Identification of WNK1 as a therapeutic target to suppress IgH/MYC expression in multiple myeloma.

Multiple myeloma (MM) remains an incurable hematological malignancy demanding innovative therapeutic strategies. Targeting MYC, the notorious yet traditionally undruggable oncogene, presents an appealing avenue. Here, using a genome-scale CRISPR-Cas9 screen, we identify the WNK lysine-deficient protein kinase 1 (WNK1) as a regulator of MYC expression in MM cells. Genetic and pharmacological inhibition of WNK1 reduces MYC expression and, further, disrupts the MYC-dependent transcriptional program. Mechanistically, WNK1 inhibition attenuates the activity of the immunoglobulin heavy chain (IgH) enhancer, thus reducing MYC transcription when this locus is translocated near the MYC locus. WNK1 inhibition profoundly impacts MM cell behaviors, leading to growth inhibition, cell-cycle arrest, senescence, and apoptosis. Importantly, the WNK inhibitor WNK463 inhibits MM growth in primary patient samples as well as xenograft mouse models and exhibits synergistic effects with various anti-MM compounds. Collectively, our study uncovers WNK1 as a potential therapeutic target in MM.

The CREB1/WNK1 axis promotes the tumorigenesis of ovarian cancer via regulating HIF-1.

The aim of this study was to explore the functions and molecular mechanisms of the WNK lysine deficient protein kinase 1 (WNK1) in the development of ovarian cancer. Firstly, loss- and gain-of-function assays were carried out and subsequently cell proliferation, apoptosis, invasion and migration were detected. Furthermore, WNK1 action on glucose uptake, lactate production and adenosine triphosphate (ATP) level were assessed. The roles of WNK1 on cisplatin resistance were explored using CCK-8, colony formation, and flow cytometry in vitro. Immunohistochemistry, Western blot and qRT-PCR were conducted to determine the protein and mRNA expression. Additionally, tumor growth in vivo was also monitored. We found that the overexpression of WNK1 predicted a bad prognosis of ovarian cancer patients. WNK1 enhanced the malignant behavior and facilitated glycolysis of ovarian cancer cells. Moreover, WNK1 increased cisplatin resistance in ovarian cancer cells. Mechanistically, we found that WNK1 expression was promoted by CREB1 at the transcriptional level. And CREB1 could facilitate ovarian cancer cells malignant behavior through target upregulating WNK1. Besides, we also showed that WNK1 facilitated the malignant behavior by accelerating HIF-1 expression. In xenograft tumor tissues, the downregulation of WNK1 significantly reduced HIF-1α expression. These data demonstrated that the CREB1/WNK1 axis could promote the tumorigenesis of ovarian cancer via accelerating HIF-1 expression, suggesting that the CREB1/WNK1 axis could be a potential target during the therapy of ovarian cancer.

WNK1 mediates amphiregulin-induced MMP9 expression and cell invasion in human extravillous trophoblast cells.

The invasion of human extravillous trophoblast (EVT) cells is a critical event required for a successful pregnancy. Amphiregulin, a ligand of the epidermal growth factor receptor (EGFR), has been shown to stimulate cell invasion in an immortalized human EVT cell line, HTR-8/SVneo. The with-no-lysine kinase 1 (WNK1) is involved in regulating cell invasion. It is known that WNK1 is expressed in the human placenta, but its role in human EVT cells remains unknown. In the present study, we show that AREG treatment phosphorylated WNK1 at Thr60 in both HTR-8/SVneo and primary human EVT cells. The stimulatory effect of AREG on WNK1 phosphorylation was mediated by the activation of PI3K/AKT, but not the ERK1/2 signaling pathway. AREG upregulated matrix metalloproteinase 9 (MMP9) but not MMP2. In addition, cell invasiveness was increased in response to the treatment of AREG. Using the siRNA-mediated knockdown approach, our results showed that the knockdown of WNK1 attenuated the AREG-induced upregulation of MMP9 expression and cell invasion. Moreover, the expression of WNK1 was downregulated in the placentas with preeclampsia, a disease resulting from insufficiency of EVT cell invasion during pregnancy. This study discovers the physiological function of WNK1 in human EVT cells and provides important insights into the regulation of MMP9 and cell invasion in human EVT cells.

WNK1 controls endosomal trafficking through TRIM27-dependent regulation of actin assembly.

The protein kinase WNK1 (with-no-lysine 1) influences trafficking of ion and small-molecule transporters and other membrane proteins as well as actin polymerization state. We investigated the possibility that actions of WNK1 on both processes are related. Strikingly, we identified the E3 ligase tripartite motif-containing 27 (TRIM27) as a binding partner for WNK1. TRIM27 is involved in fine tuning the WASH (Wiskott-Aldrich syndrome protein and SCAR homologue) regulatory complex which regulates endosomal actin polymerization. Knockdown of WNK1 reduced the formation of the complex between TRIM27 and its deubiquitinating enzyme USP7 (ubiquitin-specific protease 7), resulting in significantly diminished TRIM27 protein. Loss of WNK1 disrupted WASH ubiquitination and endosomal actin polymerization, which are necessary for endosomal trafficking. Sustained receptor tyrosine kinase (RTK) expression has long been recognized as a key oncogenic signal for the development and growth of human malignancies. Depletion of either WNK1 or TRIM27 significantly increased degradation of the epidermal growth factor receptor (EGFR) following ligand stimulation in breast and lung cancer cells. Like the EGFR, the RTK AXL was also affected similarly by WNK1 depletion but not by inhibition of WNK1 kinase activity. This study uncovers a mechanistic connection between WNK1 and the TRIM27-USP7 axis and extends our fundamental knowledge about the endocytic pathway regulating cell surface receptors.

The WNK1-ERK5 route plays a pathophysiological role in ovarian cancer and limits therapeutic efficacy of trametinib.

BACKGROUND: The dismal prognosis of advanced ovarian cancer calls for the development of novel therapies to improve disease outcome. In this regard, we set out to discover new molecular entities and to assess the preclinical effectiveness of their targeting. METHODS: Cell lines, mice and human ovarian cancer samples were used. Proteome profiling of human phosphokinases, in silico genomic analyses, genetic (shRNA and CRISPR/Cas9) and pharmacological strategies as well as an ex vivo human preclinical model were performed. RESULTS: We identified WNK1 as a highly phosphorylated protein in ovarian cancer and found that its activation or high expression had a negative impact on patients' survival. Genomic analyses showed amplification of WNK1 in human ovarian tumours. Mechanistically, we demonstrate that WNK1 exerted its action through the MEK5-ERK5 signalling module in ovarian cancer. Loss of function, genetic or pharmacological experiments, demonstrated anti-proliferative and anti-tumoural effects of the targeting of the WNK1-MEK5-ERK5 route. Additional studies showed that this pathway modulated the anti-tumoural properties of the MEK1/2 inhibitor trametinib. Thus, treatment with trametinib activated the WNK1-MEK5-ERK5 route, raising the possibility that this effect may limit the therapeutic benefit of ERK1/2 targeting in ovarian cancer. Moreover, in different experimental settings, including an ex vivo patient-derived model consisting of ovarian cancer cells cultured with autologous patient sera, we show that inhibition of WNK1 or MEK5 increased the anti-proliferative and anti-tumour efficacy of trametinib. CONCLUSIONS: The present study uncovers the participation of WNK1-MEK5-ERK5 axis in ovarian cancer pathophysiology, opening the possibility of acting on this pathway with therapeutic purposes. Another important finding of the present study was the activation of that signalling axis by trametinib, bypassing the anti-tumoural efficacy of this drug. That fact should be considered in the context of the use of trametinib in ovarian cancer.

CBX2-mediated suppression of SIAH2 triggers WNK1 accumulations to promote glycolysis in hepatocellular carcinoma.

Previous studies have highlighted the poor prognosis of liver cancer, and treatment effects are overall limited. We aimed to confirm the biological roles of SIAH2 in liver cancer and provide potential therapeutic targets. Differential analysis was conducted based on public datasets and found that SIAH2 expressed lowly in HCC samples relative to normal tissues, which was demonstrated in tumor samples via immunohistochemistry (IHC). Besides, SIAH2 overexpression could significantly suppress HCC proliferation. SIAH2 deficiency induced cell proliferation, migration and self-renewal abilities in vitro and in vivo. Mechanistically, SIAH2 could interact with WNK1, and trigger the ubiquitination and degradation of WNK1 proteins. In addition, low SIAH2 depended on elevated WNK1 proteins to drive HCC malignant features, including proliferation, migration and stemness. Meanwhile, we further found that CBX2 could regulate SIAH2 expressions. CBX2 cooperated with EZH2 to mediate the H3K27me3 enrichment on the promoter region of SIAH2 to suppress its transcriptional levels. High CBX2/EZH2 levels in HCC correlated with poor prognosis of patients. Gene set enrichment analysis (GSEA) further implicated that WNK1 correlates tightly with glycolytic process in HCC samples. WNK1 overexpression was found to notably enhance glycolytic activity, whereas WNK1 deficiency could significantly suppress the HCC glycolysis activity. Lastly, the subcutaneous tumor model further demonstrated that targeting WNK1 was effective to inhibit the in vivo tumor growth of SIAH2low HCC. Collectively, down-regulated SIAH2 expressions induced by CBX2/EZH2 could drive progression and glycolysis via accumulating WNK1 proteins, indicating that CBX2/SIAH2/WNK1 axis is a potential prognostic biomarker and therapeutic vulnerability for human HCC.

Identification of a Class of WNK Isoform-Specific Inhibitors Through High-Throughput Screening.

Introduction: WNK [with no lysine (K)] kinases are serine/threonine kinases associated with familial hyperkalemic hypertension (FHHt). WNKs are therapeutic targets for blood pressure regulation, stroke and several cancers including triple negative breast cancer and glioblastoma. Here, we searched for and characterized novel WNK kinase inhibitors. Methods: We used a ~210,000-compound library in a high-throughput screen, re-acquisition and assay, commercial specificity screens and crystallography to identify WNK-isoform-selective inhibitors. Results: We identified five classes of compounds that inhibit the kinase activity of WNK1: quinoline compounds, halo-sulfones, cyclopropane-containing thiazoles, piperazine-containing compounds, and nitrophenol-derived compounds. The compounds are strongly pan-WNK selective, inhibiting all four WNK isoforms. A class of quinoline compounds was identified that further shows selectivity among the WNK isoforms, being more potent toward WNK3 than WNK1. The crystal structure of the quinoline-derived SW120619 bound to the kinase domain of WNK3 reveals active site binding, and comparison to the WNK1 structure reveals the potential origin of isoform specificity. Discussion: The newly discovered classes of compounds may be starting points for generating pharmacological tools and potential drugs treating hypertension and cancer.

B cell-intrinsic requirement for WNK1 kinase in antibody responses in mice.

Migration and adhesion play critical roles in B cells, regulating recirculation between lymphoid organs, migration within lymphoid tissue, and interaction with CD4+ T cells. However, there is limited knowledge of how B cells integrate chemokine receptor and integrin signaling with B cell activation to generate efficient humoral responses. Here, we show that the WNK1 kinase, a regulator of migration and adhesion, is essential in B cells for T-dependent and -independent antibody responses. We demonstrate that WNK1 transduces signals from the BCR, CXCR5, and CD40, and using intravital imaging, we show that WNK1 regulates migration of naive and activated B cells, and their interactions with T cells. Unexpectedly, we show that WNK1 is required for BCR- and CD40-induced proliferation, acting through the OXSR1 and STK39 kinases, and for efficient B cell-T cell collaboration in vivo. Thus, WNK1 is critical for humoral immune responses, by regulating B cell migration, adhesion, and T cell-dependent activation.

Aberrant expression of WNK lysine deficient protein kinase 1 is associated with poor prognosis of colon adenocarcinoma.

BACKGROUNDS: WNK1 (WNK lysine deficient protein kinase 1) is a kind of protein kinase and participates in angiogenesis, having a potent tumor promoting role. WNK1 is ubiquitously expressed, and its upregulated expression has been reported in several tumor types. AIMS: Here, we aimed to investigate the correlation between WNK1 expression and colon adenocarcinoma (COAD) progression. METHODS: In the current study, WNK1 expression was evaluated by immunohistochemically in adjacent normal colonic mucosae and primary adenocarcinomas. The effect of WNK1 on overall survival (OS) and its associations with the clinicopathological parameters were analyzed in a retrospective cohort of COAD patients (n = 185). The tumor-related effects of WNK1 in COAD were further tested via cellular and mice experiments. RESULTS: According to our cohort, higher WNK1 expression was significantly associated with unfavorable prognostic factors, such as high pT stage, pN stage, as well as shorter OS. Moreover, WNK1 exhibited tumor promoting role in COAD cancer cell lines as well as in nude mice. Silencing WNK1 can significantly inhibit the proliferation of COAD both in vitro and in vivo. CONCLUSIONS: In all, WNK1 acts as a tumor promoter and may be used as a COAD prognostic biomarker.

WNK1-OSR1 Signaling Regulates Angiogenesis-Mediated Metastasis towards Developing a Combinatorial Anti-Cancer Strategy.

Lysine-deficient protein kinase-1 (WNK1) is critical for both embryonic angiogenesis and tumor-induced angiogenesis. However, the downstream effectors of WNK1 during these processes remain ambiguous. In this study, we identified that oxidative stress responsive 1b (osr1b) is upregulated in endothelial cells in both embryonic and tumor-induced angiogenesis in zebrafish, accompanied by downregulation of protein phosphatase 2A (pp2a) subunit ppp2r1bb. In addition, wnk1a and osr1b are upregulated in two liver cancer transgenic fish models: [tert x p53-/-] and [HBx,src,p53-/-,RPIA], while ppp2r1bb is downregulated in [tert x p53-/-]. Furthermore, using HUVEC endothelial cells co-cultured with HepG2 hepatoma cells, we confirmed that WNK1 plays a critical role in the induction of hepatoma cell migration in both endothelial cells and hepatoma cells. Moreover, overexpression of OSR1 can rescue the reduced cell migration caused by shWNK1 knockdown in HUVEC cells, indicating OSR1 is downstream of WNK1 in endothelial cells promoting hepatoma cell migration. Overexpression of PPP2R1A can rescue the increased cell migration caused by WNK1 overexpression in HepG2, indicating that PPP2R1A is a downstream effector in hepatoma. The combinatorial treatment with WNK1 inhibitor (WNK463) and OSR1 inhibitor (Rafoxanide) plus oligo-fucoidan via oral gavage to feed [HBx,src,p53-/-,RPIA] transgenic fish exhibits much more significant anticancer efficacy than Regorafenib for advanced HCC. Importantly, oligo-fucoidan can reduce the cell senescence marker-IL-1ß expression. Furthermore, oligo-fucoidan reduces the increased cell senescence-associated ß-galactosidase activity in tert transgenic fish treated with WNK1-OSR1 inhibitors. Our results reveal the WNK1-OSR1-PPP2R1A axis plays a critical role in both endothelial and hepatoma cells during tumor-induced angiogenesis promoting cancer cell migration. By in vitro and in vivo experiments, we further uncover the molecular mechanisms of WNK1 and its downstream effectors during tumor-induced angiogenesis. Targeting WNK1-OSR1-mediated anti-angiogenesis and anti-cancer activity, the undesired inflammation response caused by inhibiting WNK1-OSR1 can be attenuated by the combination therapy with oligo-fucoidan and may improve the efficacy.

WNK1 collaborates with TGF-ß in endothelial cell junction turnover and angiogenesis.

Angiogenesis is essential for growth of new blood vessels, remodeling existing vessels, and repair of damaged vessels, and these require reorganization of endothelial cell-cell junctions through a partial endothelial-mesenchymal transition. Homozygous disruption of the gene encoding the protein kinase WNK1 results in lethality in mice near embryonic day (E) 12 due to impaired angiogenesis. This angiogenesis defect can be rescued by endothelial-specific expression of an activated form of the WNK1 substrate kinase OSR1. We show that inhibition of WNK1 kinase activity not only prevents sprouting of endothelial cells from aortic slices but also vessel extension in inhibitor-treated embryos ex vivo. Mutations affecting TGF-ß signaling also result in abnormal vascular development beginning by E10 and, ultimately, embryonic lethality. Previously, we demonstrated cross-talk of WNK1 with TGF-ß-regulated SMAD signaling, and OSR1 was identified as a component of the TGF-ß interactome. However, molecular events jointly regulated by TGF-ß and WNK1/OSR1 have not been delineated. Here, we show that inhibition of WNK1 promotes TGF-ß-dependent degradation of the tyrosine kinase receptor AXL, which is involved in TGF-ß-mediated cell migration and angiogenesis. We also show that interaction between OSR1 and occludin, a protein associated with endothelial tight junctions, is an essential step to enable tight junction turnover. Furthermore, we show that these phenomena are WNK1 dependent, and sensitive to TGF-ß. These findings demonstrate intimate connections between WNK1/OSR1 and multiple TGF-ß-sensitive molecules controlling angiogenesis and suggest that WNK1 may modulate many TGF-ß-regulated functions.

WNK Inhibition Increases Surface Liquid pH and Host Defense in Cystic Fibrosis Airway Epithelia.

In cystic fibrosis (CF), reduced HCO3- secretion acidifies the airway surface liquid (ASL), and the acidic pH disrupts host defenses. Thus, understanding the control of ASL pH (pHASL) in CF may help identify novel targets and facilitate therapeutic development. In diverse epithelia, the WNK (with-no-lysine [K]) kinases coordinate HCO3- and Cl- transport, but their functions in airway epithelia are poorly understood. Here, we tested the hypothesis that WNK kinases regulate CF pHASL. In primary cultures of differentiated human airway epithelia, inhibiting WNK kinases acutely increased both CF and non-CF pHASL. This response was HCO3- dependent and involved downstream SPAK/OSR1 (Ste20/SPS1-related proline-alanine-rich protein kinase/oxidative stress responsive 1 kinase). Importantly, WNK inhibition enhanced key host defenses otherwise impaired in CF. Human airway epithelia expressed two WNK isoforms in secretory cells and ionocytes, and knockdown of either WNK1 or WNK2 increased CF pHASL. WNK inhibition decreased Cl- secretion and the response to bumetanide, an NKCC1 (sodium-potassium-chloride cotransporter 1) inhibitor. Surprisingly, bumetanide alone or basolateral Cl- substitution also alkalinized CF pHASL. These data suggest that WNK kinases influence the balance between transepithelial Cl- versus HCO3- secretion. Moreover, reducing basolateral Cl- entry may increase HCO3- secretion and raise pHASL, thereby improving CF host defenses.

WNK1 kinase signaling in metastasis and angiogenesis.

With-no-lysine kinases (WNKs) are a novel family of serine/threonine protein kinases participating in ion homeostasis via the WNK-OSR1/SPAK-NKCC cascade. Recent studies of WNK1 have revealed that its related signaling pathways mediated tumor-induced angiogenesis and carcinogenesis and uncovered novel roles of WNK1 in endothelial cell migration and proliferation, tumor cell proliferation, and metastasis. Herein, we review the functions of WNK1 in cancer metastasis and angiogenesis and propose WNK1 targeting as an anti-cancer strategy.

UBR5 is a novel regulator of WNK1 stability.

The with no lysine (K) 1 (WNK1) protein kinase maintains cellular ion homeostasis in many tissues through actions on ion cotransporters and channels. Increased accumulation of WNK1 protein leads to pseudohypoaldosteronism type II (PHAII), a form of familial hypertension. WNK1 can be degraded via its adaptor-dependent recruitment to the Cullin3-RBX1 E3 ligase complex by the ubiquitin-proteasome system. Disruption of this process also leads to disease. To determine if this is the primary mechanism of WNK1 turnover, we examined WNK1 protein stability and degradation by measuring its rate of decay after blockade of translation. Here, we show that WNK1 protein degradation exhibits atypical kinetics in HeLa cells. Consistent with this apparent complexity, we found that multiple degradative pathways can modulate cellular WNK1 protein amount. WNK1 protein is degraded by not only the proteasome but also the lysosome. Non-lysosomal cysteine proteases calpain and caspases also influence WNK1 degradation, as inhibitors of these proteases modestly increased WNK1 protein expression. Importantly, we discovered that the E3 ubiquitin ligase UBR5 interacts with WNK1 and its deficiency results in increased WNK1 protein. Our results further demonstrate that increased WNK1 in UBR5-depleted cells is attributable to reduced lysosomal degradation of WNK1 protein. Taken together, our findings provide insights into the multiplicity of degradative pathways involved in WNK1 turnover and uncover UBR5 as a previously unknown regulator of WNK1 protein stability that leads to lysosomal degradation of WNK1 protein.

The WNK1 kinase regulates the stability of transcription factors during wound healing of human corneal epithelial cells.

Due to its superficial anatomical localization, the cornea is continuously subjected to injuries. Damages to the corneal epithelium trigger important changes in the composition of the extracellular matrix to which the basal human corneal epithelial cells (hCECs) attach. These changes are perceived by membrane-bound integrins and ultimately lead to re-epithelialization of the injured epithelium through intracellular signalin. Among the many downstream targets of the integrin-activated signaling pathways, WNK1 is the kinase whose activity is the most strongly increased during corneal wound healing. We previously demonstrated that pharmacological inhibition of WNK1 prevents proper closure of wounded human tissue-engineered cornea in vitro. In the present study, we investigated the molecular mechanisms by which WNK1 contributes to corneal wound healing. By exploiting transcription factors microarrays, electrophoretic mobility-shift assay, and gene profiling analyses, we demonstrated that the DNA binding properties and expression of numerous transcription factors (TFs), including the well-known, ubiquitous TFs specific protein 1 (Sp1) and activator protein 1 (AP1), were reduced in hCECs upon WNK1 inhibition by WNK463. This process appears to be mediated at least in part by alteration in both the ubiquitination and glycosylation status of these TFs. These changes in TFs activity and expression impacted the transcription of several genes, including that encoding the α5 integrin subunit, a well-known target of both Sp1 and AP1. Gene profiling revealed that only a moderate number of genes in hCECs had their level of expression significantly altered in response to WNK463 exposition. Interestingly, analysis of the microarray data for these deregulated genes using the ingenuity pathway analysis software predicted that hCECs would stop migrating and proliferating but differentiate more when they are grown in the presence of the WNK1 inhibitor. These results demonstrate that WNK1 plays a critical function by orienting hCECs into the appropriate biological response during the process of corneal wound healing.

Loss of WNK1 Suppressed the Malignant Behaviors of Hepatocellular Carcinoma Cells by Promoting Autophagy and Activating AMPK Pathway.

Background: WNK lysine deficient protein kinase 1 (WNK1) has been shown to be highly expressed in hepatocellular carcinoma (HCC) samples and related to poor prognosis of HCC patients based on bioinformatics analysis. However, the specific function of WNK1 in HCC has not been analyzed. This study is aimed at exploring the function of WNK1 in HCC progression as well as its related molecular mechanism. Methods: After knockdown of WNK1 by small interference RNA, cell counting kit-8, colony formation, western blot, Transwell, and wound healing assays were employed to evaluate the biological behaviors of HCC cells. Immunofluorescent staining was applied to detect the effect of WNK1 on LC3 II. GSK690693 or si-AMPK was applied to block AMPK pathway. The expression of autophagy and AMPK pathway related molecules was examined by western blot assay. Results: WNK1 was highly expressed in HCC cell lines and loss of WNK1 inhibited HCC cell proliferation, cell cycle, migration, and invasion. Additionally, we demonstrated that loss of WNK1 promoted the autophagy and activated AMPK pathway in HCC cells. While, GSK690693 treatment or si-AMPK transfection suppressed the autophagy and promoted HCC cells proliferation. However, WNK1 knockdown counteracted the effect of GSK690693 or si-AMPK in regulating HCC cell proliferation. Finally, we demonstrated that WNK1 regulated the malignant behaviors of HCC cells by modulating autophagy and AMPK pathway. Conclusions: The above results indicated that WNK1 may be a worthwhile target to be considered for therapy of HCC.

Long non-coding RNA (lncRNA) five prime to Xist (FTX) promotes retinoblastoma progression by regulating the microRNA-320a/with-no-lysine kinases 1 (WNK1) axis.

Long non-coding RNA (lncRNA) five prime to Xist (FTX) exerts important functions in human cancer, while its role in retinoblastoma (RB) remains unclear. This study aimed to investigate the role of FTX in RB. The expression levels of FTX were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was evaluated by cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) staining and colony formation assays. Cell migration and invasion were detected by Transwell assay. The relationship among FTX, microRNA-320a (miR-320a) and with-no-lysine kinase 1 (WNK1) was also investigated. In the present study, we found that the expression levels of FTX were notably elevated in RB tissues and cancer cell lines. Overexpression of FTX exacerbated the aggressive phenotypes (cell proliferation, migration and invasion) of RB cells. Downregulation of miR-320a obviously attenuated the inhibitory effects of knockdown of FTX in RB malignant phenotypes, and knockdown of WNK1 also reversed the impacts of miR-320a inhibitor on malignant phenotypes. In vivo experiments further confirmed that knockdown of FTX efficiently prevents tumor growth in vivo. Our results revealed that FTX promoted RB progression by targeting the miR-320a/WNK1 axis (graphical abstract), suggesting that FTX might be a novel therapeutic target for RB.

Chloride sensing by WNK1 regulates NLRP3 inflammasome activation and pyroptosis.

The NLRP3 inflammasome mediates the production of proinflammatory cytokines and initiates inflammatory cell death. Although NLRP3 is essential for innate immunity, aberrant NLRP3 inflammasome activation contributes to a wide variety of inflammatory diseases. Understanding the pathways that control NLRP3 activation will help develop strategies to treat these diseases. Here we identify WNK1 as a negative regulator of the NLRP3 inflammasome. Macrophages deficient in WNK1 protein or kinase activity have increased NLRP3 activation and pyroptosis compared with control macrophages. Mice with conditional knockout of WNK1 in macrophages have increased IL-1ß production in response to NLRP3 stimulation compared with control mice. Mechanistically, WNK1 tempers NLRP3 activation by balancing intracellular Cl- and K+ concentrations during NLRP3 activation. Collectively, this work shows that the WNK1 pathway has a critical function in suppressing NLRP3 activation and suggests that pharmacological inhibition of this pathway to treat hypertension might have negative clinical implications.

Phosphorylated WNK kinase networks in recoded bacteria recapitulate physiological function.

Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria. We used this approach to identify biochemical properties of WNK kinases, a motif for SPAK substrates, and small-molecule kinase inhibitors for phosphorylated SPAK. We show that the kinase inhibitors modulate SPAK substrates in cells, alter cell volume, and reduce migration of glioblastoma cells. Our work establishes a protein-engineering platform technology that demonstrates that synthetically active WNK kinase networks can accurately model cellular systems and can be used more broadly to target networks of phosphorylated proteins for research and discovery.