San Antonio Symposium – December 4-8, 2018 P4-13-03 Elacestrant (RAD1901) demonstrates anti-tumor activity in models resistant to CDK4/6 inhibitors Hitisha Patel, Julie Tao, Heike Arlt, and Teeru Bihani Radius Health, Inc. Waltham, MA, USA

Background Results

• receptor positive (ER+) breast cancers comprise two-thirds of all breast cancers1 2,3 Elacestrant inhibited growth of a PDX model insensitive to palbociclib • Current standard of care for ER+ breast cancer involves inhibiting the ER pathway either by Inhibitors of the ER signaling pathway Elacestrant inhibited growth regardless of prior Elacestrant demonstrated long-term growth 1. Inhibiting the synthesis of estrogen (aromatase inhibitors, AI) treatment history or ESR1 status inhibition in CDK4/6 inhibitor-resistant lines WHIM43-D538G 2. Directly binding to ER and modulating its activity (SERMs; ), or aromatase aromatase CDK4/6 inhibitor-sensitive (de novo palbociclib resistance) 3. Directly binding to ER and causing receptor degradation (SERDs; ) R 1400 ER modulators (SERMs) Palbociclib CDK4/6 vehicle PgR mRNA R R R R ) Ribociclib inhibitor-sensitive Palbociclib Ribociclib Abemaciclib ) tamoxifen palbociclib o l 3

• The addition of a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor to an endocrine agent Aromatase inhibitors r

t 1.0 R e ERα n roughly doubles PFS, and this has led to the approval and use of certain CDK4/6 inhibitors in anastrozole ESR1-wt Abemaciclib 1200 fulvestrant 3 mg/dose exemestane ER ER elacestrant 30 mg/kg E2F1 o c e t combination with either AI in the first-line metastatic setting or in combination with 100 d cha n g 0.5

Control i v o l t

letrozole F 4,5 Growth inhibition, EC50 (nM) elacestrant 60 mg/kg Cyclin E1 fulvestrant, in the second-line metastatic setting ** ER target genes/proliferation 1000 re l a

( ** ESR1 ** (PgR, GREB1, TFF1) 0.0 • Understanding how treatment resistance to CDK4/6 inhibitors manifests is important in order ESR1-wt Vinculin le ib g g wild-type ic icl ose g/k g/k veh oc g/d 8 alb 0 m 0 m ER ER Rb-null model ; pRb= not detected p t 3 m t 3 t 6 to evaluate the activity of ER-targeted therapies in a CDK4/6 inhibitor-resistant setting CDK4/6 inhibitor-sensitive 0.17 800 an ran ran 50 str est est lve lac lac • Herein, we describe the activity of elacestrant, an orally administered SERD that binds to ER ER degraders (SERDs) PalbociclibR 0.19 Elacestrant fu e e Quantification of protein levels 6,7 fulvestrant R

and targets it for degradation in a dose-dependent manner , in CDK4/6 inhibitor-resistant Ribociclib 0.13 ns )

600 ) Elacestrant ) 0 . 0

0 E2F1 Cyclin E1 .

. ERα Elacestrant r volume ± SEM (mm 1.5 1.5 % growth inhibition R 1.5

0.25 * s 1

Abemaciclib s 1 s 1 settings o li n

• Orally bioavailable, ER selective small molecule l a l a u l a li n

0 li n u n c u (normalized to ctrl as 100%) * 1.0 1.0 1.0 i o ct r v n c -2 -1 012 o ct r o ct r / n c • Elacestrant is currently under clinical investigation in postmenopausal women with advanced i • Under investigation in ongoing Phase 1 clinical 400 i v v / / Tu m ed t 1 ed t ed t a z F z i NE 1 Log[elacestrant (nM) ] z

i 0.5 0.5 i trials in metastatic breast cancer patients 0.5 2

ER+ breast cancer (NCT02650817, NCT02338349) C ER CDK4/6 E C ma l ma l ma l r r

R R R r o o inhibitor-sensitive Palbociclib Ribociclib Abemaciclib o • Preclinical activity in multiple breast cancer models n n (

n 0.0 0.0

0.0 ( ESR1-mut 200 ( le ib e g g le ib e g g le ib e g g ic icl os g/k g/k ic icl os g/k g/k ic icl os g/k g/k veh oc g/d veh oc g/d veh oc g/d alb 0 m 0 m alb 0 m 0 m alb 0 m 0 m 100 D538G p t 3 m p t 3 m p t 3 m an rant 3 rant 6 an rant 3 rant 6 an rant 3 rant 6 0 10 20 30 40 50 60 str est est str est est str est est lve lac lac lve lac lac lve lac lac Growth inhibition, EC50 (nM) fu e e fu e e fu e e Control ns= not significant, *p <0.05 Days of Treatment ESR1 mutant Results (D538G) ESR1-mut 50 CDK4/6 inhibitor-sensitive 4.2 D538G PalbociclibR 5.6 Conclusions Generation and characterization of palbociclib-resistant models in wild-type and mutant ESR1 backgrounds RibociclibR 4.1 Elacestrant % growth inhibition AbemaciclibR 3.4 0 • Elacestrant induced dose-dependent long-term growth inhibition in CDK4/6 • Elacestrant (30 mg/kg) degraded ER, reduced E2F1 expression, and Cyclin E1 (normalized to ctrl as 100%) 0123 ESR1: wild-type ESR1: Mutant (D538G) ESR1: Mutant (Y537S) 8 Log[elacestrant (nM) ] inhibitor-resistant lines regardless of prior treatment history or ESR1 mutant status expression in the WHIM43 PDX model (palbociclib-resistant/Rb null ) 500 nM 500 nM 500 nM CDK4/6 • Elacestrant (30 mg/kg) demonstrated tumor growth inhibition in vivo in PDX • The characterization of CDK4/6 inhibitor resistance demonstrated that in these 50 nM 50 nM 50 nM ESR1-mut R R R Palbociclib Palbociclib Palbociclib inhibitor-sensitive Palbociclib Ribociclib Abemaciclib models that were a) previously treated with palbociclib (>100 days), or that were resistant cell lines ER, ER signaling, and importantly, ER-driven proliferation Y537S b) de novo resistant to palbociclib (WHIM43-Rb null8) was retained S R wt-LTED-palboS wt-LTED-palboR D538G-palbo D538G-palbo Y537S-palboS Y537S-palboR Growth inhibition, EC50 (nM) 100 ) • Elacestrant demonstrated growth inhibitory activity in vitro and in vivo in several • Elacestrant inhibited ER drive/signaling in palbociclib-resistant cell lines ) ) ESR1 mutant

100 % 100 Control 100 %

100 100 % 100

ion CDK4/6 inhibitor-resistant models that demonstrated

s (Y537S) ion • These data warrant the investigation of elacestrant in a post-CDK4/6 inhibitor ion s s a a a ctrl nhibi t ESR1-mut Loss of Rb Overexpression of Cyclin D1 i ctrl   ctrl nhibi t nhibi t CDK4/6 inhibitor-sensitive 15.9 patient population i i o t h o o t h t h

d 50

50 d 50 50 R ow t  Overexpression of Cyclin E1  Overexpression of CDK6 ow t r ow t Palbociclib 5.4 r r g Y537S g S S g S Palbo % Palbo Palbo % % R Overexpression of E2F1 PalboR R R  no rma liz e Palbo Palbo Ribociclib 11.1 ( no rma li ze d no rma liz e ( 0 0 ( 0

% growth inhibition R -3 -2 -1 0 1 -3 -2 -1 0 1 -3 -2 -1 0 1 Abemaciclib 16.1 Elacestrant µ Log[Palbociclib (µM)] Log[Palbociclib ( M)] Log[Palbociclib (µM)] 0 (normalized to ctrl as 100%) 0123 Log[elacestrant (nM) ] Materials and Methods

Generation of CDK4/6 inhibitor resistance: Colony formation assay: In vitro: Cells were plated at a density of 1000-10000 cells/well and allowed to grow for 2-5 wks Elacestrant inhibited ER signaling in Elacestrant inhibited growth of a PDX Resistance to CDK4/6 inhibitors was established by exposing HCC1428-LTED (wild-type ER), depending on the cell line. Treatments (control, palbociclib (500 nM), ribociclib (500 nM),

S R MCF7-Y537S, and MCF7-D538G cell lines to increasing concentrations of the respective abemaciclib (250 nM), and elacestrant (300 nM)) were performed in triplicate; media and LTED= long-term estrogen deprived; palbo= palbociclib; palbo = palbociclib-sensitive; palbo = palbociclib-resistant CDK4/6 inhibitor-resistant models model previously treated with a CDK4/6i with current maintenance of these cells at 500 nM of palbociclib and ribociclib for compound was replaced weekly. At the end of treatment, cells were fixed in ESR1-wt combination of fulvestrant + palbociclib PalboR and RiboR cells respectively, and 250 nM of abemaciclib for AbemaR cells. paraformaldehyde and stained with crystal violet for visualization. PgR Tff1 GREB1 % % % In vivo:

S S s S 10 0 10 0 10 0 s

Palbo s Palbo Palbo PDX: ST941-HI

Generation and characterization of ribociclib- and abemaciclib-resistant models in wild-type and l s s s

e 100 100 100 a a a R R eve l R ST941-HI patient-derived xenograft (PDX) fragments were implanted into athymic-nude v Quantitative Real-time PCR (RT-qPCR): l ve l l l l Palbo Palbo Palbo Palbo-naïve e r r r le l t t t A c c c mutant ESR1 backgrounds N 2500 mice. Tumors were measured twice/wk with Vernier calipers; volumes were calculated using R o o o vehicle t t t In vitro: RN A RN A m 2 d d d m

m fulvestrant (3 mg/dose) + 1 e e e 50 50 50 the formula: (L*W )*0.52. The tumors were treated with Vehicle, Fulvestrant B R 2000 At the end of treatment, cells were lysed and lysates were processed according to the E

ff 1 palbociclib (25 mg/kg) g ) ± SEM 3 T R P # (3 mg/dose/week) + palbociclib (25 mg/kg daily), and elacestrant (30 mg/kg daily). Tumors

G fulvestrant (3 mg/dose) ESR1: wild-type ESR1: Mutant (D538G) ESR1: Mutant (Y537S) m manufacturer’s instructions (1-step Cells-to-Ct kit). qPCR was performed using the 1-step o rma liz o rma liz o rma liz m N N N 1500 500 nM 500 nM 500 nM 0 0 0 ( growing in the presence of fulvestrant (3 mg/dose/week) + palbociclib (25 mg/kg daily)

e master mix and TaqMan™ probes (Applied Biosystems). The Ct values were analyzed to 0 100 nM 1000 nM 0 100 nM 1000 nM 0 100 nM 1000 nM were allowed to grow >1500 mm3 and then harvested and retransplanted into a new 50 nM 50 nM 50 nM 1000 assess relative changes in expression of PgR (progesterone receptor) mRNA, tff1 (trefoil Ribociclib Ribociclib Ribociclib elacestrant elacestrant elacestrant

Volu m cohort of mice considered passage (P1).

r 500 factor 1) mRNA, and GREB1 (growth regulated by estrogen) mRNA with GAPDH as an wt-LTED-riboS wt-LTED-riboR D538G-riboS D538G-riboR Y537S-riboS Y537S-riboR ESR1-mut D538G o internal control, using the 2-∆∆CT method. 250 nM 250 nM 250 nM PgR Tff1 GREB1 Tu m 0 % % % 0 10 20 30 40 50 PDX models: 0 25 nM 25 nM 25 nM 0 In vivo: 0 0 S S s S 10 0 1 1 Days of Treatment

s Palbo Abemaciclib Abemaciclib Abemaciclib s Palbo Palbo l s s s

e 100 100 100

a End of study tumors were pulverized with the cryoPREP™ Impactor (Covaris) and total RNA a a R R eve l R v

l Tumors were passaged as fragments into athymic nude mice (Nu (NCR)-Foxn1nu). ve l l l Palbo Palbo l Palbo e r le r r l t t t S R S R S R A c c c N wt-LTED-abema wt-LTED-abema D538G-abema D538G-abema Y537S-abema Y537S-abema A WHIM43 PDX fragments were implanted into ovariectomized mice without estradiol was extracted with the RNeasy mini kit (Qiagen). qPCR was performed using methods o R o o N

t PDX: ST941-HI t t RN A R m d d d m m 1 e 50 described above. e 50 e 50 supplementation (Horizon). Tumors were measured as described above. Elacestrant and ) ) B ) R Palbo-treated g E 100 100 100 ff 1 P T R palbociclib was administered orally, daily for the duration of study. Fulvestrant was 100 % G 100 %

100 % 2500 ion ion o rma liz s s ion o rma liz o rma liz s 7 a a a N N 0 0 N 0 vehicle administered once/week subcutaneously. ctrl ctrl nhibi t nhibi t ctrl Western blot analysis: nhibi t i i 0 100 nM 1000 nM i 0 100 nM 1000 nM 0 100 nM 1000 nM o o o

t fulvestrant (3 mg/dose) + h t h 50 t

50 h

50 ± SEM

S

d 2000 d

Ribo d elacestrant elacestrant elacestrant ) palbociclib (25 mg/kg) ow t ow t ow t 3 r r S R r S Cells were harvested 24h post-dosing (ctrl or 500 nM palbo) and protein expression was g g Ribo Ribo g Ribo elacestrant (30 mg/kg) % % R R Ribo % Ribo Proliferation assays: analyzed using antibodies as follows: ERα, PR, E2F1, CCNE1, CCNE2, CCND1, Rb, pRb, no rma liz e no rma liz e no rma liz e ( ( ESR1-mut Y537S 1500 0 0 ( 0 (m m -3 -2 -1 0 1 -3 -2 -1 0 1 -3 -2 -1 0 1 Cells were plated at a density of 5000 cells/well and treated the next day with respective CDK2, CDK4, CDK6 (Cell Signaling Technologies, Cat#13258; #3153; #3742, #4129, Log[Ribociclib (µM)] Log[Ribociclib (µM)] Log[Ribociclib µ(M)] PgR Tff1 GREB1 % % %

) #4132, #2978, #9309, #8516, #2546, #12790, #13331), p107, p130 (Abcam: ab168458, )

) treatments. Viability was measured after 7 days and data are graphed as % growth S S s S

10 0 1000 10 0 100 10 0

s Palbo

s Palbo Palbo l

100 s s 100 s

e 100 100 100

100 % ab6545) and Vinculin: Sigma-Aldrich, #v9131. a a a R R eve l R v 100 % inhibition relative to control. 100 % l ve l ion l l l s Palbo ion

ion Palbo Palbo e s r s r r le a l t t A t Volu me a a c c c r N A o ctrl R o o nhibi t N ctrl t i ctrl o t t nhibi t 500 nhibi t RN A i i o R m t o h o

50 d d d t h t

50 h

50 m m 1 e 50 e e d 50 50 d d B R ow t ow t r ow t g E S ff 1

r S r

g Abema T P

Abema R g g

S Tu m % Abema R R G % R Abema % Abema 0 o rma liz o rma liz Abema o rma liz References N no rma liz e N 0 0 N ( no rma liz e 0 no rma liz e 0 10 20 30 40 50 60 ( ( 0 0 0 -3 -2 -1 0 1 -3 -2 -1 0 1 -3 -2 -1 0 1 0 100 nM 1000 nM 0 100 nM 1000 nM 0 100 nM 1000 nM 1. Perou CM, et al. Nature. 2000;406:747-52. 4. Goel S. et al. Ther Adv Med Oncol. 2018; DOI:10:1758835918786451. 7. Bihani T, et al. Clin Cancer Res. 2017;23:4793-4804. Log[Abemaciclib (µM)] Log[Abemaciclib (µM)] Log[Abemaciclib (µM)] Days of Treatment elacestrant elacestrant elacestrant 2. Dellapasqua S, Castiglione-Gertsch M. Breast. 2005;14:555-63. 5. Shah M. et al. Oncology. 2018;32(5):216-222. 8. Wardell et al. Clin Cancer Res. 2015;21(22). LTED= long-term estrogen deprived; ribo= ribociclib; abema= abemaciclib; riboS= ribociclib-sensitive; riboR= ribociclib-resistant; abemaS= abemaciclib-sensitive; abemaR= abemaciclib-resistant #fulvestrant (3 mg/dose) data is from a separate study 3. NCCN Clinical Practice Guidelines in Oncology: Breast Cancer. NCCN.org. 2016. 6. Garner F, et al. Anticancer Drugs. 2015;26:948-56.

H Patel, N Tao, H Arlt, and T Bihani are employees of and own company stock in Radius Health, Inc. This study was sponsored by Radius Health, Inc. on behalf of its wholly owned subsidiary, Radius Pharmaceuticals, Inc. and This presentation is the intellectual property of the authors/presenter. Reprints, copies, and distribution are prohibited without permission. graphic services were provided by Phillips Gilmore Oncology Communications, Inc. and were funded by Radius Health, Inc. on behalf of its wholly owned subsidiary, Radius Pharmaceuticals, Inc.