Hyperactivation of Mtorc1 Drives Resistance to the Pan-HER Tyrosine Kinase Inhibitor Neratinib in HER2-Mutant Cancers Dhivya R

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Hyperactivation of mTORC1 drives resistance to the pan-HER tyrosine kinase inhibitor neratinib in HER2-mutant cancers Dhivya 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. ((A) Immunoblot analysis of parental and neratinib-resistant 5637 cells treated with indicated drugs Clinical background Neratinib resistant HER2-mutant cells sustain S6 phosphorylation in the combinations for 24 hours; alpelsib (PI3K-alpha specific inhibitor), everolimus (mTORC1 inhibitor), selumetinib Clinical response to neratinib in phase 2 SUMMIT ‘basket’ trial presence of neratinib (MEK1/2 inhibitor), Buparlisib (pan-PI3K inhibitor), MK2206 (AKT inhibitor). (B) Heatmaps representing 12- • Tumor genomic profiling has identified patients with cancers 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 harboring activating ERBB2 (HER2) mutations that are sensitive to NR NR A B combination every 72 hours. (D) Immunoblot analysis of neratinib treated 5637 and OVCAR8 cells A HER2 targeted therapies. transfected with indicated siRNA. (E) Growth assay of neratinib treated cells transfected with indicated B L755S V777L siRNAs. (F) Growth assay of TSC2 knockdown 5637, OVCAR8, MCF7 HER2 and HER2 cells treated n o n -m T O R a c tiv a tin g a lte ra tio n s • In the SUMMIT phase II ‘basket’ trial, a subset of patients with every 3 days with indicated concentrations of neratinib. m T O R a c tiv a tin g a lte ra tio n s 1 0 0 ERBB2-mutant cancers exhibited significant clinical benefit from

Combined neratinib and mTORC1 suppression overcomes t n

treatment with the pan-HER irreversible tyrosine kinase inhibitor e

c 5 0 r neratinib resistance e (TKI) neratinib. P • However, durable responses to neratinib are few, suggesting A B 0 mechanisms of de novo and acquired drug resistance. Thus, we C R P R S D P D sought to identify actionable mechanisms of resistance to neratinib. D Neratinib-resistant HER2-mutant cells are cross-resistant to other (A) Immunoblots of parental and neratinib-resistant 5637 and OVCAR8 cells treated with increasing C HER2 TKIs concentrations of neratinib. 24 h later, whole cell lysates 5637 HER2S310F extracellular domain mutation; OVCAR8 HER2G776V kinase domain mutation were probed for key members of ERBB pathway. (B) Immunoblot analysis of parental and neratinib- N R N R Neratinib A N e ra tin ib d o s e re s p o n s e - 5 6 3 7 c e lls L a p a tin ib d o s e re s p o n s e - 5 6 3 7 c e lls - resistant 5637 and OVCAR8 cells treated with increasing 1 5 0 1 5 0 p a re n ta l B 5 6 3 7 concentrations of neratinib for 24 hours.

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

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y (A) Growth of 5637 tumors

i l

N R c e lls ~ 8 0 0 n M l i

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b Combined neratinib and mTORC1 inhibition alone suppresses S6 a

a treated with vehicle, neratinib (40 i

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NR V V D mg/kg), everolimus (5 mg/kg) or 0 0 E 5637 phosphorylation in neratinib-resistant cells 1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2 1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2 drug combination. (B) Percent 5637 5637-NR 5637 5637-NR 5637 5637-NR lo g [N e r a tin ib ], u M lo g [L a p a tin ib ], u M 5637 5637-NR 5637 5637-NR 5637 5637-NR change in volume of individual NR L a p a tin ib d o s e re s p o n s e - O V C A R 8 N R c e lls Neratinib (1 uM) - + - + - - + - + - + - - + - + - + - - + Neratinib dose response – OVCAR8 cells A Neratinib (1 uM) - + - + - + - + - + - + - + - + - + - + - + - + + + + tumors in each treatment arm 1 5 0 1 5 0 Buparlisib (1 uM) - - + + - - + + ------

O V C A R 8 O V C A R 8 Alpelisib (1 uM) - - + + - - + + ------shown in (A). (C) H-scores for ) ) Everolimus (100 nM) t - - - + -

t O V C A R 8 N R O V C A R 8 N R ------+ + - + ------

OVCAR8 n

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

e e

c 1 0 0

c 1 0 0 pS6 levels based on IHC analysis r

r MK2206 (1 uM) ------+ + - - + + e e Selumetinib (1 uM) ------+ + IC p a re n ta l ~ 1 2 0 n M P - - - - - + + -

P 5 0 -

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y of tumors shown in (A). (D) y

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a Representative images from (C),

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OVCAR8 ERBB2 0 0 p-mTOR -4 -3 -2 -1 0 1 2 1 0 -4 1 0 -3 1 0 -2 1 0 -1 1 0 0 1 0 1 1 0 2 1 0 1 0 1 0 1 0 1 0 1 0 1 0 (S2448) F G H lo g [N e r a tin ib ], u M lo g [L a p a tin ib ], u M p-mTOR mTORC1 activation could be partly attributed to RAS pathway (S2448) (A) 12-point dose response curves of parental and neratinib-resistant 5637 and OVCAR8 cells treated with mTOR upregulation mTOR neratinib or lapatinib. 6 days post-treatment, cells were counted on a Coulter counter. Neratinib-resistant cells p-AKT were generated over a period of 6-8 months through gradual dose escalation. (B) Crystal violet stained images of (S473) p-AKT parental versus neratinib-resistant 5637 and OVCAR8 cells treated with neratinib. AKT (S473) A B 5637NR OVCAR8NR Identification of mTORC1 as a potential driver of neratinib resistance p-ERK AKT

p-S6 p-S6 (S235/6). (S235/6). A B C (A-C) Outcomes of patients enrolled in SUMMIT ‘basket’ trial based on mTOR pathway alteration status. Source:

) p-S6 p-S6 L755S S 5 6 3 7 p a re n ta l v s N R - tre a te d (S240/4) cBioPortal SUMMIT (Nature 2018). (D) Immunoblot analysis of DV90, SNUC2A, MCF7 HER2 and E (S240/4)

N -1 ( 3 .0 1 0 V777L

N E S HER2 cells treated with indicated concentrations of neratinib, everolimus or combination for 24 hours. (E)

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s S6 Viability assay to test synergy between neratinib and everolimus. Cells were treated with increasing

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

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

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d cancers progressing on neratinib.

z i l N R N R a 1 .5 1 0 -4 5 6 3 7 O V C A R 8 Enrichment score:0.35; q = 0.003

m Enrichment score:0.42; q =0.001

r T a B g s g g t i n n t M x e g o k n n o D e n e n s n o i i i e is E o ti i F ti s l g l l n l N n a r a a s p c o a 1 0 0 1 0 0 N c a m a y e o n t n n e t n p n ia j p g g g l s H u s g e i F i i p j i v r s s s s o l e e 2 e r s g t r 1 E s 5 m a  t f r t o c r in a y C a m i F ta l r r r ta C o p R N S e a g o K h I e Conclusions A n t O S lo a l k g T 2 a b i l o b m L r J s A m I e  m t 6

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

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

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

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

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v overcame resistance to neratinib. h 2 1 0 N e ra tin ib - + - + - + - + - - + - + -

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R R 1 i R R i R R R R i i i i i i d s s s s s s s s l e l o S S S S z o S S • mTORC1 activation in neratinib resistance cells was achieved, at-least in part through RAS upregulation. r i t A A A r A l D t A A E n R R n R a R R R -3 F o N o 0 1 0 H K H K N

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r Knockdown of RAS suppressed S6 phosphorylation and restored sensitivity to neratinib. T ts t g e g g B n 1 2 s n l n M e o e i i s n e e n n k s V V i li o d s o li li E g o s i n i s F n r p s s e s a t i n t n a a N o N a t t a a e t k e e n t n l p o r o n n p e g y p g g a c g g s i r s c p i i i s F e r r g o s s e s v e 2 a a o ic e e s s r NR NR h o e r s E c t t t s h t r 1 5 g a m a p o n t n V C C r t n i y C a li U (A) Immunoblot analysis of 5637 and OVCAR8 cells treated with neratinib, buparlisib (pan-PI3kinase i M m o s e e r R t 2 Y Y r h K o g t o a M r t O S n G M M e l h o o a p o p r T 2 ig r d P m L S e e m I s t v n • Patients with cancers harboring mTOR activating alterations did not exhibit clinical benefit from neratinib s i A m  t a F le a fl inhibitor), trametinib (MEK inhibitor) or everolimus (mTOR inihibitor) for 24 h. (B) Enrichment plots for RAS N o id In T h x C O pathway related genes in neratinib treated parental vs. neratinib-resistant cells (C) Active-RAS pulldown assay in compared to those without mTOR activating alterations. (A) RNA-seq based gene set enrichment analysis of pathways significantly upregulated in neratinib treated parental and neratinib resistant 5637 and OVCAR8 cells treated with neratinib for 6 hours. (D) Growth assay of parental vs. neratinib-resistant cells. (B) Connectivity map analysis to identify drugs that could potentially reverse neratinib treated cells transfected with indicated siRNA. • Addition of TORC1 inhibitors may improve the activity of irreversible HER2 TKIs against cancers with expression of resistance associated genes. (C) Enrichment plots for mTOR pathway related genes in neratinib HER2 activating mutations. treated parental vs. neratinib-resistant cells.