Robert A. Blake

SU6656, a Selective Src Family Kinase Inhibitor, Used To Probe Growth Factor Signaling

ABSTRACT The use of small-molecule inhibitors to study molecular components of cellular signal transduction pathways provides a means of analysis complementary to currently used techniques, such as antisense, dominant-negative (interfering) mutants and constitutively activated mutants. We have identified and characterized a small-molecule inhibitor, SU6656, which exhibits selectivity for Src and other members of the Src family. A related inhibitor, SU6657, inhibits many kinases, including Src and the platelet-derived growth factor (PDGF) receptor. The use of SU6656 confirmed our previous findings that Src family kinases are required for both Myc induction and DNA synthesis in response to PDGF stimulation of NIH 3T3 fibroblasts. By comparing PDGF-stimulated tyrosine phosphorylation events in untreated and SU6656-treated cells, we found that some substrates (for example, c-Cbl, and protein kinase C δ) were Src family substrates whereas others (for example, phospholipase C-γ) were not. One protein, the adaptor Shc, was a substrate for both Src family kinases (on tyrosines 239 and 240) and a distinct tyrosine kinase (on tyrosine 317, which is perhaps phosphorylated by the PDGF receptor itself). Microinjection experiments demonstrated that a Shc molecule carrying mutations of tyrosines 239 and 240, in conjunction with an SH2 domain mutation, interfered with PDGF-stimulated DNA synthesis. Deletion of the phosphotyrosine-binding domain also inhibited synthesis. These inhibitions were overcome by heterologous expression of Myc, supporting the hypothesis that Shc functions in the Src pathway. SU6656 should prove a useful additional tool for further dissecting the role of Src kinases in this and other signal transduction pathways.

A Widely Applicable, High-Throughput TR-FRET Assay for the Measurement of Kinase Autophosphorylation: VEGFR-2 as a Prototype

Homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assays represent a highly sensitive and robust high-throughput screening (HTS) method for the quantification of kinase activity. Traditional TR-FRET kinase assays detect the phosphorylation of an exogenous substrate. The authors describe the development and optimization of a TR-FRET technique that measures the autophosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) kinase and extend its applicability to a variety of other kinases. The VEGFR-2 assay demonstrated dose-dependent inhibition by compounds known to modulate the catalytic activity of this receptor. In addition, kinetic analysis of a previously characterized VEGFR-2 inhibitor was performed using the method, and results were consistent with those obtained using a different assay format. Because of the known involvement of VEGFR-2 in angiogenesis, this assay should facilitate HTS for antiangiogenic agents. In addition, this general technique should have utility for the screening for inhibitors of kinases as potential therapeutic agents for many other disease indications.

An Automated Image Capture and Quantitation Approach to Identify Proteins Affecting Tumor Cell Proliferation

To facilitate the characterization of proteins that negatively regulate tumor cell proliferation in vitro, the authors have implemented a high-throughput functional assay that measures S-phase progression of tumor cell lines. For 2 tumor cell lines—human melanoma A375 and human lung carcinoma A549—conditions were established using the cyclin-dependent kinase inhibitor, p27kip; the tumor suppressor p53, a kinase-inactive allele of the cell cycle-regulated serine/threonine kinase Aurora2; and the G1/S drug block, aphidicolin. For screening purposes, gene libraries were delivered by adenoviral infection. Cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on a Cellomics ArrayScan®II was used to quantify the effects of these treatments on cell proliferation. The assay can be used to identify novel proteins involved in proliferation and serves as a more robust, reproducible, and sensitive alternative to enzyme-linked immunosorbent assay (ELISA)-based technologies.

Abstract NG05: Not all “SERDs” are equal: Context-independent ER degradation and full ER antagonism define the next generation of ER therapeutics

At least eight new molecular entities targeting the Estrogen Receptor (ER) have recently entered clinical development for ER+ breast cancer, highlighting the profound resurgence of interest in next generation ER therapeutics. The discovery of highly prevalent mutations in ESR1, the gene that encodes ERα, in recurrent ER+ disease, adds weight to the hypothesis that many patients fail ER-targeted agents because those therapeutic agents fail to fully disable ER, and underscores the relevance of ER as a drug target on which tumors depend. Strategies for the development of this new series of therapeutics have primarily centered around creating orally bioavailable ER ligands that belong to the same class of endocrine agent as fulvestrant, a selective ER downregulator (SERD), so named because of its ER degradation function. Though the clinical activity of fulvestrant is believed to be limited by its poor drug-like properties, a recent study demonstrated superiority of fulvestrant over the aromatase inhibitor, anastrozole. This phase III trial result supports the notion that therapeutics mechanistically similar to fulvestrant, but with improved bioavailability, may have best-in-disease potential. Thus, along with attention to pharmacokinetic properties, optimization of ER degradation has emerged as a key parameter in drug discovery. In contrast, the original drug discovery campaign that identified fulvestrant focused on the identification of ER ligands that, as well as antagonizing estrogen, lacked any ER agonist potential under estrogen-deprived conditions (i.e. were full ER antagonists), using the rodent uterus as a sensitive tool to screen out ligands with estrogenic potential. The rationale at the core of this strategy was that ER ligands that achieve full suppression of ER signaling would potentially drive superior efficacy versus the ER ligands available at the time, such as tamoxifen, which although suppressive relative to estrogen, promote weak ER agonism (i.e. partial ER agonist). The observation that fulvestrant promoted ER protein degradation was a retrospective discovery that provided a compelling explanation for how it might achieve full ER suppression. GDC-0810 and AZD9496 were the first ER ligands prospectively optimized for ER degradation in breast cancer cells, to enter clinical trials. Intriguingly though, both molecules display weak ER agonism in the uterus, suggesting that ER degradation in one context does not preclude ER agonism in another. A careful evaluation of GDC-0810 and AZD9496 across a collection of ER+ breast cancer cell lines revealed that despite optimization for ER degradation in MCF7 cells, both fail to promote ER turnover in several other cell lines. Transcriptional analysis of cells treated with GDC-0810 in hormone-deprived conditions further revealed that GDC-0810 can weakly activate ER signaling in breast cancer cells in which it does not induce ER turnover; thus partial ER agonism is not restricted to the uterus. We hypothesized that ligands that inhibit the ER ligand-binding domain (LBD), but do not induce robust ER turnover would trigger weak activation of ER; keeping in mind that in addition to the ligand-activated transactivation domain within the LBD, ER contains transactivation potential in a distinct N-terminal activation function 1 (AF1) domain. To explore this idea we leveraged GDC-0927 and GNE-274. GDC-0927 is in Phase I clinical studies, and like GDC-0810 and AZD9496 was optimized for ER degradation in MCF7 cells. However, GDC-0927 displays no ER agonism in the uterus. GNE-274 is a tool compound that is structurally related to GDC-0927 but does not induce ER turnover. GDC-0927 results in ER degradation in all ER+ cell lines tested, and retains a full antagonist profile even in lines where GDC-0810 displays partial agonism. As predicted, GNE-274 which does not result in ER turnover, functions as a partial ER agonist in breast cancer cell lines and in the uterus. Despite divergent transcriptional activity, GDC-0927 and GNE-274 are both highly/equi-potent inhibitors of the LBD, measured by displacement of co-activator peptides. These data suggest that inhibition of the LBD is not sufficient to suppress activity of the entire estrogen receptor. We propose that ER ligands that inhibit the LBD but fail to increase ER turnover enable ER signaling through activation of the AF1 domain. This paradigm has been proposed for 4OH-tamoxifen, and we extend this model. GDC-0927 and fulvestrant retain ER antagonist and degradation activity against the constitutively active ER.Y537S and ER.D538G variants commonly found in metastatic breast cancer. We speculated that other ER mutations might more dramatically influence the ER antagonist and/or degradation profile of these ligands, and thus performed an ER mutagenesis screen. We discovered a series of single amino acid substitutions that switch GDC-0927 and fulvestrant from full antagonists to weak agonists. Importantly, these mutations prevent GDC-0927- and fulvestrant-mediated degradation of ER, concomitant with their functional switch. This series of alterations appear to converge on disabling helix 12 (H12) - a key functional region of the LBD responsible for co-factor recruitment - since deletion of H12 had the same consequences as the single amino acid substitutions. These data suggest that, 1) H12 plays a key role in ER degradation mediated by GDC-0927 and fulvestrant, and 2) partial agonist activity likely arises from the AF1 domain, since the key functional region of the LBD required for recruitment of co-activators is disabled. Conventional views of ER degradation imply that loss of ER protein would drive the loss of all ER activities, based on observations of SERD-mediated ER protein loss in whole cell lysates. Intriguingly though, cellular fractionation assays demonstrate that ER ligands, regardless of degradation/full antagonist status, trigger rapid engagement with the DNA. In the case of fulvestrant and GDC-0927, re-localization of ER to DNA precedes loss of ER protein evident in whole cell lysates. Even at later time-points, however, when ER protein levels in whole cell lysates are low, ER is retained at the chromatin. A surprising observation we made is that although all ER ligands assessed promote ER DNA engagement, we could distinguish the behavior of the partial agonists from full antagonists prior to loss of ER protein, using chromatin immunoprecipitation (CHIPseq) assays as well as live imaging of cells expressing mNeon-ER. Specifically, FRAP (fluorescence recovery after photobleaching) demonstrated that while partial agonists 4OH-tamoxifen and GNE-274 maintain high mobility of ER, the full antagonists fulvestrant and GDC-0927 profoundly decrease ER mobility, suggesting that the immobilization of ER is likely a general property of full antagonists. In summary, we find that full ER antagonism, defined by suppression of both the LBD and the AF1 domain, is accompanied by robust ER degradation. Notably though, ligands capable of promoting ER degradation in some cell lines are not necessarily full antagonists in all cellular contexts. We argue that the designation of “SERD”, which is ascribed to molecules that show even context-dependent ER degradation activity, does not imply that a molecule belongs to the same mechanistic/therapeutic class as fulvestrant. ER ligands that are truly devoid of partial agonism - the key driver behind the identification and development of fulvestrant - remain a small class. The observation that full ER antagonism invariably associates with ER degradation could be interpreted as degradation driving full antagonism. However, we have discovered that full antagonists can be distinguished from partial agonists prior to the loss of ER protein, suggesting that ER degradation may be a consequence of full suppression of ER, rather than a driver of full suppression. Citation Format: Jane Guan, Wei Zhou, Anneleen Daemen, Steven J. Hartman, Robert A. Blake, Amy Heidersbach, Mamie Yu, May Liang-Chu, Scott Martin, Cecile Chalouni, Irene Chen, Lori S. Friedman, Xiaojing Wang, Ciara Metcalfe. Not all “SERDs” are equal: Context-independent ER degradation and full ER antagonism define the next generation of ER therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr NG05.

Abstract 3621: Characterization of the effects of estrogen receptor alpha Y537S and D538G mutations on receptor pharmacology

The frontline therapy for estrogen receptor alpha (ERα) positive Breast Cancer (ER+BC) involves various forms of endocrine therapy, consisting of either Selective Estrogen Receptor Modulators (SERMs) or aromatase inhibitors. An emerging mechanism of ER+BC resistance to endocrine therapy, and consequently disease relapse, has been associated with a set of “hotspot” mutations in and near to helix-12 of the ERα ligand binding domain. Selective Estrogen Receptor Degraders/Down-regulators (SERDs) represent an important pharmacological strategy being applied to develop treatments for resistant ER+BC. Here, we compare 2 of the most frequent ERα hotspot mutations (Y537S and D538G), with ERα wildtype (WT) and the ability of a set of SERM/SERDs and other ERα ligands to bind, antagonize, degrade/stabilize ERα and affect cell proliferation. Common themes that emerged included the observation that the concentration of each drug required to bind, antagonize or degrade ERα Y537S or ERα D538G was typically higher than that required for ERα WT, although the extent of the shift varied between drugs and the type of measurement. An unexpected observation was that 4-hydroxy-tamoxifen (a major active metabolite of tamoxifen) stabilized nuclear ERα Y537S protein. This represents a potential mechanism that may limit the efficacy of Tamoxifen in treating ERα Y537S ER+BC. Citation Format: Steven J. Hartman, Tracy Kleinheinz, Jonathan White, Stephen Daly, Ria Goodwin, Wei Zhou, Jun Liang, Xiaojing Wang, Daniel F. Ortwine, Lori Friedman, Martin O’Rourke, Ciara Metcalfe, Robert A. Blake. Characterization of the effects of estrogen receptor alpha Y537S and D538G mutations on receptor pharmacology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3621. doi:10.1158/1538-7445.AM2017-3621