Estrogen Receptor Efficacy Research Articles

Abstract 6001: 6-Phosphofructo-2-kinase inhibition to increase the efficacy of ER and CDK4/6 inhibitors against ER+ breast cancer

Abstract Deregulation of the estrogen receptor (ER)-cyclin D1-CDK4/6 pathway is a hallmark of ER+ breast cancer that has prompted the development of CDK4/6 inhibitors. Although administration of CDK4/6 inhibitors to patients with ER+ advanced breast cancers have resulted in an improvement in progression free survival, almost all patients invariable develop resistance and relapse over time. Activation of glucose metabolism upon CDK4/6 inhibition is becoming increasingly evident. A key stimulator of glycolysis is the PFKFB3 enzyme which synthesizes fructose 2,6-bisphosphate (F2,6BP), a potent allosteric activator of the rate-limiting 6-phosphofructo-1-kinase (PFK1). F2,6BP controls flux throughout the entire glycolytic pathway and, as a result is required for the proliferation of cancer cells. In preliminary studies, we provide evidence that PFKFB3 expression and activity is increased in response to CDK4 inhibition in ER+ breast cancer cells and that a PFKFB3 inhibitor, PFK-158, increases the anti-tumor activity of anti-CDK4 targeted therapy in vitro. Importantly, we found that ER+ cancer cells with acquired resistance to palbociclib have increased levels of PFKFB3 and are highly susceptible to the effects of PFK-158. Our studies provide a clear rationale to propose the use of PFKFB3 inhibitors in combination with CDK4/6 inhibitors as an effective strategy to increase the efficiency and/or overcome resistance to CDK4/6 inhibition. Citation Format: Lilibeth Lanceta, Nadiia Lypova, Jasson Chesney, Yoannis Imbert-Fernandez. 6-Phosphofructo-2-kinase inhibition to increase the efficacy of ER and CDK4/6 inhibitors against ER+ breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6001.

Abstract PD2-07: Prediction of CDK inhibitor efficacy in ER+/HER2- breast cancer using machine learning algorithms

Abstract Background: Three cyclin dependent kinase 4/6 inhibitors (CDKIs) are approved by the Food and Drug Administration (FDA) for the treatment of patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced or metastatic breast cancer in combination with hormonal therapy. However, the choice of when to initiate CDKI therapy (in the first- or second-line setting, or with aromatase inhibitor or fulvestrant) can be clinically challenging as studies have not directly compared these two approaches, nor have studies determined the benefit of continuation of CDKI therapy with changes in hormonal therapy backbone. Methods: Patient-level data from eight randomized controlled pivotal trials submitted to the FDA for new or supplemental marketing applications were pooled. We performed exploratory analyses of the predictive power of the baseline patient and disease characteristic data to forecast outcomes based on a specific treatment pathway. Variables included were: age, race, ethnicity, country and continent of origin, height, weight, body mass index (BMI), menopause status, estrogen receptor (ER) status, progesterone receptor (PR) status, ECOG performance score, histological type, histological grade, and initial disease stage. Additionally, baseline levels at the time of enrollment of alanine aminotransferase (ALT), albumin, alkaline phosphatase, aspartate aminotransferase (AST), bilirubin, blood urea nitrogen (BUN), calcium, creatinine, hemoglobin, magnesium, potassium, sodium, lymphocytes, neutrophils, platelets, and white blood cells. Statistical and machine learning algorithms were used to build predictive models based on the clinical trial results. Results: The pooled studies included 4580 breast cancer patients. Baseline random survival forest models were constructed for patients that received a CDKI plus hormonal therapy and for patients that received hormonal therapy alone. Preliminary findings for these models produced prediction accuracies of 69.2% for the CDKI model and 70.6% for the hormonal therapy alone model. Median predicted survival times for patients were calculated in both treatment scenarios and used to identify those that were more likely to benefit from initial use of CDKIs and those less likely to benefit. Through further analysis of the patients in each group, we identified patient characteristics that are prognostic of survival. Conclusions: This exploratory analysis utilized clinical trial data to create a model where clinical and disease characteristics are entered to come up with an efficacy prediction. Future efforts will extend this model to predict adverse event development. Citation Format: Jeremy Mason, Yutao Gong, Laleh Amiri-Kordestani, Suparna Wedam, Jennifer J Gao, Harpreet Singh, Richard Pazdur, Peter Kuhn, Gideon Blumenthal, Julia A Beaver. Prediction of CDK inhibitor efficacy in ER+/HER2- breast cancer using machine learning algorithms [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-07.

Short- and Long-Term Effects of CDK4/6 Inhibition on Early-Stage Breast Cancer.

CDK4/6 inhibitors are used in the treatment of advanced estrogen receptor (ER)(+) breast cancer. Their efficacy in ER(-) and early-stage breast cancer is currently under investigation. Here, we show that palbociclib, a CDK4/6 inhibitor, can inhibit both progression of ductal carcinoma in situ (DCIS) and growth of invasive disease in both an ER(-) basal breast cancer model (MCFDCIS) and an ER(+) luminal model (MCF7 intraductal injection). In MCFDCIS cells, palbociclib repressed cell-cycle gene expression, inhibited proliferation, induced senescence, and normalized tumorspheres formed in Matrigel while the formation of acini by normal mammary epithelial cells (MCF10A) was not affected. Palbociclib treatment of mice with MCFDCIS tumors inhibited their malignant progression and reduced proliferation of invasive lesions. Transcriptomic analysis of the tumor and stromal cell compartments showed that cell cycle and senescence genes, and MUC16, an ovarian cancer biomarker gene, were repressed during treatment. Knockdown of MUC16 in MCFDCIS cells inhibited proliferation of invasive lesions but not progression of DCIS. After cessation of palbociclib treatment genes associated with differentiation, for example, P63, inflammation, IFNγ response, and antigen processing and presentation remained suppressed in the tumor and surrounding stroma. We conclude that palbociclib can prevent progression of DCIS and is antiproliferative in ER(-) invasive disease mediated in part via MUC16. Lasting effects of CDK4/6 inhibition after drug withdrawal on differentiation and the immune response could impact the approach to treatment of early-stage ER(-) breast cancer.

Exhausted CD8+ cells (Tex) to predict response to PD-1 therapy in estrogen receptor (+) hormone therapy resistant breast cancer predictive of response to immune checkpoint inhibitors after epigenetic priming.

1044Background: Immune checkpoint inhibitors have revolutionized cancer therapy, yet have limited efficacy in estrogen receptor (ER)+ breast cancer. Implicated factors include scarcity of tumor inf...

Abstract P1-09-09: Efficacy of estrogen receptor β agonists in the prevention of breast cancer progression to therapy resistance

Abstract Estrogen plays an important role in the initiation and progression of breast cancer (BCa). Approximately, 70% of breast tumors are estrogen receptor (ER) positive at the time of presentation. Endocrine therapy using aromatase inhibitors (AI), or anti-estrogen (AE) molecules are widely used for treating ER+ve BCa. However, their efficacy is limited by intrinsic and acquired therapy resistance and most patients develop resistance to these drugs. The transcriptional effects of estrogen are mediated by two ERs (ERα and ERβ) and both are expressed in normal breast tissue. Unlike ERα, ERβ functions as tumor suppressor. However, role of ERβ specific agonists in the prevention of BCa progression remains elusive. In this study, we investigated the effectiveness of two ERβ agonists (S-Equol and LY500307) in the prevention of BCa progression using endocrine therapy sensitive (MCF7-aro) and letrozole resistant (MCF7-aro-LTLT) cells. Our results demonstrated that treatment with ERβ agonists inhibit short-and long-term growth of both endocrine therapy sensitive and resistant BCa cells. In addition, ERβ agonists treatment inhibited invasion and migration of both MCF7-aro and MCF7-aro-LTLT cells. Importantly, cell cycle analysis revealed that ERβ agonists induced cell cycle arrest. Our gene microarray analysis demonstrated that both ERβ agonists significantly modulated genes involved in the cell cycle progression, DNA replication and cell death pathways. Further, gene enrichment analysis of differentially expressed genes revealed that genes involved in the cell cycle checkpoints emerged as significant pathway modulated by ERβ agonists treatment in MCF7-aro cells. Interestingly, in letrozole-resistant MCF7-aro cells, DNA replication was significantly affected by ERβ agonists treatment. Pathway analysis also identified enrichment for chemokine signaling pathways. We confirmed pathway analysis by qRT-PCR and western blot analysis. Accordingly, treatment of in vivo syngeneic xenografts with ERβ agonists significantly inhibited BCa progression. Collectively, these results from this study suggest that ERβ agonists have potential to prevent the progression of BCa progression. Citation Format: Ramasamy K, Samayoa C, Krishnegowda NK, Vadlamudi RK, Tekmal RR. Efficacy of estrogen receptor β agonists in the prevention of breast cancer progression to therapy resistance [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-09.

Abstract PD4-05: Patterns of genomic alterations in ER-positive advanced breast cancer patients treated with CDK4/6 inhibitors

Abstract Background:Cyclin D kinase inhibitors (CDK-is) have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer (MBC) when combined with aromatase inhibition or estrogen receptor (ER) antagonism. Despite the benefit of this approach, clinical resistance develops sometimes early in the treatment without any response to endocrine therapy (primary endocrine resistance) or after initial response (secondary resistance) in all patients in the metastatic setting and the molecular basis for this resistance are still largely unknown. We evaluated the pattern of genomic alterations in circulating cell-free tumor DNA (ctDNA) analysis of metastatic breast cancer patients with ER-positive tumors treated with palbociclib combined with either letrozole or fulvestrant and progressing during therapy. Methods: We conducted a retrospective study of patients with ER-positive MBC who had longitudinal assessment of their disease by ctDNA analysis. The plasma-based assay was performed utilizing Guardant360 (Guardant Health, CA), a digital NGS technology to sequence a panel of > 50 cancer genes. After tabulating number of genomic alterations detected for every patient at baseline and after CDK-i therapy, analysis was performed to identify molecular profile changes in the entire population and in individuals with early progression of disease (<6 months). Results: We analyzed data of 15 ER-positive MBC patients: 8 patients received fulvestrant/palbociclib and 7 received letrozol/palpociclib. The most common mutations before CDK-i therapy were: PIK3CA (16%), TP53 (16%), ESR1 (13%), KIT (9%), EGFR (3%), APC (3%), ERBB2 (3%), MYC (3%), PTEN (3%), RB1 (3%). After therapy with CDK-i the pattern of mutations showed stable and persistent incidence of PIK3CA, TP53 and ESR1. However, new mutations where identified: FGFR1 (6%), IDH (2%), BRCA1 (2%), BRCA2 (2%), CCNE (2%), CCND1 (2%), RAF (2%), AR (2%), ALK(2%). Also, the pattern of gene amplifications presented an increased rate of MYC and FGFR1 amp. Patients with progression of disease before 6 months of CDK-i therapy presented baseline higher number and variation of mutations compared to patients with disease controlled beyond 6 months of therapy. Conclusion: Longitudinal assessment with ctDNA analysis suggest that a genomic alteration landscape consisting of persistent detection of driver and acquired mutations along with emergent new abnormalities in regulatory genes could potentially be related to primary or secondary resistance to CDK-Is in ER+ MBC patients. Future investigation of these alterations should be conducted. Citation Format: Cruz MR, Limentani K, Taxter T, Santa-Maria CA, Behdad A, Gradishar WJ, Nagy RJ, Cristofanilli M. Patterns of genomic alterations in ER-positive advanced breast cancer patients treated with CDK4/6 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-05.

Abstract P3-07-05: CDK8 inhibition improves the efficacy of ER- and HER2-targeted drugs in breast cancer

Abstract Over 70% of breast cancer patients are estrogen receptor (ER) positive and 25% of patients over-express HER2 making these patients susceptible to therapeutic intervention with ER- and HER2-targeted therapies, respectively. However, intrinsic and acquired resistance to targeted therapies is a significant clinical issue and new therapeutic approaches aimed to preventing and overcoming resistance are urgently needed. We have previously shown that high expression of CDK8, a transcription regulating kinase, is associated with shorter relapse free survival in both ER and HER2 positive breast cancer. We have found that CDK8 inhibition by a selective small molecule inhibitor (Senexin B), by shRNA knockdown or by CRISPR/CAS9 knockout, strongly inhibits estrogen signaling in ER-positive breast cancer cells. Senexin B produces a synergistic growth inhibitory effect with an antiestrogen fulvestrant in all the tested ER-positive breast cancer cell lines in vitro and in MCF7 xenograft model in vivo. Senexin B treatment also inhibited invasive growth of MCF7 xenografts. CDK8 inhibition suppressed the emergence of estrogen independence upon long-term estrogen deprivation. A highly synergistic growth inhibitory effect occurred when Senexin B was combined with an anti-HER2 monoclonal antibody (a biosimilar of trastuzumab) or with the HER2/EGFR small molecule inhibitor lapatinib. These synergistic effects were observed in all HER2 positive breast cancer cell lines tested including those that exhibit innate and acquired resistance to HER2 targeting therapy. Furthermore, combining lapatinib with Senexin B completely abrogated the emergence of acquired lapatinib resistance. Taken together these results suggest that CDK8 inhibition, when combined with either ER- or HER2-targeted therapies, offers a rational approach to improving the efficacy of targeted drugs in breast cancer. Citation Format: McDermott MS, Chumanevich A, Liang J, Chen M, Altilia S, Hennes C, Roninson IB, Broude EV. CDK8 inhibition improves the efficacy of ER- and HER2-targeted drugs in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-05.

Inhibition of Nicotinamide Phosphoribosyltransferase Induces Apoptosis in Estrogen Receptor-Positive MCF-7 Breast Cancer Cells.

PurposeTumor cells have increased turnover of nicotinamide adenine dinucleotide (NAD+), the main coenzyme in processes including adenosine diphosphate-ribosylation, deacetylation, and calcium mobilization. NAD+ is predominantly synthesized in human cells via the salvage pathway, with the first component being nicotinamide. Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme in this pathway, and its chemical inhibition by FK866 has elicited antitumor effects in several preclinical models of solid and hematologic cancers. However, its efficacy in estrogen receptor (ER)-positive and human epidermal growth factor receptor 2-positive breast cancer cells has not been previously investigated. In this study, we aimed to deplete the NAD+ content of MCF-7 cells, a model cell line for ER-positive breast cancer, by inhibiting NAMPT in order to evaluate downstream effects on p53 and its acetylation, p21 and Bcl-2-associated X protein (BAX) expression, and finally, apoptosis in MCF-7 breast cancer cells.MethodsMCF-7 cells were cultured and treated with FK866. NAD+ levels in cells were determined colorimetrically. Levels of p53 and its acetylated form were determined by Western blotting. Expression of p21 and BAX was determined by real-time polymerase chain reaction. Finally, levels of apoptosis were assessed by flow cytometry using markers for annexin V and propidium iodide.ResultsFK866 treatment was able to increase p53 levels and acetylation, upregulate BAX and p21 expression, and induce apoptosis in MCF-7 cells. Addition of exogenous NAD+ to cells reversed these effects, suggesting that FK866 exerted its effects by depleting NAD+ levels.ConclusionResults showed that FK866 could effectively inhibit NAD+ biosynthesis and induce programmed cell death in MCF-7 cells, suggesting that NAMPT inhibitors may be useful for the treatment of ER-positive breast cancers.

Early Adaptation and Acquired Resistance to CDK4/6 Inhibition in Estrogen Receptor-Positive Breast Cancer.

Small-molecule inhibitors of the CDK4/6 cell-cycle kinases have shown clinical efficacy in estrogen receptor (ER)-positive metastatic breast cancer, although their cytostatic effects are limited by primary and acquired resistance. Here we report that ER-positive breast cancer cells can adapt quickly to CDK4/6 inhibition and evade cytostasis, in part, via noncanonical cyclin D1-CDK2-mediated S-phase entry. This adaptation was prevented by cotreatment with hormone therapies or PI3K inhibitors, which reduced the levels of cyclin D1 (CCND1) and other G1-S cyclins, abolished pRb phosphorylation, and inhibited activation of S-phase transcriptional programs. Combined targeting of both CDK4/6 and PI3K triggered cancer cell apoptosis in vitro and in patient-derived tumor xenograft (PDX) models, resulting in tumor regression and improved disease control. Furthermore, a triple combination of endocrine therapy, CDK4/6, and PI3K inhibition was more effective than paired combinations, provoking rapid tumor regressions in a PDX model. Mechanistic investigations showed that acquired resistance to CDK4/6 inhibition resulted from bypass of cyclin D1-CDK4/6 dependency through selection of CCNE1 amplification or RB1 loss. Notably, although PI3K inhibitors could prevent resistance to CDK4/6 inhibitors, they failed to resensitize cells once resistance had been acquired. However, we found that cells acquiring resistance to CDK4/6 inhibitors due to CCNE1 amplification could be resensitized by targeting CDK2. Overall, our results illustrate convergent mechanisms of early adaptation and acquired resistance to CDK4/6 inhibitors that enable alternate means of S-phase entry, highlighting strategies to prevent the acquisition of therapeutic resistance to these agents. Cancer Res; 76(8); 2301-13. ©2016 AACR.

Neoadjuvant Endocrine Therapy for Breast Cancer: More Questions Than Answers

Neoadjuvant endocrine therapy studies for breast cancer are a great opportunity to develop insights into the biologic basis for the efficacy of estrogen receptor–targeting agents. Neoadjuvant endocrine treatment is also appealing from the drug development perspective. Theoretically, a promising new adjuvant endocrine strategy could be first tested as a short-term neoadjuvant treatment against a standard medication. Improvements in tumor response rate, surgical outcomes, and evidence for enhanced efficacy at the cellular level, for example in terms of the effect on proliferation, would provide a sound basis for taking the new approach forward into the resource-demanding setting of a phase III adjuvant trial. The potential of randomized phase III neoadjuvant endocrine treatment trials was first emphasized by the results of a double-blind study by Eiermann et al (Letrozole P024) that compared 4 months of the aromatase inhibitor letrozole with tamoxifen as neoadjuvant treatment for women with hormone-receptor–positive tumors who were ineligible for breast-conserving surgery. Letrozole outperformed tamoxifen in terms of clinical and radiologic response rates as well as in the incidence of subsequent breast-conserving surgery. Interestingly, the advantage of letrozole appeared to be particularly evident in a subpopulation of tumors with estrogen-receptor–positive (ER ) and human epidermal growth factor receptor (HER) 1 and/or HER2-positive tumors, indicating that the comparison of endocrine agents in the neoadjuvant setting could provide insights into the molecular basis for differences in efficacy between endocrine agents. The enhanced efficacy of letrozole was also apparent at the level of the cell cycle, since letrozole suppressed tumor Ki67 immunohistochemical staining to a greater extent than tamoxifen. These results were consistent with the advantages of third generation aromatase inhibitors over tamoxifen in other disease settings. The Immediate Preoperative Anastrozole, tamoxifen or Combined with Tamoxifen study (IMPACT) is only the second phase III neoadjuvant endocrine trial to be reported in the peer-reviewed literature. The primary hypothesis focused on a drug development question: would the results of the neoadjuvant endocrine trial (IMPACT) mirror those of a much larger adjuvant study (the Arimidex, Tamoxifen, Alone or in Combination trial; ATAC)? In particular, the investigators wanted to prove that negative preliminary data for enhanced efficacy of anastrozole and tamoxifen in combination compared with tamoxifen alone in the preoperative setting would theoretically have prevented ATAC investigators from randomly assigning 3,000 women to the combination arm. With these primary aims in mind, 330 patients with ER breast cancer were randomly assigned to 3 months of treatment with either anastrozole, tamoxifen, or the combination of the two in a multicenter trial in the United Kingdom and Germany. Response was monitored by caliper measurements (“clinical response”) and by ultrasound. Final surgical outcomes were compared with the surgical approach that would have been initially chosen for the patient if she had not received primary systemic therapy. Unfortunately, none of these clinical end points showed any difference among the three arms and the efficacy of anastrozole was lower than postulated in the statistical design. Given these negative results, this editorial was solicited to consider whether the hypothesis regarding the neoadjuvant-adjuvant link is in jeopardy for neoadjuvant endocrine trials, or whether the IMPACT study was a valiant but flawed test of the postulate. A comparison of the conduct of the IMPACT trial versus the Letrozole P024 trial reveals some straightforward issues that support the latter conclusion. First is the question of sample size. Whereas the IMPACT and Letrozole P024 trial sizes were similar, the IMPACT study involved a three-way randomization. With fewer patients in each treatment group than the Letrozole JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 23 NUMBER 22 AUGUST 1 2005