Longitudinal single-cell analysis reveals RUNX1T1 as an early driver in treatment-induced neuroendocrine transdifferentiation

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is a lethal, castration-resistant subtype of prostate cancer. While t-NEPC typically arises from adenocarcinoma through neuroendocrine transdifferentiation after androgen pathway inhibition, the temporal dynamics and molecular drivers of this process remain poorly understood. Here, utilizing the first-in-field patient-derived xenograft (PDX) model of adenocarcinoma-to-NEPC transdifferentiation (LTL331/331R), we performed longitudinal single-cell transcriptomic sequencing (scRNA-seq) across seven timepoints spanning pre-castration to relapsed NEPC. Our analysis demonstrated 15 distinct cell clusters, including twelve adenocarcinoma clusters and two NEPC clusters (ASCL1high/FOXA2low and ASCL1low/FOXA2high clusters). Notably, we revealed a newly-discovered, early intermediate transitional cell state during t-NEPC development distinguished by epithelial-mesenchymal transition (EMT), stem cell-related, metabolically active, and HDAC-associated regulatory signatures. Analysis of this intermediate transitional cluster led to the identification of RUNX1T1 as a pivotal transcriptional regulator of NEPC transdifferentiation. Functionally, RUNX1T1 overexpression promoted AR pathway inhibition (ARPI) -induced NE transdifferentiation and increased resistance to ARPI treatment in prostate adenocarcinoma. RUNX1T1 knockdown reverses the NE transdifferentiation, inhibits NEPC cell proliferation and induces apoptosis, and cell cycle arrest. In summary, this study identifies a critical intermediate transitional cell state during t-NEPC development and reveals the heterogeneity of terminal NEPC, offering new insights into NEPC biology and emphasizing the importance of early intervention. Moreover, the discovery of RUNX1T1 as a key early driver active in both the initial and terminal phases of NEPC progression presents promising opportunities for therapeutic intervention. HighlightsO_LILongitudinal single-cell RNA sequencing of treatment-induced adeno-to-NE transdifferentiation model revealed an intermediate transitional cell state in NEPC development and progression. C_LIO_LIThe sequential emergence of ASCL1high/FOXA2low and ASCL1low/FOXA2high NEPC subclusters indicates a temporal evolution of NEPC and highlights their role in contributing to NEPC heterogeneity. C_LIO_LIRUNX1T1, a transcriptional regulator showing increased expression in both intermediate cell state and NEPCs, is identified as an early driver of NEPC development. C_LIO_LIRUNX1T1 plays important functional roles in promoting adeno-to-NE transdifferentiation in early phase of NEPC development and maintaining NE phenotype and aggressiveness in terminal NEPC. C_LIO_LITargeting RUNX1T1 or its complex could offer new therapeutic strategies for NEPC management. C_LI