Abstract B43: The CDK4 and 6 inhibitor abemaciclib induces both epigenetic and immunogenic responses in preclinical models of Ewing’s sarcoma
Authors: Dowless Michele , Lowery Caitlin , Renschler Matthew , Blosser Wayne , Stephens Jennifer , Flack Robert , Credille Kelly , Chen Mia , Dorsey Frank , Sams Lillian , Ebert Philip , Olsen Jonathan , Shackleford Terry , Houghton Peter , Stancato Louis 
Published: February 2019

Abstract
Ewing’s sarcoma (ES) is a rare but aggressive cancer typically occurring in the bone or soft tissue of children and adolescents. Current treatment regimens composed of surgery, radiation, and high-dose chemotherapy have improved outcomes for localized disease cases; however, ES continues to present a clinical challenge in the recurrent or metastatic setting. ES is often characterized by a chromosomal translocation that results in a chimeric EWS/ETS transcription factor. This fusion protein may contribute to tumorigenesis through modulation of downstream targets leading to cell cycle dysregulation, due in part to the overexpression of Cyclin D1. In addition, ES also has a high degree of DNA methylation when compared to other tumor types, which can lead to epigenetic silencing of key tumor suppressor genes. Abemaciclib is a small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4 and 6) currently under clinical evaluation for metastatic breast cancer, non-small cell lung cancer, and other adult solid tumors. Abemaciclib shows antitumor activity in preclinical models of human adult and pediatric cancer with functional Rb and high cyclin D expression, and has been reported to elicit an immune response in addition to its cell cycle effects in breast cancer. This study was designed to assess abemaciclib-induced antitumor effects, in addition to epigenetic and immunogenic changes, in preclinical ES models.
Six ES cell lines were treated with abemaciclib and the resulting changes in protein expression and methylation were evaluated by Western blot and DNA promoter methylation analysis, respectively. In addition, tumor-secreted factors from conditioned media were measured by ELISA and activation of the interferon (IFN) pathway was assessed through Western blot, qRT-PCR, and RNAseq analysis. In vivo studies of abemaciclib as a single agent or in combination with ES standard-of-care (SOC) in multiple mouse xenograft models were also performed to measure abemaciclib-mediated antitumor effects and epigenetic and immunogenic changes. Single-agent abemaciclib elicited tumor growth delay or tumor stasis, while the combination with SOC was generally superior to either monotherapy arm, often resulting in objective responses. Abemaciclib induced global DNA demethylation through the Rb-E2F-DNMT1 axis, and a ligand-independent type III IFN response. In addition, abemaciclib increased expression of the tumor cell antigen presentation protein MHC-1, and this effect was more durable than abemaciclib-dependent cell cycle effects. These data demonstrate that abemaciclib activity in ES goes beyond cell cycle arrest to include a measurable and durable immunogenic response. Therefore, the multipronged antitumor effects of abemaciclib, namely CDK4/6 inhibition, global DNA demethylation, and resulting immunogenic changes, may be a viable therapeutic option for ES patients when used in combination with appropriate chemotherapy, and additional studies to better understand this unique biology are ongoing.
Citation Format: Michele Dowless, Caitlin Lowery, Matthew Renschler, Wayne Blosser, Jennifer Stephens, Robert Flack, Kelly Credille, Mia Chen, Frank Dorsey, Lillian Sams, Philip Ebert, Jonathan Olsen, Terry Shackleford, Peter Houghton, Louis Stancato. The CDK4 and 6 inhibitor abemaciclib induces both epigenetic and immunogenic responses in preclinical models of Ewing’s sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B43.