Elucidating the Functional and Mechanistic Roles of LINE-1 Retrotransposons in Myeloid Leukemia

项目总结 反转录转座子在人类癌症中的作用历来被低估，因为它们 被认为是无关紧要的“垃圾”DNA，因为它们的高度重复和变异造成了显著的影响 技术挑战。LINE-1(或L1)反转录转座子有大约500,000个拷贝，占17% 人类基因组，但只有大约100个全长人类特异性L1元件(L1H)能够激活 逆转位。L1编码的蛋白质还可以动员非自主反转录转座子、非编码RNA和 MRNAs，导致产生三分之一的人类DNA。因此，L1被考虑的主要是 有害的，因为它们的活性会导致基因突变和染色体改变，从而导致 包括癌症在内的人类疾病。通过基于结构域的人染色质调节蛋白CRISPR筛选，我们 鉴定了MPP8，HUSH复合体的一个成分，负责沉默L1反转录转座子，是一种 急性髓系白血病(AML)细胞的选择性表观遗传依赖性。虽然对于正常来说是可有可无的 造血、MPP8缺失可能通过重新激活L1s而影响AML的启动和维持。异位L1激活 表型复制MPP8缺失，而阻断L1逆转座则取消表型。L1抑制是 与人类急性髓系白血病患者的治疗抵抗和预后不良有关。因此，当反转录转座子 在历史上被认为是作为遗传不稳定和体细胞的来源而促进癌症的作用 突变，我们的发现支持反转录转座子作为选择性基因具有意想不到的“肿瘤抑制”功能 髓系白血病的依赖性。本项目的目标是阐明细胞的功能和机制 L1反转录转座子作为AML病理生理学新的调控途径。中心假设是L1 反转录转座子在AML启动过程中调节DNA损伤反应和/或炎症信号的作用 L1活性控制髓系白血病对DNA损伤诱导剂的治疗反应。 我们的假设是在使用正交法进行广泛的初步研究的基础上提出的 在生理学相关AML模型中调节L1功能的方法和一种新发现的分子 抑制反转录转座子和致癌细胞增殖之间的联系。在这些指导下 研究发现，这些假说将通过三个具体的目标来检验：1)建立母语的功能角色 反转录转座子在髓系白血病克隆进化中的作用。2)阐明第一语言的机制作用 反转录转座子在AML发病机制中的作用。3)确定调节反转录转座子活性对 髓系白血病对DNA损伤诱导剂的治疗反应。总而言之，这些研究将 不仅阐明了逆转座子意想不到的肿瘤抑制功能的机制基础 在髓系白血病中的活性，也建立了一个新的范式，作为以前的流动DNA元件 白血病生物学中未知的调控途径，其干预可能导致抗白血病作用的改善 治疗。

PROJECT SUMMARY The role of retrotransposons in human cancer has been historically underappreciated both because they have been dismissed as unimportant ‘junk’ DNA, and because their high repetition and variation pose significant technical challenges. There are ~500,000 copies of LINE-1 (or L1) retrotransposons occupying 17% of the human genome, but only ~100 full-length human-specific L1 elements (L1Hs) are capable of active retrotransposition. L1-encoded proteins also mobilize non-autonomous retrotransposons, noncoding RNAs and mRNAs, leading to the generation of a third of human DNA. As such, L1s have been considered mostly deleterious because their activity can lead to genetic mutations and chromosomal alterations that contribute to human disorders including cancer. By domain-based CRISPR screens of human chromatin regulators, we identified MPP8, a component of the HUSH complex responsible for the silencing of L1 retrotransposons, as a selective epigenetic dependency of acute myeloid leukemia (AML) cells. While dispensable for normal hematopoiesis, MPP8 loss impaired AML initiation and maintenance by reactivating L1s. Ectopic L1 activation phenocopied MPP8 loss, whereas blocking L1 retrotransposition abrogated the phenotypes. L1 suppression is associated with therapy resistance and poor prognosis in human AML patients. Hence, while retrotransposons are historically recognized for their cancer-promoting roles as sources of genetic instability and somatic mutations, our findings support an unexpected ‘tumor-suppressive’ function for retrotransposons as a selective dependency for myeloid leukemia. The goal of this project is to elucidate the functional and mechanistic roles of L1 retrotransposons as a new regulatory pathway in AML pathophysiology. The central hypotheses are that L1 retrotransposons function to modulate DNA damage response and/or inflammatory signaling in AML-initiating cells, and that L1 activity controls therapeutic response to DNA damage-inducing agents in myeloid leukemia. Our hypotheses have been formulated on the basis of extensive preliminary studies using orthogonal approaches to modulate L1 function in physiologically relevant AML models and a newly discovered molecular link between suppression of retrotransposons and propagation of cancer-initiating cells. Guided by these findings, these hypotheses will be tested by three specific aims: 1) Establish the functional roles of L1 retrotransposons in the clonal evolution of myeloid leukemia. 2) Elucidate the mechanistic roles of L1 retrotransposons in AML pathogenesis. 3) Determine the effects of modulating retrotransposon activity on therapeutic response to DNA damage-inducing agents in myeloid leukemia. Taken together, these studies will not only elucidate the mechanistic basis for the unexpected ‘tumor-suppressive’ function for retrotransposon activity in myeloid leukemia, but also establish a new paradigm for mobile DNA elements as a previously uncharted regulatory pathway in leukemia biology, whose intervention may lead to improved anti-leukemia therapies.