Analyzing the Hypersensitivity of MMR-deficient Colorectal Cancers to mTOR Inhibition and the Response of Cancer Stem Cells

项目摘要 DNA错配修复(MMR)基因突变是Lynch综合征(LS)和MMR缺陷的原因 导致相当大比例(15%-20%)的散发性结直肠癌发生。MMR NOT的损失 不仅会增加基因组突变率，还会导致肿瘤对常规药物的抵抗力 化疗药物。目的：研究MMR缺失如何导致结直肠肿瘤的发生和影响 肠道肿瘤对治疗的反应我们开发了一种新的小鼠系，命名为VCMsh2Thu，携带 “人源化”转化生长因子βRII等位基因(清华)在MMR驱动的肠道中易受编码移码突变的影响 肿瘤发生学。VCMsh2Thu是第一个与结直肠癌(CRC)非常相似的小鼠模型 LS患者大肠癌的组织病理学特征。在初步研究中，我们发现，虽然只有雷帕霉素 对MMR-熟练(MMR+)结直肠癌细胞株有细胞抑制作用，对MMR- 缺陷型(MMR-)CRC细胞系。重要的是，雷帕霉素治疗导致了快速而持久的消退。 我们的LS小鼠模型中MMR(-)小肠和大肠肿瘤以及三个MMR(-)患者来源的肿瘤的研究 CRC异种移植(PDX)，但不是MMR(+)CRC PDX。有趣的是，癌基因的突变，如KRAS 或PI3KCA和包括APC或TrP53在内的肿瘤抑制基因到目前为止还没有影响雷帕霉素的反应 在这些PDX型号中。MMR(-)肿瘤的显著反应是由于未能有效修复 雷帕霉素诱导DNA氧化损伤导致分化肿瘤自噬细胞死亡 细胞。我们还观察到，MMR(-)肿瘤包含有治疗耐药的“癌症干细胞壁龛”，包括 雷帕霉素期间积聚的Lgr5+和Bmi1+肿瘤干细胞(CSCs)以及Paneth细胞 治疗。重要的是，对MMR(-)CRC有机物的初步研究表明，雷帕霉素是有效的 抑制P-糖蛋白转运体(P-gp)可以提供一种新策略 因为他们被消灭了。此外，与人类患者中的CRC类似，VCMsh2Thu小鼠中的CRC是 以干细胞标记物ALDH1A3的表达增加为特征，表明 ALDH1A3+干细胞在MMR-肿瘤发生中的作用一种新的ALDH1A3-Cre敲入等位基因(AC)使MMR失活 在ALDH1A3+干细胞中，研究其对肠道肿瘤形成和雷帕霉素反应的影响。基座 根据这些发现，在本应用中，我们建议研究mTOR抑制在预防和治疗中的作用。 VCMsh2Thu临床前小鼠和一组临床注释良好的MMR(-)PDX模型中的CRC的治疗。 此外，我们将测试一种涉及P-gp抑制的新策略，用于消除MMR(-)小鼠的CSCs 和人类CRC。最后，我们将对持续存在的CSC人群进行公正的分析 单细胞RNAseq(ScRNAseq)分析雷帕霉素对小鼠和人MMR(-)癌细胞的作用

Project Summary Mutations in DNA mismatch repair (MMR) genes are causative in Lynch Syndrome (LS) and defective MMR drives tumorigenesis in a significant proportion (15 - 20%) of sporadic colorectal cancers. The loss of MMR not only increases genomic mutation rates, but also results in the resistance of tumors to conventional chemotherapeutic agents. To study the how the loss of MMR causes colorectal tumorigenesis and affects the response of intestinal tumors to treatment we developed a novel mouse line, termed VCMsh2Thu, carrying a “humanized” TgfβRII allele (Thu) that is susceptible to coding frameshift mutations during MMR-driven intestinal tumorigenesis. VCMsh2Thu is the first mouse model that develops colorectal cancers (CRCs) closely mimicking the histopathologic features of CRCs in LS patients. In preliminary studies, we found that while rapamycin only had a cytostatic effect on MMR-proficient (MMR+) CRC cell lines, it exerted a strong cytotoxic effect on MMR- deficient (MMR-) CRC cell lines. Importantly, rapamycin treatment induced the rapid and persistent regression of MMR(-) small and large intestinal tumors in our LS mouse models as well as of three MMR(-) patient-derived CRC xenografts (PDXs), but not of MMR(+) CRC PDXs. Interestingly, mutations in oncogenes such as KRAS or PI3KCA and tumor suppressor genes including APC or TRP53 so far did not affect the rapamycin response in these PDX models. The striking response of MMR(-) tumors was due to the failure to efficiently repair rapamycin-induced oxidative DNA damage, which resulted in autophagic cell death of differentiated tumor cells. We also observed that MMR(-) tumors contain treatment resistant “cancer stem cell niches” consisting of Lgr5+ and Bmi1+ cancer stem cells (CSCs) as well as Paneth cells that accumulate during rapamycin treatment. Importantly, preliminary studies in MMR(-) CRC organoids showed that rapamycin is effectively exported from CSCs and that inhibition of the P-glycoprotein transporter (P-gp) could provide a novel strategy for their elimination. In addition, similar to CRCs in human patients, the CRCs in VCMsh2Thu mice are characterized by increased expression of the stem cell marker Aldh1a3, suggesting an important role for Aldh1a3+ CSCs in MMR- tumorigenesis. A novel Aldh1a3-Cre knock-in allele (AC) allows us to inactivate MMR in Aldh1a3+ stem cells and study the effects on intestinal tumorigenesis and the rapamycin response. Based on these findings, in this application we propose to study the effect of mTOR inhibition on the prevention and treatment of CRCs in VCMsh2Thu preclinical mice and a panel of clinically well-annotated MMR(-) PDX models. In addition, we will test a novel strategy involving P-gp inhibition for the elimination of CSCs in MMR(-) mouse and human CRCs. Finally, we will perform an unbiased analysis of the CSC populations that persist during rapamycin treatment in mouse and human MMR(-) CRCs by single cell RNAseq (scRNAseq) analysis.