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

项目总结/摘要 T-ALL占儿童急性淋巴细胞白血病病例的10-15%和成人急性淋巴细胞白血病病例的20-25%。激活 NOTCH 1的突变存在于约60%的T-ALL病例中并且被认为诱导白血病转化， 部分通过激活c-Myc（Myc）。Myc诱导核糖体的生物合成，从而引发核仁应激 通路这是一个进化上保守的途径，包括核糖体蛋白RPL 5的易位， RPL 11和RPL 22进入核质，在那里它们结合并改变p14 ARF（ARF）和MDM 2的活性， 其又激活p53，诱导生长停滞。为了克服这种负反馈循环，超过80%的T- ALL携带CDKN 2A（编码P16 INK 4A和ARF）的双等位基因缺失。此外，约17%的T-ALL携带 RPL 5、RPL 11或RPL 22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，并且核仁应激途径的失活对于 T-ALL的分子发病机制。为检验这一假设，提出了以下具体目标。 目标1。为了确定在正常T细胞发育过程中是否诱导核仁应激， 选择携带CDKN 2A突变的克隆。Notch信号传导在正常的早期T细胞凋亡中起重要作用。 细胞发育我们假设，在早期T细胞发育过程中诱导核仁应激， 携带CDKN 2A（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 来自野生型小鼠的祖细胞/前体细胞，并分析核仁应激的迹象。我们还将生成Arf 或Ink 4a缺陷的混合骨髓嵌合体，并评估供体嵌合性。我们预测核仁应力 在T-细胞祖细胞/前体中的突变将导致T-细胞谱系中Arf缺陷细胞的选择性扩增。 目标二。为了确定激活Notch 1突变是否诱导T细胞中的核仁应激， 祖先我们将评估在T-ALL中发现的NOTCH 1突变的强制表达对 核糖体生物发生和使用MOLM 13细胞系和原代鼠T细胞诱导核仁应激。 在平行研究中，我们还将在几种T-ALL异种移植物中敲低NOTCH 1表达。在每种情况下， 将评估核仁应力

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