PIKfyve抑制剂靶向治疗STING通路缺陷黑色素瘤的机制研究

doi:10.3969/j.issn.1674-8115.2025.09.005

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (9): 1126-1137.doi: 10.3969/j.issn.1674-8115.2025.09.005

PIKfyve抑制剂靶向治疗STING通路缺陷黑色素瘤的机制研究

杨小雨, 黄蕊, 吴以加, 张哲, 房燕(), 沈键锋()

上海交通大学医学院附属第九人民医院眼科，上海市眼眶病眼肿瘤重点实验室，上海 200011

收稿日期:2025-03-30 接受日期:2025-04-30 出版日期:2025-09-28 发布日期:2025-09-30
通讯作者: 沈键锋，教授，博士；电子信箱：jfshen@shsmu.edu.cn。
房　燕，助理研究员，博士；电子信箱：yanyan2021fang@sjtu.edu.cn。
基金资助:
国家重点研发计划(2021YFC2701103);上海交通大学医学院“双百人”项目(20191817)

Mechanistic study of targeting melanoma with STING pathway deficiencies via PIKfyve inhibitor

YANG Xiaoyu, HUANG Rui, WU Yijia, ZHANG Zhe, FANG Yan(), SHEN Jianfeng()

Department of Ophthalmology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai 200011, China

Received:2025-03-30 Accepted:2025-04-30 Online:2025-09-28 Published:2025-09-30
Contact: SHEN Jianfeng, E-mail: jfshen@shsmu.edu.cn.
FANG Yan, E-mail: yanyan2021fang@sjtu.edu.cn.
Supported by:
National Key R&D Program of China(2021YFC2701103);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20191817)

摘要/Abstract

摘要：

目的·探究干扰素基因刺激因子（stimulator of interferon genes，STING）信号通路缺陷型黑色素瘤中，联用磷脂酰肌醇激酶FYVE型锌指结构域蛋白（phosphoinositide 3-kinase，FYVE-type zinc finger containing，PIKfyve）抑制剂YM201636与STING激动剂diABZI的抗肿瘤效果及潜在机制。方法·分别从CCLE（Cancer Cell Line Encyclopedia）数据库和UniProt数据库获取人类肿瘤细胞系中STING的mRNA表达水平和蛋白表达水平数据，并进一步根据表达水平的中位数鉴定出STING mRNA高表达、蛋白低表达的黑色素瘤细胞。通过实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）和Western blotting验证人类黑色素瘤细胞中STING的表达水平。进一步使用Western blotting筛选出STING蛋白表达较低的鼠源黑色素瘤细胞YUMM1.7，并检验YM201636能否恢复YUMM1.7的STING蛋白水平。联合应用YM201636和STING激动剂diABZI，采用CCK-8法分析双药协同的肿瘤细胞杀伤效果，Western blotting检测STING下游信号分子TANK结合激酶1（TANK-binding kinase 1，TBK1）和干扰素调节因子3（interferon regulatory factor 3，IRF3）的磷酸化水平，qRT-PCR评估Ⅰ型干扰素的表达。建立小鼠黑色素瘤模型，进行YM201636和diABZI的单药或联合治疗，测量肿瘤体积并评估治疗效果。通过RNA测序和免疫荧光染色分析小鼠肿瘤组织肿瘤微环境中免疫细胞的变化。结果·数据库分析、qRT-PCR和Western blotting证实人类黑色素瘤中部分细胞系存在STING mRNA高表达、蛋白低表达的现象。YM201636显著提高了STING蛋白在YUMM1.7细胞中的表达（P<0.001），并且在联合diABZI使用时，显著增强TBK1和IRF3蛋白的磷酸化（P<0.05），提示STING通路被有效激活。同时，YM201636和diABZI联合处理显著促进Ⅰ型干扰素的产生（P<0.001），并且显著增强肿瘤细胞的生长抑制作用。在小鼠体内实验中，与单独治疗相比，YM201636和diABZI的联合治疗能显著抑制黑色素瘤的生长。肿瘤微环境的免疫特征分析显示，联合治疗组中CD4⁺T细胞和CD8⁺T细胞的浸润显著增多（P<0.05）。结论·联用PIKfyve抑制剂YM201636能在STING缺陷型黑色素瘤中恢复STING蛋白的表达，增强STING激动剂diABZI的抗肿瘤效果。

关键词: 黑色素瘤, STING激动剂, PIKfyve抑制剂

Abstract:

Objective ·To explore the antitumor effects and potential mechanisms of combining phosphoinositide 3-kinase, FYVE-type zinc finger containing (PIKfyve) inhibitor YM201636 with the stimulator of interferon genes (STING) agonist diABZI in STINGpathway-deficient melanoma. Methods ·The mRNA and protein expression levels of STING in human cancer cell lines were obtained from the Cancer Cell Line Encyclopedia (CCLE) and UniProt databases. Based on median expression values, melanoma cell lines with high STING mRNA but low protein expression were identified. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to validate STING mRNA and protein expression in human melanoma cells. The murine melanoma cell line YUMM1.7, characterized by low STING protein expression, was selected through Western blotting. The ability of YM201636 to restore STING protein expression in YUMM1.7 cells was evaluated. STING agonist diABZI was then applied in combination with YM201636 to analyze the synergistic tumor cell-killing effect through CCK-8 assay. Western blotting was used to detect the phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), and qRT-PCR was used to evaluate type Ⅰ interferon expression. A mouse melanoma model was established and treated with YM201636, diABZI, or their combination. Tumor volume was measured, and treatment efficacy was assessed. RNA sequencing and immunofluorescence staining were performed to analyze immune cell infiltration in the tumor microenvironment. Results ·Database analyses, qRT-PCR, and Western blotting confirmed that some human melanoma cell lines exhibited high STING mRNA expression but low STING protein levels. YM201636 significantly increased STING protein expression in YUMM1.7 cells (P<0.001). Combined treatment with YM201636 and diABZI significantly enhanced phosphorylation of TBK1 and IRF3 (P<0.05), indicating effective activation of the STING signaling pathway. This combination also promoted the expression of type Ⅰ interferons (P<0.001) and enhanced tumor cell killing in vitro. In vivo, the combination therapy markedly suppressed melanoma growth compared to monotherapy. Immune profiling of the tumor microenvironment revealed significantly increased infiltration of CD4⁺ T cells and CD8⁺ T cells in the combination treatment group (P<0.05). Conclusion ·The PIKfyve inhibitor YM201636 could restore STING protein expression in STING-deficient melanoma and enhance the antitumor efficacy of the STING agonist diABZI, offering a promising therapeutic strategy for tumors with defective STING signaling.

Key words: melanoma, STING agonist, PIKfyve inhibitor

中图分类号:

R739.7

杨小雨, 黄蕊, 吴以加, 张哲, 房燕, 沈键锋. PIKfyve抑制剂靶向治疗STING通路缺陷黑色素瘤的机制研究[J]. 上海交通大学学报（医学版）, 2025, 45(9): 1126-1137.

YANG Xiaoyu, HUANG Rui, WU Yijia, ZHANG Zhe, FANG Yan, SHEN Jianfeng. Mechanistic study of targeting melanoma with STING pathway deficiencies via PIKfyve inhibitor[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(9): 1126-1137.

图/表 7

表1 用于qRT-PCR检测的引物序列

Tab 1 Primer sequences for qRT-PCR

Gene	Species	Forward (5'→3')	Reverse (5'→3')
Ifna	Mouse	TGCCCAGCAGATCAAGAAGG	TCAGGGGAAATTCCTGCACC
Ifnb1	Mouse	CGTGGGAGATGTCCTCAACT	CCTGAAGATCTCTGCTCGGAC
Cxcl9	Mouse	TCGGACTTCACTCCAACACA	CCTTATCACTAGGGTTCCTCGAA
Cxcl10	Mouse	CCACGTGTTGAGATCATTGCC	TCACTCCAGTTAAGGAGCCC
β-actin	Mouse	ATCTTCCGCCTTAATACT	GCCTTCATACATCAAGTT
STING	Human	GCTGCTGTCTCCCCATTCAG	GCTGGATGCAGGTTGGAGTA
β-actin	Human	CTCCATCCTGGCCTCGCTGT	GCTGTCACCTTCACCGTTCC

图1 人类黑色素瘤中 STING mRNA和蛋白的表达Note： A. Spearman correlation between the expression of STING mRNA and STING protein in human cancer cell lines. B. Spearman correlation between the expression of STING mRNA and STING protein in human melanoma cell lines. C. qRT-PCR analysis of relative expression of STING in the OMM1, OMM2.3, MEL270, and MEL290 cell lines. D. Western blotting analysis of STING protein expression in the OMM1, OMM2.3, MEL270, and MEL290 cell lines. E. The ratio of total STING to β-actin in each cell line. ①P=0.010, ②P=0.007, ③P<0.001.

Fig 1 Expression of STING mRNA and protein in human melanoma

图2 YM201636和diABZI协同增强STING信号通路Note：A/B. Western blotting analysis of STING protein expression in B16F10 and YUMM1.7 cell lines. C/D. Western blotting analysis of STING protein expression in YUMM1.7 cell line following treatment with various concentrations of YM201636 for 24 h. E/F.Western blotting was performed to assess STING protein levels in YUMM1.7 cells treated with PBS (Ctrl), YM201636 (0.5 µmol/L, for 24 h), Bafilomycin A1 (BafA1, 30 nmol/L, for 4 h), or the combination of YM201636 and BafA1 (YM201636 pretreated for 20 h, followed by 4 h co-treatment with BafA1). G‒I. Western blotting analysis of the phosphorylation levels of IRF3 and TBK1 in YUMM1.7 cells treated with PBS (Ctrl), YM201636 (0.5 µmol/L), diABZI (1 µmol/L), or a combination of YM201636 (0.5 µmol/L) and diABZI (1 µmol/L) for 48 h. The levels of phosphorylated IRF3 (p-IRF3) and phosphorylated TBK1 (p-TBK1), as well as total IRF3 and TBK1, were assessed. J‒M. qRT-PCR was used for the detection of the expression of Ifna, Ifnb1, Cxcl9, and Cxcl10 in YUMM1.7 cell line treated with PBS, YM201636 (0.5 µmol/L), diABZI (1 µmol/L), or a combination of YM201636 (0.5 µmol/L) and diABZI (1 µmol/L) for 48 h. ①P<0.001, ②P=0.002, ③P=0.041, ④P=0.043, ⑤P=0.017, ⑥P=0.004, ⑦P=0.012, ⑧P=0.007, ⑨P=0.006, ⑩P=0.001.

Fig 2 Synergistic activation of the STING signaling pathway by YM201636 and diABZI

图3 YM201636和diABZI双药协同的肿瘤细胞杀伤效果Note：Evaluation of cell proliferation by CCK-8 assay in YUMM1.7 cells treated with diABZI alone (A), YM201636 alone (B), or the combination of YM201636 and diABZI (C) at various concentrations for 48 h. ①P=0.016, ②P=0.005, ③P=0.001.

Fig 3 Synergistic antitumor effect of YM201636 and diABZI in tumor cell killing

图4 体内模型评估diABZI与YM201636联合治疗的抗肿瘤效果Note： A. Diagram illustrating the treatment protocol with diABZI and YM201636. C57BL/6 mice received subcutaneous injections of 2.0×105 YUMM1.7 cells on day 0. Treatments included diABZI (1.0 mg/kg, intravenously), YM201636 (3.0 mg/kg, orally), the combination of both, or PBS. B. Tumor images. C‒E. Corresponding tumor volume changes (C), tumor weight (D), and body weight (E) of YUMM1.7 tumor-bearing mice treated with PBS, YM201636, diABZI or the combination of YM201636 and diABZI. F. Illustrative photos and quantitative data from Ki67 immunohistochemistry staining of tumor samples across various groups. ①P=0.048, ②P<0.001, ③P=0.002, ④P=0.004.

Fig 4 Antitumor efficacy of combined diABZI and YM201636 treatment in mice with established murine melanoma

图5 diABZI与YM201636的联合使用显著激活免疫基因Note： A‒C. Volcano plot of significant genes between the YM201636+diABZI groupand the Ctrl group (A), between the YM201636+diABZI group andthe YM201636 group (B), and between the YM201636+diABZI group and the diABZI group (C). Differentially expressed genes were defined as |log2 (fold change) | ≥1 and FDR<0.05. D‒F. GO enrichment analysis of differentially expressed genes between the treatment groups.

Fig 5 Combination of diABZI and YM201636 led to a marked upregulation of immune-associated gene expression

图6 diABZI与YM201636的联合使用重塑肿瘤免疫微环境Note： A‒H. CIBERSORT analysis of of immune cells in tumor tissues across various treatment groups (PBS, YM201636, diABZI, or the combination of YM201636 and diABZI). A. Dendritic cells (DCs). B. CD4+ T cells. C. CD8+ T cells. D. NK cells. E. Neutrophils. F. M0 macrophages. G. M1 macrophages. H. M2 macrophages. I/J. Representative images and quantitative analysis of CD4+ T cells (I) and CD8+ T cells (J) by immunohistochemical staining in tumor tissues across various groups.①P=0.009, ②P=0.005, ③P=0.002, ④P=0.008, ⑤P<0.001, ⑥P=0.044, ⑦P=0.015, ⑧P=0.032, ⑨P=0.047, ⑩P=0.003, ⑪P=0.019, ⑫P=0.014, ⑬P=0.011.

Fig 6 Combination of diABZI and YM201636 reprogrammed the tumor immune microenvironment

参考文献 27

[1]	GULEN M F, KOCH U, HAAG S M, et al. Signalling strength determines proapoptotic functions of STING[J]. Nat Commun, 2017, 8(1): 427.
[2]	WANG J, LI S X, WANG M, et al. STING licensing of type Ⅰ dendritic cells potentiates antitumor immunity[J]. Sci Immunol, 2024, 9(92): eadj3945.
[3]	WANG H, HU S Q, CHEN X, et al. cGAS is essential for the antitumor effect of immune checkpoint blockade[J]. Proc Natl Acad Sci USA, 2017, 114(7): 1637-1642.
[4]	LI L Y, YIN Q, KUSS P, et al. Hydrolysis of 2'3'-cGAMP by ENPP1 and design of nonhydrolyzable analogs[J]. Nat Chem Biol, 2014, 10(12): 1043-1048.
[5]	XIA T L, KONNO H, AHN J, et al. Deregulation of STING signaling in colorectal carcinoma constrains DNA damage responses and correlates with tumorigenesis[J]. Cell Rep, 2016, 14(2): 282-297.
[6]	FALAHAT R, BERGLUND A, PEREZ-VILLARROEL P, et al. Epigenetic state determines the in vivo efficacy of STING agonist therapy[J]. Nat Commun, 2023, 14(1): 1573.
[7]	KONNO H, YAMAUCHI S, BERGLUND A, et al. Suppression of STING signaling through epigenetic silencing and missense mutation impedes DNA damage mediated cytokine production[J]. Oncogene, 2018, 37(15): 2037-2051.
[8]	FALAHAT R, BERGLUND A, PUTNEY R M, et al. Epigenetic reprogramming of tumor cell-intrinsic STING function sculpts antigenicity and T cell recognition of melanoma[J]. Proc Natl Acad Sci USA, 2021, 118(15): e2013598118.
[9]	SHARMA G, GUARDIA C M, ROY A, et al. A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis[J]. Autophagy, 2019, 15(10): 1694-1718.
[10]	GAYLE S, LANDRETTE S, BEEHARRY N, et al. Identification of apilimod as a first-in-class PIKfyve kinase inhibitor for treatment of B-cell non-Hodgkin lymphoma[J]. Blood, 2017, 129(13): 1768-1778.
[11]	DOĞAN E, DÜZGÜN Z, YILDIRIM Z, et al. The effects of PIKfyve inhibitor YM201636 on claudins and malignancy potential of nonsmall cell cancer cells[J]. Turk J Biol, 2021, 45(1): 26-34.
[12]	GUI X, YANG H, LI T, et al. Autophagy induction via STING trafficking is a primordial function of the cGAS pathway[J]. Nature, 2019, 567(7747): 262-266.
[13]	GONUGUNTA V K, SAKAI T, POKATAYEV V, et al. Trafficking-mediated STING degradation requires sorting to acidified endolysosomes and can be targeted to enhance anti-tumor response[J]. Cell Rep, 2017, 21(11): 3234-3242.
[14]	GENTILI M, LIU B X, PAPANASTASIOU M, et al. ESCRT-dependent STING degradation inhibits steady-state and cGAMP-induced signalling[J]. Nat Commun, 2023, 14(1): 611.
[15]	CLAUDE-TAUPIN A, MOREL E. Phosphoinositides: functions in autophagy-related stress responses[J]. Biochim Biophys Acta Mol Cell Biol Lipids, 2021, 1866(6): 158903.
[16]	ZHENG C F. Protein dynamics in cytosolic DNA-sensing antiviral innate immune signaling pathways[J]. Front Immunol, 2020, 11: 1255.
[17]	SHARMA G, OJHA R, NOGUERA-ORTEGA E, et al. PPT1 inhibition enhances the antitumor activity of anti-PD-1 antibody in melanoma[J]. JCI Insight, 2020, 5(17): e133225.
[18]	RAMANJULU J M, SCOTT PESIRIDIS G, YANG J S, et al. Design of amidobenzimidazole STING receptor agonists with systemic activity[J]. Nature, 2018, 564(7736): 439-443.
[19]	MOTEDAYEN AVAL L, PEASE J E, SHARMA R, et al. Challenges and opportunities in the clinical development of STING agonists for cancer immunotherapy[J]. J Clin Med, 2020, 9(10): 3323.
[20]	AMOUZEGAR A, CHELVANAMBI M, FILDERMAN J N, et al. STING agonists as cancer therapeutics[J]. Cancers (Basel), 2021, 13(11): 2695.
[21]	HANSON M C, CRESPO M P, ABRAHAM W, et al. Nanoparticulate STING agonists are potent lymph node-targeted vaccine adjuvants[J]. J Clin Invest, 2015, 125(6): 2532-2546.
[22]	KIM D S, ENDO A, FANG F G, et al. E7766, a macrocycle-bridged stimulator of interferon genes (STING) agonist with potent pan-genotypic activity[J]. ChemMedChem, 2021, 16(11): 1740-1743.
[23]	PAN B S, PERERA S A, PIESVAUX J A, et al. An orally available non-nucleotide STING agonist with antitumor activity[J]. Science, 2020, 369(6506): eaba6098.
[24]	PAYNE S H. The utility of protein and mRNA correlation[J]. Trends Biochem Sci, 2015, 40(1): 1-3.
[25]	YANG J H, LUO Z Y, MA J Y, et al. A next-generation STING agonist MSA-2: from mechanism to application[J]. J Control Release, 2024, 371: 273-287.
[26]	XIA T L, KONNO H, AHN J, et al. Deregulation of STING signaling in colorectal carcinoma constrains DNA damage responses and correlates with tumorigenesis[J]. Cell Rep, 2016, 14(2): 282-297.
[27]	WANG L, LIANG Z D, GUO Y Z, et al. STING agonist diABZI enhances the cytotoxicity of T cell towards cancer cells[J]. Cell Death Dis, 2024, 15(4): 265.

PIKfyve抑制剂靶向治疗STING通路缺陷黑色素瘤的机制研究

Mechanistic study of targeting melanoma with STING pathway deficiencies via PIKfyve inhibitor

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