CANCER
MTOR signaling orchestrates stress-induced
mutagenesis, facilitating adaptive evolution in cancer
Arcadi Cipponi1,2, David L. Goode3,4, Justin Bedo5,6,7, Mark J. McCabe2,8, Marina Pajic1,2,
David R. Croucher1,2, Alvaro Gonzalez Rajal^1 , Simon R. Junankar1,2, Darren N. Saunders^9 ,
Pavel Lobachevsky^3 , Anthony T. Papenfuss5,6,7,10, Danielle Nessem^1 , Max Nobis1,2, Sean C. Warren1,2,
Paul Timpson1,2, Mark Cowley2,8, Ana C. Vargas^11 , Min R. Qiu2,12, Daniele G. Generali13,14,
Shivakumar Keerthikumar3,4, Uyen Nguyen^1 , Niall M. Corcoran15,16,17, Georgina V. Long18,19,20,21,
Jean-Yves Blay22,23, David M. Thomas1,2
In microorganisms, evolutionarily conserved mechanisms facilitate adaptation to harsh conditions through
stress-induced mutagenesis (SIM). Analogous processes may underpin progression and therapeutic failure in
human cancer. We describe SIM in multiple in vitro andin vivo models of human cancers under nongenotoxic
drug selection, paradoxically enhancing adaptation at a competing intrinsic fitness cost. A genome-wide
approach identified the mechanistic target of rapamycin (MTOR) as a stress-sensing rheostat mediating SIM
across multiple cancer types and conditions. These observations are consistent with a two-phase model for
drug resistance, in which an initially rapid expansion of genetic diversity is counterbalanced by an intrinsic
fitness penalty, subsequently normalizing to complete adaptation under the new conditions. This model
suggests synthetic lethal strategies to minimize resistance to anticancer therapy.
G
enetic diversity is central to adaptation
and evolution in cancer. Mutagenesis in
malignancies occurs incrementally ( 1 , 2 )
or in transient bursts ( 3 – 7 ), providing
increased cell-to-cell variation that facil-
itates adaptation to selective pressures, including
anticancer therapies. Paradoxically, mutagenesis
induced by some therapies, such as ionizing
radiation, forms a core component of treatment
for most cancers. The evolutionary effects of
mutagenesis appear to be context dependent;
they are potentially harmful under favorable
conditions yet beneficial under stressed con-
ditions. Stress-induced mutagenesis (SIM)
programs that link environmental conditions
to genetic diversity are a common atavistic
theme across the phylogenetic tree ( 8 )and
are well described in prokaryotes ( 9 )and
lower eukaryotes ( 10 – 13 ).
Here, we sought evidence for cognate pro-
grams in cancer. We first looked for accumu-
lation of DNA damage ( 14 ) in human cancers
exposed to therapies not considered directly
genotoxic. Phosphorylated histone H2AX
(g-H2AX) was used as a marker of DNA double-
strand breaks (DSBs) in pre- and posttreatment
samples taken from diverse cohorts of chemo-
and radiotherapy-naïve cancer patients. In-
creased DNA damage was consistently observed
in prostate cancer patients treated with andro-
gen deprivation therapy, in breast cancer pa-
tients treated with the aromatase inhibitor
letrozole, in melanoma patients treated with
the BRAF inhibitors dabrafenib or vemur-
afenib, and in patients with gastrointestinal
stromal tumors treated with the KIT inhibi-
tor imatinib (Fig. 1A and fig. S1). DNA DSBs
were also increased in patient-derived xeno-
graft (PDX) pancreatic cancer models treated
with the CDK4/6 inhibitor palbociclib and
the epidermal growth factor receptor (EGFR)
inhibitor erlotinib, suggesting that increased
DNA damage is a recurrent feature in human
cancers exposed to nongenotoxic therapies.
We interrogated this phenomenon further
in vitro using multiple human cancer cell lines
exposed to nongenotoxic drug selection. Drugs
specific to genetic targets in each cell line were
titrated to near-extinction conditions, consist-
ently generating one to 20 resistant colonies
per 100,000 cells (Fig. 1B and table S1), which
were analyzed for genetic and molecular fea-
tures, as well as drug sensitivity (fig. S2). All
model systems displayed increased DSBs early
during evolution (Fig. 1C), which decreased to
baseline during subsequent culture (fig. S3).
The levels of DSBs were stable in the absence
of selection.To further characterize this phenomenon, we
undertook whole-genome sequencing (average
read depth 116×) on single cell–derived clonal
populations obtained from untreated and early-
phase, drug-resistant 94T778 human liposarcoma
and SKMEL28 human melanoma lines. Phyloge-
netic trees were computed on the basis of copy
number changes, revealing in both lines distinct
clusters of untreated and drug-resistant clones
sharing a common trunk. The observed lengths
of the trunks (0.414 and 0.539 arbitrary units for
94T778 and SKMEL28 clones, respectively) were
significantly longer than the average branch
lengths of the respective parental clones (0.118
in 94T778,P= 1.8e-27, and 0.209 in SKMEL28,
P= 1.4e-6), consistent with accelerated ge-
nomic evolution induced by the therapeutic
bottleneck (Fig. 1D and figs. S4 and S5). To
formally estimate mutation rates, we expanded
clones using an equal number of generations
(20 for 94T778; 21 for SKMEL28). Taking into
account differential senescence and cell death
(figs. S6 and S7, tables S2 and S3, and materials
and methods), we quantified the percentage of
de novo single nucleotide variants (SNVs) as
the number of subclonal SNVs divided by
the number of total unique SNVs. Intraclonal
diversity, as measured by the number of de novo
SNVs, was significantly higher in the resistant
populations in both cell lines (Fig. 1E), consistent
with higher mutation rates. We sought evi-
dence for resistance-specific mutational signa-
tures but observed no consistent patterns (fig.
S8). We also performed targeted sequencing
(figs. S9 to S11) on bulk populations, which
revealed transiently increased structural and
single nucleotide variation early during drug
selection.
To further characterize the dynamics of ge-
nomic instability, the 94T778 cell line, con-
taining two amplified targetable oncogenes
(CDK4 and MDM2), was exposed to palboci-
clib or to nutlin-3a, an inhibitor of the p53–
MDM2 interaction. Cells were transduced at
both early and late time points of evolution
with a single-copy fluorescent reporter gene
(mCherry). In this system, genomic alterations
lead to loss of gene expression. In agreement
with the targeted sequencing data, drug-
resistant cell populations assayed early during
evolution demonstrated a significant loss of
fluorescence compared with baseline or late
time points (fig. S12). To approximate theRESEARCH
Cipponiet al.,Science 368 , 1127–1131 (2020) 5 June 2020 1of5
(^1) The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia. (^2) St. Vincent's Clinical School, University of New South Wales, Sydney, NSW, Australia.
(^3) Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. (^4) Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, VIC, Australia. (^5) Bioinformatics Division, Walter and
Eliza Hall Institute of Medical Research, Parkville, VIC, Australia.^6 Department of Computing and Information Systems, the University of Melbourne, Parkville, VIC, Australia.^7 Peter MacCallum
Cancer Centre, Parkville, VIC, Australia.^8 Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.^9 School of Medical Sciences, University of
New South Wales, NSW, Australia. 12 10 Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia.^11 Douglass Hanly Moir Pathology, Turramurra, NSW, Australia.
Anatomical and Molecular Oncology Pathology, SYDPATH, St. Vincent’s Hospital, Darlinghurst, NSW, Australia.^13 Department of Medical, Surgery and Health Sciences, University of Trieste,
Trieste, Italy.^14 Breast Cancer Unit and Translational Research Unit, ASST Cremona, Cremona, Italy.^15 Division of Urology, Royal Melbourne Hospital, Parkville, VIC, Australia.^16 Department of
Urology, Peninsula Health, Frankston, VIC, Australia.^17 Department of Surgery, University of Melbourne, VIC, Australia.^18 Melanoma Institute Australia, Wollstonecraft, NSW, Australia.^19 The
University of Sydney, Sydney, NSW, Australia.^20 Royal North Shore Hospital and Mater Hospital, Sydney, NSW, Australia.^21 Crown Princess Mary Cancer Centre Westmead Hospital, Sydney, NSW,
Australia.^22 Centre Leon Berard and Université Claude Bernard Lyon, Lyon, France.^23 UNICANCER, Paris, France.
*Corresponding author. Email: [email protected] (A.C.); [email protected] (D.M.T.)