FEN1-Nuclease-Targeted Therapy for Ewing Sarcoma

FEN1-核酸酶靶向治疗尤文肉瘤

• 批准号: 10368155
• 负责人: Sun Ha Choo
• 金额: $ 56.33万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 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/10368155
• 关键词: Ablation; Address; Adolescent; Affect; Animals; BRCA1 gene; Bar Codes; Biological Assay; Biological Availability; Biopsy; CRISPR library; CRISPR/Cas technology; Cell Death; Cell Line; Cells; Cessation of life; Child; Chromosome Breakage; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Connective and Soft Tissue Neoplasm; Coupled; DNA; DNA Binding Domain; DNA Damage; DNA Repair; DNA biosynthesis; Dependence; Drug Kinetics; Drug resistance; EWS-FLI1 fusion protein; EWSR1 gene; Enzymes; Ewings sarcoma; FLI1 gene; Family; Future; Genes; Genetic; Genetic Transcription; Goals; Gold; Growth; Guide RNA; Human; Hypersensitivity; In Vitro; Institutional Review Boards; Knock-out; Legal patent; Letters; Libraries; Malignant Childhood Neoplasm; Malignant Neoplasms; Measures; Mitotic; Molecular; Molecular Medicine; Mus; Nude Mice; Okazaki fragments; Oncoproteins; Operative Surgical Procedures; Patients; Pediatric Oncologist; Pediatrics; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Primitive Neuroectodermal Tumor; Principal Investigator; Process; Protocols documentation; Radiation; Reporting; Research; Research Project Grants; Resected; Resistance; Sampling; Signal Transduction; Small Interfering RNA; Specificity; Statistical Methods; Surgical Flaps; Survival Rate; Testing; Therapeutic; Transactivation; Transplantation; Tumor Cell Line; Tumor Volume; Xenograft procedure; advanced disease; aggressive therapy; brca gene; cancer cell; cell transformation; chemotherapy; cytotoxic; data mining; endonuclease; genome-wide; improved; in vivo; in vivo evaluation; inhibitor; mouse model; neoplastic cell; novel; novel therapeutics; nuclease; patient derived xenograft model; patient prognosis; preclinical evaluation; preclinical study; response; small molecule; synergism; targeted nucleases; targeted treatment; three dimensional cell culture; tumor; tumor growth; tumorigenesis; young adult

PROJECT SUMMARY Ewing Sarcoma Family of Tumors (ESFT) are bone and soft tissue cancers that affect children and adolescents. Surgery, radiation and multi-agent chemotherapy have improved patient prognosis but a therapeutic plateau has been reached for both localized and metastatic cases. Hence, there is an urgent need for novel and targeted therapeutic strategies. Towards that goal, this Multi-PI collaborative research project will investigate the hypothesis that ESFT cells are dependent on FEN1, an endonuclease that processes the 5’ flaps of Okazaki fragments during lagging strand DNA synthesis, for viability. This hypothesis is supported by: (a) Four genome- scale CRISPR library screens have independently found that ESFT cell lines are highly sensitive to FEN1- CRISPRs; (b) ESFT cells were reported to be phenotypically BRCA1-deficient, and we have shown that BRCA1/2-deficient cells are hypersensitive to FEN1 inhibition; and (c) we have found several ESFT cell lines to be sensitive to FEN1-CRISPRs, FEN1-siRNAs and two small molecular FEN1-inhibitors. A team of three principal investigators will jointly direct this project: RD Kolodner is a geneticist and an inventor of FEN1-inhibitors in documented patent applications, JYJ Wang is a cancer biologist with expertise in DNA damage response, and SH Choo is a pediatric oncologist with an ongoing IRB to conduct research with ESFT patient samples. Together, we will pursue four specific aims to investigate the ESFT-dependency on FEN1. AIM-1: To demonstrate and to quantify FEN1-essentiality in a panel of 10 ESFT cell lines by genetic ablation of FEN1 with validated siRNAs and CRISPR/CAS9, respectively. AIM-2: To determine the cytotoxic effects of small molecule FEN1 inhibitors on 10 ESFT cell lines by short-term growth and death assays and longer-term colony formation assays in 2D and 3D cultures. We will edit FEN1 in ESFT cells to express a drug-resistant FEN1R enzyme so as to demonstrate on-target effects. We will evaluate the potential synergistic interactions between FEN1-inhibitors and the chemotherapeutic drugs currently used in the clinic to treat ESFT patients. AIM-3: To investigate the mechanisms underlying ESFT dependency on FEN1 by addressing three mechanistic questions on whether (a) the EWS-FLI1 oncoprotein of ESFT induces FEN1-dependency, (b) FEN1-inhibitors cause irreversible blockade of DNA replication in ESFT cells, and (c) FEN1-inhibitors cause DNA breakage and mitotic catastrophe in ESFT cells. AIM-4: To determine the effects of FEN1 inhibitors on ESFT growth in mice. We have found that a FEN1- inhibitor (SMD154) reduced the growth of orthotopic xenografts from an ESFT cell line in athymic nude mice. The pharmacokinetics (PK) of SMD154 support its testing on orthotopic xenografts from two ESFT cell lines that show sufficiently low in vitro IC50 for this compound. We will construct 4-6 ESFT patient-derived xenografts (PDX) from biopsies and resected tumors. We will test SMD154 and future FEN1-inhibitors with optimized PKs on cell line- and patient-derived xenografts. This comprehensive preclinical study will produce a detailed understanding of the ESFT-dependency on FEN1 and pave the way towards developing FEN1-inhibitors to treat ESFT.

项目总结 尤因肉瘤家族肿瘤(ESFT)是一种影响儿童和青少年的骨和软组织癌症。 手术、放疗和多因素化疗改善了患者的预后，但治疗的平台期 无论是局部病例还是转移性病例都已取得联系。因此，迫切需要新颖和有针对性的 治疗策略。为了这个目标，这个多PI合作研究项目将研究 ESFT细胞依赖于FEN1的假说，FEN1是一种处理冈崎5‘瓣的核酸内切酶 在滞后的链DNA合成过程中的片段，以求生存。这一假说得到以下支持：(A)四个基因组- Scale CRISPR文库筛选独立发现ESFT细胞系对FEN1-1高度敏感- (B)ESFT细胞被报道为表型BRCA1缺陷，我们已经证明 BRCA1/2缺失的细胞对FEN1抑制非常敏感；以及(C)我们发现了几个ESFT细胞系， 对FEN1-CRISPR、FEN1-siRNAs和两种小分子FEN1-抑制剂敏感。一个三人的团队 主要研究人员将共同指导这一项目：科洛德纳是遗传学家，也是FEN1抑制剂的发明者。 在有文件记录的专利申请中，JYJ Wang是一位在DNA损伤反应方面拥有专业知识的癌症生物学家，以及 ShChoo是一名儿科肿瘤学家，正在进行IRB，对ESFT患者样本进行研究。一起， 我们将追求四个具体目标来调查ESFT对FEN1的依赖。目标-1：示范和 通过用有效的siRNAs基因消融FEN1来量化10个ESFT细胞系中FEN1的重要性 和CRISPR/Cas9。目的-2：测定小分子FEN1抑制剂的细胞毒作用 对10株ESFT细胞进行短期生长和死亡实验及长期集落形成实验 和3D文化。我们将在ESFT细胞中编辑FEN1以表达耐药的FEN1R酶，以便 演示目标上的效果。我们将评估FEN1-抑制剂之间潜在的协同作用 以及目前临床上用于治疗ESFT患者的化疗药物。目标-3：调查 ESFT依赖FEN1的潜在机制，通过回答关于是否(A)的三个机械性问题 ESFT的EWS-FLI1癌蛋白诱导FEN1依赖，(B)FEN1抑制剂引起不可逆转的阻断 (C)FEN1抑制剂导致ESFT细胞DNA断裂和有丝分裂灾难 细胞。目的-4：确定FEN1抑制剂对小鼠ESFT生长的影响。我们发现了一种FEN1- 抑制剂(SMD154)抑制裸鼠ESFT细胞系原位移植瘤的生长。 SMD154的药代动力学(PK)支持其在来自两个ESFT细胞系的原位异种移植瘤上的测试 对该化合物表现出足够低的体外IC50。我们将构建4-6个ESFT患者来源的异种移植物(PDX) 来自活组织检查和肿瘤切除。我们将在细胞上用优化的PKS测试SMD154和未来的FEN1-1抑制剂 线来源和患者来源的异种移植物。这项全面的临床前研究将产生一个详细的理解 减少ESFT对FEN1的依赖，并为开发FEN1抑制剂治疗ESFT铺平道路。