DNMT3A Mutations Identify a Prognostic Subgroup in Peripheral T-Cell Lymphoma

Background

Mutational profiling of angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) has revealed recurrent mutations in DNMT3A, a de novo methyltransferase. DNMT3A catalyzes the conversion of cytosine to 5-methylcytosine (5-mC) while interacting with histones and transcription factors to influence gene expression. While the DNMT3A mutational profile in PTCL entities indicates loss-of-function, hotspot change-of-function mutations (e.g., DNMT3A^R882H/C) have been observed with their frequency differing between PTCL entities. Despite the high occurrence of DNMT3A mutations in PTCLs (~30% of cases), their functional consequences have not been extensively studied. Herein, we examined DNMT3A mutations in AITL and the novel molecular subgroups of PTCL-NOS (i.e., PTCL-TBX21 and PTCL-GATA3) and observed distinct biological and prognostic significance associated with DNMT3A mutations in the PTCL-TBX21 subgroup.

Methods

PTCL-NOS cases (n = 141) were utilized following PTCL-T_FH exclusion. Using previously described molecular classification methods, cases were classified as PTCL-TBX21 (n = 80) or PTCL-GATA3 (n = 61). A separate cohort of AITL cases (n = 176) were included for comparative purposes. Clinical outcome data were assessed with the Kaplan-Meier method. Mutation data were generated from DNA-sequencing (n = 224) or RNA-sequencing methods (n = 46). Gene expression comparisons were conducted using BRB-ArrayTools. Immune-cell signatures were generated from the CIBERSORT and/or xCell computational tools. 5-mC DNA immunoprecipitation sequencing (MeDIP-Seq) was performed on available PTCL-TBX21 cases (n = 7) or healthy tonsil controls (n = 2). Four of these cases carried DNMT3A mutations (n = 3 DNMT3A^R882, n = 1 DNMT3A^Q886) while the remaining cases (n = 3) were wild type for DNMT3A. In vitro analyses of ectopic expression of the DNMT3A^R882H mutant or DNMT3A knockdown were conducted using healthy-donor CD3⁺ T-cells or the CD8⁺ T8ML1 PTCL cell line. Following corrections for false discoveries, p-values < 0.05 were considered significant.

Results

DNMT3A-mutated PTCL-TBX21 cases had an inferior overall survival, with DNMT3A mutated residues skewed toward the methyltransferase domain. In contrast to the DNMT3A mutation profile seen in AITL, PTCL-TBX21 featured DNMT3A^R882H/C mutations at a frequency (30%) similar to other hematological malignancies. Gene expression profiling revealed that DNMT3A-mutant PTCL-TBX21 cases were enriched for activated CD8⁺ T-cell gene signatures and showed association with the previously described T_H1/αβ cytotoxic T-cell lymphoma subgroup. Following MeDIP-Seq, assessment of differentially methylated regions comparing DNMT3A^R882/Q886 PTCL-TBX21 cases to wild type found hypomethylation in pathways associated with T-cell activation, TCR signaling, and T_H1 responses. In vitro analyses demonstrated that ectopic expression of the DNMT3A^R882H mutant or DNMT3A knockdown lead to enhanced proliferation and NF-κB signaling in T8ML1 cells in comparison to control vectors. In primary CD3⁺ T-cell cultures, ectopic expression of the DNMT3A^R882H mutant protein resulted in the preferential outgrowth of CD8⁺ T-cells.

Conclusions

Taken together, our findings establish mutations in DNMT3A as a novel prognostic marker in PTCL-TBX21. The integrated expression, methylation, and in vitro findings suggest that disruption of DNMT3A leads toward an activated and cytotoxic phenotype and could potentially drive oncogenic TCR signaling. Clinically, as these cases were associated with the T_H1/αβ cytotoxic T-cell lymphoma subgrouping, these findings should be taken into consideration for future treatment strategies regarding PTCL-NOS patients as current standard-of-care treatments may be particularly inadequate in the treatment of PTCLs with cytotoxic phenotype.