Modeling the novel SERD elacestrant in cultured fulvestrant-refractory HR-positive breast circulating tumor cells.

PURPOSE: Metastatic hormone receptor-positive (HR+) breast cancer initially responds to serial courses of endocrine therapy, but ultimately becomes refractory. Elacestrant, a new generation FDA-approved oral selective estrogen receptor degrader (SERD) and antagonist, has demonstrated efficacy in a subset of women with advanced HR+breast cancer, but there are few patient-derived models to characterize its effect in advanced cancers with diverse treatment histories and acquired mutations. METHODS: We analyzed clinical outcomes with elacestrant, compared with endocrine therapy, among women who had previously been treated with a fulvestrant-containing regimen from the recent phase 3 EMERALD Study. We further modeled sensitivity to elacestrant, compared with the currently approved SERD, fulvestrant in patient-derived xenograft (PDX) models and cultured circulating tumor cells (CTCs). RESULTS: Analysis of the subset of breast cancer patients enrolled in the EMERALD study who had previously received a fulvestrant-containing regimen indicates that they had better progression-free survival with elacestrant than with standard-of-care endocrine therapy, a finding that was independent estrogen receptor (ESR1) gene mutations. We modeled elacestrant responsiveness using patient-derived xenograft (PDX) models and in ex vivo cultured CTCs derived from patients with HR+breast cancer extensively treated with multiple endocrine therapies, including fulvestrant. Both CTCs and PDX models are refractory to fulvestrant but sensitive to elacestrant, independent of mutations in ESR1 and Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) genes. CONCLUSION: Elacestrant retains efficacy in breast cancer cells that have acquired resistance to currently available ER targeting therapies. Elacestrant may be an option for patients with HR+/HER2- breast cancer whose disease progressed on fulvestrant in the metastatic setting. TRANSLATIONAL RELEVANCE: Serial endocrine therapy is the mainstay of management for metastatic HR+breast cancer, but acquisition of drug resistance highlights the need for better therapies. Elacestrant is a recently FDA-approved novel oral selective estrogen receptor degrader (SERD), with demonstrated efficacy in the EMERALD phase 3 clinical trial of refractory HR+breast cancer. Subgroup analysis of the EMERALD clinical trial identifies clinical benefit with elacestrant in patients who had received prior fulvestrant independent of the mutational status of the ESR1 gene, supporting its potential utility in treating refractory HR+breast cancer. Here, we use pre-clinical models, including ex vivo cultures of circulating tumor cells and patient-derived xenografts, to demonstrate the efficacy of elacestrant in breast cancer cells with acquired resistance to fulvestrant.

Elacestrant demonstrates strong anti-estrogenic activity in PDX models of estrogen-receptor positive endocrine-resistant and fulvestrant-resistant breast cancer.

The selective oestrogen receptor (ER) degrader (SERD), fulvestrant, is limited in its use for the treatment of breast cancer (BC) by its poor oral bioavailability. Comparison of the orally bioavailable investigational SERD elacestrant, versus fulvestrant, demonstrates both drugs impact tumour growth of ER+ patient-derived xenograft models harbouring several ESR1 mutations but that elacestrant is active after acquired resistance to fulvestrant. In cell line models of endocrine sensitive and resistant breast cancer both drugs impact the ER-cistrome, ER-interactome and transcription of oestrogen-regulated genes similarly, confirming the anti-oestrogenic activity of elacestrant. The addition of elacestrant to CDK4/6 inhibitors enhances the antiproliferative effect compared to monotherapy. Furthermore, elacestrant inhibits the growth of palbociclib-resistant cells. Lastly, resistance to elacestrant involves Type-I and Type-II receptor tyrosine kinases which are amenable to therapeutic targeting. Our data support the wider clinical testing of elacestrant.

Elacestrant (RAD1901) exhibits anti-tumor activity in multiple ER+ breast cancer models resistant to CDK4/6 inhibitors.

BACKGROUND: Addition of CDK4/6 inhibitors (CDK4/6i) to endocrine therapy significantly increased progression-free survival, leading to their approval and incorporation into the metastatic breast cancer treatment paradigm. With these inhibitors being routinely used for patients with advanced estrogen receptor-positive (ER+) breast cancer, resistance to these agents and its impact on subsequent therapy needs to be understood. Considering the central role of ER in driving the growth of ER+ breast cancers, and thus endocrine agents being a mainstay in the treatment paradigm, the effects of prior CDK4/6i exposure on ER signaling and the relevance of ER-targeted therapy are important to investigate. The objective of this study was to evaluate the anti-tumor activity of elacestrant, a novel oral selective estrogen receptor degrader (SERD), in preclinical models of CDK4/6i resistance. METHODS: Elacestrant was evaluated as a single agent, and in combination with alpelisib or everolimus, in multiple in vitro models and patient-derived xenografts that represent acquired and "de novo" CDK4/6i resistance. RESULTS: Elacestrant demonstrated growth inhibition in cells resistant to all three approved CDK4/6i (palbociclib, abemaciclib, ribociclib) in both ESR1 wild-type and mutant backgrounds. Furthermore, we demonstrated that elacestrant, as a single agent and in combination, inhibited growth of patient-derived xenografts that have been derived from a patient previously treated with a CDK4/6i or exhibit de novo resistance to CDK4/6i. While the resistant lines demonstrate distinct alterations in cell cycle modulators, this did not affect elacestrant's anti-tumor activity. In fact, we observe that elacestrant downregulates several key cell cycle players and halts cell cycle progression in vitro and in vivo. CONCLUSIONS: We demonstrate that breast cancer tumor cells continue to rely on ER signaling to drive tumor growth despite exposure to CDK4/6i inhibitors. Importantly, elacestrant can inhibit this ER-dependent growth despite previously reported mechanisms of CDK4/6i resistance observed such as Rb loss, CDK6 overexpression, upregulated cyclinE1 and E2F1, among others. These data provide a scientific rationale for the evaluation of elacestrant in a post-CDK4/6i patient population. Additionally, elacestrant may also serve as an endocrine backbone for rational combinations to combat resistance.

EMERALD: Phase III trial of elacestrant (RAD1901) vs endocrine therapy for previously treated ER+ advanced breast cancer.

Elacestrant is a novel, nonsteroidal, orally bioavailable selective estrogen receptor degrader (SERD) that has demonstrated activity in patients with estrogen receptor (ER)-positive/HER2-negative breast cancer previously treated with endocrine therapies including fulvestrant and/or CDK 4/6 inhibitor therapy, and in those with ESR1 mutations (ESR1-mut) known to confer endocrine resistance. Herein, we describe the design and methodology of EMERALD, an international, multicenter, randomized, open-label, active-controlled, Phase III clinical study comparing the efficacy and safety of elacestrant to standard-of-care endocrine monotherapy treatment (fulvestrant or an aromatase inhibitor, per investigator's choice) in patients with ER-positive/HER2-negative advanced breast cancer. Primary end points are progression-free survival in ESR1-mut patients and in all patients (NCT03778931; EudraCT 2018-002990-24).

Selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders (SERDs) in cancer treatment.

Breast cancer is the most frequently diagnosed cancer in women, with estrogen receptor positive (ER+) breast cancer making up approximately 75% of all breast cancers diagnosed. Given the dependence on active ER signaling in these tumors, the predominant treatment strategy has been to inhibit various aspects of this pathway including directly antagonizing ER with the use of selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders (SERDs). Interestingly, the dependence on ER for breast cancer growth is often retained even after progression through several lines of antiestrogen therapy, making ER a bonafide biomarker for this cancer subtype and driving the continued research and development of novel ER-targeted therapeutics to treat this patient population. This, combined with the continuous discovery of mechanisms underlying endocrine resistance, is resulting in a continually evolving treatment landscape for ER+ breast cancer. This review discusses various ER antagonists investigated for the treatment of breast cancer, outlining their pharmacological and tissue-specific mechanisms of action as well as their specified use within the ER+ breast cancer setting. In addition, mechanisms of resistance to SERMs and SERDs, the use of ER antagonists in combination therapy strategies, and the ongoing development of novel drugs are also reviewed in the context of the changing clinical landscape of ER+ breast cancer. Lastly, the role of SERMs and SERDs in non-breast cancer indications is also discussed.

Elacestrant (RAD1901), a Selective Estrogen Receptor Degrader (SERD), Has Antitumor Activity in Multiple ER+ Breast Cancer Patient-derived Xenograft Models.

Purpose: Estrogen receptor-positive (ER+) breast cancers are typically treated with endocrine agents, and dependence on the ER pathway is often retained even after multiple rounds of antiestrogen therapy. Selective estrogen receptor degraders (SERD) are being developed as a strategy to more effectively target ER and exploit ER dependence in these cancers, which includes inhibiting both wild-type and mutant forms of ER. The purpose of this study was to evaluate the efficacy of a novel orally bioavailable SERD, elacestrant (RAD1901), in preclinical models of ER+ breast cancer.Experimental Design: Elacestrant was evaluated as a single agent and in combination with palbociclib or everolimus in multiple ER+ breast cancer models, including several patient-derived xenograft models.Results: Elacestrant induces the degradation of ER, inhibits ER-mediated signaling and growth of ER+ breast cancer cell lines in vitro and in vivo, and significantly inhibits tumor growth of multiple PDX models. Furthermore, we demonstrate that elacestrant in combination with palbociclib or everolimus can lead to greater efficacy in certain contexts. Finally, elacestrant exhibits significant antitumor activity both as a single agent and in combination with palbociclib in two patient-derived breast cancer xenograft models harboring ESR1 mutations.Conclusions: These data underscore the potential clinical utility of elacestrant as a single agent and as a combination therapy, for both early- and late-stage ER+ disease. Clin Cancer Res; 23(16); 4793-804. ©2017 AACR.

Effective combination therapies in preclinical endocrine resistant breast cancer models harboring ER mutations.

Although endocrine therapy is successfully used to treat patients with estrogen receptor (ER) positive breast cancer, a substantial proportion of this population will relapse. Several mechanisms of acquired resistance have been described including activation of the mTOR pathway, increased activity of CDK4 and activating mutations in ER. Using a patient derived xenograft model harboring a common activating ER ligand binding domain mutation (D538G), we evaluated several combinatorial strategies using the selective estrogen receptor degrader (SERD) fulvestrant in combination with chromatin modifying agents, and CDK4/6 and mTOR inhibitors. In this model, fulvestrant binds WT and MT ER, reduces ER protein levels, and downregulated ER target gene expression. Addition of JQ1 or vorinostat to fulvestrant resulted in tumor regression (41% and 22% regression, respectively) though no efficacy was seen when either agent was given alone. Interestingly, although the CDK4/6 inhibitor palbociclib and mTOR inhibitor everolimus were efficacious as monotherapies, long-term delayed tumor growth was only observed when co-administered with fulvestrant. This observation was consistent with a greater inhibition of compensatory signaling when palbociclib and everolimus were co-dosed with fulvestrant. The addition of fulvestrant to JQ1, vorinostat, everolimus and palbociclib also significantly reduced lung metastatic burden as compared to monotherapy. The combination potential of fulvestrant with palbociclib or everolimus were confirmed in an MCF7 CRISPR model harboring the Y537S ER activating mutation. Taken together, these data suggest that fulvestrant may have an important role in the treatment of ER positive breast cancer with acquired ER mutations.

Differential regulation of mTOR signaling determines sensitivity to AKT inhibition in diffuse large B cell lymphoma.

Agents that target components of the PI3K/AKT/mTOR pathway are under investigation for the treatment of diffuse large B cell lymphoma (DLBCL). Given the highly heterogeneous nature of DLBCL, it is not clear whether all subtypes of DLBCL will be susceptible to PI3K pathway inhibition, or which kinase within this pathway is the most favorable target. Pharmacological profiling of a panel of DLBCL cell lines revealed a subset of DLBCL that was resistant to AKT inhibition. Strikingly, sensitivity to AKT inhibitors correlated with the ability of these inhibitors to block phosphorylation of S6K1 and ribosomal protein S6. Cell lines resistant to AKT inhibition activated S6K1 independent of AKT either through upregulation of PIM2 or through activation by B cell receptor (BCR) signaling components. Finally, combined inhibition of AKT and BTK, PIM2, or S6K1 proved to be an effective strategy to overcome resistance to AKT inhibition in DLBCL.

AZD2014, an Inhibitor of mTORC1 and mTORC2, Is Highly Effective in ER+ Breast Cancer When Administered Using Intermittent or Continuous Schedules.

mTOR is an atypical serine threonine kinase involved in regulating major cellular functions, such as nutrients sensing, growth, and proliferation. mTOR is part of the multiprotein complexes mTORC1 and mTORC2, which have been shown to play critical yet functionally distinct roles in the regulation of cellular processes. Current clinical mTOR inhibitors only inhibit the mTORC1 complex and are derivatives of the macrolide rapamycin (rapalogs). Encouraging effects have been observed with rapalogs in estrogen receptor-positive (ER(+)) breast cancer patients in combination with endocrine therapy, such as aromatase inhibitors. AZD2014 is a small-molecule ATP competitive inhibitor of mTOR that inhibits both mTORC1 and mTORC2 complexes and has a greater inhibitory function against mTORC1 than the clinically approved rapalogs. Here, we demonstrate that AZD2014 has broad antiproliferative effects across multiple cell lines, including ER(+) breast models with acquired resistance to hormonal therapy and cell lines with acquired resistance to rapalogs. In vivo, AZD2014 induces dose-dependent tumor growth inhibition in several xenograft and primary explant models. The antitumor activity of AZD2014 is associated with modulation of both mTORC1 and mTORC2 substrates, consistent with its mechanism of action. In combination with fulvestrant, AZD2014 induces tumor regressions when dosed continuously or using intermittent dosing schedules. The ability to dose AZD2014 intermittently, together with its ability to block signaling from both mTORC1 and mTORC2 complexes, makes this compound an ideal candidate for combining with endocrine therapies in the clinic. AZD2014 is currently in phase II clinical trials.

Resistance to everolimus driven by epigenetic regulation of MYC in ER+ breast cancers.

Acquired resistance to PI3K/mTOR/Akt pathway inhibitors is often associated with compensatory feedback loops involving the activation of oncogenes. Here, we have generated everolimus resistance in ER+ breast cancer cells and in long-term estrogen deprived (LTED) models that mimic progression on anti-estrogens. This allowed us to uncover MYC as a driver of mTOR inhibitor resistance. We demonstrate that both everolimus resistance and acute treatment of everolimus can lead to the upregulation of MYC mRNA, protein expression and, consequently, the enrichment of MYC signatures as revealed by RNA sequencing data. Depletion of MYC resulted in resensitization to everolimus, confirming its functional importance in this setting. Furthermore, ChIP assays demonstrate that MYC upregulation in the everolimus resistant lines is mediated by increased association of the BRD4 transcription factor with the MYC gene. Finally, JQ1, a BRD4 inhibitor combined with everolimus exhibited increased tumor growth inhibition in 3D Matrigel models and an in vivo xenograft model. These data suggest that MYC plays an important role in mediating resistance to everolimus in ER+ and ER+/LTED models. Furthermore, given the regulation ofMYCby BRD4 in this setting, these data have implications for increased therapeutic potential of combining epigenetic agents with mTOR inhibitors to effectively downregulate otherwise difficult to target transcription factors such as MYC.

Synergistic induction of apoptosis by combination of BTK and dual mTORC1/2 inhibitors in diffuse large B cell lymphoma.

Diffuse large B cell lymphoma is generally treated by chemotherapy and there is an unmet medical need for novel targeted therapies or combination therapies. Using in vitro screening, we have identified the combination of ibrutinib, an inhibitor of the tyrosine kinase BTK, and AZD2014, an mTOR catalytic inhibitor, as being highly synergistic in killing ABC-subtype DLBCL cell lines. Simultaneous inhibition of BTK and mTOR causes apoptosis both in vitro and in vivo and results in tumor regression in a xenograft model. We identify two parallel mechanisms that underlie apoptosis in this setting: cooperative inhibition of cap-dependent translation, and the inhibition of an NF-κB/IL10/STAT3 autocrine loop. Combined disruption of these pathways is required for apoptosis. These data represent a rational basis for the dual inhibition of BTK and mTOR as a potential treatment for ABC-subtype DLBCL.

Cyclin D1 activity regulates autophagy and senescence in the mammary epithelium.

Overexpression of cyclin D1 is believed to endow mammary epithelial cells (MEC) with a proliferative advantage by virtue of its contribution to pRB inactivation. Accordingly, abrogation of the kinase-dependent function of cyclin D1 is sufficient to render mice resistant to breast cancer initiated by ErbB2. Here, we report that mouse cyclin D1(KE/KE) MECs (deficient in cyclin D1 activity) upregulate an autophagy-like process but fail to implement ErbB2-induced senescence in vivo. In addition, immortalized cyclin D1(KE/KE) MECs retain high rates of autophagy and reduced ErbB2-mediated transformation in vitro. However, highlighting its dual role during tumorigenesis, downregulation of autophagy led to an increase in senescence in cyclin D1(KE/KE) MECs. Autophagy upregulation was also confirmed in human mammary epithelial cells (HMEC) subjected to genetic and pharmacologic inhibition of cyclin D1 activity and, similar to our murine system, simultaneous inhibition of Cdk4/6 and autophagy in HMECs enhanced the senescence response. Collectively, our findings suggest a previously unrecognized function of cyclin D1 in suppressing autophagy in the mammary epithelium.

The protein kinase Akt1 regulates the interferon response through phosphorylation of the transcriptional repressor EMSY.

The protein kinases Akt1, Akt2, and Akt3 possess nonredundant signaling properties, few of which have been investigated. Here, we present evidence for an Akt1-dependent pathway that controls interferon (IFN)-regulated gene expression and antiviral immunity. The target of this pathway is EMSY, an oncogenic interacting partner of BRCA2 that functions as a transcriptional repressor. Overexpression of EMSY in hTERT-immortalized mammary epithelial cells, and in breast and ovarian carcinoma cell lines, represses IFN-stimulated genes (ISGs) in a BRCA2-dependent manner, whereas its knockdown has the opposite effect. EMSY binds to the promoters of ISGs, suggesting that EMSY functions as a direct transcriptional repressor. Akt1, but not Akt2, phosphorylates EMSY at Ser209, relieving EMSY-mediated ISG repression. The Akt1/EMSY/ISG pathway is activated by both viral infection and IFN, and it inhibits the replication of HSV-1 and vesicular stomatitis virus (VSV). Collectively, these data define an Akt1-dependent pathway that contributes to the full activation of ISGs by relieving their repression by EMSY and BRCA2.

Mitosis hit with an ATM transaction fee: aurora B-mediated activation of ATM during mitosis.

In this issue of Molecular Cell, Yang et al. (2011) demonstrate that Aurora B phosphorylates ATM, leading to its mitotic activation and ability to phosphorylate Bub1 and regulate the spindle checkpoint, thus maintaining genomic integrity.

Novel expression of CST1 as candidate senescence marker.

Senescent cells exhibit altered expression of numerous genes. Identifying the significance of the changes in gene expression may help advance our understanding of the senescence biology. Here, we report on the consistent and strong upregulation of CST1 expression during cellular senescence, independent of the initial trigger. CST1 expression at both the messenger RNA and protein levels was barely detected in control cells, which included early passage proliferating, quiescent, or immortal human fibroblasts and various human tumor cell lines. Immunoblotting and immunofluorescence cytochemical studies further suggest that CST1 accumulates intracellularly, within vesicular structures. We discuss these results in light of the known function of CST1 as a potent inhibitor of lysosomal cysteine proteases.

Dissecting the senescence-like program in tumor cells activated by Ras signaling.

Activated Ras signaling can induce a permanent growth arrest in osteosarcoma cells. Here, we report that a senescence-like growth inhibition is also achieved in human carcinoma cells upon the transduction of H-Ras(V12). Ras-induced tumor senescence can be recapitulated by the transduction of activated, but not wild-type, MEK. The ability for H-Ras(V12) to suppress tumor cell growth is drastically compromised in cells that harbor endogenous activating ras mutations. Notably, growth inhibition of tumor cells containing ras mutations can be achieved through the introduction of activated MEK. Tumor senescence induced by Ras signaling can occur in the absence of p16 or Rb and is not interrupted by the inactivation of Rb, p107, or p130 via short hairpin RNA or the transduction with HPV16 E7. In contrast, inactivation of p21 via short hairpin RNA disrupts Ras-induced tumor senescence. In summary, this study uncovers a senescence-like program activated by Ras signaling to inhibit cancer cell growth. This program appears to be intact in cancer cells that do not harbor ras mutations. Moreover, cancer cells that carry ras mutations remain susceptible to tumor senescence induced by activated MEK. These novel findings can potentially lead to the development of innovative cancer intervention.

Differential oncogenic Ras signaling and senescence in tumor cells.

Several studies have shown that forced expression of oncogenic H-ras can induce a senescence-like permanent growth arrest in normal cells. Here we report that expression of oncogenic H-ras in human osteosarcoma U2OS cells also resulted in a senescence-like flat and enlarged cell morphology and permanent growth arrest. In contrast to normal human fibroblasts, U2OS cells were arrested independently of the p16 and ARF tumor suppressors. Treatment with a MEK inhibitor or a p38MAPK inhibitor interrupted oncogenic H-ras-induced growth arrest in U2OS cells, suggesting that activation of MAPK pathways is important. To further determine whether this process is unique to oncogenic H-ras signaling, we examined the effect of oncogenic K-ras on normal cells and human osteosarcoma cells. Similar to oncogenic H-ras, oncogenic K-ras also induced senescence in normal fibroblasts, while transforming immortalized mouse fibroblasts. However, in contrast to oncogenic H-ras, oncogenic K-ras failed to induce a permanent growth arrest in osteosarcoma U2OS cells. Additionally, cells transduced with oncogenic K-ras exhibited distinguishable cellular changes compared to those transduced with oncogenic H-ras. In summary, we report for the first time that oncogenic H-ras signaling can trigger a senescence-like growth arrest in tumor cells, independent of the p16 and ARF tumor suppressors. This result suggests that tumor cells may harbor a senescence-like program that can be activated by ras signaling. Moreover, our study uncovered a cell type-dependent differential response to oncogenic K-ras, as compared to oncogenic H-ras.