Comprehensive Ex Vivo Transposon Mutagenesis Identifies Genes That Promote Growth Factor Independence and Leukemogenesis

Abstract

Aberrant signaling through cytokine receptors and their downstream signaling pathways is a major oncogenic mechanism underlying hematopoietic malignancies. To better understand how these pathways become pathologically activated and to potentially identify new drivers of hematopoietic cancers, we developed a high-throughput functional screening approach using ex vivo mutagenesis with the Sleeping Beauty transposon. We analyzed over 1,100 transposon-mutagenized pools of Ba/F3 cells, an IL3-dependent pro-B-cell line, which acquired cytokine independence and tumor-forming ability. Recurrent transposon insertions could be mapped to genes in the JAK/ STAT and MAPK pathways, confirming the ability of this strategy to identify known oncogenic components of cytokine signaling pathways. In addition, recurrent insertions were identified in a large set of genes that have been found to be mutated in leukemia or associated with survival, but were not previously linked to the JAK/STAT or MAPK pathways nor shown to functionally contribute to leukemogenesis. Forced expression of these novel genes resulted in IL3-independent growth in vitro and tumorigenesis in vivo, validating this mutagenesis-based approach for identifying new genes that promote cytokine signaling and leukemogenesis. Therefore, our findings provide a broadly applicable approach for classifying functionally relevant genes in diverse malignancies and offer new insights into the impact of cytokine signaling on leukemia development.

Evolution of malignant plasmacytoma cell lines from K14E7 Fancd2−/− mouse long-term bone marrow cultures

“…Malignant plasmacytoma cell lines in our studies were growth factor independent. Recent data indicates that at least 12 different genes in the JAK/STAT and MAP Kinase pathways can induce transposon mutagenesis of IL-3 factor dependent cell lines to factor independence [66]. Whether the E7 oncogene in the absence of the Fancd2 protein induced these or other genes in our LTBMC derived cell lines is not known.…”

Ex Vivo Transposon-Mediated Genetic Screens for Cancer Gene Discovery

“…Then the following “insertions in window size (bp)” were considered as significant (P<0.01) for our study [11]: 2 in 18 bp, 3 in 297 bp, 4 in 958 bp, 5 in 1895 bp, 6 in 3029 bp, 7 in 4309 bp, 8 in 5698 bp, 9 in 7175 bp, 10 in 8723 bp, 11 in 10330 bp.…”

“…Transposon insertions sites are then recovered by high-throughput sequencing. We recently applied this approach to comprehensively identify and validate genes that promote growth factor independence and transformation of murine Ba/F3 cells [11]. In this chapter, we provide a detailed protocol for performing ex vivo mutagenesis screens in these cells.…”

“…These prior studies demonstrated how the functional analysis of genes in these cells informs our understanding of the mechanisms of leukemogenesis. We performed a comprehensive analysis of genes that drive growth-factor independence and malignant transformation of Ba/F3 cells [11], which we hypothesized would reveal novel genes that promote signaling through the IL3 pathway (Figure 1) and that participate more broadly in hematopoietic malignancies and other cancers.…”

A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

“…By contrast, transposon systems, such as Sleeping Beauty (SB) and piggyBac, have become more commonly used for insertional mutagenesis due to their flexible design and reduced integration site bias. While transposon mutagenesis has been used to perform phenotypic selection in cells ex vivo (Chen et al 2016;Guo et al 2016;Kodama et al 2016), it has more frequently been employed in engineered mouse models of cancer (O'Donnell 2018), likely due to the relative inefficiency of mutagenesis when both transposon and transposase vectors must be introduced independently to cells in culture.…”