Tumor-targeted disruption of mismatch repair in microsatellite stable colorectal cancer

微卫星稳定结直肠癌中错配修复的肿瘤靶向破坏

• 批准号: 10578049
• 负责人: Judy Lieberman
• 金额: $ 24.82万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10578049
• 关键词: Affinity; CD8-Positive T-Lymphocytes; CXCL13 gene; Carcinoma; Cells; Chimera organism; Clinical; Colorectal Cancer; Colorectal Neoplasms; DNA; DNA Adduction; DNA Adducts; DNA Damage; Defect; Dependence; Disease; Epithelium; Fibroblasts; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Genetically Engineered Mouse; Graft Enhancements; Human; Immune; Immune checkpoint inhibitor; Immune response; Immunocompetent; Immunologic Deficiency Syndromes; Immunologic Surveillance; Immunologics; Immunotherapy; Implant; Infiltration; Inflammation; Inflammatory; Interferons; Intervention; Intestines; Killer Cells; LGR5 gene; Link; Macrophage; Malignant Neoplasms; Mammary Neoplasms; Metalloproteases; Methods; Microsatellite Instability; Microsatellite Repeats; Minority; Mismatch Repair; Mismatch Repair Deficiency; Modeling; Mus; Mutate; Mutation; Myeloid Cells; Neoplasm Metastasis; Neutrophil Infiltration; Operative Surgical Procedures; Organoids; Outcome; Pharmaceutical Preparations; Phenotype; Prognosis; Resistance; Small Interfering RNA; Somatic Mutation; Subcutaneous Injections; T-Lymphocyte; TACSTD1 gene; Testing; Therapeutic; Tissues; Tumor Antigens; Tumor Immunity; Tumor Tissue; Tumor-infiltrating immune cells; aggressive breast cancer; antitumor effect; aptamer; cancer cell; cancer diagnosis; cancer infiltrating T cells; cancer stem cell; checkpoint therapy; chemokine; chemotherapy; colon cancer cell line; colon cancer patients; design; immune checkpoint blockade; improved; knock-down; metastatic colorectal; monocyte; mortality; neoantigens; neutrophil; programs; promoter; recruit; repair enzyme; repaired; resistance mechanism; response; small molecule inhibitor; stem cells; subcutaneous; triple-negative invasive breast carcinoma; tumor; tumor growth; virtual

Colorectal cancer (CRC) is the third most diagnosed cancer in the US with the second highest mortality rate. Locally advanced and metastatic disease have a poor prognosis. A small subset (5-15%) of metastatic CRC patients, who bear tumors deficient in DNA mismatch repair (MMR) that have high microsatellite instability (MSI- high), can respond to immunotherapy with checkpoint blockade (CPI), but most CRC do not. We hypothesize that to induce immune responses in microsatellite stable (MSS) CRC, new strategies that go beyond CPI are needed. In MSI-high CRC tumors, tumor-infiltrating myeloid cells, fibroblasts and the tumors themselves all have inflammatory gene expression signatures and are colocalized into inflammatory hubs with adjacent tumor- reactive CD8 T cells, suggesting that MSI-high CRCs selectively provoke a robust inflammatory multicellular network within the tumor that recruits anti-tumor killer cells. Disruption of MMR in CRC mouse tumor grafts enhances tumor neoantigen expression and recruitment of tumor-specific T cells to trigger immune surveillance. We hypothesize that interventions that convert MSS CRC to an MMR-deficient MSI phenotype could enhance antitumor immunity and make resistant MSS CRC sensitive to CPI. This proposal will investigate whether tumor-targeted gene knockdown can convert MSS CRC to immune responsive MSI-high tumors. There are no known small molecule inhibitors of MMR. We will exploit a method of epithelial cancer-targeted gene knockdown that uses subcutaneous injection of aptamer-small interfering RNAs (AsiCs), which link a high affinity EpCAM aptamer for epithelial tumor targeting to small interfering RNAs, for tumor-selective gene knockdown. EpCAM, the first described tumor antigen, is ~100-1000-fold more highly expressed in epithelial cancers (including virtually all CRC) than normal epithelia, making it attractive for selective CRC tumor targeting. We previously showed that EpCAM-AsiCs that knockdown tumor-dependency genes or genes whose knockdown promotes immune recognition can strongly suppress aggressive breast cancer in orthotopic, metastatic and genetically engineered mouse tumor models and induce an effective immune response in immunologically cold tumors. In this proposal we will evaluate EpCAM-AsiCs to knockdown Mlh1, encoding a key MMR enzyme, in MSS CRC subcutaneous and caecal tumor implants of 4 mouse CRC cell lines and 2 MMR-proficient, MSS organoid lines derived from an aggressive genetically engineered mouse model, which conditionally express mutations in intestinal stem cells of four key genes that are frequently mutated in human CRC – Apc, Kras, Tgfbr2 and Trp53. We will evaluate the effect of MMR disruption on tumor growth in immunodeficient and immunocompetent mice and dissect in detail how microsatellite stability, tumor mutational burden and gene expression in the tumor and the immune response to the tumor are altered. We will also investigate whether CPI enhances the antitumor effect of tumor-targeted Mlh1 knockdown.

结直肠癌（CRC）是美国第三大诊断癌症，死亡率第二高。 局部晚期和转移性疾病预后差。转移性CRC的一个小子集（5-15%） 患有DNA错配修复（MMR）缺陷的肿瘤的患者具有高微卫星不稳定性（MSI-1）， 高），可以对检查点阻断（CPI）免疫治疗有反应，但大多数CRC没有。我们假设 为了在微卫星稳定（MSS）CRC中诱导免疫应答，超越CPI的新策略是 needed.在MSI高的CRC肿瘤中，肿瘤浸润的髓样细胞、成纤维细胞和肿瘤本身都具有 炎症基因表达特征，并与相邻肿瘤共定位于炎症中心， 反应性CD 8 T细胞，这表明MSI高CRC选择性地引起强烈的炎症性多细胞 肿瘤内的网络招募抗肿瘤杀伤细胞。CRC小鼠肿瘤移植物中MMR的破坏 增强肿瘤新抗原表达和肿瘤特异性T细胞的募集以触发免疫监视。 我们假设，将MSS CRC转化为MMR缺陷型MSI表型的干预措施可以增强 抗肿瘤免疫，使耐药MSS CRC对CPI敏感。该提案将调查是否 肿瘤靶向基因敲低可将MSS CRC转化为免疫应答性MSI高肿瘤。那里 没有已知的小分子MMR抑制剂。我们将探索一种上皮癌靶向基因治疗的方法， 使用皮下注射适体-小干扰RNA（AsiC）的敲低， EpCAM适体用于上皮肿瘤靶向小干扰RNA，用于肿瘤选择性基因敲除。 EpCAM是第一个描述的肿瘤抗原，在上皮癌中的表达高约100-1000倍 因此，它比正常上皮细胞（包括几乎所有的CRC）具有更高的生物学活性，使其对于选择性CRC肿瘤靶向具有吸引力。我们 先前表明，敲低肿瘤依赖性基因或敲低肿瘤依赖性基因的EpCAM-AsiC 促进免疫识别可以强烈抑制原位，转移性和侵袭性乳腺癌。 基因工程小鼠肿瘤模型，并诱导有效的免疫反应， 肿瘤的在本提案中，我们将评估EpCAM-AsiC敲低Mlh 1（编码关键的MMR酶）， MSS CRC皮下和盲肠肿瘤植入物，4种小鼠CRC细胞系和2种MMR-proficient MSS 来源于攻击性基因工程小鼠模型的类器官系，其条件性表达 肠干细胞中四个关键基因的突变，这四个基因在人类CRC中经常突变- Apc，Kras， Tgfbr 2和Trp 53。我们将评估MMR破坏对免疫缺陷和肿瘤生长的影响。 并详细分析了微卫星稳定性、肿瘤突变负荷和基因 在肿瘤中的表达和对肿瘤的免疫应答被改变。我们还将调查CPI是否 增强肿瘤靶向Mlh 1敲低的抗肿瘤作用。