The transcription factor FOXM1 is upregulated and overexpressed in aggressive, therapy-resistant forms of hormone receptor-positive and triple negative breast cancers, and is associated with less good patient survival. FOXM1 signaling is also a key driver in many other cancers. Here, we identify a new class of compounds effective in suppressing FOXM1 activity in breast cancers, and displaying good potency for antitumor efficacy. The compounds bind directly to FOXM1 and alter its proteolytic sensitivity, reduce the cellular level of FOXM1 protein by a proteasome- dependent process, and suppress breast cancer cell proliferation and cell cycle progression and increase apoptosis. RNA-seq and gene set enrichment analyses indicate that the compounds decrease expression of FOXM1-regulated genes and suppress gene ontologies under FOXM1 regulation. Several compounds have favorable pharmacokinetic properties and show good tumor suppression in preclinical breast tumor models. These compounds may be suitable for further clinical evaluation in targeting aggressive breast cancers driven by FOXM1.

Many ERα-positive breast cancers develop resistance to endocrine therapy via mutation of estrogen receptors (ER) whose constitutive activation is associated with shorter patient survival. Because there is now a clinical need for new antiestrogens (AE) against these mutant ER, we describe here our development and characterization of three chemically novel AE that effectively suppress proliferation of breast cancer cells and tumors. Our AE are effective against wild type and Y537S and D538G ER, the two most commonly occurring constitutively active ER. The 3 new AE suppressed proliferation and estrogen target gene expression in WT and mutant ER-containing cells and were more effective in D538G than in Y537S cells and tumors. Compared to WT ER, mutants exhibited ~10 to 20-fold lower binding affinity for AE and a reduced ability to be blocked in coactivator interaction, likely contributing to their relative resistance to inhibition by AE. Comparisons between mutant ER-containing MCF7 and T47D cells revealed that AE responses were compound, cell-type and ERα-mutant dependent. These new ligands have favorable pharmacokinetic properties and effectively suppressed growth of WT and mutant ER-expressing tumor xenografts in NOD/SCID-gamma mice after oral or subcutaneous administration; D538G tumors were more potently inhibited by AE than Y537S tumors. These studies highlight the differential responsiveness of the mutant ER to different AE and make clear the value of having a toolkit of AE for treatment of endocrine therapy-resistant tumors driven by different constitutively active ER.

An effective endocrine therapy for breast cancer is to selectively and effectively degrade the estrogen receptor (ER). Up until now, there have been largely only two molecular scaffolds capable of doing this. In this study, we have developed new classes of scaffolds that possess selective estrogen receptor degrader (SERD) and ER antagonistic properties. These novel SERDs potently inhibit MCF-7 breast cancer cell proliferation and the expression of ER target genes, and their efficacy is comparable to Fulvestrant. Unlike Fulvestrant, the modular protein-targeted chimera (PROTAC)-type design of these novel SERDs should allow easy diversification into a library of analogs to further fine-tune their pharmacokinetic properties including oral availability. This work also expands the pool of currently available PROTAC-type scaffolds that could be beneficial for targeted degradation of various other therapeutically important proteins.

To search for new antiestrogens more effective in treating breast cancers, we explored alternatives to the acrylic acid side chain used in many antiestrogens. To facilitate our search, we used a simple adamantyl ligand core that by avoiding stereochemical issues enabled rapid synthesis of acrylate ketone, ester, and amide analogs. All compounds were high affinity estrogen receptor-alpha (ERα) ligands, but displayed a range of efficacies and potencies as antiproliferative and ERα-downregulating agents. There were large differences in activity between compounds having minor structural changes, but antiproliferative and ERα-downregulating efficacies generally paralleled one another. Some compounds with side chain polar groups had particularly high affinities. The secondary carboxamides had the best cellular activities, and the 3-hydroxypropylamide was as efficacious as fulvestrant in suppressing cell proliferation and gene expression. This study has produced structurally novel antiestrogens based on a simple adamantyl core structure with acrylate side chains optimized for cellular antagonist activity.

Invariant natural killer T-cells (iNKT) are a subset of T-cells that play a regulatory role in sepsis. Following cecal ligation and puncture (CLP), iNKT cells emigrate from the liver and into the circulation and peritoneum in a manner dependent upon co-inhibitory molecule Programmed Cell Death Receptor 1 (PD-1). We hypothesized that the effect of PD-1 on iNKT cell emigration was dependent upon the direct PD-1:PD-L1 interaction and that PD-1 and PD-L1 would play a role in chemotaxis and chemokine receptor expression. Adoptive transfer of Vybrant-labelled wild type (WT) cells showed the donor iNKT cells migrated from the liver to the peritoneum following CLP, but PD-L1 deficient donor iNKT cells did not. In a chemotaxis assay, WT iNKT cells chemotaxed to CXCL12, but PD-1 and PD-L1 deficient iNKT cells did not. Using flow cytometry to evaluate chemokine receptor expression, peritoneal iNKT expression of CXCR4 increased following CLP in the WT, PD-1, and PD-L1 deficient animals, and CXCR6 increased in the WT and PD-1 deficient animals. In conclusion here we document that the hepatic emigration of iNKT cells following CLP to the peritoneum appears dependent upon the direct PD-1:PD-L1 interaction, however, while PD-1 and PD-L1 appear to play a role in chemotaxis, this is unlikely a reflection of iNKT cell chemokine receptor expression changes.

Efforts to improve estrogen receptor-α (ER)–targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.

A major risk for patients having estrogen receptor α (ERα)-positive breast cancer is the recurrence of drug-resistant metastases after initial successful treatment with endocrine therapies. Recent studies have implicated a number of activating mutations in the ligand-binding domain of ERα that stabilize the agonist conformation as a prominent mechanism for this acquired resistance. There are several critical gaps in our knowledge regarding the specific pharmacophore requirements of an antagonist that could effectively inhibit all or most of the different mutant ERs. To address this, we screened various chemotypes for blocking mutant ER-mediated transcriptional signaling and identified RU58668 as a model compound that contains structural elements that support potent ligand-induced inhibition of mutant ERs. We designed and synthesized a focused library of novel antagonists and probed how small and large perturbations in different ligand structural regions influenced inhibitory activity on individual mutant ERs in breast cancer cells. Effective inhibition derives from both non-polar and moderately polar motifs in a multifunctional side chain of the antagonists, with the nature of the ligand core making important contributions by increasing the potency of ligands possessing similar types of side chains. Some of our new antagonists potently blocked the transcriptional activity of the three most common mutant ERs (L536R, Y537S, D538G) and inhibited mutant ER-mediated cell proliferation. Supported by our molecular modeling, these studies provide new insights into the role of specific components, involving both the ligand core and multifunctional side chain, in suppressing wild-type and mutant ER-mediated transcription and breast cancer cell proliferation.

FOXM1 (Forkhead box M1) is an oncogenic transcription factor that is greatly upregulated in breast cancer and many other cancers where it promotes tumorigenesis, and cancer growth and progression. It is expressed in all subtypes of breast cancer and is the factor most associated with risk of poor patient survival, especially so in triple negative breast cancer (TNBC). Thus, new approaches to inhibiting FOXM1 and its activities, and combination therapies utilizing FOXM1 inhibitors in conjunction with known cancer drugs that work together synergistically, could improve cancer treatment outcomes. Targeting FOXM1 might prove especially beneficial in TNBC where few targeted therapies currently exist, and also in suppressing recurrent advanced estrogen receptor (ER)-positive and HER2-positive breast cancers for which treatments with ER or HER2 targeted therapies that were effective initially are no longer beneficial. We present these perspectives and future directions in the context of what is known about FOXM1, its regulation, and its key roles in promoting cancer aggressiveness and metastasis, while being absent or very low in most normal non-regenerating adult tissues. We discuss new inhibitors of FOXM1 and highlight FOXM1 as an attractive target for controlling drug-resistant and difficult-to-suppress breast cancers, and how blocking FOXM1 might improve outcomes for patients with all subtypes of breast cancer.