Pan-Cancer MLL3 Mutational Landscape and Its Association with Immunotherapy

Translocation-linked Mixed Lineage Leukemia (MLL) genes were discovered ~ 30 years ago and are homologs of Trithorax in Drosophila. MLL3 is a histone methyltransferase catalyzing the monomethylation of histone H3 lysine 4 (H3K4me1), a hallmark for gene enhancers. Although recurrent mutations of MLL3 are reported across a broad spectrum of cancers and MLL3 represents one of the most commonly mutated cancer genes, a pan-cancer-wide portrait of this high-frequency mutational event is still lacking. We report here a comprehensive analysis of MLL3 mutation and its association with molecular, cellular, and clinical features in 33 cancer types. The genetic alterations of MLL3 are primarily driven by point mutations or small INDELs rather than large-scale chromosomal perturbations and exhibit lineage-specific variations, which spontaneously yet heterogeneously impinge on the clinical behaviors of cancers and are intrinsically linked to hot tumor microenvironments, especially in uterine corpus endometrial carcinoma (UCEC) and colon adenocarcinoma (COAD), where MLL3 aberrances surprisingly predict better overall survivals and favorable responses to immunochemotherapy. Concurrent mutation of MLL3 and POLE in UCEC is associated with high immune cell infiltration and immune checkpoint gene expression, endowing the applicability of immune checkpoint inhibitors in these patients. Mouse models with Mll3-ablated colon cancer validate the immunostimulatory trait of Mll3 aberration. Our study provides a pan-cancer spectrum of the MLL3 mutational event and will contribute to the understanding of the genetic evolution and disease management of cancers, especially COAD and UCEC. Overall design: Mll3WT and Mll3KO CT26 cells for RNA-seq and CUT&Tag, and tumor tissues for single-cell RNA-seq

易位相关混合谱系白血病（Translocation-linked Mixed Lineage Leukemia, MLL）基因于约30年前被发现，是果蝇中三胸基因（Trithorax）的同源基因。MLL3为组蛋白甲基转移酶，可催化组蛋白H3赖氨酸4的单甲基化修饰（H3K4me1），该修饰是基因增强子的标志性特征。尽管已有研究在广泛的癌症谱系中报道了MLL3的复发性突变，且MLL3属于最常见的突变致癌基因之一，但目前仍缺乏针对该高频突变事件的泛癌全景分析。本研究对33种癌症类型中的MLL3突变及其与分子、细胞及临床特征的关联开展了全面分析。MLL3的遗传改变主要由点突变或小片段插入缺失（insertions and deletions, INDELs）驱动，而非大规模染色体畸变，且呈现谱系特异性差异；这些改变会自发且异质性地影响癌症的临床行为，并与热肿瘤微环境存在内在关联，尤其在子宫体子宫内膜癌（Uterine Corpus Endometrial Carcinoma, UCEC）和结肠腺癌（Colon Adenocarcinoma, COAD）中，MLL3异常竟可预测更优的总生存期以及对免疫化疗的良好响应。在子宫体子宫内膜癌中，MLL3与DNA聚合酶ε（POLE）的共突变与高免疫细胞浸润及免疫检查点基因表达水平升高相关，为该类患者应用免疫检查点抑制剂提供了理论依据。敲除Mll3的小鼠结肠癌模型验证了Mll3异常的免疫刺激特性。本研究构建了MLL3突变事件的泛癌图谱，将有助于推动癌症的遗传演化研究与疾病管理，尤其针对结肠腺癌和子宫体子宫内膜癌。整体实验设计：用于RNA测序（RNA-seq）和CUT&Tag实验的Mll3野生型（Mll3WT）与Mll3基因敲除（Mll3KO）CT26细胞，以及用于单细胞RNA测序（single-cell RNA-seq）的肿瘤组织。