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 López, 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 Gaussialuciferase 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.