Abstract Number: The Pharmacological Effects of Receptor alpha Y537S and D538G Mutations 3621 Steven J. Hartman1, Tracy Kleinheinz1, Jonathan White2, Stephen Daly2, Ria Goodwin2, Wei Zhou1, Jun Liang1, Gina Wang1, Lori Friedman1, Martin O’Rourke2, Ciara Metcalfe1, Robert A. Blake1. (1 - Genentech, South San Francisco, CA 94080; 2- Charles River Laboratories).

INTRODUCTION RESULTS 3. ERa Degradation 4. In Vitro Proliferation Effects a) MCF7 ERa Y537S knock-in vs Wildtype MCF7 The frontline therapy for alpha 1. ERa Binding a) GDC-0810 and AZD9496 AZD9496 + GDC-0810 (ERa) positive (ER BC) involves Equilibrium and Kinetic Binding Studies Key various forms of endocrine therapy, consisting of WT WT either Selective Estrogen Receptor Modulators Y537S Y537S

)

)

(SERMs) or aromatase inhibitors. An emerging nM ( nM Proliferation Proliferation Proliferation Proliferation ( + Kd mechanism of ER BC resistance to endocrine Kd Cell lines generated

D538G D538G from MCF7 parental therapy, and consequently disease relapse, has Y537S • Both GDC-0810 and AZD9496 are strong degraders of been associated with a set of “hotspot” mutations endogenous ERa WT and over-expressed ERa WT, but are weak The proliferation EC50 values of GDC-0810 and AZD9496 are higher

a WT Kd (nM) WT Kd (nM) in and near to helix-12 of the ER ligand binding a in MCF7 cells expressing ERa Y537S relative to ERa WT only. Equilibrium Ki Kinetic Kd degraders of the over-expressed Y537S and D538G ER mutants. domain. Selective Estrogen Receptor b) Degradation of ERa mutants (Over-Expressed; OE) Proliferation EC50 (MCF7) Proliferation Maximum Inhibition % (MCF7) Degraders/Down-regulators (SERDs), such as ERa Y537S and D538G Mutations Increase Drug Residence Time (1,2) Key Residence Time (T ) GDC-0810 and AZD9496 , represent an Off-Rate WT D538G Y537S 1/2 D538G Y537S important pharmacological strategy being applied

to develop treatments for resistant ER+BC. Here, 4OHT 2 2 2 2

/ MCF7 /

1 a 1 a we compare 2 of the most frequent ER hotspot GDC-0810

T D538G T Y537S a – DC50 of 4OHT in mutations (Y537S and D538G), with ERa wildtype Y537S OE and in Parental is an increase in protein (WT) and the ability of a set of SERM/SERDs and AZD9496 a level.

1 1 Negative degradation WT T /2 WT T /2 a – poor fit other ERa ligands to bind, antagonize, Time (s) values indicate stabilization. degrade/stabilize ERa and affect cell proliferation. 2.ERa Antagonism • Degradation of the D538G and Y537S ERa mutants typically requires higher drug concentrations than WT ERa.

a) In Vitro Analysis of Antagonism • The shift in EC50 between WT and mutants degradation is b) In Vitro Proliferation Effects of ERa Y537S and ERa D538G and Y537S Mutants Require Higher Concentrations of typically less than the shift in antagonism EC50. SERM/Ds To Antagonize Co-Activator Peptide Binding. (Similar results c) Degradation of ERa Y537S (CRISPR Knock-In) D538G Mutations vs ERa Wildtype (Over-Expressed in (3) MCF7 cells) Brief methods section have been reported for 4OHT and E2 ).

1. Selective Estrogen Receptor Modulators and MCF7 Degraders (SERMs, SERDs) MCF7 EC50 Y537S Y537S

D538G GDC- 0810 Y537S Y537S Tamoxifen D538G 4-Hydroxytamoxifen WT WT

AZD9496 The majority of SERM/Ds required higher concentrations to inhibit RAD-1901 proliferation in MCF7 cells over-expressing ERa Y537S (or CRISPR

2. Binding Studies WT WT knock-in) and D538G relative to WT. • The most significant relative shift in degradation EC50 Fluormone™ ES2 • TR-FRET • Fluorescein tagged between ERa WT and Y537S was seen for RAD-1901. Fluormone™ ES2 estradiol probe binding is + b) Cell-Based Analysis of Antagonism Section 3C: Tb Tb detected by TR-FRET d) 4OHT Stabilization of ERa Y537S • ERa Y537S represents CONCLUSION: 10-40% of total ERa in Key 1. In Vitro Antagonism Studies Key the Y537S knock-in cell  Y537S and D538G mutations of ERa : line  Increase the residence time of most SERM/Ds, relative to WT ERa Coactivator + E2 • TR-FRET • Degradation EC50 values • Fluorescent tagged are higher and E2 E2  Require higher concentrations to antagonize and degrade ERa PGC1a binding is maximum degradation is blocked by bound + Antagonist Tb a EX: GDC-0810 antagonist lower in the ER Y537S  Require higher concentrations to inhibit ERa dependent proliferation MCF7 relative to MCF7 ERa WT  4OHT stabilizes ERa Y537S through an unknown mechanism Cell lines generated 2. Cell-based Antagonism Studies from MCF7 parental  GDC-0810 and AZD9496 have similar in vitro profiles, achieve similar Section 3D: DMSO 1 uM Fulvestrant maximum degradation, antagonism and inhibition of proliferation. Key • 4OHT stabilizes Y537S Key: Red = GREB1 mRNA in the OE line, while Yellow = ERα mRNA Blue = Hoechst/DNA weakly degrading the Key other forms of ERa. • Each fluorescent puncta within a cell represents a single GREB1 mRNA transcript • ERα antagonist successfully down regulates GREB1, a gene known to be • 4OHT has a biphasic regulated by ERα. ACKNOWLEDGEMENTS effect on ERa levels in 3. ERa Degradation Studies Estradiol and the Y537S CRISPR line, ERa kinetic binding studies: Art Wittwer, Jim Gierse (Confluence Discovery Technologies) Diethylstilbesterol caused ERa (MCF7) increases in GREB1 in the with an increase at MCF7 Cell Parental and WT OE Line higher concentrations. while tamoxifen caused REFERENCES increases in each line. This is likely due to a 1) Joseph, J.D. et al. The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer. eLife 5 (2016). mix of stabilization of 2) 2) De Savi, C. et al. Optimization of a novel binding motif to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetra hydro-1H- Primary Secondary Fixed pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (AZD9496), a potent and orally bioavailable selective estrogen receptor downregulator and antagonist. Journal of Antibody Antibody • GDC-0810 and AZD9496 have similar antagonism profiles. + Hoechst Y537S and weak medicinal chemistry (2015). ERα a 3) Fanning, S.W. et al. somatic mutations Y537S and D538G confer breast cancer endocrine resistance by stabilizing the activating DMSO Control Fulvestrant 5nM • In most cases SERM/D antagonism EC50: WT < D538G < Y537S. degradation of WT ER . function-2 binding conformation. eLife 5 (2016).

Endocrinology. Nuclear Receptors and Endocrine Oncology Therapies. Tuesday Apr 4, 2017 8:00 AM - 12:00 PM. Halls A-C, Poster Section 25, Poster Board Number: 20