Bypassing mutant p53 in radiation therapy

放射治疗中绕过突变型 p53

基本信息

批准号：
9068886
负责人：
Hongdau Peter Liu
金额：
$ 4.86万
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/9068886
关键词：
Aftercare Apoptosis Apoptotic Biological Markers Blinded Bypass CASP2 gene 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 Genetic study Health Hela Cells Human Ionizing radiation Libraries Malignant Neoplasms Mediating Methods Modeling Mutate Mutation PTEN gene Pathway interactions Pharmaceutical Preparations Phenotype Preclinical Drug Evaluation Process Radiation Radiation Tolerance Radiation therapy Radiation-Sensitizing Agents Radioresistance Radiosensitization Regulation Research Resistance Signal Transduction Specificity TP53 gene Testing Therapeutic Tumor Suppressor Proteins Zebrafish base cancer cell cancer therapy chemical genetics chemotherapy clinically relevant improved in vivo inhibitor/antagonist irradiation knock-down mutant neoplastic cell novel novel therapeutic intervention oncology patient stratification protein complex radiosensitizing resistance mechanism response screening small molecule small molecule libraries targeted treatment tumor

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：肿瘤抑制剂TP53的突变导致辐射后的凋亡逃避，对常规癌症治疗的潜在抗性，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来绕过这些改变。我们以前发现了一个新的凋亡过程，称为“ CHK1抑制”（CS）途径，该途径可在用CHK1抑制剂处理后恢复TP53突变细胞的放射敏感性（Sidi等人Cell。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途径需要组装一个称为Piddosom的蛋白质复合物，称为Piddosom，由PIDD，RAIDD和Caspase-2组成（Ando等人，Mol.Mol Cell 2012）2。该建议的重点是（1）鉴定进一步的遗传决定因素，以预测放射疗法中CHK1抑制剂疗效，以及（2）发现针对具有TP53突变肿瘤的新型放射增敏剂。利用人类癌细胞和斑马鱼，我们研究了除突变体p53以外的癌症改变对细胞凋亡敏感性的影响。我们发现，第二个通常是突变的肿瘤抑制剂PTEN中的突变阻塞了电离辐射（IR）和CHK1抑制（CHK1I）后的凋亡的执行。此外，我们将PTEN-AKT-IAP信号传导轴确定为促进对CS凋亡的抗性的候选途径。 PTEN的丧失似乎抑制了caspase-2激活下游的CS途径，因为尽管没有影响IR+CHK1I之后，但PTEN受损的HeLa细胞无法从事凋亡。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检验这一假设，以确定哪种IAP（如果有）负责caspase-2抑制。这些发现将通过斑马鱼中的化学和遗传研究在体内扩展。为了确定在TP53突变肿瘤中恢复放射性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，以执行640 FDA批准化合物的盲型表型放射敏感筛查。在p53突变的鱼类中，与15 Gy IR处理的药物的主要筛查与15 Gy IR相结合产生了140种化合物，能够产生与CHK1I相当的表型（即辐射后4天> 75％生病或死鱼）。到目前为止，我们的次要筛选与未辐照的胚胎鉴定了6种化合物，这些化合物证明了特定的放射敏作用。这些候选放射增敏剂的遗传特异性将通过临床相关性，提出的作用机理和功效来确认并确定优先级。所提出的靶标以及细胞死亡的机制将在细胞培养和体内确认。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。