Doctor of Philosophy (PhD)
Dr. Rene Opavsky, PhD
Dr. Angie Rizzino, PhD
DNA methyltransferase 3A (DNMT3A) is a master epigenetic regulator of benign and malignant hematopoiesis. To dissect the biological consequences of homozygous and heterozygous Dnmt3a inactivation in malignant hematopoiesis, we generated Dnmt3a homozygous null (Dnmt3aΔ/Δ) and Dnmt3a heterozygous (Dnmt3a+/–) mice and compared the presentations of hematologic malignancies between cohorts. Bi-allelic inactivation of Dnmt3a results in the presentation of mature lymphoid neoplasms resembling chronic lymphocytic leukemia (CLL; B220+CD19+CD5+; 88% penetrance (37/42)) and CD8+ peripheral T-cell lymphoma (PTCL; TCRβ+CD3+CD8+CD4—; 40% penetrance (17/42)). In contrast, mono-allelic inactivation of Dnmt3a results in the presentation of CLL and PTCL at reduced penetrance (47% (14/30) & 10% (3/30), respectively) and, rarely, a mature myeloproliferative neoplasm (MPN; CD11b+Gr-1+; 10% penetrance (3/30)).
Molecular interrogation of PTCLs revealed genome-wide deregulation of DNA methylation, characterized by 10-fold greater hypomethylation than hypermethylation of promoters and enhancers. Transcription factor binding sites for AML1, NF-κB, and OCT1 were enriched in hypomethylated promoters, implicating these transcription factors in tumor pathogenesis or DNMT3A-associated DNA methylation. Whereas 71 hypomethylated genes showed an increased expression in PTCL, only 3 hypermethylated genes were silenced, suggesting cancer-specific hypomethylation more frequently affects the transcriptome than hypermethylation in lymphoma. Importantly, we observed in Dnmt3a-deficient PTCLs the downregulation of p53 protein by western blot and p53 target genes by gene set enrichment analysis. Decreased p53 protein expression occurred in pre-tumor thymocytes of 9 months old, but not 6 weeks old, Dnmt3a+/– disease-free mice, demonstrating that p53 downregulation occurs an intermediate event in tumorigenesis. Analysis of Dnmt3a+/– tumors revealed that PTCL develops without mutation or silencing of the remaining wild-type Dnmt3a allele. These data demonstrate that Dnmt3a is a haploinsufficient tumor suppressor in murine mature CD8+ PTCL and loss of p53 protein occurs as an intermediate event in tumorigenesis.
To better understand the dysregulated epigenetic events favoring the development of CLL and PTCL from B-1a and CD8+ cells in Dnmt3aΔ/Δ mice, we compared the methylomes and transcriptomes of normal B-1a and CD8+ T-cells in addition to comparison of malignant CLL and PTCL cells. We observe that whereas patterns of methylation and transcription in normal B-1a cells and CD8+ T cells are similar, methylomes and transcriptomes in malignant B-1a and CD8+ T cells are remarkably distinct, suggesting a cell-type specific function for Dnmt3a in cellular transformation.
Upchurch, Garland Michael, "DNMT3A Haploinsufficiency Provokes Hematologic Malignancy of B-Lymphoid, T-Lymphoid, and Myeloid Lineage in Mice" (2017). Theses & Dissertations. 220.
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