FoxA1/2-dependent epigenomic reprogramming drives lineage switching in lung adenocarcinoma [Bisulfite-Seq]. FoxA1/2-dependent epigenomic reprogramming drives lineage switching in lung adenocarcinoma [Bisulfite-Seq]

The ability of cancer cells to alter their identity is essential for tumor survival and progression. Loss of the pulmonary lineage specifier NKX2-1 within KRAS-driven lung adenocarcinoma (LUAD) enhances tumor progression and results in a pulmonary-to-gastric lineage switch that is dependent upon the activity of pioneer factors FoxA1 and FoxA2; however, the underlying mechanism remains largely unknown. Here, we show that FoxA1/2 reprogram the epigenetic landscape of NKX2-1-negative LUAD to facilitate a gastric differentiation program. Using sequential recombination models, we find that FoxA1/2 are required for demethylation of gastric-defining genes after Nkx2-1 deletion. FoxA1 colocalizes with TET3, an enzyme that mediates DNA demethylation, in NKX2-1-negative tumors. Deletion of Foxa1/2 results in loss of TET3 occupancy at key gastric marker genes, indicating that FoxA1/2 recruit TET3 to lineage-specific sites. H3K27ac ChIP-seq and HiChIP show that FoxA1/2 also control the activity of enhancers and promoters as well as their 3D interactions at gastric target genes following NKX2-1 loss. Furthermore, oncogenic KRAS is required for the FoxA1/2-dependent epigenetic reprogramming in NKX2-1-negative LUAD. This work demonstrates the role of FoxA1/2 in rewiring the methylation and histone landscape and cis-regulatory dynamics of NKX2-1-negative LUAD to drive cancer cell lineage switching. Overall design: To identify the FoxA1/2- and KRAS-dependent methylation changes, enzymatic methyl-seq was performed on sorted GFP+, DAPI+ tumor nuclei from each GEMM (K, KN, KNF1F2, N) To determine whether FoxA1/2 were required to maintain methylation patterns in a gastric differentiation program, enzymatic methyl-seq was performed on 3D organoid lines, KG1A and 22E, in the presence and absence of FoxA1/2.

癌细胞改变自身身份的能力，对于肿瘤的存活与进展至关重要。在KRAS驱动的肺腺癌（LUAD）中，肺谱系特异性因子NKX2-1的缺失会促进肿瘤进展，并引发依赖于先驱因子FoxA1与FoxA2活性的肺-胃谱系转换；然而，其背后的分子机制仍未被完全阐明。本研究证实，FoxA1/2可对NKX2-1阴性LUAD的表观遗传图谱进行重编程，以促进胃系分化程序的执行。通过连续重组模型，我们发现Nkx2-1缺失后，胃系特征基因的去甲基化过程依赖于FoxA1/2的存在。在NKX2-1阴性的肿瘤中，FoxA1与介导DNA去甲基化的酶TET3存在共定位现象。敲除Foxa1/2会导致TET3在关键胃系标记基因上的结合丢失，这表明FoxA1/2可将TET3招募至谱系特异性位点。H3K27ac染色质免疫共沉淀测序（ChIP-seq）与高阶染色质构象捕获测序（HiChIP）结果显示，NKX2-1缺失后，FoxA1/2还可调控胃系靶基因的增强子、启动子活性及其三维空间相互作用。此外，在NKX2-1阴性LUAD中，依赖FoxA1/2的表观遗传重编程过程需要致癌性KRAS的参与。本研究阐明了FoxA1/2如何重塑NKX2-1阴性LUAD的甲基化组、组蛋白修饰图谱以及顺式调控动态，从而推动癌细胞的谱系转换。
实验整体设计：为鉴定FoxA1/2与KRAS依赖的甲基化变化，我们对来自各基因工程小鼠模型（GEMM，包括K、KN、KNF1F2、N组）的分选GFP阳性、DAPI阳性肿瘤细胞核进行了酶解法甲基化测序（enzymatic methyl-seq）。为明确FoxA1/2是否在胃系分化程序中维持甲基化模式所必需，我们分别在FoxA1/2存在与缺失的情况下，对3D类器官系KG1A与22E进行了酶解法甲基化测序。