David W. Sant

Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment

Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.

Evaluation of somatic mutations in tibial pseudarthrosis samples in neurofibromatosis type 1

Background Tibial pseudarthrosis is associated with neurofibromatosis type 1 (NF1) and there is wide clinical variability of the tibial dysplasia in NF1, suggesting the possibility of genetic modifiers. Double inactivation of NF1 is postulated to be necessary for the development of tibial pseudarthrosis, but tissue or cell of origin of the ‘second hit’ mutation remains unclear. Methods Exome sequencing of different sections of surgically resected NF1 tibial pseudarthrosis tissue was performed and compared to germline (peripheral blood). Results A germline NF1 splice site mutation (c.61-2A>T, p.L21 M68del) was identified from DNA extracted from peripheral blood. Exome sequencing of DNA extracted from tissue removed during surgery of the tibial pseudarthrosis showed a somatic mutation of NF1 (c.3574G>T, p.E1192*) in the normal germline allele. Further analysis of different regions of the tibial pseudarthrosis sample showed enrichment of the somatic mutation in the soft tissue within the pseudarthrosis site and absence of the somatic mutation in cortical bone. In addition, a germline variant in PTPN11 (c.1658C>T, p.T553M), a gene involved in the RAS signal transduction pathway was identified, although the clinical significance is unknown. Conclusions Given that the NF1 somatic mutation was primarily detected in the proliferative soft tissue at the pseudarthrosis site, it is likely that the second hit occurred in mesenchymal progenitors from the periosteum. These results are consistent with a defect of differentiation, which may explain why the mutation is found in proliferative cells and not within cortical bone tissue, as the latter by definition contains mostly mature differentiated osteoblasts and osteocytes.

Neurofibromin deficiency-associated transcriptional dysregulation suggests a novel therapy for tibial pseudoarthrosis in NF1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in NF1. Among the earliest manifestations is tibial pseudoarthrosis and persistent nonunion after fracture. To further understand the pathogenesis of pseudoarthrosis and the underlying bone remodeling defect, pseudoarthrosis tissue and cells cultured from surgically resected pseudoarthrosis tissue from NF1 individuals were analyzed using whole‐exome and whole‐transcriptome sequencing as well as genomewide microarray analysis. Genomewide analysis identified multiple genetic mechanisms resulting in somatic biallelic NF1 inactivation; no other genes with recurring somatic mutations were identified. Gene expression profiling identified dysregulated pathways associated with neurofibromin deficiency, including phosphoinositide 3‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK) signaling pathways. Unlike aggressive NF1‐associated malignancies, tibial pseudoarthrosis tissue does not harbor a high frequency of somatic mutations in oncogenes or other tumor‐suppressor genes, such as p53. However, gene expression profiling indicates that pseudoarthrosis tissue has a tumor‐promoting transcriptional pattern, despite lacking tumorigenic somatic mutations. Significant overexpression of specific cancer‐associated genes in pseudoarthrosis highlights a potential for receptor tyrosine kinase inhibitors to target neurofibromin‐deficient pseudoarthrosis and promote proper bone remodeling and fracture healing.

cAMP signaling regulates DNA hydroxymethylation by augmenting the intracellular labile ferrous iron pool

It is widely accepted that cAMP regulates gene transcription principally by activating the protein kinase A (PKA)-targeted transcription factors. Here, we show that cAMP enhances the generation of 5-hydroxymethylcytosine (5hmC) in multiple cell types. 5hmC is converted from 5-methylcytosine (5mC) by Tet methylcytosine dioxygenases, for which Fe(II) is an essential cofactor. The promotion of 5hmC was mediated by a prompt increase of the intracellular labile Fe(II) pool (LIP). cAMP enhanced the acidification of endosomes for Fe(II) release to the LIP likely through RapGEF2. The effect of cAMP on Fe(II) and 5hmC was confirmed by adenylate cyclase activators, phosphodiesterase inhibitors, and most notably by stimulation of G protein-coupled receptors (GPCR). The transcriptomic changes caused by cAMP occurred in concert with 5hmC elevation in differentially transcribed genes. Collectively, these data show a previously unrecognized regulation of gene transcription by GPCR-cAMP signaling through augmentation of the intracellular labile Fe(II) pool and DNA hydroxymethylation.

Ascorbate induces apoptosis in melanoma cells by suppressing Clusterin expression.

Pharmacological levels of ascorbate have long been suggested as a potential treatment of cancer. However, we observed that EC50 of ascorbate was at a similar level for cultured healthy melanocytes and melanoma cells, suggesting a limit of pharmacological ascorbate in treating cancer. Loss of 5-hydroxymethylcytosine (5 hmC) is an epigenetic hallmark of cancer and ascorbate promotes 5 hmC generation by serving as a cofactor for TET methylcytosine dioxygenases. Our previous work demonstrated that ascorbate treatment at physiological level (100 μM) increased 5 hmC content in melanoma cells toward the level of healthy melanocytes. Here we show that 100 µM of ascorbate induced apoptosis in A2058 melanoma cells. RNA-seq analysis revealed that expression of the Clusterin (CLU) gene, which is related to apoptosis, was downregulated by ascorbate. The suppression of CLU was verified at transcript level in different melanoma cell lines, and at protein level in A2058 cells. The anti-apoptotic cytoplasmic CLU was decreased, while the pro-apoptotic nuclear CLU was largely maintained, after ascorbate treatment. These changes in CLU subcellular localization were also associated with Bax and caspases activation, Bcl-xL sequestration, and cytochrome c release. Taken together, this study establishes an impending therapeutic role of physiological ascorbate to potentiate apoptosis in melanoma.

Human Epicardial Fat Expresses Glucagon-Like Peptide 1 and 2 Receptors Genes

Epicardial adipose tissue (EAT) is an easily measurable visceral fat of the heart with unique anatomy, functionality, and transcriptome. EAT can serve as a therapeutic target for pharmaceutical agents targeting the fat. Glucagon-like peptide-1 (GLP-1) and GLP-2 analogues are newer drugs showing beneficial cardiovascular and metabolic effects. Whether EAT expresses GLP- 1 and 2 receptors (GLP-1R and GLP-2R) is unknown. RNA-seq analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to evaluate the presence of GLP-1R and GLP-2R in EAT and subcutaneous fat (SAT) obtained from 8 subjects with coronary artery disease and type 2 diabetes mellitus undergoing elective cardiac surgery. Immunofluorescence was also performed on EAT and SAT samples using Mab3f52 against GLP-1R. Our RNA-sequencing (RNA-seq) analysis showed that EAT expresses both GLP-1R and GLP-2R genes. qRT-PCR analysis confirmed that GLP-1R expression was low but detected by 2 different sets of intron-spanning primers. GLP-2R expression was detected in all patients and was found to be 5-fold higher than GLP-1R. The combination of accurately spliced reads from RNA-seq and successful amplification using intron-spanning primers indicates that both GLP-1R and GLP-2R are expressed in EAT. Immunofluorescence clearly showed that GLP-1R is present and more abundant in EAT than SAT. This is the first time that human EAT is found to express both GLP-1R and GLP-2R genes. Pharmacologically targeting EAT may induce beneficial cardiovascular and metabolic effects.

Whole Exome Profiling of Ocular Surface Squamous Neoplasia.

Ocular surface squamous neoplasia (OSSN) represents a spectrum of diseases ranging from mild dysplasia to invasive squamous cell carcinoma. OSSN can be successfully managed with surgical excision or with medical therapy. Recently, interferon-α-2b (IFNα-2b) treatment has been established as a standard treatment option for OSSN, eliminating the need for surgical excision. However, approximately 15% of tumors do not respond to the IFNα-2b therapy. 1 It remains unclear which tumor-specific factors may affect treatment response and/or course after treatment. This information is important as it can help individualize therapy. For example, physicians may proceed directly to surgery, or use a different agent, in patients in whom IFNα-2b is unlikely to be effective.

Phenotypic and Functional Characteristics of Human Schwann Cells as Revealed by Cell-Based Assays and RNA-SEQ

This study comprehensively addresses the phenotype, function, and whole transcriptome of primary human and rodent Schwann cells (SCs) and highlights key species-specific features beyond the expected donor variability that account for the differential ability of human SCs to proliferate, differentiate, and interact with axons in vitro. Contrary to rat SCs, human SCs were insensitive to mitogenic factors other than neuregulin and presented phenotypic variants at various stages of differentiation, along with a mixture of proliferating and senescent cells, under optimal growth-promoting conditions. The responses of human SCs to cAMP-induced differentiation featured morphological changes and cell cycle exit without a concomitant increase in myelin-related proteins and lipids. Human SCs efficiently extended processes along those of other SCs (human or rat) but failed to do so when placed in co-culture with sensory neurons under conditions supportive of myelination. Indeed, axon contact-dependent human SC alignment, proliferation, and differentiation were not observed and could not be overcome by growth factor supplementation. Strikingly, RNA-seq data revealed that ~ 44 of the transcriptome contained differentially expressed genes in human and rat SCs. A bioinformatics approach further highlighted that representative SC-specific transcripts encoding myelin-related and axon growth-promoting proteins were significantly affected and that a deficient expression of key transducers of cAMP and adhesion signaling explained the fairly limited potential of human SCs to differentiate and respond to axonal cues. These results confirmed the significance of combining traditional bioassays and high-resolution genomics methods to characterize human SCs and identify genes predictive of cell function and therapeutic value.

Vitamin C sensitizes melanoma to BET inhibitors

Bromodomain and extraterminal inhibitors (BETi) are promising cancer therapies, yet prominent side effects of BETi at effective doses have been reported in phase I clinical trials. Here, we screened a panel of small molecules targeting epigenetic modulators against human metastatic melanoma cells. Cells were pretreated with or without ascorbate (vitamin C), which promotes DNA demethylation and subsequently changes the sensitivity to drugs. Top hits were structurally unrelated BETi, including JQ1, I-BET151, CPI-203, and BI-2536. Ascorbate enhanced the efficacy of BETi by decreasing acetylation of histone H4, but not H3, while exerting no effect on the expression of BRD proteins. Histone acetyltransferase 1 (HAT1), which catalyzes H4K5ac and H4K12ac, was downregulated by ascorbate mainly via the TET-mediated DNA hydroxymethylation pathway. Loss of H4ac, especially H4K5ac and H4K12ac, disrupted the interaction between BRD4 and H4 by which ascorbate and BETi blocked the binding of BRD4 to acetylated histones. Cotreatment with ascorbate and JQ1 induced apoptosis and inhibited proliferation of cultured melanoma cells. Ascorbate deficiency as modeled in Gulo-/- mice diminished the treatment outcome of JQ1 for melanoma tumorgraft. In contrast, ascorbate supplementation lowered the effective dose of JQ1 needed to successfully inhibit melanoma tumors in mice. On the basis of our findings, future clinical trials with BETi should consider ascorbate levels in patients. Furthermore, ascorbate supplementation might help reduce the severe side effects that arise from BETi therapy by reducing the dosage necessary for treatment.Significance: This study shows that ascorbate can enhance the efficacy of BET inhibitors, providing a possible clinical solution to challenges arising in phase I trials from the dose-dependent side effects of this class of epigenetic therapy. Cancer Res; 78(2); 572-83. ©2017 AACR.

Molecular Characteristics of Conjunctival Melanoma Using Whole-Exome Sequencing

Importance Conjunctival melanoma (CM) is a highly aggressive ocular cancer for which treatment options are limited; the molecular pathogenesis is poorly understood. Objective To identify the molecular characteristics of CM using next-generation whole-exome sequencing (WES). Design, Setting, and Participants Whole-exome sequencing was performed on tumor DNA extracted from the archived specimens of 5 patients with CM who had been treated with surgical excision between 2006 and 2011. These samples were analyzed at a tertiary academic ocular oncology referral center using a customized bioinformatic pipeline. Main Outcomes and Measures Sample analyses were designed to detect driver mutations, chromosome copy number aberrations, and mutation signatures. Results The study’s 5 patients ranged in age from 51 to 77 years. Four of the 5 were female, and all were white. Mutations were detected in known oncogenes, including BRAF, NRAS, NF1, EGFR, ALK, TERT, and APC. None of the mutations associated with uveal melanoma were found. All samples demonstrated a C→T mutation signature typical of UV-induced DNA damage. The most common CNA was a gain in chromosome 6p. Conclusions and Relevance In these 5 patients, WES allowed identification of mutations that can be targeted with therapy and supported the role of UV light in CM pathogenesis. These findings indicate a need for larger studies to evaluate the diagnostic, prognostic, and therapeutic value of WES for CM.