Synthetic lethality in VHL tumor suppressor null tumors

 描述(申请人提供)：合成致命性筛选最初由Hartwell、Friend和他的同事提出，由于它们能够识别选择性杀死肿瘤细胞而保留正常组织的药物，因此对癌症治疗具有很大的希望。这一方法假定，携带特定突变的癌细胞将比没有突变的正常细胞对某些途径的操纵更加敏感。Von Hippel-Lindau(VHL)抑癌基因在约90%的肾透明细胞癌(CC-RCC)中发生突变或丢失。由于缺乏VHL的CC-RCC抵抗现有的治疗方法并经常转移，因此寻找有效的新疗法对CC-RCC患者的治疗至关重要。通过对药理活性化合物文库的筛选，我鉴定了七个化合物，它们选择性地杀死VHL缺陷的CC-RCC。重要的是，这些化合物结合和失活的分子靶标是已知的。这种方法是创新的，因为它基于一种药物筛选，旨在不仅识别化合物，而且还识别它们各自的蛋白质靶标，这些靶标专门影响VHL缺陷的CC-RCC细胞。在这项应用中，我们将重点关注两种化合物(Y-27632和RKI-1447)，它们显示出最强的合成致死性和VHL缺乏症。由于这两个化合物都被报道针对Rho相关蛋白激酶(ROCK1&2)，我们进行了体外RNA干扰实验，并证实了ROCK1的下调而不是ROCK2的下调是VHL缺陷型CC-RCC死亡的关键。我们假设，在体内靶向ROCK通路将导致对VHL缺陷肿瘤的抑制，对正常组织的毒性最小。我们进一步假设，VHL缺失下游的低氧诱导因子(HIF)通路的激活对于观察到的合成致死效应是重要的。我们将通过以下具体目标来检验这些假说：(1)评估ROCK抑制在CC-RCC原位小鼠模型中的体内疗效；(2)确定ROCK抑制在CC-RCC中是否具有缺氧诱导因子(HIF)途径的综合致命性。重要的是，所有VHL缺陷的CC-RCC患者可以分为两组：同时表达HIF1和HIF2的肿瘤和仅表达HIF2的肿瘤。如果观察到的合成致死率是由HIF2介导的，则所有VHL缺陷患者将预期对ROCK靶向治疗有反应；如果观察到的合成致死率是由HIF1+HIF2介导的，则其肿瘤缺乏HIF1的患者预计对ROCK靶向治疗的反应较差。因此，关键是要确定合成致死率是否取决于HIF，如果是，它取决于HIF1、HIF2，还是两者兼而有之。因此，当前研究的目标是解决岩石抑制剂作为候选患者治疗药物的效用，并确定将预测患者是否将从岩石抑制中受益的潜在生物标记物。 上述两个目标都有望在临床上具有重要意义，并对CC-RCC的癌症治疗产生重大影响。

 DESCRIPTION (provided by applicant): Synthetic-lethality screens, initially proposed by Hartwell, Friend and colleagues, hold great promise for cancer therapy since they enable the identification of drugs that selectively kill tumor cells while sparing normal tissue. This approac presumes that cancer cells harboring a specific mutation will be more sensitive to manipulation of certain pathways than normal cells that lack the mutation. The Von Hippel-Lindau (VHL) tumor-suppressor gene is mutated/lost in about 90% of Clear Cell Renal Cell Carcinomas (CC-RCCs). Because VHL-deficient CC-RCCs resist current therapies and frequently metastasize, identifying effective new therapies will be crucial for treatment of CC-RCC patients. By screening of the Library of Pharmacologically Active Compounds, I identified seven compounds that selectively kill VHL-deficient CC-RCC. Importantly the molecular targets that these compounds bind and inactivate are already known. This approach is innovative since it is based on a drug screen designed to identify not only compounds but also their respective protein targets that specifically affect VHL-deficient CC-RCC cells. In this application we will focus on two compounds (Y-27632 and RKI-1447) that display the strongest synthetic lethality with VHL deficiency. Since both of these compounds were reported to target Rho-associated protein kinases (ROCK1&2), we performed in vitro RNA-interference experiments and verified that down regulation of ROCK1, but not ROCK2, is critical for VHL-deficient CC-RCC death. We hypothesize that targeting the ROCK pathway in vivo will lead to suppression of VHL-deficient tumors with minimal toxicity to normal tissues. We further hypothesize that the activation of the Hypoxia-Inducible Factor (HIF) pathway downstream of VHL loss is important for the observed synthetic-lethal effect. We will test these hypotheses through the following specific aims: (1) To assess the in vivo efficacy of ROCK inhibition in an orthotopic mouse model of CC-RCC; (2) To determine if ROCK inhibition is synthetically lethal with Hypoxia-Inducible Factor (HIF) pathway in CC-RCC. Importantly, all VHL-deficient CC-RCC patients can be divided to two groups: those with tumors expressing both HIF1 and HIF2, and those with tumors expressing HIF2 only. If the observed synthetic lethality is mediated by HIF2, all VHL-deficient patients would be expected to respond to ROCK-targeted therapies; if the observed synthetic lethality is mediated by HIF1+HIF2, patients whose tumors lack HIF1 would be expected to be less responsive to ROCK-targeted therapies. It is therefore critical to determine whether the synthetic lethality depends on HIF, and if so, whether it depends on HIF1, HIF2, or both. Thus, the objective of the current study is to address the utility of ROCK inhibitors as candidate patient therapeutics and identify potential biomarkers that will predict whether patients will benefit from ROCK inhibition. Both of the above aims are expected to be clinically important and have a major impact on cancer therapies for CC-RCC.