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

描述（由申请方提供）：在免疫应答期间，B细胞在生发中心（GC）内经历快速增殖和免疫球蛋白（IG）基因重塑，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大多数B细胞恶性肿瘤。我们最近发现ATM在GC B细胞中IG类别转换重组（CSR）过程中被AIDS依赖性DNA双链断裂（DSB）激活，通过LKB 1向CREB B的已知转录共激活因子CRTC2发出信号。使用全基因组定位分析，我们确定CRTC2失活意外地抑制了控制GC B细胞增殖、自我更新和分化为分泌抗体（Ab）的浆细胞的遗传程序，同时对抗淋巴瘤发生。在人B细胞淋巴瘤样本的初步研究中，通过ATM或LKB 1抑制或通过最近在CRTC 2中鉴定的体细胞突变或遗传多态性鉴定了该途径的缺陷。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。我们的数据显示了一个新的结果的DNA损伤反应（DDR）使用B淋巴细胞作为模型系统。已知DNA损伤激活细胞DDR，其决定3种主要的细胞命运：1）具有DNA修复和周期再进入的瞬时细胞周期停滞，2）永久退出细胞周期（衰老），或3）凋亡。在这里，我们建议定义关键的分子决定因素和意想不到的DNA损伤的第四个结果的意义，这是驱动前体细胞成熟，在这种情况下，从GC B细胞的抗体分泌浆细胞。从某种意义上说，这种新的结果是细胞衰老的一种形式，因为具有潜在致瘤性DNA损伤的细胞被迫离开快速分裂的前体库，以保护宿主免受癌症的侵害。然而，这第四种DDR选择通过与分化偶联而与衰老显著不同，这导致了一种重要的新功能，即针对感染因子的Ab产生。我们确定的途径是DSB启动的ATM-> LKB 1->"X"->"Y "/CRTC 2->靶基因表达，其控制从GC B细胞向浆细胞的转变。在目标1中，我们将通过从14个成员AMPK家族中消除候选物和/或通过生物化学-质谱分析，随后在独特的人GC B细胞分化系统中进行shRNA和过表达研究来鉴定~85 kDa LKB 1直接靶向磷蛋白"X"。在目标2中，我们将确定CREB独立的CRTC2相互作用的"Y"因子，其控制介导DSB诱导分化为浆细胞的基因程序。这两个识别和功能目标对于完成这种新型信号通路并与Aim 3研究联系起来至关重要。在目标3中，我们通过分析独特的LKB 1 B谱系敲除（KO）小鼠提供临床前和临床相关性，并确定人GC B细胞淋巴瘤中的通路缺陷是否由遗传或体细胞改变引起。总的来说，我们剖析了DNA损伤的一个新的和意想不到的第四个结果-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.