Role of an Aberrant N6-Methyladenosine-LncRNA Axis in the Development and Maintenance of Drug Resistance through Regulating the Leukemia Stem Cell

酪氨酸激酶靶向（TKI）疗法已经彻底改变了白血病治疗，但TKI不能杀死 白血病干细胞（LSC），其负责繁殖和疾病复发，并且被认为是 治疗失败的根源。我们的长期目标是进一步解决如何监管LSC持久性， 并开发新的LSC消除治疗策略，以提高治愈率和生存率。短期 本研究的目的是确定N6-甲基腺苷（m6 A）-长非编码RNA（lncRNA） 轴调节LSC的干性和持久性在TKI选择过程中，并探索治疗 靶向m6 A-lncRNA轴根除TKI耐药LSC的潜力，并破译潜在的 分子机制m6 A甲基化是RNA上最常见的表位转录组修饰（即， lncRNA），并关键地调节lncRNA启动的基因表达。lncRNA异常通常与 与癌症疾病进展和耐药性有关。初步证据表明，m6 A-lncRNA轴 耐药LSC来自我们的原理证明研究，证明i）动态和可逆的m6 A 由脂肪量和肥胖相关蛋白（FTO）决定的甲基化组有助于白血病细胞避免TKI杀伤 导致TKI抗性; ii）有许多lncRNA（注释）在抗性中差异表达， 而不是敏感细胞。这些lncRNA中约50%具有m6 A基序，并且在mRNA水平上具有改变的m6 A量。 耐药细胞，总的来说，表明一个独特的lncRNA签名，是特异性的TKI耐药， 受m6 A甲基化调节; iii）在不应答的患者中这些m6 A相关lncRNA的上调 TKIs是更坏的结果的预测，并且它们的敲低损害抗性细胞生长， 对TKI敏感的抗性细胞; iv）与敏感细胞相比，TKI抗性细胞高度表达LSC标志物 （CD 117、CD 44、CD 25、CD 133），其上调与m6 A减少相关。我们的假设是， FTO-m6 A-lncRNA级联可能是控制LSC对TKI持续性的关键途径， 目标是根除持久性LSC，提高TKI治愈率。我们将通过三个目标来检验我们的假设：1） 确定FTO-m6 A轴如何调节TKI抗性中的lncRNA畸变; 2）确定是否和 动态m6 A甲基化组如何调节LSC对TKI的持久性; 3）确定是否以及如何 FTO-m6 A-lncRNA级联的药理学靶向使用临床前白血病杀死持续性LSC 模型拟议的研究在概念上是创新的，因为它针对一种新的途径（FTO-m6 A- lncRNA级联），以了解LSC对TKI的持久性，并开发消除TKI的新方案 抗性LSC。拟议的研究是重要的，因为研究结果将a）确定新的途径（即， FTO-m6 A-lncRNA级联）调节LSC持久性，加深了对LSC的分子理解， lncRNA功能; B）开发新的方法（靶向FTO-m6 A-lncRNA级联）以消除持续的 LSC改善难治性白血病患者的管理。

Tyrosine kinase targeted (TKI) therapies have revolutionized leukemia treatment, but TKIs are not able to kill leukemia stem cells (LSCs), which are responsible for propagating and disease recurrence, and believed to be the source of treatment failure. Our long-term goals are to further address how LSC persistence is regulated, and to develop new LSC-eliminating treatment strategies to improve cure rates and survival. The short-term goals of this research are to determine whether a N6-methyladenosine (m6A)--long non-coding RNA (lncRNA) axis regulates LSC stemness and persistence during TKI selection process, and to explore the therapeutic potential of targeting the m6A-lncRNA axis for eradicating TKI resistant LSCs and also decipher the underlying molecular mechanisms. The m6A methylation is the most common epitranscriptomic modification on RNAs (i.e., lncRNAs), and crucially regulates lncRNA-initiated gene expression. lncRNA abnormalities frequently associate with cancer disease progression and drug resistance. The preliminary evidence linking an m6A-lncRNA axis to resistant LSCs is from our proof of principle studies demonstrating that i) a dynamic and reversible m6A methylome determined by fat mass and obesity-associated protein (FTO) helps leukemia cells avoid TKI killing leading to TKI resistance; ii) there are many lncRNAs (annotated) that are differentially expressed in resistant versus sensitive cells. About 50% of these lncRNAs bear m6A motifs and have the changed m6A amounts in resistant cells, collectively, suggesting a unique lncRNA signature that is specific to TKI resistance and is regulated by m6A methylation; iii) upregulation of these m6A-associated lncRNAs in patients who do not respond to TKIs is predicative of worse outcomes, and knockdown of them impairs resistant cell growth and renders resistant cells sensitive to TKIs; iv) compared to sensitive ones, TKI-resistant cells highly express LSC markers (CD117, CD44, CD25, CD133) whose upregulation is associated with m6A reduction. Our hypothesis is that the FTO-m6A-lncRNA cascade may be a critical pathway to control LSC persistence to TKIs and a new druggable target to eradicate persistent LSCs improving TKI cure rates. We will test our hypothesis through three aims: 1) Determine how the FTO-m6A axis regulates lncRNA aberrations in TKI resistance; 2) Determine whether and how a dynamic m6A methylome regulates LSC persistence to TKIs; 3) Determine whether and how pharmacological targeting of the FTO-m6A-lncRNA cascade kills persistent LSCs using preclinical leukemia models. The proposed studies are conceptually innovative, because it targets a new pathway (the FTO-m6A- lncRNA cascade) in understanding LSC persistence to TKIs and in developing new regimens to eliminate TKI resistant LSCs. The proposed research is significant, because the findings will a) identify new pathways (i.e., FTO-m6A-lncRNA cascade) that regulate LSC persistence, deepening the molecular understanding of LSCs and lncRNA functions; b) develop new approaches (targeting the FTO-m6A-lncRNA cascade) to eliminate persistent LSCs improving the management of patients with refractory leukemia.