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）不同的激活模式和依赖于个别信号转导通路;具体来说，工作已经定义了显示Akt通路的激活，并依赖于Akt的生长和生存和一个单独的组的生长Akt的独立的方式。这项工作提出了两个问题：（a）哪些信号转导途径对Akt非依赖性的转录至关重要？(b)哪些突变驱动Akt的激活（以及Akt非依赖性突变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：首先，我们将开发技术，允许功能筛选搜索基因，这对多发性骨髓瘤的不同亚型的生存至关重要。为了实现这一点，我们将采用测量慢病毒shRNA载体群体中每个单独的shRNA载体的频率的技术。然后，我们将用一群这样的shRNA载体（最初靶向所有激酶）感染代表每个亚组的白血病细胞系，并在体外扩增前后测量每个载体的频率，并最终在受感染细胞移植后在体内测量。以这种方式，我们将确定在不同亚型的这些克隆扩增期间靶向特定基因的载体是否将被富集或选择，从而获得哪些途径对每个亚型是必需的信息。在第一个资助期，我们的目标是提供一个原理证明，这种功能性shRNA筛选是可行的，并提供临床重要信息。在第二种方法中，我们将使用最近开发的高通量（超深度）测序技术来筛选数千个基因，以确定是否存在潜在的致病相关（致癌）突变。作为原理证明，这种方法将首先在骨髓瘤细胞系中进行测试，并通过shRNA筛选进行平行研究。此后，将对选定的一组10份Akt依赖性和10份Akt非依赖性原发性MM样本以及来自每例患者的正常DNA进行测序，以鉴定可能驱动Akt激活或Akt非依赖性MM中其他关键信号传导途径的潜在候选突变。

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.