Rheb mutants drive constitutive mTORC1 signalling in CHO cells
Enabling cells to produce more protein is highly beneficial for the efficient production of therapeutic proteins such as monoclonal antibodies (mAbs). mAbs are most commonly produced from Chinese Hamster Ovary (CHO) cells (Kunert & Reinhart, 2016; Sharker & Rahman, 2020). Given that Rheb mutants increase protein synthesis and enhance ER capacity in HEK293 cells, we hypothesised that these mutants, when expressed in CHO cells, may also result in increased secretory mAb titres. For our initial experiments we utilised our previously reported CHO cell lines that stably expresses either the secreted luciferase reporter Gaussia Luciferase (GLuc) or Firefly Luciferase (FLuc). As it was first important to confirm that Rheb mutants can also drive hyperactive mTORC1 signalling in CHO cells, we co-transfected them with vectors encoding Rheb mutants and with vectors encoding FLAG-TSC1/2. Here we used both GLuc-CHO and FLuc-CHO cells grown in monolayer in fully supplemented Ham’s F12 medium. Cells were then starved of serum for 16 h and then transferred to D-PBS for 1 h prior to lysates being harvested for immunoblot analysis. mTORC1 activity was assessed by monitoring the phosphorylation of the well-characterized mTORC1 substrate, S6K1, at Thr389, rpS6 at Ser240 and Ser244 (a substrate of S6K1) and of 4E-BP1 at Thr37, Thr46 and Ser65 (all direct substrates of mTORC1). Phosphorylation of all effectors were slightly elevated in cells expressing Rheb[WT] compared to cells that received EV and, as expected, was almost completely abolished in cells that were co-transfected with FLAG-TSC1/2 in both GLuc-CHO (Fig. 3a) and FLuc-CHO cells (Fig. 3b).
In contrast, cells expressing Rheb[T23M] or [E40K] showed higher levels of phosphorylation of S6K1, rpS6 and 4E-BP1, even in the presence of FLAG-TSC1/2, suggesting these mutants drive hyperactivation of mTORC1 (i.e., which is insensitive to inhibition by TSC) in GLuc-CHO (Fig. 3a) and FLuc-CHO (Fig. 3b) cells. These data are consistent with our findings for expression of these mutants of Rheb in human cells (Jianling Xie et al., 2020).
We next sought to determine the effect of Rheb mutations on cell proliferation. To do this, GLuc-CHO cells were allowed to proliferate for 7 days while being counted on a haemocytometer every day in order to generate a cell growth curve. None of the Rheb mutants had any effect on cell proliferation when GLuc-CHO cells were grown in fully supplemented medium or medium supplemented with 0.5% FBS (Fig. 3c, Supplementary Fig. S2a). However, when GLuc-CHO cells were grown in medium supplemented with 1% FBS, Rheb[T23M] promoted both an increased rate of proliferation as well as supporting an increased total cell number (Supplementary Fig. S2b). This was even more pronounced when cells were grown in the absence of FBS with Rheb[T23M] promoting a marked increase in the rate of proliferation (Fig. 3d). To confirm these results, BrdU incorporation assays were performed in order to more directly quantify the effect of Rheb[T23M] on cell proliferation. Consistent with our findings for HEK293 cells (Jianling Xie et al., 2020), Rheb[T23M], [Y35N] and [E40K] each drove increased cell proliferation in both GLuc-CHO and FLuc-CHO cells compared to both Rheb[WT] and EV (Fig. 3e/f).