RAPID resistance to BET inhibitors is mediated by FGFR1 in glioblastoma.

Bromodomain and extra-terminal domain (BET) proteins are therapeutic targets in several cancers including the most common malignant adult brain tumor glioblastoma (GBM). Multiple small molecule inhibitors of BET proteins have been utilized in preclinical and clinical studies. Unfortunately, BET inhibitors have not shown efficacy in clinical trials enrolling GBM patients. One possible reason for this may stem from resistance mechanisms that arise after prolonged treatment within a clinical setting. However, the mechanisms and timeframe of resistance to BET inhibitors in GBM is not known. To identify the temporal order of resistance mechanisms in GBM we performed quantitative proteomics using multiplex-inhibitor bead mass spectrometry and demonstrated that intrinsic resistance to BET inhibitors in GBM treatment occurs rapidly within hours and involves the fibroblast growth factor receptor 1 (FGFR1) protein. Additionally, small molecule inhibition of BET proteins and FGFR1 simultaneously induces synergy in reducing GBM tumor growth in vitro and in vivo. Further, FGFR1 knockdown synergizes with BET inhibitor mediated reduction of GBM cell proliferation. Collectively, our studies suggest that co-targeting BET and FGFR1 may dampen resistance mechanisms to yield a clinical response in GBM.

Fully Customizable, Low-Cost, Multi-Contact Nerve Cuffs for Spatially Selective Neuromodulation.

Selective neuromodulation of peripheral nerves is an emerging treatment for neurological diseases that are resistant to traditional drug therapy. While nerve cuffs with multichannel stimulation can be made by many varied methods, they usually require specialized microfabrication or additive manufacturing equipment. A truly low-cost and effective method of creating a custom cuff has not been accessible to researchers to prototype new methodologies and therapies in acute studies. Here, we present an inexpensive, highly repeatable method to create multi-contact nerve cuffs that require a simple postproduction PEDOT:PSS coating to improve the tissue/electrode interface. We demonstrate spatially selective neuromodulation with the proposed cuff design on the rat sciatic by preferentially activating the tibialis anterior (TA) and the lateral gastrocnemius (LG) in longitudinal and transverse stimulation patterns. This demonstrates that the proposed cuff fabrication method was not only effective for selective neuromodulation, but it is also significantly lower in cost, fully-customizable, and easily manufactured for future selective neuromodulation studies.

Review of Seismic Risk Mitigation Policies in Earthquake-Prone Countries: Lessons for Earthquake Resilience in the United States.

This article reviews the current state of practice in seismic risk mitigation, focusing on policies in ten of the most earthquake-prone countries around the world. In particular, the review compares policies to retrofit existing buildings and mechanisms for financing seismic risk mitigation, within the context of seismic risk and design standards for each country. The goal of the review is to identify policy best practices that may be useful for national and local governments that are interested in improving their earthquake resilience. The result is a set of best practice recommendations that are organized conceptually around key stages of the seismic retrofit process: (1) risk assessment; (2) knowledge transfer; (3) setting targets; (4) implementation; and (5) monitoring. While these lessons may be valuable to any earthquake-prone country, the recommendations are framed with particular attention to the United States where seismic risk mitigation is primarily the responsibility of local governments.

Motivators and impediments to seismic retrofit implementation for wood-frame soft-story buildings: A case study in California.

Motivating property owners to mitigate seismic risks for existing buildings is a major challenge for many earthquake-prone regions. This article identifies primary factors that may affect the adoption of seismic retrofit by owners of commercial and residential buildings, assesses the influence of economic, social, regulatory, and individual factors on retrofit implementation in three California cities, and discusses potential approaches to promoting seismic retrofits. Data for three retrofit programs are utilized to create predictive models for retrofit probability. The results suggest that retrofit probability for multifamily residential buildings may increase with building height, median housing value, educational attainment, and population density in the neighborhood, but may decrease with building age, building size, land value, and housing vacancy rate in the neighborhood. The retrofit decision for commercial buildings is strongly correlated with the number of stories and rooms, land value, vacancy rate, and population density, while the retrofit decision for residential buildings is highly associated with building age, number of rooms, land value, median housing value, median contract rent, and educational attainment. Overall, promoting seismic retrofits requires careful consideration of different motivators and impediments to owner's retrofit actions for commercial and residential buildings, and for older, taller, larger buildings, which tend to be more vulnerable but are associated with higher retrofit costs. In addition, neighborhood characteristics including median housing value and vacancy rate may be strong indicators of the retrofit probability among building clusters.

XPO1 inhibition with selinexor synergizes with proteasome inhibition in neuroblastoma by targeting nuclear export of IkB.

Across many cancer types in adults, upregulation of the nuclear-to-cytoplasmic transport protein Exportin-1 (XPO1) correlates with poor outcome and responsiveness to selinexor, an FDA-approved XPO1 inhibitor. Similar data are emerging in childhood cancers, for which selinexor is being evaluated in early phase clinical studies. Using proteomic profiling of primary tumor material from patients with high-risk neuroblastoma, as well as gene expression profiling from independent cohorts, we have demonstrated that XPO1 overexpression correlates with poor patient prognosis. Neuroblastoma cell lines are also sensitive to selinexor in the low nanomolar range. Based on these findings and knowledge that bortezomib, a proteasome inhibitor, blocks degradation of XPO1 cargo proteins, we hypothesized that combination treatment with selinexor and bortezomib would synergistically inhibit neuroblastoma cellular proliferation. We observed that selinexor promoted nuclear retention of IkB and that bortezomib augmented the ability of selinexor to induce cell-cycle arrest and cell death by apoptosis. This synergy was abrogated through siRNA knockdown of IkB. The synergistic effect of combining selinexor and bortezomib in vitro provides rationale for further investigation of this combination treatment for patients with high-risk neuroblastoma.

Identification of Wee1 as a target in combination with avapritinib for gastrointestinal stromal tumor treatment.

Management of gastrointestinal stromal tumors (GISTs) has been revolutionized by the identification of activating mutations in KIT and PDGFRA and clinical application of RTK inhibitors in advanced disease. Stratification of GISTs into molecularly defined subsets provides insight into clinical behavior and response to approved targeted therapies. Although these RTK inhibitors are effective in most GISTs, resistance remains a significant clinical problem. Development of effective treatment strategies for refractory GISTs requires identification of novel targets to provide additional therapeutic options. Global kinome profiling has the potential to identify critical signaling networks and reveal protein kinases essential in GISTs. Using multiplexed inhibitor beads and mass spectrometry, we explored the majority of the kinome in GIST specimens from the 3 most common molecular subtypes (KIT mutant, PDGFRA mutant, and succinate dehydrogenase deficient) to identify kinase targets. Kinome profiling with loss-of-function assays identified an important role for G2/M tyrosine kinase, Wee1, in GIST cell survival. In vitro and in vivo studies revealed significant efficacy of MK-1775 (Wee1 inhibitor) in combination with avapritinib in KIT mutant and PDGFRA mutant GIST cell lines as well as notable efficacy of MK-1775 as a monotherapy in the engineered PDGFRA mutant line. These studies provide strong preclinical justification for the use of MK-1775 in GIST.

Kinome Profiling of Primary Endometrial Tumors Using Multiplexed Inhibitor Beads and Mass Spectrometry Identifies SRPK1 as Candidate Therapeutic Target.

Endometrial carcinoma (EC) is the most common gynecologic malignancy in the United States, with limited effective targeted therapies. Endometrial tumors exhibit frequent alterations in protein kinases, yet only a small fraction of the kinome has been therapeutically explored. To identify kinase therapeutic avenues for EC, we profiled the kinome of endometrial tumors and normal endometrial tissues using Multiplexed Inhibitor Beads and Mass Spectrometry (MIB-MS). Our proteomics analysis identified a network of kinases overexpressed in tumors, including Serine/Arginine-Rich Splicing Factor Kinase 1 (SRPK1). Immunohistochemical (IHC) analysis of endometrial tumors confirmed MIB-MS findings and showed SRPK1 protein levels were highly expressed in endometrioid and uterine serous cancer (USC) histological subtypes. Moreover, querying large-scale genomics studies of EC tumors revealed high expression of SRPK1 correlated with poor survival. Loss-of-function studies targeting SRPK1 in an established USC cell line demonstrated SRPK1 was integral for RNA splicing, as well as cell cycle progression and survival under nutrient deficient conditions. Profiling of USC cells identified a compensatory response to SRPK1 inhibition that involved EGFR and the up-regulation of IGF1R and downstream AKT signaling. Co-targeting SRPK1 and EGFR or IGF1R synergistically enhanced growth inhibition in serous and endometrioid cell lines, representing a promising combination therapy for EC.

Social and Economic Components of Resilient Multi-Hazard Building Design.

In 2017, U.S. damages from natural hazard events exceeded $300B, suggesting that current targets for building performance do not sufficiently mitigate loss. The significant costs borne by individuals, insurers, and government do not include impacts from social disruption, displacement, and subsequent economic and livelihood effects. In 2016, Congress mandated the National Institute of Standards and Technology (NIST) develop a report (NIST SP 1224) describing the research needs, implementation activities, and engineering principles necessary to improve the performance of residential and commercial buildings subjected to natural hazards. An Immediate Occupancy Performance Objective (IOPO) could help preserve building and social functions post event, minimizing physical, social, and economic disaster. The stakeholder-informed NIST report sets forth items needed for multi-hazard building design that can support enhanced resilience decision-making. This paper highlights the social and economic considerations that require additional research, particularly with regard to feasibility and potential impacts from an IOPO. These topics must be considered prior to and throughout the IOPO technical development and community implementation processes to ensure better outcomes after natural hazard events.

Functional proteomics interrogation of the kinome identifies MRCKA as a therapeutic target in high-grade serous ovarian carcinoma.

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological cancer with few effective, targeted therapies. HGSOC tumors exhibit genomic instability with frequent alterations in the protein kinome; however, only a small fraction of the kinome has been therapeutically targeted in HGSOC. Using multiplexed inhibitor beads and mass spectrometry, we mapped the kinome landscape of HGSOC tumors from patients and patient-derived xenograft models. The data revealed a prevalent signature consisting of established HGSOC driver kinases, as well as several kinases previously unexplored in HGSOC. Loss-of-function analysis of these kinases in HGSOC cells indicated MRCKA (also known as CDC42BPA) as a putative therapeutic target. Characterization of the effects of MRCKA knockdown in established HGSOC cell lines demonstrated that MRCKA was integral to signaling that regulated the cell cycle checkpoint, focal adhesion, and actin remodeling, as well as cell migration, proliferation, and survival. Moreover, inhibition of MRCKA using the small-molecule BDP9066 decreased cell proliferation and spheroid formation and induced apoptosis in HGSOC cells, suggesting that MRCKA may be a promising therapeutic target for the treatment of HGSOC.

Advancing Clinicopathologic Diagnosis of High-risk Neuroblastoma Using Computerized Image Analysis and Proteomic Profiling.

A subset of patients with neuroblastoma are at extremely high risk for treatment failure, though they are not identifiable at diagnosis and therefore have the highest mortality with conventional treatment approaches. Despite tremendous understanding of clinical and biological features that correlate with prognosis, neuroblastoma at ultra-high risk for treatment failure remains a diagnostic challenge. As a first step towards improving prognostic risk stratification within the high-risk group of patients, we determined the feasibility of using computerized image analysis and proteomic profiling on single slides from diagnostic tissue specimens. After expert pathologist review of tumor sections to ensure quality and representative material input, we evaluated multiple regions of single slides as well as multiple sections from different patients' tumors using computational histologic analysis and semiquantitative proteomic profiling. We found that both approaches determined that intertumor heterogeneity was greater than intratumor heterogeneity. Unbiased clustering of samples was greatest within a tumor, suggesting a single section can be representative of the tumor as a whole. There is expected heterogeneity between tumor samples from different individuals with a high degree of similarity among specimens derived from the same patient. Both techniques are novel to supplement pathologist review of neuroblastoma for refined risk stratification, particularly since we demonstrate these results using only a single slide derived from what is usually a scarce tissue resource. Due to limitations of traditional approaches for upfront stratification, integration of new modalities with data derived from one section of tumor hold promise as tools to improve outcomes.

Evaluation of a research use only luminex based assay for measurement of procalcitonin in serum samples.

Research use only (RUO) assays do not undergo a validation process similar to test kits used for clinical purposes. Several studies have suggested that RUO assays need to be validated prior to use in any research studies. We evaluated a research use only Luminex platform based assay for measuring serum procalcitonin levels (Bio-Plex ProTM Human Acute Phase Multiplex Assay, Bio-Rad Laboratories, Hercules, CA) for comparability with an FDA cleared assay for procalcitonin (VIDAS B.R.A.H.M.S. PCT Assay, bioMérieux, Durham, NC). We tested 1,072 serum samples collected from patients with suspected sepsis in an intensive care unit setting for the comparison. There was poor correlation of the luminex based assay (r=0.081) with the VIDAS PCT Assay in the clinically relevant measurement range (<10 ng/mL). Additionally the Bio-Plex assay showed poor precision. Mass-spectrometry analysis of material eluted from PCT beads did not reveal any identifiable procalcitonin. The results show that research use only assays need to be validated to determine their suitability for research studies.

Resistance to BET Bromodomain Inhibitors Is Mediated by Kinome Reprogramming in Ovarian Cancer.

Small-molecule BET bromodomain inhibitors (BETis) are actively being pursued in clinical trials for the treatment of a variety of cancers, but the mechanisms of resistance to BETis remain poorly understood. Using a mass spectrometry approach that globally measures kinase signaling at the proteomic level, we evaluated the response of the kinome to targeted BETi treatment in a panel of BRD4-dependent ovarian carcinoma (OC) cell lines. Despite initial inhibitory effects of BETi, OC cells acquired resistance following sustained treatment with the BETi JQ1. Through application of multiplexed inhibitor beads (MIBs) and mass spectrometry, we demonstrate that BETi resistance is mediated by adaptive kinome reprogramming, where activation of compensatory pro-survival kinase networks overcomes BET protein inhibition. Furthermore, drug combinations blocking these kinases may prevent or delay the development of drug resistance and enhance the efficacy of BETi therapy.

Identification of prolyl hydroxylation modifications in mammalian cell proteins.

Prolyl hydroxylation is a PTM that plays an important role in the formation of collagen fibrils and in the oxygen-dependent regulation of hypoxia inducible factor-α (HIF-α). While this modification has been well characterized in the context of these proteins, it remains unclear to what extent it occurs in the remaining mammalian proteome. We explored this question using MS to analyze cellular extracts subjected to various fractionation strategies. In one strategy, we employed the von Hippel Lindau tumor suppressor protein, which recognizes prolyl hydroxylated HIF-α, as a scaffold for generating hydroxyproline capture reagents. We report novel sites of prolyl hydroxylation within five proteins: FK506-binding protein 10, myosin heavy chain 10, hexokinase 2, pyruvate kinase, and C-1 Tetrahydrofolate synthase. Furthermore, we show that identification of prolyl hydroxylation presents a significant technical challenge owing to widespread isobaric methionine oxidation, and that manual inspection of spectra of modified peptides in this context is critical for validation.

Defective Tibetan PHD2 binding to p23 links high altitude adaption to altered oxygen sensing.

The Tibetan population has adapted to the chronic hypoxia of high altitude. Tibetans bear a genetic signature in the prolyl hydroxylase domain protein 2 (PHD2/EGLN1) gene, which encodes for the central oxygen sensor of the hypoxia-inducible factor (HIF) pathway. Recent studies have focused attention on two nonsynonymous coding region substitutions, D4E and C127S, both of which are markedly enriched in the Tibetan population. These amino acids reside in a region of PHD2 that harbors a zinc finger, which we have previously discovered binds to a Pro-Xaa-Leu-Glu (PXLE) motif in the HSP90 cochaperone p23, thereby recruiting PHD2 to the HSP90 pathway to facilitate HIF-α hydroxylation. We herein report that the Tibetan PHD2 haplotype (D4E/C127S) strikingly diminishes the interaction of PHD2 with p23, resulting in impaired PHD2 down-regulation of the HIF pathway. The defective binding to p23 depends on both the D4E and C127S substitutions. We also identify a PXLE motif in HSP90 itself that can mediate binding to PHD2 but find that this interaction is maintained with the D4E/C127S PHD2 haplotype. We propose that the Tibetan PHD2 variant is a loss of function (hypomorphic) allele, leading to augmented HIF activation to facilitate adaptation to high altitude.

Possible rabies exposures in Peace Corps volunteers, 2011.

We surveyed Peace Corps Medical Officers (PCMOs) to determine the frequency of and responses to possible rabies exposures of U.S. Peace Corps volunteers (PCVs). Surveys were sent to 56 PCMOs serving in countries with moderate or high rabies vaccine recommendations from the U.S. Centers for Disease Control and Prevention (CDC), of which 38 (68%) responded. Thirty-seven PCMOs reported that, of 4,982 PCVs, 140 (3%) experienced possible rabies exposures. Of these, 125 (89%) had previously received rabies vaccination, 129 (92%) presented with adequately cleansed wounds, and 106 (76%) were deemed to require and were given post-exposure prophylaxis (PEP). Of 35 respondents, 30 (86%) reported that rabies vaccine was always accessible to PCVs in their country within 24 hours. Overall, the Peace Corps is successful at preventing and treating possible rabies exposures. However, this study identified a few gaps in policy implementation. The Peace Corps should continue and strengthen efforts to provide education, preexposure vaccination, and PEP to PCVs.

The global availability of rabies immune globulin and rabies vaccine in clinics providing indirect care to travelers.

We assessed rabies vaccine (RV) and immune globulin (RIG) availability on the local market by querying US Embassy medical staff worldwide. Of 112 responses, 23% were from West, Central, and East Africa. RV and RIG availability varied by region. Possible rabies exposures accounted for 2% of all travelers' health inquiries.

The global availability of rabies immune globulin and rabies vaccine in clinics providing direct care to travelers.

BACKGROUND: Rabies, which is globally endemic, poses a risk to international travelers. To improve recommendations for travelers, we assessed the global availability of rabies vaccine (RV) and rabies immune globulin (RIG). METHODS: We conducted a 20-question online survey, in English, Spanish, and French, distributed via e-mail to travel medicine providers and other clinicians worldwide from February 1 to March 30, 2011. Results were compiled according to the region. RESULTS: Among total respondents, only 190 indicated that they provided traveler postexposure care. Most responses came from North America (38%), Western Europe (19%), Australia and South and West Pacific Islands (11%), East and Southeast Asia (8%), and Southern Africa (6%). Approximately one third of 187 respondents stated that patients presented with wounds from an animal exposure that were seldom or never adequately cleansed. RIG was often or always accessible for 100% (n = 5) of respondents in the Middle East and North Africa; 94% (n = 17) in Australia and South and West Pacific Islands; 20% (n = 1) in Tropical South America; and 56% (n = 5) in Eastern Europe and Northern Asia. Ninety-one percent (n = 158) of all respondents reported that RV was often or always accessible. For all regions, 35% (n = 58) and 26% (n = 43) of respondents felt that the cost was too high for RIG and RV, respectively. CONCLUSION: The availability of RV and RIG varied by geographic region. All travelers should be informed that RIG and RV might not be readily available at their destination and that travel health and medical evacuation insurance should be considered prior to departure. Travelers should be educated to avoid animal exposures; to clean all animal bites, licks, and scratches thoroughly with soap and water; and to seek medical care immediately, even if overseas.

Prolyl hydroxylase domain protein 2 (PHD2) binds a Pro-Xaa-Leu-Glu motif, linking it to the heat shock protein 90 pathway.

Prolyl hydroxylase domain protein 2 (PHD2, also known as Egg Laying Defective Nine homolog 1) is a key oxygen-sensing protein in metazoans. In an oxygen-dependent manner, PHD2 site-specifically prolyl hydroxylates the master transcription factor of the hypoxic response, hypoxia-inducible factor-α (HIF-α), thereby targeting HIF-α for degradation. In this report we show that the heat shock protein 90 (HSP90) co-chaperones p23 and FKBP38 interact via a conserved Pro-Xaa-Leu-Glu motif (where Xaa = any amino acid) in these proteins with the N-terminal Myeloid Nervy and DEAF-1 (MYND)-type zinc finger of PHD2. Knockdown of p23 augments hypoxia-induced HIF-1α protein levels and HIF target genes. We propose that p23 recruits PHD2 to the HSP90 machinery to facilitate HIF-1α hydroxylation. These findings identify a link between two ancient pathways, the PHD:HIF and the HSP90 pathways, and suggest that this link was established concurrent with the emergence of the PHD:HIF pathway in evolution.

From the CDC: new country-specific recommendations for pre-travel typhoid vaccination.

Typhoid fever continues to be an important concern for travelers visiting many parts of the world. This communication provides updated guidance for pre-travel typhoid vaccination from the US Centers for Disease Control and Prevention (CDC) and describes the methodology for assigning country-specific recommendations.