Sean W. Harshman

Characterization of Multiple Myeloma Vesicles by Label-Free Relative Quantitation

Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. The role that extracellular vesicles (EVs), microvesicles and exosomes, released by MM cells have in cell‐to‐cell communication and signaling in the bone marrow is currently unknown. This paper describes the proteomic content of EVs derived from MM.1S and U266 MM cell lines. First, we compared the protein identifications between the vesicles and cellular lysates of each cell line finding a large overlap in protein identifications. Next, we applied label‐free spectral count quantitation to determine proteins with differential abundance between the groups. Finally, we used bioinformatics to categorize proteins with significantly different abundances into functional groups. The results illustrate the first use of label‐free spectral counting applied to determine relative protein abundances in EVs.

Histone H1 phosphorylation in breast cancer.

Breast cancer is the second leading cause of cancer-related deaths in women. The need for new clinical biomarkers in breast cancer is necessary to further predict prognosis and therapeutic response. In this article, the LC-MS histone H1 phosphorylation profiles were established for three distinct breast cancer cell lines. The results show that the extent of H1 phosphorylation can distinguish between the different cell lines. The histone H1 from the metastatic cell line, MDA-MB-231, was subjected to chemical derivitization and LC-MS/MS analysis. The results suggest that the phosphorylation at threonine 146 is found on both histone H1.2 and histone H1.4. Cell lines were then treated with an extracellular stimulus, estradiol or kinase inhibitor LY294002, to monitor changes in histone H1 phosphorylation. The data show that histone H1 phosphorylation can increase and decrease in response to extracellular stimuli. Finally, primary breast tissues were stained for the histone H1 phosphorylation at threonine 146. Variable staining patterns across tumor grades and subtypes were observed with pT146 labeling correlating with tumor grade. These results establish the potential for histone H1 phosphorylation at threonine 146 as a clinical biomarker in breast cancer.

Proteomic characterization of circulating extracellular vesicles identifies novel serum myeloma associated markers.

UNLABELLED Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells in the bone marrow (BM). The microenvironment plays a key role in MM cell survival and drug resistance through release of soluble factors, expression of adhesion molecules and release of extracellular vesicles (EVs). The aim of this manuscript is to use proteomic profiling of EVs as a tool to identify circulating tumor associated markers in MM patients. First, we characterized the EV protein content obtained from different MM cell lines. Then, we established differences in protein abundance among EVs isolated from MM patient serum and BM and the serum of healthy donors. These data show that the Major Histocompatibility Complex Class I is highly enriched in EVs of MM cell lines and MM patients serum. Next, we show that CD44 is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R. Furthermore, CD44 was found to be differentially expressed in EVs isolated from newly diagnosed MM patients. Finally through ELISA analysis, we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as an easily accessible source for MM biomarkers. BIOLOGICAL SIGNIFICANCE Extracellular vesicles are becoming a research focus due to their roles in cancer cell biology such as immune evasion, therapeutic resistance, proliferation and metastases. While numerous studies of vesicle characterization and biology have been conducted in many cancer models, the role of EV in MM remains relatively unstudied. Here we found that EVs isolated from MM cells are enriched in MHC-1 antigen presenting complex and its binding protein β2-MG, this observation is compatible with the enhanced proteasome activity of MM cells compared to other cancers and the ability of functional MHC-1 to bind and present peptides, generated from protein degradation by the proteasome. Additionally, our experiments show that CD44 is particularly enriched in the EV fraction of corticosteroid resistant MM.1R cells and is differentially expressed in the EV fraction of MM patients. This is of high significance due to the established role of CD44 in adhesion of MM cells to BMSC and induction of IL-6, the primary cytokine for MM cell survival, secretion by the BMSC. Furthermore, ELISA assays for CD44 content from the serum of 254 newly diagnosed MM patients enrolled in a Phase 3 randomized trial show highly variable CD44 levels and those patients with >280 ng/mL serum CD44 showing a reduced overall survival time. These results suggest the potential use of CD44 as a prognostic biomarker in MM.

Evaluation of Bio-VOC Sampler for Analysis of Volatile Organic Compounds in Exhaled Breath

Monitoring volatile organic compounds (VOCs) from exhaled breath has been used to determine exposures of humans to chemicals. Prior to analysis of VOCs, breath samples are often collected with canisters or bags and concentrated. The Bio-VOC breath sampler, a commercial sampling device, has been recently introduced to the market with growing use. The main advantage for this sampler is to collect the last portion of exhaled breath, which is more likely to represent the air deep in the lungs. However, information about the Bio-VOC sampler is somewhat limited. Therefore, we have thoroughly evaluated the sampler here. We determined the volume of the breath air collected in the sampler was approximately 88 mL. When sampling was repeated multiple times, with the succeeding exhalations applied to a single sorbent tube, we observed linear relationships between the normalized peak intensity and the number of repeated collections with the sampler in many of the breath VOCs detected. No moisture effect was observed on the Tenax sorbent tubes used. However, due to the limitation in the collection volume, the use of the Bio-VOC sampler is recommended only for detection of VOCs present at high concentrations unless repeated collections of breath samples on the sampler are conducted.

Assaying pharmacodynamic endpoints with targeted therapy: flavopiridol and 17AAG induced dephosphorylation of histone H1.5 in acute myeloid leukemia.

Histone H1 is commonly used to assay kinase activity in vitro. As many promising targeted therapies affect kinase activity of specific enzymes involved in cancer transformation, H1 phosphorylation can serve as potential pharmacodynamic marker for drug activity within the cell. In this study we utilized a phosphoproteomic workflow to characterize histone H1 phosphorylation changes associated with two targeted therapies in the Kasumi‐1 acute myeloid leukemia cell line. The phosphoproteomic workflow was first validated with standard casein phosphoproteins and then applied to the direct analysis of histone H1 from Kasumi‐1 nuclear lysates. Ten H1 phosphorylation sites were identified on the H1 variants, H1.2, H1.3, H1.4, H1.5 and H1.x. LC MS profiling of intact H1s demonstrated global dephosphorylation of H1.5 associated with therapy by the cyclin‐dependent kinase inhibitor, flavopiridol and the Heat Shock Protein 90 inhibitor, 17‐(Allylamino)‐17‐demethoxygeldanamycin. In contrast, independent treatments with a nucleotide analog, proteosome inhibitor and histone deacetylase inhibitor did not exhibit decreased H1.5 phosphorylation. The data presented herein demonstrate that potential of histones to assess the cellular response of reagents that have direct and indirect effects on kinase activity that alters histone phosphorylation. As such, this approach may be a highly informative marker for response to targeted therapies influencing histone phosphorylation.

The identification of hypoxia biomarkers from exhaled breath under normobaric conditions

Pilots have reported experiencing in-flight hypoxic-like symptoms since the inception of high-altitude aviation. As a result, the need to monitor pilots, in-flight, for the onset of hypoxic conditions is of great interest to the aviation community. We propose that exhaled breath is an appropriate non-invasive medium for monitoring pilot hypoxic risk through volatile organic compound (VOC) analysis. To identify changes in the exhaled breath VOCs produced during periods of reduced O2 levels, volunteers were exposed to simulated flight profiles, i.e. sea level for 5 min, O2 levels found at elevated altitudes for 5 min or placebo and 5 min at 100% O2 recovery gas, using a modified flight mask interfaced with a reduced O2 breathing device. During the course of these test events, time series breath samples from the flight mask and pre/post bag samples were collected and analyzed by gas chromatography/mass spectrometry (GC/MS). Seven compounds (pentanal, 4-butyrolactone, 2-pentanone, 2-hexanone, 2-cyclopenten-1-one, 3-methylheptane and 2-heptanone) were found to significantly change in response to hypoxic conditions. Additionally, the isoprene, 2-methyl-1,3-butadiene, was found to increase following the overall exposure profile. This study establishes an experimental means for monitoring changes in VOCs in response to hypoxic conditions, a computational workflow for compound analysis via the Metabolite Differentiation and Discovery Lab and MatLab(©) software and identifies potential volatile organic compound biomarkers of hypoxia exposure.

The stability of Tenax TA thermal desorption tubes in simulated field conditions on the HAPSITE® ER

Due to the growing need to monitor aircraft cabin, cockpit and breathing-line air quality, functional assessment of sampling equipment for the specialised field conditions of flight need to be established for both in-flight and ground safety. In this article, we assess the reliability of Tenax TA thermal desorption tubes to perform under various relevant field sampling conditions, such as storage temperature, loading temperature, vibrational velocity, gravitational force (G Force) and altitude pressure with semi-real-time gas chromatograph-mass spectrometer (GC-MS) analysis on the field portable HAPSITE® ER (Hazardous Air Pollutants on Site Extended Range) instrument. First, we show that Tenax TA thermal desorption tubes can handle storage under extreme environmental conditions, 4–77°C, over numerous analytical test cycles. Next, we confirm that extreme loading temperature, both hot (77°C) and cold (4°C), does not affect the analytical reliability of Tenax TA thermal desorption tubes. Then, we illustrate that G Force may have a significant (p ≤ 0.0364) effect on Tenax TA performance while vibrational velocity (p ≤ 0.7265) and low ambient air pressure (p ≤ 0.1753), such as that found at high altitude, do not. Finally, several Tenax TA thermal desorption tubes were flight-tested, demonstrating that the durability of these tubes maybe insufficient for use on military cargo aircraft (p = 0.0107). The results presented here provide a rationale for additional testing of Tenax TA thermal desorption tubes for flight suitability.

Isolation and analysis of linker histones across cellular compartments

UNLABELLED Analysis of histones, especially histone H1, is severely limited by immunological reagent availability. This paper describes the application of cellular fractionation with LC-MS for profiling histones in the cytosol and upon chromatin. First, we show that linker histones enriched by cellular fractionation gives less nuclear contamination and higher histone content than when prepared by nuclei isolation. Second, we profiled the soluble linker histones throughout the cell cycle revealing phosphorylation increases as cells reach mitosis. Finally, we monitored histone H1.2-H1.5 translocation to the cytosol in response to the CDK inhibitor flavopiridol in primary CLL cells treated ex vivo. Data shows that all H1 variants translocate in response to drug treatment with no specific order to their cytosolic appearance. The results illustrate the utility of cellular fractionation in conjunction with LC-MS for the analysis of histone H1 throughout the cell. BIOLOGICAL SIGNIFICANCE This paper demonstrates the first time application of cellular fractionation to characterize cytosolic histone H1 by liquid chromatography mass spectrometry (LC-MS). Using the Ramos Burkitts lymphoma cell line, cellular fractionation was shown to give less nuclear contamination and higher histone content than preparations by nuclei isolation. Further application of the cellular fractionation approach was shown by using primary chronic lymphocytic leukemia (CLL) cells to monitor the movement of histone H1 across cellular compartments in response to the cyclin dependent kinase inhibitor flavopiridol. Collectively, these data establish a mass spectrometric method for exploration into the function of cytosolic histone H1.

Storage stability of exhaled breath on Tenax TA

Exhaled breath is coming to the forefront of non-invasive biomarker discovery efforts. Concentration of exhaled breath volatile organic compounds (VOCs) on thermal desorption (TD) tubes with subsequent analysis by gas chromatography-mass spectrometry (GC-MS) has dominated this field. As discovery experimentation increases in frequency, the need to evaluate the long-term storage stability of exhaled breath VOCs on thermal desorption adsorbent material is critical. To address this gap, exhaled breath was loaded on Tenax TA thermal desorption tubes and stored at various temperature conditions. 74 VOCs, 56 of which have been previously uncharacterized, were monitored using GC-MS over a period of 31 d. The results suggest that storage of exhaled breath at cold temperatures (4 °C) provides the most consistent retention of exhaled breath VOCs temporally. Samples were determined to be stable up to 14 d across storage conditions prior to gaining or losing 1-2 standard deviations in abundance. Through gene set enrichment analysis (GSEA), certain chemical classes were found to be positively (acids) or negatively (sulfur-containing) enriched temporally. By means of field sample collections, the effect of storage and shipping was found to be similar to those studies preformed in the laboratory at 4 °C. Collectively this study not only provides recommendations for proper storage conditions and storage length, but also illustrates the use of GSEA to exhaled breath based GC-MS data.

Spinal Muscular Atrophy Biomarker Measurements from Blood Samples in a Clinical Trial of Valproic Acid in Ambulatory Adults

Abstract Background: Clinical trials of therapies for spinal muscular atrophy (SMA) that are designed to increase the expression the SMN protein ideally include careful assessment of relevant SMN biomarkers. Objective: In the SMA VALIANT trial, a recent double-blind placebo-controlled crossover study of valproic acid (VPA) in ambulatory adult subjects with SMA, we investigated relevant pharmacodynamic biomarkers in blood samples from SMA subjects by direct longitudinal measurement of histone acetylation and SMN mRNA and protein levels in the presence and absence of VPA treatment. Methods: Thirty-three subjects were randomized to either VPA or placebo for the first 6 months followed by crossover to the opposite arm for an additional 6 months. Outcome measures were compared between the two treatments (VPA and placebo) using a standard crossover analysis. Results: A significant increase in histone H4 acetylation was observed with VPA treatment (p = 0.005). There was insufficient evidence to suggest a treatment effect with either full length or truncated SMN mRNA transcript levels or SMN protein levels. Conclusions: These measures were consistent with the observed lack of change in the primary clinical outcome measure in the VALIANT trial. These results also highlight the added benefit of molecular and pharmacodynamic biomarker measurements in the interpretation of clinical trial outcomes.