Functional validation of novel drug targets in multiple myeloma by use of a targeted shRNA library

使用靶向 shRNA 文库对多发性骨髓瘤中的新药物靶点进行功能验证

• 批准号: 370022528
• 负责人: Professorin Dr. Franziska Jundt
• 金额: --
• 依托单位: Medizinische Klinik und Poliklinik II
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/370022528?language=en
• 关键词: Functional; validation; novel; drug; targets

Despite impressive improvement in the treatment of multiple myeloma (MM) during the past decade through the use of target-specific agents, there is still a need for the identification of novel substances since in most cases MM remains an incurable disease. Most MM patients suffer from relapses even after many years due to intrinsic and acquired pharmacological resistance. It has been clearly established that aberrant Notch signaling is a master regulator of onset, progression and drug resistance in MM. Current strategies for Notch inhibition involve (1) blockade of receptor activation/cleavage through gamma secretase inhibitors (GSI) of which Notch is no exclusive target and (2) Notch1-directed antibodies. Thus far these strategies have been hampered by side effects such as skin disorders and gastrointestinal toxicities most probably due to wild-type Notch inhibition in nondiseased cells. We hypothesize that parts of these negative side effects might be avoided by selective blocking of critical downstream Notch effectors which mediate drug resistance in MM cells. The goal of this project is the identification and functional validation of yet unknown druggable Notch targets in MM by use of a high-throughput functional shRNA screening technology.In preliminary work we performed gene expression profiling employing RNAseq analysis to reveal Notch target genes both in engineered MM cells with low and inducible Notch signaling activity and in highly Notch-activated MM cells after Notch inhibition through GSI. In collaboration with Martin Eilers´s group we most recently generated a shRNA library that specifically targets Notch downstream effectors in MM cells. In this project we will now functionally screen candidate target genes for a role in MM survival and resistance to drugs such as melphalan, bortezomib and lenalidomide (WP1). Two different constitutively active forms of Notch1 (NdeltaE and NIC) will allow for identification of cytoplasmic as well as nuclear interaction partners (WP1). Functional validation of critical effectors of the shRNA screens will be achieved by use of chemical inhibitors (small molecule inhibitors), if available, and by nanogel-mediated transfer of target-specific shRNAs in MM cells (WP2). In-vivo response will be tested in two MM mouse models: (1) our newly established BALB/c-MOPC315.BM and (2) the VK*MYC model (WP3). For quantitative determination of bone disease (trabecular and cortical bone morphology, density and porosity) we use in collaboration with Bettina Willie´s group in Montreal (Canada) in-vivo microCT analysis and conventional histomorphometry (WP3). The knowledge gained from these experiments will aid in the development of novel strategies for treating MM patients with intrinsic and acquired drug resistance.

尽管在过去的十年里，通过使用靶向特异性药物治疗多发性骨髓瘤(MM)取得了令人印象深刻的进步，但仍然需要识别新的物质，因为在大多数情况下，MM仍然是一种不治之症。大多数多发性骨髓瘤患者由于内在和获得性药理耐药，即使在多年后也会复发。已明确地证实，异常的Notch信号是MM发病、进展和耐药的主要调节因素。目前抑制Notch的策略包括：(1)通过伽玛分泌酶抑制剂(GSI)阻断受体的激活/裂解，而Notch不是其唯一靶点；(2)Notch1导向的抗体。到目前为止，这些策略一直受到皮肤病和胃肠道毒性等副作用的阻碍，最可能的原因是未患病细胞中的野生型Notch抑制。我们推测，通过选择性地阻断介导MM细胞耐药的关键下游Notch效应器，可以避免这些负面副作用的一部分。该项目的目标是利用高通量功能性shRNA筛选技术鉴定和功能验证MM中未知的可用药的Notch靶点。在初步工作中，我们使用RNAseq分析进行基因表达谱分析，以揭示具有低Notch信号活性的工程化MM细胞中的Notch靶标基因，以及通过GSI抑制Notch信号活性后的高度Notch激活的MM细胞中的Notch靶标基因。在与Martin Eilers的S团队的合作中，我们最近创建了一个针对MM细胞Notch下游效应子的shRNA文库。在这个项目中，我们现在将从功能上筛选在多发性骨髓瘤存活和对马法兰、波替佐米和来那度胺(WP1)等药物耐药方面起作用的候选靶基因。两种不同的Notch1组成活性形式(NdeltaE和Nic)将允许识别细胞质和核相互作用伙伴(WP1)。ShRNA筛选的关键效应器的功能验证将通过使用化学抑制剂(如果有的话)和纳米凝胶介导的MM细胞中靶向特异性shRNA的转移(WP2)来实现。体内反应将在两个MM小鼠模型上进行测试：(1)我们新建立的BALB/c-MOPC315.BM和(2)VK*MYC模型(WP3)。为了定量确定骨骼疾病(骨小梁和皮质骨的形态、密度和孔隙率)，我们与加拿大蒙特利尔的Bettina Willie的S团队合作，使用体内显微CT分析和传统的组织形态计量学(WP3)。从这些实验中获得的知识将有助于开发治疗具有内在和获得性耐药的多发性骨髓瘤患者的新策略。