Examine the function of APOBEC3B in epithelial ovarian cancer stem cells

总结/摘要 卵巢癌是最致命的妇科恶性肿瘤，经常在晚期诊断。 卵巢癌最具侵袭性的类型之一是上皮性卵巢癌（EOC），5年生存率较低， 超过20%。近年来，PARP抑制剂（PARPi）显示出令人印象深刻的治疗效果， 维持治疗和复发情况。然而，由于PARPi的广泛应用，患者最终 对PARPi产生耐药性，使他们几乎没有治疗选择。小说的发展 克服PAPRi抗性的治疗代表了迫切的未满足的医疗需求。抗PARPi 患者通常具有富集的癌症干细胞（CSC），其具有增强的促存活和自我更新能力。 有证据表明，EOC CSCs与原发性肿瘤的生长、转移、疾病复发有关 和对化疗的耐药性，表明CSC是根除EOC的有吸引力的靶点。我们的长期目标 是通过消除这种细胞来确定新的靶点，以开发毒性更小、更有效的治疗方法 人口载脂蛋白B mRNA编辑酶催化多肽样3（APOBEC 3）蛋白是一个家族 在单链DNA上催化胞苷转化为尿苷（C-到-U）的DNA脱氨酶。这个重要的DNA 修饰在广泛的癌症类型中是丰富的，并且代表突变的唯一酶源。 我们发现APOBEC 3B（A3 B）是EOC CSC中主要的DNA突变体。C-to-U DNA突变活性 与非CSC相比，EOC CSC中A3 B表达降低。重要的是，A3 B的抑制导致 PARPi抗性、CSC频率升高和干性因子SOX 2表达增强。这些 新的见解提高了A3 B活化可能使EOC CSC对PARPi敏感的可能性。我们的核心假设 EOC CSC维持低水平A3 B以避免过度的DNA损伤，从而抵消这种效应 关于PARPi本研究的总体目标是剖析A3 B和DNA脱氨作用的机制， 调节CSC维持并促进EOC CSC中对PARPi的反应。我们将讨论我们的假设， 两个具体目的：1）确定修饰的A3 B活性是否会使CSC对PARPi的联合治疗敏感， 2)确定A3 B调节EOC CSC对PARP抑制剂反应的新机制。我们将 在患者来源的类器官模型中进行A3 B过表达研究，并全面评估变化 EOC CSC功能和对PARPi的响应。从机制上讲，我们将评估DNA损伤途径， 染色体异常和过表达A3 B的CSC中ssDNA断裂水平 治疗PARPi。此外，A3 B调节CSC对PARPi反应的分子机制 也将被审查。这项创新的研究将提高我们对A3 B如何使EOC CSCs敏感的理解， PARPi通过促进DNA损伤并发现预防卵巢癌的有价值的新治疗靶点 复发这些机制可能广泛适用于其他CSC驱动的恶性肿瘤。

SUMMARY/ABSTRACT Ovarian cancer is the most lethal gynecologic malignancy and is frequently diagnosed at an advanced-stage. One of the most aggressive types of ovarian cancer is epithelial ovarian cancer (EOC) and 5-year survival is less than 20%. In recent years, PARP inhibitors (PARPi) has shown impressive therapeutic efficacy as both maintenance therapy and in recurrent setting. However, since the wide application of PARPi, patients ultimately develop drug resistance to PARPi which leaves them with few treatment options. Thus, the development of novel therapies to overcome PAPRi resistance represents an urgent unmet medical need. The PARPi-resistant patients often have enriched cancer stem cells (CSCs) with enhanced pro-survival and self-renewal capacity. Evidence demonstrations that EOC CSCs are responsible for primary tumor growth, metastasis, disease relapse and resistance to chemotherapy, suggesting CSC is an attractive target for eradicating EOC. Our long-term goal is to identify new targets for development of less toxic and more effective therapies by eliminating this cell population. Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are a family of DNA deaminase that catalyze cytidine to uridine (C-to-U) on single-strand DNA. This important DNA modification is abundant in a wide array of cancer types and represent the only enzymatic source of mutations. We discovered that APOBEC3B (A3B) is the main DNA mutators in EOC CSC. The C-to-U DNA mutation activity and A3B expression is reduced in EOC CSCs compared to non-CSCs. Importantly, inhibition of A3B leads to PARPi resistance, elevated frequency of CSCs, and enhanced expression of stemness factor SOX2. These novel insights raise the possibility that A3B activation may sensitize EOC CSCs to PARPi. Our central hypothesis is that EOC CSCs maintain low level of A3B in order to avoid excess DNA damage thereby counters the effect of PARPi. The overall goal of this study is to dissect the mechanism through which A3B and DNA deamination regulates CSC maintenance and promote response to PARPi in EOC CSC. We will address our hypothesis in two specific aims: 1) Determine if modified A3B activity will sensitize CSCs to combination therapy with PARPi, 2) Identify the novel mechanisms by which A3B regulates response to PARP inhibitor in EOC CSCs. We will perform A3B overexpression studies in patient-derived organoid models and comprehensively evaluate changes in EOC CSC functions and the response to PARPi. Mechanistically, we will assess DNA damage pathways, chromosomal abnormalities, and ssDNA break levels in CSCs that overexpress A3B alone and in combination treatment with PARPi. In addition, the molecular mechanism of how A3B regulates CSC’s response to PARPi will also be examined. This innovative study will improve our understanding of how A3B sensitize EOC CSCs to PARPi by promoting DNA damage and uncover valuable novel therapeutic targets to preventing ovarian cancer relapse. These mechanisms may be broadly applicable to other CSC-driven malignancies.