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Rheumatoid arthritis (RA) is a widespread autoimmune disorder of the joints. Long noncoding RNAs (lncRNAs) have been reported to participate in the pathogenesis of RA by serving as competitive endogenous RNAs. LncRNA small nucleolar RNA host gene 14 (SNHG14) is involved in the development of various diseases. Here, we found that high expression of SNHG14 in RA was closely related to the disease activity. Functional assays indicated that SNHG14 knockdown obviously hampered phorbol myristate acetate-activated THP-1 (pTHP-1) cell proliferation and proinflammatory cytokines production. In mechanism, SNHG14 served as a sponge of microRNA-17-5p (miR-17-5p), and misshapen like kinase 1 (MINK1) was a target of miR-17-5p. SNHG14 depletion-induced inhibitory effects on cell proliferation and inflammatory response were reversed by MINK1 overexpression in macrophages. Moreover, SNHG14 promoted the jun N-terminal kinase (JNK) signaling via the miR-17-5p/MINK1 axis. Overall, SNHG14 boosted the process of RA by MINK1 activating the JNK pathway.

The nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3 (NLRP3) inflammasome is essential in inflammation and inflammatory disorders. Phosphorylation at various sites on NLRP3 differentially regulates inflammasome activation. The Ser725 phosphorylation site on NLRP3 is depicted in multiple inflammasome activation scenarios, but the importance and regulation of this site has not been clarified. The present study revealed that the phosphorylation of Ser725 was an essential step for the priming of the NLRP3 inflammasome in macrophages. We also showed that Ser725 was directly phosphorylated by misshapen (Msn)/NIK-related kinase 1 (MINK1), depending on the direct interaction between MINK1 and the NLRP3 LRR domain. MINK1 deficiency reduced NLRP3 activation and suppressed inflammatory responses in mouse models of acute sepsis and peritonitis. Reactive oxygen species (ROS) upregulated the kinase activity of MINK1 and subsequently promoted inflammasome priming via NLRP3 Ser725 phosphorylation. Eliminating ROS suppressed NLRP3 activation and reduced sepsis and peritonitis symptoms in a MINK1-dependent manner. Altogether, our study reveals a direct regulation of the NLRP3 inflammasome by Msn family kinase MINK1 and suggests that modulation of MINK1 activity is a potential intervention strategy for inflammasome-related diseases.

Upregulation of the developmental Wnt/planar cell polarity pathway is observed in many cancers and is associated with cancer development at early and late stages. We recently showed that PRICKLE1 and VANGL2, two core Wnt/PCP components, are overexpressed in triple negative breast cancer and associated with poor prognosis. PRICKLE1 is a cytoplasmic protein phosphorylated by the poorly described serine/threonine kinase MINK1 which triggers its localization at the plasma membrane, a key step for its function. Knockdown experiments have demonstrated that MINK1 and PRICKLE1 contribute to TNBC cell motility and spreading in vitro and in vivo. However, the identity of MINK1 substrates and the role of MINK1 enzymatic activity in this process have not yet been addressed issues.We carried out a phosphoproteomic strategy and identified novel MINK1 substrates including LL5β. LL5β is a membrane scaffold molecule that anchors microtubules at the cell cortex through its association with the plus-end MT proteins CLASPs to trigger focal adhesion disassembly. LL5β is a prominent member of the MINK1-PRICKLE1 protein complex and is directly phosphorylated by MINK1 that promotes its interaction with CLASP. Using a kinase inhibitor, we demonstrate that the enzymatic activity of MINK1 is involved in the protein complex assembly and localization, and cell migration. Analysis of gene expression data show that the concomitant up-regulation of PRICKLE1 and LL5β mRNA levels encoding MINK1 substrates is associated with a poor metastasis-free survival for TNBC patients. Altogether, our results suggest that MINK1 may represent a potential target in TNBC.

The developmental Wnt/planar cell polarity (PCP) pathway is the most recently described branch of the Wnt signaling pathways and is strongly implicated in cancer development at early and late stages (PMID: 28718442 and PMID: 30952630). Upregulation of the Wnt/PCP components is observed in many cancers and is associated with cancer progression. However, how their molecular functions are regulated remains an open question. Recent data from our laboratory showed that Prickle1 and Vangl2, two core Wnt/PCP components, are overexpressed in Triple Negative Breast Cancers (TNBC) and associated with poor prognosis (PMID: 27184734 and 26754771). Prickle1 is a cytoplasmic protein phosphorylated by the serine/threonine kinase Mink1, which triggers Prickle1 localization at the plasma membrane and regulates its activity (PMID: 22037766). Activation of this axis contributes to breast cancer cell motility and metastatic spreading in vitro and in vivo (PMID: 27184734). To extend our knowledge of the Mink1 substrates, we carried out a phosphoproteomic strategy and identified LL5β. LL5β is a membrane scaffold molecule that anchors microtubules (MTs) at the cell cortex through its association with CLASP, a plus-end MT protein, to trigger focal adhesion disassembly. We found that LL5β is a prominent member of the Prickle1-associated protein complex and harbors a consensus motif for Mink1 phosphorylation. At the molecular level, we demonstrated a two-step phosphorylation cascade carried out by Mink1 that phosphorylates sequentially Prickle1 to mediate its association with LL5β, then LL5β to promote its interaction with CLASP. Finally, analysis of gene expression data suggests that the concomitant up-regulation of Prickle1 and LL5β mRNA levels is associated with a poor metastasis-free survival for TNBC patients. In an effort to counteract the Mink1-Prickle1-LL5β prometastatic pathway, we selected a Mink1 inhibitor that recapitulates all the phenotypes observed following the knockdown of Mink1 expression. This chemical compound is currently under investigation in preclinical assays using xenografted TNBC cell lines and patient-derived xenografts in nude mice. Citation Format: Avais Daulat, Monica Silveira Wagner, Malgorzata Kowalczewska, Pascal Finetti, Rémy Castellano, Stéphane Audebert, François Bertucci, Luc Camoin, Jean-Paul Borg. The serine-threonine kinase Mink1 as a potential therapeutic target in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2290.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. The GR activity is also modulated via protein-protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on the GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studies, and X-ray crystallography to identify key residues within the ligand-binding domain of the GR, T524 and S617, whose phosphorylation results in binding of the representative 14-3-3 protein 14-3-3ζ. A kinase screen identified misshapen-like kinase 1 (MINK1) as responsible for phosphorylating T524 and Rho-associated protein kinase 1 for phosphorylating S617; cell-based approaches confirmed the importance of both GR phosphosites and MINK1 but not Rhoassociated protein kinase 1 alone in inducing GR-14-3-3 binding. Together our results provide molecular-level insight into 14-3-3-mediated regulation of the GR and highlight both MINK1 and the GR-14-3-3 axis as potential targets for future therapeutic intervention.

International audience; Determination of what is the specificity of subunits composing a protein complex is essential when studying gene variants on human pathophysiology. The pore-forming α-subunit KCNQ1, which belongs to the voltage-gated ion channel superfamily, associates to its β-auxiliary subunit KCNE1 to generate the slow cardiac potassium IKs current, whose dysfunction leads to cardiac arrhythmia. Using pharmacology, gene invalidation and single molecule fluorescence assays, we found that KCNE1 fulfils all criteria of a bona fide auxiliary subunit of the TMEM16A chloride channel, which belongs to the anoctamin superfamily. Strikingly, assembly with KCNE1 switches TMEM16A from a calcium-dependent to a voltage-dependent ion channel. Importantly, clinically relevant inherited mutations within the TMEM16A-regulating domain of KCNE1 abolish the TMEM16A modulation, suggesting that the TMEM16A-KCNE1 current may contribute to inherited pathologies. Altogether, these findings challenge the dogma of the specificity of auxiliary subunits regarding protein complexes and questions ion channel classification.

The c-Jun-N-terminal kinase (JNK) signaling pathway regulates nervous system development, axon regeneration, and neuronal degeneration after acute injury or in chronic neurodegenerative disease. Dual leucine zipper kinase (DLK) is required for stress-induced JNK signaling in neurons, yet the factors that initiate DLK/JNK pathway activity remain poorly defined. In the present study, we identify the Ste20 kinases MAP4K4, misshapen-like kinase 1 (MINK1 or MAP4K6) and TNIK Traf2- and Nck-interacting kinase (TNIK or MAP4K7), as upstream regulators of DLK/JNK signaling in neurons. Using a trophic factor withdrawal-based model of neurodegeneration in both male and female embryonic mouse dorsal root ganglion neurons, we show that MAP4K4, MINK1, and TNIK act redundantly to regulate DLK activation and downstream JNK-dependent phosphorylation of c-Jun in response to stress. Targeting MAP4K4, MINK1, and TNIK, but not any of these kinases individually, is sufficient to protect neurons potently from degeneration. Pharmacological inhibition of MAP4Ks blocks stabilization and phosphorylation of DLK within axons and subsequent retrograde translocation of the JNK signaling complex to the nucleus. These results position MAP4Ks as important regulators of the DLK/JNK signaling pathway.SIGNIFICANCE STATEMENTNeuronal degeneration occurs in disparate circumstances: during development to refine neuronal connections, after injury to clear damaged neurons, or pathologically during disease. The dual leucine zipper kinase (DLK)/c-Jun-N-terminal kinase (JNK) pathway represents a conserved regulator of neuronal injury signaling that drives both neurodegeneration and axon regeneration, yet little is known about the factors that initiate DLK activity. Here, we uncover a novel role for a subfamily of MAP4 kinases consisting of MAP4K4, Traf2- and Nck-interacting kinase (TNIK or MAP4K7), and misshapen-like kinase 1 (MINK1 or MAP4K6) in regulating DLK/JNK signaling in neurons. Inhibition of these MAP4Ks blocks stress-induced retrograde JNK signaling and protects from neurodegeneration, suggesting that these kinases may represent attractive therapeutic targets.

Several arylamide-based antiproliferative agents are known and some of them are currently FDA-approved anticancer drugs. Provoked by the need to fill the existing room with new drugs, 31 compounds constituting a series of flavone-based arylamide derivatives were synthesized and biologically evaluated. Towards extensive evaluation, sixty diverse cancer cell lines representing nine cancer diseases of various origins have been used for evaluation of their efficacy, spectrum and potency. Two compounds 2aw and 2ax emerged as effective, broad-spectrum and potent anticancer agents that outperformed masitinibt and imatininb, which are FDA-approved anticancer drugs. Kinases profiling as possible targets for the potent compound 2aw showed that it might be a hit compound offering a starting point to develop inhibitors of STE20/GCK-IV kinase family members including HGK, TNIK and MINK1 kinases. Mechanistic study showed that compounds 2aw triggers cell cycle arrest in HT29 colon cancer cells. In conclusion, this work presents compound 2aw as a new broad-spectrum anticancer agent for further development of promising treatment of diverse cancers.

The TRAF2 and NCK interacting kinase (TNIK) has been proposed to play a role in cytoskeletal organization and synaptic plasticity and has been linked, among others, to neurological disorders. However, target validation efforts for TNIK have been hampered by the limited kinase selectivity of small molecule probes and possible functional compensation in mouse models. Both issues are at least in part due to its close homology to the kinases MINK1 (or MAP4K6) and MAP4K4 (or HGK). As part of our interest in validating TNIK as a therapeutic target for neurological diseases, we set up a panel of biochemical and cellular assays, which are described herein. We then examined the activity of known amino-pyridine-based TNIK inhibitors (1, 3) and prepared structurally very close analogs that lack the ability to inhibit the target. We also developed a structurally orthogonal, naphthyridine-based TNIK inhibitor (9) and an inactive control molecule of the same chemical series. These validated small-molecule probes will enable dissection of the function of TNIK family in the context of human disease biology.

Fu et al. demonstrate that reactive oxygen species (ROS)–sensing molecule misshapen/NIK-related kinase 1 (MINK1) can specifically suppress Th 17 cell differentiation through direct phosphorylation of SMAD2. This study provides the mechanism of how ROS limit inflammatory response and unveils the potential health risk of antioxidant supplementation., T helper type 17 cells (Th17 cells) are major contributors to many autoimmune diseases. In this study, we demonstrate that the germinal center kinase family member MINK1 (misshapen/NIK-related kinase 1) negatively regulates Th17 cell differentiation. The suppressive effect of MINK1 on induction of Th17 cells is mediated by the inhibition of SMAD2 activation through direct phosphorylation of SMAD2 at the T324 residue. The importance of MINK1 to Th17 cell differentiation was strengthened in the animal model of experimental autoimmune encephalomyelitis (EAE). Moreover, we show that the reactive oxygen species (ROS) scavenger N-acetyl cysteine boosts Th17 cell differentiation in a MINK1-dependent manner and exacerbates the severity of EAE. Thus, we have not only established MINK1 as a critical regulator of Th17 cell differentiation, but also clarified that accumulation of ROS may limit the generation of Th17 cells. The contribution of MINK1 to ROS-regulated Th17 cell differentiation may suggest an important mechanism for the development of autoimmune diseases influenced by antioxidant dietary supplements.

SummaryAdenomatous polyposis coli(APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the pro-proliferative Wnt signaling pathway by promoting the degradation of β-catenin, but the extent to which APC exerts Wnt/β-catenin-independent tumor suppressive activity is unclear. To identify interaction partners and β-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed TMT mass spectrometry. Affinity enrichment-mass spectrometry revealed over 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of β-catenin. By combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an alternative APC-containing destruction complex and provide evidence for the potential contribution of MINK1 toAPCmutant phenotypes. Collectively, our results highlight the extent and importance of Wnt-independent APC functions in epithelial biology and disease.

Adenomatous polyposis coli (APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the pro-proliferative Wnt signaling pathway by promoting the degradation of &#946-catenin, but the extent to which APC exerts Wnt/&#946-catenin-independent tumor suppressive activity is unclear. To identify interaction partners and &#946-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed TMT mass spectrometry. Affinity enrichment-mass spectrometry revealed over 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of &#946-catenin. By combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an alternative APC-containing destruction complex and provide evidence for the potential contribution of MINK1 to APC mutant phenotypes. Collectively, our results highlight the extent and importance of Wntindependent APC functions in epithelial biology and disease.

International audience; Components of the evolutionarily conserved developmental planar cell polarity (PCP) pathway were recently described to play a prominent role in cancer cell dissemination. However, the molecular mechanisms by which PCP molecules drive the spread of cancer cells remain largely unknown. PRICKLE1 encodes a PCP protein bound to the promigratory serine/threonine kinase MINK1. We identify RICTOR, a member of the mTORC2 complex, as a PRICKLE1-binding partner and show that the integrity of the PRICKLE1-MINK1-RICTOR complex is required for activation of AKT, regulation of focal adhesions, and cancer cell migration. Disruption of the PRICKLE1-RICTOR interaction results in a strong impairment of breast cancer cell dissemination in xenograft assays. Finally, we show that upregulation of PRICKLE1 in basal breast cancers, a subtype characterized by high metastatic potential, is associated with poor metastasis-free survival.

In this issue of JEM, Fu et al. identified the kinase Mink1 as a novel negative regulator of Th17 cell generation. Mink1, activated by reactive oxygen species (ROS), prevents TGF-β activation of Smad2, therefore limiting Th17 cell differentiation., In this issue of JEM, Fu et al. (https://doi.org/10.1084/jem.20161120) identified the kinase Mink1 as a novel negative regulator of Th17 cell generation. Mink1, activated by reactive oxygen species (ROS), prevents TGF-β activation of Smad2, therefore limiting Th17 cell differentiation.

The major histocompatability complex (MHC) is a central receptor in the adaptive immune response and is the underlying target of several effective therapies for cancer. Druggable kinases may provide the opportunity to modulate the immune response toward MHC. However, the regulation of MHC-I by kinases is largely unstudied, even though many patients with cancer are receiving therapeutic kinase inhibitors. The entire human kinome was screened using a pooled shRNA interference-based approach in a human mesothelioma cell line to uncover kinase regulators of MHC-I. Negative and positive regulators of cell surface HLA levels were discovered. A subset of highly scoring positive and negative kinase hits were subsequently validated by additional RNAi, and pharmacologic inhibitors when available. MAP2K1 (MEK), EGFR, and RET were validated as negative regulators of HLA expression in multiple cancer types. We mapped the pathways responsible for increased HLA upon kinase inhibition. Interestingly, inhibition of the MAP Kinase pathway broadly influenced expression of other components of the antigen presentation machinery. Moreover, DDR2 and MINK1 were shown to positively regulate HLA-A*02:01. This had therapeutic relevance, as shown with a therapeutic TCR mimic antibody to a MHC/peptide complex. Druggable kinases may thus serve as immediately applicable targets for modulating immunotherapy for many diseases. Citation Format: Elliott J. Brea, Claire Oh, Eusebio Manchado, Ron Gejman, George Mo, Patrizia Mondello, Ralph Garippa, Neal Rosen, David A. Scheinberg. The regulation by kinases of the expression of human major histocompatibility class I molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4899.