Progress in Biophysics and Molecular Biology 147 (2019) 17e25

Contents lists available at ScienceDirect

Progress in Biophysics and Molecular Biology

journal homepage: www.elsevier.com/locate/pbiomolbio

The mTOR pathway: Implications for DNA replication

Noa Lamm, Samuel Rogers, Anthony J. Cesare*
Genome Integrity Unit, Children's Medical Research Institute, University of Sydney, Westmead, New South Wales, 2145, Australia

a r t i c l e i n f o a b s t r a c t

Article history: DNA replication plays a central role in genome health. Deleterious alteration of replication dynamics, or
Received 15 January 2019 “replication stress”, is a key driver of genome instability and oncogenesis. The replication stress response
Received in revised form is regulated by the ATR kinase, which functions to mitigate replication abnormalities through coordi-
1 April 2019
nated efforts that arrest the cell cycle and repair damaged replication forks. mTOR kinase regulates
Accepted 9 April 2019
signaling networks that control cell growth and metabolism in response to environmental cues and cell
Available online 13 April 2019
stress. In this review, we discuss interconnectivity between the ATR and mTOR pathways, and provide
putative mechanisms for mTOR engagement in DNA replication and the replication stress response.
Keywords:
DNA replication
Finally, we describe how connectivity between mTOR and replication stress may be exploited for cancer
Replication stress therapy.
mTOR © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
ATR license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Genome stability

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2. The canonical mTOR pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.1. Upstream and downstream targets of mTOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2. Evolutionary conservation of the TOR pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3. mTOR localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.4. Cross talk between mTOR complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.5. mTOR and cell cycle progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3. mTOR and DNA replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.1. Potential functions for mTOR in DNA replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1.1. ATR-CHK1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1.2. Ribonucleotide reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.1.3. FANCD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.1.4. Actin cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4. Targeting replication stress through mTOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
5. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Declaration of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

1. Introduction

DNA replication is a highly regulated process with profound

implications for genome stability. “Replication stress” is a general
* Corresponding author. term referring to any condition that alters DNA replication rates
E-mail address: [email protected] (A.J. Cesare).

https://doi.org/10.1016/j.pbiomolbio.2019.04.002
0079-6107/© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
18 N. Lamm et al. / Progress in Biophysics and Molecular Biology 147 (2019) 17e25

[reviewed in (Giannattasio and Branzei, 2017; Zeman and Cimprich, and Manning, 2008). Other mTORC1 regulators include the Wnt
2014; Zhang et al., 2018)]. There are many sources of replication signaling pathway (Inoki et al., 2006), the inflammatory cytokine
stress, including endogenous stress from shortages of histones or TNFa (Lee et al., 2007), the stress responsive metabolic regulator
deoxyribonucleotide triphosphates (dNTPs), hard to replicate DNA (AMPK) (Inoki et al., 2003), and regulated in development and DNA
sequences, and collisions between transcription and replication damage response 1 (REDD1) (Brugarolas et al., 2004). In contrast,
complexes [reviewed in (Giannattasio and Branzei, 2017; Zeman the upstream regulation of mTORC2 is poorly characterized
and Cimprich, 2014; Zhang et al., 2018)]. Exogenous factors, such (Cybulski and Hall, 2009). As with mTORC1, mTORC2 is responsive
as chemotherapeutics that negatively impact DNA replication, also to growth factors (e.g. IGF-1/PI3K) (Liu et al., 2015) and to the
induce replication stress. Notably, oncogene expression leads to ribosome maturation factor Nip7, although the mechanistic basis
replication stress early in cancer development through diverse for mTORC2 activation remains unclear (Zinzalla et al., 2011).
mechanisms that include depletion of the tightly regulated dNTP mTORC1 maintains the balance of anabolic processes including
pool (Bester et al., 2011). Genome instability is one of the canonical ribosome biogenesis and production of nucleotides, lipids, and
hallmarks of cancer, and replication stress is emerging as a major proteins, with catabolic processes that include cell cycle arrest, cell
driver of oncogenic genome alteration (Burrell et al., 2013; Hanahan death, and autophagy. Nucleotide production induced by mTORC1
and Weinberg, 2011). activation includes the de novo synthesis of both pyrimidines and
The replication stress response is triggered by the presence of purines through phosphorylation of p70S6K and ATF4. Specifically,
stalled replication forks. Once active, the replication stress response p70S6K activates CAD (Carbamoyl-Phosphate Synthetase 2,
evokes several physiological processes including: cell cycle arrest to Aspartate Transcarbamylase, and Dihydroorotase) an essential
prevent mitosis; inhibition of the de novo firing of replication ori- component of the de novo pyrimidine synthesis pathway (Ben-
gins and activation of dormant origins in replicons where replica- Sahra et al., 2013; Robitaille et al., 2013), whereas ATF4 activates
tion was already initiated; activation of pathways to increase the MTHFD2, to provide one-carbon units for purine synthesis (Ben-
cellular pool of dNTPs; and the stabilization, repair, and restart of Sahra et al., 2016). mTOR-dependent p70S6K activation also in-
stalled replication forks (Branzei and Foiani, 2010; Zeman and duces lipid production and glucose metabolism [reviewed thor-
Cimprich, 2014; Zhang et al., 2018). ATR (ataxia telangiectasia and oughly elsewhere (Saxton and Sabatini, 2017)].
rad3 related) is the main regulator of the replication stress Another mTORC1 anabolic role is the coordinated induction of
response, and through its effector CHK1 (checkpoint kinase 1), rRNA synthesis and ribosomal protein production to promote
controls these aforementioned processes [reviewed in (Saldivar ribosome biogenesis. rRNA synthesis requires transcription, which
et al., 2017)]. The ATR-CHK1 signaling axis is highly conserved, is regulated thorough an mTORC1 effect on Pol I and III (Kantidakis
with Mec1-Rad53 in S. cerevisiae and Rad3-Cds1 in S. pombe sharing et al., 2010; Mayer et al., 2004). However, the effect of mTORC1 on
many similarities with mammalian ATR-CHK1 both structurally and transcription is not restricted to rRNA synthesis only. Instead
functionally [reviewed in (Saldivar et al., 2017)]. mTORC1 inhibition effects global gene transcription via the regu-
The mechanistic target of rapamycin (mTOR) is an atypical lation of specific transcription factors [reviewed in (Laplante and
serine/threonine kinase belonging to the phosphatidylinositol 3- Sabatini, 2013)]. In addition to global effect on gene transcription,
kinase (PI3K)-related kinase (PIKK) family. The PIKK family in- mTORC1 globally controls protein production, including ribosomal
cludes ATR and the other major regulators of the DNA damage proteins. This is achieved through phosphorylation of two direct
response (ATM and DNA-PK), suggesting a common evolutionary targets: p70S6K and 4E-BP1(a member of the eukaryotic initiation
origin. Canonical mTOR function is to regulate cellular responses to factor 4E binding protein family). p70S6K phosphorylation is
a wide range of environmental stresses, including nutrient starva- important for translation initiation (Holz et al., 2005), while
tion, growth factor deprivation and hypoxia [reviewed in (Saxton phosphorylation of 4E-BP1 proteins enables dissociation of the
and Sabatini, 2017)]. However, recent evidence supports an translation initiation factor eIF4E and assembly of the mature 80S
expanded role for mTOR in the DNA damage and replication stress ribosome necessary for 5’ cap-dependent mRNA translation
responses. In this review, we summarize evidence linking mTOR to (Gingras et al., 1999). In addition, mTORC1 also promotes cell
DNA replication and the replication stress response and discuss growth by suppressing autophagy and protein turnover through
how this link might be exploited for cancer therapy. inhibition of several key protein targets that include: ULK1, a
regulator of autophagosome formation (Kim et al., 2011); TFEB, a
2. The canonical mTOR pathway transcription factor involved in expression of the autophagy ma-
chinery (Settembre et al., 2012); and ERK5, which regulates
2.1. Upstream and downstream targets of mTOR proteasome-dependent proteolysis (Rousseau and Bertolotti, 2016).
mTORC2 mainly controls cell proliferation, cell survival, and
mTOR is the catalytic subunit of two protein complexes, mTOR cytoskeleton rearrangements through three distinct signaling
complex 1 (mTORC1) and 2 (mTORC2) (Loewith et al., 2002; pathways. First, mTORC2 regulates the AGC (PKA/PKG/PKC) family
Sarbassov et al., 2005a). The mTORC1 and mTORC2 complexes of protein kinases including PKCa (Jacinto et al., 2004; Sarbassov
differ in their accessory subunits which mediate substrate speci- et al., 2004), PKCd (Gan et al., 2012) and PKCg (Thomanetz et al.,
ficity (Fig. 1). mTORC1 is activated in response to growth factors and 2013), all of which drive actin cytoskeleton remodeling and cell
mitogen-dependent signaling pathways through repression of the migration. Second, mTORC2 activates the PI3K/AKT signaling
inhibitory tuberous sclerosis complex (TSC). TSC is a heterotrimeric pathway that controls cell survival and proliferation through
complex consisting of TSC1, TSC2 and TBC1D7, which is converged several key substrates including the mTORC1 inhibitor TSC2
upon by several activating pathways upstream of mTOR (Dibble (Sarbassov et al., 2005b). Finally, mTORC2 activates SGK1 which is
et al., 2012). For example, the IGF-1 (insulin/insulin-like growth also a member of the AGC family and important to the cellular
factor-1) pathway induces production of PIP3 (phosphatidylinositol stress response through its role in ion transport (Garcia-Martinez
(3,4,5) trisphosphates) by PI3K (phosphoinositide 3-kinase), which and Alessi, 2008) (Fig. 1).
in turn triggers AKT-dependent phosphorylation of TSC2 that re- Notably, an in-depth understanding of the direct targets of
leases mTOR1 repression (Huang and Manning, 2008) (Fig. 1). mTORC1 and 2 were enabled by the identification of Rapamycin, a
Similarly, RTK (receptor tyrosine kinase)-dependent Ras signaling compound with antifungal, immunosuppressive and antitumor
also phosphorylates TSC2 to release and activate mTORC1 (Huang characteristics (Martel et al., 1977; Vezina et al., 1975). Rapamycin,
 N. Lamm et al. / Progress in Biophysics and Molecular Biology 147 (2019) 17e25 19

Fig. 1. The mTORC1 and mTORC2 signaling pathways. The mTORC1 and 2 complexes are illustrated with accessory proteins, based on the 5.9 Å cryo-EM structure of mTORC1 and
2 associated with the FKBP12-rapamycin complex (Yang et al., 2013). Both mTORC1 and 2 are activated in response to growth factors. AKT activation stimulates mTORC1 activation
by inhibiting the TSC complex, leading to mTORC1 derepression. As a substrate of mTORC2, AKT also functions to establish a feedback loop between the two TORC complexes. Major
pathways downstream of mTORC1 and 2 are illustrated.

and the corresponding family of derivatives that function similarly synthesis [reviewed in (Roelants et al., 2017)].
(i.e. “rapalogs”), primarily inhibit mTORC1 but will also inhibit
mTORC2 under chronic exposure (Sarbassov et al., 2006). Rapa- 2.3. mTOR localization
mycin and the rapalogs form a complex with the FK506 binding
protein-12 (FKBP-12) that binds mTOR to block mTORC1 activity mTOR protein has been found at several cellular locations
(Chung et al., 1992). This inhibits mTORC1-dependent cell cycle [reviewed in (Betz and Hall, 2013)]. There is a broad consensus that
progression, cell survival, and angiogenesis. The second generation TORC1 is found at the lysosome in diverse species from yeast to
of mTOR inhibitors much as PP242, BEZ235 and INK128 block the humans, though it has also been reported in the ER, Golgi, cell
catalytic activity of mTOR and therefore inhibit both mTORC1 and 2 membrane and mitochondria as well [reviewed in (Betz and Hall,
[reviewed in (Zheng and Jiang, 2015)]. To date, no specific mTORC2 2013)]. Components of the mTORC1 signaling pathway, including
inhibitor has been identified. mTOR itself, Raptor, and p70S6K, have been detected in the nucleus,
however it is not clear if they form a functional nuclear complex
2.2. Evolutionary conservation of the TOR pathway (Audet-Walsh et al., 2017; Rosner and Hengstschlager, 2008).
TORC2 was documented at the plasma membrane (Ebner et al.,
The role of the TOR pathway in proliferation and growth regu- 2017), endosomal vesicles (Ebner et al., 2017), mitochondria-
lation is evolutionary conserved in most eukaryotes including yeast associated ER membrane (Boulbes et al., 2011) and in the mito-
[reviewed in (Loewith and Hall, 2011)], flies (Zhang et al., 2000), chondria (Desai et al., 2002). However, several papers identified
plants (Xiong and Sheen, 2014) and mammals [reviewed in (Saxton that mTORC2 shuttles between the nucleoplasm and the cytoplasm
and Sabatini, 2017)]. The “two branches-two complexes” mode of but its specific role inside the nucleus is unknown (Rosner and
TORC substrate specificity is also conserved across eukaryotic Hengstschlager, 2008, 2012). How the distribution of TORC1 and
species [reviewed in (Wullschleger et al., 2006)]. In budding yeast 2 to various locations is regulated, and how this localization effects
there are two genes encoding TOR catalytic subunits. Yeast Tor1 can complex function, is a matter for future studies.
function as the catalytic subunit in either TORC1 or TORC2, whereas
Tor2 serves only in the TORC2 complex, [reviewed in (Loewith and 2.4. Cross talk between mTOR complexes
Hall, 2011)). Like the mammalian complex, yeast TORC1 responds
to diverse environmental cues and controls anabolic processes, and A series of complex and context-dependent feedback loops
yeast TORC2 induces actin cytoskeleton rearrangements. In exist between the mTORC complexes [reviewed in (Xie and
S. cerevisiae TORC2 also has a direct role in regulating lipid Proud, 2013)]. Both mTOR complexes are activated by insulin
20 N. Lamm et al. / Progress in Biophysics and Molecular Biology 147 (2019) 17e25

and insulin-like growth factors (IGFs). The insulin/PI3K pathway

activates AKT by stimulating its phosphorylation at AKT-Thr308
(Williams et al., 2000). AKT then phosphorylates and represses
TSC2 to activate mTORC1 (Inoki et al., 2002) and the mTORC2
subunit Sin1 on T86 in response to insulin (Humphrey et al.,
2013). However, AKT is also an mTORC2 substrate, as mTORC2
phosphorylates AKT on Ser 473 (Guertin et al., 2006). However,
AKT-pSer473 was not found to be important for mTORC1 acti-
vation. Thus, while AKT is upstream of mTORC1 and downstream
of mTORC2, it is not clear whether it mediates crosstalk between
the two mTORC complexes [reviewed in (Huang and Manning,
2009)]. mTORC1 can impair mTORC2 activation, as well as
inhibit its own activity, thorough a negative autoregulatory loop
by either phosphorylating insulin receptor substrate-1 (IRS-1)
(Um et al., 2004) or Grb10 (Hsu et al., 2011). Both IRS-1 and Grb10
are involved in signaling events upstream of PI3K, hence their
inhibition suppresses both mTOR complexes. A further inhibitory
link from mTORC1 to mTORC2 involves p70S6K. mTORC1-
dependent p70S6K phosphorylation of the mTORC2 subunit
Sin1 in response to IGF1/insulin was shown to dissociate Sin1
from the mTORC2 complex and inhibit mTORC2 activity (Liu
et al., 2013). However, an independent study identified that
Sin1 phosphorylation in response to IGF1/insulin augments
mTORC2 activity, suggesting instead a positive feedback loop
(Humphrey et al., 2013). As these studies were done in different
cell types, it is possible that the crosstalk between mTORC com-
plexes is regulated in a cell type specific manner.

2.5. mTOR and cell cycle progression

Cell cycle progression demands a large supply of nutrients to

ensure adequate energy and protein synthesis to support cell
growth. As the main coordinator of nutrient availability and Fig. 2. mTORC1 and 2 regulate the G1/S transition. mTORC1 upregulates cyclins D
metabolism, mTORC1 can regulate cell cycle progression through and E through regulation of transcription and translation. mTORC1 and 2 also regulate
its functions controlling energy homeostasis [reviewed in (Cuyas phosphorylation of the CDK inhibitor p27kip1, blocking p27kip1 nuclear localization. The
net effect of these activities promotes stabilization of the Cyclin DeCDK4/6 complex
et al., 2014)]. However, beyond its control of cellular metabolism,
and CyclinE-CDK2 required for G1/S transition.
mTORC1 can also directly affect the cyclin dependent kinases
(CDKs), their obligate cyclin binding partners, and the CDK in-
hibitors which regulate cell cycle progression (Fig. 2). 3. mTOR and DNA replication
Inhibiting mTOR activity, by rapamycin or nutrient starvation,
induces G1-phase cell cycle arrest in p53 competent cells (Foster Many lines of evidence link mTOR regulation with replication
et al., 2010). Both the 4E-BP1 and p70S6K downstream arms of dynamics (Fig. 3). First, inhibition of mTOR in p53-deficient mouse
the TORC1 signaling cascade are independently required to mediate embryo fibroblasts and cancer cells does not induce G1-arrest as
mTOR-dependent progression from G1-phase by transcriptionally observed in p53-proficient cell lines. Instead inhibiting mTOR in
and translationally regulating the G1/S transition cyclins (D-type p53-compromised tissues induces apoptosis specifically during S-
and E-type cyclins) (Averous et al., 2008; Fingar et al., 2004; Oka phase as the DNA is replicated (Huang et al., 2001, 2003). This
et al., 2013). Additionally, the CDK inhibitor p27kip1 is phosphory- suggests mTOR activity is required to effectively complete DNA
lated by AKT or SGK1 (Hong et al., 2008). Phosphorylation of p27kip1 replication.
sequesters this protein in the cytoplasm to block its nuclear func- Second, treating budding yeast with Rapamycin dramatically
tion as a CDK inhibitor, thus promoting stabilization of the cyclin increased cell lethality, specifically during S-phase, in response to
DeCDK4/6 complex and G1/S transition (Medema et al., 2000). the alkylating agent methyl-methane sulfonate (MMS) (Shen et al.,
Furthermore, in response to DNA damage or replication stress, 2007). The MMS mechanism of action includes replication stress
p53 negatively regulates mTORC1 while simultaneously inducing induction, and the observed S-phase lethality suggested a role for
cell cycle arrest via induction of p21CIP1/WAF1 (Feng et al., 2007). TORC in promoting S-phase progression in the presence of DNA
Notably, there is a bi-directional crosstalk between mTORC1 and damage. Furthermore, direct analysis of replication fork progres-
p53, as mTORC1 activates p53 in response to DNA damage via the sion in MMS treated cells revealed that TOR function includes
p70S6K pathway (Lai et al., 2010). The p53-mTORC axis can be pro- promoting replication fork stability (Shen et al., 2007). Interest-
survival or pro-cell death depending on many factors which include ingly, cells from Tuberous Sclerosis (TSC) patients, which are
the cell type, duration of stimuli, and the amount of damage characterized by exorbitant mTORC1 signaling due to the loss of
[reviewed in (Ma et al., 2018)]. In recent years several studies have function of the mTOR inhibitory TSC complex, display replication
also shown that different members in the TOR pathway also stress and asymmetric fork progression, aberrant S-phase pro-
regulate mitotic progress including mTOR itself, its regulators TSC gression, and hypersensitivity to genotoxic stress (Pai et al., 2016).
and raptor, and the mTORC1 target p70S6K [reviewed in (Cuyas Together, these data suggest that disrupting mTOR signaling, by
et al., 2014)].
 N. Lamm et al. / Progress in Biophysics and Molecular Biology 147 (2019) 17e25 21

addition, mTOR itself is transiently phosphorylated following DNA

damage in an ATR-dependent manner (Selvarajah et al., 2015; Shen
et al., 2007).
Conversely, evidence supporting mTORC1/2 regulation of ATR-
CHK1 signaling induced by replication stress was found in several
cancer cell lines (Selvarajah et al., 2015; Zhou et al., 2017). Zhou
et al. identified in rhabdomyosarcoma xenografts and cultured
rhabdomyosarcoma cells that mTORC1 suppressed spontaneous
DNA damage and replication stress by elevating CHK1 protein
levels (Zhou et al., 2017). The effect was not mediated by 4E-BP1
and cap-dependent protein translation, but rather by p70S6K-
dependent upregulation of the G1/S cyclin-dependent kinases
(CDK4 and 6) required to induce CHK1 transcription. Furthermore,
in this context the mTORC1-p70S6K axis was essential for increases
in CHK1 transcription, whereas the mTORC2-AKT axis promoted
this effect but was not essential (Zhou et al., 2017). In breast and
colorectal cancer cells, mTORC2 was found to be essential for CHK1
upregulation in response to DNA damage during S-phase. Ablating
Fig. 3. Potential mTOR functions in DNA replication. A stalled replication fork is mTORC2 prevented DNA damage-induced S and G2/M cell cycle
illustrated with the leading and lagging strand DNA polymerases, the mini-
arrest, as well as total CHK1 protein upregulation and phosphory-
chromosome maintenance (MCM) 2e7 helicase, the FANCD2 repair factor, and ATR
kinase. ATR kinase regulates the replication stress response and directly phosphory- lation, while mTORC1 was dispensable in these cell lines. Crosstalk
lates mTOR. mTORC1 and 2 participate in the replication stress through multiple between the ATR and mTOR complexes thus appears to be regu-
pathways. mTORC1 and 2 upregulate the ATR effector CHK1 to promote the replication lated with cell type specificity (Selvarajah et al., 2015).
stress response. mTORC 1 and 2 also upregulate FANCD2 transcription by blocking the
Cumulatively, the data reveal reciprocal regulation of mTOR and
nuclear localization of NF-kB, which suppresses FANCD2 expression. Additionally,
mTORC1 and 2 also upregulate RNR complex members RRM1 and 2, which catalyze ATR-CHK1 signaling in response to S-phase DNA damage. This
dNTP production during the replication stress response. interaction is essential for promoting cell cycle arrest, repair, and
survival under replication stress conditions. Moreover, both
mTORC1 and 2 affect the ATR-CHK1 pathway independently and
either inhibition or upregulation, disrupts the DNA replication through different modes of regulation. Further studies are required
program. to characterize the complicated nature of those feedback loops and
A third line of evidence comes from several yeast mutants with the unique roles that mTORC1 and 2 play in response to impaired S-
hypomorphic alleles of the replication initiation complex genes, phase progression.
CDC45 and DPB11, which exhibit perturbations in DNA replication
and an increased sensitivity to TOR inhibitors (Shen et al., 2007). 3.1.2. Ribonucleotide reductase
Additionally, mutant fission yeast cells defective for TORC2 or the Tight regulation of dNTP pools is essential to maintain replica-
downstream AGC-like kinase, Gad8, are highly sensitive to chronic tion fork speed in eukaryotic cells, and elevation of the dNTP pool is
replication stress but are insensitive to ionizing radiation a physiological outcome of the replication stress response (Zhao
(Schonbrun et al., 2013). et al., 2001). Replication stressed cells elevate their dNTP pool by
Lastly, in an unbiased screen that analyzed the translatome and ATReCHK1 dependent upregulation of RRM2 (Ribonucleoside-
transcriptome changes induced by mTORC1/2 in response to DNA diphosphate Reductase subunit M2), a subunit of the Ribonucleo-
damage in breast cancer cells, the most differentially expressed tide Reductase (RNR) complex that catalyzes dNTP production from
genes were DNA replication proteins (Silvera et al., 2017). This in- ribonucleotides (NTPs) (Buisson et al., 2015). Consistent with a
cludes members of the DNA polymerase family including Pol-E2, protective capacity in the replication stress response, high levels of
Pol-q, PolG2 and PolA2, as well as licensing and replication fac- RRM2 were shown to suppress different phenotypes associated
tors such as MCM3, MCM6, ORC6, CLSPN, PCNA and GINS2. These with ATR dysfunction and insufficiency (Chabes et al., 2003). A
genes were dependent on mTORC1/2 regulation at both the mRNA similar outcome occurs in yeast, where the replication checkpoint
and protein level. Furthermore, mTORC1/2 inhibition induced (Mec-Rad53 in S. cerevisiae and Rad3-Cds1 in S. pombe) leads to
persistent replication stress, evidenced by S-phase arrest, sustained upregulation of RNR through nucleus-to-cytoplasm redistribution
phosphorylation of CHK1, ATM and CHK2, and g-H2AX foci for- of RNR subunits and increased transcription (Bondar et al., 2004;
mation (Silvera et al., 2017). Interestingly, the global transcrip- Lee and Elledge, 2006).
tional/translational response to DNA damage was dependent on TORC1 facilitates NTP biosynthesis. This role was traditionally
both mTORC1 and 2, as independent inhibition of mTORC1 or 2 did assumed to be part of the TORC1 program to stimulate ribosome
not impair the coordinated response to DNA damage nor did it biogenesis, a process that requires a large NTP pool necessary to
induce replication stress (Silvera et al., 2017). support elevated rRNA transcription. However, TORC1 also modu-
lates RNR activity, to facilitate dNTP synthesis from the cellular NTP
3.1. Potential functions for mTOR in DNA replication pool (Shen et al., 2007). In S. cerevisiae, TORC1 induces transcription
of RNR1 and 3 following replication stress in a Rad53-dependent
3.1.1. ATR-CHK1 manner to promote cell survival at the cost of increased mutation
Several lines of evidence are congruent with bi-directional rates (Shen et al., 2007). In mammals, mTORC1 positively controls
feedback between the mTOR and the ATR-CHK1 signaling axes. In both RRM1 (ribonucleotide reductase large subunit 1) and RRM2 in
a large-scale proteomic analysis to identify ATM and ATR substrates various cancer cell lines and mouse tumor xenografts. This occurs
in human 293T cells, several proteins from the IGF1-AKT-mTOR through a transcriptional mechanism dependent on p70S6K upre-
pathway were identified, including: TSC1, AKT, 4E-BP1 and p70S6K. gulation of CDK4/6 activity, and eIF-4E cap-dependent protein
Suggesting these proteins are directly phosphorylated by ATM or translation (He et al., 2017). Thus, mTORC signaling plays a role in
ATR under DNA damage conditions (Matsuoka et al., 2007). In regulating the nucleotide pool in cancer cells and in response to
22 N. Lamm et al. / Progress in Biophysics and Molecular Biology 147 (2019) 17e25

replication stress. role in DNA replication and genome stability thorough an effect on
In S. pombe, TORC2 and its mediator Gad8 also regulate RNR actin remodeling. However, recent studies identified that nuclear F-
expression through interaction with the MluI cell cycle box-binding actin participates in drosophila and mammalian DSB repair, though
factor (MBF) transcription complex. In response to replication no link to mTORC function was explored (Caridi et al., 2018;
stress, TORC2 and Gad8 relocate to the nucleus where Gad8 binds Schrank et al., 2018). Additionally, during G1-phase, F-actin plays
the MBF transcription complex inducing the transcription of role in replication initiation through transcription regulation
several genes that participate in the G1/S transition and the repli- (Parisis et al., 2017). Importantly, under nucleotide depletion
cation stress response. This includes, cdt2þ, an adaptor subunit of induced by HU, the levels of nuclear actin and two factors that
an E3 ubiquitin ligase essential for initiating DNA replication; stimulate actin polymerization, IQGAP1(IQ Motif Containing
cdc18þ, a MCM complex loader; and cdc22þ, which encodes for the GTPase Activating Protein 1) and Rac1 GTPase (Rac family small
large subunit of the RNR complex (Cohen et al., 2016). GTPase), are significantly increased (Johnson et al., 2013). IQGAP1 is
a master regulator of actin dynamics and is a substrate of the
3.1.3. FANCD2 mTOR2 effector PKCε (Grohmanova et al., 2004), and the Rac1
FANCD2 belongs to the Fanconi Anaemia Pathway required for GTPase regulates both mTORC1 and 2 and is regulated by mTORC2
repair of double strand breaks (DSBs) and stalled replication forks (Saci et al., 2011). However, whether mTOR induces actin poly-
[reviewed in (Federico et al., 2018)]. In the replication stress merization in response to replication stress, and what role actin
response, ATR mediates the transient association of FANDC2 with dynamics play in the replication stress response, is a matter for
the MCM helicase complex at stalled replication forks to control further study.
replisome function (Lossaint et al., 2013). FANCD2 also recruits
FAN1 (Fanconi Anemia associated Nuclease 1), a 50 flap endonu- 4. Targeting replication stress through mTOR
clease to stalled replication forks to re-start DNA replication and to
prevent chromosome abnormalities (Lachaud et al., 2016). Aberrant mTOR activation contributes to the pathogenesis of
mTORC1 regulates FANCD2 through at least two different many tumor types [reviewed in (Kim et al., 2017; Meric-Bernstam
mechanisms. In paediatric rhabdomyosarcoma cells and mouse and Gonzalez-Angulo, 2009; Mossmann et al., 2018)]. mTOR itself
xenografts, mTORC1 was shown to regulate FANCD2 transcription is rarely mutated, but rather is affected by modulation of its up-
by the p70S6K signaling arm that promotes CDK4/6 activity (Shen stream regulators or downstream effectors. Oncogenic PI3K/AKT
et al., 2013). Inhibiting TORC1, or knockdown of mTOR by siRNA, signaling, which occurs upstream of mTOR, is altered by mutation,
decreased FANCD2 protein levels both in vivo and in vitro. A overexpression, or deletion in many types of cancer including:
different mechanism was shown in hematopoietic stem and pro- breast, endometrial thyroid, prostate, melanoma and glioblastoma
genitor cells where mTOR deficiency increases phosphorylation of [reviewed in (Meric-Bernstam and Gonzalez-Angulo, 2009)). p53
NF-kB and promotes its translocation to the nucleus. There, NF-kB and LKB1, negative regulators of mTOR, are commonly mutated
binds to FANCD2 promotor and suppresses FANCD2 expression. across a variety of tumor types [reviewed in (Saxton and Sabatini,
Interestingly, for the NF-kB-dependent reduction of FANCD2 2017)]. Since these oncogenic pathways converge on mTOR, it is
expression both TORC1 and 2 complexes appear to be required not surprising that an immense research effort has focused on the
(Guo et al., 2013). development of mTOR inhibitors for cancer therapy. The first gen-
eration “rapalogs” were poorly tolerated, and therefore have
3.1.4. Actin cytoskeleton limited clinical utility. However, second generation catalytic in-
One of the main functions of TORC2 is to regulate the poly- hibitors demonstrated important clinical benefits in several cancer
merization of monomeric or globular actin to filamentous actin (F- types. Unfortunately, the success of single-agent therapy in the
actin). This is mediated through TORC2 regulation of the AGC- mTOR pathway was limited and mainly resulted in disease stabi-
family of kinases (the PKC proteins in mammals and Ypk1 and lization rather than regression [reviewed in (Meric-Bernstam and
Ypk2 in yeast) (Kamada et al., 2005). Several studies in yeast have Gonzalez-Angulo, 2009)].
demonstrated that TORC2 impacts replication integrity and As outlined here, our assertion is that mTOR plays an important
genome stability through an undefined role in actin dynamics. A role in DNA replication and the replication stress response. Notably,
chemical genetic screen performed in S. cerevisiae identified a role combining classic chemotherapies that interfere with DNA repli-
for TORC2 in mediating the survival of cells exposed to replication cation, such as cisplatin, melphalan, and etoposide, with mTOR
stress, oxidative damage, or break-inducing agents like Zeocin or inhibitors proved to be highly lethal in both cell line and mouse
ionizing radiation (Shimada et al., 2013). Further characterization models (Mondesire et al., 2004; Shen et al., 2013; Silvera et al.,
identified that the functional outcomes of inhibiting TORC2 could 2017; Steelman et al., 2008; Zhou et al., 2017). For example,
be recapitulated by ablating Ypk1 and/or 2, or by preventing actin RAD001 (everolimus), a rapamycin derivative, dramatically en-
polymerization (Shimada et al., 2013). These results indicated that hances cisplatin-induced apoptosis in wild-type p53 human non-
TORC2-dependent F-actin regulation participated in the cellular small cell lung carcinoma, but not mutant p53 tumor cells
response to low levels of DNA damage. Similar results were ob- (Beuvink et al., 2005). Rapamycin was also shown to enhance
tained from studies in S. pombe where disruption of Gad8, a cisplatin-induced apoptosis in human promyelocytic leukemia cell
mediator of TORC2-dpendent actin remodeling (Ikai et al., 2011), line HL-60 and the human ovarian cancer cell line SKOV3 (Shi et al.,
resulted in hypersensitivity to DNA-damaging agents (Schonbrun 1995). Treatment with the mTOR inhibitor AZD8055 together with
et al., 2009). A follow up study suggested that the TORC2 com- melphalan showed synthetic lethality in cultured RH30 cells (Shen
plex was specifically required for cell survival under chronic DNA et al., 2013). Combinatorial mTOR inhibition with rapamycin, along
replication stress induced by hydroxyurea (HU), MMS, or campto- with the genotoxic chemotherapeutic Paclitaxel, enhanced anti-
thecin (Schonbrun et al., 2013). Furthermore, synthetic lethal in- tumor efficacy in vivo in nude mice bearing breast cancer xeno-
teractions were identified between Tor2 or gad8 mutants, and grafts (Mondesire et al., 2004). Considering the evidence described
deletions of DNA repair or replication fork re-start genes, such as in this review, a compelling model for the enhanced lethality of
mus81, mms1, or mms22, even in the absence of DNA damage- genotoxic stress combined with mTOR inhibition results from a
inducing agents (Schonbrun et al., 2013). diminished capacity in the treated cells to tolerate replication
It is currently unknown whether mammalian mTORC2 plays stress.
 N. Lamm et al. / Progress in Biophysics and Molecular Biology 147 (2019) 17e25 23

In the last two decades, significant efforts were deployed to Acknowledgements

develop therapies, such as ATR and CHK1 inhibitors, that target the
replication stress response [reviewed in (Forment and O'Connor, Noa Lamm is supported by an early career fellowship from the
2018)]. Interestingly, combinatorial CHK1 and mTOR inhibition Cancer Institute NSW. Research in the Cesare lab is supported by
showed promising results. This specific treatment induced syner- grants from the Australian NHMRC (1106241, 1104461, 1162886),
gistic cytotoxicity in p53 mutant colon cancer cell lines (Massey the Goodridge Foundation, the Sydney West Radiation Oncology
et al., 2016) and in Ewing sarcoma cell lines (Koppenhafer et al., Network, and philanthropy from Stanford Brown, Inc (Sydney,
2018). Thus, combining mTOR inhibitors with factors required to Australia).
repair replication stress-induced damage may be a promising
approach for cancer therapy. Appendix A. Supplementary data

5. Conclusions and future perspectives Supplementary data to this article can be found online at
https://doi.org/10.1016/j.pbiomolbio.2019.04.002.
Our collective knowledge of the replication stress response has
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