Recurrent secondary genomic alterations in desmoplastic small round cell tumors.

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is a rare, highly aggressive, translocation-associated soft-tissue sarcoma that primarily affects children, adolescents, and young adults, with a striking male predominance. It is characterized by t(11;22) generating a novel EWSR1-WT1 fusion gene. Secondary genomic alterations are rarely described. METHODS: Tumor tissue from 83 DSRCT patients was assayed by hybrid-capture based comprehensive genomic profiling, FoundationOne® Heme next generation sequencing analysis of 406 genes and RNA sequencing of 265 genes. Tumor mutation burden was calculated from a minimum of 1.4 Mb sequenced DNA. Microsatellite instability status was determined by a novel algorithm analyzing 114 specific loci. RESULTS: Comprehensive genomic profiling identified several genomically-defined DSRCT subgroups. Recurrent genomic alterations were most frequently detected in FGFR4, ARID1A, TP53, MSH3, and MLL3 genes. With the exception of FGFR4, where the genomic alterations predicted activation, most of the alterations in the remaining genes predicted gene inactivation. No DSRCT were TMB or MSI high. CONCLUSIONS: In summary, recurrent secondary somatic alterations in FGFR4, ARID1A, TP53, MSH3, and MLL3 were detected in 82% of DSRCT, which is significantly greater than previously reported. These alterations may have both prognostic and therapeutic implications.

Integration-defective lentiviral vector mediates efficient gene editing through homology-directed repair in human embryonic stem cells.

Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However, certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site, probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein, LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs.

A critical role for SHP2 in STAT5 activation and growth factor-mediated proliferation, survival, and differentiation of human CD34+ cells.

SHP2, a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene, plays a critical role in developmental hematopoiesis in the mouse, and gain-of-function mutations of SHP2 are associated with hematopoietic malignancies. However, the role of SHP2 in adult hematopoiesis has not been addressed in previous studies. In addition, the role of SHP2 in human hematopoiesis has not been described. These questions are of considerable importance given the interest in development of SHP2 inhibitors for cancer treatment. We used shRNA-mediated inhibition of SHP2 expression to investigate the function of SHP2 in growth factor (GF) signaling in normal human CD34(+) cells. SHP2 knockdown resulted in markedly reduced proliferation and survival of cells cultured with GF, and reduced colony-forming cell growth. Cells expressing gain-of-function SHP2 mutations demonstrated increased dependency on SHP2 expression for survival compared with cells expressing wild-type SHP2. SHP2 knockdown was associated with significantly reduced myeloid and erythroid differentiation with retention of CD34(+) progenitors with enhanced proliferative capacity. Inhibition of SHP2 expression initially enhanced and later inhibited STAT5 phosphorylation and reduced expression of the antiapoptotic genes MCL1 and BCLXL. These results indicate an important role for SHP2 in STAT5 activation and GF-mediated proliferation, survival, and differentiation of human progenitor cells.

Ubiquitous Luciferase Expression in "Firefly Rats" Does Not Alter the Pancreatic Islet Morphology, Metabolism, and Function.

"Firefly rats" ubiquitously express the luciferase reporter gene under the control of constitutively active ROSA26 promoter in inbred Lewis rats. Due to the minimal immunogenicity of luciferase, wide applications of Firefly rats have been reported in solid organ/cell transplantation studies for in vivo imaging, permitting quantitative and non-invasive tracking of the transplanted graft. ROSA26 is a non-coding gene and generally does not affect the expression of other endogenous genes. However, the effect of ubiquitous luciferase expression on islet morphology and function has not been thoroughly investigated, which is critical for the use of Firefly rats as islet donors in islet transplantation studies. Accordingly, in vivo glucose homeostasis (i.e., islet function in the native pancreas) was compared between age-matched luciferase-expressing Firefly rats and non-luciferase-expressing rats. In vivo assessments demonstrated no statistical difference between these rats in non-fasting blood glucose levels, intraperitoneal glucose tolerance tests, and glucose-stimulated serum C-peptide levels. Furthermore, islets were isolated from both rats to compare the morphology, function, and metabolism in vitro. Isolated islets from both rats exhibited similar in vitro characteristics in post-isolation islet yield, islet size, beta cell populations, insulin content per islet, oxygen consumption rate, and glucose-stimulated insulin secretion. In conclusion, ubiquitous luciferase expression in Firefly rats does not affect their islet morphology, metabolism, and function; this finding is critical and enables the use of isolated islets from Firefly rats for the dual assessment of islet graft function and bioluminescence imaging of islet grafts.

Brief report: phenotypic rescue of induced pluripotent stem cell-derived motoneurons of a spinal muscular atrophy patient.

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders in humans and is a common genetic cause of infant mortality. The disease is caused by loss of the survival of motoneuron (SMN) protein, resulting in the degeneration of alpha motoneurons in spinal cord and muscular atrophy in the limbs and trunk. One function of SMN involves RNA splicing. It is unclear why a deficiency in a housekeeping function such as RNA splicing causes profound effects only on motoneurons but not on other cell types. One difficulty in studying SMA is the scarcity of patient's samples. The discovery that somatic cells can be reprogrammed to become induced pluripotent stem cell (iPSCs) raises the intriguing possibility of modeling human diseases in vitro. We reported the establishment of five iPSC lines from the fibroblasts of a type 1 SMA patient. Neuronal cultures derived from these SMA iPSC lines exhibited a reduced capacity to form motoneurons and an abnormality in neurite outgrowth. Ectopic SMN expression in these iPSC lines restored normal motoneuron differentiation and rescued the phenotype of delayed neurite outgrowth. These results suggest that the observed abnormalities are indeed caused by SMN deficiency and not by iPSC clonal variability. Further characterization of the cellular and functional deficits in motoneurons derived from these iPSCs may accelerate the exploration of the underlying mechanisms of SMA pathogenesis.

BCR-ABL gene expression is required for its mutations in a novel KCL-22 cell culture model for acquired resistance of chronic myelogenous leukemia.

Acquired resistance through genetic mutations is a common phenomenon in several cancer therapies using molecularly targeted drugs, best exemplified by the BCR-ABL inhibitor imatinib in treating chronic myelogenous leukemia (CML). Overcoming acquired resistance is a daunting therapeutic challenge, and little is known about how these mutations evolve. To facilitate understanding the resistance mechanisms, we developed a novel culture model for CML acquired resistance in which the CML cell line KCL-22, following initial response to imatinib, develops resistant T315I BCR-ABL mutation. We demonstrate that the emergence of BCR-ABL mutations do not require pre-existing BCR-ABL mutations derived from the original patient as the subclones of KCL-22 cells can form various BCR-ABL mutations upon imatinib treatment. BCR-ABL mutation rates vary from cell clone to clone and passages, in contrast to the relatively stable mutation rate of the hypoxanthine-guanine phosphoribosyltransferase gene. Strikingly, development of BCR-ABL mutations depends on its gene expression because BCR-ABL knockdown completely blocks KCL-22 cell relapse on imatinib and acquisition of mutations. We further show that the endogenous BCR-ABL locus has significantly higher mutagenesis potential than the transduced randomly integrated BCR-ABL cDNA. Our study suggests important roles of BCR-ABL gene expression and its native chromosomal locus for acquisition of BCR-ABL mutations and provides a new tool for further studying resistance mechanisms.

Site-specific gene insertion mediated by a Cre-loxP-carrying lentiviral vector.

Retroviral vectors have been used to treat patients with the X-linked severe combined immunodeficiency disease and chronic granulomatous disease. In both cases, success has been undermined by clonal expansion of transduced cells in some patients due to insertional mutagenesis induced by random vector integration. This outcome underscores the importance of designing vectors for site-specific gene insertion to avoid unanticipated gene disruption or gene activation. In the present study, we incorporated the sequence-specific Cre protein into lentiviral virions. We demonstrated that the virion-associated Cre protein remained enzymatically active and was capable of directing site-specific insertion of a gene in the vector into a defined loxP site in the host genome. As there are loxP-like sequences throughout human genome that can be recognized by either wild-type Cre or Cre variants, our study demonstrates a new strategy of designing lentiviral-based vector for gene targeting.

Detection of CRISPR/Cas9-Generated Off-Target Effect by Integration-Defective Lentiviral Vector.

Clustered regularly interspaced short palindromic repeat (CRISPR) and other gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) show great promises for research and therapeutic applications. One major concern is the off-target effects generated by these nucleases at unintended genomic sequences. In silico methods are usually used for off-target site prediction. However, based on currently available algorithms, the predicted cleavage activity at these potential off-target sites does not always reflect the true cleavage in vivo. Here we present an unbiased screening protocol using integration-defective lentiviral vector (IDLV) and deep sequencing to map the off-target sites generated by gene editing tools.

RASSF1A is part of a complex similar to the Drosophila Hippo/Salvador/Lats tumor-suppressor network.

The Ras Association Domain Family 1A (RASSF1A) gene is one of the most frequently silenced genes in human cancer. RASSF1A has been shown to interact with the proapoptotic kinase MST1. Recent work in Drosophila has led to the discovery of a new tumor-suppressor pathway involving the Drosophila MST1 and MST2 ortholog, Hippo, as well as the Lats/Warts serine/threonine kinase and a protein named Salvador (Sav). Little is known about this pathway in mammalian cells. We report that complexes consisting of RASSF1A, MST2, WW45 (the human ortholog of Sav), and LATS1 exist in human cells. MST2 enhances the RASSF1A-WW45 interaction, which requires the C-terminal SARAH domain of both proteins. Components of this complex are localized at centrosomes and spindle poles from interphase to telophase and at the midbody during cytokinesis. Both RASSF1A and WW45 activate MST2 by promoting MST2 autophosphorylation and LATS1 phosphorylation. Mitosis is delayed in Rassf1a(-/-) mouse embryo fibroblasts and frequently results in cytokinesis failure, similar to what has been observed for LATS1-deficient cells. RASSF1A, MST2, or WW45 can rescue this defect. The complex of RASSF1A, MST2, WW45, and LATS1 consists of several tumor suppressors, is conserved in mammalian cells, and appears to be involved in controlling mitotic exit.

A Small Molecule-Controlled Cas9 Repressible System.

CRISPR-Cas9 has been developed into a powerful molecular tool for genome engineering, and it has revolutionized the field of biomedical research. Despite the tremendous potential of CRISPR-Cas9 in biomedical research, precise control of CRISPR-Cas9 over the dose and exposure time is important to expand its applications. In this study, we fused Cas9 with a peptide termed small molecule-assisted shut-off (SMASh) consisting of a protease domain and a degron domain derived from hepatitis C virus (HCV). The presence of SMASh allows tight control of the Cas9 stability via a clinically approved HCV protease inhibitor asunaprevir (ASV). We showed that the engineered Cas9 responded to ASV administration and rapidly degraded in a dose- and time-dependent manner. Cas9 degradation was reversible upon ASV removal that restored the gene editing activity. We also showed that limiting the level of Cas9 in cells increased the specificity of gene editing. The SMASh tag therefore provides an effective tool to control Cas9 stability, allowing an improvement in the accuracy, safety, and versatility of the CRISPR-Cas9 system for genome editing and gene regulation studies.

Using Gene Editing to Establish a Safeguard System for Pluripotent Stem-Cell-Based Therapies.

A major challenge in using human pluripotent stem cells (hPSCs) in therapy is the risk of teratoma formation due to contaminating undifferentiated stem cells. We used CRISPR-Cas9 for in-frame insertion of a suicide gene, iC9, into the endogenous SOX2 locus in human embryonic stem cell (ESC) line H1 for specific eradication of undifferentiated cells without affecting differentiated cells. This locus was chosen over NANOG and OCT4, two other well-characterized stem cell loci, due to significantly reduced off-target effect. We showed that undifferentiated H1-iC9 cells were induced to apoptosis by iC9 inducer AP1903, whereas differentiated cell lineages including hematopoietic cells, neurons, and islet beta-like cells were not affected. We also showed that AP1903 selectively removed undifferentiated H1-iC9 cells from a mixed cell population. This strategy therefore provides a layer of safety control before transplantation of a stem-cell-derived product in therapy.

A Human Embryonic Stem Cell Model of Aß-Dependent Chronic Progressive Neurodegeneration.

We describe the construction and phenotypic analysis of a human embryonic stem cell model of progressive Aß-dependent neurodegeneration (ND) with potential relevance to Alzheimer's disease (AD). We modified one allele of the normal APP locus to directly express a secretory form of Aß40 or Aß42, enabling expression from this edited allele to bypass the normal amyloidogenic APP processing pathway. Following neuronal differentiation, edited cell lines specifically accumulate intracellular aggregated/oligomeric Aß, exhibit a synaptic deficit, and have an abnormal accumulation of endolysosomal vesicles. Edited cultures progress to a stage of overt ND. All phenotypes appear at earlier culture times for Aß42 relative to Aß40. Whole transcriptome RNA-Seq analysis identified 23 up and 70 down regulated genes (differentially expressed genes) with similar directional fold change but larger absolute values in the Aß42 samples suggesting common underlying pathogenic mechanisms. Pathway/annotation analysis suggested that down regulation of extracellular matrix and cilia functions is significantly overrepresented. This cellular model could be useful for uncovering mechanisms directly linking Aß to neuronal death and as a tool to screen for new therapeutic agents that slow or prevent human ND.

Pancreatic human islets and insulin-producing cells derived from embryonic stem cells are rapidly identified by a newly developed Dithizone.

We developed an optimized Dipheylthiocarbazone or Dithizone (DTZ) with improved physical and chemical properties to characterize human islets and insulin-producing cells differentiated from embryonic stem cells. Application of the newly formulated iDTZ (i stands for islet) over a range of temperatures, time intervals and cell and tissue types found it to be robust for identifying these cells. Through high transition zinc binding, the iDTZ compound concentrated in insulin-producing cells and proved effective at delineating zinc levels in vitro.

Safety and Efficacy of a Lentiviral Vector Containing Three Anti-HIV Genes-CCR5 Ribozyme, Tat-rev siRNA, and TAR Decoy-in SCID-hu Mouse-Derived T Cells.

Gene therapeutic strategies show promise in controlling human immunodeficiency virus (HIV) infection and in restoring immunological function. A number of efficacious anti-HIV gene constructs have been described so far, including small interfering RNAs (siRNAs), RNA decoys, transdominant proteins, and ribozymes, each with a different mode of action. However, as HIV is prone to generating escape mutants, the use of a single anti-HIV construct would not be adequate to afford long range-viral protection. On this basis, a combination of highly potent anti-HIV genes-namely, a short hairpin siRNA (shRNA) targeting rev and tat, a transactivation response (TAR) decoy, and a CCR5 ribozyme-have been inserted into a third-generation lentiviral vector. Our recent in vitro studies with this construct, Triple-R, established its efficacy in both T-cell lines and CD34 cell-derived macrophages. In this study, we have evaluated this combinatorial vector in vivo. Vector-transduced CD34 cells were injected into severe combined immunodeficiency (SCID)-hu mouse thy/liv grafts to determine their capacity to give rise to T cells. Our results show that phenotypically normal transgenic T cells are generated that are able to resist HIV-1 infection when challenged in vitro. These important attributes of this combinatorial vector show its promise as an excellent candidate for use in human clinical trials.

KIF20A/MKLP2 regulates the division modes of neural progenitor cells during cortical development.

Balanced symmetric and asymmetric divisions of neural progenitor cells (NPCs) are crucial for brain development, but the underlying mechanisms are not fully understood. Here we report that mitotic kinesin KIF20A/MKLP2 interacts with RGS3 and plays a crucial role in controlling the division modes of NPCs during cortical neurogenesis. Knockdown of KIF20A in NPCs causes dislocation of RGS3 from the intercellular bridge (ICB), impairs the function of Ephrin-B-RGS cell fate signaling complex, and leads to a transition from proliferative to differentiative divisions. Germline and inducible knockout of KIF20A causes a loss of progenitor cells and neurons and results in thinner cortex and ventriculomegaly. Interestingly, loss of function of KIF20A induces early cell cycle exit and precocious neuronal differentiation without causing substantial cytokinesis defect or apoptosis. Our results identify a RGS-KIF20A axis in the regulation of cell division and suggest a potential link of the ICB to regulation of cell fate determination.

Inhibition of infectious human immunodeficiency virus type 1 virions via lentiviral vector encoded short antisense RNAs.

During the life cycles of most retroviruses and lentiviruses, dimerization and packaging of two copies of viral genomic RNA is required for the subsequent conversion of RNA into double stranded DNA by reverse transcriptase. For human immunodeficiency virus type 1 (HIV-1), dimerization is mediated by interactions of the stem-loop structures in the dimerization-packaging, or psi (Psi) domain. We have tethered anti-HIV gag ribozymes and small antisense RNAs to the HIV Psi domain in an HIV-1 lentiviral vector to facilitate copackaging of these replication inhibitors with HIV genomic RNAs during HIV infectious challenge. In order to maximize the base pairing of the ribozymes or antisense segments to the HIV-1 genomic target, sequences in HIV-1 were identified that are highly accessible to antisense pairing. Ribozymes or antisense RNAs designed to target these sequences were inserted in the lentiviral vector at the same relative distance to the Psi element as the HIV-1 target sites. Packaged vectors were transduced into CEM cells followed by challenges with HIV-1. Only the constructs that harbored short antisense segments complementary to HIV-1 gag produced replication incompetent HIV-1. These results demonstrate that a short stretch of antisense pairing downstream of the dimerization domain in an HIV-based vector can drive dimerization and provide a powerful approach for inhibition of HIV-1.

Downregulation of human Cdc6 protein using a lentivirus RNA interference expression vector.

Eukaryotic CDC6 gene function is required for the initiation of DNA replication and is a key regulatory protein during cell cycle progression. The human CDC6 gene is not expressed in most normal tissues, in contrast with its marked expression in proliferating cancer cells. An effective way to explore the gene functions of CDC6 is to knock-down the CDC6 messenger RNA (mRNA) and examine the phenotypic consequences. In this chapter, we describe the construction of a lentivirus vector to express a CDC6 DNA segment. The transcript is able to fold by itself because the sense and antisense regions are complementary. There is a 9-nucleotide (nt) loop region allowing for the short hairpin RNA (shRNA) to form. Cellular ribonucleases process the shRNA into a functional short interfering RNA (siRNA). Down-regulation of Cdc6 protein is confirmed by Western blots.

Effect of mutational inactivation of tyrosine kinase activity on BCR/ABL-induced abnormalities in cell growth and adhesion in human hematopoietic progenitors.

Chronic myelogenous leukemia (CML) results from transformation of a primitive hematopoietic cell by the BCR/ABL gene. The specific BCR/ABL signaling mechanisms responsible for transformation of primitive human hematopoietic cells are not well defined. Previous studies have suggested that constitutively activated tyrosine kinase activity plays an important role for in abnormal proliferation of CML progenitors but has not clearly defined its role in abnormal adhesion and migration. We established a human progenitor model of CML by ectopic expression of BCR/ABL in normal CD34+ cells using retrovirus-mediated gene transfer. CD34+ cells expressing BCR/ABL demonstrated several features characteristic of primary CML progenitors including increased proliferation in committed and primitive progenitor culture, reduced adhesion to fibronectin, and reduced chemotaxis to stroma-derived factor-1alpha. We expressed a kinase-inactive BCR/ABL gene to directly investigate the role of kinase activity in abnormal progenitor function. Abnormalities in proliferation were completely reversed, whereas defects in adhesion and migration were significantly improved but not completely reversed in cells expressing a kinase-inactive BCR/ABL. Furthermore, the BCR/ABL kinase inhibitor imatinib mesylate markedly inhibited proliferation of BCR/ABL-expressing progenitors but did not fully correct the adhesion and migration defects. Expression of BCR/ABL genes with deletions of either the COOH-terminal actin binding or proline-rich domains resulted in enhanced adhesion and chemotaxis compared with wild-type BCR/ABL but did not affect progenitor proliferation. We conclude that abnormal kinase activity is essential for abnormal proliferation and survival of CML progenitors but that abnormal adhesion and migration result from both kinase-dependent and -independent mechanisms.

Off-target effects of engineered nucleases.

Recent advances in gene editing with engineered nucleases have transformed our ability to manipulate the genome from diverse organisms for applications ranging from biomedical research to disease treatment. A major complication with these engineered nucleases is the binding of the nuclease to unintended genomic sites that share sequence homology with the on-target site. Cleavage of these off-target sites followed by DNA repair using normal cellular DNA repair mechanisms can cause gene mutation or gross chromosome rearrangement. Identification of nuclease-generated off-target sites is a daunting task due to the size and complexity of the mammalian genome. Five unbiased, genome-wide strategies have been developed to detect the off-target cleavage. Some of these strategies reach the sensitivity near the detection limit of directed deep sequencing and have sufficient precision and resolution to objectively assessing the off-target effect of any engineered nuclease. Significant progress has also been made recently to boost the nuclease targeting specificity by protein engineering to modify the structure of the nuclease and alter the interaction with its genomic target. In several studied cases, the off-target effect generated by the modified nuclease is completely eliminated. These modified nucleases significantly improve the overall fidelity of gene editing. These developments will enable gene editing tools to be applied more broadly and safely in basic research and disease treatment.