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Comprehensive screenings to clarify indirect cell–cell interactions, such as those in the tumor microenvironment, especially comprehensive assessments of supporting cells' effects, are challenging. Therefore, in this study, indirect CRISPR screening for drug resistance with cell–cell interactions was invented. The photoconvertible fluorescent protein Dendra2 was inducted to supporting cells and explored the drug resistance responsible factors of supporting cells with CRISPR screenings. Random mutated supporting cells co-cultured with leukemic cells induced drug resistance with cell–cell interactions. Supporting cells responsible for drug resistance were isolated with green-to-red photoconversion, and 39 candidate genes were identified. Knocking out C9orf89, MAGI2, MLPH, or RHBDD2 in supporting cells reduced the ratio of apoptosis of cancer cells. In addition, the low expression of RHBDD2 in supporting cells, specifically fibroblasts, of clinical pancreatic cancer showed a shortened prognosis, and a negative correlation with CXCL12 was observed. Indirect CRISPR screening was established to isolate the responsible elements of cell–cell interactions. This screening method could reveal unknown mechanisms in all kinds of cell–cell interactions by revealing live phenotype-inducible cells, and it could be a platform for discovering new targets of drugs for conventional chemotherapies. An indirect CRISPR screening system based on the photoconvertible fluorescent protein, Dendra2, enables the discovery of elements responsible for drug resistance from cell–cell interactions. [ABSTRACT FROM AUTHOR]

Mechanical forces drive critical cellular processes that are reflected in mechanical phenotypes, or mechanotypes, of cells and their microenvironment. We present here "Rupture And Deliver" Tension Gauge Tethers (RAD-TGTs) in which flow cytometry is used to record the mechanical history of thousands of cells exerting forces on their surroundings via their propensity to rupture immobilized DNA duplex tension probes. We demonstrate that RAD-TGTs recapitulate prior DNA tension probe studies while also yielding a gain of fluorescence in the force-generating cell that is detectable by flow cytometry. Furthermore, the rupture propensity is altered following disruption of the cytoskeleton using drugs or CRISPR-knockout of mechanosensing proteins. Importantly, RAD-TGTs can differentiate distinct mechanotypes among mixed populations of cells. We also establish oligo rupture and delivery can be measured via DNA sequencing. RAD-TGTs provide a facile and powerful assay to enable high-throughput mechanotype profiling, which could find various applications, for example, in combination with CRISPR screens and -omics analysis. Mechanical forces drive critical cellular processes, but methods to study such cellular forces are typically low-throughput. Here the authors present a method using "Rupture And Deliver" Tension Gauge Tethers, where flow cytometry or sequencing can be used to record the mechanical history of thousands of individual cells. [ABSTRACT FROM AUTHOR]

Purpose of Review: Patients with neurofibromatosis type 1 (NF1) are at increased risk for benign and malignant neoplasms. Recently, targeted therapy with the MEK inhibitor class has helped address these needs. We highlight recent successes with selumetinib while acknowledging ongoing challenges for NF1 patients and future directions. Recent Findings: MEK inhibitors have demonstrated efficacy for NF1-related conditions, including plexiform neurofibromas and low-grade gliomas, two common causes of NF1-related morbidity. Active investigations for NF1-related neoplasms have benefited from advanced understanding of the genomic and cell signaling alterations in these conditions and development of sound preclinical animal models. Summary: Selumetinib has become the first FDA-approved targeted therapy for NF1 following its demonstrated efficacy for inoperable plexiform neurofibroma. Investigations of combination therapy and the development of a representative NF1 swine model hold promise for translating therapies for other NF1-associated pathology. [ABSTRACT FROM AUTHOR]

Schwannomatosis (SWNTS) is a genetic cancer predisposition syndrome that manifests as multiple and often painful neuronal tumors called schwannomas (SWNs). While germline mutations in SMARCB1 or LZTR1, plus somatic mutations in NF2 and loss of heterozygosity in chromosome 22q have been identified in a subset of patients, little is known about the epigenomic and genomic alterations that drive SWNTS-related SWNs (SWNTS-SWNs) in a majority of the cases. We performed multiplatform genomic analysis and established the molecular signature of SWNTS-SWNs. We show that SWNTS-SWNs harbor distinct genomic features relative to the histologically identical non-syndromic sporadic SWNs (NS-SWNS). We demonstrate the existence of four distinct DNA methylation subgroups of SWNTS-SWNs that are associated with specific transcriptional programs and tumor location. We show several novel recurrent non-22q deletions and structural rearrangements. We detected the SH3PXD2A-HTRA1 gene fusion in SWNTS-SWNs, with predominance in LZTR1-mutant tumors. In addition, we identified specific genetic, epigenetic, and actionable transcriptional programs associated with painful SWNTS-SWNs including PIGF, VEGF, MEK, and MTOR pathways, which may be harnessed for management of this syndrome. [ABSTRACT FROM AUTHOR]

Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma. [ABSTRACT FROM AUTHOR]

The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations inDck in both Ara-C resistant cell lines. CRISPR and TALEN-based KO of Dck dramatically increased the IC50 of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance. [ABSTRACT FROM AUTHOR]

'Gain' of supernumerary copies of the 8q24.21 chromosomal region has been shown to be common in many human cancers and is associated with poor prognosis. The well-characterized myelocytomatosis (MYC) oncogene resides in the 8q24.21 region and is consistently co-gained with an adjacent 'gene desert' of approximately 2 megabases that contains the long non-coding RNA gene PVT1, the CCDC26 gene candidate and the GSDMC gene. Whether low copy-number gain of one or more of these genes drives neoplasia is not known. Here we use chromosome engineering in mice to show that a single extra copy of either the Myc gene or the region encompassing Pvt1, Ccdc26 and Gsdmc fails to advance cancer measurably, whereas a single supernumerary segment encompassing all four genes successfully promotes cancer. Gain of PVT1 long non-coding RNA expression was required for high MYC protein levels in 8q24-amplified human cancer cells. PVT1 RNA and MYC protein expression correlated in primary human tumours, and copy number of PVT1 was co-increased in more than 98% of MYC-copy-increase cancers. Ablation of PVT1 from MYC-driven colon cancer line HCT116 diminished its tumorigenic potency. As MYC protein has been refractory to small-molecule inhibition, the dependence of high MYC protein levels on PVT1 long non-coding RNA provides a much needed therapeutic target. [ABSTRACT FROM AUTHOR]

Malignant peripheral nerve sheath tumors (MPNSTs) are sarcomas of Schwann cell lineage origin that occur sporadically or in association with the inherited syndrome neurofibromatosis type 1. To identify genetic drivers of MPNST development, we used the Sleeping Beauty (SB) transposon-based somatic mutagenesis system in mice with somatic loss of transformation-related protein p53 (Trp53) function and/or overexpression of human epidermal growth factor receptor (EGFR). Common insertion site (CIS) analysis of 269 neurofibromas and 106 MPNSTs identified 695 and 87 sites with a statistically significant number of recurrent transposon insertions, respectively. Comparison to human data sets identified new and known driver genes for MPNST formation at these sites. Pairwise co-occurrence analysis of CIS-associated genes identified many cooperating mutations that are enriched in Wnt/β-catenin, PI3K-AKT-mTOR and growth factor receptor signaling pathways. Lastly, we identified several new proto-oncogenes, including Foxr2 (encoding forkhead box R2), which we functionally validated as a proto-oncogene involved in MPNST maintenance. [ABSTRACT FROM AUTHOR]

Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline. [ABSTRACT FROM AUTHOR]

We describe a system that permits conditional mobilization of a Sleeping Beauty (SB) transposase allele by Cre recombinase to induce cancer specifically in a tissue of interest. To demonstrate its potential for developing tissue-specific models of cancer in mice, we limit SB transposition to the liver by placing Cre expression under the control of an albumin enhancer/promoter sequence and screen for hepatocellular carcinoma (HCC)–associated genes. From 8,060 nonredundant insertions cloned from 68 tumor nodules and comparative analysis with data from human HCC samples, we identify 19 loci strongly implicated in causing HCC. These encode genes, such as EGFR and MET, previously associated with HCC and others, such as UBE2H, that are potential new targets for treating this neoplasm. Our system, which could be modified to drive transposon-based insertional mutagenesis wherever tissue-specific Cre expression is possible, promises to enhance understanding of cancer genomes and identify new targets for therapeutic development. [ABSTRACT FROM AUTHOR]

Retroviruses, acting as somatic cell insertional mutagens, have been widely used to identify cancer genes in the haematopoietic system and mammary gland. An insertional mutagen for use in other mouse somatic cells would facilitate the identification of genes involved in tumour formation in a wider variety of tissues. Here we report the ability of the Sleeping Beauty transposon to act as a somatic insertional mutagen to identify genes involved in solid tumour formation. A Sleeping Beauty transposon, engineered to elicit loss-of-function or gain-of-function mutations, transposed in all somatic tissues tested and accelerated tumour formation in mice predisposed to cancer. Cloning transposon insertion sites from these tumours revealed the presence of common integration sites, at known and candidate cancer genes, similar to those observed in retroviral mutagenesis screens. Sleeping Beauty is a new tool for unbiased, forward genetic screens for cancer genes in vivo. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Insertional mutagenesis has been at the core of functional genomics in many species. In the mouse, improved vectors and methodologies allow easier genome-wide and phenotype-driven insertional mutagenesis screens. The ability to generate homozygous diploid mutations in mouse embryonic stem cells allows prescreening for specific null phenotypes prior to in vivo analysis. In addition, the discovery of active transposable elements in vertebrates, and their development as genetic tools, has led to in vivo forward insertional mutagenesis screens in the mouse. These new technologies will greatly contribute to the speed and ease with which we achieve complete functional annotation of the mouse genome. [ABSTRACT FROM AUTHOR]

Presents a study that reported that cells co-purifying with mesenchymal stem cells, also known as multipotent adult progenitor cells (MAPC), differentiate, at the single cell level, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. Culture of undifferentiated mouse and rat MAPC; In vitro differentiation of single murine MAPC (mMAPC); mMAPC engraftment and differentiation in tissue-specific cells.

EVI27: is a common site of retroviral integration in BXH2 murine myeloid leukemias. Here we show that integration at Evi27 occurs in a CpG island ∼6 kb upstream from a novel gene (designated Evi27) with homology to the IL17 receptor (Il17r) and that proviral integrations result in increased expression of the Evi27 protein on the cell surface. The human EVI27 homolog was also cloned and mapped to chromosome 3p21. Multiple Evi27 isoforms were detected at the RNA and protein level in both human and mouse, indicating that Evi27 expression is complex. Some of the isoforms are shown to likely represent secreted soluble forms of the protein produced by intron incorporation or by proteolytic cleavage. In the mouse, highest Evi27 expression occurs in liver and testes with lower expression in kidney and lung. In humans, EVI27 is expressed at high levels in the kidney, with moderate levels in the liver, brain, and testes. Within hematopoietic cells, Evi27 expression is restricted. Northern and Western analysis showed that Evi27 is expressed in selected T-cell, B-cell and myeloid cell lines. These results suggest that Evi27 expression is tightly regulated during hematopoietic differentiation. Collectively, these studies identify a new member of the cytokine receptor family whose increased and uncoordinated expression may lead to myeloid leukemia by altering Evi27's normal ability to control the growth and/or differentiation of hematopoietic cells. Oncogene (2000) 19, 2098–2109 [ABSTRACT FROM AUTHOR]

Ecotropic MuLVs induce myeloid leukemia in BXH2 mice by insertional mutagenesis of cellular proto-oncogenes or tumor suppressor genes. Disease genes can thus be identified by viral tagging as common sites of viral integration in BXH2 leukemias. Previous studies showed that a frequent common integration site in BXH2 leukemias is the Nf1 tumor suppressor gene. Unexpectedly, about half of the viral integrations at Nf1 represented a previously undiscovered defective nonecotropic virus, termed MRV. Because other common integration sites in BXH2 leukemias encoding proto-oncogenes contain ecotropic rather than MRV viruses, it has been speculated that MRV viruses may selectively target tumor suppressor genes. To determine if this were the case, 21 MRV-positive BXH2 leukemias were screened for new MRV common integration sites. One new site, Mrvi1 was identified that was disrupted by MRV in two of the leukemias. Ecotropic virus did not disrupt Mrvi1 in 205 ecotropic virus-positive leukemias, suggesting that Mrvi1 is specifically targeted by MRV. Mrvi1 encodes a novel protein with homology to Jaw1, a lymphoid restricted type II membrane protein that localizes to the endoplasmic reticulum. MRV integration occurs at the 5′ end of the gene between two differentially used promoters. Within hematopoietic cells, Mrvi1 expression is restricted to megakaryocytes and some myeloid leukemias. Like Jaw1, which is downregulated during lymphoid differentiation, Mrv1 is downregulated during monocytic differentiation of BXH2 leukemias. Taken together, these data suggest that MRV integration at Mrvi1 induces myeloid leukemia by altering the expression of a gene important for myeloid cell growth and/or differentiation. Experiments are in progress to test whether Mrvi1 is a tumor suppressor gene. [ABSTRACT FROM AUTHOR]

Retroviral insertional mutagenesis in BXH2 and AKXD recombinant inbred mice induces a high incidence of myeloid or B- and T-cell leukaemia and the proviral integration sites in the leukaemias provide powerful genetic tags for disease gene identification. Some of the disease genes identified by proviral tagging are also associated with human disease, validating this approach for human disease gene identification. Although many leukaemia disease genes have been identified over the years, many more remain to be cloned. Here we describe an inverse PCR (IPCR) method for proviral tagging that makes use of automated DNA sequencing and the genetic tools provided by the Mouse Genome Project, which increases the throughput for disease gene identification. We also use this IPCR method to clone and analyse more than 400 proviral integration sites from AKXD and BXH2 leukaemias and, in the process, identify more than 90 candidate disease genes. Some of these genes function in pathways already implicated in leukaemia, whereas others are likely to define new disease pathways. Our studies underscore the power of the mouse as a tool for gene discovery and functional genomics. [ABSTRACT FROM AUTHOR]

In the original publication, corresponding author information missed indicating that Dr. Zadeh. [ABSTRACT FROM AUTHOR]

Osteosarcomas are characterized by highly disrupted genomes. Although osteosarcomas lack common fusions, we find evidence of many tumour specific gene-gene fusion transcripts, likely due to chromosomal rearrangements and expression of transcription-induced chimeras. Most of the fusions result in out-of-frame transcripts, potentially capable of producing long novel protein sequences and a plethora of neoantigens. To identify fusions, we explored RNA-sequencing data to obtain detailed knowledge of transcribed fusions, by creating a novel program to compare fusions identified by deFuse to de novo transcripts generated by Trinity. This allowed us to confirm the deFuse results and identify unusual splicing patterns associated with fusion events. Using various existing tools combined with this custom program, we developed a pipeline for the identification of fusion transcripts applicable as targets for immunotherapy. In addition to identifying candidate neoantigens associated with fusions, we were able to use the pipeline to establish a method for measuring the frequency of fusion events, which correlated to patient outcome, as well as highlight some similarities between canine and human osteosarcomas. The results of this study of osteosarcomas underscores the numerous benefits associated with conducting a thorough analysis of fusion events within cancer samples. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 nuclease system and conditional overexpression in the murine osteosarcoma cell lines K12 and K7M2. Proliferation, migration, and anchorage independent growth were evaluated. RNA sequencing and immunohistochemistry of human osteosarcoma tissue samples were used to further evaluate the potential role of the Slit-Robo pathway in osteosarcoma. The effects of Srgap2 expression modulation in the murine OS cell lines support the hypothesis that SRGAP2 may have a role as a suppressor of metastases in osteosarcoma. Additionally, SRGAP2 and other genes in the Slit-Robo pathway have altered transcript levels in a subset of mouse and human osteosarcoma, and SRGAP2 protein expression is reduced or absent in a subset of primary tumor samples. SRGAP2 and other axon guidance proteins likely play a role in osteosarcoma metastasis, with loss of SRGAP2 potentially contributing to a more aggressive phenotype. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML. [ABSTRACT FROM AUTHOR]

A correction to the article "Pluripotency of mesenchymal stem cells derived from adult marrow," which appeared in June 20, 2002 issue.

Rapid and stable genetic engineering of the regenerating liver would allow systematic, in vivo testing of contributions by many genes to regenerating tissue. Following fumaryl acetoacetate hydrolase (Fah) gene transfer to hepatocytes, selective repopulation of the liver occurs in Fah-deficient mice. This genetic correction is readily mediated with transposons. Using this approach, we show that genes with biological utility can be linked to a selectable FAH transposon cassette. First, net conversion of Fah-/- liver tissue to transgenic tissue, and its outgrowth, was monitored by bioluminescence in vivo from a luciferase gene linked to the FAH gene. Second, co-expressed shRNAs stably reduced target gene expression, indicating the potential for loss-of-function assays. Third, a mutant, gain-of-function allele of human a1-antitrypsin (hAAT) was linked to Fah and resulted in protein inclusions within hepatocytes, which are the histopathological hallmark of hAAT deficiency disorder. Finally, oncogenes linked to Fah resulted in fulminant hepatocellular carcinoma. Conclusion: co-expression with FAH is an effective technique for expression of transgenes in regenerating adult hepatocytes with applicability to genetic studies of the liver stem cell repopulation. [ABSTRACT FROM AUTHOR]