Abstract Background. The Androgen Receptor (AR) is a crucial master regulator for Prostate Cancer (PC) survival. Most metastatic PCs exhibit an AR-active phenotype- ARPC, but under treatment pressure, a subset of ARPC undergoes a lineage transition to neuroendocrine PC- NEPC or double negative PC (DNPC). LTL331 is an in vivo model that undergoes a treatment-induced lineage switch from ARPC to NEPC. Hypothesis and methods. We hypothesized that double negative (AR-/NE-) phenotype represents a de-differentiated intermediate stage from ARPC to NEPC trans-differentiation, where AR downstream gene function loss precedes the AR loss, potentially via reversible epigenetic mechanisms. We implemented both RNA FISH-IF imaging and sequencing approaches to characterize and explore the underlying mechanisms. Results. We adapted the LTL331 ARPC PDX to grow as a 2D cell strain and determined that these cells, LTL331DNPCCL exhibit features of DNPC. Notably, LTL331DNPCCL expressed high levels of AR transcripts yet low AR protein. Similarly, AR regulated genes (e. g. , KLK3, NKX3. 1) exhibited transcription-translation discordance, suggesting post-transcriptional alterations in gene regulation. Supraphysiological levels of androgens, as well as proteasome inhibition restored AR protein levels but did not rescue AR-regulated gene expression. LTL331DNPCCL cells exhibit a DNPC phenotype initially, with late passage cells activating neuroendocrine and EMT related genes including CEACAM5, FOXF1, and HGF. Conclusion. These results indicate that the transition from an ARPC state to a NEPC state includes an intermediate phenotype with maintenance of AR expression but loss of canonical AR activity, followed by complete AR loss and gain of NE characteristics. Notably, early steps in the process are regulated via post-translational regulation with a degree of reversibility. The model provides insights into the transdifferentiation process and possible treatment window for lineage reversion. Citation Format: Ruihong Wang, Dapei Li, Sander Frank, Ruth Dumpit, Armand Bankhead, Ilsa Coleman, Peter Nelson. Identification of a potential intermediate cell state in the treatment-induced lineage transition from AR-driven prostate cancer to neuroendocrine prostate cancer abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (2Suppl): Abstract nr A073.
Wang et al. (2026) studied this question.