MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou, 510060, Guangdong, China.
J Hematol Oncol. 2020 Jun 17;13(1):78. doi: 10.1186/s13045-020-00909-y.
Mixed-lineage leukemia (MLL) gene rearrangements trigger aberrant epigenetic modification and gene expression in hematopoietic stem and progenitor cells, which generates one of the most aggressive subtypes of leukemia with an apex self-renewal. It remains a challenge to directly inhibit rearranged MLL itself because of its multiple fusion partners and the poorly annotated downstream genes of MLL fusion proteins; therefore, novel therapeutic targets are urgently needed.
qRT-PCR, receiver operating characteristic (ROC), and leukemia-free survival analysis were used to validate LAMP5-AS1 (LAMP5 antisense 1) expression and evaluate its clinical value. We performed in vitro and in vivo experiments to investigate the functional relevance of LAMP5-AS1 in MLL leukemia progression and leukemia cell stemness. RNA electrophoretic mobility shift assays (EMSA), histone methyltransferase assay, RNA pull-down assay, and RNA fluorescence in situ hybridization (FISH) were used to validate the relationship between LAMP5-AS1 and the methyltransferase activity of DOT1L. The downstream ectopic target genes of LAMP5-AS1/DOT1L were validated by the chromatin immunoprecipitation (ChIP) and western blot.
We discovered that a long noncoding RNA (lncRNA) LAMP5-AS1 can promote higher degrees of H3K79 methylation, followed by upregulated expression of the self-renewal genes in the HOXA cluster, which are responsible for leukemia stemness in context of MLL rearrangements. We found that LAMP5-AS1 is specifically overexpressed in MLL leukemia patients (n = 58) than that in the MLL-wt leukemia (n = 163) (p < 0.001), and the patients with a higher expression level of LAMP5-AS1 exhibited a reduced 5-year leukemia-free survival (p < 0.01). LAMP5-AS1 suppression significantly reduced colony formation and increased differentiation of primary MLL leukemia CD34+ cells. Mechanistically, LAMP5-AS1 facilitated the methyltransferase activity of DOT1L by directly binding its Lys-rich region of catalytic domain, thus promoting the global patterns of H3K79 dimethylation and trimethylation in cells. These observations supported that LAMP5-AS1 upregulated H3K79me2/me3 and the transcription of DOT1L ectopic target genes.
This is the first study that a lncRNA regulates the self-renewal program and differentiation block in MLL leukemia cells by facilitating the methyltransferase activity of DOT1L and global H3K79 methylation, showing its potential as a therapeutic target for MLL leukemia.
混合谱系白血病（MLL）基因重排在造血干祖细胞中触发异常的表观遗传修饰和基因表达，产生最具侵袭性的白血病亚型之一，具有顶端自我更新能力。由于其多个融合伙伴和 MLL 融合蛋白下游基因注释不佳，直接抑制重排的 MLL 本身仍然是一个挑战；因此，迫切需要新的治疗靶点。
使用 qRT-PCR、接收者操作特征（ROC）和无白血病生存分析来验证 LAMP5-AS1（LAMP5 反义 1）的表达并评估其临床价值。我们进行了体外和体内实验，以研究 LAMP5-AS1 在 MLL 白血病进展和白血病细胞干性中的功能相关性。RNA 电泳迁移率变动分析（EMSA）、组蛋白甲基转移酶测定、RNA 下拉测定和 RNA 荧光原位杂交（FISH）用于验证 LAMP5-AS1 与 DOT1L 甲基转移酶活性之间的关系。LAMP5-AS1/DOT1L 的下游异位靶基因通过染色质免疫沉淀（ChIP）和 Western blot 验证。
我们发现，一种长非编码 RNA（lncRNA）LAMP5-AS1 可以促进更高程度的 H3K79 甲基化，随后上调 HOXA 簇中自我更新基因的表达，这在 MLL 重排的情况下负责白血病干性。我们发现 LAMP5-AS1 在 MLL 白血病患者（n=58）中特异性过表达，而在 MLL-wt 白血病患者（n=163）中过表达（p<0.001），并且 LAMP5-AS1 表达水平较高的患者白血病无复发生存率降低（p<0.01）。LAMP5-AS1 抑制显著降低了原代 MLL 白血病 CD34+细胞的集落形成并增加了分化。从机制上讲，LAMP5-AS1 通过直接结合其催化结构域的富含赖氨酸区域，促进细胞中 H3K79 二甲基化和三甲基化的整体模式，从而促进 DOT1L 的甲基转移酶活性。这些观察结果表明，LAMP5-AS1 上调了 H3K79me2/me3 和 DOT1L 异位靶基因的转录。
这是第一项研究表明，一种长非编码 RNA 通过促进 DOT1L 的甲基转移酶活性和全局 H3K79 甲基化来调节 MLL 白血病细胞中的自我更新程序和分化阻滞，表明其作为 MLL 白血病治疗靶点的潜力。
