p38 MAP kinase in early thymocyte development

描述（由申请人提供）：p38 MAP激酶（MAPK）通路在应激刺激（例如电离照射）下被激活，诱导DNA双链断裂（dsb），并在诱导细胞周期检查点（主要是G2/M）中起作用，以促进DNA修复。在T细胞发育过程中，胸腺中的V(D)J重组也会产生dsb。V(D) j介导的dsb（编码端和信号端）触发DNA损伤反应和DNA修复，类似于电离辐射产生的dsb所诱导的。虽然编码端连接对于功能性TCR的形成至关重要，但修复切除DNA片段侧翼的信号端对于防止DNA片段在基因组中随机整合和基因组不稳定也很重要。我们之前已经证明p38 MAPK在经历TCR2 V(D)J重组的双阴性（DN）3胸腺细胞中被激活，并有助于诱导G2/M细胞周期检查点。此外，p38 MAPK也促进DN3胸腺细胞的存活。我们最近的研究表明，p38 MAPK磷酸化GSK32的一个先前未表征的残基（Ser389），这种磷酸化使GSK32失活。GSK32失活与生存率增加有关。我们提出p38 MAPK在DN3胸腺细胞中被V(D) j介导的dsb触发的DNA损伤反应（Atm激活）激活，不仅通过诱导细胞周期checkpoint，还通过GSK32的失活促进生存，有助于修复信号端和防止信号端整合。我们将研究：1)由于V(D) j介导的DSBs引发的DNA损伤反应，DN3胸腺细胞中p38 MAPK的激活是否由ATM介导（Aim 1）；2) p38 MAPK通过磷酸化和灭活GSK32向DN3胸腺细胞提供生存信号（Aim 2）；3) p38 MAPK的激活促进了信号端修复，减少了信号端片段在基因组中的整合（Aim 3）。公共卫生相关性：DNA损伤和基因组不稳定与恶性肿瘤的发展明显相关。为了防止基因组不稳定，细胞通常会经历细胞周期检查点（G1/S和G2/M检查点），从而延迟正常周期，以便有时间进行DNA修复。虽然紫外线和电离照射以及化疗药物是DNA损伤的主要诱导剂，但在发育中的T细胞和B细胞中，当其相应的T细胞受体（TCR）或BCR基因进行重组时，基因组不稳定性持续存在。在这个过程中，称为V(D)J重组，具有非保护双链断裂（信号端）的DNA片段从基因组中切除。这些信号端DNA片段对T细胞和B细胞是危险的，因为它们可以攻击其他双工DNA，并可以随机整合到基因组中，导致潜在的恶性肿瘤。T细胞和B细胞白血病的发生频率相对较高，这些恶性肿瘤通常与染色体易位、缺失和插入有关，这些染色体易位、缺失和插入会影响特定原癌基因的表达或活性。因此，在T细胞和B细胞受体产生过程中检查点的建立对于维持基因组的稳定性非常重要。我们建议研究p38 MAP激酶通路在阻止未成熟胸腺细胞（DN3胸腺细胞）中由TCRb V(D)J重组引起的信号端片段整合到基因组中的作用，通过促进细胞周期检查点（主要是G2/M）的诱导并提供生存。p38 MAPK通路的药物抑制剂目前处于临床试验阶段。如果我们的假设是正确的，这些抑制剂应该主要避免用于T细胞发育高度活跃的婴儿。此外，可能影响这一途径的基因突变可能决定了发生T细胞恶性肿瘤的相对较高的易感性。

DESCRIPTION (provided by applicant): The p38 MAP kinase (MAPK) pathway is activated in response to stress stimuli (e.g. ionizing irradiation) that induce DNA double stranded breaks (DSBs) and plays a role in the induction of cell cycle checkpoints (primarily G2/M) to facilitate DNA repair. DSBs are also generated during the development of T cells while undergoing V(D)J recombination in the thymus. V(D)J-mediated DSBs (coding and signal ends) trigger a DNA damage response and DNA repair similar to those induced by DSBs generated by ionizing irradiation. Although joining of coding ends is critical for the formation of functional TCR, repairing of signal ends flanking the excised DNA fragment is also important to prevent integration of the DNA fragments randomly in the genome and genomic instability. We have previously shown that p38 MAPK is activated in double negative (DN)3 thymocytes undergoing V(D)J recombination of the TCR2 and contributes to the induction of a G2/M cell cycle checkpoint. In addition, p38 MAPK also provides survival to DN3 thymocytes. Our recent studies show that p38 MAPK phosphorylates GSK32 at a non-previously characterized residue (Ser389) and this phosphorylation inactivates GSK32. Inactivation of GSK32 is associated with increased survival. We propose that p38 MAPK is activated in DN3 thymocytes by the DNA damage response (Atm activation) triggered by V(D)J-mediated DSBs and contributes to repairing of signal ends and prevention of signal end integration not only by inducing a cell cycle checkpoint, but also by promoting survival through inactivation of GSK32. We will investigate whether: 1) activation of p38 MAPK in DN3 thymocytes is mediated by ATM as a result of the DNA damage response triggered by V(D)J-mediated DSBs (Aim 1); 2) p38 MAPK provides survival signals to DN3 thymocytes by phosphorylating and inactivating GSK32 (Aim 2); 3) activation of p38 MAPK promotes the repair of signal ends and diminishes integration of signal end fragments in the genome (Aim 3). Public Health Relevance: DNA damage and genomic instability are clearly associated with the development of malignancies. To prevent genomic instability, cells normally undergo cell cycle checkpoints (G1/S and G2/M checkpoints) that delay the normal cycle to allow time for DNA repair to occur. Although UV and ionizing irradiation as well as chemotherapeutic drugs are the major inducers of DNA damage, genomic instability is continuously present in developing T and B cells while undergoing recombination of their corresponding T cell receptor (TCR) or BCR genes. During this process, denominated V(D)J recombination, fragments of DNA with non-protected double stranded breaks (signal ends) are excised from the genome. These signal end DNA fragments can be a hazard for T and B cells since they can attack other duplex DNA and can randomly integrate into the genome, causing potential malignancies. The frequency of T and B cell leukemias is relatively high and often these malignancies have been associated with chromosomal translocations, deletions and insertions that affect the expression or activity of specific proto-oncogenes. The establishment of checkpoints during the generation of T and B cell receptors is therefore important to maintain genomic stability. We propose to investigate the role that the p38 MAP kinase pathway has in preventing integration in the genome of signal ended fragments resulting from TCRb V(D)J recombination in immature thymocytes (DN3 thymocytes), by promoting the induction of a cell cycle checkpoint (primarily G2/M) and also providing survival. Pharmaceutical inhibitors of the p38 MAPK pathway are currently in clinical trials. If our hypothesis is correct, these inhibitors should be avoided primarily in infants where T cell development is highly active. In addition, genetic mutations that may affect this pathway could determine a relatively higher susceptibility to develop T cell malignancies.