Abstract A30: A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation

To identify the cellular components that participate in the regulation of mTOR complex 1 (mTORC1), the amino acid-dependent, rapamycin-inhibitable complex, we carried out a genome-wide RNAi depletion screen. We employed a rabbit monoclonal antibody specific for RPS6 [Ser235P/Ser236P] and high content microscopy to quantify rpS6 phosphorylation in the pancreatic ductal adenocarcinoma cancer cell line (PDAC) MiaPaCa-2. Applying a stringent selection, we retrieved over 600 genes wherein at least two RNAi gave significant reduction in S6 phosphorylation. This cohort is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. To identify mTORC1 regulators that did not require the TSC, we examined which of the positives identified in MiaPaCa-2 cells were also required for S6 phosphorylation in TSC1 null mouse embryo fibroblasts. Among the 541 such gene products examined in TSC1 null MEFs, RNAi pools directed against 79 were found to reduce S6 phosphorylation significantly in 2 or 3 replicates. Among these, GPCRs, other transmembrane proteins and several signaling proteins comprised 30%, elements concerned with DNA structure and transcription, RNA processing and translation together comprised 28%, and the remainder distributed among proteins concerned with membrane transport and traffic, protein traffic and modification, mitochondria, metabolism and the cytoskeleton. Surprisingly, the only elements among this cohort previously associated with the maintenance of mTORC1 activity are two of the numerous subunits of the vacuolar ATPase subunits and the CUL4 subunit DDB1. RNAis against another 84 TSC1null MEF targets were observed to reduce S6 phosphorylation in only one of three replicates, however an indication that this group also bears attention is the presence of RPS6KB1 itself, as well as Rac1 and MAP4K3, a protein kinase that supports amino acid signaling of mTORC1 to RPS6KB1. Our screen confirmed previously known and proposed regulators of S6 phosphorylation and mTORC1 in addition to previously unidentified mTORC1 related proteins. Our results suggest the potential operation of feedback pathways from mRNA translation, RNA processing and DNA repair in the regulation of mTORC1. Since many cancers exhibit mTOR hyperactivation, identifying previously unappreciated proteins needed for maintenance of mTORC1 activity may provide new drug targets and lead to the development of beneficial therapies for tumors sensitive to mTOR inhibition. In addition, we found 43 genes that matched the mTOR essentiality profile in 216 different cancer cell lines represented in the Achilles dataset. These genes may help delineate a more general cellular state characterized by co-dependency of mTOR dependency and the respective P-S6 positive genes therefore providing the rationale for selecting specific proteins for further analysis as candidate drug targets. Citation Format: Angela Papageorgiou, Pablo Tamayo, Jill Mesirov, Joseph Avruch, Joseph Rapley. A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A30.