Development of Small Molecule Crm-1 Inhibitor for Pancreatic Cancer Therapy

描述(申请人提供)：胰腺癌是一种致命的疾病，每30分钟就有两名美国人丧生(年死亡率33,000人)。标准化疗和新开发的靶向治疗的失败使总体存活率(5%)的改善停滞不前，这表明迫切需要更新的策略来管理这种疾病。为了配合这项射频治疗的目标，即确定潜在的分子靶点，并测试新的治疗策略，我们打算调查治疗潜力。 我们新开发的选择性核输出抑制剂(SINE)在胰腺癌中靶向CRM-1(染色体区域维护1)的作用。CRM-1是核转运受体Importin超家族的一员，识别含有富含亮氨酸的核输出序列(NES)的蛋白质，是核外输出蛋白质的主要受体。CRM-1的靶标之一是肿瘤抑制蛋白(TSP)、前列腺癌细胞凋亡反应-4(PAR-4)。早些时候，我们曾建议将PAR-4作为胰腺癌的潜在治疗靶点，因为这种TSP的下调与总体生存率的降低直接相关。CRM-1对PAR-4的核排斥作用使癌细胞对凋亡产生抵抗。我们的初步研究结果表明，Sine(KPT-185和临床候选KPT-251)可以通过抑制CRM-1而将PAR-4锁定在胰腺癌细胞核中，从而导致癌细胞选择性凋亡和抗肿瘤作用。令我们惊讶的是，正常细胞对Sine没有反应，这似乎与低蛋白激酶A(PKA)导致的低PAR-4表达和不明显的PAR-4磷酸化有关。因此，我们假设，靶向抑制CRM-1是一种有吸引力的策略，可以在细胞核内引导TSP(特别是PAR-4)，诱导胰腺癌特异性细胞凋亡。基于我们的初步发现，为了验证我们的假设，我们提出了1.在一组表达不同的PKA和PAR-4的胰腺癌细胞系中，确定PAR-4的核定位与KPT-185和KPT-251的生长抑制和诱导凋亡活性是否存在直接关系，并确定敲除PKA是否可以通过PAR-4和其他机制导致对KPT介导的杀伤的抵抗。2.评价KPT-251(MTD的临床候选药物)对不同PAR-4表达(高PAR-4PKA与低PAR-4PKA)胰腺细胞系建立的异种移植动物模型的体内疗效。影响：CRM-1是治疗胰腺癌的可用药靶点，然而，目前还没有针对这种有害的核出口因子的药物。早先开发CRM-1抑制剂的尝试没有成功，天然产物Leptomycin B由于严重毒性在一次临床试验中失败就是例证。从那时起，该领域没有看到任何认真的尝试，开发可用于临床治疗胰腺癌的新型CRM-1抑制剂。我们新开发的KPT-Sine是高度特异的口服活性药物，具有出色的药代动力学参数，有望用于治疗致命的胰腺癌。

DESCRIPTION (provided by applicant): Pancreatic cancer is a deadly disease that takes two American lives every 30 minutes (annual mortality >33,000). Failure of standard chemotherapies and newly developed targeted therapies has stagnated any improvement on the overall dismal survival (<5%) indicating that newer strategies for the management of this disease are urgently needed. In line with the goals of this RFA i.e. to identify potential molecula targets, and to test new therapeutic strategies, we intend to investigate the therapeutic potential of targeting CRM-1 (chromosome region maintenance 1) by our newly developed selective inhibitors of nuclear export (SINE) in pancreatic cancer. CRM-1 is a member of the importin superfamily of nuclear transport receptors, recognizing proteins bearing a leucine-rich nuclear export sequence (NES) and is the major receptor for the export of proteins out of the nucleus. Among the target of CRM-1 is the tumor suppressor protein (TSP) prostate apoptosis response-4 (Par-4). Earlier we had proposed Par-4 as a potential therapeutic target in pancreatic cancer since downregulation of this TSP has been directly linked to poor overall survival. Nuclear exclusion of Par-4 by CRM-1 renders the cancer cells resistant to apoptosis. Our preliminary findings demonstrate that SINE (KPT-185 and clinical candidate KPT-251) can lock Par-4 in pancreatic cancer cell nucleus by inhibiting CRM-1 that leads to cancer cell selective apoptosis and anti-tumor effects. To our surprise normal cells did not respond SINEs and this emerges to be mechanistically co-related to low Par-4 expression and insignificant Par-4 phosphorylation due to low protein kinase A (PKA). Hence we hypothesize that targeted inhibition of CRM-1 is an attractive strategy to guide TSPs (specifically Par-4) in the nucleus, inducing pancreatic cancer specific apoptosis. Based on our provocative initial findings and to test our hypothesis we propose 1. To establish whether there is a direct relationship between Par-4 nuclear localization and growth inhibitory and apoptosis inducing activity of KPT-185 and KPT-251 in a panel of pancreatic cancer cell lines with differential PKA and Par-4 expression and establish whether knockdown of PKA can cause resistance to KPT-mediated killing through Par-4 and other mechanisms. 2. Assess the in vivo efficacy of KPT- 251 (clinical candidate at MTD) in animal xenograft mice models developed from pancreatic cell lines with differential Par-4 expression (high Par-4 PKA vs low Par-4 PKA). Impact: CRM-1 is a druggable target in pancreatic cancer, however, currently there is no existing drug targeting this detrimental nuclear exporter. Earlier attempts to develop CRM-1 inhibitor were not successful as exemplified by the failure of the natural product Leptomycin B in a single clinical trial due to severe toxicity. Sinc then the field has not witnessed any serious attempts to develop newer classes of CRM-1 inhibitors that could be used clinically for the treatment of pancreatic cancer. Our newly developed KPT-SINE's are highly specific, orally active drugs with excellent pharmacokinetic parameters and hold promise against deadly pancreatic cancer.