In a recent study published in the Journal of Experimental Medicine, researchers investigated whether bone marrow-derived cells with heterozygous loss of Dnmt3a (Dnmt3a^+/Δ), the most common genetic alteration in clonal hematopoiesis (CH), contribute to colitis-associated colon cancer (CAC) pathogenesis.

Study: Hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer. Image Credit: vetpathologist/Shutterstock.com

Background

CH, the clonal expansion of mutant hematopoietic stem cells, is associated with shorter survival in solid tumor patients, including colon cancer.

The deoxyribonucleic acid methyltransferase three alpha (DNMT3A) is frequently altered in clonal hematopoiesis, with mutations causing heterozygous loss of function.

Understanding the causal association between clonal hematopoiesis and aggressive tumor phenotypes is crucial for optimal therapeutic approaches and improved survival rates.

About the study

In the present study, researchers investigated the role of clonal hematopoiesis in C colitis-associated colon cancer pathogenesis driven by DNMT3A gene alterations.

The team used a murine model that combined CAC with experimental clonal hematopoiesis driven by Dnmt3a. The colon tissues were subjected to colonoscopy, histopathological examination, ribonucleic acid (RNA) sequencing, transcriptomic profiling, next-generation sequencing, gene set enrichment analysis (GSEA), immunofluorescence, and immunohistochemical analysis.

The Dnmt3a-driven clonal hematopoiesis cells were treated with an angiogenesis inhibitor, axitinib to evaluate the therapeutic potential of axitinib.

To investigate the impact of Dnmt3a^+/Δ within hematopoietic organs characteristic of clonal hematopoiesis on colitis-associated colon cancer development, murine animals were sacrificed for colon assessment ten weeks after initiating dextran sulfate sodium (DSS) and azoxymethane (AOM) treatment.

To determine the association between genetic alterations in the blood system and CAC severity, the team examined the presence of CH mutations among paired blood and tumor samples obtained from 66 CAC patients.

A bone marrow transplantation-based approach was used to model clonal hematopoiesis in the murine animals experimentally.

To closely simulate human clonal hematopoiesis in which mutant genetic clones contribute to mature cell counts in the blood, wild-type mice were transplanted using 10% Dnmt3a^+/Δ (the Dnmt3a^+/+ or control group) along with 90% wild-type competitor bone marrow cells which could be differentiated by the cluster of differentiation 45.1 and 45.2 leukocytic markers.

Animals diagnosed with hematopoietic diseases using flow cytometry and complete blood counts were excluded from the analysis.

Results

The heterozygous loss of Dnmt3a in the bone marrow led to an accentuated CAC phenotype, with higher tumor penetrance and increased tumor burden in the murine model combining colitis-associated colon cancer with experimental Dnmt3a-clonal hematopoiesis compared to controls.

Hematopoietic-specific Dnmt3a haploinsufficiency promoted cancer initiation and progression in colitis-associated colon cancer, in line with previous studies.

Histopathological findings indicated increased immune cell infiltration, ulcer formation, colonic epithelial injury, dysplasia, and adenocarcinoma formation. Dnmt3a gene haploinsufficiency within the bone marrow yielded advanced colitis-associated colon cancer histopathology and increased cancer burden.

Transcriptome profiling of colon tumors identified signatures of increased colitis-associated colon tumor formation among mice with Dnmt3a-driven experimental clonal hematopoiesis, with an enrichment of genes related to carcinogenesis, including angiogenesis.

In particular, increased expression of vascular endothelial growth factor (VEGF), fibroblast growth factor receptor 1 (Ffgr-1), epithelial-mesenchymal transition (EMT), angiogenesis, and β-catenin/Wnt Family Member 1 (Wnt), mitotic spindle, E2F transcription factor 1 (E2F), master regulator of cell cycle entry and proliferative metabolism (MYC) gene signaling, and gap 2 (G2)/mitosis (M) checkpoint.

In contrast, the downregulation of genes associated with adipogenesis or the metabolism of fatty acids and oxidative phosphorylation was observed.

The findings indicated increased proliferation in the epithelium of regenerating colon cells among Dnmt3a^+/Δ mice, leading to advanced colitis-associated colorectal cancer pathology.

Axitinib treatment eliminated the colon cancer-promoting effects of Dnmt3a^+/Δ bone marrow directly by decreased tumor vascular density and indirectly by normalizing aberrant Dnmt3a-CH hematopoiesis, with a decrease in circulating myeloid cells and an increase in T lymphocytes among Dnmt3a^+/Δ BM chimeras.

The findings indicated that axitinib could mitigate the unfavorable effects of clonal hematopoiesis among cancer patients.

Nine weeks after AOM/DSS therapy initiation, colonoscopic examination revealed increased colon wall opacification, numerous fibrin patches, and bleeding, indicating increased colon pathology.

In addition, the team detected larger and more numerous tumors in the accessible areas of the colon among Dnmt3a^+/Δ bone marrow chimeras compared to Dnmt3a^+/+-engrafted controls.

The modified murine endoscopic index of colitis-associated colon cancer severity (MEICS) scores were significantly elevated in the Dnmt3a^+/Δ group.

A patient with DNMT3A(R882) clonal hematopoiesis did not develop distant metastases, whereas both patients with non-R882 mutations did, indicating the possibility of different clonal hematopoiesis alterations imparting divergent functional consequences.

Conclusion

Based on the study findings, hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer through various molecular mechanisms.

This alteration in bone marrow impacts colitis-associated colon cancer pathogenesis through non-tumor-cell-autonomous pathways, suggesting potential therapeutic strategies. Further research is needed to investigate underlying mechanisms, including immunological involvement.

Journal reference:

• Feng, Y. et al. (2023) "Hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer", Journal of Experimental Medicine, 220(11). doi: 10.1084/jem.20230011. https://rupress.org/jem/article/220/11/e20230011/276202/