Nature - USA (2020-01-16)

Nature | Vol 577 | 16 January 2020 | 423

suggests that EGFR signalling is required for the escape from drug-
induced quiescence.
Inhibition of EGFR signalling, either by targeting EGFR or SHP2,
attenuated the adaptive reactivation of KRAS–GTP in KRAS(G12C)-
mutant lung cancer cells and ‘RASless’^21 mouse embryonic fibroblasts
(Extended Data Fig. 7f–i). Cotargeting EGFR or SHP2 together with
KRAS(G12C) attenuated the escape from drug-induced quiescence
(Fig. 2f) and enhanced the antiproliferative and/or antitumour effect
(Fig. 2g, Extended Data Fig. 7j). Broad suppression of receptor tyrosine

kinases by serum deprivation enhanced the antiproliferative effect of

G12Ci in several models (Extended Data Fig. 7k), which suggests that

additional growth factors may contribute in a tumour- or subpopula-

tion-specific manner. Thus, EGFR signalling coordinates—at least in

part—the heterogeneous response to the G12Ci treatment.

Although it is predominantly activated in G2M to control cell division,

AURKA has also previously been implicated in the regulation of mito-

genic signalling^22 –^25 (Fig. 3a) and acquired resistance to EGFR or PI3K

inhibition^26 ,^27. AURKA was upregulated along the adapting trajectory

(Extended Data Fig. 6) and expressed at higher levels in sorted adapt-

ing cells than in sorted quiescent cells (Fig. 3b). sgRNAs against AURKA

ranked highly in the screen (Fig. 3a). Individual knockout of AURKA

augmented the antiproliferative effect of the G12Ci and prevented

the reactivation of KRAS over time (Fig. 3c, d). Inhibitors targeting

AURKA^28 suppressed the reactivation of KRAS–GTP during the G12Ci

treatment, both in KRAS(G12C)-mutant cancer cells (Extended Data

Fig. 8a) and RASless mouse embryonic fibroblasts (Extended Data

Fig. 8b). Doxycycline-inducible AURKA expression not only enhanced

the adaptive reactivation of KRAS–GTP and CRAF–MEK–ERK signalling

during the G12Ci treatment, but also attenuated the antiproliferative

effect of treatment with G12Ci (Extended Data Fig. 8c, d).

AURKA interacts with wild-type HRAS in non-malignant cells to

enhance its interaction with CRAF^24. Here we found that AURKA also

interacts with KRAS(G12C) in cancer cells (Extended Data Fig. 8e). Treat-

ment with G12Ci or an AURKA inhibitor (AURKAi) displaced only CRAF

or only AURKA, respectively, from KRAS(G12C). Treatment with a com-

bination of G12Ci and AURKAi displaced both interactions to enhance

pCRAF and pERK inhibition, relative to treatment with each drug alone

(Extended Data Fig. 8f, g). This suggests that AURKA complexes with

KRAS to stabilize its interaction with downstream effector CRAF. As

expected, targeting AURKA prevented the escape from G12Ci-induced

quiescence (Fig. 3e). Combined inhibition of AURKA and KRAS(G12C)

had a synergistic antiproliferative effect across KRAS(G12C)-mutant

models (Extended Data Fig. 8h) and resulted in a stronger antitumour

effect in vivo, as compared to G12Ci monotherapy (Fig. 3f, Extended

Data Fig. 8i, j). Thus, the heterogeneous bypass of G12Ci-induced qui-

escence also depends on the AURKA-mediated adaptive reactivation

of KRAS.

The non-uniform response to the G12Ci treatment may occur

in a KRAS(G12C)-dependent or KRAS(G12C)-independent manner.

Unlike treatment with G12Ci, treatment with a KRASG12C-specific siRNA

(KRASG12C siRNA)^29 yielded a uniform induction of quiescence (Fig. 4a).

The KRASG12C siRNA differs from the inhibitor in that it suppresses sig-

nalling in a manner that is not dependent upon conformation, but is

otherwise susceptible to KRAS(G12C)-independent adaptive pressures.

The uniform effect of the KRASG12C siRNA suggests that reactivation

c

e

a

b

g

DC2

DC1

Clusters 3541

Inhibited Adapting

10

KRAS HBEGF AURKA

f

HBEGF

EGFR

GRB2:SOS1

KRAS

(GDP)

SHP2

KRAS

(GTP)

RAF PI3K

PTEN

G1→S

p21

RB1

–4 –2 02

MAGeCK β score

Genes (19,1

15)CDKN1APTEN

RB1

HBEGFEGFR

GRB2SHP2

NT

d

2345

p27K–, log(AU)

G12Ci, 72 h

+EGFRi

0.5 +SHP2i

Scaled density^0

1.0

2345

p27K–, log(AU)

0.5

Scaled density^0

1.0 G12Ci, 72 h

+EGF at 0 h

+EGF at 24 h

+EGF at 48 h

β-Actin

p27K–

72

H

pEGFR

EGFR

pSHP2

SHP2

4244872

Unsorted

72 G12Ci, h

L

0

P = 0.00 4

Time (d)

07142128

Tumour(% change)^500

100

400

800

–50

EGFRi

Control

Combination

G12Ci

Expression,

z 4

2

0

–2^0

2

(^42)
–1
0
1
KRAS HBEGF AURKA
Fig. 2 | Adaptation to the G12Ci treatment is dependent on EGFR signalling.
a, b, The peak (a) or mean (b) expression of genes with trajectory-specific
expression. a, Cells from the indicated clusters were projected in their
diffusion component coordinates. Cells with peak expression in the indicated
genes are shown in navy. b, Cells were grouped by cluster, ordered in
pseudotime and the mean expression was calculated for pools of 15 cells (grey)
or the entire cluster (navy). c, A genome-wide CRISPR–Cas9 screen in H358 cells
identified EGFR signalling intermediates as potential regulators of the G12Ci
treatment. NT, non-targeting sgRNAs. SHP2 is also known as PTPN11.
d, Immunoblots of extracts from G12Ci-treated and FACS-sorted H358 p27K−
cells. e, f, The cells were treated with the G12Ci for 72 h alone, in the presence of
EGF stimulation at the indicated times (e) or in the presence of the indicated
EGFR signalling inhibitors (f). g, Mice bearing xenografts of H2122 cells were
treated as shown, to determine the effect on tumour growth. Mean + s.e.m,
n = 4 mice. A two-sided t-test P value is shown. A representative of two
independent experiments is shown in d–f.
Tumour P = 0.035
(% change)
(^500)
400
800
–50
100
–100
Time (d)
014284256
G12Ci + AURKAi
G12Ci
AURKAi
Control
acb
CCNB1
AURKA
04244872 G12Ci (h)
Unsorted p27K–
72
L
72
H
GAPDH
e
RAN
TPX2
AURKA
PLK1
CCNB1:CDK1
KRAS
(GTP)
CRAF
–4–2 02
MAGeCK β score
Genes (19,1
15)
AURKARAN
CCNB1
NT
d f
AURKA
NT
DMSOG12Ci(14 d)
1
2
1
sgRNA
G12Ci alone

• AURKAi


• panAURKi
  2345
  p27K–, log(AU)
  Cells
  0
  50
  100
  150
  1
  KRAS–GTP
  sgNT
  KRAS
  pRSK
  RSK

• +sgAURKA

sgAURKA

02824487280960282448728096 G12Ci (h)

AURKA

GAPDH

Fig. 3 | AURKA is involved in the adaptive reactivation of KR AS and escape
from drug-induced quiescence. a, AURK A signalling intermediates identified
in the CRISPR–Cas9 screen as potential regulators of the response to the G12Ci
treatment. b, Immunoblots of extracts from FACS-sorted H358 p27K− cells.
c, d, KR AS(G12C)-mutant lung cancer cells (H358) that express a non-targeting
(NT) sgRNA or AURKA-specific sgRNAs were treated with the G12Ci and

analysed by immunoblotting (c) or by crystal violet staining (d). e, Cells were

treated with the indicated inhibitors for 72 h and analysed by FACS. f, Mice

bearing xenografts of H358 cells were treated with the indicated inhibitors to

determine the effect on tumour growth. Mean + s.e.m, n = 4 mice. A two-sided

t-test P value is shown. A representative of three independent experiments is

shown in b–e.