Low-copy piggyBac transposon mutagenesis in mice identifies genes driving melanoma

Abstract

Despite considerable efforts to sequence hypermutated cancers such as melanoma, distinguishing cancer-driving genes from thousands of recurrently mutated genes remains a significant challenge. To circumvent the problematic background mutation rates and identify new melanoma driver genes, we carried out a low-copy piggyBac transposon mutagenesis screen in mice. We induced eleven melanomas with mutation burdens that were 100-fold lower relative to human melanomas. Thirty-eight implicated genes, including two known drivers of human melanoma, were classified into three groups based on high, low, or backgroundlevel mutation frequencies in human melanomas, and we further explored the functional significance of genes in each group. For two genes overlooked by prevailing discovery methods, we found that loss of membrane associated guanylate kinase, WW and PDZ domain containing 2 and protein tyrosine phosphatase, receptor type, O cooperated with the v-raf murine sarcoma viral oncogene homolog B (BRAF) recurrent V600E mutation to promote cellular transformation. Moreover, for infrequently mutated genes often disregarded by current methods, we discovered recurrent mitogen-activated protein kinase kinase kinase 1 (Map3k1)-activating insertions in our screen, mirroring recurrent MAP3K1 up-regulation in human melanomas. Aberrant expression of Map3k1 enabled growth factor-autonomous proliferation and drove BRAFindependent ERK signaling, thus shedding light on alternative means of activating this prominent signaling pathway in melanoma. In summary, our study contributes several previously undescribed genes involved in melanoma and establishes an important proof-of-principle for the utility of the low-copy transposon mutagenesis approach for identifying cancer-driving genes, especially those masked by hypermutation.piggyBac PB transposon | somatic mutagenesis | low-copy genetic screen

Transposon mutagenesis identifies genetic drivers of BrafV600E melanoma

“…Our screen identified 72% (26/36) of these Pten ceRNAs 36 ; however, we did not observe a significant enrichment for these ceRNAs among our CIS genes ( P > 0.05). In a separate study, Ni et al 37 identified five CIS genes using PiggyBac transposon mutagenesis. Our screen identified three of these genes and paralogs of the other two genes.…”

MAP3K1: Genomic Alterations in Cancer and Function in Promoting Cell Survival or Apoptosis

“…Ni et al utilized a piggyBac transposon strategy that identified multiple activating insertions in introns 9 and 10 of the MAP3K1 gene. 74 These insertions were shown to result in the production of truncated MAP3K1 products lacking the N-terminal SWIM and RING domains (Fig. 1A).…”

Substrate Trapping Proteomics Reveals Targets of the βTrCP2/FBXW11 Ubiquitin Ligase

“…Specifically, multinucleated cells likely exhibit increased susceptibility to mitotic/cytokinesis abnormalities which could promote genomic instability. Consistent with this, RAPGEF2 and its interacting protein MAGI-2 were independently identified in an in vivo low-copy-number piggyBac insertional mutagenesis screen for drivers of melanoma (27). In that study, transposon mutagenesis (transpogenesis) within the RAPGEF2 locus was predicted to result in an overexpressed full-length protein.…”

“…A role for RAPGEF2 in cancer, albeit complex and tissue specific, has also emerged. First, a murine piggyBac insertional mutagenesis screen identified RAPGEF2 and its associated protein MAGI1 as "driver" genes in melanoma (27). Second, RAPGEF2 controls the migratory capacity of multiple cell types.…”