Bypassing mutant p53 in radiation therapy

放射治疗中绕过突变型 p53

基本信息

批准号：
8716978
负责人：
Hongdau Peter Liu
金额：
$ 4.77万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-15 至 2017-06-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8716978
关键词：
Aftercare Apoptosis Apoptotic Biological Markers Blinded Bypass Cancer Patient Cancerous Cell Culture Techniques Cell Cycle Arrest Cell Death Cells Cleaved cell Clinical DNA DNA Damage Embryo FDA approved Fishes Genetic Genetic Determinism Hela Cells Human Ionizing radiation Libraries Malignant Neoplasms Mediating Methods Modeling Mutate Mutation PTEN gene Pathway interactions Patients Pharmaceutical Preparations Phenotype Preclinical Drug Evaluation Process Radiation Radiation Tolerance Radiation therapy Radiation-Sensitizing Agents Radioresistance Radiosensitization Regulation Research Resistance Signal Transduction Specificity Stratification TP53 gene Testing Therapeutic Tumor Suppressor Proteins Zebrafish base cancer cell cancer therapy caspase-2 chemical genetics chemotherapy clinically relevant improved in vivo inhibitor/antagonist irradiation mutant neoplastic cell novel novel therapeutic intervention oncology protein complex public health relevance resistance mechanism response screening small molecule small molecule libraries therapeutic target tumor

项目摘要

Project Summary Mutation of the tumor suppressor TP53 leads to the evasion of apoptosis after radiation, underlying resistance to conventional cancer treatments, such as radiation and chemotherapy. Our research seeks to bypass these alterations through the discovery of parallel cell-death pathways. We previously discovered a novel apoptotic process, termed "Chk1-suppressed" (CS) pathway, which restores radiosensitivity in TP53 mutant cells after treatment with Chk1 inhibitors (Sidi et al. Cell 2008)1. Using both zebrafish and mammalian TP53-mutant models, our lab has established that the CS pathway requires the assembly of a protein complex called the PIDDosome, composed of PIDD, RAIDD, and caspase-2 (Ando et al. Mol Cell 2012)2. This proposal focuses on (1) identifying further genetic determinants predicting Chk1 inhibitor efficacy in radiotherapy and (2) discovering novel radiosensitizers against tumors with TP53 mutation. Utilizing both human cancer cells and zebrafish, we investigated the impact of cancer alterations other than mutant p53 on cellular sensitivity to CS apoptosis. We found that a mutation in PTEN, the second most commonly mutated tumor suppressor, blocks the execution of apoptosis after ionizing radiation (IR) and Chk1 inhibition (Chk1i). Furthermore, we identified the PTEN-Akt-IAP signaling axis as a candidate pathway to promote resistance to CS apoptosis. The loss of PTEN appears to inhibit the CS pathway downstream of caspase-2 activation, as HeLa cells with impaired PTEN fail to engage in apoptosis, despite an unaffected ability to cleave caspase-2 following IR+Chk1i. Our hypothesis is that one or more IAPs act downstream of Akt to directly inhibit active caspase-2. I propose to test this hypothesis using a candidate knockdown approach to determine which IAP(s), if any, is responsible for caspase-2 inhibition. The findings will be extended in vivo through both chemical and genetic studies in zebrafish. To identify novel targeted strategies for restoring radiosensitivity in TP53 mutant tumors, we exploited the high-throughput drug screening capability of zebrafish to perform a blinded phenotypic radiosensitization screen of 640 FDA-approved compounds. A primary screen of drugs treated in combination with 15 Gy IR yielded 140 compounds capable of producing phenotypes comparable to Chk1i (i.e. >75% sick or dead fish 4 days post irradiation) in p53 mutant fish. Our secondary screen of irradiated vs non-irradiated embryos so far has identified 6 compounds that demonstrate specific radiosensitizing effects. The genetic specificity of these candidate radiosensitizers will be confirmed and prioritized by clinical relevance, proposed mechanism of action, and efficacy. The proposed targets, along with the mechanism(s) of cell death will be confirmed in cell culture and in vivo. The results of these findings may aid in rapidly benefiting cancer patients by proposing a new application for an already approved and available drug.

项目摘要肿瘤抑制剂TP53的突变导致辐射后的凋亡逃避对常规癌症治疗的耐药性，例如放射线和化学疗法。我们的研究试图通过发现平行细胞死亡途径绕过这些改变。我们以前发现了一个新型凋亡过程，称为“ CHK1抑制”（CS）途径，该途径恢复了TP53中的放射敏感性用CHK1抑制剂处理后的突变细胞（Sidi等，Cell 2008）1。使用斑马鱼和哺乳动物 TP53突变模型，我们的实验室已经确定CS途径需要组装蛋白质复合物称为PIDDOSMOM，由PIDD，RAIDD和CASPASE-2组成（Ando等人Cell 2012）2。这个建议重点是（1）识别预测CHK1抑制剂在放射疗法中的疗效的进一步遗传决定因素和（2）发现针对具有TP53突变的肿瘤的新型放射增敏剂。利用人类癌细胞和斑马鱼，我们研究了癌症改变的影响其他比突变p53对CS凋亡的细胞敏感性。我们发现PTEN中的突变是第二个突变通常突变的肿瘤抑制剂阻止电离辐射（IR）和CHK1后的凋亡执行抑制（CHK1I）。此外，我们将PTEN-AKT-IAP信号轴确定为促进对CS凋亡的抵抗力。 PTEN的丢失似乎抑制了下游的CS途径 caspase-2激活，因为尽管未受影响在IR+CHK1I之后切割caspase-2的能力。我们的假设是AKT下游的一个或多个IAP作用直接抑制活性caspase-2。我建议使用候选敲低方法来检验这一假设确定哪些IAP（如果有）负责caspase-2抑制。这些发现将在体内扩展通过斑马鱼中的化学和遗传研究。为了确定恢复TP53突变肿瘤中辐射敏感性的新型靶向策略，我们利用了斑马鱼的高通量药物筛查能力执行盲表型放射敏化 640个FDA批准化合物的屏幕。与15 GY IR相结合处理的药物的主要屏幕产生的140种化合物能够产生与CHK1I相当的表型（即> 75％生病或死鱼4 辐照后天数）p53突变鱼。到目前为止已经确定了6种表现出特定放射敏作用的化合物。这些的遗传特异性候选放射激素将通过临床相关性，提出的机制确认并优先考虑动作和功效。所提出的靶标以及细胞死亡的机制将在细胞中确认文化和体内。这些发现的结果可能有助于通过提出A 已批准和可用药物的新申请。