Abstract S4-03: Targeted and selective degradation of estrogen receptor (ER) alpha by PROTACs
Authors: Flanagan JJ , Rossi AK , Anderoli M , Willard RR , Gordon D , Harling J , Churcher I , Smith I , Zinn N , Bantscheff M , Crews CM , Crew A , Coleman KG , Winkler JD , Qian Y 
Published: March 2017

ERα-positive breast cancers comprise approximately 80% of all newly diagnosed cases. Current treatment approaches targeting ER signaling include antagonizing and/or downregulating ER or reducing estrogen levels. Faslodex (fulvestrant) is the only clinically-approved agent that is both a potent ER antagonist and downregulator but has limitations given its pharmacokinetics and route of administration. Over the past several years, targeted ER therapies have focused on developing selective estrogen receptor downregulators (SERDs, i.e, GDC-0810, GDC-0927, AZD9496, RAD1901). The mechanisms involved in ER downregulation by SERD binding are not completely understood, but evidence suggests that conformational changes in the receptor upon ligand binding combined with specific co-regulator interactions destabilize the receptor making it a target for passive proteasomal degradation. We hypothesized that the complex ER pharmacology required for SERD-based passive degradation might be different across various ER-positive cell lines and that targeted degradation of the receptor by actively recruiting the ubiquitin-proteasome machinery would provide a better approach for reducing ER levels. To test this hypothesis, we developed potent molecules directed against ER using our pioneering technology—proteolysis targeting chimeras (PROTACs). PROTACs are heterobifunctional molecules that actively recruit specific E3 ligases resulting in ubiquitylation and degradation of target proteins. When testing for ER degradation using several SERDs and ER PROTACs, we discovered that both fulvestrant and ER PROTACs provided robust degradation in all ER-positive lines (&lt;1 nM 50% degradation; &gt;90% reduction) whereas other SERDs did not degrade or only modestly degraded the receptor. Importantly, MCF-7 cells were uniquely sensitive to SERD-based degradation of ER compared to other cell lines. Subcutaneous administration of fulvestrant (1mpk) or ER PROTACs (10 mpk) reduced uterine ER alpha levels in immature rats (&gt;65% reduction). PROTAC-mediated degradation of ER was also achieved in breast cancer xenografts. To further validate the PROTAC mechanism, incubation of ER-positive cells with ER PROTACs resulted in increased levels of poly-ubiquitylated ERα when compared to SERDs. Lastly, to demonstrate the specificity of PROTAC-mediated ERα degradation, we utilized a cellular expression proteomics-based approach to examine over 7,000 proteins. In this experiment, only ERα and several known proteins whose genes are regulated by ERα, were significantly reduced by PROTACs. It remains to be seen how the current class of investigational downregulators will perform in the clinic. More importantly, a better understanding of the therapeutic potential and benefit of degrading the receptor instead of inhibiting the receptor needs to be explored. To that end, we continue to develop and characterize novel ER PROTACs with the anticipation that targeted ERα degradation will provide a greater clinical benefit than receptor antagonism.

Citation Format: Flanagan JJ, Rossi AK, Anderoli M, Willard RR, Gordon D, Harling J, Churcher I, Smith I, Zinn N, Bantscheff M, Crews CM, Crew A, Coleman KG, Winkler JD, Qian Y. Targeted and selective degradation of estrogen receptor (ER) alpha by PROTACs [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 S4-03.