Harnessing Artificial MicroRNA Clusters Against Glioblastoma Epigenetic Plasticity and Resistance to Therapy

胶质母细胞瘤是成人中最常见的原发性脑癌，仍然是一种致命的疾病。尽管 尽管进行了大量的研究和临床努力，但在过去几十年中，其生存率并没有显著提高。一 这种不稳定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。这种阻力取决于 激活防御机制，以应对损害，并介导的表观遗传学的复杂，多- 控制肿瘤细胞转录状态的组分机制。我们的研究已经定义了 致癌蛋白EZH 2、BMI 1和LSD 1是这种表观遗传反应的关键组分。我们还 证明它们受特定microRNA（miR-124、miR-128和miR-137）调节，共表达 在正常大脑中，同时在胶质母细胞瘤中丢失。这三种microRNA的重新表达在 胶质母细胞瘤提供了对这三种蛋白质的强烈抑制，并导致非常相关的致敏作用 肿瘤细胞的压力，无论是在体外还是体内。这项建议的总体目标有两个方面：第一， 表征EZH 2、BMI 1和LSD 1的共激活介导肿瘤存活的机制， 建立其在这种反应中的必要作用;第二，开发一种microRNA介导的策略， 这种对肿瘤至关重要的表观遗传屏障，以支持与当前基因治疗方法协同的基因治疗方法， 标准疗法AIM 1提出实验来剖析EZH 2/BMI 1/LSD 1表观遗传的作用， 肿瘤对替莫唑胺、放射和免疫疗法的反应。AIM 2提供新颖的 设计和构建人工基因的解决方案，使microRNA的有效载荷倍增，从而 作为一个理想的基因治疗平台，多靶向这种表观遗传反应。AIM 3调查了 基于多microRNA的治疗在胶质母细胞瘤动物模型中的适用性，以解决 体内肿瘤抗性。创新：拟议的研究使用人工microRNA簇作为对照， 复杂的表观遗传肿瘤生存反应的研究是一种创新的和迄今为止尚未探索的方法。还有， 该提案介绍了利用microRNA生物学、加工和 从整合基因治疗方法的角度来看，细胞间运输。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聚类、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.