A small molecule activator of the unfolded protein response eradicates human breast tumors in mice

Abstract

Metastatic estrogen receptor α (ERα) positive breast cancer is presently incurable. Seeking to target these drug-resistant cancers, we report the discovery of a compound, called ErSO, that activates the anticipatory unfolded protein response (a-UPR) and induces rapid and selective necrosis of ERα-positive breast cancer cell lines in vitro. We then tested ErSO in vivo in several preclinical orthotopic and metastasis mouse models carrying different xenografts of human breast cancer lines or patient-derived breast tumors. In multiple orthotopic models, ErSO treatment given either orally or intraperitoneally for 14-21 days induced tumor regression without recurrence. In a cell line tail vein metastasis model, ErSO was also effective at inducing regression of most lung, bone, and liver metastases. ErSO treatment induced almost complete regression of brain metastases in mice carrying intracranial human breast cancer cell line xenografts. Tumors that did recur remained sensitive to retreatment with ErSO. ErSO was well tolerated in mice, rats, and dogs at doses above those needed for therapeutic responses and had little or no effect on normal ERα-expressing murine tissues. ErSO mediated its anticancer effects through activation of the a-UPR, suggesting that activation of a tumor protective pathway could induce tumor regression.

One Sentence Summary:

Activation of the unfolded protein response induces regression of estrogen receptor α positive breast cancer xenografts in mice.

Introduction

In the approximately 75% of breast cancers that are estrogen receptor α (ERα; ESR1) positive (1), estrogen (17β-estradiol, E₂), acting primarily through ERα, drives proliferation and survival. Consequently, targeted therapies for these tumors, termed endocrine therapies, such as aromatase inhibitors, tamoxifen and other selective ERα modulators (SERMs), and fulvestrant and other selective ERα degraders/downregulators (SERDs) all ultimately inhibit ERα function (2). Whereas these endocrine therapies reduce breast cancer recurrence and mortality, some tumor cells persist, often leading to the development of de novo or acquired resistance in primary tumors and nearly universal resistance in the metastatic setting (3-6).

The treatment of drug-resistant ERα-positive breast cancer remains a major clinical challenge and most patients with metastatic disease experience recurrence within seven years (5, 7). Although resistance mechanisms are diverse (2, 6), one major resistance mechanism is mutation of ERα, commonly ERαY537S or ERαD538G (8), leading to constitutively activated ERα that drives estrogen-independent tumor growth and increased metastatic potential (2, 6, 8-14). Moreover, patients whose tumors express the ERαY537S or ERαD538G mutations exhibit markedly shorter median survival than patients whose tumors express wild-type ERα (15).

Diverse agents are in development to target breast tumors containing wild-type and mutant ERα, including second-generation SERDs and selective estrogen receptor covalent antagonists (SERCA) (2, 16). In mouse models these agents typically induce moderate regression of primary tumors and either remain untested against metastases or have limited ability to induce regression. Combining PI3KCA inhibitors (17, 18) or CDK4/6 inhibitors with endocrine therapies shows promise (19), but often leads to development of resistance. Immunotherapy has been largely unsuccessful in ERα-positive breast cancer (2). Given that the drugs in development share mechanisms of action with current therapies, have moderate efficacy against metastatic tumors in preclinical models (16), and lead to the development of resistance, metastatic ERα-positive breast cancer will likely remain an unmet medical need in the absence of alternative therapeutic approaches.

An alternative strategy to these largely inhibition-based approaches is to use a turn-on approach to convert a tumor-selective protective pathway into a lethal, targeted anticancer response. Endocrine therapy-resistant tumors typically maintain ERα expression, suggesting a therapeutic opportunity through leveraging ERα expression (2, 8, 16, 20). A tunable tumor-protective pathway is the tumor-protective estrogen-ERα-activated anticipatory unfolded protein response (a-UPR) (21).

Here, we describe a small molecule (R)-1, referred to as ErSO, that acts through ERα to elicit strong and sustained cytotoxic activation of the a-UPR. Unlike current therapies that are largely cytostatic, ErSO does not compete with estrogens for binding to ERα and selectively kills ERα-positive breast cancer cells including those harboring known resistance-mediating ERα mutations. Evaluation of oral and injected ErSO in orthotopic cell line xenograft and patient-derived xenograft (PDX) mouse models showed it induced complete regression (average >100,000-fold regression) of ERα-positive breast tumors, regardless of ERα mutational status and without recurrence after cessation of treatment. Notably, tumors that exhibited marked regression but did recur were not ErSO-resistant and remained sensitive to retreatment with ErSO. Moreover, ErSO resulted in complete regression of most tumors in a fulvestrant-resistant, ERα-mutant, patient-derived xenograft (PDX) mouse model. In tail vein injected mouse models of metastatic breast cancer, ErSO treatment eradicated most lung, bone, and liver metastases within seven days, and induced regression in a xenograft model of brain metastases

Results

Discovery of ErSO, a small molecule that selectively kills ERα (WT and mutant)-positive cancer cells

We conducted a medicinal chemistry campaign in an attempt to identify a-UPR hyperactivators that were highly potent inducers of breast cancer cell death and identified the compound (±)-1 (Fig. S1A,B). Enantiomers of (±)-1 were separated by preparative chiral chromatography with absolute configurations determined by X-ray crystallography (see Supplementary Materials and Methods). Only enantiomer (R)-1 (named as ErSO, Fig. 1A) exhibited activity against ERα-positive breast cancer cells, whereas the opposite enantiomer (compound (S)-1) was devoid of anticancer activity (Fig. 1B-C). ErSO activity was assessed in a panel of widely used breast cancer cell lines, including those expressing wild-type ERα and those expressing the constitutively active partially therapy-resistant (22) ERαY537S and ERαD538G mutations. Cell viability experiments showed ErSO was effective (average IC₅₀ ~20 nM, range 11-43 nM) against both the breast cancer cell lines expressing wild-type ERα and the ERαY537S and ERαD538G mutations such as human breast cancer cell lines TYS and TDG (Fig. 1D, Fig. S1C). ErSO was also effective against tamoxifen- and fulvestrant-resistant breast cancer cell lines containing wild type ERα (23, 24) (Fig. S1D).

Fig. 1. ErSO, a compound that kills ERα-positive breast cancer cell lines in vitro.

(A) Chemical Structure of ErSO. (B) 24 hour dose-response curves and IC₅₀ values for each compound against MCF-7 cells. Cell viability was measured by Alamar blue fluorescence and set relative to a vehicle control and a quantitative dead control treated with Raptinal. (C) Crystal violet staining of T47D cells after 24 hour incubations with compounds, concentrations as indicated. Image is representative of 2 independent biological experiments. (D) IC₅₀ values of ErSO after 24 hour incubation against a panel of ERα-positive breast cancer cell lines. TYS, TDG and MYS, MDG refer to T47D (T) or MCF-7 (M) with ERα mutations Y537S (YS) or D538G (DG). MCF-7 parental (par.) were the cells used to develop MYS and MDG cells. Cell viability was measured by Alamar blue fluorescence and set relative to a vehicle control and a quantitative dead control treated with Raptinal. Error represents s.e.m of at least 3 independent experiments. (E-G) Long-term cell culture experiment with T47D, TDG, TYS, MCF-7 par., MDG and MYS cells. Cells were seeded at a density of 4,000 cells per well. 4 days after seeding, cell number was estimated and quantified by MTS. Vehicle treatment shown was measured after 4 days of incubation. ErSO treated cells were incubated for 2 weeks at a concentration of 1 μM. Following treatment, compound was removed by 3 PBS washes. Cells were then cultured for an additional month following the 2 weeks of treatment. If retreated, cells were retreated for 2 weeks with ErSO (1 μM). Cell number was determined using MTS from a standard curve of cell number versus absorbance for each cell line. (H) MCF-7 cells were incubated as indicated for 24 hours and then stained with annexin V-FITC and PI dyes and analyzed via flow cytometry; E2: estradiol; Raptinal (10 μM). (I) TYS cells were treated for 24h with 1 μM OHT (z-4-hydroxytamoxifen), Fulv. (Fulvestrant), AZ9496 (orally available SERD), Bril. (Brilanestrant), or ErSO. Percent viability was determined by automated Trypan Blue exclusion assay. (J) Cells were incubated with ErSO or positive control, Raptinal (Rapt., 10 μM), for 24 h, stained with annexin V-FITC and PI dyes and analyzed by flow cytometry. (K) Shown is % viable cells from automated Trypan Blue exclusion assays cells. Cells: ERα-negative MDA-MB-231 (labeled 231) cells, MDA-MB-231 (ERα in): stably transfected to express ERα, and MDA-MB-231 (ERα in with KD): MDA-MB-231 (ERα in) cells in which ERα was then depleted by both siRNA knockdown and degradation by 1 μM Fulvestrant for the last 24 hours. MDA-MB-231 cells with ERα knock-in/knockdown were treated with the indicated concentrations of ErSO for 24 hours. (B,D,H-K) Data are plotted as mean ± s.e.m.; n ≥ 3 biological replicates. (F,G) Mean ± s.e.m.; n = 8 biological replicates. (I,K) Statistics were calculated with student t-test, p values: ***: p ≤ 0.001, ****: p ≤ 0.0001, n.s. (not significant): p ≥ 0.05 See also Fig. S1,S2.

Although ErSO killed ERα-positive breast cancer cells (Fig. 1B-D, S1C), it remained possible that a small pool of cells survived and would regrow as ErSO-resistant cells. We therefore carried out long-term cell treatment-regrowth and retreatment experiments (Fig. 1E-G). The cells were either all killed by ErSO and did not regrow over a month without treatment (Fig. 1F), or, if a few cells survived and regrew, they remained completely sensitive to ErSO retreatment (Fig. 1G). Unlike SERMs and SERDs (16), ErSO activity was independent of the presence of estrogen (Fig. 1H). Notably, in contrast to current and experimental SERMs, ErSO rapidly killed ERα-positive breast cancer cells in vitro (Fig. 1C, 1I).

ErSO kills breast cancer cells in an ERα-dependent manner and binds to ERα in vitro

Providing insight into the role of ERα in ErSO action, ErSO treatment induced rapid killing of ERα positive MCF-7 human breast cancer cells, but had no effect on ERα negative MDA-MB-231 breast cancer cells in vitro (Fig. 1J). We next evaluated the effect of introducing ERα into MDA-MB-231 cells by stable transfection by using MDA-MB-231-ERα_in cells that express ERα (25, 26). Consistent with ErSO acting through ERα, treating MDA-MB-231-ERα_in cells with ErSO resulted in rapid cell death (Fig. 1K, Fig. S2A). ERα expression in MDA-MB-231-ERα_in cells was then knocked down with a combination of siRNA and SERD treatment (Fig. S2A). Notably, knockdown of ERα largely reversed ErSO induced cell death (Fig. 1K, Fig. S2A). Expression and knockdown of ERα in immortalized, non-malignant MCF-10A cells demonstrated a similar ERα dependence for ErSO-induced cell death (Fig. S2B,C).

X-ray diffraction studies did not yield a usable structure. We therefore used saturation transfer difference nuclear magnetic resonance (NMR) to test for direct interactions of ErSO with purified E₂-ERα-LBD (ligand binding domain). Saturation transfer difference-NMR exploits the transfer of magnetization from the protein to a bound small molecule (27-30). Notably, after saturation transfer difference for 5 or 1.5 seconds, ErSO, but not the inactive (S)-1 enantiomer, showed proton signals in the saturation transfer difference-NMR spectrum (Fig. S2D). Together, the biological data showing ERα is required for ErSO induced cell death (Fig. 1K, Fig. S2A-C) and the saturation transfer difference-NMR data showing enantiomer-selective interaction of ErSO with purified ERα-LBD (Fig. S2D) suggested that direct binding of ErSO to ERα triggered death of breast cancer cells.

We next assessed ErSO activity in a diverse cell line panel in vitro and observed that ErSO was highly selective and exhibited potent activity (average IC₅₀ value for cell death induction of 34 nM) against cell lines known to have ERα protein expression. ErSO was inactive (average IC₅₀ value of 12.4 μM) against cell lines lacking ERα (Fig S2E, F). Interestingly, ErSO did exhibit activity against a small number of breast cancer cell lines traditionally described as ERα-negative, specifically Hs578t, MDA-MB-453, MDA-MB-468, and BT-20 (Fig S2E,F). Indeed, in line with previous reports (31, 32), we found that BT-20 cells contained detectable ERα by Western blot analysis (Fig. S2G), even though this cell line is often referred to as ERα-negative. Given that ERα promoter methylation is known to reduce ERα expression, we examined ESR1 promotor methylation in the four breast cancer cell lines (Hs578t, MDA-MB-453, MDA-MB-468, and BT-20) and as reported (33-42) they do not have ESR1 promoter methylation (Fig. S2E). To further evaluate the potential role of ERα in the sensitivity of Hs578t cells to ErSO, we carried out ESR1 siRNA and fulvestrant pretreatment in these cells and found that this largely reversed ErSO induced cell death (Fig. S2H).

The kinetics of cell growth inhibition and cell death upon ErSO treatment was markedly different between high, low, and no ERα expressing breast cancer cell lines. Cell death was observed within 6 hours in those cell lines expressing high amounts of ERα whereas low expressors were sensitive to ErSO within 24 hours of treatment and the no ERα expressor breast cancer cell lines remained largely insensitive to ErSO (Fig. S2I). Of note, longer incubations (>24 hours) with ErSO appeared to narrow this ERα-dependent therapeutic window (Fig. S2J) with a clear time dependence of the ERα-dependent IC₅₀ of ErSO. More detailed studies using 4-day ErSO dose response experiments showed that for high ERα expressors (MCF-7 cells) ErSO treatment at 50 nM was sufficient for potent cytotoxic cell killing; in contrast, in the low ERα expressor cell lines BT-20 and MDA-MB-468, ErSO treatment up to 1,000 nM remained cytostatic (Fig. S2K). After 4 days of ErSO treatment, the Hs578t cell line did undergo cell death necrosis (Fig. S2K). An ESR1 siRNA combined with fulvestrant largely reversed ErSO-induced killing of Hs578t breast cancer cells (Fig. S2H).

ErSO treatment induces regression of ERα-positive tumors in multiple orthotopic cancer cell line and patient-derived xenograft mouse models

ErSO efficacy was evaluated against large (~300-400 mm³) tumors in orthotopic xenograft mouse model of MFC-7, a human ERα-positive breast cancer cell line. Unlike fulvestrant, ErSO, administered once daily (orally 40 mg/kg for 21 days) resulted in elimination of these tumors, with >99% reduction in all cases, and no measurable tumor burden in 4 of 6 mice (Fig. 2A, Fig. S3A,B). Notably, variable dosing studies showed that ErSO administered once a week (oral or i.v.) was sufficient for a robust response (Fig. 2B,C), with complete tumor regression at the highest dose (Fig. S3C). No significant difference in daily weight measurements was observed between vehicle-treated and ErSO-treated mice (Fig. S3D,E).

Fig. 2. ErSO treatment ablates WT and mutant ERα primary breast tumors in orthotopic cell line xenograft mouse models.

(A) Orthotopic MCF-7 tumors were established in ovariectomized Nu/J mice supplemented with a 60 day E2 pellet (0.36 mg) and grown for 28 days to ~400 mm³, randomized, and treated with vehicle daily (n = 3), vehicle weekly (n = 3) (presented as a single averaged Veh. line), Fulv. weekly (5 mg/mouse, s.c., n = 6), ErSO daily (10 or 40 mg/kg p.o., n = 6 for both arms). (B,C) Mice were dosed with ErSO as indicated (0.5-40 mg/kg p.o. or i.v.) once-per-week for three weeks (q7dx3, dosed on Day 0, 7, 14). Study conducted by South Texas Accelerated Research Therapeutics, n = 5 for each arm, some arms are copied between panels, such as Vehicle and Fulv. arms, all were performed in the same study. (D) Orthotopic TYS-luc. tumors in NSG ovariectomized mice (no E2 supplementation, 60 days to establish) were treated daily as indicated (n = 5). Tumors were quantitated using flux values from luciferase-based bioluminescent imaging at days 0, 3, 7, and 14. Also shown are representative bioluminescent images of ErSO treated mice from Day 0 and 14. (E) Summary of mouse models bioluminescent imaging. Orthotopic breast tumors were generated by injecting the indicated cell lines in Matrigel into NSG mice. Large breast tumors allowed to grow out (~2 months for TYS-luc, ~4 months for TDG-luc and ~1 month for MYS-luc and MDG-luc). Then mice were treated daily for 2 (TYS-Luc: oral, TDG-Luc: p.o. and i.p.) or 3 weeks (TYS-Luc: injected, MYS-Luc and MDG-Luc: p.o. and i.p.) with 40 mg/kg ErSO daily. No treated mice were dropped from the study. (A-C) mean ± s.e.m.; analysis compared to vehicle treated tumors utilized a two-way ANOVA with Tukey correction, p values: *: p ≤ 0.05, ***: p ≤ 0.001, ****: p ≤ 0.0001. (D) mean ± s.e.m.; n = 6 mice (vehicle), n= 5 mice (10 and 40 mg/kg ErSO p.o.) See also Fig. S3.

To investigate the ability of ErSO to induce regression of mutant ERα tumors, T47D and MCF-7 breast cancer cells harboring either the Y537S or D538G mutations [the TYS (14), TDG (14), MYS (43), MDG (43) cell lines] were stably transfected to express luciferase. Within 7 days, oral ErSO treatment of these orthotopic human cell line xenograft mice induced >10,000 fold regression of TYS-luciferase expressing breast tumors in all 5 mice, and >100,000 fold regression (to undetectable amounts) within 14 days as shown by bioluminescent imaging of luciferase as compared to vehicle treated mice (Fig. 2D,E, Fig. S3F for i.p. treatment). Analogous results were seen in mouse experiments with TDG-, MYS-, and MDG-luciferase expressing cell line-derived xenograft models. Combined data from the 4 tumor xenograft mouse models showed 38/39 tumors regressed >95%, with 18/39 regressing to undetectable amounts (Fig. 2E, Fig. S3F-L). No significant change in mouse weight was observed between the groups in these studies (Fig. S3M-O).

Given that luciferase activity in serially transplanted TYS-luciferase expressing tumors was maintained for >6 months (Fig. S4A), we next assessed whether ErSO elicited a durable response in treated mice with TYS-luciferase expressing tumors that had regressed to undetectable amounts. None of the mice (Fig. 2D) exhibited tumor recurrence for up to 6 months after cessation of ErSO treatment at which time the experiment was terminated (Fig. 3A). Similar observations were made in both TYS and TDG cell line xenograft mouse models (Fig. S4B,C). In mouse models exhibiting near complete regression after suboptimal dosing with ErSO, tumors that regrew remained fully sensitive to a second cycle of ErSO treatment with 16/17 re-treated tumors regressing >95% and 11/17 regressing to undetectable amounts (Fig. 2E, 3B, Fig. S4D-F).

Fig. 3. ErSO treatment ablates mutant ERα breast cancer cell line xenografts and a PDX mouse model.

A(A) Orthotopic TYS-luc. (TYS refers to T47D (T) with ERα mutation Y537S (YS)) tumors in NSG ovariectomized mice (no E2 supplementation, 60 days to establish) were treated daily as indicated (Fig. 2D). Tumors were quantitated using flux values from luciferase-based bioluminescent imaging monthly for 6 months after cessation of treatment. Shown are representative bioluminescent images of ErSO treated mice from 6 months with no treatment. (B) Mice with orthotopic TDG-luc. (TDG refers to T47D (T) with ERα mutations D538G (DG)) tumors were treated with ErSO. Mice whose tumors regrew after cessation of treatment (denoted by red bar) were retreated with 40 mg/kg/day i.p. (C,D) Study conducted by South Texas Accelerated Research Therapeutics. Orthotopic ST941/HI tumors (with Y537S ERα mutation, see also Fig. S4G) were treated as indicated. Dosing: Fulvestrant 5 mg/mouse, subcutaneous, q7x3; Tamoxifen 1 mg, subcutaneous , 3x weekly, x2; ErSO 10 or 40 mg/kg orally, QD for 14 days. Pre-treatment tumor volume ~150 mm³ (n = 10 for all groups). (A,B) mean ± s.e.m.; n = 4 mice (vehicle), n= 5 mice (10 and 40 mg/kg ErSO orally) (C,D) mean ± s.e.m.; n = 10 mice/treatment group. See also Fig. S4.

To evaluate the efficacy of ErSO against tumors that better mimic heterogeneous, mutant ERα human breast tumors, a hormone-independent, patient-derived xenograft called ST941/HI was used. This PDX harbored the ERαY537S mutation and is a low-expressing ERα-positive tumor (44) (Fig. S4G). In contrast to standard-of-care tamoxifen and fulvestrant treatment, 14 days of oral ErSO treatment led to regression of all ST941/HI tumors, with 6/10 mice having no measurable tumor burden even 30 days after cessation of treatment(Fig. 3C,D).

ErSO induces regression of metastatic models for human ERα-positive tumors including brain metastases

To explore the ability of ErSO to eradicate tumors growing in major metastatic sites, we next performed ex vivo imaging of lungs from mice with orthotopic human cell line-derived TYS/TDG-luciferase expressing breast tumors. We observed that 100% of TYS-luciferase expressing and TDG-luciferase expressing tumors formed lung metastases after primary tumor engraftment (14). Using this spontaneous metastasis preclinical model, we observed that ErSO-treated mice had no lung metastases, suggesting complete regression of tumors (Fig. S5A,B). Next we observed in one mouse with extensive lung metastases from tail-vein injection, daily oral ErSO treatment for 7 days resulted in complete regression without recurrence after 4 months (Fig. 4A). ErSO treatment also induced complete regression of large skeletal bone and cranial metastases from tail vein injection xenografts in 2 mice (Fig. S5C,D). Oral ErSO treatment induced complete regression of all metastases within 7 days in a mouse with multiple metastatic lesions, with no recurrence after 4 months (Fig. 4B, Fig. S5E,F). After tail vein injection of MDG-luciferase expressing tumor cells, metastatic burden was greatly reduced by ErSO treatment (Fig. 4C).

Fig. 4. ErSO induces regression in metastatic ERα-mutant human breast cancer cell line xenografts in mice.

(A,B) Mice with TYS-luc. lung (A) and multiple (B) metastatic tumors formed after tail-vein injection were treated once-a-day for 7 days with 40 mg/kg ErSO orally. Imaging 4 months after no treatment. Images are 3D-DLIT renders. (C) MDG-luc. cells were introduced by tail vein injection, and the resulting mice treated with vehicle or ErSO (40 mg/kg intraperitoneal, daily) for 14 days. Tumor burden was quantified using total flux across the mouse and plotted relative to Day 0. Representative image presented. (D,E), Mice (CD-1) were treated with the indicated doses and times, then sacrificed and their serum and brains collected. Concentrations were determined via LC/MS/MS analysis. The average blood per mouse was approximated as 58.5 ml/kg. Data plotted as mean ± s.e.m.; n = 3 mice for all groups. (F) Tumors in brain were established by intracranial implantation of MYS-luc. cells. Treatment: vehicle or ErSO (40 mg/kg intraperitoneal or orally) daily for 14 days; tumor burden quantitated by BLI. Dashed line represents 0% change. Representative ErSO-treated (intraperitoneal) image shown. (C,F) mean ± s.e.m.; n = 4 mice (vehicle), n= 5 mice (ErSO). Analysis by 2-way ANOVA with Bonferroni correction post-hoc test with p values: **: p ≤ 0.01, ***: p ≤ 0.001. See also Fig. S5.

To evaluate ErSO against a preclinical model of brain metastases (45-47), we intracranially implanted MYS-luciferase expressing breast cancer cells. ErSO is able to penetrate the mouse blood-brain barrier with a brain:serum ratio of ~42:58 (Fig. 4D,E). ErSO injected (i.p.) daily for 14 days in the preclinical brain metastatic mouse model elicited an average ~80% tumor reduction. Compared to vehicle tumors at day 14, mice treated with ErSO orally or intraperitoneal injection had tumors 7- and 180-fold smaller, respectively (Fig. 4F, Fig. S5G,H).

ErSO has drug-like properties and is tolerated in multiple species

ErSO displayed high absorption with excellent cell permeability and was a poor efflux substrate in vitro (Fig. S6A), which are both key factors for its ability to cross the blood-brain barrier (Fig. 4D,E). ErSO was stable in human, murine, canine, rat, and monkey plasma, as well as simulated gastric fluid (Fig. S6B) and when incubated with human, murine, rat, canine, and monkey liver microsomes and hepatocytes (Fig. S6B). ErSO’s major metabolite resulted from glucuronidation of the phenol, which was only minimally present (8-36%, Fig. S6C). In a general safety panel, ErSO demonstrated minimal inhibition of hERG, hCav1.2, hNav1.5, and cytochrome P450’s (Fig. S6D).

ErSO is tolerated in mice (maximum tolerated dose, MTD, of at least 150 mg/kg), as well as, rats and canines when given orally (Fig. 5A). Pharmacokinetic studies in mice, rats, and canines demonstrated that, regardless of the route of administration either intravenously or orally, ErSO achieved serum concentrations well-above the cell culture IC₅₀ (Fig. 5B, Fig. S6E). Daily dosing of ErSO for 7 days (a dosing strategy that rapidly regressed ERα-positive tumors as shown above) in healthy female mice revealed no signs of cytotoxicity in ERα-positive tissues, such as the uterus and fallopian tube glandular epithelium and mammary ductal glandular epithelium, with no loss of ERα-expressing normal cells (Fig. 5C). Unlike ERα knockout mice, ErSO did not increase 17β-estradiol amounts (Fig. 5D). Further, in a standard experiment for assessing uterine growth (48, 49), ErSO treatment (40 mg/kg i.p. daily for three days) had no significant (p > 0.05) impact on uterine tissue growth nor perirenal fat in healthy, non-tumor bearing female mice (Fig. 5E,F); however there was a statistically significant (p < 0.01) difference when measuring visceral (inguinal/gluteal) fat (Fig. 5G).

Fig. 5. ErSO is tolerated in vivo and achieves biologically relevant concentrations in mice, with minimal effects seen in ERα-positive normal murine tissues.

(A) Summary of maximum tolerated dosing experiment with ErSO (oral, single dose). Allometric scaling is relative to murine estimates. (B) Serum concentrations of ErSO after indicated doses, route of administration, and time. Concentration was determined via LC/MS/MS analysis. The average 24 hour IC₅₀ of ErSO for ERα-positive breast cancer cells is ~20-40 nM. (C) Non-ovariectomized female mice (wild type CD-1 females) were treated with ErSO (40 mg/kg orally or intraperitoneal) daily for 7 days. ERα-positive tissues were harvested, stained, and compared to ERα-knockout (KO) murine tissue (top image: uterus/fallopian tube glandular epithelium, 100X; bottom image: mammary ductal glandular epithelium, 200x). (D) Circulating amounts of estradiol (17β-estradiol) upon ErSO treatment (7 days, daily 40 mg/kg orally or intraperitoneal) as compared to amounts observed in ERα-KO mice. (E-G) Analysis of uterine weight, perirenal fat, and visceral fat in response to ErSO treatment (40 mg/kg for three doses intraperitoneal, i.p.) with and without 17β-estradiol (E2) treatment. (H,I) MDA-MB-231 mouse model with ErSO treatment (40 mg/kg orally once-a-week), plotted as individual mice tumor burden (H) and average calculated tumor doubling time (days, I); n=12 for each arm. (J) MDA-MB-231-ERain tumor model with ErSO treatment (40 mg/kg orally once-a-week), plotted as individual mice tumor burden; n = 8 for each arm. (B) mean ± s.e.m.; n = 3 mice (40 mg/kg ErSO orally), n = 1-3 mice (20 mg/kg ErSO intravenous). (C) Images are representative of n = 5 (Vehicle orally), n = 6 (Vehicle intraperitoneal), n = 9 (ErSO orally), n = 9 (ErSO intraperitoneal), and n = 3 (ERα-KO). (D), box and whisker plot of n = 5 mice (vehicle orally), n = 6 mice (vehicle intraperitoneal), n = 9 mice (ErSO orally), n = 9 mice (ErSO intraperitoneal), and n = 3 mice (ERα-KO) Data analyzed by 1-way ANOVA with a post-hoc Tukey test; p value: ***:p ≤ 0.001. (E-G), mean ± s.e.m.; n = 5 mice, Data analyzed by One-way ANOVA followed by Šidák’s multiple comparison; p value: **: p ≤ 0.01 ****:p ≤ 0.0001. (I), Data analyzed by two-tail, unpaired student t-test; n.s. (not significant): p ≥ 0.05. See also Fig. S6.

Critical for ErSO’s tolerability in vivo is its selective and ERα-dependent anticancer effect (Fig. 1K). To investigate if this selectivity is observed in vivo, we compared the effect of ErSO on breast tumors derived from MDA-MB-231 lacking ERα expression and ERα expressing MDA-MB-231-ERα_in cells. Consistent with a lack of off-target toxicity and an ERα-dependent anticancer activity, oral ErSO treatment once a week for a minimum of three total doses did not inhibit non-ERα expressing MDA-MB-231 tumor growth (Fig. 5H), with no significant (p = 0.6368) change in the calculated tumor doubling time as compared to vehicle treated mice (Fig. 5I). In contrast, oral ErSO treatment once a week for a minimum of three total doses of MDA-MB-231-ERα_in tumors largely blocked tumor growth (Fig. 5J).

ErSO kills breast cancer cells via hyperactivation of the anticipatory-UPR pathway

To initiate the a-UPR, activator-bound ERα activates Src kinase, which phosphorylates and activates PLCγ, enzymatically producing inositol triphosphate, (IP₃) that binds to and opens IP₃ receptor (IP₃R) channels in the membrane of the endoplasmic reticulum, leading to a rapid efflux of calcium stored in the lumen of the endoplasmic reticulum into the cytosol (50, 51). Consistent with ErSO exerting its cytotoxic actions by rapidly hyperactivating the a-UPR, only ErSO, but not the inactive enantiomer (S)-1, induced a rapid (<1 minute) increase in cytosolic calcium (Supplementary Movies 1-4). Moreover, 2-APB, which locks the endoplasmic reticulum IP₃R Ca²⁺ channels closed, strongly inhibited the ErSO-induced increase in cytosol Ca²⁺ (Supplementary Movies 1-4).

Consistent with dramatic and cytotoxic a-UPR hyperactivation, ErSO treatment induced the UPR marker spliced XBP1 (sp-XBP1) mRNA >1,000 fold, to amounts >15x higher than the previously reported a-UPR hyperactivator, BHPI (50) (Fig. 6A). Consistent with more robust activation of the a-UPR by ErSO, it was far more effective than BHPI in inducing rapid death of ERα positive human breast cancer cell lines in vitro (Fig. S6F). Supporting a pivotal role for a-UPR hyperactivation, breast cancer cell lines, T47D, TDG and TYS, treated with ErSO exhibited rapid inhibition of protein synthesis (Fig. 6B), thapsigargin-sensitive depletion of ATP (Fig. 6C, Fig. S6G), and activation of a-UPR protein markers (increasing p-PERK, p-eIF2α, and p-AMPK; decreased ATF6αp90; Fig. 6D,E). Notably, insertion of ERα expression into ERα negative cells was sufficient for ErSO induced ATP depletion and inhibition of protein synthesis, which was reversed by knockdown of ERα expression (Fig. 6F,G, Fig. S6H,I). ErSO did not induce a-UPR activation in ERα negative MDA-MB-231, as shown by western blot analysis of ATF6αp90 and p-eIF2α amounts (Fig. S6J). Importantly, human breast cancer cell lines MDA-MB-468, BT-20, and Hs578t which may be very low expressors of ERα and were sensitive to ErSO. These three cell lines showed markers for a-UPR activation after treatment with ErSO; consistent with the slower kinetics of cell death for these cancer cell lines after ErSO treatment, the a-UPR activation was slower and not as strong as that observed in MCF-7 cells which strongly express ERα (Fig. S2J). Transmission electron microscopy (TEM) images of MCF-7 cell lines treated with ErSO revealed features of necrotic cell death (Fig. S6K). Neither PARP-1 cleavage nor meaningful ERα degradation (beyond what is expected with cell death) were observed as a result of ErSO treatment in MCF-7 cell lines(Fig. 6H).

Fig. 6. ErSO activates the a-UPR, leading to tumor regression in breast cancer cell line xenograft mouse models.

(A) Induction of sp-XBP1 mRNA in T47D cells treated for the indicated times with 1 μM BHPI or ErSO, shown as relative fold change versus vehicle-treatment as calculated from sp-XBP1 mRNA expression measured by qRT-PCR. (B) ErSO (1 μM) effect on protein synthesis in T47D cells. Protein synthesis quantified by ³⁵S-methionine incorporation. Plotted is percent inhibition (%) relative to vehicle controls. (C) Time course of ErSO (1 μM)-mediated intracellular ATP depletion in T47D, TYS, and TDG cells. ATP determined using ATPlite Luminescence Assay. Displayed as % ATP depletion relative to vehicle control for each cell line. (D,E) Western blot analysis of a-UPR protein markers in T47D cells at the indicated times after ErSO treatment (500 nM). Images are representative of 2 independent biological replicates. (F,G) Relative ATP amounts and protein synthesis in MDA-MD-231 cells with ERα knock-in/knockdown. Cells were treated with the indicated concentrations and incubated for 1 hour. ATP amounts and protein synthesis were determined and quantified using ATPlite Luminescence Assay and ³⁵S-methionine incorporation, respectively. (H) Western blot analysis of MCF-7 cells treated for 4 hours with ErSO (at indicated concentrations), (S)-1 (1 μM), Raptinal (10 μM, positive control for apoptosis), and DMSO (vehicle negative control). Vinculin (Vin.) is a loading control. Images are representative of 2 independent biological replicates. (I) MCF-7 cells were orthotopically bilaterally grafted and tumors were grown until total tumor burden was ~400 mm³ prior to daily treatment with vehicle or ErSO (40 mg/kg oral.). Mice (n = 5 per arm) were sacrificed after 7 days and tumors harvested. Tumor burden is displayed as a sum of both tumors. (J) Western blot analysis of MCF-7 tumors after 7 days of daily ErSO (40 mg/kg oral ) treatment. Each number represents an independent mouse tumor. Vinculin (Vin.) was used as a loading control. Western blots shown are representative of 4 technical replicates. (K) Immunohistochemical (IHC) analysis of MCF-7 orthotopic model (I) H&E staining showing macrophage infiltrate (F4/80), and -CASP3 staining . Representative images of ErSO treated tumors are at 40x. (A-C) Data are plotted with individual points and the mean ± s.e.m.; n = 6 (A,C) or 5 (B) biological replicates. (F,G) Data is plotted with individual points and/or the mean ± s.e.m.; n ≥ 3 biological replicates. Statistics were calculated with student t-test, p values: ***: p ≤ 0.001, ****: p ≤ 0.0001, n.s. (not significant): p ≥ 0.05. See also Fig. S6.

ErSO treatment leads to hyperactivation of the a-UPR and necrosis of breast tumors

To assess whether ErSO’s ability to induce tumor regression was accompanied by activation of the a-UPR in vivo, MCF-7 orthotopic tumor-bearing mice were sacrificed prior to complete ErSO-mediated tumor eradication following daily oral treatment with ErSO for 7 days(Fig. 6I). Tumors from MCF-7 xenograft mice treated with ErSO for 7 days demonstrated increases in p-PERK and p-eIF2α, with no ERα degradation (Fig. 6J). Moreover, consistent with activation of the PERK arm of the UPR, ErSO rapidly and robustly inhibited intratumoral protein synthesis in a MYS-Luciferase expressing tumor model in orthotopic xenograft mice (Fig. S6L). Supporting necrosis (52, 53), IHC analysis of ErSO-treated MCF-7 orthotopic tumors demonstrated necrotic tumor regression with no increases in cleaved caspase-3 following 7 days of daily oral ErSO (Fig. 6K). Consistent with the ability of necrosis to induce immunogenic cell death (52-54) even in these immune compromised athymic mice, we observed a profound increase in macrophage infiltration (Fig. 6K).

Discussion

While improved therapies and sequential use of drugs with different targets has substantially extended survival of patients with metastatic ERα-positive breast cancer, nearly all patients still succumb to the disease (5, 7). Components of the estrogen- ERα activated a-UPR are overexpressed in aggressive ERα-positive human breast cancers and this overexpression correlates with tumor recurrence, therapy resistance, and poor outcomes (21). Weak and transient activation of the a-UPR by estrogen is cytoprotective (23); however, strong and sustained a-UPR activation in breast cancer cells is cytotoxic and triggers necrotic cell death. Since prolonged a-UPR is toxic to cancer cells (21), we exploited this pathway to target these challenging tumors. The previously described a-UPR activator, BHPI, does not rapidly induce death of many ERα-positive cancer cells in vitro (50) (Fig. S6F) and exhibits undesirable side effects in mice. We therefore executed a medicinal chemistry campaign that led to the identification of ErSO, a cytotoxic a-UPR activator. In more than 100 ErSO-treated immunocompromised mouse models with both primary and metastatic tumors, an ErSO resistant tumor was not observed. Although there are many mechanisms leading to resistance to current endocrine therapies (6), intratumoral and intertumoral heterogeneity in the large pool of surviving breast cancer cells means that acquired or de novo resistance will almost always occur (2, 55). The ability of ErSO to induce complete, or near complete tumor regression means that, even when regression is not complete, the pool of surviving tumor cells in which resistance might arise is relatively small. Notably, tumors that do recur remain completely sensitive to ErSO retreatment. For example, 4/4 primary MYS-Luciferase expressing tumors in mice regrew, but after retreatment with ErSO, 3 out of 4 MYS-Luciferase tumors in mice regressed to undetectable amounts (Fig. 2E) and the fourth tumor regressed >95%. This is consistent with recent findings in unrelated studies with cells that survive apoptosis-inducing chemotherapy in colon cancer (56) and ERα-negative breast cancer (57).

For anticancer drugs with inhibitory modes of action, loss of expression of the target represents a common resistance mechanism. Since complete knockdown of ERα largely abolishes ErSO-induced cell death and a-UPR hyperactivation (Fig. 6G,H, Fig. S6H,I), loss of ERα expression might be envisioned as a resistance mechanism. Perhaps due to oncogene addiction, even after endocrine therapy targeting ERα, loss of ERα expression is not a common resistance event in patients (2, 8, 16, 20). Moreover, other components of the a-UPR pathway leading to cell death, such as Src, PERK, and eIF2α, are overexpressed in ERα positive breast tumors (23), suggesting they play important roles in tumor physiology. Other pathway components such as IP3Rs and ATP-depleting SERCA pumps play crucial roles in normal calcium homeostasis. Therefore, the critical nature of a-UPR pathway components strongly selects against their loss in cancer cells; this trait, combined with ErSO’s activation of this pathway, is likely why ErSO-resistant tumors have not been observed.

Several human breast cancer cell lines, such as MDA-MB-468, BT-20, and Hs578t, likely expressing trace amounts of ERα exhibited some sensitivity to ErSO (Fig. S2E,F). This may be a consequence of preferential partitioning of limiting ERα into the ERα:SRC:PLCγ complex that initiates the a-UPR activation, and a-UPR pathway overexpression in breast cancer (23, 58, 59). The proteins that initiate a-UPR activation, ERα, Src and PLCγ have multiple functions in cells and only a tiny fraction of each protein is likely localized in the multiprotein complex that initiates the anticipatory UPR (58). Thus, the availability of other components of the complex will influence whether very low amounts of ERα in a cell will be sufficient to form the extremely low abundance ERα:SRC:PLCγ complex. Because the a-UPR plays an important role in tumor growth and survival, in cells with extremely low concentrations of ERα, this interplay between components of the ERα: Src:PLCγ complex likely facilitates partitioning of limiting ERα into the a-UPR activating complex, enabling sensitivity to ErSO. Although this currently cannot be assessed directly because this extremely low abundance multiprotein complex can only be immunoprecipitated in cells expressing substantial ERα (58), this type of limited availability and partitioning due to competition between complexes is well known in the control of transcription factor activity (60).

The demonstration that ErSO activates the a-UPR markers in several of these sensitive human breast cell lines, including MDA-MB-468, Hs578t (where ERα is not detectable by Western blot) and BT-20, (Fig. S2J) suggests that ErSO is acting through the same a-UPR pathway we observe in classical ErSO-sensitive, ERα-positive breast cancer cells. For example in Hs578t cells, ERα was not detected via Western blotting but siRNA-induced ERα mRNA knockdown and fulvestrant-induced degradation largely reversed sensitivity to ErSO, supporting the view that ERα is responsible for the response to ErSO in Hs578t cells (Fig. S2K). While ERα is central to ErSO action, it remains possible that in some contexts, other protein targets may interact with ErSO and activate the a-UPR, or work through pathways not yet identified to inhibit proliferation of cancer cells. While ErSO did not inhibit growth of non-ERα expressing MDA-MB-231 tumors, in cell culture its antiproliferative activity shifted slightly with time (IC₅₀: 23 μM at 24 hours to 2 μM at 120 hours), suggesting caution should be exhibited in long-term studies using ErSO. As low amounts of ERα were sufficient for ErSO to induce cancer cell death (Fig. S2H), it was critical to assess potential toxicity. Because the a-UPR is tumor protective and is already activated in breast cancers and because a-UPR components are overexpressed in breast cancer, strong and sustained a-UPR activation by ErSO is sufficient to selectively induce necrotic cell death in breast cancer cells. Consistent with this, after ErSO treatment, there was no ablation of ERα-positive murine normal tissues (Fig. 5C) nor effects on uterine weight (Fig. 5E). Human and rodent ESR1 sequences are highly conserved (61, 62) further suggesting ErSO’s selectivity for human ERα-positive cancer cells while lacking effects in normal murine tissues. Moreover, ErSO at extremely high doses was tolerated in dogs (Fig. 5A). These results suggest that the presence of ERα in normal cells is not sufficient for cell killing by ErSO. A crucial consideration in the treatment of ERα-positive breast cancer is the treatment of premenopausal patients. Because endocrine therapies compete with endogenous estrogens to enhance therapy efficacy, the controversial practice of ovarian function ablation/suppression (OFA/OFS) is necessary (7, 63, 64). In contrast, ErSO’s activity is estradiol-independent (Fig. 1H), ErSO-treatment does not ablate or alter the endocrine signaling axis (Fig. 5D), and complete tumor responses are seen even in the presence of exogenous estradiol-supplementation (Fig. 2A-C). These features suggest an alternative for the treatment of premenopausal patients, likely making OFA/OFS unnecessary.

There are several limitations to our study that specifically merit discussion. Many anticancer drugs have a primary target that they bind with high affinity and, after extended clinical use, are shown to also have secondary targets that can impact drug effectiveness and toxicity. While there is abundant evidence that the primary mode of ErSO’s action is through ERα-dependent hyperactivation of the a-UPR, the possibility remains that, there may be other a-UPR activators that play a role in ErSO’s action, or proteins acting through other pathways that impact the response to ErSO. However, the inactivity of ErSO against ERα-negative MDA-MB-231 tumors in vivo supports ErSO’s ERα-dependent activity (Fig. 5H), a tumor model where even targeted ERα degrader fulvestrant shows some activity (65).

We did not observe ErSO-resistant clones in vitro or in vivo. While this deprives us of the mechanistic insights we might derive from resistant cells, the absence of easily developed resistance is a promising property in an anticancer drug. Unlike endocrine therapies such as tamoxifen and fulvestrant whose primary sites of action are at the level of nuclear transcription, ErSO elicits robust activation of the UPR sensor, PERK kinase. Partially activated PERK kinase is known to phosphorylate and further activate PERK, establishing a sensitive feed-forward autoregulatory loop (66) resulting in inhibition of protein synthesis. Moreover, ErSO-induced ATP depletion activates AMPK, further inhibiting protein synthesis. This ErSO-mediated inhibition of protein synthesis means that in ErSO-treated cells most changes in ERα occupancy of DNA sites or in mRNA levels will not result in changes in protein amounts. Therefore, effects of ErSO on classic ERα biology, including estrogen response element activity, the ERα cistrome, ERα subcellular localization and coregulator recruitment, remain to be studied and will be important in future studies elucidating and describing the complex ERα biology in response to ErSO treatment.

While ErSO is effective in mouse models when administered orally or by injection, even once a week, careful evaluation will be required to determine the best dosing route and regimen for human trials. This is a preclinical study using breast tumors derived from human cancer cells in mice and exploring toxicity in mice and dogs. Specifically, in-depth and comprehensive analysis of ErSO-effects in a variety of contexts will be critical to further demonstrate safety prior to evaluation in humans. For example necropsy studies and other crucial investigational new drug (IND)-enabling assessments are necessary for any human translation efforts. Finally, clinical trials will be required to evaluate whether ErSO’s effectiveness in mouse xenografts and lack of toxicity in mice and dogs extends to human breast cancer patients.

Complete tumor responses against drug-sensitive tumor models, such as the MCF-7 orthotopic model, are rarely observed with SERMs and SERDs (16, 44, 67-71) and advanced, metastatic breast cancer remains a challenging setting in breast cancer treatment. However, ErSO achieves tumor eradication in both SERM/SERD-sensitive mouse models, drug-resistant primary and metastatic tumor mouse models, and importantly in a fulvestrant/tamoxifen-resistant, ERα-mutant PDX mouse model. Moreover, ErSO is effective in a model of breast cancer that has metastasized to the brain, a therapeutic challenge not addressed by current drugs and rarely explored in preclinical studies. These results indicate ErSO has potential to treat advanced, drug-resistant ERα-positive breast cancer, especially ERα-mutant metastatic disease. ERα expression is sufficient to render tumors from a triple negative breast cancer cell xenograft, in which ERα does not drive proliferation, sensitive to ErSO (Fig. 5H-J). Moreover, in some breast cancer cells traditionally considered ERα negative, extremely low levels of ERα are likely sufficient for response to ErSO. These observations broaden the potential therapeutic range of ErSO to include breast tumors with diverse drivers of proliferation that maintain very low expression of ERα.

Our data demonstrate the activity of ErSO for the treatment of ERα-positive breast cancer, namely complete regression of ERα wildtype and mutant-positive primary and metastatic tumors. While inhibiting ERα action has been the hallmark of endocrine therapy since the advent of tamoxifen (72), the ERα-dependent, a-UPR hyperactivation described here demonstrates the utility of other modalities leveraging ERα expression. This suggests more broadly that turn-on anticancer strategies, such as hyperactivation of the a-UPR with ErSO may impart new therapeutic opportunities for known targets.

Materials and Methods

Study Design:

The objective of this report was to establish ErSO’s effects on cancer cells in cell culture and in vivo. To this end, we utilized literature/industry standard cell death and mechanistic experiments to provide justifications for conclusions described herein. In most cases, we utilized multiple experimental designs and readouts to further solidify our conclusions. For example, cell death was determined by flow cytometry, alamar blue fluorescence, and trypan blue exclusion; where all three methods yielded the same conclusions. Crucially, our study was conducted by multiple research teams, all coming to similar conclusions surrounding ErSO’s effects. In most cases and when possible, experiments were carried out in at least triplicate, as noted in figure legends. Sample sizes used were in line with previous literature, our laboratories standard practices, and ErSO’s large responses; all sample sizes are reported in figure legends. Mice were randomized to treatment groups or in such a way that starting tumor volumes were similar. Mouse experiments were in accordance with approved IACUC protocols. Because many tumors’ measurements used instrument-based bioluminescent imaging and because of the rapid and dramatic tumor responses to ErSO, no blinding was used for studies herein.

Cell Lines and Culturing Conditions

All cell lines were cultured at 37°C with 5% CO₂. All ERα-positive cell lines were grown in media lacking phenol red. MCF-7 and HepG2 cells were grown in EMEM with 10% FBS and 1% penicillin-streptomycin (P/S). T47D, MDA-MB-231 parental, and MDA-MB-231-ERα cells were cultured in MEM with 10% FBS and 1% P/S. TYS and TDG cells were grown in 10% CD-FBS and 1% P/S. MCF-7 parental, MYS, and MDG cell lines were grown in DMEM supplemented with 5% FBS and 1% P/S. BT-474, ZR-75-1, PC-3, BT-549, A549, E0771, HCC-1806, HCT-116, 4T1, and MDA-MB-231 cells were cultured in RPMI-1640 with 10% FBS and 1% P/S. MDA-MB-468, CaOV3, IGROV-1, MDA-MB-453, Hs578t, BT-20, MDA-MB-436 were grown in DMEM with 10% FBS and 1% P/S. OVCAR-3 cells were grown in RPMI-1640 with 10% FBS, 0.01 mg/mL bovine insulin, and 1% P/S. PEO4 cells were incubated with 10% FBS DMEM supplemented with 10 μg/mL insulin, 1:250 diluted Dulbecco’s NEAA’s, 1 mM glutamine, and 1% P/S. K7M2, HL-60, and K-562 cell lines were cultured with IMDM supplemented with 10% FBS and 1% P/S. MCF10A cells were cultured in DMEM/F12 with 2% CD-FBS supplemented with 1:5,000 EGF, 1:1,000 insulin, 0.5 μg/mL hydrocortisone, and 0.1 μg/mL Cholera Toxin.

Cell Line Validation

Cell lines were utilized directly from ATCC stocks and/or have been further authenticated using the PowerPlex16HS Assay (Promega) as described previously:(73) 15 Autosomal Loci, X/Y at the University of Arizona Genetics Core (UAGC). >1 million cells were harvested and lysed using the cell lysis buffer (50 mM Tris, 50 mM EDTA, 25 mM sucrose, 100 mM NaCl, 1% SDS, pH 8). DNA extraction and short tandem repeats (STRs) profiling for each cell line were carried out at the UAGC. The resulting autosomal STR profiles were compared to reference databases such as ATCC, DSMZ, and JCRB.

Assessment of Cell Death via Flow Cytometry

Cells were plated into a six or twelve-well plate at necessary cell densities (100,000-500,000 cells/well) and allowed to adhere overnight. The next day, the indicated concentrations of compounds were added as DMSO stocks and allowed to incubate at 37° C for 24 hours. After the indicated incubation period, cells were harvested and resuspended in 450 μL of cold buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) premixed with Annexin V-FITC and PI dyes. Samples were analyzed on a BD Biosciences LSRII flow cytometer, and data analysis was performed using FSC Express Version5.

Trypan Blue Exclusion Assay to Assess Cell Death

Cells (300,000) were plated in six-well plates. The next day, vehicle or compound treatment was added for indicated times. Cells were then harvested in a total of 1mL and spun at 500rpm for 5 minutes. Medium was aspirated to a final volume of 100 μL and cells were resuspended. 10μL of the resuspended cell mixture was aliquoted and mixed with 10 μL of 0.4% Trypan Blue Stain prior to counting viable and dead cells utilizing a Countess II cell counter (ThermoFisher).

Alamar Blue Fluorescence for Cellular Activity (IC₅₀)

6,000 cells were seeded per well in a 96-well plate and allowed to adhere before DMSO solutions of compounds were added to each well. Final concentration of DMSO in each well is 1%, final volume: 100 μL. At the end of 24 hours, medium was aspirated and new medium (100 μL) was added. Alamar blue solution was added (10 μL of 1 mg resazurin per 10 mL PBS). After 2-4 hours incubation, fluorescence (λ_excit. = 555 nm, λ_emission. = 585 nm) was measured. The fluorescence of each well was read with a SpectraMax M3 plate reader (Molecular Devices. Percent dead was determined by comparison to a 100% dead control: 100 μM raptinal treated cells IC₅₀ was calculated using Origin Pro V10.

Protein Synthesis Inhibition Assay

Protein synthesis rates were determined by measuring ³⁵S-methionine incorporation. The method used has been fully described (50). In brief, cells were cultured at indicated times with denoted compound and concentrations. 3 μCi ³⁵S-methionine was added for the final 30 minutes of treatment. Cells were then washed with PBS, lysed with RIPA buffer, clarified, and normalized supernatants were pipetted onto Whatman 540 filter-paper discs. Following three washings with 10% trichloroacetic acid, then 5% trichloroacetic acid, and air dried. Trapped protein was solubilized, and the filters counted.

IACUC Guidelines and Protocol Numbers

All mouse model work at UIUC was conducted in accordance with UIUC IACUC guidelines and approved protocols. The following approved IACUC protocols were used for the work described here: 18075, 17062, 20032.

Mouse Xenograft Studies

Detailed descriptions of the PDX model and the diverse xenograft studies used for bioluminescent imaging of primary and metastatic tumors are in Supplementary Materials and Methods. MCF-7 Orthotopic Model-Nu/J ovariectomized mice (Jackson’s lab) were supplemented with a 60 day E2 pellet (0.36 mg, Innovative Research of America) 2-3 days prior to tumor cell inoculation. MCF-7 cells (5x10⁶, in 1:1 HBSS:matrigel) were inoculated into the mammary fat pad and allowed to establish and tumors grown to ~300-400 mm³ (28 days to establish). Mice were then randomized, and then treated with vehicle daily (n = 3), vehicle weekly (n = 3), fulv. weekly (5 mg/mouse, s.c., n = 6), ErSO daily (10 or 40 mg/kg p.o.). Vehicle arms were averaged. Tumor size was measured by caliper. Tumor images were taken at the end of the study (day 21) and are representative of all tumors (n = 6).

BBB Penetrance Study

CD-1 mice were injected with ErSO intravenously (tail vein) at doses indicated and sacrificed 5 and 15 minutes after injection (n=3 for each time point and dose). Mice were sacrificed and blood collected. Residual circulatory volume was removed via perfusion. Blood samples were centrifuged at 13,000 RCF for five min. and the supernatant was collected and stored at −80°C prior to analysis. Brains were harvested from the cranial vault, weighed, and flash frozen. Thawed brains were then homogenized in 1000 μL of cold methanol using a handheld tissue homogenizer. The resultant slurry was centrifuged twice at 13,000 RCF for 10 minutes per run and the supernatant was collected and frozen at −80°C prior to analysis. Samples were then analyzed by LC-MS/MS (Metabolomics Laboratory of the Roy J Carver Biotechnology Center UIUC) to determine ErSO concentration in both serum and brain. To calculate absolute brain:serum ratios, an approximated mouse blood volume of 58.5 mL/kg was utilized for each mouse.

Statistics:

All statistical analyses are detailed in their corresponding figure legends. All data is shown as mean ± s.e.m. Statistical analyses include two-tailed student t-test with unequal variance, two-way ANOVA with Tukey correction, two-way ANOVA with Bonferroni correction post-hoc test, and one-way ANOVA followed by Šidák’s multiple comparison. The analysis and p-value reported are presented in each figure panel’s corresponding legend.