Although nearly half of human melanomas harbor oncogenic BRAFV600E mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in BrafV600E mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAFV600E melanoma and discover new genes with potential clinical importance in human melanoma.

A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor’s major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.

30The systematic identification of genetic events driving cellular transformation and tumor progression in the absence 31 of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell 32 carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular 33 landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically 34 define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified 35 established tumor suppressor genes, as well as previously unknown oncogenic drivers of cuSCC. Functional analysis 36 confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the 37 initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely 38 heterogeneous genetic landscape of cuSCC initiation and progression, which could be harnessed to better 39 understand skin oncogenic etiology and prioritize therapeutic candidates. 40

The systematic identification of genetic events driving cellular transformation and tumor progression in the absence of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified both known tumor suppressor genes and novel oncogenic drivers of cuSCC. Functional analysis confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely heterogeneous genetic landscape of cuSCC initiation and progression, which can be harnessed to better understand skin oncogenic etiology and prioritize therapeutic candidates.

SBCapSeq is a transposon-based liquid-phase capture experimental and bioinformatic workflow optimized for Ion Torrent sequencing. SBCaptureSeq permits selective, semi-quantitative, and scalable deep sequencing of Sleeping Beauty transposon insertion sites from adaptor-ligated, barcoded Ion Torrent libraries created from populations of cells (bulk specimens) and single cells from both tumor and non-tumor genomes. This protocol includes detailed procedures for genomic DNA isolation, library preparation, capture hybridization, target sequence enrichment, sequencing, and data analysis. The SBCapSeq method, which takes about 7-10 days to perform, permits semiquantitative sequencing and has been specifically optimized for Sleeping Beauty transposon mutagenesis genetic studies.