Sebastian Bernales

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide

IntroductionThe androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer.MethodsWe examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR.ResultsIn a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis.ConclusionsAR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Cognition-Enhancing Properties of Dimebon in a Rat Novel Object Recognition Task Are Unlikely to Be Associated with Acetylcholinesterase Inhibition or N-Methyl-d-aspartate Receptor Antagonism

Dimebon (dimebolin) treatment enhances cognition in patients with Alzheimers disease (AD) or Huntingtons disease. Although Dimebon was originally thought to improve cognition and memory through inhibition of acetylcholinesterase (AChE) and the N-methyl-d-aspartate (NMDA) receptor, the low in vitro affinity for these targets suggests that these mechanisms may not contribute to its clinical effects. To test this hypothesis, we assessed whether Dimebon enhances cognition in rats and if such an action is related to either mechanism or additional candidate mechanisms. Acute oral administration of Dimebon to rats (0.05, 0.5, and 5 mg/kg) enhanced cognition in a novel object recognition task and produced Dimebon brain concentrations of 1.7 ± 0.43, 14 ± 5.1, and 172 ± 94 nM, respectively. At these concentrations, Dimebon did not alter the activity of recombinant human or rat brain AChE. Unlike the AChE inhibitors donepezil and galantamine, Dimebon did not change acetylcholine levels in the hippocampus or prefrontal cortex of freely moving rats. Dimebon displays affinity for the NMDA receptor (Ki = 105 ± 18 μM) that is considerably higher than brain concentrations associated with cognition enhancement in the novel object recognition task and 200-fold weaker than that of memantine (Ki = 0.54 ± 0.05 μM). Dimebon did not block NMDA-induced calcium influx in primary neuronal cells (IC50 > 50 μM), consistent with a lack of significant effect on this pathway. The cognition-enhancing effects of Dimebon are unlikely to be mediated by AChE inhibition or NMDA receptor antagonism, and its mechanism of action appears to be distinct from currently approved medications for AD.

Enzalutamide, an androgen receptor signaling inhibitor, induces tumor regression in a mouse model of castration-resistant prostate cancer

Enzalutamide (formerly MDV3100 and available commercially as Xtandi®), a novel androgen receptor (AR) signaling inhibitor, blocks the growth of castration‐resistant prostate cancer (CRPC) in cellular model systems and was shown in a clinical study to increase survival in patients with metastatic CRPC. Enzalutamide inhibits multiple steps of AR signaling: binding of androgens to AR, AR nuclear translocation, and association of AR with DNA. Here, we investigate the effects of enzalutamide on AR signaling, AR‐dependent gene expression and cell apoptosis.

Abstract P2-14-02: Preclinical Evaluation of Enzalutamide in Breast Cancer Models

Background: The vast majority of breast cancers express the androgen receptor (AR) with ∼80% of ER+ and 30% to50% of ER− tumors being positive for AR by immunohistochemistry (IHC). Approximately 30% of breast cancer patients with estrogen receptor positive (ER+) tumors do not respond to tamoxifen or aromatase inhibitors and AR signaling has been implicated as a possible mechanism of this insensitivity. AR overexpression has also been associated with resistance to either tamoxifen or aromatase inhibitors in cell line models. Hypothesis: ER+ breast cancers can switch from estrogen to androgen-dependent growth as they become resistant to traditional endocrine therapies and AR can serve as a therapeutic target in AR+ breast cancers, irrespective of ER status. Methods: The proliferative effect of dihydrotestosterone (DHT) and estradiol (E2) in the presence enzalutamide (formerly MDV3100) was examined in vitro and in xenograft studies using models of both ER+ and ER− breast cancer that express AR. Furthermore, AR and ER were examined by IHC in clinical specimens from breast cancer patients treated with neoadjuvant exemestane and adjuvant tamoxifen. Tumor tissue was compared to adjacent normal breast epithelium. Results: In ER+ models, bicalutamide and enzalutamide both inhibited DHT-mediated proliferation, while enzalutamide uniquely inhibited E2-mediated proliferation. In vivo, enzalutamide significantly reduced estrogen- and androgen-mediated growth of MCF7 (ER+/AR+) xenograft tumors. Remarkably, enzalutamide demonstrated an anti-proliferative effect comparable to tamoxifen. in vitro, enzalutamide inhibits E2-stimulated tumor cell proliferation and E2 bound ER activation of genes such as PR and SDF-1, despite no observed binding of enzalutamide to ER alpha or beta. In ER−/AR+ MDA-MB-453 xenografts, enzalutamide treatment resulted in decreased nuclear AR localization, increased apoptosis and tumor growth inhibition. Conclusions: Enzalutamide demonstrated significant anti-tumor activity in preclinical models of breast cancers that express AR, regardless of ER status. When enzalutamide opposes DHT, it functions by excluding AR from the nucleus, thereby reversing the anti-apoptotic effect of AR. In contrast, when opposing E2, it causes a decrease in tumor cell proliferation. Profiling of ER+ xenografts is underway to further elucidate the mechanism by which this occurs. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-14-02.

Dual Inhibiton of Bruton’s Tyrosine Kinase and Phosphoinositide-3-Kinase p110δ as a Therapeutic Approach to Treat non-Hodgkin’s B cell Malignancies

Although new targeted therapies, such as ibrutinib and idelalisib, have made a large impact on non-Hodgkin’s lymphoma (NHL) patients, the disease is often fatal because patients are initially resistant to these targeted therapies, or because they eventually develop resistance. New drugs and treatments are necessary for these patients. One attractive approach is to inhibit multiple parallel pathways that drive the growth of these hematologic tumors, possibly prolonging the duration of the response and reducing resistance. Early clinical trials have tested this approach by dosing two drugs in combination in NHL patients. We discovered a single molecule, MDVN1003 (1-(5-amino-2,3-dihydro-1H-inden-2-yl)-3-(8-fluoro-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine), that inhibits Bruton’s tyrosine kinase and phosphatidylinositol-3-kinase δ, two proteins regulated by the B cell receptor that drive the growth of many NHLs. In this report, we show that this dual inhibitor prevents the activation of B cells and inhibits the phosphorylation of protein kinase B and extracellular signal-regulated kinase 1/2, two downstream mediators that are important for this process. Additionally, MDVN1003 induces cell death in a B cell lymphoma cell line but not in an irrelevant erythroblast cell line. Importantly, we found that this orally bioavailable dual inhibitor reduced tumor growth in a B cell lymphoma xenograft model more effectively than either ibrutinib or idelalisib. Taken together, these results suggest that dual inhibition of these two key pathways by a single molecule could be a viable approach for treatment of NHL patients.

Abstract 4756: Elucidating the role of AR in breast cancer .

Background: In breast cancers, the androgen receptor (AR) is more widely expressed than estrogen receptor alpha (ER) or progesterone receptor (PR), and AR has recently emerged as a useful marker for refinement of breast cancer subtype classification. Approximately 77% of invasive breast cancer tumors are positive for AR and among the subtypes, 88% of ER+, 59% of HER2+, and 32% of triple negative breast cancers (ER-/PR-/HER2-) are positive for AR protein expression by IHC. AR expression has been associated with resistance to current endocrine therapies (tamoxifen and aromatase inhibitors) in cell line and preclinical models, and clinical studies. Hypothesis: We hypothesized that ER+ breast cancers that become resistant to traditional endocrine therapies might do so by switching from estrogen to androgen-dependence and that AR holds potential as a therapeutic target in AR+ breast cancers, whether ER+ or ER-. Methods: We quantified the percentage of cells positive for nuclear AR compared to the percentage of cells positive for ER in a cohort of tamoxifen treated patients with clinical outcome data. We also examined the effect of dihydrotestosterone (DHT) and estradiol (E2) in the presence of enzalutamide (formerly MDV3100), a novel AR inhibitor, in in vitro and in vivo models of ER+, ER-, and Her2+ breast cancer that retain AR. Results: Our clinical data indicate that a high ratio of AR to ER protein is indicative of a shorter time to relapse in patients treated with tamoxifen and a lack of response to neo-adjuvant AI treatment. In ER+ models, bicalutamide and enzalutamide both inhibit DHT-mediated proliferation, while enzalutamide uniquely inhibited E2-mediated proliferation. In xenograft tumor studies, enzalutamide significantly reduced estrogen- and androgen-mediated growth of ER+/AR+ xenograft tumors. Remarkably, enzalutamide demonstrated an anti-proliferative effect comparable to tamoxifen on tumors in E2 treated mice and inhibited the expression of classic E2-regulated genes such as SDF-1. Enzalutamide opposed DHT-stimulated proliferation of ER-/AR+ MDA-MB-453 tumors in vivo and caused decreased nuclear AR localization and increased tumor cell apoptosis. In Her2+ breast cancer cell lines, enzalutamide enhanced the response to trastuzumab and decreased the amount of both phosphorylated and total Her3. Conclusions: Enzalutamide demonstrated significant anti-tumor activity in preclinical models of breast cancers that express AR, regardless of ER status. In Her2+ breast cancer, inhibition of AR with enzalutamide may enhance response to Her2-directed therapy or overcome resistance to such agents by reducing levels of Her3. The AR:ER ratio may be a new predictor of response to traditional E2/ER directed endocrine therapy and may indicate that patients who relapse while on tamoxifen or AIs might be good candidates for AR-directed therapy. Citation Format: Nicholas C. D9Amato, Haihua Gu, Dawn R. Cochrane, Sebastian Bernales, Britta M. Jacobsen, Paul Jedlicka, Kathleen C. Torkko, Susan M. Edgerton, Ann D. Thor, Anthony D. Elias, Andrew A. Protter, Jennifer K. Richer. Elucidating the role of AR in breast cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4756. doi:10.1158/1538-7445.AM2013-4756

Abstract LB-109: MDV3100, an androgen receptor signaling inhibitor, inhibits tumor growth in breast cancer preclinical models regardless of estrogen receptor status

Background: The androgen receptor (AR) is detected by immunohistochemistry in approximately 75% of all invasive breast cancer, with ∼88% of estrogen receptor (ER) positive (ER+) tumors also expressing AR and ∼20 to 30% of ER negative (ER-) tumors also retaining AR expression. Potent inhibition of AR activity could be a therapeutic strategy in AR+ ER+ breast cancer. MDV3100 is an androgen receptor signaling inhibitor (ARSI), which inhibits AR activity via three mechanisms: 1) inhibition of androgen binding to AR, 2) inhibition of AR nuclear translocation, and 3) inhibition of nuclear AR-DNA binding; and has demonstrated an overall survival benefit in men with post-docetaxel prostate cancer. Methods: Two ER+/AR+ breast cancer cell lines, MCF7and BCK4 (recently derived from a pleurocentesis), were used to assess the proliferative effect of dihydrotestosterone (DHT) and estradiol (E2) in ovariectomized mice. MDV3100 was compared to bicalutamide and tamoxifen. Growth effects of MDV3100 on ER-/AR+ cells in tissue culture and in xenografts were also examined. Results: Both bicalutamide and MDV3100 inhibited DHT-mediated proliferation of ER+/AR+ cell lines. Although MDV3100 binds AR very effectively and does not bind ER, it inhibited E2-mediated proliferation. MDV3100 also blocked E2 mediated upregulation of AR, PR, and SDF-1. MDV3100 inhibited E2-stimulated tumor growth of MCF7 mammary xenografts as effectively as tamoxifen. With ER-/AR+ cells (MDA-MB-453), MDV3100 reduced DHT-induced nuclear translocation, cell growth in tissue culture and tumor growth in mouse orthotopic xenografts. Conclusions: MDV3100 blocked both DHT- and E2-mediated growth of breast cancer cells, whereas bicalutamide enhanced E2-mediated proliferation. MDV3100 may have unique therapeutic utility in patients with AR+ breast cancer, regardless of ER status. Funding: DOD Breast Cancer Program Idea Award BC074403, Avon Foundation for Women, and University of Colorado Cancer Center pilot project funds to JKR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-109. doi:1538-7445.AM2012-LB-109

Abstract A76: MDV3100, a novel androgen receptor signaling inhibitor, promotes cell apoptosis and tumor regression in both in vitro and in vivo models of castration-resistant prostate cancer.

Prostate tumors initially respond to androgen deprivation therapy but eventually progress despite low serum levels of testosterone. In this stage, known as castration-resistant prostate cancer (CRPC), abnormal androgen receptor (AR) signaling persists and classic anti-androgens such as bicalutamide are ineffective at controlling tumor growth. The novel AR signaling inhibitor MDV3100 has been demonstrated to inhibit AR signaling in preclinical models of CRPC. Here, we investigate further the cellular effects of MDV3100 treatment on AR signaling in in vitro models and in vivo xenograft models of CRPC. Our experiments reveal that MDV3100 (> 100 nM) inhibits dihydrotestosterone (DHT) induced AR-target gene expression in LNCaP cells and LNCaP/AR cells, a CRPC cell culture model in which AR is over-expressed. Inhibition of Prostate-Specific Antigen mRNA expression by MDV3100 was effective at concentrations 10-fold lower than with bicalutamide. As evidenced in other studies and in contrast to bicalutamide, MDV3100 did not display agonistic effects on AR signaling at 10 μM. In contrast to bicalutamide, MDV3100 (> 1 μM) inhibited androgen dependent nuclear translocation of AR, as measured with a quantitative optical based enzyme fragment complementation assay and AR-YFP localization in HEK-293 cells. Furthermore, in the presence of DHT, MDV3100 effectively reduced cell viability of LNCaP cells at concentrations as low as 0.1 μM, 10-fold lower than bicalutamide. MDV3100 also decreased cell viability of LNCaP/AR and W741C-LNcaP cells. We found that the apoptosis marker cleaved-caspase 3 was induced in LNCaP/AR cells treated with 1 M MDV3100 for 48 hours. Under these conditions, MDV3100 was more potent than bicalutamide in decreasing cell viability. Finally, we show that MDV3100 inhibited tumor growth and induced regression of tumor volume in a xenograft model of CRPC generated with LNCaP/AR cells. These results demonstrate that MDV3100, unlike anti-androgens, is effective in both in vitro and in vivo models of CRPC and that its mechanistic advantages over other anti-androgens could be explained by its ability to inhibit AR nuclear translocation and induce cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A76.