Abstract PD2-04: ERα phosphorylation at pS294: A biomarker of ligand or mutational (Y537S, D538G) activation, and a receptor target for CDK2 inhibition
Authors: Benz CC , Scott GK , Chu D , Kaur R , Muthurajah M , Rothschild D , Frazier K , Park BH 
Published: May 2016

Background: Certain ERα phosphorylation (p) sites are essential for ERα transcriptional activity; and with development of ERα p-specific antibodies, some of these sites predict endocrine responsiveness. Unlike other ERα p-sites, pS294 has been shown to be induced by ligand activation and not by cross-talking growth factor signals. With development of a new rabbit monoclonal, pS294 induction was found to be dependent on a cyclin-dependent kinase (CDK). This study aimed to identify the specific CDK mediating induction of pS294, determine if ligand-independent ERα activating mutations (Y537S, D538G) also induce pS294, and learn if specific CDK inhibitors might enhance endocrine therapeutic efficacy by suppressing pS294.

Methods: MCF7 cells, untreated (stripped media) or stimulated by estradiol (E2, 10nM) or growth factor (EGF, 5nM), were treated with either CDK-specific knockdown siRNAs or small molecule CDK inhibitors (with indicated specificities): Roscovitine (pan-CDKs); Dinaciclib (CDK1, CDK2, CDK5, CDK9); Palbociclib (CDK4, CDK6); JNJ7706621 (CDK1, CDK2); BMS265246 (CDK1, CDK2); and SNS032 (CDK2, CDK7, CDK9). Whole cell, nuclear or cytosolic lysates were either Western blotted (for ERα or specific CDKs) or first immunoprecipitated (total ERα, pS294-ERα) and then immunoblotted. RT-PCR of cellular RNA quantified pS294-ERα induced transcripts (EGF3, AREG, CXCL12 vs. GAPDH) potentially inhibited by CDK inhibitors. MCF7 overexpressing ERα activating mutations (Y537S, D538G) were produced by either transient transfection or knock-in; knock-in clones were innoculated into immunocompromised mice to assess ligand-independent xenograft tumor growth in vivo, while transfected cells and tumors were assessed for ligand-independent ERα phosphorylation.

Results: CDK2 was determined to be the primary kinase mediating ligand-dependent induction of pS294-ERα, with co-precipitation of cyclins A/E confirming the expected mechanism of CDK2 recruitment to chromatin-bound pS294-ERα. Knock-in MCF7 cells expressing either Y537S or D538G ERα rapidly formed tumors in vivo without E2 supplementation; tumors and transiently transfected cells overexpressing mutated ERα showed pS294 &gt;&gt; pS118 expression, with constitutive pS294 suppressed by Dinaciclib but not by Palbociclib. CDK1/2 inhibitors (Dinaciclib, BMS265246) but not a CDK4/6 inhibitor (Palbociclib) cooperated with tamoxifen (4-HT) to induce apoptosis in wildtype MCF7.

Conclusion: CDK2 is the primary mediator of pS294 induced by either ligand stimulation or ligand-independent mutational activation of ERα. While the CDK4/6 inhibitor Palbociclib is a recently approved adjunct to endocrine therapy, it enhances cytostatic growth arrest without affecting ERα phosphorylation or receptor induced gene expression. In contrast, CDK2 inhibitors like Dinaciclib should be explored for their ability to enhance ER-positive breast cancer cell death in combination with antiestrogens and for their ability to prevent the emergence of constitutively active ERα mutations by suppressing pS294 induction, essential for ERα mediated gene transactivation and breast tumor growth.

Citation Format: Benz CC, Scott GK, Chu D, Kaur R, Muthurajah M, Rothschild D, Frazier K, Park BH. ERα phosphorylation at pS294: A biomarker of ligand or mutational (Y537S, D538G) activation, and a receptor target for CDK2 inhibition. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD2-04.