A destabilizing Y891D mutation in activated EGFR impairs sensitivity to kinase inhibition.

EGFR tyrosine kinase inhibitors (TKIs) have transformed the treatment of EGFR-mutated non-small cell lung carcinoma (NSCLC); however, therapeutic resistance remains a clinical challenge. Acquired secondary EGFR mutations that increase ATP affinity and/or impair inhibitor binding are well-described mediators of resistance. Here we identify a de novo EGFR Y891D secondary alteration in a NSCLC with EGFR L858R. Acquired EGFR Y891D alterations were previously reported in association with resistance to first generation EGFR TKIs. Functional studies in Ba/F3 cells demonstrate reduced TKI sensitivity of EGFR L858R + Y891D, with the greatest reduction observed for first and second generation TKIs. Unlike other EGFR mutations associated with TKI resistance, Y891D does not significantly alter ATP affinity or promote steric hindrance to inhibitor binding. Our data suggest that the Y891D mutation destabilizes EGFR L858R, potentially generating a population of misfolded receptor with preserved signaling capacity but reduced sensitivity to EGFR inhibitors. These findings raise the possibility of protein misfolding as a mechanism of resistance to EGFR inhibition in EGFR-mutated NSCLC.

Making National Cancer Institute-Designated Comprehensive Cancer Center Knowledge Accessible to Community Oncologists via an Online Tumor Board: Longitudinal Observational Study.

BACKGROUND: Expert knowledge is often shared among multidisciplinary academic teams at tumor boards (TBs) across the country, but these conversations exist in silos and do not reach the wider oncology community. OBJECTIVE: Using an oncologist-only question and answer (Q&A) website, we sought to document expert insights from TBs at National Cancer Institute-designated Comprehensive Cancer Centers (NCI-CCCs) to provide educational benefits to the oncology community. METHODS: We designed a process with the NCI-CCCs to document and share discussions from the TBs focused on areas of practice variation on theMednet, an interactive Q&A website of over 13,000 US oncologists. The faculty translated the TB discussions into concise, non-case-based Q&As on theMednet. Answers were peer reviewed and disseminated in email newsletters to registered oncologists. Reach and engagement were measured. Following each Q&A, a survey question asked how the TB Q&As impacted the readers' practice. RESULTS: A total of 23 breast, thoracic, gastrointestinal, and genitourinary programs from 16 NCI-CCC sites participated. Between December 2016 and July 2021, the faculty highlighted 368 questions from their TBs. Q&As were viewed 147,661 times by 7381 oncologists at 3515 institutions from all 50 states. A total of 277 (75%) Q&As were viewed every month. Of the 1063 responses to a survey question on how the Q&A affected clinicians' practices, 646 (61%) reported that it confirmed their current practice, 163 (20%) indicated that a Q&A would change their future practice, and 214 (15%) reported learning something new. CONCLUSIONS: Through an online Q&A platform, academics at the NCI-CCCs share knowledge outside the walls of academia with oncologists across the United States. Access to up-to-date expert knowledge can reassure clinicians' practices, significantly impact patient care in community practices, and be a source of new knowledge and education.

RASGRF1 Fusions Activate Oncogenic RAS Signaling and Confer Sensitivity to MEK Inhibition.

PURPOSE: The identification of actionable oncogenic alterations has enabled targeted therapeutic strategies for subsets of patients with advanced malignancies, including lung adenocarcinoma (LUAD). We sought to assess the frequency of known drivers and identify new candidate drivers in a cohort of LUAD from patients with minimal smoking history. EXPERIMENTAL DESIGN: We performed genomic characterization of 103 LUADs from patients with ≤10 pack-year smoking history. Tumors were subjected to targeted molecular profiling and/or whole-exome sequencing and RNA sequencing in search of established and previously uncharacterized candidate drivers. RESULTS: We identified an established oncogenic driver in 98 of 103 tumors (95%). From one tumor lacking a known driver, we identified a novel gene rearrangement between OCLN and RASGRF1. The encoded OCLN-RASGRF1 chimera fuses the membrane-spanning portion of the tight junction protein occludin with the catalytic RAS-GEF domain of the RAS activator RASGRF1. We identified a similar SLC4A4-RASGRF1 fusion in a pancreatic ductal adenocarcinoma cell line lacking an activating KRAS mutation and an IQGAP1-RASGRF1 fusion from a sarcoma in The Cancer Genome Atlas. We demonstrate these fusions increase cellular levels of active GTP-RAS, induce cellular transformation, and promote in vivo tumorigenesis. Cells driven by RASGRF1 fusions are sensitive to targeting of the RAF-MEK-ERK pathway in vitro and in vivo. CONCLUSIONS: Our findings credential RASGRF1 fusions as a therapeutic target in multiple malignancies and implicate RAF-MEK-ERK inhibition as a potential treatment strategy for advanced tumors harboring these alterations. See related commentary by Moorthi and Berger, p. 2983.

Pembrolizumab for management of patients with NSCLC and brain metastases: long-term results and biomarker analysis from a non-randomised, open-label, phase 2 trial.

BACKGROUND: We did a phase 2 trial of pembrolizumab in patients with non-small-cell lung cancer (NSCLC) or melanoma with untreated brain metastases to determine the activity of PD-1 blockade in the CNS. Interim results were previously published, and we now report an updated analysis of the full NSCLC cohort. METHODS: This was an open-label, phase 2 study of patients from the Yale Cancer Center (CT, USA). Eligible patients were at least 18 years of age with stage IV NSCLC with at least one brain metastasis 5-20 mm in size, not previously treated or progressing after previous radiotherapy, no neurological symptoms or corticosteroid requirement, and Eastern Cooperative Oncology Group performance status less than two. Modified Response Evaluation Criteria in Solid Tumors (mRECIST) criteria was used to evaluate CNS disease; systemic disease was not required for participation. Patients were treated with pembrolizumab 10 mg/kg intravenously every 2 weeks. Patients were in two cohorts: cohort 1 was for those with PD-L1 expression of at least 1% and cohort 2 was patients with PD-L1 less than 1% or unevaluable. The primary endpoint was the proportion of patients achieving a brain metastasis response (partial response or complete response, according to mRECIST). All treated patients were analysed for response and safety endpoints. This study is closed to accrual and is registered with ClinicalTrials.gov, NCT02085070. FINDINGS: Between March 31, 2014, and May 21, 2018, 42 patients were treated. Median follow-up was 8·3 months (IQR 4·5-26·2). 11 (29·7% [95% CI 15·9-47·0]) of 37 patients in cohort 1 had a brain metastasis response. There were no responses in cohort 2. Grade 3-4 adverse events related to treatment included two patients with pneumonitis, and one each with constitutional symptoms, colitis, adrenal insufficiency, hyperglycaemia, and hypokalaemia. Treatment-related serious adverse events occurred in six (14%) of 42 patients and were pneumonitis (n=2), acute kidney injury, colitis, hypokalaemia, and adrenal insufficiency (n=1 each). There were no treatment-related deaths. INTERPRETATION: Pembrolizumab has activity in brain metastases from NSCLC with PD-L1 expression at least 1% and is safe in selected patients with untreated brain metastases. Further investigation of immunotherapy in patients with CNS disease from NSCLC is warranted. FUNDING: Merck and the Yale Cancer Center.

Amplification of Wild-type KRAS Imparts Resistance to Crizotinib in MET Exon 14 Mutant Non-Small Cell Lung Cancer.

PURPOSE: MET inhibitors can be effective therapies in patients with MET exon 14 (METex14) mutant non-small cell lung cancer (NSCLC). However, long-term efficacy is limited by the development of drug resistance. In this study, we characterize acquired amplification of wild-type (WT) KRAS as a molecular mechanism behind crizotinib resistance in three cases of METex14-mutant NSCLC and propose a combination therapy to target it. EXPERIMENTAL DESIGN: The patient-derived cell line and xenograft (PDX) DFCI358 were established from a crizotinib-resistant METex14-mutant patient tumor with massive focal amplification of WT KRAS. To characterize the mechanism of KRAS-mediated resistance, molecular signaling was analyzed in the parental cell line and its KRAS siRNA-transfected derivative. Sensitivity of the cell line to ligand stimulation was assessed and KRAS-dependent expression of EGFR ligands was quantified. Drug combinations were screened for efficacy in vivo and in vitro using viability and apoptotic assays. RESULTS: KRAS amplification is a recurrent genetic event in crizotinib-resistant METex14-mutant NSCLC. The key characteristics of this genetic signature include uncoupling MET from downstream effectors, relative insensitivity to dual MET/MEK inhibition due to compensatory induction of PI3K signaling, KRAS-induced expression of EGFR ligands and hypersensitivity to ligand-dependent and independent activation, and reliance on PI3K signaling upon MET inhibition. CONCLUSIONS: Using patient-derived cell line and xenografts, we characterize the mechanism of crizotinib resistance mediated by KRAS amplification in METex14-mutant NSCLC and demonstrate the superior efficacy of the dual MET/PI3K inhibition as a therapeutic strategy addressing this resistance mechanism.

ERBB Signaling Interrupted: Targeting Ligand-Induced Pathway Activation.

A patient with advanced lung adenocarcinoma harboring a CD74-NRG1 gene rearrangement, which promotes ERBB2-ERBB3 heterodimerization and activation of downstream signaling, had an exceptional therapeutic response to an experimental anti-ERBB3 antibody. This result illustrates how NRG1 rearrangements, which are observed at a low frequency in a variety of solid tumors, may represent tractable therapeutic targets. Cancer Discov; 8(6); 676-8. ©2018 AACR.See related article by Drilon et al., p. 686.

Assigning clinical meaning to somatic and germ-line whole-exome sequencing data in a prospective cancer precision medicine study.

PURPOSE: Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing (WES) data for clinical care. METHODS: One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences. RESULTS: At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach. CONCLUSION: The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies.Genet Med advance online publication 26 January 2017.

MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells.

The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A. We observed increased intracellular concentrations of methylthioadenosine (MTA, the metabolite cleaved by MTAP) in cells harboring MTAP deletions. Furthermore, MTA specifically inhibited PRMT5 enzymatic activity. Administration of either MTA or a small-molecule PRMT5 inhibitor showed a modest preferential impairment of cell viability for MTAP-null cancer cell lines compared with isogenic MTAP-expressing counterparts. Together, our findings reveal PRMT5 as a potential vulnerability across multiple cancer lineages augmented by a common "passenger" genomic alteration.

A functional landscape of resistance to ALK inhibition in lung cancer.

We conducted a large-scale functional genetic study to characterize mechanisms of resistance to ALK inhibition in ALK-dependent lung cancer cells. We identify members of known resistance pathways and additional putative resistance drivers. Among the latter were members of the P2Y purinergic receptor family of G-protein-coupled receptors (P2Y1, P2Y2, and P2Y6). P2Y receptors mediated resistance in part through a protein-kinase-C (PKC)-dependent mechanism. Moreover, PKC activation alone was sufficient to confer resistance to ALK inhibitors, whereas combined ALK and PKC inhibition restored sensitivity. We observed enrichment of gene signatures associated with several resistance drivers (including P2Y receptors) in crizotinib-resistant ALK-rearranged lung tumors compared to treatment-naive controls, supporting a role for these identified mechanisms in clinical ALK inhibitor resistance.

Genetic modifiers of EGFR dependence in non-small cell lung cancer.

Lung adenocarcinomas harboring activating mutations in the epidermal growth factor receptor (EGFR) represent a common molecular subset of non-small cell lung cancer (NSCLC) cases. EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and thus represent a dependency in NSCLCs harboring these alterations, but the genetic basis of EGFR dependence is not fully understood. Here, we applied an unbiased, ORF-based screen to identify genetic modifiers of EGFR dependence in EGFR-mutant NSCLC cells. This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. A subset of these genes can complement loss of EGFR activity across multiple EGFR-dependent models. Unbiased gene-expression profiling of cells overexpressing EGFR bypass genes, together with targeted validation studies, reveals EGFR-independent activation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT pathways. Combined inhibition of PI3K-mTOR and MEK restores EGFR dependence in cells expressing each of the 18 EGFR bypass genes. Together, these data uncover a broad spectrum of kinases capable of overcoming dependence on EGFR and underscore their convergence on the PI3K-AKT and MEK-ERK signaling axes in sustaining EGFR-independent survival.

Administration of vincristine in a patient with Machado-Joseph disease.

Chemotherapy-induced peripheral neurotoxicity is a major problem because it represents the dose-limiting side effect of a significant number of antineoplastic drugs, such as vinca alkaloids. Hereditary neuropathies usually predispose to severe vincristine neurotoxicity. Here, we report the case of a 56-year-old man with Machado-Joseph disease, also known as spinocerebellar ataxia type 3, treated with a vinca alkaloid without exacerbation of neurological symptoms.

Haploinsufficiency for ribosomal protein genes causes selective activation of p53 in human erythroid progenitor cells.

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome, a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction, but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14, the ribosomal protein gene deleted in the 5q-syndrome, or RPS19, the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect, whereas nutlin-3, a compound that activates p53 through inhibition of HDM2, selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome, we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythropoiesis in the 5q-syndrome, DBA, and perhaps other bone marrow failure syndromes.

An SGK1 site in WNK4 regulates Na+ channel and K+ channel activity and has implications for aldosterone signaling and K+ homeostasis.

The steroid hormone aldosterone is secreted both in the setting of intravascular volume depletion and hyperkalemia, raising the question of how the kidney maximizes NaCl reabsorption in the former state while maximizing K(+) secretion in the latter. Mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring increased renal NaCl reabsorption and impaired K(+) secretion. PHAII-mutant WNK4 achieves these effects by increasing activity of the Na-Cl cotransporter (NCC) and the Na(+) channel ENaC while concurrently inhibiting the renal outer medullary K(+) channel (ROMK). We now describe a functional state for WNK4 that promotes increased, rather than decreased, K(+) secretion. We show that WNK4 is phosphorylated by SGK1, a mediator of aldosterone signaling. Whereas wild-type WNK4 inhibits the activity of both ENaC and ROMK, a WNK4 mutation that mimics phosphorylation at the SGK1 site (WNK4(S1169D)) alleviates inhibition of both channels. The net result of these effects in the kidney would be increased K(+) secretion, because of both increased electrogenic Na(+) reabsorption and increased apical membrane K(+) permeability. Thus, modification at the PHAII and SGK1 sites in WNK4 impart opposite effects on K(+) secretion, decreasing or increasing ROMK activity and net K(+) secretion, respectively. This functional state for WNK4 would thus promote the desired physiologic response to hyperkalemia, and the fact that it is induced downstream of aldosterone signaling implicates WNK4 in the physiologic response to aldosterone with hyperkalemia. Together, the different states of WNK4 allow the kidney to provide distinct and appropriate integrated responses to intravascular volume depletion and hyperkalemia.

WNK4 regulates activity of the epithelial Na+ channel in vitro and in vivo.

Homeostasis of intravascular volume, Na(+), Cl(-), and K(+) is interdependent and determined by the coordinated activities of structurally diverse mediators in the distal nephron and the distal colon. The behavior of these flux pathways is regulated by the renin-angiotensin-aldosterone system; however, the mechanisms that allow independent modulation of individual elements have been obscure. Previous work has shown that mutations in WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension with hyperkalemia, due to altered activity of specific Na-Cl cotransporters, K(+) channels, and paracellular Cl(-) flux mediators of the distal nephron. By coexpression studies in Xenopus oocytes, we now demonstrate that WNK4 also inhibits the epithelial Na(+) channel (ENaC), the major mediator of aldosterone-sensitive Na(+) (re)absorption, via a mechanism that is independent of WNK4's kinase activity. This inhibition requires intact C termini in ENaC beta- and gamma-subunits, which contain PY motifs used to target ENaC for clearance from the plasma membrane. Importantly, PHAII-causing mutations eliminate WNK4's inhibition of ENaC, thereby paralleling other effects of PHAII to increase sodium balance. The relevance of these findings in vivo was studied in mice harboring PHAII-mutant WNK4. The colonic epithelium of these mice demonstrates markedly increased amiloride-sensitive Na(+) flux compared with wild-type littermates. These studies identify ENaC as a previously unrecognized downstream target of WNK4 and demonstrate a functional role for WNK4 in the regulation of colonic Na(+) absorption. These findings support a key role for WNK4 in coordinating the activities of diverse flux pathways to achieve integrated fluid and electrolyte homeostasis.

WNK3, a kinase related to genes mutated in hereditary hypertension with hyperkalaemia, regulates the K+ channel ROMK1 (Kir1.1).

The serine-threonine kinase WNK3 modulates Cl- transport into and out of cells through its regulation of SLC12A cation/Cl- cotransporters, implicating it as (one of) the long-sought Cl-/volume-sensitive kinase(s). Integrators in homeostatic systems regulate structurally diverse but functionally coupled elements. For example, the related kinase WNK4 regulates the Na-Cl co-transporter (NCC), paracellular Cl- flux, and the K+ channel ROMK1 (Kir1.1) to maintain renal NaCl and K+ homeostasis; mutations in PRKWNK4, encoding WNK4, cause a Mendelian disease featuring hypertension and hyperkalemia. It is known that WNK3 is expressed in the nephron's distal convoluted tubule (DCT) and stimulates NCC activity. Here, we show that WNK3 is also expressed in cortical and outer medullary collecting duct principal cells. Accordingly, we tested WNK3's effect on the mediators of NaCl and K+ handling in these nephron segments--the epithelial sodium channel (ENaC), paracellular Cl- flux, and ROMK1--using established model systems. WNK3 did not alter paracellular Cl- flux in tetracycline-responsive MDCK II cells, nor affect amiloride-sensitive currents when co-expressed with ENaC in Xenopus laevis oocytes. However, additional co-expression studies in oocytes revealed WNK3 inhibited the renal-specific K+ channel ROMK1 activity greater than 5.5-fold (p < .0001) by altering its plasmalemmal surface expression; WNK3 did not affect ROMK1's conductance or open/closed probability. In contrast, WNK3 had no effect on the activity of the cardiac long-QT syndrome K+ channel KCNQ1/KCNE1 when co-expressed in oocytes. Inhibition of ROMK1 is independent of WNK3's catalytic activity and is mediated by WNK3's carboxyl terminus--a mechanism distinct from its known kinase-dependent activation of NCC. A kinase-inactivating point mutation, or a missense mutation homologous to one in WNK4 that causes disease produced a gain-of-function effect, enhancing WNK3's inhibition of ROMK1 greater than 2.5-fold relative to wild type kinase (p < .0001). The magnitude and specificity of WNK3's effects at both NCC and ROMK1, its co-expression with its targets in the distal nephron, and the established in vivo effect of WNK4 at these same targets provide evidence that WNK3's action is physiologically relevant. WNK3 is likely a component of one of the mechanisms that determines the balance between renal NaCl reabsorption and K+ secretion.

WNK3 kinase is a positive regulator of NKCC2 and NCC, renal cation-Cl- cotransporters required for normal blood pressure homeostasis.

WNK1 and WNK4 [WNK, with no lysine (K)] are serine-threonine kinases that function as molecular switches, eliciting coordinated effects on diverse ion transport pathways to maintain homeostasis during physiological perturbation. Gain-of-function mutations in either of these genes cause an inherited syndrome featuring hypertension and hyperkalemia due to increased renal NaCl reabsorption and decreased K(+) secretion. Here, we reveal unique biochemical and functional properties of WNK3, a related member of the WNK kinase family. Unlike WNK1 and WNK4, WNK3 is expressed throughout the nephron, predominantly at intercellular junctions. Because WNK4 is a potent inhibitor of members of the cation-cotransporter SLC12A family, we used coexpression studies in Xenopus oocytes to investigate the effect of WNK3 on NCC and NKCC2, related kidney-specific transporters that mediate apical NaCl reabsorption in the thick ascending limb and distal convoluted tubule, respectively. In contrast to WNK4's inhibitory activity, kinase-active WNK3 is a potent activator of both NKCC2 and NCC-mediated transport. Conversely, in its kinase-inactive state, WNK3 is a potent inhibitor of NKCC2 and NCC activity. WNK3 regulates the activity of these transporters by altering their expression at the plasma membrane. Wild-type WNK3 increases and kinase-inactive WNK3 decreases NKCC2 phosphorylation at Thr-184 and Thr-189, sites required for the vasopressin-mediated plasmalemmal translocation and activation of NKCC2 in vivo. The effects of WNK3 on these transporters and their coexpression in renal epithelia implicate WNK3 in NaCl, water, and blood pressure homeostasis, perhaps via signaling downstream of vasopressin.

Regulation of diverse ion transport pathways by WNK4 kinase: a novel molecular switch.

Key components of complex physiological regulatory pathways can be uncovered through the molecular-genetic study of rare, inherited diseases. WNK kinases are a recently discovered class of serine-threonine kinases that are distinctive because of the substitution of cysteine for lysine in subdomain II of the catalytic domain. Mutations in PRKWNK1 and PRKWNK4, which encode WNK1 and WNK4, result in an inherited syndrome of hypertension and hyperkalemia. Recent physiological work has revealed that WNK4 alters the balance of NaCl reabsorption and K(+) secretion in the distal nephron by actions on both transcellular and paracellular ion-flux pathways. Additionally, WNK4 is expressed in extra-renal epithelia with prominent roles in Cl(-) handling, and it regulates transporters that are responsible for Cl(-) flux across apical and basolateral membranes. WNK kinases are components of a novel signaling pathway that is important for the control of blood pressure and electrolyte homeostasis.

A cluster of metabolic defects caused by mutation in a mitochondrial tRNA.

Hypertension and dyslipidemia are risk factors for atherosclerosis and occur together more often than expected by chance. Although this clustering suggests shared causation, unifying factors remain unknown. We describe a large kindred with a syndrome including hypertension, hypercholesterolemia, and hypomagnesemia. Each phenotype is transmitted on the maternal lineage with a pattern indicating mitochondrial inheritance. Analysis of the mitochondrial genome of the maternal lineage identified a homoplasmic mutation substituting cytidine for uridine immediately 5' to the mitochondrial transfer RNA(Ile) anticodon. Uridine at this position is nearly invariate among transfer RNAs because of its role in stabilizing the anticodon loop. Given the known loss of mitochondrial function with aging, these findings may have implications for the common clustering of these metabolic disorders.

Paracellular Cl- permeability is regulated by WNK4 kinase: insight into normal physiology and hypertension.

Paracellular ion flux across epithelia occurs through selective and regulated pores in tight junctions; this process is poorly understood. Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a disease featuring hypertension and hyperkalemia. Whereas WNK4 is known to regulate several transcellular transporters and channels involved in NaCl and K+ homeostasis, its localization to tight junctions suggests it might also regulate paracellular flux. We performed electrophysiology on mammalian kidney epithelia with inducible expression of various WNK4 constructs. Induction of wild-type WNK4 reduced transepithelial resistance by increasing absolute chloride permeability. PHAII-mutant WNK4 produced markedly larger effects, whereas kinase-mutant WNK4 had no effect. The electrochemical and pharmacologic properties of these effects indicate they are attributable to the paracellular pathway. The effects of WNK4 persist when induction is delayed until after tight-junction formation, demonstrating a dynamic effect. WNK4 did not alter the flux of uncharged solutes, or the expression or localization of selected tight-junction proteins. Transmission and freeze-fracture electron microscopy showed no effect of WNK4 on tight-junction structure. These findings implicate WNK signaling in the coordination of transcellular and paracellular flux to achieve NaCl and K+ homeostasis, explain PHAII pathophysiology, and suggest that modifiers of WNK signaling may be potent antihypertensive agents.