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

尽管在过去十年中，通过使用靶向药物，多发性骨髓瘤（MM）的治疗取得了令人印象深刻的进步，但由于在大多数情况下，MM仍然是一种无法治愈的疾病，因此仍然需要鉴定新的物质。由于固有的和获得性的药物耐药性，大多数MM患者即使在多年后也会复发。已经明确地证实，异常的Notch信号是MM发病、进展和耐药的主要调节因子。目前抑制Notch的策略包括：(1)通过γ分泌酶抑制剂（GSI）阻断受体激活/切割，而Notch并不是其唯一的靶点；(2)notch1定向抗体。到目前为止，这些策略一直受到副作用的阻碍，如皮肤疾病和胃肠道毒性，这很可能是由于野生型Notch抑制在未患病细胞中。我们假设，通过选择性阻断介导MM细胞耐药的关键下游Notch效应物，可以避免部分这些负面副作用。该项目的目标是利用高通量功能性shRNA筛选技术鉴定MM中未知的可药物Notch靶点并进行功能验证。在初步工作中，我们使用RNAseq分析进行了基因表达谱分析，以揭示Notch信号活性低和诱导的工程MM细胞以及通过GSI抑制Notch后高度激活的MM细胞中的Notch靶基因。在与Martin Eilers团队的合作中，我们最近生成了一个shRNA文库，专门针对MM细胞中的Notch下游效应物。在这个项目中，我们现在将对候选靶基因进行功能筛选，以确定其在MM存活和对美法仑、硼替佐米和来那度胺（WP1）等药物的耐药性中的作用。Notch1的两种不同组成活性形式（NdeltaE和NIC）将允许鉴定细胞质和核相互作用伙伴（WP1）。shRNA筛选的关键效应物的功能验证将通过使用化学抑制剂（小分子抑制剂）（如果有的话）和纳米凝胶介导的靶特异性shRNA在MM细胞中的转移（WP2）来实现。体内反应将在两种MM小鼠模型中进行测试：(1)我们新建立的BALB/c-MOPC315。(2) VK*MYC模型（WP3）。为了定量确定骨病（骨小梁和骨皮质形态、密度和孔隙度），我们与加拿大蒙特利尔的Bettina Willie团队合作，使用体内微ct分析和常规组织形态测定法（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MM患者的新策略。

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.