Palazestrant (OP-1250), A Complete Estrogen Receptor Antagonist, Inhibits Wild-type and Mutant ER-positive Breast Cancer Models as Monotherapy and in Combination.

The estrogen receptor (ER) is a well-established target for the treatment of breast cancer, with the majority of patients presenting as ER-positive (ER+). Endocrine therapy is a mainstay of breast cancer treatment but the development of resistance mutations in response to aromatase inhibitors, poor pharmacokinetic properties of fulvestrant, agonist activity of tamoxifen, and limited benefit for elacestrant leave unmet needs for patients with or without resistance mutations in ESR1, the gene that encodes the ER protein. Here we describe palazestrant (OP-1250), a novel, orally bioavailable complete ER antagonist and selective ER degrader. OP-1250, like fulvestrant, has no agonist activity on the ER and completely blocks estrogen-induced transcriptional activity. In addition, OP-1250 demonstrates favorable biochemical binding affinity, ER degradation, and antiproliferative activity in ER+ breast cancer models that is comparable or superior to other agents of interest. OP-1250 has superior pharmacokinetic properties relative to fulvestrant, including oral bioavailability and brain penetrance, as well as superior performance in wild-type and ESR1-mutant breast cancer xenograft studies. OP-1250 combines well with cyclin-dependent kinase 4 and 6 inhibitors in xenograft studies of ER+ breast cancer models and effectively shrinks intracranially implanted tumors, resulting in prolonged animal survival. With demonstrated preclinical efficacy exceeding fulvestrant in wild-type models, elacestrant in ESR1-mutant models, and tamoxifen in intracranial xenografts, OP-1250 has the potential to benefit patients with ER+ breast cancer.

Estrogen receptor beta increases the efficacy of antiestrogens by effects on apoptosis and cell cycling in breast cancer cells.

Clinical evidence indicates that higher levels of estrogen receptor beta (ERbeta) predicts improved disease-free and overall survival in patients treated with adjuvant tamoxifen therapy. To better understand the mechanisms in which ERbeta can modulate breast cancer therapies, we introduced ERbeta under an inducible promoter into MCF-7 breast cancer cells. In these cells, induction of ERbeta expression led to a shift in the potency and an increase in the efficacy of tamoxifen to inhibit proliferation. A similar effect on breast cancer cells was observed for two other antiestrogens, raloxifene, and fulvestrant. Induced expression of ERbeta did not enhance the antiproliferative effects of small molecule inhibitors that target the epidermal growth factor receptor, insulin growth factor receptor-1 and histone deacetylase, indicating ERbeta specifically cooperates with antiestrogens. The combination of ERbeta expression, which arrests cells in G2, and tamoxifen, which arrests cells in G1, led to a potent blockade of the cell cycle. ERbeta also increased tamoxifen-induced cell death and cooperated with tamoxifen to induce expression of the pro-apoptotic gene bik. In summary, our data indicates that ERbeta increases the efficacy of antiestrogens by effects on apoptosis and on cell cycling and, together with clinical observations, suggests ERbeta could be a valuable prognostic marker and potential therapeutic target.

Inhibition of histone deacetylase enhances the anti-proliferative action of antiestrogens on breast cancer cells and blocks tamoxifen-induced proliferation of uterine cells.

Here we report a novel potential therapeutic strategy using histone deacetylase (HDAC) inhibitors to enhance the action of hormonal therapy agents in estrogen receptor alpha (ER alpha)-positive breast cancer. HDAC inhibitors [trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA)], inhibited proliferation of MCF-7 breast cancer cells and, in combination with tamoxifen inhibited proliferation better than with either agent alone. VPA, an anti-convulsant drug with HDAC inhibitory activity, enhanced tamoxifen action at doses within the concentration range used for anti-convulsive therapy. VPA cooperated with tamoxifen in a variety of ER alpha-positive cell lines and was also effective when combined with other antiestrogens, and with aromatase inhibition. VPA enhanced antiestrogen action by promoting cell death via apoptosis without affecting cell cycling. Some of this action may be due to VPA's ability to induce the pro-apoptotic gene Bik, which is also induced by antiestrogens. Remarkably, VPA blocked the undesirable pro-proliferative action of tamoxifen on uterine endometrial cells. Our in vitro results suggest that VPA and other HDAC inhibitors have the potential to enhance hormonal therapy for ER alpha-positive breast cancer and simultaneously reverse the adverse effects of antiestrogens in the uterus.

p38 mitogen-activated protein kinase stimulates estrogen-mediated transcription and proliferation through the phosphorylation and potentiation of the p160 coactivator glucocorticoid receptor-interacting protein 1.

Nuclear hormone receptors, such as the estrogen receptors (ERs), are regulated by specific kinase signaling pathways. Here, we demonstrate that the p38 MAPK stimulates both ERalpha- and ERbeta-mediated transcription in MCF-7 breast carcinoma, Ishikawa endometrial adenocarcinoma, and human embryonic kidney 293 cells. Inhibition of this potentiation using the p38 inhibitor, RWJ67657, blocked estrogen-mediated transcription and proliferation. Activated ERs promote gene expression in part through the recruitment of the p160 class of coactivators. Because no direct p38 phosphorylation sites have been determined on either ERalpha or beta, we hypothesized that p38 could target the p160 class of coactivators. We show for the first time using pharmacological and molecular techniques that the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) is phosphorylated and potentiated by the p38 MAPK signaling cascade in vitro and in vivo. S736 was identified as a necessary site for p38 induction of GRIP1 transcriptional activation. The C terminus of GRIP1 was also demonstrated to contain a p38-responsive region. Taken together, these results indicate that p38 stimulates ER-mediated transcription by targeting the GRIP1 coactivator.

Cyclin D1 antagonizes BRCA1 repression of estrogen receptor alpha activity.

The cyclin D1 gene is frequently overexpressed in human breast cancer and is capable of inducing mammary tumorigenesis when overexpressed in transgenic mice. The BRCA1 breast tumor susceptibility gene product inhibits breast cancer cellular growth and the activity of several transcription factors. Herein, cyclin D1 antagonized BRCA1-mediated repression of estrogen receptor alpha (ERalpha)-dependent gene expression. Cyclin D1 repression of BRCA1 function was mediated independently of its cyclin-dependent kinase, retinoblastoma protein, or p160 (SRC-1) functions in human breast and prostate cancer cells. In vitro, cyclin D1 competed with BRCA1 for ERalpha binding. Cyclin D1 and BRCA1 were both capable of binding ERalpha in a common region of the ERalpha hinge domain. A novel domain of cyclin D1, predicted to form a helix-loop-helix structure, was required for binding to ERalpha and for rescue of BRCA1-mediated ERalpha transcriptional repression. In chromatin immunoprecipitation assays, 17beta-estradiol (E2) enhanced ERalpha and cyclin D1 recruitment to an estrogen response element (ERE). Cyclin D1 expression enhanced ERalpha recruitment to an ERE. E2 reduced BRCA1 recruitment and BRCA1 expression inhibited E2-induced ERalpha recruitment at 12 hours. Cyclin D1 expression antagonized BRCA1 inhibition of ERalpha recruitment to an ERE, providing a mechanism by which cyclin D1 antagonizes BRCA1 function at an ERE. As cyclin D1 abundance is regulated by oncogenic and mitogenic signals, the antagonism of the BRCA1-mediated ERalpha repression by cyclin D1 may contribute to the selective induction of BRCA1-regulated target genes.

A conserved lysine in the estrogen receptor DNA binding domain regulates ligand activation profiles at AP-1 sites, possibly by controlling interactions with a modulating repressor.

BACKGROUND: Estrogen receptors alpha and beta (ERalpha and ERbeta) differentially activate genes with AP-1 elements. ERalpha activates AP-1 targets via activation functions with estrogens (the AF-dependent pathway), whereas ERbeta, and a short version of ERalpha (ERalpha DBD-LBD) activate only with anti-estrogens (AF-independent pathway). The DNA binding domain (DBD) plays an important role in both pathways, even though neither pathway requires ERE recognition. RESULTS: Mutations of a highly conserved DBD lysine (ERalpha.K206A/G), lead to super-activation of AP-1 through activation function dependent pathways, up to 200 fold. This super-activity can be elicited either through ER AFs 1 or 2, or that of a heterologous activation function (VP16). The homologous substitution in ERbeta, K170A, or in ERalpha DBD-LBD leads to estrogen-dependent AP-1 activation and loss of the usually potent anti-estrogen effects. Each of numerous K206 substitutions in ERalpha, except K206R, eliminates anti-estrogen activation and this loss correlates perfectly with a loss of ability to titrate a repressive function from the RU486 bound progesterone receptor. CONCLUSION: We conclude that ER DBDs contain a complex regulatory function that influences ligand activation profiles at AP-1. This function, which requires the integrity of the conserved lysine, both allows for activation at AP-1 with anti-estrogens (with ERbeta and ERalpha DBD-LBD), and prevents ERalpha from becoming superactive at AP-1 with estrogens. We discuss the possibility that a repressor interaction with the DBD both mediates the AF-independent pathway and dampens the AF dependent pathway. Mutations in the conserved lysine might, by this model, disrupt the binding or function of the repressor.

ERbeta Binds N-CoR in the Presence of Estrogens via an LXXLL-like Motif in the N-CoR C-terminus.

Nuclear receptors (NRs) usually bind the corepressors N-CoR and SMRT in the absence of ligand or in the presence of antagonists. Agonist binding leads to corepressor release and recruitment of coactivators. Here, we report that estrogen receptor beta (ERbeta) binds N-CoR and SMRT in the presence of agonists, but not antagonists, in vitro and in vivo. This ligand preference differs from that of ERalpha interactions with corepressors, which are inhibited by estradiol, and resembles that of ERbeta interactions with coactivators. ERbeta /N-CoR interactions involve ERbeta AF-2, which also mediates coactivator recognition. Moreover, ERbeta recognizes a sequence (PLTIRML) in the N-CoR C-terminus that resembles coactivator LXXLL motifs. Inhibition of histone deacetylase activity specifically potentiates ERbeta LBD activity, suggesting that corepressors restrict the activity of AF-2. We conclude that the ER isoforms show completely distinct modes of interaction with a physiologically important corepressor and discuss our results in terms of ER isoform specificity in vivo.

Differential SERM effects on corepressor binding dictate ERalpha activity in vivo.

Selective estrogen receptor modulators (SERMs) show differential effects upon ERalpha activation function 1 (AF-1). Tamoxifen allows strong ERalpha AF-1 activity, whereas raloxifene allows less and ICI 182,780 (ICI) allows none. Here, we show that blockade of corepressor histone de-acetylase (HDAC) activity reverses the differential inhibitory effect of SERMs upon AF-1 activity in MCF-7 cells. This suggests that differential SERM repression of AF-1 involves HDAC-dependent corepressors. Consistent with this, ICI and raloxifene are more potent than tamoxifen in promoting ERalpha-dependent sequestration of progesterone receptor-associated corepressors. Moreover, ICI and raloxifene are more efficient than tamoxifen in promoting ERalpha binding to the corepressor N-CoR in vivo and in vitro. An ERalpha mutation (537X) that increases N-CoR binding in the presence of all SERMs blocks AF-1 activity. An ERalpha mutation (L379R) that decreases N-CoR binding increases AF-1 activity in the presence of ICI and raloxifene and reverses the effect of the 537X mutation. The 537X and L379R mutations also alter the ligand preference of ERalpha action at AP-1 sites and C3 complement, an action that also involves AF-1. Together, our results suggest that differential SERM effects on corepressor binding can explain differences in SERM effects on ERalpha activity. We propose a model for differential effects of SERMs on N-CoR binding.

Opposing action of estrogen receptors alpha and beta on cyclin D1 gene expression.

Induction of cyclin D1 gene transcription by estrogen receptor alpha (ERalpha) plays an important role in estrogen-mediated proliferation. There is no classical estrogen response element in the cyclin D1 promoter, and induction by ERalpha has been mapped to an alternative response element, a cyclic AMP-response element at -57, with possible participation of an activating protein-1 site at -954. The action of ERbeta at the cyclin D1 promoter is unknown, although evidence suggests that ERbeta may inhibit the proliferative action of ERalpha. We examined the response of cyclin D1 promoter constructs by luciferase assay and the response of the endogenous protein by Western blot in HeLa cells transiently expressing ERalpha, ERalphaK206A (a derivative that is superactive at alternative response elements), or ERbeta. In each case, ER activation at the cyclin D1 promoter is mediated by both the cyclic AMP-response element and the activating protein-1 site, which play partly redundant roles. The activation by ERbeta occurs only with antiestrogens. Estrogens, which activate cyclin D1 gene expression with ERalpha, inhibit expression with ERbeta. Strikingly, the presence of ERbeta completely inhibits cyclin D1 gene activation by estrogen and ERalpha or even by estrogen and the superactive ERalphaK206A. The observation of the opposing action and dominance of ERbeta over ERalpha in activation of cyclin D1 gene expression has implications for the postulated role of ERbeta as a modulator of the proliferative effects of estrogen.

p300 Modulates the BRCA1 inhibition of estrogen receptor activity.

We previously reported that expression of the breast cancer susceptibility gene BRCA1 strongly inhibits the transcriptional activity of the estrogen receptor (ER-alpha) in human breast and prostate cancer cell lines but only weakly inhibits ER-alpha activity in cervical cancer cells (S. Fan et al., Science (Wash. DC), 284: 1354-1356, 1999). We now report that the ability of BRCA1 to repress ER-alpha activity correlates with its ability to induce down-regulation of the cellular levels of the transcriptional coactivator p300 in breast and prostate, but not in cervical cancer cells. On the other hand, BRCA1 failed to alter the expression of the CREB binding protein (CBP), the structural and functional homologue of p300, in any of these cell types. Ectopic expression of either p300 or CBP "rescued" (i.e., reversed) the BRCA1 inhibition of ER-alpha activity, whereas two other nuclear receptor coactivators, the p300/CBP-associated factor (PCAF) and the glucocorticoid receptor-interacting protein-1 (GRIP1), failed to rescue the ER-alpha activity. The rescue function mapped to the cysteine-histidine rich domain CH3, a region of p300/CBP that we found to interact directly with the conserved COOH-terminal activation domain (AF-2) of ER-alpha. p300 and ER-alpha were also found to interact in vivo and to colocalize within the nucleus in breast cancer cells. These findings suggest that the cofactors p300 and CBP modulate the ability of the BRCA1 protein to inhibit ER-alpha signaling. They further suggest that the BRCA1 inhibition of ER-alpha activity may be attributable, at least in part, to the down-regulation of p300.

Structure-based design and synthesis of a thyroid hormone receptor (TR) antagonist.

Antagonists have been developed for several nuclear receptors but not for others, including TRs. TR antagonists may have significant clinical utility for treating hormone excess states and other conditions. A structure derived "extension hypothesis" was applied to synthesize a TR antagonist. The principal design feature was to attach an extension group to a TR agonist whose structure would perturb formation of the TR coactivator-binding surface. The compound, 3,5-dibromo-4-(3',5'-diisopropyl-4'-hydroxyphenoxy)benzoic acid, has no (TRalpha) or very weak partial (TRbeta) TR agonist activity and blocks TR binding of T3, formation of the coactivator-binding surface, and both a positive T3 response on a thyroid hormone response element and a negative T3 response on the TSHbeta promoter in cultured cells. The results suggest that 3,5-dibromo-4-(3',5'-diisopropyl-4'-hydroxyphenoxy)benzoic acid is a TR antagonist for thyroid hormone response element-mediated responses, this approach can be used more generally to generate nuclear receptor antagonists, and this compound or analogues may have medical and research utility.

Design of thyroid hormone receptor antagonists from first principles.

It is desirable to obtain TR antagonists for treatment of hyperthyroidism and other conditions. We have designed TR antagonists from first principles based on TR crystal structures. Since agonist ligands are buried in the fold of the TR ligand binding domain (LBD), we reasoned that ligands that resemble agonists with large extensions should bind the LBD, but would prevent its folding into an active conformation. In particular, we predicted that extensions at the 5' aryl position of ligand should reposition helix (H) 12, which forms part of the co-activator binding surface, and thereby inhibit TR activity. We have found that some synthetic ligands with 5' aryl ring extensions behave as antagonists (DIBRT, NH-3), or partial antagonists (GC-14, NH-4). Moreover, one compound (NH-3) represents the first potent TR antagonist with nanomolar affinity that also inhibits TR action in an animal model. However, the properties of the ligands also reveal unexpected aspects of TR behavior. While nuclear receptor antagonists generally promote binding of co-repressors, NH-3 blocks co-activator binding and also prevents co-repressor binding. More surprisingly, many compounds with extensions behave as full or partial agonists. We present hypotheses to explain both behaviors in terms of dynamic equilibrium of H12 position.