Abstract LB-74: A high-throughput RNAi sensitization screen of rapamycin identifies targets for rational drug combination strategies

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC RNAi screening affords an unbiased and rapid method to identify genes involved in particular cellular processes and is thus a powerful tool for drug target identification and validation. Rapamycin is a well defined inhibitor of mTOR, a protein being actively pursued as an anti-tumor target. In breast cancer patients, however, treatment with mTOR inhibitors has shown only modest activity suggesting that a combined approach may be required to maximize the clinical application of mTOR inhibitors in these patients. In this study, we performed a high-throughput synthetic siRNA-based RNAi screen of the human kinome plus 350 additional genes (4 siRNAs per gene) in combination with rapamycin, to identify candidate genes whose silencing potentiate the inhibitory effects of rapamycin. Screens were conducted in the ER+ breast cancer cell line MCF-7 and the triple negative breast cancer cell line MDA-MB-468. For the RNAi screen rapamycin (10 nM) or vehicle only (0.1% DMSO) was added 48 hours post siRNA transfection and cell viability was measured after a further 24 hours of incubation. By comparing the normalized effect on cell viability for each siRNA (Z-score) in the control and rapamycin screens we identified 36 candidate genes whose silencing sensitized MCF-7 cells to rapamycin and 56 that sensitized MDA-MB-468 cells. The silencing of six candidate genes sensitized both cell lines to rapamycin, AKT1, ASPA, CDKN2A, MAP3K7IP1, MAPK12 and PCTK3. The sensitization of rapamycin by silencing of AKT1 was confirmed. Rapamycin treatment increased AKT phosphorylation in the cell lines. HDAC inhibitors have been shown to facilitate the dephosphorylation of AKT; therefore, we tested the combination of rapamycin with the HDAC inhibitor MS-275. MS-275 completely blocks rapamycin-induced AKT phosphorylation, and combining it with rapamycin synergistically inhibited the growth of the MCF-7, MDA-MB-468, MDA-MB-435 and MDA-MB-231 breast cancer cell lines. We investigated if other pathways are affected by combining rapamycin with MS-275. De-regulation of the RAF-MEK-ERK pathway is frequently detected in human cancers but the high level of ERK phosphorylation is significantly reduced in cells treated with the rapamycin/MS-275 combination. To profile the unphosphorylated, mono and dual-phosphorylated isoforms of ERK1 and ERK2 we used a capillary isoelectric focusing immunoassay to show that the di-phosphorylated isoform of ERK2 was decreased. Our results highlight the potential of high-throughput siRNA screens to identify rational drug combination strategies. 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-74.