Authors:
 Sheng-Yu Ku1, Maria Mica Garcia1, Martin Bakht1, Yanqing Wang2, Yasutaka Yamada1, Loredana Puca3, Spencer Rosario2, Aram Vosoughi3, Olivier Elemento3, David W. Goodrich2, Himisha Beltran1
 
 
 Affiliations:
 1Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA, USA; 2Department of Pharmacology & Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA; 3Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, NY, USA
 
 
 Abstract Body:
 "Background: One mechanism of acquired resistance to androgen receptor (AR) targeted therapies in prostate cancer is evolution from an AR-driven luminal prostate cancer to an AR-low or AR-negative neuroendocrine prostate cancer (NEPC) phenotype. This lineage plasticity is associated with loss of RB1 and TP53 tumor suppressor genes and expression of neuroendocrine and neuronal lineage markers including Achaete-scute homolog 1 (ASCL1) and Delta-like ligand 3 (DLL3). As ASCL1 and DLL3 function to suppress Notch signaling, we sought to further investigate the role of Notch signaling in NEPC. 
 
 Methods: We evaluated the expression of Notch signaling genes by RNA-seq in a cohort of 735 samples (423 patients) including benign prostate (n= 173) localized prostate cancer (PCa, n=324), castration resistant prostate adenocarcinoma (CRPC-Adeno, n=127), and NEPC (n=111), patient-derived organoid models (n=6), and mouse models of NEPC, and generated a scoring method to calculate Notch activity. We performed immunohistochemistry to confirm protein expression in selected cases. We conducted ChIP-seq analysis of H3K27ac and H3K27me3 on Notch related genes using clinical specimens. We generated a GEMM with constitutively expressing Nicd1 in (Pten/Rb1/Trp53)-/- background (TKO). We introduced an active form of NOTCH2 in human NEPC organoids. We treated transgenic mice with resveratrol, a dietary supplement that activates Notch.
 
 Results: Gene expression profiles of NEPC showed significant downregulation of NOTCH1, NOTCH2, and HES1 compared with benign, PCa as well as CRPC-Adeno. Notch signaling score was also significantly lower in NEPC and negatively correlated with NEPC score. IHC confirmed low A, Krt8, Notch1/2, and Hes1 protein expression in NEPC together with expression of neuroendocrine markers Syp and Ascl1. ChIP-seq analysis of clinical specimens demonstrated high H3K27me3 and low H3K27ac on Notch positive regulators in NEPC but not CRPC-adeno. Nicd1-TKO GEMM exhibited a mix of phenotypes with both NE-positive and NE-negative foci. Lymph node metastatic tumors showed both Ar and Krt8 expression and lost NE markers. Ectopic expression of NOTCH2 intracellular domain (NICD2) in human NEPC organoids increased level of HES1 and downregulated expression of ASCL1 and DLL3 indicating the reactivation of Notch signaling. Moreover, NICD2 induced the luminal lineage marker, KRT8, expression and repressed several neuroendocrine markers and transcriptional factors including CHGA, INSM1, BRN2, but not SOX2 and SYP, suggesting that Notch signaling activation could partially repress neuroendocrine differentiation and restore the luminal lineage. Resveratrol treatment of TKO transgenic mice demonstrated switching from an AR-low NEPC phenotype towards an AR-positive luminal phenotype along with restoration of Notch signaling. 
 
 Conclusions: These data demonstrate that downregulation of Notch signaling is associated with the NEPC phenotype. Molecular or pharmacological Notch reactivation modulates NEPC and AR signaling. These findings underscore a potential reversible mechanism underlying acquired resistance to androgen deprivation therapy and open the door to new biomarker and therapeutic strategies."
 
 
 Funding Acknowledgments:
 PCF YIA (SK), DOD PC190250 (SK), R01CA207757 (DWG), R21CA179907 (DWG), R01CA70292 (DWG), R37CA241486 (HB), DOD PC160264 (HB)
 
 
 Corresponding Author Email:
 sheng-yu_lu@dfci.harvard.edu
himisha_beltran@dfci.harvard.edu
 
 
 Conflict of Interest:
 Nothing to Disclose

27th Annual PCF Scientific Retreat Virtual Poster Session