relative contribution of de novo versus preex-
isting genetic variation to drug resistance,
clonal population dynamics were tracked by
engineering 94T778 and the human melanoma
A375 cell line with a high-complexity DNA
barcode library ( 15 ). Although we observed
selection of preexisting genetic variation, the
high frequency of individual barcodes (94T778
line average 69.6%, range 42.9 to 93.3%; A375
line average 17.9%, range 3 to 64.7%) is
consistent with a contribution of de novo
mutagenesis to resistance (fig. S13). This is also
suggested by the late (37 weeks, T3) emergence
of subclonalTP53mutations (C277F, R273L)
induced by nutlin-3a, neither of which is visible
atweeks11(T1)or21(T2)(fig.S11).
As noted, mutagenesis may directly entail a
fitness penalty driven by the accumulation of
deleterious mutations. To study the counter-
selective effects of SIM, we examined the
clonogenic potential of both parental and
resistant lines in the absence of drug selec-
tion. We observed universally decreased clono-
genicity relative to drug-naïve controls (Fig. 1F).
Consistent with these effects, colonies obtained
from nutlin-3a–resistant 94T778 contained upto 45% senescent cells at 6 weeks, falling to 6%
at 25 weeks, compared with 1.5% in untreated
controls (fig. S14). Extinction events may ex-
plain the loss of some initially resistant clones;
for example, the C238F resistance mutation in
TP53was detected at 11 weeks under nutlin-3a
selection but was lost thereafter (fig. S11). These
data support a SIM-induced intrinsic fitness
penalty early during adaptation.
To identify common genetic pathways me-
diating SIM in human cancer, we conducted
a genome-wide functional screen using the
94T778 cell line. The presence of multipleCipponiet al.,Science 368 , 1127–1131 (2020) 5 June 2020 2of5
Fig. 1. Selection with targeted anticancer therapies results in genetic
diversity.(A) Assessment of nuclear DNA DSBs in clinical samples and in PDX
pancreatic cancer models. Shown is the fold change (mean ± SD) relative to
pretreatment samples in patients and vehicle-treated controls in PDX models.
Blue and black symbols represent negative and positive changes, respectively.
Black diamonds indicate the overall fold change (FC) with 95% confidence
intervals (95% CIs). (B) Schematic illustrating the time spans of the selection
and evolution phases of in vitro model systems. Shaded area represents
total population size, and lines represent the fate of individual clonal lineages
after bottleneck. Dashed lines indicate extinction events; solid lines indicate
evolution and clonal expansion. (C) Fold change values of DNA DSBs relative to
untreated parental cells (mean ± SD). (D) Phylogenetic analysis of CNVs
observed in single cell–derived clonal populations obtained from early-phase,
tunicamycin-resistant 94T778 and vemurafenib-resistant SKMEL28 lines. The
values below each set of parental (P) and drug-resistant (R) clones represent the
average branch length. Values below the common trunks (orange) indicate
their length.Pvalues were obtained fromZtests. (E) Quantification of de novo
SNVs generated during the expansion of the clonal populations.Pvalues were
obtained from quasibinomial logistic regression. (F) Colony formation assays
performed in the absence of drug selection revealing the impact of the adaptive
response on cellular fitness during the early phase of evolution.Pvalues were
obtained from two-tailedttest relative to drug-naïve parental cells.RESEARCH | REPORT