Novel DNA Repair Inhibitors for Cancer Therapy

项目概要/摘要。 我们寻求鉴定对癌细胞有选择性毒性的新型治疗剂， 使肿瘤对放疗或化疗敏感。我们发现一种细胞穿透性的狼疮衍生物 自身抗体（3E 10）增加癌细胞对放射和DNA靶向化疗的敏感性。 重要的是，3E 10本身对BRCA 2和PTEN缺陷型癌细胞是合成致死的，但在其他方面对BRCA 2和PTEN缺陷型癌细胞是合成致死的。 对培养物中的细胞或小鼠无毒。该抗体在人体测试时也没有显示出可检测的毒性 在狼疮患者的I期临床试验中作为假定的抗独特型疫苗。我们之前确定3E 10 是一个有效的抑制剂同源依赖性修复（HDR），我们现在已经确定了RAD 51作为 功能目标我们还发现，3E 10基于其在肿瘤组织中的作用而在体内被优先摄取。 细胞渗透机制，为进一步开发其用于癌症治疗提供了基础。这些 新的结果为增强3E 10的效力提供了基础（通过定向突变，亲和力成熟， 多价构建体），并通过鉴定合成的致死性的 相互作用（通过无偏的shRNA缺失筛选和对策展的癌细胞系的询问）和 确定与其他药物的协同作用，作为临床前动物肿瘤研究的前奏。我们预计 3E 10将对DNA修复和损伤反应途径缺陷的癌症具有合成致死性。 我们还开发了一种选择性靶向DNA修复抑制剂的策略， 酸性肿瘤微环境我们将使用pH低插入肽（pHLIP），其定向插入跨 细胞膜在低pH下，并提供货物选择性地进入体内肿瘤细胞。关注DNA-PK 非同源末端连接途径（NHEJ）的DNA修复，我们将建立在合作取得的进展， 致力于开发靶向肿瘤的反义和小分子DNA-PK抑制剂。我们接下来会合并 产生在γ位置修饰的γ PNA以增加与RNA的结合以获得有效的反义活性。这是 基于我们发表在《自然》杂志上的有希望的概念验证研究， pHLIP-PNA缀合物的活性。我们还将小分子DNA-PK抑制剂与pHLIP偶联， 利用由于正常组织毒性而尚未进入临床的有效分子， 赋予肿瘤选择性。这项工作将提供一个通用的平台，适用于其他DNA修复靶点。 我们最近发现了癌代谢物2-羟基戊二酸（2 HG）作为一种新的生物标志物， 人类恶性肿瘤中的DNA修复。我们发现，2 HG水平升高会导致BRCAness表型， 缺乏HDR，使癌细胞对PARP抑制剂和3E 10的合成致死杀伤敏感。 2 HG是由异柠檬酸脱氢酶-1和-2（IDH 1/2）突变的新变体活性产生的， 神经胶质瘤白血病和其他癌症我们将研究2 HG抑制DNA的机制 修复和识别可用于人类肿瘤治疗的漏洞。

Project Summary/Abstract. We seek to identify novel therapeutic agents that are selectively toxic to cancer cells and that specifically sensitize tumors to radiation or chemotherapy. We have discovered that a cell-penetrating, lupus-derived autoantibody (3E10) increases the sensitivity of cancer cells to radiation and to DNA-targeted chemotherapy. Importantly, 3E10, by itself, is synthetically lethal to BRCA2- and PTEN-deficient cancer cells, but is otherwise non-toxic to cells in culture or to mice. The antibody also showed no detectable toxicity in humans when tested in a phase I clinical trial in lupus patients as a putative anti-idiotype vaccine. We previously determined 3E10 to be a potent inhibitor of homology-dependent repair (HDR), and we have now identified RAD51 as the functional target. We have also found that 3E10 is preferentially taken up in tumor tissue in vivo based on its mechanism of cell penetration, providing a further basis to pursue its development for cancer therapy. These new results provide the basis to enhance the potency of 3E10 (by directed mutation, affinity maturation, and multi-valent constructs) and to rationally develop therapeutic strategies by identifying synthetic lethal interactions (via unbiased shRNA dropout screen and interrogation of curated cancer cell lines) and determining synergies with other agents, as a prelude to pre-clinical animal tumor studies. We expect that 3E10 will be synthetic lethal to cancers deficient in DNA repair and damage response pathways. We also have developed a strategy to selectively target DNA repair inhibitors to tumors by exploiting the acidic tumor microenvironment. We will use a pH low insertion peptide (pHLIP) that inserts directionally across cell membranes at low pH and delivers cargoes selectively into tumor cells in vivo. Focusing on DNA-PK in the non-homologous end-joining pathway (NHEJ) of DNA repair, we will build on advances made in collaborative work to develop tumor-targeted antisense and small molecule inhibition of DNA-PK. We will incorporate next generation γPNAs modified at the γ position to increase binding to RNA for potent antisense activity. This is based on our promising proof-of-concept studies published in Nature demonstrating the in vivo anti-tumor activity of pHLIP-PNA conjugates. We will also conjugate small molecule DNA-PK inhibitors to pHLIP, leveraging potent molecules that have not advanced to the clinic because of normal tissue toxicity, and conferring tumor selectivity. This work will provide a versatile platform to apply to other DNA repair targets. We have recently identified the oncometabolite, 2-hydroxyglutarate (2HG), as a new biomarker of deficient DNA repair in human malignancies. We found that elevated levels of 2HG confer a BRCAness phenotype of deficient HDR that renders cancer cells sensitive to synthetic lethal killing by PARP inhibitors and by 3E10. 2HG is produced by the neomorphic activity of isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations found in gliomas, leukemia, and other cancers. We will investigate the mechanism by which 2HG suppresses DNA repair and identify vulnerabilities that can be exploited for therapeutic gain in human tumors.