High-sensitivity quantification of antisense oligonucleotides for pharmacokinetic characterization.

Aim: Antisense oligonucleotides (ASOs) are a fast-growing drug modality. Pharmacokinetic characterization and accurate quantification of ASOs is critical for drug development. LC-MS and hybridization immunoassays are common methods to quantify ASOs but may lack sensitivity. In this study we aimed to develop an ASO quantification method with improved sensitivity. Methods: We developed a branched DNA approach for ASO quantification and compared it with hybridization immunoassays. Results: The branched DNA assay showed significantly improved sensitivity, with LLOQ 31.25 pg/ml in plasma, 6.4-and 16-fold higher than dual-probe hybridization electrochemiluminescence and single-probe hybridization ELISA, respectively, with adequate precision, accuracy, selectivity and specificity and acceptable matrix interference. Conclusion: Branched DNA for ASO quantification has significantly higher sensitivity and lower hemolysis interference.

Disease can be caused by genetic mutations that lead to overproduction or underproduction of an aberrant protein. Antisense oligonucleotides (ASOs) are a relatively new class of drugs. While most current drugs act at the protein level, ASOs work at the RNA level and minimize synthesis of the aberrant protein. ASOs are small synthetic nucleotides that specifically bind and modify the target RNA. Quantification of ASOs is important in drug development to understand how much of the drug is in circulation or in the body after a certain period of time. While there are methods available to quantify ASOs, they lack sensitivity. We developed a method called 'branched DNA' to quantify ASOs, and compared it with known ASO quantification methods. We found that the branched DNA method showed improved sensitivity compared with other existing methods and is a reliable method to quantify ASOs. This method may be used in clinical trials when improved sensitivity quantification is needed and thus facilitate the ASO drug development field.

Enrichment of circulating tumor-derived extracellular vesicles from human plasma.

Extracellular vesicles (EVs) are gaining considerable traction within the liquid biopsy arena, as carriers of information from cells in distant sites that may not be accessible for biopsy. Therefore, there is a need to develop methods to enrich for specific EV subtypes, based on their cells of origin. Here we describe the development of an automated method to enrich tumor-derived EVs from plasma using the CellSearch technology compared to Total EVs isolated using differential ultracentrifugation (DUC). We use a modified CellSearch protocol to enrich EpCAM+ EVs from the plasma of patients with non-small cell lung carcinoma (NSCLC) and triple negative breast cancer (TNBC). As a test case, we examined PD-L1, an immune checkpoint ligand known to be expressed in some tumor tissues, to demonstrate enrichment for EpCAM+ EVs. For this purpose, we developed two custom immunoassays utilizing the Simoa HD-1 analyzer (Quanterix) to detect PD-L1 in EVs and interrogate specific EV populations from human plasma. PD-L1 was present in Total EVs from the plasma of healthy individuals and cancer patients, since it is also expressed on several immune cells. However, EpCAM+ EVs were only detectable from the plasma of cancer patients, suggesting these are tumor-derived EVs. As low as 250 µL of plasma could be used to reliably detect PD-L1 from patient-derived EpCAM+ EVs. In summary, this report demonstrates the development of a robust tumor-derived EV enrichment method from human blood. Furthermore, this proof-of-concept study is extendable to other known cancer-specific proteins expressed on EVs exuded from tumors.

Mutational Analysis of Patients With Colorectal Cancer in CALGB/SWOG 80405 Identifies New Roles of Microsatellite Instability and Tumor Mutational Burden for Patient Outcome.

PURPOSE: CALGB/SWOG 80405 was a randomized phase III trial that found no statistically significant difference in overall survival (OS) in patients with first-line metastatic colorectal cancer treated with chemotherapy plus either bevacizumab or cetuximab. Primary tumor DNA from 843 patients has been used to discover genetic markers of OS. PATIENTS AND METHODS: Gene mutations were determined by polymerase chain reaction. Microsatellite status was determined by genotyping of microsatellites. Tumor mutational burden (TMB) was determined by next-generation sequencing. Cox proportional hazard models were used, with adjusting factors. Interaction of molecular alterations with either the bevacizumab or the cetuximab arms was tested. RESULTS: Patients with high TMB in their tumors had longer OS than did patients with low TMB (hazard ratio [HR], 0.73 [95% CI, 0.57 to 0.95]; P = .02). In patients with microsatellite instability-high (MSI-H) tumors, longer OS was observed in the bevacizumab arm than in the cetuximab arm (HR, 0.13 [95% CI, 0.06 to 0.30]; interaction P < .001 for interaction between microsatellite status and the two arms). Patients with BRAF mutant tumors had shorter OS than did patients with wild-type (WT) tumors (HR, 2.01 [95% CI, 1.49 to 2.71]; P < .001). Patients with extended RAS mutant tumors had shorter OS than did patients with WT tumors (HR, 1.52 [95% CI, 1.26 to 1.84]; P < .001). Patients with triple-negative tumors (WT for NRAS/KRAS/BRAF) had a median OS of 35.9 months (95% CI, 33.0 to 38.8 months) versus 22.2 months (95% CI, 19.6 to 24.4 months ) in patients with at least one mutated gene in their tumors (P < .001). CONCLUSION: In patients with metastatic colorectal cancer treated in first line, low TMB, and BRAF and RAS mutations are negative prognostic factors. Patients with MSI-H tumors benefited more from bevacizumab than from cetuximab, and studies to confirm this effect of MSI-H are warranted.

A transcriptional MAPK Pathway Activity Score (MPAS) is a clinically relevant biomarker in multiple cancer types.

KRAS- and BRAF-mutant tumors are often dependent on MAPK signaling for proliferation and survival and thus sensitive to MAPK pathway inhibitors. However, clinical studies have shown that MEK inhibitors are not uniformly effective in these cancers indicating that mutational status of these oncogenes does not accurately capture MAPK pathway activity. A number of transcripts are regulated by this pathway and are recurrently identified in genome-based MAPK transcriptional signatures. To test whether the transcriptional output of only 10 of these targets could quantify MAPK pathway activity with potential predictive or prognostic clinical utility, we created a MAPK Pathway Activity Score (MPAS) derived from aggregated gene expression. In vitro, MPAS predicted sensitivity to MAPK inhibitors in multiple cell lines, comparable to or better than larger genome-based statistical models. Bridging in vitro studies and clinical samples, median MPAS from a given tumor type correlated with cobimetinib (MEK inhibitor) sensitivity of cancer cell lines originating from the same tissue type. Retrospective analyses of clinical datasets showed that MPAS was associated with the sensitivity of melanomas to vemurafenib (HR: 0.596) and negatively prognostic of overall or progression-free survival in both adjuvant and metastatic CRC (HR: 1.5 and 1.4), adrenal cancer (HR: 1.7), and HER2+ breast cancer (HR: 1.6). MPAS thus demonstrates potential clinical utility that warrants further exploration.

Actin cytoskeleton-dependent regulation of corticotropin-releasing factor receptor heteromers.

Stress responses are highly nuanced and variable, but how this diversity is achieved by modulating receptor function is largely unknown. Corticotropin-releasing factor receptors (CRFRs), class B G protein-coupled receptors, are pivotal in mediating stress responses. Here we show that the two known CRFRs interact to form heteromeric complexes in HEK293 cells coexpressing both CRFRs and in vivo in mouse pancreas. Coimmunoprecipitation and mass spectrometry confirmed the presence of both CRF1R and CRF2ßR, along with actin in these heteromeric complexes. Inhibition of actin filament polymerization prevented the transport of CRF2ßR to the cell surface but had no effect on CRF1R. Transport of CRF1R when coexpressed with CRF2ßR became actin dependent. Simultaneous stimulation of cells coexpressing CRF1R+CRF2ßR with their respective high-affinity agonists, CRF+urocortin2, resulted in approximately twofold increases in peak Ca2+ responses, whereas stimulation with urocortin1 that binds both receptors with 10-fold higher affinity did not. The ability of CRFRs to form heteromeric complexes in association with regulatory proteins is one mechanism to achieve diverse and nuanced function.

Urocortin 3 expression at baseline and during inflammation in the colon: corticotropin releasing factor receptors cross-talk.

Urocortins (Ucn1-3), members of the corticotropin-releasing factor (CRF) family of neuropeptides, are emerging as potent immunomodulators. Localized, cellular expression of Ucn1 and Ucn2, but not Ucn3, has been demonstrated during inflammation. Here, we investigated the role of Ucn3 in a rat model of Crohn's colitis and the relative contribution of CRF receptors (CRF1 and CRF2) in regulating Ucn3 expression at baseline and during inflammation. Ucn3 mRNA and peptide were ubiquitously expressed throughout the GI tract in naïve rats. Ucn3 immunoreactivity was seen in epithelial cells and myenteric neurons. On day 1 of colitis, Ucn3 mRNA levels decreased by 80% and did not recover to baseline even by day 9. Next, we ascertained pro- or anti-inflammatory actions of Ucn3 during colitis. Surprisingly, unlike observed anti-inflammatory actions of Ucn1, exogenous Ucn3 did not alter histopathological outcomes during colitis and neither did it alter levels of pro-inflammatory cytokines IL-6 and TNF-α. At baseline, colon-specific knockdown of CRF1, but not CRF2 decreased Ucn3 mRNA by 78%, whereas during colitis, Ucn3 mRNA levels increased after CRF1 knockdown. In cultured cells, co-expression of CRF1+CRF2 attenuated Ucn3-stimulated intracellular Ca(2+) peak by 48% as compared to cells expressing CRF2 alone. Phosphorylation of p38 kinase increased by 250% during colitis and was significantly attenuated after Ucn3 administration. Thus, our results suggest that a balanced and coordinated expression of CRF receptors is required for proper regulation of Ucn3 at baseline and during inflammation.

Corticotropin-releasing factor receptor 2 mediates sex-specific cellular stress responses.

Although females suffer twice as much as males from stress-related disorders, sex-specific participating and pathogenic cellular stress mechanisms remain uncharacterized. Using corticotropin-releasing factor receptor 2-deficient (Crhr2-/-) and wild-type (WT) mice, we show that CRF receptor type 2 (CRF2) and its high-affinity ligand, urocortin 1 (Ucn1), are key mediators of the endoplasmic reticulum (ER) stress response in a murine model of acute pancreatic inflammation. Ucn1 was expressed de novo in acinar cells of male, but not female WT mice during acute inflammation. Upon insult, acinar Ucn1 induction was markedly attenuated in male but not female Crhr2-/- mice. Crhr2-/- mice of both sexes show exacerbated acinar cell inflammation and necrosis. Electron microscopy showed mild ER damage in WT male mice and markedly distorted ER structure in Crhr2-/- male mice during pancreatitis. WT and Crhr2-/- female mice showed similarly distorted ER ultrastructure that was less severe than distortion seen in Crhr2-/- male mice. Damage in ER structure was accompanied by increased ubiquitination, peIF2, and mistargeted localization of vimentin in WT mice that was further exacerbated in Crhr2-/- mice of both sexes during pancreatitis. Exogenous Ucn1 rescued many aspects of histological damage and cellular stress response, including restoration of ER structure in male WT and Crhr2-/- mice, but not in females. Instead, females often showed increased damage. Thus, specific cellular pathways involved in coping and resolution seem to be distinct to each sex. Our results demonstrate the importance of identifying sex-specific pathogenic mechanisms and their value in designing effective therapeutics.

Endothelin-converting enzyme-1 actions determine differential trafficking and signaling of corticotropin-releasing factor receptor 1 at high agonist concentrations.

CRF receptor 1 (CRF(1)), a key neuroendocrine mediator of the stress response, has two known agonists corticotropin-releasing factor (CRF) and urocortin 1 (Ucn1). Here we report that endothelin-converting enzyme-1 (ECE-1) differentially degrades CRF and Ucn1; ECE-1 cleaves Ucn1, but not CRF, at critical residue Arginine-34/35', which is essential for ligand-receptor binding. At near K(D) agonist concentration (30 nm), both Ucn1- and CRF-mediated Ca(2+) mobilization are ECE-1 dependent. Interestingly, at high agonist concentration (100 nm), Ucn1-mediated Ca(2+) mobilization remains ECE-1 dependent, whereas CRF-mediated mobilization becomes independent of ECE-1 activity. At high agonist concentration, ECE-1 inhibition disrupted Ucn1-, but not CRF-induced CRF(1) recycling and resensitization, but did not prolong the association of CRF(1) with ß-arrestins. RNA interference-mediated knockdown of Rab suggests that both Ucn1- and CRF-induced CRF(1) resensitization is dependent on activity of Rab11, but not of Rab4. CRF(1) behaves like a class A G protein-coupled receptor with respect to transient ß-arrestins interaction. We propose that differential degradation by ECE-1 is a novel mechanism by which CRF(1) receptor is protected from overactivation by physiologically relevant high concentrations of higher affinity ligand to mediate distinct resensitization and downstream signaling.

Phosphorylation and dephosphorylation of tyrosine 141 regulate stability and degradation of INrf2: a novel mechanism in Nrf2 activation.

INrf2-Nrf2 proteins are sensors of chemical/radiation stress. Nrf2, in response to stresses, is released from INrf2. Nrf2 is translocated into the nucleus where it binds to the antioxidant response element and coordinately activates the expression of a battery of genes that protect cells against oxidative and electrophilic stress. An autoregulatory loop between INrf2 and Nrf2 regulates their cellular abundance. Nrf2 activates INrf2 gene expression, and INrf2 serves as an adapter for degradation of Nrf2. In this report, we demonstrate that mutation of tyrosine 141 in bric-a-bric, tramtrack, broad complex domain to alanine rendered INrf2 unstable and nonfunctional. INrf2Y141A mutant degraded rapidly as compared with wild type INrf2, although it could dimerize and bind Nrf2. De novo synthesized INrf2 protein was phosphorylated at tyrosine 141. Tyrosine 141-phosphorylated INrf2 was highly stable. Treatment with hydrogen peroxide, which is an oxidizing agent, led to dephosphorylation of INrf2Y141, resulting in rapid degradation of INrf2. This resulted in stabilization of Nrf2 and activation of ARE-mediated gene expression. These results demonstrate that stress-induced dephosphorylation of tyrosine 141 is a novel mechanism in Nrf2 activation and cellular protection.

Calcium- and salt-stress signaling in plants: shedding light on SOS pathway.

As salt stress imposes a major environmental threat to agriculture, understanding the basic physiology and genetics of cell under salt stress is crucial for developing any transgenic strategy. Salt Overly Sensitive (SOS) genes (SOS1-SOS3) were isolated through positional cloning. Since sos mutants are hypersensitive to salt, their characterization resulted in the discovery of a novel pathway, which has helped in our understanding the mechanism of salt-stress tolerance in plants. Genetic analysis confirmed that SOS1-SOS3 function in a common pathway of salt tolerance. This pathway also emphasizes the significance of Ca2+ signal in reinstating cellular ion homeostasis. SOS3, a Ca2+ sensor, transduces the signal downstream after activating and interacting with SOS2 protein kinase. This SOS3-SOS2 complex activates the Na+/H+ antiporter activity of SOS1 thereby reestablish cellular ion homeostasis. Recently, SOS4 and SOS5 have also been characterized. SOS4 encodes a pyridoxal (PL) kinase that is involved in the biosynthesis of pyridoxal-5-phosphate (PLP), an active form of vitamin B6. SOS5 has been shown to be a putative cell surface adhesion protein that is required for normal cell expansion. Under salt stress, the normal growth and expansion of a plant cell becomes even more important and SOS5 helps in the maintenance of cell wall integrity and architecture. In this review we focus on the recent advances in salt stress and SOS signaling pathway. A broad coverage of the discovery of SOS mutants, structural aspect of these genes and the latest developments in the field of SOS1-SOS5 has been described.

Calcium signaling network in plants: an overview.

Calcium ion (Ca(2+)) is one of the very important ubiquitous intracellular second messenger molecules involved in many signal transduction pathways in plants. The cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) have been found to increased in response to many physiological stimuli such as light, touch, pathogenic elicitor, plant hormones and abiotic stresses including high salinity, cold and drought. This Ca(2+) spikes normally result from two opposing reactions, Ca(2+) influx through channels or Ca(2+) efflux through pumps. The removal of Ca(2+) from the cytosol against its electrochemical gradient to either the apoplast or to intracellular organelles requires energized 'active' transport. Ca(2+)-ATPases and H(+)/Ca(2+) antiporters are the key proteins catalyzing this movement. The increased level of Ca(2+) is recognised by some Ca(2+)-sensors or calcium-binding proteins, which can activate many calcium dependent protein kinases. These kinases regulate the function of many genes including stress responsive genes, resulted in the phenotypic response of stress tolerance. Calcium signaling is also involved in the regulation of cell cycle progression in response to abiotic stress. The regulation of gene expression by cellular calcium is also crucial for plant defense against various stresses. However, the number of genes known to respond to specific transient calcium signals is limited. This review article describes several aspects of calcium signaling such as Ca(2+) requiremant and its role in plants, Ca(2+) transporters, Ca(2+)-ATPases, H(+)/ Ca(2+)-antiporter, Ca(2+)-signature, Ca(2+)-memory and various Ca(2+)-binding proteins (with and without EF hand).

Further Characterization of Calcineurin B-Like Protein and Its Interacting Partner CBL-Interacting Protein Kinase from Pisum sativum.

The recently discovered calcium sensor calcineurin B-like proteins (CBLs), and their interacting partners CBL-interacting protein kinases (CIPKs), have emerged as a key network in response to stress and calcium signaling in plants. The studies on CBL and CIPK are so far mainly restricted to Arabidopsis and work on dissecting this pathway in higher plants is inadequate. Our recent studies revealed that calcium sensor CBL from pea gets phosphorylated by pea CIPK. Furthermore, the transcript levels of both the CBL and CIPK from pea were coordinately upregulated in response to various stresses including high salinity, cold, wounding, salicyclic acid and calcium but not to abscisic acid and dehydration. Here we report the results on the computational analysis of EF hands of PsCBL protein, which indicate that it contains all the functional domains required for calcium binding activity. We have also focused on homology based computational modeling of PsCBL and PsCIPK proteins using AtCBL2 and Chk1 as templates respectively, which suggested the high degree of conservation between AtCBL2, PsCBL and OsCBL and between Chk1, PsCIPK and AtCIPK18. Furthermore, the direct interaction of PsCBL and PsCIPK mutant proteins was studied by the yeast 2-hybrid system, which confirmed that FISL domain is an important interaction module required for PsCBL and PsCIPK interaction.

Cloning and characterization of CBL-CIPK signalling components from a legume (Pisum sativum).

The studies on calcium sensor calcineurin B-like protein (CBL) and CBL interacting protein kinases (CIPK) are limited to Arabidopsis and rice and their functional role is only beginning to emerge. Here, we present cloning and characterization of a protein kinase (PsCIPK) from a legume, pea, with novel properties. The PsCIPK gene is intronless and encodes a protein that showed partial homology to the members of CIPK family. The recombinant PsCIPK protein was autophosphorylated at Thr residue(s). Immunoprecipitation and yeast two-hybrid analysis showed direct interaction of PsCIPK with PsCBL, whose cDNA and genomic DNA were also cloned in this study. PsCBL showed homology to AtCBL3 and contained calcium-binding activity. We demonstrate for the first time that PsCBL is phosphorylated at its Thr residue(s) by PsCIPK. Immunofluorescence/confocal microscopy showed that PsCBL is exclusively localized in the cytosol, whereas PsCIPK is localized in the cytosol and the outer membrane. The exposure of plants to NaCl, cold and wounding co-ordinately upregulated the expression of PsCBL and PsCIPK genes. The transcript levels of both genes were also coordinately stimulated in response to calcium and salicylic acid. However, drought and abscisic acid had no effect on the expression of these genes. These studies show the ubiquitous presence of CBL/CIPK in higher plants and enhance our understanding of their role in abiotic and biotic stress signalling.

Cold, salinity and drought stresses: an overview.

World population is increasing at an alarming rate and is expected to reach about six billion by the end of year 2050. On the other hand food productivity is decreasing due to the effect of various abiotic stresses; therefore minimizing these losses is a major area of concern for all nations to cope with the increasing food requirements. Cold, salinity and drought are among the major stresses, which adversely affect plants growth and productivity; hence it is important to develop stress tolerant crops. In general, low temperature mainly results in mechanical constraint, whereas salinity and drought exerts its malicious effect mainly by disrupting the ionic and osmotic equilibrium of the cell. It is now well known that the stress signal is first perceived at the membrane level by the receptors and then transduced in the cell to switch on the stress responsive genes for mediating stress tolerance. Understanding the mechanism of stress tolerance along with a plethora of genes involved in stress signaling network is important for crop improvement. Recently, some genes of calcium-signaling and nucleic acid pathways have been reported to be up-regulated in response to both cold and salinity stresses indicating the presence of cross talk between these pathways. In this review we have emphasized on various aspects of cold, salinity and drought stresses. Various factors pertaining to cold acclimation, promoter elements, and role of transcription factors in stress signaling pathway have been described. The role of calcium as an important signaling molecule in response to various stress signals has also been covered. In each of these stresses we have tried to address the issues, which significantly affect the gene expression in relation to plant physiology.