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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

Next generation sequencing copy number plots for Case #2

Antibodies, compounds, oligonucleotides, and other reagents used in the study

Supplementary Table from RASGRF1 Fusions Activate Oncogenic RAS Signaling and Confer Sensitivity to MEK Inhibition

Supplementary Data from RASGRF1 Fusions Activate Oncogenic RAS Signaling and Confer Sensitivity to MEK Inhibition

Supplementary Figure from 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

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.

Oncogenic mutations in the epidermal growth factor receptor (EGFR) are identified in a subset of non-small cell lung carcinomas (NSCLC). These alterations lead to constitutive EGFR activation and upregulation of pathways promoting cell survival and proliferation. The development of small molecule inhibitors of EGFR has transformed the care of patients with advanced EGFR-mutant NSCLC. We review here the clinical development and activity of the third-generation EGFR inhibitor osimertinib in the advanced and adjuvant settings. As with other targeted therapies, resistance to osimertinib is common and limits the efficacy of this agent. Here we present an overview of reported acquired resistance mechanisms and potential therapeutic strategies to overcome resistance. The heterogeneity of resistance mechanisms (including secondary EGFR mutations and diverse EGFR-independent alterations) presents a major therapeutic challenge in EGFR-mutant NSCLC.

Summary 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.

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. 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. 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. 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

NRG1 rearrangements are oncogenic drivers that are enriched in invasive mucinous adenocarcinomas (IMAs) of the lung. The oncoprotein binds ERBB3-ERBB2 heterodimers and activates downstream signaling, supporting a therapeutic paradigm of ERBB3/ERBB2 inhibition. As proof of concept, a durable response was achieved with anti-ERBB3 monoclonal antibody therapy (GSK2849330) in an exceptional responder with an NRG1-rearranged IMA on a phase 1 trial (NCT01966445). In contrast, response was not achieved with anti-ERBB2 therapy (afatinib) in four NRG1-rearranged IMA patients (including the index patient post-GSK2849330). While in vitro data supported the use of either ERBB3 or ERBB2 inhibition, these clinical results were consistent with more profound anti-tumor activity and downstream signaling inhibition with anti-ERBB3 versus anti-ERBB2 therapy in an NRG1-rearranged patient-derived xenograft model. Analysis of 8,984 and 17,485 tumors in The Cancer Genome Atlas and MSK-IMPACT datasets, respectively, identified NRG1 rearrangements with novel fusion partners in multiple histologies, including breast, head and neck, renal, lung, ovarian, pancreatic, prostate, and endometrial cancers.

9057 Background: In the registrational phase 2 CodeBreaK 100 trial, sotorasib demonstrated a response rate of 37.1% with median duration of 10.0 months, a median progression-free survival of 6.8 months, and a tolerable safety profile in patients with pretreated KRAS p.G12C mutated NSCLC. Patients received a median of 2 prior lines of therapy. Here, we report PRO measures of health-related quality of life (QoL), physical functioning, and key lung cancer symptoms from this trial. Methods: Eligible patients had KRAS p.G12C mutated advanced NSCLC and received prior standard therapies. Sotorasib was given at an oral daily dose of 960 mg with 21-day treatment cycles until disease progression. Disease-related symptoms and health-related QoL were evaluated as exploratory endpoints on day 1 of each cycle from baseline to discontinuation, using the European Organization for Research and Treatment of Cancer Quality-of-life Questionnaire Core 30 (EORTC QLQ-C30) and its lung cancer module, EORTC QLQ-LC13. The single item, 5-point scale GP5, of the Functional Assessment of Cancer Therapy-General version was used to evaluate the impact of side effects. Predefined analyses included change from baseline using descriptive statistics and mixed model for repeated measures for global health status/QoL, physical functioning, and key lung cancer symptoms of cough, dyspnea and chest pain. Results: Of 126 patients enrolled, compliance rates for each of the questionnaires were high throughout the study ( > 70%). Data up to cycle 11 (where n > 20) are presented. EORTC QLQ-C30 global health status/QoL and physical functioning were maintained over time (least-square mean changes ranged from -3.5 to 0.2 and 0.1 to 3.9, respectively). EORTC QLQ-C30 symptoms of fatigue, nausea/vomiting, pain, dyspnea, insomnia, appetite loss, and constipation were stable or improved. Similarly, key lung cancer-related symptoms, as measured by EORTC QLQ-LC13, remained stable or improved from baseline, with the greatest least-square mean change of -11.2 (95% Cl: -16.2, -6.1) for cough, -4.9 (95% Cl: -10.3, 0.4) for chest pain, and -3.4 (95% Cl: -7.8, 1.0) for dyspnea. Most patients reported on the GP5 that they were “not at all” (54.2%-79.2%) or “a little bit” (8.3%-33.3%) bothered by side effects from sotorasib, with 0%-7.4% reporting being bothered as “quite a bit” and 0% as “very much”. Conclusions: In patients from the single-arm phase 2 trial of sotorasib, PRO measures suggested maintenance or improvement of global health status/QoL, physical functioning, and the severity of key lung cancer-related symptoms, including cough, dyspnea, and chest pain. Self-reported side effect bother was minimal. These data, together with the encouraging efficacy and safety profiles, strongly support the use of sotorasib in this population. Clinical trial information: NCT03600883.

SummaryWe 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.

272 Background: Expert knowledge is often shared among academic oncologists at tumor boards (TBs) at National Cancer Institute Designated Cancer Centers (NCI-CCs), but not documented or made accessible to community oncologists. Using an oncologist-only question and answer (Q&A) website, we sought to disseminate expert insights from TBs at NCI-CCs to provide educational benefit to the oncology community. Methods: A process was designed with faculty at 11 NCI-CCs to document and share discussions from TBs focused on areas of clinical complexity and practice variation on theMednet.org, an interactive Q&A website of over 8,700 US oncologists. One faculty member from each TB was selected as a site leader. She or he distilled discussions about patient management from the TB into a question that addressed the clinical situation being discussed. After the question was posted, faculty at the participating NCI-CCs were asked to answer the question on theMednet. Answers were peer reviewed, indexed, stored and disseminated via email newsletters to registered oncologists. Community engagement was measured by Q&A page views, upvotes of Q&A, and poll participation. Results: A total of 15 Breast, Thoracic, and Gastrointestinal programs from 11 NCI-CCs participated. Between 12/2016 and 5/2019, faculty highlighted 146 questions from their TBs. Q&A were viewed 43,291 times by 3,585 oncologists including 2,264 community oncologists. One hundred and eighty-four answers are posted by 56 academic physicians and peer reviewed by 76 academic physicians. One hundred and eighty-five publications were cited. Community oncologists upvoted Q&A 808 times and voted in 45 polls related to the questions 1,667 times. Viewership of NCI-CC Q&A increased by 419% over time. Q&A were repeatedly searched and viewed, with 90% of all TB Q&A viewed every month. Conclusions: Via the online Q&A theMednet platform, NCI-CC providers effectively made expert knowledge easily accessible to community oncologists across the US. Timely access to evidence based recommendations from expert faculty can inform future practice choices in the community. [Table: see text]

Tumors put in a vulnerable position Cancer cells often display alterations in metabolism that help fuel their growth. Such metabolic “rewiring” may also work against the cancer cells, however, by creating new vulnerabilities that can be exploited therapeutically. A variety of human tumors show changes in methionine metabolism caused by loss of the gene coding for 5-methylthioadenosine phosphorylase (MTAP). Mavrakis et al. and Kryukov et al. found that the loss of MTAP renders cancer cell lines sensitive to growth inhibition by compounds that suppress the activity of a specific arginine methyltransferase called PRMT5. Conceivably, drugs that inhibit PRMT5 activity could be developed into a tailored therapy for MTAP-deficient tumors. Science , this issue pp. 1208 and 1214

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.

9069Background: Non-small cell lung cancers (NSCLC) harboring METex14 activating mutations can respond dramatically to treatment with MET TKIs, but the mechanisms of acquired resistance to these th...

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 β- and γ-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.

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− cotransporter (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 hyperkalaemia. 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 coexpressed with ENaC in Xenopus laevis oocytes. However, additional coexpression studies in oocytes revealed WNK3 inhibited the renal-specific K+ channel ROMK1 activity greater than 5.5-fold (P < 0.0001) 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 coexpressed 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 < 0.0001). The magnitude and specificity of WNK3's effects at both NCC and ROMK1, its coexpression 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 probably a component of one of the mechanisms that determines the balance between renal NaCl reabsorption and K+ secretion.

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.

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.

The WNK kinases are a recently discovered family of serine-threonine kinases that have been shown to play an essential role in the regulation of electrolyte homeostasis. This review focuses on the recent evidence elucidating the functions of these kinases in normal and disease physiology.Mutations in WNK1 and WNK4 have been shown to cause pseudohypoaldosteronism type II, a disease featuring hypertension with hyperkalemia. Recent work has demonstrated that WNK4 is a potent inhibitor of diverse epithelial transporters including the thiazide-sensitive sodium chloride co-transporter (NCCT) and the renal outer medullary potassium ion channel. In addition, WNK4 activity promotes paracellular chloride ion flux. Importantly, mutations in WNK4 that cause disease have divergent effects on these transport pathways. WNK4 mutations relieve the inhibition of NCCT, increase the inhibition of the renal outer medullary potassium ion channel, and further increase paracellular chloride ion flux. These findings can explain the observed physiological abnormalities in patients with pseudohypoaldosteronism type II, and support a model in which WNK4 is a molecular switch that can alter the balance between chloride ion reabsorption and potassium ion secretion. The WNK kinases are also found in diverse epithelia throughout the body that are involved in chloride ion flux, suggesting that these kinases may play a general role in the regulation of chloride ion flux.The WNK kinases define a previously unrecognized signaling pathway that is essential for the integrated regulation of electrolyte homeostasis. Their function has implications for understanding the coordinated regulation of electrolyte homeostasis and blood pressure, and identifies WNKs as dynamic regulators of the paracellular flux pathway.

Mutations in the serine-threonine kinase WNK4 [with no lysine (K) 4] cause pseudohypoaldosteronism type II, a Mendelian disease featuring hypertension with hyperkalemia. In the kidney, WNK4 regulates the balance between NaCl reabsorption and K + secretion via variable inhibition of the thiazide-sensistive NaCl cotransporter and the K + channel ROMK. We now demonstrate expression of WNK4 mRNA and protein outside the kidney. In extrarenal tissues, WNK4 is found almost exclusively in polarized epithelia, variably associating with tight junctions, lateral membranes, and cytoplasm. Epithelia expressing WNK4 include sweat ducts, colonic crypts, pancreatic ducts, bile ducts, and epididymis. WNK4 is also expressed in the specialized endothelium of the blood–brain barrier. These epithelia and endothelium all play important roles in Cl – transport. Because WNK4 is known to regulate renal Cl – handling, we tested WNK4's effect on the activity of mediators of epithelial Cl – flux whose extrarenal expression overlaps with WNK4. WNK4 proved to be a potent inhibitor of the activity of both the Na + -K + -2Cl – cotransporter (NKCC1) and the Cl – /base exchanger SLC26A6 (CFEX) (>95% inhibition of NKCC1-mediated 86 Rb influx, P < 0.001; >80% inhibition of CFEX-mediated [ 14 C] formate uptake, P < 0.001), mediators of Cl – flux across basolateral and apical membranes, respectively. In contrast, WNK4 showed no inhibition of pendrin, a related Cl – /base exchanger. These findings indicate a general role for WNK4 in the regulation of electrolyte flux in diverse epithelia. Moreover, they reveal that WNK4 regulates the activities of a diverse group of structurally unrelated ion channels, cotransporters, and exchangers.

A key question in systems biology is how diverse physiologic processes are integrated to produce global homeostasis1. Genetic analysis can contribute by identifying genes that perturb this integration. One system orchestrates renal NaCl and K+ flux to achieve homeostasis of blood pressure and serum K+ concentration (refs. 2,3). Positional cloning implicated the serine-threonine kinase WNK4 in this process4; clustered mutations in PRKWNK4, encoding WNK4, cause hypertension and hyperkalemia (pseudohypoaldosteronism type II, PHAII5) by altering renal NaCl and K+ handling. Wild-type WNK4 inhibits the renal Na-Cl cotransporter (NCCT); mutations that cause PHAII relieve this inhibition6. This explains the hypertension of PHAII but does not account for the hyperkalemia. By expression in Xenopus laevis oocytes, we show that WNK4 also inhibits the renal K+ channel ROMK. This inhibition is independent of WNK4 kinase activity and is mediated by clathrin-dependent endocytosis of ROMK, mechanisms distinct from those that characterize WNK4 inhibition of NCCT. Most notably, the same mutations in PRKWNK4 that relieve NCCT inhibition markedly increase inhibition of ROMK. These findings establish WNK4 as a multifunctional regulator of diverse ion transporters; moreover, they explain the pathophysiology of PHAII. They also identify WNK4 as a molecular switch that can vary the balance between NaCl reabsorption and K+ secretion to maintain integrated homeostasis.

Mutations in the serine-threonine kinases WNK1 and WNK4 [ w ith n o lysine ( K ) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl − dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a “mirror image” of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na–Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCT-mediated Na + influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ( 22 Na-influx reduced 50%, P < 1 × 10 −9 , surface expression reduced 75%, P < 1 × 10 −14 in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.

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.

An understanding of driver genetic events in tumorigenesis has led to the development of targeted therapies with clinical benefit for subsets of patients with melanoma, non-small cell lung cancer, and other malignancies. The identification of new cancer vulnerabilities may provide additional therapeutic opportunities for malignancies refractory to standard treatments and may expand the use of targeted therapies to a broader patient population. To identify new cancer vulnerabilities and associated genetic features, we have integrated insights about recurrent genetic alterations (from the Cancer Cell Line Encyclopedia) with cancer cell line dependencies identified from genome-scale RNA interference (from Project Achilles). Using this approach, we find that cancer cell lines harboring a highly-recurrent alteration (homozygous deletion of the gene encoding methylthioadenosine phosphorylase or MTAP) show selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77, core components of the methylosome. Homozygous loss of MTAP occurs as a passenger event in cancer due to its proximity to the commonly-deleted tumor suppressor gene CDKN2A. MTAP is lost in >40% of glioblastomas and in 25-30% of melanomas, urothelial carcinomas, and pancreatic adenocarcinomas (in addition to other cancers). MTAP cleaves methylthioadenosine (MTA) to generate precursor substrates for methionine and adenine salvage pathways, which are compromised with MTAP loss. While MTAP deletion in cancer has long been recognized as a potential vulnerability (as tumors with MTAP loss are dependent on de novo purine synthesis), therapeutic strategies to exploit MTAP loss have not been successfully implemented. Our findings credential PRMT5 as a previously-unrecognized potential therapeutic target in MTAP-deleted cancers. We have performed mechanistic studies to confirm a role for MTAP in modulating sensitivity to genetic depletion of PRMT5 or WDR77. Furthermore, we propose a mechanism by which homozygous MTAP deletion confers a selective dependency on PRMT5 and WDR77. We show that loss of MTAP leads to increased intracellular levels of MTA, which in turn specifically inhibits the catalytic activity of PRMT5. Since PRMT5 activity is essential for cell viability, MTAP-deleted cells may have some degree of basal inhibition of PRMT5 activity that confers heightened sensitivity to further reduction in PRMT5 activity with gene depletion. Given the frequency of homozygous MTAP loss in cancer, our findings suggest a potential role for PRMT5 inhibition in multiple malignancies, either as a single agent or possibly in combination with other therapies. As MTAP loss is not mutually exclusive of oncogenic drivers in cancer (such as activating BRAF mutations in melanoma or EGFR mutations in lung cancer), it may theoretically be possible to combine PRMT5 inhibition with targeting of oncogenic drivers in an effort to augment anti-tumor activity and possibly delay or prevent the emergence of drug resistance. Citation Format: Frederick H. Wilson. PRMT5 as a therapeutic target in MTAP-deleted cancers [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr IA24.

Hypertension is a major public health problem of largely unknown cause. Here, we identify two genes causing pseudohypoaldosteronism type II, a Mendelian trait featuring hypertension, increased renal salt reabsorption, and impaired K + and H + excretion. Both genes encode members of the WNK family of serine-threonine kinases. Disease-causing mutations in WNK1 are large intronic deletions that increase WNK1 expression. The mutations in WNK4 are missense, which cluster in a short, highly conserved segment of the encoded protein. Both proteins localize to the distal nephron, a kidney segment involved in salt, K + , and pH homeostasis. WNK1 is cytoplasmic, whereas WNK4 localizes to tight junctions. The WNK kinases and their associated signaling pathway(s) may offer new targets for the development of antihypertensive drugs.

Publisher Summary The molecular genetic discovery that mutations in WNK1 and WNK4 cause pseudohypoaldosteronism type II (PHAII), has unraveled the complex mechanism by which the kidney regulates the balance between salt reabsorption and potassium secretion. Logical targets include the sodium and chloride reabsorption pathways mediated by Na-Cl cotransporter (NCC) and epithelial sodium channel (ENaC), the mediators and regulators of distal nephron potassium secretion, including renal outer medullary potassium channel (ROMK), the BK potassium channel, and ENaC, the paracellular chloride flux pathway, and the apical H+-ATPase involved in distal proton secretion. The marked impairment in renal potassium secretion is the most consistent feature of PHAII and suggests several potential targets. The potassium channel ROMK is the major mediator of renal potassium secretion in the connecting tubule (CNT)/CCD under normal circumstances and is one possible target of WNK4. ENaC, the major pathway for electrogenic sodium reabsorption in the CNT and CCD, is another potential target of the WNKs. ENaC activity produces the lumen-negative potential and is required for normal potassium secretion, as revealed by inherited and acquired alteration in ENaC activity. States featuring increased ENaC activity often cause hypokalemia owing to the increased lumen-negative potential but mutations that result in loss of ENaC function lead to impaired production of a lumen-negative potential and often profound hyperkalemia.

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.

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− cotransporter (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 hyperkalaemia. 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 coexpressed with ENaC in Xenopus laevis oocytes. However, additional coexpression studies in oocytes revealed WNK3 inhibited the renal-specific K+ channel ROMK1 activity greater than 5.5-fold (P < 0.0001) 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 coexpressed 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 < 0.0001). The magnitude and specificity of WNK3's effects at both NCC and ROMK1, its coexpression 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 probably a component of one of the mechanisms that determines the balance between renal NaCl reabsorption and K+ secretion.

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.

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.

Mutations in WNK1 and WNK4 , genes encoding members of a novel family of serine–threonine kinases, have recently been shown to cause pseudohypoaldosteronism type II (PHAII), an autosomal dominant disorder featuring hypertension, hyperkalemia, and renal tubular acidosis. The localization of these kinases in the distal nephron and the Cl − dependence of these phenotypes suggest that these mutations increase renal Cl − reabsorption. Although WNK4 expression is limited to the kidney, WNK1 is expressed in many tissues. We have examined the distribution of WNK1 in these extrarenal tissues. Immunostaining using WNK1-specific antibodies demonstrated that WNK1 is not present in all cell types; rather, it is predominantly localized in polarized epithelia, including those lining the lumen of the hepatic biliary ducts, pancreatic ducts, epididymis, sweat ducts, colonic crypts, and gallbladder. WNK1 is also found in the basal layers of epidermis and throughout the esophageal epithelium. The subcellular localization of WNK1 varies among these epithelia. WNK1 is cytoplasmic in kidney, colon, gallbladder, sweat duct, skin, and esophagus; in contrast, it localizes to the lateral membrane in bile ducts, pancreatic ducts, and epididymis. These epithelia are all notable for their prominent role in Cl − flux. Moreover, these sites largely coincide with those involved in the pathology of cystic fibrosis, a disease characterized by deranged epithelial Cl − flux. Together with the known pathophysiology of PHAII, these findings suggest that WNK1 plays a general role in the regulation of epithelial Cl − flux, a finding that suggests the potential of new approaches to the selective modulation of these processes.

We isolated and characterized a novel K-Cl cotransporter, KCC3, from human placenta. The deduced protein contains 1,150 amino acids. KCC3 shares 75–76% identity at the amino acid level with human, pig, rat, and rabbit KCC1 and 67% identity with rat KCC2. KCC3 is 40 and 33% identical to two Caenorhabditis elegans K-Cl cotransporters and ∼20% identical to other members of the cation-chloride cotransporter family (CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Cl cotransporter (NCC). Hydropathy analysis indicates a typical KCC topology with 12 transmembrane domains, a large extracellular loop between transmembrane domains 5 and 6 (unique to KCCs), and large NH2and COOH termini. KCC3 is predominantly expressed in kidney, heart, and brain, and is also expressed in skeletal muscle, placenta, lung, liver, and pancreas. KCC3 was localized to chromosome 15. KCC3 transiently expressed in human embryonic kidney (HEK)-293 cells fulfilled three criteria for increased expression of K-Cl cotransport: stimulation of cotransport by swelling, treatment with N-ethylmaleimide, or treatment with staurosporine.

The first of these Studies was concerned chiefly with the history of Ostia during the period when the city was still growing and its prosperity increasing. Even so, during the period already considered, the prosperity of Ostia, though real, was to this extent artificial, in that it depended upon factors over which the citizens themselves had no control. Ostia was the port of Rome, and nothing else, and in consequence any lowering of the standard of living in, or reduction of imports into the capital city must have had immediate and marked repercussions upon her prosperity. She even lacked to a great extent those reserves of wealth which in other cities might be drawn upon to tide over bad times. The typical citizen of Ostia came to the city in the hope of making his fortune there; but when he had made it, he usually preferred to retire to some more pleasant town, such as Tibur, Tusculum, Velitrae, or Rome itself, where he could enjoy his leisure. Few families seem to have remained in the city for more than two, or, at the most, three generations. Whilst therefore fortunes were made in Ostia, wealth was not accumulated there.

Serious excavation of the archaeological remains of Ostia really began only in the early years of the present century. Spasmodic work upon the site was, it is true, undertaken during the eighteenth and nineteenth centuries, but the earliest excavators, De Norogna, Volpato, La Piccola, Hamilton and Fagan, were little more than treasure-hunters, more intent upon gaining possession of some artistic masterpiece than upon elucidating the problems of the past. Even the distinguished archaeologists of the nineteenth century, Visconti, Lanciani and Gatti, worked at Ostia only spasmodically, and often failed to publish adequate accounts of their discoveries. Nor were serious attempts made to protect from the ravages ofrainand frost the buildings unearthed.

The building with which this study is concerned occupies the eastern half of Region ii, 2, just inside the city gate at Ostia. Two specific statements have been made concerning it, that it commenced as magazzini or horrea in the republican era, and that it was converted into baths in the late third century A.D.; these were the suggestions of the excavators, and have never yet been questioned. They are points of considerable importance, because this building would thus be the only example of republican horrea yet discovered in Ostia, and the conversion of horrea into baths or shops, which the theory implies, would be important for the economic history of Ostia, whether the reason for the change was the concentration of horrea elsewhere or merely the decline of the city. The second statement, too, would point to building activity in Ostia at a time when no other big building was being put up. This paper is an attempt to prove that at no time was the building used as horrea, and that the conversion to baths is to be placed not in the third, but in the late first, or very early second century A.D. Five main periods will be distinguished, of which the appended table gives a summary.

In this laboratory current research procedures require that simultaneous measurements of blood lipoproteins and blood lipids be accomplished in an effort to estimate the risk of coronary disease. It has been suggested that little additional information is gained by the multiple determinations. These studies report (1) the extent of correlation between ultracentrifugally determined serum lipoproteins, (2) the errors of measurement involved in all determinations, and (3) age-specific values for all serum parameters in the same population. It was found that although a high correlation exists between a linear combination of ultracentrifugally determined lipoproteins and the serum concentrations of cholesterol and phospholipid, the prediction of lipoprotein levels from the purely chemical measurements is not entirely satisfactory. Therefore, for the time being at least, all measurements will have to be accomplished.