Abstract 1187: An integrated DNA interactome and transcriptome profiling reveals a PU.1/enhancer RNA-mediated feedforward regulatory loop regulating myeloid cell function and leukemia relapse

Abstract The purine-rich box1 protein PU.1 plays essential roles in the development and functions of blood cells. Disruptions in the precise expression patterns of PU.1 are linked to a variety of blood cancers including acute myeloid leukemia (AML). Various studies have attributed the regulatory role of PU.1, as a transcription factor, to its control over myeloid coding genes. However, the role of PU.1 in regulating noncoding genes that give rise to long noncoding RNAs, which are increasingly reported to regulate normal and malignant hematopoiesis, remains largely overlooked. In this study, we systematically identified non-coding RNA target genes of PU.1 and examined the role of LOUP (a myeloid-associated long non-coding RNA) in both physiological and malignant conditions using interdisciplinary approaches that include multiomics analyses, cell differentiation, and functional assays, and PU.1 binding motif disruption in cell and animal models. We initially compiled a comprehensive catalog of putative and verified enhancers from databases and published sources. Next, we interrogated this enhancer catalog with genome-wide PU.1 occupancy and PU.1-mediated noncoding transcriptome profiles in myeloid cells. In doing so, we identified a repertoire of PU.1-mediated noncoding RNAs that are associated with enhancers. Among these, we were surprised to find LOUP, which we had previously identified as an RNA inducer of PU.1. The genomic region within the LOUP locus occupied by PU.1 is characterized by the epigenetic features of a myeloid-specific superenhancer. Targeted disruption of the PU.1-binding motifs resulted in downregulation of LOUP promoter activity and LOUP expression. Depletion of LOUP reduced expression of monocyte/macrophage cell markers as well as the transcriptional program associated with monocyte/macrophage differentiation. Further, LOUP depletion reduced macrophage engulfment of fluorescently labeled latex beads while altering the transcriptional program associated with phagocytosis, suggesting that LOUP is essential for normal microbial clearance by macrophages. Finally, in accordance with its known role in promoting AML cell differentiation and inhibiting cell growth, our findings indicate that LOUP is downregulated in relapsed AML patient samples as well as de novo AML cells harboring oncogenic fusion proteins. In summary, our results indicate that LOUP and PU.1 function as partner regulatory biomolecules within an enhancer-mediated feedforward regulatory loop that promotes their mutual expression. The PU.1/LOUP axis regulates monocytic and macrophage differentiation and function and is associated with leukemia pathophysiology. Our findings suggest that this feedforward regulatory loop could serve as a potential target hub for therapeutic interventions. Citation Format: Asm Waliullah, Kevin Qiu, Barbara Dziegielewska, Minh L. Tran, Andrew Pan, Leran Wang, Tuan Nguyen, Junyan Zhang, Golam Mohi, Daniel G. Tenen, Jeffrey W. Craig, Bon Q. Trinh. An integrated DNA interactome and transcriptome profiling reveals a PU.1/enhancer RNA-mediated feedforward regulatory loop regulating myeloid cell function and leukemia relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1187.