Proteasome Inhibition and ER Stress

描述（由申请人提供）：基于蛋白酶体抑制剂（PI）的联合化疗目前正在评估胰腺癌和其他实体恶性肿瘤的治疗。我们的假设是PIs在癌细胞中引起内质网（ER）应激，这种应激介导细胞杀伤。此外，我们已经获得了初步证据，表明pi对细胞死亡的影响是高度异质性的，并且与它们是否诱导eIF21磷酸化有关，eIF21是未折叠蛋白反应（UPR）的一个组成部分，介导抑制全局蛋白合成。具体来说，在对pi诱导的细胞凋亡具有相对抗性的细胞系中，pi促进了eIF21磷酸化的强烈磷酸化，但在最敏感的细胞系中，它们不能这样做（或减弱翻译）。确定这种异质性的生化基础可以前瞻性地识别最有可能对基于pi的联合化疗有反应的肿瘤，并应该为治疗干预提供新的靶点。我们还希望更好地定义一种最有前途的基于pi的联合方案，即硼替佐米与组蛋白去乙酰化酶（HDAC）抑制剂的联合，所诱导的细胞凋亡的分子机制。我们已经获得了良好的初步证据，表明HDAC抑制剂通过破坏被称为聚合体的细胞保护结构来促进蛋白酶体抑制剂介导的细胞凋亡，而聚合体似乎具有减轻内质网应激的功能。基因沉默研究已经证明，导致聚合体破坏的HDAC是HDAC6，并且更有选择性的HDAC6抑制剂可能会产生与泛HDAC抑制剂（如SAHA）相当或更好的肿瘤细胞杀伤效果，而且毒性更小。为了直接检验我们的假设，我们提出以下具体目标。(1)明确硼替佐米诱导eIF21磷酸化的分子机制。我们将验证pi通过诱导分子伴侣（HSP70?）的表达来抑制PERK激活的假设，该分子伴侣（HSP70?）在药物敏感细胞中阻断PERK同质聚集；(2)确定内质网应激在pi诱导的细胞凋亡中的作用。在这里，我们将评估ROS、Ca2+、JNK、Noxa和caspase-4对pi诱导的细胞凋亡的贡献；(3)：确定PIs与HDAC抑制剂联合治疗异种移植物的毒性和抗肿瘤效果。我们将比较PIs与SAHA（一种泛HDAC抑制剂）、tubacin （hdac6选择性）或SNDX-275 （I型HDAC特异性）联合治疗在体外和来自敏感和耐药细胞系的原位肿瘤中的效果。我们还将研究是否可以在这些动物的外周血中测量药物-靶标相互作用和生物反应的药效学标志物，并在胰腺癌患者的II期临床试验中应用这些方法来测量硼替佐米加SAHA治疗的效果。公共卫生相关性：临床试验已经证明，常规和研究性癌症治疗的效果是明显不同的，目前正在努力将治疗与对它们的反应性相关的特定分子特征相匹配，以更好地利用它们的效果（“个性化医疗”）。这里提出的研究的总体目标是更好地了解两种有前途的研究癌症疗法（蛋白酶体抑制剂和组蛋白去乙酰化酶抑制剂）诱导细胞杀伤的机制，以便在临床前模型中注意到这些药物的有希望的效果可以在患者中得到最好的利用。我们将直接比较两种蛋白酶体抑制剂和三种HDAC抑制剂的效果，这些抑制剂要么已经获得fda批准，要么正在临床试验中进行评估，要么正在开发用于人类临床应用。在我们的第三个特定目标中，我们将开发和应用药效学分析来测量药物-靶标相互作用，以及在患者II期临床试验背景下有效药物靶向的生物学后果。

DESCRIPTION (provided by applicant): Proteasome inhibitor (PI)-based combination chemotherapy is currently being evaluated for the treatment of pancreatic cancer and other solid malignancies. Our hypothesis is that PIs cause endoplasmic reticular (ER) stress in cancer cells and this stress mediates cell killing. Furthermore, we have obtained preliminary evidence that the effects of PIs on cell death are highly heterogeneous and are linked to whether or not they induce phosphorylation of eIF21, a component of the unfolded protein response (UPR) that mediates suppression of global protein synthesis. Specifically, PIs promote strong phosphorylation of eIF21 phosphorylation in the cell lines that are relatively resistant to PI-induced apoptosis, but they fail to do so (or attenuate translation) in the cell lines that are most sensitive. Identifying the biochemical basis for this heterogeneity could enable the prospective identification of tumors that are most likely to respond to PI-based combination chemotherapy and should yield new targets for therapeutic intervention. We also wish to better define the molecular mechanisms involved in the apoptosis that is induced by one of the most promising PI-based combination regimens, namely, the combination of bortezomib plus histone deacetylase (HDAC) inhibitors. We have obtained good preliminary evidence that HDAC inhibitors promote proteasome inhibitor-mediated apoptosis by disrupting cytoprotective structures known as aggresomes that appear to function to alleviate ER stress. Gene silencing studies have demonstrated that the HDAC responsible for aggresome disruption is HDAC6, and it is possible that more selective HDAC6 inhibitors will yield comparable or better tumor cell killing than pan HDAC inhibitors (like SAHA) with less toxicity. To directly test our hypotheses we propose the following Specific Aims. (1) Define the molecular mechanisms that control bortezomib-induced phosphorylation of eIF21. We will test the hypothesis that PIs inhibit PERK activation by inducing the expression of a molecular chaparone (HSP70?) that blocks PERK homoaggregation in drug-sensitive cells; (2) Determine role of ER stress in PI-induced apoptosis. Here we will assess the contributions of ROS, Ca2+, JNK, Noxa, and caspase-4 ot PI-induced apoptosis; (3): Determine the toxicity and anti-tumor efficacy of combination therapy with PIs and HDAC inhibitors in xenografts. We will compare the effects of combination therapy with PIs plus SAHA (a pan HDAC inhibitor), tubacin (HDAC6-selective), or SNDX-275 (type I HDAC-specific) in vitro and in orthotopic tumors derived from sensitive and resistant cell lines. We will also investigate whether or not pharmacodynamic markers of drug-target interaction and biological response can be measured in the peripheral blood of these animals and apply these methods to measure the effects of therapy with bortezomib plus SAHA within the context of a Phase II clinical trial in patients with pancreatic cancer. PUBLIC HEALTH RELEVANCE: Clinical trials have demonstrated that the effects of conventional and investigational cancer therapies are remarkably heterogeneous, and efforts are currently underway to match therapies to the specific molecular features associated with responsiveness to them in an effort to better exploit their effects ("personalized medicine"). The overall goal of the research proposed here is to better understand the mechanisms involved in cell killing induced by two promising classes of investigational cancer therapies (proteasome inhibitors and histone deacetylase inhibitors) so that the promising effects of these drugs that have been noted in preclinical models can be best exploited in patients. We will be directly comparing the effects of two proteasome inhibitors and three HDAC inhibitors that are either already FDA-approved, being evaluated in clinical trials, or being developed for clinical application in humans, and in our third Specific Aim we will develop and apply pharmacodynamic assays to measure drug-target interactions and the biological consequences of effective drug targeting within the context of a Phase II clinical trial in patients.