Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish

Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700–6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.     

Integration Profile and Safety of an Adenovirus Hybrid-Vector Utilizing Hyperactive Sleeping Beauty Transposase for Somatic Integration

We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB) transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We found that in all mice which received the hyperactive SB transposase, transgene expression levels were stabilized in a dose-dependent manner and that the highest vector dose was accompanied by fatalities in mice. To analyze potential genotoxic side-effects due to somatic integration into host chromosomes, we performed a genome-wide integration site analysis using linker-mediated PCR (LM-PCR) and linear amplification-mediated PCR (LAM-PCR). Analysis of genomic DNA samples obtained from HSB5 treated female and male mice revealed a total of 1327 unique transposition events. Overall the chromosomal distribution pattern was close-to-random and we observed a random integration profile with respect to integration into gene and non-gene areas. Notably, when using the LM-PCR protocol, 27 extra-chromosomal integration events were identified, most likely caused by transposon excision and subsequent transposition into the delivered adenoviral vector genome. In total, this study provides a careful evaluation of the safety profile of adenovirus/Sleeping Beauty transposase hybrid-vectors. The obtained information will be useful when designing future preclinical studies utilizing hybrid-vectors in small and large animal models.     

Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells

Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5′TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.     

Sequencing methods and datasets to improve functional interpretation of sleeping beauty mutagenesis screens

Background

Animal models of cancer are useful to generate complementary datasets for comparison to human tumor data. Insertional mutagenesis screens, such as those utilizing the Sleeping Beauty (SB) transposon system, provide a model that recapitulates the spontaneous development and progression of human disease. This approach has been widely used to model a variety of cancers in mice. Comprehensive mutation profiles are generated for individual tumors through amplification of transposon insertion sites followed by high-throughput sequencing. Subsequent statistical analyses identify common insertion sites (CISs), which are predicted to be functionally involved in tumorigenesis. Current methods utilized for SB insertion site analysis have some significant limitations. For one, they do not account for transposon footprints – a class of mutation generated following transposon remobilization. Existing methods also discard quantitative sequence data due to uncertainty regarding the extent to which it accurately reflects mutation abundance within a heterogeneous tumor. Additionally, computational analyses generally assume that all potential insertion sites have an equal probability of being detected under non-selective conditions, an assumption without sufficient relevant data. The goal of our study was to address these potential confounding factors in order to enhance functional interpretation of insertion site data from tumors.

Results

We describe here a novel method to detect footprints generated by transposon remobilization, which revealed minimal evidence of positive selection in tumors. We also present extensive characterization data demonstrating an ability to reproducibly assign semi-quantitative information to individual insertion sites within a tumor sample. Finally, we identify apparent biases for detection of inserted transposons in several genomic regions that may lead to the identification of false positive CISs.

Conclusion

The information we provide can be used to refine analyses of data from insertional mutagenesis screens, improving functional interpretation of results and facilitating the identification of genes important in cancer development and progression.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1150) contains supplementary material, which is available to authorized users.     

精子特异性 Sleeping Beauty 转座酶转基因小鼠模型的建立

目的：建立精子特异性表达Sleeping Beauty （ SB）转座酶转基因小鼠模型,为研究SB转座子在小鼠中的应用提供工具。方法克隆精子特异性启动子用以驱动SB转座酶基因的表达,建立精子特异性表达SB转座酶的载体,利用显微注射方法建立以C57BL/6J为背景的精子特异性表达SB转座酶的转基因小鼠。 PCR鉴定首建鼠的基因型,western blot（WB）和免疫组织化学（IHC）检测SB转座酶基因在小鼠生殖腺睾丸中的表达情况,筛选睾丸中高表达SB转座酶的转基因小鼠。结果显微注射方式获得了5只首建小鼠,其中3只能稳定传代,利用WB和IHC成功的筛选出一株在精子中高表达SB转座酶的转基因小鼠。结论成功建立了精子特异性高表达SB转座酶转基因小鼠模型,为将SB转座子作为一种基因工程工具应用于小鼠基因修饰模型的建立提供非常重要的工具资源。     

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR^1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10^th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application^4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2^nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats^9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)¹². In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR⁺ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-21¹³. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.     

NMR structural analysis of Sleeping Beauty transposase binding to DNA

The Sleeping Beauty (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon thus far in clinical trials for human gene therapy. In the absence of atomic level structural information, the development of SB transposon relied primarily on the biochemical and genetic homology data. While these studies were successful and have yielded hyperactive transposases, structural information is needed to gain a mechanistic understanding of transposase activity and guides to further improvement. We have initiated a structural study of SB transposase using Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) spectroscopy to investigate the properties of the DNA‐binding domain of SB transposase in solution. We show that at physiologic salt concentrations, the SB DNA‐binding domain remains mostly unstructured but its N‐terminal PAI subdomain forms a compact, three‐helical structure with a helix‐turn‐helix motif at higher concentrations of NaCl. Furthermore, we show that the full‐length SB DNA‐binding domain associates differently with inner and outer binding sites of the transposon DNA. We also show that the PAI subdomain of SB DNA‐binding domain has a dominant role in transposase's attachment to the inverted terminal repeats of the transposon DNA. Overall, our data validate several earlier predictions and provide new insights on how SB transposase recognizes transposon DNA.     

Clonal Expansion Analysis of Transposon Insertions by High-Throughput Sequencing Identifies Candidate Cancer Genes in a PiggyBac Mutagenesis Screen

Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10⁶ reads corresponding to >10⁴ insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.     

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth^1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect^1,2, mammalian^3-5, and human cells6 including primary human T cells^7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency^9,10.Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer¹¹. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved¹². We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene⁷. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes¹³. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model¹⁴. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized¹⁵.Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens¹⁴, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed⁷. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)¹⁵. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.     

Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells

The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into ‘safe harbor’ sites in the genomes of autologous, patient-derived iPS cells.     

Generation of an inducible and optimized piggyBac transposon system

Genomic studies in the mouse have been slowed by the lack of transposon-mediated mutagenesis. However, since the resurrection of Sleeping Beauty (SB), the possibility of performing forward genetics in mice has been reinforced. Recently, piggyBac (PB), a functional transposon from insects, was also described to work in mammals. As the activity of PB is higher than that of SB11 and SB12, two hyperactive SB transposases, we have characterized and improved the PB system in mouse ES cells. We have generated a mouse codon-optimized version of the PB transposase coding sequence (CDS) which provides transposition levels greater than the original. We have also found that the promoter sequence predicted in the 5′-terminal repeat of the PB transposon is active in the mammalian context. Finally, we have engineered inducible versions of the optimized piggyBac transposase fused with ERT2. One of them, when induced, provides higher levels of transposition than the native piggyBac CDS, whereas in the absence of induction its activity is indistinguishable from background. We expect that these tools, adaptable to perform mouse-germline mutagenesis, will facilitate the identification of genes involved in pathological and physiological processes, such as cancer or ES cell differentiation.     

一种包含转座子Sleeping Beauty和PiggyBac的新型多功能载体的构建

DNA转座子作为一种遗传学工具对脊椎动物的转基因、突变体产生、癌基因发现和基因治疗方面都有巨大的贡献. 目前,哺乳动物中应用最为广泛、活性最高的DNA转座子为重构于鲑鱼的Sleeping Beauty (SB)转座子和来源于甘蓝蠖度尺蛾 (cabbage looper moth Trichoplusia ni)的PiggyBac (PB)转座子. 本研究中,我们成功构建了包含PB和SB两种转座子的杂合转座载体,命名为PBSBD. 在杂合转座载体中融入了基因捕获框及loxp/Frt元件,用以实现转座过程中的基因捕获和条件性敲除. 在HepG2细胞中通过检测报告基因的表达情况及阳性克隆的定位,对构建的杂合转座载体PBSBD进行了活性的初步验证. 结果表明,PBSBD能够有效被2种转座酶识别,并能检测到报告基因的表达. 本研究所构建的杂合转座载体PBSBD结合2种转座酶,可以应用于大规模筛选突变基因和研究基因功能. 并且该杂合转座载体还可以利用SB转座酶的邻近转座特性,结合载体内所包含的loxp/Frt元件用以邻近区域DNA片段的条件性敲除,研究大片段DNA在生物体中的作用.     

Genetic control of renal tumorigenesis by the mouse Rtm1 locus

Background

The genetic basis of susceptibility to renal tumorigenesis has not yet been established in mouse strains. Mouse lines derived by bidirectional phenotypic selection on the basis of their maximal (AIRmax) or minimal (AIRmin) acute inflammatory responsiveness differ widely in susceptibility to spontaneous and urethane-induced renal tumorigenesis. To map the functional loci modulating renal tumor susceptibility in these mice, we carried out a genome-wide genetic linkage study, using SNP arrays, in an (AIRmax x AIRmin)F2 intercross population treated with a single urethane dose at 1 week of age and phenotyped for renal tumors at 35 weeks of age.

Results

AIRmax mice did not develop renal tumors spontaneously nor in response to urethane, whereas in AIRmin mice renal tumors formed spontaneously (in 52% of animals) and after urethane induction (89%). The tumors had a papillary morphology and were positive for alpha-methylacyl-CoA racemase and negative for CD10. By analysis of 879 informative SNPs in 662 mice, we mapped a single quantitative trait locus modulating the incidence of renal tumors in the (AIRmax x AIRmin)F2 intercross population. This locus, which we named Renal tumor modifier QTL 1 (Rtm1), mapped to chromosome 17 at 23.4 Mb (LOD score = 15.8), with SNPs rs3696835 and rs3719497 flanking the LOD score peak. The A allele of rs3719497 from AIRmin mice was associated with a 2.5-fold increased odds ratio for renal tumor development. The LOD score peak included the Tuberous sclerosis 2 (Tsc2) gene which has already been implicated in kidney disease: loss of function by germline retroviral insertion is associated with spontaneous renal tumorigenesis in the Eker rat, and heterozygous-null Tsc2^(+/-) mice develop renal cystadenomas.

Conclusions

We mapped Rtm1 as a single major locus modulating renal tumorigenesis in a murine intercross population. Thus, the AIR mouse lines can be considered a new genetic model for studying the role of germline and somatic molecular alterations in kidney neoplastic disease.     

Enhancement of Sleeping Beauty transposition by CpG methylation: possible role of heterochromatin formation

The Sleeping Beauty (SB) transposase is the most active transposase in vertebrate cells, and the SB transposon system has been used as a tool for insertional mutagenesis and gene delivery. Previous studies have indicated that the frequency of chromosomal transposition is considerably higher in mouse germ cells than in mouse embryonic stem cells, suggesting the existence of unknown mechanisms that regulate SB transposition. Here, we demonstrated that CpG methylation of the transposon region enhances SB transposition. The transposition efficiencies of a methylated transposon and an unmethylated transposon which had been targeted in the same genomic loci by recombination-mediated cassette exchange in mouse erythroleukemia cells were compared, and at least a 100-fold increase was observed in the methylated transposon. CpG methylation also enhanced transposition from plasmids into the genome. Chromatin immunoprecipitation assays revealed that histone H3 methylated at lysine-9, a hallmark of condensed heterochromatin, was enriched at the methylated transposon, whereas the unmethylated transposon formed a relaxed euchromatin structure, as evidenced by enrichment of acetylated histone H3 and reporter gene expression. Possible roles of heterochromatin formation in the transposition reaction are discussed. Our findings indicate a novel relationship between CpG methylation and transposon mobilization.     

The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells

Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrates are transpositionally inactive due to the accumulation of mutations in their transposase genes. A novel open reading frame-trapping method was used to isolate uninterrupted transposase coding regions from the genome of the frog species Rana pipiens. The isolated clones were ~90% identical to a predicted transposase gene sequence from Xenopus laevis, but contained an unpredicted, ~180 bp region encoding the N-terminus of the putative transposase. None of these native genes was found to be active. Therefore, a consensus sequence of the transposase gene was derived. This engineered transposase and the transposon inverted repeats together constitute the components of a novel transposon system that we named Frog Prince (FP). FP has only ~50% sequence similarity to Sleeping Beauty (SB), and catalyzes efficient cut-and-paste transposition in fish, amphibian and mammalian cell lines. We demonstrate high-efficiency gene trapping in human cells using FP transposition. FP is the most efficient DNA-based transposon from vertebrates described to date, and shows ~70% higher activity in zebrafish cells than SB. Frog Prince can greatly extend our possibilities for genetic analyses in vertebrates.     

Going non-viral: the Sleeping Beauty transposon system breaks on through to the clinical side

Molecular medicine has entered a high-tech age that provides curative treatments of complex genetic diseases through genetically engineered cellular medicinal products. Their clinical implementation requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective and economically viable manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are prevalent in ongoing pre-clinical and translational research. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here we review several recent refinements of the system, including the development of optimized transposons and hyperactive SB variants, the vectorization of transposase and transposon as mRNA and DNA minicircles (MCs) to enhance performance and facilitate vector production, as well as a detailed understanding of SB’s genomic integration and biosafety features. This review also provides a perspective on the regulatory framework for clinical trials of gene delivery with SB, and illustrates the path to successful clinical implementation by using, as examples, gene therapy for age-related macular degeneration (AMD) and the engineering of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy.     

Comparative Oncogenomics Implicates the Neurofibromin 1 Gene (NF1) as a Breast Cancer Driver

Identifying genomic alterations driving breast cancer is complicated by tumor diversity and genetic heterogeneity. Relevant mouse models are powerful for untangling this problem because such heterogeneity can be controlled. Inbred Chaos3 mice exhibit high levels of genomic instability leading to mammary tumors that have tumor gene expression profiles closely resembling mature human mammary luminal cell signatures. We genomically characterized mammary adenocarcinomas from these mice to identify cancer-causing genomic events that overlap common alterations in human breast cancer. Chaos3 tumors underwent recurrent copy number alterations (CNAs), particularly deletion of the RAS inhibitor Neurofibromin 1 (Nf1) in nearly all cases. These overlap with human CNAs including NF1, which is deleted or mutated in 27.7% of all breast carcinomas. Chaos3 mammary tumor cells exhibit RAS hyperactivation and increased sensitivity to RAS pathway inhibitors. These results indicate that spontaneous NF1 loss can drive breast cancer. This should be informative for treatment of the significant fraction of patients whose tumors bear NF1 mutations.