Sleeping Beauty Insertional Mutagenesis Research Articles

RBFOX2 deregulation promotes pancreatic cancer progression and metastasis through alternative splicing

RNA splicing is an important biological process associated with cancer initiation and progression. However, the contribution of alternative splicing to pancreatic cancer (PDAC) development is not well understood. Here, we identify an enrichment of RNA binding proteins (RBPs) involved in splicing regulation linked to PDAC progression from a forward genetic screen using Sleeping Beauty insertional mutagenesis in a mouse model of pancreatic cancer. We demonstrate downregulation of RBFOX2, an RBP of the FOX family, promotes pancreatic cancer progression and liver metastasis. Specifically, we show RBFOX2 regulates exon splicing events in transcripts encoding proteins involved in cytoskeletal remodeling programs. These exons are differentially spliced in PDAC patients, with enhanced exon skipping in the classical subtype for several RBFOX2 targets. RBFOX2 mediated splicing of ABI1, encoding the Abelson-interactor 1 adapter protein, controls the abundance and localization of ABI1 protein isoforms in pancreatic cancer cells and promotes the relocalization of ABI1 from the cytoplasm to the periphery of migrating cells. Using splice-switching antisense oligonucleotides (AONs) we demonstrate the ABI1 ∆Ex9 isoform enhances cell migration. Together, our data identify a role for RBFOX2 in promoting PDAC progression through alternative splicing regulation.

Forward Genetic Screen Implicates Drivers of Leukemic Progression in a Novel Model of Trp53 R270H myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a highly variable clinical course. The most catastrophic complication of MDS is transformation to secondary acute myeloid leukemia (sAML). Notably, mutations in TP53 confer the single highest risk of transformation to sAML and death. However, some patients with TP53 mutated MDS do not develop sAML, suggesting that additional genetic events cooperate with TP53 mutations to transform MDS to sAML. Understanding the mechanisms of transformation of MDS to sAML could provide targets for therapeutic intervention. To model the genetics of MDS, we crossed mice bearing Trp53 R270H ( Trp53 is the murine TP53 gene) and deletion of genes syntenic with human chromosome 5q (del(5q)). To discover how additional mutations contribute to disease progression, we utilized Sleeping Beauty (SB) transposon mutagenesis in Trp53 R270H/del(5q) mice. SB transposase mobilized SB mutagenic T2/Onc transposons which randomly insert within the genome. T2/Onc transposons are designed to induce gain or loss of function alterations depending on the site and orientation of insertion with respect to targeted genes. We used the Mx1-Cre transgene to activate SB transposase and T2/Onc transposition in hematopoietic progenitors. Trp53 R270H anddel(5q)(or cytogenetically normal, CN) mice were crossed to SB mice to generate donor mice of the following genotypes: Trp53 R270H/del(5q)/ SB, Trp53 R270H/CN /SB, Trp53 WT/del(5q)/ SB, Trp53 WT/CN/ SB mice, and mice without SB transposition, (no transposition, NT: Trp53 R270H/del(5q) /NT). Bone marrow cells were transplanted into recipients, and SB insertional mutagenesis was activated using pI-pC to activate Cre. Mice receiving Trp53 WT/CN/ SB bone marrow developed more frequent T-cell leukemia (n=3/10) than myeloid leukemia (n=1/10). In contrast, mice receiving Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB bone marrow developed predominantly myeloid leukemia (n=14/28) more commonly than T-cell leukemia (1/28). Mixed phenotype leukemia was seen in 7/28 of these mice. Together, these data demonstrate a strong bias towards myeloid disease in SB-mutagenized Trp53 R270H bone marrow. To identify genes with SB insertions, we performed RNA sequencing to detect SB T2/Onc transposon-endogenous genefusion transcripts. Among Trp53 WT/CN/ SB leukemias, the most common recurrent SB fusions involved Notch1 and Ikzf1 as has previously reported for SB-associated T-cell leukemias . Among Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB leukemias, the most common recurrent SB-fusions involved Erg, Eras and Il2rb with Erg fusions detected 85% of Trp53 R270H leukemias (n=17/20). SB inserted upstream of Erg promoter indicating that these fusions likely upregulate expression of Erg. Indeed, Erg levels are significantly higher in leukemias that express SB-Erg fusions relative to leukemias that do not (p<0.0023). ERG is not recurrently mutated in human AML, but the ERG gene locus is commonly amplified, especially TP53 mutant AML. ERG is known to support normal hematopoietic stem cell self-renewal. Notably, Erg-insertions were also detected in a model of MDS expressing stabilized cyclin E with SB-mediated progression to erythroleukemia (Loeb 2019). Using gene set enrichment analysis, we found that hematopoietic stem cell and leukemic stem cell signatures are enriched in Erg-SB fusion leukemias. In our analyses of two independent data sets (TCGA and BEAT AML), stem cell signatures are also among the most highly enriched pathways in human AMLs expressing high ERG levels. Furthermore, in a human AML single cell RNA sequencing dataset (van Galen 2019), we found that ERG expression is highest in AML cells with the most immature stem and progenitor-like features. Together, these findings implicate a role for ERG as a driver of progression of MDS to AML by enhancing aberrant self-renewal. In summary, we present a novel murine model of Trp53/del(5q) MDS. In this model, Erg upregulation is associated with progression to AML and upregulation of leukemia stem cell gene expression profiles. These data implicate ERG as a major contributor to progression of MDS to secondary AML in the setting of mutant p53. Understanding the mechanisms of disease progression and self-renewal in myeloid malignancies with p53 mutations is critical to define effective therapeutic strategies in these rapidly fatal, treatment resistant diseases.

Sleeping Beauty insertional mutagenesis screen identifies the pro-metastatic roles of CNPY2 and ACTN2 in hepatocellular carcinoma tumor progression

A forward genetic Sleeping Beauty (SB) insertional mutagenesis screen, followed by high-throughput transcriptome sequencing, was used to identify driver genes responsible for hepatocellular carcinoma (HCC)-associated metastasis. Using RNA-sequencing (RNA-seq) to identify transposon-endogenous transcriptome fusion genes, the phylogenetic lineage between the parental liver tumor and secondary metastasis can be determined to provide mechanistic insight to genetic changes involved in the metastatic evolution process. In the current study, two novel candidate genes were identified to be potentially involved in HCC-associated metastatic progression, canopy FGF signaling regulator 2 (Cnpy2) and actinin alpha 2 (Actn2). Transposon-Cnpy2 fusion transcripts were identified in both primary liver tumors and lung metastases. Its significant association with clinicopathological characteristics and correlated gene enrichment in metastasis-related mechanisms suggest its potential role in modulating local invasion and angiogenesis. Other known driver genes for human HCC that can also promote metastatic progression include epidermal growth factor receptor (Egfr) and RNA imprinted and accumulated in nucleus (Rian). Metabolic pathway related gene carbamoyl phosphate synthetase (Cps1) was identified to play an important role in early HCC development, while cell junction-related pathway gene Rac family small GTPase 1 (Rac1) was identified to take part in both HCC and pro-metastatic progression. Importantly, actinin alpha 2 (Actn2) was identified exclusively in the secondary metastasis site and its role in HCC-related metastatic process was elucidated using in vitro approaches. ACTN2-overexpression in human liver cancer cells displayed enhanced cellular motility and invasion abilities, indicating its possible function in later stage of metastasis, such as extravasation and lung colonization.

Abstract A37: SB insertional mutagenesis identifies new metastasis-promoting tumor suppressor genes in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease with poor patient outcome. While acquired oncogenic activation of KRAS is an initiating event, disease progression requires functional perturbation of additional genes and cellular pathways. Identifying key molecular events that cooperate with oncogenic KRAS in human tumors to drive disease progression is a challenge due in part to the high degree of inter- and intratumor heterogeneity in PDAC genomes. We have taken a comparative genomics approach to identify progression drivers using a mouse model of metastatic PDAC driven by Sleeping Beauty (SB) insertional mutagenesis in the context of oncogenic Kras. Statistical enrichment analysis of SB insertions defined hundreds of cooperating tumor-suppressor genes in SB PDAC tumors. We hypothesized that recurrently mutated TSGs in pancreatic tumors drive metastatic disease. Enrichment analysis of high-read depth insertions from SB PDAC tumors defined 48 drivers predicted to contribute to disease progression. Thirty-two of these genes were statistically enriched in a population-based analysis of SB insertions in metastases isolated from a subset of the animals from the primary cohort. We demonstrated significant increases in primary tumor burden and metastatic spread by single-gene knockdown of novel TSGs TCF12, FNDC3B, or RBFOX2 in orthotopic human PDAC mouse models. TCF12 encodes a basic Helix-loop-helix (bHLH) transcription factor that partners with other bHLH transcription factors in different cellular contexts, including TWIST, a transcription factor involved in epithelial-to-mesenchymal transition (EMT); PTF1A, a pancreas-specific transcription factor with essential roles in pancreas development and cancer; and SMAD3, a TGFβ-related transcription factor involved in cellular migration. TCF12 resides in a chromosomal region (15q21) frequently lost in pancreatic cancer, and decreased TCF12 expression is associated with poor patient prognosis. Using human PDAC expression data from ICGC, we identified 209 genes that exhibit coregulated gene expression with TCF12. A subset of these genes function in Wnt or TGF-Beta signaling pathways and show consistent decreases in expression upon TCF12 knockdown in orthotopic tumors. Ongoing experiments aim to define the TCF12 transcriptional targets involved in metastatic progression. Citation Format: Michelle Maurin, Devin J. Jones, Mik A. Black, Justin Y. Newberg, Nancy A. Jenkins, Neal G. Copeland, Karen M. Mann. SB insertional mutagenesis identifies new metastasis-promoting tumor suppressor genes in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A37.

Abstract 673: The role of MYPT1/2, ASPP2 and MYH9 in invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancer cases in women. One of the characteristics of this type of cancer is the loss of intercellular adhesion which is facilitated by inactivating mutations in E-cadherin. However the loss of E-cadherin alone is not enough to induce ILC. An in vivo Sleeping Beauty insertional mutagenesis screen was performed to identify genes that together with E-cadherin loss contribute to ILC development in mice. In around 85% of the tumors that were analyzed during this screen one of four hits were observed, namely MYPT1, MYPT2, ASPP2 and MYH9. Interestingly these four hits appear to be mutually exclusive indicating a shared mechanism of action. To investigate how these genes might affect tumorigenesis we first looked at the location of the transposon insertions. This revealed that for MYPT1, MYPT2 and ASPP2 the transposons appeared to localize to a specific region in the gene indicating that these insertions could result in a truncation variant. In the case of MYH9 no clear localization of insertions was found which in combination with the recently published data indicates a loss of function. Northern blot analysis revealed the presence of truncation variants for MYPT1/2 and ASPP2 which for MYPT1 were confirmed to also result in truncated protein variants. In order to identify the tumorigenic potential of truncated MYPT1 and ASPP2 we overexpressed them in spontaneously immortalized mammary epithelial cell lines HC11 and NMuMG. We are using these cell lines to analyse differences in cell proliferation, anchorage independent growth, sensitivity to apoptosis in vitro and tumor formation in vivo. To further analyse the hits in an E-cadherin negative setting we made use of primary mammary epithelial cells (MECs) isolated from genetically engineered mice with mammary gland specific E-cadherin loss. These MECs normally do not grow past several passages in vitro. However, these MECs when we overexpress truncated MYPT1 or ASPP2, or use shRNA mediated knockdown of MYH9 can be passaged and grown in vitro consistently. Finally we are generating conditional mouse models with mammary gland specific E-cadherin loss in combination of expression of the truncated variants of MYPT1 or ASPP2, or loss of MYH9. We will to monitor whether these mice are prone to developing ILC. Citation Format: Koen Schipper, Julian de Ruiter, Sjors Kas, Eva Schut, Marco Koudijs, David Addams, Jos Jonkers. The role of MYPT1/2, ASPP2 and MYH9 in invasive lobular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 673.

PTPS-02FOXR2: AN ONCOGENE IN MEDULLOBLASTOMA

Medulloblastoma is the most common malignant brain tumor in children. There continues to be significant morbidity associated with current therapy, which includes surgery, chemotherapy and radiation. More knowledge about drivers of this tumor will allow development of more targeted therapies. Our lab has previously identified FOXR2 as a driver for MB using Sleeping Beauty insertional mutagenesis. We have begun to examine the role of FOXR2 in the role of medulloblastoma formation by over expression and transcription activator like effector nuclease mediated knock-out in cellular models, RNA sequencing and reverse phase protein array (RPPA) of these cells, and allograft mouse models. Overexpression of FOXR2 in a non-tumor forming, immortalized mouse cerebellar cell line (C17.2) results in significantly increased soft agar colony formation but no differences in proliferation. This is similar to the Koso et al, 2014 findings that examined the effects of FOXR2 overexpression in granule neuron progenitor cells. RNA sequencing of the C17.2 cells with foxr2 overexpression have identified upstream regulators which are activated such as HIF1A, IFG1, TP53, ERRB and MYC that have previously been identified to be upregulated in medulloblastoma. The exact mechanism of FOXR2 has yet to be elucidated and additional RNA sequencing of the knock out cell lines in comparison to the wild type will be conducted to further our understanding. RPPA has also been completed and analysis is being carried out currently. In addition to our in vitro findings we have examined the level of expression of 45 human medulloblastoma samples via staining of tissue microarray and from those samples there appears to be a subset of patients in the SHH subgroup which express FOXR2 at high levels. Our findings support that FOXR2 is an oncogene in a number of patients with SHH subtype medulloblastoma and may be a target for development of therapeutic targets.

Abstract 2893: Sleeping Beauty uncovers cooperating driver genes in a preclinical mouse model of myeloid leukemia

Abstract Myeloid leukemia is associated with few predominant mutations and translocations in humans. In order to uncover additional loci that drive the disease, we developed an aggressive, fully-penetrant mouse model of myeloid leukemia using Sleeping Beauty insertional mutagenesis. Animals developed distinct foci of expanded splenic red pulp as early as 30 days of age and had a median survival of 70 days. SB insertions sequenced from 168 spleens of mice with end-stage disease revealed more than 470 statistically defined candidate cancer genes (CCGs), 87 of which have orthologs mutated in human AML. Pathway enrichment analysis using the CCGs confirmed that MAP kinase and Jak-Stat signaling play an important role in the disease. Twenty-five CCGs were defined as trunk drivers based on high sequence read representation in multiple tumors from the population. Among the trunk drivers was a suite of transcription factors that act downstream of MAP kinase signaling, including Ets- family member genes Erg, Ets1 and Fli1. We sequenced SB insertions from single cells isolated from a single tumor to interrogate cooperating relationships among trunk drivers. Hierarchical clustering of SB insertions revealed intra-tumor heterogeneity and identified three distinct subclonal populations. Importantly, Erg and Ghr anchored one subpopulation that was distinct from a second subpopulation anchored by Ets1 and Notch1. These gene relationships observed in single cells confirmed the population-based statistical analysis of cooperativity between Erg and Ghr and Ets1 and Notch1 from the cohort of 168 tumors. Mutual-exclusivity for Erg and Ets1 in driving myeloid leukemia was supported by microarray expression analysis in a subset of the tumors. RNA-seq analysis confirmed that SB insertions drive expression of many CCGs including trunk divers Erg, Ghr, Ets1 and Notch1. Chimeric transcripts for the four driver genes contained the SB splice donor and downstream wild type exons encoding the functional domains of the translated proteins, suggesting that SB drives the mis-expression of these proteins to induce myeloid leukemia. We have identified new CCGs that contribute to myeloid disease progression and gained insight into both inter- and intra- tumor heterogeneity of this tumor type. Importantly, SB-driven myeloid leukemia is a robust, transplantable genetic model that offers a unique platform for preclinical testing. Transposon insertion profiles provide a powerful means to stratify tumors for targeted therapies. Finally, SB can be harnessed to uncover loci that confer therapy response or resistance. Citation Format: Karen M. Mann, Justin Newberg, Nicholas Navin, David J. Adams, Nancy Jenkins, Neal Copeland, Michael B. Mann. Sleeping Beauty uncovers cooperating driver genes in a preclinical mouse model of myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2893. doi:10.1158/1538-7445.AM2015-2893

BRAF inhibitor resistance mediated by the AKT pathway in an oncogenic BRAF mouse melanoma model

BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors elicit a transient anti-tumor response in ∼ 80% of BRAF(V600)-mutant melanoma patients that almost uniformly precedes the emergence of resistance. Here we used a mouse model of melanoma in which melanocyte-specific expression of Braf(V618E) (analogous to the human BRAF(V600E) mutation) led to the development of skin hyperpigmentation and nevi, as well as melanoma formation with incomplete penetrance. Sleeping Beauty insertional mutagenesis in this model led to accelerated and fully penetrant melanomagenesis and synchronous tumor formation. Treatment of Braf(V618E) transposon mice with the BRAF inhibitor PLX4720 resulted in tumor regression followed by relapse. Analysis of transposon insertions identified eight genes including Braf, Mitf, and ERas (ES-cell expressed Ras) as candidate resistance genes. Expression of ERAS in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thymoma viral oncogene homolog 1), a phenotype reverted by combinatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associated with phosphorylation/inactivation of BAD, and that the resistance of hepatocyte growth factor-treated human melanoma cells to PLX4720 can be reverted by treatment with the BAD-like BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD pathway in resistance to BRAF inhibition and illustrate an in vivo approach for finding drug resistance genes.

In Vivo Identification of Tumor- Suppressive PTEN ceRNAs in an Oncogenic BRAF-Induced Mouse Model of Melanoma

We recently proposed that competitive endogenous RNAs (ceRNAs) sequester microRNAs to regulate mRNA transcripts containing common microRNA recognition elements (MREs). However, the functional role of ceRNAs in cancer remains unknown. Loss of PTEN, a tumor suppressor regulated by ceRNA activity, frequently occurs in melanoma. Here, we report the discovery of significant enrichment of putative PTEN ceRNAs among genes whose loss accelerates tumorigenesis following Sleeping Beauty insertional mutagenesis in a mouse model of melanoma. We validated several putative PTEN ceRNAs and further characterized one, the ZEB2 transcript. We show that ZEB2 modulates PTEN protein levels in a microRNA-dependent, protein coding-independent manner. Attenuation of ZEB2 expression activates the PI3K/AKT pathway, enhances cell transformation, and commonly occurs in human melanomas and other cancers expressing low PTEN levels. Our study genetically identifies multiple putative microRNA decoys for PTEN, validates ZEB2 mRNA as a bona fide PTEN ceRNA, and demonstrates that abrogated ZEB2 expression cooperates with BRAF(V600E) to promote melanomagenesis.

Harnessing transposons for cancer gene discovery

Recently, it has become possible to mobilize the Tc1/mariner transposon, Sleeping Beauty (SB), in mouse somatic cells at frequencies high enough to induce cancer. Tumours result from SB insertional mutagenesis of cancer genes, thus facilitating the identification of the genes and signalling pathways that drive tumour formation. A conditional SB transposition system has also been developed that makes it possible to limit where SB mutagenesis occurs, providing a means to selectively model many types of human cancer. SB mutagenesis has already identified a large collection of known cancer genes in addition to a plethora of new candidate cancer genes and potential drug targets.

Abstract 2199: A murine insertional mutagenesis screen to identify oncogenes and tumor suppressor genes involved in Schwann cell tumorigenesis

Abstract Introduction: Schwann cell tumors can occur sporadically, or in association with inherited tumor pre-disposition syndromes, such as Neurofibromatosis type 1 and type 2. A common feature of Schwann cell neoplasia is overexpression of the epidermal growth factor receptor (EGFR) and loss of p53 pathway function. To gain more insight into the genetic basis of neoplastic Schwann cell diseases we are using the conditional Sleeping Beauty (SB) transposon-based somatic mutagenesis system. Methods: To create mutations in Schwann cells and their precursors, five transgenes were introduced into mice. The first transgene conditionally expresses the Sleeping Beauty (SB11) transposase enzyme when Cre recombinase is also expressed1,2. The second transgene is an oligodendritic and Schwann cell specific Cre-recombinase controlled by the CNP (3’-cyclic nucleotide 3’-phosphodiesterase) promoter. The third transgene is a concatomer of oncogenic transposons (T2/Onc) capable of activating and/or disrupting endogenous gene expression. Since loss of p53 function and/or overexpression of epidermal growth factor receptor (EGFR) are associated with Malignant Peripheral Nerve Sheath Tumors (MPNSTs), CNP-EGFR and conditional dominant negative p53R270H alleles are also included3,4. Mice possessing all five transgenes experience transposition and insertional mutations in Schwann cell lineage cells. Results: SB is expressed and active in the Schwann cell lineage with variegated expression in other tissue types. Mice that harbor both a conditional dominant negative p53 allele and CNP-EGFR transgene develop nervous tissue tumors at 70% penetrance. These tumors are primarily neurofibromas, but in addition, there are Schwannomas, oculomotor nerve tumors, and dermal MPNSTs. Preliminary data indicate that MPNSTs develop more frequently in mice that are also undergoing SB transposon mutagenesis. Initial analysis of transposon insertion sites showed over 10 common sites of transposon insertion (CIS), implicating new genes in the pathogenesis of Schwann cell tumors. Among the CIS were the Neurofibromatosis type 1 and type 2 genes (Nf1 and Nf2), validating the screen. The Pten gene was also often targeted, consistent with the hypothesis that loss of Pten activity could play a major role in Schwann cell tumor progression. These and other CIS-associated genes will be discussed. Conclusions: Expression of EGFR at high levels cooperates with loss of function mutations in the p53 gene to induce Schwann cell tumors. SB insertional mutagenesis can contribute to Schwann cell tumorigenesis and identify new candidates in tumor progression. 1. Keng, V.W., et al. (2009). Nat Biotechnol. 3:264-74 2. Starr, T.K., et al. (2009). Science. 323:1747-50 3. Rasheed, B.K., et al. (1994). Cancer Res. 54:1324-1330 4. Perry, A., et al. (2002). Jour. Neuropathol. Exp. Neurol. 61:702-709 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2199.

Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system

Transposons have provided important genetic tools for functional genomic screens in lower eukaryotes but have proven less useful in higher eukaryotes because of their low transposition frequency. Here we show that Sleeping Beauty (SB), a member of the Tc1/mariner class of transposons, can be mobilized in mouse somatic cells at frequencies high enough to induce embryonic death and cancer in wild-type mice. Tumours are aggressive, with some animals developing two or even three different types of cancer within a few months of birth. The tumours result from SB insertional mutagenesis of cancer genes, thus facilitating the identification of genes and pathways that induce disease. SB transposition can easily be controlled to mutagenize any target tissue and can therefore, in principle, be used to induce many of the cancers affecting humans, including those for which little is known about the aetiology. The uses of SB are also not restricted to the mouse and could potentially be used for forward genetic screens in any higher eukaryote in which transgenesis is possible.