FGFR基因变异及FGFR受体抑制剂的研究进展

FGFR信号通路参与正常细胞的增殖、迁移和抗凋亡。FGFR基因变异包括激活突变、基因易位和基因扩增，从而导致FGFR信号通路异常激活，驱动肿瘤细胞增殖、存活及促进血管生成，促使肿瘤进展。目前已经在10余种恶性肿瘤中发现FGFR基因变异，不同瘤种的FGFR1～4亚型及其基因变异类型均有所不同。近期，Pemigatinib获得国家药品监督管理局（National Medical Products Administration,NMPA）批准。需明确不同瘤种FGFR基因变异与肿瘤治疗和预后的关系，探索FGFR基因检测方法以识别具有临床价值的基因变异，进而有效提高患者的临床获益。全文总结了近几年相关领域进展，以期呈现目前FGFR抑制剂在晚期肿瘤治疗中的研究进展和FGFR基因变异病理检测现状。     

肿瘤治疗新靶点:碱性成纤维细胞生长因子及其受体信号

碱性成纤维细胞生长因子（bFGF）是一个促细胞分裂、增殖及血管生成的多肽生长因子,肿瘤组织中bFGF及其受体过度表达为抗肿瘤治疗提供潜在靶目标,研制靶向性抗bFGF抗体、FGFR抑制剂等抑制肿瘤细胞增殖和新血管形成,阻断肿瘤生长的营养和转移途径,成为抗肿瘤热点。本文就bFGF及其受体信号在肿瘤的发生、发展和转移中的作用及临床转化研究进行综述。      

抗成纤维细胞生长因子及其受体信号通路药物在肿瘤治疗中应用的研究进展

成纤维细胞生长因子受体（FGFR）是一种受体酪氨酸激酶（RTK），与其配体成纤维细胞生长因子（FGF）相结合，激活下游的信号转导通路，参与调控细胞的正常生理活动。当FGFR基因发生扩增、突变或者融合等异常改变时，就会导致下游细胞信号通路的异常激活，促进细胞的增殖、迁移、侵袭及上皮-间质转化，进而促进肿瘤的发展。同时，FGFR在多种肿瘤中均呈高表达，因此FGF/FGFR可作为肿瘤治疗的重要靶点。根据药物作用机制，可以将抗FGFR信号通路药物分为两大类，分别为FGFR-酪氨酸激酶抑制剂（TKI）和阻断FGF/FGFR的单克隆抗体。目前，已有多种针对FGF/FGFR的靶向药物进入临床试验阶段，在肿瘤治疗中取得较好的临床效果，有的靶向药物获批用于临床肿瘤的治疗，为肿瘤的精准治疗带来了新的曙光。     

靶向FGFR4的抗肿瘤药物研究进展

 成纤维细胞生长因子受体4（FGFR4）通过启动STAT3、MAPK和PI3K/AKT等信号级联,调节细胞的增殖、分化和转移。其基因突变、过表达或其配体分子过表达等异常信号转导在某些肿瘤的发生发展中发挥着重要的作用,表明FGFR4是此类肿瘤潜在的治疗靶标。靶向FGFR4的抗肿瘤药物或方法主要包括小分子抑制剂、单克隆抗体、配体捕获蛋白、短链RNA寡核苷酸适配体（shRNA）。本文回顾FGFR4异常的肿瘤及靶向FGFR4抗肿瘤药物的研究进展。     

FGFl9和FGFR4在肿瘤中作用的研究进展

FGFl9和FGFR4的表达与肿瘤发生有关，FGFR4基因单核苷酸多态性与肿瘤的发生、侵袭转移和预后密切相关。通过阻断FGFl9和FGFR4的相互作用可以抑制肿瘤生长，这为肿瘤的生物治疗提供了有效的靶标。作者总结国内外对FGFl9和FGFR4在肿瘤中作用的研究进展，综述其在肿瘤的发生、浸润、转移、预后和治疗上的作用。     

FGFR4基因状态与晚期非小细胞肺癌患者临床病理特征及预后的关系

目的:研究FGFR4基因与晚期非小细胞肺癌（non-small cell lung cancer，NSCLC）患者临床病理特征及预后的关系。方法:采用高通量测序技术筛选肿瘤标本中的FGFR4基因突变状态。通过卡方检验分析FGFR4基因突变状态与临床病理特征的相关性。用Logistic分析其疗效。用Kaplan-Meier曲线和COX模型评估患者生存。结果:在163例晚期非小细胞肺癌患者中FGFR4基因突变患者共有48例（29.4%）。FGFR4基因突变状态与患者的性别、年龄、吸烟史、病理类型、ECOG评分、T分期、是否有淋巴结转移、EGFR及ALK基因状态无明显相关性（P>0.05）。FGFR4野生型患者的一线疗效明显优于突变型的患者（突变型vs野生型，P<0.001），FGFR4野生型患者的疾病控制率（DCR）达68.6%。生存分析显示，突变型患者的中位OS和中位PFS短于野生型的患者（中位OS:16个月vs 43个月，P<0.001；中位PFS:8个月vs 14个月，P<0.001）。多因素结果显示，ECOG 2~3分（HR=2.353，95%CI:1.259~4.398，P=0.007）、有淋巴结转移（HR=3.754，95%CI:2.310~6.101，P＜0.001）、FGFR4基因突变（HR=2.517，95%CI:1.521~4.165，P＜0.001）是影响患者生存的独立危险因素。结论:FGFR4基因影响晚期非小细胞肺癌的预后，野生型患者的生存时间明显长于突变型患者。     

免疫检查点抑制剂耐药机制及耐药后治疗策略的研究进展

免疫检查点抑制剂(Immune-checkpoint blockers,ICBs)治疗已在多种肿瘤中获得了较好的疗效,患者生活质量明显改善.然而,并不是所有接受ICBs治疗的病人在病灶控制、症状改善和生存时间等方面均获益,很多患者对首次使用ICBs治疗一段时间后出现的耐药现象,是目前临床面临的难题.本综述列举了原发性耐药和获得性耐药可能的原因.原发性耐药包括肿瘤微环境、癌细胞本身及其他相关因素;获得性耐药包括非经典的免疫共抑制分子继发性过表达、抗原提呈信号通路异常及T细胞活化杀伤功能异常等.并阐述了可能存在的多种新型联合治疗方式,包括联合放化疗及联合靶向治疗等措施.     

PTEN信号转导通路与肿瘤的多药耐药

基因调控、信号转导通路异常均可引起细胞增殖失控,导致肿瘤发生.肿瘤细胞对化疗药物耐药是肿瘤患者死亡的主要原因.细胞内药物有效浓度的降低、DNA损伤的修复障碍、基因的突变及异常表达、信号转导通路的异常等均参与了肿瘤细胞的多药耐药.张力蛋白同源10号染色体缺失的磷酸酶基因(phosphatase and tension homology deleted on chromosome ten gene,PTEN)是具有磷酸酶活性的抑癌基因,在多种肿瘤细胞中异常表达,主要通过抑制P13K/Akt/mTOR(mammalian target of rapamycin,mTOR)等多种信号转导通路参与细胞的增殖、凋亡及化疗耐药.因此,上调野生型PTEN的表达,或使用PBK/Akt/mTOR信号通路抑制剂,可逆转肿瘤细胞的多药耐药,提高传统化疗的疗效.     

RNA干扰FGFR3基因表达诱导骨肉瘤细胞凋亡及下调Wnt信号通路的 研究

目的:探讨RNA干扰人成纤维细胞生长因子受体3（FGFR3）基因表达对骨肉瘤细胞凋亡及Wnt信号通路的影响。方法:以LipofectamineTM 2000为载体，参照其转染说明将设计合成的2个针对FGFR3的特异性siRNA及阴性对照siRNA转染人骨肉瘤MG63细胞，同时设置空白对照组，转染48 h，Western blotting检测FGFR3、β-catenin、Survivin和Bax蛋白的表达；流式细胞术检测细胞凋亡率；二氯二氢荧光素二乙酸酯（H2DCFHDA）探针检测活性氧（ROS）含量。结果:2个FGFR3 siRNA转染MG63细胞后FGFR3蛋白表达均明显受到抑制，与空白对照组及NC组比较差异具有统计学意义（P<0.05）。MG63细胞转染FGFR3 siRNA后细胞凋亡率明显升高，ROS含量明显升高，β-catenin和Survivin蛋白的表达明显降低，Bax蛋白的表达明显升高，与NC组比较差异均具有统计学意义（P<0.05）。结论:RNA干扰FGFR3基因表达可诱导骨肉瘤细胞凋亡，机制可能与细胞内ROS含量升高及Wnt信号通路下调有关。     

表皮生长因子受体通路在乳腺癌耐药机制中的研究及应用

表皮生长因子受体在多种肿瘤中存在过度表达.在三苯氧胺耐药(TAM-R)的乳腺癌细胞中表皮生长因子受体(EGFR)的过度表达预示着肿瘤预后不良.研究乳腺癌TAM-R的机制及寻找克服耐药的方案十分重要.已有大量临床前研究显示EGFR信号通路的激活导致乳腺癌细胞产生TAM-R,临床上已有应用EGFR抑制剂于TAM-R乳腺癌的报道,但并非所有患者都能从中获益.EGFR抑制剂的原发或继发耐药是临床上遇到的新难题.     

Discovery and identification of new non-ATP competitive FGFR1 inhibitors with therapeutic potential on non-small-cell lung cancer

Fibroblast growth factor receptor (FGFR) tyrosine kinases have been regarded as a target for cancer treatment, and there is much interest in inhibiting FGF/FGFR signaling by small molecules as a therapeutic approach to cancer. Generally, inhibitors mimics ATP structure and block the binding between ATP and FGFR kinase. Here, two novel, non-ATP-competitive, selective, irreversible FGFR1 inhibitors, A114 and A117, were identified via kinase inhibitory assay from 156 synthetic bisaryl-1,4-dien-3-one derivatives. A “DFG-OUT” inactive conformation binding mode with FGFR1 was predicted by molecular docking. A114 and A117 showed significant anti-tumor activity both in vitro and in vivo via targeting FGFR1.     

FGFR1 activation is an escape mechanism in human lung cancer cells resistant to afatinib,a pan-EGFR family kinase inhibitor

Most NSCLC patients with EGFR mutations benefit from treatment with EGFR-TKIs, but the clinical efficacy of EGFR-TKIs is limited by the appearance of drug resistance. Multiple kinase inhibitors of EGFR family proteins such as afatinib have been newly developed to overcome such drug resistance. We established afatinib-resistant cell lines after chronic exposure of activating EGFR mutation-positive PC9 cells to afatinib. Afatinib-resistant cells showed following specific characteristics as compared to PC9: [1] Expression of EGFR family proteins and their phosphorylated molecules was markedly downregulated by selection of afatinib resistance; [2] Expression of FGFR1 and its ligand FGF2 was alternatively upregulated; [3] Treatment with anti-FGF2 neutralizing antibody blocked enhanced phosphorylation of FGFR in resistant clone; [4] Both resistant clones showed collateral sensitivity to PD173074, a small-molecule FGFR-TKIs, and treatment with either PD173074 or FGFR siRNA exacerbated suppression of both afatinib-resistant Akt and Erk phosphorylation when combined with afatinib; [5] Expression of twist was markedly augmented in resistant sublines, and twist knockdown specifically suppressed FGFR expression and cell survival. Together, enhanced expression of FGFR1 and FGF2 thus plays as an escape mechanism for cell survival of afatinib-resistant cancer cells, that may compensate the loss of EGFR-driven signaling pathway.     

Mutation in the FGFR1 tyrosine kinase domain or inactivation of PTEN is associated with acquired resistance to FGFR inhibitors in FGFR1‐driven leukemia/lymphomas

Stem cell leukemia/lymphoma syndrome (SCLL) is driven by constitutive activation of chimeric FGFR1 kinases generated by chromosome translocations. We have shown that FGFR inhibitors significantly suppress leukemia and lymphoma development in vivo, and cell viability in vitro. Since resistance to targeted therapies is a major reason for relapse, we developed FGFR1‐overexpressing mouse and human cell lines that are resistant to the specific FGFR inhibitors AZD4547 and BGJ398, as well as non‐specific inhibitors, such as ponatinib, TKI258 and E3810. Two mutually exclusive mechanisms for resistance were demonstrated; an activating V561M mutation in the FGFR1 kinase domain and mutational inactivation of PTEN resulting in increased PI3K/AKT activity. Ectopic expression of PTEN in the PTEN‐mutant cells resensitizes them to FGFR inhibitors. Treatment of resistant cells with BGJ398, in combination with the BEZ235 PI3K inhibitor, shows an additive effect on growth in vitro and prolongs survival in xenograft models in vivo. These studies provide the first direct evidence for both the involvement of the FGFR1 V561M mutation and PTEN inactivation in the development of resistance in leukemias overexpressing chimeric FGFR1. These studies also provide a potential strategy to treat leukemias and lymphomas driven by FGFR1 activation that become resistant to FGFR1 inhibitors.     

FGFR1 Cooperates with EGFR in Lung Cancer Oncogenesis,and Their Combined Inhibition Shows Improved Efficacy

Introduction

There is substantial evidence for the oncogenic effects of fibroblast growth factor receptor 1 (FGFR1) in many types of cancer, including lung cancer, but the role of this receptor has not been addressed specifically in lung adenocarcinoma.

Multiple receptor tyrosine kinase activation attenuates therapeutic efficacy of the fibroblast growth factor receptor 2 inhibitor AZD4547 in FGFR2 amplified gastric cancer

Fibroblast growth factor receptor 2 (FGFR2)-targeted therapy has attracted considerable attention as novel anticancer agents in gastric cancer (GC). However, intrinsic or acquired drug resistance has emerged as a major challenge to their clinical use. In this study, we demonstrated that several receptor tyrosine kinase (RTK), including EGFR, HER3 and MET, activations contributed to AZD4547 (a selective FGFR2 inhibitor) hyposensitivity in FGFR2 amplified GC cells. The rescue effect was abrogated by inhibiting these RTKs with their targeted tyrosine kinase inhibitors (TKIs). In addition, synergy in growth inhibition was observed when the GC cells were treated with a combination of AZD4547 and cetuximab (an EGFR monoclonal antibody) both in vitro and in vivo. More importantly, tissue microarray analysis revealed that these resistance-conferring RTKs were highly expressed in FGFR2 positive GC patients. Taken together, these observations demonstrated RTKs including EGFR, HER3 and MET activations as novel mechanisms of hyposensitivity to AZD4547. It will be clinically valuable to investigate the involvement of RTK-mediated signaling in intrinsicor acquired resistance to FGFR2 TKIs in GC. A combination targeted therapeutic strategy may be recommended for treating FGFR2 amplified GC patients with these RTK activations.     

The future of bladder cancer therapy: Optimizing the inhibition of the fibroblast growth factor receptor

Therapeutic options for metastatic bladder cancer (BC) have seen minimal evolution over the past 30 years, with platinum-based chemotherapy remaining the mainstay of standard of care for metastatic BC. Recently, five immune checkpoint inhibitors (ICIs) have been approved by the FDA as second-line therapy, and two ICIs are approved as first-line treatment in selected patients. Molecular alterations of muscle-invasive bladder cancer (MIBC) have been reported by The Cancer Genome Atlas. About 15% of patients with MIBC have molecular alterations in the fibroblast growth factor (FGF) axis. Several ongoing trials are testing novel FGF receptor (FGFR) inhibitors in patients with FGFR genomic aberrations. Recently, erdafitinib, a pan-FGFR inhibitor, was approved by the FDA in patients with metastatic BC who have progressed on platinum-based chemotherapy. We reviewed the literature over the last decade and provide a summary of current knowledge of FGF signaling, and the prognosis associated with FGFR mutations in BC. We cover the role of FGFR inhibition with non-selective and selective tyrosine kinase inhibitors as well as novel agents in metastatic BC. Efficacy and safety data including insights from mechanism-based toxicity are reported for selected populations of metastatic BC with FGFR aberrations. Current strategies to managing resistance to anti-FGFR agents is addressed, and the importance of developing reliable biomarkers as the therapeutic landscape moves towards an individualized therapeutic approach.     

Molecular subclasses of hepatocellular carcinoma predict sensitivity to fibroblast growth factor receptor inhibition

A recent gene expression classification of hepatocellular carcinoma (HCC) includes a poor survival subclass termed S2 representing about one‐third of all HCC in clinical series. S2 cells express E‐cadherin and c‐myc and secrete AFP. As the expression of fibroblast growth factor receptors (FGFRs) differs between S2 and non‐S2 HCC, this study investigated whether molecular subclasses of HCC predict sensitivity to FGFR inhibition. S2 cell lines were significantly more sensitive (p < 0.001) to the FGFR inhibitors BGJ398 and AZD4547. BGJ398 decreased MAPK signaling in S2 but not in non‐S2 cell lines. All cell lines expressed FGFR1 and FGFR2, but only S2 cell lines expressed FGFR3 and FGFR4. FGFR4 siRNA decreased proliferation by 44% or more in all five S2 cell lines (p < 0.05 for each cell line), a significantly greater decrease than seen with knockdown of FGFR1‐3 with siRNA transfection. FGFR4 knockdown decreased MAPK signaling in S2 cell lines, but little effect was seen with knockdown of FGFR1‐3. In conclusion, the S2 molecular subclass of HCC is sensitive to FGFR inhibition. FGFR4‐MAPK signaling plays an important role in driving proliferation of a molecular subclass of HCC. This classification system may help to identify those patients who are most likely to benefit from inhibition of this pathway.     

Targeting FGFR in non-small cell lung cancer: implications from the landscape of clinically actionable aberrations of FGFR kinases

Objective:Dysfunction in fibroblast growth factor receptor (FGFR) signaling has been reported in diverse cancer types, including non-small cell lung cancer (NSCLC). The frequency of FGFR aberrations in Chinese NSCLC patients is therefore of great clinical significance.Methods:A total of 10,966 NSCLC patients whose tumor specimen and/or circulating cell-free DNA (cfDNA) underwent hybridization capture-based next-generation sequencing were reviewed. Patients’ clinical characteristics and treatment histories were also evaluated.Results:FGFR aberrations, including mutations, fusions, and gene amplifications, were detected in 1.9% (210/10,966) of the population. FGFR abnormalities were more frequently observed in lung squamous cell carcinomas (6.8%, 65/954) than lung adenocarcinomas (1.3%, 128/9,596). FGFR oncogenic mutations were identified in 19 patients (∼0.17%), of which, 68% were male lung squamous cell carcinoma patients. Eleven out of the 19 patients (58%) had concurrent altered PI3K signaling, thus highlighting a potential combination therapeutic strategy of dual-targeting FGFR and PI3K signaling in such patients. Furthermore, FGFR fusions retaining the intact kinase domain were identified in 12 patients (0.11%), including 9 FGFR3-TACC3, 1 FGFR2-INA, 1 novel FGFR4-RAPGEFL1, and 1 novel fusion between the FGFR1 and SLC20A2 5′-untranslated regions, which may have caused FGFR1 overexpressions. Concomitant EGFR mutations or amplifications were observed in 6 patients, and 4 patients received anti-EGFR inhibitors, in whom FGFR fusions may have mediated resistance to anti-EGFR therapies. FGFR amplification was detected in 24 patients, with the majority being FGFR1 amplifications. Importantly, FGFR oncogenic mutations, fusions, and gene amplifications were almost always mutually exclusive events.Conclusions:We report the prevalence of FGFR anomalies in a large NSCLC population, including mutations, gene amplifications, and novel FGFR fusions.     

Depletion of CLK2 sensitizes glioma stem-like cells to PI3K/mTOR and FGFR inhibitors

The Cdc2-like kinases (CLKs) regulate RNA splicing and have been shown to suppress cell growth. Knockdown of CLK2 was found to block glioma stem-like cell (GSC) growth in vivo through the AKT/FOXO3a/p27 pathway without activating mTOR and MAPK signaling, suggesting that these pathways mediate resistance to CLK2 inhibition. We identified CLK2 binding partners using immunoprecipitation assays and confirmed their interactions in vitro in GSCs. We then tested the cellular viability of several signaling inhibitors in parental and CLK2 knockdown GSCs. Our results demonstrate that CLK2 binds to 14-3-3τ isoform and prevents its ubiquitination in GSCs. Stable CLK2 knockdown increased PP2A activity and activated PI3K signaling. Treatment with a PI3K/mTOR inhibitor in CLK2 knockdown cells led to a modest reduction in cell viability compared to drug treatment alone at a lower dose. However, FGFR inhibitor in CLK2 knockdown cells led to a decrease in cell viability and increased apoptosis. Reduced expression of CLK2 in glioblastoma, in combination with FGFR inhibitors, led to synergistic apoptosis induction and cell cycle arrest compared to blockade or either kinase alone.     

Identification and In-Depth Analysis of the Novel FGFR2-NDC80 Fusion in a Cholangiocarcinoma Patient: Implication for Therapy

Fibroblast growth factor receptor 2 (FGFR2) fusions have emerged as a new therapeutic target for cholangiocarcinoma in clinical practice following the United States Food and Drug Administration (FDA) approval of Pemigatinib in May 2020. FGFR2 fusions can result in a ligand-independent constitutive activation of FGFR2 signaling with a downstream activation of multiple pathways, including the mitogen-activated protein (MAPK) cascade. Until today, only a limited number of fusion partners have been reported, of which the most prevalent is BicC Family RNA Binding Protein (BICC1), representing one-third of all detected FGFR2 fusions. Nonetheless, in the majority of cases rare or yet unreported fusion partners are discovered in next-generation sequencing panels, which confronts clinicians with a challenging decision: Should a therapy be based on these variants or should the course of treatment follow the (limited) standard regime? Here, we present the case of a metastasized intrahepatic cholangiocarcinoma harboring a novel FGFR2-NDC80 fusion, which was discussed in our molecular tumor board. The protein NDC80 kinetochore complex component (NDC80) is an integral part of the outer kinetochore, which is involved in microtubule binding and spindle assembly. For additional therapeutic guidance, an immunohistochemical analysis of the predicted fusion and downstream effector proteins was performed and compared to cholangiocarcinoma samples of a tissue microarray. The FGFR2-NDC80 fusion resulted in strong activation of the FGFR2 signaling pathway. These supporting results led to a treatment recommendation of Pemigatinib. Unfortunately, the patient passed away before the commencement of therapy.