Systemic shRNA screens and high throughout sequencing to investigate pathway dependence in multiple myeloma

这个中心项目是基于观察，已经证明不同的多发性骨髓瘤(MM)在单个信号转导通路的激活模式和依赖方面存在差异；具体地说，该工作定义了显示Akt通路激活并依赖Akt生长和生存的MMS，以及一组单独的以Akt不依赖的方式生长的MMS。这项工作提出了两个问题：(A)哪些信号转导通路对Akt非依赖性MMS至关重要？(B)哪些突变驱动Akt的激活(以及Akt非依赖性MMS中的其他关键信号通路)？为了解决这些问题，这个Z项目将采取两种策略：第一，我们将开发允许功能筛查搜索对不同亚型多发性骨髓瘤生存至关重要的基因的技术。为了实现这一点，我们将采用测量慢病毒shRNA载体种群中每个单独shRNA载体的频率的技术。然后，我们将用一组这样的shRNA载体(最初针对所有的激酶)感染代表每个亚群的白血病细胞系，并测量每个载体在体外扩增前后的频率，最终在受感染细胞移植后的体内。通过这种方式，我们将确定针对特定基因的载体在不同MMS的克隆扩增过程中是否会得到丰富或选择，从而获得哪些途径对每个亚型是必不可少的信息。在第一个资助期，我们的目标是提供一项原则证明，证明这种功能性shRNA筛查是可行的，并提供临床上有意义的信息。在第二种方法中，我们将使用最近开发的高通量(超深)测序技术来筛选数千个基因，以寻找潜在致病相关(致癌)突变的存在。作为原则的证明，这种方法将首先在通过shRNA筛选平行研究的骨髓瘤细胞系中进行测试。此后，将对一组选定的10个Akt依赖和10个Akt非依赖的原发MM样本以及每个患者的正常DNA进行测序，以确定可能驱动Akt或其他关键信号通路在Akt非依赖性MM中激活的潜在候选突变。

This central project is based on observations, that have demonstrated that different multiple myelomas (MM) differ in the activation pattern of and dependence on individual signal transduction pathways; specifically, the work has defined MMs that show activation of the Akt pathway and depend on Akt for growth and survival and a separate group of MMs that grow in an Akt-independent manner. The work raises two questions: (a) Which signal transduction pathways are critical for Akt-independent MMs? (b) Which mutations drive activation of Akt (and other critical signaling pathways in Akt-independent MMs)? To address these questions, this Z-project will pursue two strategies: first, we will develop technologies that allow functional screens to search for genes, which are critical for survival of different subtypes of multiple myeloma. To achieve this, we will adopt techniques that measure the frequency of each individual shRNA vector in a population of lentiviral shRNA vectors. We will then infect leukemic cell lines representing each subgroup with a population of such shRNA vectors (initially targeting all kinases) and measure the frequency of each vector before and after expansion in vitro and, ultimately, in vivo after transplantation of infected cells. In this manner, we will determine whether vectors targeting specific genes will be enriched or selected against during these clonal expansion of different MMs and thereby obtain information which pathways are essential for each subtype. In the first funding period, we aim to provide a proof-of-principle that such functional shRNA screens are feasible and provide clinically significant information. In a second approach, we will use the recently developed high throughput (ultra deep) sequencing technology to screen thousands of genes for the presence of potentially pathogenetically relevant (oncogenic) mutations. As a proof-of-principle, this approach will first be tested in myeloma cell lines that are investigated in parallel by shRNA screens. Thereafter, a selected set of 10 Akt-dependent and 10 Akt-independent primary MM samples as well as normal DNA from each of the patients will be sequenced to identify potential candidate mutations that may drive activation of Akt or other critical signaling pathways in Akt-independent MM.