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

描述（由申请人提供）：在免疫应答过程中，B细胞经历生发中心（GCs）内免疫球蛋白（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反应相关的基因毒性应激也促进了大多数B细胞恶性肿瘤。我们最近发现，在GC B细胞的IG类开关重组（CSR）过程中，由aids依赖性DNA双链断裂（dsb）激活的ATM通过LKB1发出信号，使CRTC2失活，CRTC2是一种已知的CREB转录共激活因子。通过全基因组定位分析，我们确定CRTC2失活意外地抑制了控制GC B细胞增殖、自我更新和向抗体（Ab）分泌浆细胞分化的遗传程序，同时反对淋巴瘤发生。通过ATM或LKB1抑制或最近发现的CRTC2体细胞突变或遗传多态性，在人类B细胞淋巴瘤样本的初步研究中发现了这一途径的缺陷。预计这些通路改变会导致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。我们的数据显示了使用B淋巴细胞作为模型系统的DNA损伤反应（DDR）的新结果。众所周知，DNA损伤激活了细胞DDR，它决定了3种主要的细胞命运：1)DNA修复和周期重新进入的短暂细胞周期阻滞，2)细胞周期永久退出（衰老），或3)细胞凋亡。在这里，我们建议定义DNA损伤的关键分子决定因素和意想不到的第四种结果的意义，这是驱动前体细胞成熟，在这种情况下，从GC B细胞到分泌ab的浆细胞。从某种意义上说，这种新结果是细胞衰老的一种形式，因为具有潜在致瘤性DNA损伤的细胞被迫脱离快速分裂的前体库，以保护宿主免受癌症的侵害。然而，第四种DDR选择与衰老有很大的不同，它与分化耦合，导致一个重要的新功能，抗感染因子的Ab生产。我们发现的途径是dsb启动的ATM->LKB1->“X”->“Y”/CRTC2->靶基因表达，控制从GC B细胞向浆细胞的转变。在Aim 1中，我们将通过从AMPK家族的14个成员中剔除候选蛋白和/或通过生物化学-质谱分析鉴定~85 kDa LKB1直接靶蛋白“X”，然后在独特的人GC B细胞分化系统中进行shRNA和过表达研究。在Aim 2中，我们将确定creb独立的crtc2相互作用的“Y”因子(s)，该因子控制介导dsb诱导的向浆细胞分化的基因程序。这两个鉴定和功能目标是完成这一新的信号通路并与Aim 3研究联系起来的必要条件。在Aim 3中，我们通过分析一种独特的LKB1 B谱系敲除（KO）小鼠提供临床前和临床相关性，并确定人类GC B细胞淋巴瘤的通路缺陷是由遗传还是体细胞改变引起的。总之，我们剖析了DNA损伤的一个新的和意想不到的第四结果-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.