Acquired FGFR and FGF alterations confer resistance to estrogen receptor (ER) targeted therapy in ER+ metastatic breast cancer

Beyond acquired mutations in the estrogen receptor (ER), mechanisms of resistance to ER-directed therapies in ER+ breast cancer have not been clearly defined. We conducted a genome-scale functional screen spanning 10,135 genes to investigate genes whose overexpression confer resistance to selective estrogen receptor degraders. Pathway analysis of candidate resistance genes demonstrated that the FGFR, ERBB, insulin receptor, and MAPK pathways represented key modalities of resistance. In parallel, we performed whole exome sequencing in paired pre-treatment and post-resistance biopsies from 60 patients with ER+ metastatic breast cancer who had developed resistance to ER-targeted therapy. The FGFR pathway was altered via FGFR1, FGFR2, or FGF3/FGF4 amplifications or FGFR2 mutations in 24 (40%) of the post-resistance biopsies. In 12 of the 24 post-resistance tumors exhibiting FGFR/FGF alterations, these alterations were not detected in the corresponding pre-treatment tumors, suggesting that they were acquired or enriched under the selective pressure of ER-directed therapy. In vitro experiments in ER+ breast cancer cells confirmed that FGFR/FGF alterations led to fulvestrant resistance as well as cross-resistance to the CDK4/6 inhibitor palbociclib, through activation of the MAPK pathway. The resistance phenotypes were reversed by FGFR inhibitors and, to a lesser extent, MEK inhibitors, suggesting potential treatment strategies. Examination of the transcriptional output (mRNA) of the FGFR activation, with T47D cells perturbed to overexpress FGFR pathway activation including FGFR1, FGFR2 (WT, K660N, M538I and N550K), and FGF3, as well as GFP and parental as a control. DMSO, fulvestrant (100 nM), palbociclib (1 uM), FIIN-3 (100 nM), and trametinib (500 nM) as single agent and in combinations for 24 hours. All experimental conditions were done in 6 replicates

除雌激素受体（Estrogen Receptor, ER）的获得性突变之外，ER阳性（ER+）乳腺癌对ER靶向治疗的耐药机制尚未得到明确阐释。本研究开展了覆盖10135个基因的全基因组功能筛选，旨在鉴定过表达可赋予细胞对选择性雌激素受体降解剂（selective estrogen receptor degraders, SERDs）耐药性的基因。对候选耐药基因的通路富集分析显示，成纤维细胞生长因子受体（FGFR）、ERBB、胰岛素受体及丝裂原活化蛋白激酶（MAPK）通路是耐药产生的关键机制。与此同时，我们对60例经ER靶向治疗后出现耐药的ER阳性转移性乳腺癌患者的配对治疗前及耐药后活检组织进行了全外显子组测序。在24例（占比40%）耐药后活检组织中，FGFR通路通过FGFR1、FGFR2或FGF3/FGF4基因扩增，或FGFR2突变发生了改变。在24例存在FGFR/FGF异常的耐药后肿瘤中，有12例的配对治疗前肿瘤未检测到此类异常，提示这些异常是在ER靶向治疗的选择压力下获得或富集的。ER阳性乳腺癌细胞的体外实验证实，FGFR/FGF异常可通过激活MAPK通路导致氟维司群耐药，以及对细胞周期蛋白依赖性激酶4/6（CDK4/6）抑制剂帕博西尼的交叉耐药。FGFR抑制剂可逆转该耐药表型，MEK抑制剂也可部分逆转，这提示了潜在的治疗策略。为检测FGFR激活后的转录产物（mRNA），本研究对T47D细胞进行基因扰动以激活FGFR通路，具体包括过表达FGFR1、FGFR2（野生型WT、K660N、M538I及N550K突变体）及FGF3，并以GFP转染及亲本细胞作为对照。实验设置了单药及联合给药组，分别为二甲基亚砜（DMSO）、氟维司群（100 nM）、帕博西尼（1 μM）、FIIN-3（100 nM）及曲美替尼（500 nM），处理细胞24小时。所有实验条件均设置6次生物学重复。