The Role of Hottip Lncrna m6a Modification in AML Genome Organization and Leukemogenesis

Abstract

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with the dysregulation of HOX genes. MLLr+ AML is characterized by aberrant expression of HOX genes and the HOX cofactor MEIS1. HOTTIP, a HOXA-associated long non-coding RNA (lncRNA), is significantly up-regulated in MLLr+ AML. Aberrant overexpression of Hottip dramatically perturbs hematopoietic stem cell (HSC) self-renewal leading to mouse AML-like disease through maintaining a leukemic profiling. Recent studies show that N6-methyladenosine (m6A), as the most prevalent internal modification in eukaryotic coding and non-coding RNAs, plays important roles in normal and malignant hematopoiesis. There are several potential m6A modified sites in HOTTIP exon regions and 3'UTR region by re-analyzing m6A-methylated RNA immunoprecipitation sequencing (m6A-meRIP-seq) data in leukemia. However, it is still unclear whether HOTTIP m6A modification plays a critical role in AML genome organization and AML leukemogenesis. Our HOTTIP ChIRP-MASS Spectrometry (ChIRP-MS) data shows that HOTTIP directly interacts with m6A methyltransferase METTL3/METTL14 and CTCF/cohesin chromatin organization proteins in MOLM13 AML cells. To investigate whether HOTTIP lncRNA is methylated by METTL3, we perform m6A-eCLIP-seq and METTL3-eCLIP-seq in MOLM13 cells. Our data indicates that there are several m6A modified sites co-bound with METTL3 in HOTTIP exon and 3'UTR regions. Our m6A-eCLIP-qPCR data further confirms that depletion of METTL3 significantly decreases HOTTIP m6A modification levels at HOTTIP RNA m6A core motif GGACU regions (g1, g2 and g3) by comparing shScramble and shMETTL3 cells. It suggests that HOTTIP lncRNA contains specific m6A modified sites methylated by METTL3. Next, to further examine whether and which HOTTIP m6A sites is required for the interaction of HOTTIP RNA with CTCF protein, we have performed in vitro RNA methylation and RNA binding protein assay by synthesizing biotinylated HOTTIP RNA fragments in g1, g2 and g3 regions. Subsequently, these biotin-HOTTIP-g1/g2/g3 RNAs are methylated with METTL3/METTL14 recombinant proteins and then incubated with GST control or GST-CTCF fusion in vitro. Our data shows that m6A modification of HOTTIP-g2 or HOTTIP-g3 RNA fragment enhances its interaction with GST-CTCF protein, suggesting that HOTTIP m6A modification plays a critical role in coordinating with CTCF-mediated genome organization. To investigate the function of HOTTIP m6A modification in AML leukemogenesis, we have generated CRISPR-dCas13-ALKBH5 (dALK) system to specifically demethylate HOTTIP m6A sites (g1, g2 and g3) by co-transfecting gRNA vector and the catalytically inactive Cas13 (dCas13) fused with m6A demethylase AlkB homolog 5 (ALKBH5) vector. Targeting dALK to specific m6A site in HOTTIP lncRNA results in site-specific loss of m6A modification. Although loss of HOTTIP m6A modification never affects the stability and transcription of HOTTIP in dALK clones, loss of HOTTIP m6A modification significantly affects the transcription of leukemic signature genes HOXA9/13 and WNT target (β-catenin and MYC etc) in dALK-g2 or dALK-g3 cells. Additionally, our HOTTIP ChIRP-qPCR shows that loss of HOTTIP m6A modification affects HOTTIP binding at WNT target loci (β-catenin and MYC loci) in dALK-g2 or dALK-g3 cells. Critically, our data find that loss of HOTTIP m6A modification significantly impairs cell proliferation and cell cycle in dALK-g2 or dALK-g3 cells compared with ctrl cells. To further elucidate whether HOTTIP m6A modification is required for AML leukemogenesis in vivo, we xenograft ctrl and dALK clones into NOD-scid IL2Rgammanull (NSG) mice. Our data indicates that mice transplanted with ctrl or dALK-g1 cells dies around 40 days after transplantation, while those receiving dALK-g2 or dALK-g3 cells survives significantly longer up to 55 days. Critically, we also find that ctrl recipient mice have significantly higher human CD45+ cell chimerism in the BM than mice receiving dALK-g2 or dALK-g3 AML cells. Thus, loss of the specific HOTTIP m6A modification site reduces the AML leukemic burden in vivo. In summary, this research will provide a prospective insight for the role of HOTTIP-m6A-CTCT axis in AML genome organization and leukemogenesis.