Basis for Lymphomagenesis in Akt2 Transgenic Mice

摘要 AKT在肿瘤的发生中起核心作用，在人类淋巴瘤中经常被激活。V-AKT是一种 诱导小鼠胸腺T细胞淋巴瘤的逆转录病毒携带的癌基因和转基因小鼠 在未成熟T细胞中特异性表达组成活性形式Akt(Myr-Akt)发育胸腺 淋巴瘤。来自Myr-Akt2小鼠模型的多发性胸腺淋巴瘤被发现含有一种新的反转 6号染色体，inv(6)，T细胞受体β链基因(Tcrb)和Dss1基因的断裂点。一个 没有检测到融合蛋白，但重排将Tcrb增强子放置在几个基因和 持续上调的非编码RNA。上调其中一个候选基因的编码 转录因子Dlx5最近在几种常见的人类癌症中被报道，这表明 当表达异常时，这种同源框基因可能是致癌的。这样做的广泛、长期目标是 该项目旨在提高我们对Akt2在淋巴肿瘤发生中的作用的理解，并通过以下方式阐明机制 其中Dlx5的上调与Akt2信号融合，促进肿瘤的形成。具体目标是： 1)评估上调的Dlx5与淋巴瘤形成的相关性。我们将确定细胞增殖是否 和/或带有inv(6)的胸腺淋巴瘤细胞的活性被Dlx5基因敲除所抑制。此外，骨骼 将进行骨髓嵌合体实验以确定Dlx5基因在非恶性骨髓中是否被敲除 来自inv(6)阳性淋巴瘤高发细胞系的细胞可抑制受体肿瘤的形成 野生型产仔仔。我们还将检查是否转基因小鼠在胸腺中过表达Dlx5 患上自发性T细胞淋巴瘤。2)使用直接遗传方法，测试Dlx5是否合作 Akt2参与胸腺肿瘤的发生发展。MYR-Akt2小鼠将与Dlx5转基因小鼠杂交或有条件 Dlx5基因敲除小鼠，以确定肿瘤的发展分别是加速还是抑制。我们还将 通过评估细胞生长，确定Akt2与Dlx5在肿瘤发生中的合作机制， 单独表达D1x5和Myr-Akt2的转基因小鼠原代胸腺细胞的增殖和存活 而不是结合在一起。3)确定两种淋巴瘤是否都需要Akt2信号的过度激活 已建立的肿瘤的发展和维持。荧光原位杂交、聚合酶链式反应和磁性 将使用共振显微成像分析来确定在肿瘤期间出现inv(6)的时间 然后，将使用早期干预策略来确定是否抑制Akt2信号 预防或延缓淋巴瘤的发展。此外，还将开发可诱导的MyR-Akt2模型 确定在已确诊的胸腺淋巴瘤中Akt2的失活是否会导致肿瘤消退。这个项目将 加深对Akt2介导的淋巴肿大的理解以及Akt2与一种新的 可能的癌基因Dlx5，其激活也可能与多种人类癌症的发生有关。

ABSTRACT AKT plays a central role in tumorigenesis and is frequently activated in human lymphoma. v-akt is an oncogene harbored by a retrovirus that induced thymic T-cell lymphomas in mice, and transgenic mice expressing constitutively active forms of Akt (Myr-Akt) specifically in immature T cells develop thymic lymphomas. Multiple thymic lymphomas from a Myr-Akt2 mouse model were found to harbor a novel inversion of chromosome 6, inv(6), with breakpoints in the T-cell receptor beta chain locus (Tcrb) and Dss1 gene. A fusion protein was not detected, but the rearrangement places the Tcrb enhancer near several genes and noncoding RNAs that are consistently up regulated. Up regulation of one of these candidate genes, encoding the transcription factor Dlx5, has recently been reported in several common human cancers, suggesting that this homeobox gene may be oncogenic when expressed aberrantly. The broad, long-term objective of this project is to improve our understanding of the role of Akt2 in lymphomagenesis and elucidate mechanisms by which up regulation of Dlx5 converges with Akt2 signaling to promote tumor formation. The specific aims are: 1) Assess the relevance of up-regulated Dlx5 to lymphoma formation. We will determine if cell proliferation and/or viability of thymic lymphoma cells with the inv(6) are inhibited by knock down of Dlx5. In addition, bone marrow chimera experiments will be carried out to determine if knock down of Dlx5 in non-malignant marrow cells from a founder line with high incidence of inv(6)-positive lymphomas inhibits tumor formation in recipient wild-type littermates. We will also examine if transgenic mice engineered to overexpress Dlx5 in the thymus develop spontaneous T-cell lymphomas. 2) Using a direct genetic approach, test whether Dlx5 cooperates with Akt2 in thymic tumor development. Myr-Akt2 mice will be crossed with Dlx5 transgenic mice or conditional Dlx5 knockout mice to determine if tumor development is accelerated or inhibited, respectively. We will also identify mechanisms by which Akt2 cooperates with Dlx5 in tumorigenesis by assessing cell growth, proliferation and survival in primary thymocytes from transgenic mice expressing Dlx5 or Myr-Akt2 alone versus in combination. 3) Determine if hyperactivation of Akt2 signaling is required for both lymphoma development and maintenance of established tumors. Fluorescence in situ hybridization, PCR, and magnetic resonance microimaging analyses will be employed to establish when the inv(6) arises during tumor development; then an early intervention strategy will be used to determine if inhibition of Akt2 signaling prevents or delays the development of lymphoma. In addition, an inducible Myr-Akt2 model will be developed to ascertain if inactivation of Akt2 in established thymic lymphomas results in tumor regression. This project will enhance our understanding of Akt2-mediated lymphomagenesis and cooperation between Akt2 and a novel putative oncogene, Dlx5, whose activation may also contribute to the pathogenesis of various human cancers.