正常的免疫系統運作可偵測及消滅體內之癌細胞，或是清除有害之外來物質及病原體。然而，過度活躍的免疫反應會異常地攻擊自身組織與器官而造成自體免疫疾病。此外，免疫力低下造成無法清除體內癌細胞而導致癌症生成；慢性或反覆性發炎卻也會促進癌症形成。美國國家衛生研究院指出全美至少2350萬人罹患自體免疫疾病，遠高於其他慢性疾病。我國自體免疫疾病為重大傷病中位列第三名的疾病，患者飽受病痛之苦，必須終生治療與用藥，承受藥物引發的副作用。因長年缺乏有效、經濟的醫療對策，故治療自體免疫疾病是醫界長久以來的挑戰。

我們研究團隊於2011年發表的Nature Immunology論文中指出，MAP4K3 (又名GLK)蛋白激酶藉由磷酸化PKCθ可活化NF-κB訊息傳遞及控制自體免疫疾病。蛋白激酶GLK為控制T淋巴細胞活化與自體免疫反應的重要分子。GLK基因剔除小鼠呈現減低之T細胞免疫反應，並增長40%壽命。結合基礎及臨床醫學研究，我們發現GLK過量表現於八種自體免疫疾病，包含: 全身性紅斑狼瘡(systemic lupus erythematosus, SLE)、類風溼性關節炎(rheumatoid arthritis)、成人史迪爾氏病(adult-onset Still's disease)。GLK過量表現於T淋巴細胞中與自體免疫疾病嚴重性高度相關。然而，GLK過量表現於SLE病患中的成因，多年來仍懸而未解。我們以深度次世代基因定序分析SLE病患之臨床檢體，發現高達四成的SLE病患帶有GLK基因之變異，這些基因變異造成GLK之mRNA或是蛋白質過度地穩定而量增，導致自體免疫疾病。我們以Machine Learning (AI)運算結合三種統計法，分析臨床檢體轉錄體學，發現UHRF1P過量表現於T淋巴細胞中，乃是自體免疫疾病全身紅斑性狼瘡(SLE)致病關鍵。我們證明「假基因UHRF1P」實為可對抗泛素酶且保護GLK的蛋白分子。研究也揭示UHRF1P為SLE的新穎治療標靶。

Th17細胞所產生的IL-17A發炎性細胞激素為自體免疫疾病的致病因子。我們研究揭示GLK過量表現所誘發之AhR-RORγt蛋白質複合體，正是IL-17A誘發多種自體免疫疾病的致病關鍵。研究證實GLK訊息傳遞路徑，及其所誘發之AhR-RORγt蛋白質複合體，為自體免疫疾病深具潛力的治療標靶。我們接續研究發現，除了誘發IL-17A發炎性細胞激素之外，GLK會抑制調節性T細胞(Treg cell)的分化與活性。因此，調降GLK可以抑制IL-17A產生並且同時提高調節性T細胞，達到加乘性抑制自體免疫反應的功效，優於單獨使用IL-17A抑制劑之效用。

除了自體免疫疾病，我們分析新冠肺炎病患檢體之單細胞RNA定序結果，意外發現新冠病毒的棘蛋白(spike protein)會誘發蛋白激酶GLK高量表現。GLK會磷酸化ACE2蛋白(新冠病毒受體)，使ACE2躲過泛素酶UBR4誘發的泛素化降解機制，造成細胞表面的ACE2蛋白穩定增加。同時，GLK過量表現亦會誘發裝載著ACE2蛋白之胞外小體(exosomes)釋出，傳遞予其他原本ACE2表現量不高的細胞，這些接收了ACE2蛋白的細胞更容易被新冠病毒所感染。研究成果證實蛋白激酶GLK過量表現為COVID-19致病機轉之關鍵因子，同時說明新冠病毒快速引發臨床症狀且容易傳播的原因。

此外，我們在癌症相關的研究證實，蛋白激酶GLK過量表現會形成「GLK-IQGAP1蛋白質複合體」，GLK會磷酸化IQGAP1細胞骨架調控蛋白，造成肺癌細胞移行。此連鎖反應是造成肺癌細胞遠端轉移的致病關鍵。分析臨床檢體發現，肺癌腫瘤組織中的GLK-IQGAP1蛋白質複合體越高，則病患存活率越低，而GLK誘發IQGAP1磷酸化越高，則病患存活率下降更是明顯。因此，GLK-IQGAP1蛋白質複合體及GLK誘發之IQGAP1磷酸化程度，將能作為癌症復發轉移的預測指標，也極具潛力作為治療標靶，有助於開發新型癌症精準醫療策略。

歷經多年努力，我們研發出GLK小分子抑制劑(verteporfin，IC50= 1.15 nM)，此抑制劑在動物模式中大幅減緩自體免疫疾病、發炎性疾病、癌症轉移、SARS-CoV-2病毒感染，而且GLK小分子抑制劑可抑制SLE病患T淋巴細胞所分泌之IL-17A產生。特別的是，抑制GLK會延長小鼠之壽命。此發明將可望應用於治療自體免疫疾病、發炎性疾病、老化，以及癌症。

（103年度TBF學術講座、國家衛生研究院免疫醫學研究中心　譚澤華特聘研究員暨主任）

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MAP4K3 (GLK) is a novel therapeutic target for autoimmune disease, inflammatory disease, cancer, and aging

Tuning inflammation is the key to orchestrating host immune responses to threats from pathogen infection or host cell damage. Hyperactivation/dysregulation of immune responses induces inflammatory diseases or autoimmune diseases. National Institutes of Health (NIH) of the USA indicates that between 23.5 and 50.0 million people in America live with one or more autoimmune diseases; the prevalence of autoimmune disease is much higher than that of other chronic diseases. In Taiwan, autoimmune disease is the third among the top 10 catastrophic illnesses. Autoimmune diseases patients such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and ankylosing spondylitis need to receive treatments throughout their life; therefore, estimated at more than $100 billion U.S. dollars per year for health care costs in the USA.

For the past two decades, our research team has been studying the signaling pathways and immune functions of T cells and other immune cells. Most importantly, our translational research using biochemical approaches, transgenic/knockout mice, and clinical samples has revealed novel biomarkers and therapeutic targets for several human diseases including SLE, RA, COVID-19, and cancer.

Our 2011 Nature Immunology paper reported that MAP4K3 (also named GLK) positively regulates T-cell activation and IKK/NF-κB signaling through phosphorylating and activating PKC-θ in T-cell receptor (TCR) signaling. TCR signaling activates GLK by inducing its direct interaction with the proximal signaling molecule SLP-76. We generated and characterized GLK-deficient mice and found that the activation of PKCθ/IKK and the differentiation of Th1/Th17 are reduced in GLK-deficient T cells. GLK-deficient mice also show impaired production of antigen-specific antibodies and cytokines after immunization. Interestingly, GLK-deficient mice display an increased 40% life-span, indicating that inhibition of GLK prevents aging. Using autoimmune disease mouse models, we found that GLK-deficient mice are resistant to the induction of the IL-17A-mediated experimental autoimmunity and display lower Th17 responses. Using clinical samples from autoimmune diseases patients, we further demonstrate that the frequencies of GLK-positive peripheral T cells are highly correlated with disease severity of SLE, adult-onset Still’s disease (AOSD), and RA. These findings indicate that GLK-positive T cell is a biomarker and therapeutic target for multiple IL-17A-mediated autoimmune diseases.

In the past decade, it was unclear how GLK is upregulated in SLE patients. To understand the underlying mechanism of GLK overexpression in SLE patients, our research team performed next-generation sequencing (deep sequencing) using genomic DNAs from 181 SLE patients and 250 controls from two independent cohorts. Remarkably, 39.2% SLE patients harbor GLK germline or somatic variants. The SLE patients with higher frequencies of GLK variants show increased disease severity. Thus, GLK gene variant is a risk of autoimmune diseases.

To identify novel intracellular molecules from the T cells of SLE patients as diagnostic biomarkers and therapeutic targets for SLE, peripheral blood T cells were freshly isolated from whole bloods of SLE patients and healthy controls. The total RNAs of purified T cells were subjected to transcriptomics. Analysis of the T-cell transcriptomics data of two independent human SLE patient cohorts by three AI machine learning models revealed a novel gene, UHRF1P, as a new SLE biomarker. T-cell-specific UHRF1P transgenic mice manifest the induction of IL-17A and autoimmune inflammation. Mechanistically, UHRF1P prevents the E3 ligase UHRF1-induced Lys48-linked ubiquitination and degradation of GLK. Consistently, IL-17A induction and autoimmune phenotypes of UHRF1P transgenic mice are obliterated by GLK knockout. Furthermore, UHRF1P protein is indeed overexpressed in the peripheral blood T cells of human SLE patients. Collectively, UHRF1P overexpression in T cells antagonizes the E3 ligase function of parental UHRF1 and induces IL-17A-mediated autoimmune responses.

Overproduction of the proinflammatory cytokine IL-17A is a key causal factor of autoimmune diseases. Thus, IL-17A is an emerging therapeutic target for autoimmune and inflammatory diseases. Our research team has studied the mechanism of IL-17A overproduction using several knockout mice and biochemical approaches. We found that overexpression of the kinase GLK induces the AhR-RORγt complex and subsequent IL-17A overproduction, leading to autoimmune diseases. Most importantly, the AhR-RORγt complex specifically induces IL-17A overproduction but not other cytokines. Conversely, inhibition of the AhR-RORγt complex will not affect the production of other cytokines and maintain normal functions without any side effects. Taken together, the GLK-induced AhR-RORγt heterodimer is a therapeutic target for SLE.

In contrast to Th17-mediated inflammatory responses, Treg cells (CD4⁺Foxp3⁺ T cells) suppress excessive innate or adaptive immune responses by modulating the activity or proliferation of multiple immune cells. Downregulation or mutation of the Treg master transcription factor Foxp3 results in impaired Treg development and uncontrolled immune responses, leading to the induction of autoimmune diseases (such as SLE and RA). We also found that GLK-deficient Treg cells show enhanced suppressive function compared to that of wild-type Treg cells. Moreover, in vitro differentiation of GLK-deficient T cells is enhanced compared to that of wild-type Treg cells. Conversely, Treg suppressive function and Treg differentiation are reduced by T-cell-specific GLK transgene (Lck-GLK Tg). We demonstrated that GLK-IKKβ signaling induces FoxO1 phosphorylation and nuclear export, inhibiting Foxp3 transcription and Treg differentiation.

Besides autoimmune diseases, our research team has discovered GLK as a key to COVID-19 pathogenesis. The major entry receptor of SARS-CoV-2 on epithelial cells is angiotensin-converting enzyme 2 (ACE2). SARS-CoV-2 infection of airway epithelium is quite efficient, while ACE2 levels are generally low in airway epithelial cells of healthy individuals, had puzzled scientists. We analyzed single-cell RNA sequencing (scRNA-seq) data from COVID-19 patients and surprisingly found that the induction of GLK levels in epithelial cells is correlated with COVID-19 severity and with increased extracellular exosome pathways. Mechanistically, the spike protein of SARS-CoV-2 induces GLK mRNA levels in the infected epithelial cells. GLK phosphorylates and stabilizes ACE2 by blocking UBR4-mediated proteasomal degradation of ACE2. This is the first report that reveals the novel phosphorylation residues, ubiquitination residues, kinase, and E3 ubiquitin ligase of the ACE2 protein in COVID-19 patients. In addition, GLK overexpression in epithelial cells induces the release of ACE2-containing exosomes, resulting in increased ACE2 protein levels and enhanced SARS-CoV-2 infection of otherwise less susceptible cells. The GLK-phosphorylated ACE2 and ACE2-containing exosomes are also increased in the sera of COVID-19 patients. This mechanism provides an explanation for why ACE2 levels are very low in airway epithelial cells of normal individuals, whereas SARS-CoV-2 infection of airway epithelium is quite efficient.

In addition to inflammatory diseases, our team reported that GLK overexpression in lung epithelial cells is correlated with human lung cancer recurrence and poor prognosis. The scaffold protein IQGAP1 is identified as a GLK-interacting protein. Super-resolution live-cell imaging showed that GLK and IQGAP1 co-localize at the leading edge of filopodia in migrating cells. GLK directly phosphorylates IQGAP1, enhancing Cdc42 activation and subsequent cell migration. GLK-induced cell migration and lung cancer metastasis in mice are abolished by IQGAP1 knockout. Consistently, human NSCLC patient tissues display increased phospho-IQGAP1 levels, which correlate with poor survival of NSCLC patients. Collectively, GLK promotes lung cancer metastasis by binding to, phosphorylating, and activating IQGAP1.

To develop GLK inhibitors, we screened one hundred thousand (101,200) compounds to identify GLK inhibitors using both cell-based luciferase reporter assays and in vitro kinase assays. We have also identified two small-molecule GLK inhibitors, including verteporfin (IC50= 1.15 nM) and alexidine dihydrochloride. These two GLK inhibitors were granted US patent in 2022. The GLK inhibitor verteporfin suppresses the AhR-RORγt complex and IL-17A production in the T cells of human SLE patents. The GLK inhibitor verteporfin also attenuates disease severity of autoimmune mouse models. Thus, GLK inhibitors are potential therapeutic drugs for autoimmune diseases such as SLE and RA. Remarkably, GLK overexpression in the T cell contributes to autoimmune disease by both inducing IL-17A production and inhibiting Treg. Conversely, GLK inhibitor mitigates autoimmune disease by both blocking IL-17A production and inducing Treg. It is double whammy; thus, GLK inhibitor would be more efficient than IL-17A blockage alone. Furthermore, GLK inhibitor also suppresses lung cancer metastasis and SARS-CoV-2 pseudovirus infection in mice. Most interestingly, GLK-deficient mice show prolonged lifespan by 40%. Collectively, GLK is a novel therapeutic target for aging, cancer recurrence, inflammatory diseases, and autoimmune diseases.



(2014 TBF Chair in Biotechnology, Immunology Research Center, National Health Research Institutes, Distinguished Investigator, Professor Tse-Hua Tan)