Durable response to first-line PARP inhibition in BRCA-mutated metastatic cholangiocarcinoma: case report.

Background: Cholangiocarcinoma (CCA) is an increasingly prevalent malignancy worldwide, with poor outcomes even when diagnosed at an early stage. While recent trials have shown benefit from the addition of immunotherapy to standard-of-care chemotherapy, the improvement in overall survival is modest. Multiple novel therapies for advanced CCA targeting actionable genetic alterations have been approved in recent years; BRCA1/2 mutations are identified in up to 5% of CCA patients and may be an additional target for novel treatment approaches. While BRCA mutations have been shown in clinical trials to predict response to poly(ADP-ribose) polymerase (PARP) inhibitors in several solid tumors including breast, ovarian, prostate, and pancreas, no similar large-scale trials have been published in CCA to date. We report here a durable response to PARP inhibitor monotherapy in BRCA-mutated extrahepatic CCA; to our knowledge, this is the second report of first-line PARP inhibitor monotherapy and the first reported use of the second-generation PARP inhibitor talazoparib in this setting. Case Description: We report the case of a 79-year-old man with metastatic extrahepatic CCA harboring a somatic BRCA1 mutation who declined chemotherapy and was instead treated in the first-line metastatic setting with the PARP inhibitor talazoparib; he experienced a complete radiographic response six months into treatment and has remained on talazoparib for over three years without evidence of disease recurrence. Conclusions: This case adds to a growing list of retrospective studies supporting the clinical activity of PARP inhibitors in BRCA-mutated extrahepatic CCA. However, prospective data are clearly needed prior to adoption of this strategy in clinical practice. Fortunately, multiple trials investigating novel combination therapies utilizing PARP inhibitors in CCA are underway.

Unravelling the role of transient redox partner complexes in P450 electron transfer mechanics.

The molecular evolution of cytochromes P450 and associated redox-driven oxidative catalysis remains a mystery in biology. It is widely believed that sterol 14α-demethylase (CYP51), an essential enzyme of sterol biosynthesis, is the ancestor of the whole P450 superfamily given its conservation across species in different biological kingdoms. Herein we have utilized X-ray crystallography, molecular dynamics simulations, phylogenetics and electron transfer measurements to interrogate the nature of P450-redox partner binding using the naturally occurring fusion protein, CYP51-ferredoxin found in the sterol-producing bacterium Methylococcus capsulatus. Our data advocates that the electron transfer mechanics in the M. capsulatus CYP51-ferredoxin fusion protein involves an ensemble of ferredoxin molecules in various orientations and the interactions are transient. Close proximity of ferredoxin, however, is required to complete the substrate-induced large-scale structural switch in the P450 domain that enables proton-coupled electron transfer and subsequent oxygen scission and catalysis. These results have fundamental implications regarding the early evolution of electron transfer proteins and for the redox reactions in the early steps of sterol biosynthesis. They also shed new light on redox protein mechanics and the subsequent diversification of the P450 electron transfer machinery in nature.

The NIMH intramural healthy volunteer dataset: A comprehensive MEG, MRI, and behavioral resource.

The NIMH Healthy Research Volunteer Dataset is a collection of phenotypic data characterizing healthy research volunteers using clinical assessments such as assays of blood and urine, mental health assessments, diagnostic and dimensional measures of mental health, cognitive and neuropsychological functioning, structural and functional magnetic resonance imaging (MRI), along with diffusion tensor imaging (DTI), and a comprehensive magnetoencephalography battery (MEG). In addition, blood samples of healthy volunteers are banked for future analyses. All data collected in this protocol are broadly shared in the OpenNeuro repository, in the Brain Imaging Data Structure (BIDS) format. In addition, task paradigms and basic pre-processing scripts are shared on GitHub. There are currently few open access MEG datasets, and multimodal neuroimaging datasets are even more rare. Due to its depth of characterization of a healthy population in terms of brain health, this dataset may contribute to a wide array of secondary investigations of non-clinical and clinical research questions.

Circulating Tumor DNA as a Biomarker of Radiographic Tumor Burden in SCLC.

INTRODUCTION: Blood-based next-generation sequencing assays of circulating tumor DNA (ctDNA) have the ability to detect tumor-associated mutations in patients with SCLC. We sought to characterize the relationship between ctDNA mean variant allele frequency (VAF) and radiographic total-body tumor volume (TV) in patients with SCLC. METHODS: We identified matched blood draws and computed tomography (CT) or positron emission tomography (PET) scans within a prospective SCLC blood banking cohort. We sequenced plasma using our previously developed 14-gene SCLC-specific ctDNA assay. Three-dimensional TV was determined from PET and CT scans using MIM software and reviewed by radiation oncologists. Univariate association and multivariate regression analyses were performed to evaluate the association between mean VAF and total-body TV. RESULTS: We analyzed 75 matched blood draws and CT or PET scans from 25 unique patients with SCLC. Univariate analysis revealed a positive association between mean VAF and total-body TV (Spearman's ρ = 0.292, p < 0.01), and when considering only treatment-naive and pretreatment patients (n = 11), there was an increase in the magnitude of association (ρ = 0.618, p = 0.048). The relationship remained significant when adjusting for treatment status and bone metastases (p = 0.046). In the subgroup of patients with TP53 variants, univariate analysis revealed a significant association (ρ = 0.762, p = 0.037) only when considering treatment-naive and pretreatment patients (n = 8). CONCLUSIONS: We observed a positive association between mean VAF and total-body TV in patients with SCLC, suggesting mean VAF may represent a dynamic biomarker of tumor burden that could be followed to monitor disease status.

Limitations of Screening for Depression as a Proxy for Suicide Risk in Adult Medical Inpatients.

BACKGROUND: Medically ill hospitalized patients are at elevated risk for suicide. Hospitals that already screen for depression often use depression screening as a proxy for suicide risk screening. Extant research has indicated that screening for depression may not be sufficient to identify all patients at risk for suicide. OBJECTIVE: The present study aims to determine the effectiveness of a depression screening tool, the Patient Health Questionnaire-9, in detecting suicide risk among adult medical inpatients. METHODS: Participants were recruited from inpatient medical/surgical units in 4 hospitals as part of a larger validation study. Participants completed the Patient Health Questionnaire-9 and 2 suicide risk measures: the Ask Suicide-Screening Questions and the Adult Suicidal Ideation Questionnaire. RESULTS: The sample consisted of 727 adult medical inpatients (53.4% men; 61.8% white; mean age 50.1 ± 16.3 years). A total of 116 participants (116 of 727 [16.0%]) screened positive for suicide risk and 175 (175 of 727 [24.1%]) screened positive for depression. Of the 116 patients who screened positive for suicide risk, 36 (31.0%) screened negative for depression on the Patient Health Questionnaire-9. Of 116, 73 (62.9%) individuals who were at risk for suicide did not endorse item 9 (thoughts of harming oneself or of being better off dead) on the Patient Health Questionnaire-9. CONCLUSION: Using depression screening tools as a proxy for suicide risk may be insufficient to detect adult medical inpatients at risk for suicide. Asking directly about suicide risk and using validated tools is necessary to effectively and efficiently screen for suicide risk in this population.

Collagen IVα345 dysfunction in glomerular basement membrane diseases. II. Crystal structure of the α345 hexamer.

Our recent work identified a genetic variant of the α345 hexamer of the collagen IV scaffold that is present in patients with glomerular basement membrane diseases, Goodpasture's disease (GP) and Alport syndrome (AS), and phenocopies of AS in knock-in mice. To understand the context of this "Zurich" variant, an 8-amino acid appendage, we developed a construct of the WT α345 hexamer using the single-chain NC1 trimer technology, which allowed us to solve a crystal structure of this key connection module. The α345 hexamer structure revealed a ring of 12 chloride ions at the trimer-trimer interface, analogous to the collagen α121 hexamer, and the location of the 170 AS variants. The hexamer surface is marked by multiple pores and crevices that are potentially accessible to small molecules. Loop-crevice-loop features constitute bioactive sites, where pathogenic pathways converge that are linked to AS and GP, and, potentially, diabetic nephropathy. In Pedchenko et al., we demonstrate that these sites exhibit conformational plasticity, a dynamic property underlying assembly of bioactive sites and hexamer dysfunction. The α345 hexamer structure is a platform to decipher how variants cause AS and how hypoepitopes can be triggered, causing GP. Furthermore, the bioactive sites, along with the pores and crevices on the hexamer surface, are prospective targets for therapeutic interventions.

TSPO-targeted PET and Optical Probes for the Detection and Localization of Premalignant and Malignant Pancreatic Lesions.

PURPOSE: Pancreatic cancer is among the most aggressive malignancies and is rarely discovered early. However, pancreatic "incidentalomas," particularly cysts, are frequently identified in asymptomatic patients through anatomic imaging for unrelated causes. Accurate determination of the malignant potential of cystic lesions could lead to life-saving surgery or spare patients with indolent disease undue risk. Current risk assessment of pancreatic cysts requires invasive sampling, with attendant morbidity and sampling errors. Here, we sought to identify imaging biomarkers of high-risk pancreatic cancer precursor lesions. EXPERIMENTAL DESIGN: Translocator protein (TSPO) expression, which is associated with cholesterol metabolism, was evaluated in premalignant and pancreatic cancer lesions from human and genetically engineered mouse (GEM) tissues. In vivo imaging was performed with [18F]V-1008, a TSPO-targeted PET agent, in two GEM models. For image-guided surgery (IGS), V-1520, a TSPO ligand for near-IR optical imaging based upon the V-1008 pharmacophore, was developed and evaluated. RESULTS: TSPO was highly expressed in human and murine pancreatic cancer. Notably, TSPO expression was associated with high-grade, premalignant intraductal papillary mucinous neoplasms (IPMNs) and pancreatic intraepithelial neoplasia (PanIN) lesions. In GEM models, [18F]V-1008 exhibited robust uptake in early pancreatic cancer, detectable by PET. Furthermore, V-1520 localized to premalignant pancreatic lesions and advanced tumors enabling real-time IGS. CONCLUSIONS: We anticipate that combined TSPO PET/IGS represents a translational approach for precision pancreatic cancer care through discrimination of high-risk indeterminate lesions and actionable surgery.

Structure and physiological function of the human KCNQ1 channel voltage sensor intermediate state.

Voltage-gated ion channels feature voltage sensor domains (VSDs) that exist in three distinct conformations during activation: resting, intermediate, and activated. Experimental determination of the structure of a potassium channel VSD in the intermediate state has previously proven elusive. Here, we report and validate the experimental three-dimensional structure of the human KCNQ1 voltage-gated potassium channel VSD in the intermediate state. We also used mutagenesis and electrophysiology in Xenopus laevisoocytes to functionally map the determinants of S4 helix motion during voltage-dependent transition from the intermediate to the activated state. Finally, the physiological relevance of the intermediate state KCNQ1 conductance is demonstrated using voltage-clamp fluorometry. This work illuminates the structure of the VSD intermediate state and demonstrates that intermediate state conductivity contributes to the unusual versatility of KCNQ1, which can function either as the slow delayed rectifier current (IKs) of the cardiac action potential or as a constitutively active epithelial leak current.

Colon adenocarcinoma with metastases to the scaphoid: a case report and review of the literature.

Metastases to the hand and wrist are extremely rare, with <250 cases described in the literature. We present a case of acrometastasis of colon adenocarcinoma to the scaphoid in an 81-year-old male. Adenocarcinoma of the colon metastasizes to bone in an estimated 10% of cases; however, we are unaware of reports of this tumor metastasizing to the scaphoid or to any of the other carpal bones. We were able to identify only two cases of scaphoid metastases in the literature. This case highlights the potential for metastatic disease and other lesions to develop in the scaphoid and carpus.

On-target Resistance to the Mutant-Selective EGFR Inhibitor Osimertinib Can Develop in an Allele-Specific Manner Dependent on the Original EGFR-Activating Mutation.

PURPOSE: The third-generation EGFR inhibitor, osimertinib, is the first mutant-selective inhibitor that has received regulatory approval for the treatment of patients with EGFR-mutant lung cancer. Despite the development of highly selective third-generation inhibitors, acquired resistance remains a significant clinical challenge. Recently, we and others have identified a novel osimertinib resistance mutation, G724S, which was not predicted in in vitro screens. Here, we investigate how G724S confers resistance to osimertinib.Experimental Design: We combine structure-based predictive modeling of G724S in combination with the 2 most common EGFR-activating mutations, exon 19 deletion (Ex19Del) and L858R, with in vitro drug-response models and patient genomic profiling. RESULTS: Our simulations suggest that the G724S mutation selectively reduces osimertinib-binding affinity in the context of Ex19Del. Consistent with our simulations, cell lines transduced with Ex19Del/G724S demonstrate resistance to osimertinib, whereas cells transduced with L858R/G724S are sensitive to osimertinib. Subsequent clinical genomic profiling data further suggest G724S occurs with Ex19Del but not L858R. Furthermore, we demonstrate that Ex19Del/G724S retains sensitivity to afatinib, but not to erlotinib, suggesting a possible therapy for patients at the time of disease relapse. CONCLUSIONS: Altogether, these data suggest that G724S is an allele-specific resistance mutation emerging in the context of Ex19Del but not L858R. Our results fundamentally reframe the problem of targeted therapy resistance from one focused on the "drug-resistance mutation" pair to one focused on the "activating mutation-drug-resistance mutation" trio. This has broad implications across clinical oncology.

Mechanisms of KCNQ1 channel dysfunction in long QT syndrome involving voltage sensor domain mutations.

Mutations that induce loss of function (LOF) or dysfunction of the human KCNQ1 channel are responsible for susceptibility to a life-threatening heart rhythm disorder, the congenital long QT syndrome (LQTS). Hundreds of KCNQ1 mutations have been identified, but the molecular mechanisms responsible for impaired function are poorly understood. We investigated the impact of 51 KCNQ1 variants with mutations located within the voltage sensor domain (VSD), with an emphasis on elucidating effects on cell surface expression, protein folding, and structure. For each variant, the efficiency of trafficking to the plasma membrane, the impact of proteasome inhibition, and protein stability were assayed. The results of these experiments combined with channel functional data provided the basis for classifying each mutation into one of six mechanistic categories, highlighting heterogeneity in the mechanisms resulting in channel dysfunction or LOF. More than half of the KCNQ1 LOF mutations examined were seen to destabilize the structure of the VSD, generally accompanied by mistrafficking and degradation by the proteasome, an observation that underscores the growing appreciation that mutation-induced destabilization of membrane proteins may be a common human disease mechanism. Finally, we observed that five of the folding-defective LQTS mutant sites are located in the VSD S0 helix, where they interact with a number of other LOF mutation sites in other segments of the VSD. These observations reveal a critical role for the S0 helix as a central scaffold to help organize and stabilize the KCNQ1 VSD and, most likely, the corresponding domain of many other ion channels.

Pharmacological blockade of ASCT2-dependent glutamine transport leads to antitumor efficacy in preclinical models.

The unique metabolic demands of cancer cells underscore potentially fruitful opportunities for drug discovery in the era of precision medicine. However, therapeutic targeting of cancer metabolism has led to surprisingly few new drugs to date. The neutral amino acid glutamine serves as a key intermediate in numerous metabolic processes leveraged by cancer cells, including biosynthesis, cell signaling, and oxidative protection. Herein we report the preclinical development of V-9302, a competitive small molecule antagonist of transmembrane glutamine flux that selectively and potently targets the amino acid transporter ASCT2. Pharmacological blockade of ASCT2 with V-9302 resulted in attenuated cancer cell growth and proliferation, increased cell death, and increased oxidative stress, which collectively contributed to antitumor responses in vitro and in vivo. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in oncology, representing a new class of targeted therapy and laying a framework for paradigm-shifting therapies targeting cancer cell metabolism.

Structural and biochemical differences between the Notch and the amyloid precursor protein transmembrane domains.

γ-Secretase cleavage of the Notch receptor transmembrane domain is a critical signaling event for various cellular processes. Efforts to develop inhibitors of γ-secretase cleavage of the amyloid-ß precursor C99 protein as potential Alzheimer's disease therapeutics have been confounded by toxicity resulting from the inhibition of normal cleavage of Notch. We present biochemical and structural data for the combined transmembrane and juxtamembrane Notch domains (Notch-TMD) that illuminate Notch signaling and that can be compared and contrasted with the corresponding traits of C99. The Notch-TMD and C99 have very different conformations, adapt differently to changes in model membrane hydrophobic span, and exhibit different cholesterol-binding properties. These differences may be exploited in the design of agents that inhibit cleavage of C99 while allowing Notch cleavage.

Optimized Translocator Protein Ligand for Optical Molecular Imaging and Screening.

Translocator protein (TSPO) is a validated target for molecular imaging of a variety of human diseases and disorders. Given its involvement in cholesterol metabolism, TSPO expression is commonly elevated in solid tumors, including glioma, colorectal cancer, and breast cancer. TSPO ligands capable of detection by optical imaging are useful molecular tracers for a variety of purposes that range from quantitative biology to drug discovery. Leveraging our prior optimization of the pyrazolopyrimidine TSPO ligand scaffold for cancer imaging, we report herein a new generation of TSPO tracers with superior binding affinity and suitability for optical imaging and screening. In total, seven candidate TSPO tracers were synthesized and vetted in this study; the most promising tracer identified (29, Kd = 0.19 nM) was the result of conjugating a high-affinity TSPO ligand to a fluorophore used routinely in biological sciences (FITC) via a functional carbon linker of optimal length. Computational modeling suggested that an n-alkyl linker of eight carbons in length allows for positioning of the bulky fluorophore distal to the ligand binding domain and toward the solvent interface, minimizing potential ligand-protein interference. Probe 29 was found to be highly suitable for in vitro imaging of live TSPO-expressing cells and could be deployed as a ligand screening and discovery tool. Competitive inhibition of probe 29 quantified by fluorescence and 3H-PK11195 quantified by traditional radiometric detection resulted in equivalent affinity data for two previously reported TSPO ligands. This study introduces the utility of TSPO ligand 29 for in vitro imaging and screening and provides a structural basis for the development of future TSPO imaging ligands bearing bulky signaling moieties.

Triple-Row Modification of the Suture-Bridge Technique for Arthroscopic Rotator Cuff Repair.

Recent advances to improve outcomes in rotator cuff repair include using arthroscopic double-row suture-bridge techniques in an effort to reconstruct the rotator cuff footprint and improve fixation. However, when using this technique for larger tears, it can be difficult to get the lateral portion of the rotator cuff into an anatomic position. This report describes a triple-row modification of the suture-bridge technique that results in significantly more footprint contact area and contact pressure compared with the double-row and standard suture-bridge techniques. Maximizing the rotator cuff footprint contact area exposes more of the tendon to bone and may improve the healing potential.

Structural basis for KCNE3 modulation of potassium recycling in epithelia.

The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K(+)) channel to enable K(+) recycling coupled to transepithelial chloride ion (Cl(-)) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K(+) recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated "up" state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the "CF gender gap."

2-Amino-4-bis(aryloxybenzyl)aminobutanoic acids: A novel scaffold for inhibition of ASCT2-mediated glutamine transport.

Herein, we report the discovery of 2-amino-4-bis(aryloxybenzyl)aminobutanoic acids as novel inhibitors of ASCT2(SLC1A5)-mediated glutamine accumulation in mammalian cells. Focused library development led to two novel ASCT2 inhibitors that exhibit significantly improved potency compared with prior art in C6 (rat) and HEK293 (human) cells. The potency of leads reported here represents a 40-fold improvement over our most potent, previously reported inhibitor and represents, to our knowledge, the most potent pharmacological inhibitors of ASCT2-mediated glutamine accumulation in live cells. These and other compounds in this novel series exhibit tractable chemical properties for further development as potential therapeutic leads.

Oxidative cyclizations in orthosomycin biosynthesis expand the known chemistry of an oxygenase superfamily.

Orthosomycins are oligosaccharide antibiotics that include avilamycin, everninomicin, and hygromycin B and are hallmarked by a rigidifying interglycosidic spirocyclic ortho-δ-lactone (orthoester) linkage between at least one pair of carbohydrates. A subset of orthosomycins additionally contain a carbohydrate capped by a methylenedioxy bridge. The orthoester linkage is necessary for antibiotic activity but rarely observed in natural products. Orthoester linkage and methylenedioxy bridge biosynthesis require similar oxidative cyclizations adjacent to a sugar ring. We have identified a conserved group of nonheme iron, α-ketoglutarate-dependent oxygenases likely responsible for this chemistry. High-resolution crystal structures of the EvdO1 and EvdO2 oxygenases of everninomicin biosynthesis, the AviO1 oxygenase of avilamycin biosynthesis, and HygX of hygromycin B biosynthesis show how these enzymes accommodate large substrates, a challenge that requires a variation in metal coordination in HygX. Excitingly, the ternary complex of HygX with cosubstrate α-ketoglutarate and putative product hygromycin B identified an orientation of one glycosidic linkage of hygromycin B consistent with metal-catalyzed hydrogen atom abstraction from substrate. These structural results are complemented by gene disruption of the oxygenases evdO1 and evdMO1 from the everninomicin biosynthetic cluster, which demonstrate that functional oxygenase activity is critical for antibiotic production. Our data therefore support a role for these enzymes in the production of key features of the orthosomycin antibiotics.

Automated structure refinement for a protein heterodimer complex using limited EPR spectroscopic data and a rigid-body docking algorithm: a three-dimensional model for an ankyrin-CDB3 complex.

We report here specialized functions incorporated recently in the rigid-body docking software toolkit TagDock to utilize electron paramagnetic resonance derived (EPR-derived) interresidue distance measurements and spin-label accessibility data. The TagDock package extensions include a custom methanethiosulfonate spin label rotamer library to enable explicit, all-atom spin-label side-chain modeling and scripts to evaluate spin-label surface accessibility. These software enhancements enable us to better utilize the biophysical data routinely available from various spin-labeling experiments. To illustrate the power and utility of these tools, we report the refinement of an ankyrin:CDB3 complex model that exhibits much improved agreement with the EPR distance measurements, compared to model structures published previously.