A Fourth Outcome: DNA Damage and the Differentiation of B Cells

描述(申请人提供)：在免疫反应期间，B细胞在生发中心(GC)内经历免疫球蛋白(IG)基因的快速增殖和重塑，以产生记忆B细胞和浆细胞。不幸的是，与GC反应相关的遗传毒性应激也促进了大多数B细胞恶性肿瘤的发生。我们最近发现，在GC B细胞的IG类开关重组(CSR)过程中，由AID依赖的DNA双链断裂(DSB)激活的ATM通过LKB1发出信号，灭活CREB的转录辅助激活因子CRTC2。利用全基因组定位分析，我们确定CRTC2失活出人意料地抑制了控制GC B细胞增殖、自我更新和分化为抗体(Ab)分泌浆细胞的遗传程序，同时反对淋巴肿大。在对人类B细胞淋巴瘤样本的初步研究中，通过ATM或LKB1抑制或最近发现的CRTC2的体细胞突变或遗传多态，发现了这一途径的缺陷。这些途径的改变预计会导致GC B细胞增殖增加和浆细胞分化受损，这将在体外和体内进行测试。我们的数据显示了以B淋巴细胞为模型系统的DNA损伤反应(DDR)的新结果。众所周知，DNA损伤激活了细胞内的DDR，它决定了三种主要的细胞命运：1)DNA修复和周期重入导致的短暂的细胞周期停滞，2)永久退出细胞周期(衰老)，或3)细胞凋亡。在这里，我们建议定义关键的分子决定因素和DNA损伤的第四个意想不到的结果的意义，这是驱动前体细胞成熟，在这种情况下，从GC B细胞到分泌抗体的浆细胞。在某种意义上，这一新结果是细胞衰老的一种形式，因为具有潜在致癌DNA损伤的细胞被迫离开快速分裂的前体池，以保护宿主免受癌症的伤害。然而，这第四种DDR选择与衰老有很大的不同，因为它与分化相结合，这导致了一种基本的新功能，即产生针对感染性病原体的抗体。我们确定的途径是DSB启动的ATM-&gt；LKB1-&gt；“X”-&gt；“Y”/CRTC2-&gt；控制GC B细胞向浆细胞转变的靶基因表达。在目标1中，我们将通过从AMPK家族14个成员中剔除候选蛋白和/或通过生化-质谱分析，然后在一个独特的人GC B细胞分化系统中进行shRNA和过表达研究，来鉴定~85 kDa LKB1直接靶向磷蛋白“X”。在目标2中，我们将确定CREB非依赖的CRTC2相互作用的“Y”因子(S)，它控制着一个基因程序，该基因程序介导了DSB诱导的浆细胞分化。这两个识别和功能目标是完成这一新的信号通路并与Aim 3研究相联系所必需的。在目标3中，我们通过对一只独特的LKB1 B系基因敲除(KO)小鼠的分析，提供临床前和临床相关性，并确定人类GC B细胞淋巴瘤的途径缺陷是由遗传还是躯体改变引起的。总体而言，我们剖析了DNA损伤的第四个新的、意想不到的结果--细胞分化。

DESCRIPTION (provided by applicant): During an immune response, B cells undergo rapid proliferation and remodeling of immunoglobulin (IG) genes within germinal centers (GCs) to generate memory B and plasma cells. Unfortunately, genotoxic stress associated with the GC reaction also promotes most B cell malignancies. We recently discovered that ATM, activated by AID-dependent DNA double stranded breaks (DSBs) during IG class switch recombination (CSR) in GC B cells, signals through LKB1 to inactivate CRTC2, a known transcriptional co-activator of CREB. Using genome-wide location analysis, we determined that CRTC2 inactivation unexpectedly represses a genetic program that controls GC B cell proliferation, self-renewal, and differentiation into antibody (Ab)-secreting plasma cells while opposing lymphomagenesis. Defects in this pathway were identified in pilot studies of human B cell lymphoma samples by ATM or LKB1 repression or by a recently identified somatic mutation or genetic polymorphism in CRTC2. These pathway alterations are predicted to result in increased GC B cell proliferation and impaired plasma cell differentiation, which will be tested here in vitro and in vivo. Our data show a new outcome for the DNA damage response (DDR) using B lymphocytes as the model system. It is known that DNA damage activates a cellular DDR, which determines 3 main cell fates: 1) transient cell cycle arrest with DNA repair and cycle reentry, 2) permanent exit from the cell cycle (senescence), or 3) apoptosis. Here, we propose to define key molecular determinants and the significance of an unexpected fourth outcome for DNA damage, which is to drive precursor cell maturation, in this case from a GC B cell to an Ab-secreting plasma cell. In a sense, this new outcome is a form of cell senescence, in that a cell with potentially tumorigenic DNA damage is forced out of a rapidly dividing precursor pool to protect the host from cancer. However, this fourth DDR option differs significantly from senescence by coupling with differentiation, which leads to an essential new function, Ab production against infectious agents. The pathway we identified is DSB-initiated ATM->LKB1->"X"->"Y"/CRTC2-> target gene expression that controls the transition from a GC B cell to a plasma cell. In Aim 1, we will identify ~85 kDa LKB1 direct target phosphoprotein "X" by candidate elimination from the 14 member AMPK family and/or by biochemistry- mass spectrometry analysis, followed by shRNA and over-expression studies in a unique human GC B cell differentiation system. In Aim 2, we will identify CREB-independent CRTC2-interacting "Y" factor(s) that control a gene program that mediates DSB-induced differentiation into plasma cells. These two identification and function aims are essential to complete this novel signaling pathway and to link with Aim 3 studies. In Aim 3, we provide pre-clinical and clinical relevance by analysis of a unique LKB1 B-lineage knockout (KO) mouse and we determine whether pathway defects in human GC B cell lymphomas result from inherited or somatic alterations. Overall, we dissect a new and unexpected fourth outcome for DNA damage- cell differentiation.