Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE.
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA.
Blood. 2022 Sep 15;140(11):1278-1290. doi: 10.1182/blood.2021015019.
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 (OS), with DNMT3A-mutated residues skewed toward the methyltransferase domain and dimerization motif (S881-R887). Transcriptional profiling demonstrated significant enrichment of activated CD8+ T-cell cytotoxic gene signatures in the DNMT3A-mutant PTCL-TBX21 cases, which was further validated using immunohistochemistry. Genomewide methylation analysis of DNMT3A-mutant vs wild-type (WT) PTCL-TBX21 cases demonstrated hypomethylation in target genes regulating interferon-γ (IFN-γ), T-cell receptor signaling, and EOMES (eomesodermin), 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, and -V716, vs 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 sequencing(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 and thus may further refine pathological heterogeneity in PTCL-NOS and suggest alternative treatment strategies for this subset.
外周 T 细胞淋巴瘤（PTCLs）是异质性 T 细胞肿瘤，常伴有表观遗传失调。我们研究了常见的 PTCL 实体中的新的 DNA 甲基转移酶 3A（DNMT3A）突变，包括血管免疫母细胞性 T 细胞淋巴瘤和在未特指的 PTCL-NOS 中确定的新型分子亚型，指定为 PTCL-GATA3 和 PTCL-TBX21。PTCL-TBX21 中存在 DNMT3A 突变的病例总生存（OS）较差，DNMT3A 突变残基偏向于甲基转移酶结构域和二聚化结构域（S881-R887）。转录谱分析显示，DNMT3A 突变的 PTCL-TBX21 病例中存在显著富集的活化 CD8+ T 细胞细胞毒性基因特征，使用免疫组化进一步验证。DNMT3A 突变型与野生型（WT）PTCL-TBX21 病例的全基因组甲基化分析显示，靶基因的低甲基化可调节干扰素-γ（IFN-γ）、T 细胞受体信号和 EOMES（eomesodermin），EOMES 是细胞毒性效应细胞的主转录调节剂。在具有 Dnmt3a 缺失的 PTCL 小鼠模型（体内）中观察到类似的发现，并通过在 CD8+ T 细胞系中外源性表达 DNMT3A 突变体（DNMT3A-R882、-Q886 和 -V716，与 WT 相比）在体外进一步验证，导致 T 细胞激活和 EOMES 上调。此外，在原代 CD3+ T 细胞培养物中稳定、异位表达 DNMT3A 突变体导致 CD8+ T 细胞的优先生长，而 CD8+ T 细胞的生长具有 DNMT3AR882H 突变。CD3+T 细胞的单细胞 RNA 测序（RNA-seq）分析显示 CD8+ T 细胞亚群极化的差异，与 DNMT3A 突变的 PTCL-TBX21 中的发现相呼应，并验证了与 DNMT3AR882H 突变相关的细胞毒性和 T 细胞记忆转录程序。我们的研究结果表明，DNMT3A 突变定义了 PTCL-TBX21 中的细胞毒性亚群，具有预后意义，因此可能进一步细化 PTCL-NOS 的病理异质性，并为该亚群提出替代治疗策略。
