ID OF MOLECULES THAT SUBSTITUTE FOR ACTIVE NOTCH1 IN T LINEAGE LEUKEMOGENESIS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 NOTCH 1编码一种跨膜受体，对T细胞发育至关重要，在约55%的人T系急性淋巴细胞白血病（T-ALL）病例中发生突变。 Notch 1蛋白的激活需要γ-分泌酶蛋白酶的切割，其释放Notch 1胞内结构域（NICD），然后易位到细胞核并上调转录。 Notch 1的突变形式是γ-分泌酶依赖性的，并且已经开发了几种γ-分泌酶抑制剂（GSI）。 我们在Mx 1-cre; LSL-KrasG 12 D小鼠中利用逆转录病毒插入突变（RIM）产生了一大批遗传和生化多样性T-ALL。 在该模型中，Notch 1频繁突变，并且可以使用识别仅在γ-分泌酶切割后暴露的瓦尔1477的特异性抗体来检测NICD。 我们已经在20种肿瘤衍生细胞系中彻底询问了Notch 1依赖性，发现对GSI的敏感性与NICD的存在相关，并且GSI治疗降低了NCID水平。 这项工作目前正在PNAS出版。 有趣的是，我们鉴定了3种细胞系，它们强烈表达由NICD特异性抗体识别的蛋白质，但具有GSI抗性。 这种蛋白质在GSI暴露后不会消失。 进一步的分析显示，这些细胞系不表达Notch 1 mRNA或具有Notch 1靶基因的上调，表明Notch 1未被激活。 重要的是，我们从未检测到这种蛋白质，并希望使用质谱法来确定其身份。 未来的实验将涉及1）使用shRNA敲低T-ALL细胞中的蛋白质，以评估其在肿瘤发生中的作用，以及2）筛选人类T-ALL患者样本中编码该蛋白质的基因突变。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. NOTCH1 encodes a transmembrane receptor that is critical for T cell development and is mutated in ~55% of human T lineage acute lymphoblastic leukemia (T-ALL) cases. Activation of the Notch1 protein requires cleavage by the gamma-secretase protease, which releases the Notch1 intracellular domain (NICD) that then translocates to the nucleus and upregulates transcription. Mutated forms of Notch1 are gamma-secretase dependent and several gamma-secretase inhibitors (GSIs) have been developed. We harnessed retroviral insertional mutagenesis (RIM) in Mx1-cre; LSL-KrasG12D mice to generate a large panel of genetically and biochemically diverse T-ALLs. In this model, Notch1 is frequently mutated and NICD can be detected using a specific antibody that recognizes Val 1477 that is exposed only after gamma-secretase cleavage. We have thoroughly interrogated Notch1 dependence in 20 tumor-derived cell lines and found that sensitivity to GSI correlates with the presence of NICD and GSI treatment reduces NCID levels. This work is currently in press at PNAS. Interestingly, we identified 3 cell lines that robustly express a protein recognized by the NICD specific antibody, but are GSI-resistant. This protein does not disappear following GSI exposure. Further analysis revealed that these lines do not express Notch1 mRNA or have upregulation of Notch1 target genes, indicating that Notch1 is not activated. Importantly, we never detect this protein and would like to use mass spectrometry to determine its identity. Future experiments would involve 1) using shRNA to knockdown the protein in T-ALL cells to assess its role in tumorigenesis, and 2) screening human T-ALL patient samples for mutations in the gene that encodes this protein.