Synthetic lethality in VHL tumor suppressor null tumors

 描述（由申请人提供）：最初由Hartwell，Friend及其同事提出的合成致死性筛选对癌症治疗有很大的希望，因为它们能够鉴定选择性杀死肿瘤细胞同时保留正常组织的药物。这种方法假定携带特定突变的癌细胞将比缺乏突变的正常细胞对某些途径的操纵更敏感。Von Hippel-Lindau（VHL）肿瘤抑制基因在约90%的透明细胞肾细胞癌（CC-RCC）中突变/丢失。由于VHL缺陷的CC-RCC抵抗当前的治疗并且经常转移，因此确定有效的新疗法对于CC-RCC患者的治疗将是至关重要的。通过筛选药理活性化合物库，我鉴定了7种选择性杀死VHL缺陷型CC-RCC的化合物。重要的是，这些化合物结合和抑制的分子靶标是已知的。这种方法是创新的，因为它是基于药物筛选设计，以确定不仅化合物，而且其各自的蛋白质目标，具体影响VHL缺陷CC-RCC细胞。在本申请中，我们将重点关注两种化合物（Y-27632和RKI-1447），这两种化合物在VHL缺陷时显示出最强的合成致死性。由于这两种化合物都被报道靶向Rho相关蛋白激酶（ROCK1和2），我们进行了体外RNA干扰实验，并验证了ROCK1而不是ROCK2的下调对VHL缺陷型CC-RCC死亡至关重要。我们假设，在体内靶向ROCK通路将导致VHL缺陷型肿瘤的抑制，对正常组织的毒性最小。我们进一步假设，VHL损失下游的缺氧诱导因子（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的来检验这些假设：（1）评估ROCK抑制在CC-RCC原位小鼠模型中的体内功效;（2）确定ROCK抑制在CC-RCC中是否与缺氧诱导因子（HIF）通路一起是合成致死的。重要的是，所有VHL缺陷的CC-RCC患者可以分为两组：肿瘤同时表达HIF 1和HIF 2的患者，以及肿瘤仅表达HIF 2的患者。如果观察到的合成致死性由HIF 2介导，则预期所有VHL缺陷患者对ROCK靶向治疗有反应;如果观察到的合成致死性由HIF 1 + HIF 2介导，则预期肿瘤缺乏HIF 1的患者对ROCK靶向治疗的反应较低。因此，确定合成致死性是否取决于HIF至关重要，如果是，它是否取决于HIF 1，HIF 2或两者。因此，本研究的目的是解决ROCK抑制剂作为候选患者治疗剂的效用，并鉴定将预测患者是否将从ROCK抑制中获益的潜在生物标志物。 上述两个目标预计将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.