CRISPR targeting of FOXL2 c.402C>G mutation reduces malignant phenotype in granulosa tumor cells and identifies anti-tumoral compounds

FOXL2 is a transcription factor that plays a key role in sex determination, ovary development and maintenance. Mutations related to this gene have been described in syndromes involving premature ovarian failure and granulosa cell tumors. This kind of rare cancer (less than 5% of diagnosed ovarian cancers) has been causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we have used CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results indicate that this Cas9-mediated strategy allows the specific elimination of the mutation with no activity on the wild type allele. Granulosa cells depleted on FOXL2 c.402C>G show a reduced malignant phenotype. Specifically, we detect changes in cell proliferation, invasion, and cell death levels. In addition, we show that granulosa tumor cells become more susceptible to Dasatinib and Ketoconazole treatments when FOXL2 c.402C>G allele is eliminated. Our transcriptomic and proteomic analyses indicate that CRISPR-modified granulosa tumor cells significantly change their expression signature towards a wild type like phenotype. Finally, this expression signature has led us to discover new compounds with antiproliferative and proapoptotic effects on granulosa cell tumor cells. Our results demonstrate the potential of CRISPR for specifically targeting and eliminating a granulosa cell tumor-causing mutation, as well as its therapeutic potential for the treatment of this rare ovarian cancer.

叉头框转录因子L2（FOXL2）是一种转录因子，在性别决定、卵巢发育与维持过程中发挥关键作用。该基因的相关突变已在早发性卵巢功能不全与颗粒细胞瘤（granulosa cell tumors, GCTs）相关的综合征中被报道。这类罕见癌症（占确诊卵巢癌的比例不足5%）在97%的成人病例（成人颗粒细胞瘤，Adult Granulosa Cell Tumors, AGCTs）中，与FOXL2基因c.402C>G、p.C134W突变存在明确的因果关联。本研究采用CRISPR技术，在颗粒细胞瘤细胞中特异性消除FOXL2 c.402C>G突变。研究结果显示，该Cas9介导的编辑策略可特异性清除目标突变，且对野生型等位基因无编辑活性。缺失FOXL2 c.402C>G突变的颗粒细胞瘤细胞，其恶性表型显著减弱。具体而言，我们检测到细胞增殖、侵袭与细胞死亡水平均发生显著改变。此外，我们发现当FOXL2 c.402C>G突变等位基因被消除后，颗粒细胞瘤细胞对达沙替尼（Dasatinib）与酮康唑（Ketoconazole）的药物敏感性显著提升。转录组与蛋白质组分析结果表明，经CRISPR编辑的颗粒细胞瘤细胞，其基因表达特征向野生型表型发生显著重塑。基于该表达特征，我们筛选得到一批对颗粒细胞瘤细胞具有抗增殖与促凋亡活性的新型化合物。本研究结果证实，CRISPR技术可特异性靶向并消除导致颗粒细胞瘤的致病变异，同时展现出治疗这类罕见卵巢癌的治疗潜力。