altogether, these findings suggest that eIF4A1 is a viable target for tumor radiosensitization. Citation Format: Stacey L. Lehman, Theresa Wechsler, Kayla R. Schwartz, Amy Wahba, Lauren E. Brown, John A. Porco, Jerry Pelletier, Kevin Camphausen, Philip J. Tofilon. Inhibition of the translation initiation factor eIF4A1 enhances tumor cell radiosensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6507.