Abstract B34: PD-0332991/palbociclib facilitates the physical interaction between RB1 and BIN1 to increase the vulnerability of pediatric osteosarcoma cells to PARP inhibition
Authors: Folk Watson P. , Homlar Kelly , Sakamuro Daitoku 
Published: February 2019

Abstract
Osteosarcoma (OS) is a sporadic bone malignancy that is diagnosed mainly in childhood and adolescence. Conventional genotoxic chemotherapy such as cisplatin and doxorubicin has been used as first-line therapy for pediatric OS patients. However, OS cells eventually acquire chemoresistance with poor prognosis. Once OS cells acquire chemoresistance, little or no therapeutic option is available. It is therefore critical to better understand genetic, epigenetic, and signaling alterations in OS cells, by which a novel mechanism-based therapeutic strategy to halt the development of OS may be established. Although there is no single genetic event leading to the development of OS, increased mortality, metastasis, and chemoresistance are frequently observed in pediatric OS patients deficient in the retinoblastoma 1 tumor suppressor protein (RB1), compared to RB1-positive OS cases. These facts suggest that the activation and maintenance of RB1 function may improve the efficacy of OS chemotherapy.
In this study, we found that, particularly when RB1 is hypophosphorylated, RB1 physically interacts with the proapoptotic tumor suppressor BIN1 in the human OS cell line U2OS for growth suppression. The adenovirus type 5 E1A oncoprotein disrupted the physical RB1/BIN1 complex, which implies that the protein complex is functionally meaningful as potential anticancer machinery. Acting through the coiled-coil effector domain of BIN1, BIN1 physically interacted with the C pocket domain of RB1 (RB1-C) in vitro. Consistent with this, heterologous expression of BIN1 hindered the effect of serum stimulation on phosphorylation of RB1-C at serine 807/811, which is mediated by cyclin-dependent kinases 4 and 6 (CDK4/6). The transcriptional repression caused by a recombinant BIN1 protein fused with the DNA-binding domain of the yeast transcription factor GAL4 was counterbalanced by suberoylanilide hydroxamic acid (SAHA), a chemical inhibitor of histone deacetylases (HDAC), only in the presence of RB1. This suggests that the BIN1-dependent gene repression greatly requires an HDAC activity employed by RB1. As predicted, in vitro BIN1-induced cancer suppression was compromised by the cotransfection of RB1 short-hairpin RNA. Of note, PD-0332991/palbociclib, a U.S. FDA-approved chemical inhibitor of CDK4/6, which is known to induce hypophosphorylated RB1, stabilized the RB1/BIN1 complex in U2OS cells and markedly increased the susceptibility to olaparib, an FDA-approved inhibitor of poly(ADP-ribose) polymerase (PARP), in a manner dependent on BIN1 and RB1. Because U2OS cells do not carry the molecular features with BRCA1/2-deficient tumors or “BRCAness,” our study suggests a novel BRCAness-independent synthetic lethal possibility activated by the combination of PD-0332991/palbociclib and olaparib in pediatric OS cells.
Citation Format: Watson P. Folk, Kelly Homlar, Daitoku Sakamuro. PD-0332991/palbociclib facilitates the physical interaction between RB1 and BIN1 to increase the vulnerability of pediatric osteosarcoma cells to PARP inhibition [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 B34.