1. Supplementary Figure S13
Figure S13. H3B-5942 effects in ST941 subcutaneous xenografts in female nude mice. Changes in tumor PGR and NPY1R (pharmacodynamic markers of ERa) at indicated time points along with tumor and plasma H3B-5942 concentrations after a single oral dose of H3B-5942 at the indicated doses. Data represent the mean ± SEM (N=3).
Supplementary Figure S13

2. ERa direct target genes
Vehicle
QDx1
(24h) 1 2 3
(48h)
(72h)
QDx2
QDx3
PGR
NPY1R
Figure S14. H3B-5942 suppresses ERa activity in an ERaY537S/WT breast cancer PDX model (ST941) subcutaneously grown in female nude mice. A, Heatmap showing suppression in expression of a panel of ERa direct target genes6 in tumors collected at various time points following a single oral dose (QDx1, 24, 48 and 72 hours post-dose collections), 24 hours post-second oral daily dose (QDx2), and 24 hours post-third oral daily dose (QDx3) of H3B-5942 at 200 mg/kg. Two well-characterized ERa target genes, PGR and NPY1R, are highlighted. N=2 for vehicle treatment, N=3 for H3B-5942 treatment. (B) Changes in tumor PGR (red solid) and NPY1R (red open) expression as assessed by qPCR analysis at indicated time points along with tumor H3B-5942 concentrations after oral dosing of H3B-5942 at indicated schedules (green diamond). Data presented as mean ± SEM.
Supplementary Figure S14

3. Supplementary Figure S15
Figure S15. Antitumor activity of H3B-5942, tamoxifen, and fulvestrant in the ERaWT MCF7 xenograft model. A, H3B-5942 was administered orally QD at 1, 3, 10 and 30 mg/kg in female nude mice carrying MCF7 tumor xenografts. Tamoxifen was dosed SC Q2D at 1 mg/mouse whereas fulvestrant was dosed SC QW at 5 mg/mouse. Data represent the mean tumor volume ± SEM (N=8, control; N=6, treatment groups). *p<0.05, **p<0.01 versus vehicle control on day 17 (multiple unpaired t-tests with significance determined using the Holm-Sidak method). B, Waterfall plot showing individual tumor responses on day 17 following H3B-5942, tamoxifen or fulvestrant treatment. Data presented as percent change in tumor volume on day 17 relative to day 0 (first day of treatment).
Supplementary Figure S15

4. Supplementary Figure S16
Figure S16. H3B-5942 was screened for activity across multiple ERa positive and negative PDX models of breast cancer. H3B-5942 orally dosed daily at 200 mg/kg showed efficacy in ERa-positive ST986, ST2177 and ST897 models while TGI was not observed in ERa negative PDX models ST1248, ST996, and ST1337. N=1 for vehicle treatment; N=2 for H3B-5942 treatment (ST897, N=1 for TGI calculation). Data represent the mean tumor volume ± SEM.
Supplementary Figure S16

5. A B C ERa PR Ki67 Supplementary Figure S17 Vehicle 3 mg/kg 10 mg/kg
Tamoxifen
Fulvestrant
H3B-5942
ERa PR
Ki67 C
Figure S17. Antitumor activity of H3B-5942, tamoxifen, and fulvestrant in the ERaY537S/WT ST941 PDX model. A, Waterfall plot showing individual tumor responses on day 35 following H3B-5942, tamoxifen or fulvestrant administration. Data presented as percent change in tumor volume on day 35 relative to day 0 (first day of treatment). B, IHC analysis of ERa, PR and Ki67 and (C) gene expression analysis of ERa target genes PKIB, NPY1R and SERPINA3 in endpoint ST941 tumors from efficacy study presented in (A). Representative images are shown for IHC analysis in (B) and expression analysis of 4 samples/group presented as dot plot in (C). Data are expressed as mean ± SD. *p<0.05, **p<0.01, versus DMSO control (two-sided t-test).
Supplementary Figure S17

6. Supplementary Figure S18
Figure S18. SoCs tamoxifen and fulvestrant, and H3B-5942 were profiled in the ERaY537S/- WHIM20 PDX model of breast cancer. A, Tamoxifen subcutaneously dosed at 1 mg/mouse Q2D, and fulvestrant subcutaneously dosed at 5 mg/mouse QW showed no significant activity. N=8 for vehicle control and tamoxifen-treated groups, N=7 for the fulvestrant-treated group. B, H3B-5942 orally dosed daily at 100 mg/kg showed significant TGI (p<0.05) whereas tamoxifen subcutaneously dosed at 1 mg/mouse Q2D did not significantly induce TGI. N=9 for all groups. Data represent the mean tumor volume ± SEM. * p<0.05 versus vehicle control (Dunnet’s multiple comparison test). All doses and regimens were well tolerated.
Supplementary Figure S18

7. Supplementary Figure S19
Figure S19. H3B-5942 is well tolerated in a number of ERaWT and ERaMUT breast cancer models. Percent body weight change at indicated days following oral dosing of H3B-5942, and subcutaneous dosing of tamoxifen and fulvestrant at indicated doses. Data represent the mean tumor volume ± SEM. QD, once daily dose. Q2D, every other day dosing. QW, once weekly dosing. PO, Per os (oral dosing). SC, subcutaneous dosing.
Supplementary Figure S19