Abstract Aberrations in non-coding DNA regions, particularly those located in regulatory elements, are increasingly implicated as a hallmark of prostate cancer. Yet, mapping enhancers that underpin tumor-specific transcription remains challenging. Here, we developed an integrative workflow to prioritize prostate cancer-specific enhancers (PSEs) by analyzing 204 H3K27ac ChIP-seq datasets from prostate tumor and normal tissues, alongside prostate cell lines. We connected the differentially activated enhancer landscape to three-dimensional (3D) chromatin organization in prostate cancer by identifying key oncogenic topologically associating domains (TADs) from Hi-C datasets. We selected a previously uncharacterized but high-priority locus at chr6q24.1 for in-depth study. To define cancer-specific 3D architecture at chr6q24.1, we generated Region-Capture Micro-C (RCMC) maps at nucleosome resolution in RWPE-1 (normal) and 22Rv1 (cancer) cells. This revealed highly nested enhancer-promoter (E-P) interactions, which we termed multi-connected enhancer hubs, that were prominent in cancer but absent in normal, exceeding the sensitivity of conventional Hi-C for enhancer-centered contacts. CRISPR/Cas9 deletion of individual enhancers across the locus, followed by multi-omic profiling, revealed two distinct enhancer classes. Central PSEs (cPSEs) function as core regulatory organizers, whose deletions weakened activities of other PSEs, collapsed locus-wide chromatin interactions, reduced target gene expression, and impaired cancer cell proliferation without broadly altering CTCF/cohesin architecture. In contrast, redundant PSEs (rPSEs) are buffered by neighboring rescuing enhancers to preserve transcription via compensatory rewiring that strengthens alternative E-P contacts. Our data also suggest that these two classes of enhancer behaviors are associated with differential activity of FOXA1, a pioneer transcription factor in prostate cancer. Together, our study revealed cancer-specific, multi-connected enhancer hubs essential for prostate tumorigenesis and uncovered the regulatory hierarchy of enhancers, providing a framework to functionally characterize and validate oncogenic non-coding DNA regions that sustain prostate cancer phenotypes. This study advances our understanding of non-coding regulatory regions and offers future opportunities for developing novel precision epigenome-based clinical interventions for prostate cancer. Citation Format: Huan Cao, Zexun Wu, Baixi Ji, Seolyn Yang, Leonardo Gonzalez-Smith, Andrew Vu, Suhn K. Rhie. Decoding 3D enhancer architecture identifies hierarchical oncogenic regulatory programs in 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 7241.
Cao et al. (2026) studied this question.