Harnessing artificial microRNA clusters against glioblastoma epigenetic plasticity and resistance to therapy

胶质母细胞瘤是成人最常见的原发脑癌，仍然是一种致命的疾病。尽管 尽管进行了密集的研究和临床努力，但在过去的几十年里，它的存活率并没有显著提高。一个 这种顽固性的主要原因是肿瘤能够逃避目前可用的毒性 治疗，包括替莫唑胺(TMZ)、放射和免疫调节。这一阻力取决于 激活防御机制以响应损伤，并通过表观遗传介导的复杂，多- 控制肿瘤细胞转录状态的组成机制。我们的研究已经明确了 致癌蛋白EZH2、BMI1和LSD1是这种表观遗传反应的关键成分。我们还有 证明它们受特定的microRNAs(miR-124、miR-128和miR-137)调控，共同表达 在正常脑中，在胶质母细胞瘤中同时消失。这三种microRNAs在大肠杆菌中的再表达 胶质母细胞瘤对这三种蛋白有很强的抑制作用，并导致非常相关的致敏作用。 无论是在体外还是在体内，肿瘤细胞的压力都很大。这项建议的总体目标有两个：第一， 描述EZH2、BMI1和LSD1共同激活介导肿瘤生存的机制， 确定其在这一反应中的必要作用；第二，开发一种微RNA介导的策略来减缓 这种表观遗传屏障对肿瘤至关重要，以支持与电流协同的基因治疗方法 标准疗法。AIM1提出实验来剖析EZH2/BMI1/LSD1表观遗传学的作用 肿瘤对替莫唑胺、放射治疗和免疫治疗反应的复杂性。AIM2提供了新颖的 设计和构建人工基因的解决方案，使microRNAs的有效负载成倍增加，从而 作为多靶点这种表观遗传反应的理想基因治疗平台。AIM3调查 基于多种微RNA的治疗在胶质母细胞瘤动物模型中的适用性，以解决关键问题 体内肿瘤耐药性的研究。创新：拟议的研究使用人造microRNA簇进行控制 在复杂的表观遗传肿瘤生存反应中，是一种创新的、迄今未被探索的方法。另外， 这项建议介绍了利用microRNAs生物学、加工和 从综合基因疗法的角度看细胞间的贩运。长期目标： 我们寻求通过应用以下原则对胶质母细胞瘤患者的护理产生有意义的影响 MicroRNA聚集性、表观遗传干扰以及与其他疗法的协同作用。

Glioblastoma is the most common primary brain cancer in adults and remains a deadly disease. Despite intense research and clinical efforts, its survival has not significantly improved for the past several decades. A major reason for this recalcitrant nature is the tumor’s ability to escape toxicity from currently available therapies, including Temozolomide (TMZ), radiation, and immune-modulation. This resistance depends on the activation of defense mechanisms in response to damage, and is mediated epigenetically by a complex, multi- component machinery that governs the transcriptional status of the tumor cells. Our studies have defined oncogenic proteins EZH2, BMI1 and LSD1 as crucial components of this epigenetic response. We have also demonstrated that they are regulated by specific microRNAs (miR-124, miR-128 and miR-137), co-expressed in normal brain, and simultaneously lost in glioblastoma. The re-expression of these three microRNAs in glioblastoma provides a strong inhibition against the three proteins and results in a very relevant sensitization of tumor cells to stress, both in vitro and in vivo. The overall goal of this proposal is two-fold: first, to characterize the mechanism whereby the co-activation of EZH2, BMI1 and LSD1 mediates tumor survival, establishing its necessary role in this response; Second, to develop a microRNA-mediated strategy to abate this epigenetic shield that is vital to the tumor, to support a gene therapy approach synergistic with current standard therapies. AIM1 proposes experiments to dissect the role of the EZH2/BMI1/LSD1 epigenetic complex in tumor responses against Temozolomide, radiation, and immunotherapy. AIM2 provides novel solutions to design and construct artificial genes that multiply the payload of microRNAs, and which thus function as an ideal gene therapy platform for multi-targeting this epigenetic response. AIM3 investigates the applicability of multi-microRNA-based therapy in animal models of glioblastoma, to address the vital problem of tumor resistance in vivo. INNOVATION: The proposed study to use artificial microRNA clusters for the control of the complex epigenetic tumor survival response is an innovative and heretofore unexplored approach. Also, this proposal introduces strategies to exploit the unique features of microRNAs biology, processing and intercellular trafficking in the perspective of an integrated gene therapy approach. LONG-TERM OBJECTIVE: We seek to meaningfully impact the care of glioblastoma patients through the application of principles of microRNA clustering, epigenetic interference, and synergism with other therapies.