Hyperactivation of mTORC1 drives resistance to the pan-HER tyrosine kinase inhibitor neratinib in HER2-mutant cancersDhivya R. Sudhan1, Angel Guerrero-Zotano4, Helen Won8, Paula González Ericsson5, Alberto Servetto1,Kyung-min Lee1, Luigi Formisano4, Yan Guo7, Qi Liu6, Lisa N. Kinch3, Teresa Dugger4, James Koch4,

Richard E. Cutler, Jr.9, Alshad S. Lalani9, Richard Bryce9, Alan Auerbach9, Ariella B. Hanker1,2, Carlos L.Arteaga1,2

UTSW Simmons Comprehensive Cancer Center1, Department of Internal Medicine2, Howard Hughes Medical Institute3, University of Texas Southwestern Medical Center, Dallas TX; Department of Medicine4, Breast Cancer Program5, Vanderbilt-Ingram Cancer

Center; Center for Quantitative Sciences6, Vanderbilt University Medical Center, Nashville, TN; Comprehensive Cancer Center7, University of New Mexico, Albuquerque, NM; Memorial Sloan Kettering Cancer Center8, New York, NY; Puma Biotechnology

Inc.9,Los Angeles, CA.

Clinical background

Conclusions

B

• Tumor genomic profiling has identified patients with cancers

harboring activating ERBB2 (HER2) mutations that are sensitive to

HER2 targeted therapies.

• In the SUMMIT phase II ‘basket’ trial, a subset of patients with

ERBB2-mutant cancers exhibited significant clinical benefit from

treatment with the pan-HER irreversible tyrosine kinase inhibitor

(TKI) neratinib.

• However, durable responses to neratinib are few, suggesting

mechanisms of de novo and acquired drug resistance. Thus, we

sought to identify actionable mechanisms of resistance to neratinib.

Clinical response to neratinib in phase 2 SUMMIT ‘basket’ trial

• Neratinib-resistant HER2-mutant cells remained cross-resistant to other HER2 targeting agents. RNAseq

revealed significant enrichment of mTORC1 pathway in neratinib-resistant cells.

• Addition of the TORC1 inhibitor everolimus to neratinib as well as knockdown of RHEB or RPTOR

overcame resistance to neratinib.

• mTORC1 activation in neratinib resistance cells was achieved, at-least in part through RAS upregulation.

Knockdown of RAS suppressed S6 phosphorylation and restored sensitivity to neratinib.

• Patients with cancers harboring mTOR activating alterations did not exhibit clinical benefit from neratinib

compared to those without mTOR activating alterations.

• Addition of TORC1 inhibitors may improve the activity of irreversible HER2 TKIs against cancers with

HER2 activating mutations.

.

Combined neratinib and mTORC1 inhibition alone suppresses S6

phosphorylation in neratinib-resistant cells5637 5637-NR

Neratinib (1 uM)

Alpelisib (1 uM)

+ +

++

-

--

- + +

++

-

--

-

5637 5637-NR

+ +-- + +--

5637

+ +- + +--

5637-NR

-

- --- - --- - -- - ----

Everolimus (100 nM) ---- ---- - ++- - ++- - -- - ----

Selumetinib (1 uM) ---- ---- - --- - --- - ++ - ++--

p-ERBB2

(Y1248)

p-mTOR

(S2448)

p-S6

(S235/6).

mTOR

p-AKT

(S473)

p-S6

(S240/4)

AKT

p-ERK

S6

5637 5637-NR

Neratinib (1 uM)

Buparlisib (1 uM)

+ +

++

-

--

- + +

++

-

--

-

5637 5637-NR

+ +-- + +--

5637

+ +- + +--

5637-NR

-

- --- - --- - -- - ----

Everolimus (100 nM) ---- ---- - ++- - ++- - -- - ----

MK2206 (1 uM) ---- ---- - --- - --- - ++ - ++--

p-ERBB2

(Y1248)

p-mTOR

(S2448)

p-S6

(S235/6).

mTOR

p-AKT

(S473)

p-S6

(S240/4)

ERBB2

AKT

b-Actin

mTORC1 activation could be partly attributed to RAS pathway

upregulation

Neratinib-resistant HER2-mutant cells are cross-resistant to other

HER2 TKIs

(A) 12-point dose response curves of parental and neratinib-resistant 5637 and OVCAR8 cells treated with

neratinib or lapatinib. 6 days post-treatment, cells were counted on a Coulter counter. Neratinib-resistant cells

were generated over a period of 6-8 months through gradual dose escalation. (B) Crystal violet stained images of

parental versus neratinib-resistant 5637 and OVCAR8 cells treated with neratinib.

B

56

37

56

37

NR

Neratinib

OV

CA

R8

OV

CA

R8

NR

-

5637 HER2S310F extracellular domain mutation; OVCAR8 HER2G776V kinase domain mutation

1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2

0

5 0

1 0 0

1 5 0

L a p a t in ib d o s e re s p o n s e - 5 6 3 7N R

c e lls

lo g [L a p a tin ib ] , u M

Via

bil

ity

(P

erc

en

t)

p a re n ta l

N R 5 6 3 7

IC 5 0 : p a re n ta l ~ 1 5 0 n M

N R c e lls > 5 0 0 0 n M

1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2

0

5 0

1 0 0

1 5 0

N e ra t in ib d o s e re s p o n s e - 5 6 3 7N R

c e lls

lo g [N e ra t in ib ] , u M

Via

bil

ity

(P

erc

en

t)

5 6 3 7

5 6 3 7 N R

IC 5 0 p a re n ta l 1 4 n M

N R c e lls ~ 8 0 0 n M

Neratinib dose response – OVCAR8NR cells

1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2

0

5 0

1 0 0

1 5 0

lo g [N e ra t in ib ] , u M

Via

bil

ity

(P

erc

en

t)

O V C A R 8

O V C A R 8 N R

IC 5 0 p a re n ta l ~ 1 2 0 n M

N R c e lls > 1 M

1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2

0

5 0

1 0 0

1 5 0

L a p a t in ib d o s e re s p o n s e - O V C A R 8N R

c e lls

lo g [L a p a tin ib ], u M

Via

bil

ity

(P

erc

en

t)

O V C A R 8

O V C A R 8 N R

A

B

Neratinib resistant HER2-mutant cells sustain S6 phosphorylation in the

presence of neratinib

(A) Immunoblots of parental and neratinib-resistant 5637

and OVCAR8 cells treated with increasing

concentrations of neratinib. 24 h later, whole cell lysates

were probed for key members of ERBB pathway.

(B) Immunoblot analysis of parental and neratinib-

resistant 5637 and OVCAR8 cells treated with increasing

concentrations of neratinib for 24 hours.

Identification of mTORC1 as a potential driver of neratinib resistance

(A) RNA-seq based gene set enrichment analysis of pathways significantly upregulated in neratinib treated

parental vs. neratinib-resistant cells. (B) Connectivity map analysis to identify drugs that could potentially reverse

expression of resistance associated genes. (C) Enrichment plots for mTOR pathway related genes in neratinib

treated parental vs. neratinib-resistant cells.

((A) Immunoblot analysis of parental and neratinib-resistant 5637 cells treated with indicated drugs

combinations for 24 hours; alpelsib (PI3K-alpha specific inhibitor), everolimus (mTORC1 inhibitor), selumetinib

(MEK1/2 inhibitor), Buparlisib (pan-PI3K inhibitor), MK2206 (AKT inhibitor). (B) Heatmaps representing 12-

point dose response assays of 5637NR and OVCAR8NR cells treated with indicated single agent or drug

combination. (C) Representative images of cells seeded in a 12 well plate, treated with indicated drug

combination every 72 hours. (D) Immunoblot analysis of neratinib treated 5637NR and OVCAR8NR cells

transfected with indicated siRNA. (E) Growth assay of neratinib treated cells transfected with indicated

siRNAs. (F) Growth assay of TSC2 knockdown 5637, OVCAR8, MCF7 HER2L755S and HER2V777L cells treated

every 3 days with indicated concentrations of neratinib.

A B 5637NR

Enrichment score:0.42; q =0.001

OVCAR8NR

Enrichment score:0.35; q = 0.003

B CA

TN

F

sig

nalin

g v

ia N

FkB

Allo

gra

ft r

eje

ct i

on

Inf l

am

mato

ry r

esp

on

se

mT

OR

C1 s

ign

alin

g

E2F

targ

ets

Kra

s s

ign

alin

g

IL2 S

tat5

sig

nalin

g

Co

mp

lem

en

t

Ch

ole

ste

rol h

om

eo

sta

sis E

MT

Hyp

oxia

Ap

ical ju

nct i

on

INF

resp

on

se

IL6 J

ak S

tat

sig

nalin

g

1 .5

2 .0

2 .5

3 .0

1 0 -4

1 0 -3

1 0 -2

1 0 -1

5 6 3 7 p a re n ta l v s N R - tre a te d

No

rm

ali

ze

d e

nric

hm

en

t s

co

re

(N

ES

)

FD

R q

va

lue

N E S

F D R q v a lu e

E2F

targ

ets

G2 M

ch

eckp

oin

t

MY

C t

arg

ets

V1

MY

C t

arg

ets

V2

Sp

erm

ato

gen

esis

Ch

ole

ste

rol h

om

eo

sta

sis

Kra

s s

ign

alin

g

Oxid

at i

ve p

ho

sp

ho

ryla

t io

n

Mit

ot i

c s

pin

dle

An

dro

gen

resp

on

se

Po

r tein

secre

t io

n

Inf l

am

mato

ry r

esp

on

se

mT

OR

C1 s

ign

alin

g

IL2 S

tat5

sig

nalin

g

TN

F

sig

nalin

g v

ia N

FkB

EM

T

UV

resp

on

se

0

1

2

3

4

1 0 -3

1 0 -2

1 0 -1

O V C A R 8 p a re n ta l v s N R - tre a te d

No

rm

ali

ze

d e

nric

hm

en

t s

co

re

(N

ES

)

FD

R q

va

lue

N E S

F D R q v a lu e

Combined neratinib and mTORC1 suppression overcomes

neratinib resistance

C D

B

C R P R S D P D

0

5 0

1 0 0

Pe

rc

en

t

m T O R a c tiv a tin g a lte ra tio n s

n o n -m T O R a c tiv a tin g a lte ra tio n s

(A-C) Outcomes of patients enrolled in SUMMIT ‘basket’ trial based on mTOR pathway alteration status. Source:

cBioPortal SUMMIT (Nature 2018). (D) Immunoblot analysis of DV90, SNUC2A, MCF7 HER2L755S and

HER2V777L cells treated with indicated concentrations of neratinib, everolimus or combination for 24 hours. (E)

Viability assay to test synergy between neratinib and everolimus. Cells were treated with increasing

concentrations of single agents or drug combinations. Staining intensities were quantified colorimetrically and

combination indices were determined using Chou-Talalay test. (F-H) mTOR pathway mutations acquired in

cancers progressing on neratinib.

A

B

A

C

D E F

A B

(A) Growth of 5637NR tumors

treated with vehicle, neratinib (40

mg/kg), everolimus (5 mg/kg) or

drug combination. (B) Percent

change in volume of individual

tumors in each treatment arm

shown in (A). (C) H-scores for

pS6 levels based on IHC analysis

of tumors shown in (A). (D)

Representative images from (C),

DC

0

5 0

1 0 0

Re

lati

ve

ce

ll n

um

be

r

(pe

rc

en

t)

- - - -+ + + +N era tin ib

(200 nM )

Co

ntr

ol

siR

NA

HR

AS

siR

NA

KR

AS

siR

NA

NR

AS

siR

NA

5 6 3 7N R

0

5 0

1 0 0

Re

lati

ve

ce

ll n

um

be

r

(pe

rc

en

t)

- - - -+ + + +N e ra t in ib

Co

ntr

ol

siR

NA

HR

AS

siR

NA

KR

AS

siR

NA

NR

AS

siR

NA

O V C A R 8N R

(A) Immunoblot analysis of 5637NR and OVCAR8NR cells treated with neratinib, buparlisib (pan-PI3kinase

inhibitor), trametinib (MEK inhibitor) or everolimus (mTOR inihibitor) for 24 h. (B) Enrichment plots for RAS

pathway related genes in neratinib treated parental vs. neratinib-resistant cells (C) Active-RAS pulldown assay in

parental and neratinib resistant 5637 and OVCAR8 cells treated with neratinib for 6 hours. (D) Growth assay of

neratinib treated cells transfected with indicated siRNA.

A

CD

E

F G H