FEN1-Nuclease-Targeted Therapy for Ewing Sarcoma

FEN1-核酸酶靶向治疗尤文肉瘤

• 批准号: 10209659
• 负责人: Sun Ha Choo
• 金额: $ 57.44万
• 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/10209659
• 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; Cleaved cell; 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; Prognosis; 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/antagonist; mouse model; neoplastic cell; novel; novel therapeutics; nuclease; patient derived xenograft model; 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.

项目摘要 尤因肉瘤家族（ESFT）是影响儿童和青少年的骨骼和软组织癌。 手术，辐射和多药化疗的患者预后有所改善，但治疗高原具有 因此，迫切需要新颖和针对性 治疗策略。为了实现这一目标，这个多PI合作研究项目将调查 假设ESFT细胞取决于Fen1，这是一种处理冈崎5'襟翼的核酸内切酶 滞后链DNA合成期间的碎片，以实现生存能力。该假设得到：（a）四个基因组 - 比例CRISPR库屏幕独立发现ESFT细胞系对Fen1-高度敏感 crisprs; （b）据报道ESFT细胞在表型上是BRCA1缺陷，我们已经表明 BRCA1/2缺陷细胞对Fen1抑制作用过敏； （c）我们发现了几个ESFT细胞系 对Fen1-Crisprs，Fen1-SiRNA和两个小分子Fen1抑制剂敏感。一支由三个团队组成的团队 首席研究人员将共同指导该项目：RD Kolodner是遗传学家和FEN1抑制剂的清单 在有记录的专利申请中，JYJ Wang是一名癌症生物学家，在DNA损伤反应方面具有专业知识，并且 SH Choo是一名儿科肿瘤学家，持续使用IRB，可以使用ESFT患者样品进行研究。一起， 我们将追求四个特定的目标，以研究FEN1的ESFT依赖性。 AIM-1：展示和 通过经过验证的siRNA的遗传消融，在10个ESFT细胞系中量化FEN1-肯定性 和CRISPR/CAS9。 AIM-2：确定小分子Fen1抑制剂的细胞毒性作用 在10个ESFT细胞系上，通过短期生长和死亡测定和2D中的长期菌落形成测定 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 展示靶向效应。我们将评估Fen1抑制剂之间的潜在协同相互作用 以及目前在诊所中用于治疗ESFT患者的化学治疗药物。 AIM-3：调查 ESFT对Fen1的依赖的机制通过解决（a）是否是否是否解决了Fen1的机制 ESFT的EWS-FLI1癌蛋白诱导FEN1依赖性，（B）FEN1抑制剂引起不可逆的阻塞 ESFT细胞中的DNA复制，（c）Fen1抑制剂引起DNA断裂和ESFT有丝分裂灾难 细胞。 AIM-4：确定FEN1抑制剂对小鼠ESFT生长的影响。我们发现fen1- 抑制剂（SMD154）降低了无胸腺裸鼠ESFT细胞系的原位异种移植物的生长。 SMD154的药代动力学（PK）支持其对两种ESFT细胞系的原位异种移植物的测试 对于该化合物显示足够低的体外IC50。我们将构建4-6个ESFT患者衍生的Xenographictic（PDX） 来自活检和切除的肿瘤。我们将在细胞上使用优化的PKS测试SMD154和将来的FEN1抑制剂 线和患者衍生的Xenographictics。这项全面的临床前研究将产生详细的理解 Fen1上的ESFT依赖性，并为开发Fen1抑制剂治疗ESFT的道路​​铺平了道路。