Somatic IRF4 mutations drive disease-defining expression signatures and thymic tropism in primary mediastinal large B-cell lymphoma [RNA-seq]

Disease-defining signatures in lymphomas, driven by intricate molecular mechanisms, have advanced molecular taxonomies, refined classification, and may guide clinical management; however, the role of these signatures in driving disease hallmarks including subtype-specific organotropism remains largely unexplored. Primary mediastinal large B-cell lymphoma (PMBCL) is an exemplary lymphoma characterized by disease manifestations in the thymic niche, unique genetic alterations and immune escape. Here, we identified IRF4-C99R mutations uniquely occurring in PMBCL through mutational meta-analysis of large-scale datasets. Our functional studies, integrating multi-omics approaches with genome editing in PMBCL cells, revealed that IRF4-C99R contributes to a differentiation block phenotype. Specifically, we showed that IRF4-C99R reduces its binding to the ISRE motif within PRDM1, which encodes a key transcriptional regulator of B-cell differentiation, resulting in decreased PRDM1 expression. Additionally, IRF4-C99R suppresses TNIK, a key IFNγ pathway regulator, by impairing ISRE motif binding, thereby reducing IFNγ signaling and increasing thymus and activation-regulated chemokine (TARC) expression, which drives TARC-mediated chemotaxis of T regulatory cells. We also revealed that IRF4-C99R upregulates Ephrin Type-B Receptor 1 (EPHB1) through non-canonical AICE motif binding, and showed that overexpression of EPHB1 in an immunocompetent syngeneic lymphoma model influenced organotropism to favor thymic localization, without affecting tumor burden in other organs. IRF4-C99R mutation-induced phenotypes were validated in primary PMBCL tissues using single-nuclei RNA sequencing, confirming that the molecular mechanisms observed in vitro align with the pathophysiology of PMBCL in patients. Together, these findings demonstrate how a single genetic mutation orchestrates the coordinated regulation of hallmark traits including thymus-specific tropism in PMBCL. RNA-seq profiling of DLBCL-derived cell line DOHH2 ectopically expressing WT IRF4 or IRF4-C99R, and IRF4-C99R-mutated PMBCL-derived cell lines U-2940 and Karpas-1106p, untreated or stimulated with Ephrin-B2.

由复杂分子机制驱动的淋巴瘤疾病特征（disease-defining signatures），推动了分子分类学的进步，优化了淋巴瘤分型方案，并可为临床管理提供指导；然而，此类特征在驱动疾病标志性表型（包括亚型特异性器官嗜性（organotropism））方面的作用，仍在很大程度上未被探索。原发性纵隔大B细胞淋巴瘤（Primary mediastinal large B-cell lymphoma, PMBCL）是一类典型淋巴瘤，以病变定位于胸腺微环境、携带独特遗传改变及免疫逃逸表型为主要特征。本研究通过对大规模数据集进行突变荟萃分析，鉴定出仅在PMBCL中特异性出现的IRF4-C99R突变。我们的功能研究将多组学（multi-omics）分析策略与PMBCL细胞的基因组编辑技术相结合，证实IRF4-C99R可诱导分化阻滞表型。具体而言，实验证实IRF4-C99R会减弱其与PRDM1基因内含有的ISRE基序的结合能力——PRDM1编码B细胞分化的关键转录调控因子——最终导致PRDM1的表达水平下调。此外，IRF4-C99R通过损害ISRE基序结合，抑制关键IFNγ通路调控因子TNIK，从而削弱IFNγ信号通路活性，并上调胸腺活化调节趋化因子（thymus and activation-regulated chemokine, TARC）的表达，进而介导调节性T细胞的趋化迁移。我们还发现，IRF4-C99R可通过非经典AICE基序结合上调Ephrin B型受体1（Ephrin Type-B Receptor 1, EPHB1）；进一步研究证实，在免疫健全的同基因淋巴瘤模型中过表达EPHB1，可促进肿瘤的胸腺定向嗜性，而不会对其他器官的肿瘤负荷产生影响。我们利用单细胞核RNA测序（single-nuclei RNA sequencing）技术，在原发性PMBCL组织中验证了IRF4-C99R突变诱导的表型，证实体外实验观察到的分子机制与患者体内PMBCL的病理生理学特征相符。综上，本研究阐明了单个遗传突变如何协同调控PMBCL的多种标志性特征，其中包括胸腺特异性嗜性。本数据集包含以下样本的RNA测序（RNA-seq）谱数据：弥漫性大B细胞淋巴瘤（DLBCL）来源细胞系DOHH2，该细胞系分别异位过表达野生型IRF4或IRF4-C99R；以及携带IRF4-C99R突变的PMBCL来源细胞系U-2940与Karpas-1106p，上述细胞均经Ephrin-B2刺激或未接受处理。