Novel DNA Repair Inhibitors for Cancer Therapy

项目摘要/摘要。 我们寻求寻找对癌细胞有选择性毒性的新型治疗剂，并特别 使肿瘤对放射或化疗敏感。我们发现了一种穿透细胞的狼疮来源 自身抗体(3E10)增加了癌细胞对辐射和DNA靶向化疗的敏感性。 重要的是，3E10本身对BRCA2和PTEN缺陷的癌细胞是合成致死的，但不是这样 对培养中的细胞或小鼠无毒。该抗体在测试时也没有在人体内检测到毒性。 在狼疮患者的I期临床试验中，作为假定的抗独特型疫苗。我们之前确定了3E10要 是同源依赖修复(HDR)的有效抑制因子，我们现在已经确定RAD51是 功能目标。我们还发现，3E10在体内的肿瘤组织中优先被摄取，基于其 细胞穿透机制，为进一步研究其在肿瘤治疗中的应用提供了基础。这些 新的结果为提高3E10(通过定向突变、亲和力成熟和 多价结构)，并通过识别合成致死物来合理地开发治疗策略 相互作用(通过无偏见的shRNA缺失筛选和对经过策划的癌细胞株的询问)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。我们期待着 3E10将对DNA修复和损伤反应途径缺乏的癌症产生合成致死作用。 我们还开发了一种策略，通过利用DNA修复抑制剂来选择性地将DNA修复抑制物靶向肿瘤 酸性肿瘤微环境。我们将使用一种pH低的插入肽(PHLIP)，它可以定向插入到 细胞在低pH值下被膜，并选择性地将货物运送到体内的肿瘤细胞中。关注DNA-PK DNA修复的非同源末端连接途径(NHEJ)，我们将建立在协作方面的进展 致力于开发肿瘤靶向的反义和小分子抑制DNA-PK。我们将合并NEXT 世代γ核酸在γ位置进行修饰，以增加与RNAs的结合，从而具有强大的反义活性。这是 基于我们在《自然》杂志上发表的有希望的概念验证研究，证明了体内抗肿瘤 PHLIP-PNA偶联物的活性。我们还将小分子DNA-PK抑制剂偶联到PHLIP上， 利用由于正常组织毒性而尚未进入临床的有效分子，以及 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。 我们最近发现了一种新的生物标志物--2-羟基戊二酸(2HG)。 人类恶性肿瘤中的DNA修复。我们发现，2HG水平的升高赋予了BRCAness表型 HDR缺陷，使癌细胞对PARP抑制剂和3E10的合成致命杀伤敏感。 2HG是由异柠檬酸脱氢酶-1和异柠檬酸脱氢酶-2(IDH1/2)突变的新构象活性产生的 胶质瘤、白血病和其他癌症。我们将研究2HG抑制DNA的机制 修复和识别可用于治疗人类肿瘤的漏洞。

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.