Graduation Date

Winter 12-17-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cancer Research

First Advisor

Javeed Iqbal

Abstract

Peripheral T-cell lymphoma (PTCL) is heterogenous group of mature T-cell neoplasms characterized by distinctive transcriptional and genetic lesions. Herein, we investigated DNMT3A mutations in angioimmunoblastic T-cell lymphoma (AITL, n = 176) and novel molecular subtypes (i.e., PTCL-GATA3, n = 61 and PTCL-TBX21, n = 80) within PTCL- NOS and observed significant biological and prognostic differences associated with DNMT3A mutations. DNMT3A-mutated PTCL-TBX21 cases showed inferior overall survival (OS; p < 0.005), with DNMT3A mutations (DNMT3A-MT) skewed toward the methyltransferase domain and in the dimerization domain (S881-R887). Transcriptional profiling demonstrated significant enrichment of activated CD8+ T-cell cytotoxic gene signatures in the tumor microenvironment of DNMT3A-MT PTCL-TBX21 cases. Genome-wide methylation analysis of DNMT3A-R882/Q886 versus wild-type in PTCL-TBX21 cases demonstrated hypomethylation in target genes regulating T-cytotoxic genes, TCR signaling, and correlated with EOMES expression, a master transcriptional regulator of cytotoxic T-cells. We further validated these observations in vitro by ectopic expression of DNMT3A-MTs (R882H, Q886Stop, V716D) in a CD8+ T-cell line resulting in TCR activation and EOMES upregulation. Furthermore, stable ectopic expression of the DNMT3A mutants in primary CD3+ T-cell cultures resulted in the preferential outgrowth of CD8+ T-cells in cells with DNMT3A-R882H mutation. Sc-RNA-seq analysis of these CD3+ T-cells validated the cytotoxic transcriptional program associated with DNMT3A-R882H. With the tumor microenvironment and TCR signaling playing vital roles in PTCL prognosis, we incorporated murine models to mimic loss-of-function in previously described TCR regulators (i.e., CAV1 and DNMT3A). For CAV1, we observed that Cav1-deficiency, but not heterozygosity, is associated with persistent splenomegaly and that both aged Cav1+/- and Cav1-/- mice present with decreased CD19+CD22+ B cells and secondary-follicle atrophy. For DNMT3A, we observed that Dnmt3a-/- in Tet2-/--IDH2R172K-derived tumors resulted in a more aggressive disease featuring CD3+ T-cell rich tumors with gene expression profiles indicating enhanced T-cell activation. In summary, our findings indicate that DNMT3A mutations define a cytotoxic subset in PTCL-TBX21 with prognostic significance, and that further insight into the microenvironmental regulators in PTCL-NOS may provide insight on alternative treatment strategies for PTCL-NOS patients.

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