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15-20%。由于TNBC缺乏雌激素、孕激素和Her2/neu受体的表达，因此针对雌激素受体和Her2/neu的治疗方法无法治疗，因此TNBC的治疗面临着临床挑战。tnbc与较短的复发和死亡时间相关。携带种系BRCA1突变的个体中约70%的BrCas为三阴性，TNBC中BRCA1突变的发生率为16-42%。brca1相关的BrCas具有异常的DNA修复，并且由于TNBC与brca1相关的BrCa具有一些组织学特征，因此DNA修复途径被认为在TNBC的发展和治疗反应中发挥重要作用。在美国，以铂为基础的疗法治疗tnbc已经发生了转变。铂化合物引起DNA交联断裂（ICLs）。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通路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，从而赋予DNA损伤耐受性，并且可以是无错误或容易出错的。促进TNBC进展和治疗耐药的机制尚不清楚，迄今为止还没有确定预防和治疗的关键靶点。在这项应用中，我们提出靶向PRR途径的主要成分Rad6，将通过阻止获得抗性和克服Pt抗性，有利于Pt处理的tnbc。Rad6基因编码一种泛素（Ub）偶联酶，其催化活性对PRR功能至关重要。Rad6B在BrCa中过表达。Rad6B在正常乳腺细胞中的组成性过表达可诱导非整倍体和顺铂（CDDP）抗性，而Rad6B抑制可使CDDP敏感。Rad6表达与CDDP敏感性之间的关系与PRR活性直接相关。PRR活性是易出错还是无错误取决于PCNA分别被Rad6单泛素化还是多泛素化。Rad6还通过促进FancD2泛素化（ICL修复的一个关键事件）来调节FA通路的激活。我们已经确定了一个Rad6的小分子抑制剂（SMI），其目标是其Ub偶联活性。Rad6 SMI处理MDA-MB-231 TNBC细胞可减弱CDDP诱导的PCNA和FancD2泛素化，增强CDDP敏感性。我们假设Rad6是刺激pt诱导的FA/BRCA修复通路的主要参与者。我们提出，抑制Rad6将通过使PRR失活，从而破坏PRR与FA通路的串扰，使tnbc对Pt治疗敏感。我们将通过以下两个具体目的来验证这一假设：(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，确定Rad6干预治疗BRCA1野生型和BRCA1突变型TNBC细胞的疗效。

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.