Abstract 4962: Spatial transcriptomics reveals distinct ASCL1 and ASCL2 gene expression patterns in advanced prostate cancer

Abstract Background: ASCL1 and ASCL2 are bHLH transcription factors aberrantly reactivated in advanced prostate cancer. ASCL1 drives neuroendocrine differentiation by activating neuronal genes to promote androgen receptor (AR) independence. ASCL2, regulated by Wnt/β-catenin signaling, maintains stem-like programs and facilitates epithelial to neuroendocrine transition under androgen deprivation. Both factors promote lineage plasticity leading to castration-resistant and neuroendocrine prostate cancer (NEPC). This study examines their distinct gene expression patterns to elucidate how ASCL1 and ASCL2 contribute to lineage transitions and identify potential targets to block transdifferentiation. Methods: Spatial transcriptomic profiling was performed on a Gleason 10 prostate cancer specimen using the 10x Genomics Visium platform. Regions with ASCL1-high, ASCL2-high, or ASCL1/ASCL2-low expression were analyzed for differential gene expression using Loupe Browser, heatmaps, volcano and violin plots, and pathway enrichment via Gene Set Enrichment Assay (GSEA). A patient derived (PDX) model derived from the same patient was subjected to castration, and intact (n=4) and relapsed (n=6) tumors underwent RNA-seq and GSEA. A spontaneously immortalized prostate cancer cell line, UCDCaP, was derived from the same patient, and its castration-resistant derivative, UCDCaP-CR, was established through serial castration-relapse cycles in mouse passages. Key genes were validated by qRT-PCR and Western blotting. Results: ASCL1 and ASCL2 were expressed in a mutually exclusive manner in prostate cancer patient tissues. Compared with ASCL2-high regions, ASCL1-high areas showed higher expression of WNT5A, FOLH1 (PSMA), KRT15, and PROX1, but lower levels of AR, AKR1C3, MAOA, and MET. GSEA indicated enrichment of E2F, Myc, DNA repair, translation, and neural lineage pathways in ASCL1-high regions, while interferon signaling, androgen response, extracellular matrix, and epithelial-mesenchymal transition (EMT) pathways were enriched in ASCL2-high regions. Castration-relapsed PDX tumors exhibited increased AR, AKR1C3, ASCL1, and ASCL2, with reduced FOXA1, FOXJ1, DUSP1, and ALDH1A3. Parallel analyses in UCDCaP-CR cells showed upregulated ASCL2 and neuroendocrine markers, and downregulated ASCL1, PTEN, AR response, and P53 pathways. Conclusions: ASCL1 and ASCL2 define distinct molecular states in prostate cancer. ASCL1 is linked to neural lineage and proliferative pathways, whereas ASCL2 is associated with androgen response and EMT programs. Their mutual exclusivity suggests divergent mechanisms of lineage plasticity. The transition toward ASCL2 dominance in castration-relapsed models, accompanied by neuroendocrine marker upregulation and loss of AR and P53 signaling, highlights their dynamic interplay in driving therapy resistance and neuroendocrine differentiation. Citation Format: Fan Wei, Joy C. Yang, Kenneth Iczkowski, Marc A. Dall'Era, Chengfei Liu. Spatial transcriptomics reveals distinct ASCL1 and ASCL2 gene expression patterns in advanced prostate cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4962.