Investigate FOXA2 mediated DNA demethylation during NEPC transformation by Nanopore Long Read sequencing.

Emerging evidence suggests that the epigenetic state, including the DNA methylation, chromatin accessibility and histone modification, is substantially remodeled, leading to transcriptional reprogramming during the transformation of castration resistant adenocarcinoma (CRPC-Adeno) to neuroendocrine (CRPC-NE) lineage and LUAD to LUAD-SCLC lineage . However, the underlying mechanism is largely unknown. Here, we found FOXA2 transdifferentiates prostate adenocarcinoma away from an AR+/AR gene signature positive (AR+/ARS+) luminal cell lineage to an embryonic and neuroendocrine-lineage by inducing a population of AR+/ARS- transient cells through NKX2-1. Mechanistically, as a pioneer factor, FOXA2 primes NE lineage enhancers by inducing regional chromatin accessibility and DNA demethylation at early stage of transformation. Whereas, once the expression of NKX2-1 is induced by FOXA2, FOXA2 interacts with NKX2-1, mediates gene promoter and enhancer looping and cooperatively recruits P300/CBP complex to NE lineage enhancers, thereby activates NE lineage gene transcription. Interestingly, similar mechanism is also observed in SCLC cancer, suggesting FOXA2 and NKX2-1 are general regulators orchestrating the epigenetic landscape of NE lineage cancer. Finally, therapeutic targeting P300/CBP with CCS1477 which is in clinical trial for lethal PCa significantly impairs NEPC tumor growth. To examine whether FOXA2 overexpression mediated regional DNA demethylation during NEPC transformation, we performed directed methylation with long-read sequencing (DiMeLo-seq) which enables us to analyze DNA CpG methylation and FOXA2 binding simultaneously on the same single DNA molecule in D2, D14 and D28 LNCaP+FOXA2 cells. In addition, to validate the findings in LNCaP+FOXA2 model, we performed Reduced representation methylation sequencing (RRMS) in LuCaP CRPC PDX (LuCaP35CR, LuCaP77CR and LuCaP147) and LuCaP NEPC PDX (LuCaP93, LuCaP145.2 and LuCaP173.1).