Low-copy piggyBac transposon mutagenesis in mice identifies genes driving melanoma Thomas K. Nia,1, Sean F. Landrettea, Robert D. Bjornsonb, Marcus W. Bosenbergc, and Tian Xua,d,2 aDepartment of Genetics, Howard Hughes Medical Institute, and Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06510; bDepartment of Computer Science and Yale Center for Genome Analysis, Yale University, New Haven, CT 06511; cDepartment of Dermatology, Yale University School of Medicine, New Haven, CT 06510; and dInstitute of Developmental Biology and Molecular Medicine, Fudan–Yale Center for Biomedical Research, School of Life Science, Fudan University, Shanghai 200433, China Edited by James E. Cleaver, University of California, San Francisco, CA, and approved August 9, 2013 (received for review July 30, 2013) Despite considerable efforts to sequence hypermutated cancers to human cancer sequencing (17–19). Prevailing high-copy SB such as melanoma, distinguishing cancer-driving genes from insertional mutagenesis screens for cancer-gene discovery mo- thousands of recurrently mutated genes remains a significant bilize hundreds of transposons per cell to generate hundreds of challenge. To circumvent the problematic background mutation insertional mutations, and this strategy has proven highly effi- rates and identify new melanoma driver genes, we carried out cient at inducing tumors in mice. Although high insertional a low-copy piggyBac transposon mutagenesis screen in mice. We mutation rates present problems for distinguishing drivers from induced eleven melanomas with mutation burdens that were 100- passengers similar to those seen in human cancers, high-copy fold lower relative to human melanomas. Thirty-eight implicated screens use large cohorts of mice to achieve adequate statistical genes, including two known drivers of human melanoma, were power for subsequent analysis. Although low specificity remains classified into three groups based on high, low, or background- a serious issue, high-copy screens have identified several dozen level mutation frequencies in human melanomas, and we further genes whose human orthologs are frequently mutated in co- explored the functional significance of genes in each group. For lorectal and pancreatic cancers (20–22). two genes overlooked by prevailing discovery methods, we found For hypermutated cancers, the utility of such cross-species that loss of membrane associated guanylate kinase, WW and PDZ comparisons is limited due to the rampant mutation rates in- domain containing 2 and protein tyrosine phosphatase, receptor herent in high-copy experimental mouse screens. In theory, the type, O cooperated with the v-raf murine sarcoma viral oncogene efficiency of PB transposition would permit screens for tumori- homolog B (BRAF) recurrent V600E mutation to promote cellular genesis to be performed using greatly reduced numbers of muta- transformation. Moreover, for infrequently mutated genes often genic transposons. The development of such low-copy mutagenesis disregarded by current methods, we discovered recurrent mito- systems would circumvent the insertional hypermutation produced gen-activated protein kinase kinase kinase 1 (Map3k1)-activating by high-copy experimental mouse screens, thereby minimizing insertions in our screen, mirroring recurrent MAP3K1 up-regula- passenger mutations and enriching for potentially causative muta- tion in human melanomas. Aberrant expression of Map3k1 en- tions. The virtue of such an approach would permit smaller cohorts abled growth factor-autonomous proliferation and drove BRAF- of mice to be used and enable effective cross-species comparisons to independent ERK signaling, thus shedding light on alternative further facilitate the identification of driver genes in human cancer. means of activating this prominent signaling pathway in mela- We were intrigued by the potential of using limited numbers of noma. In summary, our study contributes several previously unde- transposons per cell for inducing tumors, as this approach has scribed genes involved in melanoma and establishes an important not previously been exploited. As proof-of-concept for the low- proof-of-principle for the utility of the low-copy transposon muta- genesis approach for identifying cancer-driving genes, especially Significance those masked by hypermutation. piggyBac PB transposon | somatic mutagenesis | low-copy genetic screen Passenger mutation rates are highly elevated in many human cancers, posing a significant hurdle for the identification of cancer-driving genes. In this study, we screened for melanomas he sequencing of human cancer exomes has led to the fi in mice using a unique, low-copy transposon mutagenesis Tidenti cation of new candidate driver genes with the un- system that can induce tumors with few somatic mutations. derstanding that genes promoting cancer growth (drivers) are We show that our experimental system accurately recapitulated more frequently mutated compared with genes not involved in the genetic basis of human melanomas with only five somatic cancer (passengers) (1). In addition, exome sequencing has un- mutations, thus circumventing hundreds of unrelated passengers. covered the existence of hypermutated tumors, which possess – Using cross-species comparative analyses and functional studies greatly elevated background mutation rates (2 4). Nearly all in human cells, we identified three previously undescribed genes melanomas and lung cancers are hypermutated, with an average – involved in melanoma and several dozen candidate genes. Our of 433 and 256 nonsilent mutations per tumor, respectively (5 study demonstrates that the low-copy transposon mutagenesis 13). In these cancers, disproportionate increases in passenger approach can facilitate the identification of cancer-driving genes mutations obfuscate the differential mutation frequencies used that are masked by high passenger mutation rates. to distinguish drivers from passengers. Uncontrolled inflation of false-positive drivers obscures the identities of true drivers (low Author contributions: T.K.N. and T.X. designed research; T.K.N. and S.F.L. performed re- specificity) whereas overly stringent filters compromise the de- search; R.D.B. and M.W.B. contributed new reagents/analytic tools; T.K.N., M.W.B., and tection of true drivers (low sensitivity) (14). A productive balance T.X. analyzed data; and T.K.N. and T.X. wrote the paper. between sensitivity and specificity remains to be achieved. Given The authors declare no conflict of interest. these considerable challenges, efforts to identify genes important This article is a PNAS Direct Submission. fi for hypermutated cancers would bene t from experimental 1Present address: Department of Developmental, Molecular and Chemical Biology, Tufts cancer screens that can substantially reduce passenger mutations. University, Boston, MA 02111. Since the mobilization of piggyBac (PB) and Sleeping Beauty 2To whom correspondence should be addressed. E-mail: [email protected]. (SB) transposons in mice (15, 16), transposon somatic muta- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. genesis screens have become important functional complements 1073/pnas.1314435110/-/DCSupplemental. E3640–E3649 | PNAS | Published online September 3, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1314435110 Downloaded by guest on September 26, 2021 copy mutagenesis methodology, we set our sights on identifying transcription units or genes (hereafter referred to as insertional PNAS PLUS new candidate genes for melanoma, the most hypermutated of mutations), and the rest were intergenic. We noted that very few human cancers. Melanomas often harbor driver mutations in insertional mutations accounted for the vast majority of se- BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog quencing reads within each tumor. The preponderance of reads (NRAS), cyclin-dependent kinase inhibitor 2A (CDKN2A), and from these insertions likely reflects their clonal abundance in phosphatase and tensin homolog (PTEN), as well as frequent the melanomas and thus identifies them as potential driver amplifications but rare protein-coding mutations in micro- mutations. Random sampling of one-third of all insertional phthalmia-associated transcription factor (MITF) and v-kit Hardy- mutations by genomic PCR confirmed the clonal abundance Zuckerman 4 feline sarcoma viral oncogene homolog (KIT)(23). of mutations (Fig. S1). In total, 45 insertional mutations in 38 However, knowledge of new driver genes remains limited. Using genes (37 protein-coding, 1 noncoding RNA) were identified, cor- a low-copy PB mutagenesis system (seven-copy) in mice, we report responding to an average of 4.1 insertional mutations per tumor the induction of melanomas with 100-fold lower mutation rates (Table S1). compared with human melanomas, the validation of Cdkn2a and Importantly, among the identified genes are two bona fide Mitf as driver genes, and the identification of 36 previously melanoma driver genes, Mitf and Cdkn2a. MITF is a melanocyte undescribed candidate genes. By cross-species comparative anal- lineage-restricted oncogene that is frequently amplified in hu- ysis and experiments in human cells, we show that MAGI2, man melanomas (23). Consistent with the alterations in human PTPRO,andMAP3K1 have functional significance to human melanomas, we found that mouse melanomas harboring in- melanoma and are either overlooked or undetected by prevailing sertional mutations in Mitf were up-regulated approximately cancer-gene