Targeting Rad6 Postreplication DNA Repair to Treat Triple Negative Breast Cancer

描述(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(TNBCs)约占这些癌症的15%-20%。TNBC的治疗是一个临床挑战，因为它们不能用针对雌激素受体和Her2/neu的治疗，因为它们缺乏雌激素、孕激素和Her2/neu受体的表达。TNBCs的复发和死亡时间较短。在携带种系BRCA1突变的个体中，大约70%的BRCA是三阴性的，而在TNBC中BRCA1突变的发生率从16%到42%不等。BRCA1相关的BRCA具有异常的DNA修复，由于TNBCs与BRCA1相关的BRCA有一些共同的组织学特征，DNA修复通路被认为在TNBC的发展和治疗反应中发挥着重要作用。在美国，已经有了用基于铂的疗法来治疗TNBCs的转变。PT化合物导致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)途径的激活。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而赋予对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进展和治疗耐药的机制尚不清楚，到目前为止还没有确定对预防和治疗有用的关键靶点。在这一应用中，我们认为靶向PrR途径的主成分Rad6将通过防止获得电阻和克服铂电阻而有益于经铂处理的TNBCs。Rad6基因编码一种泛素(Ub)结合酶，其催化活性是PRR功能所必需的。RAD6B在BRCA中高表达。在正常乳腺细胞中过表达Rad6B会导致非整倍体和顺铂(CDDP)耐药，而抑制Rad6B则会增加顺铂的敏感性。这种Rad6表达与CDDP敏感性之间的关系与PRR活性直接相关。PRR活性是否容易出错取决于增殖细胞核抗原分别是被Rad6单泛素化还是多泛素化。RAD6还通过促进FANCD2泛素化来调节FA途径的激活，FANCD2泛素化是ICL修复的关键事件。我们已经确定了一种针对Rad6的Ub结合活性的小分子抑制剂(SMI)。用RAD6 SMI处理MDA-MB-231 TNBC细胞可减弱CDDP诱导的增殖细胞核抗原和FANCD2泛素化，并增强CDDP的敏感性。我们推测，Rad6在铂诱导的FA/BRCA修复通路的刺激中起主要作用。我们认为，抑制RAD6将通过使PRR失活，从而破坏PRR与FA通路的串扰，从而使TNBCs对铂治疗敏感。我们将通过以下两个具体目标来验证这一假说：(1)确定Rad6在BRCA1野生型和BRCA1突变型TNBC细胞ICL修复中的功能作用。(2)利用体内和新型体外三维培养平台，研究RAD6干预对BRCA1野生型和突变型TNBC细胞的治疗作用。

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.