Gene expression analyses of GFP+ sorted, lineage depleted (Lin-) bone marrow (BM) cells from wild-type or VavCre+β-Catenin-KO mice infected with pMIG-GFP (control) or pMIG-N1IC-GFP retrovirus.. Mus musculus

Notch activation is instrumental in the development of most T-cell acute lymphoblastic leukemia (T-ALL) cases, yet Notch mutations alone are not sufficient to recapitulate the full human disease in animal models. Using multiple in vivo and in vitro T-ALL models we here demonstrate that β-Catenin is essential for Notch-driven T-cell leukemic initiation. Transcriptome analyses of leukemic initiating cells revealed a switch in β-Catenin activity that was Notch-context dependent. Moreover, ChIP-seq coupled with RNA-Seq in human Notch-active T-ALL showed that leukemic β-Catenin was independent of canonical LEF/TCF partners, and instead depended on direct association with Notch or ZBTB33/Kaiso for gene activation. The functional relevance of this mechanism is exemplified by the MYC 3´enhancer that requires β-Catenin and Notch1 recruitment to induce MYC expression. Finally, we demonstrate that pharmacological inhibition of β-Catenin with PKF115-584 prevented and partially reverted leukemogenesis induced by active Notch1. These microarray data show the transcriptional activities of N1IC and β-Catenin in wild-type or leukemic initiating cell (LIC) contexts in N1IC-transduced adult Lineage-depleted BM cells. Overall design: Study is based on biological triplicates. Total RNA from 500-2500 transduced BM Lin- GFP+ sorted cells was isolated with RNEasy Mini or Micro kits (QIAgen). RNA quality was assessed with the Bioanalyzer 2100 PicoChip (Agilent Technologies, Palo Alto, CA) and only RNA samples with RIN >6 was used for microarrays. RNAs were amplified using the Ovation™ Pico WTA System (NuGEN Technologies, San Carlos, CA) and sense transcript cDNA (ST-cDNA) was generated using the WT-Ovation™ Exon Module (NuGEN Technologies). After, ST-cDNA was fragmented and labelled with the FL-Ovation™ cDNA Biotin Module V2 (NuGEN Technologies), and the biotinylated cDNA were hybridized to Affymetrix GeneChip® Mouse Gene 1.0 ST arrays as biological triplicates. Following hybridization, the arrays were washed, stained and scanned to generate CEL files for each array. CEL files were analyzed in R (v3.1.2) with the oligo (v1.30.0) and limma (v3.22.4) packages from Bioconductor, data were normalized using RMA (Robust Multi-array Average) and expression values log2-transformed.

Notch激活在大多数T细胞急性淋巴细胞白血病（T-cell acute lymphoblastic leukemia, T-ALL）病例的发生发展中发挥关键作用，但仅靠Notch突变不足以在动物模型中重现完整的人类疾病状态。本研究借助多种体内（in vivo）与体外（in vitro）T-ALL模型，证实β-连环蛋白（β-Catenin）是Notch驱动的T细胞白血病起始过程所必需的分子。对白血病起始细胞的转录组分析显示，β-连环蛋白的活性呈现出依赖于Notch环境的动态转换。此外，对人类Notch活性型T-ALL开展的染色质免疫共沉淀测序（ChIP-seq）结合RNA测序（RNA-Seq）分析表明，白血病相关的β-连环蛋白并不依赖经典的LEF/TCF伴侣蛋白，而是通过与Notch或ZBTB33/Kaiso直接结合来实现基因激活。这一机制的功能相关性可通过MYC 3'增强子得到印证：该增强子需要β-连环蛋白与Notch1的共同募集，才能诱导MYC的表达。最后，本研究证实，使用PKF115-584对β-连环蛋白进行药理学抑制，可有效预防并部分逆转由活性Notch1诱导的白血病发生。

本项微阵列数据展示了在转导了N1IC的成年谱系耗竭骨髓（BM）细胞中，野生型或白血病起始细胞（LIC）环境下N1IC与β-连环蛋白的转录活性。

整体实验设计：本研究基于三次生物学重复的实验设置。从500~2500个经转导的骨髓谱系阴性（Lin-）、GFP阳性的分选细胞中提取总RNA，采用RNEasy Mini或Micro试剂盒（QIAgen）完成RNA提取。使用Agilent 2100生物分析仪（Agilent Technologies, Palo Alto, CA）的PicoChip芯片评估RNA质量，仅RIN值＞6的RNA样本可用于后续微阵列实验。通过"Ovation™ Pico WTA System"（NuGEN Technologies, San Carlos, CA）对RNA进行扩增，并借助"WT-Ovation™ Exon Module"（NuGEN Technologies）生成正义转录cDNA（ST-cDNA）。随后对ST-cDNA进行片段化处理，使用"FL-Ovation™ cDNA Biotin Module V2"（NuGEN Technologies）进行生物素标记，再将生物素化的cDNA与Affymetrix GeneChip® 小鼠基因1.0 ST芯片进行杂交，每组设置三次生物学重复。杂交完成后，对芯片进行洗涤、染色与扫描，生成每张芯片的CEL文件。使用R软件（v3.1.2）中的Bioconductor包oligo（v1.30.0）与limma（v3.22.4）对CEL文件进行分析，数据通过RMA（稳健多阵列平均法，Robust Multi-array Average）进行标准化，并将表达值进行log2转换。