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.

项目概要 DNA 错配修复 (MMR) 基因突变是林奇综合征 (LS) 和 MMR 缺陷的病因 在相当大比例（15-20%）的散发性结直肠癌中，MMR 的丧失不会导致肿瘤发生。 不仅增加了基因组突变率，而且还导致肿瘤对常规肿瘤产生抵抗力 研究 MMR 的缺失如何导致结直肠肿瘤发生并影响 肠道肿瘤对治疗的反应 我们开发了一种新型小鼠品系，称为 VCMsh2Thu，携带 “人源化”TgfβRII 等位基因 (Thu) 在 MMR 驱动的肠道过程中容易编码移码突变 VCMsh2Thu 是第一个与结直肠癌 (CRC) 非常相似的小鼠模型。 在初步研究中，我们发现仅使用雷帕霉素即可观察 LS 患者 CRC 的组织病理学特征。 对 MMR 丰富 (MMR+) CRC 细胞系具有细胞抑制作用，对 MMR- 具有强烈的细胞毒作用 重要的是，雷帕霉素治疗诱导了缺陷（MMR-）CRC细胞系的快速且持续的消退。 我们的 LS 小鼠模型中的 MMR(-) 小肠和大肠肿瘤以及三种 MMR(-) 患者来源的肿瘤 CRC 异种移植物 (PDX)，但不是 MMR(+) CRC PDX。 或 PI3KCA 和肿瘤抑制基因（包括 APC 或 TRP53）迄今为止不影响雷帕霉素反应 在这些 PDX 模型中，MMR(-) 肿瘤的显着反应是由于未能有效修复。 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 我们还观察到 MMR(-) 肿瘤含有治疗耐药性的“癌症干细胞生态位”。 Lgr5+ 和 Bmi1+ 癌症干细胞 (CSC) 以及雷帕霉素期间积累的潘氏细胞 重要的是，MMR(-) CRC 类器官的初步研究表明雷帕霉素是有效的。 从 CSC 输出，抑制 P-糖蛋白转运蛋白 (P-gp) 可以提供一种新的策略 此外，与人类患者的 CRC 类似，VCMsh2Thu 小鼠中的 CRC 也被消除。 其特征是干细胞标记物 Aldh1a3 的表达增加，这表明干细胞标记物 Aldh1a3 的重要作用 Aldh1a3+ CSCs 在 MMR-肿瘤发生中的作用 一种新的 Aldh1a3-Cre 敲入等位基因 (AC) 使我们能够灭活 MMR。 Aldh1a3+ 干细胞并研究对肠道肿瘤发生和雷帕霉素反应的影响。 基于这些发现，在本申请中，我们建议研究 mTOR 抑制对预防和治疗的影响。 VCMsh2Thu 临床前小鼠的 CRC 治疗和一组临床注释良好的 MMR(-) PDX 模型。 此外，我们将测试一种涉及 P-gp 抑制的新策略，以消除 MMR(-) 小鼠中的 CSC 最后，我们将对持续存在的 CSC 群体进行公正的分析。 通过单细胞 RNAseq (scRNAseq) 分析雷帕霉素治疗小鼠和人类 MMR(-) CRC。