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Genetic tools and mutagenesis strategies based on transposable elements are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of their inherent capacity to insert into DNA, transposons can be developed into powerful tools for chromosomal manipulations. Transposon-based forward mutagenesis screens have numerous advantages including high throughput, easy identification of mutated alleles, and providing insight into genetic networks and pathways based on phenotypes. For example, the Sleeping Beauty transposon has become highly instrumental to induce tumors in experimental animals in a tissue-specific manner with the aim of uncovering the genetic basis of diverse cancers. Here, we describe a battery of mutagenic cassettes that can be applied in conjunction with transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain-of-function mutagenesis for functional gene annotation in vertebrate models, including zebrafish, mice, and rats. [ABSTRACT FROM AUTHOR]

Large-scale genomic efforts to study human cancer, such as the cancer gene atlas (TCGA), have identified numerous cancer drivers in a wide variety of tumor types. However, there are limitations to this approach, the mutations and expression or copy number changes that are identified are not always clearly functionally relevant, and only annotated genes and genetic elements are thoroughly queried. The use of complimentary, nonbiased, functional approaches to identify drivers of cancer development and progression is ideal to maximize the rate at which cancer discoveries are achieved. One such approach that has been successful is the use of the Sleeping Beauty ( SB ) transposon-based mutagenesis system in mice. This system uses a conditionally expressed transposase and mutagenic transposon allele to target mutagenesis to somatic cells of a given tissue in mice to cause random mutations leading to tumor development. Analysis of tumors for transposon common insertion sites (CIS) identifies candidate cancer genes specific to that tumor type. While similar screens have been performed in mice with the PiggyBac (PB) transposon and viral approaches, we limit extensive discussion to SB . Here we discuss the basic structure of these screens, screens that have been performed, methods used to identify CIS. [ABSTRACT FROM AUTHOR]

Although rare, synovial sarcoma (SS) is one of the most common soft tissue sarcomas affecting young adults. To investigate potential tumor markers related to synovial sarcoma prognosis, we carried out a single-institution retrospective analysis of 103 patients diagnosed with SS between 1980 and 2009. Clinical outcome data were obtained from medical records, and archived tissue samples were used to evaluate the relationship between progression-free survival (PFS) and several prognostic factors, including tumor expression of FGFR3 and FGFR4. No associations were found between PFS and gender, body mass index, tumor site, SS18-SSX translocation, or FGFR4 expression. As seen in previous studies, age at diagnosis (<35, 63% versus ≥35 years, 31% 10-year PFS; P = .033), histologic subtype (biphasic, 75% versus monophasic 34% 10-year PFS; P = .034), and tumor size (≤5 cm, 70% versus >5 cm, 22% 10-year PFS; P < .0001) were associated with PFS in SS patients. In addition, in a subset of patients with available archived tumor samples taken prior to chemotherapy or radiation (n = 34), higher FGFR3 expression was associated with improved PFS (P = .030). To the best of our knowledge, this is the largest study of SS to date to suggest a potential clinical role for FGFR3. While small numbers make this investigation somewhat exploratory, the findings merit future investigation on a larger scale. © 2013 Elsevier Inc. All rights reserved. [ABSTRACT FROM AUTHOR]

Investigational therapy can be successfully undertaken using viral- and nonviral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently, the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR+ T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease. [ABSTRACT FROM AUTHOR]

The stable introduction of therapeutic transgenes into human cells can be accomplished using viral and nonviral approaches. Transduction with clinical-grade recombinant viruses offers the potential of efficient gene transfer into primary cells and has a record of therapeutic successes. However, widespread application for gene therapy using viruses can be limited by their initially high cost of manufacture at a limited number of production facilities as well as a propensity for nonrandom patterns of integration. The ex vivo application of transposon-mediated gene transfer now offers an alternative to the use of viral vectors. Clinical-grade DNA plasmids can be prepared at much reduced cost and with lower immunogenicity, and the integration efficiency can be improved by the transient coexpression of a hyperactive transposase. This has facilitated the design of human trials using the Sleeping Beauty (SB) transposon system to introduce a chimeric antigen receptor (CAR) to redirect the specificity of human T cells. This review examines the rationale and safety implications of application of the SB system to genetically modify T cells to be manufactured in compliance with current good manufacturing practice (cGMP) for phase I/II trials. [ABSTRACT FROM AUTHOR]

Although the basic functions of the prostate gland are conserved among mammals, its morphology varies greatly among species. Comparative studies between mouse and human are important because mice are widely used to study prostate cancer, a disease that occurs in a region-restricted manner within the human prostate. An informatics-based approach was used to identify prostate-specific human genes as candidate markers of region-specific identity that might distinguish prostatic ducts prone to prostate cancer from ducts that rarely give rise to cancer. Subsequent analysis of normal and cancerous human prostates demonstrated that the genes microseminoprotein-β ( MSMB) and transglutaminase 4 ( TGM4) were expressed in distinct groups of ducts in the normal human prostate, and only MSMB was detected in areas of prostate cancer. The mouse orthologs of MSMB and TGM4 were then used for expression studies in mice along with the mouse ventrally expressed gene spermine binding protein ( SBP). All three genes were informative markers of region-specific epithelial identity with distinct expression patterns that collectively accounted for all ducts in the mouse prostate. Together with the human data, this suggested that MSMB expression defines an anatomical domain in the mouse prostate that is molecularly most similar to human prostate cancers. Computer-assisted serial section reconstruction was used to visualize the complete expression domains for MSMB, SBP, and TGM4 in the mouse prostate. This showed that MSMB is expressed in prostatic ducts that comprise 21% of the mouse dorso-lateral prostate. Finally, the expression of MSMB, SBP, and TGM4 was evaluated in a mouse prostate cancer model created by the prostate epithelium-specific deletion of the tumor suppressor PTEN. MSMB and TGM4 were rapidly and dramatically down-regulated in response to PTEN deletion suggesting that this model of prostate cancer includes a more rapid de-differentiation of the prostatic epithelium than is observed in organ-confined human prostate cancers. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a common and deadly form of leukemia. New therapies are desperately needed. The availability of appropriate disease model systems is crucial to development of effective treatment. A significant progress in generating and characterizing such model systems has been made in the past decade. Here, the relative benefits of cell lines, transgenic mice and xenografted primary AML model will be compared, along with speculation on new models suitable for AML drug discovery. [Copyright &y& Elsevier]

Methods for cancer gene discovery include identification of viral oncogenes, identification of genes associated with recurrent chromosomal aberrations, and screens for genes capable of the transformation of cells in culture. In recent years, the completed genome sequence of human and model organisms has markedly enhanced cancer gene identification. Whole genome, high-throughput screens have been facilitated by the advent of new technologies such as murine leukemia virus-based mutagenesis, Sleeping Beauty-based mutagenesis, RNA interference, exon re-sequencing, and high-resolution methods for detecting chromosomal amplifications and deletions; these, in turn, have led to the identification of novel tumor suppressors and oncogenes. The identification of genes that are altered by mutation or expression and which are directly involved in tumor initiation and maintenance will be instrumental for understanding cancer phenotypic variation and for identifying crucial therapeutic targets. [Copyright &y& Elsevier]

WNT/β-catenin signaling is critical to the development of many cancer types. A paper by Mo and colleagues in a recent issue of Cell shows that autocrine CXCL12/CXCR4 chemokine signaling activates β-catenin signaling in a rare peripheral nerve sarcoma. Together with the availability of small molecules targeting CXCR4, this finding suggests new avenues for cancer therapy. [Copyright &y& Elsevier]

Radiation therapy (RT) has been a primary treatment modality in cancer for decades. Increasing evidence suggests that RT can induce an immunosuppressive shift via upregulation of cells such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). MDSCs inhibit antitumor immunity through potent immunosuppressive mechanisms and have the potential to be crucial tools for cancer prognosis and treatment. MDSCs interact with many different pathways, desensitizing tumor tissue and interacting with tumor cells to promote therapeutic resistance. Vascular damage induced by RT triggers an inflammatory signaling cascade and potentiates hypoxia in the tumor microenvironment (TME). RT can also drastically modify cytokine and chemokine signaling in the TME to promote the accumulation of MDSCs. RT activation of the cGAS-STING cytosolic DNA sensing pathway recruits MDSCs through a CCR2-mediated mechanism, inhibiting the production of type 1 interferons and hampering antitumor activity and immune surveillance in the TME. The upregulation of hypoxia-inducible factor-1 and vascular endothelial growth factor mobilizes MDSCs to the TME. After recruitment, MDSCs promote immunosuppression by releasing reactive oxygen species and upregulating nitric oxide production through inducible nitric oxide synthase expression to inhibit cytotoxic activity. Overexpression of arginase-1 on subsets of MDSCs degrades L-arginine and downregulates CD3ζ, inhibiting T-cell receptor reactivity. This review explains how radiation promotes tumor resistance through activation of immunosuppressive MDSCs in the TME and discusses current research targeting MDSCs, which could serve as a promising clinical treatment strategy in the future. [ABSTRACT FROM AUTHOR]

In the paper by Ishida et al. in this issue of Cancer Cell , the authors report the results of targeted inactivation of a Rap1-specific GTPase-activating protein (GAP) gene, called SPA-1, in mice. Rap1 hyperactivation was observed in hematopoietic cells, which led over time to features associated with symptoms typical of human myeloid dyslastic and myeloid proliferative diseases. The authors present additional data showing that the level of Rap1 activation is important for regulating myelopoiesis and that, in the right context, can deliver an oncogenic signal. [Copyright &y& Elsevier]

Objective: We previously showed that Vav promoter-tetracycline transactivator (Vav-tTA)–driven tetracycline-regulated element (TRE)-NRAS(V12) expression resulted in mastocytosis development in mice. To investigate which hematopoietic cells express TRE-driven transgenes when combined with Vav-tTA, we assayed hematopoietic cells, including bone marrow–derived mast cells (BMMC) and CD34-positive hematopoietic progenitor cells (HPC) as well as myeloid and lymphoid lineages. To determine if suppression of NRAS(V12) expression early in life would delay mastocytosis we treated developing and juvenile mice with doxycycline (Dox). Materials and Methods: Vav-tTA–driven luciferase expression was assayed by live mouse imaging and relative light unit measurement before or after treating Vav-tTA and TRE-luciferase (TRE-Luc) cotransgenic mice with Dox. Magnetic cell sorting and fluorescence-activating cell sorting methods were used to sort hematopoietic cells. To suppress TRE-mediated luciferase or NRAS(V12) expression in Vav-tTA cotransgenic mice, we added Dox to the drinking water. Results: B cells in the bone marrow and T cells in the thymus expressed Vav-tTA–driven luciferase at much higher levels than in myeloid cells, BMMC, and CD34-positive HPC, which showed relatively low levels. Dox treatment completely eliminated the luciferase expression from all hematopoietic cells. Repression of TRE-NRAS(V12) expression early in life was sufficient to increase the latency of mastocytosis development. Conclusion: The Vav-tTA transgenic line will be very useful for conditional transgene expression in developing B and T cells. Vav-tTA–driven NRAS(V12) expression is sufficient for mastocytosis development, but not for myeloid leukemia. Lymphoid cells are resistant to NRAS(V12) transformation despite high level of expression. [Copyright &y& Elsevier]

Abstract Non-alcoholic fatty liver disease (NAFLD) is becoming an epidemic disease in adults and children worldwide. Importantly, there are currently no approved treatments available for NAFLD. This study aims to investigate the potential applications of sodium tanshinone IIA sulfonate (STS) on improving the NAFLD condition using both in vitro and in vivo approaches. The results showed that STS markedly inhibited lipid accumulation in oleic acid (OA) and palmitic acid (PA) treated HepG2 and primary immortalized human hepatic (PIH) cells. STS suppressed lipogenesis by inhibiting expression of sterol regulatory element binding transcription factor 1 (SREBF1), fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD). In addition, STS reduced inflammation in cells treated with OA-PA, shown by decreased transcriptional levels of tumor necrosis factor (TNF), transforming growth factor beta 1 (TGFB1) and interleukin 1 beta (IL1B). Consistently, protective effects on hepatic steatosis in db/db mice were observed after STS administration, demonstrated by decreased lipid accumulation in mouse hepatocytes. This protective effect might be associated with STS induced activation of sirtuin 1 (SIRT1)/protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1) pathways. Our findings suggest a potential therapeutic role for STS in the treatment of NAFLD. [ABSTRACT FROM AUTHOR]

Parthenolide ( PTL ) is a sesquiterpene lactone natural product with anti-proliferative activity to cancer cells. Selective eradication of leukemic stem cells (LSCs) over healthy hematopoietic stem cells (HSCs) by PTL has been demonstrated in previous studies, which suggests PTL and related molecules may be useful for targeting LSCs. Eradication of LSCs is required for curative therapy. Chemical optimizations of PTL to improve potency and pharmacokinetic parameters have focused largely on the α-methylene-γ-butyrolactone, which is essential for activity. Conversely, we evaluated modifications to the C1–C10 olefin and benchmarked new inhibitors to PTL with respect to inhibitory potency across a panel of cancer cell lines, ability to target drug-resistant acute myeloid leukemia (AML) cells, efficacy for inhibiting clonal growth of AML cells, toxicity to healthy bone marrow cells, and efficiency for promoting intracellular reactive oxygen species (ROS) levels. Cyclopropane 4 was found to possess less toxicity to healthy bone marrow cells, enhanced potency for the induction of cellular ROS, and similar broad-spectrum anti-proliferative activity to cancer cells in comparison to PTL . [ABSTRACT FROM AUTHOR]

Abstract: Inactivation of the NF1 tumor suppressor causes myeloproliferative diseases. NF1 encodes a GTPase activating protein (GAP) for Ras. Myeloid cells with loss of NF1 have high levels of Ras-GTP, functionally equivalent to the effects of RAS oncogenes. We investigated the effects of the NF1 GAP-related domain (GRD) in proliferation, apoptosis and Ras-GTP levels in Nf1-negative acute myeloid leukemia (AML) cells. In AML cells, with cooperating mutations, the expression of the neurofibromin GRD causes significant reductions of N- and K-Ras-GTP levels, which is not incompatible with AML cell survival, but which is strongly selected against due to suppression of proliferation. [Copyright &y& Elsevier]

Objective: Chemoresistance remains a major clinical obstacle to curative chemotherapy of acute myeloid leukemia (AML), but the molecular mechanisms underlying resistance to chemotherapeutic agents used in AML are largely unknown. We have attempted to investigate genetic mechanisms causing resistance to Ara-C [1-β-D-arabinofuranosyl-cytosine (cytarabine)], one mainstay in AML chemotherapy for decades. Material and Methods: Highly Ara-C-resistant murine BXH-2 strain AML cell lines were generated, and their molecular changes were compared to their sensitive parental lines. The causative changes were confirmed using a genetic approach. Results: We derived nine highly Ara-C-resistant murine BXH-2 strain AML sublines via in vitro selection. p21Cip1 was dramatically downregulated and p53 protein accumulation induced by Ara-C treatment was impaired in one resistant line. In this line, repeated Ara-C exposure had selected for cells that harbor a genomic deletion affecting the splicing of Trp53 mRNA. This deletion produces an aberrant Trp53 mRNA, in which exon 4 is skipped, producing a protein lacking parts of both the transactivation and DNA-binding domains. Retroviral transduction of the sensitive parental cells with a dominant-negative Trp53 cDNA caused changes in the protein levels of p21Cip1, BAX, and cleaved caspase-3, but not bcl-XL, and rendered the cells more resistant to Ara-C. Unexpectedly, we found that pifithrin-α (PFTα), a compound that has been proposed to regulate p53 protein activity, induced apoptosis in both Ara-C-sensitive and -resistant lines, and decreased Ara-C resistance in cells with either normal or mutant Trp53 genes. Conclusions: These data indicate that Trp53 loss-of-function could partly explain the acquisition of AML chemoresistance, and suggest that PFTα could be useful in treatment of relapsed AML. [Copyright &y& Elsevier]

The Sleeping Beauty transposon system (SB) has been shown to mediate nonviral integration of expression constructs resulting in long-term gene expression in several mammalian targets. Often, however, it is difficult to discern long-term expression resulting from transposition vs nonhomologous chromosomal recombination or maintenance of plasmid DNA in an extrachromosomal form. We have designed a system to silence expression from nontransposed sequences, making it possible to determine more specifically the amount of expression resulting from transposition. A transposon plasmid, pT2F/Cage (carrying a murine erythropoietin (Epo) gene transcriptionally regulated by the ubiquitously expressed CAGS promoter), was engineered to contain LoxP sites positioned so as to interrupt expression upon Cre-mediated recombination. Upon transposition these sites become segregated, thus protecting the expression construct from Cre-mediated recombination and subsequent silencing. Interferon-inducible Mx1Cre mice were administered pT2F/Cage with or without transposase-encoding plasmid. At 2 to 4 weeks postinjection, in the absence of SB transposase, Cre induction reduced Epo expression to about 1% of that seen in the group that was administered transposase-encoding plasmid, which maintained Epo levels near those of the uninduced groups. Southern hybridization analysis and plasmid rescue of transfected tissue supported the efficient Cre-mediated silencing of nontransposed sequences. These results indicate a substantial level of DNA-mediated expression not associated with transposition, but which can be quantitatively distinguished from transposition by its sensitivity to Cre recombinase. The results also provide additional evidence for the effectiveness of the Sleeping Beauty transposon system as an in vivo DNA-mediated gene transfer strategy for achieving long-term expression.Molecular Therapy (2006) 13, 617–624; doi: 10.1016/j.ymthe.2005.10.015 [ABSTRACT FROM AUTHOR]

Sleeping Beauty (SB) is a DNA transposon capable of mediating gene insertion and long-term expression in vertebrate cells when co-delivered with a source of transposase. In all previous reports of SB-mediated gene insertion in somatic cells, the transposase component has been provided by expression of a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to transposon re-mobilization and reintegration. We addressed this potential problem by supplying the transposase-encoding molecule in the form of mRNA. We show that transposase-encoding mRNA can effectively mediate transposition in vitro in HT1080 cells and in vivo in mouse liver following co-delivery with a recoverable transposon or with a luciferase transposon. We conclude that in vitro-transcribed mRNA can be used as an effective source of transposase for SB-mediated transposition in mammalian cells and tissues.Molecular Therapy (2006) 13, 625–630; doi: 10.1016/j.ymthe.2005.10.014 [ABSTRACT FROM AUTHOR]

Abstract: Glioblastoma is a fatal brain tumor that becomes highly vascularized by secreting proangiogenic factors and depends on continued angiogenesis to increase in size. Consequently, a successful antiangiogenic therapy should provide long-term inhibition of tumor-induced angiogenesis, suggesting long-term gene transfer as a therapeutic strategy. In this study a soluble vascular endothelial growth factor receptor (sFlt-1) and an angiostatin–endostatin fusion gene (statin-AE) were codelivered to human glioblastoma xenografts by nonviral gene transfer using the Sleeping Beauty (SB) transposon. In subcutaneously implanted xenografts, co-injection of both transgenes showed marked anti-tumor activity as demonstrated by reduction of tumor vessel density, inhibition or abolition of glioma growth, and increase in animal survival (P = 0.003). Using luciferase-stable engrafted intracranial gliomas, the anti-tumor effect of convection-enhanced delivery of plasmid DNA into the tumor was assessed by luciferase in vivo imaging. Sustained tumor regression of intracranial gliomas was achieved only when statin-AE and sFlt-1 transposons were coadministered with SB-transposase-encoding DNA to facilitate long-term expression. We show that SB can be used to increase animal survival significantly (P = 0.008) by combinatorial antiangiogenic gene transfer in an intracranial glioma model. [Copyright &y& Elsevier]

Gene therapy has the potential to become an effective component of cancer treatment by transferring genes that cause immunomodulation or tumor cell death or that inhibit angiogenesis into tumor cells or tumor-associated stroma. Viral vectors have been the primary gene transfer vehicles used for intratumoral gene transfer to date. Plasmid-based vectors may be safer and more scalable than viral vectors. However, attempts at plasmid-based intratumoral gene transfer have been met with transient expression and poor gene transfer efficiency. Here we report integration and long-term expression of reporter genes in human glial tumors, growing in nude mice, using the Sleeping Beauty (SB) transposon system. A two-plasmid system was used, in which linear polyethylenimine was complexed with a GFP, NEO, or luciferase transposon plasmid and a SB transposase-expressing plasmid. SB-mediated transposition led to chromosomal integration of the NEO transgene in roughly 8% of tumor cells. SB-mediated insertions were cloned from the genomes of glial tumor cells to provide molecular proof of transposase-mediated integration. Luciferase studies showed that SB facilitated long-term expression of the transgene in glial tumors. SB-mediated intratumoral gene transfer is a novel, nonviral technique that could be used to augment conventional therapy for glioblastoma or other cancers. [Copyright &y& Elsevier]

Gene transfer to the lung could provide important new treatments for chronic and acquired lung diseases such as cystic fibrosis, α1-antitrypsin deficiency, emphysema, and cancer. DNA-mediated gene transfer to the lung has been previously demonstrated, but anticipated effectiveness has been limited by low gene transfer efficiencies and by transient expression of the transgene. Here, we combine plasmid-based gene transfer with the integrating capacity of the nonviral Sleeping Beauty (SB) transposon vector system to mediate gene insertion and long-term gene expression in mouse lung. We observed transgene expression after 24 h in lungs of all animals injected with the luciferase transposon (pT/L), but expression for up to 3 months required codelivery of a plasmid encoding the Sleeping Beauty transposase. We also observed long-term expression in pT/L-injected animals transgenic for SB transposase. Transgene expression was localized to the alveolar region of the lung, with transfection including mainly type II pneumocytes. We used a linker-mediated PCR technique to recover transposon flanking sequences, demonstrating transposition of pT/L into mouse chromosomal DNA of the lung. [Copyright &y& Elsevier]

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70–80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy. [Copyright &y& Elsevier]

LC-1 (also known as DMAPT or dimethylamino-parthenolide), a prodrug of parthenolide, was tested for anti-proliferative activity against glioma. LC-1 was found to have low micromolar cytotoxic activity against three glioma cell lines and was also found to be brain penetrant in healthy mice (2.1–3.0 brain-to-plasma ratio). In a syngeneic GL261 murine glioma model, LC-1 slowed tumor growth kinetics and extended the survival time of tumor-bearing mice in comparison to the vehicle control. Consequently, LC-1 represents a promising lead compound for further development as a glioma therapy. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OSA) is a heterogeneous and aggressive solid tumor of the bone. We recently identified the colony stimulating factor 1 receptor (Csf1r) gene as a novel driver of osteosarcomagenesis in mice using the Sleeping Beauty (SB) transposon mutagenesis system. Here, we report that a CSF1R-CSF1 autocrine/paracrine signaling mechanism is constitutively activated in a subset of human OSA cases and is critical for promoting tumor growth and contributes to metastasis. We examined CSF1R and CSF1 expression in OSAs. We utilized gain-of-function and loss-of-function studies (GOF/LOF) to evaluate properties of cellular transformation, downstream signaling, and mechanisms of CSF1R-CSF1 action. Genetic perturbation of CSF1R in immortalized osteoblasts and human OSA cell lines significantly altered oncogenic properties, which were dependent on the CSF1R-CSF1 autocrine/paracrine signaling. These functional alterations were associated with changes in the known CSF1R downstream ERK effector pathway and mitotic cell cycle arrest. We evaluated the recently FDA-approved CSF1R inhibitor Pexidartinib (PLX3397) in OSA cell lines in vitro and in vivo in cell line and patient-derived xenografts. Pharmacological inhibition of CSF1R signaling recapitulated the in vitro genetic alterations. Moreover, in orthotopic OSA cell line and subcutaneous patient-derived xenograft (PDX)-injected mouse models, PLX3397 treatment significantly inhibited local OSA tumor growth and lessened metastatic burden. In summary, CSF1R is utilized by OSA cells to promote tumorigenesis and may represent a new molecular target for therapy. Unlabelled Image • Sleeping Beauty mutagenesis identified CSF1R as a potential driver of OSA. • Oncogenic CSF1R is expressed and is constitutively active in a subset of OSA. • PLX3397 treatment effectively reduced local OSA tumor growth and metastasis. [ABSTRACT FROM AUTHOR]

An abstract of the article "Target-Site Preferences of Sleeping Beauty Transposons in an Intron of the B-raf Oncogene," by Jason B. Bell, Christopher S. Hackett, Aron M. Geurts, Corey M. Carlson, Lara S. Collier, Geyi Liu, David A. Largaespada and Perry B. Hackett is presented.

To expand the repertoire of transposable elements available for gene transfer in higher vertebrates, we investigated the properties of the Tol2 transposon in mammalian cells and in vivo. Tol2 is a member of the hAT family of DNA transposons and is derived from an active element found in the tyrosinase gene of medaka fish (Oryzias latipes). Tol2 can transpose in mouse ES cells, suggesting its potential use as a gene transfer reagent in higher vertebrates.We first tested the gene transfer activity of Tol2 in cultured human HeLa and HT1080 cell lines by G418 resistant colony forming assay and found that it is comparable to that of Sleeping Beauty. Furthermore, inceasing the amount of transposase-expressing plasmid resulted in increased transposition activity until a plateau was reached. In contrast to Sleeping Beauty, overexpression inhibition was not observed for Tol2. We also tested the effect of transposon size on Tol2 transposition and found that elements of up to 10 kilobases in size transpose as efficiently as smaller 2 kilobase transposons.The Tol2 transposable element routinely used to date contains a significant portion of the transposase open reading frame complete with multiple splice sites, promoter and polyadenylation cassettes. As these DNA elements pose a potential insertional mutagenesis risk in gene therapy applications, we sought to identify minimal sequences required for Tol2 transposition. In contrast to a previously published report, we found that internal sequences are not necessary for transposition, and we were able to reduce the size of the element by about 3 kilobases, creating Tol2mini.To investigate the activity of Tol2 in vivo, we coinjected a luciferase-expressing Tol2 transposon along with transposase-expressing plasmid rapidly and in high volume into the tail vein of wild type mice. We observed long term expression of the luciferase transgene in mouse liver, suggesting genomic integration of the transgene. Increasing the dose transposase led to increase in transposition efficiency without overexpression inhibition.Finally, we used both full length Tol2 and Tol2mini to correct the genetic defect in a mouse model of hereditary tyrosinemia type 1 by rapid, high volume co-injection with a transposase-encoding plasmid and transposon encoding fumaryl acetoacetate hydrolase and firefly luciferase. Liver repopulation was quantitatively monitored over time by increasing luciferase activity, measured as light emitted from the liver (please see abstract by Wilber et al), and required co-administration of transposase-encoding plasmid.Our results suggest that the Tol2 transposable element has excellent potential as a gene therapy tool and may be particularly suitable for applications where large cDNAs or cell type-specific promoters are required for full therapeutic potential. In addition, the apparent lack of overexpression inhibition suggests that Tol2 can be readily optimized for a variety of gene therapy applications.Molecular Therapy (2006) 13, S147–S147; doi: 10.1016/j.ymthe.2006.08.446 [ABSTRACT FROM AUTHOR]

Retroviruses and transposons are mobile genetic elements with the ability to integrate genetic information into chromosomes for human gene therapy. However, integration-site preferences for mobile elements are poorly understood. We analyzed the insertion sites of several mobile elements to detect patterns of integration that can lead to the prediction of preferred integration sites. Based on our findings, we developed an automated method, ProTIS©, capable of faithfully predicting which base pairs in chromosomal DNA are preferred sites for Sleeping Beauty (SB) transposons. The preference for integration into certain sites is based upon structural characteristics of sequences flanking the dinucleotide insertion site. For any gene, chromosome, or whole genome, ProTIS© can generate a profile of predicted integration events. We have described an in vitro assay of transposition that uses a mathematical description of DNA deformability, Vstep, which allowed us to identify shared structural patterns among several preferred integration sites for SB transposons. For the SB transposon system we established the following equation that allows prediction of the relative likelihood of integration of an SB transposon into defined lengths of sequence:Likelihood of Integration = [10(# preferred sites) + 5(# semi-preferred sites) + (# basal sites)]ProTIS is valid for integration of SB transposons into limited and extended regions of genomic DNA up to 3.2 Mbp. We used a similar approach to analyze DNA integration patterns of other mobile genetic elements including retroviruses, lentiviruses and insect transposons. We analyzed 11,791 piggyBac integration events into its target TTAA sites and 5,070 integrations of P-elements, which do not have defined target sites. We were unable to find consistent Vstep pattern shared among either the piggyBac or P-element integration sites. We examined 695 MLV, 1371 HIV-1, 148 SIV and 551 ASLV integration sites for Vstep patterns that would aid in predicting integration preferences. Although we found symmetric patterns that overlapwith the base pairs involved in the target site duplication for most viral vectors, their patterns are based on the same compilations used to identify unique, weak consensus sequences for the various viruses and therefore cannot be used to generate algorithms for predicting integration sites based on structure rather than sequence. Our data suggest that as the density of integrations into a defined sequence increases our approach using ProTIS will have utility for human gene therapy applications for 'randomly' integrating vectors.Molecular Therapy (2006) 13, S200–S200; doi: 10.1016/j.ymthe.2006.08.591 [ABSTRACT FROM AUTHOR]

An abstract of the article "Combinatorial Antiangiogenic Gene Therapy by Nonviral Gene Transfer Using the Sleeping Beauty Transposon Causes Tumor Regression and Improves Survival in Mice Bearing Intracranial Human Glioblastoma," by John R. Ohlfest and colleagues is presented.

An abstract of the article "Direct Comparison of Integration Efficiency and Stable Gene Expression Mediated by the Sleeping Beauty Transposon System and by the PhiC31 Phage Integrase System in Cultured Human Fibroblasts and in Various Stem Cell Targets," by Andy Wilber, Fernando Montoya, Aron M. Geurts, David A. Largaespada, Uma Lakshmipathy and R. Scott McIvor is presented.