Clonal hematopoiesis (CH), defined as an expanded hematopoietic stem cell (HSC) clone with somatic mutations in the absence of blood malignancy, is common in aging and associated with increased severity of inflammatory non-hematologic disorders. The co-occurrence of CH in patients with solid tumors correlates with poor prognosis and a more aggressive phenotype of the malignancy. This leads to the notion that CH mutations alter anti-cancer immunity, and that CH-derived monocytes and macrophages may contribute to a proinflammatory tissue microenvironment. Being innate immune cells, monocytes and macrophages can mount a heightened inflammatory and defense responses to secondary challenges (“trained immunity”) that are known to accentuate tumor control. On the other hand, excessive or prolonged stress stimulus may lead to its suppression (“immune tolerance”). The effect of DNMT3A mutations found in CH on innate immune responses has not been explored.

Using colitis-associated colon cancer (CAC) as a model, we previously reported that CH driven by heterozygous loss of Dnmt3a, mimicking the most common genetic alteration in human CH, promoted initiation and progression of solid tumors in the context of severe chronic inflammation. We hypothesized that Dnmt3a-CH tilts myeloid responses toward immune tolerance rather than trained immunity, compromising tumor control.

To assess the impact of Dnmt3a-CH to tumor immune microenvironment (TME), we created a mouse model with 10% test BM cells (Dnmt3a+/– or WT control) marked with CD45.2 mixed with 90% CD45.1+CD45.2+ WT competitor and engrafted into pre-conditioned CD45.1+ recipients prior to CAC induction by the azoxymethane (AOM)/dextran sulfate sodium (DSS) method. Mice with Dnmt3a+/– BM developed more numerous, larger CAC tumors, solidifying the prooncogenic role of Dnmt3a-CH. Flow cytometry revealed preferential infiltration of Dnmt3a+/– BM-derived cells in the TME, particularly immune-suppressive M2-like macrophages. Increased abundance of Dnmt3a+/– anti-inflammatory M2-like macrophages was confirmed by scRNA-seq on FACS-sorted tumor-infiltrating donor-derived CD45.2 cells from mice non-competitively transplanted with Dnmt3a+/– compared to WT control BM. Cytokine profiling in whole tumor lysates revealed reduced pro-inflammatory IL-1β and TNF-α indicating a switch to a tolerogenic immune state in Dnmt3a+/– BM chimerae.

Repeated inflammatory insults can cause a switch to immune tolerance. To test this, mice transplanted with Dnmt3a+/– and WT control BM were subjected to 3 bouts of DSS-induced bacterial colitis or kept untreated. Despite developing more pronounced colitis as evidenced by greater shortening of the colon and increased weight loss, Dnmt3a+/–-engrafted recipients showed decreased pro-inflammatory cytokine levels in serum and elevated levels of immunosuppressive IL-10. Strikingly, although in vitro differentiated BM-derived macrophages (BMDMs) from untreated Dnmt3a+/– mice showed higher phagocytic activity compared to WT controls, this function was dramatically impaired after DSS challenge in alignment with an immune tolerance phenotype. Similar results were observed in human THP-1 cells CRISPR-edited to disrupt one DNMT3A allele. After stimulation with high-dose lipopolysaccharide (LPS) to mimic an inflammatory challenge associated with endotoxemia secondary to colitis, DNMT3A-disrupted THP-1 macrophages exhibited significantly dampened phagocytosis compared to WT controls. RNA-seq analysis revealed the DNMT3A+/- THP-1 showed reduced expression of gene sets and pathways related to inflammation and innate immune defense.

These observations demonstrate that tolerogenic reprogramming of Dnmt3a-CH macrophages after an inflammatory insult establishes a tumor-permissive immune microenvironment in CAC. Indeed, myeloid-specific heterozygous Dnmt3a loss (by LysM-Cre) was sufficient to promote tumor initiation, while depletion of phagocytes in vivo using clodronate liposomes during CAC induction reversed Dnmt3a-CH-induced increase in colon tumor burden.

In conclusion, DNMT3A-driven CH enables colitis-associated tumorigenesis by priming myelopoiesis towards innate immune tolerance under excessive inflammation, creating an immunosuppressive TME and corrupting tumor control. Targeting innate immune tolerance in individuals with DNMT3A-CH may represent a strategy to mitigate the risk of inflammation-associated cancers.

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2025
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