CRISPR-DAV: CRISPR NGS data analysis and visualization pipeline.

SUMMARY: The simplicity and precision of CRISPR/Cas9 system has brought in a new era of gene editing. Screening for desired clones with CRISPR-mediated genomic edits in a large number of samples is made possible by next generation sequencing (NGS) due to its multiplexing. Here we present CRISPR-DAV (CRISPR Data Analysis and Visualization) pipeline to analyze the CRISPR NGS data in a high throughput manner. In the pipeline, Burrows-Wheeler Aligner and Assembly Based ReAlignment are used for small and large indel detection, and results are presented in a comprehensive set of charts and interactive alignment view. AVAILABILITY AND IMPLEMENTATION: CRISPR-DAV is available at GitHub and Docker Hub repositories: https://github.com/pinetree1/crispr-dav.git and https://hub.docker.com/r/pinetree1/crispr-dav/. CONTACT: xuning.wang@bms.com.

Identification and validation of G protein-coupled receptors modulating flow-dependent signaling pathways in vascular endothelial cells.

Vascular endothelial cells are exposed to mechanical forces due to their presence at the interface between the vessel wall and flowing blood. The patterns of these mechanical forces (laminar vs. turbulent) regulate endothelial cell function and play an important role in determining endothelial phenotype and ultimately cardiovascular health. One of the key transcriptional mediators of the positive effects of laminar flow patterns on endothelial cell phenotype is the zinc-finger transcription factor, krüppel-like factor 2 (KLF2). Given its importance in maintaining a healthy endothelium, we sought to identify endothelial regulators of the KLF2 transcriptional program as potential new therapeutic approaches to treating cardiovascular disease. Using an approach that utilized both bioinformatics and targeted gene knockdown, we identified endothelial GPCRs capable of modulating KLF2 expression. Genetic screening using siRNAs directed to these GPCRs identified 12 potential GPCR targets that could modulate the KLF2 program, including a subset capable of regulating flow-induced KLF2 expression in primary endothelial cells. Among these targets, we describe the ability of several GPCRs (GPR116, SSTR3, GPR101, LGR4) to affect KLF2 transcriptional activation. We also identify these targets as potential validated targets for the development of novel treatments targeting the endothelium. Finally, we highlight the initiation of drug discovery efforts for LGR4 and report the identification of the first known synthetic ligands to this receptor as a proof-of-concept for pathway-directed phenotypic screening to identify novel drug targets.

Enhanced antitumor immunity by a novel small molecule HPK1 inhibitor.

BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) has been demonstrated as a negative intracellular immune checkpoint in mediating antitumor immunity in studies with HPK1 knockout and kinase dead mice. Pharmacological inhibition of HPK1 is desirable to investigate the role of HPK1 in human immune cells with therapeutic implications. However, a significant challenge remains to identify a small molecule inhibitor of HPK1 with sufficient potency, selectivity, and other drug-like properties suitable for proof-of-concept studies. In this report, we identified a novel, potent, and selective HPK1 small molecule kinase inhibitor, compound K (CompK). A series of studies were conducted to investigate the mechanism of action of CompK, aiming to understand its potential application in cancer immunotherapy. METHODS: Human primary T cells and dendritic cells (DCs) were investigated with CompK treatment under conditions relevant to tumor microenvironment (TME). Syngeneic tumor models were used to assess the in vivo pharmacology of CompK followed by human tumor interrogation ex vivo. RESULTS: CompK treatment demonstrated markedly enhanced human T-cell immune responses under immunosuppressive conditions relevant to the TME and an increased avidity of the T-cell receptor (TCR) to recognize viral and tumor-associated antigens (TAAs) in significant synergy with anti-PD1. Animal model studies, including 1956 sarcoma and MC38 syngeneic models, revealed improved immune responses and superb antitumor efficacy in combination of CompK with anti-PD-1. An elevated immune response induced by CompK was observed with fresh tumor samples from multiple patients with colorectal carcinoma, suggesting a mechanistic translation from mouse model to human disease. CONCLUSION: CompK treatment significantly improved human T-cell functions, with enhanced TCR avidity to recognize TAAs and tumor cytolytic activity by CD8+ T cells. Additional benefits include DC maturation and priming facilitation in tumor draining lymph node. CompK represents a novel pharmacological agent to address cancer treatment resistance.

'Cold shock' increases the frequency of homology directed repair gene editing in induced pluripotent stem cells.

Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20-40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1-20%) can be enhanced two- to ten-fold in both iPSCs and HEK293 cells by 'cold shocking' cells at 32 °C for 24-48 hours following transfection. This method can also increases the proportion of loci that have undergone complete sequence conversion across the donor sequence, or 'perfect HDR', as opposed to partial sequence conversion where nucleotides more distal to the CRISPR cut site are less efficiently incorporated ('partial HDR'). We demonstrate that the structure of the single-stranded DNA oligo donor can influence the fidelity of HDR, with oligos symmetric with respect to the CRISPR cleavage site and complementary to the target strand being more efficient at directing 'perfect HDR' compared to asymmetric non-target strand complementary oligos. Our protocol represents an efficient method for making CRISPR-mediated, specific DNA sequence changes within the genome that will facilitate the rapid generation of genetic models of human disease in iPSCs as well as other genome engineered cell lines.

An Analytical Comparison of Dako 28-8 PharmDx Assay and an E1L3N Laboratory-Developed Test in the Immunohistochemical Detection of Programmed Death-Ligand 1.

AIM: Nivolumab, a fully human immunoglobulin G4 programmed death-1 (PD-1) immune checkpoint inhibitor antibody, has activity in melanoma, non-small-cell lung cancer (NSCLC), renal cell carcinoma (RCC), and Hodgkin lymphoma. Nivolumab is approved in the USA and EU for advanced melanoma, NSCLC, and RCC, and relapsed Hodgkin lymphoma in the USA. Programmed death-ligand 1 (PD-L1), a PD-1 ligand, is expressed on mononuclear leukocytes, myeloid cells, and tumor cells. PD-L1 is being investigated as a potential biomarker to predict the association of tumor PD-L1 expression with nivolumab efficacy. METHODS: Bristol-Myers Squibb and Dako previously reported on an automated PD-L1 immunohistochemical (IHC) assay that detects cell surface PD-L1 in formalin-fixed, paraffin-embedded, human tumor tissue specimens using Dako's Autostainer Link 48. The primary antibody for this assay is a rabbit monoclonal antihuman PD-L1 antibody, clone 28-8. Another rabbit monoclonal antihuman PD-L1 antibody, clone E1L3N, was compared with 28-8 for specificity and sensitivity using an identical detection method followed by vendor-recommended detection methods. RESULTS: Using PD-L1 null clones of L2987 and ES-2 tumor cell lines, both antibodies were specific for detection of PD-L1 on the plasma membrane, although E1L3N also stained cytoplasm in ES-2 knockout cells. Using the identical method, E1L3N was slightly more sensitive than 28-8 based on staining intensities. Using manufacturer-recommended detection methods and predefined scoring criteria for plasma membrane staining of tumor and immune cells, 28-8 demonstrated significantly improved detection compared with E1L3N. CONCLUSIONS: Epitope retrieval and highly sensitive detection reagents are key determinants in IHC detection of PD-L1.

Quantitative PCR tissue expression profiling of the human SGLT2 gene and related family members.

SGLT2 (for "Sodium GLucose coTransporter" protein 2) is the major protein responsible for glucose reabsorption in the kidney and its inhibition has been the focus of drug discovery efforts to treat type 2 diabetes. In order to better clarify the human tissue distribution of expression of SGLT2 and related members of this cotransporter class, we performed TaqMan™ (Applied Biosystems, Foster City, CA, USA) quantitative polymerase chain reaction (PCR) analysis of SGLT2 and other sodium/glucose transporter genes on RNAs from 72 normal tissues from three different individuals. We consistently observe that SGLT2 is highly kidney specific while SGLT5 is highly kidney abundant; SGLT1, sodium-dependent amino acid transporter (SAAT1), and SGLT4 are highly abundant in small intestine and skeletal muscle; SGLT6 is expressed in the central nervous system; and sodium myoinositol cotransporter is ubiquitously expressed across all human tissues.

Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes.

Acetyl coenzyme A (acetyl-CoA) carboxylase isozyme 1 (ACC1) and acetyl-CoA carboxylase isozyme 2 (ACC2) are critical for de novo fatty acid synthesis and for the regulation of beta-oxidation. Emerging evidence indicates that one or both isozymes might be therapeutic targets for the treatment of obesity, type 2 diabetes, and dyslipidemia. One of the major obstacles in the field is the lack of readily-available source of recombinant human ACC enzymes to support systematic drug discovery efforts. Here, we describe an efficient and optimal protocol for expressing and isolating recombinant mammalian ACCs with high yield and purity. The resultant human ACC2, human ACC1, and rat ACC2 possess high specific activities, are properly biotinylated, and exhibit kinetic parameters very similar to the native ACC enzymes. We believe that the current study paves a road to a systematic approach for drug design revolving around the ACC inhibition mechanism.

A general liquid chromatography/mass spectroscopy-based assay for detection and quantitation of methyltransferase activity.

Methyltransferases form a large class of enzymes, most of which use S-adenosylmethionine as the methyl donor. In fact, S-adenosylmethionine is second only to ATP in the variety of reactions for which it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to specific enzymes and/or substrates. In this work we describe a sensitive liquid chromatography/mass spectroscopy-based methyltransferase assay. The assay monitors the conversion of S-adenosylmethionine to S-adenosylhomocysteine and can be applied to any methyltransferase and substrate of interest. We used the well-characterized enzyme catechol O-methyltransferase to demonstrate that the assay can monitor activity with a variety of substrates, can identify new substrates, and can be used even with crude preparation of enzyme. Furthermore, we demonstrate the utility of the assay for kinetic characterization of enzymatic activity.

Identification of acyl coenzyme A:monoacylglycerol acyltransferase 3, an intestinal specific enzyme implicated in dietary fat absorption.

Acyl coenzyme A:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol using 2-monoacylglycerol and fatty acyl coenzyme A. This enzymatic reaction is believed to be an essential and rate-limiting step for the absorption of fat in the small intestine. Although the first MGAT-encoding cDNA, designated MGAT1, has been recently isolated, it is not expressed in the small intestine and hence cannot account for the high intestinal MGAT enzyme activity that is important for the physiology of fat absorption. In the current study, we report the identification of a novel MGAT, designated MGAT3, and present evidence that it fulfills the criteria to be the elusive intestinal MGAT. MGAT3 encodes a approximately 36-kDa transmembrane protein that is highly homologous to MGAT1 and -2. In humans, expression of MGAT3 is restricted to gastrointestinal tract with the highest level found in the ileum. At the cellular level, recombinant MGAT3 is localized to the endoplasmic reticulum. Recombinant MGAT3 enzyme activity produced in insect Sf9 cells selectively acylates 2-monoacylglycerol with higher efficiency than other stereoisomers. The molecular identification of MGAT3 will facilitate the evaluation of using intestinal MGAT as a potential point of intervention for antiobesity therapies.

Expression and characterization of human transient receptor potential melastatin 3 (hTRPM3).

Transient receptor potential (TRP) cation-selective channels are an emerging class of proteins that are involved in a variety of important biological functions including pain transduction, thermosensation, mechanoregulation, and vasorelaxation. Utilizing a bioinformatics approach, we have identified the full-length human TRPM3 (hTRPM3) as a member of the TRP family. The hTRPM3 gene is comprised of 24 exons and maps to human chromosome 9q-21.12. hTRPM3 is composed of 1555 amino acids and possesses the characteristic six-transmembrane domain of the TRP family. hTRPM3 is expressed primarily in kidney and, at lesser levels, in brain, testis, and spinal cord as demonstrated by quantitative RT-PCR and Northern blotting. In situ hybridization in human kidney demonstrated that hTRPM3 mRNA expression is predominantly found in the collecting tubular epithelium. Heterologous expression of hTRPM3 in human embryonic kidney cells (HEK 293) showed that hTRPM3 is localized to the cell membrane. hTRPM3-expressing cells exhibited Ca2+ concentration-dependent Ca2+ entry. Depletion of intracellular Ca2+ stores by lowering extracellular Ca2+ concentration and treatment with the Ca2+-ATPase inhibitor thapsigargin or the muscarinic receptor agonist carbachol further augmented hTRPM3-mediated Ca2+ entry. The nonselective Ca2+ channel blocker, lanthanide gadolinium (Gd3+), partially inhibited hTRPM3-mediated Ca2+ entry. These results are consistent with the hypothesis that hTRPM3 mediates a Ca2+ entry pathway that apparently is distinct from the endogenous Ca2+ entry pathways present in HEK 293 cells.