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\begin{document}

\title{Constitutively-active Rheb mutants {[}T23M{]} and {[}E40K{]} drive
increased production and secretion of recombinant protein in Chinese
hamster ovary cells}



\author[1]{Stuart De Poi}%
\author[1]{Jianling Xie}%
\author[2]{Christopher Mark Smales}%
\author[1]{Christopher Proud}%
\affil[1]{South Australian Health and Medical Research Institute}%
\affil[2]{University of Kent}%


\vspace{-1em}



  
  \date{June 10, 2024}


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\selectlanguage{english}
\begin{abstract}
Monoclonal antibodies are high value agents used for disease therapy
(`biologic drugs') or as diagnostic tools which are widely used in the
health care sector. They are generally manufactured in mammalian cells,
in particular Chinese hamster ovary (CHO) cells cultured in defined
media, and are harvested from the medium. Rheb is a small GTPase which,
when bound to GTP, activates mechanistic target of rapamycin complex 1
(mTORC1), a protein kinase that drives anabolic processes including
protein synthesis and ribosome biogenesis. Here we show that certain
constitutively-active mutants of Rheb drive faster protein synthesis in
CHO cells and increase the expression of proteins involved in the
processing of secreted proteins via the endoplasmic reticulum, which
expands in response to expression of Rheb. Active Rheb mutants, in
particular Rheb{[}T23M{]}, drive increased cell number under serum-free
conditions similar to those used in the biotechnology industry.
Rheb{[}T23M{]} also enhances the expression of the reporter protein
luciferase and, especially strongly, the secreted Gaussia luciferase.
Moreover, Rheb{[}T23M{]} markedly (2-3 fold) enhances the amount of this
luciferase and of a model immunoglobulin into the medium. Our data
clearly demonstrate that expressing Rheb{[}T23M{]} in CHO cells provides
a simple approach to promoting cell growth in defined medium and the
production of secreted proteins of high commercial value%
\end{abstract}%



\sloppy


\textbf{Keywords}

Recombinant proteins; biologic drug; mTORC1; defined medium; Rheb

\textbf{Introduction}

Mammalian cell culture, most notably Chinese Hamster Ovary (CHO) cells,
are commonly used to produce recombinant proteins such as monoclonal
antibodies (mAbs) by the biopharmaceutical industry for use in both
therapy and diagnosis (Goulet \& Atkins, 2020). Such proteins offer
enormous advances in disease therapy (or diagnosis) and are products of
high commercial value. Over the last several decades there have been
significant advances in the efficiency of CHO cells to produce these
recombinant proteins (Budge et al., 2020; Kunert \& Reinhart, 2016;
Sharker \& Rahman, 2020; Srirangan, Loignon, \& Durocher, 2020).
However, the rate of protein production remains a significant
bottleneck, particularly for many of the novel format based
biotherapeutics now in development, and therefore there remains
substantial interest in further advancing the productivity of CHO cells
(Budge et al., 2020; Sharker \& Rahman, 2020).

The rate of protein synthesis (mRNA Translation) is one important
limiting determinant of recombinant protein production (Godfrey et al.,
2017; Khoo \& Al-Rubeai, 2009; O'Callaghan et al., 2010; Roobol et al.,
2020; Smales et al., 2004). One key regulator of protein synthesis is
the mechanistic target of rapamycin complex 1 (mTORC1) (X. Wang \&
Proud, 2006). mTORC1 is a protein kinase that is activated by a variety
of upstream signals, most notably amino acids (Kim, Goraksha-Hicks, Li,
Neufeld, \& Guan, 2008; Sancak et al., 2008) and growth factors (Inoki,
Li, Zhu, Wu, \& Guan, 2002; B. D. Manning, Tee, Logsdon, Blenis, \&
Cantley, 2002), and acts as a master regulator of anabolic processes
including protein synthesis (Proud, 2019) and ribosomal biogenesis
(Iadevaia, Liu, \& Proud, 2014; Saxton \& Sabatini, 2017), both of which
are critical for efficient protein production. This is achieved through
phosphorylation of a number of downstream effectors such as ribosomal
protein S6 kinase 1 (S6K1) (Chung, Kuo, Crabtree, \& Blenis, 1992),
eukaryotic elongation factor 2 kinase (eEF2K) (X. Wang et al., 2014),
eIF4E binding protein 1 (4E-BP1) (Beretta, Gingras, Svitkin, Hall, \&
Sonenberg, 1996), and Maf1 (Michels et al., 2010), a regulator of
ribosomal RNA transcription. mTORC1 signalling is thus potentially a
major positive regulator of efficient, high-level production of
recombinant proteins in mammalian cells. Increased phosphorylation of
4E-BP1, which permits increased translation initiation, has indeed been
shown to increase production of interferon-\selectlanguage{greek}γ \selectlanguage{english}(Kaur et al., 2007). We
have also previously shown that the ratio of eIF4E to 4E-BP1 correlates
to higher cell productivity (Joss\selectlanguage{ngerman}é, Xie, Proud, \& Smales, 2016). In
addition to this, mTORC1 drives other anabolic pathways that contribute
to cell growth and faster protein production, including lipid synthesis
(Caron, Richard, \& Laplante, 2015)and ribosome biogenesis (Iadevaia et
al., 2014).

mTORC1 is activated by the small GTPase Rheb (Ras homologue enriched in
brain) when it is in its GTP-bound form; its conversion to the inactive
GDP-bound state is promoted by the tuberous sclerosis complex which
includes the proteins TSC1 and TSC2, the latter acting as a
GTPase-activator protein (GAP) for Rheb (Garami et al., 2003; Inoki,
Zhu, \& Guan, 2003; Tee, Manning, Roux, Cantley, \& Blenis, 2003). In
turn, the ability of TSC1/2 to impair Rheb function is inhibited by
signalling events activated by hormones, mitogenic stimuli and growth
factors (Huang \& Manning, 2008; Inoki et al., 2002; B.D. Manning \&
Cantley, 2003; Zhang et al., 2003). We have recently discovered that
several mutants of Rheb (which occur in certain human cancers) are
resistant to the GAP activity of TSC2 and are thus `constitutively
active', promoting high levels of mTORC1 activity in human cells
(Jianling Xie et al., 2020).

The folding, assembly and maturation of most secreted proteins occurs,
with the assistance of chaperones, within the endoplasmic reticulum
(ER). Homeostatic control of the ER is mediated by the unfolded protein
response (UPR) (Preissler \& Ron, 2019). In response to protein
disequilibrium in the ER, the protein kinase RNA-like ER kinase (PERK)
undergoes homodimerization and becomes active (Cui, Li, Ron, \& Sha,
2011). PERK then phosphorylates eukaryotic initiation factor 2\selectlanguage{greek}α \selectlanguage{english}(eIF2\selectlanguage{greek}α\selectlanguage{english})
at serine-51 (Harding, Zhang, Bertolotti, Zeng, \& Ron, 2000; Harding,
Zhang, \& Ron, 1999). eIF2\selectlanguage{greek}α \selectlanguage{english}is a component of the heterotrimeric
initiation factor eIF2 which is required to deliver the initiator
methionyl-tRNA to the 43S preinitiation complex in order to initiate
mRNA translation (Merrick \& Pavitt, 2018), a process that requires the
hydrolysis of eIF2-bound GTP to GDP (Kapp \& Lorsch, 2004). In order to
facilitate subsequent initiation events, the guanine exchange factor
(GEF) eIF2B binds eIF2 and catalyses the exchange of the GDP for GTP to
regenerate active eIF2.GTP. When eIF2\selectlanguage{greek}α \selectlanguage{english}is phosphorylated in response to
upstream stress, eIF2 is unable to be released from eIF2B and thus is no
longer able to initiate translation (Bogorad, Lin, \& Marintchev, 2018).
This has two contrasting consequences which in combination mediate
homeostasis of the ER. Firstly, it leads to global inhibition of protein
synthesis, decreasing the `load' of new proteins to be folded within the
ER (Wek, 2018). However, secondly, certain mRNAs utilise their upstream
open reading frames (uORFs) to undergo preferentially translation in
response to P-eIF2\selectlanguage{greek}α \selectlanguage{english}mediated inhibition of general protein synthesis
(Harding, Novoa, et al., 2000). One such protein is activating
transcription factor 4 (ATF4). Translation of ATF4 is thus selectively
upregulated in response to phosphorylation of eIF2\selectlanguage{greek}α. \selectlanguage{english}ATF4 is a
transcription factor that drives the expression of genes responsible for
protein homeostasis which are collectively called ER quality control
genes (ERQC) (Preissler \& Ron, 2019). ERQC genes consist mainly of
chaperones and other protein folding genes; thus upregulation of ATF4
counters ER stress by increasing the protein folding capacity of the
cell (Shaffer et al., 2004; Sriburi, Jackowski, Mori, \& Brewer, 2004;
M. Wang \& Kaufman, 2016).

Here we show that two such Rheb mutants, T23M and E40K, drive
constitutive mTORC1 signalling in CHO cells and enhance the production
of recombinant protein including, importantly, its secretion from the
cells. Manipulation of mTORC1 signalling by these Rheb mutants therefore
has the clear potential to enhance the production of proteins of high
commercial value.

\textbf{Materials and Methods}

\emph{Cell Culture, Vectors and Transfection}

HEK293 cells were cultured in Dulbecco's modified Eagle medium (DMEM)
containing 10\% foetal bovine serum and 1\% penicillin/streptomycin at
37\selectlanguage{ngerman}°C with 5\% (v/v) CO\textsubscript{2}. Firefly Luciferase (FLuc)
and\emph{Gaussia} Luciferase (GLuc) expressing Chinese Hamster Ovary
(CHO) cells were cultured in Ham's F12 medium containing 10\% foetal
bovine serum and 1\% penicillin/streptomycin at 37°C with 5\% (v/v)
CO\textsubscript{2}. EXPI-CHO cells were acquired from ThermoFisher
Scientific (SA, Australia) and cultured in Expi-CHO\selectlanguage{english} Expression Medium.
All cells were regularly tested for mycoplasma contamination.
pRK7-FLAG-Rheb vectors were used as previously described (Jianling Xie
et al., 2020). peGFP-N1 neomycin selection vector was a kind gift from
Dr. Timothy Sargeant. To create stable transfection vectors, DNA
sequence encoding FLAG-Rheb was amplified from pRK7-FLAG-Rheb vectors
via PCR with the addition of 5' \emph{EcoRI} and 3'
\emph{BamHI}restriction sites. Amplicons were cloned into peGFP-N1
neomycin selection vector via the \emph{EcoRI} and \emph{BamHI}
restriction sites. Transfection into HEK293, FLuc-CHO and GLuc-CHO was
performed using Lipofectamine 3000 (ThermoFisher Scientific, SA,
Australia) according to the manufacturer's instructions. Transfections
into EXPI-CHO\selectlanguage{english} was performed via the Expifectamine\selectlanguage{english} transfection system
as per the manufacturer's instructions.

\emph{Immunofluorescence}

HEK293 cells were seeded into 2 cm x 1 cm chamber slides at a density of
50,000 cells/slide 24 h prior to transfection. Cells were grown until
they reached 80\% confluency. Medium was then removed, and cells washed
3x in PBS before being fixed in 1 mL of 4\% (v/v) paraformaldehyde in
PBS for 15 min. Cells were then washed 3x with PBS before being
permeabilized with 1 mL of freshly made 0.05\% (v/v) Triton X-100 in PBS
for 5 min. Cells were washed 3x with PBS and blocked for 1 h in 10\%
(v/v) normal donkey serum. Cells were washed 3x with PBS before 400 \selectlanguage{greek}µ\selectlanguage{english}L
of PBS containing 2\% (v/v) normal donkey serum and 1/200 dilutions of
indicated primary antibodies was added. Cells were left in primary
antibody at 4\selectlanguage{ngerman}°C overnight. Primary antibodies were removed, and cells
washed 3x in PBS before secondary antibody solution of 400 \selectlanguage{greek}µ\selectlanguage{english}L PBS
containing 2\% (v/v) normal donkey serum and 1/200 dilutions of donkey
anti-rabbit AF488, AF594 and AF647 were added to each sample. Cells were
incubated under foil at room temperature for 1 h before secondary
antibodies were removed and cells washed 3x with PBS. Cells were mounted
with 400 \selectlanguage{greek}µ\selectlanguage{english}L of VectaShield Hardset containing DAPI and cover slips
carefully applied. Cells were incubated overnight in a 5\% (v/v)
CO\textsubscript{2} incubator before being sealed with nail polish.
Images were collected using a Leica TCS SP8X/MP (Leica Microsystems,
NSW, Australia) confocal microscope and processed with ImageJ software
package.

\emph{SUnSET Assay}

To perform surface sensing of translation (SUnSET) assays, cells were
grown to 70-80\% confluency in fully supplemented medium. Medium was
then replaced for Earle's Balanced Salts Solution (EBSS). For one well,
medium was replaced with EBSS containing 1 \selectlanguage{greek}µ\selectlanguage{english}M cycloheximide (CHX) as a
positive control. After 30 min, puromycin was added to the cells to a
final concentration of 1 \selectlanguage{greek}µ\selectlanguage{english}M and allowed to incorporate for 30 min. Cell
lysates were then harvested for SDS-PAGE immunoblot analysis with
detection of incorporated puromycin via an anti-puromycin antibody.

\emph{SDS-PAGE and Immunoblot Analysis}

SDS-PAGE/Western blot analysis was carried out as previously described
(J. Xie et al., 2019). Briefly, cells were lysed in ice-cold lysis
buffer containing 1\% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl pH 7.5,
2.5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM sodium
orthovanadate, 1 mM dithiothreitol (DTT) and 1 mM \selectlanguage{greek}β-\selectlanguage{english}glycerophosphate
supplemented with protease inhibitor cocktail. Protein concentrations
were determined via Bradford assay and normalized. Equal aliquots of
protein (20 \selectlanguage{greek}µ\selectlanguage{english}g) were denatured in Laemmli loading buffer, heated at 95\selectlanguage{ngerman}°C
for 3 min and separated by SDS-PAGE using gels containing 7-13\%
acrylamide and 0.1\%-0.36\% bis-acrylamide as required. Proteins were
transferred to nitrocellulose membranes, which were blocked and
incubated with primary antibody as indicated (Supplementary Table S1).
The relevant fluorescently-conjugated secondary antibody was applied and
signals were imaged using a LiCor Odyssey® CLx imager (Millennium
Science, VIC, Australia).

\emph{RT-qPCR}

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
was performed by first extracting RNA via phenol-chloroform extraction
using TriReagent (Sigma-Aldrich, NSW, Australia) as per the
manufacturer's instructions (Jianling Xie et al., 2020). Contaminating
DNA removal and reverse transcription were then performed on equal
amounts of RNA using QuantiNova\selectlanguage{english} Reverse Transcription Kit. cDNA diluted
1:10 in nuclease free water was used as a template for qPCR reactions
with SYBR Green PCR Master Mix (Sigma-Aldrich). For a list of primers
used for qPCR reactions, see Supplementary Table S2.

\emph{Bromodeoxyuridine (BrdU) Assay}

Was performed as previously described (Jianling Xie et al., 2020). BrdU
Cell Proliferation Assay Kit (Cell Signalling Technology, Danvers, MA
\#6813) was performed as per the manufacturer's instructions. Cells were
plated at 10,000 cells per well in a 96-well plate 2 h prior to addition
of 10 \selectlanguage{greek}µ\selectlanguage{english}l of 10x BrdU solution. 2 h after addition of BrdU, cells were
fixed/denatured for 30 min. BrdU detection antibody was diluted 1:100
and added to cells for 1 h. Cells were washed 3x in wash buffer before
addition of HRP-linked secondary antibody for 30 min. Cells were washed
3x before addition of 3,3',5,5'-tetramethylbenzidine. After 30 min, STOP
solution was added and absorbance determined at 450 nm.

\emph{Firefly and} Gaussia \emph{Luciferase Assays}

Firefly luciferase assay was performed using Steady-Glo\selectlanguage{ngerman}® Luciferase
Assay System (Promega, NSW Sydney) as per the manufacturer's
instructions. To prepare cells, FLuc-CHO cells were seeded in 6-well
plates and transfected with vector encoding Rheb mutants. Cells were
grown for 24 h and then seeded into 96-well plates. Cells were allowed
to grow for 24 h before Firefly Luciferase assay was performed. 100 \selectlanguage{greek}µ\selectlanguage{english}L
of assay reagent was added to each well and 5 min allowed for cell
lysis. Luminescence was then measured using GloMax\selectlanguage{ngerman}® Discover microplate
reader (Promega Australia, NSW, Australia)

\emph{Gaussia} luciferase (GLuc) assays were performed using the
BioLux®\emph{Gaussia} Luciferase Assay Kit (New England Biolabs,
Ipswich, MA) as per the manufacturer's instructions. Cells were prepared
as per the Firefly luciferase assay. GLuc Assay solution was prepared by
mixing 1:1000 BioLux® GLuc Substrate and BioLux® GLuc Assay buffer. 20
\selectlanguage{greek}µ\selectlanguage{english}L of cell growth medium was transferred to a black 96-well plate and 50
\selectlanguage{greek}µ\selectlanguage{english}L GLuc Assay solution added. Luminescence was read using a GloMax\selectlanguage{ngerman}®
Discover microplate reader with a 5 second integration.

\emph{Cell Growth Curves}

Cell growth curves were produced by seeding cells at a density of 1000
cells per well in 6-well plates. Cells were allowed to grow for 24 h
before medium was replaced as indicated. Cells were counted on a
haemocytometer every 24 h for 7 days. For GLuc-CHO cells, cells were
counted every 4 h from 24 h post-seeding to 60 h post seeding.

\emph{IgG Production and Secretion Assay}

ExpiCHO-S\selectlanguage{english} cells stably expressing Rheb{[}WT{]}, {[}T23M{]} or
{[}E40K{]} were seeded at a density of 4 x 10\textsuperscript{6}cells/mL
in 125 mL shaker flasks and allowed to grow for 16 h to a density of 10
x 10\textsuperscript{6} cells/mL. Cells were then diluted to 6 x
10\textsuperscript{6} cells/mL and transfected with pcDNA3.2 vector
encoding heavy and light chains of rabbit IgG1 at 2:1 light:heavy chain
ratio via ExpiFectamine\selectlanguage{english} CHO Transfection Kit according to the
manufacturer's instructions. ExpiFectamine\selectlanguage{english} CHO/plasmid complexes were
prepared by diluting 12 \selectlanguage{greek}µ\selectlanguage{english}g plasmid in 1 mL OptiPRO medium and 80 \selectlanguage{greek}µ\selectlanguage{english}L of
ExpiFectamine CHO Reagent in 920 \selectlanguage{greek}µ\selectlanguage{english}L OptiPRO medium. Diluted
ExpiFectamine reagent was added to diluted DNA and incubated at room
temperature for 5 min before ExpiFectamine CHO/DNA complex was added to
cells. Cells were incubated at 37\selectlanguage{ngerman}°C and 8\% CO\textsubscript{2} on an
orbital shaker at 125 rpm at 19 mm shaking diameter. After 24 h, 150 \selectlanguage{greek}µ\selectlanguage{english}L
of ExpiCHO Enhancer and 6 mL of ExpiCHO Feed were added to cells. 20
\selectlanguage{greek}µ\selectlanguage{english}L of medium were removed each day in order to determine rate of
secretion. 10-days post transfection, cells were pelleted via
centrifugation at 1000 rpm for 10 min and supernatant collected.

IgG concentration in growth medium was determined via Easy-Titre Rabbit
IgG Assay Kit as per the manufacturer's instructions. Growth medium
containing rabbit IgG1 were diluted 1:1000 in PBS. 20 \selectlanguage{greek}µ\selectlanguage{english}L of Anti-IgG
Sensitized Beads were transferred to a 96-well plate and 20 \selectlanguage{greek}µ\selectlanguage{english}L of
diluted cell medium containing rabbit IgG added. Plates were mixed on a
plate mixer for 5 min before 100 \selectlanguage{greek}µ\selectlanguage{english}L of blocking buffer was added to each
well. Absorbance was measured at 405 nm and IgG concentration calculated
from a standard curve of know IgG concentration generated from 1:2
serial dilutions starting at 500 ng/mL.

\textbf{Results}

Rheb-T23M and E40K drive mild endoplasmic reticulum stress through
increased protein synthesis

In a recent study (Jianling Xie et al., 2020), we showed that certain
mutations in Rheb (which arise in human cancers) are able to drive
hyperactive mTORC1 signalling in mouse or human cells.

As mTORC1 is well known to promote multiple steps in mRNA translation
(protein synthesis), we assessed whether these Rheb mutants can drive
increased protein synthesis. To do this we employed surface sensing of
translation western blot assay (SUnSET-WB (Schmidt, Clavarino, Ceppi, \&
Pierre, 2009)). In the SUnSET-WB technique cells are treated with a low
concentration of puromycin (1 \selectlanguage{greek}µ\selectlanguage{english}M), a compound which acts as a structural
analogue of aminoacyl-tRNA (specifically tyrosyl-tRNA) and can therefore
be incorporated into nascent polypeptides. Incorporated puromycin can
then be detected by western blot with puromycin-specific antibodies.
Thus SUnSET-WB is a radioactive-free assay to measure rates of protein
synthesis. For our initial experiments, we elected to utilise HEK293
cells as they are both a common cell line used for cell signalling
studies, and ones in which we have previously shown Rheb mutations drive
constitutive mTORC1 signalling. HEK293 cells transiently transfected
with vectors encoding FLAG-Rheb{[}WT{]}, {[}T23M{]}, {[}Y35N{]} or
{[}E40K{]} or an empty vector (EV) were transferred to Dulbecco's
phosphate buffered saline (D-PBS) for 30 min prior to the addition of 1
\selectlanguage{greek}µ\selectlanguage{english}M puromycin for an additional 30 min. One well of untransfected cells
was pre-treated with 50 \selectlanguage{greek}µ\selectlanguage{english}g/mL of cycloheximide, a potent inhibitor of
protein synthesis, for 30 minutes prior to the addition of puromycin to
provide a `negative control' for any immunostaining that is not due to
ongoing protein synthesis. Cell lysates were then harvested for western
blot analysis. Rheb{[}T23M{]} and {[}E40K{]} each stimulated a large
increase in puromycin incorporation compared to either Rheb{[}WT{]} or
EV (Fig. 1a; quantified in Fig. 1b). Interestingly, despite
Rheb{[}Y35N{]} being known to drive hyperactive mTORC1 signalling, it
did not increase puromycin incorporation, in line with the fact that
Rheb mutants differ in their downstream effects (Jianling Xie et al.,
2020).

It has been shown that increases in overall protein synthesis can
overload the protein folding capacity of the endoplasmic reticulum (ER)
resulting in ER stress and activation of the unfolded protein response
(Sriburi et al., 2004). This process has been observed in response to
increased mTORC1 activity (Appenzeller-Herzog \& Hall, 2012; Dong et
al., 2015) and results in an expansion of the ER and therefore increased
protein folding capacity (Shaffer et al., 2004; Sriburi et al., 2004; M.
Wang \& Kaufman, 2016). We hypothesised that Rheb-mutants may drive mild
ER stress resulting in increased ER volume and protein folding capacity.
To test this, we first studied several proteins involved in both the
ATF4 arm of the UPR and proteins involved in protein folding. Cells
stably expressing plasmids encoding Rheb{[}WT{]} or mutants of Rheb
showed increased expression of ATF4 compared to the empty vector (Fig.
1c). Interestingly, there was no significantly greater change in ATF4
protein expression in cells expressing Rheb mutants compared to WT.
However, Rheb{[}T23M{]} and {[}E40K{]} did promote an increase, or
tended to cause an increase, in classical UPR markers or ER resident
proteins including PERK, BiP/Grp78, IRE-1\selectlanguage{greek}α, \selectlanguage{english}PDI and ERO1-1\selectlanguage{greek}α \selectlanguage{english}(Fig. 1c;
quantified in Supplementary Figure 1). Calnexin did not change. There
was also an increase in the protein folding markers ERO1-L\selectlanguage{greek}α \selectlanguage{english}(Fig. 1c;
quantified in Supplementary Fig. S1).

To assess whether these changes reflected increased levels of the
corresponding mRNAs, we performed RT-qPCR for the mRNAs encoding BiP
(\emph{HSPA5} ; Fig. 1d), PDI (\emph{PDI} ; Fig. 1e), IRE1\selectlanguage{greek}α \selectlanguage{english}(\emph{ERN1}
; Fig. 1f), and ATF4 (\emph{ATF4} ; Fig. 1g) whose levels were increased
by mutant Rheb expression. Increases in mRNA encoding both UPR and
protein folding proteins correlated with protein increases with the
notable exception of ATF4 mRNA which was significantly higher in cells
expressing Rheb mutants compared to both the EV and Rheb{[}WT{]} (Fig.
1d-g). To determine if these observed changes are associated with an
increase in ER volume, we performed immunofluorescence on HEK293 cells
stably expressing Rheb mutants or WT as well as an empty vector. To
image the ER, we chose to probe with an anti-calnexin antibody as
calnexin is an ER surface protein and there was no significant change in
the level of calnexin protein with the different Rheb mutants (Fig. 1b)
(so that any alterations seen in the extent of the ER would be
independent of changes in its overall levels). Volume was calculated
based on a \selectlanguage{greek}β-\selectlanguage{english}actin counter stain. Both Rheb{[}T23M{]} and {[}E40K{]}
promote a significant increase in ER volume compared to Rheb{[}WT{]} and
EV. Rheb{[}Y35N{]} did not promote an increase in ER volume (Fig. 2a;
quantified Fig.2b). These data suggest the Rheb{[}T23M{]} and {[}E40K{]}
mutants can each promote increase protein synthesis which, in turn,
drives a mild ER stress resulting in increased ER volume and concomitant
protein folding capacity.

These data for HEK293 cells prompted us to extend our studies to CHO
cells, the dominant type of cells used for in industry for producing
recombinant proteins, particularly as an expanded ER is reported to
enhance the ability of CHO cells to produce secretory recombinant
proteins (Budge et al., 2020).

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 \emph{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).

Rheb{[}T23M{]} and {[}E40K{]} drive increased protein synthesis and
secretion dependent on ER stress

To test whether Rheb mutants drive an increase in recombinant protein
production in CHO cells, we transfected FLuc-CHO cells with vectors
encoding Rheb mutants and performed firefly luciferase assays every 48 h
for 7 days. In order to determine if the protein production capacity
of\emph{individual} cells was increased (and to take into account
possible differences in cell number due to the effect of Rheb mutants on
cell proliferation), the firefly luciferase assays were normalised to
the number of cells present as counted on a haemocytometer. Cells
expressing Rheb{[}T23M{]} showed a small yet significant increase in
Firefly luciferase (Fig. 4a/b) indicating an increase in protein
production. Interestingly, other Rheb mutants did not significantly
increase the accumulation of firefly luciferase suggesting that while
they promote constitutive mTORC1 activity, this does not result in
universal upregulation of protein production.

Next, we sought to determine the effect of Rheb mutants on the output of
a secreted protein, which is the key parameter for the production of
recombinant proteins of commercial interest, which are harvested from
the culture medium. To do this, we transfected GLuc-CHO cells with
vectors encoding Rheb mutants and performed \emph{Gaussia} luciferase
assays on the growth media. As with the firefly luciferase assays,
results were normalised to cell number to give an indication of
accumulated \emph{Gaussia} luciferase secretion on a per-cell basis. In
contrast to firefly luciferase production, Rheb{[}T23M{]}, {[}Y35N{]}
and {[}E40K{]} all promoted a significant increase in GLuc secretion as
calculated in this way (Fig. 4c). Cells expressing these mutants showed
an approximately 3-fold increase in GLuc secretion after 7 days; no
difference was seen in amounts of intracellular GLuc (not shown),
consistent with Rheb mutants boosting secretion of the additional GLuc.
As expected, treatment with the potent mTOR inhibitor AZD8055 (Chresta
et al., 2010) reversed the effect of mutant Rheb (Fig. 4c/d). These data
suggest that, as well as increasing protein synthesis, Rheb mutants may
also increase cells' secretory capacity, consistent with the findings
for HEK293 cells. As increased ER volume is known to increase secretory
capacity (Budge et al., 2020; M. Wang \& Kaufman, 2016), we also
performed the GLuc assay using cells treated with a combination of the
ER stress response inhibitors iPERK (Axten et al., 2012), a compound
that inhibits the kinase activity of PERK, and ISRIB (Sidrauski et al.,
2013), a compound that interferes with the ability of phosphorylated
eIF2 to inhibit the guanine exchange of eIF2B (Sidrauski, McGeachy,
Ingolia, \& Walter, 2015). Inhibition of the ER stress response
dramatically decreased the secretion of GLuc from cells expressing Rheb
mutants (Fig. 4e) indicating Rheb mediated ER expansion is crucial for
increased GLuc secretion. Consistent with this, western blot analysis of
GLuc-CHO cells transiently expressing Rheb mutants or an empty vector
show that Rheb{[}T23M{]} and {[}E40K{]} significantly drive increased
expression of the ATF4 arm of the UPR (Fig. 4f).

Rheb{[}T23M{]} and {[}E40K{]} drive increased mAb secretion under
conditions similar to those used in industry

While GLuc-CHO cells act as a good reporter system for interrogating the
effect of Rheb mutants on protein secretion, there are several
limitations that make these cells unsuitable for use as an industrial
research tool. First, to be useful in an industrial context, CHO cells
must be grown in suspension and in chemically defined, serum-free media.
Second, while GLuc may be a secreted protein, it is not a model
biotherapeutic protein and lacks glycosylation. To address these issues,
we acquired EXPI-CHO cells and modified them to stably express
Rheb{[}WT{]}, {[}T23M{]}, {[}Y35N{]} or {[}E40K{]} (Supplementary Fig.
S3). We then performed SDS-PAGE western blot analysis on lysates
harvested from these cells to confirm the hyperactivity of mTORC1
signalling and ER stress phenotype observed in HEK293 and GLuc-CHO cells
was present in these cells. Consistent with GLuc-CHO cells, in EXPI-CHO
cells stably expressing Rheb mutants or Rheb{[}WT{]}, {[}T23M{]} and
{[}E40K{]} drove increased phosphorylation of rpS6 at Ser240/244,
indicative of activated mTORC1, as well as increased phosphorylation of
4E-BP1 at Thr37 and Thr46 suggesting that Rheb{[}T23M{]} and {[}E40K{]}
drive hyperactive mTORC1 signalling in these cells (Fig. 5a).

In addition, Rheb{[}T23M{]} and {[}E40K{]} each drove increased
expression of the ATF4 arm of the UPR as well as of ER stress markers
such as BiP (Fig. 5b). Interestingly, we were unable to detect any
differences in protein synthesis via SUnSET assay (Fig. 5c). This is
likely because these cells stably express Rheb mutants and therefore
increased protein synthesis likely results in increased total protein
amounts. As the amounts of cell lysate loaded onto the immunoblot gels
are normalised to total protein, such normalisation will effectively
`eliminate' any effect on the rate of protein synthesis. Cells
expressing Rheb{[}T23M{]} or {[}E40K{]} also showed significantly faster
cell proliferation as assessed by counting cells on a haemocytometer
every 24 h for 7 days (Fig. 5d) as well as in a BrdU assay for DNA
synthesis (Fig. 5e)

To determine the effect of Rheb mutants on mAb production and secretion
in these cells, we transfected cells with a vector encoding the heavy
and light chains of rabbit IgG1 and grew the cells in serum-free,
chemically-defined medium for 10 days with the cells being fed on day 2.
This more closely mimics the type of conditions the biopharmaceutical
industry would use when using a transient expression approach and CHO
cells to produce a recombinant IgG. We found that after 10 days of
growth, Rheb{[}T23M{]} and {[}E40K{]} promoted a 2-3 fold increase in
IgG1 yield (Fig. 5f). Testing accumulated antibody in the growth medium
every 24 h over the course of the experiment revealed that duration of
antibody secretion was similar in all cases, but that the rate of
release of secreted IgG1 was markedly increased in cells expressing
Rheb{[}T23M{]} or {[}E40K{]}. This suggests that Rheb{[}T23M{]} and
{[}E40K{]} promote both increased production and, very importantly,
secretion of a recombinant antibody.

\textbf{Discussion}

It is clear that any increase in IgG titre in medium is beneficial to
the biopharmaceutical industry in terms of enhancing the production of
the desired product and particularly its levels in the cell medium from
which is it harvested. Here we show that Rheb mutations we previously
identified are capable of increasing IgG titres in CHO cells grown in
suspension in chemically-defined, serum-free medium replicating
industrial growth conditions.

Consistent with our previous study (Jianling Xie et al., 2020), we found
that the exogenous expression of the Rheb mutants Rheb{[}T23M{]},
{[}Y35N{]} and {[}E40K{]} drive constitutive mTORC1 signalling through
insensitivity to TSC-GAP activity and thus rendering mTORC1 insensitive
to removal of upstream activators. We show that in HEK293 cells,
Rheb{[}T23M{]} and {[}E40K{]} drive an increased rate of protein
synthesis consistent with previous studies (Jianling Xie et al., 2020)
as well as the known function of mTORC1 as a key regulator of protein
synthesis (Proud, 2019) and ribosome biogenesis (Iadevaia et al., 2014).
mTORC1 also promotes mitochondrial function (de la Cruz L\selectlanguage{ngerman}ópez, Toledo
Guzmán, Sánchez, \& García Carrancá, 2019). A recent paper has also
shown the mTORC1 signaling can stimulate ATF4 expression independently
of the UPR and thereby upregulate a subset of ATF4 target genes
(Torrence et al., 2020). As a key activator of mTORC1 signalling, the
expression of an active Rheb mutant such as T23M can drive multiple
anabolic pathways, so that this single manipulation can lead to enhanced
cell growth and proliferation and faster protein synthesis. This
obviates the need to modify multiple genes thereby offering a
substantial advantage for cell engineering.

We unexpectedly found that Rheb{[}Y35N{]}, despite driving constitutive
mTORC1 signalling, did not increase protein synthesis. This is
consistent with our finding that, while Rheb{[}T23M and {[}E40K{]}
increased the levels of ATF4, Rheb{[}Y35N{]} did not. Rheb{[}T23M{]} and
{[}E40K{]} also resulted in an increase in markers of the integrated
stress response and expansion of the endoplasmic reticulum (ER).

These data suggest that the increase in protein synthesis driven by
constitutive activation of mTORC1 increases the protein folding load on
the ER. As unfolded proteins accumulate in the ER, the integrated stress
response becomes activated; in the short term, this drives increased
expression of proteins required for protein folding and expansion of the
ER. If this expansion is enough to accommodate the increased protein
folding requirements, cells can maintain increased protein synthesis
without progressing into apoptosis which can occur during prolonged
activation of the UPR (Shaffer et al., 2004; Sriburi et al., 2004; M.
Wang \& Kaufman, 2016). This mechanism allows cells to produce more
protein without adverse effects. The fact we only observe this
phenomenon in cells expressing Rheb{[}T23M{]} and {[}E40K{]} but not
Rheb{[}Y35N{]} is surprising and suggests that mTORC1 activation alone
is not sufficient to support a long-term increase in protein synthesis.
We previously showed through proteomic analysis of NIH3T3 cells stably
expressing Rheb{[}T23M{]} and {[}E40K{]} that additional pathways are
activated by these mutations. Cells expressing Rheb{[}T23M{]} showed
increased reliance on anerobic glycolysis while cells expressing
Rheb{[}E40K{]} showed increased autophagic flux (Jianling Xie et al.,
2020). It is possible that activation of these pathways does not occur
in Rheb{[}Y35N{]} so that cells expressing Rheb{[}Y35N{]} are not able
to sustain the energy or nutrient requirements to sustain prolonged
increases in protein synthesis.

We show that this mechanism of mTORC1-driven activation of the UPR is
required for increased secretion of the reporter
\emph{Gaussia}luciferase from CHO cells stably expressing GLuc when
grown in monolayer. This hypothesis is supported by the observation that
both inhibition of mTOR and inhibition of the ATF4 arm of the UPR
decreased GLuc secretion. We also observed a significant upregulation of
ATF4 in cells expressing Rheb{[}T23M{]} or {[}E40K{]}. We show that this
is also true for CHO cells grown in suspension in chemically-defined,
serum-free medium. We found that stable expression of Rheb{[}T23M{]} or
{[}E40K{]} resulted in around a 2-3 fold increase in IgG1 secretion
compared to Rheb{[}WT{]} overexpression or cells expressing only their
endogenous Rheb.

These results clearly point to a potential approach to significant
increasing the efficiency of production of commercially valuable
proteins. A further development of this approach would be to generate
cell lines stably expressing Rheb{[}T23M{]} or with the chromosomal
copies of the Rheb gene mutated appropriately. Since all cells in the
population would then be expressing mutant Rheb which is not the case
for transient transfection), an even greater augmentation of protein
output would be expected. The ability to double product output over the
same time-frame without increasing nutrient input promises to make
production of currently expensive treatments more efficient and may
therefore lead to significant reductions in the cost of treatment, thus
allowing more people being able to benefit from these highly effective
treatments and/or at a lower cost. Our findings may thus be of direct
value to the burgeoning `biological drug' sector.

\textbf{Acknowledgements}

Our initial studies in the UK were supported by Project Grants from the
UK Biotechnology \& Biological Sciences Research Council (BB/J007714/1
to CGP and BB/J006408/1 to CMS). Subsequent financial support was
provided by the South Australian Health \& Medical Research Institute,
SDP gratefully acknowledges funding through the {[}Australian{]}
Research Training Program.

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\textbf{Figure 1: Overexpression of Rheb promotes protein synthesis and
increases ER stress markers. (A)} HEK293 cells were transfected with
indicated vectors (EV = empty vector) and grown for 48 h prior to
harvest. Lysates were harvested for SDS-PAGE western blot analysis with
the indicated antibodies. SuNSET assays were then performed; the growth
medium was changed for EBSS 90 min prior to addition of 30 \selectlanguage{greek}µ\selectlanguage{english}M puromycin
for 30 min. Samples indicated `CHX' were pre-treated with cycloheximide,
to inhibit ongoing protein synthesis. \textbf{(B)} Quantification of
data from (A). \textbf{(C)} Lysates of cells that had been transfected
with the indicated vectors were analysed by SDS-PAGE and immunoblot
using the indicated antibodies. \textbf{(D-G)} RT-qPCR analysis for
known ER stress markers was performed on cDNA extracted from HEK293
cells expressing the indicated Rheb mutants or an empty vector (EV).
Figures represent mean \selectlanguage{ngerman}± s.d for 3 independent experiments. All graphs
represent mean ± s.d for 3 independent experiments. The data were
analysed via Student's t-test compared to EV. *: P \textless{} 0.05; **
P \textless{} 0.01; *** P \textless{} 0.001.

\textbf{Figure 2: Rheb-T23M and E40K drive increased ER volume.(A)}
Immunofluorescence of HEK293 cells transiently expressing the indicated
Rheb mutants or EV. Cells were transfected with indicated vectors 24 h
prior to fixation and addition of the indicated primary antibodies.
\textbf{(B)} Quantification of (A) for the ratio of area containing
Calnexin : area containing actin. Graph represent mean ± s.d for 3
independent experiments. The data were analysed via Student's t-test. *:
P \textless{} 0.05; ** P \textless{} 0.01.

\textbf{Figure 3: Rheb mutants drive constitutive mTORC1 signalling in
GLuc-CHO cells.} \textbf{(A)} SDS-PAGE and western blot analysis of
lysates harvested from GLuc-CHO cells transiently expressing the
indicated Rheb mutants or transfected with an empty vector (EV); some
were co-transfected with FLAG-TSC1/2, as indicated. The growth medium
was replaced with medium lacking FBS for 16 h followed by D-PBS for 60
min immediately prior to harvesting. \textbf{(B)} As in (A) for FLuc-CHO
cells. \textbf{(C)} GLuc-CHO cells expressing the indicated Rheb mutants
or an empty vector (EV) were grown in fully supplemented media with cell
number recorded every 24 h for 7 days. \textbf{(D)} As in (C) with cells
grown in media lacking FBS. Figures are representative of mean ± S.D for
3 independent experiments. \textbf{(E)} BrdU incorporation assay of
FLuc-CHO cells expressing the indicated Rheb mutants or EV. Cells were
allowed to grow in fully supplemented media for 24 h before addition of
BrdU for 1 h. \textbf{(F)} As in (E) for GLuc-CHO cells. All graphs
represent mean ± s.d for 3 independent experiments. The data were
analysed via Student's t-test compared to EV. *: P \textless{} 0.05; ***
P \textless{} 0.001.

\textbf{Figure 4: Rheb mutants promote increased secretion of Gaussia
Luciferase.} \textbf{(A)} CHO cells stably expressing Firefly luciferase
(FLuc) were transfected with the indicated Rheb mutants or an empty
vector (EV); FLuc assays were then performed every 48 h for 7 days.
Results are normalized to cell number. \textbf{(B)} Quantification of
data in \textbf{(A)} for day 7. \textbf{(C)} CHO cells stably expressing
Gaussia Luciferase (GLuc) were transfected with the indicated Rheb
mutants or an empty vector (EV) and treated with either DMSO (solid
line) or 1 \selectlanguage{greek}µ\selectlanguage{english}M AZD8055 (dashed line) for the duration of the experiment.
GLuc assays performed on growth media every 48 h for 7 days. Results are
normalized to cell number. \textbf{(D)} Quantification of data
in\textbf{(C)} for day 7. \textbf{(E)} As in (C), but cells were treated
with a cocktail of 1 \selectlanguage{greek}µ\selectlanguage{english}M iPERK and 1 \selectlanguage{greek}µ\selectlanguage{english}M ISRIB (dashed line). Experiments
shown (C) and (E) were performed simultaneously but are presented
separately for clarity. \textbf{(F)} SDS-PAGE western blot analysis of
cell lysates harvested from GLuc-CHO cells transiently transfected with
the indicated Rheb mutants or an empty vector (EV). All graphs represent
mean \selectlanguage{ngerman}± s.d for 3 independent experiments. The data were analysed via
Student's t-test. *: P \textless{} 0.05; ** P \textless{} 0.01; *** P
\textless{} 0.001.

\textbf{Figure 5: Rheb T23M and E40K drive increased secretion of mAb
from CHO-S cells.} \textbf{(A)} SDS-PAGE western blot analysis of
lysates harvested from Expi-CHO cells stably over-expressing the
indicated Rheb mutant or transfected with empty vector (EV).\textbf{(B)}
SDS-PAGE western blot analysis of lysates harvested from Expi-CHO cells
stably over-expressing the indicated Rheb mutant or transfected with
empty vector (EV). \textbf{(C)} EXPI-CHO cells stably expressing the
indicated Rheb mutant or endogenous Rheb (Endo) were grown for 48 h
prior treatment with 30 \selectlanguage{greek}µ\selectlanguage{english}Mt puromycin for 30 m. Lysates were then
harvested for SuNSET assays. \textbf{(D)} Expi-CHO cells stably
expressing the indicated Rheb mutants or endogenous Rheb (Endo) were
allowed to proliferate with cell number recorded every 24 h for 7 days.
\textbf{(E)} BrdU incorporation assay of Expi-CHO cells stably
expressing the indicated Rheb mutants or endogenous Rheb (Endo). Cells
were allowed to proliferate for 24 h before addition of BrdU for 1
h.\textbf{(F)} IgG yield from Expi-CHO cells stably expressing the
indicated Rheb mutant or endogenous Rheb after 10 days of
expression.\textbf{(G)} Time course of IgG secretion from Expi-CHO cells
stably expressing the indicated Rheb mutant. Figure represents n=1. All
other figures are representative of 3 independent experiments. All
graphs represent mean \selectlanguage{ngerman}± s.d for 3 independent experiments. The data were
analysed via Student's t-test compared to Endo. *: P \textless{} 0.05;
** P \textless{} 0.01; *** P \textless{} 0.001.

\textbf{Supplementary Figure 1: Mutant Rheb drives an increase in levels
of proteins of the ER stress response} . \textbf{Related to Figure
1}Quantification of SDS-PAGE western blot data for Fig. 1a. All graphs
represent mean ± s.d for 3 independent experiments. The data were
analysed via Student's t-test compared to Endo. *: P \textless{} 0.05.

\textbf{Supplementary Figure 2: Rheb-T23M displays increased
proliferation on media containing reduced FBS. Related to Figure 3.
(A)}GLuc-CHO cells expressing the indicated Rheb mutants or an empty
vector (EV) were grown in media supplemented with 1\% FBS with cell
number recorded every 24 h for 7 days. \textbf{(B)} f-CHO cells
expressing the indicated Rheb mutants or EV were grown in media
supplemented with 0.5\% FBS with cell number recorded every 24 h for 7
days. All graphs represent mean ± s.d for 3 independent experiments. The
data were analysed via Student's t-test compared to Endo. *** P
\textless{} 0.001.

\textbf{Supplementary Figure 3: Characterization of Expi-CHO cells
stably over expressing Rheb mutants. Related to Figure 5.} Sanger
sequencing results of Expi-CHO monoclonal cell lines transfected with
vector encoding both the indicated Rheb mutant and NeoR to grant
resistance to G-418. Cells were grown for 6 weeks in the presence of 1
\selectlanguage{greek}µ\selectlanguage{english}M G-418 before DNA was extracted and sequenced using primers specific
to the inserted plasmid.\selectlanguage{english}
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