WO2023045266A1 - Fgfr及其相关信号通路抑制剂在制备治疗fgfr2突变型肿瘤的药物中的应用 - Google Patents

Fgfr及其相关信号通路抑制剂在制备治疗fgfr2突变型肿瘤的药物中的应用 Download PDF

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WO2023045266A1
WO2023045266A1 PCT/CN2022/079122 CN2022079122W WO2023045266A1 WO 2023045266 A1 WO2023045266 A1 WO 2023045266A1 CN 2022079122 W CN2022079122 W CN 2022079122W WO 2023045266 A1 WO2023045266 A1 WO 2023045266A1
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fgfr2
mutation
related diseases
diseases caused
inhibiting
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WO2023045266A9 (zh
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邓初夏
雷海鹏
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澳门大学
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  • the present disclosure relates to the field of biomedicine, in particular, to the application of FGFR and its related signaling pathway inhibitors in the preparation of drugs for treating FGFR2 mutant tumors.
  • the present disclosure provides the application of a reagent for inhibiting a signaling pathway, wherein the signaling pathway is selected from at least one of the STAT3 signaling pathway and the MAPK signaling pathway, in the preparation of a medicament for preventing and/or treating related diseases caused by FGFR2 mutations kind.
  • the FGFR2 mutation is FGFR2 S252W.
  • the related diseases caused by FGFR2 mutation include: at least one of breast cancer, liver cancer, cholangiocarcinoma, gastric cancer, endometrial cancer, lung cancer, Apert syndrome and Crouzon syndrome.
  • the FGFR2 mutation comprises at least one of FGFR2 S252W, FGFR2 N549K, FGFR2 N549H, FGFR2 E565A, FGFR2 K569M, FGFR2 L617V, FGFR2 K647R, FGFR2 V564F, and FGFR2 amplification or fusion.
  • the agent for inhibiting the signaling pathway is selected from at least one of a STAT3 inhibitor and an ERK inhibitor.
  • the final concentration of the STAT3 inhibitor is 0.1-5 ⁇ M.
  • the final concentration of the ERK inhibitor is 1-10 ⁇ M.
  • the STAT3 inhibitor when the agent for inhibiting the signaling pathway includes a STAT3 inhibitor, includes: at least one of C188-9, FLLL32, S3I-201, HJC0152, InS3-54A18 and NSC-368262 kind;
  • the agent for inhibiting the signaling pathway includes an ERK inhibitor
  • the ERK inhibitor includes at least one of U0126, BVD-523, CC-90003, GDC-0994, KO-947, LTT462, LY3214996 and MK-8353 .
  • the present disclosure provides a use of a reagent for inhibiting a signaling pathway, wherein the signaling pathway is selected from at least one of the STAT3 signaling pathway and the MAPK signaling pathway, in a drug for preventing and/or treating related diseases caused by FGFR2 mutations kind.
  • the FGFR2 mutation comprises at least one of FGFR2 S252W, FGFR2 N549K, FGFR2 N549H, FGFR2 E565A, FGFR2 K569M, FGFR2 L617V, FGFR2 K647R, FGFR2 V564F, and FGFR2 amplification or fusion.
  • the agent for inhibiting the signaling pathway is selected from at least one of STAT3 inhibitors and ERK inhibitors.
  • the present disclosure provides a method of treating related diseases caused by FGFR2 mutations, comprising:
  • the related diseases caused by FGFR2 mutation include: at least one of breast cancer, liver cancer, cholangiocarcinoma, gastric cancer, endometrial cancer, lung cancer, Apert syndrome and Crouzon syndrome.
  • the present disclosure provides the use of FGFR2 inhibitors in the preparation of drugs for preventing and/or treating related diseases caused by FGFR2 mutations.
  • the FGFR2 mutation is FGFR2 S252W.
  • the FGFR2 mutation comprises at least one of FGFR2 N549K, FGFR2 N549H, FGFR2 E565A, FGFR2 K569M, FGFR2 L617V, FGFR2 K647R, FGFR2 V564F, and FGFR2 amplification or fusion.
  • the present disclosure provides the application of FGFR2 inhibitors and immune checkpoint inhibitors in the preparation of drugs for preventing and/or treating related diseases caused by FGFR2 mutations.
  • the FGFR2 mutation is FGFR2 S252W.
  • the FGFR2 mutation comprises at least one of FGFR2 N549K, FGFR2 N549H, FGFR2 E565A, FGFR2 K569M, FGFR2 L617V, FGFR2 K647R, FGFR2 V564F, and FGFR2 amplification or fusion.
  • the immune checkpoint includes any one of PD-1 and PD-L1.
  • the present disclosure provides the use of a STAT3 inhibitor in the preparation of a medicament for inhibiting or reversing EMT promoted by FGFR2 mutation.
  • the FGFR2 mutation comprises at least one of FGFR2 S252W, FGFR2 N549K, FGFR2 N549H, FGFR2 E565A, FGFR2 K569M, FGFR2 L617V, FGFR2 K647R, FGFR2 V564F, and FGFR2 amplification or fusion.
  • the present disclosure provides the use of a STAT3 inhibitor for inhibiting or reversing epithelial-mesenchymal transition, EMT, promoted by FGFR2 mutations.
  • the FGFR2 mutation comprises at least one of FGFR2 S252W, FGFR2 N549K, FGFR2 N549H, FGFR2 E565A, FGFR2 K569M, FGFR2 L617V, FGFR2 K647R, FGFR2 V564F, and FGFR2 amplification or fusion.
  • the present disclosure provides a method for treating related diseases caused by epithelial-mesenchymal transition (EMT) promoted by FGFR2 mutation, comprising:
  • a therapeutically effective amount of a STAT3 inhibitor is administered to a subject in need thereof.
  • the present disclosure provides the application of FGFR2 inhibitors in the preparation of drugs for preventing and/or treating related diseases caused by low expression of YY1.
  • the low expression of YY1 is caused by FGFR2 mutation.
  • the FGFR2 mutation comprises at least one of FGFR2 S252W, FGFR2 N549K, FGFR2 N549H, FGFR2 E565A, FGFR2 K569M, FGFR2 L617V, FGFR2 K647R, FGFR2 V564F, and FGFR2 amplification or fusion.
  • the present disclosure provides the use of FGFR2 inhibitors for preventing and/or treating related diseases caused by low expression of YY1.
  • the low expression of YY1 is caused by FGFR2 mutation
  • the FGFR2 mutation comprises at least one of FGFR2 S252W, FGFR2 N549K, FGFR2 N549H, FGFR2 E565A, FGFR2 K569M, FGFR2 L617V, FGFR2 K647R, FGFR2 V564F, and FGFR2 amplification or fusion.
  • the present disclosure provides a method for treating related diseases caused by low expression of YY1, including:
  • a therapeutically effective amount of a FGFR2 inhibitor is administered to a subject in need thereof.
  • the present disclosure provides the application of the reagent for inhibiting or reversing the low expression of YY1 in the preparation of a medicament for preventing and/or treating related diseases caused by FGFR2 mutation.
  • the FGFR2 mutation comprises at least one of FGFR2 S252W, FGFR2 N549K, FGFR2 N549H, FGFR2 E565A, FGFR2 K569M, FGFR2 L617V, FGFR2 K647R, FGFR2 V564F, and FGFR2 amplification or fusion.
  • the present disclosure provides a FGFR2 inhibitor, a combination of an FGFR2 inhibitor and an immune checkpoint, or a reagent for inhibiting or reversing the low expression of YY1, for use in medicines for preventing and/or treating related diseases caused by FGFR2 mutations.
  • the FGFR2 mutation comprises at least one of FGFR2 S252W, FGFR2 N549K, FGFR2 N549H, FGFR2 E565A, FGFR2 K569M, FGFR2 L617V, FGFR2 K647R, FGFR2 V564F, and FGFR2 amplification or fusion.
  • the present disclosure provides a method of treating related diseases caused by FGFR2 mutations, comprising:
  • a therapeutically effective amount of a FGFR2 inhibitor, a combination of a FGFR2 inhibitor and an immune checkpoint, or an agent for inhibiting or reversing the low expression of YY1 is administered to a subject in need thereof.
  • the related diseases caused by FGFR2 mutation include: at least one of breast cancer, liver cancer, cholangiocarcinoma, gastric cancer, endometrial cancer, lung cancer, Apert syndrome and Crouzon syndrome.
  • the present disclosure provides a method of screening anticancer drugs, comprising:
  • Fig. 1 is that FGFR2 activation in embodiment 1 strengthens mammary gland branching morphogenesis and promotes mammary gland tumorigenesis;
  • A-C is 3 months (A and B) and 6 months (C) the fourth pair of mouse mammary gland panorama staining figure, each Three mice were used for each genotype;
  • D is a representative H&E staining picture of wild-type and mutant mammary glands.
  • Fig. 2 is that FGFR2 activation promotes the occurrence of triple-negative breast cancer in embodiment 2;
  • A shows the pathological characteristics of mutant mouse mammary gland tumors by H&E staining;
  • B is that immunofluorescence (IF) shows K14 and /or K18-positive proportion;
  • C is the proportion of TNBC and other tumor subtypes revealed by immunohistochemistry (IHC);
  • D is the schematic diagram of paraffin section immunofluorescence (IF), K18 (green) and K14 (red), DAPI (ie 4',6-diamidino-2-phenylindole) stains the nucleus;
  • E stands for ER (estrogen receptor), PR (progesterone receptor), HER2 (human epidermal growth factor 2), ki67 (cell proliferation marker) immunohistochemical (IHC) staining picture, in which ER, PR, HER2, and ki67 are indicators representing cell proliferation;
  • F is flow cytometry analysis of K14 and K18 in Fg
  • Fig. 3 is that FGFR2 activation regulates EMT in embodiment 3;
  • A-B is that RNA-Seq shows the differential expression of gene between Fgfr2-WT and Fgfr2-S252W mammary gland, through pathway enrichment (A) and string analysis (B);
  • C is Using WB to analyze the changes of key molecules in the mouse mammary gland signaling pathway, such as ERK1/2 (Thr202/Tyr204), STAT3 (Tyr705), AKT (Ser473), c-Jun (Ser73), mTOR (Ser2448);
  • D is using real-time RT -PCR analysis of fgfr2-mediated changes in wild-type and mutant breast Fgf;
  • E is starvation treatment of Fgfr2-WT and Fgfr2-S252W breast cell lines for 12 hours, then given bFGF treatment, and WB analysis of FRS2 and ERK1 using antibodies /2.
  • F is a representative WB analysis showing the expression levels of EMT-related genes in Fgfr2-WT and Fgfr2-S252W breast tumors
  • G is a representative WB analysis showing MDA-MB-231 Changes of EMT markers in cells after inhibiting FGFR2, STAT3 or ERK
  • H is a representative WB analysis of signaling pathway molecules after transfection of FGFR2-WT and FGFR2-S252W in STAT3 knockout MDA-MB-231 cells Change
  • P value was carried out using GraphPad Prism 7 software, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001;
  • Figure 4 shows that the activation of FGFR2 in Example 4 negatively regulates BRCA1 through the FRS2 ⁇ /STAT3/MAPK signaling pathway;
  • A-B is the expression of Brca1 in Fgfr2-WT and Fgfr2-S252W tumors detected by RT-PCR and WB;
  • C-D is RT- PCR detection of BRCA1 mRNA transcription levels in MDA-MB-231 and MCF7 cells after transfection of FGFR2-WT and FGFR2-S252W;
  • E is BRCA1 reporter gene after transfection of FGFR2-WT and FGFR2-S252W in MDA-MB-231 cells Changes in luciferase activity;
  • F is the binding site region of transcription factor YY1;
  • G-H is the ChIP analysis shows that FGFR2-S252W and YY1 competitively bind to the BRCA1 promoter;
  • I-J is FGFR2 mainly regulates YY1 through the FG
  • FIG. 6 shows that FGFR2-induced STAT3 and ERK signals promote PD-L1 expression in Example 6;
  • signalling pathway refers to a phenomenon in which a signal transmits a message from the outside of the cell to the inside of the cell when a certain response occurs in the cell, and the cell responds according to the message.
  • STAT short for signal transducer and activator of transcription
  • SH2 and SH3 domains that bind to specific phosphorylated tyrosine-containing peptides.
  • STAT When STAT is phosphorylated, it aggregates into homologous or heterodimer activated transcriptional activators, enters the nucleus and binds to a specific site of the target gene promoter sequence to promote its transcription.
  • MAPK mitogen-activated protein kinase
  • mitogen-activated protein kinase mitogen-activated protein kinase
  • activated serine-threonine protein kinase It is an important transmitter that transmits signals from the cell surface to the interior of the nucleus.
  • FGFR fibroblast growth factor receptor
  • FGFR2 fibroblast growth factor receptor
  • EMT epithelial-mesenchymal transition
  • immune checkpoint refers to a series of molecules expressed on immune cells that can regulate the degree of immune activation, and can play an important role in the occurrence of textile autoimmunity.
  • YY1 is a widely distributed transcription factor belonging to the GLI-Kruppel class of zinc finger proteins. This protein is involved in the repression and activation of various promoters. YY1 can directly introduce histone deacetylase and histone acetyltransferase into the promoter, thereby activating or repressing the promoter, so that the histone modification acts on YY1.
  • the present disclosure provides the application of a reagent for inhibiting a signaling pathway selected from the STAT3 signaling pathway and the MAPK signaling pathway in the preparation of a medicament for preventing and/or treating related diseases caused by FGFR2 mutations at least one.
  • FGFR2 is one of four membrane-bound receptor tyrosine kinases (RTKs) that mediate the signaling of about 22 fibroblast growth factors (FGFs).
  • RTKs membrane-bound receptor tyrosine kinases
  • FGFs fibroblast growth factors
  • the gene mutation of FGFR2 can activate the downstream signaling pathways of FGFR2, such as PI3K-AKT, MAPK and mTOR signaling pathways.
  • Treatment herein may refer to partial inhibition or complete cure.
  • the agent for inhibiting the signal pathway is selected from at least one of STAT3 inhibitor (STAT3i) and ERK inhibitor (ERKi).
  • STAT3i STAT3 inhibitor
  • ERKi ERK inhibitor
  • FGFR2 mutations may cause a series of related diseases, such as enhancing the occurrence of mammary gland branching and promoting the formation of mammary gland tumors.
  • FGFR-S252W up-regulates the expression level of immune checkpoint PD-L1 through STAT3 signaling pathway and MAPK signaling pathway, and inhibits STAT3 signaling Any one of the pathways and the MAPK signaling pathway can achieve the result of inhibiting the high expression of PD-L1.
  • Agents that inhibit STAT3 signaling pathway include but are not limited to STAT3 inhibitors, and agents that inhibit MAPK signaling pathway include but are not limited to ERK1/2 inhibitors. It should be noted that the effect of combined treatment with STAT3 inhibitors and ERK inhibitors is stronger than that of either of them alone.
  • the FGFR2 mutation is the FGFR2 S252W point mutation.
  • the FGFR2 mutation includes FGFR2 S252W point mutation, FGFR2 N549K point mutation, FGFR2 N549H point mutation, FGFR2 E565A point mutation, FGFR2 K569M point mutation, FGFR2 L617V point mutation, FGFR2 K647R point mutation, FGFR2 V564F point mutation and FGFR2 At least one of amplification or fusion.
  • related diseases caused by FGFR2 mutation include: at least one of breast cancer, liver cancer, cholangiocarcinoma, gastric cancer, endometrial cancer, lung cancer, Apert syndrome and Crouzon syndrome.
  • breast cancer includes triple negative breast cancer.
  • the present disclosure does not limit the types of STAT3 inhibitors and ERK inhibitors, as long as the expression level of STAT3 and/or ERK can be inhibited, including mRNA level and/or protein level, or the function of STAT3 and/or ERK can be inhibited, such as using Reagents for knocking down or knocking out STAT3 and/or ERK genes belong to STAT3 inhibitors and/or ERK inhibitors.
  • the concentration (final concentration) of the STAT3 inhibitor is 0.1-5 ⁇ M, may be 0.1-4 ⁇ M, 1-4 ⁇ M, 1.5-3.5 ⁇ M or 2-3 ⁇ M, such as 0.1 ⁇ M, 0.5 ⁇ M, 1 ⁇ M, 1.5 ⁇ M ⁇ M, 2 ⁇ M, 2.5 ⁇ M, 3 ⁇ M, 3.5 ⁇ M, 4 ⁇ M, 4.5 ⁇ M or 5 ⁇ M.
  • the concentration of the ERK inhibitor is 1-10 ⁇ M, may be 1-5 ⁇ M, 5-10 ⁇ M or 2-8 ⁇ M, such as 1 ⁇ M, 2 ⁇ M, 3 ⁇ M, 4 ⁇ M, 5 ⁇ M, 6 ⁇ M, 7 ⁇ M, 8 ⁇ M, 9 ⁇ M or 10 ⁇ M. Under the limitation of the above concentration range, it has better effect.
  • the STAT3 inhibitor includes: at least one of C188-9, FLLL32, S3I-201, HJC0152, InS3-54A18 and NSC-368262.
  • the agent for inhibiting the signaling pathway includes an ERK inhibitor
  • the ERK inhibitor includes at least one of U0126, BVD-523, CC-90003, GDC-0994, KO-947, LTT462, LY3214996 and MK-8353 .
  • Combination therapy with STAT3 inhibitors and ERK inhibitors has a synergistic effect compared to using either of them alone.
  • Embodiments of the present disclosure also provide the use of agents for inhibiting signaling pathways, wherein the signaling pathways are selected from STAT3 signaling pathways and MAPK signaling pathways, in medicines for preventing and/or treating related diseases caused by FGFR2 mutations at least one.
  • Embodiments of the present disclosure also provide a method for treating related diseases caused by FGFR2 mutations, including:
  • a therapeutically effective amount of an agent for inhibiting a signaling pathway is administered to a subject in need thereof.
  • the related diseases caused by FGFR2 mutation include: at least one of breast cancer, liver cancer, cholangiocarcinoma, gastric cancer, endometrial cancer, lung cancer, Apert syndrome and Crouzon syndrome.
  • Embodiments of the present disclosure also provide applications of FGFR2 inhibitors in the preparation of drugs for preventing and/or treating related diseases caused by FGFR2 mutations.
  • FGFR2 mutation and related diseases caused by it are the same as those described in any of the foregoing embodiments, and the subsequent embodiments are the same, and will not be repeated here.
  • Embodiments of the present disclosure also provide applications of FGFR2 inhibitors and immune checkpoint inhibitors in the preparation of drugs for preventing and/or treating related diseases caused by FGFR2 mutations. Combination therapy of FGFR2 inhibitors and immune checkpoint inhibitors has better therapeutic effect.
  • the immune checkpoint includes any one of PD-1 and PD-L1.
  • Embodiments of the present disclosure also provide the use of STAT3 inhibitors in the preparation of drugs for inhibiting or reversing epithelial-mesenchymal transition (EMT) promoted by FGFR2 mutations.
  • EMT epithelial-mesenchymal transition
  • the embodiment of the present disclosure also provides the application of the FGFR2 inhibitor in the preparation of a medicament for preventing and/or treating related diseases caused by the low expression of YY1.
  • the low expression of YY1 is caused by FGFR2 mutation.
  • Embodiments of the present disclosure also provide a use of a STAT3 inhibitor for inhibiting or reversing EMT promoted by FGFR2 mutation.
  • Embodiments of the present disclosure also provide a method for treating related diseases caused by epithelial-mesenchymal transition (EMT) promoted by FGFR2 mutation, including:
  • a therapeutically effective amount of a STAT3 inhibitor is administered to a subject in need thereof.
  • the embodiment of the present disclosure also provides the application of the agent for inhibiting or reversing the low expression of YY1 in the preparation of a medicament for preventing and/or treating related diseases caused by FGFR2 mutation.
  • Embodiments of the present disclosure also provide the use of FGFR2 inhibitors in the prevention and/or treatment of related diseases caused by low expression of YY1
  • Embodiments of the present disclosure also provide a method for treating related diseases caused by low expression of YY1, including:
  • a therapeutically effective amount of a FGFR2 inhibitor is administered to a subject in need thereof.
  • the embodiment of the present disclosure also provides the application of the agent that promotes the combination of YY1 and BRCA1 promoter in the preparation of drugs for preventing and/or treating related diseases caused by FGFR2 mutation.
  • Embodiments of the present disclosure also provide FGFR2 inhibitors, combinations of FGFR2 inhibitors and immune checkpoints, or agents for inhibiting or reversing the low expression of YY1, which are used in drugs for the prevention and/or treatment of related diseases caused by FGFR2 mutations use.
  • Embodiments of the present disclosure also provide a method for treating related diseases caused by FGFR2 mutations, including:
  • a therapeutically effective amount of a FGFR2 inhibitor, a combination of a FGFR2 inhibitor and an immune checkpoint, or an agent for inhibiting or reversing the low expression of YY1 is administered to a subject in need thereof.
  • the related diseases caused by FGFR2 mutation include: at least one of breast cancer, liver cancer, cholangiocarcinoma, gastric cancer, endometrial cancer, lung cancer, Apert syndrome and Crouzon syndrome.
  • Embodiments of the present disclosure also provide a method for screening anticancer drugs, including:
  • FGF/FGFR2 signaling drives the formation of triple-negative breast cancer (TNBC), and that FGFR2-STAT3 promotes epithelial-mesenchymal transition (EMT), and also found that FGFR2 is inhibited by MAPK-YY1 BRCA1 to accelerate tumor formation.
  • FGFR2 also regulates the expression of PD-L1 through STAT3-MAPK.
  • Example 1 Fgfr2 mutation enhances mammary gland branching morphogenesis and promotes mammary gland tumor formation.
  • Fgfr2-S252W mice Nine months later, mammary tumor development in Fgfr2-S252W mice was observed. During the 22-month period of continuous observation, mammary gland tumors appeared in 22 of 73 mice (30%), but mammary gland tumors were also observed in 3 control mice (MMTV-Cre) (G in Figure 1). Thus, Fgfr2 mutations randomly induce mammary tumorigenesis after prolonged latency. Notably, of the 22 mice with mammary tumors, 5 were found to have tumors in the lung, liver, chest cavity, or lymph nodes at necropsy (HJ in Figure 1), which we believed to be metastatic from breast cancer tumor.
  • Example 2 Fgfr2 activation promotes the development of triple negative breast tumors.
  • Mammary tumors associated with Fgfr2 mutations were characterized by pathological analysis of mammary tumor tissues from Fgfr2-S252W mice. It was mainly invasive ductal carcinoma (IDC), accounting for 75% (24/32), and the rest were invasive lobular carcinoma, lobular carcinoma in situ, and ductal carcinoma in situ (A in Figure 2). Immunofluorescence (IF) showed that 65.625% (21/32) of breast tumors were basaloid carcinomas, and 34.375% (11/32) of other tumors were tubular tumors (B in Figure 2, D in Figure 2).
  • IDC invasive ductal carcinoma
  • IF Immunofluorescence
  • the molecular subtypes of breast cancer showed that 4/32 (12.5%) were tubular (Luminal) type A (positive estrogen receptor and/or progesterone receptor, HER2 negative, low Ki-67 protein level), 8/32 ( 25%) were tubular type B (estrogen receptor and/or progesterone receptor positive, HER2 positive or HER2 negative, 2/32 (6.25%) were HER2 enriched, 18/32 (56.25%) were triple negative Breast cancer (TNBC) (C in Figure 2, E in Figure 2). Therefore, most of the tumors induced by Fgfr2-S252W mice were TNBC (E in Figure 2).
  • the cell population was sorted by K14/K18 expression to detect the tumor sphere-forming characteristics of the cells, and it was found that only K14-positive basal-like cells were able to form tumor spheres (F in Figure 2). Therefore, basal-like cells are more tumorigenic in this mutant mouse (G-I in Figure 2).
  • Example 3 Fgfr2 activates STAT3-MAPK signal to regulate EMT.
  • this example analyzes the transcriptional profiles in mammary glands of WT and Fgfr2-S252W mice at 6 months.
  • 671 differentially expressed genes (DEGs) were identified. Among them, 359 were up-regulated and 312 were down-regulated.
  • the protein-protein interaction network is shown in Figure 3B. The two groups of genes were positively correlated with inflammatory factors (green boxes) and growth factors/EMT (red boxes), respectively.
  • GSEA Gene set enrichment analysis
  • Transcriptional profiling revealed significant upregulation of Fgf2 and Fgf10 in mutated mammary glands and two other Fgfs: Fgf3 and Fgf7 in tumors (Fig. 3B, 3D). Studies have shown that FGFR2-S252W enhances its ligand-binding ability.
  • Fgfr2-S252W and WT mammary epithelial cell lines were treated with FGF2 (bFGF), and downstream signals including FRS2 ⁇ , ERK1/2, AKT and EMT were detected.
  • FGF2 FGF2
  • downstream signals including FRS2 ⁇ , ERK1/2, AKT and EMT were detected.
  • the MAPK-ERK signal responded rapidly, but gradually disappeared after 2 hours, but the STAT3 signal was activated after 24 hours (E in Figure 3).
  • the levels of EMT-associated proteins Snail, CDH2, MMP9 and Vim were also higher in Fgfr2-S252W than in wild-type mammary epithelial cells. Therefore, FGF ligands can further enhance the Fgfr2-S252W-mediated signaling pathway and play an important role in Fgfr2-S252W mammary tumorigenesis.
  • the CRISPR/Cas9 system was used to knock out STAT3 in MDA-MB-231 cells, and then Fgfr2-WT and Fgfr2-S252W were transfected into the knockout cell line, and the knockout of STAT3 significantly inhibited the expression of Fgfr2 up-regulated proteins. expression (H in Figure 3), confirming that STAT3 is a major mediator of Fgfr2 signaling in ERK activation and regulates EMT gene expression.
  • Example 4 FGFR2 activates FRS2 ⁇ /STAT3/MAPK signaling pathway to regulate BRCA1 and YY1.
  • MCF7 and MDA-MB-231 cells were transfected with FGFR2-WT and FGFR2-S252W, and it was found that ectopic expression of FGFR2, especially the down-regulation of BRCA1 mRNA level by FGFR2-S252W was more obvious ( Figure 4 C-D), and FGFR2 signal negatively regulated the expression of BRCA1 transcription.
  • a luciferase reporter gene was constructed, which included 1704 bases (between -1460bp and +244bp) of the regulatory region of the human BRCA1 reporter gene.
  • This example also creates a sequence deletion construct (type IV vector) that gradually truncates at a distance of about 40 bp from the 0.24 kb region.
  • the region between -201bp to -162bp (39bp) maintains luciferase activity, as all type IV constructs lacking this region have much lower luciferase activity.
  • the vector containing the 39 base pairs alone (V-vector) also showed low luciferase activity. Therefore, other regions within the 0.24 kb fragment may also be required for the positive regulation of BRCA1 transcription.
  • FGFR2-WT or FGFR2-S252W were co-transfected to investigate whether FGFR2 signaling inhibits luciferase activity in the 0.24 kilobase reporter. From FGFR2 to the FGFR2-S252W structure, a graded decrease in BRCA1 expression could be observed (Fig. 4, E).
  • YY1 positively regulates the expression of BRCA1 by binding to the consensus site of BRCA1 in the 39 base pair region between -162bp and -201bp (Fig. and protein levels were reduced. This finding was associated with significantly lower levels of BRCA1.
  • 60 breast cancer samples were also analyzed by IHC and found that FGFR2 was negatively correlated with YY1 and BRCA1, suggesting that YY1 may be responsible for the reduction of BRCA1 after FGFR2 activation.
  • chromatin immunoprecipitation (ChIP) analysis was used to verify whether FGFR2 prevents YY1 from binding to the BRCA1 promoter.
  • Expression of FGFR2-S252W blocked the binding of YY1 to the BRCA1 promoter (G in Figure 4).
  • YY1-activated BRCA1 reporter gene transcription was also blocked (Fig. 4, H).
  • FGFR2-S252W was transfected into five different breast cancer cell lines. Western blot showed that the low expression of YY1 and BRCA1 was observed in four cell lines (MDA-MB-231, MCF7, T47D and MDA-MB-468) after transfection of FGFR2-S252W.
  • FGFR2-S252W was transfected into MDA-MB-231 cells, and it was found that it significantly reduced the mRNA (I-J in Figure 4) and protein (K in Figure 4) levels of YY1 and BRCA1, and this inhibitory effect was very large
  • FGFR inhibitors BGJ398 and AZD4547
  • STAT3 C188-9
  • ERK U0126
  • FGFR2 may regulate YY1/BRCA1 through STAT3 and ERK/MAPK signaling pathways.
  • knockdown of STAT3 restored YY1 and BRCA1 protein levels repressed by FGFR2-S252W (L in Figure 4).
  • two breast cancer patient-derived organoids were used for validation and they were treated with bFGF (50 ng/mL).
  • the data showed that the bFGF treatment group increased cell proliferation (M in Figure 4) and organoids also grew larger (N-O in Figure 4, K in Figure 4), but had no significant effect on cell morphology.
  • the data also showed that STAT3 and MAPK were activated while BRCA1 and YY1 were significantly decreased (P in Fig. 4).
  • FGFR2 regulates BRCA1 in breast tissue and tumors through YY1, and this process is mediated by STAT/ERK signaling.
  • Fgfr2-S252W mice were crossed with mammary gland-specific knockout Brca1 (Brca1co/co; mmtv-cre, or Brca1-mko) mice to generate the double mutant Fgfr2-S252W; Brca1-MKO. Relatively more extensive branching morphogenesis was observed in the mammary glands of Fgfr2-S252W and Brca1-MKO mice than in each single mutation-carrying parent (Fig. 5, A-B).
  • this example evaluated the expression of proteins related to the FGFR2 signaling pathway in the tumors of FGFR2-S252W, FGFR2-S252W; Brca1-MKO and Brca1-MKO mice.
  • FGF3, FGF7, and pFRS2 levels were higher in Fgfr2-S252W;Brca1-MKO tumors.
  • the levels of pSTAT3, pERK1/2 and p-cJun were also higher in tumors from double mutant mice than in FGFR-S252W or Brca1-MKO tumors (Figure 5, E-F).
  • immunohistochemistry revealed relatively high levels of the immune checkpoint protein PD-L1 in Fgfr2-S252W tumors.
  • PD-L1 was more significantly upregulated in FGFR-S252W and Brca1-MKO double mutant tumors (Fig. 5, F).
  • Example 6 Fgfr2 regulates PD-L1 through STAT3-MAPK.
  • the association of FGFR2 and PD-L1 was investigated using immunohistochemistry in a tissue array of 415 human breast cancer samples.
  • the expression level of FGFR2 was divided into three grades: high, medium and low. Among them, 149 cases were high (149/415; 36%), 169 cases were low (169/415; 41%), and 97 cases were low (97/415; 23%) (A-B in Fig. 6).
  • PD-L1 expression was detected in 121 samples (121/415; 29%). Among them, there were 41 cases of high expression (41/415; 9.8%), 43 cases of medium expression (43/415; 10.36%), and 36 cases of low expression (37/415; (8.92%) (A, C in Fig. 6 ). The expression of FGFR2 was positively correlated with the expression of PD-L1 (D-E in Figure 6).
  • FGFR2-induced PD-L1 expression in this example, a cell line (231-S252W) stably expressing FGFR2-S252W was constructed in MDA-MB-231 cells and treated with FGFR inhibitors. Both inhibitors inhibited the expression of PD-L1 and downregulated the expression of pSTAT3 and pERK1/2 (G in Figure 6). Therefore, STAT3 and ERK may be involved in fgfr2-mediated regulation of PD-L1.
  • 231-S252W cells were treated with STAT3 inhibitor (C188-9) and ERK inhibitor (U0126), and it was confirmed that both drugs inhibited the expression of PD-L1 in a dose-dependent manner (Fig. 6 H and Fig. 6 I) .
  • C188-9 at concentrations above 1 ⁇ M downregulates the expression of pSTAT3, pERK1/2 and PD-L1. Therefore, STAT3 may regulate PD-L1 through ERK1/2.
  • U0126 began to inhibit pERK and PD-L1 at a concentration of 1 mM, and down-regulated pSTAT3 at a concentration of 20 ⁇ M (I in Figure 6). Therefore, ERK signaling may regulate STAT3 through a feedback mechanism.
  • Fgfr2-S252W; Brcal-MKO tumors had significantly fewer activated CD8+ T cells than Fgfr2-S252W or Brca1-MKO tumors (K-N in Figure 6).
  • Fgfr2-S252W;Brca1-MKO tumors had an imbalance in the ratio of M1:M2 macrophages compared to Fgfr2-S252W or Brca1-MKO breast tumors (K-N in Figure 6).
  • M2 macrophages help create an immunosuppressive environment that prevents T cell activation.
  • upregulation of multiple signaling pathways triggered by FGFR2 activation cooperates with Brca1 deficiency to create an immune microenvironment that enhances tumor progression.
  • Example 7 Blocking the FGFR signaling pathway can inhibit the growth of brca1-deficient tumors.
  • Example 8 Establishment of a tumor slice culture platform for rapid evaluation of the efficacy of anticancer drugs.
  • Fgfr2-S252W and Fgfr2-S252W; Brca1-1-tumors are sensitive to FGFRi, but the speed of animal experiments is slow. Therefore, this example uses fresh tumor tissues to establish a rapid evaluation platform for evaluating the efficacy of anti-tumor drugs.
  • the prepared Fgfr2-S252W mouse mammary gland tumor tissue slices were cultured on an air-liquid interface system. The survival rate of the sliced tissues was about 90% in the first 5 days, and dropped to about 80% after 6-7 days.
  • tumor slice culture technology is a good platform for screening targeted therapy.
  • Immune checkpoint blockade (ICB) targeting PD-1 and/or PD-L1 holds promise as a means of treating cancer. However, it is only effective in 20% of patients, and the tumor slice platform of the present disclosure can rapidly evaluate the efficacy of this immunotherapy approach. This example studies whether this three-dimensional slice culture method can be used to evaluate the efficiency of ICB mediated by anti-PD-1 and anti-PD-L1 antibodies. At 4 days after treatment, both anti-PD-1 and anti-PD-L1 killed cancer cells in a dose-dependent manner.
  • This disclosure provides the application of combined immune checkpoint inhibitors and FGFR and its related signaling pathway inhibitors in the preparation of drugs for the treatment of FGFR2 mutant tumors, and provides research directions and approaches for the research of FGRR2 signaling pathways and the development of related drugs. It has wide application value.

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Abstract

FGFR及其相关信号通路抑制剂在制备治疗FGFR2突变型肿瘤的药物中的应用。FGF/FGFR2信号驱动三阴性乳腺癌的形成，并伴随FGFR2-STAT3促进上皮间质转化，还发现FGFR2通过MAPK-YY1抑制BRCA1来加速肿瘤的形成。此外，FGFR2还通过STAT3-MAPK调控PD-L1的表达。

Description

FGFR及其相关信号通路抑制剂在制备治疗FGFR2突变型肿瘤的药物中的应用
相关申请的交叉引用
本公开要求于2021年09月23日提交中国专利局的申请号为“202111117003.X”名称为“FGFR及其相关信号通路抑制剂在制备治疗FGFR2突变型肿瘤的药物中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及生物医药领域，具体而言，涉及FGFR及其相关信号通路抑制剂在制备治疗FGFR2突变型肿瘤的药物中的应用。
背景技术
现在，女性乳腺癌已经超过肺癌，成为全世界最常见的癌症。在2020年12月15日，世界卫生组织国际癌症研究机构(IARC)发布了2020年全球最新癌症数据显示，乳腺癌是女性第一大癌症致死原因，预计到2020年将导致超过68万例死亡，因此对于寻找新型乳腺癌的治疗方法迫在眉睫。
发明内容
本公开提供了用于抑制信号通路的试剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用，所述信号通路选自STAT3信号通路和MAPK信号通路中的至少一种。
在一些实施方式中，所述FGFR2突变为FGFR2 S252W。
在一些实施方式中，由FGFR2突变导致的相关疾病包括：乳腺癌、肝癌、胆管癌、胃癌、子宫内膜癌、肺癌、Apert综合征和Crouzon综合征中的至少一种。
在一些实施方式中，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
在一些实施方式中，用于抑制信号通路的试剂选自STAT3抑制剂以及ERK抑制剂中的至少一种。
在一些实施方式中，所述STAT3抑制剂的终浓度为0.1～5μM。
在一些实施方式中，所述ERK抑制剂的终浓度为1～10μM。
在一些实施方式中，当所述抑制信号通路的试剂包括STAT3抑制剂时，所述STAT3抑制剂包括：C188-9、FLLL32、S3I-201、HJC0152、InS3-54A18以及NSC-368262中的至少一种；
当所述抑制信号通路的试剂包括ERK抑制剂时，所述ERK抑制剂包括U0126、BVD-523、CC-90003、GDC-0994、KO-947、LTT462、LY3214996以及MK-8353中的至少一种。
本公开提供一种用于抑制信号通路的试剂在用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的用途，所述信号通路选自STAT3信号通路和MAPK信号通路中的至少一种。
在一些实施方式中，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
在一些实施方式中，所述用于抑制信号通路的试剂选自STAT3抑制剂以及ERK抑制剂中的至少一种。
本公开提供一种治疗由FGFR2突变导致的相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的用于抑制信号通路的试剂；
在一些实施方式中，所述由FGFR2突变导致的相关疾病包括：乳腺癌、肝癌、胆管癌、胃癌、子宫内膜癌、肺癌、Apert综合征和Crouzon综合征中的至少一种。
本公开提供了FGFR2抑制剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用。
在一些实施方式中，所述FGFR2突变为FGFR2 S252W。
在一些实施方式中，所述FGFR2突变包括FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
本公开提供了FGFR2抑制剂和免疫检查点抑制剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用。
在一些实施方式中，所述FGFR2突变为FGFR2 S252W。
在一些实施方式中，所述FGFR2突变包括FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。在一些实施方式中，所述免疫检查点包括PD-1和PD-L1中的任意一种。
本公开提供了STAT3抑制剂在制备用于抑制或逆转由FGFR2突变促进的上皮间质转化EMT的药物中的应用。
在一些实施方式中，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。本公开提供STAT3抑制剂在用于抑制或逆转由FGFR2突变促进的上皮间质转化EMT中的用途。
在一些实施方式中，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
本公开提供一种治疗由FGFR2突变促进的上皮间质转化EMT导致的相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的STAT3抑制剂。
本公开提供了FGFR2抑制剂在制备用于预防和/或治疗由YY1低表达导致的相关疾病的药物中的应用。
在一些实施方式中，所述YY1低表达是由FGFR2突变引发的。
在一些实施方式中，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。本公开提供FGFR2抑制剂在用于预防和/或治疗由YY1低表达导致的相关疾病中的用途。
在一些实施方式中，所述YY1低表达是由FGFR2突变引发的；
在一些实施方式中，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
本公开提供一种治疗由YY1低表达导致的相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的FGFR2抑制剂。
本公开提供了用于抑制或逆转YY1低表达的试剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用。
在一些实施方式中，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
本公开提供了FGFR2抑制剂、FGFR2抑制剂和免疫检查点的组合或者用于抑制或逆转YY1低表达的试剂，用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的用途。
在一些实施方式中，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
本公开提供了一种治疗由FGFR2突变导致的相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的FGFR2抑制剂、FGFR2抑制剂和免疫检查点的组合或者用于抑制或逆转YY1低表达的试剂。
在一些实施方式中，所述由FGFR2突变导致的相关疾病包括：乳腺癌、肝癌、胆管癌、胃癌、子宫内膜癌、肺癌、Apert综合征和Crouzon综合征中的至少一种。
本公开提供了一种筛选抗癌药物的方法，包括：
制备肿瘤切片样本，并将其进行三维组织培养；
使用FGFR抑制剂处理经三维组织培养的所述肿瘤切片样本；
评价所述肿瘤切片样本的细胞活力以筛选所述抗癌药物。
附图说明
为了更清楚地说明本公开实施方式的技术方案，下面将对实施方式中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为实施例1中FGFR2激活增强乳腺分支形态发生并促进乳腺肿瘤发生；其中，A-C为3个月(A和B)和6个月(C)第四对小鼠乳腺全景染色图，每个基因型用3只小鼠；D为野生型和突变型乳腺代表性H&E染色图片。E-F为野生型和突变型乳腺中CD24和CD29流式细胞术分析，每个基因型用6只小鼠；G为野生型和突变型小鼠乳腺随时间变化成瘤数；H-J为突变型小鼠发生肿瘤(H)，并转移到肺(I)和肝脏(J)；数据代表平均SEM,n＝6；P值使用GraphPad Prism 7软件进行；*p<0.05，**p<0.01，***p<0.001，****p<0.0001；
图2为实施例2中FGFR2激活促进三阴性乳腺癌的发生；其中，A为H&E染色展示突变型小鼠乳腺肿瘤的病理学特征；B为免疫荧光(IF)显示Fgfr2-S252W肿瘤中K14和/或K18阳性占比；C为免疫组化(IHC)揭露突变型肿瘤TNBC及其他亚型肿瘤的占比；D为石蜡切片免疫荧光(IF)示意图，K18(绿色)和K14(红色)，DAPI(即4',6-二脒基-2-苯基吲哚)是染细胞核；E为ER(雌激素受体)、PR(孕激素受体)、HER2(人表皮生长因子2)、ki67(细胞增殖标记物)免疫组化(IHC)染色图片，其中ER、PR、HER2、ki67为代表细胞增殖的指标；F为Fgfr2-S252W乳腺中K14和K18流式细胞术分析，只有K14+细胞可以形成肿瘤球；G-I为肿瘤成球率(MFE)分析(G)，分别转染FGFR2-WT(H)和FGFR2-S252W(I)的MDA-MB-231细胞MFE的图片；P值使用GraphPad Prism 7软件进行，*p<0.05，**p<0.01，***p<0.001，****p<0.0001；
图3为实施例3中FGFR2激活调控EMT；其中，A-B为RNA-Seq显示Fgfr2-WT和Fgfr2-S252W乳腺之间基因的差异表达，通过pathway enrichment(A)和string analysis(B)；C为用WB分析小鼠乳腺信号通路关键分子的变化，如ERK1/2(Thr202/Tyr204)、STAT3(Tyr705)、AKT(Ser473)、c-Jun(Ser73)、mTOR(Ser2448)；D为利用实时RT-PCR分析fgfr2介导的野生型和突变型乳腺Fgf的变化；E为将Fgfr2-WT和Fgfr2-S252W乳腺细胞系进行饥饿处理12小时，之后给予bFGF处理，并使用抗体进行WB分析FRS2、ERK1/2、AKT和EMT标记物的变化；F为具有代表性的WB分析显示EMT相关基因在Fgfr2-WT和Fgfr2-S252W乳腺肿瘤中表达水平；G为代表性的WB分析显示MDA-MB-231细胞中EMT标记物在抑制FGFR2、STAT3或ERK后的改变；H为具有代表性的WB分析在敲除STAT3的MDA-MB-231细胞中转染FGFR2-WT和FGFR2-S252W之后信号通路分子的变化；P值使用GraphPad Prism 7软件进行，*p<0.05，**p<0.01，***p<0.001，****p<0.0001；
图4为实施例4中FGFR2的激活通过FRS2α/STAT3/MAPK信号通路负调控BRCA1；其中，A-B为通过RT-PCR和WB检测Fgfr2-WT和Fgfr2-S252W肿瘤中Brca1的表达；C-D为RT-PCR检测转染FGFR2-WT和FGFR2-S252W后MDA-MB-231和MCF7细胞中BRCA1 mRNA的转录水平；E为BRCA1报告基因在MDA-MB-231细胞中转染FGFR2-WT和FGFR2-S252W后荧光素酶活性变化；F为转录因子YY1结合位点区域；G-H为ChIP分析显示，FGFR2-S252W与YY1竞争性结合到BRCA1启动子上；I-J为FGFR2主要通过FGFR2/FRS2/MAPK信号通路调控YY1和BRCA1；K为通过免疫印迹法检测抑制FGFR/FRS2/STAT3/MAPK信号通路后，BRCA1和YY1的表达得以恢复，验证FGFR2通过FGFR/FRS2/STAT3/MAPK信号通路调控BRCA1和YY1；L为免疫印迹法分析敲除STAT3后转染FGFR2-WT和FGFR2-S252W后YY1/BRCA1的表达；M-P为MTT连续7天测量两种3D细胞系(3D-#T1和3D-#T2)经bFGF处理后的生长曲线及测量3D细胞系(N,O)的大小，western blot检测部分下游信号的表达(P)；P值使用GraphPad Prism 7软件进行；*p<0.05,**p<0.01,***p<0.001,****p<0.0001；
图5为实施例5中Fgfr2激活和敲除Brca1双突变加速乳腺肿瘤的形成；其中，A-B为各种基因型小鼠整个乳腺及H&E染色；C为各种基因型小鼠乳腺肿瘤统计分析，(n＝数量的老鼠)；D为各种基因型小鼠乳腺肿瘤分子亚型分析，(n＝肿瘤数量)；E为Western blot对各种基因型的肿瘤组织中相关标记物分析；F为应用免疫组化染色方法检测PD-L1和pSTAT3在各种基因型肿瘤中的表达；G为Western blot检测BRCA1异位表达对231-S252W细胞的影响；H为将两个BRCA1表达质粒转染231-WT和231-S252W细胞中观察细胞增值变化；P值使用GraphPad Prism 7软件进行。*p<0.05,**p<0.01,***p<0.001,****p<0.0001；
图6为实施例6中FGFR2诱导的STAT3和ERK信号促进PD-L1表达；其中，A-F为分析FGFR2在人乳腺癌组织阵列中的表达以及PD-L1、p-STAT3和p-ERK1/2相关性，其中，A为FGFR2和PD-L1免疫组化代表性图片；B-D为FGFR2和PD-L1在BC组织的分析结果，E为FGFR2表达与PD-L1表达的相关性分析，F为FGFR2和p-STAT3及p-ERK1/2免疫组化代表性图片；G-I为在MDA-MB-231细胞中转染FGFR2-S252W后用不同浓度的FGFR，STAT3和ERK抑制剂处理后，通过WB分析PD-L1表达变化；J为FGFR2激活通过STAT3-ERK信号促进PD-L1的表达；K-L为分析各种基因型肿瘤中CD8+T细胞和活化的CD8+T的情况；M-N为M1/M2巨噬细胞(F4/80,CD11b和CD206)在各种基因型肿瘤中的情况；P值使用GraphPad Prism 7软件进行。*p<0.05,**p<0.01,***p<0.001,****p<0.0001；
图7为实施例7中建立快速评估抗癌药物疗效的肿瘤切片培养平台；其中，A-B为在Fgfr2-S252W肿瘤细胞用shRNA敲低brca1，用RT-PCR(A)和WB(B)检测Brca1敲低水平；C为评估 BGJ398对所示小鼠细胞系增殖的影响；D-F为体内评估BGJ398对肿瘤生长/重量的影响(n＝6)，其中D为实验组与对照组最后肿瘤大小照片，E-F为BGJ398对肿瘤总体积/重量的影响；G为H&E和免疫组化切片染色分析；H为WB分析FRS2、ERK1/2、STAT3标记物的变化；I-J为评估三维肿瘤切片培养系统中免疫治疗的可行性及有效性；M为免疫组化染色分析；P值使用GraphPad Prism 7软件进行；*p<0.05,**p<0.01,***p<0.001,****p<0.0001。
具体实施方式
术语定义：
在利用实施方式和实施例描述本发明之前，应理解本文所用术语仅用于描述本公开的目的，并不旨在限制本发明的范围。
如本文所用，术语“信号通路”是指指当细胞里要发生某种反应时，信号从细胞外到细胞内传递了一种信息，细胞要根据这种信息来做出反应的现象。
如本文所用，术语“STAT”，全称为信号传导及转录激活蛋白(signal transducer and activator of transcription)，是一种能与DNA结合的蛋白质的家族。含有SH2和SH3结构域，可与特定的含磷酸化酪氨酸的肽段结合。当STAT被磷酸化后，发生聚合成为同源或异源二聚体形式的活化的转录激活因子，进入胞核内与靶基因启动子序列的特定位点结合，促进其转录。
如本文所用，术语“MAPK”全称为丝裂原活化蛋白激酶(mitogen-activated protein kinase)是一组能被不同的细胞外刺激，如细胞因子、神经递质、激素、细胞应激及细胞黏附等激活的丝氨酸-苏氨酸蛋白激酶。是将信号从细胞表面传导到细胞核内部的重要传递者。
如本文所用，术语“FGFR”全称为“成纤维生长因子受体”，属于受体型蛋白酪氨酸激酶，已知的FGFR主要包括4种类型，成纤维细胞生长因子受体被广泛认为是一类抗肿瘤的重要药物靶标。其中“FGFR2”为其中的一种类型。
如本文所用，术语“EMT”全称为“上皮间质转化(EMT)”，是上皮细胞来源的恶性肿瘤细胞获得迁移和侵袭能力的重要生物学过程，激活上皮间质转化(EMT)是癌细胞转移的关键过程，在此过程中，上皮细胞获得间充质细胞的特征，细胞运动性和迁移能力增强。
如本文所用，术语“免疫检查点”是指在免疫细胞上表达，能调节免疫激活程度的一系列分子，能够对纺织自身免疫作用的发生发挥重要作用。
如本文所用，术语“YY1”是一种广泛分布的转录因子，属于GLI-Kruppel类锌指蛋白。该蛋白参与抑制和激活多种启动子。YY1可将组蛋白脱乙酰酶和组蛋白乙酰转移酶直接导入启动子，从而激活或抑制启动子，从而使组蛋白修饰作用于YY1。
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
首先，本公开提供了用于抑制信号通路的试剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用，所述信号通路选自STAT3信号通路和MAPK信号通路中的至少一种。
其中，FGFR2是介导约22种成纤维细胞生长因子(FGFs)信号的四种膜结合受体酪氨酸激酶(RTKs)之一。FGFR2的基因突变可以激活FGFR2的下游信号通路，例如，PI3K-AKT，MAPK及mTOR信号通路等。
本文中的“治疗”可以指部分抑制或完全治愈。
可选地，用于抑制信号通路的试剂选自STAT3抑制剂(STAT3i)以及ERK抑制剂(ERKi)中的至少一种。
FGFR2突变可能引发一系列相关病症，如，增强乳腺分支形态的发生，促进乳腺肿瘤的形成，FGFR-S252W通过STAT3信号通路以及MAPK信号通路上调免疫检查点PD-L1的表达水平，而抑制STAT3信号通路以及MAPK信号通路中的任意一种，能够达到抑制PD-L1高表达的结果。抑制STAT3信号通路的试剂包括但不限于STAT3抑制剂，抑制MAPK信号通路的试剂包括但不限于ERK1/2抑制剂。需要说明的是，同时采用STAT3抑制剂与ERK抑制剂进行联合治疗的效果，比单独采用其中任意一种要强。
可选地，所述FGFR2突变为FGFR2 S252W点突变。
可选地，所述FGFR2突变包括FGFR2 S252W点突变、FGFR2 N549K点突变、FGFR2 N549H点突变、FGFR2 E565A点突变、FGFR2 K569M点突变、FGFR2 L617V点突变、FGFR2 K647R点突变、FGFR2 V564F点突变以及FGFR2扩增或融合中的至少一种。
可选地，由FGFR2突变导致的相关疾病包括：乳腺癌、肝癌、胆管癌、胃癌、子宫内膜癌、肺癌、Apert综合征和Crouzon综合征中的至少一种。可选地，乳腺癌包括三阴性乳腺癌。
本公开不对STAT3抑制剂以及ERK抑制剂的种类进行限定，只要是能够达到抑制STAT3和/或ERK的表达水平，包括mRNA水平和/或蛋白水平，或者达到抑制STAT3和/或ERK功能，诸如用于敲低或敲除STAT3和/或ERK基因的试剂，即属于STAT3抑制剂和/或ERK抑制剂。
可选地，所述STAT3抑制剂的作用浓度(终浓度)为0.1～5μM，可以为0.1～4μM、1～4μM、1.5～3.5μM或2～3μM，诸如0.1μM、0.5μM、1μM、1.5μM、2μM、2.5μM、3μM、3.5μM、4μM、4.5μM或5μM。可选地，所述ERK抑制剂的作用浓度为1～10μM，可以为1～5μM、5～10μM或2～8μM，诸如1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、9μM或10μM。在上述浓度范围的限定下，具有更好的作用效果。
可选地，当所述抑制信号通路的试剂包括STAT3抑制剂时，所述STAT3抑制剂包括：C188-9、FLLL32、S3I-201、HJC0152、InS3-54A18和NSC-368262中的至少一种。当所述抑制信号通路的试剂包括ERK抑制剂时，所述ERK抑制剂包括U0126、BVD-523、CC-90003、GDC-0994、KO-947、LTT462、LY3214996以及MK-8353中的至少一种。
采用STAT3抑制剂和ERK抑制剂进行联合治疗，相对于单独采用其中任意一种而言，具有协同增效的作用。
本公开实施方式还提供了用于抑制信号通路的试剂在用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的用途，所述信号通路选自STAT3信号通路和MAPK信号通路中的至少一种。
本公开实施方式还提供了一种治疗由FGFR2突变导致的相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的用于抑制信号通路的试剂。
在一些实施方式中，由FGFR2突变导致的相关疾病包括：乳腺癌、肝癌、胆管癌、胃癌、子宫内膜癌、肺癌、Apert综合征和Crouzon综合征中的至少一种。
本公开实施方式还提供了FGFR2抑制剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用。
FGFR2突变及其导致的相关疾病同前述任意实施方式所述，后续实施方式同此，不在赘述。
本公开实施方式还提供了FGFR2抑制剂和免疫检查点抑制剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用。FGFR2抑制剂和免疫检查点抑制剂的联合治疗具有更好的治疗效果。
可选地，所述免疫检查点包括PD-1和PD-L1中的任意一种。
本公开实施方式还提供了STAT3抑制剂在制备用于抑制或逆转由FGFR2突变促进的上皮间质转化EMT的药物中的应用。
本公开实施方式还提供了FGFR2抑制剂在制备用于预防和/或治疗由YY1低表达导致的相关疾病的药物中的应用。
可选地，所述YY1低表达是由FGFR2突变引发的。
本公开实施方式还提供了STAT3抑制剂在用于抑制或逆转由FGFR2突变促进的上皮间质转化EMT中的用途。
本公开实施方式还提供了一种治疗由FGFR2突变促进的上皮间质转化EMT导致的相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的STAT3抑制剂。
本公开实施方式还提供了用于抑制或逆转YY1低表达的试剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用。
本公开实施方式还提供了FGFR2抑制剂在用于预防和/或治疗由YY1低表达导致的相关疾病中的用途
本公开实施方式还提供了一种治疗由YY1低表达导致的相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的FGFR2抑制剂。
本公开实施方式还提供了促进YY1与BRCA1启动子结合的试剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用。
本公开实施方式还提供了FGFR2抑制剂、FGFR2抑制剂和免疫检查点的组合或者用于抑制或逆转YY1低表达的试剂，用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的用途。
本公开实施方式还提供了一种治疗由FGFR2突变导致的相关疾病的方法，包括：
向有此需要的受试者给药治疗有效量的FGFR2抑制剂、FGFR2抑制剂和免疫检查点的组合或者用于抑制或逆转YY1低表达的试剂。
在一些实施方式中，由FGFR2突变导致的相关疾病包括：乳腺癌、肝癌、胆管癌、胃癌、子宫内膜癌、肺癌、Apert综合征和Crouzon综合征中的至少一种。
本公开实施方式还提供了一种筛选抗癌药物的方法，包括：
制备肿瘤切片样本，并将其进行三维组织培养；
使用FGFR抑制剂处理经三维组织培养的所述肿瘤切片样本；
评价所述肿瘤切片样本的细胞活力以筛选所述抗癌药物。
本公开通过研究FGFR2对于乳腺癌的作用机制，发现FGF/FGFR2信号驱动三阴性乳腺癌(TNBC)的形成，并伴随FGFR2-STAT3促进上皮间质转化(EMT)，还发现FGFR2通过MAPK-YY1抑制BRCA1来加速肿瘤的形成。此外，FGFR2还通过STAT3-MAPK调控PD-L1的表达。基于上述发现，本公开提供了联合免疫检查点抑制剂和FGFR及其相关信号通路抑制剂在制备治疗FGFR2突变型肿瘤的药物中的应用，为FGRR2信号通路的研究以及相关药物的开发提供了研究方向和途径。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例
实施例1：Fgfr2突变增强乳腺分支形态发生，促进乳腺肿瘤的形成。
应用loxp系统在乳腺组织中特异性的激活Fgfr2，Fgfr2-S252W小鼠乳腺的全景图与野生型(WT)小鼠相比，Fgfr2-S252W小鼠乳腺分支更密集(图1中A-C)。组织切片显示突变小鼠的细胞数 量相对更多(图1中D)。流式细胞术显示乳腺上皮细胞中CD29 HiCD24 Med群体相对增加(图1中E-F)，乳腺干细胞的这些特征表明，Fgfr2的激活增强了乳腺分支形态的发生和上皮细胞的增殖，从而增加了乳腺干细胞的数量，促进肿瘤的形成。9个月后，观察到Fgfr2-S252W小鼠乳腺肿瘤的发生。在持续观察的22个月时间里，在73只老鼠中有22只(30％)出现乳腺肿瘤，但也有3只对照小鼠(MMTV-Cre)也观察到了乳腺肿瘤(图1中G)。因此，Fgfr2突变在长时间的潜伏后随机诱导乳腺肿瘤的发生。值得注意的是，在22只患乳腺肿瘤的小鼠中，有5只在尸检时发现有肺、肝、胸腔或淋巴结的肿瘤(图1中H-J)，我们认为它们是从乳腺癌转移过来的肿瘤。
实施例2：Fgfr2激活促进三阴性乳腺肿瘤的发展。
通过病理分析Fgfr2-S252W小鼠乳腺肿瘤组织，揭示与Fgfr2突变相关的乳腺肿瘤的特征。主要是为浸润性导管癌(IDC)，占比75％(24/32),其余为浸润性小叶癌、原位小叶癌和导管原位癌(图2中A)。免疫荧光(IF)显示65.625％(21/32)的乳腺肿瘤表现为类基底样癌，其他肿瘤为管状肿瘤34.375％(11/32)(图2中B，图2中D)。
乳腺癌分子亚型显示，4/32(12.5％)为管状(Luminal)A型(雌激素受体和/或孕激素受体阳性，HER2阴性，Ki-67蛋白水平低)，8/32(25％)为管状B型(雌激素受体和/或孕激素受体阳性，HER2阳性或HER2阴性，2/32(6.25％)为HER2富集型，18/32(56.25％)为三阴性乳腺癌(TNBC)(图2中C，图2中E)。因此，Fgfr2-S252W小鼠诱导的肿瘤大部分为TNBC(图2中E)。
然后，通过K14/K18表达对细胞群进行分类，以检测细胞形成肿瘤球形特性，结果发现只有K14表达阳性的基底样细胞能够形成肿瘤球体(图2中F)。因此，基底样细胞在该突变小鼠中具有更强的致瘤性(图2中G-I)。
实施例3：Fgfr2激活STAT3-MAPK信号调控EMT。
为研究Fgfr2诱导肿瘤发生的分子机制，本实施例分析了6个月时WT和Fgfr2-S252W小鼠乳腺中的转录谱。鉴定出671个差异表达基因(DEGs)。其中，359个表达上调，312个表达下调。生物信息学分析揭示了肿瘤的发生可能与上皮细胞分化、增殖、炎症反应、MAPK级联反应、趋化因子等通路相关(图3中A)。蛋白与蛋白相互作用网络如图3中B所示。两组基因分别与炎症因子(绿框)和生长因子/EMT(红框)呈正相关。
基因集富集分析(GSEA)显示，Fgfr2-S252W乳腺中的乳腺癌通路和MAPK信号通路被高度激活。Western blot分析MAPK/ERK、PI3K/AKT/mTOR和JNK/c-JUN通路中的多个相关基因，FRS2α、GRB2、ERK1/2、c-JUN、AKT(Ser473)、mTOR和STAT3磷酸化水平显著上调(图3中C)。
转录谱显示突变的乳腺中的Fgf2和Fgf10以及肿瘤中的另外两个Fgfs:Fgf3和Fgf7显著上调(图3中B，3中D)。研究表明，FGFR2-S252W增强了其配体结合能力。
用FGF2(bFGF)处理Fgfr2-S252W和WT乳腺上皮细胞系，并检测下游信号包括FRS2α、ERK1/2、AKT和EMT等。其中MAPK-ERK信号迅速响应，但2小时后逐渐消失，但STAT3信号则在24小时后被激活(图3中E)。Fgfr2-S252W中EMT相关蛋白Snail、CDH2、MMP9和Vim的水平也高于野生型乳腺上皮细胞。因此，FGF配体可以进一步增强Fgfr2-S252W介导的信号通路，并在Fgfr2-S252W乳腺肿瘤发生中发挥重要作用。
Fgfr2-S252W乳腺肿瘤中上皮细胞标记物E-cadherin的表达低于Fgfr2-WT乳腺肿瘤，也从免疫荧光(IF)得到佐证(图3中F)。
然后，通过抑制STAT3或ERK信号评估FGFR2-S252W调控EMT的机制。在转入FGFR2-S252W的样本中，蛋白pFRS2(Tyr196)、pSTAT3(Tyr705)、pERK1/2、N-Cad和Snail的水平显著升高(图3中G)。然而，使用BGJ398或AZD4547抑制Fgfr2后，这些反应完全逆转。C188-9抑制STAT3可 显著抑制蛋白pERK1/2、N-Cad和Snail。U0126抑制ERK对pSTAT3和Snail水平的影响较轻，但对N-Cad的影响与STAT3i相似。这些数据表明STAT3是Fgfr2信号通路调控网络的主要分子。
进一步地，使用CRISPR/Cas9系统在MDA-MB-231细胞中敲除STAT3，然后将Fgfr2-WT和Fgfr2-S252W转染到敲除的细胞系中，STAT3敲除后显著抑制了Fgfr2上调蛋白的表达(图3中H)，证实STAT3是ERK激活中Fgfr2信号的主要中介，并调节EMT基因表达。
实施例4：FGFR2激活FRS2α/STAT3/MAPK信号通路调控BRCA1和YY1。
Fgfr2-S252W中大多数肿瘤为TNBC。因此，本实施例检测了Brca1的表达，因为它的缺失会触发TNBC癌的形成。RT-PCR显示，FGFR2-S252W乳腺和肿瘤中的Brca1 mRNA水平低于对照组(图4中A)。Brca1蛋白水平的表达同样在蛋白质印迹法(Western blotting)得到验证(图4中B)。因此，FGFR2信号下调了BRCA1。
用MCF7和MDA-MB-231细胞转染FGFR2-WT和FGFR2-S252W，发现异位表达FGFR2，特别是FGFR2-S252W下调BRCA1 mRNA水平更为明显(图4中C-D)，FGFR2信号负调控BRCA1的转录。
本实施例构建了一个荧光素酶报告基因，包含了人类BRCA1报告基因调控区的1704个碱基(介于-1460bp和+244bp之间)。所有包含β启动子(I型载体)或缺乏β启动子且包含-201bp以上区域(II型载体)的报告基因的荧光素酶活性都显著低于包含α启动子的0.24千碱基(III型载体)。因此，β启动子和-201bp以上区域负调控BRCA1的转录，而0.24千碱基片段正调控BRCA1的表达。
本实施例还创建了一个序列缺失结构(IV型载体)，逐渐截断距离0.24kb区域约40bp的距离。在-201bp到-162bp(39bp)之间的区域维持荧光素酶活性，因为所有IV型结构体缺乏这个区域，其荧光素酶活性都要低得多。然而，单独包含该39个碱基对的载体(V型载体)也显示出低荧光素酶活性。因此，0.24千碱基片段中的其他区域可能也需要BRCA1转录的正调控。然后，共转染FGFR2-WT或FGFR2-S252W以研究FGFR2信号是否抑制0.24千碱基报告中的荧光素酶活性。从FGFR2到FGFR2-S252W结构中，可以观察到BRCA1表达的分级降低(图4中E)。
YY1通过结合BRCA1在-162bp和-201bp之间的39个碱基对区域的共识位点，正调控BRCA1的表达(图4中F)，在FGFR2突变小鼠的乳腺和肿瘤中，YY1的转录和蛋白水平都降低了。这一发现与BRCA1水平显著降低相关。还通过IHC分析了60例乳腺癌样本，发现FGFR2与YY1和BRCA1呈负相关，表明YY1可能是FGFR2激活后BRCA1减少的原因。
接下来，使用染色质免疫沉淀(ChIP)分析来验证FGFR2是否阻止YY1与BRCA1启动子的结合。FGFR2-S252W的表达阻断了YY1与BRCA1启动子的结合(图4中G)。YY1激活的BRCA1报告基因转录也被阻断(图4中H)。为了阐明FGFR2-S252W调控BRCA1的分子机制，将FGFR2-S252W转染到五种不同的乳腺癌细胞系中。蛋白质印迹法显示转染FGFR2-S252W后在四种细胞系(MDA-MB-231,MCF7,T47D和MDA-MB-468)中均观察到YY1和BRCA1的低表达。
进一步地，将FGFR2-S252W转染到MDA-MB-231细胞中，发现其显著降低YY1和BRCA1的mRNA(图4中I-J)和蛋白(图4中K)水平，这种抑制作用在很大程度上可以被FGFR抑制剂(BGJ398和AZD4547)、STAT3(C188-9)或ERK(U0126)(图4中I-4K)逆转。这些数据提示FGFR2对YY1/BRCA1的调控可能通过STAT3和ERK/MAPK信号通路。在MDA-MB-231细胞中，敲除STAT3可恢复被FGFR2-S252W抑制的YY1和BRCA1蛋白水平(图4中L)。
更进一步地，使用了两个乳癌患者衍生的类器官(PDOs)进行验证，并用bFGF(50ng/mL)处理它们。数据显示，加入bFGF处理组细胞增殖增加(图4中M)和类器官球也长得更大(图4中N-O， 图4中K)，但对细胞形态没有明显影响。此外，数据还显示STAT3和MAPK激活而BRCA1和YY1显著降低(图4中P)。
综上所述，FGFR2通过YY1调控乳腺组织和肿瘤中的BRCA1，这一过程是由STAT/ERK信号介导。
实施例5：Fgfr2跟Brca1双突变老鼠加速肿瘤的形成。
将Fgfr2-S252W小鼠与乳腺特异性敲除Brca1(Brca1co/co；mmtv-cre，或Brca1-mko)小鼠杂交，生成双突变体Fgfr2-S252W；Brca1-MKO。在Fgfr2-S252W和Brca1-MKO小鼠的乳腺中观察到相对更广泛的分支形态发生，比在每一个单一携带突变的亲本中观察到的要更多(图5中A-B)。
大约6个月时，-S252W；Brca1-MKO小鼠开始出现乳腺肿瘤，中位时间为10个月左右。相比之下，-S252W和Brca1-MKO小鼠分别在15个月和21个月的中位时间出现乳腺肿瘤(图5中C)。因此，Fgfr2-S252W和Brca1-MKO小鼠的肿瘤发生速度明显快于每单一一个携带单基因突变的亲本。TNBC在Fgfr2-S252W；Brca1-MKO小鼠中的发生率为62.5％(19/32)(图5中D)。因此，Fgfr2激活和Brca1缺失显著增强了乳腺肿瘤的发生，同时也增加了TNBC的形成。
为了阐明肿瘤加速发生的原因，本实施例评估了FGFR2-S252W、FGFR2-S252W；Brca1-MKO和Brca1-MKO小鼠肿瘤中与FGFR2信号通路相关的蛋白的表达。FGF3、FGF7和pFRS2水平在Fgfr2-S252W；Brca1-MKO肿瘤中表达更高。来自双突变小鼠的肿瘤中pSTAT3、pERK1/2和p-cJun的水平也高于FGFR-S252W或者Brca1-MKO肿瘤(图5中E-F)。这些数据表明，BRCA1可能通过抑制这些FGFR2信号通路来延缓细胞生长。
进一步地，我们在231-S252W细胞中异位表达BRCA1，发现它确实抑制了FGFR2-S252W触发的增强pSTAT3和pAKT(图5中G)，并显著抑制MDA-MB-231和231-S252W细胞的增殖(图5中H)。总之，这些数据表明，双突变肿瘤比单突变肿瘤激活更多的致癌信号，从而加速了肿瘤的发生。
值得注意的是，免疫组化(IHC)显示Fgfr2-S252W肿瘤中免疫检查点蛋白PD-L1水平相对较高。PD-L1在FGFR-S252W和Brca1-MKO双突变肿瘤中上调更明显(图5中F)。
实施例6：Fgfr2通过STAT3-MAPK调控PD-L1。
使用免疫组化方法在415例人类乳腺癌样本的组织阵列中研究FGFR2和PD-L1的相关性。
FGFR2表达水平分为高、中、低三个档次。其中高有149例(149/415；36％)、低有169例(169/415；41％)、低有97例(97/415；23％)(图6中A-B)。
121份样本中检测到PD-L1的表达(121/415；29％)。其中，高表达有41例(41/415；9.8％)，中等表达有43例(43/415；10.36％)，低表达36例(37/415；(8.92％)(图6中A，C)。FGFR2表达与PD-L1表达呈正相关(图6中D-E)。
综上所述，这些数据表明FGFR2激活与乳腺癌中的PD-L1表达呈正相关。此外，在人体组织阵列中检测了FGFR2的活性，结果显示FGFR2表达与pSTAT3和pERK1/2呈正相关(图6中F)。
为阐明FGFR2诱导PD-L1表达的分子机制，本实施例在MDA-MB-231细胞中构建稳定表达FGFR2-S252W细胞株(231-S252W)并用FGFR抑制剂处理。这两种抑制剂均抑制PD-L1的表达，并下调pSTAT3和pERK1/2的表达(图6中G)。因此，STAT3和ERK可能参与fgfr2介导的PD-L1调控。
用STAT3抑制剂(C188-9)和ERK抑制剂(U0126)处理231-S252W细胞，证实这两种药物都以剂量依赖的方式抑制PD-L1的表达(图6中H和图6中I)。C188-9(浓度超过1μM时)下调pSTAT3、pERK1/2和PD-L1的表达。因此，STAT3可能通过ERK1/2调控PD-L1。U0126在浓度 为1mM时开始抑制pERK和PD-L1，在浓度为20μM时下调pSTAT3(图6中I)。因此，ERK信号可能通过反馈机制调控STAT3。这些实验结果表明，STAT3和ERK1/2共同参与了FGFR2对PD-L1的调控。
进一步地，在不同浓度下用STAT3i和ERKi共同处理细胞，发现两种抑制剂联合产生的效果比单独使用一种抑制剂更强。此外，1μM STAT3i和5μMERKi显著抑制PD-L1表达(图6中J)。据报道PD-L1高表达限制抗肿瘤免疫应答。考虑到Fgfr2-S252W中的PD-L1的高表达，检测了Brca1-MKO和Fgfr2-S252W小鼠肿瘤中相关的免疫分子的变化。不同标记物的流式细胞术显示Fgfr2-S252W；Brcal-MKO肿瘤中激活的CD8+T细胞明显少于Fgfr2-S252W或Brca1-MKO肿瘤(图6中K-N)。Fgfr2-S252W；Brca1-MKO肿瘤与Fgfr2-S252W或Brca1-MKO乳腺肿瘤相比，M1:M2巨噬细胞比例失衡(图6中K-N)。M2巨噬细胞帮助产生免疫抑制环境，阻止T细胞的激活。因此，FGFR2激活引发的多信号通路上调与Brca1缺陷协作，创造了一个增强肿瘤进展的免疫微环境。
实施例7：阻断FGFR信号通路可抑制brca1功能缺失的肿瘤增长。
通过shRNA敲降技术在Fgfr2-S252W乳腺肿瘤细胞中敲低Brca1(图7中A-B)。在细胞增殖实验中，所有Fgfr2-S252W和Fgfr2-S252W+shBrca1的细胞系对FGFR抑制剂(FGFRi)BGJ398敏感程度几乎相同(图7中C)。
然后，将这些细胞植入裸鼠的第4个乳腺脂肪垫中，建立异体肿瘤。当肿瘤形成后，比较了BGJ398治疗组和未治疗组小鼠的肿瘤生长情况。在没有BGJ398的情况下，Fgfr2-S252W+shBrca1-1和Fgfr2-S252W+shBrca1-2肿瘤比Fgfr2-S252W肿瘤生长速度更快，在BGJ398的干预下显著抑制所有肿瘤的生长(图7中D-E)。在药物处理的情况下，没有任何治疗组导致小鼠体重显著下降(图7中F)。在BGJ398的作用下，肿瘤的增殖显著降低，细胞凋亡明显增加(图7中G)。这些数据证实FGFR2信号促进并维持brca1缺陷的肿瘤进展。此外，FGFRi抑制STAT3-ERK活性以及Snail和N-cadherin的表达(图7中H)。因此，抑制FGFR2对肿瘤的生长有深远的影响。
实施例8：建立快速评价抗癌药物疗效的肿瘤切片培养平台。
开发快速、可靠的模型来验证单个和联合抗肿瘤药物的治疗效果至关重要。Fgfr2-S252W和Fgfr2-S252W；Brca1-1-肿瘤对FGFRi敏感，但是动物体内试验速度缓慢，因此，本实施例利用新鲜的肿瘤组织建立一个快速评估抗肿瘤药物药效评价平台。制备Fgfr2-S252W小鼠乳腺肿瘤组织切片放入气液界面系统上进行培养，前5天切片组织存活率约为90％，6-7天后下降至80％左右。肿瘤组织用不同浓度的BGJ398处理4天后，比较其组织细胞活力,BGJ398治疗组以剂量递增的方式使细胞凋亡。因此肿瘤切片培养技术是筛选靶向治疗的良好平台。
免疫检查点阻断(ICB)以PD-1和/或PD-L1为靶点，有望作为一种治疗癌症的手段。然而，它只对20％的患者有效，本公开的肿瘤切片平台可以快速评估这种免疫治疗方法的疗效。本实施例研究了这种三维切片培养的方法是否可以用来评估抗PD-1和抗PD-L1抗体介导的ICB的效率。在治疗后4天，抗PD-1和抗PD-L1均以剂量依赖的方式杀死癌细胞。
然后，探讨FGFR2抑制剂联合ICB治疗的效果，结果发现FGFR2抑制剂浓度15μM、30μM联合PD-L1显著增强疗效(图7中I和图7中K)。5μg PD-1和FGFR抑制剂组合的疗效远远大于2.5g PD-1和FGFR抑制剂组合的疗效(图7中J-L)。
通过免疫组化染色检测IFNγ、CD8+、裂解的胱天蛋白酶(即含半胱氨酸的天冬氨酸蛋白水解酶-3)(cleaved caspase)3、Ki67和PD-L1蛋白水平，以揭示FGFR抑制剂和免疫治疗抑制肿瘤生长的机制。从图7中M可以看出，培养4天后CD8、Ki67、PD-L1水平均未下降。然而与对照组相比，抗PD-L1组PD-L1和Ki67水平显著降低，INF-γ和CD8水平显著升高。因此，肿瘤切片培养系统可以快速评估FGFR抑制剂和PD-1/PD-L1阻断剂联合应用对FGF/FGFR驱动肿瘤患者的疗 效。
以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开提供了联合免疫检查点抑制剂和FGFR及其相关信号通路抑制剂在制备治疗FGFR2突变型肿瘤的药物中的应用，为FGRR2信号通路的研究以及相关药物的开发提供了研究方向和途径，具有广泛的应用价值。

Claims (20)

  1. 用于抑制信号通路的试剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用，其特征在于，所述信号通路选自STAT3信号通路和MAPK信号通路中的至少一种。
  2. 根据权利要求1所述的用于抑制信号通路的试剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用，其特征在于，所述FGFR2突变为FGFR2 S252W；
    优选地，由FGFR2突变导致的相关疾病包括：乳腺癌、肝癌、胆管癌、胃癌、子宫内膜癌、肺癌、Apert综合征和Crouzon综合征中的至少一种。
  3. 根据权利要求1所述的用于抑制信号通路的试剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用，其特征在于，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
  4. 根据权利要求1所述的用于抑制信号通路的试剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用，其特征在于，用于抑制信号通路的试剂选自STAT3抑制剂以及ERK抑制剂中的至少一种。
  5. 根据权利要求3所述的用于抑制信号通路的试剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用，其特征在于，所述STAT3抑制剂的终浓度为0.1～5μM；
    优选地，所述ERK抑制剂的终浓度为1～10μM；
    优选地，当所述抑制信号通路的试剂包括STAT3抑制剂时，所述STAT3抑制剂包括：C188-9、FLLL32、S3I-201、HJC0152、InS3-54A18以及NSC-368262中的至少一种；
    当所述抑制信号通路的试剂包括ERK抑制剂时，所述ERK抑制剂包括U0126、BVD-523、CC-90003、GDC-0994、KO-947、LTT462、LY3214996以及MK-8353中的至少一种。
  6. 用于抑制信号通路的试剂在用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的用途，其特征在于，所述信号通路选自STAT3信号通路和MAPK信号通路中的至少一种；
    优选地，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种；
    优选地，所述用于抑制信号通路的试剂选自STAT3抑制剂以及ERK抑制剂中的至少一种。
  7. 一种治疗由FGFR2突变导致的相关疾病的方法，其特征在于，包括：
    向有此需要的受试者给药治疗有效量的用于抑制信号通路的试剂；
    优选地，所述由FGFR2突变导致的相关疾病包括：乳腺癌、肝癌、胆管癌、胃癌、子宫内膜癌、肺癌、Apert综合征和Crouzon综合征中的至少一种。
  8. FGFR2抑制剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用；
    优选地，所述FGFR2突变为FGFR2 S252W；
    优选地，所述FGFR2突变包括FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
  9. FGFR2抑制剂和免疫检查点抑制剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用；
    优选地，所述FGFR2突变为FGFR2 S252W；
    优选地，所述FGFR2突变包括FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
  10. 根据权利要求9所述的FGFR2抑制剂和免疫检查点抑制剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用，其特征在于，所述免疫检查点包括PD-1和PD-L1中的任意一种。
  11. STAT3抑制剂在制备用于抑制或逆转由FGFR2突变促进的上皮间质转化EMT的药物中的应用；
    优选地，所述FGFR2突变为FGFR2 S252W；
    优选地，所述FGFR2突变包括FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
  12. STAT3抑制剂在用于抑制或逆转由FGFR2突变促进的上皮间质转化EMT中的用途；
    优选地，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
  13. 一种治疗由FGFR2突变促进的上皮间质转化EMT导致的相关疾病的方法，包括：
    向有此需要的受试者给药治疗有效量的STAT3抑制剂。
  14. FGFR2抑制剂在制备用于预防和/或治疗由YY1低表达导致的相关疾病的药物中的应用；
    优选地，所述YY1低表达是由FGFR2突变引发的；
    优选地，所述FGFR2突变为FGFR2 S252W；
    优选地，所述FGFR2突变包括FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
  15. FGFR2抑制剂在用于预防和/或治疗由YY1低表达导致的相关疾病中的用途；
    优选地，所述YY1低表达是由FGFR2突变引发的；
    优选地，所述FGFR2突变为FGFR2 S252W；
    优选地，所述FGFR2突变包括FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
  16. 一种治疗由YY1低表达导致的相关疾病的方法，其特征在于，包括：
    向有此需要的受试者给药治疗有效量的FGFR2抑制剂。
  17. 用于抑制或逆转YY1低表达的试剂在制备用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的应用；
    优选地，所述FGFR2突变为FGFR2 S252W；
    优选地，所述FGFR2突变包括FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
  18. FGFR2抑制剂、FGFR2抑制剂和免疫检查点的组合或者用于抑制或逆转YY1低表达的试剂，用于预防和/或治疗由FGFR2突变导致的相关疾病的药物中的用途；
    优选地，所述FGFR2突变包括FGFR2 S252W、FGFR2 N549K、FGFR2 N549H、FGFR2 E565A、FGFR2 K569M、FGFR2 L617V、FGFR2 K647R、FGFR2 V564F以及FGFR2扩增或融合中的至少一种。
  19. 一种治疗由FGFR2突变导致的相关疾病的方法，其特征在于，包括：
    向有此需要的受试者给药治疗有效量的FGFR2抑制剂、FGFR2抑制剂和免疫检查点的组合或者用于抑制或逆转YY1低表达的试剂；
    优选地，所述由FGFR2突变导致的相关疾病包括：乳腺癌、肝癌、胆管癌、胃癌、子宫内膜癌、肺癌、Apert综合征和Crouzon综合征中的至少一种。
  20. 一种筛选抗癌药物的方法，其特征在于，包括：
    制备肿瘤切片样本，并将其进行三维组织培养；
    使用FGFR抑制剂处理经三维组织培养的所述肿瘤切片样本；
    评价所述肿瘤切片样本的细胞活力以筛选所述抗癌药物。
PCT/CN2022/079122 2021-09-23 2022-03-03 Fgfr及其相关信号通路抑制剂在制备治疗fgfr2突变型肿瘤的药物中的应用 WO2023045266A1 (zh)

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