Prostate adenocarcinoma transition to neuroendocrine small cell tumors requires ASCL1

Most patients with prostate adenocarcinoma develop resistance to therapies targeting the androgen receptor (AR). Consequently, a portion of these patients develop AR-indifferent neuroendocrine prostate cancer (NEPC), a rapidly progressing cancer with limited therapies and poor survival outcomes. Current research to understand the transition to NEPC suggests a model of lineage plasticity, where AR-dependent luminal tumors progress towards an AR-independent neuroendocrine (NE) lineage. Genetic analysis of human NEPC showed a frequent loss of RB1 and TP53, and in experimental models, loss of both genes facilitates the transition to a NE lineage. Transcriptomics has shown the lineage transcription factors ASCL1 and NEUROD1 are present in NEPC. In this study, we used genetically engineered mouse models harboring Cre induced loss of Rb1 and Trp53 with MycT58A overexpression (RPM), to model prostate adenocarcinoma with NEPC by establishing prostate organoids that are subsequently used to generate subcutaneous allograft tumors. These tumors are heterogeneous and display adenocarcinoma, squamous, and NE features. ASCL1 and NEUROD1 are expressed within the NE defined regions, with ASCL1 being more predominant than NEUROD1. Genetic loss of ASCL1 in this model does not decrease tumor growth or tumor formation, however, there is a notable decrease in NE identity and an increase in cells with basal-like cell identity. This study provides a new in vivo model to study the progression of prostate cancer to NEPC and establishes the requirement for ASCL1 in driving neuroendocrine differentiation. Overall design: Organoids from prostates of Genetically Engineered Mouse Models (GEMMs) harboring Rb1flox/flox, Trp53flox/flox, and CAG-LSLMycT58A (RPM: JAX #029971) or Rb1flox/flox, Trp53flox/flox, and CAG-LSLMycT58A with Ascl1flox/flox (RPMA), infected with lentivirus expressing Cre recombinase, were maintained in culture. RNA was isolated from multiple RPM or RPMA organoids for bulk-RNA sequencing. Nuclei were isolated from subcutaneous allograft tumors derived from RPM and RPMA prostate organoids and submitted for single-nuclei RNA sequencing.