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.

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.

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.

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.

TagDock: an efficient rigid body docking algorithm for oligomeric protein complex model construction and experiment planning.

We report here new computational tools and strategies to efficiently generate three-dimensional models for oligomeric biomolecular complexes in cases where there is limited experimental restraint data to guide the docking calculations. Our computational tools are designed to rapidly and exhaustively enumerate all geometrically possible docking poses for an oligomeric complex, rather than generate detailed, atomic-resolution models. Experimental data, such as interatomic distance measurements, are then used to select and refine docking poses that are consistent with the experimental restraints. Our computational toolkit is designed for use with sparse data sets to generate intermediate-resolution docking models, and utilizes distance difference matrix analysis to identify further restraint measurements that will provide maximum additional structural refinement. Thus, these tools can be used to help plan optimal residue positions for probe incorporation in labor-intensive biophysical experiments such as chemical cross-linking, electron paramagnetic resonance, or Förster resonance energy transfer spectroscopy studies. We present benchmark results for docking the collection of all 176 heterodimer protein complexes from the ZDOCK database, as well as a protein homodimer with recently collected experimental distance restraints, to illustrate the toolkit's capabilities and performance, and to demonstrate how distance difference matrix analysis can automatically identify and prioritize additional restraint measurements that allow us to rapidly optimize docking poses.

Loop-loop interactions regulate KaiA-stimulated KaiC phosphorylation in the cyanobacterial KaiABC circadian clock.

The Synechococcus elongatus KaiA, KaiB, and KaiC proteins in the presence of ATP generate a post-translational oscillator that runs in a temperature-compensated manner with a period of 24 h. KaiA dimer stimulates phosphorylation of KaiC hexamer at two sites per subunit, T432 and S431, and KaiB dimers antagonize KaiA action and induce KaiC subunit exchange. Neither the mechanism of KaiA-stimulated KaiC phosphorylation nor that of KaiB-mediated KaiC dephosphorylation is understood in detail at present. We demonstrate here that the A422V KaiC mutant sheds light on the former mechanism. It was previously reported that A422V is less sensitive to dark pulse-induced phase resetting and has a reduced amplitude of the KaiC phosphorylation rhythm in vivo. A422 maps to a loop (422-loop) that continues toward the phosphorylation sites. By pulling on the C-terminal peptide of KaiC (A-loop), KaiA removes restraints from the adjacent 422-loop whose increased flexibility indirectly promotes kinase activity. We found in the crystal structure that A422V KaiC lacks phosphorylation at S431 and exhibits a subtle, local conformational change relative to wild-type KaiC. Molecular dynamics simulations indicate higher mobility of the 422-loop in the absence of the A-loop and mobility differences in other areas associated with phosphorylation activity between wild-type and mutant KaiCs. The A-loop-422-loop relay that informs KaiC phosphorylation sites of KaiA dimer binding propagates to loops from neighboring KaiC subunits, thus providing support for a concerted allosteric mechanism of phosphorylation.

An allosteric mechanism for drug block of the human cardiac potassium channel KCNQ1.

The intracellular aspect of the sixth transmembrane segment within the ion-permeating pore is a common binding site for many voltage-gated ion channel blockers. However, the exact site(s) at which drugs bind remain controversial. We used extensive site-directed mutagenesis coupled with molecular modeling to examine mechanisms in drug block of the human cardiac potassium channel KCNQ1. A total of 48 amino acid residues in the S6 segment, S4-S5 linker, and the proximal C-terminus of the KCNQ1 channel were mutated individually to alanine; alanines were mutated to cysteines. Residues modulating drug block were identified when mutant channels displayed <50% block on exposure to drug concentrations that inhibited wild-type current by ≥90%. Homology modeling of the KCNQ1 channel based on the Kv1.2 structure unexpectedly predicted that the key residue modulating drug block (F351) faces away from the permeating pore. In the open-state channel model, F351 lines a pocket that also includes residues L251 and V254 in S4-S5 linker. Docking calculations indicated that this pocket is large enough to accommodate quinidine. To test this hypothesis, L251A and V254A mutants were generated that display a reduced sensitivity to blockage with quinidine. Thus, our data support a model in which open state block of this channel occurs not via binding to a site directly in the pore but rather by a novel allosteric mechanism: drug access to a side pocket generated in the open-state channel configuration and lined by S6 and S4-S5 residues.

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.

Determination of structural models of the complex between the cytoplasmic domain of erythrocyte band 3 and ankyrin-R repeats 13-24.

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.

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.

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.

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.

Structural dynamics and single-stranded DNA binding activity of the three N-terminal domains of the large subunit of replication protein A from small angle X-ray scattering.

Replication protein A (RPA) is the primary eukaryotic single-stranded DNA (ssDNA) binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial interdomain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments with two multidomain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high-affinity ssDNA binding domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA-bound state and therefore freely available to serve as a protein recruitment module.

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.

The evolutionarily conserved residue A653 plays a key role in HERG channel closing.

Human ether-a-go-go-related gene (HERG) encodes the rapid, outwardly rectifying K(+) current I(Kr) that is critical for repolarization of the cardiac action potential. Congenital HERG mutations or unintended pharmaceutical block of I(Kr) can lead to life-threatening arrhythmias. Here, we assess the functional role of the alanine at position 653 (HERG-A653) that is highly conserved among evolutionarily divergent K(+) channels. HERG-A653 is close to the 'glycine hinge' implicated in K(+) channel opening, and is flanked by tyrosine 652 and phenylalanine 656, which contribute to the drug binding site. We substituted an array of seven (I, C, S, G, Y, V and T) amino acids at position 653 and expressed individual variants in heterologous systems to assess changes in gating and drug binding. Substitution of A653 resulted in negative shifts of the V(1/2) of activation ranging from -23.6 (A653S) to -62.5 (A653V) compared to -11.2 mV for wild-type (WT). Deactivation was also drastically altered: channels with A653I/C substitutions exhibited delayed deactivation in response to test potentials above the activation threshold, while A653S/G/Y/V/T failed to deactivate under those conditions and required hyperpolarization and prolonged holding potentials at -130 mV. While A653S/G/T/Y variants showed decreased sensitivity to the I(Kr) inhibitor dofetilide, these changes could not be correlated with defects in channel closure. Homology modelling suggests that in the closed state, A653 forms tight contacts with several residues from the neighbouring subunit in the tetramer, playing a key role in S6 helix packing at the narrowest part of the vestibule. Our study suggests that A653 plays an important functional role in the outwardly rectifying gating behaviour of HERG, supporting channel closure at membrane potentials negative to the channel activation threshold.

The chimpanzee brain shows human-like perisylvian asymmetries in white matter.

Modern neuroimaging technologies allow scientists to uncover interspecies differences and similarities in hemispheric asymmetries that may shed light on the origin of brain asymmetry and its functional correlates. We analyzed asymmetries in ratios of white to grey matter in the lateral aspect of the lobes of the brains of chimpanzees. We found marked leftward asymmetries for all lobar regions. This asymmetry was particularly pronounced in the frontal region and was found to be related to handedness for communicative manual gestures as well as for tool use. These results point to a continuity in asymmetry patterns between the human and chimpanzee brain, and support the notion that the anatomical substrates for lateralization of communicative functions and complex manipulative activities may have been present in the common hominid ancestor.

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.