Enhancers That Free Stalled Polymerase Drive Tumor-Feeding Macrophages Across Cancers

Tumor-associated macrophages, or TAMs, have long been recognized as double agents inside cancer. Some of them rally cytotoxic T cells and mount inflammatory attacks on malignant cells, while others quietly remodel tissue, sprout blood vessels, and suppress immunity so that tumors can grow and spread. A new pan-cancer study published in the Journal of Advanced Research has now mapped, at single-cell resolution, the epigenetic circuitry that steers macrophages toward the tumor-supportive state, and in doing so has uncovered a surprising mechanical detail: the decisive enhancers appear to work largely by releasing RNA polymerase II that sits paused at the start of pro-tumorigenic genes.

The research team, led by investigators including Xin Gao and Deqing Hu of Tianjin Medical University, assembled an enormous chromatin accessibility dataset drawn from 208 single-cell ATAC sequencing samples. These samples spanned healthy adult and fetal tissues, peripheral blood from healthy donors and patients with systemic lupus erythematosus, precancerous familial adenomatous polyposis lesions, and tumors from colorectal, clear cell renal cell, ovarian, and endometrial cancers. After computational integration and batch correction using the ArchR framework, the myeloid compartment resolved into fourteen clusters, five of which were macrophage populations with strikingly distinct tissue distributions and functional signatures.

Two of those populations stood out. Macro-SPP1 cells, defined by expression of the secreted phosphoprotein SPP1, were concentrated in cancerous tissues and showed elevated activity of genes such as IL10, PPARG, INHBA, and IL4I1, along with strong wound healing, hypoxia, and angiogenesis signatures. Macro-C1QC cells, enriched in precancerous polyp tissue, carried markers of tissue-resident macrophages such as FOLR2 and MRC1 alongside anti-inflammatory programs. Motif analysis of their open chromatin revealed candidate regulators: NF-κB-related and bZIP factors dominated the accessible regulatory regions of Macro-SPP1, whereas bHLH and C2H2 zinc finger factors marked Macro-C1QC.

To connect these accessibility patterns to gene output, the researchers integrated the chromatin data with a published pan-cancer single-cell transcriptome of myeloid cells and computed peak-to-gene links, correlations between the accessibility of regulatory elements and the expression of their target genes. Super-enhancer analysis then identified the most densely wired regulatory hubs in each population. In Macro-SPP1, the top super-enhancer-linked genes included chemokines and SOD2, and the full set of 162 high-confidence super-enhancer-regulated genes was enriched for inflammatory response, IL-10 signaling, angiogenesis, wound healing, and HIF-1 signaling. When the team scored these genes in bulk tumor data from The Cancer Genome Atlas, high signature scores predicted poor overall survival in 18 of 29 cancer types. Notably, the predicted fraction of M2-like macrophage infiltration did not track with prognosis, suggesting that the specific enhancer-driven transcriptional program, rather than the sheer number of suppressive macrophages, is what matters for patient outcomes.

Pseudotime trajectory analysis then addressed the question of where these cells come from. The results supported two divergent routes to a pro-tumorigenic identity. Macro-SPP1 cells appeared to descend from peripheral monocytes through an intermediate Mono-NLRP3 stage, while Macro-C1QC cells seemed to arise from resident Macro-LYVE1 macrophages already embedded in tissue. Along the monocyte route, early activation of inflammatory chemokines, IL1B, and THBS1 was followed later by IL10, with NF-κB and AP-1 family factors initiating the process and PPARG and NFAT5 driving the terminal state. The tissue-resident route instead involved CCL3 and CCL4 chemokines and factors such as PRDM1, HES1, ETV5, and MAF. The identification of NFAT5, a transcription factor best known for mediating adaptation to hyperosmotic stress, as a key regulator alongside PPARG and the chromatin-binding protein MECP2 was particularly intriguing, hinting that TAMs activate a broad stress-adaptation program to survive the metabolically hostile tumor microenvironment.

To test these candidates experimentally, the team used the standard THP-1 monocyte model, differentiating cells into macrophages and polarizing them toward inflammatory M1 states with interferon-gamma and lipopolysaccharide, or suppressive M2 states with interleukin-4. NFAT5, PPARG, and MECP2 were all upregulated during M2 polarization, and CRISPR-Cas9 knockdown of any of the three significantly reduced the M2 population, as measured by diminished expression of markers such as CD206 and CD163, while leaving M1 differentiation largely intact. The same results held when macrophages were exposed to conditioned medium from colorectal cancer cells, a more tumor-like stimulus, reinforcing the link between these factors and pro-tumorigenic polarization.

The most conceptually significant part of the study concerned how the relevant enhancers actually work. Enhancers can activate genes in two ways: by opening promoter chromatin so that the transcription preinitiation complex can assemble, or by releasing RNA polymerase II that has initiated transcription but stalled just downstream of the transcription start site. Classifying TAM-associated genes by whether their expression tracked promoter or enhancer accessibility, the researchers found that 71.2 percent of genes linked to Macro-SPP1 regulatory elements were so-called type E genes, whose promoters remained constitutively accessible across all cell states while only their enhancers changed. These type E genes, which included CCL2, HIF1A, CD274, and NFAT5, were also significantly enriched for fine-mapped cancer-risk variants from genome-wide association studies, unlike regulatory elements linked to other macrophage populations.

Constitutively open promoters with dynamically regulated enhancers are precisely the configuration in which pause-release operates, a paradigm well established in embryonic development. Re-analysis of RNA polymerase II ChIP-seq data from mouse macrophages confirmed the prediction: type E genes showed pronounced polymerase pausing in resting macrophages, and that pausing dropped sharply upon interleukin-4 stimulation, the signal that drives alternative, tumor-supportive polarization. The team then dissected the gene HIPK2, a type E gene whose product dampens NF-κB signaling. Disrupting individual HIPK2 enhancers with CRISPR-Cas9 reduced HIPK2 expression, lowered M2 marker expression at both RNA and protein levels, and, critically, increased polymerase occupancy at the transcription start site while depleting it from the gene body, the molecular fingerprint of enhanced pausing. Disrupting an upstream enhancer of NFAT5 produced a parallel loss of M2 polarization.

Taken together, the study sketches a sequential regulatory cascade for the monocyte-derived TAM route: AP-1 factors respond first to tumor infiltration, NF-κB drives an inflammatory phase, and that inflammation is then resolved into a pro-repair, immunosuppressive phenotype through PPARG and NFAT5 acting at super-enhancers that liberate paused polymerase at genes governing wound healing, angiogenesis, and immune suppression. The authors propose that blocking this pause-release mechanism with epigenetic or transcriptional inhibitors could strip tumors of their macrophage allies and potentially overcome resistance to checkpoint immunotherapy, a strategy conceptually distinct from simply depleting macrophages or blocking their recruitment.

The work carries caveats that the authors themselves acknowledge. The polymerase pausing data derive from mouse macrophages rather than human TAMs, so direct ChIP-seq in patient samples will be needed for definitive validation, and the survival analysis relies on bulk tumor data that cannot unambiguously assign gene expression to specific cell types. The in vitro polarization systems, while informative, are simplified stand-ins for the complex tumor microenvironment. Even so, by unifying single-cell chromatin maps across cancers, trajectory modeling, GWAS variant enrichment, and mechanistic perturbation experiments, the study delivers one of the most complete pictures to date of how human tumors reprogram macrophages at the level of cis-regulatory DNA, and it nominates a concrete set of enhancers, transcription factors, and polymerase dynamics as targets for the next generation of macrophage-directed cancer therapies.

Subject of Research: Epigenetic regulation of tumor-associated macrophage differentiation via enhancer-controlled RNA polymerase II pausing

Article Title: Pan-cancer epigenetic landscape of human tumor-associated macrophages reveals crucial enhancers governing their heterogenous formation by Pol II pausing modulation

Article References: Xiong, C., Ning, H., Wang, X., Xie, Y., Liu, Z., Hu, D., & Gao, X. (2026). Pan-cancer epigenetic landscape of human tumor-associated macrophages reveals crucial enhancers governing their heterogenous formation by Pol II pausing modulation. Journal of Advanced Research, 88, 929-945. https://doi.org/10.1016/j.jare.2026.01.048

Image Credits: AI Generated

DOI: 10.1016/j.jare.2026.01.048

Keywords: tumor-associated macrophages, enhancers, RNA polymerase II pausing, single-cell ATAC-seq, super-enhancers, NFAT5, PPARG, tumor microenvironment, macrophage polarization, cancer immunotherapy, epigenetics, pan-cancer analysis