FEN1-Nuclease-Targeted Therapy for Ewing Sarcoma

项目摘要 尤文肉瘤家族肿瘤（ESFT）是影响儿童和青少年的骨和软组织癌症。 手术、放疗和多药化疗改善了患者的预后，但治疗平台期 局部和转移性病例均已达到。因此，迫切需要一种新颖的、有针对性的 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细胞依赖于FEN 1，FEN 1是一种加工冈崎5 '瓣的内切核酸酶， 在滞后链DNA合成期间的片段，用于活力。这一假设得到以下方面的支持：（a）四个基因组- 大规模的CRISPR文库筛选已经独立地发现ESFT细胞系对FEN 1高度敏感。 CRISPR;（B）据报道ESFT细胞在表型上是BRCA 1缺陷的，我们已经证明， BRCA 1/2缺陷型细胞对FEN 1抑制高度敏感;（c）我们发现了几种ESFT细胞系， 对FEN1-CRISPR、FEN1-siRNA和两种小分子FEN1抑制剂敏感。一个三人小组 主要研究者将共同指导该项目：RD Kolodner是一位遗传学家，也是FEN 1抑制剂的发明者 在专利申请文件中，JYJ Wang是一位癌症生物学家，擅长DNA损伤反应， SH Choo是一名儿科肿瘤学家，正在进行IRB对ESFT患者样本进行研究。在一起， 我们将追求四个具体目标来研究ESFT对FEN 1的依赖性。目标1：演示和 通过用经验证的siRNA对FEN 1进行基因消融来量化一组10个ESFT细胞系中的FEN 1-必需性 和CRISPR/CAS9。目的-2：确定小分子FEN 1抑制剂的细胞毒作用 通过短期生长和死亡试验以及2D中的长期集落形成试验对10种ESFT细胞系进行了研究。 3D文化我们将在ESFT细胞中编辑FEN 1以表达耐药FEN 1 R酶，以便 显示出目标效果。我们将评估FEN 1抑制剂之间的潜在协同相互作用 以及目前临床上用于治疗ESFT患者的化疗药物。目的3：研究 ESFT依赖于FEN 1的潜在机制，通过解决三个机制问题，即（a） ESFT的EWS-FLI 1癌蛋白诱导FEN 1依赖性，（B）FEN 1抑制剂引起不可逆阻断 在ESFT细胞中的DNA复制，和（c）FEN 1-抑制剂引起ESFT中的DNA断裂和有丝分裂灾难 细胞目的-4：研究FEN 1抑制剂对小鼠ESFT生长的影响。我们已经发现，一个FEN1- 抑制剂（SMD 154）减少了来自ESFT细胞系的原位异种移植物在无胸腺裸鼠中的生长。 SMD 154的药代动力学（PK）支持其在来自两种ESFT细胞系的原位异种移植物上的测试， 显示该化合物体外IC 50足够低。我们将构建4 - 6个ESFT患者来源的异种移植物（PDX） 活组织检查和切除的肿瘤。我们将测试SMD 154和未来的FEN 1抑制剂与优化的PK细胞上 来源于细胞系和患者的异种移植物。这项全面的临床前研究将产生详细的了解 ESFT对FEN 1的依赖性，并为开发FEN 1抑制剂治疗ESFT铺平了道路。

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.