ASCL1 represses a latent osteogenic program in small cell lung cancer in multiple cells of origin [ChIP-Seq]

ASCL1 is a neuroendocrine-lineage-specific oncogenic driver of small cell lung cancer (SCLC), highly expressed in a significant fraction of tumors. However, ~25% of human SCLC are ASCL1-low and associated with low-neuroendocrine fate and high MYC expression. Using genetically-engineered mouse models (GEMMs), we show that alterations in Rb1/Trp53/Myc in the mouse lung induce an ASCL1+ state of SCLC in multiple cell types. Genetic depletion of ASCL1 in MYC-driven SCLC dramatically inhibits tumor initiation, but surprisingly converts tumors to a SOX9+ mesenchymal/neural-crest-stem-like state that has the capacity to differentiate into RUNX2+bone tumors. ASCL1 represses SOX9 expression, as well as WNT and NOTCH developmental pathways, consistent with human gene expression data. SCLC demonstrates remarkable cell fate plasticity with ASCL1 repressing the emergence of non-endodermal stem-like fates that have the capacity for bone differentiation. Here, we perform ChIP on RPM tumors against ASCL1, NEUROD1, and H3k27Ac to view binding profiles and identify target genes of ASCL1 and NEUROD1. Overall design: Individual lung tumors from mice were micro-dissected, flash frozen and stored at -80°C until chromatin immunoprecipitation

ASCL1是小细胞肺癌（small cell lung cancer, SCLC）的神经内分泌谱系特异性致癌驱动因子，在相当比例的肿瘤中呈高表达。然而约25%的人类SCLC为ASCL1低表达亚型，伴随低神经内分泌分化表型及高MYC表达。本研究借助基因工程小鼠模型（genetically-engineered mouse models, GEMMs）证实，小鼠肺部Rb1、Trp53与Myc的遗传改变可在多种细胞类型中诱导出SCLC的ASCL1阳性状态。在MYC驱动的SCLC中敲除ASCL1可显著抑制肿瘤起始，但出人意料的是，会将肿瘤转化为SOX9阳性的间充质/神经嵴干细胞样表型，该表型具备分化为RUNX2阳性骨源性肿瘤的能力。ASCL1可抑制SOX9的表达，以及WNT与NOTCH发育通路，这与人类基因表达数据相一致。SCLC具有显著的细胞命运可塑性，ASCL1可抑制具备骨分化能力的非内胚层干细胞样表型的出现。本研究针对RPM肿瘤开展染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验，靶向ASCL1、NEUROD1及H3K27Ac，以解析结合谱并鉴定ASCL1与NEUROD1的靶基因。实验整体设计：将小鼠的单个肺部肿瘤进行显微解剖、快速冷冻，并保存于-80℃直至开展染色质免疫沉淀实验。