LAUSR

SMG1 is a member of the PI-3 kinase-like kinase family of proteins, which includes ataxia telangiectasia mutated (ATM), Dna-dependent protein kinase (DNA-PK) and mTOR. Our previous work demonstrated that SMG1 loss in mice increased cancer development, particularly blood cancers. The mechanism by which SMG1 loss enhances cancer development is currently unknown. Here we show that SMG1 acts to negatively regulate mTORC2 activity. In cells from mice lacking one or both alleles of SMG1 or following siRNA knockdown of SMG1, phosphorylation of Akt or Protein kinase C alpha is significantly increased. Using downregulation of SMG1 followed by treatment with IGF1 or serum to induce signaling from mTORC2 and 1 respectively, downstream reporters of mTORC1 (P-S6 Kinase Thr389) and mTORC2 (P-AktSer473) were upregulated. Upregulation of mTORC1 is at least partly due to feedback from mTORC2 as P-S6KThr389 upregulation can be partially inhibited by Akt inhibition. Phosphorylation of both can be completely inhibited by the mTOR inhibitor INK128. Phosphorylation of mTOR at Ser2481, a marker of mTORC2 complex stability, increased at both baseline and upon growth factor stimulation after SMG1 knockdown. No effect of SMG1 knockdown was seen upstream of mTORC2. Additionally, overexpression of SMG1 but not a kinase dead mutant limited mTORC2 signaling. SMG1 can be immunoprecipitated with members of the mTORC2 complex including mTOR, Rictor and Sin1.1 but no Raptor or other Sin1 isoforms are detected in these immunoprecipitations. This indicates that SMG1 can inhibit mTORC2 activity by interacting with Sin1.1 containing complexes. The unique exon present in Sin1.1 contains a putative SMG1 phosphorylation site. We are currently investigating whether SMG1-mediated phosphorylation at this site mediates inhibition of mTORC2 activity. We have further examined the relationship between SMG1 expression and mTOR activity in patients with chronic lymphocytic leukemia (CLL). We recruited 70 patients from Liverpool Hospital and isolated circulating tumor cells from their blood. We analyzed SMG1 expression level and extent of Akt Ser473 phosphorylation in these cells. SMG1 expression inversely correlated with phosphorylation of Akt, indicating that SMG1 also acts to negatively regulate mTORC2 in cancer cells. Overall, we have identified a novel mechanism for regulation of mTORC2 activity by SMG1, which may explain why SMG1 loss enhances cancer development. Citation Format: Alexander James, Patricia Rebeiro, Uda Ho, C. Soon Lee, Nicole Caixeiro, Martin F. Lavin, Tara L. Roberts. Suppressor of morphogenesis in genitalia 1 (SMG1) is a novel negative regulator of mammalian target of rapamycin complex 2 (mTORC2) [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A16.