LAUSR

Peripheral T-cell lymphomas (PTCLs) are heterogenous T-cell neoplasms often associated with epigenetic dysregulation. We investigated de novo DNA methyltransferase 3A (DNMT3A) mutations in common PTCL entities, including angioimmunoblastic T-cell lymphoma and novel molecular subtypes identified within PTCL-not otherwise specified (PTCL-NOS) designated as PTCL-GATA3 and PTCL-TBX21. DNMT3A-mutated PTCL-TBX21 cases showed inferior overall-survival, with DNMT3A mutated residues skewed towards the methyltransferase domain and dimerization motif (S881-R887). Transcriptional profiling demonstrated significant enrichment of activated CD8+ T-cell cytotoxic gene signatures in the DNMT3A-mutantPTCL-TBX21 cases, which was further validated using immunohistochemistry. Genome-wide methylation analysis of DNMT3A-mutantversus wild-type PTCL-TBX21 cases demonstrated hypomethylation in target genes regulating IFN-g, TCR signaling and EOMES, a master transcriptional regulator of cytotoxic effector cells. Similar findings were observed in a murine model of PTCL with Dnmt3a loss (in-vivo) and further validated in-vitro by ectopic expression of DNMT3A-mutants (DNMT3A-R882, -Q886, -V716, versus WT) in CD8+T-cell line, resulting in T-cell 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 with DNMT3AR882H mutation. Single-cell-RNA-seq analysis of CD3+ T-cells revealed differential CD8+ T-cell subset polarization, mirroring findings in DNMT3A-mutated PTCL-TBX21 and validating the cytotoxic and T-cell memory transcriptional programs associated with the DNMT3AR882H mutation. Our findings indicate that DNMT3A mutations define a cytotoxic subset in PTCL-TBX21 with prognostic significance, thus may further refine pathological heterogeneity in PTCL-NOS and suggest alternative treatment strategies for this subset.