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Cancer-associated alterations in DNA methylation include hypermethylation of focal CpG islands within promoters, as well as hypomethylation of large stretches of gene-poor chromosomal domains, so called Partially Methylated Domains (PMDs). While CpG island hypermethylation is associated with silencing of tumor suppressor genes, the functional consequences of hypomethylation are uncertain. PMDs may be cause or consequence of cancer cell proliferation, and they have been associated with transcriptional de-repression of repetitive sequences, as well as histone modification-triggered silencing of rare protein encoding genes within these domains. In studying Circulating Tumor Cells (CTCs) isolated from blood specimens of patients with prostate cancer, we mapped PMDs at the single cell level, identifying a set of 40 “core PMDs” that are shared across cancer cells, within and across different individuals, as well as in prostate cancer cell lines. By analyzing single nuclei from micro-dissected early-stage prostate tumors, we find that hypomethylation of these core PMDs occurs early during prostate tumorigenesis. Core PMDs are highly enriched for immune-related genes, many of which are co-located within a single chromosomal locus targeted for hypomethylation-associated silencing. Nested between adjacent PMDs, we also identify domains that have retained DNA methylation (Preserved Methylation Islands; PMIs), which have sharply demarcated borders, spanning one or a small number of genes. In contrast to PMDs, PMIs are enriched for cell proliferation-associated genes. Together, these observations suggest that hypomethylation-associated silencing of immune-related genes, with sparing of adjacent proliferation-associated genes, is an early event in prostate cancer development. These large-scale chromosomal modifications are detectable within CTCs and may present diagnostic opportunities. Citation Format: Daniel A. Haber, Hongshan Guo, Ben S. Wittner, Joanna A Vuille, Michael S. Lawrence, Chin-Lee Wu, Mehmet Toner, David T. Miyamoto, Shyamala Maheswaran. Hypomethylation of immune-related loci contribute to prostate tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr IA010.

Summary Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3′ UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.