Howard Hughes Medical Institute, Department of Genetics, Ludwig Center, Harvard Medical School, Boston, MA 02115, USA; Division of Genetics, Brigham and Women's Hospital, Boston, MA 02115, USA.
Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.
Mol Cell. 2019 Mar 7;73(5):885-899.e6. doi: 10.1016/j.molcel.2018.12.008. Epub 2019 Jan 24.
BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.
BRCA1 或 BRCA2 的失活会导致乳腺癌和卵巢癌，但也会使多聚（ADP-核糖）聚合酶（PARP）抑制剂变得脆弱。为了寻找其他在 BRCA2 缺陷背景下抑制具有合成致死性的靶点，我们使用针对 DNA 修复的短发夹 RNA（shRNA）和基于 CRISPR（成簇的规则间隔的短回文重复序列）的文库筛选了两对 BRCA2 同基因细胞系。我们发现 BRCA2 缺陷细胞对包括碱基切除修复、ATR 信号和剪接在内的多条途径存在选择性依赖性。我们鉴定了 APEX2 和 FEN1 作为与 BRCA1 和 BRCA2 功能丧失均具有合成致死性的基因。BRCA2 缺陷细胞需要无嘌呤内切酶活性和 PCNA 结合结构域的 Ape2（APEX2），而不是 Ape1（APEX1）。此外，BRCA2 缺陷细胞需要 5' 发夹内切酶而不是 Fen1 的 5'-3' 外切酶活性，并且化学抑制 Fen1 可选择性靶向 BRCA 缺陷细胞。最后，我们开发了一个微同源介导的末端连接（MMEJ）报告，并表明 Fen1 参与 MMEJ，强调了在同源重组（HR）缺陷背景下 MMEJ 作为旁系修复途径的重要性。
