Comparative Analysis of Multiplex Platforms for Detecting Vitreous Biomarkers in Diabetic Retinopathy.

Purpose: To evaluate the feasibility of using the Proximity Extension Assay (PEA) platform to detect biomarkers in vitreous and to compare the findings with results obtained with an electrochemiluminescent (ECL) sandwich immunoassay. Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic controls were tested using two different proteomics platforms. Forty-one assays were completed with the ECL platform and 459 with the PEA platform. Spearman's rank correlation coefficient (rs ) was used to determine the direction and strength of the relationship between protein levels detected by both platforms. Results: Three hundred sixty-six PEA assays detected the tested protein in at least 25% of samples, and the difference in protein abundance between PDR and controls was statistically significant for 262 assays. Seventeen ECL assays yielded a detection rate ≥ 25%, and the difference in protein concentration between PDR and controls was statistically significant for 13 proteins. There was a subset of proteins that were detected by both platforms, and for those the Spearman's correlation coefficient was higher than 0.8. Conclusions: PEA is suitable for the analysis of vitreous samples, showing a strong correlation with the ECL platform. The detection rate of PEA panels was higher than the panels tested with ECL. The levels of several proinflammatory and angiogenic cytokines were significantly higher in PDR vitreous compared to controls. Translational Relevance: This study provides new information on the yields of small-volume assays that can detect proteins of interest in ocular specimens, and it identifies patterns of cytokine dysregulation in PDR.

Targeting cellular heterogeneity with CXCR2 blockade for the treatment of therapy-resistant prostate cancer.

Hormonal therapy targeting androgen receptor (AR) is initially effective to treat prostate cancer (PCa), but it eventually fails. It has been hypothesized that cellular heterogeneity of PCa, consisting of AR+ luminal tumor cells and AR- neuroendocrine (NE) tumor cells, may contribute to therapy failure. Here, we describe the successful purification of NE cells from primary fresh human prostate adenocarcinoma based on the cell surface receptor C-X-C motif chemokine receptor 2 (CXCR2). Functional studies revealed CXCR2 to be a driver of the NE phenotype, including loss of AR expression, lineage plasticity, and resistance to hormonal therapy. CXCR2-driven NE cells were critical for the tumor microenvironment by providing a survival niche for the AR+ luminal cells. We demonstrate that the combination of CXCR2 inhibition and AR targeting is an effective treatment strategy in mouse xenograft models. Such a strategy has the potential to overcome therapy resistance caused by tumor cell heterogeneity.

Assessment of Diagnostic Outcomes of RNA Genetic Testing for Hereditary Cancer.

Importance: Performing DNA genetic testing (DGT) for hereditary cancer genes is now a well-accepted clinical practice; however, the interpretation of DNA variation remains a challenge for laboratories and clinicians. Adding RNA genetic testing (RGT) enhances DGT by clarifying the clinical actionability of hereditary cancer gene variants, thus improving clinicians' ability to accurately apply strategies for cancer risk reduction and treatment. Objective: To evaluate whether RGT is associated with improvement in the diagnostic outcome of DGT and in the delivery of personalized cancer risk management for patients with hereditary cancer predisposition. Design, Setting, and Participants: Diagnostic study in which patients and/or families with inconclusive variants detected by DGT in genes associated with hereditary breast and ovarian cancer, Lynch syndrome, and hereditary diffuse gastric cancer sent blood samples for RGT from March 2016 to April 2018. Clinicians who ordered genetic testing and received a reclassification report for these variants were surveyed to assess whether RGT-related variant reclassifications changed clinical management of these patients. To quantify the potential number of tested individuals who could benefit from RGT, a cohort of 307 812 patients who underwent DGT for hereditary cancer were separately queried to identify variants predicted to affect splicing. Data analysis was conducted from March 2016 and September 2018. Main Outcomes and Measures: Variant reclassification outcomes following RGT, clinical management changes associated with RGT-related variant reclassifications, and the proportion of patients who would likely be affected by a concurrent DGT and RGT multigene panel testing approach. Results: In total, 93 if 909 eligible families (10.2%) submitted samples for RGT. Evidence from RGT clarified the interpretation of 49 of 56 inconclusive cases (88%) studied; 26 (47%) were reclassified as clinically actionable and 23 (41%) were clarified as benign. Variant reclassifications based on RGT results changed clinical management recommendations for 8 of 18 patients (44%) and 14 of 18 families (78%), based on responses from 18 of 45 clinicians (40%) surveyed. A total of 7265 of 307 812 patients who underwent DGT had likely pathogenic variants or variants of uncertain significance potentially affecting splicing, indicating that approximately 1 in 43 individuals could benefit from RGT. Conclusions and Relevance: In this diagnostic study, conducting RNA testing resolved a substantial proportion of variants of uncertain significance in a cohort of individuals previously tested for cancer predisposition by DGT. Performing RGT might change the diagnostic outcome of at least 1 in 43 patients if performed in all individuals undergoing genetic evaluation for hereditary cancer.

Quantitative Analysis of BRCA1 and BRCA2 Germline Splicing Variants Using a Novel RNA-Massively Parallel Sequencing Assay.

Clinical genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming widespread. However, the interpretation of variants of unknown significance (VUS) in HBOC genes, such as the clinically actionable genes BRCA1 and BRCA2, remain a challenge. Among the variants that are frequently classified as VUS are those with unclear effects on splicing. In order to address this issue we developed a high-throughput RNA-massively parallel sequencing assay-CloneSeq-capable to perform quantitative and qualitative analysis of transcripts in cell lines and HBOC patients. This assay is based on cloning of RT-PCR products followed by massive parallel sequencing of the cloned transcripts. To validate this assay we compared it to the RNA splicing assays recommended by members of the ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium. This comparison was performed using well-characterized lymphoblastoid cell lines (LCLs) generated from carriers of the BRCA1 or BRCA2 germline variants that have been previously described to be associated with splicing defects. CloneSeq was able to replicate the ENIGMA results, in addition to providing quantitative characterization of BRCA1 and BRCA2 germline splicing alterations in a high-throughput fashion. Furthermore, CloneSeq was used to analyze blood samples obtained from carriers of BRCA1 or BRCA2 germline sequence variants, including the novel uncharacterized alteration BRCA1 c.5152+5G>T, which was identified in a HBOC family. CloneSeq provided a high-resolution picture of all the transcripts induced by BRCA1 c.5152+5G>T, indicating it results in significant levels of exon skipping. This analysis proved to be important for the classification of BRCA1 c.5152+5G>T as a clinically actionable likely pathogenic variant. Reclassifications such as these are fundamental in order to offer preventive measures, targeted treatment, and pre-symptomatic screening to the correct individuals.

TMEM110 regulates the maintenance and remodeling of mammalian ER-plasma membrane junctions competent for STIM-ORAI signaling.

The stromal interaction molecule (STIM)-ORAI calcium release-activated calcium modulator (ORAI) pathway controls store-dependent calcium entry, a major mechanism of physiological calcium signaling in mammalian cells. The core elements of the pathway are the regulatory protein STIM1, located in the endoplasmic reticulum (ER) membrane, the calcium channel ORAI1 in the plasma membrane, and sites of close contact between the ER and the plasma membrane that permit the two proteins to interact. Research on calcium signaling has centered on STIM1, ORAI1, and a few proteins that directly modulate STIM-ORAI function. However, little is known about proteins that organize ER-plasma membrane junctions for STIM-ORAI-dependent calcium signaling. Here, we report that an ER-resident membrane protein identified in a previous genome-wide RNAi screen, transmembrane protein 110 (TMEM110), regulates the long-term maintenance of ER-plasma membrane junctions and the short-term physiological remodeling of the junctions during store-dependent calcium signaling.

DNA damage triggers Golgi dispersal via DNA-PK and GOLPH3.

The response to DNA damage, which regulates nuclear processes such as DNA repair, transcription, and cell cycle, has been studied thoroughly. However, the cytoplasmic response to DNA damage is poorly understood. Here, we demonstrate that DNA damage triggers dramatic reorganization of the Golgi, resulting in its dispersal throughout the cytoplasm. We further show that DNA-damage-induced Golgi dispersal requires GOLPH3/MYO18A/F-actin and the DNA damage protein kinase, DNA-PK. In response to DNA damage, DNA-PK phosphorylates GOLPH3, resulting in increased interaction with MYO18A, which applies a tensile force to the Golgi. Interference with the Golgi DNA damage response by depletion of DNA-PK, GOLPH3, or MYO18A reduces survival after DNA damage, whereas overexpression of GOLPH3, as is observed frequently in human cancers, confers resistance to killing by DNA-damaging agents. Identification of the DNA-damage-induced Golgi response reveals an unexpected pathway through DNA-PK, GOLPH3, and MYO18A that regulates cell survival following DNA damage.

HNF4α antagonists discovered by a high-throughput screen for modulators of the human insulin promoter.

Hepatocyte nuclear factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic ß cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and were proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.

Phenothiazine neuroleptics signal to the human insulin promoter as revealed by a novel high-throughput screen.

A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic beta-cell. A cell line from human islets in which the expression of insulin and other beta-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of beta-cell differentiated function.

GOLPH3 bridges phosphatidylinositol-4- phosphate and actomyosin to stretch and shape the Golgi to promote budding.

Golgi membranes, from yeast to humans, are uniquely enriched in phosphatidylinositol-4-phosphate (PtdIns(4)P), although the role of this lipid remains poorly understood. Using a proteomic lipid-binding screen, we identify the Golgi protein GOLPH3 (also called GPP34, GMx33, MIDAS, or yeast Vps74p) as a PtdIns(4)P-binding protein that depends on PtdIns(4)P for its Golgi localization. We further show that GOLPH3 binds the unconventional myosin MYO18A, thus connecting the Golgi to F-actin. We demonstrate that this linkage is necessary for normal Golgi trafficking and morphology. The evidence suggests that GOLPH3 binds to PtdIns(4)P-rich trans-Golgi membranes and MYO18A conveying a tensile force required for efficient tubule and vesicle formation. Consequently, this tensile force stretches the Golgi into the extended ribbon observed by fluorescence microscopy and the familiar flattened form observed by electron microscopy.