Bypassing mutant p53 in radiation therapy

描述（申请人提供）：肿瘤抑制因子TP53突变导致放疗后逃避细胞凋亡，对常规癌症治疗，如放疗和化疗产生耐药性。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“Chk1抑制”（CS）途径，该途径在Chk1抑制剂治疗后恢复TP53突变细胞的放射敏感性（Sidi等）。细胞2008)1。通过使用斑马鱼和哺乳动物的tp53突变模型，我们的实验室已经确定CS通路需要一种名为PIDDosome的蛋白质复合物的组装，PIDDosome由PIDD、RAIDD和caspase-2组成（Ando等）。《Mol Cell》（2012）本提案的重点是(1)确定预测Chk1抑制剂在放疗中的疗效的进一步遗传决定因素；(2)发现针对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p53突变体以外的癌症改变对细胞CS凋亡敏感性的影响。我们发现，在电离辐射（IR）和Chk1抑制（Chk1i）后，PTEN（第二常见突变的肿瘤抑制因子）的突变阻断了细胞凋亡的执行。此外，我们发现PTEN-Akt-IAP信号轴是促进CS细胞凋亡抗性的候选途径。PTEN的缺失似乎抑制了caspase-2激活下游的CS途径，因为PTEN受损的HeLa细胞无法参与凋亡，尽管在IR+Chk1i后切割caspase-2的能力不受影响。我们的假设是一种或多种IAPs作用于Akt的下游，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，以确定哪种IAP(s)（如果有的话）对caspase-2抑制负责。这些发现将通过斑马鱼的化学和遗传研究在体内得到扩展。为了确定恢复TP53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批准的化合物进行了盲法表型放射增敏筛选。与15 Gy IR联合处理的药物的初步筛选产生了140种化合物，这些化合物能够在p53突变鱼中产生与Chk1i相似的表型（即辐射后4天内有75%的鱼生病或死亡）。到目前为止，我们对辐照和未辐照胚胎的二次筛选已经确定了6种显示出特定放射致敏效应的化合物。这些候选放射增敏剂的遗传特异性将通过临床相关性、拟议的作用机制和疗效来确认和优先考虑。提出的靶点以及细胞死亡的机制将在细胞培养和体内得到证实。这些发现的结果可能有助于迅速使癌症患者受益

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.