critical drug targets in these cells allowed us
to identify shared genetic determinants of
resistance. Cells were engineered with a whole-
genome short hairpin RNA (shRNA) library
and subjected to five different selective con-
ditions (Fig. 2A): (i) culture for 2 weeks with-
out selection to estimate selective drift; (ii)
equivalently titrated near-extinction selective
conditions using nutlin-3a;(iii) palbociclib;
(iv) tunicamycin targeting the unfolded pro-
tein response; and (v) incrementally increas-
ing concentrations of nutlin-3a (to determine
the relevance of selective stringency).
Initially, the screen identified condition-
specific enrichment of shRNAs for genes known
to confer resistance, as well as previously un-
known genes, in these important pathways
(“solution”genes). For examples of known
resistance genes, shRNAs targetingTP53and
RB1were among the top-ranked genes for the
nutlin-3a and palbociclib conditions, respec-
tively (table S4 and fig. S15).Thisprovided
evidence that the assay was performing as
expected. We next looked for genes with
shRNAs that were enriched across all near-
extinction conditions (“facilitator”genes). Fa-
cilitator genes were associated with lower
levels of enrichment compared with solution
genes (table S5), which was perhaps due to an
indirect role in mediating resistance through
subsequent stochastic events, or possibly to
counterselection. Top-ranked among facilita-
tors were the genes encoding the Chaperonin
Containing TCP1 Subunit 3 (CCT3) and the
RNA-processing factor Heterogeneous Nuclear
Ribonucleoprotein L (HNRNPL)( 16 ).CCT3 hasan important role in telomere maintenance
( 17 , 18 ) and genome stability ( 19 ), and hnRNP
protein–mediated alternative splicing has a role
in cancer progression ( 20 ). The next most en-
riched gene was that coding for the mechanistic
target of rapamycin (MTOR)(tableS6andfig.
S16). In addition, the analysis revealed the en-
richment for shRNAs directed to positive reg-
ulators of MTOR and the depletion of hairpins
targeting the negative regulatorPTEN(Fig. 2B).
The MTOR signaling pathway is widely ex-
pressed in human tissues and functions as an
evolutionarily conserved sensor of environ-
mental and endogenous stress ( 21 – 23 ). The
94T778, SKMEL28, and human breast cancer
SKBR3 cell lines were engineered with MTOR-
directed shRNAs and were exposed to tunica-
mycin, vemurafenib, and the EGFR/HER2Cipponiet al.,Science 368 , 1127–1131 (2020) 5 June 2020 3of5
Fig. 2. Whole-genome RNAi screen identifies MTOR as a common evolutionary
capacitor in cancer.(A) Schematic diagram of the genome-wide RNAi screen.
(B) Enrichment (left panel) and depletion (right panel) of hairpins targeting
components of MTOR signaling. Genes are ranked byqvalues, with genes in red
and light red being highly significant and approaching significance, respectively.
The dashed line indicates the 0.05 statistical significance threshold. (C)Silencing
of MTOR signaling imposes a fitness penalty in normal culture conditions (white
bars) but fosters adaptability in response to pharmacologic pressures (gray bars).
Pvalues were obtained from two-tailedttest versus nonsilencing (NS) controls.
(D) Silencing of MTOR affects therapeutic responsiveness to palbociclib in PDX
pancreatic cancer models.Pvalues were obtained from two-tailedttest versus first
time point after the end of the second treatment cycle.RESEARCH | REPORT