The selective advantage of mismatch repair loss in colonic stem cells

项目摘要 林奇综合征(LS)是一种遗传性疾病，患者易患结直肠、子宫内膜、卵巢和 其他癌症。LS是由DNA错配修复(MMR)基因的遗传突变引起的，其中的缺陷 也是15%-30%的散发性结直肠癌的基础。MMR功能的丧失与1000倍的 突变率的增加可能会增加重要癌基因和肿瘤抑制基因突变的风险。 MMR途径还激活细胞周期检查点和细胞死亡，以响应外源DNA损伤， 然而，这种损伤反应在预防肿瘤发生中的作用尚不清楚。我们假设 失去这种MMR依赖的损伤反应的结肠干细胞(CSCs)将获得比 邻近的精通MMR的细胞，特别是在突变环境中，例如可能在结肠中发现的。 我们预测，在DNA损伤增加的情况下，失去MMR将提高存活率，有利于这些 干细胞占据的竞争中的细胞。最终，这将导致更多的 高度可变的肠道细胞，增加了癌症表型的外显性。检验这一假说 由于缺乏合适的模式系统，以前一直存在问题。然而，人类最近的发展 结肠器官和肠样模型现在允许我们研究MMR丢失对CSC动力学的影响 以下目的：1)确定人类胚胎干细胞(HESCs)中MMR功能的丧失是否会导致 没有或存在外源DNA损伤剂的直接优势。我们将使用 CRISPR/Cas9介导的基因编辑敲除hESCs中的MMR基因并检测其生长和 损害反应以及确定这些反应背后的机制。2)确定是否 MMR缺陷型CSCs在结肠类器官和肠样组织中具有选择性优势。为此， 我们将区分MMR熟练和缺陷的HESCs为结肠类器官。作为一个补充模式，我们 还将从人类成人结肠组织样本中创建MMR敲除肠样。使用这两个系统，我们将 比较对外源DNA损伤剂或致癌压力的反应。我们还将创建混合的 含有MMR熟练和缺陷细胞的有机化合物，并测试MMR缺陷细胞是否有生长 或在存在或不存在DNA损伤的情况下随着时间的推移而获得生存优势。3)确定MMR损耗是否 从而在体内对CSCs具有选择性优势。我们将使用一种可诱导的干细胞特异性基因敲除小鼠 建立Msh2嵌合型肠隐窝模型及检测MMR缺陷型CSCs是否优于野生型 CSCS。总之，这些目的利用新的方法来研究MMR功能丧失的机制 有助于肿瘤的发生，提供可能有助于解释疾病外显的信息，同时指导 LS相关癌症的诊断、预防和治疗。

Project Summary Lynch syndrome (LS) is a hereditary disease that predisposes patients to colorectal, endometrial, ovarian and other cancers. LS is caused by inherited mutations in the DNA mismatch repair (MMR) genes, defects in which also underlie 15-30% of sporadic colorectal cancers. Loss of MMR function is associated with a 1,000-fold increase in mutation rate likely increasing the risk of mutation to important oncogenes and tumor suppressors. The MMR pathway also activates cell cycle checkpoints and cell death in response to exogenous DNA damage, however, the role of this damage response in preventing tumorigenesis is not known. We hypothesize that colonic stem cells (CSCs) that lose this MMR-dependent damage response will gain a selective advantage over neighboring MMR-proficient cells, particularly in a mutagenic environment such as may be found in the colon. We predict that loss of MMR will enhance survival under conditions of increased DNA damage, favoring these cells in a competition for stem cell niche occupancy. Ultimately, this will lead to the production of more hypermutable intestinal cells, increasing the penetrance of the cancer phenotype. Testing this hypothesis has been problematic previously due to lack of a suitable model system. However, the recent development of human colonic organoid and enteroid models now allow us to study the effects of MMR loss on CSC dynamics via the following aims: 1) Determine whether loss of MMR function in human embryonic stem cells (hESCs) leads to an immediate advantage in the absence or presence of exogenous DNA damaging agents. We will use CRISPR/Cas9-mediated gene editing to knock out the MMR genes in hESCs and examine their growth and damage response as well as determine the mechanism underlying those responses. 2) Determine whether MMR-deficient CSCs have a selective advantage in colonic organoids and colonic enteroids. For this purpose, we will differentiate MMR-proficient and deficient hESCs into colonic organoids. As a complementary model, we will also create MMR knock out enteroids from human adult colon tissue samples. Using both systems, we will compare the response to exogenous DNA damaging agents or oncogenic stress. We will also create mixed organoids containing MMR-proficient and deficient cells and test whether the MMR-deficient cells have a growth or survival advantage over time in the presence or absence of DNA damage. 3) Determine whether MMR loss leads to a selective advantage for CSCs in vivo. We will use an inducible, stem cell specific knockout mouse model of Msh2 to create mosaic intestinal crypts and test whether MMR-deficient CSCs outcompete wild-type CSCs. Together, these aims utilize novel approaches to study the mechanism by which loss of MMR function contributes to tumorigenesis providing information that may help explain disease penetrance while guiding the diagnosis, prevention and treatment of LS-associated cancers.