Targeting sideroflexin 4, a mitochondrial inner membrane protein involved iniron sulfur cluster biogenesis, to enhance the efficacy of DNA-damaging drugs inovarian cancer

摘要 今天被诊断出患有卵巢癌的女性长期生存机会只有很小的改善 与40年前确诊的女性相比两个不同但相关的观察可能提供一个 改善卵巢癌患者的前景的机会。两者都涉及DNA损伤和修复。1)的 最有效和广泛使用的药物，铂化合物（顺铂和卡铂），失去了它们的活性。 随着时间的推移有效性-耐药性的发展。耐药性的一个重要（但不是唯一）原因 是卵巢癌修复顺铂DNA加合物引起的损伤的获得性能力。2)不同的 一类诱导DNA损伤的药物，PARP抑制剂，最近已被FDA批准用于 有DNA修复缺陷的患者，如BRACA 1/2的生殖系或获得性突变。不幸的是， 具有DNA修复基因缺陷的患者仅代表卵巢癌患者的一小部分。 我们认为，靶向线粒体蛋白铁黄素4（SFXN 4）可能提供一种途径，解决 这两个问题。我们发现，降低SFXN 4的水平会破坏铁硫簇的形成， 卵巢癌细胞的线粒体和胞浆。我们认为Fe-S团簇的损失将具有主要的 影响卵巢癌的细胞后果。首先，铁硫蛋白的减少将增加氧化应激 以及通过细胞铁的获取和重新分配造成DNA损伤。第二，因为关键酶 参与NER（核苷酸切除修复）和HRR（同源重组修复）的蛋白质需要Fe-S簇 对于它们的功能和稳定性，靶向SFXN 4将降低这些修复途径的DNA活性。给定 减少SFXN 4既能诱导DNA损伤又能抑制DNA修复，我们进一步探讨了这种可能性， 假设SFXN 4破坏可能增强铂基化合物的功效， PARP抑制剂对没有DNA修复缺陷的卵巢癌的有效性：即，使这些 癌症对铂类药物和PARP抑制剂非常敏感，比如BRCA突变细胞。在试点 实验中，我们确实观察到抑制SFXN 4 1）抑制DNA修复蛋白; 2）增强 顺铂敏感性; 3）卵巢癌细胞对PARP抑制剂敏感。 我们提出了三个具体的目的来研究SFXN 4在卵巢癌中的作用。在目标1中，我们探讨如何 以及SFXN 4的减少在多大程度上增加了氧化应激，限制了DNA修复并增强了DNA修复。 损害在目标2中，我们研究了靶向SFXN 4在增强铂类和非铂类药物的有效性方面的作用。 基于化合物和PARP抑制剂的细胞培养物和小鼠模型。在目标3中，我们确定精确的 SFXN 4在Fe-S簇生物发生中发挥作用的分子位点和机制，并将其直接联系起来 影响NER和HRR途径中DNA修复蛋白的减少。 影响：靶向SFXN 4可能代表了增强铂类药物疗效的机会， 将PARP抑制剂的使用扩展到大多数具有正常DNA修复功能的卵巢癌患者。

ABSTRACT A woman diagnosed today with an ovarian cancer has only a minimally improved chance of long term survival compared to a woman diagnosed 40 years ago. Two different yet related observations may provide an opportunity to improve the outlook of ovarian cancer patients. Both involve DNA damage and repair. 1) The most effective and widely used drugs, the platinum compounds (cisplatin and carboplatin), lose their effectiveness over time – drug resistance develops. An important (but not the only) reason for drug resistance is an acquired ability of ovarian cancers to repair the damage caused by cisplatin DNA adducts. 2) A different class of drugs that induce DNA damage, the PARP inhibitors, has recently been approved by the FDA for patients with DNA repair defects, such as germline or acquired mutations in BRACA1/2. Unfortunately, patients with defects in DNA repair genes represent only a small fraction of ovarian cancer patients. We propose that targeting the mitochondrial protein sideroflexin 4 (SFXN4) may provide a path to addressing both these problems. We discovered that reducing the levels of SFXN4 disrupted Fe-S cluster formation in the mitochondria and cytosol of ovarian cancer cells. We propose that the loss of Fe-S clusters will have major cellular consequences affecting ovarian cancer. First, reduction of Fe-S proteins will increase oxidative stress and DNA damage via the acquisition and redistribution of cellular iron. Second, because critical enzymes involved in NER (nucleotide excision repair) and HRR (homologous recombination repair) require Fe-S clusters for their function and stability, targeting SFXN4 will reduce the activity of DNA these repair pathways. Given the possibility that reduction of SFXN4 could both induce DNA damage and inhibit DNA repair, we further hypothesize that disruption of SFXN4 might enhance the efficacy of platinum-based compounds and expand the effectiveness of PARP inhibitors to ovarian cancers without defects in DNA repair: i.e., render these cancers exquisitely sensitive, like BRCA-mutant cells, to platinum-based drugs and PARP inhibitors. In pilot experiments, we indeed observed that inhibiting SFXN4 1) inhibited DNA repair proteins; 2) enhanced sensitivity to cisplatin; 3) sensitized ovarian cancer cells to PARP inhibitors. We propose three Specific Aims to study the effects of SFXN4 in ovarian cancer. In Aim 1, we explore how and to what extent reduction of SFXN4 increases oxidative stress, limits DNA repair and enhances DNA damage. In Aim 2, we examine the role of targeting SFXN4 in enhancing the effectiveness of both platinum- based compounds and PARP inhibitors in cell cultures and mouse models. In Aim 3 we determine the precise molecular site and mechanism by which SFXN4 functions in Fe-S cluster biogenesis, and directly link this effect to the reduction of DNA repair proteins in NER and HRR pathways. Impact: targeting SFXN4 may represent an opportunity to enhance the efficacy of platinum-based drugs and extend the use of PARP inhibitors to the majority of ovarian cancer patients with normal DNA repair function.