Selective Modulation Of Ires-Mediated Translation In Malignant Cells

There are two distinct modes of protein synthesis in the cell. The conventional mechanism of cap-dependent ribosomal scanning is utilized for general protein synthesis, whereas internal ribosome entry site (IRES)-mediated translation appears to be reserved for synthesis of those gene products most critical for cell proliferation and survival, providing a fail-safe or emergency system to ensure that such essential polypeptides can be manufactured when needed, particularly under conditions of stress when general protein synthesis is compromised. To date, there has been no means, experimental or therapeutic, by which to selectively modulate IRES function; rather, IRES-mediated translation has typically been studied by inhibiting general protein synthesis and attributing the remaining translational activity to the IRES. To address this problem, a cell-based high-throughput screen of 135,000 compounds was performed to identify small molecules capable of selectively inhibiting translation mediated through the IGF1R IRES. The identification of these compounds allows us, for the first time, to selectively perturb IRES-mediated translation in its native context, and assess the consequences. Data obtained using the bicistronic reporter system demonstrate that the lead compounds and their structural analogs selectively inhibit second cistron translation (mediated through the IRES). Western analyses show that, at sufficient concentration (≥ 5 μg/ml), the IRES inhibitors completely block de novo IGF1R protein synthesis in genetically-unmodified breast tumor cells, confirming activity against the endogenous IRES. The IRES inhibitors function in a manner which is essentially reciprocal to rapamycin, and can be clearly distinguished from cycloheximide. Spectrum of activity of the small molecule IRES inhibitors extends beyond IGF1R to a subset of potential IRES targets, including c-myc. The IRES inhibitors differentially modulate synthesis of the two c-Myc protein isoforms, bringing about a dramatic decrease in p64, the dominant (oncogenic) isoform, while transiently stimulating synthesis of p67, the minor (growth-inhibitory) isoform. RNase protection analyses show no discernible changes in c-myc mRNA, confirming that the effects of the IRES inhibitor are exerted at the translational level. Sustained IRES inhibition has profound, detrimental effects on human tumor cells, inducing massive (u003e99%) cell death and complete loss of clonogenic survival in tumor models as disparate as triple negative breast carcinoma, glioblastoma, and acute myeloid leukemia. Beyond a critical threshold of dose and duration of exposure, there is no evidence of recovery, even with prolonged incubation in absence of the compound. Together, these findings support the conclusion that IRES-mediated translation is of fundamental importance to the survival of malignant cells, and may represent a critical point of vulnerability which could be exploited therapeutically. Citation Format: Christos Vaklavas, Zheng Meng, Hyoungsoo Choi, William E. Grizzle, Kurt R. Zinn, Scott W. Blume. Selective modulation of IRES-mediated translation in malignant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4363. doi:10.1158/1538-7445.AM2015-4363