Basis for Lymphomagenesis in Akt2 Transgenic Mice

描述（由申请人提供）：AKT在肿瘤发生中起核心作用，在人类淋巴瘤中经常被激活。v-akt是一种由逆转录病毒携带的致癌基因，可诱导小鼠胸腺T细胞淋巴瘤，在未成熟T细胞中特异性表达组成型活性Akt （Myr-Akt）的转基因小鼠可发展为胸腺淋巴瘤。来自Myr-Akt2小鼠模型的多发性胸腺淋巴瘤被发现含有6号染色体inv(6)的新反转，在t细胞受体β链位点（Tcrb）和Dss1基因中具有断点。没有检测到融合蛋白，但是重排将Tcrb增强子放置在几个持续上调的基因和非编码rna附近。这些候选基因之一编码转录因子Dlx5，最近在几种常见的人类癌症中被报道上调，这表明这种同源盒基因在异常表达时可能致癌。该项目的长期目标是提高我们对Akt2在淋巴瘤发生中的作用的理解，并阐明Dlx5上调与Akt2信号汇合促进肿瘤形成的机制。具体目的是：1)评估Dlx5上调与淋巴瘤形成的相关性。我们将确定携带inv(6)的胸腺淋巴瘤细胞的细胞增殖和/或活力是否通过敲低Dlx5被抑制。此外，将进行骨髓嵌合体实验，以确定来自inv(6)阳性淋巴瘤高发的始祖系的非恶性骨髓细胞中Dlx5的敲除是否会抑制受体野生型幼崽的肿瘤形成。我们还将研究在胸腺中过度表达Dlx5的转基因小鼠是否会发生自发性t细胞淋巴瘤。2)采用直接遗传学方法，检测Dlx5是否与Akt2在胸腺肿瘤的发生发展中有协同作用。Myr-Akt2小鼠将分别与Dlx5转基因小鼠或条件Dlx5基因敲除小鼠杂交，以确定肿瘤的发展是否被加速或抑制。我们还将通过评估表达Dlx5或Myr-Akt2单独或联合表达的转基因小鼠的原代胸腺细胞的细胞生长、增殖和存活，来确定Akt2与Dlx5在肿瘤发生中的合作机制。3)确定Akt2信号的过度激活对于淋巴瘤的发展和肿瘤的维持是否都是必需的。荧光原位杂交、PCR和磁共振微成像分析将用于确定inv(6)在肿瘤发展过程中何时出现；那么早期干预策略将用于确定Akt2信号的抑制是否可以预防或延迟淋巴瘤的发展。此外，将开发一个诱导型Myr-Akt2模型，以确定在已建立的胸腺淋巴瘤中，Akt2失活是否会导致肿瘤消退。该项目将增强我们对Akt2介导的淋巴瘤发生以及Akt2与一种新的推定致癌基因Dlx5之间的合作的理解，该基因的激活也可能有助于各种人类癌症的发病机制。公共卫生相关性：细胞酶AKT2在大多数人类癌症中过度活跃，促进肿瘤细胞存活和对化疗的耐药性。我们在小鼠模型中的研究发现了一种新的基因（Dlx5），当表达异常时，它与激活的Akt2一起诱导胸腺侵袭性淋巴瘤。人类DLX5最近被证明在一些淋巴瘤以及更常见的肿瘤（如肺癌）中表达水平升高，这表明DLX5可能作为人类akt2相关恶性肿瘤的新治疗靶点。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The cellular enzyme AKT2 is hyperactive in most human cancers and promotes tumor cell survival and resistance to chemotherapy. Our studies in a mouse model have uncovered a novel gene (Dlx5) that, when expressed abnormally, works together with activated Akt2 to induce aggressive lymphomas of the thymus. Human DLX5 has recently been shown to be expressed at elevated levels in some lymphomas as well as more common tumors such as lung cancers, suggesting that DLX5 may serve as a new therapeutic target in human AKT2-related malignancies.