Comprehensive in Vitro Characterization of the Mechanism of Action of EPI-7386, an Androgen Receptor N-Terminal Domain Inhibitor

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Comprehensive in vitro characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor

Nan Hyung Hong1, Shihua Sun2, Peter Virsik1, Alessandra Cesano1, Han-Jie Zhou1, Elahe A. Mostaghel3,4,5, Stephen R. Plymate2,4, Ronan Le Moigne1

1 ESSA Pharma Corp, South San Francisco , CA 2 University of Washington, Division of Gerontology and Geriatric Medicine, Seattle, WA 3 Fred Hutchinson Cancer Research Center, Clinical Research Division, Seattle, WA 4 VA Puget Sound Health Care System, Seattle, WA 5 University of Washington, Medical Oncology Division, Seattle, WA

Contact: [email protected]

Presented at AACR Annual Meeting 2021 Background

v As a key driver of prostate cancer, androgen receptor (AR) signaling has been a major target of prostate cancer therapy v All current anti-androgens function through the ligand-binding aniten domain (LBD) of the AR v Anti-androgen resistance mechanisms generally develop at the LBD, due to mutations in the LBD itself and the expression of constitutively active splice variants of AR that lack the LBD v Anitens are small molecules capable of targeting the AR N- terminal domain that can inhibit transcriptional activity of the AR even in the presence of LBD-driven anti-androgen resistance Figure 1. Anitens are first-in-class NTD inhibitors of the androgen receptor. Structure of the androgen receptor (AR) and mechanism of inhibition. The AR comprises three main functional domains: the LBD, involved v EPI-7386 is a new generation of aniten which exhibits high in binding with androgens, the DBD, and the NTD, which orchestrate the potency, low metabolism, on-target specificity transactivation of the receptor. It was previously demonstrated that the first- generation aniten and its stereoisomers specifically bind to the transactivation unit 5 (Tau5) of AR NTD to block essential protein-protein interactions required for the transcriptional activity of AR1-3.

1De Mol et al., ACS Chem Biol, 2016, 2Andersen et al., Cancer Cell, 2010, 3CONFIDENTIALSadar, Cancer Res, 2011 2 EPI-7386 interacts with both AR-FL and AR-V7

AR-FL (+100 nM DHT) in LNCaP cells AR-V7 (-DHT) in LNCaP95 cells A D

EPI-7386 B 3 µM EPI-7386 C E 30 µM EPI-7386 F DMSO DMSO 1.5 1.0 1.0 1.0 1.0 0µM 0µM 41°C 39°C /I /I /I /I x x I

x CETSA EC x I 0.5 50 I I 0.5 0.5 0.5 CETSA EC50 : 6.4 µM : 4.3 µM

0.0 0.0 0.0 40 44 48 52 -7 -6 -5 -4 40 45 50 55 -9 -8 -7 -6 -5 -4 °C [M] °C [M]

Figure 2. Cellular Thermal Shift Assay (CETSA) of AR target engagement by EPI-7386. AR target engagement by EPI-7386 induced a decrease in AR thermal stability. (A-C) AR-FL target engagement in LNCaP cells was determined in the presence of 100 nM DHT and (D-F) AR-V7 in LNCaP95 cells was determined in the absence of DHT. (A, D) Representative Western Blot showing thermostable AR-FL in LNCaP cells (A) or AR-V7 in LNCaP95 (D) following heat shock in the presence (+) or absence (-) of EPI-7386. (B, E) Melt and shift curve of AR-FL in LNCaP cells (B) or AR-V7 in LNCaP95 cells (E) treated with EPI-7386 (blue) at the indicated concentration and DMSO control (black). (C, F) Isothermal (47℃) dose-response curve of AR-FL in LNCaP cells and AR-V7 in LNCaP95 cells. Potency of target engagement (CETSA EC50) was calculated from multiple independent experiments (n≥9). * AR-V7 specific antibody (clone RM7; RevMab) was used to detect AR-V7 in LNCaP95. ** No AR-V7 thermal shift was induced by DHT unlike AR-FL, confirming assay specificity to AR-V7 lacking LBD

CONFIDENTIAL 3 EPI-7386 is active in the AR splice variant (ARv567es) expressing cell line

A B C CWR AD1 Luc_9-21-20_controlCWR onlyCWR-R1 AD1-AD1 Luc_9-21-20 CWR D567 Luc_6-19-20_controlCWRCWR alone D567-R1 Luc_6-19-20_treatment-D567 CWR-R1-D567 : expressing only AR-FL 6000 6000 : expressing only ARv567es 20000 20000 EPI-7386 : expressing only ARv567es CWR D567 MTT_4-29-20_noR1881_%CWR D567 MTT_4-29-20_w/R1881_%_% 400 Enzalutamide 300 4000 4000 120 120 300 100 100 200 80 80 200 2000 2000 60 60 100 40 40 Viability (%) Viability RL (Firefly/Protein) RL (Firefly/Protein) RL (Firefly/Protein) 100 20 20 0 0 0 0 0 0 5 5 5 5 10 20 10 10 10 10 2.5 2.5 2.5 2.5 1.11 0.37 3.33 1.25 1.25 1.25 1.25 0.123

0.156 0.313 0.625 0.156 0.313 0.625 DMSO 0.156 0.313 0.625 0.156 0.313 0.625 DMSO DMSO DMSO EPI-7386 [µM] Enzalutamide [µM] EPI-7386 [µM] Enzalutamide [µM] 10 uM Enza EPI-7386 [µM] 10 uM EPI-7386 10 nM R1881 10 10 nM R1881 10 - R1881 + 1nM R1881

Figure 3. Effect of EPI-7386 against AR splice variant driven transcriptional activity and cell growth. (A,B) AR-driven transcriptional activity was assessed using a probasin promoter (ARR2PB) driven luciferase reporter in CWR-R1-AD1 cells expressing only AR-FL in the presence of 5%FBS (A) or in CWR-R1-D567 cells expressing only AR-V567es under serum free condition (B). (C) Cell viability analysis of CWR-R1-D567 with MTT assay showing relative viable cell % to DMSO control after 72 hr treatment of EPI-7386 or enzalutamide in the absence or presence of R1881. * CWR-R1-D567 cells were derived from the CWR-R1-AD1 cells by TALEN-mediated deletion of AR exons 5-74

4NyquistCONFIDENTIAL et al., PNAS, 2013 4 EPI-7386 inhibits AR genomic binding

PROMOTER (0-3 kb) 5`-UTR A + 1nM R1881 B EXON C FKBP5 TMPRSS2 KLK3 CHRNA2 SLC45A3 53.30% INTRON 10 !M 10 !M PROMOTER3`-UTR (0-3 kb) 5`-UTRDOWNSTREAM (0-3 kb) DMSO 1 nM R1881 EPI-7386 Enza DMSO EXONDIST INTERGENIC (525) INTRON [0-327] [0-42] [0-20] [0-131] [0-50] 3`-UTR PROMOTERPROMOTER (0-3 kb) (0-3 kb) DMSO DOWNSTREAM5`-UTR (0-3 kb) 5`-UTR 1nM R1881 DISTEXON INTERGENIC EXON 1nM R1881 (31510) INTRON INTRONPROMOTER3`-UTR (0-3 kb) 3`-UTR5`-UTRDOWNSTREAM (0-3 kb)10μM EPI-7386 10!M EPI-7386 Total=100 DOWNSTREAMEXONDIST INTERGENIC (0-3 kb) (9121) DISTINTRON INTERGENIC R1881 10µM Enza

3`-UTR + DOWNSTREAM (0-3 kb) 10!MEnza Total=100.1 DIST INTERGENIC (10249) Copy of Copy of Copy of Copy of KLK2, FKBP5, TMPRSS2 0 25 50 75 100 E 30 Total=100.1 DMSO R1881 Percentage (%) 20 Total=100 5 uM EPI-7386 5 uM Enza D Total=100 6 % of Target % of Background treatment De novo Motif P-value Best match Sequences with Motif Sequences with Motif G A TTT DMSO CAC 1e-92 66.85% 18.42% FOXA1(Forkhead) 4 TATTTGTCGA 20 A A CTC G GT R1881 G T A AT T 1e-202 19.04% 2.88% GRE/ARE(NR) GG T A ACT C GA A T CG GAGAC C 10 2 R1881 AGA + ATGTAA C 1e-479 70.55% 19.51% FOXA1(Forkhead) C A T GAC T C T T CCA T 0 10 µM EPI-7386 Binding Events Detected per 1000 cells -5 0 5 -5 0 5-5 0 5-5 0 5 R1881 G TTA G T C C + AGT 1e-444 68.70% 19.45% FOXA1(Forkhead) T T G 0 A G CT C C T AA A Distance from center (Kb) 10 µM Enza Untr12 KLK3 KLK2 FKBP5 TMPRSS2 Untr12 KLK3 KLK2 FKBP5 TMPRSS2

6h 24h

Figure 4. ChIP-seq analysis of AR genomic distribution. (A-D) Cells were treated with DMSO, R1881, EPI-7386 or enzalutamide(Enza) in the presence of R1881 as indicated for 24 h. (A) Heatmap view of AR ChIP-seq signals around AR peaks (± 5 kb) detected in LNCaP cells with treatments as indicated. (B) Bar chart showing genomic distribution of AR- binding peaks including promoters (withing 3 kb upstream of TSS), 5’ UTRs, exons, introns, 3’UTRs, downstream (within 3 kb downstream of the gene), and intergenic regions. Total peak numbers are also noted. (C) Gene track view of AR ChIP-seq data at FKBP5, TMPRSS2, KLK3, CHRNA2, and SLC45A3 loci, visualized by Integrative Genomics Viewer (IGV). (D) The top enriched motifs present in AR ChIP-seq peaks of each condition identified using Homer. (E) ChIP-qPCR validation in LNCaP cells with AR antibody. Cells were treated with DMSO, 1 nM R1881, EPI-7386, or enzalutamide in the presence of 1nM R1881 for 6 or 24 h. Untr12, primer that binds to gene dessert area in chromosome 12, was used for negative control locus.

CONFIDENTIAL 5 EPI-7386 shows superior activity than enzalutamide in the AR-V7 expressing LNCaP95 cells by modulating both AR-FL and AR-V7 driven gene expression

+/- R1881 FC3_padj<0.01 A downregulated upregulated 7.5uM Enzalutamide N/A C Z-score -4 2

- R1881 7.5uM EPI-7386 982 959

7.5uM Enzalutamide 97 / 59 NUCB2 LAMC1 GCLC 7.5uM EPI-7386 776 1032 GULP1

+ R1881 PFKFB2 HOMER2 AKAP12 0 500 1000 1500 2000 2500 NCOA1 RHOU Number of genes ELL2 HIF1A w/ 1nM R1881 PRKCD KLK3 KLK2 FASN B HOXB13 Withoutno R1881 R1881 With 1nM R1881 C19orf48 CROT Vehicle 5 uM ENZ 5 uM EPI-7386 GNAI1 Vehicle 5 uM ENZ 5 uM EPI-7386 PPAP2A R1881 7.5 uM ENZ 7.5 uM EPI-7386 7.5 uM ENZ 7.5 uM EPI-7386 SLC45A3 FKBP5 60 F2RL1 2.0 45 ATAD 2 30 GUCY1A3 LPAR3 GRIN3A 1.5 18 AD AM7 ETV1 15 CBR4 STEAP2 SNX1 1.0 12 MAPK8 RTN4 9 DOPEY2 MBOAT2 STK39 0.5 6 UBE2E3 PRKD1 3 STEAP1 Relative mRNA expression Relative mRNA expression 0.0 0 5 uM EPI-7386 7.5 uM EPI-7386 Vehicle 5 or 7.5 uM enzalutamide

KLK3 KLK2 KLK3 KLK2 FKBP5 NKX3-1 FKBP5 NKX3-1 CHRNA2 CHRNA2 TMPRSS2 SLC45A3 TMPRSS2 SLC45A3

Figure 5. Evaluation of the effect of EPI-7386 on transcriptome in LNCaP95 cells. RNAseq analysis of cells treated with EPI-7386 or enzalutamide (ENZ) for 24 h in the presence or absence of 1 nM R1881. N=3 (A) Number of genes significantly up- or down-regulated following treatments. Fold change (FC) >3, pAdj<0.01. (B) Relative expression levels of representative AR-regulated genes in the absence of R1881 (left) or presence of R1881 (right). (C) Heatmap showing expression of AR-V7 regulated genes following treatments in the absence of R1881.

CONFIDENTIAL 6 EPI-7386 inhibits AR transcriptional activity similarly to enzalutamide but with a few notable differences, and combination with enzalutamide exhibits a broader & deeper inhibition of

R1881-responsive genes Z-score androgen-responsive gene expression -5 9 significantly modulated by treatment EPI-7386 Down EPI-7386 UP (FC>3, FDR<0.01) vs R1881 vs R1881 STEAP 4 ENZ Down ENZ UP CCDC141 SLC3 8 A4 SLC2 A3 TUB A3 D TMEM1 0 0 SNAI2 ANDROGEN RESPONSE ESTROGEN RESPONSE EARLY Downregulated Upregulated TTN F5 EPI-7386 Enzalutamide TB X 1 5 C E CSGALNACT1 A G2M CHECKPOINT UV RESPONSE DN HPGD ORM1 FKBP5 NKX3-1 CHRNA2 513 563 ALPK2 TMPRSS2 STEAP4 CHRNA2 E2F TARGETS EPITHELIAL MESENCHYMAL TRANSITION 5/5uM Combo activated(25) DOCK8 KLK3 - OR5 B2 STEAP4 NPPC KLK3 PLA2G4E 10 MTORC1 SIGNALING INTERFERON GAMMA RESPONSE 274 177 PDE3A 10 5uM EPI-7386 TMC C 3 FK BP 5 EMP 1

CHOLESTEROL HOMEOSTASIS TNFA SIGNALING VIA NFKB R1881 ADAM7 CHRNA2 FKBP5 TMPRSS2 269 216 PGC NKX3-1 5uM ENZ OP RK 1 8 AMIGO2 8 MITOTIC SPINDLE P53 PATHWAY SLC6 A2 0 MAN1 A1 7.5uM EPI-7386 435 520 VWA2 MYC TARGETS V1 INFLAMMATORY REPONSE SI

) ST8 SIA4

) PCED1B 6 6 ABCA1 7.5uM ENZ 312 289 CCDC83 MYC TARGETS V2 EPI-7386 Down INTERFERON ALPHA RESPONSE EPI-7386 UP LRRC31 pAdj

pAdj SLC3 0 A1 0 vs R1881 PTPRB vs R1881 ACKR3 ESTROGEN RESPONSE EARLY ENZ Down IL2 STAT5 SIGNALING ENZ UP ABCA12 4 CERK 4 0 300 600 900 1200 repressed(25)

- PLCB2 SP TLC3 Log10( GLYCOLYSIS HYPOXIA Log10(

- SYTL2 - LRRN1 Number of genes FFAR2 ESTROGEN RESPONSEANDROGEN LATE RESPONSE MYOGENESISESTROGEN RESPONSE EARLY TINAGL1 2 2 ADM

R1881 MMP 1 0 Z-score MET HYPOXIAG2M CHECKPOINT UV RESPONSE DN R1881-responsive genes IL6 JAK STAT3 SIGNALING -5 9 0 0 TNFA SIGNALING VIA NFKB E2F TARGETSsignificantly modulated by treatment EPITHELIALAPOPTOSIS MESENCHYMAL TRANSITION R1881 only 7.5 μM 7.5 μM Vehicle 5/5 μM F EPI-7386 ENZ Combo -5 0 5 -10 -5 0 5 (FC>3, FDR<0.01) MTORC10 SIGNALING5 10 15 INTERFERON0 GAMMA2 RESPONSE4 6 5 uM single8 vs 5/5uM combo : broader inhibition STEAP 4 log2 Fold Change CCDC141 log2 Fold Change SLC3 8 A4 EPI-7386 Down EPI-7386-Log10(pAdj) UP -Log10(pAdj) 5uM vs 7.5 uM EPI-7386 : shows dose response while 7.5 uM Enza does not SLC2 A3 CHOLESTEROL HOMEOSTASIS TNFA SIGNALING VIA NFKB TUB A3 D Differentially expressed genes with fold change > 2 and FDR <0.01 are labled in purple vs R1881 vs R1881 dramatically change the # genes TMEM1 0 0 ENZ Down ENZ UP SNAI2 and green MITOTIC SPINDLEDownregulated Upregulated P53 PATHWAY TTN F5 TB X 1 5 CSGALNACT1 ANDROGEN RESPONSE ESTROGEN RESPONSE EARLYMYC TARGETS V1 INFLAMMATORY REPONSE HPGD D ORM1 CHRNA2 Androgen responseG2M CHECKPOINT UV RESPONSE DNMYC TARGETS513 V2 563 INTERFERON ALPHA RESPONSE ALPK2 B 5/5uM Combo activated(25) DOCK8

- OR5 B2 NPPC E2F TARGETS EPITHELIAL MESENCHYMALESTROGEN TRANSITION RESPONSE EARLY IL2 STAT5 SIGNALING*R1881-responsive genes = Genes that are modulated by R1881 stimulation at least 2 fold with FDR PLA2G4E R1881 EPI-7386 Enzalutamide 274 177 PDE3A 5uM EPI-7386 <0.01 as compared to Vehicle control. TMC C 3 MTORC1 SIGNALING INTERFERON GAMMA RESPONSE FK BP 5 GLYCOLYSIS HYPOXIA EMP 1

0.8 0.0 0.0 R1881 ADAM7 NES 2.64 -0.1 NES -2.38 CHOLESTEROL HOMEOSTASISNES -2.55 TNFA SIGNALING VIA NFKB5uM ENZ 269 216 PGC ESTROGEN RESPONSE LATE MYOGENESIS OP RK 1 0.6 p < 0.001 -0.2 p < 0.001 -0.2 p < 0.001 AMIGO2 MITOTIC SPINDLE SLC6 A2 0 -0.3 P53 PATHWAY MAN1 A1 0.4 -0.4 -0.4 7.5uM EPI-7386HYPOXIA435 520 IL6 JAK STAT3 SIGNALING VWA2 MYC TARGETS V1 INFLAMMATORY REPONSE SI -0.5 ST8 SIA4 0.2 -0.6 PCED1B -0.6 TNFA SIGNALING VIA NFKB APOPTOSIS ABCA1 MYC TARGETS V2 INTERFERON ALPHA RESPONSE7.5uM ENZ 312 289 CCDC83 0.0 -0.7 LRRC31 -0.8 SLC3 0 A1 0 PTPRB Enrichment(ES)score ESTROGEN RESPONSE EARLY IL2 STAT5 SIGNALING 0 5 10 15 0 2 4 6 8 ACKR3 ABCA12 CERK GLYCOLYSIS HYPOXIA 0 300 600 900 1200 repressed(25)

-Log10(pAdj) - -Log10(pAdj) PLCB2 EPI-7386 Down EPI-7386 UP SP TLC3 4 4 4 SYTL2 ESTROGEN RESPONSE LATE vs R1881 MYOGENESIS vs R1881 LRRN1 2 2 2 ENZ Down NumberENZ UP of genes FFAR2 TINAGL1 0 0 0 HYPOXIA IL6 JAK STAT3 SIGNALING ADM

R1881 MMP 1 0 -2 -2 -2 MET TNFA SIGNALING VIA NFKB APOPTOSIS Signal2Noise -4 -4 -4 ANDROGEN RESPONSE ESTROGEN RESPONSE EARLY 0 2000 4000 6000 8000 0 2000 4000 6000 8000 10000 0 1000 2000 3000 4000 5000 6000 7000 R1881 only 7.5 μM 7.5 μM Vehicle 5/5 μM G2M CHECKPOINT0 5 10 15 UV RESPONSE0 2 DN 4 6 8 -Log10(pAdj) -Log10(pAdj) EPI-7386 ENZ Combo E2F TARGETS EPITHELIAL MESENCHYMAL5 uM single TRANSITIONvs 5/5uM combo : broader inhibition MTORC1 SIGNALING INTERFERON5uM vsGAMMA 7.5 uM RESPONSE EPI-7386 : shows dose response while 7.5 uM Enza does not dramatically change the # genes CHOLESTEROL HOMEOSTASIS TNFA SIGNALING VIA NFKB Figure 6. Transcriptomic analysisMITOTICin SPINDLELNCaP cells. RNA-seq analysis of LNCaPP53 PATHWAYcells treated with EPI-7386 or enzalutamide (ENZ) as a single agent and in combination for 24 h in the presence of 1 nM R1881. N=MYC3 (A) TARGETSVolcano V1 plots of the differentiallyINFLAMMATORYexpressed REPONSEgenes between 7.5 µM EPI-7386 or 7.5 µM ENZ treatment vs R1881 only. Significantly down- MYC TARGETS V2 INTERFERON ALPHA RESPONSE *R1881-responsive genes = Genes that are modulated by R1881 stimulation at least 2 fold with FDR regulated genes are in purpleESTROGENand significantly RESPONSE EARLY up-regulated genes are in greenIL2 STAT5. FC SIGNALING>2, pAdj <0.01. (B) GSEA plots showing enrichment of androgen response gene signature. (C,D) <0.01 as compared to Vehicle control. Hallmark pathway enrichment analysisGLYCOLYSISfor down- (C) and up-regulated genes (D) followingHYPOXIA treatments. (E,F) Broader and deeper inhibition of AR pathway by combination treatment of EPI-7386 with ENZ. (E) NumberESTROGEN RESPONSEof R1881 LATE-responsive genes significantly up- MYOGENESISor down-regulated following treatment. FC >3, FDR <0.01. (F) Heatmap showing top 50 R1881- HYPOXIA IL6 JAK STAT3 SIGNALING responsive genes that are modulatedTNFA SIGNALINGmore VIA NFKBthan 3-fold by single agent and combinationAPOPTOSIStreatment. 0 5 10 15 0 2 4 6 8 -Log10(pAdj) -Log10(pAdj) *LNCaP cells are resistant to apalutamide and darolutamide due to T878A mutation, andCONFIDENTIAL not tested in this model 7 EPI-7386 combination with ‘lutamides exhibits a broader & deeper inhibition of AR- regulated gene expression in AR amplified VCaP cells

EPI alone dose response_fkbp5_slc45a3Vehicle EPIR1881 alone dose response_fkbp5_slc45a3 EPI alone dose response 10 uM EPI A 15 uM EPI Vehicle 20 uM EPI 60 Copy of Fold change2_FDR<0.01 combo R1881 Vehicle

Relative mRNA expression Relative mRNA 10 uM EPI C Z-score combo 8 60 R1881 -8 8

15 uM EPI expression Relative mRNA 10 uM EPI Down-regulated Up-regulated D Vehicle 10 uM EPI 2 uM DAR + 10 uM EPI 20 uM EPI 15 uM EPI 6 40 20 uM EPI Vehicle 10 uM EPI 2 uM DAR + 10 R1881uM EPI 2 uM DAR 5 uM APA + 10 uM EPI 40 20uM EPI-7386 818 1451 R1881 2 uM DAR 5 uM APA + 10 uM EPI 5 uM APA 5 uM ENZ + 10 uM EPI 5 uM APA 5 uM ENZ + 10 uM EPI 5 uM ENZ 4 15uM EPI-7386 417 680 combo-1 20 5 uM ENZ 12 20 10uM EPI-7386 206 / 268 12 60 Vehicle

2 activated(50) - 10 50 R1881 0 500 1000 1500 2000 2500 Relative mRNA expression 0 10 40 expression Relative mRNA 10 uM EPI FKBP5 SLC45A3 ACSL3 Number of genes 0 0 2 uM DAR KLK3 TMPRSS2 NKX3-1 CHRNA2 FKBP5 SLC45A3 ACSL3 R1881 8 20 5 uM APA 8 5 uM ENZ 6 15 2 uM DAR + 10 uM EPI 6 5 uM APA + 10 uM EPI

4 5 uM ENZ + 10 uM EPI B 10 4

1011 1310 repressed(50) Relative mRNA expression 5uM APA/10uM EPI - 2

5uM ENZ/10uM EPI 993 1502 Relative mRNA expression 2 5 2uM DAR/10uM EPI 1009 1480 R1881 10uM EPI-7386 207 / 268 0 KLK3 TMPRSS2 NKX3-1 CHRNA2 ACSL3 5uM APA 673 1012 0 0 Down-regulated Vehicle R1881 10μM EPI KLK3 TMPRSS2 NKX3-1 CHRNA2 ACSL3 SLC45A3 FKBP5 5uM ENZ 648 954 Up-regulated 5μM ENZ 5μM ENZ 5μM 5μM ENZ 5μM DAR 2μM APA 5μM APA 5μM 2uM DAR 604 841 DAR 2μM APA 5μM DAR 2μM APAcombo ENZ combo APAcombo ENZ combo APAcombo ENZ combo DAR DAR combo DAR combo DAR combo 0 1000 2000 3000 ‘lutadmide single EPI-7386 + ‘lutadmide Number of genes

Figure 7. Transcriptomic analysis in VCaP cells. VCaP cells were treated with EPI-7386 (EPI), enzalutamide (ENZ), apalutamide (APA), or darolutamide (DAR) as a single agent and in combination for 24 h in the presence of 1 nM R1881. N=3 (A) Bar graph showing dose response activity of EPI-7386 against androgen-responsive genes from RNAseq. (B) Number of genes significantly up- or down-regulated following treatments compared to R1881 stimulation only condition. FC >2, pAdj <0.01 (C) Heatmap comparing expression level of top 50 R1881-activated or -repressed genes across VCaP cells treated with single agent or combination. (D) Relative expression levels of representative AR-regulated genes in the presence of R1881, showing deeper inhibition by combination treatment.

CONFIDENTIAL 8 Summary

EPI-7386 is a second-generation AR NTD inhibitor (aniten) with the following characteristics:

• Engagement with both AR-FL and AR-V7 in cells • Active against both full-length AR and multiple AR splice variants (AR-V7 and ARv567es) • Strong displacement of R1881 (androgen)-induced genomic AR binding • On-target activity against the transcriptional activity of the AR, overall, similarly to enzalutamide but with a few notable qualitative and quantitative differences • Complementarity with the second generation of ‘lutamides in inhibiting the AR-associated transcriptional activity, with broader and deeper inhibition of the AR pathway

Ø A Phase 1 dose escalation clinical trial (NCT04421222) of EPI-7386 in men with mCRPC progressing on standard of care therapies including second generation anti-androgens is underway. Ø Early signs of biological activity and declining PSA levels in a multi-refractory patient (01-002) in the initial 200 mg cohort were observed (2021 ASCO-GU, poster #119). Ø Currently, 800 mg cohort is being dosed.

CONFIDENTIAL 9