The SAGA complex maintains the oncogenic gene expression program in MYCN-amplified neuroblastoma [ChIP-Seq]

Pediatric cancers are frequently driven by fusion or amplification events that result in aberrant transcription factor activity. As transcription factors themselves remain challenging to target, an emerging therapeutic approach for these cancers is to target epigenetic complexes that help maintain oncogenic transcriptional programs. It is therefore critical to identify the complete set of epigenetic modulators maintaining the oncogenic epigenetic landscape of pediatric cancers. Here, we used functional genomic screens to identify epigenetic complexes critical for viability in cell line models of MYCN-amplified neuroblastoma, a disease of dysregulated development driven by an aberrant oncogenic transcriptional program. We identified multiple genes within the transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex as selective dependencies in MYCN-amplified neuroblastoma. Integrating ChIP-seq, ATAC-seq, and RNA-seq with targeted protein-degradation and gene editing tools, we characterized the DNA recruitment sites of the SAGA complex in neuroblastoma, and the consequences of SAGA complex lysine acetyltransferase (KAT) activity loss on histone acetylation and gene expression. We demonstrate that loss of SAGA KAT activity suppresses MYC and MYCN gene expression programs and impairs cell cycle progression. Further, we showed that the SAGA complex is pharmacologically targetable with a KAT2A/KAT2B proteolysis targeting chimera molecule that demonstrated significant activity in vitro and in vivo. Our findings expand our understanding of the histone modifying complexes that maintain the oncogenic transcriptional state in this disease and suggest therapeutic potential for inhibitors of SAGA KAT activity in MYCN-amplified neuroblastoma. Overall design: Investigate alterations in TADA2B binding after TADA2B loss (degradation dTAG v1 TADA2B vs. DMSO at 6 hours) in KELLY, NGP and NB1 neuroblastoma cells, one replicate per condition. Investigate alterations in MYCN, H3K27ac and H3K9ac binding after TADA2B loss (degradation dTAG v1 TADA2B vs. DMSO at 6 hours) in KELLY neuroblastoma cells, one replicate per condition. Investigate alterations in MYCN, H3K27ac and H3K9ac binding after KAT2A/KAT2B targeting (PROTAC GSK-699-1 vs. DMSO at 6 hours) in KELLY neuroblastoma cells, two replicates per condition.

儿童癌症通常由融合或扩增事件驱动，导致转录因子活性异常。由于转录因子本身仍难以靶向，针对此类癌症的新兴治疗策略是靶向有助于维持致癌转录程序的表观遗传复合物。因此，鉴定维持儿童癌症致癌表观遗传景观的完整表观遗传调控因子集合至关重要。
本研究通过功能基因组筛选，在MYCN扩增型神经母细胞瘤的细胞系模型中鉴定对细胞存活至关重要的表观遗传复合物。MYCN扩增型神经母细胞瘤是一类由异常致癌转录程序驱动的发育失调疾病。我们在转录辅激活因子Spt-Ada-Gcn5-乙酰转移酶（Spt-Ada-Gcn5-acetyltransferase, SAGA）复合物中鉴定出多个基因，它们是MYCN扩增型神经母细胞瘤的选择性依赖因子。
我们将染色质免疫共沉淀测序（ChIP-seq）、转座酶可及性测序（ATAC-seq）及RNA测序（RNA-seq）数据与靶向蛋白质降解、基因编辑工具相结合，解析了SAGA复合物在神经母细胞瘤中的DNA招募位点，以及SAGA复合物赖氨酸乙酰转移酶（lysine acetyltransferase, KAT）活性缺失对组蛋白乙酰化与基因表达的影响。
研究证实，SAGA KAT活性缺失会抑制MYC与MYCN基因表达程序，并损害细胞周期进程。进一步实验表明，SAGA复合物可通过KAT2A/KAT2B蛋白降解靶向嵌合体（proteolysis targeting chimera, PROTAC）分子实现药理学靶向，该分子在体外与体内均展现出显著活性。
本研究的发现深化了我们对维持该疾病致癌转录状态的组蛋白修饰复合物的认知，并提示SAGA KAT活性抑制剂在MYCN扩增型神经母细胞瘤中的治疗潜力。
实验整体设计：
1. 在KELLY、NGP及NB1神经母细胞瘤细胞中，探究TADA2B缺失（dTAG v1 TADA2B降解 vs. 6小时DMSO处理）后TADA2B结合的变化，每组设置1个生物学重复。
2. 在KELLY神经母细胞瘤细胞中，探究TADA2B缺失（dTAG v1 TADA2B降解 vs. 6小时DMSO处理）后MYCN、H3K27ac及H3K9ac结合的变化，每组设置1个生物学重复。
3. 在KELLY神经母细胞瘤细胞中，探究靶向KAT2A/KAT2B（PROTAC GSK-699-1 vs. 6小时DMSO处理）后MYCN、H3K27ac及H3K9ac结合的变化，每组设置2个生物学重复。