inhibitor lapatinib, respectively. All cell lines
showed a reduction in MTOR protein levels
and a reduced phosphorylation of the down-
stream kinase p70-S6K (fig. S17A). In addi-
tion, we observed a transcriptional repression
of genes positively regulated by MTOR and
an increased expression of genes repressed by
MTOR ( 24 ) (fig. S17B). As expected,MTOR
silencing impaired clonogenicity in the absence
of selection, but paradoxically increased clono-
genicity under drug selection (Fig. 2C and
supplementary materials), recapitulating the
models described earlier. This effect was not
caused by altered drug sensitivity in short-term
assays (fig. S18). To exclude off-target effects
from gene knock-down, these experiments
were validated using shRNAs targeting the
3 ′untranslated region ofMTOR. The stress-
induced adaptive potential was abrogated
using an RNA interference (RNAi)–resistant
open reading frame of the gene (fig. S19). In vivo,
repression of MTOR accelerated emergence
of palbociclib resistance in a PDX pancreatic
cancer model (Fig. 2D) despite enhancing the
effect of palbociclib in short-term ex vivo assays
(fig. S20). Consistent with previously described
kinetics of mutagenesis and impaired intrinsic
fitness, a time course analysis of MTOR and
p70-S6K phosphorylation levels revealed a sig-
nificant reduction early during evolution and
a restoration to pretreatment levels late during
adaptation (fig. S21).
Because inhibition ofMTORimpairs the
DNA-damage response ( 25 , 26 ), we examined
DNA DSBs in 94T778 cells engineered with
MTOR-specific shRNAs. The analysis of con-
focal microscopy image data revealed elevated
levels of DSBs comparable to those seen in cells
spontaneously resistantto nutlin-3a (fig. S22A).
Furthermore, the selective MTOR inhibitor
torkinib (pp242) delayed the repair of ioniz-
ing radiation–induced DNA DSBs, assessed
as the number ofg-H2AX (fig. S22B) and 53BP1
(fig. S22C) foci. To quantitate the effects of
MTOR silencing on mutagenesis, we under-
took whole-genome sequencing on single cell–
derived clonal populations obtained from
94T778 cells expressing nonsilencing or MTOR-
specific shRNAs. We founda significant increase
in intraclonal diversity in MTOR-silenced cells
(figs.S23toS25,tablesS2andS3,andmaterials
and methods). No MTOR-specific mutational
signatures were evident in 94T778 clonal
populations (fig. S8). In our lines, pharmaco-
logic inhibition of MTOR repressed expres-
sion of the homologous recombination (HR)
and Fanconi anemia pathways and selectively
repressed high-fidelity, but not error-prone
DNApolymerases(Fig.3AandtableS7).
To ascertain whether similar transcriptional
changes are elicited across a broader range
of cancer types, we interrogated the LINCS
L1000 database, a transcriptome dataset of
100 lines exposed to ~400 drugs across mul-tiple doses and time points ( 27 ). Almost iden-
tical patterns were seen in multiple cell lines
exposed to three different MTOR inhibitors
(fig. S26), as well as in 94T778 cells develop-
ing spontaneous drug resistance, as described
earlier (Fig. 3B and table S8). In the LINCS
L1000 database, multiple cytostatic drugs sim-
ilarly repress accurate DNA repair (fig. S27).
This suggests that inhibiting cell proliferation
may affect genetic diversity through coor-
dinated regulation of multiple components of
DNA repair. To test whether altered DNA re-
pair would enhance adaptation, we reinterro-
gated the genome-wide screen and found
specific enrichment for shRNAs targeting HR
genes (table S9). To determine whether the
repression of HR directly facilitates drug re-
sistance, we engineered 94T778, SKBR3, and
SKMEL28 cells with shRNAs targetingBRCA1,
BRCA2,andPALB2and subjected the cells to
selection. Recapitulating both spontaneous drug
resistance models and silencing of MTOR, im-
pairment of HR reduced clonogenicity in the
absence of selection and increased clonoge-
nicity under pharmacologic pressure (Fig. 3C
and supplementary materials). The induction
of a defective DNA-damage response by non-
genotoxic cytostatic therapies suggests synthetic
lethality-based strategies. In a preliminary proof-
of-concept experiment, we assessed the impact
of palbociclib combined with a poly(ADP-ribose)
polymerase inhibitor (rucaparib) in aCipponiet al.,Science 368 , 1127–1131 (2020) 5 June 2020 4of5
Fig. 3. Inhibition of MTOR fosters adaptive mutagenesis by repressing
accurate DNA repair.(A) Transcriptional changes induced by torkinib (pp242).
Pvalues were obtained from one-sided Wilcoxon rank-sum test and are shown in
table S7. (B) Transcriptional changes observed during the early phase of the
adaptive evolution in 94T778 cells exposed to palbociclib (P), nutlin-3a (N), and
tunicamycin (T) sampled at 10 weeks for P and N and at 9 weeks after bottleneck
for the T condition.Pvalues were obtained from one-sided Wilcoxon rank-sum
test and are shown in table S8. (C) Effect of silencing of HR genes in the absence
(white bars) or presence of pharmacologic pressures (gray bars).Pvalues were
obtained from two-tailedttest versus NS control shRNA. (D) Effect of palbociclib and
rucaparib combination therapy on tumor growth inhibition (bar plot, 30 days) and
overall survival (Kaplan–Meier plot) in a PDX pancreatic cancer model.Pvalues for
tumor growth were obtained from two-tailedttest; a log-rank test on palbociclib +
rucaparib versus palbociclib monotherapy was used in the Kaplan–Meier plot.RESEARCH | REPORT