Molecular Pathways in Suppression and Development of T Lineage Lymphomas

描述（由申请人提供）：染色体DMA双链断裂（DSBs）是由多种因素诱导的，并且在每个细胞中不断发生。dsb的适当修复对于维持基因组稳定性和防止细胞转化至关重要。我们最近证明，共济失调毛细血管扩张突变（ATM）蛋白是细胞DSB反应的主要调节因子，在染色体DSB修复过程中需要维持修复复合体中断裂的DNA末端。ATM的直接底物是H2AX，这是一种核心组蛋白变体，在dsb周围的染色质中被磷酸化。我们已经证明H2ax的两个等位基因拷贝的表达对于无差错DSB修复、维持基因组稳定性和抑制癌症是必不可少的。值得注意的是，H2AX基因的杂合性缺失（LOH）在多种人类肿瘤中普遍出现，这表明H2AX在人类中具有类似的剂量依赖性功能。在本应用中，我们拟阐明H2AX在ATM及相关激酶下游的作用机制，以促进无差错DSB修复，维持基因组稳定性，抑制恶性转化。要研究的具体假设是，H2AX的一个关键功能是将断裂的DNA链固定在一起，并确保DNA末端得到适当修复。在Specific Aim 1中，我们将验证我们的假设，即H2AX在V(D)J重组过程中起作用，在G1到S过渡时将断裂的DNA链结合在一起，从而防止抗原受体位点易位。我们将利用新的细胞系和原代胸腺细胞在G1期细胞的特定基因组位置诱导DSB中间体，并在持续的细胞周期进程中监测它们的修复。在Specific Aim 2中，我们将验证我们的假设，即其他激酶下游的ATM独立H2AX功能对于正常DSB修复、预防基因组不稳定和抑制癌症至关重要。我们将利用缺乏H2AX和ATM的细胞系和胸腺来阐明ATM独立的H2AX功能的作用。在Specific Aim 3中，我们将验证我们的假设，即H2AX在正常和癌基因驱动的增殖过程中诱导的复制相关dsb中起作用，以防止驱动恶性转化的p53缺失。我们将直接评估H2AX对p53缺失肿瘤的抑制能力。相关性：我们的研究将有助于更好地理解基因组完整性维持的分子机制，以及这些缺陷如何导致恶性转化。这些知识和由此产生的动物模型将有助于开发更有效的人类癌症诊断和/或治疗工具。因此，我们提出的研究将对人类疾病产生广泛的影响。

DESCRIPTION (provided by applicant): Chromosomal DMA double strand breaks (DSBs) are induced by a variety of factors and occur constantly in every cell. The proper repair of DSBs is essential for maintaining genomic stability and preventing cellular transformation. We recently demonstrated that the Ataxia Telangiectasia Mutated (ATM) protein, which is the master regulator of the cellular DSB response, is required during chromosomal DSB repair to maintain broken DNA ends in repair complexes. An immediate substrate of ATM is H2AX, a core histone variant that is phosphorylated in chromatin around DSBs. We have demonstrated that expression of both allelic copies of H2ax is essential for error-free DSB repair, maintanence of genomic stability, and suppression of cancer. Notably, deletion or loss of heterozygosity (LOH) of the H2AX gene appears commonly in a wide variety of human tumors, suggesting similar dosage-dependent H2AX functions in man. In this application, we propose to elucidate mechanisms by which H2AX functions downstream of ATM and related kinases to promote error-free DSB repair, maintain genomic stability, and suppress malignant transformation. The specific hypothesis to be investigated is that one critical function of H2AX is to hold broken DNA strands together and ensure that DNA ends are properly repaired. In Specific Aim 1, we will test our hypothesis that H2AX functions during V(D)J recombination to hold together broken DNA strands upon G1 to S transition and, thereby, prevent antigen receptor locus translocations. We will employ novel cell lines and primary thymocytes to induce DSB intermediates at specific genomic locations in G1 phase cells and monitor their repair during continued cell cycle progression. In Specific Aim 2, we will test our hypothesis that ATM independent H2AX functions downstream of other kinases are essential for normal DSB repair, prevention of genomic instability, and suppression of cancer. We will utilize cell lines and thymocyes deficient for H2AX and ATM to elucidate the roles of ATM independent H2AX functions. In Specific Aim 3, we will test our hypothesis that H2AX functions at replication-associated DSBs induced during normal and oncogene-driven proliferation to prevent p53 deletions that drive malignant transformation. We will directly evaluate the ability of H2AX to suppress tumors with p53 deletions. Relevance: Our studies will lead to a greater understanding of the molecular mechanisms through which genomic integrity is maintained and how defects in these can lead to malignant transformation. This knowledge and the animal models generated will contribute to the development of more effective diagnostic and/or therapeutic tools for human cancers. Consequently, our proposed studies will have broad implications for human disease.