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

分析 MMR 缺陷型结直肠癌对 mTOR 抑制的超敏性以及癌症干细胞的反应

• 批准号: 10380891
• 负责人: WINFRIED EDELMANN
• 金额: $ 39.68万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10380891
• 关键词: ABCB1 gene; Affect; Alleles; Cell Death; Clinical; Code; Colon Carcinoma; Colorectal Cancer; Colorectal Neoplasms; Cytostatics; DNA; Defect; Development; Endometrium; Etiology; Failure; Frameshift Mutation; Gene Expression; Gene Mutation; Genomics; Goals; Hereditary Nonpolyposis Colorectal Neoplasms; Human; Hypersensitivity; Intestinal Neoplasms; Intestines; KRAS2 gene; Knock-in; LGR5 gene; Malignant Neoplasms; Mismatch Repair; Molecular; Mus; Mutation; Oncogenes; Organoids; Ovary; Paneth Cells; Pathway interactions; Patients; Population; Prevention; Prevention strategy; Resistance; Resolution; Role; SDZ RAD; Sirolimus; Small Intestines; Syndrome; System; TP53 gene; Testing; Tissues; Tumor Suppressor Genes; Tumor Suppressor Proteins; Xenograft Model; Xenograft procedure; base; cancer stem cell; chemotherapeutic agent; colon cancer cell line; colon cancer patients; colon tumorigenesis; colorectal cancer treatment; cytotoxic; efficacy study; efficacy testing; gene repair; intestinal tumorigenesis; mTOR inhibition; mammalian genome; mouse model; neoplastic cell; novel; novel strategies; oxidative DNA damage; patient derived xenograft model; pre-clinical; refractory cancer; repaired; resistance mechanism; response; single-cell RNA sequencing; stem cell biomarkers; stem cell population; stem cells; treatment response; tumor; tumor growth; tumor xenograft; tumorigenesis

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.

项目摘要 DNA不匹配修复（MMR）基因的突变是属和缺陷MMR 在零星结直肠癌的显着比例（15-20％）中驱动肿瘤发生。 MMR的损失 仅增加基因组突变率，但也导致肿瘤对常规的抗性 化学治疗剂。研究MMR的丧失如何引起结直肠肿瘤的发生并影响 肠道肿瘤对治疗的反应我们开发了一条新型的小鼠系，称为VCMSH2THU，带有A “人性化”TGFβRII等位基因（THU），在MMR驱动的肠道期间易受编码的框架突变 肿瘤发生。 VCMSH2THU是第一个开发结直肠癌（CRC）的鼠标模型 LS患者CRC的组织病理学特征。在初步研究中，我们发现虽然雷帕霉素仅 对MMR促进（MMR+）CRC细胞系具有细胞抑制作用，对MMR-的细胞毒性作用很强 缺陷（MMR-）CRC细胞系。重要的是，雷帕霉素治疗引起了快速和持续的回归 LS小鼠模型中的MMR（ - ）小肠肿瘤以及三个MMR（ - ）患者衍生 CRC异种移植物（PDXS），但不是MMR（+）CRC PDX。有趣的是，癌基因的突变（例如KRAS） 到目前为止 在这些PDX模型中。 MMR（ - ）肿瘤的惊人反应是由于无法有效修复 雷帕霉素诱导的氧化DNA损伤，导致分化肿瘤的自噬细胞死亡 细胞。我们还观察到MMR（ - ）肿瘤包含耐药性的“癌症干细胞壁ni”，由 LGR5+和BMI1+癌症干细胞（CSC）以及在雷帕霉素期间积累的Paneth细胞 治疗。重要的是，在MMR（ - ）CRC器官中的初步研究表明雷帕霉素有效 从CSC出口并抑制P-糖蛋白转运蛋白（P-GP）可以提供一种新型策略 为了消除。此外，与人类患者类似的CRC类似，VCMSH2THU小鼠中的CRC是 特征是干细胞标记物aldh1a3的表达增加，这表明 MMR-肿瘤发生中的ALDH1A3+ CSC。一种新颖的Aldh1a3-cre敲入等位基因（AC）使我们能够失活的MMR 在ALDH1A3+干细胞中，研究对肠道肿瘤发生和雷帕霉素反应的影响。基于 在这些发现中，在本申请中，我们建议研究MTOR抑制对预防和 CRC在VCMSH2THU临床前小鼠和一组临床上通知的MMR（ - ）PDX模型中的处理。 此外，我们将测试涉及P-gp抑制的新策略，以消除MMR（ - ）小鼠中的CSC。 和人类CRC。最后，我们将对CSC人群进行公正的分析 通过单细胞RNASEQ（SCRNASEQ）分析，在小鼠和人MMR（ - ）CRC中处理雷帕霉素。