1. A B MCF7-ERaWT MCF7-ERaY537S MCF7-ERaY537N MCF7-ERaY537C MCF7-ERaD538G
MCF7-ERaE380Q
Figure S20. H3B-5942 in combination with CDK4/6 inhibitors leads to enhanced potency in breast cancer lines in vitro. A, Curve-shift examples for the CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib in combination with 1 mM H3B-5942 from the screen in the ST941 PDX-derived ERaY537S/WT line are shown (boxes= CDK4/6 inhibitor treatment alone; circles= CDK4/6 inhibitors in combination with H3B-5942). B, Combinations of H3B-5942 plus CDK4/6 inhibitor palbociclib were confirmed as synergistic in vitro in MCF7 cells engineered to overexpress ERaWT and indicated ERa mutants. H3B-5942 was tested in combination with palbociclib over the range of 2.5 µM to 25 pM for 6 days and then assessed for viability/proliferation (n=2). Data were normalized to untreated controls and to data at time zero (T0) where 100% and 0% equal uninhibited growth and stasis, respectively. Analysis of synergy used the Loewe Model, and the results were graphed in Combenefit software using the dose-response surface view of synergy/antagonism.
Supplementary Figure S20

2. A B STDEV (FOLD) Supplementary Figure S21
Compound
Fold
Everolimus
8.6
8.1
Temsirolimus
7.8
7.5
Sirolimus
7.4
AZD8055
5.8
PKI-402
Torkinib
5.6 KU
5.1 PF
4.9
Omipalisib
Amgen PI3K-mTOR
4.8
WYE mTOR Inh
4.6
WYE
4.2
3.8
NVP-BGT
2.9
STDEV (FOLD) B
Figure S21. H3B-5942 in combination with mTOR inhibitors leads to enhanced potency and efficacy in PDX-ERaY537S/WT line in vitro. A, To find synergistic drug combinations, a reference compound collection was profiled in dose-response +/- 1 µM H3B-5942 in vitro against the PDX-ERaY537S/WT line for 6 days. Results for all combinations were plotted as the fold standard deviation of the difference in the area under the curves. Inset shows the top 10% of the test, and results for the mTOR inhibitors tested are summarized in the table. B, Curve-shift examples for the mTOR inhibitors everolimus, sirolimus, and temsirolimus in combination with 1 mM H3B-5942 are shown (boxes= mTOR inhibitor treatment alone; circles= mTOR inhibitors in combination with H3B-5942).
Supplementary Figure S21

3. A Supplementary Figure S22 MCF7-ERaWT MCF7-ERaY537S 100 nM 250 nM
α-tubulin
Cyclin D1
p-Rb
DMSO
H3B-5942
Palbociclib
Combo Rb
100 nM
250 nM
MCF7-ERaWT
MCF7-ERaY537S A
Supplementary Figure S22

4. Supplementary Figure S22
Figure S22. H3B-5942 in combination with palbociclib improves growth suppression of MCF7-ERaWT and MCF7- ERaY537S lines over single-agent treatments. A-B, Combination treatment improves suppression of (A) phosphorylated Rb (p-Rb) and (B) proliferation markers MKI67 and CDC25A over single-agent treatments alone following 5 days of continuous treatment. C, Five-day combination treatment improves growth suppression of MCF7-ERaWT and MCF7- ERaY537S lines over single-agent treatments.
Supplementary Figure S22

5. Supplementary Figure S23
Supplementary Figure S23. H3B-5942 in combination with CDK4/6 inhibitor palbociclib improves efficacy over single agents alone in the ERaWT MCF7 xenograft model and in the ERaY537S/WT ST941 PDX model. A, Daily oral dosing of H3B-5942 at 3 mg/kg with palbociclib at 75 mg/kg improves efficacy over single agent administration alone in the MCF7 xenograft model. N=7 for H3B-5942 treatment group, N=8 for all other groups. B, Daily oral dosing of H3B-5942 at 10 mg/kg with palbociclib at 75 mg/kg improves efficacy over single agent administration alone in the ERaY537S/WT ST941 PDX model. N=6 for all groups. Efficacy data in (A-B) presented as waterfall plots depicting percent change in individual tumor volumes relative to day 0 (first day of dosing) on days 17 and 35 in the MCF7 and ST941 models, respectively. C-D, Body weights following single agent or combination treatments at indicated doses in the (C) MCF7 and (D) ST941 models. Data represent the mean BW ± SEM
Supplementary Figure S23