Bypassing mutant p53 in radiation therapy

描述（由申请人提供）：肿瘤抑制因子TP 53的突变导致放射后细胞凋亡的逃避，潜在的对常规癌症治疗如放射和化学疗法的抗性。我们的研究试图通过发现平行的细胞死亡途径来绕过这些改变。我们之前发现了一种新的凋亡过程，称为“Chk 1抑制”（CS）途径，其在用Chk 1抑制剂处理后恢复TP 53突变细胞的放射敏感性（Sidi et al. Cell 2008）1。使用斑马鱼和哺乳动物TP 53突变模型，我们的实验室已经确定CS途径需要组装称为PIDDosome的蛋白质复合物，由PIDD，RAIDD和半胱天冬酶-2组成（Ando et al. Mol Cell 2012）2。该建议的重点是（1）确定进一步的遗传决定因素预测Chk 1抑制剂在放射治疗中的疗效和（2）发现新的放射增敏剂对TP 53突变的肿瘤。 利用人类癌细胞和斑马鱼，我们研究了除了突变型p53以外的癌症改变对CS凋亡的细胞敏感性的影响。我们发现，第二个最常见的突变肿瘤抑制因子PTEN的突变，阻断电离辐射（IR）和Chk 1抑制（Chk 1 i）后细胞凋亡的执行。此外，我们确定了PTEN-Akt-IAP信号传导轴作为促进CS凋亡抗性的候选途径。PTEN的缺失似乎抑制了caspase-2激活的CS途径下游，因为具有受损的PTEN的HeLa细胞不能参与凋亡，尽管IR+ Chk 1 i后切割caspase-2的能力不受影响。我们的假设是一个或多个IAP作用于Akt下游，直接抑制活性caspase-2。我建议使用候选敲除方法来测试这一假设，以确定哪些IAP（如果有的话）负责caspase-2抑制。这些发现将通过斑马鱼的化学和遗传研究在体内扩展。 为了确定恢复TP 53突变肿瘤放射敏感性的新靶向策略，我们利用斑马鱼的高通量药物筛选能力对640种FDA批准的化合物进行盲法表型放射增敏筛选。对与15戈伊IR组合处理的药物的初步筛选产生了140种能够在p53突变鱼中产生与Chk 1 i相当的表型的化合物（即照射后4天>75%的病鱼或死鱼）。到目前为止，我们对辐照与非辐照胚胎的二次筛选已经确定了6种化合物，它们表现出特定的放射增敏作用。这些候选放射增敏剂的遗传特异性将通过临床相关性、拟定的作用机制和疗效进行确认和优先排序。将在细胞培养物和体内证实所提出的靶点沿着细胞死亡的机制。这些发现的结果可能有助于通过提出一种新的治疗方法来迅速使癌症患者受益。 已批准和可用药物的新申请。

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.