Anti-MLLT10 (aa 965-1027) polyclonal antibody

Background: 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. Methods: qRT-PCR, receiver operating characteristic (ROC), and leukemia-free survival analysis were used to validate LAMP5-AS1 (LAMP5antisense 1) expression and evaluate its clinical value. We performed in vitro and in vivo experiments to investigate the functional relevance of LAMP5-AS1 inMLLleukemia 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. Results: 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 theHOXAcluster, which are responsible for leukemia stemness in context ofMLLrearrangements. We found that LAMP5-AS1 is specifically overexpressed inMLLleukemia patients (n= 58) than that in theMLL-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 primaryMLLleukemia 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. Conclusions: This is the first study that a lncRNA regulates the self-renewal program and differentiation block inMLLleukemia cells by facilitating the methyltransferase activity of DOT1L and global H3K79 methylation, showing its potential as a therapeutic target forMLLleukemia.

Mixed lineage leukemia (MLL/KMT2A) rearrangements MLL-r) are one of the most frequent chromosomal aberrations in acute myeloid leukemia. We evaluated the function of Meningioma 1 (MN1), a co-factor of HOXA9 and MEIS1, in human and murine MLL-rearranged leukemia by CRISPR-Cas9 mediated deletion of MN1. MN1 was required for in vivo leukemogenicity of MLL positive murine and human leukemia cells. Loss of MN1 inhibited cell cycle and proliferation, promoted apoptosis and induced differentiation of MLL-rearranged cells. Expression analysis and chromatin immunoprecipitation with sequencing from previously reported data sets demonstrated that MN1 primarily maintains active transcription of HOXA9 and HOXA10, which are critical downstream genes of MLL, and their target genes like BCL2, MCL1 and Survivin. Treatment of MLL-rearranged primary leukemia cells with antiMN1 siRNA significantly reduced their clonogenic potential in contrast to normal CD34 - hematopoietic progenitor cells, suggesting a therapeutic window for MN1 targeting. In summary, our findings demonstrate that MN1 plays an essential role in MLL fusion leukemias and serve as a therapeutic target in MLL-rearranged acute myeloid leukemia.