Project Details
Projekt
Systemic shRNA screens and high throughout sequencing to investigate pathway dependence in multiple myeloma
Subject Area
Hematology, Oncology
Term
from 2009 to 2014
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 100308792
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.
DFG Programme
Clinical Research Units
