The Impact of Nucleolar Stress on Thymocyte Development and T Cell Acute Lymphoblastic Leukemia Transformation

项目摘要/摘要 T-ALL占儿童急性淋巴细胞白血病的10%-15%，占成人急性淋巴细胞白血病的20%-25%。正在激活 NOTCH1突变存在于约60%的T-ALL病例中，并被认为诱导白血病转化， 部分是通过激活c-Myc(Myc)。MYC诱导核糖体生物合成，触发核仁应激 路径。这是一条进化上保守的途径，包括核糖体蛋白RPL5， RPL11和RPL22与核质结合并改变p14ARF(ARF)和MDM2的活性， 这反过来激活了P53，导致生长停滞。为了克服这种负反馈循环，超过80%的T- 它们都带有CDKN2A的双等位基因缺失(编码P16INK4A和ARF)。此外，约17%的T-ALL携带 RPL5、RPL11或RPL22的突变。基于这一遗传证据，我们假设Notch信号 在T-ALL中诱导核仁应激，而核仁应激途径的失活是 T-ALL的分子发病机制。为了检验这一假设，我们提出了以下具体目标。 目的1.确定在正常T细胞发育过程中核仁应激的诱导 选择携带CDKN2A突变的克隆。Notch信号在正常早期T-T细胞中起重要作用 细胞发育。我们假设，在T细胞发育早期诱导核仁应激选择 携带CDKN2A(或RPL基因)突变的T细胞克隆。为了验证这一假设，我们将分离T细胞 野生型小鼠的祖细胞/前体细胞，并分析核仁压力的迹象。我们还将生成Arf 或Ink4a缺乏混合骨髓嵌合体，并评估供者嵌合体。我们预测核仁压力 在T细胞中，祖细胞/前体细胞将导致T细胞谱系中Arf缺陷细胞的选择性扩增。 目的2.确定激活Notch1突变是否会引起T细胞的核仁应激 祖先。我们将评估在T-ALL中发现的NOTCH1突变的强制表达对 使用MOLM13细胞系和原代小鼠T细胞的核糖体生物发生和核仁应激的诱导。 在平行研究中，我们还将下调NOTCH1在几个T-ALL异种移植中的表达。在每一种情况下， 将对核仁应力进行评估

Project Summary/Abstract T-ALL represents 10-15% of pediatric and 20-25% of adult cases of acute lymphoblastic leukemia. Activating mutations of NOTCH1 are present in ~60% of cases of T-ALL and are thought to induce leukemic transformation, in part, by activating c-Myc (Myc). Myc induces ribosome biogenesis, which can trigger the nucleolar stress pathway. This is an evolutionarily conserved pathway that includes the translocation of ribosome proteins, RPL5, RPL11, and RPL22 to the nuclear cytoplasm where they bind and alter the activity of p14ARF (ARF) and MDM2, which in turn activates p53, inducing a growth arrest. To overcome this negative feedback loop, over 80% of T- ALL carry biallelic deletions of the CDKN2A (encoding for P16INK4A and ARF). In addition, ~17% of T-ALL carry mutations of RPL5, RPL11, or RPL22. Based on this genetic evidence, we hypothesize that Notch signaling induces nucleolar stress in T-ALL and that inactivation of the nucleolar stress pathway is essential to the molecular pathogenesis of T-ALL. The following specific aims are proposed to test this hypothesis. Aim 1. To determine whether induction of nucleolar stress during normal T-cell development selects for clones carrying mutations in CDKN2A. Notch signaling plays an essential role in normal early T- cell development. We hypothesize that induction of nucleolar stress during early T-cell development selects for T-cell clones carrying mutations of CDKN2A (or RPL genes). To test this hypothesis, we will isolate T-cell progenitors/precursors from wildtype mice and analyze for signs of nucleolar stress. We also will generate Arf or Ink4a deficient mixed bone marrow chimeras and assess donor chimerism. We predict that nucleolar stress in T-cell progenitors/precursors will result in the selective expansion of Arf deficient cells in the T-cell lineage. Aim 2. To determine whether activating Notch1 mutations induce nucleolar stress in T cell progenitors. We will assess the impact of the enforced expression of NOTCH1 mutations found in T-ALL on ribosome biogenesis and induction of nucleolar stress using the MOLM13 cell line and primary murine T-cells. In parallel studies, we also will knock-down NOTCH1 expression in several T-ALL xenografts. In each case, nucleolar stress will be assessed