Exposure to ionizing radiation results in injuries of hematopoietic, gastrointestinal and respiratory systems which are the leading causes responsible for morbidity and mortality. Gastrointestinal injury occurs as an acute radiation syndrome. To help inform on the natural history of the radiation-induced injury of the partial body irradiation model, we quantitatively profiled the proteome of jejunum from non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing over a time period of three weeks. Jejunum was analyzed by liquid chromatography-tandem mass spectrometry and pathway and gene ontology analysis were performed. 3245 unique proteins were quantified out of more than 3700 proteins identified in this study. A total of 289 proteins of the quantified proteins showed significant and consistent responses across at least three time points post-irradiation, of which 263 proteins showed strong upregulations while 26 proteins showed downregulations. Bioinformatic analysis suggests significant pathway and upstream regulator perturbations post-high dose irradiation and shed light on underlying mechanisms of radiation damage. Canonical pathways altered by radiation included GP6 Signaling Pathway, Acute Phase Response Signaling, LXR/RXR Activation, and Intrinsic Prothrombin Activation Pathway. Additionally, we observed dysregulation of proteins of the retinoid pathway and retinoic acid, an active metabolite of vitamin A, as quantified by liquid chromatography-tandem mass spectrometry. Correlation of changes in protein abundance with a well-characterized histological endpoint, corrected crypt number, was used to evaluate biomarker potential. These data further define the natural history of the gastrointestinal acute radiation

High-dose radiation exposure results in organ-specific sequelae that occurs in a time-and dosedependent manner. The partial body irradiation with minimal bone marrow sparing model was developed to mimic intentional or accidental radiation exposures in humans where bone marrow sparing is likely and permits the concurrent analysis of coincident short-and long-term damage to organ systems. To help inform on the natural history of the radiation-induced injury of the partial body irradiation model, we quantitatively profiled the plasma proteome of non-human primates following 12 Gy partial body irradiation with 2.5% bone marrow sparing with 6 MV LINACderived photons at 0.80 Gy min −1 over a time period of three weeks. The plasma proteome was analyzed by liquid chromatography-tandem mass spectrometry. A number of trends were identified in the proteomic data including pronounced protein changes as well as protein changes that were consistently upregulated or downregulated at all time points and dose levels interrogated. Pathway and gene ontology analysis were performed; bioinformatic analysis revealed significant pathway and biological process perturbations post high dose irradiation and shed light on underlying mechanisms of radiation damage. Additionally, proteins were identified that had the greatest potential to serve as biomarkers for radiation exposure.

Increased airway smooth muscle (ASM) cell mass and secretory functions are characteristics of airway inflammatory diseases, such as asthma. To date, there are no effective therapies to combat ASM cell proliferation, which contributes to bronchoconstriction and airway obstruction. Growth factors such as platelet‐derived growth factor (PDGF) and the activation of the ERK1/2 are major regulators of ASM cell proliferation and airway remodeling in asthma. However, given the ubiquitous expression and multiple functions of ERK1/2, complete inhibition of ERK1/2 using ATP‐competitive inhibitors may lead to unwanted off‐target effects. Alternatively, we have identified compounds that are designed to target substrate docking sites and act as function‐selective inhibitors of ERK1/2 signaling. Here, we show that both function‐selective and ATP‐competitive ERK1/2 inhibitors are effective at inhibiting PDGF‐mediated proliferation, collagen production, and IL‐6 secretion in ASM cells. Proteomic analysis revealed that both types of inhibitors had similar effects on reducing proteins related to TGF‐β and IL‐6 signaling that are relevant to airway remodeling. However, function‐selective ERK1/2 inhibitors caused fewer changes in protein expression compared with ATP‐competitive inhibitors. These studies provide a molecular basis for the development of function‐selective ERK1/2 inhibitors to mitigate airway remodeling in asthma with defined regulation of ERK1/2 signaling.—Defnet, A. E., Huang, W., Polischak, S., Yadav, S. K., Kane, M. A., Shapiro, P., Deshpande, D. A. Effects of ATP‐competitive and function‐selective ERK inhibitors on airway smooth muscle cell proliferation. FASEB J. 33, 10833–10843 (2019). http://www.fasebj.org

As described in the previous chapter, most kinase inhibitors that have been developed for use in the clinic act by blocking ATP binding; however, there is growing interest in identifying compounds that target kinase activities and functions without interfering with the conserved features of the ATP-binding site. This chapter will highlight alternative approaches that exploit unique kinase structural features that are being targeted to identify more selective and potent inhibitors. The figure below, adapted from (Sammons et al., Molecular Carcinogenesis 58:1551–1570, 2019), provides a graphical description of the various approaches to manipulate kinase activity. In addition to the type I and II inhibitors, type III kinase inhibitors have been identified to target sites adjacent to the ATP-binding site in the catalytic domain. New information on kinase structure and substrate-binding sites has enabled the identification of type IV kinase inhibitor compounds that target regions outside the catalytic domain. The combination of targeting unique allosteric sites outside the catalytic domain with ATP-targeted compounds has yielded a number of novel bivalent type V kinase inhibitors. Finally, emerging interest in the development of irreversible compounds that form selective covalent interactions with key amino acids involved in kinase functions comprise the class of type VI kinase inhibitors.

Extracellular signal-regulated kinase-1/2 (ERK1/2) pathway inhibitors are important therapies for treating many cancers. However, acquired resistance to most protein kinase inhibitors limits their ability to provide durable responses. Approximately 50% of malignant melanomas contain activating mutations in BRAF, which promotes cancer cell survival through the direct phosphorylation of the mitogen-activated protein kinase MAPK/ERK 1/2 (MEK1/2) and the activation of ERK1/2. Although the combination treatment with BRAF and MEK1/2 inhibitors is a recommended approach to treat melanoma, the development of drug resistance remains a barrier to achieving long-term patient benefits. Few studies have compared the global proteomic changes in BRAF/MEK1/2 inhibitor-resistant melanoma cells under different growth conditions. The current study uses high-resolution label-free mass spectrometry to compare relative protein changes in BRAF/MEK1/2 inhibitor-resistant A375 melanoma cells grown as monolayers or spheroids. While approximately 66% of proteins identified were common in the monolayer and spheroid cultures, only 6.2 or 3.6% of proteins that significantly increased or decreased, respectively, were common between the drug-resistant monolayer and spheroid cells. Drug-resistant monolayers showed upregulation of ERK-independent signaling pathways, whereas drug-resistant spheroids showed primarily elevated catabolic metabolism to support oxidative phosphorylation. These studies highlight the similarities and differences between monolayer and spheroid cell models in identifying actionable targets to overcome drug resistance.

Vitamin A deficiency has been shown to exacerbate allergic asthma. Previous studies have postulated that retinoic acid (RA), an active metabolite of vitamin A and high-affinity ligand for RA receptor (RAR), is reduced in airway inflammatory condition and contributes to multiple features of asthma including airway hyperresponsiveness and excessive accumulation of airway smooth muscle (ASM) cells. In this study, we directly quantified RA and examined the molecular basis for reduced RA levels and RA-mediated signaling in lungs and ASM cells obtained from asthmatic donors and in lungs from allergen-challenged mice.Levels of RA and retinol were significantly lower in lung tissues from asthmatic donors and house dust mite (HDM)-challenged mice compared to non-asthmatic human lungs and PBS-challenged mice, respectively. Quantification of mRNA and protein expression revealed dysregulation in the first step of RA biosynthesis consistent with reduced RA including decreased protein expression of retinol dehydrogenase (RDH)-10 and increased protein expression of RDH11 and dehydrogenase/reductase (DHRS)-4 in asthmatic lung. Proteomic profiling of nonasthmatic and asthmatic lungs also showed significant changes in the protein expression of AP-1 targets consistent with increased AP-1 activity. Further, basal RA levels and RA biosynthetic capabilities were decreased in asthmatic human ASM cells. Treatment of human ASM cells with all-trans RA (ATRA) or the RARγ-specific agonist (CD1530) resulted in the inhibition of mitogen-induced cell proliferation and AP-1-dependent transcription. These data suggest that RA metabolism is decreased in asthmatic lung and that enhancing RAR signaling using ATRA or RARγ agonists may mitigate airway remodeling associated with asthma.

Chronic pulmonary diseases, including chronic obstructive pulmonary disease (COPD) and asthma, are major causes of death and reduced quality of life. Characteristic of chronic pulmonary disease is excessive lung inflammation that occurs in response to exposure to inhaled irritants, chemicals, and allergens. Chronic inflammation leads to remodeling of the airways that includes excess mucus secretion, proliferation of smooth muscle cells, increased deposition of extracellular matrix proteins and fibrosis. Protein kinases have been implicated in mediating inflammatory signals and airway remodeling associated with reduced lung function in chronic pulmonary disease. This review will highlight the role of protein kinases in the lung during chronic inflammation and examine opportunities to use protein kinase inhibitors for the treatment of chronic pulmonary diseases.

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