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

尽管在过去的十年中，通过使用靶向特异性药物，多发性骨髓瘤（MM）的治疗有了令人印象深刻的改善，但仍然需要鉴定新的物质，因为在大多数情况下，MM仍然是一种不治之症。大多数MM患者甚至在多年后由于内在和获得性药理学抗性而复发。已经清楚地确定，异常Notch信号传导是MM中的发作、进展和耐药性的主要调节因子。Notch抑制的当前策略涉及（1）通过γ分泌酶抑制剂（GSI）阻断受体活化/裂解，其中Notch不是唯一的靶标，和（2）Notch 1定向的抗体。到目前为止，这些策略受到副作用的阻碍，如皮肤病和胃肠道毒性，最有可能是由于野生型Notch抑制非病变细胞。我们假设，这些负面副作用的一部分可能会避免选择性阻断关键下游Notch效应介导MM细胞的耐药性。本项目的目标是通过高通量的功能性shRNA筛选技术来鉴定和功能验证MM中尚未发现的可药用Notch靶点。在前期工作中，我们使用RNAseq分析来揭示Notch靶基因，无论是在具有低和诱导型Notch信号活性的工程MM细胞中，还是在通过GSI抑制Notch后高度Notch激活的MM细胞中。通过与Martin Eilers的团队合作，我们最近创建了一个特异性靶向MM细胞中Notch下游效应子的shRNA文库。在这个项目中，我们将从功能上筛选候选靶基因，以确定其在MM生存和对美法仑、硼替佐米和来那度胺（WP 1）等药物耐药性中的作用。Notch 1的两种不同的组成型活性形式（NdeltaE和NIC）将允许鉴定细胞质以及核相互作用伴侣（WP 1）。将通过使用化学抑制剂（小分子抑制剂）（如果可用）和通过纳米凝胶介导的MM细胞中靶特异性shRNA转移（WP 2）来实现shRNA筛选的关键效应子的功能验证。将在两种MM小鼠模型中测试体内反应：（1）我们新建立的BALB/c-MOPC 315.BM和（2）VK*MYC模型（WP 3）。为了定量测定骨疾病（骨小梁和皮质骨形态、密度和孔隙度），我们与加拿大蒙特利尔的Bettina Willie小组合作，使用体内microCT分析和常规组织形态测定法（WP 3）。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性的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.