Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe)) and presenilin 1 (PSEN1(M146V)) and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.

Adenosine A2A receptor null chondrocyte transcriptome resembles that of human osteoarthritic chondrocytes.

Adenosine signaling plays a critical role in the maintenance of articular cartilage and may serve as a novel therapeutic for osteoarthritis (OA), a highly prevalent and morbid disease without effective therapeutics in the current market. Mice lacking adenosine A2A receptors (A2AR) develop spontaneous OA by 16 weeks of age, a finding relevant to human OA since loss of adenosine signaling due to diminished adenosine production (NT5E deficiency) also leads to development of OA in mice and humans. To better understand the mechanism by which A2AR and adenosine generation protect from OA development, we examined differential gene expression in neonatal chondrocytes from WT and A2AR null mice. Analysis of differentially expressed genes was analyzed by KEGG pathway analysis, and oPOSSUM and the flatiron database were used to identify transcription factor binding enrichment, and tissue-specific network analyses and patterns were compared to gene expression patterns in chondrocytes from patients with OA. There was a differential expression of 2211 genes (padj<0.05). Pathway enrichment analysis revealed that pro-inflammatory changes, increased metalloprotease, reduced matrix organization, and homeostasis are upregulated in A2AR null chondrocytes. Moreover, stress responses, including autophagy and HIF-1 signaling, seem to be important drivers of OA and bear marked resemblance to the human OA transcriptome. Although A2AR null mice are born with grossly intact articular cartilage, we identify here the molecular foundations for early-onset OA in these mice, further establishing their role as models for human disease and the potential use of adenosine as a treatment for human disease.

Selective inhibition of protein arginine methyltransferase 5 blocks initiation and maintenance of B-cell transformation.

Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)-induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV(+) lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas.

Sorafenib and 2-Deoxyglucose Synergistically Inhibit Proliferation of Both Sorafenib-Sensitive and -Resistant HCC Cells by Inhibiting ATP Production.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths globally. Sorafenib is the only first-line systemic drug for advanced HCC, but it has very limited survival benefits because patients treated with sorafenib either suffer from side effects or show disease progression after initial response. Thus, there is an urgent need to develop novel strategies for first-line and second-line therapies. The association between sorafenib resistance and glycolysis prompted us to screen several drugs with known antiglycolytic activity to identify those that will sensitize cells to sorafenib. We demonstrate that the combination of glycolytic inhibitor 2-deoxyglucose (2DG) and sorafenib drastically inhibits viability of sorafenib-sensitive and -resistant cells. However, the combination of other antiglycolytic drugs like lonidamine, gossypol, 3-bromopyruvate, and imatinib with sorafenib does not show synergistic effect. Cell cycle analysis revealed that the combination of 2DG and sorafenib induced cell cycle arrest at G0/G1. Mechanistic investigation suggests that the cell cycle arrest is due to depletion of cellular ATP that activates AMP-activated protein kinase (AMPK), which, in turn, inhibits mammalian target of rapamycin (mTOR) to induce cell cycle arrest. This study provides strong evidence for the therapeutic potential of the combination of sorafenib and 2DG for HCC.

Indole-3-carbinol inhibits tumorigenicity of hepatocellular carcinoma cells via suppression of microRNA-21 and upregulation of phosphatase and tensin homolog.

A major obstacle to successful treatment of hepatocellular carcinoma (HCC) is its high resistance to cytotoxic chemotherapy due to overexpression of multidrug resistance genes. Activation of the AKT pathway is known to be involved in chemoresistance in HCC; however, the underlying mechanisms modulating the AKT pathway by chemopreventive agents remain unclear. In the present study, we found that indole-3-carbinol (I3C) treatment for tumor cells repressed the AKT pathway by increasing the expression of phosphatase and tensin homolog (PTEN) in HCC xenograft tumor and HCC cell lines. qRT-PCR data showed that the expression of miR-21 and miR-221&222 was significantly reduced by I3C in HCC cells in vitro and in vivo. Reactivation of the AKT pathway via restoration of miR-21 was reversed by I3C. Ectopic expression of miR-21 mediated-accelerated wound healing was abrogated by I3C. Moreover, reducing the expression of miR-21 by anti-miR decreased the resistance of HCC cells to I3C. These results provide experimental evidences that I3C could function as a miR-21 regulator, leading to repression of the PTEN/AKT pathway and opening a new avenue for eradication of drug-resistant cells, thus potentially helping to improve the therapeutic outcome in patients diagnosed with HCC.

Characterization of a subpopulation of developing cortical interneurons from human iPSCs within serum-free embryoid bodies.

Production and isolation of forebrain interneuron progenitors are essential for understanding cortical development and developing cell-based therapies for developmental and neurodegenerative disorders. We demonstrate production of a population of putative calretinin-positive bipolar interneurons that express markers consistent with caudal ganglionic eminence identities. Using serum-free embryoid bodies (SFEBs) generated from human inducible pluripotent stem cells (iPSCs), we demonstrate that these interneuron progenitors exhibit morphological, immunocytochemical, and electrophysiological hallmarks of developing cortical interneurons. Finally, we develop a fluorescence-activated cell-sorting strategy to isolate interneuron progenitors from SFEBs to allow development of a purified population of these cells. Identification of this critical neuronal cell type within iPSC-derived SFEBs is an important and novel step in describing cortical development in this iPSC preparation.

Reciprocal regulation of microRNA-122 and c-Myc in hepatocellular cancer: role of E2F1 and transcription factor dimerization partner 2.

UNLABELLED: c-Myc is a well-known oncogene frequently up-regulated in different malignancies, whereas liver-specific microRNA (miR)-122, a bona fide tumor suppressor, is down-regulated in hepatocellular cancer (HCC). Here we explored the underlying mechanism of reciprocal regulation of these two genes. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and northern blot analysis demonstrated reduced expression of the primary, precursor, and mature miR-122 in c-MYC-induced HCCs compared to the benign livers, indicating transcriptional suppression of miR-122 upon MYC overexpression. Indeed, chromatin immunoprecipitation (ChIP) assay showed significantly reduced association of RNA polymerase II and histone H3K9Ac, markers of active chromatin, with the miR-122 promoter in tumors relative to the c-MYC-uninduced livers, indicating transcriptional repression of miR-122 in c-MYC-overexpressing tumors. The ChIP assay also demonstrated a significant increase in c-Myc association with the miR-122 promoter region that harbors a conserved noncanonical c-Myc binding site in tumors compared to the livers. Ectopic expression and knockdown studies showed that c-Myc indeed suppresses expression of primary and mature miR-122 in hepatic cells. Additionally, Hnf-3ß, a liver enriched transcription factor that activates miR-122 gene, was suppressed in c-MYC-induced tumors. Notably, miR-122 also repressed c-Myc transcription by targeting transcriptional activator E2f1 and coactivator Tfdp2, as evident from ectopic expression and knockdown studies and luciferase reporter assays in mouse and human hepatic cells. CONCLUSION: c-Myc represses miR-122 gene expression by associating with its promoter and by down-regulating Hnf-3ß expression, whereas miR-122 indirectly inhibits c-Myc transcription by targeting Tfdp2 and E2f1. In essence, these results suggest a double-negative feedback loop between a tumor suppressor (miR-122) and an oncogene (c-Myc).

Genome-wide methylation profiling in decitabine-treated patients with acute myeloid leukemia.

The outcome of older (≥ 60 years) acute myeloid leukemia (AML) patients is poor, and novel treatments are needed. In a phase 2 trial for older AML patients, low-dose (20 mg/m(2) per day for 10 days) decitabine, a DNA hypomethylating azanucleoside, produced 47% complete response rate with an excellent toxicity profile. To assess the genome-wide activity of decitabine, we profiled pretreatment and post treatment (day 25/course 1) methylomes of marrow samples from patients (n = 16) participating in the trial using deep-sequencing analysis of methylated DNA captured by methyl-binding protein (MBD2). Decitabine significantly reduced global methylation compared with pretreatment baseline (P = .001). Percent marrow blasts did not correlate with global methylation levels, suggesting that hypomethylation was related to the activity of decitabine rather than to a mere decrease in leukemia burden. Hypomethylation occurred predominantly in CpG islands and CpG island-associated regions (P ranged from .03 to .04) A significant concentration (P < .001) of the hypomehtylated CpG islands was found in chromosome subtelomeric regions, suggesting a differential activity of decitabine in distinct chromosome regions. Hypermethylation occurred much less frequently than hypomethylation and was associated with low CpG content regions. Decitabine-related methylation changes were concordant with those previously reported in distinct genes. In summary, our study supports the feasibility of methylome analyses as a pharmacodynamic endpoint for hypomethylating therapies.

Clinical and pharmacodynamic activity of bortezomib and decitabine in acute myeloid leukemia.

We recently reported promising clinical activity for a 10-day regimen of decitabine in older AML patients; high miR-29b expression associated with clinical response. Subsequent preclinical studies with bortezomib in AML cells have shown drug-induced miR-29b up-regulation, resulting in loss of transcriptional activation for several genes relevant to myeloid leukemogenesis, including DNA methyltransferases and receptor tyrosine kinases. Thus, a phase 1 trial of bortezomib and decitabine was developed. Nineteen poor-risk AML patients (median age 70 years; range, 32-84 years) enrolled. Induction with decitabine (20 mg/m(2) intravenously on days 1-10) plus bortezomib (escalated up to the target 1.3 mg/m(2) on days 5, 8, 12, and 15) was tolerable, but bortezomib-related neuropathy developed after repetitive cycles. Of previously untreated patients (age ≥ 65 years), 5 of 10 had CR (complete remission, n = 4) or incomplete CR (CRi, n = 1); 7 of 19 overall had CR/CRi. Pharmacodynamic analysis showed FLT3 down-regulation on day 26 of cycle 1 (P = .02). Additional mechanistic studies showed that FLT3 down-regulation was due to bortezomib-induced miR-29b up-regulation; this led to SP1 down-regulation and destruction of the SP1/NF-κB complex that transactivated FLT3. This study demonstrates the feasibility and preliminary clinical activity of decitabine plus bortezomib in AML and identifies FLT3 as a novel pharmacodynamic end point for future trials.

Regulation of glucose metabolism in hepatocarcinogenesis by microRNAs.

In the past decade, considerable effort has been made in elucidating the mechanism underlying the high level of aerobic glycolysis in cancer cells. While some recent studies have attempted to address this issue, the potential role of microRNAs in this process has not been explored until recently. These studies have demonstrated involvement of just five deregulated miRNAs in glucose metabolism in hepatocarcinogenesis. This review discusses the metabolic significance of these miRNAs in hepatoceullular carcinoma, their targets in glycolysis, gluconeogenesis, and pentose phosphate pathways, and provides an insight into the therapeutic potential of targeting specific miRNAs.

Methylation of the PTPRO gene in human hepatocellular carcinoma and identification of VCP as its substrate.

We have previously reported that the gene encoding protein tyrosine phosphatase receptor type-O (PTPRO) is suppressed by promoter methylation in a rat model of hepatocellular carcinoma (HCC) and it functions as tumor suppressor in leukemia and lung cancer. Here, we explored the methylation and expression of PTPRO as well as its function in human HCC. MassARRAY analysis of primary human HCC and matching liver samples (n = 24) revealed significantly higher (P = 0.004) methylation density at the promoter CGI in tumors. Combined bisulfite restriction analysis (COBRA) of another set of human HCC samples (n = 17) demonstrated that the CGI was methylated in 29% of tumors where expression of PTPRO was lower than that in corresponding matching livers. A substrate-trapping mutant of PTPRO that stabilizes the bound substrates was used to identify its novel substrate(s). VCP/p97 was found to be a PTPRO substrate by mass spectrometry of the peptides pulled down by the substrate-trapping mutant of PTPRO. Tyrosyl dephosphorylation of VCP following ectopic expression of wild-type PTPRO in H293T and HepG2 cells confirmed that it is a bona fide substrate of PTPRO. Treatment of PTPRO overexpressing HepG2 cells with Doxorubicin, a DNA damaging drug commonly used in therapy of primary HCC, sensitized these cells to this potent anticancer drug that correlated with dephosphorylation of VCP. Taken together, these results demonstrate methylation and downregulation of PTPRO in a subset of primary human HCC and establish VCP as a novel functionally important substrate of this tyrosine phosphatase that could be a potential molecular target for HCC therapy.