PDF(Pubmed)
Abstract：
BACKGROUND: Mixed-lineage leukemia (MLL) fusion gene caused by chromosomal rearrangement is a dominant oncogenic driver in leukemia. Due to having diverse MLL rearrangements and complex characteristics, MLL leukemia treated by currently available strategies is frequently associated with a poor outcome. Therefore, there is an urgent need to identify novel therapeutic targets for hematological malignancies with MLL rearrangements.
METHODS: qRT-PCR, western blot, and spearman correction analysis were used to validate the regulation of LAMP5-AS1 on LAMP5 expression. In vitro and in vivo experiments were conducted to assess the functional relevance of LAMP5-AS1 in MLL leukemia cell survival. We utilized chromatin isolation by RNA purification (ChIRP) assay, RNA pull-down assay, chromatin immunoprecipitation (ChIP), RNA fluorescence in situ hybridization (FISH), and immunofluorescence to elucidate the relationship among LAMP5-AS1, DOT1L, and the LAMP5 locus. Autophagy regulation by LAMP5-AS1 was evaluated through LC3B puncta, autolysosome observation via transmission electron microscopy (TEM), and mRFP-GFP-LC3 puncta in autophagic flux.
RESULTS: The study shows the crucial role of LAMP5-AS1 in promoting MLL leukemia cell survival. LAMP5-AS1 acts as a novel autophagic suppressor, safeguarding MLL fusion proteins from autophagic degradation. Knocking down LAMP5-AS1 significantly induced apoptosis in MLL leukemia cell lines and primary cells and extended the survival of mice in vivo. Mechanistically, LAMP5-AS1 recruits the H3K79 histone methyltransferase DOT1L to LAMP5 locus, directly activating LAMP5 expression. Importantly, blockade of LAMP5-AS1-LAMP5 axis can represses MLL fusion proteins by enhancing their degradation.
CONCLUSIONS: The findings underscore the significance of LAMP5-AS1 in MLL leukemia progression through the regulation of the autophagy pathway. Additionally, this study unveils the novel lncRNA-DOT1L-LAMP5 axis as promising therapeutic targets for degrading MLL fusion proteins.
METHODS: qRT-PCR, western blot, and spearman correction analysis were used to validate the regulation of LAMP5-AS1 on LAMP5 expression. In vitro and in vivo experiments were conducted to assess the functional relevance of LAMP5-AS1 in MLL leukemia cell survival. We utilized chromatin isolation by RNA purification (ChIRP) assay, RNA pull-down assay, chromatin immunoprecipitation (ChIP), RNA fluorescence in situ hybridization (FISH), and immunofluorescence to elucidate the relationship among LAMP5-AS1, DOT1L, and the LAMP5 locus. Autophagy regulation by LAMP5-AS1 was evaluated through LC3B puncta, autolysosome observation via transmission electron microscopy (TEM), and mRFP-GFP-LC3 puncta in autophagic flux.
RESULTS: The study shows the crucial role of LAMP5-AS1 in promoting MLL leukemia cell survival. LAMP5-AS1 acts as a novel autophagic suppressor, safeguarding MLL fusion proteins from autophagic degradation. Knocking down LAMP5-AS1 significantly induced apoptosis in MLL leukemia cell lines and primary cells and extended the survival of mice in vivo. Mechanistically, LAMP5-AS1 recruits the H3K79 histone methyltransferase DOT1L to LAMP5 locus, directly activating LAMP5 expression. Importantly, blockade of LAMP5-AS1-LAMP5 axis can represses MLL fusion proteins by enhancing their degradation.
CONCLUSIONS: The findings underscore the significance of LAMP5-AS1 in MLL leukemia progression through the regulation of the autophagy pathway. Additionally, this study unveils the novel lncRNA-DOT1L-LAMP5 axis as promising therapeutic targets for degrading MLL fusion proteins.
摘要：
背景：由染色体重排引起的混合谱系白血病（MLL）融合基因是白血病的主要致癌驱动因素。由于具有多样化的MLL重排和复杂的特征，通过目前可用的策略治疗的MLL白血病通常与不良结果相关。因此,迫切需要确定具有MLL重排的血液恶性肿瘤的新治疗靶标。
方法：qRT-PCR，westernblot,和spearman校正分析用于验证LAMP5-AS1对LAMP5表达的调节。进行了体外和体内实验以评估LAMP5-AS1在MLL白血病细胞存活中的功能相关性。我们通过RNA纯化（ChIRP）测定法利用染色质分离，RNA下拉法,染色质免疫沉淀（ChIP），RNA荧光原位杂交(FISH),和免疫荧光来阐明LAMP5-AS1、DOT1L、和LAMP5基因座。通过LC3Bpuncta评估LAMP5-AS1的自噬调节，通过透射电子显微镜（TEM）观察自溶体，和自噬通量中的mRFP-GFP-LC3斑点。
结果：该研究表明LAMP5-AS1在促进MLL白血病细胞存活中的关键作用。LAMP5-AS1作为一种新型的自噬抑制因子，保护MLL融合蛋白免受自噬降解。敲除LAMP5-AS1可显著诱导MLL白血病细胞系和原代细胞凋亡，延长小鼠体内生存期。机械上,LAMP5-AS1将H3K79组蛋白甲基转移酶DOT1L招募到LAMP5基因座，直接激活LAMP5表达。重要的是,阻断LAMP5-AS1-LAMP5轴可以通过增强MLL融合蛋白的降解来抑制它们。
结论：这些发现强调了LAMP5-AS1通过调节自噬途径在MLL白血病进展中的重要性。此外,这项研究揭示了新的lncRNA-DOT1L-LAMP5轴作为降解MLL融合蛋白的有希望的治疗靶标。
方法：qRT-PCR，westernblot,和spearman校正分析用于验证LAMP5-AS1对LAMP5表达的调节。进行了体外和体内实验以评估LAMP5-AS1在MLL白血病细胞存活中的功能相关性。我们通过RNA纯化（ChIRP）测定法利用染色质分离，RNA下拉法,染色质免疫沉淀（ChIP），RNA荧光原位杂交(FISH),和免疫荧光来阐明LAMP5-AS1、DOT1L、和LAMP5基因座。通过LC3Bpuncta评估LAMP5-AS1的自噬调节，通过透射电子显微镜（TEM）观察自溶体，和自噬通量中的mRFP-GFP-LC3斑点。
结果：该研究表明LAMP5-AS1在促进MLL白血病细胞存活中的关键作用。LAMP5-AS1作为一种新型的自噬抑制因子，保护MLL融合蛋白免受自噬降解。敲除LAMP5-AS1可显著诱导MLL白血病细胞系和原代细胞凋亡，延长小鼠体内生存期。机械上,LAMP5-AS1将H3K79组蛋白甲基转移酶DOT1L招募到LAMP5基因座，直接激活LAMP5表达。重要的是,阻断LAMP5-AS1-LAMP5轴可以通过增强MLL融合蛋白的降解来抑制它们。
结论：这些发现强调了LAMP5-AS1通过调节自噬途径在MLL白血病进展中的重要性。此外,这项研究揭示了新的lncRNA-DOT1L-LAMP5轴作为降解MLL融合蛋白的有希望的治疗靶标。
