Graduation Date

Summer 8-18-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cancer Research

First Advisor

Dr. Rene Opavsky, PhD

Second Advisor

Dr. Angie Rizzino, PhD

Abstract

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.

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