Targeting Rad6 Postreplication DNA Repair to Treat Triple Negative Breast Cancer

描述（由申请人提供）：乳腺癌（BrCa）是美国女性癌症死亡的第二大原因，三阴性乳腺癌（TNBC）占这些癌症的约15-20%。TNBC的治疗带来了临床挑战，因为它们不能用靶向雌激素受体和Her 2/neu的疗法治疗，因为它们缺乏雌激素、孕酮和Her 2/neu受体的表达。TNBC与较短的复发和死亡时间相关。携带生殖系BRCA 1突变的个体中约70%的BrCas是三阴性的，TNBC中BRCA 1突变的发生率为16- 42%。BRCA 1相关的BrCa具有异常的DNA修复，并且由于TNBC与BRCA 1相关的BrCa具有几种组织学特征，因此DNA修复途径被认为在TNBC的发展和治疗反应中起重要作用。在美国，已经转向用基于铂（Pt）的疗法治疗TNBC。铂化合物引起DNA交联断裂（ICL）。ICL的修复需要BRCA/Fanconi贫血（FA）网络和复制后DNA修复（PRR）途径的活性。PRR途径通过使细胞在面对损伤时完成DNA复制以避免有丝分裂灾难来赋予对DNA损伤的耐受性，并且可以是无错误的或容易出错的。导致TNBC进展和治疗耐药性的机制尚未得到很好的理解，迄今为止尚未确定可用于预防和治疗的关键靶标。在本申请中，我们提出靶向Rad 6（PRR途径的主要组分）将通过防止获得抗性和克服Pt抗性而有益于用Pt处理的TNBC。Rad 6基因编码泛素（Ub）结合酶，其催化活性对PRR功能至关重要。Rad 6 B在BrCa中过表达。正常乳腺细胞中Rad 6 B的组成性过表达诱导非整倍体和顺铂（CDDP）抗性，而Rad 6 B抑制赋予CDDP敏感性。Rad 6表达和CDDP敏感性之间的这种关系与PRR活性直接相关。PRR活性是易错的还是无错的取决于PCNA是被Rad 6单泛素化还是多泛素化。Rad 6还通过促进FancD 2泛素化（ICL修复的关键事件）来调节FA途径活化。我们已经确定了一个小分子抑制剂（SMI）的Rad 6，其目标是它的Ub共轭活性。用Rad 6 SMI处理MDA-MB-231 TNBC细胞减弱CDDP诱导的PCNA和FancD 2泛素化，并增强CDDP敏感性。我们假设Rad 6是刺激Pt诱导的FA/BRCA修复途径的主要参与者。我们提出，抑制Rad 6将通过使PRR失活并随后破坏PRR与FA通路的串扰而使TNBC对Pt治疗敏感。我们将用以下两个具体目标来检验这一假设：（1）确定Rad 6在BRCA 1野生型和BRCA 1突变型TNBC细胞中的ICL修复中的功能作用。(2)使用体内和新型体外三维培养平台确定Rad 6干预在治疗BRCA 1野生型和BRCA 1突变TNBC细胞中的治疗效用。

DESCRIPTION (provided by applicant): Breast cancer (BrCa) is the second leading cause of cancer mortality in women in the U.S., and triple negative breast cancers (TNBCs) comprise ~15-20% of these cancers. Treatment of TNBC poses a clinical challenge because they are not treatable with therapies targeting estrogen receptor and Her2/neu as they lack expression of estrogen, progesterone, and Her2/neu receptors. TNBCs are associated with a shorter time to recurrence and death. Approximately 70% of BrCas in individuals carrying a germline BRCA1 mutation are triple negative, and the incidence of BRCA1 mutations in TNBC ranges from 16-42%. BRCA1-associated BrCas have aberrant DNA repair, and since TNBCs share several histologic features with BRCA1-related BrCa, DNA repair pathways are thought to play a significant role in TNBC development and therapy response. In the U.S., there has been a shift to treating TNBCs with platinum (Pt)-based therapies. Pt compounds cause DNA crosslink breaks (ICLs). Repair of ICLs require activities of BRCA/Fanconi anemia (FA) network and postreplication DNA repair (PRR) pathways. PRR pathway confers tolerance to DNA damage by enabling cells to complete DNA replication in the face of damage in order to avoid mitotic catastrophe, and can be error-free or error-prone. The mechanisms contributing to progression and therapy resistance in TNBC are not well understood and no key targets useful for prevention and treatment have thus far been identified. In this application, we propose that targeting Rad6, a principal component of the PRR pathway, will be beneficial to TNBCs treated with Pt by preventing acquisition of resistance and overcoming Pt resistance. The Rad6 gene encodes an ubiquitin (Ub) conjugating enzyme, and its catalytic activity is essential for PRR function. Rad6B is overexpressed in BrCa. Constitutive overexpression of Rad6B in normal breast cells induces aneuploidy and cisplatin (CDDP) resistance, whereas Rad6B suppression confers CDDP sensitivity. This relationship between Rad6 expression and CDDP sensitivity is directly related to PRR activity. Whether the PRR activity is error-prone or error-free is dependent upon whether PCNA is mono- or poly-ubiquitinated, respectively, by Rad6. Rad6 also regulates FA pathway activation by promoting FancD2 ubiquitination, a critical event for ICL repair. We have identified a small molecule inhibitor (SMI) of Rad6 that targets its Ub conjugating activity. Treatment of MDA-MB-231 TNBC cells with Rad6 SMI attenuates CDDP-induced PCNA and FancD2 ubiquitination, and enhances CDDP sensitivity. We hypothesize that Rad6 is a major player in stimulation of Pt-induced FA/BRCA repair pathway. We propose that inhibiting Rad6 will sensitize TNBCs to Pt therapy by inactivating PRR and consequent disruption of PRR crosstalk with the FA pathway. We will test this hypothesis with the following two specific aims: (1) Determine the functional role of Rad6 in ICL repair in BRCA1 wild type and BRCA1 mutant TNBC cells. (2) Determine the therapeutic utility of Rad6 intervention in treatment of BRCA1 wild type and BRCA1 mutant TNBC cells using in vivo and a novel in vitro three-dimensional culture platform.