DOT1L controls NK cell maturation and lineage integrity [RNA-seq D0 & D2]. DOT1L controls NK cell maturation and lineage integrity [RNA-seq D0 & D2]

Epigenetic modifiers are promising targets to improve therapies in patients with cancer. Targeting the methyltransferase DOT1L with small molecule inhibitors has shown promising effects on the control of cancerous cells. However, these small molecules are given systemically and may have a profound impact on non-cancerous cells, such as the cells of the immune system. In the innate immune system, natural killer (NK) cells are a critical subset of cells with important roles in controlling transformed cells and tumour inflammation. Previous studies have shown that NK cells can convert into ILC1-like cells in a TGFβ-rich tumour microenvironment (TME). Additionally, cancer patients with acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL) have increased frequencies of ILC1 cells in peripheral blood mononuclear cells (PBMCs), which show reduced production of proinflammatory cytokines and decreased granzyme B production. In this study, we identify DOT1L as a critical regulator of NK cell activation and lineage integrity. We generated NKp46-conditional DOT1L knockout mice (DOT1L.Ncr1) and observed increased frequencies of ILC1-like cells (CD49b+ CD49a+, CD62L-) and reduced frequencies of NK cells (CD49b+ CD49a-, CD62L+) in these mice. While the absence of DOT1L increases the sensitivity to TGFβ, the increased expression of CD49a is highly intrinsic and only partially dependent on TGFβ signalling. Functionally, the increased presence of ILC1-like cells in the tumour microenvironment of DOT1L.Ncr1 mice leads to decreased tumour control. Our assessment of the transcriptional program reveals alternative uses of transcription factors, such as the AP-1 family or SMAD2/3, to maintain NK cell activation and lineage integrity. Our findings provide evidence for a previously unknown role of DOT1L in NK cell biology, and demonstrate the importance of maintaining NK cell lineage integrity for effective tumour control. These findings have significant implications for the development of improved therapies for cancer or other NK cell-lineage dependent malignancies. Overall design: Splenic NK cells of either WT or DOT1L.Ncr1 mice at steady-state (d0) or 2 days after infection with MCMV (d2) were sorted for NK1.1+ CD3/TCRβ- Ly49H+ cells prior to isolation of RNA

表观遗传调控因子（Epigenetic modifiers）是改善癌症患者治疗方案的极具潜力的靶点。靶向甲基转移酶DOT1L的小分子抑制剂已在控制癌细胞方面展现出良好效果。然而，这类小分子药物需全身给药，可能会对非癌细胞（如免疫细胞）产生显著影响。在先天免疫系统中，自然杀伤（natural killer, NK）细胞是一类关键的细胞亚群，在监视转化细胞及调控肿瘤炎症过程中发挥重要作用。既往研究表明，NK细胞在富含转化生长因子β（transforming growth factor-β, TGFβ）的肿瘤微环境（tumour microenvironment, TME）中可转化为类ILC1细胞。此外，患有急性髓系白血病（acute myeloid leukemia, AML）或慢性淋巴细胞白血病（chronic lymphocytic leukemia, CLL）的癌症患者，其外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）中ILC1细胞的频率升高，这类细胞的促炎细胞因子产生能力以及颗粒酶B（granzyme B）的表达均有所降低。本研究鉴定出DOT1L是调控NK细胞活化及谱系完整性的关键因子。我们构建了NKp46条件性敲除DOT1L的小鼠（DOT1L.Ncr1），并观察到该类小鼠体内类ILC1细胞（CD49b+ CD49a+, CD62L-）的频率升高，而NK细胞（CD49b+ CD49a-, CD62L+）的频率降低。尽管DOT1L缺失会增强细胞对TGFβ的敏感性，但CD49a的表达上调具有高度内在性，且仅部分依赖于TGFβ信号通路。功能实验结果显示，DOT1L.Ncr1小鼠肿瘤微环境中类ILC1细胞的增多会削弱肿瘤控制效果。我们对转录程序的分析揭示了转录因子的另类调控模式：例如利用AP-1家族或SMAD2/3来维持NK细胞的活化状态与谱系完整性。本研究结果证实了DOT1L在NK细胞生物学中此前未被发现的重要作用，并证明维持NK细胞谱系完整性对于实现有效的肿瘤控制至关重要。这些发现为开发更优化的癌症治疗方案或其他NK细胞谱系相关恶性肿瘤的治疗策略提供了重要依据。实验设计概述：分别提取稳态下（d0）或感染小鼠巨细胞病毒（murine cytomegalovirus, MCMV）2天后（d2）的野生型（WT）与DOT1L.Ncr1小鼠的脾脏NK细胞，通过分选获取NK1.1+ CD3/TCRβ- Ly49H+细胞，随后提取RNA。